JP2002058474A - Medium for detecting thermophilic bacterium of genus clostridium - Google Patents
Medium for detecting thermophilic bacterium of genus clostridiumInfo
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- JP2002058474A JP2002058474A JP2000248970A JP2000248970A JP2002058474A JP 2002058474 A JP2002058474 A JP 2002058474A JP 2000248970 A JP2000248970 A JP 2000248970A JP 2000248970 A JP2000248970 A JP 2000248970A JP 2002058474 A JP2002058474 A JP 2002058474A
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- medium
- detecting
- bacteria
- bacterium
- thermophilic
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Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、レトルト缶飲料製
品に対する変敗指標菌である高温性Clostridium属細菌
を簡易かつ迅速に検出する培地に関するものである。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a medium for easily and rapidly detecting thermophilic Clostridium bacterium which is a deterioration indicator bacterium for retort can beverage products.
【０００２】[0002]
【従来の技術】コーヒーやしるこ等のレトルト缶飲料
は、通常、出荷前に１２０℃から１２５℃で２０分から
４０分程度の加熱殺菌が行われるが、このような殺菌に
よっても死滅しない細菌がある。かかる細菌は一般に高
温菌と呼ばれているが、この細菌は４０℃以下の温度下
においては増殖することもなく、何ら問題とはならな
い。2. Description of the Related Art Normally, retort canned beverages such as coffee and syrup are sterilized by heating at 120 ° C. to 125 ° C. for about 20 minutes to 40 minutes before shipping, but there are bacteria that do not die even by such sterilization. . Such a bacterium is generally called a thermophilic bacterium. However, this bacterium does not grow at a temperature of 40 ° C. or less, and does not cause any problem.
【０００３】しかし、ホットベンダーで加温販売される
可能性のあるレトルト缶入り飲料にこのような菌が混入
していた場合には、ホットベンダー内の温度がそのよう
な細菌の最適増殖温度（５５℃から６０℃付近）にあた
るために、細菌が増殖し、飲料に変敗を起こしてしまう
可能性があり、そのような事態が生じないように事前に
対策を講じておく必要がある。[0003] However, when such bacteria are mixed in a retort canned beverage which is likely to be heated and sold by a hot bender, the temperature in the hot bender becomes the optimal growth temperature of such bacteria ( (Around 55 ° C. to 60 ° C.), bacteria may multiply and cause deterioration of the beverage, and it is necessary to take measures in advance to prevent such a situation from occurring.
【０００４】ここで、このような高温における変敗を防
止する際の指標となる菌（変敗指標菌）として、嫌気性
の高温性Clostridium属細菌がある。この菌は発育に不
適当な環境下では芽胞を形成して活動を停止している
が、発育条件が整うと通常の菌体に戻って増殖を始める
ため、その存否につき出荷前に綿密な検査を行うことは
製品管理上極めて重要である。このようなことから、事
前に変法ＴＧＣ培地を使った混釈法や試験管による検査
法によって高温性Clostridium属細菌を検出し、当該菌
が検出されたものを出荷工程から排除するようにしてい
る。Here, an anaerobic thermophilic Clostridium bacterium is an example of a bacterium that serves as an index for preventing deterioration at such a high temperature (a deterioration indicator bacterium). This bacterium forms a spore and stops its activity in an environment unsuitable for growth.However, when the growth conditions are met, the bacterium returns to normal cells and begins to proliferate. Is very important for product management. For this reason, thermophilic Clostridium bacteria are detected in advance by a pour method using a modified TGC medium or a test method using a test tube, and the detected bacteria are excluded from the shipping process. I have.
【０００５】しかしながら、高温性Clostridium属細菌
については増殖速度が遅いことに加え、乳入り飲料にお
いて検出を行おうとした場合には、乳成分の拡散等によ
って菌体の確認をすることが極めて困難となるため、上
記従来の検出法によっては迅速に検出することができな
かった。より具体的に言えば、通常の菌についてはその
検出期間が一般の細菌の２日程度であるのに対し、高温
性Clostridium属細菌の場合には５日から７日間と検出
期間としては極めて長期間を要していた。However, in addition to the slow growth rate of the thermophilic Clostridium genus bacteria, it is extremely difficult to confirm the bacterial cells due to the diffusion of milk components and the like when attempting to detect the milk-containing beverage. Therefore, rapid detection was not possible by the above-mentioned conventional detection method. More specifically, the detection period of ordinary bacteria is about 2 days for general bacteria, whereas that of thermophilic Clostridium bacteria is 5 to 7 days, which is an extremely long detection period. It took time.
【０００６】[0006]
【発明が解決しようとする課題】このように、従来法に
よれば、高温性Clostridium属細菌の検出には長期間を
要し、また、例えば乳入り飲料については判別が困難と
いうように、飲料の種類によっては菌体の判別が困難な
場合がある、というような問題もあった。As described above, according to the conventional method, it takes a long time to detect a thermophilic Clostridium bacterium, and for example, it is difficult to discriminate a beverage containing milk. There is also a problem that it may be difficult to discriminate the cells depending on the type of the microorganism.
【０００７】本発明は、以上のような問題を解決すべく
なされたものであり、その目的は、レトルト缶の飲料製
品に対する変敗指標菌である高温性Clostridium属細菌
を、簡易かつ迅速に判別できるような培地を提供するこ
とにある。SUMMARY OF THE INVENTION The present invention has been made to solve the above problems, and an object of the present invention is to easily and quickly identify thermophilic Clostridium bacteria which are deterioration indicators for beverage products in retort cans. It is to provide a medium that can be used.
【０００８】[0008]
【課題を解決するための手段】以上のような目的を達成
するために本発明者らが鋭意研究を行った結果、ニュー
トラルレッドを検出用培地に含ませることによって、高
温性Clostridium属細菌検出を簡易に行うことができる
ようになり、かつ、同培地にピルビン酸を含ませること
によって高温性Clostridium属細菌の検出を簡易かつ迅
速に行うことができるようにし、本発明を完成するに至
ったのである。Means for Solving the Problems As a result of intensive studies conducted by the present inventors to achieve the above object, the detection of thermophilic Clostridium bacteria by including neutral red in the detection medium was carried out. As it became possible to easily perform the detection of thermophilic Clostridium genus bacteria by including pyruvic acid in the same medium, the present invention was completed. is there.
【０００９】即ち、本発明によれば、ニュートラルレッ
ドが高温性Clostridium属細菌によって代謝され、それ
が「変色」という結果となって表れるため、視認が容易
であり、乳入り飲料のような視認が困難なものについて
も適確かつ簡易に検出を行うことができるようになるの
である。また、ピルビン酸は高温性Clostridium属細菌
の増殖を活性化するために、これを培地中に添加するこ
とによって実用上問題を生じない程度にまで検出速度を
高めることができるようになる。That is, according to the present invention, neutral red is metabolized by a thermophilic Clostridium bacterium and appears as a result of "discoloration", so that it is easy to visually recognize, and it is easy to recognize such as a milk-containing beverage. Even difficult ones can be detected accurately and easily. In addition, since pyruvic acid activates the growth of thermophilic Clostridium bacteria, its addition to a culture medium can increase the detection rate to a level that does not cause a practical problem.
【００１０】このような本発明は、それを一般化する
と、菌体の代謝によって変色する色素と細菌増殖促進物
とを検出用培地に含ませることにより、高温性Clostrid
ium属細菌検出を簡易迅速に検出することを可能にした
手法として把握することができる。そして、より詳しく
説明すると、本発明は、従来法による培地、例えば変法
ＴＧＣ培地に、ニュートラルレッドのような菌の増殖に
伴う菌体の代謝により分解される色素とピルビン酸のよ
うな細菌の増殖を促進する物とを添加することにより、
色素の分解による培地の色の変化を確認するだけで（菌
体の増殖を確認するよりも早く）菌体の確認ができると
いうものである。[0010] The present invention, when generalized, comprises a pigment which changes its color by the metabolism of bacterial cells and a bacterial growth promoter in a detection medium to provide a high-temperature Clostrid.
It can be grasped as a technique that enables simple and quick detection of ium bacteria. More specifically, the present invention relates to a method in which a pigment, which is decomposed by metabolism of cells accompanying the growth of bacteria such as neutral red, and a bacterium such as pyruvate are added to a conventional medium, for example, a modified TGC medium. By adding a substance that promotes growth,
Just confirming the change in the color of the medium due to the decomposition of the pigment allows the cells to be confirmed (faster than the confirmation of the proliferation of the cells).
【００１１】より具体的に本発明の内容を示すと、それ
は以下のようなものである。More specifically, the contents of the present invention are as follows.
【００１２】（１） 高温性Clostridium属細菌を検出
するための培地であって、菌体の代謝によって変色する
色素と、当該菌体の増殖を促進させる細菌増殖促進物
と、を含むことを特徴とする高温性Clostridium属細菌
検出用培地。(1) A medium for detecting a thermophilic Clostridium bacterium, which is characterized by containing a pigment that changes color by metabolism of the cells and a bacterial growth promoter that promotes the growth of the cells. A medium for detecting thermophilic Clostridium bacteria.
【００１３】本明細書において「含む」としたのは、本
発明による変色工程等を阻害しない限り、色素や細菌増
殖促進物以外のものが培地中に含まれていても良いこと
を表明するためである。このような本発明は、対象菌が
増殖すると、菌体の代謝によって色素が分解され、これ
により培地が変色することによって菌体を判別するもの
であるところ、菌を培養した後に色素を添加したので
は、かかる変色反応が確認できなくなってしまうからで
ある。[0013] In the present specification, the term "comprising" means that a substance other than the pigment and the bacterial growth promoting substance may be contained in the medium as long as the discoloration step and the like according to the present invention are not inhibited. It is. In the present invention, when the target bacterium proliferates, the pigment is decomposed by the metabolism of the cells, and the cells are discriminated by the discoloration of the medium, whereby the pigment was added after culturing the bacteria. This is because such a discoloration reaction cannot be confirmed.
【００１４】なお、添加した色素の変化により菌の存否
を確認する方法は、特開昭５９−２６９９号公報や特開
昭５７−６８７９６号公報においても明らかにされてい
るが、これらはいずれも細菌検出用培地のｐＨの変化に
伴う色の変化により菌の存否を確認するものである。従
って、菌体による代謝によって生じる色の変化から細菌
を判別する本発明とは明らかに異なるものである。A method for confirming the presence or absence of a bacterium based on a change in the added dye is disclosed in JP-A-59-2699 and JP-A-57-68796. The presence or absence of a bacterium is confirmed by a color change accompanying a change in the pH of the bacterium detection medium. Therefore, the present invention is clearly different from the present invention in which bacteria are distinguished from a change in color caused by metabolism by cells.
【００１５】（２） 高温性Clostridium属細菌を検出
するための培地であって、ニュートラルレッドを含むこ
とを特徴とする高温性Clostridium属細菌検出用培地。(2) A medium for detecting a thermophilic Clostridium bacterium, which is characterized by containing neutral red.
【００１６】「ニュートラルレッド」はｐＨ指示薬とし
て広く知られているが、本明細書においては菌体の代謝
によって変色する色素として用いている。即ち、「ニュ
ートラルレッド」がｐＨの変化に伴って変色するからで
はなく、培地中で高温性Clostridium属細菌が増殖する
と、菌体の代謝によって「ニュートラルレッド」が分解
され、これに伴って培地が黄色に変色することから本発
明に有効であるということになる。[0016] "Neutral red" is widely known as a pH indicator, but is used herein as a pigment that changes color by metabolism of bacterial cells. In other words, rather than because "neutral red" changes color with a change in pH, when thermophilic Clostridium spp. Grows in the medium, "neutral red" is decomposed by metabolism of the cells, and the medium is consequently degraded. Since the color changes to yellow, it is effective for the present invention.
【００１７】（３） 高温性Clostridium属細菌を検出
するための培地であって、更に、当該菌体の増殖を促進
させる細菌増殖促進物を含むことを特徴とする（２）記
載の高温性Clostridium属細菌検出用培地。(3) A high-temperature Clostridium according to (2), which is a medium for detecting a thermophilic Clostridium bacterium, further comprising a bacterial growth promoting substance for promoting the growth of the cells. Medium for detecting genus bacteria.
【００１８】ニュートラルレッドのほか、更に、細菌増
殖促進物をも添加するのは、芽胞形成菌である高温性Cl
ostridium属細菌の増殖が他の一般細菌より増殖が遅い
ことによる。即ち、飲料製造の現場においては、ニュー
トラルレッドの添加によって、簡易かつ確実に菌体の確
認ができるというだけでは不十分で、細菌増殖促進物を
添加して菌の増殖を早めることによって検査速度を加速
させる必要があることによる。[0018] In addition to neutral red, a bacterial growth promoter is also added.
This is because the growth of ostridium bacteria is slower than other common bacteria. In other words, at the site of beverage production, it is not enough that the addition of neutral red allows simple and reliable confirmation of the cells, and it is not enough to increase the test speed by adding a bacterial growth promoter to accelerate the growth of the bacteria. Due to the need to accelerate.
【００１９】（４） 前記が細菌増殖促進物ピルビン酸
またはピルビン酸塩であることを特徴とする（３）記載
の高温性Clostridium属細菌検出用培地。(4) The medium for detecting a thermophilic Clostridium bacterium according to (3), wherein said medium is a bacterial growth promoting substance pyruvate or pyruvate.
【００２０】ピルビン酸はエネルギー代謝の中間生成物
であるが、本発明における細菌増殖促進物として有効で
ある。なお、Clostridium属細菌はグルコース、フルク
トース、キシロースの３種の糖の資化性があるが、後述
する実施例３に示す通り、これらと比較しても、ピルビ
ン酸が好適である。Pyruvate is an intermediate product of energy metabolism, and is effective as a bacterial growth promoter in the present invention. Although Clostridium bacteria have assimilation of three kinds of sugars, glucose, fructose, and xylose, pyruvic acid is more preferable as compared with these as shown in Example 3 described later.
【００２１】しかし、ピルビン酸は冷蔵保存が必要とさ
れる粘性の高い液体であり、また、培地調整時にリン酸
水素二カリウムの添加によってｐＨの調整が必要とな
る。このような手順を簡素化するために、ピルビン酸塩
で代用することもできる。However, pyruvic acid is a highly viscous liquid that needs to be kept refrigerated, and the pH must be adjusted by adding dipotassium hydrogen phosphate when adjusting the medium. In order to simplify such a procedure, pyruvate can be substituted.
【００２２】（５） ３０ｇ／Ｌ程度の変法ＴＧＣ培地
中に、１〜４０ｍｇ／ｌの濃度のニュートラルレッド、
１〜３０ｍｍｏｌ／ｌの濃度のピルビン酸若しくはピル
ビン酸塩を含むものからなることを特徴とする高温性Cl
ostridium属細菌検出用培地。(5) Neutral red at a concentration of 1 to 40 mg / l in a modified TGC medium of about 30 g / L,
High temperature Cl comprising pyruvate or pyruvate at a concentration of 1 to 30 mmol / l.
Medium for detecting ostridium bacteria.
【００２３】（６） レトルト缶の飲料製品中の高温性
Clostridium属細菌の検出用培地であることを特徴とす
る（１）から（５）いずれか記載の高温性Clostridium
属細菌検出用培地。(6) High temperature property in retort can beverage products
The thermophilic Clostridium according to any one of (1) to (5), which is a medium for detecting Clostridium bacteria.
Medium for detecting genus bacteria.
【００２４】レトルト缶飲料はホットベンダーで加温販
売されることがあるため、芽胞形成菌である高温性Clos
tridium属細菌の検出が必要とされる飲料製品である。[0024] Since retort canned beverages are sometimes sold by heating with a hot bender, the thermophilic Clos, which is a spore-forming bacterium, is used.
This beverage product requires detection of tridium bacteria.
【００２５】（７） 低酸性飲料中の高温性Clostridiu
m属細菌検出用培地であることを特徴とする（１）から
（５）いずれか記載の高温性Clostridium属細菌検出用
培地。(7) High temperature Clostridiu in low acid beverage
The medium for detecting a thermophilic Clostridium bacterium according to any one of (1) to (5), which is a medium for detecting a genus m bacterium.
【００２６】ｐＨ４.６以下の酸性飲料においては、高
温性Clostridium属細菌の耐熱性は低下し、また発育も
抑制されるためにあまり問題とはならないが、低酸性飲
料においては当該菌の増殖が可能であることから、低酸
性飲料におけるかかる菌の検出は極めて重要である。な
お、低酸性飲料には、紅茶、烏龍茶等の茶系飲料、その
他、コーヒー、ココア、缶入りしるこやスープなど様々
な飲料が含まれるIn acidic beverages having a pH of 4.6 or less, the heat resistance of the thermophilic Clostridium bacterium is reduced and the growth thereof is suppressed, so this is not a serious problem. Because of its potential, detection of such bacteria in low-acid beverages is extremely important. In addition, low-acid beverages include tea-based beverages such as black tea and oolong tea, as well as various beverages such as coffee, cocoa, canned syrup and soup.
【００２７】（８） 乳入り低酸性飲料製品中の高温性
Clostridium属細菌の検出用培地であることを特徴とす
る（１）から（５）いずれか記載の高温性Clostridium
属細菌検出用培地。(8) High temperature properties in milk-containing low-acid beverage products
The thermophilic Clostridium according to any one of (1) to (5), which is a medium for detecting Clostridium bacteria.
Medium for detecting genus bacteria.
【００２８】本明細書において「乳入り飲料」と言うと
きには、乳成分を含有するあらゆる飲料が含まれる。乳
成分が含まれていればその拡散のため、従来法によるCl
ostridium属細菌の検出は困難となってしまうが、本発
明によれば、乳成分の有無にかかわらず簡易迅速に菌の
検出が可能となる。As used herein, the term "milk-containing beverage" includes any beverage containing a milk component. If milk components are present, the diffusion of Cl
Although it becomes difficult to detect the genus ostridium, according to the present invention, it is possible to detect the bacterium simply and quickly regardless of the presence or absence of a milk component.
【００２９】（９） 高温性Clostridium属細菌の検出
のためにニュートラルレッドを使用する方法。(9) A method using neutral red for detecting thermophilic Clostridium bacteria.
【００３０】[0030]
【発明の実施の形態】基礎となる培地の調整は常法によ
る。例えば変法ＴＧＣ培地や鉄サルファイト培地などを
用いることができる。また菌の測定は、高温性Clostrid
ium属細菌が嫌気性であることを考慮する必要がある。
そのため、例えば、平板培養法ではなく半流動培地法で
行う方法がある。BEST MODE FOR CARRYING OUT THE INVENTION Adjustment of a base medium is carried out by a conventional method. For example, a modified TGC medium or iron sulfite medium can be used. In addition, measurement of bacteria
It is necessary to consider that ium bacteria are anaerobic.
Therefore, for example, there is a method in which a semi-solid medium method is used instead of a plate culture method.
【００３１】以下、実施例を挙げて更に詳細に説明する
が、本発明の本質が菌体の代謝によって変色する色素と
細菌増殖促進物とを培地中に含ませるものであることは
明らかであるから、その本質部分が変更されない限りに
おいて種々の変更を行うことが可能である。従って、本
発明は以下の実施例だけに限定されるものではない。Hereinafter, the present invention will be described in more detail with reference to Examples. It is apparent that the essence of the present invention is to include a pigment which changes its color by the metabolism of cells and a bacterial growth promoter in a medium. Therefore, various changes can be made as long as the essential part is not changed. Therefore, the present invention is not limited only to the following examples.
【００３２】[0032]
【実施例１】従来の変法ＴＧＣ培地に、ニュートラルレ
ッド（ＮＲ）、ブロモチモールブルー（ＢＴＢ）、ブロ
モクレゾールパープル（ＢＣＰ）、クロルフェノールレ
ッド（ＣＰＲ）フェノールレッド（ＰＲ）といった５種
類のｐＨ指示薬を添加して、Clostoridium.thermohydro
sulfuricum及びClostoridium.thermaceticum発芽処理後
の胞子液１０３cfu/mlを１ml摂取した。各ｐＨ指示薬の
添加は、培地調整時に添加する方法と、５日間の培養後
に添加する方法との２通りを行って比較した。その結果
は以下の表１に示す通りである。Example 1 Five kinds of pH indicators such as neutral red (NR), bromothymol blue (BTB), bromocresol purple (BCP), chlorophenol red (CPR) and phenol red (PR) were added to a conventional modified TGC medium. Add Clostoridium.thermohydro
1 ml of 10 3 cfu / ml of spore solution after germination treatment of sulfuricum and Clostoridium. thermoaceticum was taken. The addition of each pH indicator was compared in two ways: a method of adding during pH adjustment of the medium and a method of adding after 5 days of culture. The results are as shown in Table 1 below.
【００３３】[0033]
【表１】 [Table 1]
【００３４】Clostoridium.thermaceticumは成長が遅
く、５日間ではｐＨが十分に下がらないため、培養後に
色素を添加して菌を判別するというのは不可能であっ
た。また、培地調整時に添加した一部のｐＨ指示薬につ
いては、ｐＨの変化があまりないにもかかわらず色が変
化していたことから、この変化はｐＨの変化によるもの
ではなく、菌の代謝等によって色素が分解されたことに
よるものであると考えられる。このことから、色素は培
地調整時に添加することが必要であることが明らかとな
った。Since Clostoridium thermoaceticum grows slowly and its pH does not drop sufficiently within 5 days, it was impossible to add a dye after culturing to discriminate bacteria. In addition, some pH indicators added during the preparation of the culture medium had changed color even though there was not much change in pH, so this change was not due to pH change but due to metabolism of bacteria. This is considered to be due to the decomposition of the dye. From this, it became clear that the dye had to be added at the time of adjusting the medium.
【００３５】[0035]
【実施例２】変法ＴＧＣ培地に色素を添加して培地を調
整し、その後、Clostoridium.thermohydrosulfuricum及
びClostoridium.thermaceticum発芽処理後の芽胞液１０
３〜１０−１cfu/mlと缶入りコーヒー（乳入り）を１ml
摂取し、ＭＰＮ法による検出感度及び判定期間の検討を
行った。Example 2 A dye was added to a modified TGC medium to adjust the medium, and then the spore fluid after germination of Clostoridium. Thermohydrosulfuricum and Clostoridium.
3 to 10 -1 cfu / ml and 1 ml of canned coffee (with milk)
After ingestion, the detection sensitivity and the determination period by the MPN method were examined.
【００３６】ここで、ＭＰＮ法とは、まず、菌液を希釈
していき各希釈段階毎に５本の試験管に摂取する。そし
て、細菌の培養後に何本の試験管に菌が増殖したかを見
ることにより、統計的に菌の濃度を算出する方法であ
る。検出感度等を比較するのに有効な手法である。以下
の表２に各種色素を細菌培養前に添加した場合における
ＭＰＮ法による比較結果を示す。なお、この表におい
て、例えば４／５とあるのは、５本摂取したうち４本に
菌が生えていることを意味する。Here, in the MPN method, first, a bacterial solution is diluted and ingested into five test tubes for each dilution step. Then, it is a method of calculating the concentration of the bacterium statistically by observing the number of test tubes in which the bacterium has grown after the cultivation of the bacterium. This is an effective method for comparing detection sensitivity and the like. Table 2 below shows comparison results by the MPN method when various dyes were added before bacterial culture. In this table, for example, "4/5" means that four out of five ingested bacteria grow.
【００３７】[0037]
【表２】 [Table 2]
【００３８】Clostoridium.thermohydrosulfuricumにお
いては、ニュートラルレッド、クロルフェノールレッ
ド、フェノールレッドの３種につき、従来法と同等の結
果が得られた。また、Clostoridium.thermac eticumで
は、ニュートラルレッドについてのみ、判定が可能だっ
た。このことから、Clostoridium属細菌の検出用の培地
に添加する色素として、ニュートラルレッドを使用する
ことが有効であることが明らかとなった。In Clostoridium thermohydrosulfuricum, the same result as the conventional method was obtained for three types of neutral red, chlorophenol red and phenol red. In addition, in Clostoridium.thermaceticum, judgment was possible only for neutral red. From this, it became clear that it is effective to use neutral red as a dye to be added to a medium for detecting Clostoridium bacteria.
【００３９】[0039]
【実施例３】Clostoridium.thermohydrosulfuricumは３
日間の培養で判定が可能であったがClostoridium.therm
aceticumの培養には４日間を要したことから、この菌の
増殖を早めるべく、グルコース、フルクトース、キシロ
ース、ピルビン酸及びピルビン酸塩を培地に添加し、菌
の増殖への貢献度を比較した。その結果、フルクトー
ス、キシロースを添加しても効果はなく、また、グルコ
ースを添加しても若干成長が良くなっただけであった。
これに対し、ピルビン酸を添加した場合には、培養が促
進された。このことから、細菌増殖促進のための培地改
良にはピルビン酸が有効であることが明らかとなった。Example 3 Clostoridium.thermohydrosulfuricum is 3
It was possible to judge by culturing for days, but Clostoridium.therm
Since cultivation of aceticum required 4 days, glucose, fructose, xylose, pyruvate and pyruvate were added to the medium in order to accelerate the growth of the bacterium, and the contribution to the growth of the bacterium was compared. As a result, the addition of fructose and xylose had no effect, and the addition of glucose only improved the growth slightly.
On the other hand, when pyruvic acid was added, the culture was promoted. From this, it became clear that pyruvic acid was effective in improving the medium for promoting bacterial growth.
【００４０】[0040]
【実施例４】ピルビン酸の最適添加濃度を検討するた
め、変法ＴＧＣ培地にニュートラルレッドを添加し、以
下の組成でピルビン酸を添加し、リン酸水素２カリウム
でｐＨを調整したExample 4 In order to examine the optimum concentration of pyruvic acid, neutral red was added to a modified TGC medium, pyruvic acid was added with the following composition, and the pH was adjusted with dipotassium hydrogen phosphate.
【００４１】 変法TGC 変法TGC+pyruvate 0.63g/l+K2HPO42g/l 変法TGC+pyruvate 1.26g/l+K2HPO43g/l 変法TGC+pyruvate 5.04g/l+K2HPO418g/lModified TGC Modified TGC + pyruvate 0.63 g / l + K 2 HPO 4 2 g / l Modified TGC + pyruvate 1.26 g / l + K 2 HPO 4 3 g / l Modified TGC + pyruvate 5.04 g / l + K 2 HPO 4 18g / l
【００４２】この培地にClostoridium.thermohydrosulf
uricum及びClostoridium.thermaceticumを摂取したとこ
ろ、以下の表３に示す結果が得られた。In this medium, Clostoridium.thermohydrosulf
When uricum and Clostoridium. thermoaceticum were ingested, the results shown in Table 3 below were obtained.
【００４３】[0043]
【表３】 [Table 3]
【００４４】[0044]
【実施例５】３０ｇ／Ｌの変法ＴＧＣ培地中に、１％の
ニュートラルレッド溶液を１ｍｌ添加し、ピルビン酸塩
を下記の組成で調整した。Example 5 1 ml of a 1% neutral red solution was added to 30 g / L modified TGC medium, and pyruvate was adjusted to the following composition.
【００４５】 変法TGC 変法TGC+0.55g/l sodium pyruvate 変法TGC+0.8g/l sodium pyruvate 変法TGC+1.1g/l sodium pyruvateModified TGC Modified TGC + 0.55 g / l sodium pyruvate Modified TGC + 0.8 g / l sodium pyruvate Modified TGC + 1.1 g / l sodium pyruvate
【００４６】この培地に、control、各濃度をそれぞれ
５本づつ添加し、Clostoridium.thermaceticumの胞子液
１０３cfu/mlを摂取したところ、以下の表４に示す結果
を得ることができた。[0046] To this medium, control, each concentration was added five increments respectively, were ingested spores was 10 3 cfu / ml of Clostoridium.Thermaceticum, it was possible to obtain the results shown in Table 4 below.
【００４７】[0047]
【表４】 [Table 4]
【００４８】この結果、３０ｇ／Ｌの変法ＴＧＣ培地中
に、１％のニュートラルレッド溶液を１ｍｌ添加した場
合には、ピルビン酸塩の添加量は０．８ｇが適切である
ことがわかった。As a result, it was found that when 1 ml of a 1% neutral red solution was added to 30 g / L of the modified TGC medium, 0.8 g of pyruvate was appropriately added.
【００４９】[0049]
【比較例１】Clostoridium.thermohydrosulfuricum、Cl
ostoridium.thermaceticum、Bacillus.stearothermophi
lus ATCC7953を試験菌株とし、従来の培地と改良培地の
検出感度及び判定期間の比較をＭＰＮ５本法を用いて行
ったところ、以下の表５に示す結果が得られた。Comparative Example 1 Clostoridium.thermohydrosulfuricum, Cl
ostoridium.thermaceticum, Bacillus.stearothermophi
When lus ATCC7953 was used as a test strain and the comparison of the detection sensitivity and the judgment period between the conventional medium and the improved medium was performed using the MPN5 method, the results shown in Table 5 below were obtained.
【００５０】[0050]
【表５】 [Table 5]
【００５１】Bacillus.stearothermophilusについては
色素の変化現象がみられないことから、本発明に係る培
地による検出はこの菌にとって不適であることが明らか
となった。一方、Clostoridium.thermohydrosulfuricum
及びClostoridium.thermaceticumについては色素の変
化がみられ、しかも７２時間の培養でこの変化がみられ
たことから、従来法と比べ、飛躍的に短時間で菌体の確
認ができることが明らかとなった。No change in pigment was observed in Bacillus stearothermophilus, indicating that the detection using the medium according to the present invention was inappropriate for this bacterium. Meanwhile, Clostoridium.thermohydrosulfuricum
And Clostoridium.thermaceticum showed a change in pigment, and this change was observed after 72 hours of cultivation, indicating that bacterial cells could be confirmed in a remarkably short time compared to the conventional method. .
【００５２】[0052]
【比較例２】比較例１同様の実験を他の飲料によって行
ったところ、以下の結果が得られた。Comparative Example 2 The same experiment as in Comparative Example 1 was conducted with other beverages, and the following results were obtained.
【００５３】[0053]
【表６】 [Table 6]
【００５４】[0054]
【表７】 [Table 7]
【００５５】[0055]
【表８】 [Table 8]
【００５６】[0056]
【表９】 [Table 9]
【００５７】[0057]
【表１０】 [Table 10]
【００５８】この結果から、他のレトルト缶飲料におい
ても７２時間の培養での細菌の検出ができることが明ら
かとなった。From these results, it was clarified that bacteria can be detected in other canned beverages after 72 hours of culture.
【００５９】[0059]
【比較例３】実際に製品を作る場合にはレトルト殺菌を
しているため、菌体は損傷しているものと考えられるこ
とから、１２１℃２０分間の熱処理を行った後、菌を試
験管に摂取したところ、以下の表１１に示す結果が得ら
れた。[Comparative Example 3] When a product is actually manufactured, retort sterilization is performed, and it is considered that the cells are damaged. As a result, the results shown in Table 11 below were obtained.
【００６０】[0060]
【表１１】 [Table 11]
【００６１】この結果、レトルト殺菌の有無にかかわら
ず、７２時間で菌を検出できることが明らかとなった。As a result, it was revealed that bacteria could be detected in 72 hours regardless of the presence or absence of retort sterilization.
【００６２】[0062]
【発明の効果】以上のような本発明によれば、レトルト
缶飲料中の高温性Clostridium属細菌が増殖したことを
培地の色の変化を見ることにより、短期間（３日間）で
容易に判別することができる。According to the present invention as described above, the growth of thermophilic Clostridium bacteria in a retort can beverage can be easily determined in a short period (three days) by observing the change in the color of the medium. can do.
Claims (9)
めの培地であって、菌体の代謝によって変色する色素
と、当該菌体の増殖を促進させる細菌増殖促進物と、を
含むことを特徴とする高温性Clostridium属細菌検出用
培地。1. A medium for detecting a thermophilic Clostridium bacterium, which comprises a pigment that changes color by metabolism of the cells, and a bacterial growth promoter that promotes the growth of the cells. Medium for detecting high temperature Clostridium bacteria.
めの培地であって、ニュートラルレッドを含むことを特
徴とする高温性Clostridium属細菌検出用培地。2. A medium for detecting a thermophilic Clostridium bacterium, which is characterized by containing neutral red.
めの培地であって、更に、当該菌体の増殖を促進させる
細菌増殖促進物を含むことを特徴とする請求項２記載の
高温性Clostridium属細菌検出用培地。3. The thermophilic Clostridium genus according to claim 2, which is a medium for detecting a thermophilic Clostridium bacterium, further comprising a bacterial growth promoting substance that promotes the growth of the cells. Medium for detecting bacteria.
ピルビン酸塩であることを特徴とする請求項３記載の高
温性Clostridium属細菌検出用培地。4. The medium for detecting a thermophilic Clostridium bacterium according to claim 3, wherein the bacterial growth promoting substance is pyruvate or pyruvate.
１〜４０ｍｇ／ｌの濃度のニュートラルレッド、１〜３
０ｍｍｏｌ／ｌの濃度のピルビン酸若しくはピルビン酸
塩を含むものからなることを特徴とする高温性Clostrid
ium属細菌検出用培地。5. In a modified TGC medium of about 30 g / L,
Neutral red at a concentration of 1 to 40 mg / l, 1 to 3
High temperature Clostrid characterized by comprising pyruvate or pyruvate at a concentration of 0 mmol / l.
Medium for detecting bacteria of the genus ium.
idium属細菌の検出用培地であることを特徴とする請求
項１から５いずれか記載の高温性Clostridium属細菌検
出用培地。6. High temperature Clostr in a retort can beverage product.
The medium for detecting thermophilic Clostridium bacteria according to any one of claims 1 to 5, which is a medium for detecting bacteria of the genus idium.
菌検出用培地であることを特徴とする請求項１から５い
ずれか記載の高温性Clostridium属細菌検出用培地。7. The medium for detecting a thermophilic Clostridium bacterium according to any one of claims 1 to 5, which is a medium for detecting a thermophilic Clostridium bacterium in a low-acid beverage.
idium属細菌の検出用培地であることを特徴とする請求
項１から５いずれか記載の高温性Clostridium属細菌検
出用培地。8. A high temperature Clostr in a low acid beverage product containing milk.
The medium for detecting thermophilic Clostridium bacteria according to any one of claims 1 to 5, which is a medium for detecting bacteria of the genus idium.
にニュートラルレッドを使用する方法。9. A method of using neutral red for detecting thermophilic Clostridium bacteria.
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| JP4675669B2 (en) * | 2005-04-28 | 2011-04-27 | アサヒ飲料株式会社 | Medium for detecting thermophilic Clostridium bacteria, method for shortening detection period, and method for detecting bacteria |
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