JP2001099803A - High-sensitivity method for determining histamine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a high-sensitivity method for determining a histamine which can quickly and accurately determine the histamine of a concentration much lower than 0.5 ppm. SOLUTION: A histamine oxidase is reacted to a sample solution containing a histamine, and the generated H2O2 is detected by a peroxidase electrode. A concentration of the histamine is determined accordingly. The peroxidase electrode utilized is obtained by uniformly kneading and burying a graphite powder, a liquid paraffin, an electron carrier and peroxidase to one end part of a cylindrical insulator (peroxidase/carbon paste electrode) to be used as a substrate sensitive part, forming a peroxidase immobilization film to a surface of the substrate response part, and coating an outer face of the film with a permeable membrane.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for quantifying histamine with high sensitivity, and more particularly to a method for quantifying histamine with high sensitivity, which can rapidly and accurately quantify a histamine concentration as low as 0.5 ppm or less.

[0002]

2. Description of the Related Art Conventionally, a rapid histamine quantification method is known, which comprises a combination of a microorganism-derived enzyme amine oxidase (AO) reagent that specifically oxidizes histamine and a method for measuring dissolved oxygen (DO). (Patent No. 2,717,745, U.
SP 5,565,329). This method is extremely rapid and has a higher A
It has a very high correlation (correlation r = 0.999) to a public analysis method such as the OAC method. The detailed principle,
For characteristics, see the following academic report. Academic Report 1: "Journal of Food Science" Vol.59, p519
-522,1994. Academic Report 2: "Food Hygiene Magazine" Volume 37, 107-113
Page, 1996.

[0003] However, in this method, the dissolved oxygen in the water saturated with the oxygen in the air is used as a reference, and the dissolved oxygen is converted to a histamine concentration using the stoichiometric relation of the following formula (1) to determine the amount. Thus, there is an advantage that quantification can be performed using oxygen in the air without using a standard solution of histamine which is an intoxicating component, but has the following disadvantages.

[0004]

Embedded image

At room temperature, the concentration of saturated dissolved oxygen in water (D
O) is about 8 mg / liter, so using the stoichiometric relation of equation (1) (a relation in which 1 mol of oxygen is consumed when 1 mol of histamine is decomposed), this DO is
When converted to histamine (Hm) concentration, it is about 28 mg / liter. The calculation formula is shown below. 8 mg x [111
_{(Hm) / 32 (O 2} ) ] = about 28 mg. That is, the dynamic range of the DO meter used for the histamine quantification method is 0 to 28 mg / liter, that is, 0 to 28 p.
pm, and the decrease in histamine by the amine oxidase reaction can be confirmed by calculation as 1/5 of the full range.
0, ie at least about 0.5 mg / liter. That is, in the above method, the lower limit of quantification of histamine is about 0.5 mg / liter, and it is impossible to further lower the lower limit of quantification. For this reason, in the conventional method, the lower limit of quantification of histamine is about 0.5 mg for 100 g of fish meat (a 10-fold dilution is usually used for measurement) (see the above academic report 2).
(See "Summary Orchids"), and it was thought that it could not be quantified with higher sensitivity.

[0006] On the other hand, the allowable value of histamine in seafood by the US FDA ｛Federal Register, Vol.60, Aug.3, p.3975
4-39756, Compliance Policy Guide (CPG) of Histamine
(1995)｝ is less than 50 ppm (5 mg / 100 g), so this method can be used for the purpose of screening test for freshness and safety of fish meat in the United States. However, in the cited documents considered for the above-mentioned allowable value determination,
The histamine level of fresh fish immediately after catch is 2 ppm (0.2 mg).
/ 100 g) or less, and the histamine level of canned tuna that is classified as acceptable by the FDA is 6 ppm (0.6 m
g / 100g) is stated in the Federal Register.
The development of a method for accurate measurement has been desired.

[0007]

SUMMARY OF THE INVENTION The present invention has an object
It is an object of the present invention to provide a highly sensitive histamine quantification method capable of quantifying histamine at a concentration lower than g / liter, that is, 0.5 ppm.

[0008]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to solve the above problems, and as a result, histamine oxidase is reacted with a histamine-containing sample solution, and H ₂ O ₂ generated is electrically transferred to a peroxidase electrode. By chemically detecting and quantifying the histamine concentration, about 0.5
It has been discovered that it is possible to quantify histamine at concentrations even lower than mg / l, ie 0.5 ppm.

Further, histamine oxidase is reacted with the histamine-containing sample solution, and the reaction solution is heated, a chelating agent is added to the reaction solution, or after both treatments, the reaction solution is added to the histamine-containing sample solution. When the generated H ₂ O ₂ is electrochemically detected with a peroxidase electrode, it has been found that, besides the above, the reliability of the measured value of the histamine concentration is extremely improved.

[0010] In the above histamine quantification method,
As a peroxidase electrode, graphite powder, liquid paraffin, electron carrier and peroxidase are uniformly kneaded and embedded in one end of a cylindrical insulator,
When this part is used as a substrate-sensitive part, a peroxidase-immobilized membrane is formed on the surface thereof, and an electrode whose outer surface is further coated with a dialysis membrane is used. It has been discovered that measurements can be made quickly and accurately.

[0011] Further, in the method for quantifying histamine,
As a peroxidase electrode, graphite powder, liquid paraffin, electron carrier and peroxidase are uniformly kneaded and embedded in one end of a cylindrical insulator,
When this part is used as a substrate sensitive part, the surface of which is contacted with a polyethyleneimine solution to form a film, a peroxidase-immobilized membrane is formed, and the electrode whose outer surface is coated with a dialysis membrane is used as described above. In addition, they found that the reliability of histamine measurements was further improved.

The present invention has been completed based on these findings, that is, (1) H ₂ produced by reacting histamine oxidase with a histamine-containing sample solution.
A highly sensitive histamine quantification method characterized by detecting O ₂ with a peroxidase electrode and quantifying the histamine concentration. (2) The present invention further comprises reacting histamine oxidase with a histamine-containing sample solution. Is heated, or a chelating agent is added to the reaction solution, or after both treatments, H ₂ O ₂ generated in the reaction solution is detected by a peroxidase electrode to quantify the histamine concentration. (3) The present invention provides a method for quantifying histamine by reacting a histamine-containing sample solution with histamine oxidase.
This is a highly sensitive histamine quantification method characterized by detecting ₂ O ₂ with the following peroxidase / carbon paste electrode and quantifying the histamine concentration. (Peroxidase carbon paste electrode) Graphite powder, liquid paraffin, electron carrier and peroxidase are uniformly kneaded and embedded at one end of a cylindrical insulator, and this part is used as a substrate sensitive part, and a peroxidase immobilized film is formed on the surface. And an electrode whose outer surface is coated with a dialysis membrane. (4) Further, the present invention is characterized in that histamine oxidase is reacted with a histamine-containing sample solution, and H ₂ O ₂ produced is detected by the following peroxidase-carbon paste electrode to quantify the histamine concentration. This is a highly sensitive quantitative method. (Peroxidase carbon paste electrode) Graphite powder, liquid paraffin, electron carrier and peroxidase are uniformly kneaded and embedded at one end of a cylindrical insulator, and this portion is used as a substrate sensitive portion, and the surface thereof is contacted with a polyethyleneimine solution. Thereafter, a peroxidase-immobilized membrane was formed, and the outer surface thereof was further covered with a dialysis membrane.

[0013]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.

Examples of the histamine-containing sample solution include any sample solution containing histamine. For example, a sample of fish and shellfish or meat is extracted by a trichloroacetic acid extraction method using a conventional method.
A sample solution prepared by a methanol extraction method, a neutral phosphate buffer heating extraction method, or the like is preferable.
7, No. 2, 109-113 "Improvement of fish meat histamine determination method using oxygen sensor and comparison with AOAC method"
reference).

Although any enzyme can be used as histamine oxidase, a research report by Adachi [H. YAM
ADA, O.D. Adachi and K.S. Ogata,
Agr. Biol. Chem. Vol. 29, NO.
9, p. 649-654 (1963)], an enzyme is extracted from cells belonging to Aspergillus niger cultured in a medium using an amine as a nitrogen source, and is subjected to ammonium sulfate precipitation, DEAE-cellulose column chromatography and the like. After purification, it is preferably purified by affinity chromatography and hydroxyapatite column chromatography, purified by dialysis and concentration using glycerin, and suspended in an approximately 2 M aqueous solution of ammonium sulfate. This enzyme is a copper-containing amine oxidase (enzyme number EC1.
4.3.6) and has an activity of 10 ⁵ U / ml as measured by the Tabor method.

Other histamine oxidases include those of the genus Arthrobacter (eg, Arthrobacter).
er globiformis IFO12137 strain)
Histamine oxidase produced by culturing a histamine oxidase-producing bacterium belonging to the group described above in a medium to produce and accumulate histamine oxidase, and collecting the histamine oxidase (see Japanese Patent Application Laid-Open No. 5-236952, a novel histamine oxidase and a method for producing the same).

When a predetermined amount of such histamine oxidase is added to a predetermined amount of the histamine-containing sample solution and mixed well, the reaction of the above formula (1) proceeds rapidly under the reaction conditions described below. H ₂ O ₂ is generated in the liquid.

As a specific example of this apparatus, a comparison (standard)
An Ag / AgCl electrode and a counter electrode (C
A device capable of electrochemically detecting H ₂ O ₂ by voltammetry for measuring a current corresponding to a voltage by a three-electrode system using a Pt electrode as E) and a peroxidase electrode of the present invention as a working electrode (WE). Is mentioned.

As the peroxidase electrode used herein, a conventionally known peroxidase electrode can be mentioned, and a peroxidase-carbon paste electrode developed by the present inventors is particularly preferable.

The above-mentioned peroxidase / carbon paste electrode comprises graphite powder: liquid paraffin: electron carrier (eg, ferrocene): peroxidase (peroxidase) in a ratio of 10: 4 to 6: 0.
Kneaded at a weight ratio of 5 to 1.5: 0.01 to 1.0 and embedded in one end (tip) of a tubular insulator 1 (for example, a hollow glass tube) as shown in FIG. The substrate-sensitive portion 2 was used, and its exposed surface (tip surface) was placed at 0.
After soaking in a 1-3% polyethylenimine solution for 1 minute or more, especially for 3 to 30 minutes, forming a film and washing, a 0.1 to 100 unit / μl peroxidase solution for 1 minute or more, particularly 3 minutes. After immersion for about 30 minutes, the membrane was washed to form a peroxidase enzyme membrane 3 immobilized on the substrate-sensitive part, and the outer surface thereof was further covered with a dialysis membrane 4 of 1 to 40 μm and fixed with an O-ring 5 or the like.
Is connected to a substrate sensitive part 2 and the other end is used as a working electrode by connecting to an apparatus capable of electrochemically detecting H ₂ O ₂ (not shown for simplicity of the drawing).

When polyethyleneimine is brought into contact with (adsorbed to) the surface of the peroxidase electrode, a reduction current proportional to the addition of H ₂ O ₂ can be stably obtained under an applied voltage (for example, around 0 mV). It is preferable because it is possible.

Further, as a component of the peroxidase electrode,
It is preferable to use an electron carrier (for example, ferrocene) because, similarly to the above, a proportional reduction current can be obtained with the addition of H ₂ O ₂ .
As the electron carrier, ferrocene as described above, p-
Benzoquinone, potassium ferricyanide, dibromoid salicylic acid, dichlorophenyl indophenol (DCIP), phenazine methosulfate (P
MS) and the like.

Hereinafter, the present invention will be described more specifically with reference to Reference Examples and Examples.

Reference Example 1 (Preparation of Peroxidase-Carbon Paste Electrode Used for Histamine Determination Method of the Present Invention) In FIG. 1, a tubular insulator 1 was a hollow glass tube having an outer diameter of 6 mm and an inner diameter of 3 mm, and Graphite powder: liquid paraffin: ferrocene
ne): 1 peroxidase
The mixture was kneaded at a ratio of 0: 5: 1: 0.1 (weight ratio) and buried to a position 50 mm deep from the lower end opening surface, and this portion was used as the substrate sensitive portion 2, and the exposed surface (lower end surface) was 0.1 mm. 6
(Weight)% Polyethylenimine P
After immersing in a -70 solution for 10 minutes to form a film and washing,
The substrate was immersed in a unit / μl peroxidase solution for 10 minutes, washed to form a peroxidase enzyme membrane 3 immobilized on the substrate-sensitive part, and the outer surface was further polished to 20 μm.
It was covered with a dialysis membrane 4 of μm and fixed with an O-ring 5 to prepare a peroxidase / carbon paste electrode (treated with polyethyleneimine).

Reference Example 2 (Preparation of Polarographic Hydrogen Peroxide Measuring Device Incorporating Peroxidase Prepared in Reference Example 1) Referring to FIG. 2, a conventionally known polarographic voltage applying device (A) and a potential difference measuring device were used. The device (B) and the current measuring device (C) were electrically connected as shown to form a circuit.
The above-mentioned circuit is associated with the above-mentioned peroxidase / carbon paste electrode as a WE (working electrode), an Ag / AgCl electrode as a RE (reference electrode), and a Pt electrode as a CE (counter electrode), as shown in FIG. A polarographic hydrogen peroxide measuring device for measuring a current corresponding to a voltage was prepared by an electrode system. D indicates a reaction cell. The measurement of H ₂ O ₂ was carried out in this reaction cell (capacity 4
ml) in 0.1 M phosphate buffer (pH 7.5), 1 mM
・ During EDTA (performed in the cell, constant applied voltage (for example, E = −
This is performed by electrochemically detecting the reduction current at 0.02 V vs. Ag / AgCl).

Reference Example 3 (Preparation of Polarographic Hydrogen Peroxide Measurement Apparatus as Control)
In the preparation of the peroxidase-carbon paste electrode of Reference Example 1 described above, "immerse in a 0.6% (by weight)% polyethylenimine P-70 solution for 10 minutes".
A peroxidase / carbon paste electrode for comparison (without polyethyleneimine treatment) was prepared in exactly the same manner except that no treatment was performed, and this was used in the same manner as in Reference Example 2 to obtain a control polarographic hydrogen peroxide. A measuring device was created.

Reference Example 4 (Measurement of H ₂ O ₂ Using Two Types of Polarographic Hydrogen Peroxide Measurement Apparatus Prepared in Reference Examples 2 and 3) FIG. 2
In the polarographic hydrogen peroxide measurement device shown in
A 0.1 M phosphate buffer (pH 7.5), 1 mM EDTA, and 37 ° C. were placed in a reaction cell (volume: 4 ml), and adjusted to a constant applied voltage of −20 mV.
_{A 2} O ₂ aqueous solution was added (injected) at an appropriate interval (about 2 minutes) at 5 to 30 µl, and the change in response current was measured. For comparison, a change in response current was similarly measured using a control polarographic hydrogen peroxide measuring device. The results are summarized in FIG.

From the results of FIG. 3, as in the comparative example,
When a peroxidase-carbon paste electrode not subjected to polyethyleneimine treatment is used, when a small amount of H ₂ O ₂ solution is added, a steady current cannot be obtained as shown by a broken line. When an imine-treated electrode is used, a steady current can be obtained with good reproducibility as shown by the solid line.

Reference Example 5 (Measurement of H ₂ O ₂ using the polarographic hydrogen peroxide measuring device obtained in Reference Example 2) Reaction cell (capacity: 4
ml), 0.1 M phosphate buffer (pH 7.5), 1
mM EDTA, 37 ° C, constant applied voltage -25mV
After adjusting to 50 μM · H ₂ O ₂ aqueous solution,
30 μl was added at appropriate intervals (about 2 minutes), and the change in response current was measured. FIG. 4 shows the results. From this result, it can be seen that a current response appears quickly with the addition of H ₂ O ₂ indicated by the arrow, and reaches a substantially steady value within 2 minutes. Further, the added volume of H ₂ O ₂ was changed from 10 μl to 30 μl.
It was found that the response of the reduction current also changed correspondingly.

Reference Example 6 (Measurement of H ₂ O ₂ Using Polarographic Hydrogen Peroxide Measuring Apparatus Obtained in Reference Example 2, Preparation of Calibration Curve) In a reaction cell (capacity: 4 ml) of the apparatus, 0.1 μm was added. 1M phosphate buffer (p
H7.5) 1 mM EDTA, at 37 ° C., and after adjusting to a constant applied voltage of −25 mV, a reduction current response when an H ₂ O ₂ aqueous solution was added thereto and the concentration of H ₂ O ₂ was changed. A calibration curve was created based on the above. In addition, as the detection limit,
A signal obtained by adding three times the standard deviation to a blank signal was used. The result is shown in FIG. From the results of FIG. 5, it can be seen that a good proportional relationship is exhibited in the H ₂ O ₂ concentration range of 100 to 1500 nM, and the detection limit is 20 nM.
It was found that detection at the level was easy. In addition, even when this measurement test was repeated every day, it was confirmed that the operation was stable and reproducible within two weeks.

Example 1 Test for confirming the time required for the histamine oxidase reaction using the polarographic hydrogen peroxide measuring device obtained in Reference Example 2) Examination of the histamine oxidase reaction time in the enzyme analysis method for histamine by the end point method did. 20
0.1 M sodium phosphate buffer containing μM histamine (pH 7.5, 0.015 unit / m 2 at 37 ° C.)
When 1 histamine oxidase was added, the amount of increase in the steady-state current value corresponding to H ₂ O ₂ generated in 100 μl of the enzyme reaction mixture was measured. The result is shown in FIG. The results in FIG. 6 show that the reaction reaches the end point in about 3 minutes.

Example 2 (Elimination test of catalase activity contained in trace amount in histamine oxidase) In a polarographic hydrogen peroxide measuring apparatus obtained in Reference Example 2, 0.
1 M sodium phosphate buffer (pH 7.5), 1 mM
EDTA and 37 ° C. were charged, and the applied voltage was adjusted to −10 mV. Then 0.1 M containing 20 μM histamine
Sodium phosphate buffer (pH 7.5) at 37 ° C
Histamine oxidase at 0.015 unit / ml was added to carry out an enzymatic reaction. The reaction solution was divided into six sections a) to f). As shown in FIG. 7, EDTA was added as a chelating agent to the sections b), d) and f) so as to have a final concentration of 1M. ), C), and e) are heated (80 ° C., required time) under the conditions shown in FIG. 7 without adding EDTA to each of the sections, and the obtained pieces are separated by a predetermined amount at predetermined intervals. The sample was placed in a reaction cell, and the change in response current was measured. FIG. 7 shows the result.

From the results shown in FIG. 7, it is presumed that the steady-state current decreases shortly after the reaction, and that histamine oxidase exhibits a weak catalase activity when the substrate histamine is decomposed and consumed to some extent. a),
As in the categories c) and e), it can be seen that the heating at 80 ° C. suppresses the catalase activity contained in the histamine oxidase in a trace amount, and is completely suppressed by maintaining the same temperature for 3 minutes or more. Also, as in the categories b), d) and f), 1 mM EDT was added to the histamine oxidase reaction solution.
It can be seen that even when A was added, the catalase activity was completely suppressed.

Example 3 A 0.1 M sodium phosphate buffer (pH 7.5), 1 mM EDTA, and the like were placed in the reaction cell (capacity: 4 ml) of the polarographic hydrogen peroxide measuring device obtained in Reference Example 2 above.
After adding 37 ° C. and adjusting to a constant applied voltage of −20 mV, histamine oxidase was added to a histamine standard solution having an initial concentration of 20 to 80 μM, and an enzymatic reaction was carried out at 37 ° C. for 3 minutes. The response current when a fixed amount of "mixture" was added to the test solution was examined. The result is shown in FIG.
Shown in From this result, it can be seen that a current response appears quickly with the addition of the enzyme reaction mixture indicated by the arrow, and reaches a substantially steady value within 2 minutes. It was also found that when the histamine concentration was changed to 20 to 80 µM, the response of the reduction current also changed correspondingly. On the other hand, in FIG. 8, it was confirmed that there was no current response even when the unreacted enzyme solution was added (arrow a).

From the results of Example 3 and FIG. 8, it can be seen that the histamine oxidase enzyme reaction solution has a current response similar to that of the H ₂ O ₂ aqueous solution, and the enzyme unreacted solution has no current response. It can be seen that the current response of the liquid is attributable (derived) to H ₂ O ₂ generated during the enzymatic reaction. Also, when the amount of the reaction solution to be added is changed,
It can be seen that the response of the reduction current changes correspondingly.

Example 4 (Measurement of histamine concentration in histamine oxidase reaction solution using polarographic hydrogen peroxide measuring device obtained in Reference Example 2, preparation of calibration curve) In a reaction cell (capacity: 4 ml) of the same device , 0.1 M sodium phosphate buffer (pH
7.5) 1 mM EDTA and 37 ° C. were added, and a constant applied voltage of −20 mV was adjusted.
Histamine oxidase was added to an 80 μM histamine standard solution, an enzymatic reaction was carried out at 37 ° C. for 3 minutes, a fixed amount of the resulting reaction mixture was added to the test solution, and the reduction current of the histamine concentration was changed. A calibration curve was created based on the response. FIG. 9 shows the result.

From the results shown in FIG. 9, it was confirmed that the histamine concentration in the histamine-containing sample solution and the response current showed a good proportional relationship in the range of histamine concentration of 100 to 1000 nM. Was found to be detectable at several tens of nM level.

Example 5 (Quantitative Method of Histamine in Tuna Fish Meat Sample)
A histamine-containing sample solution (histamine extract) was prepared from the tuna fish meat stored on the day based on the phosphoric acid heating extraction method of Nomura et al. That is, 5 g of fish meat is crushed and
After suspending in 1M phosphate buffer (pH 7.9), 90
Filling up to 50 ml after heating extraction for 15 minutes
Then, the supernatant obtained by centrifugation was used as a sample solution. Further, 5% of the sample extract was added to 0.1 M phosphate buffer (pH 7.9), histamine was further added to 50 μM, and histamine oxidase was added to 0.03 unit / ml. Perform enzyme reaction for 2-7 minutes at
Next, the mixture was heated at 80 ° C. for 3 minutes to prepare a reaction mixture.
In a reaction cell (volume 4 ml) of the polarographic hydrogen peroxide measuring device obtained in Reference Example 2, 0.1 M sodium phosphate buffer (pH 7.5), 1 mM EDTA, 37
After the temperature was adjusted to a constant applied voltage of −20 mV, a certain amount of the “reaction mixture” was added thereto, and the current value was measured. The result is shown in FIG. From the results shown in FIG. 10, it can be seen that the enzyme reaction reaches the end point in a short time of 7 minutes, and histamine can be quantified.

Example 6 In Example 5, histamine was quantified using the standard addition method in consideration of the effect of enzyme inhibition, and the histamine oxidase reaction condition was 0.03 unit / m
The reaction was carried out at 37 ° C for 7 minutes to reach equilibrium, and then heat treatment was carried out at 80 ° C for 3 minutes. Histamine was quantified by the standard addition method. The result is shown in FIG. For comparison, quantification was similarly performed for a sample solution to which histamine oxidase was not added.

From the results shown in FIG. 11, when the sample solution containing no enzyme was used (see the arrow in FIG. 11), no current response was observed, and no substance which directly gave a current response was present in the extracted sample. It turns out that can be confirmed. In addition, such a remarkable current response is observed in the enzyme reaction solution, and it can be seen that the current response changes linearly (proportionally) according to the histamine concentration in each sample changed according to the standard addition method (FIG. 11).
See the graph in the inset of). From the above results, it can be seen that according to the present invention, the amount of histamine in an actual sample such as raw fish meat can be quantified accurately and with high sensitivity without requiring any pretreatment other than the enzyme reaction.

[0041]

According to the present invention, a very small amount of histamine concentration of 0.5 ppm or less can be quickly and accurately determined. When a peroxidase / carbon paste electrode not treated with polyethyleneimine is used, the response current is unstable when attempting to determine a trace amount of H ₂ O _2. However, when the electrode treated with polyethyleneimine is used, the response current is unstable. The response current reaches a steady state, and a current with good reproducibility can be obtained. The polarographic hydrogen peroxide measuring device obtained in the present invention has a feature that it operates stably and with good reproducibility within two weeks even when the measurement test is repeated every day. Moreover, the electrochemical reaction by the peroxidase electrode reaches the end point in about 3 minutes,
Histamine quantification can be processed in a very short time. Histamine in raw fish meat such as tuna can be quantified with high sensitivity without requiring any pretreatment other than enzymatic reaction. As shown in the Examples, the present invention is suitable for rapid quantification of histamine, which is an international evaluation item of freshness and safety of fish meat. Furthermore, because of its extremely high quantitative sensitivity, diagnosis of food allergy, which has recently become a major social problem, and medical applications for its countermeasures are expected. It should be noted that a voltage corresponding to a voltage was determined by a three-electrode method using an Ag / AgCl electrode as a reference electrode (RE), a Pt electrode as a counter electrode (CE), and a peroxidase / carbon paste electrode of the present invention as a working electrode (WE). The apparatus for electrochemical measurement for measuring H ₂ O ₂ by voltammetry (polarography) for measuring the current flowing is small in size and light in weight, and therefore, the manufacturing cost is relatively low and economical. . Therefore, it can be effectively used as a means of "in situ" measurement in a fishing boat, a fishery processing plant, or a histamine poisoning accident site.

[Brief description of the drawings]

FIG. 1 is a schematic vertical sectional view showing a peroxidase-carbon paste electrode used in the histamine quantification method of the present invention.

FIG. 2 is a schematic explanatory view showing a polarographic hydrogen peroxide measuring device using the same electrode.

FIG. 3 is a diagram showing the relationship between the presence or absence of a polyoxyethylene imine treatment on the surface of the electrode and the response current.

FIG. 4 is a diagram showing a result of a response current when an H ₂ O ₂ solution is added stepwise.

FIG. 5 is a diagram showing a relationship between H ₂ O ₂ concentration and response current (hydrogen peroxide calibration curve).

FIG. 6 shows the relationship between histamine oxidase reaction time and response current.

FIG. 7 is a graph showing the effect of heating the histamine oxidase reaction-finished solution and the effect of adding EDTA to the reaction solution in the histamine quantification method of the present invention.

FIG. 8 is a diagram showing the results of response current when a histamine oxidase enzyme reaction solution was added stepwise.

FIG. 9 is a graph showing the relationship between histamine concentration and response current (histamine calibration curve).

FIG. 10 shows the relationship between histamine degradation reaction time and response current of histamine oxidase in tuna fish meat extract.

FIG. 11 is a graph showing the relationship between histamine concentration in tuna fish meat and response current (calibration curve).

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Tubular insulator (hollow glass tube) 2 ... Substrate sensitive part 3 ... Peroxidase enzyme membrane 4 ... Dialysis membrane 5 ... O-ring 6 ... Electric wire A ... Polarographic voltage applying device B ... Potential difference measuring device C ... Current measuring device D ... Reaction cell WE ... Working electrode (peroxidase / carbon paste electrode) RE ... Reference electrode (Ag / AgCl electrode) CE ... Counter electrode (Pt electrode)

Claims (4)

[Claims] 1. A highly sensitive quantification method for histamine, which comprises reacting histamine oxidase with a histamine-containing sample solution, detecting the generated H ₂ O ₂ with a peroxidase electrode, and quantifying the histamine concentration. 2. A histamine oxidase is reacted with a histamine-containing sample solution, and the reaction solution is subjected to heat treatment, a chelating agent is added to the reaction solution, or both treatments are performed. sensitive method for the determination of histamine, which comprises quantifying the histamine concentration and the resulting H ₂ O ₂ was detected by peroxidase electrode. 3. A highly sensitive quantification method for histamine, which comprises reacting histamine oxidase with a histamine-containing sample solution, detecting H ₂ O ₂ produced by a peroxidase-carbon paste electrode described below, and quantifying the histamine concentration. . (Peroxidase carbon paste electrode) Graphite powder, liquid paraffin, electron carrier and peroxidase are uniformly kneaded and embedded at one end of a cylindrical insulator, and this part is used as a substrate sensitive part, and a peroxidase immobilized film is formed on the surface. And an electrode whose outer surface is coated with a dialysis membrane. 4. reacted histamine oxidase histamine-containing sample solution, the resulting H ₂ O ₂ was detected by the following HRP-carbon paste electrode sensitive method for the determination of histamine, which comprises quantifying the histamine concentration . (Peroxidase carbon paste electrode) Graphite powder, liquid paraffin, electron carrier and peroxidase are uniformly kneaded and embedded at one end of a cylindrical insulator, and this portion is used as a substrate sensitive portion, and the surface thereof is contacted with a polyethyleneimine solution. Thereafter, a peroxidase-immobilized membrane was formed, and the outer surface thereof was further covered with a dialysis membrane.