JP2011004712A - Culture medium for detecting bacillus cereus group - Google Patents
Culture medium for detecting bacillus cereus group Download PDFInfo
- Publication number
- JP2011004712A JP2011004712A JP2009153847A JP2009153847A JP2011004712A JP 2011004712 A JP2011004712 A JP 2011004712A JP 2009153847 A JP2009153847 A JP 2009153847A JP 2009153847 A JP2009153847 A JP 2009153847A JP 2011004712 A JP2011004712 A JP 2011004712A
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- Prior art keywords
- medium
- bacillus cereus
- culture medium
- group
- cereus group
- Prior art date
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Abstract
Description
本発明は、セレウス菌グループ検出用培地に関する。 The present invention relates to a culture medium for detecting Bacillus cereus group.
セレウス菌(Bacillus cereus)は、グラム陽性の有芽胞桿菌であり、一般的に土壌や河川等の自然界に広く分布している。本細菌による汚染範囲は、土壌と関わりの深い食品である穀類、香辛料等をはじめとし、これらを使用することで交差汚染される食品、例えば焼きそば、スパゲッティー等の麺類、おにぎり、焼き飯等の米飯類、グラタン、ピザ、魚介類及びその加工品、食肉及びその加工品、食品原材料、菓子類等、広範囲な食品、環境、臨床材料等の多岐にわたっている。
本細菌による汚染はときには腐敗、変敗の原因となる。また、本細菌は嘔吐型毒素及び下痢型毒素を産生することが知られており、食中毒の原因となることもある。
従って本細菌の制御は食品衛生及び安全の点からも重要である（非特許文献１）。
Bacillus cereus is a gram-positive spore gonococcus and is generally widely distributed in nature such as soil and rivers. The range of contamination by this bacterium includes cereals and spices that are closely related to soil, foods that are cross-contaminated by using these, such as noodles such as fried noodles, spaghetti, rice balls such as rice balls and fried rice , Gratin, pizza, seafood and processed foods thereof, meat and processed foods thereof, food ingredients, confectionery, etc.
Contamination by this bacterium sometimes causes rot and deterioration. In addition, this bacterium is known to produce vomiting toxins and diarrhea toxins, and may cause food poisoning.
Therefore, control of this bacterium is important from the viewpoint of food hygiene and safety (Non-patent Document 1).
一般的にセレウス菌の検出に用いられている培地としては、NGKG寒天培地やMYP寒天培地（非特許文献２及び３）等が知られている。これらの培地には、セレウス菌の性状のひとつである卵黄反応（色素変化）を検出原理のひとつとして利用するため、卵黄が含有されている。これら卵黄含有培地調製方法は、予め卵黄以外の寒天等の培地原料を滅菌し溶解する工程、５０℃程度にまで冷却保温する工程、次いで、この培地に更に採取卵黄を無菌的に添加、混合する工程、そして、このように混合した培地原料をシャーレに分注し、固化させる工程からなっている。
このように、卵黄を添加する際に少なくとも３工程が必要で、手順が煩雑であるが、これは、卵黄成分の熱変性を防ぐためであり、特に冷却保温工程では、採取卵黄を添加する際の培地温度が高すぎると卵黄成分が変性し、一方低すぎても固化等を引き起こするため、この温度の管理が重要であり、熟練的な経験が必要とされている。また、検出原理に用いている採取卵黄の状態は採卵用鶏の種差や個体差、飼育環境等で大きく変動し易いため、採取卵黄の状態によって培地性能も左右されてしまうので、培養後の卵黄反応を有するコロニーの鑑別に経験則的なものが必要とされている。
As a medium generally used for detection of Bacillus cereus, NGKG agar medium, MYP agar medium (Non-patent Documents 2 and 3) and the like are known. These media contain egg yolk in order to use the yolk reaction (pigment change), which is one of the properties of Bacillus cereus, as one of the detection principles. These egg yolk-containing medium preparation methods include a step of previously sterilizing and dissolving medium raw materials such as agar other than egg yolk, a step of cooling and keeping the temperature to about 50 ° C., and then aseptically adding and mixing the collected egg yolk to this medium. It consists of a step and a step of dispensing the medium raw material thus mixed into a petri dish and solidifying it.
As described above, at least three steps are required when adding egg yolk, and the procedure is complicated. This is to prevent heat denaturation of the egg yolk component. If the temperature of the medium is too high, the egg yolk component is denatured. On the other hand, if the temperature is too low, solidification or the like is caused. Therefore, management of this temperature is important, and skilled experience is required. In addition, the state of the collected egg yolk used in the detection principle is likely to fluctuate greatly depending on the species, individual differences, breeding environment, etc. of the egg-collecting chickens, so the medium performance is also affected by the condition of the collected egg yolk. There is a need for empirical rules for distinguishing colonies with reactions.
また、卵黄反応を利用せず、微生物におけるフォスファチジルイノシトール特異的ホスポリーパーゼＣの産生能を利用した検出用培地が知られており、この産生能を有する微生物の一つとしてセレウス菌グループが挙げられている（特許文献１）。そして、フォスファチジルイノシトール特異的ホスポリーパーゼＣの基質である5-ブロモ-4-クロロ-3-インドキシル myo-イノシトール-1-フォスフェートを培地に含有させ、この基質分解によって蛍光・発色することでセレウス菌グループを検出している。しかしながら、この酵素反応を促進させるため、未加熱血清（アルブミン）を添加する際に、上記NGKG寒天培地等の調製の際と同様に少なくとも３工程が必要で、手順が煩雑である。しかも、このグループに属するBacillus anthracisにはこの生産能がないため非蛍光・非発色のコロニーを形成し鑑別が難しい。更に、セレウス菌グループには、食中毒細菌や病原菌も存在しているので、公衆衛生上の観点から、セレウス菌グループについて、常時検出試験を行なう必要があるにも拘らず、この基質は非常に高価であるので価格面で普及が難しく適した検出培地とは言い難い。 In addition, a detection medium that utilizes the ability to produce phosphatidylinositol-specific phospolylipase C in microorganisms without using the yolk reaction is known, and one of the microorganisms having this ability is the Bacillus cereus group. (Patent Document 1). Then, 5-bromo-4-chloro-3-indoxyl myo-inositol-1-phosphate, which is a substrate for phosphatidylinositol-specific phospolipase C, is contained in the medium, and fluorescence and color are developed by this substrate degradation. Detects Bacillus cereus group. However, in order to promote this enzyme reaction, when unheated serum (albumin) is added, at least three steps are required as in the preparation of the above NGKG agar medium and the procedure is complicated. Moreover, since Bacillus anthracis belonging to this group does not have this production ability, it forms non-fluorescent and non-colored colonies and is difficult to distinguish. Furthermore, since there are food poisoning bacteria and pathogenic bacteria in the Bacillus cereus group, this substrate is very expensive from the viewpoint of public health, although it is necessary to always carry out a detection test for the Bacillus cereus group. Therefore, it is difficult to say that it is a suitable detection medium that is difficult to spread in terms of price.
本発明は、検体中のセレウス菌グループの存在を正確に簡単に鑑別でき、しかも培地は安価で調製も簡便であり、更に一般的な食品や飲料、水等の検査や製造工程検査等幅広く利用できるセレウス菌グループ検出用培地及びこれを用いたセレウス菌グループ検出方法を提供することに関する。 The present invention can accurately and easily discriminate the presence of the Bacillus cereus group in a sample, and the medium is inexpensive and easy to prepare. Further, it is widely used for inspection of general foods, beverages, water, etc. and manufacturing process inspections. The present invention relates to a culture medium for detecting Bacillus cereus group and a method for detecting Bacillus cereus group using the same.
本発明者は、斯かる実情に鑑み、セレウス菌グループ検出に特化した培地及びその検出方法を種々検討した結果、卵黄反応を利用しなくとも、培地中に、ポリミキシンＢ、トリメトプリム及びリンコマイシン系抗生物質、α−グルコシダーゼの基質となる５−ブロモ−４−クロロ−３−インドキシル−α−Ｄ−グルコピラノシドを含有させて４成分を併用させることにより、セレウス菌グループ以外の微生物の生育を阻害しつつ、セレウス菌グループの発育に伴ってコロニー及びその周辺が発色し、セレウス菌グループを蛍光下又は目視にて簡単に判別することができ、当該α−グルコシダーゼ基質は安価であるため、一般的な食品や飲料、水等の検査や製造工程検査にも幅広く利用することができることを見出し、本発明を完成させるに至った。 In view of such circumstances, the present inventor has conducted various studies on a culture medium specialized for the detection of Bacillus cereus group and its detection method. As a result, polymyxin B, trimethoprim, and lincomycin system can be used in the culture medium without using the yolk reaction. Inhibiting the growth of microorganisms other than the Bacillus cereus group by containing 5-bromo-4-chloro-3-indoxyl-α-D-glucopyranoside, which is a substrate for antibiotics and α-glucosidase, in combination with four components However, as the Bacillus cereus group develops, the colonies and their surroundings develop color, and the Bacillus cereus group can be easily distinguished under fluorescence or visually, and the α-glucosidase substrate is inexpensive, so And found that it can be widely used for inspection of foods, beverages, water, etc. and manufacturing process inspections, leading to the completion of the present invention. .
すなわち、本発明は、検出可能な遊離性基を有するα−グルコシダーゼ基質、ポリミキシンＢ、トリメトプリム及びリンコマイシン系抗生物質を含有するセレウス菌グループ検出用培地を提供するものである。
また、本発明は、上記セレウス菌グループ検出用培地培地に、検体を接種して培養した後、当該培地上の検出可能なコロニーを判定することを特徴とするセレウス菌の検出方法を提供するものである。
That is, the present invention provides a Bacillus cereus group detection medium containing an α-glucosidase substrate having a detectable free group, polymyxin B, trimethoprim and a lincomycin antibiotic.
In addition, the present invention provides a method for detecting Bacillus cereus, characterized in that a detectable colony on the medium is determined after inoculating and culturing the specimen on the Bacillus cereus group detection medium. It is.
本発明のセレウス菌グループ検出用培地を用いれば、種々の微生物が混在している検体中のセレウス菌グループの存在を的確に効率よく簡単に鑑別ができ、しかも当該培地は安価で調製も簡便である。よって、本発明の培地は、一般的な食品や飲料、水等の検査や製造工程検査にも幅広く利用できる。 By using the Bacillus cereus group detection medium of the present invention, the presence of the Bacillus cereus group in a sample in which various microorganisms are mixed can be accurately and easily distinguished, and the medium is inexpensive and easy to prepare. is there. Therefore, the culture medium of the present invention can be widely used for inspection of general foods, beverages, water, etc. and manufacturing process inspection.
本発明にて検出する対象のセレウス菌グループとは、Bacillus cereus及びこれと遺伝的に近縁関係にあり、生化学的性状も類似する菌である。セレウス菌グループとしては、例えば、Bacillus cereus、Bacillus thuringiensis、Bacillus mycoides及びBacillus anthracis等が挙げられる（非特許文献１参照。）。このうち、Bacillus cereus、Bacillus thuringiensisが、本発明の培地にて鑑別し易いので、好ましい。 The Bacillus cereus group to be detected in the present invention is Bacillus cereus and bacteria that are genetically related to this and have similar biochemical properties. Examples of the Bacillus cereus group include Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, Bacillus anthracis, and the like (see Non-Patent Document 1). Of these, Bacillus cereus and Bacillus thuringiensis are preferable because they can be easily distinguished using the medium of the present invention.
本発明の培地に用いる、検出可能な遊離性基を有するα−グルコシダーゼ基質は、セレウス菌グループが発育する段階で特異的に産出する酵素（α−グルコシダーゼ）と反応して検出可能な遊離性基を遊離する酵素基質であれば特に限定されるものではない。例えば、前記α−グルコシダーゼ基質としては、目視又は蛍光存在下で認識できる色原体化合物又は蛍光性化合物を遊離し得るα−グルコピラノシド又はその塩が挙げられる。この塩としては、ナトリウム、カリウム等のアルカリ金属等が挙げられる。 The α-glucosidase substrate having a detectable free group used in the medium of the present invention is a free group detectable by reacting with an enzyme (α-glucosidase) specifically produced at the stage of development of the Bacillus cereus group. There is no particular limitation as long as it is an enzyme substrate that liberates. For example, the α-glucosidase substrate includes α-glucopyranoside or a salt thereof that can liberate a chromogenic compound or a fluorescent compound that can be recognized visually or in the presence of fluorescence. Examples of the salt include alkali metals such as sodium and potassium.
具体的な前記α−グルコシダーゼ基質としては、５−ブロモ−４−クロロ−３−インドキシル−α−Ｄ−グルコピラノシド（Ｘ−α−グルコシド、青色）、５−ブロモ−６−クロロ−３−インドキシル−α−Ｄ−グルコピラノシド（ＭＡＧＥＮＴＡ−α−グルコピラノシド、赤紫）、５−ブロモ−６−クロロ−３−インドリル−α−Ｄ−グルコピラノシド（赤紫）、６−クロロ−３−インドリル−α−Ｄ−グルコピラノシド（ピンク）、５−ブロモ−３−インドリル−α−Ｄ−グルコピラノシド、４−メチル−ウンベリフェリル−α−Ｄ−グルコピラノシド、ｏ−ニトロフェニル−α−Ｄ−グルコピラノシド、フェニル−α−Ｄ−グルコピラノシド、３−ニトロフェニル−α−Ｄ−グルコピラノシド、４−ニトロフェニル−α−Ｄ−グルコピラノシド、３−インドキシル−α−グルコピラノシドトリハイドレート、ｎ−ヘプティル−α−Ｄ−グルコピラノシド、５−ブロモ−４−クロロ−３−インドキシル−２−アセトアミド−２−デオキシ−α−Ｄ−グルコピラノシド等が挙げられ、このうち鑑別性の点から５−ブロモ−４−クロロ−３−インドキシル−α−Ｄ−グルコピラノシド（以下、「Ｘ−α−Ｇｌｃ」とも云う）が好ましい。 Specific examples of the α-glucosidase substrate include 5-bromo-4-chloro-3-indoxyl-α-D-glucopyranoside (X-α-glucoside, blue), 5-bromo-6-chloro-3-india. Xyl-α-D-glucopyranoside (MAGENTA-α-glucopyranoside, red purple), 5-bromo-6-chloro-3-indolyl-α-D-glucopyranoside (red purple), 6-chloro-3-indolyl-α- D-glucopyranoside (pink), 5-bromo-3-indolyl-α-D-glucopyranoside, 4-methyl-umbelliferyl-α-D-glucopyranoside, o-nitrophenyl-α-D-glucopyranoside, phenyl-α- D-glucopyranoside, 3-nitrophenyl-α-D-glucopyranoside, 4-nitrophenyl-α-D-glucopyranoside 3-indoxyl-α-glucopyranoside trihydrate, n-heptyl-α-D-glucopyranoside, 5-bromo-4-chloro-3-indoxyl-2-acetamido-2-deoxy-α-D-glucopyranoside, etc. Of these, 5-bromo-4-chloro-3-indoxyl-α-D-glucopyranoside (hereinafter also referred to as “X-α-Glc”) is preferable from the viewpoint of distinguishability.
酵素基質の培地中の含有量は、特に限定されないが、０．００１〜１０ｇ／Ｌ、特に０．０１〜１ｇ／Ｌであるのが、検出し易い点で好ましい。 The content of the enzyme substrate in the medium is not particularly limited, but is preferably 0.001 to 10 g / L, particularly 0.01 to 1 g / L from the viewpoint of easy detection.
本発明の培地に用いるセレウス菌グループ以外の微生物（以下、「他の微生物」とも云う）の発育阻止物質としては、少なくともポリミキシンＢ、トリメトプリム及びリンコマイシン系抗生物質を併用することが必要である。これによって、多くのセレウス菌グループ以外の、グラム陽性菌（特にスタフィロコッカス、エンテロコッカス）、グラム陰性菌（特に腸内細菌群、非発酵菌）の発育を阻止することができ、セレウス菌グループの判別が容易となる。なお、当該発育阻止物質にはその塩も含まれる。 It is necessary to use at least polymyxin B, trimethoprim and lincomycin antibiotics as growth inhibitory substances for microorganisms other than the Bacillus cereus group used in the culture medium of the present invention (hereinafter also referred to as “other microorganisms”). As a result, the growth of Gram-positive bacteria (especially Staphylococcus and Enterococcus) and Gram-negative bacteria (especially enterobacteria and non-fermenting bacteria) can be prevented. Discrimination becomes easy. The growth inhibitory substances include salts thereof.
ポリミキシンＢとしては、具体的にはポリミキシンＢ硫酸塩等が挙げられる。ポリミキシンＢの培地中の含有量は、特に限定されないが、１万〜１０００万ｕｎｉｔ／Ｌ、より１万〜１００万ｕｎｉｔ／Ｌであるのが好ましく、特に５万〜５０万ｕｎｉｔ／Ｌであるのが好ましい。
またトリメトプリムとしては、具体的にはトリメトプリム及びこの乳酸塩等が挙げられる。トリメトプリムの培地中の含有量は、特に限定されないが、０．０１〜５００ｍｇ／Ｌ、０．１〜５０ｍｇ／Ｌ、特に１〜５ｍｇ／Ｌであるのが好ましい。
また、リンコマイシン系抗生物質としては、例えば、リンコマイシン、クリンダマイシン及びこれらの塩等が挙げられ、具体的には、リンコマイシン塩酸塩、クリンダマイシン塩酸塩等が挙げられる。これらは単独で又は２種以上を混合して用いてもよい。リンコマイシン系抗生物質の培地中の含有量は、特に限定されないが、０．００１〜１００ｍｇ／Ｌ、より０．０１〜１０ｍｇ／Ｌであるのが好ましく、特に０．５〜２．５ｍｇ／Ｌであるのが好ましい。
Specific examples of polymyxin B include polymyxin B sulfate. The content of polymyxin B in the medium is not particularly limited, but is preferably 10,000 to 10,000,000 units / L, more preferably 10,000 to 1,000,000 units / L, and particularly 50,000 to 500,000 units / L. Is preferred.
Specific examples of trimethoprim include trimethoprim and its lactate. The content of trimethoprim in the medium is not particularly limited, but is preferably 0.01 to 500 mg / L, 0.1 to 50 mg / L, particularly 1 to 5 mg / L.
Examples of lincomycin antibiotics include lincomycin, clindamycin, and salts thereof, and specific examples include lincomycin hydrochloride and clindamycin hydrochloride. You may use these individually or in mixture of 2 or more types. The content of the lincomycin antibiotic in the medium is not particularly limited, but is preferably 0.001 to 100 mg / L, more preferably 0.01 to 10 mg / L, and particularly preferably 0.5 to 2.5 mg / L. Is preferred.
本発明において、更に培地中に含有させる上記以外の他の微生物の発育阻止物質としては、セレウス菌グループに対する発育阻害作用は弱く、他の微生物に対する発育阻害作用があるものが好ましく、培地中の発育阻止物質の含有量を調整することによってこれら微生物の発育を調整できるものが望ましい。
この上記以外の他の微生物の発育阻止物質のうち、抗真菌剤及び／又はグリシンを培地中に含有させるのが、更に広範囲の微生物（特に、酵母等の真菌類やセレウス菌グループ以外のBacillus属菌）の発育を阻止することができ、よりセレウス菌グループの判別が容易となる点から、好ましい。
抗真菌剤としては、例えば、アンホテリシンＢ、ナタマイシン等が挙げられ、アンホテリシンＢが好ましい。これらは単独で又は２種以上を混合して用いてもよい。
抗真菌剤（特にアンホテリシンＢ）の培地中の含有量は、特に限定されないが、０．０１〜１００ｍｇ力価／Ｌ、より０．１〜１０ｍｇ力価／Ｌであるのが好ましく、特に０．１〜５ｍｇ力価／Ｌであるのが好ましい。
グリシンとしては、グリシン又はその塩が挙げられ、その塩とは上記アルカリ金属塩等が挙げられる。
グリシンの培地中の含有量は、特に限定されないが、１〜５０ｇ／Ｌ、より５〜３０ｇ／Ｌであるのが好ましく、特に５〜２０ｇ／Ｌであるのが好ましい。
In the present invention, the other microorganism growth inhibitor other than those mentioned above contained in the medium is preferably one having a weak growth inhibitory action against the Bacillus cereus group and having a growth inhibitory action against other microorganisms. What can adjust the growth of these microorganisms by adjusting the content of the blocking substance is desirable.
Of these other microorganism growth inhibitory substances, the inclusion of an antifungal agent and / or glycine in the medium is a broader range of microorganisms (especially, fungi such as yeast and Bacillus genus other than the Bacillus cereus group). It is preferable from the viewpoint that the growth of the fungus) can be inhibited and the discrimination of the Bacillus cereus group becomes easier.
Examples of the antifungal agent include amphotericin B and natamycin, and amphotericin B is preferable. You may use these individually or in mixture of 2 or more types.
The content of the antifungal agent (particularly amphotericin B) in the medium is not particularly limited, but is preferably 0.01 to 100 mg titer / L, more preferably 0.1 to 10 mg titer / L, 1-5 mg titer / L is preferred.
Examples of glycine include glycine or a salt thereof, and examples of the salt include the alkali metal salts described above.
The content of glycine in the medium is not particularly limited, but is preferably 1 to 50 g / L, more preferably 5 to 30 g / L, and particularly preferably 5 to 20 g / L.
更に、糖アルコール類及び／又は無機塩類を培地中に含有させるのが好ましい。
ここで、糖アルコール類としては、例えば単糖又はオリゴ糖の糖アルコールが挙げられ、エリスリトール、キシリトール、ソルビトール、マンニトール、マルチトール等が挙げられる。このうち、マンニトールを使用するのが、セレウス菌グループの判別が容易になるので、好ましい。これらは単独で又は２種以上を混合して用いてもよい。
糖類の培地中の含有量は、特に限定されないが、１〜５０ｇ／Ｌ、より５〜３０ｇ／Ｌであるのが好ましく、特に５〜２０ｇ／Ｌであるのが好ましい。
また、無機塩類としては、塩化ナトリウム、チオ硫酸ナトリウム等の無機酸金属塩；クエン酸鉄アンモニウム、クエン酸ナトリウム等の有機酸金属塩等が挙げられる。このうち、前記無機酸金属塩が好ましく、そのうち、塩化ナトリウムが、セレウス菌グループの判別が容易になるので、好ましい。これらは単独で又は２種以上を混合して用いてもよい。
無機塩類の培地中の含有量は、特に限定されないが、０．１〜２０ｇ／Ｌ、より１〜１０ｇ／Ｌであるのが好ましく、特に３〜８ｇ／Ｌであるのが好ましい。
Furthermore, it is preferable to contain sugar alcohols and / or inorganic salts in the medium.
Examples of sugar alcohols include monosaccharide or oligosaccharide sugar alcohols such as erythritol, xylitol, sorbitol, mannitol, and maltitol. Of these, the use of mannitol is preferable because it makes it easy to distinguish the Bacillus cereus group. You may use these individually or in mixture of 2 or more types.
The content of the saccharide in the medium is not particularly limited, but is preferably 1 to 50 g / L, more preferably 5 to 30 g / L, and particularly preferably 5 to 20 g / L.
Examples of inorganic salts include inorganic acid metal salts such as sodium chloride and sodium thiosulfate; organic acid metal salts such as ammonium iron citrate and sodium citrate. Of these, the inorganic acid metal salts are preferable, and among them, sodium chloride is preferable because it makes it easy to distinguish the Bacillus cereus group. You may use these individually or in mixture of 2 or more types.
The content of the inorganic salt in the medium is not particularly limited, but is preferably 0.1 to 20 g / L, more preferably 1 to 10 g / L, and particularly preferably 3 to 8 g / L.
本発明の培地は、上記培地成分の他、炭素源、窒素源、ミネラル、ビタミン類等の菌体栄養成分やｐＨ調整剤等の培地成分を配合してもよい。
例えば、炭素源としては、フルクトース、ラクトース、サッカロース等から選ばれる１種以上のもの；窒素源としては、タンパク質分解物、酵母エキス、肉エキス、魚肉エキス等から選ばれる１種以上のもの：このミネラル源としては、銅、亜鉛、マグネシウム、コバルト等から選ばれる１種以上のもの：ビタミン類としては、ニコチン酸、パントテネート、ビオチン、リボフラビン、葉酸等から選ばれる１種以上のものが挙げられる。
The medium of the present invention may contain medium nutrient components such as carbon sources, nitrogen sources, minerals and vitamins, and medium components such as pH adjusters in addition to the above-mentioned medium components.
For example, the carbon source is one or more selected from fructose, lactose, saccharose, etc .; the nitrogen source is one or more selected from proteolysate, yeast extract, meat extract, fish extract, etc .: As the mineral source, one or more selected from copper, zinc, magnesium, cobalt and the like: As the vitamins, one or more selected from nicotinic acid, pantothenate, biotin, riboflavin, folic acid and the like can be mentioned.
また、ｐＨ調整剤に用いる成分としては、例えば、しゅう酸、酢酸、フマル酸、リンゴ酸、乳酸、グルコン酸、酒石酸等の有機酸塩；リン酸、塩酸、硫酸等の無機塩；炭酸ナトリウム、炭酸水素ナトリウム等の炭酸塩；水酸化ナトリウム等の水酸化物；アンモニア又はアンモニア水；クエン酸アミン類；低級アルカノールアミン類；アルギニン、リジン等の塩基性アミノ酸等が挙げられる。これらは単独で又は２種以上を混合して用いてもよい。このとき、培地のｐＨを５〜８、特に６．５〜７．７になるように調整できるものが好ましい。 Examples of components used in the pH adjuster include organic acid salts such as oxalic acid, acetic acid, fumaric acid, malic acid, lactic acid, gluconic acid and tartaric acid; inorganic salts such as phosphoric acid, hydrochloric acid and sulfuric acid; sodium carbonate, Examples thereof include carbonates such as sodium hydrogen carbonate; hydroxides such as sodium hydroxide; ammonia or ammonia water; citrate amines; lower alkanolamines; basic amino acids such as arginine and lysine. You may use these individually or in mixture of 2 or more types. At this time, what can adjust the pH of a culture medium to 5-8, especially 6.5-7.7 is preferable.
更に、本発明の培地に、固体又は半固体培地にするため、ゼラチン、寒天、キサンタンガム、ローカストビーンガム、グアーガム、カラギーナン等の天然由来のもの又はヒドロキシエチルセルロース等の合成由来のもの等の固体化成分やゲル化成分を含有させてもよい。これらは単独で又は２種以上を混合して用いてもよい。 Furthermore, in order to obtain a solid or semi-solid medium in the medium of the present invention, solidified components such as gelatin, agar, xanthan gum, locust bean gum, guar gum, carrageenan and other naturally derived substances and synthetically derived substances such as hydroxyethyl cellulose Or a gelling component. You may use these individually or in mixture of 2 or more types.
また、本発明の培地に、繊維質吸水性シート等の繊維質液体吸収素材を使用して、液体培地を吸収させ、これを簡易培地として用いてもよい。この繊維としては、例えば、植物、動物等由来の天然繊維、化学合成、ガラス繊維等由来の化学繊維等が挙げられ、また繊維をシート状にした不織布が好ましい。
簡易培地としては、例えば、特開昭５７−５０２２００号公報、特開平３−１５３７９号公報、特開平２−６５７９８号公報、特開平６−１８１７４１号公報、特開平９−１９２８２号公報及び特開２０００−３２５０７２公報に記載の調製方法にて作製された簡易培地が挙げられる。
簡易培地の具体的な一例としては、（ａ）水及びアルコールに可溶な接着剤、（ｂ）水に可溶でアルコールに不溶なゲル化剤、及び（ｃ）菌体栄養成分を含有する培地組成物を、該ゲル化剤より大きいメッシュを有する繊維状吸水性シートに担持させた簡易培地（特開平９−１９２８２号公報）や（ａ）水及びアルコールに可溶な接着剤０．０１〜０．４重量％、（ｂ）水に可溶でアルコールに不溶なゲル化剤、及び（ｃ）菌体栄養成分を含有するアルコール懸濁液を、防水性平板上に載置された該ゲル化剤の粒径より大きいメッシュを有する繊維質吸水性シートに含浸させ、これをアルコールの急速な蒸発を抑制しつつ乾燥して、吸水性シートを防水性平板に固着させてなる簡易培地（特開２０００−３２５０７２公報）等が挙げられる。
上記水及びアルコールに可溶な接着剤としては、ヒドロキシプロピルセルロース、ポリビニルピロリドン等が挙げられる。また、上記水に可溶でアルコールに不溶なゲル化剤としては、上記の固体化成分やゲル化成分の例示物等が挙げられる。当該ゲル化剤の平均粒径は０．５〜５０μｍが好ましい。
Moreover, the liquid culture medium may be absorbed into the culture medium of the present invention by using a fibrous liquid absorbent material such as a fibrous water absorbent sheet, and this may be used as a simple culture medium. Examples of the fibers include natural fibers derived from plants, animals, and the like, chemical fibers derived from chemical synthesis, glass fibers, and the like, and a nonwoven fabric in which the fibers are formed into a sheet shape is preferable.
Examples of the simple culture medium include, for example, JP-A-57-502200, JP-A-3-15379, JP-A-2-65798, JP-A-6-181741, JP-A-9-19282, and JP-A-9-19282. The simple culture medium produced with the preparation method of 2000-325072 gazette is mentioned.
Specific examples of the simple medium include (a) an adhesive that is soluble in water and alcohol, (b) a gelling agent that is soluble in water and insoluble in alcohol, and (c) a cell nutrient component. A simple culture medium (Japanese Patent Laid-Open No. 9-19282) in which a medium composition is supported on a fibrous water-absorbent sheet having a mesh larger than the gelling agent, or (a) an adhesive 0.01 soluble in water and alcohol -0.4% by weight, (b) a gelling agent that is soluble in water and insoluble in alcohol, and (c) an alcohol suspension containing cell nutrients is placed on a waterproof plate. A simple culture medium in which a fibrous water-absorbent sheet having a mesh larger than the particle size of the gelling agent is impregnated and dried while suppressing rapid evaporation of alcohol, and the water-absorbent sheet is fixed to a waterproof flat plate ( JP, 2000-325072, A) etc. are mentioned.
Examples of the water- and alcohol-soluble adhesive include hydroxypropyl cellulose and polyvinyl pyrrolidone. In addition, examples of the gelling agent that is soluble in water and insoluble in alcohol include the above-mentioned solidifying components and examples of gelling components. The average particle diameter of the gelling agent is preferably 0.5 to 50 μm.
本発明の培地の形態は、特に限定されず、例えば、液体培地、寒天培地及びシート状簡易培地等が挙げられる。このときの作製方法としては、例えば、上記各培地組成成分に精製水等を添加し混合攪拌した後、オートクレーブ等で滅菌し、これを滅菌シャーレ等に分注し、冷却又は放冷する方法；上記各培地成分にアルコールや精製水等を添加し混合攪拌した後、繊維質液体吸収素材を収納した容器（プラスチック、ガラス等）に分注し、ガンマ線照射滅菌する方法等が挙げられる。アルコールとしては、例えばエタノール、２−プロパノール等が挙げられる。 The form of the culture medium of this invention is not specifically limited, For example, a liquid culture medium, an agar culture medium, a sheet-like simple culture medium, etc. are mentioned. As a preparation method at this time, for example, after adding purified water or the like to each of the above-mentioned medium composition components, mixing and stirring, sterilizing with an autoclave or the like, dispensing this into a sterile petri dish or the like, and cooling or allowing to cool; Examples include a method of adding alcohol, purified water, and the like to each medium component described above, mixing and stirring, and dispensing the mixture into a container (plastic, glass, etc.) containing a fibrous liquid absorbent material and sterilizing with gamma rays. Examples of the alcohol include ethanol and 2-propanol.
本発明のセレウス菌グループの検出方法は、上記で得られた検出用培地に、検体を接種して所定の条件にて培養した後、当該培地上の検出可能なコロニーを判定する。
ここで、「検出可能なコロニー」とは、目視又は蛍光存在下で確認できる特定の色調を有する形成されたコロニーを云い、当該コロニーを判別し、セレウス菌グループの存在を判定する。
In the method for detecting a Bacillus cereus group according to the present invention, the detection medium obtained above is inoculated with a sample and cultured under predetermined conditions, and then a detectable colony on the medium is determined.
Here, the “detectable colony” refers to a formed colony having a specific color tone that can be confirmed visually or in the presence of fluorescence, distinguishing the colony, and determining the presence of a Bacillus cereus group.
検体としては、特に限定されないが、食品懸濁液、調理場や調理器具等の環境ふき取り検体あるいは増菌用培地で培養した培養液、土壌懸濁液、河川水、飲料水等が用いられる。
この検体は、そのまま又はこれを濃縮或いは希釈して本発明の培地に接種して培養してもよい。このとき、接種の際、１０〜１０-10倍程度に、検体を濃縮又は希釈するのが、コロニー数測定の点から好ましい。
また、接種法は、特に限定されないが、平板塗抹法、画線塗抹法、メンブレンフィルター法（検体ろ過後のフィルターを培地に乗せて培養する方法）が、好ましい。
The sample is not particularly limited, and food suspensions, environmentally wiped samples such as cooking places and cooking utensils, culture broths cultured in an enrichment medium, soil suspension, river water, drinking water, and the like are used.
This specimen may be cultured as it is or after inoculating or concentrating it in the medium of the present invention. At this time, it is preferable from the viewpoint of colony count to concentrate or dilute the specimen to about 10 to 10 −10 times at the time of inoculation.
The inoculation method is not particularly limited, but a flat plate smear method, a streak smear method, and a membrane filter method (a method in which a filter after specimen filtration is cultured on a medium) is preferable.
培養温度は、特に限定されないが、セレウス菌グループの生育に適した温度である２５〜４２℃、特に３０〜３８℃が好ましい。また、培養日数は、特に限定されないが、１８〜３６時間、特に１８〜３０時間であるのが好ましい。培養は、静置して好気的に行なうのが好ましい。 The culture temperature is not particularly limited, but is preferably 25 to 42 ° C, particularly 30 to 38 ° C, which is a temperature suitable for growth of the Bacillus cereus group. Moreover, although culture | cultivation days are not specifically limited, It is preferable that it is 18 to 36 hours, especially 18 to 30 hours. Cultivation is preferably performed by standing and aerobic.
培地中に形成されたセレウス菌グループのコロニー（その周辺も含む）の色は、用いる発色酵素基質によって異なるが、Ｘ−α−Ｇｌｃの場合には、薄青〜青、青緑の色調であり、斯かる色調の存在によってセレウス菌グループのコロニーであると判別する一方で、斯かる色調の存在が認められない場合には、セレウス菌グループのコロニーではないと判別する。このようにして、検体中のセレウス菌グループの存在の有無やコロニー数を判定する。
ここで、蛍光下とは、蛍光灯の照射下での目視を含む意味であるが、例えばＸ−α−Ｇｌｃの場合の特定の波長（３８０〜５７０ｎｍ）で検出（吸光度測定）を行なってもよい。吸光度測定の場合には、菌体数と吸光度が比例するので、吸光度が高いほどセレウス菌グループは多く存在すると判定したり、標準物と比較して、セレウス菌グループの存在量を定量してもよい。一方、吸光度がコントロール（被検体無添加）と比較してほとんど同じ場合にはセレウス菌グループが存在しないと判定してもよい。
The color of the colony of the Bacillus cereus group (including its surroundings) formed in the medium varies depending on the chromogenic enzyme substrate used, but in the case of X-α-Glc, the color tone is light blue to blue and blue-green. On the other hand, if it is determined that the color tone is present, it is determined that it is not a colony of the Bacillus cereus group. In this way, the presence / absence of the Bacillus cereus group in the specimen and the number of colonies are determined.
Here, under fluorescence is meant to include visual observation under illumination of a fluorescent lamp. For example, even when detection (absorbance measurement) is performed at a specific wavelength (380 to 570 nm) in the case of X-α-Glc. Good. In the case of absorbance measurement, since the number of cells and the absorbance are proportional, it can be determined that the higher the absorbance, the greater the number of the Bacillus cereus group, or the amount of the Bacillus cereus group can be quantified compared to the standard. Good. On the other hand, if the absorbance is almost the same as that of the control (no subject added), it may be determined that the Bacillus cereus group does not exist.
なお、セレウス菌グループのうち、Bacillus cereus、Bacillus thuringiensis、Bacillus mycoides及びBacillus anthracisの４種を区別する場合には、本発明の培地にて検出後、非特許文献１等公知の区別方法にて行なえばよい。 In the case of distinguishing four types of Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, and Bacillus anthracis from the Bacillus cereus group, it can be performed by a known differentiation method such as Non-Patent Document 1 after detection with the medium of the present invention. That's fine.
以下に実施例を挙げて本発明を具体的に説明するが、本発明はこれら実施例に限定されるものではない。 EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.
試験例１：本発明寒天培地
〔培地の作製〕
各培地組成を表１〜３に示した。
本発明培地の調製方法：１リットル使用量の培地組成物を1Lの精製水に加え、100℃、20分間加温溶解し、良く撹拌後、プラスチックシャーレ(90φmm)に20mLずつ分注して培地が固まるまで静置した。
NGKG寒天培地の調製方法：1リットル使用量の培地組成物を900mLの精製水に溶解し、121℃、15分間高圧蒸気滅菌後、約50℃にて保温した。無菌的に採取した20mLの卵黄を80mLの滅菌生理食塩水に加え、良く混合後その全量を予め滅菌保温したNGKG寒天培地へ加え良く撹拌後、プラスチックシャーレに20mLずつ分注して培地が固まるまで静置した。
トリプトソイ寒天（ＴＳＡ）培地の調製方法：１リットル使用量の培地組成物を1Lの精製水に加え、121℃、15分間高圧蒸気滅菌し良く撹拌後、プラスチックシャーレ(90φmm)に20mLずつ分注して培地が固まるまで静置した。
Test Example 1: Agar medium of the present invention [Preparation of medium]
Each medium composition is shown in Tables 1-3.
Preparation method of the medium of the present invention: Add 1 L of the medium composition to 1 L of purified water, dissolve by heating at 100 ° C. for 20 minutes, stir well, then dispense 20 mL each into a plastic petri dish (90 φmm). Let stand until it hardens.
Preparation method of NGKG agar medium: 1 liter of the medium composition was dissolved in 900 mL of purified water, autoclaved at 121 ° C. for 15 minutes and then kept at about 50 ° C. Add 20 mL of egg yolk collected aseptically to 80 mL of sterile physiological saline, mix well, add the whole volume to NGKG agar medium that has been pre-sterilized and kept warm, and then dispense 20 mL each into a plastic petri dish until the medium solidifies. Left to stand.
Preparation method of trypsoy agar (TSA) medium: Add 1 liter of medium composition to 1 L of purified water, autoclaved at 121 ° C for 15 minutes under high pressure steam sterilization, and dispense 20 mL each into a plastic petri dish (90 mm). The medium was allowed to stand until solidified.
〔菌株の供試〕
供試菌株はトリプトソイブイヨンで24時間前培養したものを用い、これを滅菌0.05％寒天加生理食塩水で10倍段階希釈しミクロプランター法（ミクロプランター（佐久間製作所））及びMiles-Misra法（新 細菌培地学講座−上− ＜第二販＞ 182−192頁 株式会社近代出版 １９８６年）により本発明培地に接種した。
[Testing of strains]
The test strain used was pre-cultured with tryptic soy bouillon for 24 hours, and this was diluted 10-fold with sterile 0.05% agar physiological saline and microplanter method (Microplanter (Sakuma Seisakusho)) and Miles-Misra method ( New Bacteriological Studies Course-Top-<Second Sales> 182-192 Modern Publishing Co., Ltd. (1986)) inoculated the medium of the present invention.
〔培養結果〕
表４に示すように、ミクロプランター法を用いて各菌株を供試し35℃、24時間培養したところ、本発明培地ではB.cereusの良好な発育及び青色の発色を認めた。また、B.cereusと生化学的性状が同一でセレウス菌グループの１種ある、B.thuringiensisにおいてもB.cereus同様、良好な発育及び青色の発色を認めた。その他の菌株については発育が抑制される、あるいは発育しても無色のコロニーであった。
[Culture results]
As shown in Table 4, each strain was tested using the microplanter method and cultured at 35 ° C. for 24 hours. As a result, good growth of B. cereus and blue color development were observed in the culture medium of the present invention. B. thuringiensis, which has the same biochemical properties as B. cereus and is a member of the Bacillus cereus group, also showed good growth and blue coloration as in B. cereus. Other strains were growth-suppressed or colorless colonies even when grown.
表５に示すように、Miles-Misra法により各菌株を供試し35℃、24時間培養したところ、本発明培地ではB.cereus及びB.thuringiensisにおいて、良好な発育及び青色の発色を認めた。
よって、本発明培地は、簡便に調製でき、かつ得られた本発明培地を用いれば、セレウス菌グループを他の微生物と明確に簡単に判別することができた。
As shown in Table 5, when each strain was tested and cultured at 35 ° C. for 24 hours by the Miles-Misra method, good growth and blue color development were observed in B.cereus and B.thuringiensis.
Therefore, the culture medium of the present invention can be easily prepared, and if the obtained culture medium of the present invention is used, the Bacillus cereus group can be clearly distinguished from other microorganisms.
試験例２
〔培地の作製〕
本発明の簡易培地の調製方法としては、表６に示す本発明簡易培地組成物をエタノール1000mLに懸濁し、このエタノール懸濁液1mLをコットンシート（50φmm）を格納した容器（50φmm）に分注した後、２段に重ね、非開放空間で徐々に一夜乾燥した後、蓋をした。
本培地を乾燥剤とともにアルミ包材に密封包装した後、表面線量１０〜２０ｋＧｙのガンマ線照射を行って滅菌した。
また、対照に用いたNGKG寒天培地は試験例1と同様の方法で作製した。培地組成を表６に示した。
Test example 2
[Preparation of medium]
As a method for preparing the simple medium of the present invention, the simple medium composition of the present invention shown in Table 6 is suspended in 1000 mL of ethanol, and 1 mL of this ethanol suspension is dispensed into a container (50 φmm) containing a cotton sheet (50 φmm). After that, it was piled up in two steps, gradually dried overnight in a non-open space, and then covered.
The medium was hermetically packaged in an aluminum wrapping material together with a desiccant and then sterilized by gamma irradiation with a surface dose of 10 to 20 kGy.
Further, the NGKG agar medium used as a control was prepared in the same manner as in Test Example 1. The medium composition is shown in Table 6.
〔菌株の供試〕
表７に示す供試菌株はトリプトソイブイヨンで24時間前培養したものを用い、これを滅菌生理食塩水で10倍段階希釈し、１mLを本発明簡易培地及び滅菌空シャーレに接種した。また0.1mLをNGKG寒天培地へ接種しコンラージ棒を用いて塗抹を行った。滅菌空シャーレには予め滅菌し、保温したトリプトソイ寒天培地にて混釈培養を行った。
[Testing of strains]
The test strains shown in Table 7 were pre-cultured for 24 hours in tryptic soy bouillon, which was diluted 10-fold with sterilized physiological saline, and 1 mL was inoculated into the simple medium of the present invention and a sterilized empty petri dish. Moreover, 0.1 mL was inoculated on the NGKG agar medium and smeared using a conage rod. Sterile empty petri dishes were sterilized in advance and cultured in a tryptic soy agar medium.
〔培養結果〕
表７に示すように、本発明簡易培地に、各菌株を供試し35℃、24時間培養したところ、本発明簡易培地ではB.cereusの良好な発育及び青色の発色を認めた。また、B.cereusと生化学的性状が同一である、B.thuringiensisにおいてもB.cereus同様、良好な発育及び青色の発色を認めた。その他の菌株については発育が抑制される、あるいは発育しても無色のコロニーであった。
よって、本発明培地は、簡便に調製でき、かつ得られた本発明培地を用いれば、セレウス菌グループを他の微生物と明確に簡単に鑑別することができた。
[Culture results]
As shown in Table 7, when each strain was tested in the simple medium of the present invention and cultured at 35 ° C. for 24 hours, good growth of B. cereus and blue color development were observed in the simple medium of the present invention. In addition, B. thuringiensis, which has the same biochemical properties as B. cereus, also showed good growth and blue color development as in B. cereus. Other strains were growth-suppressed or colorless colonies even when grown.
Therefore, the medium of the present invention can be easily prepared, and the obtained medium of the present invention can be used to clearly distinguish the Bacillus cereus group from other microorganisms.
以上のことから、検査試料中のセレウス菌グループの有無を的確に効率よく簡単に鑑別できる。しかも当該培地は安価で調製も簡便であった。このため、当該倍培地を、一般的な食品や飲料、水等の検査や製造工程検査等幅広く利用できる。 From the above, the presence or absence of the Bacillus cereus group in the test sample can be identified accurately, efficiently and simply. Moreover, the medium was inexpensive and easy to prepare. For this reason, the said culture medium can be utilized widely, such as a test | inspection of general food, a drink, water, and a manufacturing process test | inspection.
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