JP5757549B2 - Coloring reaction and / or fluorescence coloring reaction enhancement by egg yolk - Google Patents
Coloring reaction and / or fluorescence coloring reaction enhancement by egg yolk Download PDFInfo
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- JP5757549B2 JP5757549B2 JP2009239271A JP2009239271A JP5757549B2 JP 5757549 B2 JP5757549 B2 JP 5757549B2 JP 2009239271 A JP2009239271 A JP 2009239271A JP 2009239271 A JP2009239271 A JP 2009239271A JP 5757549 B2 JP5757549 B2 JP 5757549B2
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- 238000006243 chemical reaction Methods 0.000 title description 7
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Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
本発明は、基質に酵素を作用させる発色法における発色増強方法に関する。より詳細には、ホスファターゼ、β−グルコシダーゼ、Ｎ−アセチル−β−ヘキソサミニダーゼ、ホスホリパーゼなどの作用により基質が加水分解され、発色団化合物および／または蛍光発色団化合物を遊離する発色法における、発色増強方法およびそれに用いる試薬に関する。 The present invention relates to a coloring enhancement method in a coloring method in which an enzyme is allowed to act on a substrate. More specifically, in a chromogenic method in which a substrate is hydrolyzed by the action of phosphatase, β-glucosidase, N-acetyl-β-hexosaminidase, phospholipase, etc. to release a chromophore compound and / or a fluorescent chromophore compound. The present invention relates to a coloring enhancement method and a reagent used therefor.
酵素の作用により基質が加水分解され、発色団化合物および／または蛍光発色団化合物を遊離する反応は、目視または装置により発色や蛍光を簡易かつ高感度に測定可能であるため、生化学、免疫学、分子生物学、微生物学など幅広い分野において、酵素活性検出を目的として応用されている。特に微生物学分野では、酵素活性を測定することにより微生物を検出または識別する方法に応用され、コロニー所見の鑑別のような熟練を要さず、煩雑な微生物確認試験を省略できる簡易で迅速な方法として汎用されている。例えば、特許文献１には、発色基質である５−ブロモ−６−クロロ−３−インドキシルホスフェートを培地に含有させ、スタフィロコッカス・アウレウス（Staphylococcus aureus、以下、黄色ブドウ球菌という。）のホスファターゼ活性を検出する方法が記載されている。また、特許文献２には、５−ブロモ−４−クロロ−３−インドキシル−β−グルコシドを培地に含有させてエンテロコッカス（Enterococcus）属細菌のβ−グルコシダーゼ活性を検出する方法、特許文献３には、５−ブロモ−４−クロロ−３−インドリル−Ｎ−アセチル−β−Ｄ−グルコサミニドを培地に含有させてカンジダ・アルビカンス（Candida albicans）のＮ−アセチル−β−Ｄ−グルコサミニダーゼ活性を検出する方法、さらに特許文献４には、５−ブロモ−４−クロロ−３−インドリルホスファチジルミオイノシトールを培地に含有させてリステリア・モノサイトジェネス（Listeria monocytogenes）およびリステリア・イヴァノヴィ（Listeria ivanovii）のホスファチジルイノシトール特異的ホスホリパーゼＣ（phosphatidylinositol-specific phospholipase C、以下、PI-PLCという。）活性を検出する方法が記載されている。 The reaction that hydrolyzes the substrate due to the action of the enzyme and releases the chromophore compound and / or the fluorescent chromophore compound can be easily and highly sensitively measured for color development and fluorescence by visual or instrumentation. In a wide range of fields such as molecular biology and microbiology, it is applied for the purpose of detecting enzyme activity. Especially in the field of microbiology, it is applied to a method for detecting or distinguishing microorganisms by measuring enzyme activity, and does not require skill such as differentiation of colony findings, and can be a simple and rapid method that can omit complicated microorganism confirmation tests. As a general purpose. For example, Patent Document 1 includes 5-bromo-6-chloro-3-indoxyl phosphate, which is a chromogenic substrate, in a medium, and a phosphatase of Staphylococcus aureus (hereinafter referred to as Staphylococcus aureus). A method for detecting activity is described. Patent Document 2 discloses a method for detecting β-glucosidase activity of bacteria belonging to the genus Enterococcus by containing 5-bromo-4-chloro-3-indoxyl-β-glucoside in a medium. Contains 5-bromo-4-chloro-3-indolyl-N-acetyl-β-D-glucosaminide in the medium to detect N-acetyl-β-D-glucosaminidase activity of Candida albicans Methods, and further, in US Pat. No. 5,836,075, include 5-bromo-4-chloro-3-indolylphosphatidyl myo-inositol in the medium to produce Listeria monocytogenes and Listeria ivanovii phosphatidylinositol. Specific phospholipase C (phosphatidylinositol-specific phospholipase C) That PI-PLC.) A method of detecting is described activity.
しかし、発色基質や蛍光基質の価格が非常に高価なため、酵素基質を含む培地は高コストであるという問題がある。そのため、特に食品衛生や環境検査における黄色ブドウ球菌測定など、検査数の多い現場では、酵素基質を含有する、より低コストの培地が求められている。 However, since the price of the chromogenic substrate and the fluorescent substrate is very expensive, there is a problem that the medium containing the enzyme substrate is expensive. For this reason, a low-cost medium containing an enzyme substrate is demanded in the field where the number of examinations is large, such as the measurement of Staphylococcus aureus in food hygiene and environmental examinations.
また、メチシリン耐性黄色ブドウ球菌（Methicillin-Resistant Staphylococcus aureus、以下、MRSAという。）、バンコマイシン耐性腸球菌（Vancomycin-Resistant Enterococci）などの院内感染原因菌や黄色ブドウ球菌などの食中毒原因菌の検査においては、短時間で感度良く菌を検出することが重要である。 In addition, in the examination of nosocomial pathogenic bacteria such as methicillin-resistant Staphylococcus aureus (hereinafter referred to as MRSA) and vancomycin-resistant enterococci and food poisoning pathogens such as Staphylococcus aureus It is important to detect bacteria with high sensitivity in a short time.
特許文献５には、乳化安定化剤（TWEEN（登録商標）20）と溶媒（ジメチルスルホキシド（DMSO））を培地に添加して基質溶解性を高めることにより、発色または蛍光強度を増強して対象微生物の検出感度を上昇させることや基質含量を低減した培地を提供することが記載されている。 In Patent Document 5, an emulsion stabilizer (TWEEN (registered trademark) 20) and a solvent (dimethyl sulfoxide (DMSO)) are added to a medium to enhance substrate solubility, thereby enhancing color development or fluorescence intensity. It is described that the detection sensitivity of microorganisms is increased and a medium having a reduced substrate content is provided.
一方、卵黄を添加した培地上でスタフィロコッカス（Staphylococcus)属細菌(以下、ブドウ球菌という。)、バチルス(Bacillus)属細菌、クロストリジウム(Clostridium）属細菌などを培養すると、増殖した菌体の周辺に白濁環が生じることが知られており（いわゆる卵黄反応）、前記の菌を検出するために卵黄添加培地が利用されている（例えば、特許文献６）。さらに、黄色ブドウ球菌による卵黄反応を識別しやすくするために、黄色ブドウ球菌以外のブドウ球菌のコロニーを着色する発色基質を培地に含有させる方法が開示されている（特許文献７）。しかし、検出対象菌のコロニーを着色する酵素基質培地に卵黄を添加して発色を増強させた例は報告されていない。 On the other hand, when culturing Staphylococcus bacteria (hereinafter referred to as staphylococci), Bacillus bacteria, Clostridium bacteria, etc. on a medium supplemented with egg yolk, It is known that a white turbid ring is formed (so-called egg yolk reaction), and an egg yolk-added medium is used to detect the above-mentioned bacteria (for example, Patent Document 6). Furthermore, in order to make it easy to identify the yolk reaction caused by Staphylococcus aureus, a method is disclosed in which a chromogenic substrate for coloring colonies of staphylococci other than Staphylococcus aureus is contained in the medium (Patent Document 7). However, no example has been reported in which egg yolk is added to an enzyme substrate medium for coloring colonies of detection target bacteria to enhance color development.
本発明は、上記の現状に鑑みてなされたものであり、酵素活性の検出または識別において、高感度、迅速、低コスト化を可能にする、酵素基質の発色増強方法およびそれに用いる試薬を提供することを目的としている。 The present invention has been made in view of the above-described present situation, and provides a method for enhancing color development of an enzyme substrate and a reagent used therefor that enable high sensitivity, rapidity, and cost reduction in detection or identification of enzyme activity. The purpose is that.
本発明者は、上記課題を解決するため鋭意検討した結果、発色基質であるホスフェート化合物、β−グルコピラノシド化合物、Ｎ−アセチル−β−ヘキソサミニド化合物、ホスファチジルミオイノシトール化合物、ミオイノシトールホスフェート化合物およびコリンホスフェート化合物から選択される１種以上の化合物を含有する培地に卵黄を添加すると、培養後の検出対象菌コロニーの発色が増強されることを見出した。さらに、本発明者は、卵黄の添加による検出対象菌コロニーの発色増強が、蛍光基質である前記の化合物を含有する培地においても認められることを見出し、本発明を完成させるに至った。 As a result of diligent studies to solve the above-mentioned problems, the present inventor has developed a phosphate compound, β-glucopyranoside compound, N-acetyl-β-hexosaminide compound, phosphatidyl myo-inositol compound, myo-inositol phosphate compound and choline phosphate compound that are chromogenic substrates It has been found that when egg yolk is added to a medium containing one or more compounds selected from the above, the color development of the colonies to be detected after culture is enhanced. Furthermore, the present inventor has found that the color development enhancement of the colonies to be detected by addition of egg yolk is also observed in a medium containing the above-mentioned compound as a fluorescent substrate, and has completed the present invention.
すなわち、本発明は以下のような構成からなるものである。
（１）１種以上の基質に１種以上の酵素を作用させ発色団化合物および／または蛍光発色団化合物を遊離する発色法において、発色増強剤として卵黄または卵黄から調製された溶液を用いることを特徴とする発色増強方法。
（２）酵素がホスファターゼ、β−グルコシダーゼ、Ｎ−アセチル−β−ヘキソサミニダーゼおよびホスホリパーゼから選択されることを特徴とする上記（１）に記載の方法。
（３）卵黄が鳥類由来であることを特徴とする上記（１）または（２）に記載の方法。
（４）微生物の検出または識別における使用のための上記（１）から（３）のいずれか１項に記載の方法。
（５）上記（１）から（４）のいずれか１項に記載の方法に使用するための発色増強剤であって、卵黄または卵黄から調製された溶液を有効成分として含む発色増強剤。
（６）１種以上の基質に１種以上の酵素を作用させ発色団化合物および／または蛍光発色団化合物を遊離する発色法に用いる試薬であって、発色増強剤として卵黄または卵黄から調製された溶液を含むことを特徴とする発色試薬。
（７）酵素がホスファターゼ、β−グルコシダーゼ、Ｎ−アセチル−β−ヘキソサミニダーゼおよびホスホリパーゼから選択されることを特徴とする上記（６）に記載の試薬。
（８）卵黄が鳥類由来であることを特徴とする上記（６）または（７）に記載の試薬。
（９）微生物の検出または識別における使用のための上記（６）から（８）のいずれか１項に記載の試薬。
（１０）上記（９）に記載の試薬を用いてホスファターゼ活性、β−グルコシダーゼ活性、Ｎ−アセチル−β−ヘキソサミニダーゼ活性およびホスホリパーゼ活性から選択される酵素活性を有する微生物を検出または識別する方法。
That is, the present invention has the following configuration.
(1) In a color development method in which one or more enzymes are allowed to act on one or more substrates to release a chromophore compound and / or a fluorescent chromophore compound, the use of a solution prepared from egg yolk or egg yolk as a color enhancer A method for enhancing color development.
(2) The method according to (1) above, wherein the enzyme is selected from phosphatase, β-glucosidase, N-acetyl-β-hexosaminidase and phospholipase.
(3) The method according to (1) or (2) above, wherein the egg yolk is derived from birds.
(4) The method according to any one of (1) to (3) above, for use in detection or identification of microorganisms.
(5) A color enhancement agent for use in the method according to any one of (1) to (4) above, comprising a yolk or a solution prepared from egg yolk as an active ingredient.
(6) A reagent used in a color development method in which one or more enzymes are allowed to act on one or more substrates to release a chromophore compound and / or a fluorescent chromophore compound, prepared from egg yolk or egg yolk as a color enhancer A coloring reagent comprising a solution.
(7) The reagent according to (6) above, wherein the enzyme is selected from phosphatase, β-glucosidase, N-acetyl-β-hexosaminidase and phospholipase.
(8) The reagent according to (6) or (7) above, wherein the egg yolk is derived from birds.
(9) The reagent according to any one of (6) to (8), for use in detection or identification of microorganisms.
(10) Detect or identify a microorganism having an enzyme activity selected from phosphatase activity, β-glucosidase activity, N-acetyl-β-hexosaminidase activity and phospholipase activity using the reagent described in (9) above. Method.
本発明の発色増強方法および試薬を用いることにより、従来法に比べ測定に必要な酵素基質含量を低減し、試薬を低コスト化することが可能となる。また、従来法に比べ判定時間の短縮や高感度化も可能になる。 By using the color development enhancing method and reagent of the present invention, it is possible to reduce the enzyme substrate content necessary for the measurement and to reduce the cost of the reagent as compared with the conventional method. In addition, the determination time can be shortened and the sensitivity can be increased as compared with the conventional method.
本発明は、酵素の作用により基質が加水分解され、発色団化合物および／または蛍光発色団化合物を遊離する発色法において、発色増強剤として卵黄全体、または卵黄全体から調製された溶液を用いることを特徴とする。 In the color development method in which a substrate is hydrolyzed by the action of an enzyme to release a chromophore compound and / or a fluorescent chromophore compound, the present invention uses a whole egg yolk or a solution prepared from the whole egg yolk as a color enhancer. Features.
本発明で対象とする酵素は加水分解酵素であり、特にホスファターゼ、β−グルコシダーゼ、Ｎ−アセチル−β−ヘキソサミニダーゼおよびホスホリパーゼから選択することが好ましい。なお、Ｎ−アセチル−β−ヘキソサミニダーゼにはＮ−アセチル−β−ガラクトサミニダーゼおよびＮ−アセチル−β−グルコサミニダーゼが含まれ、ホスホリパーゼにはPI-PLCおよびホスファチジルコリン特異的ホスホリパーゼＣ（phosphatidylcholine-specific phospholipase C、以下、PC-PLCという。）が含まれる。また、本発明で対象とする酵素の由来生物は特に限定されない。ヒト、他の動物、植物、微生物などに由来する天然の酵素、遺伝子組み換え技術などにより人工的に合成された酵素などが対象酵素として挙げられる。 The enzyme to be used in the present invention is a hydrolase, and is particularly preferably selected from phosphatase, β-glucosidase, N-acetyl-β-hexosaminidase and phospholipase. N-acetyl-β-hexosaminidase includes N-acetyl-β-galactosaminidase and N-acetyl-β-glucosaminidase, and phospholipases include PI-PLC and phosphatidylcholine-specific phospholipase C (phosphatidylcholine- specific phospholipase C, hereinafter referred to as PC-PLC). Moreover, the origin organism of the enzyme made into object by this invention is not specifically limited. Examples of the target enzyme include natural enzymes derived from humans, other animals, plants, microorganisms, and the like, enzymes artificially synthesized by genetic recombination techniques, and the like.
本発明で用いる酵素基質には発色基質および蛍光基質が含まれ、本発明における発色には、発色団化合物の遊離による発色と蛍光発色団化合物の遊離による蛍光発色が含まれる。本発明で用いる発色基質は、酵素によって加水分解されうるように発色団化合物と結合しているホスフェート化合物、β−グルコピラノシド化合物、Ｎ−アセチル−β−ヘキソサミニド化合物、ホスファチジルミオイノシトール化合物、ミオイノシトールホスフェート化合物またはコリンホスフェート化合物であれば、特に限定されない。なお、ミオイノシトールホスフェート化合物には、ミオイノシトール−１−ホスフェート化合物などが含まれる。結合している発色団化合物の具体例としては、５−ブロモ−４−クロロ−３−インドキシル、５−ブロモ−３−インドキシル、５−ブロモ−６−クロロ−３−インドキシル、６−クロロ−３−インドキシル、５−ヨード−３−インドキシル、３−インドキシル、Ｎ−メチルインドキシルなどのインドキシル誘導体、２−クロロ−４−ニトロフェニル、２−ニトロフェニル、３−ニトロフェニル、４−ニトロフェニル、フェノールフタレインなどのフェニル誘導体が挙げられる。一方、本発明で用いる蛍光基質は、酵素によって加水分解されうるように蛍光発色団化合物と結合しているホスフェート化合物、β−グルコピラノシド化合物、Ｎ−アセチル−β−ヘキソサミニド化合物、ホスファチジルミオイノシトール化合物、ミオイノシトールホスフェート化合物またはコリンホスフェート化合物であれば、特に限定されない。結合している蛍光発色団化合物の具体例としては、７−ブロモ−Ｎ−（２−メトキシフェニル）−３−ヒドロキシ−２−ナフタレンカルボキシアミド（ナフトールAS−BI）、３−ヒドロキシ−２−ナフトアニリド（ナフトールAS）、６−ブロモ−２−ナフチル、１−ナフチル、２−ナフチルなどのナフチル誘導体、４−メチルウンベリフェリル、４−ブロモメチル−７−メトキシクマリン、４−ブロモメチル−７−アセトキシクマリン、７−ヒドロキシクマリン−６−イル、７−ヒドロキシクマリン、７−エトキシクマリン、７−メトキシクマリンなどのクマリン誘導体、８−ヒドロキシキノリンなどのキノリン誘導体、２−（５−クロロ−２−ヒドロキシフェニル）−６−クロロ−４（３Ｈ）−キナゾリノン（ELF-97）などのキナゾリノン誘導体が挙げられる。また、本発明では、基質を単独で用いることも可能であるが、検出目的に応じて適宜組み合わせて用いることも可能である。 The enzyme substrate used in the present invention includes a chromogenic substrate and a fluorescent substrate, and the color development in the present invention includes color development due to liberation of the chromophore compound and fluorescence color development due to the liberation of the fluorescent chromophore compound. The chromogenic substrate used in the present invention is a phosphate compound, β-glucopyranoside compound, N-acetyl-β-hexosaminide compound, phosphatidyl myo-inositol compound, myo-inositol phosphate compound bonded to a chromophore compound so that it can be hydrolyzed by an enzyme. Or if it is a choline phosphate compound, it will not specifically limit. The myo-inositol phosphate compound includes a myo-inositol-1-phosphate compound. Specific examples of bonded chromophore compounds include 5-bromo-4-chloro-3-indoxyl, 5-bromo-3-indoxyl, 5-bromo-6-chloro-3-indoxyl, 6- Indoxyl derivatives such as chloro-3-indoxyl, 5-iodo-3-indoxyl, 3-indoxyl, N-methylindoxyl, 2-chloro-4-nitrophenyl, 2-nitrophenyl, 3-nitrophenyl , Phenyl derivatives such as 4-nitrophenyl and phenolphthalein. On the other hand, the fluorescent substrate used in the present invention is a phosphate compound, β-glucopyranoside compound, N-acetyl-β-hexosaminide compound, phosphatidyl myo-inositol compound, myo, which is bonded to a fluorescent chromophore compound so that it can be hydrolyzed by an enzyme. Any inositol phosphate compound or choline phosphate compound may be used. Specific examples of the bound fluorescent chromophore compound include 7-bromo-N- (2-methoxyphenyl) -3-hydroxy-2-naphthalenecarboxamide (naphthol AS-BI), 3-hydroxy-2-naphthanilide (Naphthol AS), 6-bromo-2-naphthyl, 1-naphthyl, naphthyl derivatives such as 2-naphthyl, 4-methylumbelliferyl, 4-bromomethyl-7-methoxycoumarin, 4-bromomethyl-7-acetoxycoumarin, Coumarin derivatives such as 7-hydroxycoumarin-6-yl, 7-hydroxycoumarin, 7-ethoxycoumarin, 7-methoxycoumarin, quinoline derivatives such as 8-hydroxyquinoline, 2- (5-chloro-2-hydroxyphenyl)- Quinazolinone induction such as 6-chloro-4 (3H) -quinazolinone (ELF-97) And the like. In the present invention, the substrate can be used alone, but can be used in appropriate combination according to the purpose of detection.
本発明で用いる卵黄は、鶏、合鴨、アヒル、七面鳥、ウズラ、ダチョウなどの鳥類由来のものであれば、特に限定されない。卵黄は、周知の方法により取り出す。即ち、卵の外殻を消毒し、予め滅菌したピンセットや卵黄取り出し器具を用いて卵黄を無菌的に取り出す。取り出した卵黄は、必要に応じて無菌フィルターや、電子線照射などによる滅菌操作を適宜行った後、本発明の発色増強剤として用いる。 The egg yolk used in the present invention is not particularly limited as long as it is derived from birds such as chicken, duck, duck, turkey, quail, and ostrich. Egg yolk is removed by a well-known method. That is, the outer shell of the egg is sterilized and the egg yolk is aseptically removed using a pre-sterilized tweezers or egg yolk extracting device. The extracted egg yolk is used as a color enhancer of the present invention after appropriately performing a sterilization operation by a sterile filter or electron beam irradiation as necessary.
本発明では、卵黄をそのまま発色増強剤として用いることができるが、卵黄全体から調製された溶液もまた発色増強剤として用いることができる。卵黄全体から調製された溶液とは、卵黄全体を水、塩化ナトリウム溶液または緩衝液で希釈した溶液のことである。希釈に用いる塩化ナトリウム溶液は、目的とする酵素の検出に適した種々の濃度とすることができる。また、緩衝液は、リン酸ナトリウム緩衝液、リン酸カリウム緩衝液など、目的とする酵素の検出に適した種々の緩衝液を種々の濃度で適用することができる。発色反応液中の卵黄量は実施例に示した様に、５０％卵黄液として０．１〜２００ｍＬ／Ｌとすることが好ましく、３〜１００ｍＬ／Ｌとすることがさらに好ましい。 In the present invention, egg yolk can be used as a color enhancement agent as it is, but a solution prepared from the whole egg yolk can also be used as a color enhancement agent. A solution prepared from whole egg yolk is a solution obtained by diluting whole egg yolk with water, sodium chloride solution or buffer solution. The sodium chloride solution used for dilution can have various concentrations suitable for detection of the target enzyme. As the buffer solution, various buffer solutions suitable for detection of the target enzyme, such as sodium phosphate buffer solution and potassium phosphate buffer solution, can be applied at various concentrations. As shown in the Examples, the amount of egg yolk in the coloring reaction solution is preferably 0.1 to 200 mL / L, more preferably 3 to 100 mL / L as a 50% egg yolk solution.
本発明で検出対象となる標的微生物は、加水分解酵素、特にホスファターゼ、β−グルコシダーゼ、Ｎ−アセチル−β−ヘキソサミニダーゼ、ホスホリパーゼから選択される酵素を有するものであれば限定されない。ホスファターゼを有する微生物の具体例としては、黄色ブドウ球菌およびクロストリジウム・パーフリンゲンス（Clostridium perfringens、以下、ウェルシュ菌という。）など、β−グルコシダーゼを有する微生物の具体例としては、エンテロコッカス属細菌、クレブシエラ（Klebsiella）属細菌およびエンテロバクター（Enterobacter）属細菌など、Ｎ−アセチル−β−ヘキソサミニダーゼを有する微生物の具体例としては、カンジダ・アルビカンスなど、PI-PLCを有する微生物の具体例としては、リステリア・モノサイトジェネス、リステリア・イヴァノヴィなどのリステリア属病原性細菌、黄色ブドウ球菌、ウェルシュ菌、クロストリジウム・ノビイ（Clostridium novyi）、レジオネラ（Legionella）属細菌、ヘリコバクター・ピロリ（Helicobacter pylori）、トリパノソーマ・ブルセイ（Trypanosoma brucei）、アスペルギルス・フミガタス（Aspergillus fumigatus）、バチルス・セレウス（Bacillus cereus）、バチルス・チューリンゲンシス（Bacillus thuringiensis）およびバチルス・ミコイデス（Bacillus mycoides）など、PC-PLCを有する微生物の具体例としては、リステリア・モノサイトジェネス、黄色ブドウ球菌、ウェルシュ菌、クロストリジウム・ノビイ、バチルス・セレウス、バチルス・チューリンゲンシス、バチルス・アンスラシス（Bacillus anthracis）、シュードモナス・アエルギノーサ（Pseudomonas aeruginosa）、ヘリコバクター・ピロリ、レジオネラ・ニューモフィラ（Legionella pneumophila）、カンジダ・アルビカンスおよびアスペルギルス・フミガタスなどが挙げられる。本発明では、酵素を単独で検出することも可能であるが、１つの反応系内で複数の酵素を同時に検出または識別することも可能である。 The target microorganism to be detected in the present invention is not limited as long as it has an enzyme selected from a hydrolase, particularly phosphatase, β-glucosidase, N-acetyl-β-hexosaminidase, and phospholipase. As specific examples of microorganisms having phosphatase, specific examples of microorganisms having β-glucosidase such as Staphylococcus aureus and Clostridium perfringens (hereinafter referred to as Clostridium perfringens) include Enterococcus bacteria, Klebsiella ( Specific examples of microorganisms having N-acetyl-β-hexosaminidase such as Klebsiella bacteria and Enterobacter bacteria include specific examples of microorganisms having PI-PLC such as Candida albicans, Listeria pathogenic bacteria such as Listeria monocytogenes, Listeria Ivanovi, Staphylococcus aureus, Clostridium novyi, Clostridium novyi, Legionella, Helicobacter pylori, Tripa Specific microorganisms with PC-PLC such as Trypanosoma brucei, Aspergillus fumigatus, Bacillus cereus, Bacillus thuringiensis and Bacillus mycoides Examples include Listeria monocytogenes, Staphylococcus aureus, Clostridium perfringens, Clostridium novii, Bacillus cereus, Bacillus thuringiensis, Bacillus anthracis, Pseudomonas aeruginosa, Helicobacter pylori, Examples include Legionella pneumophila, Candida albicans, and Aspergillus fumigatus. In the present invention, it is possible to detect an enzyme alone, but it is also possible to simultaneously detect or identify a plurality of enzymes in one reaction system.
本発明の発色増強剤を含む試薬は、培地である場合と培地以外である場合を想定することができる。培地である場合には、標的微生物の培養に適した種々の基礎培地、酵素基質、選択剤、その他の成分が含有されている培地に本発明の発色増強剤を添加して用いる。本発明の発色増強剤は発色増強作用を有する他の物質と組み合わせて使用することも可能である。通常の微生物培養に使用されうる成分が共存していても、本発明の作用が影響されることはない。 The reagent containing the coloring enhancer of the present invention can be assumed to be a medium or a medium other than a medium. In the case of a medium, the coloring enhancer of the present invention is added to a medium containing various basal media, enzyme substrates, selective agents, and other components suitable for culturing the target microorganism. The color enhancement agent of the present invention can also be used in combination with other substances having a color enhancement effect. Even if components that can be used for normal microorganism culture coexist, the action of the present invention is not affected.
本発明の発色増強剤を含む試薬が培地である場合は、標的微生物の存在が疑われる試料を接種して培養後、発色を調べることで対象微生物を検出することができる。一方、発色増強剤を含む試薬が培地以外である場合は、標的酵素の存在が疑われる試料を前処理せずに試薬に接種し、適温で反応させた後、発色を調べることで対象酵素を検出することができる。また、試薬への接種の前に濃縮などの前処理操作を適宜行うことも可能である。 When the reagent containing the coloring enhancer of the present invention is a culture medium, the target microorganism can be detected by inoculating a sample suspected of the presence of the target microorganism and examining the color development after culturing. On the other hand, if the reagent containing the coloring enhancer is other than the medium, inoculate the sample without pretreatment with a sample suspected of the presence of the target enzyme, react at a suitable temperature, and then examine the color development to determine the target enzyme. Can be detected. It is also possible to appropriately perform a pretreatment operation such as concentration before inoculating the reagent.
本発明の発色増強剤を含む試薬が培地である場合、培地の形態は特に限定されない。液体、半流動、固形、シート状などのいずれの形態もとりうるが、検出のしやすさなどの観点から、固形培地が好ましく、より好ましくは平板固形培地の形態である。固形培地の固化剤としては、寒天、アガロースなど通常使用されているものが挙げられる。 When the reagent containing the color enhancement agent of the present invention is a medium, the form of the medium is not particularly limited. Any form such as liquid, semi-fluid, solid, and sheet can be used. From the viewpoint of easy detection, a solid medium is preferable, and a flat solid medium is more preferable. Examples of the solidifying agent for the solid medium include commonly used ones such as agar and agarose.
本発明に使用される試料は、酵素を含む可能性のある試料であれば特に限定されない。ヒト、他の動物の生体由来、食品、環境由来の検体、それらの培養液などが試料として挙げられる。これらの試料は、抽出、濃縮などの前処理を行っても良い。 The sample used for this invention will not be specifically limited if it is a sample which may contain an enzyme. Examples of samples include humans, other animal living organisms, foods, environmental specimens, culture solutions thereof, and the like. These samples may be subjected to pretreatment such as extraction and concentration.
以下に本発明の実施例を示すが、本発明はこれら実施例により限定されるものではない。
なお、各実施例で使用した供試菌株一覧を以下の表１に示す。
Examples of the present invention are shown below, but the present invention is not limited to these examples.
The list of test strains used in each example is shown in Table 1 below.
（Ａ．５−ブロモ−３−インドキシルホスフェートを使用した黄色ブドウ球菌のホスファターゼ活性検出）
酵素基質として５−ブロモ−３−インドキシルホスフェートを使用した黄色ブドウ球菌のホスファターゼ活性検出において、卵黄液添加による発色増強作用を調べた。
(A. Detection of phosphatase activity of Staphylococcus aureus using 5-bromo-3-indoxyl phosphate)
In the detection of phosphatase activity of Staphylococcus aureus using 5-bromo-3-indoxyl phosphate as an enzyme substrate, the coloring enhancement effect by adding egg yolk liquid was examined.
（１）培地の準備
以下に示した培地成分を秤量し、精製水に懸濁後、ｐHを７．０±０．１に調整し、１２１℃で１５分間高圧滅菌した。
(1) Preparation of medium The following medium components were weighed and suspended in purified water, pH was adjusted to 7.0 ± 0.1, and autoclaved at 121 ° C. for 15 minutes.
（２）卵黄液添加量
５０％卵黄液の最終添加量を５ｍＬ／Ｌとした。
(2) Egg yolk addition amount The final addition amount of 50% egg yolk was 5 mL / L.
（３）酵素基質添加濃度
卵黄液添加培地の酵素基質最終濃度を８０ｍｇ／Ｌとし、対照として卵黄液を添加せず、酵素基質最終濃度を８０ｍｇ／Ｌ、１５０ｍｇ／Ｌとした培地も調製した。
(3) Enzyme Substrate Addition Concentration The enzyme substrate final concentration of the egg yolk liquid addition medium was 80 mg / L, and as a control, the egg yolk liquid was not added, and the enzyme substrate final concentrations were 80 mg / L and 150 mg / L.
（４）酵素基質および卵黄液の添加
（１）で高圧滅菌後、５０℃に冷却した後、濾過滅菌した酵素基質液および卵黄液を添加して撹拌した。その後、培地を２０ｍＬずつシャーレに分注して固化した。
(4) Addition of enzyme substrate and egg yolk liquid After autoclaving in (1), after cooling to 50 ° C., the enzyme substrate liquid and egg yolk liquid sterilized by filtration were added and stirred. Thereafter, 20 mL of the medium was dispensed into a petri dish and solidified.
（５）菌の接種と培養
前培養した菌株を滅菌生理食塩水に懸濁し、ＭｃＦａｒｌａｎｄ Ｎｏ．１の菌液を作製した。その後、前記菌液を画線し、３５℃で好気培養を行い、発色強度を調べた。
(5) Bacterial inoculation and culture The precultured strain was suspended in sterile physiological saline, and McFarland No. 1 bacterial solution was prepared. Thereafter, the bacterial solution was streaked and subjected to aerobic culture at 35 ° C. to examine the color intensity.
（６）結果
酵素基質添加量８０ｍｇ／ＬにおけるＥＫＮ３０８８株の１４、２０、２４時間培養後の発色状況を卵黄液添加の有無で比較した写真を図１〜３に、また、全菌株の１２、１４、１６、１８、２０、２２、２４時間後の５−ブロモ−３−インドキシルホスフェートの分解による青色発色強度を表３に示す。発色強度は目視により、培養時間ごとに一定の任意スケールで表した。
(6) Results Photographs comparing the color development status of the EKN3088 strain after culturing for 14, 20, and 24 hours at an enzyme substrate addition amount of 80 mg / L with or without addition of egg yolk solution are shown in FIGS. Table 3 shows the blue color intensity due to decomposition of 5-bromo-3-indoxyl phosphate after 14, 16, 18, 20, 22, and 24 hours. The color intensity was visually represented on a constant arbitrary scale for each incubation time.
図１〜３は、それぞれシャーレ２枚のうち左が卵黄液添加なし、右が卵黄液添加ありである。１４、２０、２４時間培養のいずれにおいても、卵黄液添加ありで明らかな発色増強が認められていた。また、表３に示した様に、検討したすべての菌株において酵素基質添加量８０ｍｇ／Ｌ、１４時間以上の培養で卵黄液添加による発色増強作用が認められ、卵黄液添加ありの発色強度は、卵黄液添加なし酵素基質添加量１５０ｍｇ／Ｌにおける発色と比べて同等以上であった。このことから、発色増強剤としての卵黄液添加により、培地に添加する酵素基質量を５０％以下に低減できることが示唆された。さらに、酵素基質添加量８０ｍｇ／Ｌにおいて発色が認められるまでに要した培養時間は、卵黄液添加ありの方が卵黄液添加なしに比べて２〜４時間短かった。発色が認められるまでの時間は、卵黄液添加なし酵素基質添加量１５０ｍｇ／Ｌに比べても短時間であった。このことから、発色増強剤としての卵黄液添加により、判定までの培養時間を短縮できることが示唆された。 1 to 3, in each of the two petri dishes, the left is without addition of egg yolk liquid, and the right is with addition of egg yolk liquid. In any of the cultures for 14, 20, and 24 hours, a clear color enhancement was observed with the addition of egg yolk. In addition, as shown in Table 3, in all the strains examined, an enzyme substrate addition amount of 80 mg / L, a color enhancement effect due to the addition of egg yolk was observed in culture for 14 hours or more, and the color intensity with the addition of egg yolk was Compared with the color development at the enzyme substrate addition amount of 150 mg / L without addition of egg yolk liquid, it was equivalent or better. From this, it was suggested that the enzyme group mass added to a culture medium can be reduced to 50% or less by adding egg yolk liquid as a color enhancement agent. Furthermore, the culture time required for color development to be observed at the enzyme substrate addition amount of 80 mg / L was 2 to 4 hours shorter when the egg yolk solution was added than when the egg yolk solution was not added. The time until color development was recognized was a short time as compared with the enzyme substrate addition amount of 150 mg / L without addition of egg yolk. From this, it was suggested that the incubation time until determination can be shortened by adding egg yolk liquid as a color enhancer.
（Ｂ．５−ブロモ−６−クロロ−３−インドキシルホスフェートを使用した黄色ブドウ球菌のホスファターゼ活性検出）
酵素基質として５−ブロモ−６−クロロ−３−インドキシルホスフェートを使用した黄色ブドウ球菌のホスファターゼ活性検出において、卵黄液添加による発色増強作用を調べた。
以下に示す実施例１．Ｂから実施例７における酵素基質および卵黄液の添加、菌の接種操作は、実施例１．Ａと同様に行った。
(B. Detection of phosphatase activity of Staphylococcus aureus using 5-bromo-6-chloro-3-indoxyl phosphate)
In the detection of phosphatase activity of Staphylococcus aureus using 5-bromo-6-chloro-3-indoxyl phosphate as an enzyme substrate, the color enhancement effect by adding egg yolk fluid was examined.
Example 1 shown below. The addition of enzyme substrate and egg yolk liquid in Example 7 to B and the inoculation procedure of the bacteria were carried out in Example 1. Same as A.
（１）培地の準備
実施例１．Ａと同様とした。
(1) Preparation of culture medium Example 1. Same as A.
（２）卵黄液添加量
実施例１．Ａと同様とした。
(2) Amount of egg yolk liquid added Example 1. Same as A.
（３）酵素基質添加濃度
実施例１．Ａと同様とした。
(3) Enzyme substrate addition concentration Example 1. Same as A.
（４）結果
全菌株の１２、１４、１６、１８、２０、２２、２４時間培養後の５−ブロモ−６−クロロ−３−インドキシルホスフェートの分解による赤色発色強度を表４に示す。発色強度は目視により、培養時間ごとに一定の任意スケールで表した。
(4) Results Table 4 shows the red color intensity due to the decomposition of 5-bromo-6-chloro-3-indoxyl phosphate after culturing for 12, 14, 16, 18, 20, 22, and 24 hours. The color intensity was visually represented on a constant arbitrary scale for each incubation time.
表４に示す様に、検討したすべての菌株において酵素基質添加量８０ｍｇ／Ｌ、１６時間以上の培養で卵黄液添加による発色増強作用が認められ、卵黄液添加ありの発色強度は卵黄液添加なし酵素基質添加量１５０ｍｇ／Ｌにおける発色と比べて同等以上であった。このことから、発色増強剤としての卵黄液添加により、培地に添加する酵素基質量を５０％以下に低減できることが示唆された。また、酵素基質添加量８０ｍｇ／Ｌにおいて発色が認められるまでに要した培養時間は、卵黄液添加ありの方が４〜８時間短かった。発色が認められるまでの時間は、卵黄液添加なし酵素基質添加量１５０ｍｇ／Ｌに比べても短時間であった。このことから、発色増強剤としての卵黄液添加により、判定までの培養時間を短縮できることが示唆された。 As shown in Table 4, in all the strains examined, an enzyme substrate addition amount of 80 mg / L, a color enhancement effect due to addition of egg yolk solution was observed in culture for 16 hours or more, and the color intensity with addition of egg yolk solution was not added with egg yolk solution Compared with the color development at the enzyme substrate addition amount of 150 mg / L, it was equivalent or better. From this, it was suggested that the enzyme group mass added to a culture medium can be reduced to 50% or less by adding egg yolk liquid as a color enhancement agent. In addition, the incubation time required for color development to be observed at the enzyme substrate addition amount of 80 mg / L was 4 to 8 hours shorter when the egg yolk solution was added. The time until color development was recognized was a short time as compared with the enzyme substrate addition amount of 150 mg / L without addition of egg yolk. From this, it was suggested that the incubation time until determination can be shortened by adding egg yolk liquid as a color enhancer.
（５−ブロモ−６−クロロ−３−インドキシル−β−Ｄ−グルコピラノシドを使用したエンテロコッカス属細菌のβ−グルコシダーゼ活性検出）
酵素基質として５−ブロモ−６−クロロ−３−インドキシル−β−Ｄ−グルコピラノシドを使用したエンテロコッカス・フェカリス（Enterococcus faecalis）、エンテロコッカス・フェシウム(Enterococcus faecium)のβ−グルコシダーゼ活性検出において、卵黄液添加による発色増強作用を調べた。
(Detection of β-glucosidase activity of Enterococcus bacteria using 5-bromo-6-chloro-3-indoxyl-β-D-glucopyranoside)
Addition of egg yolk solution in detecting β-glucosidase activity of Enterococcus faecalis and Enterococcus faecium using 5-bromo-6-chloro-3-indoxyl-β-D-glucopyranoside as an enzyme substrate The color development enhancing effect of was investigated.
（１）培地の準備
実施例１．Ａと同様とした。
(1) Preparation of culture medium Example 1. Same as A.
（２）卵黄液添加量
実施例１．Ａと同様とした。
(2) Amount of egg yolk liquid added Example 1. Same as A.
（３）酵素基質添加濃度
卵黄液添加培地の酵素基質最終濃度を５０ｍｇ／Ｌとし、対照として卵黄液を添加せず、酵素基質最終濃度を５０ｍｇ／Ｌとした培地も調製した。
(3) Enzyme substrate addition concentration A medium was prepared in which the final enzyme substrate concentration of the egg yolk liquid addition medium was 50 mg / L, the egg yolk liquid was not added, and the final enzyme substrate concentration was 50 mg / L as a control.
（４）結果
全菌株の２４、４０時間培養後の５−ブロモ−６−クロロ−３−インドキシル−β−Ｄ−グルコピラノシドの分解による赤色発色強度を表５に示す。発色強度は目視により、培養時間ごとに一定の任意スケールで表した。
(4) Results Table 5 shows the red color intensity by decomposition of 5-bromo-6-chloro-3-indoxyl-β-D-glucopyranoside after culturing for 24 and 40 hours for all strains. The color intensity was visually represented on a constant arbitrary scale for each incubation time.
表５に示す様に、β−グルコシダーゼ活性検出においても卵黄液添加による発色増強作用が認められ、また、発色までに要する培養時間が短縮されることが示唆された。 As shown in Table 5, in the detection of β-glucosidase activity, a color enhancement effect by adding egg yolk fluid was recognized, and it was suggested that the culture time required for color development was shortened.
（５−ブロモ−４−クロロ−３−インドキシル−Ｎ−アセチル−β−Ｄ−ガラクトサミニドを使用したカンジダ・アルビカンスのＮ−アセチル−β−ガラクトサミニダーゼ活性検出）
酵素基質として５−ブロモ−４−クロロ−３−インドキシル−Ｎ−アセチル−β−Ｄ−ガラクトサミニドを使用したカンジダ・アルビカンスのＮ−アセチル−β−ガラクトサミニダーゼ活性検出において、卵黄液添加による発色増強作用を調べた。
(Detection of N-acetyl-β-galactosaminidase activity of Candida albicans using 5-bromo-4-chloro-3-indoxyl-N-acetyl-β-D-galactosaminide)
In Candida albicans N-acetyl-β-galactosaminidase activity detection using 5-bromo-4-chloro-3-indoxyl-N-acetyl-β-D-galactosaminide as an enzyme substrate, color development by addition of egg yolk The potentiating effect was examined.
（１）培地の準備
以下に示した培地成分を秤量し、精製水に懸濁後、ｐHを７．０±０．１に調整し、１２１℃で１５分間高圧滅菌した。
(1) Preparation of medium The following medium components were weighed and suspended in purified water, pH was adjusted to 7.0 ± 0.1, and autoclaved at 121 ° C. for 15 minutes.
（２）卵黄液添加量
５０％卵黄液の最終添加量を５、５０ｍＬ／Ｌとした。
(2) Egg yolk addition amount The final addition amount of 50% egg yolk was set to 5, 50 mL / L.
（３）酵素基質添加濃度
卵黄液添加培地の酵素基質最終濃度を１００ｍｇ／Ｌとし、対照として卵黄液を添加せず、酵素基質最終濃度を１００ｍｇ／Ｌとした培地も調製した。
(3) Enzyme substrate addition concentration A culture medium was prepared in which the final enzyme substrate concentration in the egg yolk liquid addition medium was 100 mg / L, and no egg yolk liquid was added as a control, and the final enzyme substrate concentration was 100 mg / L.
（４）結果
各菌株の１５、２０、２２、４５時間培養後の５−ブロモ−４−クロロ−３−インドキシル−Ｎ−アセチル−β−Ｄ−ガラクトサミニドの分解による青緑色発色強度を表７に示す。発色強度は目視により、培養時間ごとに一定の任意スケールで表した。
(4) Results Table 7 shows the blue-green color intensity by the decomposition of 5-bromo-4-chloro-3-indoxyl-N-acetyl-β-D-galactosaminide after culturing for 15, 20, 22, and 45 hours of each strain. Shown in The color intensity was visually represented on a constant arbitrary scale for each incubation time.
表７に示す様に、Ｎ−アセチル−β−ガラクトサミニダーゼ活性検出においても卵黄液添加による発色増強作用が認められ、また、発色までに要する培養時間が短縮されることが示唆された。 As shown in Table 7, in the detection of N-acetyl-β-galactosaminidase activity, a color enhancement effect by adding egg yolk fluid was observed, and it was suggested that the culture time required until color development was shortened.
（５−ブロモ−４−クロロ−３−インドキシル−Ｎ−アセチル−β−Ｄ−グルコサミニドを使用したカンジダ・アルビカンスのＮ−アセチル−β−グルコサミニダーゼ活性検出）
酵素基質として５−ブロモ−４−クロロ−３−インドキシル−Ｎ−アセチル−β−Ｄ−グルコサミニドを使用したカンジダ・アルビカンスのＮ−アセチル−β−グルコサミニダーゼ活性検出において、卵黄液添加による発色増強作用を調べた。
(Detection of N-acetyl-β-glucosaminidase activity of Candida albicans using 5-bromo-4-chloro-3-indoxyl-N-acetyl-β-D-glucosaminide)
In Candida albicans N-acetyl-β-glucosaminidase activity detection using 5-bromo-4-chloro-3-indoxyl-N-acetyl-β-D-glucosaminide as an enzyme substrate, color development enhancing effect by adding egg yolk solution I investigated.
（１）培地の準備
実施例３と同様とした。
(1) Preparation of medium The same as in Example 3.
（２）卵黄液添加量
実施例３と同様とした。
(2) Egg yolk liquid addition amount It was the same as that of Example 3.
（３）酵素基質添加濃度
実施例３と同様とした。
(3) Enzyme substrate addition concentration The same as in Example 3.
（４）結果
各菌株の１５、２０、２２、４５時間培養後の５−ブロモ−４−クロロ−３−インドキシル−Ｎ−アセチル−β−Ｄ−グルコサミニドの分解による青緑色発色強度を表８に示す。発色強度は目視により、培養時間ごとに一定の任意スケールで表した。
(4) Results Table 8 shows the blue-green color intensity by the decomposition of 5-bromo-4-chloro-3-indoxyl-N-acetyl-β-D-glucosaminide after culturing for 15, 20, 22, and 45 hours of each strain. Shown in The color intensity was visually represented on a constant arbitrary scale for each incubation time.
表８に示す様に、Ｎ−アセチル−β−グルコサミニダーゼ活性検出においても卵黄液添加による発色増強作用が認められ、また、発色までに要する培養時間が短縮されることが示唆された。 As shown in Table 8, in the detection of N-acetyl-β-glucosaminidase activity, a color enhancement effect by adding egg yolk fluid was observed, and it was suggested that the culture time required until color development was shortened.
（５−ブロモ−４−クロロ−３−インドキシル−ミオイノシトール−１−ホスフェートを使用したリステリア属細菌のPI-PLC活性検出）
酵素基質として５−ブロモ−４−クロロ−３−インドキシル−ミオイノシトール−１−ホスフェート，アンモニウム塩を使用したリステリア・モノサイトジェネス、リステリア・イヴァノヴィのPI-PLC活性検出において、卵黄液添加による発色増強作用を調べた。
(Detection of PI-PLC activity of Listeria bacteria using 5-bromo-4-chloro-3-indoxyl-myoinositol-1-phosphate)
Color detection by addition of egg yolk fluid in detection of PI-PLC activity of Listeria monocytogenes and Listeria Ivanovi using 5-bromo-4-chloro-3-indoxyl-myoinositol-1-phosphate, ammonium salt as enzyme substrate The potentiating effect was examined.
（１）培地の準備
以下に示した培地成分を秤量し、精製水に懸濁後、ｐHを７．０±０．１に調整し、１２１℃で１５分間高圧滅菌した。
(1) Preparation of medium The following medium components were weighed and suspended in purified water, pH was adjusted to 7.0 ± 0.1, and autoclaved at 121 ° C. for 15 minutes.
（２）卵黄液添加量
実施例３と同様とした。
(2) Egg yolk liquid addition amount It was the same as that of Example 3.
（３）酵素基質添加濃度
実施例３と同様とした。
(3) Enzyme substrate addition concentration The same as in Example 3.
（４）結果
各菌株の１２、１６、２０、３６、４５時間培養後の５−ブロモ−４−クロロ−３−インドキシル−ミオイノシトール−１−ホスフェートの分解による青緑色発色強度を表１０に示す。発色強度は目視により、培養時間ごとに一定の任意スケールで表した。
(4) Results Table 10 shows the blue-green color intensity by decomposition of 5-bromo-4-chloro-3-indoxyl-myoinositol-1-phosphate after culturing for 12, 16, 20, 36, and 45 hours of each strain. Show. The color intensity was visually represented on a constant arbitrary scale for each incubation time.
表１０に示す様に、PI-PLC活性検出においても卵黄液添加による発色増強作用が認められ、また、発色までに要する培養時間が短縮されることが示唆された。 As shown in Table 10, in PI-PLC activity detection, a color enhancement effect by adding egg yolk fluid was also observed, and it was suggested that the culture time required for color development was shortened.
（卵黄由来種の検討）
酵素基質として５−ブロモ−３−インドキシルホスフェートを使用した黄色ブドウ球菌のホスファターゼ活性検出において、鶏、合鴨、七面鳥、ウズラ、ダチョウ由来の卵黄液添加による発色増強作用を調べた。
(Examination of egg yolk-derived species)
In detecting the phosphatase activity of Staphylococcus aureus using 5-bromo-3-indoxyl phosphate as an enzyme substrate, the color-enhancing effect by adding egg yolk liquid derived from chicken, duck, turkey, quail and ostrich was examined.
（１）培地の準備
実施例１．Ａと同様とした。
(1) Preparation of culture medium Example 1. Same as A.
（２）卵黄液添加量
実施例１．Ａと同様とした。
(2) Amount of egg yolk liquid added Example 1. Same as A.
（３）酵素基質添加濃度
卵黄液添加培地の酵素基質最終濃度を８０ｍｇ／Ｌとし、対照として卵黄液を添加せず、酵素基質最終濃度を８０ｍｇ／Ｌとした培地も調製した。
(3) Enzyme Substrate Addition Concentration The enzyme substrate final concentration of the egg yolk liquid addition medium was 80 mg / L, and as a control, a medium was prepared with the enzyme substrate final concentration of 80 mg / L without adding egg yolk liquid.
（４）結果
２４時間培養後の５−ブロモ−３−インドキシルホスフェートの分解による青色発色強度を表１１に示す。発色強度は目視により、任意スケールで表した。
(4) Results Table 11 shows the blue color intensity due to the decomposition of 5-bromo-3-indoxyl phosphate after 24 hours of culture. The color development intensity was visually represented on an arbitrary scale.
表１１に示す様に、検討したすべての種の卵黄液添加培地において発色増強作用が認められた。このことから、鳥類由来の卵黄であれば、発色増強作用を示すことが示唆された。 As shown in Table 11, a color development enhancing effect was observed in all the yolk liquid-added media examined. From this, it was suggested that the egg yolk derived from birds exhibits a color development enhancing action.
（卵黄添加量の検討）
酵素基質として５−ブロモ−３−インドキシルホスフェートを使用した黄色ブドウ球菌のホスファターゼ活性検出において、発色増強作用を示す卵黄液添加量を調べた。
(Examination of egg yolk addition amount)
In the detection of phosphatase activity of Staphylococcus aureus using 5-bromo-3-indoxyl phosphate as an enzyme substrate, the amount of egg yolk liquid added exhibiting a color development enhancing effect was examined.
（１）培地の準備
実施例１．Ａと同様とした。
(1) Preparation of culture medium Example 1. Same as A.
（２）卵黄液添加量
５０％卵黄液の最終添加量を０．１、１、２、３、１００、２００、５００ｍＬ／Ｌとした。
(2) Egg yolk addition amount The final addition amount of 50% egg yolk was 0.1, 1, 2, 3, 100, 200, 500 mL / L.
（３）酵素基質添加濃度
卵黄液添加培地の酵素基質最終濃度を８０ｍｇ／Ｌとし、対照として卵黄液を添加せず、酵素基質最終濃度を８０ｍｇ／Ｌとした培地も調製した。
(3) Enzyme Substrate Addition Concentration The enzyme substrate final concentration of the egg yolk liquid addition medium was 80 mg / L, and as a control, a medium was prepared with the enzyme substrate final concentration of 80 mg / L without adding egg yolk liquid.
（４）結果
各添加量の卵黄液について、１４、１８、２４時間培養後の５−ブロモ−３−インドキシルホスフェートの分解による青色発色強度を表１２に示す。発色強度は目視により、培養時間ごとに一定の任意スケールで表した。
(4) Results Table 12 shows the blue color intensity due to the decomposition of 5-bromo-3-indoxyl phosphate after incubation for 14, 18, and 24 hours for each added amount of egg yolk. The color intensity was visually represented on a constant arbitrary scale for each incubation time.
表１２に示す様に、卵黄液０．１〜２００ｍＬ／Ｌ添加において、発色増強作用が認められ、いずれの菌株においても、３〜１００ｍＬ／Ｌ添加の場合に特に強い発色増強作用が認められた。 As shown in Table 12, a color enhancement effect was observed when adding 0.1 to 200 mL / L of egg yolk liquid, and a particularly strong color enhancement effect was observed when adding 3 to 100 mL / L in any strain. .
（液体培地による検討）
酵素基質として５−ブロモ−３−インドキシルホスフェートを使用した黄色ブドウ球菌のホスファターゼ活性検出において、液体培地における発色増強作用を調べた。
(Examination using liquid medium)
In detecting the phosphatase activity of Staphylococcus aureus using 5-bromo-3-indoxyl phosphate as an enzyme substrate, the color development enhancing action in a liquid medium was examined.
（１）培地の準備
以下に示した培地成分を秤量し、精製水に懸濁後、ｐHを７．０±０．１に調整し、１２１℃で１５分間高圧滅菌した。
(1) Preparation of medium The following medium components were weighed and suspended in purified water, pH was adjusted to 7.0 ± 0.1, and autoclaved at 121 ° C. for 15 minutes.
（２）卵黄液添加量
実施例１．Ａと同様とした。
(2) Amount of egg yolk liquid added Example 1. Same as A.
（３）酵素基質添加濃度
卵黄液添加培地の酵素基質最終濃度を０、８０、１６０、２４０、３２０、４００ｍｇ／Ｌとし、各酵素基質濃度について対照として卵黄液を添加しない培地も調製した。
(3) Enzyme substrate addition concentration The enzyme substrate final concentration of the egg yolk liquid addition medium was set to 0, 80, 160, 240, 320, and 400 mg / L, and a medium in which no egg yolk liquid was added as a control for each enzyme substrate concentration was also prepared.
（４）酵素基質および卵黄液の添加
（１）で高圧滅菌後、室温まで冷却した後、滅菌小試験管に５ｍＬずつ分注した。その後、濾過滅菌した酵素基質液および卵黄液を添加して撹拌した。
(4) Addition of enzyme substrate and egg yolk liquid After autoclaving in (1), after cooling to room temperature, 5 mL each was dispensed into a sterile small test tube. Thereafter, the enzyme substrate solution and egg yolk solution sterilized by filtration were added and stirred.
（５）菌の接種と培養
前培養した菌株を滅菌生理食塩水に懸濁し、ＭｃＦａｒｌａｎｄ Ｎｏ．１の菌液を作製した。その後、前記菌液を培地に１０μＬ接種し、３５℃で好気培養を行い、発色強度を調べた。
(5) Bacterial inoculation and culture The precultured strain was suspended in sterile physiological saline, and McFarland No. 1 bacterial solution was prepared. Thereafter, 10 μL of the bacterial solution was inoculated into the medium, and aerobic culture was performed at 35 ° C. to examine the color intensity.
（６）結果
ＥＫＮ３０８８株の１４、１８、２４時間培養後の５−ブロモ−３−インドキシルホスフェートの分解による青色発色強度を表１４に示す。発色強度は目視により、培養時間ごとに一定の任意スケールで表した。
(6) Results Table 14 shows the blue color development intensity due to the decomposition of 5-bromo-3-indoxyl phosphate after culturing EKN3088 strain for 14, 18, and 24 hours. The color intensity was visually represented on a constant arbitrary scale for each incubation time.
液体培地においても、表１４に示す様に、酵素基質濃度１６０ｍｇ／Ｌ以上で卵黄液添加による発色増強作用が認められた。また、卵黄液添加あり酵素基質添加量１６０ｍｇ／Ｌにおける発色は、いずれの培養時間においても卵黄液添加なし酵素基質添加量４００ｍｇ／Ｌにおける発色よりも強度が上であった。このことから、発色増強剤としての卵黄液添加により、培地に添加する酵素基質量を４０％以下に低減できることが示唆された。さらに、発色が認められるまでに要した培養時間は、酵素基質添加量１６０ｍｇ／Ｌにおいて卵黄液添加ありの方が卵黄液添加なしに比べて１０時間短く、酵素基質添加量２４０ｍｇ／Ｌ以上では、卵黄液添加ありの方が４時間短かった。このことから、発色増強剤としての卵黄液添加により、判定までの培養時間を短縮できることが示唆された。 Also in the liquid medium, as shown in Table 14, a coloring enhancement effect by adding egg yolk liquid was observed at an enzyme substrate concentration of 160 mg / L or more. In addition, the color development at 160 mg / L with the addition of enzyme yolk was higher in intensity than the color development at 400 mg / L with no addition of enzyme yolk at any culture time. From this, it was suggested that the enzyme group mass added to a culture medium can be reduced to 40% or less by adding egg yolk liquid as a color enhancement agent. Furthermore, the incubation time required until color development was observed was 10 hours shorter when the egg yolk liquid was added at the enzyme substrate addition amount of 160 mg / L than when no egg yolk solution was added, and at the enzyme substrate addition amount of 240 mg / L or more, The one with egg yolk addition was 4 hours shorter. From this, it was suggested that the incubation time until determination can be shortened by adding egg yolk liquid as a color enhancer.
（４−メチルウンベリフェリルホスフェートを使用した黄色ブドウ球菌のホスファターゼ活性検出）
酵素基質として４−メチルウンベリフェリルホスフェートを使用した黄色ブドウ球菌のホスファターゼ活性検出において、卵黄液添加による発色増強作用を調べた。
(Detection of phosphatase activity of Staphylococcus aureus using 4-methylumbelliferyl phosphate)
In the detection of phosphatase activity of Staphylococcus aureus using 4-methylumbelliferyl phosphate as an enzyme substrate, the coloring enhancement effect by adding egg yolk liquid was examined.
（１）培地の準備
実施例８と同様とした。
(1) Preparation of medium The same as in Example 8.
（２）卵黄液添加量
実施例１．Ａと同様とした。
(2) Amount of egg yolk liquid added Example 1. Same as A.
（３）酵素基質添加濃度
卵黄液添加培地の酵素基質最終濃度を０、１００、２００、４００ｍｇ／Ｌとし、各酵素基質濃度について対照として卵黄液を添加しない培地も調製した。
(3) Enzyme substrate addition concentration The enzyme substrate final concentration of the egg yolk liquid addition medium was 0, 100, 200, and 400 mg / L, and a medium in which no egg yolk liquid was added was also prepared as a control for each enzyme substrate concentration.
（４）酵素基質および卵黄液の添加
実施例８と同様とした。
(4) Addition of enzyme substrate and egg yolk liquid The same as in Example 8.
（５）菌の接種と培養
実施例８と同様とした。
(5) Inoculation and culture of bacteria The same as in Example 8.
（６）結果
各菌株の４、６、８、１０、１２時間培養後の４−メチルウンベリフェリルホスフェートの分解による蛍光強度（３６５ｎｍで観察）を表１５に示す。蛍光強度は目視により、培養時間ごとに一定の任意スケールで表した。
(6) Results Table 15 shows the fluorescence intensity (observed at 365 nm) due to degradation of 4-methylumbelliferyl phosphate after culturing for 4, 6, 8, 10, 12 hours of each strain. The fluorescence intensity was visually represented on a constant arbitrary scale for each culture time.
表１５に示した様に、蛍光基質においても８時間培養（２株とも）および１０時間培養（２株中１株）において、卵黄液添加による発色増強作用が認められ、培地に添加する酵素基質量を低減できることが示唆された。また、卵黄液添加ありの場合は、検討した２株とも８時間培養により蛍光発色が認められたのに対し、卵黄液添加なしの場合は、ＥＫＮ４９７６株において８時間培養では蛍光発色が認められず、このことから、卵黄液添加により判定までの培養時間を短縮できることが示唆された。 As shown in Table 15, in the fluorescent substrate, in 8 hours culture (both strains) and 10 hours culture (1 strain out of 2 strains), the color development enhancing effect was observed by adding egg yolk liquid, and the enzyme group added to the medium It was suggested that the mass can be reduced. In addition, when the yolk liquid was added, both of the two strains examined showed fluorescent coloration after 8 hours of culture, whereas when no egg yolk liquid was added, the EKN4976 strain did not show fluorescent coloration when cultured for 8 hours. From this, it was suggested that the incubation time until determination can be shortened by adding egg yolk liquid.
（卵黄液添加による検出感度に対する影響）
酵素基質として５−ブロモ−３−インドキシルホスフェートを使用した黄色ブドウ球菌のホスファターゼ活性検出において、検出感度に対する卵黄液添加の影響を調べた。
(Influence on detection sensitivity by adding egg yolk liquid)
In detection of phosphatase activity of Staphylococcus aureus using 5-bromo-3-indoxyl phosphate as an enzyme substrate, the influence of addition of egg yolk solution on detection sensitivity was examined.
（１）培地の準備
実施例８と同様とした。
(1) Preparation of medium The same as in Example 8.
（２）卵黄液添加量
実施例１．Ａと同様とした。
(2) Amount of egg yolk liquid added Example 1. Same as A.
（３）酵素基質添加濃度
卵黄液添加培地の酵素基質最終濃度を１６０ｍｇ／Ｌとし、対照として卵黄液を添加せず、酵素基質最終濃度を１６０ｍｇ／Ｌとした培地も調製した。
(3) Enzyme substrate addition concentration A medium was prepared in which the final enzyme substrate concentration of the egg yolk liquid addition medium was 160 mg / L, the egg yolk liquid was not added, and the final enzyme substrate concentration was 160 mg / L as a control.
（４）酵素基質および卵黄液の添加
実施例８と同様とした。
(4) Addition of enzyme substrate and egg yolk liquid The same as in Example 8.
（５）菌の接種と培養
前培養した菌株を滅菌生理食塩水に懸濁し、ＭｃＦａｒｌａｎｄ Ｎｏ．１の菌液を作製した。その後、１０倍連続希釈液を作製し、１０0〜１０-6希釈の菌液をそれぞれ培地に１０μＬ接種し、３５℃で好気培養を行い、発色強度を調べた。
(5) Bacterial inoculation and culture The precultured strain was suspended in sterile physiological saline, and McFarland No. 1 bacterial solution was prepared. Thereafter, 10-fold serial dilutions were prepared, 10 μL of 10 0 to 10 −6 dilution of the bacterial solution was inoculated on each medium, and aerobic culture was performed at 35 ° C. to examine the color intensity.
（６）結果
ＥＫＮ３０８８株の１８、２０、２４時間培養後の５−ブロモ−３−インドキシルホスフェートの分解による青色発色強度を表１６に示す。発色強度は目視により、培養時間ごとに一定の任意スケールで表した。
(6) Results Table 16 shows the intensity of blue color developed by the decomposition of 5-bromo-3-indoxyl phosphate after culturing EKN3088 strain for 18, 20, and 24 hours. The color intensity was visually represented on a constant arbitrary scale for each incubation time.
表１６に示した様に、卵黄液添加培地では１０-6希釈菌液接種、１８時間培養において発色が認められたが、卵黄液添加なしの場合は１０0希釈菌液接種、２４時間培養でも発色が認められなかった。このことから、卵黄液添加により検出感度が改善されることが示された。 As shown in Table 16, liquid egg yolk 10 -6 dilution bacterial suspension inoculation in supplemented medium, although color in 18 hours of culture was observed, 10 0 diluted bacterial suspension inoculation for no egg yolk liquid addition, in 24-hour culture Color development was not recognized. From this, it was shown that detection sensitivity is improved by adding egg yolk liquid.
本発明の発色増強方法および試薬は、発色増強剤として卵黄または卵黄から調製された溶液を用いることにより、従来法に比べ測定に必要な酵素基質含量を低減し、試薬を低コスト化することが可能となる。また、従来法に比べ判定時間の短縮や高感度化も可能になる。そのため、本発明の方法および試薬は、臨床をはじめ、食品、環境などの幅広い分野の微生物検出において有用である。 The color development enhancing method and reagent of the present invention can reduce the enzyme substrate content required for the measurement and reduce the cost of the reagent compared to the conventional method by using egg yolk or a solution prepared from egg yolk as the color enhancement agent. It becomes possible. In addition, the determination time can be shortened and the sensitivity can be increased as compared with the conventional method. Therefore, the method and reagent of the present invention are useful for detecting microorganisms in a wide range of fields including clinical, food, and environment.
Claims (4)
発色増強剤として鳥類由来の卵黄全体、または鳥類由来の卵黄全体から調製された溶液を培地に添加して用いること、および、
微生物の検出または識別のために使用すること、
を特徴とする発色増強方法。 A chromophore compound and / or a fluorescent chromophore by allowing one or more enzymes selected from phosphatase, β-glucosidase , N-acetyl-β-galactosaminidase and N-acetyl-β-glucosaminidase to act on one or more substrates In the color development method for liberating compounds,
Adding the whole egg yolk derived from birds as a color enhancement agent, or a solution prepared from whole egg yolk derived from birds to the medium , and
Use for the detection or identification of microorganisms,
A color enhancement method characterized by the above.
発色増強剤として鳥類由来の卵黄全体、または鳥類由来の卵黄全体から調製された溶液を含むこと、および、
微生物の検出または識別のために使用すること、
を特徴とする発色培地。 A chromophore compound and / or a fluorescent chromophore by allowing one or more enzymes selected from phosphatase, β-glucosidase , N-acetyl-β-galactosaminidase and N-acetyl-β-glucosaminidase to act on one or more substrates A medium used in a color development method for releasing a compound,
Including a whole egg yolk derived from birds as a color enhancer or a solution prepared from whole egg yolk derived from birds ; and
Use for the detection or identification of microorganisms,
A chromogenic medium characterized by
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