JP5758093B2 - Bacteria detection method - Google Patents
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- JP5758093B2 JP5758093B2 JP2010188930A JP2010188930A JP5758093B2 JP 5758093 B2 JP5758093 B2 JP 5758093B2 JP 2010188930 A JP2010188930 A JP 2010188930A JP 2010188930 A JP2010188930 A JP 2010188930A JP 5758093 B2 JP5758093 B2 JP 5758093B2
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
本発明は、腸内細菌科菌群の細菌検出方法、特に食品を対象とした腸内細菌科菌群の細菌検出方法に関する。 The present invention relates to a method for detecting bacteria in the Enterobacteriaceae family, and more particularly to a method for detecting the bacteria in the Enterobacteriaceae family for food.
食品の品質管理項目の一つとして腸内細菌科菌群の検査項目がある。腸内細菌科菌群は一般に無芽胞のグラム陰性桿菌で好気性又は通性嫌気性の細菌群であって、食中毒の原因菌とされている病原性大腸菌やサルモネラ属菌などを腸内細菌科菌群の一群として包括的に把握することによって効率的なリスク管理を行っている。腸内細菌科菌群には、病原性大腸菌やサルモネラ属菌などの食中毒に関連する細菌が含まれるため、食品中において腸内細菌科菌群の存在を一定程度以下に管理することが求められている。 One of the quality control items of food is an inspection item of Enterobacteriaceae family. Enterobacteriaceae are generally non-spore gram-negative bacilli and are aerobic or facultative anaerobic bacteria, and pathogenic Escherichia coli and Salmonella are considered to cause food poisoning. Efficient risk management is performed by comprehensively grasping as a group of fungi. The Enterobacteriaceae group includes bacteria related to food poisoning such as pathogenic Escherichia coli and Salmonella, so it is required to manage the presence of Enterobacteriaceae in foods to a certain extent. ing.
腸内細菌科菌群の細菌の検出方法としてＩＳＯ（２１５２８−１：２００４）に定める方法がある。この方法は、ＢＰＷ培地（緩衝ペプトン水培地）により試料中に存在する細菌を非選択的に培養する工程と、その後にＥ.Ｅ.ブロスにより腸内細菌科菌群を選択的に培養する工程と、ＶＲＢＧ培地によりＥ.Ｅ.ブロス中の培養菌を分離平板上で生育させて判定する工程とからなる方法である。しかしながら、この方法では試験開始から判定までに、少なくとも７２時間以上を要する。 There is a method defined in ISO (21528-1: 2004) as a method for detecting bacteria in the Enterobacteriaceae family. In this method, a step of non-selectively cultivating bacteria present in a sample with a BPW medium (buffered peptone water medium), and a step of selectively culturing Enterobacteriaceae group using EE broth. And a step of growing and cultivating a culture in EE broth on a separate plate using a VRBG medium. However, this method requires at least 72 hours from the start of the test to determination.
特許文献１（特開平１１−１７８５９７号公報）や特許文献２（特開２００１−１７８４９６号公報）には、容器内に液体培地と二酸化炭素の呈色指示薬とを、液体培地を遮断する二酸化炭素透過膜を隔てて収容した検査具が開示されている。当該検査具は販売名を「センシメディア（登録商標）」として商品化が図られ、腸内細菌科菌群の細菌を検出するための培地が収容された検査具が上市されている（非特許文献１）。 Patent Document 1 (Japanese Patent Application Laid-Open No. 11-178597) and Patent Document 2 (Japanese Patent Application Laid-Open No. 2001-178596) disclose a liquid medium and a carbon dioxide color indicator in a container, and carbon dioxide that blocks the liquid medium. An inspection tool that contains a permeable membrane is disclosed. The test tool is commercialized under the trade name “Sensimedia (registered trademark)”, and a test tool that contains a medium for detecting bacteria of the Enterobacteriaceae family is on the market (non-patented) Reference 1).
この検査具では、液体培地中で細菌が増殖することによって発生した二酸化炭素が二酸化炭素の透過膜を透過して、呈色指示薬と反応することで呈色指示薬が呈色する。その呈色の濃度を検出し、検査対象試料を用いて求められた所定濃度の呈色に至るまでの時間と、菌数が既知である標準試料を用いて求められた所定濃度の呈色に至るまでの時間との対比から、検査対象試料中の菌数が推定される。この検査具を用いた場合には、培養の開始から２４時間程度で菌数が求められ、菌数の限度値が設定されている場合には、推定陽性・陰性の判定が可能となる。 In this inspection tool, the color indicator is colored by the carbon dioxide generated by the growth of bacteria in the liquid medium permeating the carbon dioxide permeable membrane and reacting with the color indicator. The color density is detected, and the time to color development at the predetermined concentration obtained using the sample to be inspected and the coloration at the predetermined concentration obtained using a standard sample with a known number of bacteria The number of bacteria in the sample to be examined is estimated from the comparison with the time taken to reach the target. When this test tool is used, the number of bacteria is obtained in about 24 hours from the start of the culture, and when a limit value for the number of bacteria is set, it is possible to determine an estimated positive / negative.
例えば、牛乳専用に調製された腸内細菌科菌群用の検査具の場合には、腸内細菌科菌群を代表する数種類の標準菌株について、標準菌株ごとに液体培地に添加された菌数と所定濃度の呈色に至る時間との関係が予め求められており、試料を用いた場合における所定濃度の呈色に至るまでの時間から試料中の菌数が求められる。また、測定の開始より１２時間が経過した際に所定濃度の呈色が認められない、つまり呈色の変化が認められない場合には、菌数がゼロであると判断される。 For example, in the case of an inspection device for Enterobacteriaceae group prepared exclusively for milk, for several standard strains representing the Enterobacteriaceae group, the number of bacteria added to the liquid medium for each standard strain And the time required for color development at a predetermined concentration are obtained in advance, and the number of bacteria in the sample is obtained from the time required for color formation at a predetermined concentration when a sample is used. In addition, when 12 hours have passed since the start of the measurement, no coloration at a predetermined concentration is observed, that is, no change in coloration is observed, it is determined that the number of bacteria is zero.
また、この検査具では、細菌の増殖に伴って生じる二酸化炭素を利用しているために、検査対象である細菌以外の増菌を可能な限り抑制する必要がある。このために、例えば、特許文献２に記載されている大腸菌群用の検査具には、大腸菌群など検査対象である細菌の増殖に必要な成分の他に、他の細菌群の増菌を抑制する物質として、塩化ナトリウムが添加された液体培地や塩化ナトリウムに加えて胆汁酸又は／及び胆汁酸塩が添加された液体培地が用いられている。また、非特許文献１に記載された牛乳専用の検査具には、他の細菌群の増菌を抑制する物質として、塩化ナトリウムと抗生物質であるバンコマイシンが添加された液体培地が用いられている。
In addition, since this inspection tool uses carbon dioxide generated with the growth of bacteria, it is necessary to suppress as much as possible the increase of bacteria other than the bacteria to be inspected. For this reason, for example, the test tool for coliform group described in
しかしながら、非特許文献１に記載された腸内細菌科菌群用の検査具では、増菌処理後の培養液を試料としてとして加えた場合には、エンテロコッカス属の細菌などの腸内細菌科菌群以外の細菌（非腸内細菌科菌群の細菌）によっても呈色が観察され、腸内細菌科菌群が推定陽性（偽陽性）であると判断される場合があった。また、腸内細菌科菌群の細菌の存在によって培地が変色し、当該変色の有無によっても腸内細菌科菌群の存否が判断できる。ところが、非腸内細菌科菌群の細菌の存在によって呈色指示薬の呈色による結果と培地の変色による結果とが一致しない場合があった。この場合には、腸内細菌科菌群による呈色であるのかどうか、さらに別の方法にて確定の判定を行わなければならず、製品の出荷管理上、出荷可能となるまでの保管期間が延びるという問題もあった。その一方、ＩＳＯ法では陽性として判断されるが、非特許文献１に記載された腸内細菌科菌群用の検査具では陰性として判断されるようでは、出荷検査のための方法として採用することができない。 However, in the inspection device for Enterobacteriaceae group described in Non-Patent Document 1, when the culture solution after the enrichment treatment is added as a sample, Enterobacteriaceae such as Enterococcus bacteria Coloration was also observed by bacteria other than the group (bacteria of the non-enterobacteriaceae group), and the enterobacteriaceae group was sometimes judged to be positive (false positive). In addition, the medium changes color due to the presence of bacteria of the Enterobacteriaceae group, and the presence or absence of the Enterobacteriaceae group can also be determined by the presence or absence of the discoloration. However, due to the presence of bacteria of the non-enterobacteriaceae group, the result of coloration of the color indicator may not match the result of color change of the medium. In this case, whether or not the color is due to enterobacteriaceae group must be determined by another method, and the storage period until the product can be shipped in the shipping management of the product. There was also a problem of extension. On the other hand, if it is judged as positive in the ISO method, but it is judged as negative in the test device for Enterobacteriaceae family described in Non-Patent Document 1, it should be adopted as a method for shipping inspection. I can't.
また、特許文献２に記載の検査具は、目的とする細菌による所定量の二酸化炭素を排出するまでの時間が他の細菌による排出に要する時間に比べて短くなるような培地が用いられている。例えば大腸菌群の細菌であるＯ−１５７用の培地を用いた場合には、Ｏ−１５７の検出から遅れて、他の大腸菌などが検出されることになる。また、サルモネラ属菌用の培地を用いた場合には、サルモネラ属菌の検出から遅れて、他の大腸菌などが検出されることになる。従って、大腸菌群の細菌とサルモネラ属菌を腸内細菌科菌群として包括的に検出する検査具として用いるのは妥当ではない。
In addition, the inspection tool described in
本発明は上記背景技術に基づいてなされたものであって、本発明は食品中の腸内細菌科菌群の検出において、腸内細菌科菌群の細菌の有無を正しく判断できるだけでなく、しかも短時間で判定できる方法を提供することを目的とする。 The present invention has been made based on the above background art, and the present invention can not only correctly determine the presence or absence of bacteria in the Enterobacteriaceae family in the detection of Enterobacteriaceae in foods, An object is to provide a method capable of determining in a short time.
本発明に係る検出方法に用いられる検査具は、前記の検査具において、前記液体培地は腸内細菌科菌群の細菌増菌用成分と細菌増殖抑制成分である胆汁酸及び／又は胆汁酸塩とブリリアントグリーンを含み、他の細菌増殖抑制成分を実質的に含まないリン酸緩衝液からなる液体培地を用いた検査具である。
The inspection tool used in the detection method according to the present invention is the above-mentioned inspection tool, wherein the liquid medium is a bacterial and / or bile salt that is a component for bacterial enrichment of the Enterobacteriaceae family and a component for inhibiting bacterial growth. And a brilliant green, and a test device using a liquid medium composed of a phosphate buffer solution substantially free from other bacterial growth inhibitory components.
本発明によると、エンテロコッカス属の細菌による偽陽性の判定が少なくなり、主に乳製品などの食品における腸内細菌科菌群の細菌の存否を迅速に判断できる。 According to the present invention, false positive determinations due to Enterococcus bacteria are reduced, and the presence or absence of bacteria belonging to the family Enterobacteriaceae in foods such as dairy products can be quickly determined.
本発明の検査具は、密閉可能な容器内に、増菌用の培地を遮断する二酸化炭素透過膜で隔てて培地収容部と指示薬収容部とを備え、前記容器は外側から前記指示薬収容部の内部を見通せる透明部を備える。培地収容部には増菌用の培地が収容され、指示薬収容部には二酸化炭素の呈色指示薬が収容される。 The inspection tool of the present invention includes a medium storage part and an indicator storage part separated from each other by a carbon dioxide permeable membrane that blocks the culture medium for enrichment in a sealable container, and the container is provided from the outside of the indicator storage part. It has a transparent part that can be seen through. A medium for enrichment is accommodated in the medium accommodating part, and a color indicator for carbon dioxide is accommodated in the indicator accommodating part.
透明部は指示薬収容部の呈色が観察されればよく、容器全体が透明であっても容器の一部が透明であってもよい。また、透明部は無色であることが好ましい。呈色の変化を確実に観察するためである。容器は、例えば、図１に示すように、全体が透明となったプラスチック製の容器本体１に、キャップ７が螺合により脱着可能に取り付けられ、培地５及び呈色指示薬６が収納された後には、検査対象試料を入れるまで密閉状態で保管される。
The transparent portion only needs to observe the color of the indicator accommodating portion, and the entire container may be transparent or a part of the container may be transparent. The transparent part is preferably colorless. This is for reliably observing a change in coloration. For example, as shown in FIG. 1, after the
培地収容部２と指示薬収容部３は、増菌用の培地を遮断する二酸化炭素透過膜４で隔てられ、培地収容部２で発生した二酸化炭素が当該透過膜４を透過して指示薬収容部３に収容された呈色指示薬６と反応する。例えば、図１に示すように、指示薬収容部３を二酸化炭素透過膜４から構成した密閉した袋とし、指示薬収容部３で占められた空間以外の容器内空間を培地収容部２とすることや、図示はしないが、容器内部を二酸化酸素透過膜で上下に分割し、一方の空間を培地収容部とし、他方の空間を指示薬収容部とすることができる。また、図１に示すように、袋状からなる指示薬収容部３を構成した場合には、培地収容部２に収容された培地５の上面の高さよりも高い位置に指示薬収容部３に収容した呈色指示薬６の上面の高さが位置するように培地を収容するのが好ましい。培地５の着色に影響を受けずに呈色指示薬６の呈色を観察できるからである。また、容器内部を透過膜で上下に分割した場合には、二酸化炭素透過膜よりも下方を指示薬収容部とするのが好ましい。容器を開封して培地に試料を加えるためである。なお、図１に示す容器本体１の底の近くには、指示薬収容部３の下端部を支持する支持部材８が備えられている。この支持部材８はキャップ７の内面に設けられた支持部材（図示せず）と対をなして、呈色指示薬６の上面高さが培地５の上面高さよりも高い位置に位置するように指示薬収容部３を保持する。
The culture
二酸化炭素透過膜４は、二酸化炭素は透過するが、イオン交換を生じずに培地と呈色指示薬を確実に分離できるものであれば、その材質は特に問われない。例えば、未延伸ポリプロピレン、延伸ポリプロピレン、低密度ポリエチレン、高密度ポリエチレンなどフィルムが用いられる。その他、シリコンゴムやポリアルキルスルホンからなる均質膜、ポリテトラフルオロエチレンやポリスルホンからなる多孔質膜、ポリプロピレンとシリコンとの複合膜、ポリプロピレンとポリアルキルスルホンとの複合膜を用いることができる。 The material of the carbon dioxide permeable membrane 4 is not particularly limited as long as it can permeate carbon dioxide but can reliably separate the culture medium and the color indicator without causing ion exchange. For example, films such as unstretched polypropylene, stretched polypropylene, low density polyethylene, and high density polyethylene are used. In addition, a homogeneous film made of silicon rubber or polyalkylsulfone, a porous film made of polytetrafluoroethylene or polysulfone, a composite film of polypropylene and silicon, or a composite film of polypropylene and polyalkylsulfone can be used.
呈色指示薬は二酸化炭素と反応して呈色する指示薬であれば特に制約されず、二酸化炭素の反応剤とｐＨ指示薬との混合液、例えば、水酸化ナトリウムとチモールフタレインの水溶液、水酸化ナトリウムとフェノールフタレインの水溶液、水酸化カルシウム水溶液、ヘモグロビンや赤血球、血液などのヘムを含む物質の水溶液の他、メチルバイオレットやメチルオレンジ、コンゴーレッド、メチルレッド、ブロムチモールブルー、フェノールレッド、クレゾールレッド、チモールブルーなど各種呈色指示薬の水溶液が挙げられる。なお、呈色は着色又は脱色のいずれであってもよい。 The color indicator is not particularly limited as long as it is a color indicator that reacts with carbon dioxide, and is a mixture of a carbon dioxide reactant and a pH indicator, for example, an aqueous solution of sodium hydroxide and thymolphthalein, sodium hydroxide. And phenolphthalein aqueous solution, calcium hydroxide aqueous solution, hemoglobin, red blood cells, aqueous solution of substances containing heme such as blood, methyl violet, methyl orange, congo red, methyl red, bromthymol blue, phenol red, cresol red, An aqueous solution of various color indicators such as thymol blue can be mentioned. In addition, coloring may be either colored or decolored.
検査対象試料に細菌が存在すると、増菌により二酸化炭素が生成される。本発明の検査具は、この二酸化炭素と指示薬収容部の呈色指示薬との反応により生じた呈色を利用する。すなわち、同じ培養条件下では、検査対象試料中の細菌数は、所定濃度の呈色を生じるに要する時間と相関関係がある。検査対象試料を検査具に直接加える場合には、検査対象試料中の細菌数が多ければ、所定濃度の呈色を生じるに要する時間は短くなり、少ないほど長くなる。従って、予め菌数が既知である数種類の試料（標準試料）を用いて、所定濃度の呈色を生じるに要した時間を測定し、菌数と時間との関係（感度特性）を求めておき、検査対象試料を用いて測定した所定濃度の呈色を生じるに要した時間と対比することにより、検査対象試料中の菌数を求めることができる。また、菌数が既知である数種類の試料を用いて、一定時間に生じる呈色濃度（濃度変化）を測定しておき、その時間を経過した際に、検査対象試料を用いて測定した呈色濃度から、おおよその菌数を求めることもできる。従って、例えば、希釈して理論上１×１０0の菌数を含む試料を用いて、測定開始から１８時間あるいは２４時間など一定時間経過後に呈色の濃度変化を予め測定しておき、検査対象試料を用いて測定した濃度変化が予め測定した前記濃度変化よりも小さい場合には、理論上１×１０0以下、すなわち当該細菌は検出されないという判定が行える。 If bacteria are present in the sample to be examined, carbon dioxide is generated by enrichment. The inspection tool of the present invention uses the color produced by the reaction between the carbon dioxide and the color indicator in the indicator container. That is, under the same culture conditions, the number of bacteria in the test sample is correlated with the time required to produce a predetermined concentration of color. When the sample to be inspected is directly added to the inspection tool, the time required for producing the color at a predetermined concentration is shortened if the number of bacteria in the sample to be inspected is large, and the time is increased as the number is decreased. Therefore, using several types of samples (standard samples) with a known number of bacteria in advance, the time required to produce a color at a predetermined concentration is measured, and the relationship between the number of bacteria and time (sensitivity characteristics) is obtained. The number of bacteria in the test sample can be determined by comparing with the time required to produce the color of the predetermined concentration measured using the test sample. In addition, using several types of samples with known numbers of bacteria, the color density (concentration change) generated over a certain period of time is measured, and when that time has elapsed, the coloration measured using the sample to be examined The approximate number of bacteria can also be determined from the concentration. Thus, for example, by using a sample containing a number of bacteria theoretically 1 × 10 0 diluted, measured in advance concentration change of color after a certain like 18 hours or 24 hours from the start of measurement time, the inspection target If concentration changes measured by using a sample is smaller than the change in density was measured in advance, theoretically 1 × 10 0 or less, that the bacteria can be performed determines that it is not detected.
このような検査具として、例えば特許文献１や特許文献２に記載された種々の検査具が例示される。本発明の検査具は、このような検査具であって、偽陽性とされる細菌、すなわち、非腸内細菌科菌群による偽陽性の判断を少なくしたものである。
Examples of such inspection tools include various inspection tools described in Patent Document 1 and
本発明の検査具においては、増菌用の培地として、腸内細菌科菌群の細菌増菌用成分と細菌増殖抑制成分である胆汁酸及び／又は胆汁酸塩とブリリアントグリーンを含み、他の細菌増殖抑制成分を実質的に含まない緩衝系溶液からなる液体培地が用いられる。 In the inspection tool of the present invention, the medium for enrichment includes bacterial enrichment components of the Enterobacteriaceae family and bile acids and / or bile salts and brilliant green that are bacterial growth inhibitory components, A liquid medium consisting of a buffer system solution substantially free from bacterial growth inhibitory components is used.
増菌用の培地成分として、大腸菌群増菌用の培地成分や腸内細菌科菌群増菌用の培地成分、サルモネラ属菌増菌用の培地成分として一般的に用いられる成分、例えば、乳糖、ブドウ糖、トリプトース、酵母エキス、カゼインペプトンなどの各種ペプトンが例示され、これらの１種若しくは２種以上が用いられる。 As a medium component for enrichment, a medium component for enrichment of Escherichia coli group, a medium component for enrichment of Enterobacteriaceae group, a component commonly used as a medium component for enrichment of Salmonella, for example, lactose Various peptones such as glucose, tryptose, yeast extract and casein peptone are exemplified, and one or more of these are used.
細菌増殖抑制成分は、腸内細菌科菌群以外の細菌、例えばセレウス菌や黄色ブドウ球菌などの増殖を抑制する成分である。本発明では、胆汁酸及び／又は胆汁酸塩とブリリアントグリーンのみが用いられ、塩化ナトリウムなどこれ以外の細菌増殖抑制成分は実質的に含まない。胆汁酸や胆汁酸塩、ブリリアントグリーンは、ラウリル硫酸のような選択剤よりも、エンテロコッカス属菌などの腸内細菌科菌群以外の細菌に対する抑制能が強いとされているからである。ここで、実質的に含まないとは、腸内細菌科菌群の細菌の増殖を抑制しない程度に含まないことを意味し、例えば、胆汁酸塩の一部には少数の腸内細菌を抑制する成分が含まれたり、酵母エキスやペプトン中に含まれる塩化ナトリウムが混入したりする場合があるが、このような場合には、実質的に含まれていないと言える。従って、胆汁酸や胆汁酸塩は、精製された胆汁酸や胆汁酸塩、特にウシの胆汁酸を用いるのが好ましい。これらの細菌増殖抑制成分は、例えば１８〜２４時間の培養時間で非腸内細菌科菌群の細菌の増殖によって呈色反応を生じさせないように、培地中に配合される。なお、判定を行うまでの培養時間によって、この配合量は調整される。 The bacterial growth inhibitory component is a component that inhibits the growth of bacteria other than the Enterobacteriaceae family, such as Bacillus cereus and Staphylococcus aureus. In the present invention, only bile acids and / or bile salts and brilliant green are used, and other bacterial growth inhibitory components such as sodium chloride are not substantially contained. This is because bile acids, bile salts, and brilliant green are considered to have a greater ability to suppress bacteria other than the Enterobacteriaceae group such as Enterococcus than selection agents such as lauryl sulfate. Here, “substantially free” means that it does not contain so much that it does not inhibit the growth of bacteria of the Enterobacteriaceae family. For example, some bile salts contain a small number of enteric bacteria. In some cases, sodium chloride contained in yeast extract or peptone may be mixed, but in such a case, it can be said that it is not substantially contained. Therefore, it is preferable to use purified bile acids and bile salts, particularly bovine bile acids, as bile acids and bile salts. These bacterial growth-inhibiting components are blended in the medium so as not to cause a color reaction due to growth of bacteria of the non-enterobacteriaceae family, for example, in a culture time of 18 to 24 hours. In addition, this compounding quantity is adjusted with the culture | cultivation time until it determines.
これらの成分はリン酸緩衝液に溶解される。リン酸緩衝液はリン酸とリン酸塩とから構成される緩衝液であり、リン酸水素二ナトリウムとリン酸二水素カリウムなどのようにｐＫａ（酸解離定数）の異なる２種以上のリン酸塩を用いて構成されたり、リン酸とリン酸ニナトリウムなどのようにリン酸とその塩を用いて構成される。リン酸塩は、ナトリウム塩、カリウム塩、アンモニウム塩などいずれの塩でも差し支えない。このとき、液体培地のｐＨが７±０.５、好ましくは７.２±０.２に調整される。 These components are dissolved in phosphate buffer. The phosphate buffer is a buffer composed of phosphoric acid and phosphate, and two or more kinds of phosphoric acids having different pKa (acid dissociation constant) such as disodium hydrogen phosphate and potassium dihydrogen phosphate. It is comprised using a salt, and is comprised using phosphoric acid and its salt like phosphoric acid and disodium phosphate. The phosphate may be any salt such as sodium salt, potassium salt or ammonium salt. At this time, the pH of the liquid medium is adjusted to 7 ± 0.5, preferably 7.2 ± 0.2.
このように腸内細菌科菌群用の増殖培地と非腸内細菌科菌群の増殖抑制成分を含む培地として、培地１Ｌ中にペプトン１０±１ｇ、ブドウ糖５±０.５ｇ、リン酸水素二ナトリウム（無水物）６.５±０.６ｇ、リン酸二水素カリウム２±０.２ｇ、精製ウシ胆汁酸２０±２ｇ、ブリリアントグリーン１３±２ｍｇを含む液体培地が好適に用いられる。この培地には、Ｅ.Ｅ.ブロス（Ｅ.Ｅ.ブイヨン）として市販されている培地（例えば、ＯＸＯＩＤ社製のＥ.Ｅブロスなど）をそのまま用いることができる。もっとも、検査対象試料が溶液である場合には、添加する試料の量に応じて、試料を添加した後の最終濃度が上記濃度となるようにした高濃度の組成の培地を、培地収納部に収容するのが好ましい。培地収容部には、１×１０8〜１×１０0ｃｆｕの細菌数が検出可能な程度の量の培地が収容される。培地の収容量は、容器当たり１〜２０ｍｌ、好ましくは４〜５ｍｌである。 Thus, as a medium containing a growth medium for Enterobacteriaceae group and a growth-inhibiting component of non-Enterobacteriaceae group, 10 ± 1 g of peptone, 5 ± 0.5 g of glucose, 2 hydrogen phosphates in 1 L of the medium. A liquid medium containing 6.5 ± 0.6 g of sodium (anhydride), 2 ± 0.2 g of potassium dihydrogen phosphate, 20 ± 2 g of purified bovine bile acid, and 13 ± 2 mg of brilliant green is preferably used. As this medium, a medium commercially available as EE broth (EE broth manufactured by OXOID, etc.) can be used as it is. Of course, if the sample to be inspected is a solution, a medium with a high concentration composition in which the final concentration after addition of the sample is the above concentration is added to the medium storage unit according to the amount of the sample to be added. It is preferable to accommodate. The medium containing unit contains a quantity of medium that can detect the number of bacteria of 1 × 10 8 to 1 × 10 0 cfu. The capacity of the medium is 1 to 20 ml, preferably 4 to 5 ml per container.
本発明の細菌検出方法は上記の検査具を用いる方法であって、上記検査具に収容された液体培地に検査対象試料を添加する工程と、前記容器を密閉して培養を開始し、培養の開始から定められた時間経過時に前記指示薬収納部の呈色の濃度を測定する工程を備え、定められた呈色の濃度との対比により、細菌の存否を判断する方法である。すなわち、本発明の検査方法では、一定時間培養した後に生じた呈色指示薬の呈色の濃度と予め定められた標準となる呈色の濃度との対比によって細菌の有無を判定する。 The method for detecting bacteria according to the present invention is a method using the above-described test tool, the step of adding a sample to be tested to the liquid medium contained in the test tool, and the culture is started by sealing the container, The method includes a step of measuring the color density of the indicator storage unit when a predetermined time elapses from the start, and determining the presence or absence of bacteria by comparison with the predetermined color density. That is, in the inspection method of the present invention, the presence or absence of bacteria is determined by comparing the color density of the color indicator generated after culturing for a certain period of time with the standard color density that is determined in advance.
その検出方法には、検査対象試料を検査具の培地収容部に直接入れる場合と、検査対象試料を予め非選択培地や水などの適当な媒体に溶かした溶液を培地収容部に入れる場合とがあり、検査対象試料の種類などによって適宜選択できる。また、本発明の検査方法においては、いわゆる前培養（増菌処理）として検査対象試料中の細菌を非選択培地で培養した後に、当該培養液を検査具の培地収容部に入れるのが好ましい。検査対象試料の製造工程などにおいて、何らかの要因で損傷を受けた損傷菌を回復させ、検査対象試料中に存在する腸内細菌科菌群の検出を確実に行うためである。 In the detection method, there are a case where the sample to be examined is directly put into the medium container of the inspection tool and a case where a solution prepared by previously dissolving the sample to be examined in an appropriate medium such as a non-selective medium or water is put into the medium container. Yes, it can be appropriately selected depending on the type of sample to be inspected. In the inspection method of the present invention, it is preferable to cultivate bacteria in the sample to be inspected in a non-selective medium as so-called pre-culture (enrichment treatment), and then put the culture solution into the medium container of the inspection tool. This is because, in the manufacturing process of the sample to be inspected or the like, the damaged bacteria damaged for some reason are recovered, and the enterobacteriaceae group present in the sample to be inspected is reliably detected.
非選択培地として、例えば、トリプトンソーヤブイヨン培地やＢＰＷ培地など公知の非選択培地が用いられる。ＩＳＯ法による検査結果と高い相関を得るためにＢＰＷ培地が好ましく用いられる。培養は多種類の細菌が増殖できる条件下、好ましくは３７±１℃の好気性条件下で、１８〜２４時間で行えばよい。 As the non-selective medium, for example, a known non-selective medium such as tryptone soy broth medium or BPW medium is used. A BPW medium is preferably used in order to obtain a high correlation with the test result by the ISO method. Cultivation may be performed for 18 to 24 hours under conditions that allow various types of bacteria to grow, preferably under aerobic conditions of 37 ± 1 ° C.
検査対象試料には、食品（製品及び原材料などを含む）のみならず各種の食品の製造工程（機器表面、床面、壁面など）における拭き取りサンプルである環境試料なども該当する。また、食品の中でも、特に、牛乳、発酵乳、粉乳（乳を加熱して粉末にしたもの、全脂粉乳、脱脂粉乳、ホエイ粉など）、乳幼児用調製粉乳（栄養成分などが調製された乳幼児に与えられる粉乳：いわゆる粉ミルク）などの乳製品やベビーフードが好ましい対象であり、さらに望ましくはこれらの食品の中でも、粉乳、乳幼児用調製粉乳、ベビーフードが望ましい対象であると言える。 Samples to be inspected include not only food (including products and raw materials) but also environmental samples that are wiped samples in various food manufacturing processes (equipment surfaces, floor surfaces, wall surfaces, etc.). Among foods, in particular, milk, fermented milk, powdered milk (milk heated to powder, whole milk powder, skim milk powder, whey powder, etc.), infant formula infants (nutrients etc. are prepared) Dairy products such as powdered milk: so-called powdered milk) and baby foods are preferable targets, and more desirably among these foods, powdered milk, infant formula and baby food are preferable targets.
水、乳製品などの食品中の大腸菌群の細菌の検出に用いられる選択培地としてラウリル硫酸ブイヨン（ＬＳＴ培地）が知られているが、このＬＳＴ培地では、非腸内細菌科菌群の菌であるエンテロコッカス属菌を抑制できない（エンテロコッカス属菌の発育が中程度/良好である。）。しかしながら、Ｅ.Ｅ.ブロスでは、胆汁酸や胆汁酸塩とブリリアントグリーンによりエンテロコッカス属菌を抑制できる。そして、胆汁酸や胆汁酸塩では、エンテロコッカス属菌の一部に対して抑制能が十分ではないが、ブリリアントグリーンを加える（併用する）ことで、抑制能が十分に発揮される。ここで、例えば検査対象試料が牛乳の場合には、エンテロコッカス属菌は殺菌されるため、エンテロコッカス属菌への抑制能を勘案しなくても良いが、検査対象試料が粉乳、乳幼児用調製粉乳、ベビーフードなどの場合には、エンテロコッカス属菌が残存している可能性があり、エンテロコッカス属菌への抑制能は必要となる。そこで、検査対象試料が粉乳、乳幼児用調製粉乳、ベビーフードなどの場合には、ラウリル硫酸ブイヨンなどと比べて、Ｅ.Ｅ.ブロスを使用すること、特にブリリアントグリーンを使用することが優れていると言える。 A lauryl sulfate broth (LST medium) is known as a selective medium used for the detection of coliform bacteria in foods such as water and dairy products. Cannot suppress certain Enterococcus spp. (The growth of Enterococcus spp. Is moderate / good). However, in EE broth, enterococcus can be suppressed by bile acids, bile salts and brilliant green. In addition, bile acids and bile salts do not have sufficient inhibitory ability against some Enterococcus spp., But by adding (in combination with) brilliant green, the inhibitory ability is sufficiently exerted. Here, for example, when the sample to be examined is milk, enterococcus genus bacteria are sterilized, so there is no need to consider the ability to inhibit enterococcus, but the sample to be examined is milk powder, infant formula, In the case of baby food and the like, enterococcus spp. May remain, and the ability to suppress enterococcus spp. Is required. Therefore, when the sample to be examined is powdered milk, infant formula, baby food, etc., it is superior to use EE broth, especially brilliant green, compared to lauryl sulfate broth. It can be said.
本発明の細菌検出方法により検査され、出荷の是非を判断された食品（牛乳、発酵乳、粉乳、乳幼児用調製粉乳などの乳製品やベビーフードなど）は、衛生的な環境下で適正に製造された製品とみなされる。 Foods (milk products, fermented milk, powdered milk, dairy products such as infant formulas, baby foods, etc.) that have been tested by the bacteria detection method of the present invention and judged to be suitable for shipment are properly manufactured in a sanitary environment. Is considered a product.
検査対象試料の使用量は食品の種類や推定される非腸内細菌科菌群の細菌数、培地の使用量などによっても適宜調整される。検査対象試料の使用量が多いほど、腸内細菌科菌群の細菌を検出する確率が高くなり、検出精度の観点からは好ましいが、検査対象試料を検査具に直接加える場合には、検査対象試料の使用量が多いと、非腸内細菌科菌群が多量に存在した際に、偽陽性として判断される傾向にある。従って、１〜２０ｍｌの培地を収容した検査具であれば、０.０１〜１０ｇの個体試料又は０.０１〜１０ｍｌの液体試料が使用される。また、前培養した場合には、１〜２０ｍｌの培地に対し０.１〜１ｍｌの培養液が使用される。さらに望ましくは、４ｍｌの液体培地に対して０.１ｍｌの培養液が使用される。なお、前培養（増菌処理）してから、その一部を検査具に加える場合には、検査対象試料の使用量を多くする（増やす）ことが可能である。 The amount of the sample to be examined is appropriately adjusted depending on the type of food, the estimated number of bacteria of the non-enterobacteriaceae family, the amount of medium used, and the like. The greater the amount of the sample to be inspected, the higher the probability of detecting bacteria in the Enterobacteriaceae family, which is preferable from the viewpoint of detection accuracy, but when adding the sample to be inspected directly to the inspection tool, When the amount of the sample used is large, it tends to be judged as a false positive when a large amount of non-enterobacteriaceae group exists. Therefore, in the case of an inspection tool that accommodates 1 to 20 ml of medium, 0.01 to 10 g of an individual sample or 0.01 to 10 ml of a liquid sample is used. When pre-cultured, 0.1 to 1 ml of culture solution is used for 1 to 20 ml of medium. More desirably, 0.1 ml of culture solution is used for 4 ml of liquid medium. In addition, when a part is added to a test tool after pre-culture (enrichment process), it is possible to increase (increase) the usage-amount of a test object sample.
検査対象試料又は培養液が加えられた容器は密閉され、３７℃付近で一定時間、好ましくは１８〜２４時間培養される。細菌が存在すると呈色指示薬が呈色し、呈色の濃度が時間とともに変化する。培養を終えた段階で呈色の濃度が測定される。 The container to which the sample to be inspected or the culture solution is added is sealed and cultured at around 37 ° C. for a fixed time, preferably 18 to 24 hours. When bacteria are present, the color indicator is colored, and the concentration of the color changes with time. The color density is measured at the end of the incubation.
対比される基準となる呈色の濃度は種々の方法によって定められる。例えば、培地に添加される検査対象試料又は培養液の代わりに、同量の無菌である培地や水を加えて培養して得られた呈色の濃度を基準にする方法、培養開始直後の提示指示薬による呈色の濃度を基準にする方法、さらには、菌数が既知である試料を培地に加えて培養して得られた呈色の濃度を基準にする方法が例示される。この中では、簡便に判定できる観点から、培養開始直後の提示指示薬による呈色の濃度を基準にする方法が好ましく採用される。呈色の変化を観察するだけでよいからである。 The density of the color to be compared is determined by various methods. For example, instead of the test sample or culture solution added to the medium, a method based on the color concentration obtained by culturing with the same amount of sterile medium or water, presentation immediately after the start of culture Examples thereof include a method based on the color density of the indicator, and a method based on the color density obtained by adding a sample having a known number of bacteria to the medium and culturing. Among these, from the viewpoint of easy determination, a method based on the color concentration of the presentation indicator immediately after the start of culture is preferably employed. This is because it is only necessary to observe the color change.
そして、これらの基準となる呈色の濃度と、検査対象試料を用いて得られた呈色の濃度とを対比し、検査対象試料を用いて得られた呈色の濃度が基準となる呈色の濃度よりも同じか低いと判断されると、基準となる呈色の濃度が得られた菌数とほぼ同じか、それよりも少ない菌数の細菌が検査対象試料中に存在すること、若しくは細菌が検査対象試料中に存在しないと判定できる。このように本発明の細菌検出方法によれば、測定開始から１８〜２４時間あるいは前培養を含めると４８時間以内で、検査対象試料中の腸内細菌科菌群の細菌の有無を判断できる。 Then, the reference color density is compared with the color density obtained using the test sample, and the color density obtained using the test sample is the reference color. If it is determined that the concentration of the reference color is approximately the same as or less than the number of bacteria obtained, or that the number of bacteria is less than that in the test sample, or It can be determined that bacteria are not present in the test sample. Thus, according to the bacteria detection method of the present invention, it is possible to determine the presence or absence of bacteria of the Enterobacteriaceae group in the sample to be examined within 18 to 24 hours from the start of measurement or within 48 hours including pre-culture.
もっとも、特許文献１や特許文献２に記載されているように、検査対象試料を検査具に直接加える場合には、検査具による培養の開始から所定の呈色濃度が観察される時間を測定することによって、検査対象試料中に存在する菌数を測定できるのは言うまでもない。
However, as described in Patent Document 1 and
次に、本発明について下記の実施例に基づいて説明する。なお、下記の実施例はあくまでも例示であって、本発明は下記の実施例に限られるものではない。 Next, the present invention will be described based on the following examples. In addition, the following Example is an illustration to the last, Comprising: This invention is not limited to the following Example.
（市販品を用いた検出）
乳児用の調製粉乳（商品名：ほほえみ（明治乳業（株）製））に、非腸内細菌科菌群の細菌を接種したものを検査対象試料として、市販の検査具であるセンシメディア腸内細菌用（ＳＭＬ０１６）及び同試料２０ｍｌ添加用（ＳＭＬ０１６−２０）を用いて、試料中の細菌の検出を行った。非腸内細菌科菌群の細菌として、調製粉乳の原料などから分離されたエンテロコッカス属菌（Enterococcus sp.）、セレウス菌（Bacillus cereus）、緑濃菌（Pseudomonas aerigonosa）の３つの菌種を用いた。
(Detection using commercial products)
Sensimedia Intestine, a commercially available test tool, is prepared by inoculating infant formula milk (trade name: Hohoemi (manufactured by Meiji Dairies Co., Ltd.)) with nonintestinal bacteria family bacteria. Bacteria in the sample were detected using bacteria (SML016) and 20 ml sample (SML016-20). As bacteria of the non-enterobacteriaceae group, three types of bacteria, Enterococcus sp., Bacillus cereus, and Pseudomonas aerigonosa, isolated from raw material of formula milk are used. It was.
調製粉乳１０ｇに上記細菌を含む菌液（生理食塩水で希釈した菌液）１０μＬを接種して、ＢＰＷ培地（メルク社製、カタログ番号1.07228.0500）９０ｍｌを加えてから、３７℃、１８〜２４時間で培養を行い、前培養（増菌処理）を行った。これとは別に、細菌を含む菌液をＳＭＡ寒天培地（標準寒天培地：栄研化学社製）により、３７℃、４８時間で培養を行い、菌液に含まれる菌数を計測した。 After inoculating 10 g of the bacterial solution (bacterial solution diluted with physiological saline) containing the above bacteria into 10 g of the prepared milk powder, 90 ml of BPW medium (Merck, catalog number 1.07228.0500) was added, and then 37 ° C, Culturing was performed for 24 hours, and pre-culture (enrichment treatment) was performed. Separately, the bacterial solution containing bacteria was cultured on an SMA agar medium (standard agar medium: manufactured by Eiken Chemical Co., Ltd.) at 37 ° C. for 48 hours, and the number of bacteria contained in the bacterial solution was measured.
次に、表１に示す量の前培養液（ＢＰＷ培養液：１８時間で培養した培養液と２４時間で培養した培養液）及び滅菌精製水を検査具の培地収容部に加えて、３７℃、２４時間で培養して、呈色指示薬の呈色の変化及び培地の色の変化を観察した。また、対比として、菌液を加えなかったサンプル（表中、番号４で示される）及びＢＰＷ培地を加えなかった検査具（表中、番号５で示される）についても同様に試験した。さらに、２４時間の培養後の前培養液中の菌数を、ＶＲＢＧ寒天培地（ＯＸＯＩＤ社製、カタログ番号０４８５）及びＳＭＡ寒天培地を用いて測定した。なお、使用した市販の検査具では、牛乳をそれぞれ２.２ｍｌ及び２０ｍｌを加えるように培地成分が調整されているので、試験番号Ａを除いては、牛乳の替わりに水と前培養液を添加した。
Next, the amount of the preculture solution (BPW culture solution: the culture solution cultured for 18 hours and the culture solution cultured for 24 hours) and sterilized purified water in the amounts shown in Table 1 and the sterilized purified water are added to the culture medium storage part of the inspection tool, and The cells were cultured for 24 hours, and the change in color of the color indicator and the change in the color of the medium were observed. Further, as a comparison, a sample (indicated by number 4 in the table) to which the bacterial solution was not added and an inspection tool (indicated by
これらの結果を表１に示す。表中、「センサー」として示されたカラムは呈色指示薬の呈色（黄変）による判定を、「培養液」として示されたカラムは培地の変色による判定を示し、「＋」は陽性（変化があった）を、「−」は陰性（変化がなかった）、「＋／−」は判定できない場合を示す。 These results are shown in Table 1. In the table, the column indicated as “sensor” indicates the determination based on the coloration (yellowing) of the color indicator, the column indicated as “culture medium” indicates the determination based on the color change of the medium, and “+” indicates positive ( "-" Indicates a negative (no change), and "+/-" indicates a case where it cannot be determined.
市販されている検査具を用いた場合には、非腸内細菌科菌であるエンテロコッカス属菌が偽陽性として判断される場合があった（表中に太線の枠囲みで示される。）。また、エンテロコッカス属菌やＩＳＯ法による検出でも陽性として判断されることがあるシュードモナス属菌は、呈色による判定では陽性として判断されるが、変色による判定では陰性として判断され、呈色指示薬の呈色に基づく判定と培地の変色に基づく判定が異なるケースが見られた。 When a commercially available test tool was used, Enterococcus spp., Which is a non-enterobacteriaceae, was sometimes judged as a false positive (indicated by a bold frame in the table). In addition, Enterococcus spp. And Pseudomonas spp. That may be judged as positive even when detected by the ISO method are judged as positive in the judgment based on coloration, but are judged negative in the judgment based on discoloration. There were cases where the judgment based on the color and the judgment based on the discoloration of the medium were different.
（本発明の検査具を用いた検出）
次に市販の検査具であるセンシメディア腸内細菌用（ＳＭＬ０１６：容器容積約１６ｍｌ）に使用されている培地の代わりに、市販のＥ.Ｅ.ブロス（ＯＸＯＩＤ社製 カタログ番号ＣＭ０３１７）を用いて、上記と同様の試験を行った。その結果を表２に示す。
(Detection using the inspection tool of the present invention)
Next, instead of the culture medium used for commercially available sensimedia enterobacteria (SML016: container volume of about 16 ml), commercially available EE broth (catalog number CM0317 manufactured by OXOID) was used. The same test as described above was performed. The results are shown in Table 2.
この試験では、呈色指示薬の呈色に基づく場合には、非腸内細菌科菌であるエンテロコッカス属菌が陽性として判定されるケース（表中に太線の枠囲みで示される）もあったが、培養液による変色に基づく場合には、すべて陰性として判断された。以上のように、増殖抑制成分として塩化ナトリウムとバンコマイシンを用いる場合（市販品を用いる場合）に比べて、胆汁酸とブリリアントグリーンを用いる場合の方が良好な結果が得られた。 In this test, there was a case where Enterococcus spp., Which is a non-enterobacteriaceae, is judged as positive based on the color of the color indicator (indicated by a bold frame in the table). All cases were judged as negative when the color change was caused by the culture medium. As described above, better results were obtained when bile acids and brilliant green were used than when sodium chloride and vancomycin were used as growth inhibitory components (when commercial products were used).
実施例１において、Ｅ.Ｅ.ブロスを増菌用の培地として使用できる可能性が見いだせたので、さらに詳細な検討を行った。実施例１の結果から、前培養液を１ｍｌ添加した場合において、エンテロコッカス属菌が偽陽性として判断される可能性が考えられた。そこで、前培養液の添加量を、培地の成分濃度にほとんど影響を与えないと考えられる０.１ｍｌに設定し、呈色指示薬の呈色を観察しやすくするために培地の収容量を４ｍｌとして、実施例１と同様の実験を行った。なお、実施例２では、市販の検査具と比較すべく、センシメディアＳＭＬ０１６に用いられている容器（容積約１６ｍｌ）にＥ.Ｅ.ブロスを添加した。また、実施例２では、非腸内細菌科菌群の菌であるセレウス菌（Bacillus cereus）に替えて、腸内細菌科菌群の菌であるエンテロバクター属菌（Enterobacter cloacae）を用いた。その結果を表３及び表４に示す。表３は市販の検査具を用いた結果、表４は本発明の検査具を用いた結果である。対比として、ＩＳＯ法（ISO21528-1）による検出も行った。 In Example 1, since the possibility that EE broth can be used as a medium for enrichment was found, further detailed examination was performed. From the result of Example 1, it was considered that when 1 ml of the preculture was added, Enterococcus was judged as a false positive. Therefore, the amount of the pre-culture solution added is set to 0.1 ml, which is considered to have little effect on the component concentration of the medium, and the amount of the medium contained is 4 ml in order to make it easy to observe the coloration of the color indicator. The same experiment as in Example 1 was performed. In Example 2, EE broth was added to a container (volume of about 16 ml) used for Sensimedia SML016 for comparison with a commercially available inspection tool. Moreover, in Example 2, Enterobacter cloacae which is a microbe of the enterobacteriaceae group was used instead of Bacillus cereus which is a microbe of the nonenterobacteriaceae group. The results are shown in Tables 3 and 4. Table 3 shows the results of using commercially available inspection tools, and Table 4 shows the results of using the inspection tools of the present invention. As a comparison, detection by ISO method (ISO21528-1) was also performed.
これらの結果によると、市販の検査具を用いた場合には、ＩＳＯ法で検出された腸内細菌科菌であるエンテロバクター属菌が検出されなかったが、本発明の検査具においては約１０ｇの粉乳製品中に１ｃｆｕのエンテロバクター属菌でも検出できることが確認された。また、培地の呈色では検出できなかったエンテロバクター属菌は呈色指示薬の呈色によって判定できた。その一方、非腸内細菌科菌であるエンテロコッカス属菌では、呈色指示薬の呈色や培地の色の変化に基づく判定において双方とも陰性の判定が得られた。さらに、前培養は１８時間でも２４時間でも同じ判定が得られ、１８〜２４時間の前培養で十分に検出できることが確認された。 According to these results, when a commercially available inspection tool was used, Enterobacter spp. Which are enterobacteriaceae detected by the ISO method was not detected, but in the inspection tool of the present invention, about 10 g It was confirmed that even 1 cfu Enterobacter spp. In addition, Enterobacter spp. That could not be detected by coloration of the medium could be determined by coloration of the color indicator. On the other hand, both Enterococcus spp., Which are non-enterobacteriaceae, were negative in the determination based on the color of the color indicator and the change in the color of the medium. Furthermore, the same determination was obtained for the preculture for 18 hours or 24 hours, and it was confirmed that the preculture for 18 to 24 hours can be sufficiently detected.
実施例２において、Ｅ.Ｅ.ブロス４ｍｌに対して０.１ｍｌの前培養液を添加することで良好な結果が得られた。そこで、実施例２で使用されたＥ.Ｅ.ブロス４ｍｌを入れた本発明の検査具を用いて、菌選択性の確認試験を行った（「センシメディア法」と表記。以下同じ）。表５に示す腸内細菌科菌（ＥＢ菌）及び非腸内細菌科菌（非ＥＢ菌）について、ＢＰＷ培地により２４時間で前培養を行い、その０.１ｍｌを検査具の培地に添加して、３７℃、２４時間で培養を行った。調製粉乳１０ｇ中に表５に示す菌数の菌が含まれるように菌を接種した。なお、サンプルの一部については、明確に呈色の変化が観察されるまでの時間も測定した。その結果を表５に示した。表５に示すように、偽陰性を示す腸内細菌科菌（Ｎｏ１５）も見られたが、腸内細菌科菌３０種類のうち２９種類は陽性と判断され、ＩＳＯ法と相違がなかった。一方、ＩＳＯ法では非腸内細菌科菌も２９種類中５種類は偽陽性と判断されたが、Acinetobacter baumannii/calcoaceticusにおいて、本発明の検査具を用いた場合には、陰性として正しく判定された。また、培養開始から３時間程度で呈色の変化が観察される菌株も存在した。このように、本発明の検査具にて２４時間程度で培養すれば、ほぼ腸内細菌科菌群の菌は陽性として判定することができ、場合によっては２、３時間程度で陽性として判定できる。 In Example 2, good results were obtained by adding 0.1 ml of preculture to 4 ml of EE broth. Therefore, a fungus selectivity confirmation test was conducted using the test tool of the present invention containing 4 ml of EE broth used in Example 2 (indicated as “Sensimedia method”, the same applies hereinafter). Enterobacteriaceae (EB) and non-Enterobacteriaceae (non-EB) shown in Table 5 are pre-cultured in BPW medium for 24 hours, and 0.1 ml thereof is added to the medium of the test tool. The culture was performed at 37 ° C. for 24 hours. Bacteria were inoculated so that the number of bacteria shown in Table 5 was contained in 10 g of the prepared milk powder. For some of the samples, the time until a clear color change was observed was also measured. The results are shown in Table 5. As shown in Table 5, although Enterobacteriaceae (No15) showing false negatives was also found, 29 out of 30 types of Enterobacteriaceae were judged to be positive and were not different from the ISO method. On the other hand, 5 out of 29 types of non-enterobacteriaceae were determined to be false positives by the ISO method, but in Acinetobacter baumannii / calcoaceticus, when the test device of the present invention was used, it was correctly determined as negative. . There was also a strain in which a change in color was observed in about 3 hours from the start of culture. As described above, when cultured in the test tool of the present invention for about 24 hours, bacteria of the Enterobacteriaceae family can be determined as positive, and in some cases, can be determined as positive in about 2 to 3 hours. .
粉乳中に含まれる可能性のある腸内細菌科菌であるサカザキ菌（Cronobacter sakazakii（Enterobactor Sakazakii（E.sakazakii）））を対象にして、調製粉乳における検出感度を調べた。表６に示す菌数のサカザキ菌を１０ｇの調製粉乳（商品名：ほほえみ（明治乳業（株）製））に接種し、Ｅ.Ｅ.ブロス４ｍｌを入れた本発明の検査具を用いて、同菌の検出を試みた。ＢＰＷ培地により１８〜２４時間で前培養を行い、その０.１ｍｌを培地に添加し、３７℃、２４時間で培養を行った。その結果を表６に示す。 The detection sensitivity in formula milk powder was investigated for Sakazaki fungus (Cronobacter sakazakii (Enterobactor Sakazakii (E. sakazakii))), an enterobacteriaceae that may be contained in milk powder. Using the inspection tool of the present invention inoculating 10 g of prepared powdered milk (trade name: Hohoemi (manufactured by Meiji Dairies Co., Ltd.)) with 4 ml of EE broth Attempts were made to detect the bacteria. Pre-culture was performed in BPW medium for 18 to 24 hours, 0.1 ml of the medium was added to the medium, and the culture was performed at 37 ° C. for 24 hours. The results are shown in Table 6.
本発明の検査具を用いてもＩＳＯ法と同じ結果が得られ、陽性の場合には、前培養の後から４時間程度の短時間で判定することができた。また、調製粉乳１０ｇ中に１ｃｆｕのサカザキ菌が存在すれば、調製粉乳中のサカザキ菌を検出することができた。 Even when the test tool of the present invention was used, the same result as the ISO method was obtained, and in the case of being positive, the determination could be made in a short time of about 4 hours after the preculture. Moreover, if 1 cfu of Sakazaki bacteria were present in 10 g of the prepared milk powder, the Sakazaki bacteria in the prepared milk powder could be detected.
次に、共存菌存在下におけるサカザキ菌の検出感度を調べた。１０ｇの調製粉乳（商品名：ほほえみ（明治乳業（株）製））に表７及び表８に示す菌数のサカザキ菌（E.sakazakii JCM1233T）と枯草菌（Bacillus subtilis）又はエンテロコッカス属菌（Enterococcus durans）を接種し、Ｅ.Ｅ.ブロス４ｍｌを入れた本発明の検査具を用いて、サカザキ菌の検出を試みた。ＢＰＷ培地により１８〜２４時間で前培養を行い、その０.１ｍｌを培地に添加し、３７℃、２４時間で培養を行った。その結果を表７及び表８に示す。 Next, the detection sensitivity of Sakazaki bacteria in the presence of coexisting bacteria was examined. 10 g of prepared milk powder (trade name: Hohoemi (manufactured by Meiji Dairies Co., Ltd.)) durans) and an inspection tool of the present invention containing 4 ml of EE broth was used to detect Sakazaki bacteria. Pre-culture was performed in BPW medium for 18 to 24 hours, 0.1 ml of the medium was added to the medium, and the culture was performed at 37 ° C. for 24 hours. The results are shown in Tables 7 and 8.
本発明の検査具を用いてもＩＳＯ法と同じ結果が得られ、陽性の場合には４時間程度の短時間で判定することができた。また、調製粉乳１０ｇ中に１ｃｆｕのサカザキ菌が存在すれば、共存菌の存在下でも検出することができた。なお、エンテロコッカス属菌が共存した場合には、共存菌の存在により呈色の変化が観察されるまでの時間が長くなる傾向にあるが、２４時間で培養を行うことにより確実に検出できると判断される。 Even when the test tool of the present invention was used, the same result as the ISO method was obtained, and in the case of being positive, the determination could be made in a short time of about 4 hours. In addition, if 1 cfu of Sakazaki bacteria was present in 10 g of the prepared milk powder, it could be detected even in the presence of the coexisting bacteria. When Enterococcus coexists, the time until color change is observed due to the presence of the coexisting bacteria tends to be longer, but it can be reliably detected by culturing in 24 hours. Is done.
以上のように、既に上市されているＥ.Ｅ.ブロスを培地とした検査具を使用することによって、２４時間程度の培養時間で腸内細菌科菌群の細菌を検出することができる。
特に、乳児用調製粉乳やベビーフードなどで問題となるサカザキ菌は、ＢＰＷ培地により１８〜２４時間で前培養を行うことによって、粉乳１０ｇ当たり１ｃｆｕの細菌を確実に検出できる。このように、本発明によると前培養から遅くとも４８時間以内で腸内細菌科菌群の検査を終えることができる。
As described above, by using a commercially available test tool using EE broth as a medium, bacteria of the Enterobacteriaceae family can be detected in a culture time of about 24 hours.
In particular, Sakazaki bacteria, which are problematic in infant formulas, baby foods, etc., can reliably detect 1 cfu of bacteria per 10 g of milk powder by pre-culturing with BPW medium in 18 to 24 hours. Thus, according to the present invention, the examination of the Enterobacteriaceae family can be completed within 48 hours at the latest from the preculture.
本発明によると、短時間で食品中の腸内細菌科菌群の細菌の存否を判断できる検査具及び検査方法が提供される。 ADVANTAGE OF THE INVENTION According to this invention, the test | inspection tool and test | inspection method which can judge the presence or absence of the bacteria of the enterobacteriaceae group in a foodstuff in a short time are provided.
１ 全体が透明である容器本体
２ 培地収容部
３ 指示薬収容部
４ 二酸化炭素透過膜
DESCRIPTION OF SYMBOLS 1 Container body which is transparent as a whole 2 Medium storage part 3 Indicator storage part 4 Carbon dioxide permeable membrane
Claims (8)
前記容器は外側から前記指示薬収容部の内部を見通せる透明部を備え、前記培地収容部に腸内細菌科菌群の細菌増菌用成分と細菌増殖抑制成分である胆汁酸及び／又は胆汁酸塩とブリリアントグリーンを含み、他の細菌増殖抑制成分を実質的に含まないリン酸緩衝液からなる液体培地が収容され、前記指示薬収容部に二酸化酸素の呈色指示薬が収容された細菌検査具を用いて食品中の腸内細菌科群の細菌を検出する細菌検出方法であって、
前記収容された液体培地に、検査対象試料を添加する工程と、
前記容器を密閉して培養を開始し、培養の開始から定められた時間経過時に前記指示薬収納部の呈色の濃度を測定する工程を備え、
予め定められた呈色の濃度との対比により細菌の存否を判断する細菌検出方法。 In a sealable container, it is equipped with a medium container and an indicator container separated by a carbon dioxide permeable membrane that blocks the medium for enrichment,
The container includes a transparent portion through which the inside of the indicator accommodating portion can be seen from the outside, and a bile acid and / or bile salt that is a component for bacterial growth of the Enterobacteriaceae family and a component for inhibiting bacterial growth in the medium accommodating portion. And a brilliant green containing a liquid medium composed of a phosphate buffer substantially free of other bacterial growth inhibitory components, and using a bacterial test tool in which an oxygen dioxide color indicator is contained in the indicator containing portion A method for detecting bacteria in the Enterobacteriaceae family in food,
Adding a sample to be examined to the stored liquid medium ;
Sealing the container and starting the culture, comprising the step of measuring the concentration of the color of the indicator container when a predetermined time has elapsed from the start of the culture,
A bacteria detection method for determining the presence or absence of bacteria by contrast with a predetermined color concentration.
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