JP4968816B2 - Method for forming spores of Bacillus bacteria - Google Patents
Method for forming spores of Bacillus bacteria Download PDFInfo
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- JP4968816B2 JP4968816B2 JP2006063414A JP2006063414A JP4968816B2 JP 4968816 B2 JP4968816 B2 JP 4968816B2 JP 2006063414 A JP2006063414 A JP 2006063414A JP 2006063414 A JP2006063414 A JP 2006063414A JP 4968816 B2 JP4968816 B2 JP 4968816B2
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- bacillus
- culture
- culture solution
- dissolved oxygen
- spore
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
本発明は、バチルス属細菌の芽胞を形成する方法に関する。 The present invention relates to a method for forming spores of Bacillus bacteria.
バチルス属細菌は、植物病虫害防除や動物への整腸作用，成長促進作用や感染症予防治療効果等を有することが報告されている。このため、バチルス属細菌を農園芸用殺虫及び殺菌剤として用いたり、家畜や家禽、魚介類用の飼料に整腸剤として配合する試みがなされている。このとき、農薬製剤や飼料等の製品中のバチルス属細菌の菌数が多いほど、より高い効果が得られることになるが、製品への加工工程や、製品の流通時、店頭での陳列時において、製品中のバチルス属細菌の生菌数がかなり減少してしまうという問題があった。 Bacillus bacteria have been reported to have plant pest control, animal intestinal regulation, growth promotion, infection prevention and treatment effects, and the like. For this reason, attempts have been made to use Bacillus bacteria as agricultural and horticultural insecticides and fungicides, or to blend feeds for livestock, poultry and seafood as intestinal agents. At this time, the higher the number of Bacillus bacteria in products such as agricultural chemical formulations and feed, the higher the effect will be. However, when processing the product, distributing the product, and displaying it at the store However, there is a problem that the viable count of Bacillus bacteria in the product is considerably reduced.
バチルス属細菌の生菌数を維持するためには、耐熱性や耐乾燥性などの環境変化に耐性を持つ芽胞を形成させ安定化を図ることが望ましい。バチルス属細菌の芽胞を形成する方法や、芽胞を含有する生物農薬や飼料に関しては、数種の報告がある。 In order to maintain the viable count of Bacillus bacteria, it is desirable to form and stabilize spores that are resistant to environmental changes such as heat resistance and drought resistance. There have been several reports on methods for forming spores of Bacillus bacteria and biopesticides and feeds containing spores.
生物農薬について、例えば、特許文献１には、植物病原菌と拮抗するバチルス ズブチリスＮＣＩＢ１２３７６株菌およびＮＣＩＢ１２６１６株菌から選ばれるいずれか１種又は２種の細菌の培養物から胞子を乾燥重量で５０重量％以上含むように調製された胞子画分を含有する農園芸用殺菌剤組成物が開示されている。また、特許文献２には、植物病原菌と拮抗するバチルス ズブチリスに属する細菌の胞子及び保湿剤を含有する農園芸用殺菌剤組成物が開示されている。さらに、クワの炭疽病や白紋羽病の防除微生物として、バチルス アミロリクエファシエンスが知られている（例えば特許文献３、非特許文献１、非特許文献２参照）。飼料に関して、例えば、特許文献４には、バチルス セレウスの芽胞を有効成分とする動物用下痢治療剤が開示されている。また、特許文献５には、１〜５重量％のコーンスティープリカー及び炭素源を含有する培地において、バチルス属細菌を炭素源が消費し尽くされるまで培養し、その後２４〜７２時間通気することを特徴とするバチルス属細菌の芽胞化方法、並びに得られた芽胞を用いた飼料や添加剤を提供する方法が開示されている。 Regarding biological agrochemicals, for example, Patent Document 1 discloses that 50% by weight of spore from a culture of any one or two bacteria selected from Bacillus subtilis NCIB 12376 and NCIB 12616 strains that antagonize phytopathogenic fungi. An agricultural and horticultural fungicide composition containing a spore fraction prepared to include the above is disclosed. Patent Document 2 discloses an agricultural and horticultural fungicide composition containing spores of bacteria belonging to Bacillus subtilis that antagonize phytopathogenic fungi and a moisturizing agent. Furthermore, Bacillus amyloliquefaciens is known as a microorganism for controlling mulberry anthrax and white crest feather disease (see, for example, Patent Document 3, Non-Patent Document 1, and Non-Patent Document 2). Regarding feed, for example, Patent Document 4 discloses a therapeutic agent for diarrhea for animals comprising Bacillus cereus spores as active ingredients. Patent Document 5 states that in a medium containing 1 to 5% by weight of corn steep liquor and a carbon source, Bacillus bacteria are cultured until the carbon source is consumed, and then aerated for 24 to 72 hours. Disclosed are a characteristic method for spore-forming Bacillus bacteria, and a method for providing a feed or additive using the obtained spore.
しかしながら、細菌の増殖は、栄養分を多く与えることが必要である一方、芽胞を形成させるには栄養分を不足させたり、生育環境を悪化させることが必要であり、上述の特許文献等に記載された従来の方法では高濃度のバチルス属細菌芽胞を調製することは困難であった。 However, while the growth of bacteria requires a lot of nutrients, it is necessary to make the nutrients deficient or to deteriorate the growth environment in order to form spores. It was difficult to prepare a high concentration of Bacillus spore by conventional methods.
また、バチルス属細菌の株によっては、上述の特許文献等に記載された従来の方法を用いても芽胞化が不十分なものもある。このため、より広範な種類の株に適用し得る芽胞形成方法の開発が要求されている。従って本発明の目的は、バチルス属細菌の芽胞化を促進させ、芽胞化したバチルス属細菌を高濃度で、かつ効率良く得ることが可能な方法を提供することにある。 In addition, some strains of the genus Bacillus may have insufficient spore formation even when conventional methods described in the above-mentioned patent documents are used. Therefore, development of a spore formation method that can be applied to a wider variety of strains is required. Accordingly, an object of the present invention is to provide a method capable of promoting the spore formation of Bacillus bacteria and efficiently obtaining the spore-formed Bacillus bacteria at a high concentration.
本発明者らは、上記の課題を解決するため鋭意研究を行った結果、各種炭素源や窒素源を含有する培地でバチルス属細菌を培養する過程で、培地中の溶存酸素を一定時間１０％以下にまで低下させることにより、バチルス属細菌の芽胞化を促進させ、芽胞化したバチルス属細菌を高濃度で、かつ効率良く得ることが可能となることを見出し、本発明を完成した。 As a result of intensive studies to solve the above problems, the present inventors have determined that the dissolved oxygen in the medium is 10% for a certain period of time in the process of culturing Bacillus bacteria in a medium containing various carbon sources and nitrogen sources. The inventors have found that by reducing to the following, the spore formation of Bacillus bacteria can be promoted, and the spore-formed Bacillus bacteria can be efficiently obtained at a high concentration, and the present invention has been completed.
すなわち本発明は、（１）溶存酸素率が１０％以下の培養液でバチルス属細菌を培養する工程（Ｃ）と、工程（Ｃ）の後に、溶存酸素率が１０％より多い培養液でバチルス属細菌を培養する工程（Ｄ）とを含むことを特徴とするバチルス属細菌の芽胞を形成する方法や、（２）溶存酸素率が１０％以下の培養液で培養するバチルス属細菌が、対数増殖期（Ｌｏｇ Ｐｈａｓｅ）以降のバチルス属細菌であることを特徴とする上記（１）に記載のバチルス属細菌の芽胞を形成する方法や、（３）溶存酸素率が１０％以下の培養液で培養するバチルス属細菌が、対数増殖期（Ｌｏｇ Ｐｈａｓｅ）及び静止期（Ｓｔａｔｉｏｎａｒｙ Ｐｈａｓｅ）のバチルス属細菌であることを特徴とする上記（１）に記載のバチルス属細菌の芽胞を形成する方法や、（４）工程（Ｃ）の前に、溶存酸素率が１０％より多い培養液でバチルス属細菌を培養してバチルス属細菌を増殖させる工程（Ａ）、及び／又は、培養液の溶存酸素率を１０％以下にする工程（Ｂ）を、さらに含むことを特徴とする上記（１）〜（３）のいずれかに記載のバチルス属細菌の芽胞を形成する方法や、（５）培養液への通気量及び／又は培養液の撹拌速度を調節することによって、培養液の溶存酸素率を１０％以下にすることを特徴とする上記（１）〜（４）のいずれかに記載のバチルス属細菌の芽胞を形成する方法や、（６）培養液が、酵母エキス、コーンスティープリカー、大豆ペプトン、及び濃縮焼酎かすからなる群から選ばれるいずれか１種以上の成分を含有することを特徴とする上記（１）〜（５）のいずれかに記載のバチルス属細菌の芽胞を形成する方法や、（７）培養液が、酵母エキス、コーンスティープリカー、大豆ペプトン、及び濃縮焼酎かすからなる群から選ばれるいずれか２種以上の成分を含有することを特徴とする上記（６）に記載のバチルス属細菌の芽胞を形成する方法や、（８）培養液が、酵母エキスを０．２〜１０．０重量％、コーンスティープリカーを０．５〜２０．０重量％、大豆ペプトンを０．２〜１０．０重量％、濃縮焼酎かすを０．２〜２０．０重量％含有することを特徴とする上記（７）に記載のバチルス属細菌の芽胞を形成する方法や、（９）培養液が、糖類をさらに含有することを特徴とする上記（６）〜（８）のいずれかに記載のバチルス属細菌の芽胞を形成する方法や、（１０）培養液が、糖類を０．０５〜５．０重量％含有することを特徴とする上記（９）に記載のバチルス属細菌の芽胞を形成する方法や、（１１）バチルス属細菌が、バチルス ズブチリス（Bacillus subtilis）、バチルス アミロリクエファシエンス（Bacillus amyloliquefaciens）、バチルス パミルス（Bacillus pumils）、バチルス レンタス（Bacillus lentus）、バチルス ラテロスポルス（Bacillus laterosporus）、バチルス アルベイ（Bacillus alvei）、バチルス ポピリエ（Bacillus popilliae）、及びバチルス リチェニフォルミス（Bacillus licheniformis）からなる群から選ばれるいずれかであることを特徴とする上記（１）〜（１０）のいずれかに記載のバチルス属細菌の芽胞を形成する方法や、（１２）バチルス属細菌が、バチルス ズブチリス（Bacillus subtilis）ＦＥＲＭ ＢＰ−１０２４４又はその変異株であることを特徴とする上記（１）〜（１１）のいずれかに記載のバチルス属細菌の芽胞を形成する方法に関する。 That is, the present invention includes (1) a step (C) of culturing a Bacillus bacterium in a culture solution having a dissolved oxygen rate of 10% or less, and a Bacterium in a culture solution having a dissolved oxygen rate of more than 10% after step (C). A method of forming a spore of a Bacillus genus bacterium characterized by comprising a step (D) of culturing a genus bacterium, and (2) a Bacillus bacterium cultivated in a culture solution having a dissolved oxygen ratio of 10% or less, logarithmically The method for forming a spore of a Bacillus bacterium according to (1) above, which is a Bacillus bacterium after the growth phase (Log Phase), or (3) a culture solution having a dissolved oxygen ratio of 10% or less The method for forming a Bacillus spore according to (1) above, wherein the Bacillus bacterium to be cultured is a log phase growth phase (Station Phase) or stationary phase (Stage phase) bacterium. (4) Before step (C), the step (A) of culturing Bacillus bacteria in a culture solution having a dissolved oxygen ratio of more than 10% to proliferate the Bacillus bacteria, and / or dissolved oxygen in the culture solution The method for forming a spore of a Bacillus genus bacterium according to any one of (1) to (3) above, further comprising a step (B) of setting the rate to 10% or less, and (5) a culture solution The Bacillus according to any one of (1) to (4) above, wherein the dissolved oxygen ratio of the culture solution is adjusted to 10% or less by adjusting the aeration amount and / or the stirring speed of the culture solution. (6) The culture solution contains any one or more components selected from the group consisting of yeast extract, corn steep liquor, soybean peptone, and concentrated shochu lees. In any of (1) to (5) above (7) The culture solution contains any two or more components selected from the group consisting of yeast extract, corn steep liquor, soybean peptone, and concentrated shochu lees. The method for forming a spore of a Bacillus bacterium described in (6) above, or (8) a culture solution comprising 0.2 to 10.0% by weight of yeast extract and 0.5% of corn steep liquor Bacillus bacteria according to (7) above, containing 2 to 20.0% by weight, 0.2 to 10.0% by weight of soybean peptone, and 0.2 to 20.0% by weight of concentrated shochu lees A method for forming a spore of (9), or (9) a method for forming a spore of a Bacillus bacterium according to any one of (6) to (8) above, wherein the culture solution further contains a saccharide, (10) The culture solution contains 0.05 to 5 sugars. The method for forming a spore of a Bacillus genus bacterium according to (9) above, wherein (11) the bacterium belonging to the genus Bacillus is Bacillus subtilis, Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens, Bacillus pumils, Bacillus lentus, Bacillus laterosporus, Bacillus alvei, Bacillus popilliae, and chen form licilis lichen The method for forming a spore of a bacterium belonging to the genus Bacillus according to any one of the above (1) to (10), wherein (12) the bacterium belonging to the genus Bacillus is a Bacillus subtilis ( Bacillus subtilis) FERM BP-10244 or mutants thereof A method for forming spores Bacillus bacterium according to any one of the above (1) to (11), characterized in that.
本発明のバチルス属細菌の芽胞を形成する方法は、バチルス属細菌の芽胞化を促進させ、芽胞化したバチルス属細菌を高濃度で、かつ効率良く得ることが可能となる。 The method for forming a spore of a Bacillus bacterium according to the present invention promotes the spore formation of a Bacillus bacterium, and makes it possible to efficiently obtain the spore-formed Bacillus bacterium at a high concentration.
本発明のバチルス属細菌の芽胞を形成する方法は、溶存酸素率が１０％以下の培養液でバチルス属細菌を培養する工程（Ｃ）と、工程（Ｃ）の後に、溶存酸素率が１０％より多い培養液でバチルス属細菌を培養する工程（Ｄ）とを含んでいる限り特に制限はない。バチルス属細菌を培養する過程において工程（Ｃ）及び工程（Ｄ）を設けると、培養液中の生細胞のバチルス属細菌は自身が生産するプロテアーゼ等により死滅せずに、効率的に芽胞が形成される。本明細書における溶存酸素率とは、例えば、単位体積当たりの培養液に溶存している酸素の量を、培養液と同温度の単位体積当たりの水に１気圧条件下で溶解し得る酸素の量に対する百分率（％）で表した数値（％）をいう。溶液酸素率を産出するために用いる溶存酸素量は、例えば、卓上型培養装置MDL1000型（ＭＤＬ−６Ｃ）（株式会社丸菱バイオエンジ社製）等の機器を用いて測定することができる。 The method for forming spores of the genus Bacillus of the present invention includes a step (C) of culturing Bacillus bacterium in a culture solution having a dissolved oxygen rate of 10% or less, and a dissolved oxygen rate of 10% after step (C). There is no particular limitation as long as it includes the step (D) of culturing Bacillus bacteria with a larger amount of culture solution. When the step (C) and the step (D) are provided in the process of culturing Bacillus bacteria, the viable Bacillus bacteria in the culture solution are not killed by proteases produced by themselves, and spores are efficiently formed. Is done. The dissolved oxygen ratio in the present specification refers to, for example, the amount of oxygen dissolved in a culture solution per unit volume of oxygen that can be dissolved in water per unit volume at the same temperature as the culture solution under 1 atm. The value (%) expressed as a percentage (%) to the quantity. The amount of dissolved oxygen used to produce the solution oxygen ratio can be measured using, for example, a device such as a desktop culture device MDL1000 (MDL-6C) (manufactured by Maruhishi Bioengineer Co., Ltd.).
溶存酸素率が１０％以下の培養液でバチルス属細菌を培養する工程（Ｃ）における培養液の溶存酸素率は、１０％以下であれば特に制限されないが、バチルス属細菌の芽胞化をより促進させる観点から、８％以下であることが好ましく、５％以下であることがより好ましく、３％以下であることがさらに好ましい。 The dissolved oxygen rate of the culture solution in the step (C) of culturing Bacillus bacteria in a culture solution having a dissolved oxygen rate of 10% or less is not particularly limited as long as it is 10% or less, but further promotes the spore formation of Bacillus bacteria. From the viewpoint of making it possible, it is preferably 8% or less, more preferably 5% or less, and even more preferably 3% or less.
工程（Ｃ）におけるバチルス属細菌は、遅滞期（Ｌａｇ Ｐｈａｓｅ）のバチルス属細菌等、対数増殖期（Ｌｏｇ Ｐｈａｓｅ）以降のバチルス属細菌でなくてもよいが、芽胞化したバチルス属細菌を高濃度で、かつ効率良く得る観点から、対数増殖期（Ｌｏｇ Ｐｈａｓｅ）以降のバチルス属細菌、すなわち、対数増殖期（Ｌｏｇ Ｐｈａｓｅ）及び静止期（Ｓｔａｔｉｏｎａｒｙ Ｐｈａｓｅ）のバチルス属細菌であることが好ましく、対数増殖期（Ｌｏｇ Ｐｈａｓｅ）の後半から静止期（Ｓｔａｔｉｏｎａｒｙ Ｐｈａｓｅ）のバチルス属細菌であることがより好ましい。 The Bacillus bacterium in the step (C) may not be a Bacillus bacterium after the logarithmic growth phase (Log Phase), such as a Bacillus bacterium in the lag phase (Lag Phase). In addition, from the viewpoint of obtaining efficiently, it is preferable that the bacterium belongs to the genus Bacillus after the logarithmic growth phase (Log Phase), that is, the bacterium belonging to the genus Bacillus in the logarithmic growth phase (Log Phase) and stationary phase (Stationary Phase). More preferably, the bacterium belongs to the genus Bacillus from the second half of the phase (Log Phase) to the stationary phase (Stationary Phase).
本発明のバチルス属細菌の芽胞を形成する方法は、工程（Ｃ）の後に工程（Ｄ）を有する。工程（Ｄ）を設けないと、バチルス属細菌の胞子形成率は著しく低下する。工程（Ｄ）における培養液の溶存酸素率は、１０％より多ければ特に制限はされないが、１５％以上であることが好ましく、２０％以上であることがより好ましい。 The method for forming a spore of a Bacillus genus bacterium of the present invention has a step (D) after the step (C). If the step (D) is not provided, the sporulation rate of Bacillus bacteria will be significantly reduced. The dissolved oxygen content of the culture solution in the step (D) is not particularly limited as long as it is greater than 10%, but is preferably 15% or more, and more preferably 20% or more.
工程（Ｄ）におけるバチルス属細菌は、バチルス属細菌の芽胞形成をより促進する観点から、静止期（Ｓｔａｔｉｏｎａｒｙ Ｐｈａｓｅ）以降のバチルス属細菌であることが好ましい。ここでいう静止期とは、静止期のすべての期間である必要はなく、静止期の一部の期間をも含む。 The Bacillus bacteria in the step (D) are preferably Bacillus bacteria after the stationary phase from the viewpoint of further promoting the spore formation of the Bacillus bacteria. The stationary phase here does not need to be the entire period of the stationary period, but includes a part of the stationary period.
本発明に用いるバチルス属細菌は、本発明のバチルス属細菌の芽胞を形成する方法を適用し得る限り特に制限されないが、バチルス ズブチリス（Bacillus subtilis）、バチルス アミロリクエファシエンス（Bacillus amyloliquefaciens）、バチルス パミルス（Bacillus pumils）、バチルス レンタス（Bacillus lentus）、バチルス ラテロスポルス（Bacillus laterosporus）、バチルス アルベイ（Bacillus alvei）、バチルス ポピリエ（Bacillus popilliae）、バチルス リチェニフォルミス（Bacillus licheniformis）であることが好ましく、バチルス ズブチリス（Bacillus subtilis）、バチルス アミロリクエファシエンス（Bacillus amyloliquefaciens）、バチルス リチェニフォルミス（Bacillus licheniformis）等を好ましく例示することができる。 The Bacillus bacterium used in the present invention is not particularly limited as long as the method for forming a spore of the Bacillus bacterium of the present invention can be applied, but Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pamilus. Bacillus pumils, Bacillus lentus, Bacillus laterosporus, Bacillus alvei, Bacillus popilliae, Bacillus licheniformis, Bacillus licheniformis, Bacillus licheniformis, (Bacillus subtilis), Bacillus amyloliquefaciens, Bacillus licheniformis and the like can be preferably exemplified.
また、本発明により得られたバチルス属細菌の芽胞を農園芸用殺菌や殺虫剤に応用する場合には、特許第３５５４５９２号公報や特許弟３５２７５５７号公報に開示されているバチルス ズブチリスＮＣＩＢ１２３７６株、ＮＣＩＢ１２６１６株、ＦＥＲＭ Ｐ−１４６４７株、ＦＥＲＭ Ｐ−１４６４６株、及びこれらの変異株； 特開２００２−２９３７０８号公報、特開２００２−２９１４６７号公報及び特開２００１−１４９０６６号公報に開示されているバチルス ポピリエ ＦＥＲＭ Ｐ−１６８１８株、ＦＥＲＭ Ｐ−１８２５０株、及びこれらの変異株； 特開平１１−２４６３２４号公報に開示されているバチルス アミロリクエファシエンスＦＥＲＭ Ｐ−１６６４１株、バチルス ズブチルス Ｄ７４７株、及びこれらの変異株； さらには、本発明者が発見したＦＥＲＭ ＢＰ−１０２４４株（独立行政法人産業技術総合研究所 特許生物寄託センター（宛名：茨城県つくば市東１丁目１番地１ 中央第６）に平成１７年２月１５日付けで寄託）、ＦＥＲＭ ＡＢＰ−１０２４５株、及びこれらの変異株；などを特に好ましく例示することができる。また、本発明により得られたバチルス属細菌の芽胞を動物等の飼料に応用する場合には、ＷＯ９８／４５４０２に開示されているバチルス パミルスＡ−１株、バチルス パミルスＡ−４株、バチルス レンタスＡ−２株、バチルス レンタスＡ−３株、及びこれらの変異株などを特に好ましく例示することができる。本明細書における「変異株」とは、その親株から誘導しうる変異株をいう。 Moreover, when applying the spores of the genus Bacillus obtained by the present invention to agricultural and horticultural sterilization and insecticides, Bacillus subtilis NCIB12376, NCIB12616 disclosed in Japanese Patent No. 3554592 and Japanese Patent No. 35275557 are disclosed. Strains, FERM P-14647 strain, FERM P-14646 strain, and mutants thereof; Bacillus sporiye disclosed in JP 2002-293708, JP 2002-291467, and JP 2001-149066 FERM P-16818, FERM P-18250, and mutants thereof; Bacillus amyloliquefaciens FERM P-16641, Bacillus subtilis D747, and mutations thereof disclosed in JP-A-11-246324 ; In addition, FERM BP-10244 strain discovered by the present inventor (incorporated administrative agency National Institute of Advanced Industrial Science and Technology patent biological deposit center (address: 1st, 1st, 1st East, Tsukuba City, Ibaraki Prefecture, February 6), February 2005; (Deposited on the 15th), FERM ABP-10245 strain, and mutants thereof, and the like can be particularly preferably exemplified. In addition, when the spores of Bacillus genus bacteria obtained by the present invention are applied to animal feed, the Bacillus pamilus A-1 strain, Bacillus pamilus A-4 strain, Bacillus lentus A disclosed in WO98 / 45402. -2 strain, Bacillus lentus A-3 strain, and mutants thereof can be particularly preferably exemplified. As used herein, “mutant strain” refers to a mutant strain that can be derived from its parent strain.
本発明のバチルス属細菌の芽胞を形成する方法における培養液としては、いずれかのバチルス属細菌が利用できる窒素源と炭素源を含んでいる限り特に制限はされない。 The culture solution in the method for forming a spore of a Bacillus bacterium of the present invention is not particularly limited as long as it contains a nitrogen source and a carbon source that can be used by any Bacillus bacterium.
バチルス属細菌が利用できる窒素源として、例えば、酵母エキス；大豆ペプトン等の各種のペプトン；硝酸塩、アンモニウム塩等の無機窒素源；コーンスティープリカー（以下、「ＣＳＬ」という）、大豆、ポテト，ビート等由来の各種アミノ酸やペプチド；魚肉由来のアミノ酸やペプチド；大豆ホエー；濃縮焼酎かす（焼酎を製造する過程で生じる）；などを例示することができる。これらの中でも、バチルス属細菌の生育及び芽胞形成の観点から、酵母エキス、ＣＳＬ、大豆ペプトン、濃縮焼酎かすからなる群から選ばれるいずれか１種以上を培養液が含有することが好ましく、前述の群から選ばれるいずれか２種以上を培養液が含有することがより好ましく、前述の群から選ばれるいずれか３種以上を培養液が含有することがさらに好ましく、前述の群の４種すべてを培養液が含有することがさらにより好ましい。前述の群の窒素源のいずれか１種、２種、３種又は４種を含有する場合、それぞれの窒素源の含有量は特に制限されないが、酵母エキスは培養液に対して０．２〜１０．０重量％であることが好ましく、０．５〜５．０重量％であることがより好ましく、ＣＳＬは培養液に対して０．５〜２０．０重量％であることが好ましく、１．０〜５．０重量％であることがより好ましく、大豆ペプトンは培養液に対して０．２〜１０．０重量％であることが好ましく、０．５〜５重量％であることがより好ましく、濃縮焼酎かすは培養液に対して０．２〜２０．０重量％であることが好ましく、１．０〜１０．０重量％であることがより好ましい。 Nitrogen sources that can be used by Bacillus bacteria include, for example, yeast extract; various peptones such as soybean peptone; inorganic nitrogen sources such as nitrate and ammonium salt; corn steep liquor (hereinafter referred to as “CSL”), soybean, potato, beet For example, various amino acids and peptides derived from, etc .; amino acids and peptides derived from fish meat; soybean whey; concentrated shochu lees (generated in the process of producing shochu); and the like. Among these, from the viewpoint of growth and spore formation of Bacillus bacteria, the culture solution preferably contains at least one selected from the group consisting of yeast extract, CSL, soybean peptone, and concentrated shochu lees. More preferably, the culture solution contains any two or more selected from the group, more preferably any three or more selected from the aforementioned group, and the culture solution contains all four of the aforementioned groups. Even more preferably, the culture solution contains. When any one, two, three, or four of the nitrogen sources in the aforementioned group is contained, the content of each nitrogen source is not particularly limited, but the yeast extract is 0.2 to It is preferably 10.0% by weight, more preferably 0.5 to 5.0% by weight, and CSL is preferably 0.5 to 20.0% by weight with respect to the culture medium. More preferably, the content is 0.0 to 5.0% by weight, and the soybean peptone is preferably 0.2 to 10.0% by weight and more preferably 0.5 to 5% by weight with respect to the culture solution. Preferably, the concentrated shochu lees is preferably 0.2 to 20.0% by weight, more preferably 1.0 to 10.0% by weight, based on the culture broth.
バチルス属細菌の生育及び芽胞形成の観点から、より具体的には、酵母エキスとして、ＳＫ酵母エキスＳ−２（日本製紙ケミカル株式会社製）を好ましく挙げることができ、ＣＳＬとして、コーンスティープリカー（日本食品化工株式会社）を好ましく挙げることができ、濃縮焼酎かすとして、濃縮焼酎かす液（霧島酒造株式会社製）を好ましく挙げることができ、大豆ペプトンとして、バクテリオンＳＳＰ（マルハ株式会社）を好ましく挙げることができる。 More specifically, from the viewpoint of growth of Bacillus bacteria and spore formation, SK yeast extract S-2 (manufactured by Nippon Paper Chemical Co., Ltd.) can be preferably cited as the yeast extract, and corn steep liquor ( Nihon Shokuhin Kako Co., Ltd.) can be preferably mentioned, and as the concentrated shochu lees, the concentrated shochu lees liquor (manufactured by Kirishima Sake Brewery Co., Ltd.) can be preferably mentioned. Can be mentioned.
なお、バチルス属細菌の芽胞化をより促進させる観点から、ＣＳＬや濃縮焼酎かすの中でも、不溶物を除去したＣＳＬや濃縮焼酎かすが好ましい。 In addition, from the viewpoint of further promoting the spore formation of Bacillus bacteria, among CSL and concentrated shochu, CSL and concentrated shochu from which insoluble matter has been removed are preferable.
ＣＳＬや濃縮焼酎かすの不溶物の除去方法としては、ＣＳＬや濃縮焼酎かすをアルカリで処理し、遠心分離や膜ろ過後上清を回収する方法や、ＣＳＬや濃縮焼酎かすにクエン酸を添加して、その後、ＣＳＬや濃縮焼酎かすを微アルカリに戻し、遠心分離や膜ろ過後上清を回収する方法が挙げられる。より具体的には、ＣＳＬや濃縮焼酎かすの原液に蒸留水を添加し希釈し、さらにアルカリ剤を添加してｐＨ７．０に調整した後、遠心分離や膜ろ過等により上清を回収する方法や、ＣＳＬや濃縮焼酎かすの原液をクエン酸処理、アルカリ処理、加熱処理した後、遠心分離や膜ろ過等をして上清を回収する方法が挙げられる。 Methods for removing CSL and concentrated shochu lees insolubles include treating CSL and concentrated shochu lees with alkali, collecting the supernatant after centrifugation or membrane filtration, or adding citric acid to CSL or concentrated shochu lees. Then, the method of returning CSL and concentrated shochu lees to a slight alkali and collecting the supernatant after centrifugation or membrane filtration can be mentioned. More specifically, a method in which distilled water is added to a stock solution of CSL or concentrated shochu lees to dilute, and an alkali agent is added to adjust to pH 7.0, and then the supernatant is recovered by centrifugation, membrane filtration, or the like. In addition, there may be mentioned a method in which a CSL or concentrated shochu lees stock solution is treated with citric acid, alkali, and heat, and then the supernatant is collected by centrifugation or membrane filtration.
ＣＳＬや濃縮焼酎かすの不溶物の除去量及び処理ＣＳＬや濃縮焼酎かすの培養液への添加量は、特に制限されないが、菌の増殖や芽胞化、集菌時の作業性等に影響することもあるため、ＣＳＬや濃縮焼酎かす全量に対して好ましくは３〜１０重量％、より好ましくは４〜７重量％の不要物（乾物重量において）を除去することができ、また、このＣＳＬや濃縮焼酎かすを培養液全量に対して好ましくは０．５〜２０．０重量％添加することができる。
上記のアルカリ剤としては、水酸化ナトリウム、水酸化カリウム、アンモニア水等、特に制限はされないが、ＣＳＬ溶液や濃縮焼酎かす溶液のｐＨをおおよそ６．５〜８．０程度にまで上昇させると、ＣＳＬや濃縮焼酎かすの不溶物の除去を効率よく行うことができるため好ましい。
The amount of insoluble matter removed from CSL and concentrated shochu lees and the amount of treated CSL and concentrated shochu lees added to the culture solution are not particularly limited, but may affect the growth and spore formation of bacteria, workability during collection, etc. Therefore, it is possible to remove 3 to 10% by weight, more preferably 4 to 7% by weight of unwanted matter (in terms of dry matter weight) with respect to the total amount of CSL and concentrated shochu. Shochu lees can be added in an amount of preferably 0.5 to 20.0% by weight based on the total amount of the culture solution.
The alkali agent is not particularly limited, such as sodium hydroxide, potassium hydroxide, aqueous ammonia, etc., but when the pH of the CSL solution or concentrated shochu solution is increased to about 6.5 to 8.0, This is preferable because the insoluble matter of CSL and concentrated shochu can be efficiently removed.
バチルス属細菌が利用できる炭素源は、バチルス属細菌が資化しうるものである限り特に制限されないが、バチルス属細菌の増殖の促進や芽胞化促進等の観点から、グルコース、ラクトース、ガラクトース、マルトース等の糖類であることが好ましく、グルコース及びラクトースであることがより好ましい。培養液中の糖類の含有量は特に制限されないが、培養液に対して０．０５〜５．０重量％であることが好ましい。 The carbon source that can be used by the bacterium belonging to the genus Bacillus is not particularly limited as long as the bacterium belonging to the genus Bacillus can be assimilated. However, from the viewpoint of promoting the growth of the bacterium belonging to the genus Bacillus or promoting spore formation, glucose, lactose, galactose, maltose, etc. Of saccharides, more preferably glucose and lactose. The content of saccharides in the culture solution is not particularly limited, but is preferably 0.05 to 5.0% by weight with respect to the culture solution.
本発明の工程（Ｃ）の培養液のｐＨは、バチルス属細菌が生育できるｐＨ領域であれば特に限定されない。培養温度もバチルス属細菌が生育できる温度領域であれば特に限定されないが、２５℃〜３５℃の範囲内に調整することが、培養効果面で好ましい。 The pH of the culture solution in the step (C) of the present invention is not particularly limited as long as it is a pH range in which Bacillus bacteria can grow. The culture temperature is not particularly limited as long as it is a temperature range in which Bacillus bacteria can grow, but it is preferably adjusted in the range of 25 ° C to 35 ° C in terms of the culture effect.
工程（Ｃ）におけるその他の培養条件は、培養液の溶存酸素率が１０％以下である限り、特に制限されず、培養液に通気してもよいし、培養液を撹拌してもよいし、培養液に通気しながら培養液を撹拌してもよいが、培養液全体の溶存酸素量の偏りを減少させる観点から、培養液を撹拌することが好ましい。 Other culture conditions in the step (C) are not particularly limited as long as the dissolved oxygen ratio of the culture solution is 10% or less, and the culture solution may be aerated or the culture solution may be stirred. The culture solution may be agitated while aerated in the culture solution. However, it is preferable to agitate the culture solution from the viewpoint of reducing the deviation of the dissolved oxygen amount in the whole culture solution.
工程（Ｃ）における培養時間は、培地の組成や濃度並びに培養する菌株により異なるため一概には言えず、またバチルス属細菌の芽胞化を促進させることができる限り特に制限はされないが、３０分間から１０時間程度、より好ましくは、１〜５時間とすることができる。 The culturing time in the step (C) varies depending on the composition and concentration of the medium and the strain to be cultured, so it cannot be said unconditionally, and is not particularly limited as long as it can promote spore formation of Bacillus bacteria, but from 30 minutes About 10 hours, more preferably 1 to 5 hours.
工程（Ｄ）の培養液は、工程（Ｃ）の培養液と同様である。工程（Ｄ）における培養時間は、培地の組成や濃度並びに培養する菌株により異なるため一概には言えず、特に制限されないが、適宜調整することができる。 The culture solution in step (D) is the same as the culture solution in step (C). The culture time in the step (D) varies depending on the composition and concentration of the medium and the strain to be cultured, so it cannot be generally specified and is not particularly limited, but can be appropriately adjusted.
工程（Ｃ）における溶存酸素率の低い培養液（例えば１０％以下）の調製方法は、特に制限されず、例えば、溶存酸素率の低い水に、窒素源及び炭素源等を添加することにより製造してもよいし、溶存酸素率が１０％以上の培養液でバチルス属細菌を培養している途中で、例えば培養液への通気量及び／又は培養液の撹拌速度を調節すること等によって、培養液への酸素の供給量を、培養液中のバチルス属細菌の酸素の消費量より少なくすることにより、溶存酸素率の低い培養液を得てもよいが、溶存酸素率が１０％以上の培養液でバチルス属細菌を培養している途中で、例えば培養液への通気量及び／又は培養液の撹拌速度を調節すること等によって、培養液への酸素の供給量を、培養液中のバチルス属細菌の酸素の消費量より少なくすることにより、溶存酸素率の低い培養液を得る方法等が作業効率の観点から好ましい。 The method for preparing a culture solution having a low dissolved oxygen rate (for example, 10% or less) in step (C) is not particularly limited, and for example, it is produced by adding a nitrogen source, a carbon source, etc. to water having a low dissolved oxygen rate. In the middle of culturing Bacillus bacteria in a culture solution having a dissolved oxygen ratio of 10% or more, for example, by adjusting the aeration rate to the culture solution and / or the stirring speed of the culture solution, etc. A culture solution having a low dissolved oxygen ratio may be obtained by reducing the amount of oxygen supplied to the culture solution to be less than the amount of oxygen consumed by Bacillus bacteria in the culture solution. During the cultivation of Bacillus bacteria in the culture solution, the amount of oxygen supplied to the culture solution is adjusted by adjusting the aeration rate to the culture solution and / or the stirring speed of the culture solution, for example. Less than oxygen consumption of Bacillus bacteria And by a method such as to obtain a low dissolved oxygen rate culture is preferable from the viewpoint of working efficiency.
工程（Ｄ）における、溶存酸素率が１０％より多い培養液の調製方法や、工程（Ｄ）における培養液の溶存酸素率を１０％より多く維持するは特に制限されないが、例えば、培地に通気することや、培地を撹拌すること等により実現することができる。 In the step (D), there is no particular limitation on the method for preparing the culture solution having a dissolved oxygen ratio of more than 10%, and maintaining the dissolved oxygen ratio of the culture solution in the step (D) more than 10%. This can be realized by, for example, stirring the culture medium.
本発明のバチルス属細菌の芽胞を形成する方法は、工程（Ｃ）と工程（Ｄ）とを含んでいる限り特に制限はされないが、工程（Ｃ）の前に、溶存酸素率が１０％より多い培養液でバチルス属細菌を培養してバチルス属細菌を増殖させる工程（Ａ）、及び／又は、培養液の溶存酸素率を１０％以下にする工程（Ｂ）をさらに含んでいてもよい。これらの中でも、工程（Ａ）、工程（Ｂ）、工程（Ｃ）及び工程（Ｄ）からなる群から選ばれるいずれか３工程以上の工程の組み合わせであって、かつ工程（Ｃ）及び工程（Ｄ）を含む組み合わせが好ましく、工程（Ａ）〜工程（Ｄ）の４工程を含むことがより好ましい。
ここで、工程（Ａ）と工程（Ｂ）を含んでいる場合は、この順序でこれらの工程を含んでいることが好ましい。また、工程（Ａ）と工程（Ｂ）を含んでいる場合、工程（Ａ）と工程（Ｂ）とを個別的に含んでいてもよいし、バチルス属細菌を増殖させながら培養液の溶存酸素率を１０％以下に低下させる、すなわち工程（Ａ）と工程（Ｂ）とを同時並行的に含んでいてもよい。
The method for forming a spore of a Bacillus bacterium of the present invention is not particularly limited as long as it includes the step (C) and the step (D), but before the step (C), the dissolved oxygen ratio is more than 10%. The method may further include a step (A) of culturing Bacillus bacteria in a large amount of culture solution to proliferate the Bacillus bacteria and / or a step (B) of reducing the dissolved oxygen ratio of the culture solution to 10% or less. Among these, it is a combination of three or more steps selected from the group consisting of step (A), step (B), step (C) and step (D), and step (C) and step ( The combination including D) is preferable, and it is more preferable to include four steps of step (A) to step (D).
Here, when the process (A) and the process (B) are included, it is preferable that these processes are included in this order. Moreover, when the process (A) and the process (B) are included, the process (A) and the process (B) may be included individually, or dissolved oxygen in the culture solution while growing Bacillus bacteria. The rate may be reduced to 10% or less, that is, the step (A) and the step (B) may be included simultaneously.
工程（Ａ）における培養時間は、培地の組成や濃度並びに培養する菌株により異なるため一概には言えず、特に制限されないが、バチルス属細菌の芽胞を多量に得る観点からは、バチルス属細菌が十分に増殖するのに必要な培養時間であることが好ましく、例えば、対数増殖期（Ｌｏｇ Ｐｈａｓｅ）以降に達するまでに必要な時間、すなわち、対数増殖期（Ｌｏｇ Ｐｈａｓｅ）、及び静止期（Ｓｔａｔｉｏｎａｒｙ Ｐｈａｓｅ）のいずれかに達するまでに必要な時間等であることが好ましい。 The culturing time in step (A) varies depending on the composition and concentration of the medium and the strain to be cultured, and is not particularly limited. However, from the viewpoint of obtaining a large amount of Bacillus spore, the Bacillus bacterium is sufficient. It is preferable that the culture time is required for growth in the growth phase, for example, the time required to reach after the logarithmic growth phase (Log Phase), ie, the logarithmic growth phase (Log Phase), and stationary phase (Stationary Phase). It is preferable that the time is required to reach any of the above.
工程（Ａ）、工程（Ｂ）の培養液は、工程（Ｃ）の培養液と同様である。工程（Ａ）での培養条件は特に制限されないが、工程（Ａ）はバチルス属細菌を増殖させることを主目的としているため、工程（Ａ）での培養条件はバチルス属細菌の増殖に適した条件であることが好ましく、例えば、培養温度は、２５℃〜３５℃の範囲内に調整することが好ましい。また、工程（Ａ）の培養液における溶存酸素率は、好気性細菌であるバチルス属細菌が増殖しうる限り特に制限はないが、１５％以上であることが好ましく、２０％以上であることがより好ましい。バチルス属細菌を培養液で培養すると、遅滞期（Ｌａｇ Ｐｈａｓｅ）を過ぎ、対数増殖期（Ｌｏｇ Ｐｈａｓｅ）に入った頃から培養液中の溶存酸素率が徐々に低下し始め、その後、急激に溶存酸素率が低下するのが通常である。したがって、工程（Ａ）における培養は、バチルス属細菌が増殖し得る限り通気培養や撹拌培養でなくてもよいが、バチルス属細菌の増殖効率の観点から、通気及び／又は撹拌培養であることが好ましい。 The culture solution in step (A) and step (B) is the same as the culture solution in step (C). Although the culture conditions in the step (A) are not particularly limited, the step (A) mainly aims to grow Bacillus bacteria, so the culture conditions in the step (A) are suitable for the growth of Bacillus bacteria. For example, the culture temperature is preferably adjusted within the range of 25 ° C to 35 ° C. The dissolved oxygen rate in the culture solution of step (A) is not particularly limited as long as Bacillus bacteria which are aerobic bacteria can grow, but it is preferably 15% or more, and preferably 20% or more. More preferred. When Bacillus bacteria are cultured in a culture solution, the dissolved oxygen ratio in the culture solution begins to gradually decrease after the lag phase (Log Phase) has passed and the log growth phase (Log Phase) has been entered, and then rapidly dissolved. It is normal for the oxygen rate to decrease. Therefore, the culture in the step (A) may not be aeration culture or stirring culture as long as Bacillus bacteria can grow, but from the viewpoint of the growth efficiency of Bacillus bacteria, it may be aeration and / or stirring culture. preferable.
工程（Ｂ）における培養液の溶存酸素率を１０％以下にする手段は、特に制限されず、培養液中の酸素等の気体を脱気する等の手段を用いてもよいが、上述のように、バチルス属細菌を増殖する工程で、バチルス属細菌が培養液中の酸素を消費し、培養液中の溶存酸素率が低下することを利用することが、作業効率の観点から好ましい。より具体的にいえば、バチルス属細菌が増殖し、培養液中の溶存酸素率を維持するためには、通気量及び／又は撹拌速度を一定程度上昇させる必要があるような状況下において、通気量及び／又は撹拌速度をそこまでは上昇させない（維持又は低下させることを含む）、すなわち通気量及び／又は撹拌速度を調節することにより、培養液の溶存酸素率を１０％以下にすることが好ましい。また、同様の手段によって、工程（Ｃ）の培養において、培養液の溶存酸素率を低く（例えば１０％以下に）維持することができる。 The means for reducing the dissolved oxygen ratio of the culture broth in the step (B) to 10% or less is not particularly limited, and means such as degassing a gas such as oxygen in the culture broth may be used. In addition, it is preferable from the viewpoint of work efficiency that the Bacillus bacterium consumes oxygen in the culture solution and the dissolved oxygen ratio in the culture solution decreases in the step of growing the Bacillus bacterium. More specifically, in order to allow Bacillus bacteria to grow and maintain the dissolved oxygen rate in the culture solution, the aeration rate and / or stirring rate must be increased to a certain degree. The amount of dissolved oxygen in the culture solution can be reduced to 10% or less by adjusting the amount of aeration and / or stirring speed without increasing (including maintaining or decreasing) the volume and / or stirring speed. preferable. Moreover, the dissolved oxygen rate of a culture solution can be maintained low (for example, 10% or less) in the culture in the step (C) by the same means.
なお、工程（Ｂ）、工程（Ｃ）や工程（Ｄ）の培養液中のバチルス属細菌が増加する場合、減少する場合、維持される場合のいずれの場合も、工程（Ｂ）、工程（Ｃ）や工程（Ｄ）に含まれる。 In addition, when the Bacillus genus bacteria in the culture solution of a process (B), a process (C), or a process (D) increase, when decreasing, in any case of maintaining, a process (B), a process ( C) and step (D).
また、工程（Ａ）〜工程（Ｄ）における培養液は、工程（Ａ）〜工程（Ｄ）のいずれか１工程以上の工程の終了後に集菌して、新たな培養液及び／又は新たな培養容器を用いてもよいし、工程（Ａ）〜工程（Ｄ）のいずれか１工程以上の工程で同じ培養液及び／又は培養容器をそのまま用いてもよいが、作業効率の観点から、各工程で同じ培養液及び培養容器をそのまま用いることが好ましい。また、いずれの工程においても、培養液を適宜補充してもよい。工程（Ａ）〜工程（Ｄ）における培養液の溶存酸素率は、各工程で培養している培養液全体の溶存酸素率を意味しているので、補充後の培養液全体の溶存酸素率が各工程の数値範囲に含まれていさえすれば、後から補充する培養液の溶存酸素率は各工程の溶存酸素率の数値範囲外であってもよい。 Moreover, the culture solution in the step (A) to the step (D) is collected after the completion of any one or more steps of the step (A) to the step (D), and a new culture solution and / or a new one is obtained. A culture vessel may be used, and the same culture solution and / or culture vessel may be used as they are in any one or more of steps (A) to (D). It is preferable to use the same culture solution and culture vessel as they are in the process. Moreover, in any process, you may replenish a culture solution suitably. Since the dissolved oxygen rate of the culture solution in the steps (A) to (D) means the dissolved oxygen rate of the whole culture solution cultured in each step, the dissolved oxygen rate of the whole culture solution after supplementation is As long as it is included in the numerical range of each step, the dissolved oxygen rate of the culture solution to be replenished later may be outside the numerical range of the dissolved oxygen rate of each step.
本発明のバチルス属細菌の芽胞を形成する方法は、本発明を損なわない限り、工程（Ａ）、及び工程（Ｂ）以外にも、任意の工程を含んでいてもよい。ここでいう任意の工程は、工程（Ａ）〜工程（Ｄ）のいずれかと同時に行うような工程であってもよく、バチルス属細菌の芽胞の形成を促進する他の工程（手段）も含まれる。 The method for forming a spore of a Bacillus genus bacterium of the present invention may include an optional step in addition to the step (A) and the step (B) as long as the present invention is not impaired. The optional step mentioned here may be a step that is performed simultaneously with any one of the steps (A) to (D), and includes other steps (means) for promoting the formation of spores of Bacillus bacteria. .
工程（Ｃ）や工程（Ｄ）等の後、遠心分離や膜ろ過等により、バチルス属細菌の芽胞を得ることができる。 After the step (C), the step (D), etc., spores of Bacillus bacteria can be obtained by centrifugation, membrane filtration, or the like.
本発明のバチルス属細菌の芽胞を形成する方法においては、培養液（液体培地）に代えて、固体培地を用いることもできる。この場合、例えば、固体培地で培養して増殖させたバチルス属細菌を、密閉容器内に移し、この密閉容器中の酸素濃度を酸素吸収剤等を用いて低下させて一定時間培養することにより、本発明の方法における工程（Ｃ）と同様の効果が得られる。 In the method for forming the Bacillus spore of the present invention, a solid medium can be used instead of the culture solution (liquid medium). In this case, for example, by transferring the Bacillus bacterium cultivated and grown in a solid medium into a sealed container, and reducing the oxygen concentration in the sealed container using an oxygen absorbent or the like and culturing for a certain period of time, The same effect as in step (C) in the method of the present invention can be obtained.
バチルス属細菌の芽胞を用いれば、製品の製造時、製品の流通時、製品の保存時におけるバチルス属細菌の生存率が高く、雑菌汚染の可能性が低く、かつ菌体を高濃度に含有する各種製剤を製造することができる。このような製品は、製品としての安定性、保存性、効果の面で優れている。 When using Bacillus spore, the viability of Bacillus is high during product production, product distribution, and product storage, and the possibility of contamination is low, and it contains a high concentration of cells. Various formulations can be produced. Such a product is excellent in terms of stability, storage stability, and effect as a product.
本発明の方法により得られたバチルス属細菌の芽胞の用途は、特に制限されないが、例えば、農園芸用の殺菌剤、殺虫剤、動物用下痢治療剤等の、植物や動物等の病害防除剤や、家畜、家禽，魚介類用の飼料及び添加剤等に利用することができる。ここでいう病害とは、本発明の方法により芽胞の得られるいずれかのバチルス属細菌によって防除し得る病害である限り特に制限されない。 The use of the spore of the genus Bacillus obtained by the method of the present invention is not particularly limited. For example, a disease control agent for plants and animals, such as agricultural and horticultural fungicides, insecticides, animal diarrhea treatment agents and the like. It can also be used for feeds and additives for livestock, poultry, and seafood. The disease referred to here is not particularly limited as long as it is a disease that can be controlled by any Bacillus bacterium from which spores are obtained by the method of the present invention.
本発明の植物病害の防除剤におけるバチルス属細菌の菌の濃度は、特に制限されるものではないが、１０００〜２０００倍に希釈した際に、菌体濃度に換算して、１×１０１１ 〜１×１０２ ｃｆｕ／ｍｌ、好ましくは１×１０９〜１×１０４ ｃｆｕ／ｍｌの範囲を好適に例示することができる。また、本発明の植物病害の防除剤には、通常使用される担体、界面活性剤、分散剤、補助剤、酸化防止剤、着色剤、滑剤、紫外線吸収剤、耐電防止剤等をさらに配合することができる。 Although the density | concentration of the microbe of the Bacillus genus bacteria in the plant disease control agent of this invention is not restrict | limited in particular, when it dilutes 1000-2000 times, it converts into cell density | concentration and is 1 * 10 < 11 >-. A range of 1 × 10 2 cfu / ml, preferably 1 × 10 9 to 1 × 10 4 cfu / ml can be suitably exemplified. In addition, the plant disease control agent of the present invention is further blended with commonly used carriers, surfactants, dispersants, adjuvants, antioxidants, colorants, lubricants, ultraviolet absorbers, antistatic agents, and the like. be able to.
固体の担体としては、例えば、炭酸カルシウムや塩化カリウム、硫酸ナトリウム、硫酸カルシウム、硫酸アンモニウム等の無機塩類、クエン酸、リンゴ酸、ステアリン酸等の有機酸及びそれらの塩、乳糖、ショ糖等の糖類等、アルミナ粉、シリカゲル、ゼオライト、ヒドロキシアパタイト、リン酸ジルコニウム、リン酸チタン、酸化チタン、酸化亜鉛、ハイドロタルサイト、カオリナイト、モンモリロナイト、タルク、クレー、珪藻土、ベントナイト、ホワイトカーボン、カオリン、バーミキュライト等を挙げることができる。 Examples of the solid carrier include inorganic salts such as calcium carbonate, potassium chloride, sodium sulfate, calcium sulfate, and ammonium sulfate, organic acids such as citric acid, malic acid, and stearic acid, and salts thereof, and sugars such as lactose and sucrose. Alumina powder, silica gel, zeolite, hydroxyapatite, zirconium phosphate, titanium phosphate, titanium oxide, zinc oxide, hydrotalcite, kaolinite, montmorillonite, talc, clay, diatomaceous earth, bentonite, white carbon, kaolin, vermiculite, etc. Can be mentioned.
界面活性剤および分散剤としては、通常の農薬用製剤に使用できるものであれば特に限定されるものではなく、具体的には、非イオン性界面活性剤としてはソルビタン脂肪酸エステル（Ｃ12〜18），ＰＯＥソルビタン脂肪酸エステル（Ｃ12〜18），ショ糖脂肪酸エステルなどの糖エステル型で界面活性剤、ＰＯＥ脂肪酸エステル（Ｃ12〜18），ＰＯＥ樹脂酸エステル，ＰＯＥ脂肪酸ジエステル（Ｃ12〜18）などの脂肪酸エステル型界面活性剤、ＰＯＥアルキルエーテル（Ｃ12〜18）等のアルコール型界面活性剤、ＰＯＥアルキル（Ｃ8〜12）フェニルエーテル，ＰＯＥジアルキル（Ｃ8〜12）フェニルエーテル，ＰＯＥアルキル（Ｃ8〜12）フェニルエーテルホルマリン縮合物などのアルキルフェノール型界面活性剤、ポリオキシエチレン・ポリオキシプロピレンブロックポリマー，アルキル（Ｃ12〜18）ポリオキシエチレン・ポリオキシプロピレンブロックポリマーエーテルなどのポリオキシエチレン・ポリオキシプロピレンブロックポリマー型界面活性剤、ＰＯＥアルキルアミン（Ｃ12〜18），ＰＯＥ脂肪酸アミド（Ｃ12〜18）などのアルキルアミン型界面活性剤、ＰＯＥ脂肪酸ビスフェニルエーテルなどのビスフェノール型界面活性剤、ＰＯＡベンジルフェニル（またはフェニルフェニル）エーテル，ＰＯＡスチリルフェニル（またはフェニルフェニル）エーテルなどの多芳香環型界面活性剤、ＰＯＥエーテルおよびエステル型シリコンおよびフッ素系界面活性剤などのシリコン系、フッ素系界面活性剤、ＰＯＥヒマシ油，ＰＯＥ硬化ヒマシ油などの植物油型界面活性剤、アニオン性界面活性剤としてはアルキルサルフェート（Ｃ12〜18，Ｎａ，ＮＨ4，アルカノールアミン），ＰＯＥアルキルエーテルサルフェート（Ｃ12〜18，Ｎａ，ＮＨ4，アルカノールアミン），ＰＯＥアルキルフェニルエーテルサルフェート（Ｃ12〜18，ＮＨ4，アルカノールアミン，Ｃａ），ＰＯＥベンジル（またはスチリル）フェニル（またはフェニルフェニル）エーテルサルフェート（Ｎａ，ＮＨ4，アルカノールアミン），ポリオキシエチレン，ポリオキシプロピレンブロックポリマーサルフェート（Ｎａ，ＮＨ4，アルカノールアミン）などのサルフェート型界面活性剤、パラフィン（アルカン）スルホネート（Ｃ12〜22，Ｎａ，Ｃａ，アルカノールアミン），ＡＯＳ（Ｃ14〜16，Ｎａ，アルカノールアミン），ジアルキルスルホサクシネート（Ｃ8〜12，Ｎａ，Ｃａ，Ｍg），アルキルベンゼンスルホネート（Ｃ12，Ｎａ，Ｃａ，Ｍｇ，ＮＨ4 ，アルキルアミン，アルカノール，アミン，シクロヘキシルアミン），モノまたはジアルキル（Ｃ3〜6）ナフタレンスルホネート（Ｎａ，ＮＨ4 ，アルカノールアミン，Ｃａ，Ｍｇ），ナフタレンスルホネート・ホルマリン縮合物（Ｎａ，ＮＨ4），アルキル（Ｃ8〜12）ジフェニルエーテルジスルホネート（Ｎａ，ＮＨ4），リグニンスルホネート（Ｎａ，Ｃa），ＰＯＥアルキル（Ｃ8〜12）フェニルエーテルスルホネート（Ｎａ），ＰＯＥアルキル（Ｃ12〜18）エーテルスルホコハク酸ハーフエステル（Ｎａ）などのスルホネート型界面活性剤、カルボン酸型脂肪酸塩（Ｃ12〜18，Ｎａ，Ｋ，ＮＨ4，アルカノールアミン），Ｎ-メチル-脂肪酸サルコシネート（Ｃ12〜18，Ｎａ），樹脂酸塩（Ｎａ，Ｋ）などＰＯＥアルキル（Ｃ12〜18）エーテルホスフェート（Ｎａ，アルカノールアミン）、ＰＯＥモノまたはジアルキル（Ｃ8〜12）フェニルエーテルホスフェート（Ｎａ，アルカノールアミン），ＰＯＥベンジル（またはスチリル）化フェニル（またはフェニルフェニル）エーテルホスフェート（Ｎａ，アルカノールアミン），ポリオキシエチレン・ポリオキシプロピレンブロックポリマー（Ｎａ，アルカノールアミン），ホスファチジルコリン・ホスファチジルエタノールイミン（レシチン），アルキル（Ｃ8〜12）ホスフェートなどのホスフェート型界面活性剤、カチオン性界面活性剤としてはアルキルトリメチルアンモニウムクロライド（Ｃ12〜18），メチル・ポリオキシエチレン・アルキルアンモニウムクロライド（Ｃ12〜18），アルキル・Ｎ-メチルピリジウムブロマイド（Ｃ12〜18），モノまたはジアルキル（Ｃ12〜18）メチル化アンモニウムクロライド，アルキル（Ｃ12〜18）ペンタメチルプロピレンジアミンジクロライドなどのアンモニウム型界面活性剤、アルキルジメチルベンザルコニウムクロライド（Ｃ12〜18），ベンゼトニウムクロライド（オクチルフェノキシエトキシエチルジメチルベンジルアンモニウムクロライド）などのベンザルコニウム型界面活性剤、両性界面活性剤としてはジアルキル（Ｃ8〜12）ジアミノエチルベタイン、アルキル（Ｃ12〜18）ジメチルベンジルベタイン等のベタイン型界面活性剤、ジアルキル（Ｃ8〜12）ジアミノエチルグリシン、アルキル（Ｃ12〜18）ジメチルベンジルグリシンなどのグリシン型界面活性剤等を例示することができる。これらは、１種単独で、または２種以上を混合して用いることができる。 The surfactant and the dispersant are not particularly limited as long as they can be used in ordinary agricultural chemical preparations. Specifically, as the nonionic surfactant, sorbitan fatty acid ester (C 12-18) is used. ), POE sorbitan fatty acid ester (C 12-18 ), sugar ester type such as sucrose fatty acid ester, surfactant, POE fatty acid ester (C 12-18 ), POE resin acid ester, POE fatty acid diester (C 12-18 ) Fatty acid ester type surfactants, alcohol type surfactants such as POE alkyl ether (C 12-18 ), POE alkyl (C 8-12 ) phenyl ether, POE dialkyl (C 8-12 ) phenyl ether, POE alkylphenol type surfactants such as alkyl (C 8 to 12) phenyl ether formalin condensates, polyoxyethylene Po Polyoxypropylene-block polymers, alkyl (C 12 to 18) polyoxyethylene-polyoxypropylene block polymer surfactants such as polyoxyethylene-polyoxypropylene block polymer ether, POE alkylamine (C 12 to 18), POE fatty Alkylamine type surfactants such as amide (C 12-18 ), bisphenol type surfactants such as POE fatty acid bisphenyl ether, POA benzylphenyl (or phenylphenyl) ether, POA styrylphenyl (or phenylphenyl) ether, etc. Polyaromatic surfactants, silicon-based surfactants such as POE ether and ester-type silicones and fluorosurfactants, fluorosurfactants, vegetable oil-type surfactants such as POE castor oil and POE hydrogenated castor oil, Alkyl sulfates as the emission surfactant (C 12~18, Na, NH 4 , alkanolamine), POE alkyl ether sulfates (C 12~18, Na, NH 4 , alkanolamine), POE alkylphenyl ether sulfate (C 12 to 18, NH 4, alkanolamine, Ca), POE benzyl (or styryl) phenyl (or phenyl phenyl) ether sulfates (Na, NH 4, alkanolamine), polyoxyethylene, polyoxypropylene block polymer sulfates (Na, Sulfate surfactants such as NH 4 , alkanolamine), paraffin (alkane) sulfonate (C 12-22 , Na, Ca, alkanolamine), AOS (C 14-16 , Na, alkanolamine), dialkylsulfosa Kushineto (C 8~12, Na, Ca, Mg), alkyl benzene sulphonate (C 12, Na, Ca, Mg, NH 4, alkyl amines, alkanol, amine, cyclohexylamine), mono- or di (C 3 to 6) naphthalene Sulfonate (Na, NH 4 , alkanolamine, Ca, Mg), naphthalene sulfonate-formalin condensate (Na, NH 4 ), alkyl (C 8-12 ) diphenyl ether disulfonate (Na, NH 4 ), lignin sulfonate (Na, Sulfonate type surfactants such as Ca), POE alkyl (C 8-12 ) phenyl ether sulfonate (Na), POE alkyl (C 12-18 ) ether sulfosuccinic acid half ester (Na), carboxylic acid type fatty acid salts (C 12 ~ 18, Na, K, NH 4, alkanolamine), POE alkyl (C 12-18 ) ether phosphate (Na, alkanolamine) such as N-methyl-fatty acid sarcosinate (C 12-18 , Na), resinate (Na, K), POE mono- or dialkyl (C 8-12 ) Phenyl ether phosphate (Na, alkanolamine), POE benzyl (or styryl) phenyl (or phenylphenyl) ether phosphate (Na, alkanolamine), polyoxyethylene / polyoxypropylene block polymer (Na, alkanolamine), phosphatidylcholine · phosphatidylethanolamine imine (lecithin), alkyl (C 8 to 12) phosphate type surfactants such as phosphates, as the cationic surfactant alkyltrimethylammonium chloride (C 12 to 18), methyl Polyoxyethylene alkyl ammonium chloride (C 12 to 18), alkyl-N-methylpyridinium bromide (C 12 to 18), mono- or di (C 12 to 18) methyl ammonium chloride, alkyl (C 12 to 18) Ammonium type surfactants such as pentamethylpropylenediamine dichloride, benzalkonium type surfactants such as alkyldimethylbenzalkonium chloride (C 12-18 ), benzethonium chloride (octylphenoxyethoxyethyldimethylbenzylammonium chloride), amphoteric interfaces activator dialkyl (C 8 to 12) diamino ethyl betaine as, alkyl (C 12 to 18) betaine-type surfactants dimethylbenzyl betaine, dialkyl (C 8 to 12) diamino ethyl glycine, alkyl (C 12 to 18 ) Glycine-type surfactants such as dimethylbenzylglycine can be exemplified. These can be used individually by 1 type or in mixture of 2 or more types.
補助剤として、例えば、カルボキシメチルセルロース、ポリエチレングリコール、アラビアゴム、澱粉等を挙げることができる。 Examples of the auxiliary agent include carboxymethyl cellulose, polyethylene glycol, gum arabic, starch and the like.
本発明の植物病害の防除剤は、通常の農薬のとり得る形態、例えば、粉剤、水和剤、乳剤、フロアブル剤、粒剤等の形態を採用することができる。 The plant disease control agent of the present invention can adopt forms that can be taken by ordinary agricultural chemicals, such as powders, wettable powders, emulsions, flowables, granules and the like.
本発明の植物病害の防除方法は、上記本発明の植物病害防除剤を用いて植物病害を防除する方法であれば、特に制限されるものではなく、本発明の植物病害防除剤を通常の化学農薬と同様、各種農園芸作物等の植物体や土壌に散布処理等する方法を挙げることができる。また、特開２００１−３０２４０７記載のように、製剤を施設内に送風する送風装置の送風口付近に設置し、送風口から送出される空気とともに農薬を散布することもできる。散布処理に当たっては、本発明の植物病害防除剤を適当量の水等で希釈して使用することができ、散布量としては、バチルス属細菌の菌体濃度に換算して、通常１×１０１１〜１×１０２ ｃｆｕ／ｍｌ、好ましくは１×１０９ 〜１×１０４ ｃｆｕ／ｍｌの範囲とすることができる。 The method for controlling a plant disease of the present invention is not particularly limited as long as it is a method for controlling a plant disease using the plant disease control agent of the present invention. As with agricultural chemicals, there can be mentioned a method of spraying on plants and soil such as various agricultural and horticultural crops. Further, as described in JP-A No. 2001-302407, it can be installed near the blower port of a blower that blows the preparation into the facility, and the pesticide can be sprayed together with the air sent from the blower port. In the spraying treatment, the plant disease control agent of the present invention can be diluted with an appropriate amount of water or the like, and the spraying amount is usually 1 × 10 11 in terms of the concentration of Bacillus bacteria. ˜1 × 10 2 cfu / ml, preferably 1 × 10 9 to 1 × 10 4 cfu / ml.
動物用下痢治療剤等の動物の病害防除剤や、家畜、家禽，魚介類用の飼料等の製造法は、特に制限されないが、例えば、本発明の方法により得られたバチルス属細菌の芽胞を、動物用の試料に混合するなどして製造することができる。本発明の動物の病害の防除剤におけるバチルス属細菌の菌の濃度は、対象とする動物や用いるバチルス属細菌の種類等に左右されるため、特に制限されず、適宜調整することができる。 Methods for producing animal disease control agents such as diarrhea therapeutic agents for animals and feeds for livestock, poultry, and seafood are not particularly limited. For example, spores of Bacillus bacteria obtained by the method of the present invention are used. It can be produced by mixing with a sample for animals. The concentration of the bacterium belonging to the genus Bacillus in the animal disease control agent of the present invention depends on the target animal, the type of the bacterium belonging to the genus Bacillus, and the like, and is not particularly limited and can be adjusted as appropriate.
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。 EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.
酵母エキス（日本製紙ケミカル株式会社製 商品名：ＳＫ酵母エキスＳ−２）１重量％、ＣＳＬ（日本食品化工株式会社製）２重量％、グルコース（和光純薬株式会社製）０．２重量％を含む培地５リットル（ｐＨ７．０に調整）を１０リットル容のジャーファーメンターに入れ、１２１℃で１５分間オートクレーブ滅菌した。その後、バチルス ズブチリス ＦＥＲＭ ＢＰ−１０２４４菌株を前培養した菌懸濁液１０ｍｌを、前述のオートクレーブ滅菌した培地に植菌し、通気量５．０ＳＬ/ｍｌ、攪拌速度３５０ｒｐｍ、培養温度３０℃で、本培養を行った。培養開始２３．５時間後に、攪拌速度を２５０ｒｐｍに変更した。 Yeast extract (Nippon Paper Chemical Co., Ltd., trade name: SK Yeast Extract S-2) 1% by weight, CSL (Nippon Food Chemical Co., Ltd.) 2% by weight, glucose (Wako Pure Chemical Industries, Ltd.) 0.2% by weight 5 liters of medium containing pH (adjusted to pH 7.0) was placed in a 10 liter jar fermenter and autoclaved at 121 ° C. for 15 minutes. Thereafter, 10 ml of the bacterial suspension obtained by pre-culturing Bacillus subtilis FERM BP-10244 strain was inoculated into the aforementioned autoclave-sterilized medium, the aeration rate was 5.0 SL / ml, the stirring speed was 350 rpm, and the culture temperature was 30 ° C. Culture was performed. After 23.5 hours from the start of the culture, the stirring speed was changed to 250 rpm.
表１に、実施例１の培養過程の培養液（培養１日後及び２日後）のｐＨ及び生菌数、並びに培養２日後の菌体を集菌し、凍結乾燥物としたものの重量及び生菌数を示す。 Table 1 shows the pH and the number of viable bacteria of the culture solution (1 day and 2 days after cultivation) and the weight and viable bacteria of the lyophilized product collected from the cells after 2 days of cultivation. Indicates a number.
培養１日後の生菌数は１．４×１０９ｃｆｕ／ｍｌ、ｐＨは８．９であった。培養２日後には、生菌数が１．２×１０９ｃｆｕ／ｍｌ、ｐＨが９．１となった。培養２日後の菌体を集菌し、凍結乾燥することにより得られた凍結乾燥物は、５．３ｇ、生菌数は、７．１×１０１１ｃｆｕ／ｇであった。胞子形成は、培養１日後には３割程度、培養２日後には７割程度に観察された。胞子形成は、Dorner染色法により確認した。すなわち、（１）数滴の蒸留水を入れた試験管に菌体を懸濁して濃厚な液を作り、（２）この菌液にほぼ同量のZiehlの石炭酸フクシン液を加えてよく混和し、沸騰している湯浴中に試験管を入れ、１０分間以上加熱し、（３）加熱染色した１滴をスライドグラスにとり、１白金耳のニグロシン溶液とよく混和し、速やかにできるだけ薄く広げ、（４）そのまま風乾して検鏡した。
生細胞は白く抜けて見え、胞子は赤く染まるため、この方法により胞子形成を確認することができる。
The viable cell count after 1 day of culture was 1.4 × 10 9 cfu / ml, and the pH was 8.9. After 2 days of culture, the viable cell count was 1.2 × 10 9 cfu / ml and the pH was 9.1. The lyophilized product obtained by collecting the cells after 2 days of culture and freeze-drying them was 5.3 g, and the viable cell count was 7.1 × 10 11 cfu / g. Sporulation was observed in about 30% after 1 day of culture and in about 70% after 2 days of culture. Sporulation was confirmed by the Dorner staining method. That is, (1) make a concentrated solution by suspending the cells in a test tube containing a few drops of distilled water, and (2) add almost the same amount of Ziehl's fumarate of carboxylic acid to this solution. Put a test tube in a boiling water bath and heat for 10 minutes or longer. (3) Take 1 drop of heat-stained sample on a slide glass and mix well with Nigrosine solution in 1 platinum ear. (4) Air-dried as it was and microscopically examined.
Viable cells appear white and the spores are dyed red, so that spore formation can be confirmed by this method.
また、この培養の培養期間中の温度、ｐＨ、溶存酸素率（ＤＯ）、攪拌速度のチャート図を図１〜３に示す。図１に示されているように、培養開始５時間後から溶存酸素率が徐々に低下し始め、７時間後から急激に低下した。９時間後から１２時間後まで、溶存酸素率は０％となった後、１２時間４５分後から、急激に上昇した。
［比較例１］
Moreover, the charts of temperature, pH, dissolved oxygen ratio (DO), and stirring speed during the culture period of this culture are shown in FIGS. As shown in FIG. 1, the dissolved oxygen ratio began to gradually decrease after 5 hours from the start of culture, and rapidly decreased after 7 hours. From 9 hours to 12 hours later, the dissolved oxygen ratio became 0%, and then rapidly increased from 12 hours 45 minutes later.
[Comparative Example 1]
培養開始後の攪拌速度を４００ｒｐｍとしたこと（培養開始２３．５時間後に攪拌速度を２５０ｒｐｍに変更したことは実施例１と同様）以外は、実施例１と同条件で培養を行った。 Culturing was performed under the same conditions as in Example 1 except that the stirring speed after the start of culture was 400 rpm (same as Example 1 that the stirring speed was changed to 250 rpm 23.5 hours after the start of culture).
表２に、比較例１の培養過程の培養液（培養１日後及び２日後）のｐＨ及び生菌数、並びに培養２日後の菌体を集菌し、凍結乾燥物としたものの重量及び生菌数を示す。 Table 2 shows the pH and the number of viable bacteria in the culture solution of Comparative Example 1 (1 day and 2 days after cultivation), and the weight and viable bacteria of the lyophilized product collected from the cells after 2 days of cultivation. Indicates a number.
培養１日後の生菌数は１．０×１０９ｃｆｕ／ｍｌ、ｐＨは８．９であった。培養２日後になると生菌数は１．０×１０９ｃｆｕ／ｍｌ、ｐＨは９．０となり、実施例１の場合（培養開始時の撹拌速度が３５０ｒｐｍ）と比較し、若干低い生菌数を示した。凍結乾燥物は、５．２５ｇであり、実施例１とほとんど変わらなかったが、生菌数は、４．８×１０１１ｃｆｕ／ｇとなり、実施例１の場合に比べて約３０％程度少なくなった。胞子形成は、培養１日後には１割程度、培養２日後には６割程度に観察された。 The viable cell count after 1 day of culture was 1.0 × 10 9 cfu / ml, and the pH was 8.9. After 2 days of culture, the viable cell count was 1.0 × 10 9 cfu / ml and the pH was 9.0, which was slightly lower than that in Example 1 (the stirring speed at the start of the culture was 350 rpm). showed that. The lyophilized product was 5.25 g, which was almost the same as that in Example 1, but the viable cell count was 4.8 × 10 11 cfu / g, which was about 30% less than that in Example 1. became. Sporulation was observed in about 10% after 1 day of culture and about 60% after 2 days of culture.
また、この培養の培養期間中の温度、ｐＨ、溶存酸素率（ＤＯ）、攪拌速度のチャート図を図４〜７に示す。図４に示されているように、培養開始５時間３０分後から溶存酸素率が徐々に低下し始め、７時間後から急激に低下した。９時間後から１１時間後まで、最低の溶存酸素率を示したが、３０％程度を示していた。
［比較例２］
Moreover, the charts of temperature, pH, dissolved oxygen ratio (DO), and stirring speed during the culture period of this culture are shown in FIGS. As shown in FIG. 4, the dissolved oxygen rate began to gradually decrease after 5 hours and 30 minutes from the start of culture, and rapidly decreased after 7 hours. From 9 hours to 11 hours later, the lowest dissolved oxygen ratio was shown, but it was about 30%.
[Comparative Example 2]
培養開始後の攪拌速度を４５０ｒｐｍとしたこと（培養開始２３．５時間後に攪拌速度を２５０ｒｐｍに変更したことは実施例１と同様）以外は、実施例１と同条件で培養を行った。 Culturing was carried out under the same conditions as in Example 1 except that the stirring speed after the start of culture was 450 rpm (the stirring speed was changed to 250 rpm after 23.5 hours from the start of culture as in Example 1).
表３に、比較例２の培養過程の培養液（培養１日後及び２日後）のｐＨ及び生菌数、並びに培養２日後の菌体を集菌し、凍結乾燥物としたものの重量及び生菌数を示す。 Table 3 shows the pH and the number of viable bacteria in the culture solution (1 day and 2 days after culturing) in the culture process of Comparative Example 2, and the weight and viable bacteria of the lyophilized product collected from the cells after 2 days of culturing. Indicates a number.
培養１日後の生菌数は１．１×１０９ｃｆｕ／ｍｌ、ｐＨは８．３であった。培養２日後になると生菌数は７．５×１０８ｃｆｕ／ｍｌ、ｐＨは８．６となり、実施例１の場合（培養開始時の撹拌速度が３５０ｒｐｍ）と比較し、特に２日後は生菌数の低下が目立った。凍結乾燥物は、５．６５ｇと３５０ｒｐｍの場合より多くなったが、生菌数は、２．５×１０１１ｃｆｕ／ｇと約６５％程度少なくなった。胞子形成は、培養１日後には１割程度、培養２日後には３割程度に観察された。 The viable cell count after 1 day of culture was 1.1 × 10 9 cfu / ml, and the pH was 8.3. After 2 days of culture, the viable cell count was 7.5 × 10 8 cfu / ml, and the pH was 8.6. Compared to the case of Example 1 (the stirring speed at the start of the culture was 350 rpm), especially after 2 days, The decrease in the number of bacteria was conspicuous. The amount of lyophilized product was larger than that at 5.65 g and 350 rpm, but the viable cell count was about 65% less at 2.5 × 10 11 cfu / g. Sporulation was observed in about 10% after 1 day of culture and about 30% after 2 days of culture.
また、この培養の培養期間中の温度、ｐＨ、溶存酸素率（ＤＯ）、攪拌速度のチャート図を図８〜１１に示す。図８に示されているように、培養開始５時間３０分後から溶存酸素率が低下し始め、８．５時間後から１２時間後まで、最低の溶存酸素率を示したが、５０％程度を示していた。
［比較例３］
Moreover, the charts of temperature, pH, dissolved oxygen ratio (DO), and stirring speed during the culture period of this culture are shown in FIGS. As shown in FIG. 8, the dissolved oxygen ratio started to decrease after 5 hours and 30 minutes from the start of culture, and the minimum dissolved oxygen ratio was exhibited from 8.5 hours to 12 hours, but about 50%. Was showing.
[Comparative Example 3]
培養開始後の攪拌速度を５５０ｒｐｍとしたこと、及び培養開始２３．５時間後の攪拌速度を２００ｒｐｍとしたこと以外は、実施例１と同条件で培養を行った。ただし、機器操作上、培養開始４時間後まで攪拌速度が３５０ｒｐｍとなった。また、溶存酸素率も実測地より若干高い値を示した。 Culturing was performed under the same conditions as in Example 1 except that the stirring speed after the start of culture was 550 rpm and the stirring speed after 23.5 hours after the start of culture was 200 rpm. However, the stirring speed became 350 rpm until 4 hours after the start of the culture in terms of equipment operation. Moreover, the dissolved oxygen ratio also showed a slightly higher value than the measured site.
表４に、比較例３の培養過程の培養液（培養１日後及び２日後）のｐＨ及び生菌数、並びに培養２日後の菌体を集菌し、凍結乾燥物としたものの重量及び生菌数を示す。 Table 4 shows the pH and number of viable bacteria in the culture solution (1 day and 2 days after culturing) of the culture process of Comparative Example 3, and the weight and viable bacteria of the lyophilized product collected from the cells after 2 days of culturing. Indicates a number.
培養１日後の生菌数は１．３×１０９ｃｆｕ／ｍｌ、ｐＨは８．９であった。培養２日になると生菌数は６．６×１０８ｃｆｕ／ｍｌ、ｐＨは９．０となり、実施例１の場合（培養開始時の撹拌速度が３５０ｒｐｍ、培養開始２３．５時間後の攪拌速度が２５０ｒｐｍ）の場合と比較し、特に２日後は生菌数の低下が目立った。凍結乾燥物は、４．０ｇ、生菌数は、４．３×１０１０ｃｆｕ／ｇと約９４％程度少なくなった。胞子形成は、培養１日後には全く認められず、培養１．５日後には２割程度観察されたが、２日後には染色状態が良くなく、明らかでなかった。 The viable cell count after 1 day of culture was 1.3 × 10 9 cfu / ml, and the pH was 8.9. After 2 days of culture, the viable cell count was 6.6 × 10 8 cfu / ml, and the pH was 9.0. In the case of Example 1 (the stirring speed at the start of the culture was 350 rpm, the stirring after 23.5 hours after the start of the culture) Compared with the case where the speed was 250 rpm), the decrease in the number of viable bacteria was particularly noticeable after 2 days. The freeze-dried product was 4.0 g and the viable cell count was 4.3 × 10 10 cfu / g, which was about 94% less. Spore formation was not observed at all after 1 day of culture and was observed about 20% after 1.5 days of culture, but the staining was not good after 2 days and was not clear.
また、この培養の培養期間中の温度、ｐＨ、溶存酸素率（ＤＯ）、攪拌速度のチャート図を図１２〜１４に示す。図１２に示されているように、培養開始５時間後から溶存酸素率が低下し始め、その後８０％程度が３時間程度継続した。 In addition, charts of temperature, pH, dissolved oxygen ratio (DO), and stirring speed during the culture period of this culture are shown in FIGS. As shown in FIG. 12, the dissolved oxygen ratio began to decrease after 5 hours from the start of the culture, and then about 80% continued for about 3 hours.
上記の実施例１、比較例１〜３の培養における溶存酸素率と培養時間の関係、２日後の培養液中の生菌数、その培養液から得られた胞子の重量、胞子の生菌数、胞子形成率を表５に示す。 Relationship between dissolved oxygen ratio and culture time in the culture of Example 1 and Comparative Examples 1 to 3, the number of viable bacteria in the culture solution after 2 days, the weight of the spore obtained from the culture solution, the number of viable bacteria of the spore Table 5 shows the spore formation rate.
表５等の結果から分かるように、培養液の攪拌速度を上げ、対数増殖期（Ｌｏｇ Ｐｈａｓｅ）後半から静止期（Ｓｔａｔｉｏｎａｒｙ Ｐｈａｓｅ）等の増殖期後半から終了期における溶存酸素率を低下させるにつれて、芽胞形成率が上昇することが示され、また、対数増殖期（Ｌｏｇ Ｐｈａｓｅ）後半から静止期（Ｓｔａｔｉｏｎａｒｙ Ｐｈａｓｅ）等の増殖期後半から終了期の一定時間に溶存酸素率を１０％以下とすることにより、培養液中の生菌数を低下させることなく、効率良く芽胞を形成させ、高濃度で芽胞菌体を採取できることが明らかとなった。 As can be seen from the results of Table 5 and the like, as the stirring rate of the culture solution is increased and the dissolved oxygen ratio in the logarithmic growth phase (Log Phase) from the latter half to the stationary phase (Stationary Phase) to the end phase is decreased, It is shown that the rate of spore formation is increased, and the dissolved oxygen ratio should be 10% or less from the latter half of the logarithmic growth phase (Log Phase) to the stationary phase (Stationary Phase) and the like for a certain period of time from the late growth phase to the end phase. Thus, it was revealed that spores can be efficiently formed and spore cells can be collected at a high concentration without reducing the number of viable bacteria in the culture solution.
培養開始９時間後から３時間のみ溶存酸素率が０％となるように撹拌速度を適宜変更し、その他は撹拌速度を４５０ｒｐｍとしたこと以外は、実施例１と同条件で培養を行った。 Culturing was carried out under the same conditions as in Example 1 except that the stirring speed was appropriately changed so that the dissolved oxygen ratio was 0% only for 3 hours after 9 hours from the start of the culture, and that the stirring speed was 450 rpm.
表６に、実施例２の培養過程の培養液（培養１日後及び２日後）のｐＨ及び生菌数、並びに培養２日後の菌体を集菌し、凍結乾燥物としたものの重量及び生菌数を示す。 Table 6 shows the pH and number of viable bacteria in the culture solution (1 day and 2 days after cultivation) and the weight and viable bacteria of the lyophilized product collected from the cells after 2 days of cultivation. Indicates a number.
培養１日後の生菌数は１．１×１０９ｃｆｕ／ｍｌ、ｐＨは８．３であった。培養２日後になると生菌数は１．１×１０９ｃｆｕ／ｍｌ、ｐＨは８．７となった。凍結乾燥物は、６．８ｇ、生菌数は、４．５×１０１１ｃｆｕ／ｇであった。 The viable cell count after 1 day of culture was 1.1 × 10 9 cfu / ml, and the pH was 8.3. After 2 days of culture, the viable cell count was 1.1 × 10 9 cfu / ml and the pH was 8.7. The lyophilized product was 6.8 g, and the viable cell count was 4.5 × 10 11 cfu / g.
また、この培養の培養期間中の温度、ｐＨ、溶存酸素率（ＤＯ）、攪拌速度のチャート図を図１５〜１８に示す。
［比較例４］
In addition, charts of temperature, pH, dissolved oxygen ratio (DO), and stirring speed during the culture period of this culture are shown in FIGS.
[Comparative Example 4]
培養開始８．５時間後から３６．５時間後までのみ溶存酸素率が０％となるように撹拌速度を適宜変更し、その他は撹拌速度を４５０ｒｐｍとしたこと以外は、実施例１と同条件で培養を行った。 The same conditions as in Example 1 except that the stirring speed was appropriately changed so that the dissolved oxygen ratio was 0% only after 8.5 hours to 36.5 hours from the start of the culture, and the stirring speed was 450 rpm. Was cultured.
表７に、比較例４の培養過程の培養液（培養１日後及び２日後）のｐＨ及び生菌数、並びに培養２日後の菌体を集菌し、凍結乾燥物としたものの重量及び生菌数を示す。 Table 7 shows the pH and the number of viable bacteria in the culture solution (1 day and 2 days after culturing) in the cultivation process of Comparative Example 4, and the weight and viable bacteria of the lyophilized product collected from the cells after 2 days of culturing. Indicates a number.
培養１日後の生菌数は２．２×１０８ｃｆｕ／ｍｌ、ｐＨは７．６と実施例２に比べて低かった。培養２日後になると生菌数は２．５×１０８ｃｆｕ／ｍｌ、ｐＨは８．７と低いままであった。凍結乾燥物は、１０．２ｇと多かったが、生菌数は、１．３×１０１１ｃｆｕ／ｇと少なかった。 The viable cell count after 1 day of culture was 2.2 × 10 8 cfu / ml, and the pH was 7.6, which was lower than in Example 2. After 2 days of culture, the viable cell count remained as low as 2.5 × 10 8 cfu / ml and pH was 8.7. The lyophilized product was a large 10.2 g, but the viable cell count was a small 1.3 × 10 11 cfu / g.
また、培養期間中の温度、ｐＨ、溶存酸素率（ＤＯ）、攪拌速度のチャート図を図１９〜２２に示す。 Moreover, the charts of temperature, pH, dissolved oxygen ratio (DO), and stirring speed during the culture period are shown in FIGS.
上記の実施例２及び比較例４の培養における溶存酸素率と培養時間の関係、２日後の培養液中の生菌数、その培養液から得られた胞子の重量、胞子の生菌数、全胞子量を表８に示す。 Relationship between dissolved oxygen ratio and culture time in the culture of Example 2 and Comparative Example 4 above, the number of viable bacteria in the culture solution after 2 days, the weight of spores obtained from the culture solution, the number of viable spores, Table 8 shows the amount of spores.
表８等の結果から分かるように、あくまでも、「対数増殖期（Ｌｏｇ Ｐｈａｓｅ）後半から静止期（Ｓｔａｔｉｏｎａｒｙ Ｐｈａｓｅ）等の増殖期後半から終了期の一定時間」について溶存酸素濃度を１０％以下とすることは、芽胞の形成に好ましいが、対数増殖期後半から静止期以降の培養についても、溶存酸素を０％としたままとするのは、菌体の死滅を促進させ、かつ芽胞の形成にとって好ましくないことが示された。 As can be seen from the results in Table 8, etc., the dissolved oxygen concentration is set to 10% or less for “a certain period of time from the latter half of the logarithmic growth phase (Log Phase) to the stationary phase (Stationary Phase) to the end phase”. This is preferable for the formation of spores, but it is preferable for the culture from the latter half of the logarithmic growth phase to the resting phase to keep the dissolved oxygen at 0% in order to promote the killing of the cells and the formation of the spores. Not shown.
工業的なスケールでも本発明の効果が得られることを確かめるため、以下の実験を行った。
酵母エキス（日本製紙ケミカル株式会社製 商品名：ＳＫ酵母エキスＳ−２）４重量％、グルコース（和光純薬株式会社製）０．２重量％を含む培地６５リットル（ｐＨ７．０に調整）を１００リットル容のジャーファーメンターに入れ、１２１℃で１５分間オートクレーブ滅菌した。その後、実施例１と同じ菌株の前培養液３００ｍｌを前述のオートクレーブした培地に植菌し、培養を開始した。培養開始後経時的に菌濃度（Ａ６６０）を測定し、菌体の増殖を確認した。培養開始４時間後当たりから急速に菌体の増殖が確認されたため、培養開始６時間後から１５時間後まで、溶存酸素濃度が１００分の数ｐｐｍとなるように攪拌速度を保った。この培養における培養液の温度、撹拌速度（ＡＧＩ）、溶存酸素濃度（ｐｐｍ）、ｐＨ、通気量、菌体量（Ｏ．Ｄ．６６０）の経時的な推移を表９に示す。
In order to confirm that the effect of the present invention can be obtained even on an industrial scale, the following experiment was performed.
65 liters of medium (adjusted to pH 7.0) containing 4% by weight of yeast extract (manufactured by Nippon Paper Chemical Co., Ltd., trade name: SK yeast extract S-2) and 0.2% by weight of glucose (manufactured by Wako Pure Chemical Industries, Ltd.) The mixture was placed in a 100 liter jar fermenter and autoclaved at 121 ° C. for 15 minutes. Thereafter, 300 ml of the preculture solution of the same strain as in Example 1 was inoculated into the aforementioned autoclaved medium, and the culture was started. The bacterial concentration (A660) was measured over time after the start of culture to confirm the growth of the bacterial cells. Since the growth of the bacterial cells was confirmed rapidly from about 4 hours after the start of the culture, the stirring speed was kept from 6 hours to 15 hours after the start of the culture so that the dissolved oxygen concentration was a few hundredths of a ppm. Table 9 shows changes over time in the temperature, stirring speed (AGI), dissolved oxygen concentration (ppm), pH, aeration rate, and bacterial cell volume (OD660) in this culture.
溶存酸素濃度が１００分の数ｐｐｍとなるように保った時間を、培養開始６時間後から培養最終の８７時間後までとしたこと以外は、実施例３と同条件で培養を行った。この培養における培養液の温度、撹拌速度（ＡＧＩ）、溶存酸素濃度（ＤＯ：ｐｐｍ）、ｐＨ、通気量、菌体量（Ｏ．Ｄ．６６０）の経時的な推移を表１０に示す。 Culturing was performed under the same conditions as in Example 3, except that the dissolved oxygen concentration was maintained at a few hundredths of a ppm from 6 hours after the start of culture to 87 hours after the end of culture. Table 10 shows the changes over time in the temperature, stirring speed (AGI), dissolved oxygen concentration (DO: ppm), pH, aeration rate, and bacterial cell volume (OD660) in this culture.
また、上記の実施例３及び比較例５の培養における溶存酸濃度と培養時間の関係、その培養液から得られた胞子の重量、胞子の生菌数、全胞子量を表１１に示す。 Table 11 shows the relationship between the dissolved acid concentration and the culture time in the culture of Example 3 and Comparative Example 5, the weight of spores obtained from the culture, the number of viable spores, and the total amount of spores.
表９及び表１１の結果から分かるように、実施例３の培養では、培養中の菌体量は、Ａ６６０が２６．１になるまで増殖し、最終的（８７時間培養時点）にはＡ６６０は２０．６であった。採取された凍結乾燥物は、２０５ｇ／６５リットル培地、生菌数は、１．２×１０１２ｃｆｕ／ｇであった。 As can be seen from the results of Table 9 and Table 11, in the culture of Example 3, the amount of cells in the culture grew until A660 reached 26.1, and finally A660 was 20.6. The collected lyophilized product was a 205 g / 65 liter medium, and the viable cell count was 1.2 × 10 12 cfu / g.
表１０及び表１１の結果から分かるように、比較例５の培養では、培養開始１４時間後にはＡ６６０が１９．３になるまで増殖したが、その後菌体量は減少し、最終的にはＡ６６０は８．６５にまで減少した。採取された凍結乾燥物は、１３３ｇ／６５リットル培地、生菌数は、３．０×１０１１ｃｆｕ／ｇであり、凍結乾燥物の量、生菌数共に実施例３より少なかった。 As can be seen from the results of Table 10 and Table 11, in the culture of Comparative Example 5, A660 grew to 19.3 after 14 hours from the start of the culture, but the amount of cells decreased thereafter, and finally A660. Decreased to 8.65. The collected lyophilized product was 133 g / 65 liter medium, and the viable cell count was 3.0 × 10 11 cfu / g. The amount of the lyophilized product and the viable cell count were less than those in Example 3.
実施例３及び比較例５の培養結果から分かるように、培養スケールや培地組成を変更しても、実施例１、実施例２や比較例４と同様のことがいえることが明らかとなった。 As can be seen from the culture results of Example 3 and Comparative Example 5, it was clarified that the same thing as Example 1, Example 2 and Comparative Example 4 could be said even if the culture scale and medium composition were changed.
酵母エキス（日本製紙ケミカル株式会社製 商品名：ＳＫ酵母エキスＳ−２）０．５重量％、濃縮焼酎かす液（霧島酒造株式会社製）５．０重量％、グルコース（和光純薬株式会社製）０．２重量％を含む培地５リットル（ｐＨ７．０に調整）を１０リットル容のジャーファーメンターに入れ、１２１℃で１５分間オートクレーブ滅菌した。その後、バチルス ズブチリス ＦＥＲＭ ＢＰ−１０２４４菌株を前培養した菌懸濁液１０ｍｌを、前述のオートクレーブした培地に植菌し、通気量５．０ＳＬ/ｍｌ、攪拌速度３５０ｒｐｍ、培養温度３０℃で本培養を行った。なお、培養開始２４時間後に攪拌速度を２５０ｒｐｍに変更した。 Yeast extract (Nippon Paper Chemical Co., Ltd., trade name: SK yeast extract S-2) 0.5% by weight, concentrated shochu liquor (Kirishima Shuzo Co., Ltd.) 5.0% by weight, glucose (Wako Pure Chemical Industries, Ltd.) ) 5 liters of medium containing 0.2 wt% (adjusted to pH 7.0) was placed in a 10 liter jar fermenter and autoclaved at 121 ° C. for 15 minutes. Thereafter, 10 ml of the bacterial suspension in which Bacillus subtilis FERM BP-10244 strain was pre-cultured was inoculated into the aforementioned autoclaved medium, and the main culture was performed at an aeration rate of 5.0 SL / ml, a stirring speed of 350 rpm, and a culture temperature of 30 ° C. went. The stirring speed was changed to 250 rpm 24 hours after the start of the culture.
表１２に、実施例４の培養過程の培養液（培養１日後及び２日後）のｐＨと生菌数、並びに培養２日後の菌体を集菌し、凍結乾燥物としたものの重量と生菌数を示す。 Table 12 shows the pH and the number of viable bacteria in the culture solution (1 day and 2 days after cultivation) in Example 4 and the weight and viable bacteria of the lyophilized product collected from the cells after 2 days of cultivation. Indicates a number.
培養１日後の生菌数は２．１×１０９ｃｆｕ／ｍｌ、ｐＨは８．８であった。培養２日後には、生菌数が２．４×１０９ｃｆｕ／ｍｌ、ｐＨが９．２となった。凍結乾燥物は、８．６５ｇ、生菌数は、１．２×１０１２ｃｆｕ／ｇであった。胞子形成は、培養１日後には１割程度、培養２日後には９割程度に観察された。 The viable cell count after 2.1 days of culture was 2.1 × 10 9 cfu / ml, and the pH was 8.8. After 2 days of culture, the viable cell count was 2.4 × 10 9 cfu / ml and the pH was 9.2. The freeze-dried product was 8.65 g, and the viable cell count was 1.2 × 10 12 cfu / g. Sporulation was observed in about 10% after 1 day of culture and about 90% after 2 days of culture.
培地を、濃縮焼酎かす液（霧島酒造株式会社製）５．０重量％、グルコース（和光純薬株式会社製）０．２重量％を含む培地に変更したこと以外は、実施例４と同条件で培養を行った。 The same conditions as in Example 4 except that the medium was changed to a medium containing 5.0% by weight of concentrated shochu liquor (Kirishima Shuzo Co., Ltd.) and 0.2% by weight of glucose (Wako Pure Chemical Industries, Ltd.). Was cultured.
表１３に、実施例５の培養過程の培養液（培養１日後及び２日後）のｐＨと生菌数、並びに培養２日後の菌体を集菌し、凍結乾燥物としたものの重量と生菌数を示す。 Table 13 shows the pH and the number of viable bacteria in the culture solution (1 day and 2 days after cultivation) and the weight of the lyophilized product collected from the lyophilized product and the viable bacteria. Indicates a number.
培養１日後の生菌数は２．３×１０９ｃｆｕ／ｍｌ、ｐＨは８．２であった。培養２日後には、生菌数が８．５×１０８ｃｆｕ／ｍｌ、ｐＨが９．３となった。ＳＫ酵母エキスＳ−２と混用した実施例４の場合と比較すると、培養２日後の生菌数は低くなった。凍結乾燥物は、３．６５ｇ、生菌数は、９．０×１０１１ｃｆｕ／ｇであった。ＳＫ酵母エキスＳ−２と混用した実施例４の場合と比較すると、生菌数は、２５％減、採取量は５８％減と大幅に少なくなった。 The viable cell count after 1 day of culture was 2.3 × 10 9 cfu / ml, and the pH was 8.2. After 2 days of culture, the viable cell count was 8.5 × 10 8 cfu / ml and the pH was 9.3. Compared to the case of Example 4 mixed with SK yeast extract S-2, the viable cell count after 2 days of culture was low. The lyophilized product was 3.65 g, and the viable cell count was 9.0 × 10 11 cfu / g. Compared with the case of Example 4 mixed with SK yeast extract S-2, the number of viable bacteria was reduced by 25%, and the amount collected was significantly reduced by 58%.
培地を、ＳＫ酵母エキスＳ−２（日本製紙ケミカル株式会社製）１．０重量％、グルコース（和光純薬株式会社製）０．２重量％を含む培地に変更したこと以外は、実施例４と同条件で培養を行った。 Example 4 except that the medium was changed to a medium containing 1.0% by weight of SK yeast extract S-2 (manufactured by Nippon Paper Chemical Co., Ltd.) and 0.2% by weight of glucose (manufactured by Wako Pure Chemical Industries, Ltd.). Culturing was performed under the same conditions.
表１４に、実施例６の培養過程の培養液（培養１日後及び２日後）のｐＨと生菌数、並びに培養２日後の菌体を集菌し、凍結乾燥物としたものの重量と生菌数を示す。 Table 14 shows the pH and the number of viable bacteria in the culture solution (1 day and 2 days after culturing) of the culture process of Example 6 and the weight and viable bacteria of the lyophilized product collected from the cells after 2 days of culturing. Indicates a number.
培養１日後の生菌数は５．１×１０８ｃｆｕ／ｍｌ、ｐＨは８．６であった。培養２日後には、生菌数が６．２×１０８ｃｆｕ／ｍｌ、ｐＨが８．８となった。ＳＫ酵母エキスＳ−２の濃度は高いにも関わらず、濃縮焼酎かすと混用した実施例４の場合と比較して、生菌数は極めて低く、芽胞形成も遅かった。
さらにもう１日培養を継続したところ、生菌数は、４．５×１０８ｃｆｕ／ｍｌ、ｐＨは８．７となった。凍結乾燥物は、２．５２ｇ、生菌数は、６．９×１０１１ｃｆｕ／ｇであり、濃縮焼酎かすと混用した実施例４の場合と比較して、採取量・生菌数共に大幅に少なかった。
The viable cell count after 1 day of culture was 5.1 × 10 8 cfu / ml, and the pH was 8.6. After 2 days of culture, the viable cell count was 6.2 × 10 8 cfu / ml and the pH was 8.8. Although the concentration of SK yeast extract S-2 was high, the number of viable bacteria was extremely low and spore formation was also slow compared to Example 4 mixed with concentrated shochu lees.
When the culture was continued for another day, the viable cell count was 4.5 × 10 8 cfu / ml and the pH was 8.7. The freeze-dried product is 2.52 g, and the viable cell count is 6.9 × 10 11 cfu / g. Compared with the case of Example 4 mixed with concentrated shochu lees, both the amount collected and the viable cell count are greatly increased. There were few.
酵母エキス（日本製紙ケミカル株式会社製 商品名：ＳＫ酵母エキスＳ−２）１．０重量％、ＣＳＬ（日本食品加工株式会社製）２．０重量％、グルコース（和光純薬株式会社製）０．２重量％を含む培地５リットル（ｐＨ７．０に調整）を１０リットル容のジャーファーメンターに入れ、１２１℃で１５分間オートクレーブ滅菌した。その後、バチルス ズブチリス ＦＥＲＭ ＢＰ−１０２４４菌株を前培養した菌懸濁液１０ｍｌを、前述のオートクレーブした培地に植菌し、通気量５．０ＳＬ/ｍｌ、攪拌速度３５０ｒｐｍ、培養温度３０℃で本培養を行った。なお、培養開始２４時間後に攪拌速度を２５０ｒｐｍに変更した。 Yeast extract (trade name: SK yeast extract S-2 manufactured by Nippon Paper Chemical Co., Ltd.) 1.0% by weight, CSL (manufactured by Nippon Food Processing Co., Ltd.) 2.0% by weight, glucose (manufactured by Wako Pure Chemical Industries, Ltd.) 0 5 liters of medium containing 2 wt% (adjusted to pH 7.0) was placed in a 10 liter jar fermenter and autoclaved at 121 ° C. for 15 minutes. Thereafter, 10 ml of the bacterial suspension in which Bacillus subtilis FERM BP-10244 strain was pre-cultured was inoculated into the aforementioned autoclaved medium, and the main culture was performed at an aeration rate of 5.0 SL / ml, a stirring speed of 350 rpm, and a culture temperature of 30 ° C. went. The stirring speed was changed to 250 rpm 24 hours after the start of the culture.
表１５に、実施例７の培養過程の培養液（培養１日後及び２日後）のｐＨと生菌数、並びに培養２日後の菌体を集菌し、凍結乾燥物としたものの重量と生菌数を示す。 Table 15 shows the pH and the number of viable bacteria in the culture solution (1 day and 2 days after culturing) and the weight and viable bacteria of the lyophilized product collected after 2 days of culturing. Indicates a number.
培養１日後の生菌数は９．１×１０８ｃｆｕ／ｍｌ、ｐＨは９．２であった。培養２日後には生菌数が１．３×１０９ｃｆｕ／ｍｌ、ｐＨが９．４となった。凍結乾燥物は、６．５５ｇ、生菌数は、８．０×１０１１ｃｆｕ／ｇであった。胞子形成は、培養１日後には６割程度、培養２日後には９割程度に観察された。 The viable cell count after 1 day of culture was 9.1 × 10 8 cfu / ml, and the pH was 9.2. After 2 days of culture, the viable cell count was 1.3 × 10 9 cfu / ml and the pH was 9.4. The freeze-dried product was 6.55 g, and the viable cell count was 8.0 × 10 11 cfu / g. Sporulation was observed in about 60% after 1 day of culture and about 90% after 2 days of culture.
培地を、ＣＳＬ（日本食品加工株式会社製）２．０重量％、グルコース（和光純薬株式会社製）０．２重量％を含む培地に変更したこと以外は、実施例４と同条件で培養を行った。 Culture under the same conditions as in Example 4 except that the medium was changed to a medium containing 2.0% by weight of CSL (manufactured by Nippon Food Processing Co., Ltd.) and 0.2% by weight of glucose (manufactured by Wako Pure Chemical Industries, Ltd.). Went.
表１６に、実施例８の培養２日後の菌体を集菌し、凍結乾燥物としたものの重量と生菌数を示す。 Table 16 shows the weight and viable cell count of the lyophilized product obtained by collecting the cells after 2 days of culture in Example 8.
凍結乾燥物は、１．１７ｇ、生菌数は、１．８×１０１１ｃｆｕ／ｇであり、実施例７の場合と比較して量・生菌数共に非常に少なかった。 The lyophilized product was 1.17 g, and the viable cell count was 1.8 × 10 11 cfu / g, which was much smaller in both amount and viable cell count than in Example 7.
上記実施例４〜８の結果から、窒素源を２種以上組み合わせて使用すると、窒素源を１種のみ使用する場合に比べて、より大量かつ高生菌数の芽胞菌体を採取することができることが明らかとなった。 From the results of Examples 4 to 8 above, when two or more nitrogen sources are used in combination, a larger amount of spore cells can be collected in a larger amount than when only one nitrogen source is used. Became clear.
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| PL2547348T3 (en) * | 2010-03-17 | 2015-11-30 | Bayer Cropscience Lp | Method for using a bacillus subtilis strain for prophylaxis and treatment of gastro-intestinal conditions |
| KR102545863B1 (en) | 2015-04-09 | 2023-06-20 | 가부시키가이샤 에스디에스 바이오텍크 | Sporulation method of bacillus bacterium |
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| CN113621518B (en) * | 2021-08-23 | 2023-02-28 | 广西南宁佰奥吉生物科技有限公司 | Method for breeding spore bacteria for alcohol decomposition |
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