WO2019049966A1

WO2019049966A1 - Histamine detection method and kit

Info

Publication number: WO2019049966A1
Application number: PCT/JP2018/033142
Authority: WO
Inventors: 一彦下地; 悠子一柳
Original assignee: キッコーマン株式会社
Priority date: 2017-09-08
Filing date: 2018-09-07
Publication date: 2019-03-14
Also published as: JPWO2019049966A1; JP7227139B2

Abstract

It was discovered that fading over time can be significantly suppressed by adding a surfactant to the reaction system in a method for detecting histamine using color development utilizing histamine dehydrogenase as an index.

Description

Histamine detection method and kit

The present invention relates to a method for detecting histamine utilizing a color reaction, and in particular to an improved method for preventing fading over time. The invention also relates to a kit for use in the method.

Histamine is a chemical transmitter of allergic reactions that occur in the body, so if you take a food that has a large amount of histamine, it becomes allergic like poisoning. The symptoms are reddening of the face, etc. in a few minutes to a few hours after a meal, followed by the onset of itching, urticaria or eczema. Rarely, urticaria spreads all over the body, causing bronchitis and blood pressure drop, and it may become severe. Therefore, there has been a strong demand for development of a histamine determination method capable of measuring histamine concentration simply and quickly in food processing plants, food hygiene monitoring agencies, clinical laboratories and the like.

As a method for quantifying histamine, a fluorometric method, a chromatography method using thin phase chromatography or paper chromatography, a high performance liquid chromatography (HPLC) method, an antigen-antibody reaction method, an enzyme method and the like are known (for example, Non Patent Literature 1).

The fluorescence analysis method is an official method published in the Official Methods of Analysis of AOAC International, and is one of the commonly used methods (for example, Non-Patent Document 2). The principle is that a fluorescent dye is produced by the condensation action of histamine and o-phthalaldehyde and histamine as a fluorescent reagent, and the intensity of the fluorescence is measured with a fluorescence spectrophotometer. However, this method needs to remove the interference component from the sample before condensation, and can not avoid the trouble and time for performing cation or anion exchange resin column treatment and the like. In addition, generally, when fish meat rots, biogenic amines such as cadaverine and putrescine are also produced almost simultaneously with the formation of histamine, and there is also the inconvenience that it is necessary to separate and remove these biogenic amines in the determination of histamine.

A great deal of research has been done on histamine determination by chromatography. Although thin phase chromatography and paper chromatography can measure a large number of samples simultaneously with a relatively inexpensive measuring device, it has been pointed out that their quantitativeness is not sufficient, and gas chromatography has a non-volatile like histamine. Direct quantification of the functional amines is not possible, and it has been pointed out that it is inconvenient to convert to heptafluorobutyryl derivatives etc. and then to quantify.

On the other hand, quantification by high performance liquid chromatography (HPLC) is introduced in Japan as a best method in the book of food analysis etc. (for example, non-patent document 3). However, in addition to the complexity of the pretreatment procedure and the need for advanced equipment and technical capabilities for measurement, the chromatography operation usually takes about 30 to 60 minutes, so many analyzes Is unsuitable.

The antigen-antibody reaction method is one of the measurement methods widely used as a commercially available kit, but the operation procedure is many, and the reaction time is strict and the measurement needs to be quick.

In the enzyme method, an enzyme electrode method using an electrode coated with a monoamine oxidase-immobilized film is known (for example, Non-Patent Document 4), but it is a method of measuring the local dissolved oxygen concentration (DO) of a test solution As a result, it has the disadvantage that the accuracy is not high (about 8% relative error).

As another enzyme method, a dissolved oxygen electrode method using an enzyme having a histamine oxidase activity derived from a microorganism is known (for example, Non-Patent Document 5). This method does not require complicated interference substance removing operation or calibration operation using a histamine standard solution as compared with the conventional fluorescence analysis method or HPLC method, and the response time of the electrode is one and a half minutes. As a simple and rapid method, and as a highly accurate method (with a relative error of 0.98%), it has already obtained a high internal evaluation from the FDA (for example, Non-Patent Document 6). However, also in this method, in consideration of the fact that the output current of the oxygen electrode and the saturated dissolved oxygen concentration are easily influenced by the temperature (temperature dependent type), the entire process including the air saturation operation of the test solution is carried out. It should be done under constant temperature (37 ° C). Therefore, a large-sized reaction apparatus equipped with a thermostatted hot water jacket for keeping the temperature of the working cell constant is required, and there were problems such as the difficulty of movement and the difficulty of securing a power supply corresponding to power consumption. . In addition, the action cell to which the DO electrode is attached must be kept in a liquid-tight manner during quantitative operation, because accurate measurement of DO becomes difficult if air bubbles (air) are mixed in. Also, the action cell Were inconvenient because they had to be used repeatedly and had to be cleaned each time (i.e. the disposable action cell could not be used).

A method using histamine dehydrogenase has been developed as a method for measuring histamine with an enzyme that has solved these problems (for example, Patent Document 1). This method is based on the presence of tetrazolium-based electron carriers (eg phenazine methosulfate, meldola blue etc.) and tetrazolium-based reducing chromogenic reagents (eg MTT, Nitro-TB, WST-8 etc.) in a histamine-containing sample. This is a colorimetric method of measuring the pigment produced by adding histamine dehydrogenase to perform enzyme action and measuring the pigment formed, and pretreatment such as fractionation operation of histamine from the sample and removal operation of troublesome interfering substances (impurities) That it is unnecessary, the time required for quantification is short, it can be measured using a simple device, the action cell does not have to be operated under liquid tight conditions (ie it is operated in open system) It has many advantages, such as what can be done).

JP 2001-157597

However, in the method of detecting histamine with color development as an indicator using histamine dehydrogenase, when multi-specimen color development is to be simultaneously measured using a multi-well plate or the like, it is generated for operation in each well. At the time of detection of a dye, it is also assumed that a long time (for example, one hour or more) elapses from the start of a coloring reaction. For this reason, it is desirable to be able to quantify histamine efficiently regardless of the time from the initiation of the color reaction to the measurement.

Therefore, the inventor first examined whether or not the color fading occurred after the color reaction started in the method, and the extent thereof. As a result, it was revealed that although the color fading does not matter in a relatively short time after the initiation of the coloring reaction, the color fading occurs with time thereafter (Example 3 described later). From this fact, when trying to quantify a histamine concentration using a color development as an index after a predetermined time has elapsed using this method, discoloration at the time of detection may be a problem.

Therefore, this invention aims at providing the method which can prevent discoloration over time in the method of detecting histamine on the basis of coloring using histamine dehydrogenase as a parameter | index.

As a result of intensive studies to solve the above problems, the present inventors have found that, in a method for detecting histamine using histamine dehydrogenase as a marker of coloring, a surfactant, in particular, a surfactant represented by the following formula (1) It has been found that the addition of a surfactant having a structure can significantly suppress fading over time.

(Wherein, R represents a saturated or unsaturated hydrocarbon group, n is an integer of 0 to 10, and M represents a counter ion).

In addition, when the above-mentioned surfactant is added to the reaction system, the positive correlation between the histamine concentration and the coloring intensity is extremely high even after a long time has passed since the initiation of the color reaction, which means that the histamine concentration can be quantified with high accuracy. I also found it possible.

Furthermore, in the sample containing the interfering substance (impurity), the surfactant was found to also have the effect of suppressing the influence on the color development, and the present invention was completed.

That is, the present invention relates to a method for detecting histamine with coloring as an indicator using histamine dehydrogenase, wherein the use of a surfactant improves the influence of fading over time or the interfering substance on coloring, and the method More specifically, with regard to the kit used in the method, the following is provided:

[1] A method for detecting histamine in a sample, comprising
A method of causing histamine dehydrogenase to act on histamine in the sample in the presence of a tetrazolium salt, an electron carrier, and a surfactant, and detecting histamine in the sample using as a formazan dye generated by reduction of a tetrazolium salt as an indicator.

[2] A method for detecting histamine in a sample, comprising
In the presence of a tetrazolium salt, an electron carrier, and a surfactant, histamine in the sample is allowed to act on histamine dehydrogenase, and histamine in the sample is detected using formazan dye produced by reduction of the tetrazolium salt as an indicator. Including
The method whose said surfactant is a compound which has a structure of following formula (1).

[3] The method according to [2], wherein the compound having a structure of the formula (1) is a polyoxyethylene alkyl ether sulfate.

[4] The method according to [3], wherein the polyoxyethylene alkyl ether sulfate is polyoxyethylene lauryl ether triethanolamine sulfate or polyoxyethylene lauryl ether sodium sulfate.

[5] A tetrazolium salt is 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium monosodium salt The method according to any one of [1] to [4].

[6] The method according to any one of [1] to [5], wherein the electron carrier is 1-methoxy-5-phenazine methosulfate.

[7] A kit for use in the method of any one of [1] to [6], which comprises the following (a) to (d):
(A) histamine dehydrogenase (b) tetrazolium salt (c) electron carrier (d) surfactant.

[8] A method of detecting histamine in a sample by using histamine dehydrogenase in the presence of tetrazolium salt and an electron carrier and causing histamine dehydrogenase to act on the formazan dye produced by reduction of the tetrazolium salt as an indicator. Method to suppress fading of formazan dye by adding surfactant to.

[9] A kit for use in the method described in [8], which comprises the following (a) to (d):
(A) histamine dehydrogenase (b) tetrazolium salt (c) electron carrier (d) surfactant.

According to the present invention, in a method of detecting histamine using coloring as an indicator using histamine dehydrogenase, it is possible to significantly suppress fading over time, and moreover, it is possible to obtain a high correlation between histamine concentration and coloring intensity. . In addition, in the sample containing the interfering substance, the influence on the color development can also be suppressed. Therefore, according to the present invention, it becomes possible to quantify the histamine concentration with high accuracy regardless of the elapsed time from the initiation of the color reaction to the measurement.

It is a graph which shows the result of having measured the fading over time after the reaction start in the method of detecting histamine on the basis of coloring using histamine dehydrogenase. The study was carried out with histamine at each concentration (1 ppm to 4 ppm). It is a graph which shows the result of having measured the fading over time after the reaction start in the case of adding Emar 20C to a reaction system in the method of detecting histamine by making coloring into a parameter | index using a histamine dehydrogenase. The study was carried out with histamine at each concentration (1 ppm to 4 ppm). It is a graph which shows the result of having measured the color development intensity five minutes after the reaction start in the case of adding various surfactant to a reaction system in the method of detecting histamine on the basis of color development using histamine dehydrogenase. The study was carried out with histamine at each concentration (1 ppm to 4 ppm). It is a graph which shows the result of having measured the color development intensity 15 minutes after the reaction start in the case of adding various surfactant to a reaction system in the method of detecting histamine on the basis of coloring using histamine dehydrogenase. The study was carried out with histamine at each concentration (1 ppm to 4 ppm). It is a graph which shows the result of having measured the color development intensity 30 minutes after the reaction start in the case of adding various surfactant to a reaction system in the method of detecting histamine on the basis of coloring using histamine dehydrogenase. The study was carried out with histamine at each concentration (1 ppm to 4 ppm). It is a graph which shows the result of having measured the color development intensity 60 minutes after the reaction start in the case of adding various surfactant to a reaction system in the method of detecting histamine using a coloring as a parameter | index using a histamine dehydrogenase. The study was carried out with histamine at each concentration (1 ppm to 4 ppm). It is a graph which shows the result of having measured the color development intensity 100 minutes after reaction start at the time of adding various surfactant to a reaction system in the method of detecting histamine using a color development as a parameter | index using histamine dehydrogenase. The study was carried out with histamine at each concentration (1 ppm to 4 ppm).

-Histamine detection method-
The present invention is a method for detecting histamine in a sample, wherein histamine in the sample is allowed to act on histamine in the presence of a tetrazolium salt, an electron carrier, and a surfactant to produce a reduction of a tetrazolium salt. Provided is a method of detecting histamine in a sample using formazan dye as an indicator.

The present invention also relates to a method for detecting histamine in a sample by causing histamine dehydrogenase in the sample to act on histamine in the presence of a tetrazolium salt and an electron carrier and using a formazan dye produced by reduction of the tetrazolium salt as an indicator. Also provided is a method of inhibiting fading of formazan dye by adding a surfactant to the reaction system.

"Histamine" in the present invention is an active amine produced by degradation of histidine which is an essential amino acid by bacteria, and is considered to be a causative agent of allergy-like food poisoning.

In the present invention, the "sample" that can contain histamine is not particularly limited, and examples thereof include liquid and solid foods, in vivo substances such as urine and plasma, and living tissues. With regard to allergy-like food poisoning, for example, there are detection examples of histamine in fermented foods such as fish and processed products thereof, soy sauce, fish sauce, wine, and cheese.

The sample may be subjected to quantitative determination as it is or after extraction and filtration depending on its type. If necessary, pretreatment such as grinding, centrifugation, or solvent addition may be performed before extraction, and post treatment such as concentration, dilution, or pH adjustment may be performed after extraction. For extraction or dilution, for example, water or a buffer solution can be used. For extraction, for example, it is preferable to use a chelating agent such as EDTA.disodium (Japanese Patent Laid-Open No. 2004-129597), and heat treatment may be performed if necessary.

In the present invention, histamine dehydrogenase is caused to act on histamine in the sample in the presence of a tetrazolium salt, an electron carrier and a surfactant. Since a formazan pigment | dye is produced | generated from a tetrazolium salt by this, histamine can be detected by making the color development into a parameter | index, and the density | concentration can be quantified.

The “histamine dehydrogenase” in the present invention is an enzyme that acts on histamine in the presence of an electron acceptor to generate 4-imidazolylacetaldehyde and ammonia by an oxidative deamination reaction. The "histamine dehydrogenase" used in the present invention is not particularly limited, but those derived from, for example, Rhizobium bacteria are preferable. Histamine dehydrogenase derived from Rhizobium bacteria is described in the literature (Japanese Patent Application Laid-Open Nos. 2001-157579 and 2003-289864), and it is a food with high specificity to histamine and reduced freshness. It does not act on biogenic amines such as cadaverine and putrescine which are produced simultaneously with histamine. By using such highly specific histamine dehydrogenase, it becomes possible to efficiently detect histamine without separating biological amines other than histamine. In the reaction system, histamine dehydrogenase is usually used at a concentration of 0.02 to 2 U / ml.

The "tetrazolium salt" used as a reducing type coloring reagent is not particularly limited as long as it is reduced in the presence of an electron carrier to form a formazan dye. A preferred example is WST-8 [2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium monosodium Salt], WST-1 [2- (4-iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium monosodium salt], WST-3 [ 2- (4-Eodophenyl) -3- (2,4-dinitrophenyl) -5- (2,4-disulfenyl) -2H-tetrazolium monosodium salt], WST-4 [2-benzothiazole- 3- (4-Carboxy-2-methoxyphenyl) -5- [4- (2-sulfoethylcarbomoyl) phenyl] -2H-tetrazolium (inner salt)], WST-5 [2,2'-diben Thiazolyl-5,5'-bis [4-di (2-sulfoethyl) carbamoylphenyl] -3,3 '-(3,3'-dimethoxy-4.4'-biphenylene) ditetrazolium disodium salt], WST -9 [2- (4-nitrophenyl) -5-phenyl-3- [4- (4-sulfophenylazo) -2-sulfophenyl] -2H-tetrazolium monosodium salt], MTT [3- (4,5 -Dimethyl-thiazol-2-yl) -2,5-diphenyltetrazolium bromide], NITRO-TB [3,3 '-[3,3'-dimethoxy- (1,1'-biphenyl) -4,4' And -diyl] -bis [2- (4-nitrophenyl) -5-phenyl-2H-tetrazolium chloride]. In the reaction system, the tetrazolium salt is usually used at a concentration of 5 to 5000 μM.

The “electron carrier” is not particularly limited as long as it reduces a tetrazolium salt to form a formazan dye, and examples thereof include phenazine methosulfate, 1-methoxy-5-methylphenazinium sulfate and merdra blue. Be In the reaction system, the electron carrier is usually used at a concentration of 0.2 to 100 μM.

The present invention is characterized by using a "surfactant" in the reaction system. The "surfactant" is not particularly limited as long as it can suppress fading of formazan dye.

A preferred embodiment of the surfactant is 10% or less (e.g., 9% or less, 8% or less, 7% or less, or 20% or less of the color fading at 90 minutes after the color reaction start, as compared with the maximum color intensity after the color reaction start). 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0%). The rate of fading can be calculated from the value obtained by measuring the absorbance at 470 nm with time using a histamine solution of a predetermined concentration (for example, 4 ppm) as described in Example 3.

A preferred class of surfactants from the viewpoint of the anti-fading effect is an anionic surfactant or a nonionic surfactant.

As the anionic surfactant, one having a structure of the following formula (1) is particularly preferable.

The saturated or unsaturated hydrocarbon group preferably has 8 to 20 carbon atoms. As such a hydrocarbon group, a linear or branched alkyl group or alkenyl group such as octyl group, isooctyl group, decyl group, lauryl group, myristyl group, palmityl group, stearyl group, isostearyl group, oleyl group, docosyl And the like, and among them, a saturated alkyl group having a carbon number of 11 to 13, particularly a lauryl group is preferable.

The number n of ethylene glycol groups is an integer of 0 to 10, preferably 1 to 10 (eg, 2 to 5).

Examples of the counter ion include alkali metal ions, primary to tertiary ammonium ions, and ammonium ions. Examples of the alkali metal ion include sodium ion, lithium ion, potassium ion and the like, and examples of the primary to tertiary ammonium ions include mono, di or triethanol ammonium ion.

The surfactant having the structure of the formula (1) is disclosed, for example, in the literature (Japanese Patent Application Laid-Open No. 08-073891). Preferred commercially available surfactants are polyoxyethylene alkyl ether sulfates, such as polyoxyethylene lauryl ether triethanolamine tribasic or polyoxyethylene lauryl ether sodium sulphate. Specific commercial products of polyoxyethylene alkyl ether sulfate include, for example, Emar 20 T, Emar 20 C, Emar E-27 C, Emar 270 J, Emar 20 CM (all are Kao Corporation) and the like. Not limited to

On the other hand, as specific commercial products of preferable commercially available nonionic surfactants, for example, Reodore Super TW-S120 (Kao Corporation), Emulgen B-66 (Kao Corporation), Emalgen 420 (Kao Corporation) And Nymin F-215 (Nippon Oil Co., Ltd.), BL-9EX (Nippon Chemicals Co., Ltd.) and the like, but not limited thereto.

In addition to suppression of fading, the positive correlation between histamine concentration and coloring intensity is extremely high even after a long time from the initiation of the color reaction, and further, in the sample containing the interfering substance, the influence on the color development is From the viewpoint of being able to be suppressed, as surfactant classification, anionic surfactants are particularly preferable, and among them, the above-mentioned surfactants having the structure of the formula (1) are most preferable.

The surfactant is usually used at a concentration of 0.008% to 10%, preferably 0.01 to 5%, in the reaction system. Although surfactant can be used alone, you may use it in mixture of 2 or more types.

The temperature at which the histamine dehydrogenase is allowed to act in the reaction system is usually 20 to 70 ° C., preferably 30 to 50 ° C. The action time is not particularly limited as long as it is a time sufficient to degrade histamine in the sample, but it is usually 1 to 120 minutes. In the present invention, it was found that the addition of the surfactant suppresses the color fading even when the action time is long. Therefore, according to the present invention, it is possible to efficiently quantify histamine concentration even after a relatively short reaction time within 20 minutes or after a relatively long reaction time exceeding 60 minutes. Is possible.

The pH in the reaction system may be adjusted without adjustment, but is preferably adjusted to pH 8 to 10 using an appropriate pH adjusting agent such as an acid or alkali such as hydrochloric acid, sulfuric acid, nitric acid, sodium hydroxide or potassium hydroxide. In addition, in the reaction system, an appropriate buffer can be used. As the buffer, for example, Tris-hydrochloride, phosphate such as potassium phosphate, acetate and the like can be mentioned.

In the measurement of the coloring intensity of the formazan dye produced by the above reaction, for example, when WST-8 is used as the reduced color developing reagent, the absorbance in the vicinity of 470 nm is usually used, and WST-1 is used. Usually uses the absorbance around 438 nm, usually uses the absorbance around 433 nm when using WST-3, and typically uses the absorbance around 550 nm when using WST-4, When using WST-5, the absorbance at around 550 nm is usually used, and when using WST-9, the absorbance at around 479 nm is usually used, and when using MTT, usually around 565 nm The absorbance is used. When NITRO-TB is used, the absorbance around 530 nm is usually used.

The coloring intensity of the formazan dye can be measured by a spectrophotometer. The use of a microplate reader is preferred for the detection of multiple analytes. As a measuring device, for example, a commercially available device such as an absorbance meter B (manufactured by Kyoritsu Riken Chemical Co., Ltd.) or TriStar 2 S LB 942 Multimode Reader (Berthold) can be used.

-Histamine detection kit-
The present invention also provides a kit for use in the above method, which comprises the following (a) to (d).
(A) histamine dehydrogenase (b) tetrazolium salt (c) electron carrier (d) surfactant.

In the kit of the present invention, the histamine dehydrogenase, the tetrazolium salt and the electron carrier may be a lyophilized product or a solution preparation in which they are dissolved. In addition, histamine dehydrogenase can be used repeatedly, for example, by using it as a standard bound to a solid phase carrier. The tetrazolium salt and the electron carrier may be used as a color reagent preparation in which both of them are mixed. The surfactant may be a single liquid preparation, or may be a liquid preparation mixed with a tetrazolium salt and / or an electron carrier.

Further, in the kit of the present invention, a standard solution (histamine solution of a specific concentration) used for quantifying histamine, a buffer solution used for dissolving or diluting histamine dehydrogenase, and an extract used for extracting histamine from a sample May be included.

Each preparation is suitably used in combination at the time of use such that the concentrations of histamine dehydrogenase, tetrazolium salt, electron carrier and surfactant in the final reaction system become appropriate concentrations (described above).

In the kit of the present invention, instructions for the kit can be further included. The instructions can include, for example, information on the application, measurement principle, features, performance, configuration, usage in measurement, etc. of the kit.

Hereinafter, the present invention will be more specifically described based on examples, but the present invention is not limited to the following examples.

Example 1 Preparation Example of a Chromogenic Reagent for Histamine Detection The following components were dissolved in purified water at the following concentrations to prepare a chromogenic reagent for histamine detection.

Note 1: 1-methoxy-5-methylphenazinium sulfate note 2: 2 (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl)- 2H-tetrazolium monosodium salt.

[Example 2] Preparation Example of Enzyme Reagent for Histamine Detection The following components were dissolved in purified water at the following concentrations or units, respectively, to prepare an enzyme reagent for histamine detection.

Example 3 Detection of Histamine of Known Concentration Using a Coloring Reagent for Histamine Detection, and an Enzyme Reagent for Histamine Detection Emar 20C (Kao Co., Ltd.) was used as sodium alkyl sulfate used in the color detection reagent for histamine detection.

First, 50 μl each of 1 ppm, 2 ppm, 3 ppm and 4 ppm solutions of histamine are dispensed into the wells of a 96-well microplate, and then 50 μl each of a coloring solution for histamine detection (containing no Emar 20 C and no Emar 20 C 2%) Each aliquot was aliquoted, 50 μl aliquots of the enzyme reagent for histamine detection were dispensed at room temperature, and absorbance (Es) at 470 nm was measured with a plate reader over time.

For the color development blank, 50 μl of 750 mM tris (hydroxymethyl) aminomethane (pH 9.0) was added instead of the enzyme reagent for histamine detection, and the absorbance (Eb) at 470 nm was measured in the same manner as described above.

From the obtained absorbance, the value of Es-Eb was taken as the histamine detection absorbance. A TriStar2S LB942 Multimode Reader (Berthold) was used as a microplate reader.

FIG. 1 shows the absorbance over time when using a sodium alkyl sulfate-free reagent for histamine detection. It turned out to fade over time.

FIG. 2 shows the absorbance over time when using a histamine detection reagent containing 2% of sodium alkyl sulfate (Emar 20C). By containing Emar 20C, it turned out that discoloration is suppressed.

[Example 4] Search for a substance that enhances positive correlation between histamine concentration and coloring intensity Instead of sodium alkyl sulfate as a coloring reagent for histamine detection, Cortamine 60 W (Kao Co., Ltd.), Cortamine as a cationic surfactant 86 W (Kao Corporation), as an anionic surfactant, Emar 20 T, as a non-ionic surfactant, Leodol Super TW-S 120 (Kao Corporation), Emulgen B-66 (Kao Corporation), Emulgen 420 (Kao Co., Ltd.), Niminen F-215 (Nippon Oil Co., Ltd.), BL-9EX (Nippon Chemicals Co., Ltd.) containing 2% of each reagent, and a cationic surfactant, hexadecyltrimethylammonium bromide (TTAB, Prepare a reagent containing 0.2% of Sigma Aldrich), and As a control reagent which water was added, histamine 1 ppm, 2 ppm, 3 ppm, it was detected histamine solutions of 4ppm by the method of Example 3.

(1) Absorbance of histamine detection reagent after 5 minutes of action FIG. 3 shows the result of detection of histamine of known concentration after 5 minutes of reaction. The absorbance after the reaction was high, passed through the origin, and Emar 20T-containing reagent was confirmed as a reagent without variation. The rheodol super TW-S120 passed the origin and had no variation, but had a lower absorbance.

(2) Absorbance of histamine detection reagent after 15 minutes of action FIG. 4 shows the result of detection of histamine of known concentration 15 minutes after the reaction. The absorbance after reaction was high, passed through the origin, and Emar 20T-containing reagent was confirmed as a reagent without variation.

(3) Absorbance of histamine detection reagent after 30 minutes of action FIG. 5 shows the result of detection of histamine of known concentration after 30 minutes of reaction. The absorbance after reaction was high, passed through the origin, and Emar 20T-containing reagent was confirmed as a reagent without variation.

(4) Absorbance of histamine detection reagent after 60 minutes of action FIG. 6 shows the result of detection of histamine of known concentration after 60 minutes of reaction. The absorbance after reaction was high, passed through the origin, and Emar 20T-containing reagent was confirmed as a reagent without variation.

(5) Absorbance of histamine detection reagent 100 minutes after action FIG. 7 shows the result of detection of histamine of known concentration 100 minutes after the reaction. The absorbance after reaction was high, passed through the origin, and Emar 20T-containing reagent was confirmed as a reagent without variation.

[Example 5] Confirmation of the effect of components in various samples on histamine detection system Using histamine detection reagent, histamine by the components in various samples of soy sauce, red wine, white wine, milk (not adjusted), fish scale The impact on the detection of

The chromogenic reagent for histamine detection of Example 1 (without surfactant, Emar 20T, containing 2%) and the enzyme reagent for histamine detection of Example 2 were used. Each sample was diluted 25 times with 0.1 M EDTA.2Na (pH 8.0) to make a test solution A, and 100 ppm of known concentration of histamine was added to each sample to prepare 0.1 M EDTA.2 Na (pH 8.K). Test solution B diluted 25 times with 0). Purified water was used for the color development blank. As a measuring instrument, a JCA-BM1650 automatic analyzer manufactured by JEOL Ltd. was used, and the measurement wavelength was 451 nm of main wavelength and 751 nm of sub wavelength. After reacting 25 μl of each test solution with 25 μl of the color reagent for detection shown in Example 1 for 5 minutes at 37 ° C., 25 μl of enzyme solution for detection, 750 mM tris (hydroxymethyl) aminomethane for test solution blank 25 μl (pH 9.0) was added, and the absorbance after 5 minutes was measured. The absorbance of each sample was calculated using the absorbance of the test solution as “Es” and the absorbance of the test solution blank as “Eb”.

Absorbance of each sample = Es-Eb
The absorbance when the histamine 100 ppm standard solution is diluted and detected by the method of Example 5 is around 0.4. From the measurement results shown in Table 3, it was confirmed that Emar 20T had an effect of suppressing the negative influence on the detection by the components contained in the detection of histamine in white wine and fish scale.

Example 6 Detection of Histamine in Various Samples and Addition Recovery Test The histamine detection reagent was used to detect histamine in various samples of soy sauce, red wine, white wine, and fish sauce. Furthermore, an added recovery test was performed to add known concentrations of histamine to the detection system and to evaluate the recovery rate.

The chromogenic reagent for histamine detection of Example 1 (without surfactant, Emar 20T, containing 2%) and the enzyme reagent for histamine detection of Example 2 were used. Each sample was diluted 25 times with 0.1 M EDTA.2Na (pH 8.0) to make a test solution A, and 100 ppm of known concentration of histamine was added to each sample to prepare 0.1 M EDTA.2 Na (pH 8.K). Test solution B diluted 25 times with 0). Purified water was used for the color development blank. As a measuring instrument, a JCA-BM1650 automatic analyzer manufactured by JEOL Ltd. was used, and the measurement wavelength was 451 nm of main wavelength and 751 nm of sub wavelength. After reacting 25 μl of each test solution with 25 μl of the color reagent for detection shown in Example 1 for 5 minutes at 37 ° C., 25 μl of enzyme solution for detection, 750 mM tris (hydroxymethyl) aminomethane for test solution blank 25 μl (pH 9.0) was added, and the absorbance after 5 minutes was measured. The detected histamine concentration (ppm) was determined using a known concentration of histamine standard solution (Kikkoman Biochemifa). After measuring the absorbance of the test solution (Es), the absorbance of the test solution blank (Eb), the absorbance of the histamine standard solution (Estd), and the absorbance of the coloring blank (Ec), the histamine concentration was determined according to the following formula.

Histamine concentration (ppm) in sample = (Es-Eb) / (Estd-Ec) x 4 x 25
In the formula, "4" indicates that the standard solution has a histamine concentration of 4 ppm, and "25" indicates a dilution factor due to the sample being diluted 25 times.

Addition recovery rate (%) = (B-A) / 100 x 100
It is divided by 100 because the added histamine concentration is 100 ppm.

From the measurement results shown in Table 4, it was confirmed that the addition recovery was improved by adding Emar 20T to white wine and fish sauce.

[Example 7] Detection of histamine in soy sauce and addition recovery test Since soy sauce contains substances that negatively affect the detection system, it is possible to detect histamine in soy sauce only by diluting the sample. It was difficult to do. In the past, the soy sauce was diluted and then histamine was detected after pretreatment using a solid-phase column (Sep-Pak Accell Plus CM Plus Short Cartridge: Waters). Therefore, it was examined whether histamine could be detected without pretreatment with a solid phase column.

(1) Detection of Histamine in Soy Sauce by Using Solid Phase Column and Addition Recovery Test As a histamine dilution liquid and column washing liquid, 20 mM phosphate buffer solution (pH 6.0) was prepared. In addition, as an elution solution, a 20 mM phosphate buffer (pH 7.0) containing 175 mM NaCl was prepared.

First, 1 ml of purified water was added to 0.1 ml of soy sauce, and the diluted solution was adjusted to 20 ml to make a 200-fold diluted sample (A). Next, 1 ml of 100 ppm histamine solution was added to 0.1 ml of soy sauce, and the diluted solution was adjusted to 20 ml to make a 200-fold diluted sample with histamine added (B). A solid phase column Sep-Pak Accell Plus CM Short Cartridge, 360 mg Sorbent per Cartridge, 37-55 μm Particle Size, 50 / pk [WAT020550] (Waters) was attached to a 10 ml thermo syringe (Terumo). 10 ml of diluted sample was added to the syringe and the solution was passed through the column. The eluted solution was discarded. Next, 10 ml of washing solution was added to the syringe and the solution was passed through the column. The eluted solution was discarded. 10 ml of eluate was added to the syringe and the solution was passed through the column. All eluted solutions were collected and used as test solutions (A, B).

At 37 ° C., 500 μl of each test solution and 500 μl of the color reagent for detection (without sodium alkyl sulfate) shown in Example 1 are mixed, then 500 μl of enzyme solution for detection, 750 mM Tris (hydroxy) for test solution blank 500 μl of methyl) aminomethane (pH 9.0) was added, and the absorbance after 15 minutes was measured using a spectrophotometer (absorbance meter B: Kyoritsu Scientific Research Institute). The detected histamine concentration (ppm) was determined using a known concentration of histamine standard solution (Kikkoman Biochemifa). After measuring the absorbance of the test solution (Es), the absorbance of the test solution blank (Eb), the absorbance of the histamine standard solution (Estd), and the absorbance of the coloring blank (Ec), the histamine concentration was determined according to the following formula.

Histamine concentration (ppm) in the sample = (Es-Eb) / (Estd-Ec) x 4 x 200
In the formula, "4" indicates that the histamine concentration of the standard solution is 4 ppm, and "200" indicates a dilution factor due to the sample being diluted 200 times.

Recovery rate of addition (%) = (B-A) / 100 x 100
It is divided by 100 because the added histamine concentration is 100 ppm.

(2) Detection of histamine in soy sauce using Emar 20T-containing coloring solution and addition recovery test Add 0.1 ml of purified water to 0.1 ml of soy sauce and make it 20 ml with 0.1 M EDTA · 2Na (pH 8.0). It was considered as a double dilution sample (A). Next, 1 ml of 100 ppm histamine solution was added to 0.1 ml of soy sauce to make 20 ml with 0.1 M EDTA · 2Na (pH 8.0), and used as a 200-fold diluted sample with histamine added (B).

At 37 ° C., 500 μl of each test solution and 500 μl of the color reagent for detection (containing sodium alkyl sulfate 2%) shown in Example 1 are mixed, then 500 μl of enzyme solution for detection, 750 mM Tris for test solution blank The absorbance after 15 minutes of adding 500 μl of (hydroxymethyl) aminomethane (pH 9.0) was measured using a spectrophotometer (absorbance meter B: Kyoritsu Scientific Research Institute, Inc.). The detected histamine concentration (ppm) was determined using a known concentration of histamine standard solution (Kikkoman Biochemifa Co., Ltd.). After measuring the absorbance of the test solution (Es), the absorbance of the test solution blank (Eb), the absorbance of the histamine standard solution (Estd), and the absorbance of the coloring blank (Ec), the histamine concentration was determined according to the following formula.

(3) Detection of histamine in soy sauce and results of addition and recovery test As shown in Table 5, the detection result of histamine in soy sauce using Emar 20T-containing color detection reagent was the detection result using solid phase column Matched with. Therefore, it was found that the inclusion of Emar 20T in the histamine detection reagent makes it possible to measure soy sauce without using a solid phase column.

[Example 8] The effect of surfactant concentration on the detection and recovery of added histamine in fish scale The effect on the detection and recovery of loaded histamine in fish scale was verified using Emar 20T at each concentration as a surfactant. . The measurement of the absorbance in the detection of histamine was performed by the method described in Example 5. Also, the addition and recovery test was conducted by the method described in Example 6. In addition, in the measurement of the light absorbency, the ratio (%) to the light absorbency (0.400) of the histamine 100 ppm standard solution was also calculated.

As a result, as shown in Table 6, when 0.008 to 10% of Emar 20T was used, both the absorbance ratio and the addition recovery rate to the standard solution became 90% or more, and preferable results were obtained. When 0.01 to 5% of Emar 20T was used, both the ratio of absorbance to the standard solution and the recovery rate of addition were 95% or more, and more preferable results were obtained.

As described above, according to the present invention, when detecting histamine using the color development of formazan dye as an index, it is possible to suppress the color fading over time, and moreover, the positive correlation between the histamine concentration and the color development intensity is Extremely expensive. Furthermore, in the sample containing the interfering substance, the influence on the color development can be suppressed. This makes it possible to quantify histamine concentration with high accuracy. Since histamine contained in food is a cause of allergy-like poisoning, the present invention can be used not only in research but also in a wide range of industrial fields including food industry and medical industry.

Claims

A method of detecting histamine in a sample comprising
In the presence of a tetrazolium salt, an electron carrier, and a surfactant, histamine in the sample is allowed to act on histamine dehydrogenase, and histamine in the sample is detected using formazan dye produced by reduction of the tetrazolium salt as an indicator. Including
The method whose said surfactant is a compound which has a structure of following formula (1).

(Wherein, R represents a saturated or unsaturated hydrocarbon group, n is an integer of 0 to 10, and M represents a counter ion).
The method according to claim 1, wherein the compound having the structure of formula (1) is a polyoxyethylene alkyl ether sulfate.
The method according to claim 2, wherein the polyoxyethylene alkyl ether sulfate is polyoxyethylene lauryl ether triethanolamine tribasic or polyoxyethylene lauryl ether sodium sulfate.
The tetrazolium salt is 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium monosodium salt. The method according to any one of 1 to 3.
The method according to any one of claims 1 to 4, wherein the electron carrier is 1-methoxy-5-phenazine methosulfate.
A kit for use in the method according to any one of claims 1 to 5, comprising the following (a) to (d):
(A) histamine dehydrogenase (b) tetrazolium salt (c) electron carrier (d) surfactant having the structure of formula (1)