JP3652388B2 - Medium for isolation of Bacillus cereus - Google Patents
Medium for isolation of Bacillus cereus Download PDFInfo
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- JP3652388B2 JP3652388B2 JP23886394A JP23886394A JP3652388B2 JP 3652388 B2 JP3652388 B2 JP 3652388B2 JP 23886394 A JP23886394 A JP 23886394A JP 23886394 A JP23886394 A JP 23886394A JP 3652388 B2 JP3652388 B2 JP 3652388B2
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Description
【０００１】
【産業上の利用分野】
本発明はセレウス菌 (Bacillus cereus) 分離用培地に係り、香辛料を含む広 義の食品、環境及び臨床材料からのセレウス菌の分離を行なう場合に使用され
る。
【０００２】
【従来の技術】
セレウス菌はグラム陽性有芽胞桿菌であり、土壌細菌の一種であって自然界に汎く分布している。従って土壌と関わりの深い食品、殊に穀類、豆類、香辛料等を汚染し、又水を汚染するので、この細菌による汚染範囲はスパゲッティー、焼きそば等の麺類、米飯類、グラタン、ピザ類、魚介類、食肉及び食肉製品、澱粉等の原料素材、菓子類、脱脂粉乳、豆腐、ソース類、香辛料等の広義の食品、一般環境、臨床材料等の多岐にわたる。
尚、この細菌は蛋白質や多糖類等の高分子物質に対して高い分解能を有し、食品を腐敗・変敗させる雑菌と考えられてきたが、下痢を主徴とし或いは悪心・嘔吐を主徴とする食中毒症状を起こさせることが判明し、昭和 57 年に厚生省が食中毒菌と認定するに至っている。
このセレウス菌は農作物をはじめ、一般食品内では芽胞型として存在し、好条件が与えられると旺盛に増殖する細菌であり、集団食中毒の原因ともなるので、この細菌の検出は衛生学的品質管理上極めて重要である。
【０００３】
従来から国内で行われているセレウス菌の検査には、分離用培地として NGKG 培地又は PBCW 培地が使用されてきた [神保等「東京衛研年報」29 - 1、第 158- 162 頁 (1978 年) 及び寺山等「食衛誌」第 19 巻第 1 号第 98 - 104 頁(1978 年)]。
これらの培地の組成は下記の表 1 に示される通りである。
【０００４】
【表１】
【発明が解決しようとする課題乃至発明の目的】
セレウス菌以外の Bacillus 属細菌には、例えば B. subtilis、B. licheniformis 等が存在するが、上記の PBCW 培地はこれらの細菌に対する 抑制性が弱い点に課題があり、一方 NGKG 培地は選択性においては優れているが、卵黄反応の確認が比較的困難である点に課題がある。
従って、本発明の目的はセレウス菌以外のバチルス属細菌を抑制し、環境細菌である大腸菌 (Escherichia coli)、黄色ブドウ球菌 (Streptococcus aureus)、緑膿菌 (Pseudomonas aeruginosa) からも鑑別でき且つ卵黄反応の確認が容易である、セレウス菌分離用培地を提供することにある。
【０００６】
【課題を解決し、目的を達成する手段及び作用】
本発明者等はセレウス菌の分離に関して従来の NGKG 培地及び PBCW 培地を用いて検討を行い、その問題点を解明し、セレウス菌を特異的に鑑別する培地について鋭意検討を重ねた結果、PBCW 培地を基本とし、これにグリシンを 1 重量% 以上添加すれば B. subtilis 等のセレウス菌以外の Batillus 属細菌を抑制し 得ること並びに卵黄濃度を PBCW 培地と同一の 5% に設定すれば、卵黄濃度が2% の NGKG 培地と異なり卵黄反応の確認が容易となることを見い出して本発明 を完成するに至った。
【０００７】
従って、本発明によるセレウス属分離用培地は、重量基準でペプトン 0.5 -2.5%、塩化ナトリウム 0.1 - 0.7%、グリシン 1.0 - 5.0%、肉エキス 0.01 -0.8%、ラクトース 0.5 - 2.0%、フェノールレッド 0.003 - 0.008%、硫酸ポリミキシン B 50 単位/ml 及び寒天 1.3 - 2.0% を含有し、卵黄濃度が 5 - 7% であることを特徴としている。
【０００８】
具体的には、重量基準でペプトンが 2.0%、塩化ナトリウムが 0.5%、グリシンが 1.0%、肉エキスが 0.5%、ラクトースが 1.0%、フェノールレッドが 0.005%、硫酸ポリミキシン B が 50 単位/ml 及び寒天が 2.0% であり、卵黄濃 度が 5% であることができる。尚、肉エキスは酵母エキスに代替することができる。
【０００９】
【実施例等】
次に、製造例、参考試験例及び試験例に関連して本発明を更に詳細に且つ具体的に説明する。
製造例
下記の表 2 に示される原材料及び量割合で本発明による培地 (以下「PBCWG培地」と称する) を調製した。
【表２】
参考試験例 1
既知の PBCW 培地及び NGKG 培地を用い、11 種類のセレウス菌について、そ の発育性を検討した。即ち、トリプトソーヤブイヨンを用い、36℃ にて 24 時 間培養した菌液を 10-5 - 10-7 に稀釈し、その 0.1ml を各培地に滴下した後に、コンラージ棒で塗抹し (1ml 当り 10-6 - 10-8 稀釈)、発育性と卵黄反応を調べた。
結果は下記の表 3 に示される通りであり、発育性に大差はなかったが、卵黄 反応に関しては、NGKG 培地の場合に判定が若干困難であった。
【表３】
参考試験例 2
ＮＧＫＧ 培地 (卵黄不含の場合の pH は 6.8 ± 0.1 であるが、卵黄液を添加す ると pH は低下する傾向がある) における卵黄液濃度と pH 変化による卵黄反応について検討した。即ち、トリプトソーヤブイヨンを用い、36℃ にて 24 時間 培養した菌液を 10-6 に稀釈し、その 0.1ml を各培地に滴下した後に、コンラ ージ棒で塗抹して (1ml 当り 10-7 稀釈) 調べた。
結果は下記の表 4 に示される通りであり、卵黄液濃度を 5% に設定し、卵黄 液添加後の pH が 6.8 程度となるように pH 調整を行うことにより卵黄反応が より明確になることが判明した。
【表４】
参考試験例 3
PBCW 培地を対象とし、セレウス菌分離の選択性を高めるために、グリシンの 添加効果に関する検討を行った。即ち、PBCW 培地にグリシンを 0 - 3% 添加し た培地を調製し、参考試験例 2 と同様の方法で評価した。
結果は下記の表 5 に示される通りであり、グリシンを 1% 又はそれ以上添加 することにより、セレウスの発育に影響を及ぼすことなしに、多くの食品から分離される B. subtilis の発育を抑制し得ることが判明した。
【表５】
参考試験例 4
PBCW 培地と NGKG 培地を対象とし、製造上問題となる滅菌条件の相違による pH と色差の変化について検討した。滅菌条件は 121℃ で 15 分、30 分又は 60分或いは 132℃ で 15 分又は 30 分に設定した。
結果は下記の表 6 に示される通りであり、殺菌条件が 121℃ で 60 分及び
132℃ で 30 分の場合に PBCW 培地では pH が若干低下する傾向が認められた
が、両培地共に色差への影響は殆ど認められなかった。
【表６】
参考試験例 5
滅菌条件の異なる PBCW 培地及び NGKG 培地を対象とし、細菌の発育性を検討した。
結果は下記の表 7 に示される通りであり、NGKG 培地においては滅菌条件を
121℃、30 分又はそれ以上に設定すると P. aeruginosa に対する抑制性の激減 することが判明した。
【表７】
参考試験例 6
種々の温度条件で滅菌した普通寒天培地を対象として硫酸ポリミキシン B に よる細菌発育の抑制効果について検討した。
結果は下記の表 8 に示される通りであり、硫酸ポリミキシン B を添加しない場合には何れの培地においても供試菌株は増殖した。尚、ポリミキシン B を添 加後に滅菌処理した培地 (前添加) において、E. coli は何れの滅菌条件でも発育しなかったが、P. aeruginosa に関しては滅菌条件が 121℃、30 分又はそれ 以上の培地において生育し、参考試験例 5 と同様な傾向を示した。但し、予め 滅菌処理した培地に硫酸ポリミキシン B を添加した (後添加) 場合には、P.aeruginosa の発育も抑制された。
【表８】
試験例 1 及び比較試験例 1
セレウス菌 (B. cereus, ATCC 19637) と他の細菌との混合菌液を使用し、PBCW 培地、NGKG 培地、5-NGKG 培地 (NGKG 培地の卵黄液濃度を 5% に且つ pH を 6.8 ± 0.1 に設定したもの) 及び製造例による PBCWG 培地を対象としてセ レウスに対する選択性及び鑑別性を検討した。即ち、トリプトソーヤブイヨンを用い、36℃ にて 24 時間培養した菌液を稀釈し、セレウス菌については 10-6
に稀釈し、他の細菌については 10-1 - 10-8 に稀釈し、各稀釈液を 1ml 宛採取し、殺菌済み試験管に入れ混合したものを 0.1ml 宛各平板培地に接種し、コン ラージ 棒で塗抹して調べた。
結果は下記の表 9 に示されている通りであり、セレウス菌以外の菌種に対し て抑制性が弱かったのは PBCW 培地であり、他の 3 種類の培地はほぼ同等の抑 制力を示し、セレウス菌に対する鑑別性が良好であり且つ選択性に優れていたのは 5-NGKG 培地と PBCWG 培地であり、NGKG 培地における卵黄反応は弱いことが判明した。
【表９】
試験例 2 及び比較試験例 2
試験例 1 及び比較試験例 1 に示されている結果から、従来の NGKG 培地及びPBCW 培地よりも 5-NGKG 培地及び PBCWG 培地の方がセレウス菌の選択培地として明らかに優れているものと考えられたが、5-NGKG 培地と PBCWG 培地との間で何れが優れているかを決定するのは困難である。従って、両培地を大量に調製して (100 リットル)、安定性に関する検討を行った。
両培地の組成及び配合量は下記の表 10 に示される通りであった。
【表１０】
上記の両培地は硫酸ポリメキシン B 及び卵黄液以外の原材料を混合し、121℃において 15 分間処理して滅菌した後に、濾過滅菌した硫酸ポリメキシン B 及 び卵黄液を添加し、次いで pH 調整を行うことにより調製された。
5 ケ 月間にわたる経時試験を実施した処、両培地の物理化学的性状は、5-NGKG培地において pH 及び色差の変化が大きかったが、PBCWG 培地の場合には下記の表 11 に示される通り、顕著な変化は認められなかった。
尚、細菌の培養試験を実施した処、5-NGKG 培地の場合にはセレウス菌以外の 細菌に対する抑制性が経時的に低下し、卵黄反応も減弱する傾向が認められた
が、PBCWG 培地の場合には、下記の表 12 及び 13 に示される通りであり、そのような傾向は認められなかった。
従って、これらの安定性試験の結果として PBCWG 培地が改良 NGKG 培地であ る 5-NGKG 培地よりもセレウス選択培地として優れていることが判明した。
【表１１】
【表１２】
【表１３】
【発明の効果】
本発明による培地は、従来の NGKG 培地や PBCW 培地と比較する場合に、セレウス菌の選択性、卵黄反応の確認容易性及び保存安定性において優れている。従って、本発明による培地を使用すれば、何時でも容易にセレウス菌を分離検出することができる。[0001]
[Industrial application fields]
The present invention relates to a culture medium for Bacillus cereus separation, and is used when separating Bacillus cereus from a broad range of foods, spices, and environmental and clinical materials including spices.
[0002]
[Prior art]
Bacillus cereus is a gram-positive spore gonococcus, a kind of soil bacterium that is widely distributed in nature. Therefore, it contaminates foods closely related to soil, especially cereals, beans, spices, etc., and also pollutes water, so the range of contamination by bacteria is noodles such as spaghetti and fried noodles, cooked rice, gratin, pizzas, seafood , Meat and meat products, raw materials such as starch, confectionery, skim milk powder, tofu, sauces, broad foods such as spices, general environment, clinical materials, etc.
Although this bacterium has a high resolution with respect to high-molecular substances such as proteins and polysaccharides and has been considered to be a miscellaneous bacteria that rots and degrades food, it is mainly characterized by diarrhea or nausea / vomiting. In 1982, the Ministry of Health and Welfare approved it as a food poisoning bacterium.
The Bacillus cereus is a spore type in agricultural products and general foods. It is a bacterium that grows vigorously when given favorable conditions, and can cause mass food poisoning. It is extremely important.
[0003]
Traditionally, NGKG medium or PBCW medium has been used as a culture medium for Bacillus cereus in Japan [Jinbo et al., Tokyo Eiken Annual Report 29-1; pp. 158-162 (1978) ), And Terayama et al., “The Edibles Magazine,” Volume 19, Issue 1, pages 98-104 (1978)].
The composition of these media is shown in Table 1 below.
[0004]
[Table 1]
[Problem to be Solved by the Invention]
The Bacillus genus bacteria other than Bacillus cereus, such as B. Subtilis, B. Although licheniformis like exist, the above PBCW medium has problems in that a weak inhibitory against these bacteria, whereas NGKG medium in selectivity Is excellent, but there is a problem in that it is relatively difficult to confirm the yolk reaction.
Therefore, the object of the present invention is to suppress Bacillus bacteria other than Bacillus cereus, and can be distinguished from the environmental bacteria Escherichia coli , Streptococcus aureus , Pseudomonas aeruginosa and the yolk reaction. It is an object of the present invention to provide a culture medium for separating Bacillus cereus that can be easily confirmed.
[0006]
[Means and functions for solving the problems and achieving the objects]
The present inventors conducted studies on the isolation of Bacillus cereus using conventional NGKG medium and PBCW medium, elucidated the problems, and as a result of intensive investigations on a medium that specifically distinguishes Bacillus cereus, PBCW medium was basic, this is set to be as well as yolk concentration may inhibit Batillus bacteria other than Bacillus cereus B. subtilis or the like be added glycine 1% by weight or more PBCW medium the same 5%, egg yolk concentration However, unlike the 2% NGKG medium, it was found that the yolk reaction could be easily confirmed, and the present invention was completed.
[0007]
Therefore, according to the present invention, the culture medium for separating cereus is peptone 0.5-2.5%, sodium chloride 0.1-0.7%, glycine 1.0-5.0%, meat extract 0.01-0.8%, lactose 0.5-2.0%, phenol red 0.003 on a weight basis. -It contains 0.008%, polymyxin sulfate B 50 units / ml and agar 1.3-2.0%, and the yolk concentration is 5-7%.
[0008]
Specifically, peptone 2.0%, sodium chloride 0.5%, glycine 1.0%, meat extract 0.5%, lactose 1.0%, phenol red 0.005%, polymyxin B sulfate 50 units / ml on a weight basis and Agar can be 2.0% and egg yolk concentration can be 5%. The meat extract can be replaced with a yeast extract.
[0009]
[Examples]
Next, the present invention will be described in more detail and specifically with reference to production examples, reference test examples, and test examples.
Production Example A medium (hereinafter referred to as “PBCWG medium”) according to the present invention was prepared in the raw materials and the amount ratios shown in Table 2 below.
[Table 2]
Reference test example 1
Using known PBCW medium and NGKG medium, the growth of 11 types of Bacillus cereus was examined. That is, using a triple-soya broth, the bacterial solution cultured in 24-hour at 36 ° C. 10 -5 - diluted to 10-7, the 0.1ml after dropwise to each culture, smeared with Conradi stick (1ml 10 -6-10 -8 dilution), and the growth and egg yolk response were examined.
The results are as shown in Table 3 below, and there was no significant difference in growth, but the egg yolk reaction was slightly difficult to determine in the case of NGKG medium.
[Table 3]
Reference test example 2
The egg yolk concentration in the NGKG medium (pH without egg yolk is 6.8 ± 0.1, but the pH tends to decrease when egg yolk is added) and the egg yolk reaction due to pH change were examined. That is, dilute the bacterial solution that has been cultured at 36 ° C for 24 hours with Tryptosoya broth to 10 -6 , add 0.1 ml to each medium, and then smear with a condensate stick (10 per ml). -7 dilution) Investigated.
The results are as shown in Table 4 below. The egg yolk reaction can be made clearer by setting the yolk concentration to 5% and adjusting the pH to about 6.8 after adding the yolk. There was found.
[Table 4]
Reference test example 3
In order to improve the selectivity of Bacillus cereus isolates on PBCW medium, we examined the effect of glycine addition. That is, a medium in which 0-3% glycine was added to the PBCW medium was prepared and evaluated in the same manner as in Reference Test Example 2.
The results are shown in Table 5 below, and the addition of 1% or more of glycine suppresses the growth of B. subtilis isolated from many foods without affecting the growth of Cereus. Turned out to be possible.
[Table 5]
Reference test example 4
Using PBCW medium and NGKG medium, changes in pH and color difference due to differences in sterilization conditions, which is a problem in manufacturing, were examined. Sterilization conditions were set at 121 ° C for 15 minutes, 30 minutes or 60 minutes, or 132 ° C for 15 minutes or 30 minutes.
The results are shown in Table 6 below, and the sterilization conditions are as follows:
In the case of 30 minutes at 132 ° C, there was a tendency for pH to slightly decrease in PBCW medium, but there was almost no effect on the color difference in both mediums.
[Table 6]
Reference test example 5
Bacterial growth was examined in PBCW medium and NGKG medium with different sterilization conditions.
The results are as shown in Table 7 below.
It was found that the inhibition of P. aeruginosa drastically decreased when set at 121 ° C for 30 minutes or longer.
[Table 7]
Reference test example 6
We investigated the effect of polymyxin B sulfate on bacterial growth in normal agar medium sterilized under various temperature conditions.
The results are as shown in Table 8 below, and the test strains grew in any medium when polymyxin B sulfate was not added. Note that E. coli did not grow under any sterilization conditions in the medium sterilized after addition of polymyxin B (pre-addition), but for P. aeruginosa , the sterilization conditions were 121 ° C for 30 minutes or more. It grew in the medium and showed the same tendency as in Reference Test Example 5. However, the growth of P. aeruginosa was also suppressed when polymyxin B sulfate was added to the previously sterilized medium (post addition).
[Table 8]
Test Example 1 and Comparative Test Example 1
Using a mixed bacterial solution of Bacillus cereus ( B. cereus , ATCC 19637) and other bacteria, PBCW medium, NGKG medium, 5-NGKG medium (NGKG medium egg yolk concentration 5% and pH 6.8 ± 0.1 And PBCWG media from the production examples were studied for selectivity and discrimination against cereus. That is, using a triple-soya broth, diluted bacteria solution was incubated for 24 hours at 36 ° C., for Bacillus cereus 10-6
Diluted in, for other bacteria 10 -1 - diluted to 10 -8, each dilution was 1ml addressed collected, a mixture placed in a sterilized test tube was inoculated into 0.1ml addressed to each plate medium, con I smeared it with a large stick and examined it.
The results are as shown in Table 9 below.PBCW medium was less inhibitory against species other than Bacillus cereus, and the other three mediums showed almost the same inhibitory power. It was shown that 5-NGKG medium and PBCWG medium had good discrimination against Bacillus cereus and excellent selectivity, and the yolk reaction in NGKG medium was weak.
[Table 9]
Test Example 2 and Comparative Test Example 2
From the results shown in Test Example 1 and Comparative Test Example 1, 5-NGKG medium and PBCWG medium are clearly superior as selective media for Bacillus cereus than conventional NGKG medium and PBCW medium. However, it is difficult to determine which is better between 5-NGKG medium and PBCWG medium. Therefore, both media were prepared in large quantities (100 liters), and stability studies were conducted.
The composition and blending amount of both media were as shown in Table 10 below.
[Table 10]
Both media above should be mixed with raw materials other than polymexin B sulfate and egg yolk, treated at 121 ° C for 15 minutes, sterilized, then filtered and sterilized with polymexine B sulfate and egg yolk, and then pH adjusted. It was prepared by.
After a five-month aging test, the physicochemical properties of both media showed significant changes in pH and color difference in the 5-NGKG media, but as shown in Table 11 below for the PBCWG media, There was no noticeable change.
In addition, in the case of the bacterial culture test, in the case of 5-NGKG medium, the inhibitory effect against bacteria other than Bacillus cereus decreased with time, and the yolk reaction tended to decrease, but in the case of PBCWG medium As shown in Tables 12 and 13 below, no such tendency was observed.
Therefore, as a result of these stability tests, it was found that the PBCWG medium was superior to the 5-NGKG medium, which is an improved NGKG medium, as a Cereus selection medium.
[Table 11]
[Table 12]
[Table 13]
【The invention's effect】
The medium according to the present invention is superior in the selectivity of Bacillus cereus, the ease of confirmation of egg yolk reaction, and storage stability when compared with conventional NGKG medium and PBCW medium. Therefore, if the culture medium according to the present invention is used, Bacillus cereus can be easily separated and detected at any time.
Claims (3)
０．７％、グリシン 1.0 - 5.0%、肉エキス 0.01 - 0.8%、ラクトース 0.5 - 2.0%、フェノールレッド 0.003 - 0.008%、硫酸ポリミキシン B 50 単位/ml 及び寒天1.3 - 2.0% を含有し、卵黄濃度が 5 - 7% であることを特徴とする、セレウス 菌分離用培地。Peptone 0.5-2.5% by weight, sodium chloride 0.1-
Contains 0.7%, glycine 1.0-5.0%, meat extract 0.01-0.8%, lactose 0.5-2.0%, phenol red 0.003-0.008%, polymyxin B 50 units / ml and agar 1.3-2.0%, egg yolk concentration A medium for isolation of Bacillus cereus, characterized in that the content is 5-7%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP23886394A JP3652388B2 (en) | 1994-10-03 | 1994-10-03 | Medium for isolation of Bacillus cereus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP23886394A JP3652388B2 (en) | 1994-10-03 | 1994-10-03 | Medium for isolation of Bacillus cereus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0898678A JPH0898678A (en) | 1996-04-16 |
| JP3652388B2 true JP3652388B2 (en) | 2005-05-25 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP23886394A Expired - Lifetime JP3652388B2 (en) | 1994-10-03 | 1994-10-03 | Medium for isolation of Bacillus cereus |
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1234873A1 (en) * | 2001-02-20 | 2002-08-28 | Deutsches Ressourcenzentrum für Genomforschung GmbH | Indicator medium for the detection of contaminants in gene libraries |
| SK287315B6 (en) | 2006-06-02 | 2010-06-07 | Biotika, A. S. | A method for polymyxin B isolation from fermented soil |
| SK287293B6 (en) | 2006-06-15 | 2010-05-07 | Biotika, A. S. | A method for fermentation of polymyxin B by means of productive microorganism Bacillus polymyxa |
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1994
- 1994-10-03 JP JP23886394A patent/JP3652388B2/en not_active Expired - Lifetime
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| JPH0898678A (en) | 1996-04-16 |
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