JP4601148B2 - Selective medium for detection of heat-resistant acidophilic bacteria and detection method - Google Patents
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Description
【０００１】
【発明の属する技術分野】
本発明は、耐熱性好酸性菌を検出するための選択培地に関するものであり、より詳しくは、果汁中または果汁を含む飲料中等で増殖性を有する耐熱性好酸性菌を検出するための選択培地に関するものである。
【０００２】
【従来の技術】
果実から得られた果汁を利用した飲料においては、その液性が酸性領域にあるため、一般的には細菌が生育し難い。しかしながら、このような酸性条件下においても生育することができる細菌種が存在することが知られていた。
【０００３】
また、前述したような酸性条件下において生育する細菌であって、且つ高温条件下であっても生育可能な細菌が存在することが知られており、耐熱性好酸性菌と総称されている。耐熱性好酸性菌は果実飲料を加熱処理したとしても死滅しないため、果実飲料の製造過程や製造後の品質管理の際にこのような細菌種の有無を高感度に検出する方法が望まれていた。
【０００４】
これまでに、前述したような細菌を検出する方法としては、検査すべき溶液（果実飲料等）を予め熱処理（通常７０〜８０℃）した後、好酸性菌用培地にて４５℃程度の比較的高温で生育を確認し、さらに果汁または果汁を含む飲料中での増殖性を確認するためにこのような果汁または果汁を含む飲料等に植菌し、増殖の有無を確認するという方法が採られていた。しかしながら、この方法は手間と時間がかかり、耐熱性好酸性菌の迅速な検出は困難であった。
【０００５】
これに対して、特開平８−１４０６９７号公報には、耐熱性好酸性菌を検出するための酵母エキス、グルコース及び澱粉からなる培地、及び、前記培地を用いた耐熱性好酸性菌の検出方法が記載されている。
【０００６】
【発明が解決しようとする課題】
しかしながら、特開平８−１４０６９７号公報に記載の培地を用いた場合でも耐熱性好酸性菌の検出感度は必ずしも十分ではなく、また、培養日数が約３日間と時間がかかるという問題があった。
【０００７】
本発明は、上記従来技術の有する課題に鑑みてなされたものであり、果汁中や果汁を含む飲料中に存在する耐熱性好酸性菌を高感度且つ短時間に検出するために用いられる選択培地及び前記選択培地を用いた耐熱性好酸性菌の検出方法を提供することを目的とする。
【０００８】
【課題を解決するための手段】
本発明者らは、上記目的を達成すべく鋭意研究を重ねた結果、栄養成分が実質的に酵母エキス、グルコース及びペプトンのみからなり且つｐＨが４．０±０．１に調整された選択培地を用いることにより、比較的短時間の培養で耐熱性好酸性菌を高感度に検出することが可能であることを見出し、本発明を完成するに至った。
【０００９】
すなわち、本発明の選択培地は、耐熱性好酸性菌検出用の選択培地であって、前記選択培地が溶媒と栄養成分としての酵母エキス、グルコース及びペプトンとを含有し、酵母エキス、グルコース及びペプトンの合計量が前記選択培地に含まれる全栄養成分の総重量の９０重量％以上であり、且つ前記選択培地のｐHが４．０±０．１であることを特徴とする選択培地である。本発明の選択培地においては、前記選択培地が実質的に酵母エキス、グルコース及びペプトンのみからなることが好ましい。
【００１０】
また、本発明の耐熱性好酸性菌の検出方法は、上記の選択培地に検査試料を供給する第１の工程と、前記検査試料が供給された選択培地を耐熱性好酸性菌が繁殖可能な条件下で静置する第２の工程と、前記静置された選択培地中の耐熱性好酸性菌の有無を検出する第３の工程と、を含むことを特徴とするものである。
【００１１】
【発明の実施の形態】
以下、本発明の好適な実施形態について詳細に説明する。
【００１２】
本発明の選択培地は、耐熱性好酸性菌検出用の選択培地であって、前記選択培地が溶媒と栄養成分としての酵母エキス、グルコース及びペプトンとを含有し、酵母エキス、グルコース及びペプトンの合計量が前記選択培地に含まれる全栄養成分の総重量の９０重量％以上であり、且つ前記選択培地のｐHが４．０±０．１であることを特徴とする選択培地である。ここで、選択培地とは、微生物の集団の中からある特定の性質を示す細胞を選択的に増殖させる培地をいい、特定の微生物以外の増殖を抑える物質を添加したり、栄養要求性の違いを利用したりすることによって目的とする微生物のみを選択的に増殖させることを可能とするものである。
【００１３】
本発明の選択培地は、培養する耐熱性好酸性菌の栄養成分として酵母エキス、グルコース及びペプトンを含有するものである。ここで、前記酵母エキス、グルコース及びペプトンの合計量は本発明の選択培地に含まれる全栄養成分の総重量の９０重量％以上を占めるものであり、９５重量％以上を占めることがより好ましく、本発明の選択培地に含有される栄養成分が実質的に酵母エキス、グルコース及びペプトンのみからなることが特に好ましい。
【００１４】
また、本発明の選択培地は、前記酵母エキス、グルコース及びペプトン以外にこれらの成分を溶解する溶媒が含まれる。このような溶媒としては、例えば、水や緩衝液が挙げられるが、水を用いることが好ましい。
【００１５】
また、前記酵母エキスとは、ビール酵母またはパン酵母の自己消化または低温浸出によって得た水溶性成分を低温乾燥して粉末化したもので、アミノ酸、ビタミン、その他の栄養成分を豊富に含み、多くの種類の培地に栄養素を補うために添加される。本発明の選択培地において、酵母エキスの配合量は特に制限されないが、１０００ｍｌの溶媒に対して２．０〜３．０ｇであることが好ましく、２．５ｇであることがより好ましい。前記酵母エキスの配合量が２．０ｇより少ない場合、栄養分が不足し、耐熱性好酸性菌の増殖性が低くなる傾向にあり、３．０ｇより多い場合、栄養分が過剰となり耐熱性好酸性菌以外の一般細菌等が増殖可能となる傾向にある。
【００１６】
また、グルコースは、微生物培養用培地の炭素源として用いられる。本発明の選択培地において、グルコースの配合量は特に制限されないが、１０００ｍｌの溶媒に対して０．５〜１．５ｇであることが好ましく、１．０ｇであることがより好ましい。前記グルコースの配合量が０．５ｇより少ない場合、栄養分が不足し、耐熱性好酸性菌の増殖性が低くなる傾向にあり、１．５ｇより多い場合、栄養分が過剰となり耐熱性好酸性菌以外の一般細菌等が増殖可能となる傾向にある。
【００１７】
さらに、ペプトンとは、牛乳カゼイン、獣肉、大豆タンパク質等をペプシン、トリプシン、パパイン等のプロテアーゼまたは酸で加水分解して乾燥、粉末化したものであり、オリゴペプチドやアミノ酸を主な成分とするものである。ペプトンの中でもカゼインペプトンと獣肉ペプトンの混合物はポリペプトンと呼ばれ、一般的な微生物培養用培地において窒素源として使用される。本発明の選択培地において、ペプトンはポリペプトンであることが好ましい。また、ペプトンの配合量は特に制限されないが、１０００ｍｌの溶媒に対して４．５〜５．５ｇであることが好ましく、５．０ｇであることがより好ましい。前記ペプトンの配合量が４．５ｇより少ない場合、栄養分が不足し、耐熱性好酸性菌の増殖性が低くなる傾向にあり、５．５ｇより多い場合、栄養分が過剰となり耐熱性好酸性菌以外の一般細菌等が増殖可能となる傾向にある。
【００１８】
また、本発明の選択培地には、酵母エキス、グルコース及びペプトン以外に微量成分として有機栄養成分または無機栄養成分（硫酸マグネシウム等）を含有していてもよい
本発明の選択培地は、そのｐＨが４．０±０．１の範囲にあることを特徴とする。前記培地のｐＨがこの範囲にある場合には、果汁中または果汁を含む飲料中等に存在する耐熱性好酸性菌の増殖性が顕著に高い。一方、前記培地のｐＨが上記下限より低い場合及び上記上限より高い場合には耐熱性好酸性菌の増殖性が著しく低下する傾向にある。
【００１９】
また、本発明の選択培地の形状としては液体、半流動体、固体のいずれであってもよいが、耐熱性好酸性菌の検出が容易である固体、中でも平板固形培地であることが好ましい。前記選択培地の形状が液体の場合、上記の酵母エキス、グルコース及びペプトンを水等の溶媒に溶解し、ｐＨを調整した後、オートクレーブ滅菌等により滅菌し、使用すればよい。
【００２０】
前記のｐＨの調整は微生物培養用培地のｐＨ調整に使用される酸であれば特に制限されないが、例えば、塩酸または硫酸が挙げられ、中でも塩酸であることが好ましい。この場合、前記選択培地のｐＨが４．０±０．１に調節可能であれば用いられる塩酸の濃度は特に制限されない。
【００２１】
また、前記選択培地の形状が固体の場合、前記液体の選択培地に固化剤、例えば寒天、ゼラチン、アガロースを添加した後、滅菌、固化し、使用すればよい。
この場合、前記固化剤は寒天であることが好ましく、添加量は１０００ｍｌの溶媒に対して１３〜２０ｇであることが好ましい。
【００２２】
また、前記選択培地の形状が半流動体の場合、前記液体の選択培地に固化剤、例えば寒天、ゼラチン、アガロースを添加した後、滅菌、固化し、使用すればよい。この場合、前記固化剤は寒天であることが好ましく、添加量は１０００ｍｌの溶媒に対して８〜１２ｇであることが好ましい。
【００２３】
次に、前述した本発明の選択培地を用いて耐熱性好酸性菌を検出する方法について説明する。
【００２４】
本発明の耐熱性好酸性菌の検出方法は、前述の選択培地に検査試料を塗抹または添加する第１の工程と、前記検査試料が塗抹または添加された選択培地を静置する第２の工程と、前記静置された選択培地中の耐熱性好酸性菌の有無を検出する第３の工程と、からなることを特徴とする耐熱性好酸性菌の検出方法である。
【００２５】
先ず、本発明にかかる検査対象である果汁または果汁を含む飲料について説明する。
【００２６】
前記検査対象となる検査試料は、果汁、例えば、アップル果汁、レモン果汁、グレープ果汁、パイナップル果汁及びグレープフルーツ果汁や、これらを含む飲料であり、中でもアップル果汁であることが好ましい。ここで、このような果汁を含む飲料とは、例えば、果汁を水または無機塩類溶液等で希釈した飲料の他、野菜汁、発酵乳、乳類が含有された飲料であってもよい。
【００２７】
前記検査試料は、本発明の選択培地に供給し、耐熱性好酸性菌が繁殖可能な条件下で静置すればよいが、耐熱性菌のみを効率よく分離するために前記検査試料を予め熱処理しておいてもよい。前記熱処理の条件としては、７０〜８０℃で５〜１５分間行うことが好ましい。
【００２８】
また、このような果汁または果汁を含む飲料等から耐熱性好酸性菌を検出するにあたり、これらの原液を検査試料として用いることができるが、前記検査試料に含まれる菌類の濃度が低い場合等には必要に応じてメンブランフィルターを用いて前記検査試料をろ過することにより前記検査試料に含有される細菌をメンブランフィルター上に捕捉した後、検査試料としてもよい。また、前記検査試料を遠心分離することにより前記検査試料に含有される細菌を集菌し、検査試料としてもよい。
【００２９】
次に、本発明にかかる第１の工程について説明する。本発明にかかる第１の工程は、前述の選択培地に検査試料を供給する工程である。
【００３０】
本発明にかかる第１の工程において、用いる選択培地が液体である場合には選択培地に前記の検査試料をそのまま、またはメンブランフィルターや遠心分離によって濃縮した検査試料を少量の培地や水に懸濁して前記選択培地に添加すればよい。また、用いる選択培地が固形である場合には、検査試料をそのまま、または濃縮したものを白金耳等で塗抹すればよい。
【００３１】
次に、本発明にかかる第２の工程について説明する。本発明にかかる第２の工程は、前記検査試料が供給された選択培地を耐熱性好酸性菌が繁殖可能な条件下で静置する工程である。静置する際の条件は、検出対象である耐熱性好酸性菌が増殖可能である条件であれば特に制限されないが、温度が３０〜７０℃であることが好ましく、４０〜６０℃であることがより好ましい。温度が前記下限より低い場合、または前記上限より高い場合には耐熱性好酸性菌の増殖速度が低下する傾向にある。また、静置する時間は、検出対象である耐熱性好酸性菌が検出可能であれば特に制限されないが、１０〜５０時間であることが好ましく、２０〜４０時間であることがより好ましい。静置する時間が１０時間より短い場合には耐熱性好酸性菌が検出可能なまで増殖せず、検出が困難な傾向にあり、５０時間より長い場合には耐熱性好酸性菌が増殖しすぎ、正確な菌体数の測定が困難になる傾向にある。
【００３２】
本発明にかかる第３の工程は、前記静置された選択培地中の耐熱性好酸性菌の有無を検出する工程である。
【００３３】
前述した静置後の選択培地が液体である場合、選択培地中に増殖した耐熱性好酸性菌の検出方法は特に制限されないが、例えば、静置後の選択培地の濁度を吸光光度計等で測定すればよい。また、選択培地が固体である場合、選択培地上に増殖した耐熱性好酸性菌の検出方法は特に制限されないが、例えば、コロニーの有無を目視で検出すればよい。
【００３４】
【実施例】
以下、実施例及び比較例により本発明の内容を具体的に説明するが、本発明は以下の実施例に限定されるものではない。
【００３５】
実施例１及び比較例１〜３
先ず、本発明の選択培地（ＹＰＧ培地）を調製した。ＹＰＧ培地の組成を表１に示す。表１の組成物を混合し、塩酸を用いてｐＨを４．０に調整した。混合した溶液はオートクレーブ滅菌（１２１℃、１５分）し、以下の実験に用いた。
【００３６】
【表１】
本発明の選択培地（ＹＰＧ培地）（実施例１）、ＹＳＧ培地（特開平８−１４０６９７号公報に記載の培地）（比較例１）、ＰＤＢ培地（比較例２）及びＴＡ基礎培地（比較例３）を準備した（全て液体培地）。ここで、ＴＡ基礎培地は表２に示す組成を有し、ｐＨは塩酸を用いて４．０に調整した。また、ＰＤＢ培地は表３に示す組成を有し、ｐＨは塩酸を用いて４．０に調整した。さらに、ＹＳＧ培地は表４に示す組成を有し、ｐＨは塩酸を用いて４．０に調整した。
【００３８】
【表２】
【表３】
【表４】
このようにして調製した培地を準備し、それぞれに１×１０2個の耐熱性好酸性菌４種類（ＳＡ−６(Ａｌｉｃｙｃｌｏｂａｃｉｌｌｕｓ ｓｐ．（サッポロビール飲料（株）保有株）、ＪＣＭ５２６０（＝ＡＴＣＣ２７００９）（Ａｌｉｃｙｃｌｏｂａｃｉｌｌｕｓ ａｃｉｄｏｃａｌｄａｒｉｕｓ）、ＤＳＭ２４９８（Ａｌｉｃｙｃｌｏｂａｃｉｌｌｕｓ ａｃｉｄｏｔｅｒｒｅｓｔｒｉｓ）及びＩＦＯ１５３１０（＝ＤＳＭ４００６）（Ａｌｉｃｙｃｌｏｂａｃｉｌｌｕｓｃｙｃｌｏｈｅｐｔａｎｉｃｕｓ））を接種した。５０℃で２４時間静置培養後、それぞれの菌体数をカウントした。得られた結果を表５に示す。
【００４２】
【表５】
表５に示された結果より、ＹＰＧ培地を用いた場合、ＴＡ基礎培地、ＰＤＢ培地またはＹＳＧ培地を用いた場合と比較して耐熱性好酸性菌の増殖性が高いことが確認された。特に、果汁中での増殖能が非常に高く、検出の必要性が高い菌株であるＤＳＭ２４９８（Ａｌｉｃｙｃｌｏｂａｃｉｌｌｕｓ ａｃｉｄｏｔｅｒｒｅｓｔｒｉｓ）においては、本発明の選択培地を用いた場合にＹＳＧ培地の１０倍以上の感度で検出可能であることが確認された。従って、本発明の選択培地が果汁中または果汁を含む飲料中において高い増殖性を示す耐熱性好酸性菌を選択的に検出するために非常に有効であることが確認された。
【００４４】
実施例２
（ｐＨ調整に用いる塩酸及び硫酸の耐熱性好酸性菌の増殖に及ぼす影響）
ＹＰＧ培地に耐熱性好酸性菌であるＳＡ−６(Ａｌｉｃｙｃｌｏｂａｃｉｌｌｕｓ ｓｐ．（サッポロビール飲料（株）保有株）、ＪＣＭ５２６０（Ａｌｉｃｙｃｌｏｂａｃｉｌｌｕｓ ａｃｉｄｏｃａｌｄａｒｉｕｓ）、ＤＳＭ２４９８（Ａｌｉｃｙｃｌｏｂａｃｉｌｌｕｓ ａｃｉｄｏｔｅｒｒｅｓｔｒｉｓ）及びＩＦＯ１５３１０（Ａｌｉｃｙｃｌｏｂａｃｉｌｌｕｓ ｃｙｃｌｏｈｅｐｔａｎｉｃｕｓ）の４種類をそれぞれ１×１０2菌体ずつ接種し、５０℃で１２時間静置培養後、菌体数をカウントした。得られた結果を表６に示す。
【００４５】
【表６】
実施例３
（ｐＨ調整に用いる塩酸及び硫酸の耐熱性好酸性菌の増殖に及ぼす影響）
ｐＨの調整に硫酸を用いた以外は実施例２と同様にして４種類の耐熱性好酸性菌の菌体数をカウントした。得られた結果を表６に示す。
【００４７】
表６に示された結果より、本発明の選択培地のｐＨ調製に塩酸を用いた場合、硫酸を用いた場合より耐熱性好酸性菌の増殖性は１．５〜３倍程度高いことが確認された。
【００４８】
【発明の効果】
以上説明したように、本発明の選択培地及び検出方法によれば、果汁中や果汁を含む飲料中等に存在する耐熱性好酸性菌を高感度且つ短時間に検出することが可能となる。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a selective medium for detecting heat-resistant acidophilic bacteria, and more specifically, a selective medium for detecting heat-resistant acidophilic bacteria having growth properties in fruit juice or beverages containing fruit juice. It is about.
[0002]
[Prior art]
In beverages using fruit juice obtained from fruits, since the liquidity is in the acidic region, bacteria are generally difficult to grow. However, it has been known that there are bacterial species that can grow even under such acidic conditions.
[0003]
In addition, it is known that there are bacteria that grow under acidic conditions as described above and that can grow even under high temperature conditions, and are collectively called heat-resistant acidophilic bacteria. Since heat-resistant acidophilic bacteria do not die even when heat-treated fruit drinks, a method for detecting the presence or absence of such bacterial species with high sensitivity during the production process of fruit drinks and quality control after production is desired. It was.
[0004]
Until now, as a method for detecting bacteria as described above, after a heat treatment (usually 70 to 80 ° C.) of a solution to be examined (fruit beverage or the like) in advance, a comparison is made at about 45 ° C. in a medium for acidophilic bacteria. In order to confirm growth at a high temperature, and to confirm the growth in fruit juice or beverages containing fruit juice, inoculate such juice or beverages containing fruit juice and confirm the presence or absence of growth. It was done. However, this method is time-consuming and time-consuming, and it is difficult to quickly detect heat-resistant acidophilic bacteria.
[0005]
In contrast, JP-A-8-140697 discloses a yeast extract for detecting heat-resistant acidophilic bacteria, a medium comprising glucose and starch, and a method for detecting heat-resistant acidophilic bacteria using the medium. Is described.
[0006]
[Problems to be solved by the invention]
However, even when the medium described in JP-A-8-140697 is used, the detection sensitivity of heat-resistant acidophilic bacteria is not always sufficient, and there is a problem that it takes about 3 days to culture.
[0007]
The present invention has been made in view of the above-described problems of the prior art, and is a selective medium used for detecting heat-resistant acidophilic bacteria present in fruit juice and beverages containing fruit juice with high sensitivity and in a short time. Another object of the present invention is to provide a method for detecting heat-resistant acidophilic bacteria using the selective medium.
[0008]
[Means for Solving the Problems]
As a result of intensive studies to achieve the above-mentioned object, the present inventors have made a selective medium whose nutritional components are substantially composed only of yeast extract, glucose and peptone, and whose pH is adjusted to 4.0 ± 0.1. As a result, it was found that heat-resistant acidophilic bacteria can be detected with high sensitivity in a relatively short time of cultivation, and the present invention has been completed.
[0009]
That is, the selective medium of the present invention is a selective medium for detecting heat-resistant acidophilic bacteria, and the selective medium contains a solvent and yeast extract, glucose and peptone as nutrient components, and the yeast extract, glucose and peptone. Is a selection medium characterized in that the total amount of is not less than 90% by weight of the total weight of all nutrients contained in the selection medium, and the pH of the selection medium is 4.0 ± 0.1. In the selective medium of the present invention, it is preferable that the selective medium consists essentially of yeast extract, glucose and peptone.
[0010]
In addition, the method for detecting heat-resistant acidophilic bacteria of the present invention includes the first step of supplying a test sample to the selective medium, and the heat-resistant acidophilic bacteria can propagate on the selective medium supplied with the test sample. A second step of standing under conditions, and a third step of detecting the presence or absence of heat-resistant acidophilic bacteria in the stationary selective medium.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, preferred embodiments of the present invention will be described in detail.
[0012]
The selective medium of the present invention is a selective medium for detecting heat-resistant acidophilic bacteria, wherein the selective medium contains a solvent and yeast extract, glucose and peptone as nutrient components, and the total of yeast extract, glucose and peptone. The selection medium is characterized in that the amount is 90% by weight or more of the total weight of all nutrient components contained in the selection medium, and the pH of the selection medium is 4.0 ± 0.1. Here, the selective medium refers to a medium that selectively grows cells exhibiting a specific property from a population of microorganisms, and a substance that suppresses growth other than a specific microorganism is added, or there is a difference in auxotrophy. It is possible to selectively proliferate only the target microorganism.
[0013]
The selective medium of the present invention contains yeast extract, glucose and peptone as nutrient components of the heat-resistant acidophilic bacterium to be cultured. Here, the total amount of the yeast extract, glucose and peptone occupies 90% by weight or more of the total weight of all nutrients contained in the selective medium of the present invention, more preferably 95% by weight or more, It is particularly preferred that the nutritional component contained in the selective medium of the present invention consists essentially of yeast extract, glucose and peptone.
[0014]
The selective medium of the present invention contains a solvent that dissolves these components in addition to the yeast extract, glucose, and peptone. Examples of such a solvent include water and a buffer solution, but it is preferable to use water.
[0015]
The yeast extract is obtained by pulverizing water-soluble components obtained by self-digestion or low-temperature leaching of brewer's yeast or baker's yeast, and is rich in amino acids, vitamins and other nutritional components. It is added to supplement the nutrients of this type of medium. In the selective medium of the present invention, the blending amount of the yeast extract is not particularly limited, but is preferably 2.0 to 3.0 g, more preferably 2.5 g with respect to 1000 ml of the solvent. When the blending amount of the yeast extract is less than 2.0 g, nutrients are insufficient and the growth of the thermostable acidophilic bacteria tends to be low, and when it is more than 3.0 g, the nutrients are excessive and the thermostable acidophilic bacteria are reduced. Other general bacteria tend to be able to grow.
[0016]
In addition, glucose is used as a carbon source for the culture medium for microorganisms. In the selective medium of the present invention, the blending amount of glucose is not particularly limited, but is preferably 0.5 to 1.5 g, more preferably 1.0 g with respect to 1000 ml of the solvent. When the amount of glucose is less than 0.5 g, nutrients are insufficient, and the growth of heat-resistant acidophilic bacteria tends to be low. When it is more than 1.5 g, the nutrients are excessive and other than heat-resistant acidophilic bacteria. Tend to be able to grow general bacteria.
[0017]
Furthermore, peptone is a product obtained by hydrolyzing milk casein, animal meat, soy protein, etc. with protease or acid such as pepsin, trypsin, papain, etc., dried and powdered, and mainly composed of oligopeptides and amino acids. It is. Among peptones, a mixture of casein peptone and animal peptone is called polypeptone, and is used as a nitrogen source in a general culture medium for microorganisms. In the selective medium of the present invention, the peptone is preferably polypeptone. Moreover, the blending amount of peptone is not particularly limited, but is preferably 4.5 to 5.5 g, more preferably 5.0 g with respect to 1000 ml of the solvent. When the amount of the peptone is less than 4.5 g, nutrients are insufficient, and the growth of heat-resistant acidophilic bacteria tends to be low. When it is more than 5.5 g, the nutrients are excessive and other than heat-resistant acidophilic bacteria. Tend to be able to grow general bacteria.
[0018]
In addition, the selective medium of the present invention may contain an organic nutrient component or an inorganic nutrient component (magnesium sulfate, etc.) as a trace component in addition to yeast extract, glucose and peptone. It is characterized by being in the range of 4.0 ± 0.1. When the pH of the culture medium is in this range, the growth ability of heat-resistant acidophilic bacteria present in fruit juice or beverages containing fruit juice is remarkably high. On the other hand, when the pH of the culture medium is lower than the lower limit and higher than the upper limit, the growth ability of heat-resistant acidophilic bacteria tends to be significantly reduced.
[0019]
Moreover, the shape of the selective medium of the present invention may be any of liquid, semi-fluid, and solid, but it is preferably a solid that can easily detect heat-resistant acidophilic bacteria, particularly a flat solid medium. When the shape of the selective medium is liquid, the yeast extract, glucose and peptone described above are dissolved in a solvent such as water, adjusted to pH, and then sterilized by autoclave sterilization or the like.
[0020]
The adjustment of the pH is not particularly limited as long as it is an acid used for adjusting the pH of the culture medium for microorganisms. For example, hydrochloric acid or sulfuric acid is exemplified, and hydrochloric acid is particularly preferable. In this case, the concentration of hydrochloric acid used is not particularly limited as long as the pH of the selective medium can be adjusted to 4.0 ± 0.1.
[0021]
If the selective medium is solid, a solidifying agent such as agar, gelatin, or agarose may be added to the liquid selective medium and then sterilized, solidified, and used.
In this case, the solidifying agent is preferably agar, and the addition amount is preferably 13 to 20 g with respect to 1000 ml of the solvent.
[0022]
When the selection medium is semi-liquid, a solidifying agent such as agar, gelatin, or agarose may be added to the liquid selection medium, followed by sterilization, solidification, and use. In this case, the solidifying agent is preferably agar, and the addition amount is preferably 8 to 12 g with respect to 1000 ml of the solvent.
[0023]
Next, a method for detecting heat-resistant acidophilic bacteria using the above-described selective medium of the present invention will be described.
[0024]
The method for detecting a thermostable acidophilic bacterium of the present invention includes a first step of smearing or adding a test sample to the aforementioned selective medium, and a second step of allowing the selective medium on which the test sample is smeared or added to stand still. And a third step of detecting the presence or absence of heat-resistant acidophilic bacteria in the stationary selective medium, wherein the method is for detecting heat-resistant acidophilic bacteria.
[0025]
First, the fruit juice or the drink containing fruit juice which is the test object according to the present invention will be described.
[0026]
The test sample to be inspected is fruit juice, for example, apple fruit juice, lemon fruit juice, grape fruit juice, pineapple fruit juice and grapefruit fruit juice, and beverages containing these, among which apple fruit juice is preferable. Here, the beverage containing such fruit juice may be, for example, a beverage containing vegetable juice, fermented milk, and milk in addition to a beverage obtained by diluting fruit juice with water or an inorganic salt solution.
[0027]
The test sample may be supplied to the selective medium of the present invention and allowed to stand under conditions where heat-resistant acidophilic bacteria can propagate. However, in order to efficiently isolate only the heat-resistant bacteria, the test sample is previously heat-treated. You may keep it. The heat treatment is preferably performed at 70 to 80 ° C. for 5 to 15 minutes.
[0028]
In addition, when detecting heat-resistant acidophilic bacteria from such fruit juice or beverages containing fruit juice, these stock solutions can be used as a test sample, but when the concentration of fungi contained in the test sample is low, etc. If necessary, the test sample may be filtered using a membrane filter to capture bacteria contained in the test sample on the membrane filter, and then used as the test sample. In addition, the test sample may be centrifuged to collect bacteria contained in the test sample and may be used as the test sample.
[0029]
Next, the first step according to the present invention will be described. The first step according to the present invention is a step of supplying a test sample to the aforementioned selective medium.
[0030]
In the first step according to the present invention, when the selective medium to be used is liquid, the test sample is suspended in the selective medium as it is, or the test sample concentrated by membrane filter or centrifugation is suspended in a small amount of medium or water. May be added to the selective medium. Moreover, when the selective culture medium to be used is solid, what is necessary is just to smear the test sample as it is or concentrated with a platinum loop etc.
[0031]
Next, the 2nd process concerning this invention is demonstrated. The second step according to the present invention is a step of allowing the selective medium supplied with the test sample to stand under conditions where heat-resistant acidophilic bacteria can propagate. The conditions for standing still are not particularly limited as long as the heat-resistant acidophilic bacteria to be detected can grow, but the temperature is preferably 30 to 70 ° C, and preferably 40 to 60 ° C. Is more preferable. When the temperature is lower than the lower limit or higher than the upper limit, the growth rate of heat-resistant acidophilic bacteria tends to decrease. In addition, the standing time is not particularly limited as long as the heat-resistant acidophilic bacterium as a detection target can be detected, but it is preferably 10 to 50 hours, and more preferably 20 to 40 hours. If the standing time is shorter than 10 hours, the thermostable acidophilic bacteria do not grow until they can be detected and tend to be difficult to detect, and if longer than 50 hours, the heat resistant acidophilic bacteria grow too much. It tends to be difficult to accurately measure the number of cells.
[0032]
The third step according to the present invention is a step of detecting the presence or absence of heat-resistant acidophilic bacteria in the stationary selective medium.
[0033]
When the selective medium after standing is a liquid, the method for detecting heat-resistant acidophilic bacteria grown in the selective medium is not particularly limited. For example, the turbidity of the selective medium after standing is measured using an absorptiometer or the like. You can measure with. In addition, when the selective medium is solid, the method for detecting heat-resistant acidophilic bacteria grown on the selective medium is not particularly limited. For example, the presence or absence of colonies may be detected visually.
[0034]
【Example】
EXAMPLES Hereinafter, although the content of this invention is demonstrated concretely by an Example and a comparative example, this invention is not limited to a following example.
[0035]
Example 1 and Comparative Examples 1-3
First, the selective medium (YPG medium) of the present invention was prepared. The composition of the YPG medium is shown in Table 1. The compositions in Table 1 were mixed and the pH was adjusted to 4.0 using hydrochloric acid. The mixed solution was sterilized by autoclave (121 ° C., 15 minutes) and used for the following experiments.
[0036]
[Table 1]
Selection medium (YPG medium) of the present invention (Example 1), YSG medium (medium described in JP-A-8-140697) (Comparative Example 1), PDB medium (Comparative Example 2) and TA basal medium (Comparative Example) 3) was prepared (all liquid medium). Here, the TA basal medium had the composition shown in Table 2, and the pH was adjusted to 4.0 using hydrochloric acid. The PDB medium had the composition shown in Table 3, and the pH was adjusted to 4.0 using hydrochloric acid. Further, the YSG medium had the composition shown in Table 4, and the pH was adjusted to 4.0 using hydrochloric acid.
[0038]
[Table 2]
[Table 3]
[Table 4]
The thus-prepared medium was prepared, and 4 types of 1 × 10 2 heat-resistant acidophilic bacteria (SA-6 (Alycyclobacillus sp.), JCM5260 (= ATCC27009), respectively. (Alyclobacterium acidocaldarius), DSM2498 (Alyocilacillus acidoterrestris), and IFO15310 (= DSM4006) (Alyocilocillus cycloheptanicus), which were obtained after incubation at 50 ° C. for 24 hours. Shown in
[0042]
[Table 5]
From the results shown in Table 5, it was confirmed that when the YPG medium was used, the growth resistance of the thermostable acidophilic bacteria was higher than when the TA basal medium, PDB medium or YSG medium was used. In particular, DSM2498 (Alyicobacillus acidoterrestris), which has a very high growth ability in fruit juice and has a high necessity for detection, is detected with a sensitivity 10 times or more that of YSG medium when the selective medium of the present invention is used. It was confirmed that it was possible. Therefore, it was confirmed that the selective medium of the present invention is very effective for selectively detecting heat-resistant acidophilic bacteria exhibiting high growth in fruit juice or beverages containing fruit juice.
[0044]
Example 2
(Effects of hydrochloric acid and sulfuric acid used for pH adjustment on the growth of heat-resistant acidophilic bacteria)
In the YPG medium, heat-resistant acidophilic bacteria such as SA-6 (Alycyclobacillus sp. (Sapporo Beer Beverage Co., Ltd.), JCM5260 (Alycyclobacillus acidocaldarius), DSM2498 (Alyocilobilis acidocterrestris) and Icilobillicus acidoterstris), and 4 types of DSM2498 (Alyocilocillus acidocterrestris) and Icicobacillus acidocterrestris, respectively. 1 × 10 2 cells were inoculated and the number of cells was counted after static culture at 50 ° C. for 12 hours, and the obtained results are shown in Table 6.
[0045]
[Table 6]
Example 3
(Effects of hydrochloric acid and sulfuric acid used for pH adjustment on the growth of heat-resistant acidophilic bacteria)
The number of cells of four types of heat-resistant acidophilic bacteria was counted in the same manner as in Example 2 except that sulfuric acid was used for pH adjustment. The obtained results are shown in Table 6.
[0047]
From the results shown in Table 6, it was confirmed that when hydrochloric acid was used to adjust the pH of the selective medium of the present invention, the growth of thermotolerant acidophilic bacteria was about 1.5 to 3 times higher than when sulfuric acid was used. It was done.
[0048]
【The invention's effect】
As described above, according to the selective medium and the detection method of the present invention, it is possible to detect heat-resistant acidophilic bacteria present in fruit juice or beverages containing fruit juice with high sensitivity and in a short time.
Claims (5)
前記検査試料が供給された選択培地を耐熱性好酸性菌が繁殖可能な条件下で静置する第２の工程と、
前記静置された選択培地中の耐熱性好酸性菌の有無を検出する第３の工程と、
を含むことを特徴とする耐熱性好酸性菌の検出方法。A first step of supplying a test sample to the selective medium according to any one of claims 1 to 3 ,
A second step of allowing the selective medium supplied with the test sample to stand under conditions where heat-resistant acidophilic bacteria can propagate;
A third step of detecting the presence or absence of heat-resistant acidophilic bacteria in the stationary selective medium;
A method for detecting a heat-resistant acidophilic bacterium, comprising:
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| JPH08140697A (en) * | 1994-11-17 | 1996-06-04 | Kirin Beverage Kk | Selective culture medium for detecting thermotolerant acid-fast bacillus and its detection |
| JPH08140696A (en) * | 1994-11-17 | 1996-06-04 | Kirin Beverage Kk | Detection of thermotolerant acid-fast bacillus having proliferating property in fruit juice |
| JPH11243945A (en) * | 1998-03-03 | 1999-09-14 | Kirin Beverage Corp | New bacteria of genus alicyclobacillus |
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