JP2004041104A - Method for inspecting contamination with thermophilic acidophilic sporulation bacteria - Google Patents

Method for inspecting contamination with thermophilic acidophilic sporulation bacteria Download PDF

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JP2004041104A
JP2004041104A JP2002204308A JP2002204308A JP2004041104A JP 2004041104 A JP2004041104 A JP 2004041104A JP 2002204308 A JP2002204308 A JP 2002204308A JP 2002204308 A JP2002204308 A JP 2002204308A JP 2004041104 A JP2004041104 A JP 2004041104A
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spore
temperature
thermophilic
eosinophilic
range
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Japanese (ja)
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Keiichi Goto
後藤 慶一
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Mitsui Norin Co Ltd
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Mitsui Norin Co Ltd
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Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for rapidly and easily inspecting the (non)contamination with thermophilic acidophilic sporulation bacteria for various foods/drinks including fruits juice drinks and/or materials thereof. <P>SOLUTION: The method for inspecting whether a food/drink or material(s) thereof is contaminated with the thermophilic acidophilic sporulation bacteria comprises the following procedure. a liquid specimen is prepared from a test object and applied onto a plurality of agar medium sheets adjusted to pH3-5 each comprising an assimilation component substantially comprising yeast extract, starch and glucose and agar-agar as the solidification component, and the resulting sheets are simultaneously cultured at 40-50°C and 60-70°C for 14-24 h respectively, and the bacterial colony growth statuses at 40-50°C and 60-70°C are compared visually. By this method, in case of contamination, the bacteria can also be identified. <P>COPYRIGHT: (C)2004,JPO

Description

【0001】
【発明の属する技術分野】
本発明は、果汁入り飲食品をはじめとする各種飲食品やその原材料についての高温性好酸性芽胞形成細菌汚染の有無を迅速かつ簡便に検査する方法に関する。
【0002】
【従来の技術】
1984年に透明アップルジュースの大規模な微生物汚染事故がドイツで発生し、この原因となった細菌が高温性好酸性芽胞形成細菌、即ち、アリサイクロバチルス(Alicyclobacillus)属細菌の一つであるアリサイクロバチルス・アシドテレストリス(Alicyclobacillus acidoterrestris)(以下、acidoterrestrisと記載する)であることが判明して以来、グアイアコールやハロフェノールなどの悪臭を生成するこの細菌が果汁飲料の変敗を起こす細菌として問題視されている。また、果汁飲料には、acidoterrestrisの他にも、これと同じ高温性好酸性芽胞形成細菌であるアリサイクロバチルス・アシドカルダリウス(Alicyclobacillus acidocaldarius)(以下、acidocaldariusと記載する)およびアリサイクロバチルス・ゲノミック・スピーシーズ1(Alicyclobacillus genomic species 1)(以下、Alicyclobacillus genomic species 1と記載する)が混入することが多数報告されている。
果汁飲料から分離された高温性好酸性芽胞形成細菌には、上記の細菌の他にも、アリサイクロバチルス・アシディフィラス(Alicyclobacillus acidiphilus)やアリサイクロバチルス・ハーバリウス(Alicyclobacillus herbarius)などがあるが、その報告例は各1例のみであることから、これらの細菌が果汁飲料に混入することは極めて稀であると考えられている。
以上の点から、果汁飲料やその原料となる原果汁が高温性好酸性芽胞形成細菌に汚染されている場合、そこに混入している細菌は、acidoterrestrisacidocaldariusおよびAlicyclobacillus genomic species 1の少なくとも1種であると判定するのが一般的である。
【0003】
例えば、果汁飲料や原果汁のような酸性溶液中では細菌は増殖しにくいことから、古くは果汁飲料の製造工程における殺菌手段としては、カビや酵母や一般的な細菌を殺菌対象にしたパストル殺菌(90℃程度の加熱殺菌)で充分であるとされてきた。しかし、高温性好酸性芽胞形成細菌はこのような殺菌条件下でも生き延びることができるので、原果汁などにacidoterrestrisが混入していたり、果汁飲料の製造工程のいずれかの段階でacidoterrestrisが混入してしまったりした場合には上記のような汚染事故を招くことになる。従って、果汁飲料についてのacidoterrestrisによる汚染事故を未然に防ぐためには高温性好酸性芽胞形成細菌汚染の有無を検査する方法の確立が重要である。
【0004】
【発明が解決しようとする課題】
従来、このような検査は、例えば、原果汁の受け入れ検査や果汁飲料の品質検査として、微生物操作に関する事項についてある程度の知識を有する者が5〜10日あるいはそれ以上の日数をかけて行っていた。そのため、果汁飲料の製造に要する時間がそれだけ長くなり、出荷が遅くなるといった問題や検査結果として検査対象が高温性好酸性芽胞形成細菌に汚染されている場合に迅速に対応できないといった問題があった。
従って、果汁飲料や原果汁についての高温性好酸性芽胞形成細菌汚染の有無の検査を1日でも早く、また、誰もが簡単に行うことができる方法が待望されている。このことは、果汁飲料や原果汁に限って論じられることではなく、高温性好酸性芽胞形成細菌汚染の可能性があり、特にacidoterrestrisが混入して増殖する恐れのあるあらゆる飲食品やその原材料についても同様である。
そこで本発明は、果汁入り飲食品をはじめとする各種飲食品やその原材料についての高温性好酸性芽胞形成細菌汚染の有無を迅速かつ簡便に検査する方法を提供することを目的とする。
【0005】
【課題を解決するための手段】
上記の点に鑑みて本発明者が鋭意検討を行った結果、acidoterrestrisの至適増殖温度域と、acidocaldariusおよびAlicyclobacillus genomic species 1の至適増殖温度域との間には差異があることから、この差異を利用することにより、果汁入り飲食品をはじめとする各種飲食品やその原材料についての高温性好酸性芽胞形成細菌汚染の有無、特にacidoterrestrisの混入の有無を迅速かつ簡便に検査できることを見出した。
【0006】
本発明は上記の知見に基づいてなされたものであり、本発明の高温性好酸性芽胞形成細菌汚染の検査方法は、請求項1記載の通り、飲食品またはその原材料が高温性好酸性芽胞形成細菌に汚染されているか否かを検査するに際し、検査対象から液状検体を調製し、調製した液状検体を、資化成分の組成が実質的に酵母エキス、澱粉、グルコースからなり、固化成分として寒天を含むpH3〜5に調整した寒天培地複数枚に塗抹し、40〜50℃の範囲内の温度と60〜70℃の範囲内の温度の2種類の温度で同時に14〜24時間培養した後、40〜50℃の範囲内の温度での菌コロニー生育状況と60〜70℃の範囲内の温度での菌コロニー生育状況を目視比較することで高温性好酸性芽胞形成細菌の混入の有無と種類の鑑別を行うことを特徴とする。
また、本発明の別の高温性好酸性芽胞形成細菌汚染の検査方法は、請求項2記載の通り、飲食品またはその原材料から採取された高温性好酸性芽胞形成細菌の菌株を、資化成分の組成が実質的に酵母エキス、澱粉、グルコースからなり、固化成分として寒天を含むpH3〜5に調整した寒天培地複数枚に添加し、40〜50℃の範囲内の温度と60〜70℃の範囲内の温度の2種類の温度で同時に14〜24時間培養した後、40〜50℃の範囲内の温度での菌コロニー生育状況と60〜70℃の範囲内の温度での菌コロニー生育状況を目視比較することで高温性好酸性芽胞形成細菌の種類の鑑別を行うことを特徴とする。
また、請求項3記載の検査方法は、請求項1または2記載の検査方法において、検査対象が酸性飲食品またはその原材料であることを特徴とする。
また、請求項4記載の検査方法は、請求項3記載の検査方法において、検査対象が果汁入り飲食品または原果汁であることを特徴とする。
また、請求項5記載の検査方法は、請求項1乃至4のいずれかに記載の検査方法において、鑑別対象となる高温性好酸性芽胞形成細菌がacidoterrestrisであることを特徴とする。
また、請求項6記載の検査方法は、請求項1乃至5のいずれかに記載の検査方法において、18〜20時間培養することを特徴とする。
【0007】
【発明の実施の形態】
本発明における高温性好酸性芽胞形成細菌汚染の検査対象は、高温性好酸性芽胞形成細菌汚染の可能性があるあらゆる飲食品やその原材料である。飲食品としては、例えば、酸性飲食品が挙げられる。ここで酸性飲食品とはpH5.5以下の食品および飲料を意味する。酸性飲食品の代表例としては、果汁入り飲食品が挙げられる。ここで果汁入り飲食品とは果汁が最終濃度として0.01重量%以上含まれる食品および飲料を意味し、100%果汁飲料を含む果汁入り清涼飲料、果汁入りのゼリーやヨーグルトやガムや飴などが例示される。果汁入り飲食品の原材料としては、原果汁が挙げられるが、これは、例えば、果実から搾取した果汁またはその水分を除去して濃縮したものであり、濃縮果汁還元して使用されたりするものである。代表的に例示される果汁としては、オレンジ果汁、アップル果汁、グレープフルーツ果汁、グレープ果汁、パイン果汁、レモン果汁、ピーチ果汁、ベリー果汁、マンゴ果汁などが挙げられる。酸性飲食品としては、果汁入り飲食品の他にも、炭酸飲料、果実飲料、酸性紅茶飲料、スポーツ飲料、トマトジュースや人参ジュースなどの各種野菜ジュース、栄養ドリンク、乳性飲料、ヨーグルト、果肉入りゼリー、リキュール、ジャム、マーマレード、ドレッシングなどが挙げられる。酸性飲食品の原材料としては、果実や野菜の他にも、糖類(ブドウ糖や果糖などの単糖類、砂糖や麦芽糖などの二糖類、果糖・ブドウ糖液糖、デキストリン、各種オリゴ糖、蜂蜜、糖アルコールなど)、酸味料、着色料、香料、増粘多糖類、ゲル化剤、脱脂粉乳、加糖乳糖、乳酸菌飲料、発酵乳、ビタミン類などが挙げられる。また、穀類、いも及びでんぷん類、豆類、種実類、茶類、きのこ類、藻類、魚介類、肉類、卵類、乳類、油脂類、菓子類、し好飲料類、調味料及び香辛料類、調味加工食品などの飲食品や、これら飲食品の中で原材料を使用して製造されるものにおける当該原材料が本発明における高温性好酸性芽胞形成細菌汚染の検査対象となる。
【0008】
本発明の検査方法においては、まず、後述する寒天培地に塗抹するための液状検体を検査対象となる飲食品やその原材料から調製する。果汁飲料や原果汁などのように検査対象自体が液状のものは、それ自体を検体としてもよいし、検査対象を水に懸濁希釈したり溶解希釈したりしてなるものを検体としてもよい。検査対象が液状のものでない場合は、例えば、検査対象を細かく砕いて水に懸濁したり、ホモジナイザーを使用してホモジネートを調製したりし、これらから得られる上清を検体とすればよい。
検査対象が糖度の高いもの、例えば、糖度が40〜60°Brix程度の原果汁や糖類である場合、検査対象を水に懸濁希釈したり溶解希釈したりして糖度が18°Brix以下の検体を調製することが望ましい。高温性好酸性芽胞形成細菌は、糖度が高い環境下においては増殖しないか増殖してもその速度が極めて遅い。従って、糖度が18°Brixを超えた検体の場合、検体に高温性好酸性芽胞形成細菌が混入していても、これを培養したところで菌コロニーが充分に生育せず、結果として混入の事実を的確に把握できない恐れがあるからである。また、検査対象が酸性アルコール入り飲料や果実酒や果汁入りアルコール飲料などのようなアルコール度の高いものである場合、検査対象を水に懸濁希釈したり溶解希釈したりしてアルコール度が6%以下の検体を調製することが望ましい。高温性好酸性芽胞形成細菌は、アルコール度が高い環境下においては増殖しないか増殖してもその速度が極めて遅い。従って、アルコール度が6%を超えた検体の場合、検体に高温性好酸性芽胞形成細菌が混入していても、これを培養したところで菌コロニーが充分に生育せず、結果として混入の事実を的確に把握できない恐れがあるからである。
なお、検体を調製する際に使用する水は滅菌水であることが望ましい。
【0009】
本発明の検査方法においては、液状検体に高温性好酸性芽胞形成細菌が混入している場合、当該高温性好酸性芽胞形成細菌を培養するために、資化成分の組成が実質的に酵母エキス、澱粉、グルコースからなり、固形成分として寒天を含むpH3〜5に調整した寒天培地を使用し、この寒天培地に液状検体を画線接種するなどして塗抹する。この寒天培地は、特開平8−140697号公報に記載されている公知のものであり、例えば、果汁飲料や原果汁に混入することが知られている3種類の高温性好酸性芽胞形成細菌、即ち、acidoterrestrisacidocaldarius、およびAlicyclobacillus genomic species 1のすべてを各々の至適増殖温度域で良好に増殖せしめるという優れた特性を有する。この寒天培地の具体例としては、イーストエキストラクト(Difco社製)2.0g/L、スターチ(Difco社製)2.0g/L、D−グルコース1.0g/Lからなる成分組成を有し、硫酸を用いてpH3.7に調整してから高圧蒸気滅菌したものに、高圧蒸気滅菌した寒天を最終濃度が1.5%になるように添加し、よく混合して平板にしたものが挙げられる(このようにして調製された寒天培地をYSG寒天培地という)。
【0010】
なお、液状検体を上記の寒天培地に塗抹する前に、高温性好酸性芽胞形成細菌の発芽を誘導して増殖を促すことを目的として液状検体に対して70〜80℃で5〜25分間の加熱処理を行ってもよい。
【0011】
高温性好酸性芽胞形成細菌の培養時間は14〜24時間とする。この時間範囲において、acidoterrestrisは、40〜50℃では盛んに増殖して菌コロニーが生育するが、60〜70℃では増殖しないか増殖してもその速度が極めて遅いため菌コロニーの良好な生育は認められない。一方、acidocaldariusおよびAlicyclobacillus genomic species 1は、40〜50℃では増殖しないか増殖してもその速度が極めて遅いため菌コロニーの良好な生育は認められないが、60〜70℃では盛んに増殖して菌コロニーが生育する。本発明においては、この現象を利用して、40〜50℃での菌コロニー生育状況と60〜70℃での菌コロニー生育状況、望ましくは、43〜47℃での菌コロニー生育状況と63〜67℃での菌コロニー生育状況を目視比較し、高温性好酸性芽胞形成細菌の混入の有無と種類の鑑別を行う。
【0012】
高温性好酸性芽胞形成細菌の培養時間が14時間未満であると、検体に高温性好酸性芽胞形成細菌が混入していたとしても、培養時間が短すぎることにより菌コロニーが充分に生育しないことがある一方、培養時間が24時間を超えると、培養時間が24時間以下の場合には増殖しないか増殖してもその速度が極めて遅い温度域であっても増殖が進行することがあり、いずれの場合であっても結果として的確な細菌の種類の鑑別が困難になる。なお、培養時間は18〜20時間とすることが望ましく、18時間とすることがより望ましい。
【0013】
ある検体について、40〜50℃で菌コロニーが生育した場合には、その検体にはacidoterrestrisが混入していたと判定することができる。その検体について、60〜70℃で菌コロニーが生育しなかった場合、その検体にはacidoterrestrisのみが混入していたことになり、60〜70℃で菌コロニーが生育した場合、acidoterrestrisの他にも、acidocaldariusおよび/またはAlicyclobacillus genomic species 1が混入していたことになる。従って、ある検体について、40〜50℃で菌コロニーが生育した場合には、60〜70℃での菌コロニーの生育状況がいかなるものであっても、その検体には微生物汚染事故を起こす恐れのあるacidoterrestrisが混入していることになる。よって、本発明によれば、acidoterrestrisacidocaldarius、およびAlicyclobacillus genomic species 1の3種類の高温性好酸性芽胞形成細菌の中からacidoterrestrisの鑑別を行うことができる。
【0014】
なお、本発明によれば、メンブランフィルタなどの微生物捕捉用フィルタに検査対象から調製した液状検体をろ過し、液状検体に高温性好酸性芽胞形成細菌が混入していた場合にはフィルタ上に高温性好酸性芽胞形成細菌を捕捉し、捕捉した高温性好酸性芽胞形成細菌を、資化成分の組成が実質的に酵母エキス、澱粉、グルコースからなり、固化成分として寒天を含むpH3〜5に調整した寒天培地を使用して3日間程度培養することで菌株を採取し、当該菌株を前記の寒天培地と同じ寒天培地複数枚に添加し、40〜50℃の範囲内の温度と60〜70℃の範囲内の温度の2種類の温度で同時に14〜24時間培養した後、40〜50℃の範囲内の温度での菌コロニー生育状況と60〜70℃の範囲内の温度での菌コロニー生育状況を目視比較することで高温性好酸性芽胞形成細菌の種類の鑑別を行うことにより、高温性好酸性芽胞形成細菌汚染の有無を迅速かつ簡便に検査することもできる。
【0015】
【実施例】
以下、実施例をあげて本発明をさらに詳細に説明するが、本発明は以下の記載の限定して解釈されるものではない。
【0016】
実験1:
acidoterrestrisacidocaldarius、およびAlicyclobacillus genomic species 1のそれぞれについて表1に記載した菌株を準備し、各菌株をYSG寒天培地に画線接種し、30℃、35℃、40℃、45℃、50℃、55℃、60℃、65℃の各温度条件で同時に培養した。18時間経過後、菌コロニーの生育状況を目視確認した。その結果を表1に示す。
【0017】
【表1】

Figure 2004041104
【0018】
表1において、+は生育良好、−は生育なし、w−は極微弱に生育、wは微弱に生育、w+はマイクロコロニーを意味する。
表1から明らかなように、acidoterrestrisは、45℃±5℃で菌コロニーの生育が確認できたが、60℃以上では確認できなかった。
一方、acidocaldariusおよびAlicyclobacillus genomic species 1は、45℃±5℃で菌株コロニーの生育が微弱であるか生育が確認できなかったが、60℃以上では確認できた。
以上の結果から、45℃での培養による菌コロニーの生育状況と65℃での培養による菌コロニーの生育状況を目視比較することで、acidoterrestrisacidocaldarius、およびAlicyclobacillus genomic species 1の3種類の高温性好酸性芽胞形成細菌の中からacidoterrestrisの鑑別を行うことができることがわかった。
【0019】
実験2:
果汁飲料として市販のオレンジジュース(糖度:約10°Brix)に、acidoterrestris菌株としてPB1を添加したサンプルA、acidocaldarius菌株として3Bを添加したサンプルB、Alicyclobacillus genomic species 1菌株としてDSM11983を添加したサンプルCを調製し、各サンプルをそのまま液状検体としてYSG寒天培地2枚にそれぞれ画線接種し、一方を45℃で、他方を65℃で同時に18時間培養した。18時間経過後、菌コロニーの生育状況を確認した結果、サンプルAについては、45℃での培養で菌コロニーの生育が確認されたが、65℃での培養では確認されなかった。一方、サンプルBとサンプルCについては、45℃での培養では菌コロニーの生育が確認されなかったが、65℃での培養では確認された。以上の結果から、45℃での培養による菌コロニーの生育状況と65℃での培養による菌コロニーの生育状況を目視比較することで、acidoterrestrisacidocaldarius、およびAlicyclobacillus genomic species 1の3種類の高温性好酸性芽胞形成細菌の中からacidoterrestrisの鑑別を行うことができることがわかった。
【0020】
【発明の効果】
本発明によれば、果汁入り飲食品をはじめとする各種飲食品やその原材料についての高温性好酸性芽胞形成細菌汚染の有無を迅速かつ簡便に検査する方法が提供される。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for quickly and easily examining the presence or absence of high-temperature eosinophilic spore-forming bacterial contamination of various foods and drinks including fruit juice-containing foods and drinks and raw materials thereof.
[0002]
[Prior art]
In 1984, a large-scale microbial contamination accident of transparent apple juice occurred in Germany, and the bacterium responsible for this was thermophilic eosinophilic spore-forming bacterium, an ant belonging to the genus Alicyclobacillus. cyclo acidoterrestris (Alicyclobacillus acidoterrestris) (hereinafter, a. to as acidoterrestris) since been found to be, this bacterium to produce a bad odor, such as guaiacol and halo phenol cause spoilage of fruit juice drinks bacteria As a problem. In addition, the fruit juice beverages, A. Besides acidoterrestris, which as the same high-temperature resistant acidophilic spore-forming bacterium Alicyclobacillus acidocaldarius (Alicyclobacillus acidocaldarius) (hereinafter, A. to as acidocaldarius) and Alicyclobacillus, genomic sp 1 (Alicyclobacillus It has been reported that a large number of genomic species 1) (hereinafter, referred to as Alicyclic genomic species 1) are mixed.
The thermophilic eosinophilic spore-forming bacteria isolated from the fruit juice beverage include, in addition to the above-mentioned bacteria, Alicyclobacillus acidifilus and Alicyclobacillus herbarius . Since there is only one report for each case, it is considered that these bacteria are extremely rarely mixed into the fruit juice beverage.
In view of the above, if the original fruit juice as a fruit juice beverage and the raw material is contaminated with thermophilic acidophilic spore-forming bacteria, bacteria are mixed there is, A. acidoterrestris, A. In general, it is determined to be at least one of A. acidiccalarius and Aliclobacillus genomic species 1.
[0003]
For example, bacteria are difficult to grow in acidic solutions such as fruit juice drinks and raw juices.Therefore, as a sterilization means in the manufacturing process of fruit juice drinks, pasteur sterilization targeting molds, yeasts, and general bacteria has long been used. (Heat sterilization at about 90 ° C.) has been found to be sufficient. However, since thermophilic eosinophilic spore-forming bacteria can survive under such sterilizing conditions, A. acidoterrestris or has been mixed, A at any stage of the manufacturing process of juice. In the case where acidoterrestris is mixed in, the above-described contamination accident is caused. Therefore, the A. It is important to establish a method for examining the presence or absence of thermophilic eosinophilic spore-forming bacteria in order to prevent contamination accidents caused by C. acidoterrestris .
[0004]
[Problems to be solved by the invention]
Conventionally, such an inspection, for example, as an acceptance inspection of raw juice or a quality inspection of a juice beverage, a person having a certain degree of knowledge on microbial operations was performed over 5 to 10 days or more. . Therefore, there is a problem that the time required for manufacturing the fruit juice beverage becomes longer and the shipment is delayed, and there is a problem that it is not possible to quickly respond when the test object is contaminated with thermophilic eosinophilic spore-forming bacteria as a test result. .
Therefore, there is a long-awaited demand for a method in which a test for the presence of high-temperature eosinophilic spore-forming bacterial contamination of a fruit juice beverage or raw juice can be performed as soon as possible even by one day, and can be easily performed by anyone. This not be discussed only in fruit juices or raw juice, with a chance of thermophilic acidophilic spore-forming bacterial contamination, especially A. The same applies to any food or drink and its raw materials that may be multiplied by C. acidoterrestris .
Accordingly, an object of the present invention is to provide a method for quickly and simply testing the presence or absence of high-temperature eosinophilic spore-forming bacterial contamination of various foods and drinks including fruit juice-containing foods and drinks and raw materials thereof.
[0005]
[Means for Solving the Problems]
The present inventors have in view of the above intensive studies, A. and the optimal growth temperature range of acidoterrestris, A. There is a difference between the optimal growth temperature range of Acidocaldarius and Aliclobacillus genomic species 1, and by utilizing this difference, it is possible to improve the high-temperature properties of various foods and drinks, including juice-containing foods and drinks, and raw materials thereof. The presence or absence of acid spore-forming bacterial contamination, particularly A. It has been found that the presence or absence of acidoterrestris can be quickly and simply examined.
[0006]
The present invention has been made based on the above findings, and the method for testing thermophilic eosinophilic spore-forming bacteria contamination according to the present invention is characterized in that the food or drink or the raw material thereof is thermophilic eosinophilic spore-forming. When testing for bacterial contamination, a liquid sample is prepared from the test object, and the prepared liquid sample is composed of yeast extract, starch, and glucose, and the agar is used as a solidifying component. After spreading on a plurality of agar media adjusted to pH 3 to 5 containing, and culturing at the same temperature for 14 to 24 hours at two temperatures of 40 to 50 ° C and 60 to 70 ° C, The presence and type of contamination with thermophilic eosinophilic spore-forming bacteria by visually comparing the bacterial colony growth at a temperature within the range of 40 to 50 ° C. and the bacterial colony growth at a temperature within the range of 60 to 70 ° C. That the discrimination of And butterflies.
In addition, another method for testing thermophilic eosinophilic spore-forming bacteria according to the present invention comprises, as described in claim 2, a thermophilic acidophilic spore-forming bacterial strain collected from a food or drink or a raw material thereof, assimilating component. Is substantially composed of yeast extract, starch and glucose, and added to a plurality of agar media adjusted to pH 3 to 5 containing agar as a solidifying component, and added to a temperature in the range of 40 to 50 ° C and 60 to 70 ° C. After simultaneously culturing at two temperatures within the range for 14 to 24 hours, the bacterial colony growth at a temperature within a range of 40 to 50 ° C and the bacterial colony at a temperature within a range of 60 to 70 ° C. Is characterized by visually distinguishing thermophilic eosinophilic spore-forming bacteria.
According to a third aspect of the present invention, in the inspection method of the first or second aspect, the inspection target is an acidic food or drink or a raw material thereof.
According to a fourth aspect of the present invention, there is provided the inspection method according to the third aspect, wherein the inspection target is a juice-containing food or drink or an original juice.
The test method according to claim 5 is the test method according to any one of claims 1 to 4, wherein the thermophilic eosinophilic spore-forming bacterium to be discriminated is A. acidoterrestris .
A test method according to claim 6 is characterized in that, in the test method according to any one of claims 1 to 5, culturing is performed for 18 to 20 hours.
[0007]
BEST MODE FOR CARRYING OUT THE INVENTION
The test object of the thermophilic acidophilic spore-forming bacterium contamination in the present invention is any food or drink or a raw material thereof which may be contaminated with the thermophilic acidophilic spore-forming bacterium. Examples of the food and drink include acidic food and drink. Here, the acidic food and drink means foods and beverages having a pH of 5.5 or less. Representative examples of acidic foods and drinks include foods and drinks containing fruit juice. Here, the juice-containing food / beverage means foods and beverages containing fruit juice at a final concentration of 0.01% by weight or more, such as soft drinks containing juice containing 100% juice drink, jelly, yogurt, gum, candy and the like containing fruit juice. Is exemplified. Raw materials for juice-containing foods and drinks include raw juices, which are, for example, juices extracted from fruits or those obtained by removing the water content of the juices, and are used by reducing the concentrated juices. is there. Representative juices include orange juice, apple juice, grapefruit juice, grape juice, pine juice, lemon juice, peach juice, berry juice, mango juice and the like. As acidic foods and drinks, in addition to foods and drinks containing fruit juice, carbonated drinks, fruit drinks, acidic tea drinks, sports drinks, various vegetable juices such as tomato juice and carrot juice, nutritional drinks, milky drinks, yogurt, and pulp Jelly, liqueur, jam, marmalade, dressing and the like. Raw materials for acidic foods and drinks include fruits and vegetables, as well as sugars (monosaccharides such as glucose and fructose, disaccharides such as sugar and maltose, fructose / glucose liquid sugar, dextrin, various oligosaccharides, honey, and sugar alcohols). And the like, acidulants, coloring agents, flavors, thickening polysaccharides, gelling agents, skim milk powder, sweetened lactose, lactic acid beverages, fermented milk, vitamins and the like. In addition, cereals, potatoes and starches, beans, seeds and nuts, teas, mushrooms, algae, fish and shellfish, meat, eggs, milk, oils and fats, confectionery, delicious beverages, seasonings and spices, Foods and drinks such as seasoned processed foods and the like, and raw materials among these foods and drinks manufactured using raw materials, are to be tested for thermophilic eosinophilic spore-forming bacterial contamination in the present invention.
[0008]
In the test method of the present invention, first, a liquid specimen to be smeared on an agar medium described later is prepared from food or drink to be tested or raw materials thereof. If the test object itself is liquid, such as a fruit juice beverage or raw juice, the test object itself may be used as a sample, or a sample obtained by suspending or diluting the test object in water or dissolving it may be used as the sample. . When the test target is not a liquid, for example, the test target may be finely crushed and suspended in water, or a homogenate may be prepared using a homogenizer, and the supernatant obtained from these may be used as a sample.
When the test target is a high sugar content, for example, a raw fruit juice or a saccharide having a sugar content of about 40 to 60 ° Brix, the test target is suspended or diluted by dissolving in water and the sugar content is 18 ° Brix or less. It is desirable to prepare the specimen. Thermophilic eosinophilic spore-forming bacteria do not grow or grow very slowly in an environment with a high sugar content. Therefore, in the case of a sample having a sugar content exceeding 18 ° Brix, even if the sample is mixed with a thermophilic acidophilic spore-forming bacterium, a bacterial colony does not grow sufficiently when the sample is cultured. This is because there is a risk that it cannot be accurately grasped. In addition, when the test object is a highly alcoholic beverage such as a beverage containing acidic alcohol, an alcoholic beverage containing fruit liquor or fruit juice, the test subject is suspended or dissolved and diluted in water to have an alcohol content of 6 or less. % Or less of the sample is desirable. Thermophilic eosinophilic spore-forming bacteria do not grow or grow very slowly in an environment with a high alcohol content. Therefore, in the case of a sample having an alcohol content of more than 6%, even if a thermophilic eosinophilic spore-forming bacterium is mixed in the sample, the bacterial colony does not grow sufficiently when the sample is cultured, and the fact This is because there is a risk that it cannot be accurately grasped.
In addition, it is desirable that the water used when preparing the specimen is sterile water.
[0009]
In the test method of the present invention, when a thermophilic eosinophilic spore-forming bacterium is mixed in a liquid specimen, the composition of the assimilable component is substantially a yeast extract in order to culture the thermophilic eosinophilic spore-forming bacterium. An agar medium adjusted to pH 3 to 5 containing agar as a solid component and containing starch as a solid component is used, and a liquid sample is streaked on the agar medium and smeared. This agar medium is a known one described in JP-A-8-140697, and includes, for example, three kinds of thermophilic acidophilic spore-forming bacteria which are known to be mixed in juice drinks and raw juices, That is, A. acidoterrestris, A. It has an excellent property that all of Acidocaldarius and Alicyclobacillus genomic species 1 can be satisfactorily grown in the respective optimum growth temperature ranges. Specific examples of the agar medium include a component composition consisting of 2.0 g / L of yeast extract (manufactured by Difco), 2.0 g / L of starch (manufactured by Difco), and 1.0 g / L of D-glucose. Agar, which was adjusted to pH 3.7 with sulfuric acid and then sterilized by high-pressure steam, was added with agar subjected to high-pressure steam sterilization so that the final concentration became 1.5%, and mixed well to form a plate. (The agar medium thus prepared is referred to as a YSG agar medium).
[0010]
In addition, before smearing the liquid specimen on the agar medium, the liquid specimen was subjected to 70 to 80 ° C for 5 to 25 minutes for the purpose of inducing the germination of the thermophilic acidophilic spore-forming bacteria and promoting the proliferation. Heat treatment may be performed.
[0011]
The culture time of the thermophilic eosinophilic spore-forming bacteria is 14 to 24 hours. In this time range, A. Acidoterrestris proliferates vigorously at 40 to 50 ° C. to grow bacterial colonies, but does not grow at 60 to 70 ° C. or grows at a very low rate, so that good growth of bacterial colonies is not observed. On the other hand, A. acidocalidarius and Alicyclobacillus genomic species 1 do not grow at 40 to 50 ° C. or grow at a very low rate, so that good growth of bacterial colonies is not observed. However, bacterial colonies grow vigorously at 60 to 70 ° C. Grow. In the present invention, utilizing this phenomenon, the bacterial colony growth at 40 to 50 ° C. and the bacterial colony at 60 to 70 ° C., preferably the bacterial colony at 43 to 47 ° C. and 63 to 47 ° C. The growth status of the bacterial colonies at 67 ° C. is visually compared, and the presence or absence and type of the thermophilic eosinophilic spore-forming bacteria are discriminated.
[0012]
If the culture time of the thermophilic acidophilic spore-forming bacteria is less than 14 hours, even if the thermophilic acidophilic spore-forming bacteria are mixed in the sample, the bacterial colonies will not grow sufficiently due to the culture time being too short. On the other hand, if the culture time exceeds 24 hours, if the culture time is 24 hours or less, the growth may not proceed even if the growth rate is extremely low even if the growth rate is extremely low. As a result, it is difficult to accurately identify the type of bacteria. The culturing time is desirably 18 to 20 hours, and more desirably 18 hours.
[0013]
When a bacterial colony grew at 40 to 50 ° C. for a certain sample, the sample contained A. It can be determined that A. acidosterestris has been mixed. When no bacterial colony grew at 60 to 70 ° C. for the sample, the sample contained A. acidoterrestris was contaminated, and when a bacterial colony grew at 60 to 70 ° C., A. a . In addition to the acidoterrestris, A. This means that acidoidarius and / or Alicyclobacillus genomic species 1 were contaminated. Therefore, when a bacterial colony grows at 40 to 50 ° C. for a certain sample, the sample may cause a microbial contamination accident regardless of the growth condition of the bacterial colony at 60 to 70 ° C. A. acidterrestris . Therefore, according to the present invention, A. acidoterrestris, A. acidocaldarius, and Alicyclobacillus genomic species 1 of A among the three types of thermophilic acidophilic spore-forming bacteria. acidosterrestris can be identified.
[0014]
According to the present invention, the liquid sample prepared from the test object is filtered to a filter for capturing microorganisms such as a membrane filter, and when the thermophilic eosinophilic spore-forming bacteria are mixed in the liquid sample, the high-temperature The thermophilic eosinophilic spore-forming bacterium is captured, and the captured thermophilic acidophilic spore-forming bacterium is adjusted to a pH of 3 to 5 in which the composition of the assimilation component is substantially composed of yeast extract, starch, and glucose, and contains agar as a solidifying component. The strain was collected by culturing for about 3 days using the agar medium thus obtained, and the strain was added to a plurality of agar mediums identical to the agar medium described above, and a temperature in the range of 40 to 50 ° C and 60 to 70 ° C. After culturing at the same temperature for 14 to 24 hours at two different temperatures, the bacterial colony growth at a temperature of 40 to 50 ° C. and the bacterial colony growth at a temperature of 60 to 70 ° C. Look at the situation By performing the type discrimination of thermophilic acidophilic spore-forming bacteria by compare, can be examined for thermophilic acidophilic spore-forming bacterial contamination quickly and easily.
[0015]
【Example】
Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention should not be construed as being limited to the following description.
[0016]
Experiment 1:
A. acidoterrestris, A. acidocardarius and Alicyclobacillus genomic specifications 1 were prepared as described in Table 1, and each strain was streaked on a YSG agar medium, 30 ° C, 35 ° C, 40 ° C, 45 ° C, 50 ° C, 55 ° C, The cells were cultured simultaneously at each temperature condition of 60 ° C and 65 ° C. After 18 hours, the growth status of the bacterial colonies was visually checked. Table 1 shows the results.
[0017]
[Table 1]
Figure 2004041104
[0018]
In Table 1, + means good growth,-means no growth, w- means extremely weak growth, w means weak growth, and w + means microcolonies.
As is clear from Table 1, A. acidoterrestris is growth of bacteria colonies was confirmed at 45 ° C. ± 5 ° C., was not confirmed at 60 ° C. or higher.
On the other hand, A. In acidoidarius and Alicyclobacillus genomic specifications 1, the growth of strain colonies was weak or could not be confirmed at 45 ° C. ± 5 ° C., but could be confirmed at 60 ° C. or more.
From the above results, by visually comparing the growth status of the bacterial colonies by culturing at 45 ° C. and the bacterial colonies by culturing at 65 ° C., A. acidoterrestris, A. acidocaldarius, and Alicyclobacillus genomic species 1 of A among the three types of thermophilic acidophilic spore-forming bacteria. It was found that the identification of C. acidosterrestris could be performed.
[0019]
Experiment 2:
Commercially available orange juice as a fruit juice drink (sugar content: about 10 ° Brix) to, A. A. samples to which PB1 was added as an A. acidoterrestris strain. Sample B was 3B added as acidocaldarius strain, Alicyclobacillus genomic species 1 and DSM11983 as strains Sample C was prepared with the addition, each sample as each inoculated streaked on two YSG agar as liquid specimen, at one of 45 ° C. The other was cultured at 65 ° C. simultaneously for 18 hours. After a lapse of 18 hours, the growth status of the bacterial colonies was confirmed. As a result, the growth of the bacterial colonies was confirmed for the sample A by culturing at 45 ° C., but was not confirmed by culturing at 65 ° C. On the other hand, for Samples B and C, growth of bacterial colonies was not confirmed in the culture at 45 ° C, but was confirmed in the culture at 65 ° C. From the above results, by visually comparing the growth status of the bacterial colonies by culturing at 45 ° C. and the bacterial colonies by culturing at 65 ° C., A. acidoterrestris, A. acidocaldarius, and Alicyclobacillus genomic species 1 of A among the three types of thermophilic acidophilic spore-forming bacteria. It was found that the identification of C. acidosterrestris could be performed.
[0020]
【The invention's effect】
According to the present invention, there is provided a method for quickly and simply testing the presence or absence of high-temperature eosinophilic spore-forming bacterial contamination of various foods and drinks including fruit juice-containing foods and drinks and raw materials thereof.

Claims (6)

飲食品またはその原材料が高温性好酸性芽胞形成細菌に汚染されているか否かを検査するに際し、検査対象から液状検体を調製し、調製した液状検体を、資化成分の組成が実質的に酵母エキス、澱粉、グルコースからなり、固化成分として寒天を含むpH3〜5に調整した寒天培地複数枚に塗抹し、40〜50℃の範囲内の温度と60〜70℃の範囲内の温度の2種類の温度で同時に14〜24時間培養した後、40〜50℃の範囲内の温度での菌コロニー生育状況と60〜70℃の範囲内の温度での菌コロニー生育状況を目視比較することで高温性好酸性芽胞形成細菌の混入の有無と種類の鑑別を行うことを特徴とする高温性好酸性芽胞形成細菌汚染の検査方法。When testing whether food or drink or its raw materials are contaminated with thermophilic eosinophilic spore-forming bacteria, prepare a liquid specimen from the test object, and use the prepared liquid specimen as a mixture of substantially yeast-based components. Smeared on a plurality of agar media containing extract, starch and glucose and containing agar as a solidifying component and adjusted to pH 3-5, two types of temperature in the range of 40-50 ° C and temperature in the range of 60-70 ° C After culturing at the same temperature for 14 to 24 hours at the same temperature, the bacterial colony growth at a temperature within the range of 40 to 50 ° C. and the bacterial colony growth at a temperature within the range of 60 to 70 ° C. are visually compared to obtain a high temperature. A method for testing the contamination of thermophilic eosinophilic spore-forming bacteria, which comprises determining the presence or absence of the acidophilic eosinophilic spore-forming bacteria and the type thereof. 飲食品またはその原材料から採取された高温性好酸性芽胞形成細菌の菌株を、資化成分の組成が実質的に酵母エキス、澱粉、グルコースからなり、固化成分として寒天を含むpH3〜5に調整した寒天培地複数枚に添加し、40〜50℃の範囲内の温度と60〜70℃の範囲内の温度の2種類の温度で同時に14〜24時間培養した後、40〜50℃の範囲内の温度での菌コロニー生育状況と60〜70℃の範囲内の温度での菌コロニー生育状況を目視比較することで高温性好酸性芽胞形成細菌の種類の鑑別を行うことを特徴とする高温性好酸性芽胞形成細菌汚染の検査方法。A thermophilic eosinophilic spore-forming bacterium strain collected from food or drink or its raw material was adjusted to a pH of 3 to 5 in which the composition of the assimilation component substantially consisted of yeast extract, starch, and glucose, and contained agar as a solidification component. After adding to a plurality of agar media and culturing at the same temperature for 2 to 24 hours at a temperature in the range of 40 to 50 ° C and a temperature in the range of 60 to 70 ° C for 14 to 24 hours, The type of thermophilic acidophilic spore-forming bacteria is distinguished by visually comparing the status of bacterial colony growth at a temperature with the status of bacterial colony growth at a temperature within the range of 60 to 70 ° C. An inspection method for acid spore-forming bacterial contamination. 検査対象が酸性飲食品またはその原材料であることを特徴とする請求項1または2記載の検査方法。The inspection method according to claim 1, wherein the inspection target is an acidic food or drink or a raw material thereof. 検査対象が果汁入り飲食品または原果汁であることを特徴とする請求項3記載の検査方法。4. The test method according to claim 3, wherein the test object is a food or drink containing fruit juice or a raw juice. 鑑別対象となる高温性好酸性芽胞形成細菌がアリサイクロバチルス・アシドテレストリス(Alicyclobacillus acidoterrestris)であることを特徴とする請求項1乃至4のいずれかに記載の検査方法。The test method according to any one of claims 1 to 4, wherein the thermophilic eosinophilic spore-forming bacterium to be discriminated is Alicyclobacillus acidoterrestris . 18〜20時間培養することを特徴とする請求項1乃至5のいずれかに記載の検査方法。The test method according to any one of claims 1 to 5, wherein the culture is performed for 18 to 20 hours.
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US7767413B2 (en) 2005-04-18 2010-08-03 Microbio Kabushiki Kaisha Reagent, medium, and method for detection of alicyclobacilus acidoterrestris
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