JP3535083B2 - Medium for detecting thermophilic Clostridium bacteria - Google Patents
Medium for detecting thermophilic Clostridium bacteriaInfo
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- JP3535083B2 JP3535083B2 JP2000248970A JP2000248970A JP3535083B2 JP 3535083 B2 JP3535083 B2 JP 3535083B2 JP 2000248970 A JP2000248970 A JP 2000248970A JP 2000248970 A JP2000248970 A JP 2000248970A JP 3535083 B2 JP3535083 B2 JP 3535083B2
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- medium
- bacterium
- detecting
- genus
- thermophilic
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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。TECHNICAL FIELD The present invention relates to a medium for easily and rapidly detecting a thermophilic Clostridium genus bacterium, which is a deterioration indicator bacterium for retort canned 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 Retort canned beverages such as coffee and shiruko are usually heat-sterilized at 120 ° C. to 125 ° C. for about 20 to 40 minutes before shipment, but some bacteria do not die even by such sterilization. . Such a bacterium is generally called a thermophilic bacterium, but this bacterium does not grow at a temperature of 40 ° C. or lower 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 a bacterium is mixed in a beverage in a retort can which may be heated and sold by a hot vendor, the temperature in the hot vendor is the optimum growth temperature of such bacteria ( Since the temperature is around 55 ° C. to 60 ° C.), bacteria may grow and cause deterioration of the beverage, and it is necessary to take measures beforehand to prevent such a situation.
【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。An anaerobic thermophilic Clostridium bacterium is an example of a bacterium that serves as an index for preventing such deterioration at high temperature (degradation index bacterium). This bacterium forms spores and stops its activity in an environment unsuitable for growth, but once the growth conditions are met, it returns to normal cells and begins to proliferate. It is extremely important to manage the product. Therefore, the thermophilic Clostridium genus bacteria should be detected in advance by the pour-in method using the modified TGC medium or the test method using a test tube, and the detected bacteria should be excluded from the shipping process. There is.
【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 thermophilic Clostridium bacteria, when it is attempted to detect it in a milk-containing beverage, it is extremely difficult to confirm the bacterial cells due to the diffusion of milk components. Therefore, the conventional detection method described above cannot be used for rapid detection. More specifically, the detection period for ordinary bacteria is about 2 days for general bacteria, whereas for thermophilic Clostridium bacteria it is extremely long as 5 to 7 days. It took a long 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 genus bacterium, and it is difficult to discriminate a milk-containing beverage, for example. There is also a problem that it may be difficult to distinguish the bacterial cells depending on the type.
【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。The present invention has been made to solve the above problems, and an object thereof is to easily and quickly determine a thermophilic Clostridium genus bacterium, which is a deterioration indicator bacterium for beverage products in retort cans. It is to provide such a medium.
【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 in order to achieve the above-mentioned objects, by incorporating neutral red in a detection medium, detection of thermophilic Clostridium bacteria can be performed. It becomes possible to easily perform, and by allowing the same medium to contain pyruvic acid, it becomes possible to easily and quickly detect the thermophilic Clostridium genus bacterium, so that the present invention has been 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 bacterium belonging to the genus Clostridium, which results in "discoloration". Even difficult things can be detected accurately and easily. Further, since pyruvic acid activates the growth of thermophilic Clostridium bacteria, its addition in the medium makes it possible to increase the detection rate to such an extent that there is no 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。When the present invention as described above is generalized, a thermophilic Clostrid is added to the detection medium by including a pigment that discolors due to the metabolism of cells and a bacterial growth promoter.
It can be grasped as a method that enables simple and rapid detection of ium bacteria. And, more specifically, the present invention provides a conventional medium, for example, a modified TGC medium, with a pigment such as neutral red which is decomposed by the metabolism of cells accompanying the growth of a bacterium and a bacterium such as pyruvic acid. By adding a substance that promotes growth,
The cells can be confirmed simply by confirming the change in the color of the medium due to the decomposition of the pigment (faster than the confirmation of the growth 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 content of the present invention is 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 genus bacterium, which comprises a pigment that is discolored by the metabolism of the bacterium and a bacterial growth promoter that promotes the growth of the bacterium. 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。The term "comprising" is used in the present specification to indicate that a medium other than a pigment or a bacterial growth promoter may be contained in the medium as long as it does not impair the discoloration process according to the present invention. Is. In the present invention, when the target bacterium grows, the pigment is decomposed by the metabolism of the bacterium and the medium is discolored to distinguish the bacterium, and the dye is added after culturing the bacterium. This is because the 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 bacteria by the change in the added dye has been clarified in JP-A-59-2699 and JP-A-57-68796, both of which are disclosed. The presence or absence of bacteria is confirmed by the change in color associated with the change in pH of the bacteria detection medium. Therefore, the present invention is distinctly different from the present invention in which bacteria are discriminated from the change in color caused by metabolism by bacterial 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 genus bacterium, which contains neutral red, and is a medium for detecting a thermophilic Clostridium genus bacterium.
【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。"Neutral red" is widely known as a pH indicator, but in the present specification, it is used as a pigment that changes color by the metabolism of cells. That is, not because "Neutral Red" discolors with a change in pH, but when a thermophilic Clostridium genus bacterium grows in the medium, "Neutral Red" is decomposed by the metabolism of the bacterial cells, and the medium is accompanied by this. 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 medium for detecting a thermophilic Clostridium genus bacterium, which further comprises a bacterial growth promoter that promotes the growth of the bacterium, and the thermophilic Clostridium described in (2). 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。In addition to Neutral Red, a bacterial growth promoter is also added to the thermophilic Cl which is a spore-forming bacterium.
This is because the growth of ostridium bacteria is slower than that of other general bacteria. That is, in the field of beverage production, it is not enough to simply and surely confirm the bacterial cells by adding neutral red, and the inspection speed can be increased by adding a bacterial growth promoter to accelerate the bacterial growth. It depends on 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 genus bacterium according to (3), characterized in that the bacterial growth promoting agent is pyruvic acid 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, which is an intermediate product of energy metabolism, is effective as a bacterial growth promoter in the present invention. Although Clostridium bacteria have the ability to assimilate three types of sugars, glucose, fructose, and xylose, pyruvic acid is preferable even when compared with these as shown in Example 3 below.
【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 stored under refrigeration, and its pH must be adjusted by adding dipotassium hydrogen phosphate when adjusting the medium. Pyruvate may be substituted to simplify such a procedure.
【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 having a concentration of 1 to 40 mg / l in a modified TGC medium of about 30 g / L,
High temperature Cl, characterized by comprising pyruvic acid or pyruvate in 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 beverage products in retort cans
High temperature Clostridium according to any one of (1) to (5), which is a culture 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] Retort canned beverages may be heated and sold by a hot vendor, and therefore, the thermophilic Clos that is a spore-forming bacterium.
It is a beverage product that requires the 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 beverages
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 an acidic beverage having a pH of 4.6 or less, the heat resistance of the bacterium belonging to the genus Clostridium is decreased and the growth is suppressed, which is not a serious problem. As possible, the detection of such bacteria in low acid beverages is extremely important. Note that low-acidic beverages include tea-based beverages such as black tea and oolong tea, as well as various beverages such as coffee, cocoa, canned shiruko 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 property in milk-containing low-acid beverage products
High temperature Clostridium according to any one of (1) to (5), which is a culture 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。The term "milk-containing beverage" as used herein includes all beverages containing dairy ingredients. If dairy ingredients are included, they will diffuse, so Cl
Although it becomes difficult to detect bacteria of the genus ostridium, the present invention enables simple and rapid detection of bacteria regardless of the presence or absence of milk components.
【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 of using neutral red for the detection of 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 The preparation of a base medium is carried out by a conventional method. For example, modified TGC medium and iron sulfite medium can be used. In addition, the measurement of bacteria is based on the high temperature Clostrid
It is necessary to consider that the ium bacteria are anaerobic.
Therefore, for example, there is a method in which the semi-fluid medium method is used instead of the 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, but it is clear that the essence of the present invention is to include in the medium a pigment that discolors due to the metabolism of bacterial cells and a bacterial growth promoter. Therefore, various changes can be made as long as the essential part is not changed. Therefore, the present invention is not limited 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 types 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 fluid after germination treatment with sulfuricum and Clostridium.thermaceticum was ingested. The addition of each pH indicator was compared by performing two methods, a method of adding when adjusting the medium and a method of adding after 5 days of culture. The results are 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.thermaceticum grows slowly and the pH does not drop sufficiently after 5 days, it was impossible to discriminate the bacteria by adding a pigment after culturing. In addition, for some pH indicators added during medium adjustment, the color changed even though there was little change in pH, so this change was not due to pH change, but due to bacterial metabolism, etc. It is considered that this is because the pigment was decomposed. From this, it became clear that the pigment needs 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] Pigment fluid was added to a modified TGC medium to adjust the medium, and then the spore fluid 10 after germination treatment with Clostoridium.thermohydrosulfuricum and Clostoridium.thermaceticum 10
3 to 10 -1 cfu / ml and 1 ml canned coffee (with milk)
After ingestion, the detection sensitivity by the MPN method and the determination period 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, the bacterial solution is diluted and ingested into 5 test tubes at each dilution step. Then, it is a method of statistically calculating the concentration of bacteria by observing how many test tubes the bacteria have grown after culturing the bacteria. This is an effective method for comparing detection sensitivities and the like. Table 2 below shows the comparison results by the MPN method when various dyes were added before bacterial culture. In this table, for example, "4/5" means that 4 out of 5 ingested bacteria are inoculated.
【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 results as in the conventional method were obtained for three types of neutral red, chlorophenol red and phenol red. In Clostoridium.thermac eticum, it was possible to judge only for neutral red. From this, it became clear that it is effective to use neutral red as a pigment added to the medium for detecting Clostoridium sp.
【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 one day, but Clostoridium.therm
Since it took 4 days to culture aceticum, glucose, fructose, xylose, pyruvic acid and pyruvate were added to the medium in order to accelerate the growth of this 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 slightly improved the growth.
On the other hand, when pyruvic acid was added, the culture was promoted. From this, it became clear that pyruvic acid is effective for 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 addition 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 0.63g / l + K 2 HPO 4 2g / l Modified TGC + pyruvate 1.26g / l + K 2 HPO 4 3g / l Modified TGC + pyruvate 5.04g / 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。Clostoridium.thermohydrosulf was added to this medium.
Ingestion of uricum and Clostoridium.thermaceticum resulted in the results shown in Table 3 below.
【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 1% neutral red solution was added to 30 g / L of modified TGC medium to prepare pyruvate with 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 pyruvate[0045] Modified TGC Modified TGC + 0.55g / l sodium pyruvate Modified TGC + 0.8g / l sodium pyruvate Modified TGC + 1.1g / 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。When 5 control and 5 concentrations were added to this medium and 10 3 cfu / ml of spore fluid of Clostoridium.thermaceticum was ingested, the results shown in Table 4 below could be obtained.
【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 the 1% neutral red solution was added to the modified TGC medium of 30 g / L, the appropriate amount of pyruvate added was 0.8 g.
【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 detection sensitivity and the determination period of the conventional medium and the improved medium were compared using this method of MPN5, 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。Since Bacillus. Stearothermophilus did not show a pigment change phenomenon, it was revealed that the detection by the medium according to the present invention is not suitable for this bacterium. On the other hand, Clostoridium.thermohydrosulfuricum
And Clostoridium.thermaceticum showed a change in the pigment, and this change was observed after 72 hours of culturing. Therefore, it became clear that the cells could be confirmed in a significantly shorter time than 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 When the same experiment as in Comparative Example 1 was conducted with other beverages, 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 revealed that bacteria can be detected in other retort 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] Since the retort sterilization is carried out when the product is actually produced, it is considered that the bacterial cells are damaged. Therefore, after heat treatment at 121 ° C for 20 minutes, the bacterial cells are tested in a test tube. When ingested, 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 became clear that the bacteria can 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。EFFECT OF THE INVENTION According to the present invention as described above, it is possible to easily discriminate that a thermophilic Clostridium bacterium in a retort can beverage has proliferated in a short period of time (3 days) by observing a change in color of the medium. can do.
───────────────────────────────────────────────────── フロントページの続き (56)参考文献 特開 平７−147953（ＪＰ，Ａ) ロシア国特許出願公開2084521（ＲＵ, Ａ) Ｊ．Ｄａｉｒｙ Ｓｃｉ．（1990）, Ｖｏｌ．73，Ｎｏ．３，ｐ．719−725 ＦＥＭＳ Ｍｉｃｒｏｂｉｏｌｏｇｙ Ｒｅｖｉｅｗｓ（1995），Ｖｏｌ. 16，ｐ．151−162 Ｂｉｏｃｈｅｍ Ｃｅｌｌ Ｂｉｏｌ （1989），Ｖｏｌ．67，Ｎｏ．10，ｐ. 735−739 (58)調査した分野(Int.Cl.7，ＤＢ名) C12N 1/00 C12Q 1/00 ＭＥＤＬＩＮＥ（ＳＴＮ) ＢＩＯＳＩＳ／ＷＰＩ（ＤＩＡＬＯＧ) ＪＳＴＰｌｕｓ（ＳＴＮ)─────────────────────────────────────────────────── ─── Continuation of the front page (56) Reference JP-A-7-147953 (JP, A) Russian patent application publication 2084521 (RU, A) J. Dairy Sci. (1990), Vol. 73, No. 3, p. 719-725 FEMS Microbiology Reviews (1995), Vol. 16, p. 151-162 Biochem Cell Biol (1989), Vol. 67, No. 10, p.735-739 (58) Fields surveyed (Int.Cl. 7 , DB name) C12N 1/00 C12Q 1/00 MEDLINE (STN) BIOSIS / WPI (DIALOG) JSTPlus (STN)
Claims (10)
属細菌を検出するための検出用培地であって、ニュート
ラルレッドと、菌体の増殖促進に有効な量のピルビン酸
またはピルビン酸塩と、が、前記細菌培養前に添加され
ているものであることを特徴とする飲料中の高温性Ｃｌ
ｏｓｔｒｉｄｉｕｍ属細菌検出用培地。1. A thermophilic Clostridium in a beverage.
A detection medium for detecting a genus bacterium, wherein neutral red and an amount of pyruvic acid or pyruvate effective for promoting the growth of bacterial cells are added before the bacterial culture.
Thermophilic Cl in beverages, characterized in that those are
A medium for detecting an ostridium bacterium.
ｇ／ｌの濃度で含まれていることを特徴とする請求項１
記載の飲料中の高温性Ｃｌｏｓｔｒｉｄｉｕｍ属細菌検
出用培地。2. The neutral red has a length of 1 to 40 m.
2. The composition is contained at a concentration of g / l.
A medium for detecting thermophilic Clostridium bacteria in the beverage as described.
１〜３０ｍｍｏｌ／ｌの濃度で含まれていることを特徴
とする請求項１記載の飲料中の高温性Ｃｌｏｓｔｒｉｄ
ｉｕｍ属細菌検出用培地。3. The pyruvic acid or pyruvic acid salt is
The high temperature Clostrid in the beverage according to claim 1, characterized in that it is contained at a concentration of 1 to 30 mmol / l.
Medium for detecting ium bacterium.
項１から３いずれか記載の飲料中の高温性Ｃｌｏｓｔｒ
ｉｄｉｕｍ属細菌検出用培地。4. The thermophilic Clostr in the beverage according to claim 1, wherein the medium is a modified TGC medium.
Medium for detecting bacterium of genus idium.
ｓｔｒｉｄｉｕｍ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４いずれか記載の高温性Ｃｌｏｓｔ
ｒｉｄｉｕｍ属細菌検出用培地。5. High temperature Clo in beverage products in retort cans.
A thermophilic Clost according to any one of claims 1 to 4, which is a culture medium for detecting a bacterium of the genus stridium.
A medium for detecting a bacterium of the genus ridium.
ｉｕｍ属細菌検出用培地であることを特徴とする請求項
１から４いずれか記載の高温性Ｃｌｏｓｔｒｉｄｉｕｍ
属細菌検出用培地。6. A high temperature Clostrid in a low acid beverage.
5. A thermophilic Clostridium according to any one of claims 1 to 4, which is a medium for detecting a bacterium belonging to the genus ium.
Medium for detecting genus bacteria.
ｓｔｒｉｄｉｕｍ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４いずれか記載の高温性Ｃｌｏｓｔ
ｒｉｄｉｕｍ属細菌検出用培地。7. A high-temperature Clo in a low-acid beverage product containing milk.
A thermophilic Clost according to any one of claims 1 to 4, which is a culture medium for detecting a bacterium of the genus stridium.
A medium for detecting a bacterium of the genus ridium.
菌は嫌気性であり、前記培地は半流動培地であることを
特徴とする請求項１から７いずれか記載の高温性Ｃｌｏ
ｓｔｒｉｄｉｕｍ属細菌検出用培地。 8. The thermophilic Clostridium spp.
The fungus is anaerobic and the medium is a semi-solid medium.
The high temperature Clo according to any one of claims 1 to 7.
A medium for detecting a bacterium of the genus stridium .
Ｃｌｏｓｔｏｒｉｄｉｕｍ . ｔｈｅｒｍｏｈｙｄｒｏｓ
ｕｌｆｕｒｉｃｕｍ及び／又はＣｌｏｓｔｏｒｉｄｉｕ
ｍ . ｔｈｅｒｍａｃｅｔｉｃｕｍであることを特徴とす
る請求項１から８いずれか記載の高温性Ｃｌｏｓｔｒｉ
ｄｉｕｍ属細菌検出用培地。 9. The Clostridium bacterium comprises:
Clostoridium. Thermohydros
ulfuricum and / or Clostoridi
characterized in that it is m .
9. The high temperature Clostri according to any one of claims 1 to 8.
Medium for detecting bacterium of genus dium.
ｍ属細菌の検出のために、ニュートラルレッド、及び、Neutral red for the detection of m bacteria and
菌体の増殖促進に有効な量のピルビン酸またはピルビンPyruvate or pyruvin in an amount effective for promoting the growth of bacterial cells
酸塩、を使用する方法。A method of using an acid salt.
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| ＦＥＭＳ Ｍｉｃｒｏｂｉｏｌｏｇｙ Ｒｅｖｉｅｗｓ（１９９５），Ｖｏｌ．１６，ｐ．１５１−１６２ |
| Ｊ．Ｄａｉｒｙ Ｓｃｉ．（１９９０），Ｖｏｌ．７３，Ｎｏ．３，ｐ．７１９−７２５ |
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