JP2011041547A - New bacillus subtilis strain and method for using the same - Google Patents
New bacillus subtilis strain and method for using the same Download PDFInfo
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本発明は、雑味が少なく、風味が良好で、かつ、製造後の経時的な風味の劣化が少ない納豆等の発酵食品を製造可能な新規枯草菌株や、その利用に関するものである。 The present invention relates to a novel Bacillus subtilis strain capable of producing fermented foods such as natto with little miscellaneous taste, good flavor, and little deterioration in flavor over time after production, and use thereof.
枯草菌バシラス・サチリス（Bacillus subtilis）の一種である納豆菌は、様々な用途に用いられ、例えば納豆の製造（特許文献１及び２参照）のほか、家畜の飼料や植物の肥料の製造（特許文献３及び４参照）にも用いられる。特に納豆は、納豆菌による整腸効果や、ナットウキナーゼによる血栓予防効果や、サポニンやレシチンやリノール酸による動脈硬化予防効果や、ビタミンＫ２による骨粗鬆症・骨の老化防止効果等があると考えられているため、近年高まっている健康志向を背景に、消費者から引き続き高い注目を集めている。 Bacillus subtilis, Bacillus subtilis, is used for various purposes. For example, in addition to the production of natto (see Patent Documents 1 and 2), the production of livestock feed and plant fertilizer (patent) (Ref. 3 and 4). In particular, natto is considered to have an intestinal effect by natto bacteria, a thrombus prevention effect by nattokinase, an arteriosclerosis prevention effect by saponin, lecithin and linoleic acid, and an osteoporosis / bone aging prevention effect by vitamin K 2. Therefore, it continues to attract high attention from consumers against the background of increasing health consciousness in recent years.
納豆は、蒸煮大豆に納豆菌を接種し、それを発酵室の中で所定時間発酵、熟成させて製造されるものである。納豆の製造過程では、蒸煮大豆に含まれる糖、アミノ酸、粘質物、香り等の成分が複雑に変化していく。また、納豆製造後の保存時にも、前述の成分はさらに変化していくため、納豆の成分の変化の程度に基づいて、納豆の賞味期限が設定されている。 Natto is produced by inoculating steamed soybeans with natto bacteria and fermenting and aging them in a fermentation chamber for a predetermined time. In the natto manufacturing process, components such as sugar, amino acids, mucilage, and aroma contained in steamed soybeans change in a complex manner. Moreover, since the above-mentioned components further change during storage after natto production, the expiration date for natto is set based on the degree of change in the components of natto.
また、納豆の風味、成分、物性等は、用いる納豆菌の種類によっても影響を受ける。そのため、従来の市販納豆菌株（たとえば、宮城野菌、成瀬菌、高橋菌等）以外の新たな納豆菌株の分離やその利用が試みられている。例えば、新規な納豆菌を用いて製造した、臭いの少ない納豆（特許文献５参照）、柔らかい納豆（特許文献１及び２参照）、免疫調節作用納豆（特許文献６参照）が知られている。 The flavor, ingredients, physical properties, etc. of natto are also affected by the type of natto bacteria used. For this reason, attempts have been made to isolate and use new natto strains other than conventional commercially available natto strains (eg, Miyagino, Naruse, Takahashi, etc.). For example, natto with low odor (see Patent Document 5), soft natto (see Patent Documents 1 and 2), and immunomodulating natto (see Patent Document 6) manufactured using a novel natto bacterium are known.
そしてある種の納豆や納豆菌を、特定保健用食品や医薬品（特に整腸剤）として利用することが行なわれている。 Some types of natto and natto bacteria are used as foods for specified health use and pharmaceuticals (especially intestinal preparations).
本発明の課題は、雑味が少なく、風味が良好で、かつ、製造後の経時的な風味の劣化が少ない納豆等の発酵食品を製造可能な新規枯草菌株や、かかる枯草菌株を含有する発酵食品製造用スターターや、かかるスターターを用いた発酵食品の製造方法や、かかる製造方法により製造した発酵食品等を提供することにある。 An object of the present invention is to provide a novel Bacillus subtilis strain capable of producing fermented foods such as natto with little miscellaneous taste, good flavor, and little deterioration in flavor over time after production, and fermentation containing such Bacillus strain. The object is to provide a starter for food production, a method for producing fermented food using such a starter, a fermented food produced by such a production method, and the like.
本発明者らは、上記課題を解決するために適したバシラス・サチリス菌株を取得するべく、栃木県、東京都、千葉県、茨城県、富山県、京都府及び三重県にて採取した土壌や稲わら等合計55点の試料からスクリーニングを行った。その結果、栃木県の稲わらの試料より分離したバシラス・サチリス菌株の中に、上記課題を解決するのに適した菌株（TFC 4102株と命名。）が存在することを見い出した。すなわち、かかるTFC 4102株を利用して製造した納豆は、雑味が少なく、風味が良好で、かつ、製造後の経時的な風味の劣化が少ないことを見い出し、本発明を完成した。 In order to obtain a Bacillus subtilis strain suitable for solving the above problems, the present inventors have collected soils collected in Tochigi Prefecture, Tokyo, Chiba Prefecture, Ibaraki Prefecture, Toyama Prefecture, Kyoto Prefecture, and Mie Prefecture. Screening was conducted from a total of 55 samples such as rice straw. As a result, we found that a Bacillus subtilis strain (named TFC 4102 strain) suitable for solving the above problems exists among the Bacillus subtilis strains isolated from the rice straw samples of Tochigi Prefecture. That is, it was found that natto produced using such TFC 4102 strain has less miscellaneous taste, good flavor, and little deterioration in flavor over time after production, and thus completed the present invention.
すなわち、本発明は、（１）枯草菌バシラス・サチリス（Bacillus subtilis）TFC 4102株（ＮＩＴＥ Ｐ−７１２）又はその変異株や、（２）上記（１）に記載の枯草菌株を含有することを特徴とする発酵食品製造用スターターや、（３）発酵食品が納豆であることを特徴とする上記（２）に記載の発酵食品製造用スターターに関するものである。 That is, the present invention includes (1) a Bacillus subtilis TFC 4102 strain (NITE P-712) or a mutant strain thereof, and (2) a Bacillus strain described in (1) above. The present invention relates to a starter for producing a fermented food, or (3) a starter for producing a fermented food according to (2) above, wherein the fermented food is natto.
また、本発明は、（４）上記（２）又は（３）に記載の発酵食品製造用スターターによって、発酵し得る食材を発酵させることを特徴とする発酵食品の製造方法や、（５）発酵し得る食材が大豆であり、発酵食品が納豆であることを特徴とする上記（４）に記載の発酵食品の製造方法や、（６）上記（４）又は（５）に記載の製造方法により製造した発酵食品や、（７）上記（６）に記載の発酵食品にさらに加工を加えて製造した加工食品に関するものである。 The present invention also provides (4) a method for producing a fermented food, characterized in that the fermentable food is fermented by the fermented food production starter described in (2) or (3) above, and (5) fermentation. The method for producing fermented food according to (4) above, wherein (6) the method for producing according to (4) or (5) above is characterized in that the food that can be used is soybean and the fermented food is natto. The present invention relates to a fermented food produced and (7) a processed food produced by further processing the fermented food described in (6) above.
本発明の枯草菌株（バシラス・サチリス TFC 4102株）やその変異株によれば、雑味が少なく、風味が良好で、かつ、製造後の経時的な風味の劣化が少ない納豆等の発酵食品を製造することができる。また、本発明の枯草菌株は、前述のような性質を有する納豆等の発酵食品を製造するためのスターターなどとして好適に用いることができる。 According to the Bacillus subtilis strain of the present invention (Bacillus subtilis TFC 4102 strain) and its mutant strains, fermented foods such as natto, which have less miscellaneous taste, good flavor, and little flavor deterioration over time after production. Can be manufactured. Moreover, the Bacillus subtilis strain of the present invention can be suitably used as a starter for producing fermented foods such as natto having the properties described above.
本発明における枯草菌バシラス・サチリス TFC 4102株は、本発明者らが栃木県にて採取した稲わらから分離したものであり、かかる菌株は、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（〒２９２−０８１８ 千葉県木更津市かずさ鎌足２−５−８）に受託番号ＮＩＴＥ Ｐ−７１２として寄託されている。本発明の枯草菌株としては、上記のTFC 4102株の他、雑味が少なく、風味が良好で、かつ、製造後の経時的な風味の劣化が少ない納豆を製造し得る限り、TFC 4102株の変異株も含まれる。以下、TFC 4102株と、かかる変異株を併せて、単に「本発明の枯草菌株」とも表示する。 The Bacillus subtilis TFC 4102 strain in the present invention was isolated from rice straw collected by the present inventors in Tochigi Prefecture. Deposited as NITE P-712 at Kazusa Kamashichi 2-5-8, Kisarazu City, Chiba Prefecture 292-0818. As the Bacillus subtilis strain of the present invention, in addition to the above-mentioned TFC 4102 strain, as long as it can produce natto with less miscellaneous taste, good flavor, and little deterioration in flavor over time after production, TFC 4102 strain Mutants are also included. Hereinafter, the TFC 4102 strain and such mutant strains are also simply referred to as “the Bacillus subtilis strain of the present invention”.
上記のTFC 4102株の変異株としては、TFC 4102株を変異処理して得られる枯草菌株であって、雑味が少なく、風味が良好で、かつ、製造後の経時的な風味の劣化が少ない納豆を製造し得る枯草菌株である限り特に制限されない。ある枯草菌株が、雑味が少なく、風味が良好で、かつ、製造後の経時的な風味の劣化が少ない納豆を製造し得るかどうかは、その枯草菌株を用いて製造した納豆が、宮城野菌（宮城野納豆製造所販売）の菌液と成瀬菌（成瀬発酵化学研究所販売）の菌液を体積比で１：９（＝宮城野菌：成瀬菌）で混合した菌液を用いて製造した納豆と比較して、雑味が少なく、風味が良好で、かつ、製造後の経時的な風味の劣化が少ないかどうかを調べることによって確認することができ、より具体的には、後述の実施例４の「納豆の製造、及び、製造した納豆の官能評価」と同様の実験において、上記の宮城野菌と成瀬菌の混合菌液を用いて製造した納豆と比較して、雑味が少なく、風味が良好で、かつ、製造後の経時的な風味の劣化が少ないかどうかを調べることによって確認することができる。 As a mutant of the above TFC 4102 strain, it is a Bacillus subtilis strain obtained by mutating the TFC 4102 strain, which has less miscellaneous taste, good flavor, and little deterioration in flavor over time after production. There is no particular limitation as long as it is a Bacillus subtilis strain that can produce natto. Whether a certain Bacillus subtilis strain can produce natto with little miscellaneous taste, good flavor, and little deterioration in flavor over time after production, natto produced using the Bacillus subtilis strain is Natto manufactured by using a bacterial solution in which the bacterial solution of Miyagino Natto Factory is mixed with the bacterial solution of Naruse Bacteria (sales of Naruse Fermentation Chemistry Laboratories) at a volume ratio of 1: 9 (= Miyagino fungus: Naruse fungus). Compared to the above, it can be confirmed by examining whether there is little miscellaneous taste, good flavor, and little deterioration in flavor over time after production, more specifically, Examples described later In the same experiment as in “Manufacture of natto and sensory evaluation of manufactured natto” in No. 4, there is less miscellaneous taste and flavor compared to natto manufactured using the above mixed bacterial solution of Miyagino and Naruse. Is good and there is little deterioration in flavor over time after production. It can be confirmed by the bell.
本明細書における風味とは、旨味、後味、納豆として違和感のある異味、苦味、甘味、香りを意味し、より具体的には、旨味の強弱、旨味の質の良悪、異味の強弱、苦味の強弱、甘味の強弱、香りの強弱、香りの質の良悪を意味する。また、本明細書における雑味とは、上記の風味に関連して、質の悪い後味、異味、苦味を意味する。 The flavor in the present specification means an unpleasant taste, bitterness, sweetness and aroma as umami, aftertaste and natto, and more specifically, umami strength, quality of umami, quality of taste, bitterness, bitterness. It means the intensity of sweetness, the strength of sweetness, the strength of fragrance, and the quality of fragrance. The miscellaneous taste in the present specification means poor aftertaste, off-taste and bitterness in relation to the above-mentioned flavor.
本発明の枯草菌株は、上述のような納豆等の発酵食品を製造し得るため、納豆等の発酵食品製造用スターター（以下、単に「本発明のスターター」とも表示する。）として用いることができる。かかる本発明のスターターとしては、本発明の枯草菌株それ自体又は本発明の枯草菌株を含有する組成物である限り特に制限されないが、本発明の枯草菌株の他に適当な固体担体や液体担体、本発明の枯草菌株の品質保持を目的とする物質をさらに含有していてもよい。 Since the Bacillus subtilis strain of the present invention can produce fermented foods such as natto as described above, it can be used as a starter for producing fermented foods such as natto (hereinafter also simply referred to as “starter of the present invention”). . The starter of the present invention is not particularly limited as long as it is the Bacillus subtilis strain of the present invention itself or a composition containing the Bacillus subtilis strain of the present invention. In addition to the Bacillus subtilis strain of the present invention, a suitable solid carrier or liquid carrier, A substance intended to maintain the quality of the Bacillus subtilis strain of the present invention may further be contained.
本明細書における「発酵食品」とは、発酵し得る食材（以下、「本発明における食材」とも表示する。）を本発明のスターターによって発酵させた食品を意味する。発酵し得る食材としては、黄大豆、黒大豆、小豆、インゲン豆、グリーンピース等のマメ科植物の種子の他、トウモロコシ、米、玄米等のイネ科植物の種子や、銀杏の種子、チーズ等を好適に例示することができ、中でも、マメ科植物の種子をより好適に例示することができ、さらに中でも黄大豆、黒大豆を最も好適に例示することができる。なお、本発明における食材が黄大豆や黒大豆である場合の上記の発酵食品は、納豆と呼ばれる。 The “fermented food” in the present specification means a food obtained by fermenting a fermentable food (hereinafter also referred to as “food in the present invention”) with the starter of the present invention. Fermentable ingredients include seeds of legumes such as yellow soybeans, black soybeans, red beans, kidney beans, green peas, seeds of gramineous plants such as corn, rice, brown rice, ginko seeds, cheese, etc. Among them, legume seeds can be more preferably exemplified, and yellow soybeans and black soybeans can be most preferably exemplified. In addition, said fermented food in case the foodstuff in this invention is yellow soybean or black soybean is called natto.
本発明のスターターは、発酵食品の製造に用いることができる。本発明における発酵食品の製造方法（以下、単に「本発明の製造方法」とも表示する。）としては、本発明のスターター及び本発明における食材を用いること以外は、納豆の製法として通常用いられる方法に従って行なうことができ、本発明のスターターによって、本発明における食材を発酵させる工程を含んでいる限り特に制限されないが、本発明における食材を蒸煮する工程（Ａ）、及び、蒸煮した本発明における食材に本発明のスターターを接種し、発酵させる工程（Ｂ）を含んでいることが好ましい。 The starter of this invention can be used for manufacture of fermented food. As a method for producing fermented food according to the present invention (hereinafter also simply referred to as “the method for producing the present invention”), a method usually used as a method for producing natto, except that the starter of the present invention and the food according to the present invention are used. Is not particularly limited as long as it includes the step of fermenting the foodstuff according to the present invention by the starter of the present invention, but the step (A) of steaming the foodstuff according to the present invention and the steamed foodstuff according to the present invention It is preferable to include the step (B) of inoculating and fermenting the starter of the present invention.
上記の工程（Ａ）としては、本発明における食材を蒸煮する工程である限り特に制限はされないが、水又は湯に浸漬した本発明における食材を水切りした後、それを適当な条件で蒸煮する工程を好適に例示することができる。蒸煮の適当な条件としては、120〜135℃の範囲内の温度、及び、1.0〜2.0kg／cm2の範囲内の圧力が保持されるように蒸煮釜内に加圧蒸気を吹き込み続け、通常15〜60分間、好ましくは20〜40分間維持する条件を好適に例示することができる。 The step (A) is not particularly limited as long as it is a step of cooking the food in the present invention, but after draining the food in the present invention soaked in water or hot water, steaming it under appropriate conditions Can be preferably exemplified. Appropriate conditions for steaming are to keep blowing steam into the steamer so that a temperature in the range of 120 to 135 ° C and a pressure in the range of 1.0 to 2.0 kg / cm 2 are maintained. The conditions of maintaining for 15 to 60 minutes, preferably 20 to 40 minutes can be suitably exemplified.
上記の工程（Ｂ）としては、蒸煮した本発明における食材に本発明のスターターを接種し、発酵させる工程である限り特に制限はされないが、蒸煮した本発明における食材に本発明のスターターを適量接種し、適当な条件下で発酵させる工程を好適に例示することができる。本発明のスターターの接種量としては、蒸煮して得た本発明における食材1ｇあたりの本発明の菌株の胞子数が1.0×103個〜1.0×105個となるような接種量を好適に例示することができ、また、発酵の条件としては、酸素存在下、室温37〜48℃で高湿度の条件を好適に例示することができる。 The step (B) is not particularly limited as long as it is a step of inoculating and fermenting the starter of the present invention to the cooked food according to the present invention, but inoculating an appropriate amount of the starter of the present invention to the steamed food according to the present invention. And the process of fermenting on suitable conditions can be illustrated suitably. The inoculation amount of the starter of the present invention is preferably an inoculation amount such that the number of spores of the strain of the present invention per 1 g of the food material obtained by steaming is 1.0 × 10 3 to 1.0 × 10 5 As a condition for fermentation, a high humidity condition at room temperature of 37 to 48 ° C. in the presence of oxygen can be preferably exemplified.
上記の本発明の製造方法により、本発明の発酵食品を製造することができる。本発明の発酵食品の中でも、マメ科植物の種子を本発明のスターターによって発酵させて製造した発酵食品を好適に例示することができ、黄大豆又は黒大豆を本発明のスターターによって発酵させて製造した発酵食品である納豆をより好適に例示することができる。 The fermented food of the present invention can be produced by the production method of the present invention. Among the fermented foods of the present invention, fermented foods produced by fermenting legume seeds with the starter of the present invention can be preferably exemplified, and produced by fermenting yellow soybeans or black soybeans with the starter of the present invention. The fermented food natto can be illustrated more suitably.
また、本発明には、本発明の発酵食品にさらに加工を加えて製造した加工食品も含まれる。かかる加工としては、特に制限されないが、乾燥、粉末化、他の食材との混合、加熱、本発明のスターター以外の微生物によるさらなる発酵等を例示することができる。本発明の加工食品には、本発明の発酵食品（好ましくは納豆）を乾燥させた食品（乾燥食品）；かかる乾燥食品を粉末化した食品（粉末化食品）；本発明の発酵食品、乾燥食品又は粉末化食品を味噌と混合した食品（味噌納豆等）；本発明の発酵食品、乾燥食品又は粉末化食品を麹で発酵させた食品（麹納豆等）；の他、本発明の発酵食品、乾燥食品又は粉末化食品を用いて加工した菓子（せんべい、クッキー、ケーキ等）、麺類（パスタ、うどん、ラーメン、そば等）、その他の加工食品；を例示することができる。 The present invention also includes a processed food produced by further processing the fermented food of the present invention. Examples of such processing include, but are not limited to, drying, pulverization, mixing with other ingredients, heating, further fermentation by microorganisms other than the starter of the present invention, and the like. The processed food of the present invention includes a food obtained by drying the fermented food of the present invention (preferably natto) (dried food); a food obtained by pulverizing such a dried food (powdered food); Or a food obtained by mixing a powdered food with miso (miso natto, etc.); a fermented food of the present invention, a food obtained by fermenting a dried food or a powdered food with koji (such as koji natto); and the fermented food of the present invention, Examples include confectionery (rice crackers, cookies, cakes, etc.) processed with dried foods or powdered foods, noodles (pasta, udon, ramen, soba etc.), and other processed foods.
また、本発明の枯草菌株は、ペットや家畜、魚類等の飼料となり得る食材に含有させて飼料（以下、「本発明の飼料」とも表示する。）として用いることもできる。かかる本発明の飼料における前述の食材は、本発明の枯草菌株によって発酵されていてもよく、また、発酵されていなくてもよい。また、本発明の枯草菌株は、ヒトの食材となり得る食材に含有させて食品（以下、「本発明の食品」とも表示する。）として用いることもできる。かかる本発明の食品における前述の食材は、本発明の枯草菌株によって発酵させる必要はないが、結果的に発酵が生じた食品も、本発明の食品に含まれる。なお、枯草菌バシラス・サチリスは、その摂取時に腸内の乳酸産生細菌（ビフィドバクテリウム属細菌、ラクトバシラス属細菌、エンテロコッカス属細菌等）の菌濃度上昇や、下痢改善等の整腸作用を有することが知られているので、本発明の枯草菌株も整腸作用を有していると考えられる。したがって、前述の本発明の飼料や本発明の食品は、ヒトや家畜等に対して整腸作用を発揮することが期待される。また、本発明の枯草菌株単独でも、整腸剤（以下、「本発明の整腸剤」とも表示する。）として用いることもできる。かかる本発明の整腸剤としては、本発明の枯草菌株を含有している限り特に制限されない。 The Bacillus subtilis strain of the present invention can also be used as a feed (hereinafter also referred to as “the feed of the present invention”) by being contained in foods that can be used as feed for pets, livestock, fish and the like. The aforementioned food material in the feed of the present invention may be fermented by the Bacillus subtilis strain of the present invention or may not be fermented. The Bacillus subtilis strain of the present invention can also be used as a food (hereinafter also referred to as “the food of the present invention”) by being contained in a food that can be a human food. The above-mentioned ingredients in the food of the present invention do not need to be fermented by the Bacillus subtilis strain of the present invention, but the foods resulting in fermentation are also included in the food of the present invention. Bacillus subtilis Bacillus subtilis has an intestinal action such as an increase in the concentration of lactic acid-producing bacteria (bifidobacterium, lactobacillus, enterococcus, etc.) in the intestine and diarrhea improvement when ingested. Therefore, it is considered that the Bacillus subtilis strain of the present invention also has an intestinal regulating action. Therefore, the feed of the present invention and the food of the present invention are expected to exert an intestinal regulating action on humans, livestock and the like. The Bacillus subtilis strain of the present invention alone can also be used as an intestinal adjuster (hereinafter also referred to as “the intestinal adjuster of the present invention”). The intestinal regulating agent of the present invention is not particularly limited as long as it contains the Bacillus subtilis strain of the present invention.
また、本発明の枯草菌株は、肥料作製用添加剤（以下、「本発明の肥料作製用添加剤」とも表示する。）として用いることもできる。本発明の枯草菌株を含有する本発明の肥料作製用添加剤を、肥料の原材料となり得る材料に添加すると、かかる材料が速やかに発酵して分解し、好適な肥料を作製することができる。上記の肥料の原材料となり得る材料としては、有機物又は有機物を含有する組成物である限り特に制限されないが、落ち葉、稲わら、刈り草、家畜の糞、油かす、米ぬか、骨粉、草木灰、生ごみ、食品残さ、汚泥等を好適に例示することができる。なお、本明細書における「肥料」には、堆肥やコンポストも含まれる。 The Bacillus subtilis strain of the present invention can also be used as an additive for producing fertilizer (hereinafter also referred to as “additive for producing fertilizer of the present invention”). When the additive for producing a fertilizer of the present invention containing the Bacillus subtilis strain of the present invention is added to a material that can be a raw material of the fertilizer, the material can be rapidly fermented and decomposed to produce a suitable fertilizer. The material that can be used as a raw material for the above fertilizer is not particularly limited as long as it is an organic substance or a composition containing an organic substance, but fallen leaves, rice straw, cut grass, livestock dung, oil cake, rice bran, bone meal, grass ash, garbage Food residues, sludge and the like can be suitably exemplified. The “fertilizer” in this specification includes compost and compost.
また、本発明の枯草菌株は、土壌・水質改良剤（以下、「本発明の土壌・水質改良剤」とも表示する。）として用いることもできる。本発明の枯草菌株を含有する本発明の土壌・水質改良剤を、土壌に添加すると、土壌中の有機物の分解が促進されて土壌中の養分が増加する。また、本発明の土壌・水質改良剤を、水を含む環境に添加すると、水中の有機物の分解が促進されて、その環境の水質を改善することができる。 The Bacillus subtilis strain of the present invention can also be used as a soil / water quality improver (hereinafter also referred to as “the soil / water quality improver of the present invention”). When the soil / water quality improving agent of the present invention containing the Bacillus subtilis strain of the present invention is added to the soil, the decomposition of organic matter in the soil is promoted and the nutrients in the soil increase. Moreover, when the soil / water quality improving agent of the present invention is added to an environment containing water, decomposition of organic substances in water is promoted, and the water quality of the environment can be improved.
また、納豆を発酵し得る枯草菌を用いて納豆を製造し、かかる納豆からタンパク質分解酵素サチライシン（subtilisin、別名ズブチリシン、納豆菌のズブチリシンについてはナットウキナーゼとも呼ばれる）を抽出し、抽出したサチライシンを、その血栓溶解作用に着目していわゆる健康食品として利用することが既に知られている。したがって、本発明の枯草菌株や、かかる枯草菌株から抽出したサチライシンを食品や医薬品の作用成分としてとして用いることもできる。本発明における食品や医薬品としては、本発明の枯草菌株、又は、本発明の枯草菌株から抽出したサチライシンを含有している限り特に制限されない。本発明の食品や医薬品の剤型としては特に制限されず、乾燥粉末剤、カプセル剤、錠剤等を例示することができる。 Also, natto is produced using Bacillus subtilis that can ferment natto, and the proteolytic enzyme subtilisin (also called subtilisin, also known as nattokinase for natto subtilisin) is extracted from the natto, and the extracted subtilisin is It is already known to use it as a so-called health food by paying attention to the thrombolytic action. Therefore, the Bacillus subtilis strain of the present invention and subtilisin extracted from the Bacillus subtilis strain can also be used as an active ingredient of foods and pharmaceuticals. The food or pharmaceutical product in the present invention is not particularly limited as long as it contains the Bacillus subtilis strain of the present invention or subtilisin extracted from the Bacillus subtilis strain of the present invention. The dosage form of the food or pharmaceutical product of the present invention is not particularly limited, and examples thereof include dry powders, capsules and tablets.
なお、本明細書中において「本発明の枯草菌株」を用いる場合には、本発明の枯草菌株の培養物を用いる場合も含まれる。かかる培養物としては、本発明の枯草菌株の培養液を好適に例示することができ、発酵食品製造用スターターとして使用する場合には、中でも本発明の枯草菌株の芽胞を含む培養液（芽胞液）と、その芽胞を遠心分離にて回収後、滅菌蒸留水を用いて菌体洗浄することを数回繰り返したのち、芽胞を滅菌蒸留水に懸濁した芽胞蒸留水懸濁液を特に好適に例示することができる。本発明の枯草菌株の芽胞を含む培養液（芽胞液）の調製は、例えば、2×ＳＧ培地（Nutrient broth：1.6％（W／V）、塩化カリウム：0.2％（W／V）、硫酸マグネシウム：0.05％（W／V）の水溶液1リットルをpH7.0に調整し、121℃，15分間の条件でオートクレーブ滅菌した後、別途滅菌した１M硝酸カルシウム水溶液1mL、0.1mM塩化マンガン水溶液1mL、1mM硫酸鉄溶液1mL、50%（W／V）グルコース水溶液2mLを加えたもの）等の芽胞形成培養液を用いて、本発明の枯草菌株（栄養細胞型）を35℃、4日間培養するなどにより可能である。以下、実施例により本発明をより具体的に説明するが、本発明の技術的範囲はこれらの例示に限定されるものではない。 In addition, when using "the Bacillus subtilis strain of this invention" in this specification, the case where the culture of the Bacillus subtilis strain of this invention is used is also included. As such a culture, the culture solution of the Bacillus subtilis strain of the present invention can be preferably exemplified. When used as a starter for producing fermented foods, a culture solution containing the spores of the Bacillus subtilis strain of the present invention (spore solution) ), And the spore collected by centrifugation and then washed several times with sterile distilled water, and then a spore-distilled water suspension obtained by suspending the spore in sterile distilled water is particularly suitable. It can be illustrated. Preparation of the culture solution (spore solution) containing the spores of the Bacillus subtilis strain of the present invention is, for example, 2 × SG medium (Nutrient broth: 1.6% (W / V), potassium chloride: 0.2% (W / V), magnesium sulfate. : 1 liter of 0.05% (W / V) aqueous solution is adjusted to pH 7.0, autoclaved at 121 ° C for 15 minutes, then sterilized with 1 mL of 1M calcium nitrate aqueous solution, 0.1mM manganese chloride aqueous solution 1mL, 1mM By culturing the Bacillus subtilis strain (vegetative cell type) of the present invention at 35 ° C. for 4 days using a spore-forming culture solution such as 1 mL of iron sulfate solution and 2 mL of 50% (W / V) glucose aqueous solution) Is possible. EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.
［バシラス・サチリス菌株の分離］
納豆の製造に用い得る新規な納豆菌を分離するために、栃木県、東京都、千葉県、茨城県、富山県、京都府及び三重県にて、土壌や稲わら等、合計55点の試料を採取した。これらの各試料を、80℃に加熱したトリプチケースソイブイヨン培地にそれぞれ添加して10分間静置した後、それらの各培地を35℃に冷却して24時間培養した。その後、各培地から１白金耳量の培養液をそれぞれトリプチケースソイ寒天培地上に平板塗抹し、35℃、24時間培養した。培養して得られたコロニーの一部を、トリプチケースソイブイヨン寒天培地上へ再度平板塗抹し、35℃、24時間培養するという過程を数回繰り返して、コロニーの形態からバシラス・サチリスと推定された25株を純粋分離した。
[Isolation of Bacillus subtilis strain]
A total of 55 samples such as soil and rice straw were collected in Tochigi, Tokyo, Chiba, Ibaraki, Toyama, Kyoto and Mie prefectures in order to isolate new natto bacteria that can be used in the production of natto. Were collected. Each of these samples was added to a trypticase soy bouillon medium heated to 80 ° C. and allowed to stand for 10 minutes, and then each medium was cooled to 35 ° C. and cultured for 24 hours. Thereafter, 1 platinum loop of the culture solution from each medium was plated on a trypticase soy agar medium and cultured at 35 ° C. for 24 hours. A part of the colony obtained by culturing was again smeared on Trypticase soy bouillon agar, and the process of culturing at 35 ° C for 24 hours was repeated several times. From the colony morphology, Bacillus subtilis was estimated. 25 strains were purely isolated.
次に、純粋分離したこの25株の中から、納豆食品製造用スターターとして好適に用い得る菌株を選抜するために、これら25株をスターターとして、以下の方法にて納豆の試作を行なった。まず、これら25株のそれぞれについて、トリプチケースソイブイヨン培地を用いて35℃、2日間培養し、各菌株の菌懸濁液を調製した。 Next, in order to select a strain that can be suitably used as a starter for natto food production from the 25 isolates that were purely isolated, natto was prototyped by the following method using these 25 strains as a starter. First, each of these 25 strains was cultured at 35 ° C. for 2 days using trypticase soy bouillon medium to prepare a bacterial suspension of each strain.
一方、水に浸漬した大豆（品種：スズマル）を加圧釜にて蒸煮し、蒸煮大豆を得た。この蒸煮大豆に、前述の菌懸濁液をそれぞれ接種した。各菌懸濁液の接種量は、蒸煮大豆1gあたりの各菌の個数がおよそ1.0×104個となるような量とした。各菌懸濁を接種した各蒸煮大豆をそれぞれポリスチレンペーパー（ＰＳＰ）容器（蓋付き）に充填した後、その蒸煮大豆の表面を有孔ポリエチレンフィルムで覆い、さらにＰＳＰ容器の蓋をかぶせた。蓋をしたこのＰＳＰ容器を、室温約40℃に設定した室内に静置し、約20時間発酵させた。得られた各発酵物を確認し、納豆として好適な性質を備えた発酵物が得られた菌株を選抜した。選抜したかかる菌株をTFC 4102株と命名した。このTFC 4102株は、栃木県にて採取した稲わらの試料から分離した菌株であった。 On the other hand, soybeans immersed in water (variety: Suzumaru) were steamed in a pressure kettle to obtain steamed soybeans. The steamed soybeans were each inoculated with the aforementioned bacterial suspension. The amount of each bacterial suspension inoculated was such that the number of each bacterial per g of steamed soybean was approximately 1.0 × 10 4 . Each steamed soybean inoculated with each bacterial suspension was filled in a polystyrene paper (PSP) container (with a lid), and then the surface of the steamed soybean was covered with a perforated polyethylene film, and the lid of the PSP container was further covered. The PSP container with the lid was allowed to stand in a room set at room temperature of about 40 ° C. and fermented for about 20 hours. Each fermented product obtained was confirmed, and a strain from which a fermented product having suitable properties as natto was obtained was selected. The selected strain was named TFC 4102 strain. This TFC 4102 strain was a strain isolated from a rice straw sample collected in Tochigi Prefecture.
［TFC 4102株の16S rRNA遺伝子の部分塩基配列の解析］
実施例１で分離したTFC 4102株が、枯草菌バシラス・サチリスであるかどうかを確認するため、16S rRNA遺伝子のヌクレオチド配列の解析を試みた。具体的には以下の方法で解析を行なった。
[Analysis of partial nucleotide sequence of 16S rRNA gene of TFC 4102 strain]
In order to confirm whether the TFC 4102 strain isolated in Example 1 was Bacillus subtilis Bacillus subtilis, an analysis of the nucleotide sequence of the 16S rRNA gene was attempted. Specifically, the analysis was performed by the following method.
上記TFC 4102株をトリプチケースソイ培地で培養後、その菌体を分離し、その菌体からDNeasy Plant Mini Kit（キアゲン社製）を用いてゲノムDNAを抽出した。かかるゲノムDNAを鋳型とし、以下の表１に記載のうち、「16S 10Fと16S 1541R」のプライマーを用いて、GeneAmp PCRシステム9700（アプライドバイオシステムジャパン社製）にてPCR反応を行った。なお、表１のプライマーのうち、プライマー名の最後がＦであるプライマーはフォワードプライマーであることを示し、プライマー名の最後がＲであるプライマーはリバースプライマーであることを示す。かかるPCRの反応条件としては、PCR酵素初期活性化95℃，15分間、増幅（変性95℃，1分間、アニーリング52℃，1分間、伸長72℃，2分間）30サイクル、最終伸長72℃，5分間であり、PCR酵素として、HotStarTaq Master Mix Kit（キアゲン社製）を用いた。次に、かかるPCR増幅産物をQIAquick PCR Purification Kit（キアゲン社製）を用いて精製した。続いて、表１の各プライマーを用いてサイクルシークエンス反応をBigDye Terminator v3.1 Cycle Sequencing Kit（アプライドバイオシステムジャパン社製）を用いて行い、生成物をDyeEx 2.0 Spin Kit（キアゲン社製）を用いて精製後、Hi-Di Formamide（アプライドバイオシステムジャパン社製）を用いて、塩基配列解析用の試料を調製した。 After culturing the above TFC 4102 strain in trypticase soy medium, the cells were isolated, and genomic DNA was extracted from the cells using DNeasy Plant Mini Kit (Qiagen). Using such genomic DNA as a template, PCR reaction was performed with GeneAmp PCR system 9700 (manufactured by Applied Biosystems Japan) using the primers “16S 10F and 16S 1541R” in Table 1 below. Of the primers in Table 1, a primer whose name ends with F is a forward primer, and a primer whose name ends with R is a reverse primer. The PCR reaction conditions were as follows: PCR enzyme initial activation 95 ° C., 15 minutes, amplification (denaturation 95 ° C., 1 minute, annealing 52 ° C., 1 minute, extension 72 ° C., 2 minutes) 30 cycles, final extension 72 ° C. It was 5 minutes, and HotStarTaq Master Mix Kit (manufactured by Qiagen) was used as the PCR enzyme. Next, the PCR amplification product was purified using QIAquick PCR Purification Kit (Qiagen). Subsequently, a cycle sequencing reaction was performed using each primer in Table 1 using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems Japan), and the product was used using DyeEx 2.0 Spin Kit (Qiagen). After purification, a sample for base sequence analysis was prepared using Hi-Di Formamide (Applied Biosystems Japan).
調製した解析用試料10種について、ABI PRISM 310 Genetic Analyzer（アプライドバイオシステムジャパン社製）を用いて、それらのヌクレオチド配列を決定した。各試料の増幅領域は他のいずれかの試料の増幅領域と一部重複しているので、重複が生じないように、断片のヌクレオチド配列を整列させて、16S rRNA遺伝子のヌクレオチド配列（配列番号11）を得た。この16S rRNA遺伝子のヌクレオチド配列を、DDBJ（DNA data bank of Japan）におけるBLAST・FAST解析にかけたところ、枯草菌バシラス・サチリスの16S rRNA遺伝子配列として既に登録されていた配列の１つと100％一致した。したがって、TFC 4102株はバシラス・サチリス（Bacillus subtilis）に属する微生物と判断した。 The nucleotide sequences of the 10 analysis samples prepared were determined using ABI PRISM 310 Genetic Analyzer (Applied Biosystems Japan). Since the amplification region of each sample partially overlaps with the amplification region of any other sample, the nucleotide sequences of the fragments were aligned to prevent duplication, and the nucleotide sequence of the 16S rRNA gene (SEQ ID NO: 11 ) When the nucleotide sequence of this 16S rRNA gene was subjected to BLAST / FAST analysis in DDBJ (DNA data bank of Japan), it was 100% identical with one of the sequences already registered as the 16S rRNA gene sequence of Bacillus subtilis Bacillus subtilis. . Therefore, TFC 4102 strain was judged to be a microorganism belonging to Bacillus subtilis.
［TFC 4102株とNBRC 3013株との比較］
微生物株保存機関の納豆菌株であるバシラス・サチリスNBRC 3013株（以前のIFO 3013株と同等）と、TFC 4102株との間で、ゲノム構造に相違があるかどうかを調べるために、改変RAPD法によるバンドパターンの比較を行なった。具体的には以下の方法で行なった。
[Comparison between TFC 4102 and NBRC 3013]
To examine whether there is a difference in the genome structure between the Bacillus subtilis NBRC 3013 strain (equivalent to the previous IFO 3013 strain) and the TFC 4102 strain, the natto strain of the microbial strain storage organization, the modified RAPD method Comparison of band patterns was performed. Specifically, the following method was used.
まず、バシラス・サチリスNBRC 3013株を、独立行政法人 製品評価技術基盤機構 バイオテクノロジー本部 生物遺伝資源部門 遺伝資源保存課より購入した。TFC 4102株及びNBRC 3013株から、DNeasy Plant Mini Kit（キアゲン社製）を用いてゲノムDNAを抽出した。かかるそれぞれのゲノムDNAを鋳型とし、後述の表２に記載のR1〜R12プライマー（配列番号12〜23）のうちの１つのプライマーと、挿入配列IS4Bsu1に対するIS4Bsu1 1280Fプライマー（配列番号24）との組み合わせ（合計12組）を用いて、GeneAmp PCRシステム9700（アプライドバイオシステムジャパン社製）にてPCR反応を行った。 First, the Bacillus subtilis NBRC 3013 strain was purchased from the Genetic Resource Conservation Division, Biotechnology Headquarters, Biotechnology Headquarters, National Institute of Technology and Evaluation. Genomic DNA was extracted from TFC 4102 strain and NBRC 3013 strain using DNeasy Plant Mini Kit (Qiagen). Using each of these genomic DNAs as a template, a combination of one of the R1 to R12 primers (SEQ ID NOs: 12 to 23) described in Table 2 and an IS4Bsu1 1280F primer (SEQ ID NO: 24) for the inserted sequence IS4Bsu1 PCR reaction was performed using GeneAmp PCR system 9700 (Applied Biosystems Japan) using a total of 12 pairs.
かかるPCRの条件は、PCR酵素初期活性化95℃，15分間、増幅（変性95℃，1分間、アニーリング36℃，1分間、伸長72℃，3分間）50サイクル、最終伸長72℃，5分間であり、PCR酵素として、HotStarTaq Master Mix Kit（キアゲン社製）を用いた。各PCR反応による増幅産物を常法にしたがってアガロースゲルにて電気泳動した。その電気泳動像を図１に示す。また、NBRC 3013株に代えてTFC 4102株を用いて同様の操作を行って得られた電気泳動像を図２に示す。図１及び図２における両端のレーンＭは、分子量マーカー（DynaMarker 100 bp ladder、バイオダイナミクス研究所社製）を泳動したレーンであり、下端のバンドから順に、100bp，200bp，300bp，400bp，500bp，600bp，700bp，800bp，900bp，1000bp，1500bp，2000bp，3000bpを示す。また、図１及び図２におけるレーン1は、プライマーとしてR1とIS4Bsu1 1280Fとを用いたサンプルを、レーン2は、プライマーとしてR2とIS4Bsu1 1280Fとを用いたサンプルを、レーン3は、プライマーとしてR3とIS4Bsu1 1280Fとを用いたサンプルを、レーン4は、プライマーとしてR4とIS4Bsu1 1280Fとを用いたサンプルを、レーン5は、プライマーとしてR5とIS4Bsu1 1280Fとを用いたサンプルを、レーン6は、プライマーとしてR6とIS4Bsu1 1280Fとを用いたサンプルを、レーン7は、プライマーとしてR7とIS4Bsu1 1280Fとを用いたサンプルを、レーン8は、プライマーとしてR8とIS4Bsu1 1280Fとを用いたサンプルを、レーン9は、プライマーとしてR9とIS4Bsu1 1280Fとを用いたサンプルを、レーン10は、プライマーとしてR10とIS4Bsu1 1280Fとを用いたサンプルを、レーン11は、プライマーとしてR11とIS4Bsu1 1280Fとを用いたサンプルを、レーン12は、プライマーとしてR12とIS4Bsu1 1280Fとを用いたサンプルを泳動したレーンである。 The PCR conditions are: PCR enzyme initial activation 95 ° C, 15 minutes, amplification (denaturation 95 ° C, 1 minute, annealing 36 ° C, 1 minute, extension 72 ° C, 3 minutes) 50 cycles, final extension 72 ° C, 5 minutes As a PCR enzyme, HotStarTaq Master Mix Kit (manufactured by Qiagen) was used. Amplified products from each PCR reaction were electrophoresed on an agarose gel according to a conventional method. The electrophoresis image is shown in FIG. Further, FIG. 2 shows an electrophoresis image obtained by performing the same operation using the TFC 4102 strain instead of the NBRC 3013 strain. Lanes M at both ends in FIG. 1 and FIG. 2 are lanes on which molecular weight markers (DynaMarker 100 bp ladder, manufactured by Biodynamics Laboratories) were migrated, and in order from the bottom band, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600bp, 700bp, 800bp, 900bp, 1000bp, 1500bp, 2000bp, 3000bp are shown. 1 and 2, lane 1 is a sample using R1 and IS4Bsu1 1280F as primers, lane 2 is a sample using R2 and IS4Bsu1 1280F as primers, and lane 3 is R3 as a primer. Samples using IS4Bsu1 1280F, lane 4 is a sample using R4 and IS4Bsu1 1280F as primers, lane 5 is a sample using R5 and IS4Bsu1 1280F as primers, lane 6 is R6 as a primer Lane 7 is a sample using R7 and IS4Bsu1 1280F as primers, lane 8 is a sample using R8 and IS4Bsu1 1280F as primers, lane 9 is a primer Samples using R9 and IS4Bsu1 1280F, Lane 10 is a sample using R10 and IS4Bsu1 1280F as primers, Lane 11 is R11 and IS as primers Samples using 4Bsu1 1280F are lanes, and lane 12 is a lane in which samples using R12 and IS4Bsu1 1280F as primers are migrated.
図１の電気泳動像と図２の電気泳動像を比較すると分かるように、レーン2において、両像間のバンドパターンに明らかな差異が認められた。これにより、NBRC 3013株とTFC 4102株とは、ゲノム配列が異なることが強く示唆された。 As can be seen by comparing the electrophoretic image of FIG. 1 and the electrophoretic image of FIG. 2, in Lane 2, a clear difference was observed in the band pattern between the two images. This strongly suggested that the NBRC 3013 strain and the TFC 4102 strain differ in genome sequence.
なお、上記の実験において、プライマーIS4Bsu1 1280Fに代えて、プライマーIS4Bsu1 145R（配列番号25）を使用することや、上記の実験において、プライマーIS4Bsu1 1280FとIS4Bsu1 145Rを混合添加して使用することも可能である。これらの場合、得られる電気泳動像は図１及び２とは異なる（データ未掲載）。また、PCR酵素として他製品、例えば、AmpliTaq Gold PCR Master Mix（アプライドバイオシステムズ社製）やSensiZyme Hotstart Taq Premix（RBC Bioscience社製）を使用すると、同一のプライマーを使用しても、PCR酵素にHotStarTaq Master Mix Kit（キアゲン社製）を用いた場合の電気泳動像（図１及び２）とは異なる電気泳動像が得られる（データ未掲載）。 In the above experiment, it is possible to use the primer IS4Bsu1 145R (SEQ ID NO: 25) instead of the primer IS4Bsu1 1280F, and in the above experiment, the primers IS4Bsu1 1280F and IS4Bsu1 145R can be mixed and used. is there. In these cases, the obtained electrophoretic images are different from those in FIGS. 1 and 2 (data not shown). In addition, when other products such as AmpliTaq Gold PCR Master Mix (Applied Biosystems) or SensiZyme Hotstart Taq Premix (RBC Bioscience) are used as PCR enzymes, even if the same primers are used, HotStarTaq An electrophoretic image different from the electrophoretic image (FIGS. 1 and 2) obtained when Master Mix Kit (Qiagen) is used (data not shown).
［納豆の製造、及び、製造した納豆の官能評価］
TFC 4102株を用いて製造した納豆が、従来の市販納豆菌製造用スターターを用いて製造した納豆と比較してどのようなものであるかを調べるために、納豆の製造及びそれにより得られた納豆の官能評価を試みた。具体的には以下の方法で行なった。
[Manufacture of natto and sensory evaluation of manufactured natto]
In order to investigate what natto produced using TFC 4102 strain compared to natto produced using a conventional starter for producing commercial natto bacteria, natto was produced and obtained thereby. I tried sensory evaluation of natto. Specifically, the following method was used.
まず、TFC 4102株を２×ＳＧ培地で培養し、TFC 4102株の芽胞液を作製した。一方、水に浸漬した大豆（品種：スズマル）を加圧釜にて蒸煮し、蒸煮大豆を得た。この蒸煮大豆に、前述の芽胞液をそれぞれ接種した。芽胞液の接種量は、蒸煮大豆１ｇあたりの各芽胞の個数がおよそ1.0×104個となるような量とした。芽胞液を接種した蒸煮大豆をポリスチレンペーパー（ＰＳＰ）容器（蓋付き）に充填した後、その蒸煮大豆の表面を有孔ポリエチレンフィルムで覆い、さらにＰＳＰ容器の蓋をかぶせた。蓋をしたこのＰＳＰ容器を、室温を約40℃に設定した室内に静置し、約20時間発酵させた。発酵終了後に冷却して、納豆を得た。 First, the TFC 4102 strain was cultured in 2 × SG medium to prepare a spore solution of the TFC 4102 strain. On the other hand, soybeans immersed in water (variety: Suzumaru) were steamed in a pressure kettle to obtain steamed soybeans. This steamed soybean was inoculated with the aforementioned spore fluid. The amount of inoculated spore solution was such that the number of each spore per 1 g of steamed soybean was approximately 1.0 × 10 4 . After the steamed soybean inoculated with the spore solution was filled in a polystyrene paper (PSP) container (with a lid), the surface of the steamed soybean was covered with a perforated polyethylene film, and the lid of the PSP container was further covered. The PSP container with the lid was allowed to stand in a room whose room temperature was set to about 40 ° C. and fermented for about 20 hours. After completion of fermentation, it was cooled to obtain natto.
また、上記の納豆の製造方法において、TFC 4102株の芽胞液に代えて、市販の２種類の納豆菌株の菌液を混合した混合菌液（以下、単に「市販納豆菌株の混合菌液」とも表示する。）を用いて、納豆を製造した。前述の混合菌液としては、宮城野菌（宮城野納豆製造所販売）の菌液と成瀬菌（成瀬発酵化学研究所販売）の菌液を体積比で１：９（＝宮城野菌：成瀬菌）で混合した菌液（以下、「市販納豆菌株の混合菌液」とも表示する。）を用いた。 In addition, in the above natto production method, instead of the spore solution of TFC 4102 strain, a mixed bacterial solution obtained by mixing two commercially available natto strains (hereinafter simply referred to as “combined bacterial solution of commercial natto strain”) Natto was manufactured using As the above-mentioned mixed bacterial solution, the bacterial solution of Miyagino fungus (sales of Miyagino Natto Factory) and the fungus of Naruse fungus (sales of Naruse Fermentation Chemistry Laboratory) is 1: 9 by volume ratio (= Miyagi fungus: Naruse fungus) A mixed bacterial solution (hereinafter also referred to as “mixed bacterial solution of commercially available natto strain”) was used.
前述のようにして得られた２種類の納豆の風味を、17名のパネルに評価させた。評価対象としては、後述の表３に示す評価項目を用いた。また、評価基準としては、市販納豆菌株の混合菌液を用いて得られた納豆の場合を３点（基準点）とし、TFC 4102株の菌液を用いて得られた納豆の風味を１点〜５点の間（評点の基準は後述の表３参照）で評価させた。TFC 4102株の菌液を用いて得られた納豆の最終的な評点としては、17名のパネルのその評価項目に関する評点の平均を用いた。これらの官能評価の結果を表３に示す。 A panel of 17 people evaluated the flavors of the two types of natto obtained as described above. As evaluation targets, the evaluation items shown in Table 3 to be described later were used. In addition, as an evaluation standard, natto obtained using a mixed microbial solution of commercially available natto strains was given 3 points (reference point), and 1 natto flavor obtained using the TFC 4102 strain was used. Evaluation was made between ˜5 points (see Table 3 below for the criteria of the score). As the final score of natto obtained using the TFC 4102 strain, the average of the scores on the evaluation items of 17 panelists was used. The results of these sensory evaluations are shown in Table 3.
表３から分かるように、TFC 4102株の菌液を用いて製造した納豆は、市販納豆菌株の混合菌液を用いて製造した納豆と比較して、食感、旨味、後味、苦味、香りの点で高い評価を示し、特に、雑味（後味の質の悪さ、異味の強さ、苦味の強さ）は弱かった。 As can be seen from Table 3, natto manufactured using the TFC 4102 strain has a texture, umami, aftertaste, bitterness, and fragrance compared to natto manufactured using a mixed strain of commercially available natto strains. In particular, it showed a high evaluation, and in particular, the miscellaneous taste (poor aftertaste quality, strength of taste, strength of bitterness) was weak.
また、TFC 4102株の菌液を用いて製造した納豆について、製造後の風味の経時的な変化の様子を調べるために、製造から１週間経過後に３名のパネルによりその納豆の風味を評価した。一方、市販納豆菌株の混合菌液を用いて製造した納豆についても、製造から１週間経過後の風味を同様に評価した。その結果、TFC 4102株の菌液を用いて製造し製造後１週間経過した納豆は、上記の市販納豆菌株の混合菌液を用いて製造し製造後１週間経過した納豆と比較して、後味、異味、苦味、香りの点で良好であった。すなわち、TFC 4102株の菌液を用いて製造した納豆は、市販納豆菌株の混合液を用いて製造した納豆と比較して、製造後の品質変化が少ないことが判明した。 In addition, for natto produced using the TFC 4102 strain, the flavor of the natto was evaluated by a panel of three people one week after the production in order to examine the changes over time in the flavor after production. . On the other hand, for natto produced using a mixed bacterial solution of commercially available natto strains, the flavor after one week from the production was similarly evaluated. As a result, natto produced using the bacterial solution of TFC 4102 strain and one week after production was compared with natto produced using the above mixed bacterial solution of commercially available natto strain and one week after production. It was good in terms of taste, bitterness and aroma. That is, it was found that natto produced using the TFC 4102 strain had less quality change after production compared to natto produced using a mixture of commercially available natto strains.
本発明は、発酵食品の製造分野、発酵食品を用いた加工食品の製造分野、発酵食品やいわゆる健康食品の分野、医薬品の分野、肥料や飼料の分野、土壌・水質改良剤の分野に利用することができる。 The present invention is used in the field of fermented food production, the field of processed foods using fermented foods, the field of fermented foods and so-called health foods, the field of pharmaceuticals, the field of fertilizers and feeds, and the field of soil and water quality improvers. be able to.
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| JPN6008036626; ＳＵＮＧ，Ｍ．-Ｈ． ｅｔ ａｌ．: '"Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ ｖａｒ． ｃｈｕｎｇｋｏｏｋｊａｎｇ １６Ｓ ｒｉｂｏｓｏｍａｌ ＲＮＡ ｇｅｎｅ， ｃｏｍｐｌｅｔｅ ｓｅｑｕｅｎｃｅ"' ＧｅｎＢａｎｋ， Ａｃｃｅｓｓｉｏｎ ＡＦ２３３５７９ , 20010302 * |
| JPN6008036629; ＡＳＨＩＵＣＨＩ， Ｍ． ｅｔ ａｌ．: 'Ｉｓｏｌａｔｉｏｎ ｏｆ Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ （ｃｈｕｎｇｋｏｏｋｊａｎｇ）， ａ ｐｏｌｙ-ｇａｍｍａ-ｇｌｕｔａｍａｔｅ ｐｒｏｄｕｃｅｒ ｗｉｔｈ ｈｉｇｈ ｇｅｎｅｔｉｃ ｃ' Ａｐｐｌ． Ｍｉｃｒｏｂｉｏｌ． Ｂｉｏｔｅｃｈｎｏｌ． Ｖｏｌ．５７, 2001, ｐｐ．７６４-７６９ * |
| JPN6008036632; ＳＥＯＮＧ-ＨＹＵＮ， Ｃ． ｅｔ ａｌ．: '"Ｌｅｖａｎ-ｐｒｏｄｕｃｉｎｇ Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ ＢＳ ６２ ａｎｄ ｉｔｓ ｐｈｙｌｏｇｅｎｙ ｂａｓｅｄ ｏｎ ｉｔｓ １６Ｓ ｒＤＮＡ ｓｅｑｕｅｎｃｅ"' Ｊ． Ｍｉｃｒｏｂｉｏｌ． Ｂｉｏｔｅｃｈｎｏｌ． Ｖｏｌ．１１， Ｎｏ．３, 2001, ｐｐ．４２８-４３４ * |
| JPN6008036636; 木内幹ら: '"Ｓｔｕｄｉｅｓ ｏｎ ｍａｎｕｆａｃｔｕｒｉｎｇ ｍｅｔｈｏｄｓ ｏｆ ｆｅｒｍｅｎｔｅｄｓｏｙｂｅａｎ ｆｏｏｄｓ ａｎｄ ｉｔｓ ｆｏｏｄ ｃｕｌｔｕｒｅ ｉｎ ｓｏｕｔｈ-ｅａｓｔ ａｓｉａ．' 共立女子大学総合文化研究所年報 Ｎｏ．９, 2003, ｐｐ．１９-３５ * |
| JPN6014005914; 細井知弘: '納豆菌スターターの開発' 平成１８年度農林総合研究センター年報 , 200709, ｐ．４１, 財団法人東京都農林水産振興財団 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2013078289A (en) * | 2011-10-04 | 2013-05-02 | Takano Foods Kk | Method of producing bacillus natto and natto for suppressing effect of temperature irregularity in natto fermentation chamber |
| JP2018519337A (en) * | 2015-06-30 | 2018-07-19 | ジンゴル ナ | Method for producing fermented mineral raw pills having a function of improving constipation and fatigue recovery, and fermented mineral raw pills produced thereby |
| CN108192834A (en) * | 2017-12-11 | 2018-06-22 | 青岛寿纳豆食品有限公司 | One plant of bacterial strain for being used to prepare odorless natto |
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