JPS6250120B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for simply and quickly measuring inosine and hypoxanthine, which are produced as the freshness of fish meat, livestock meat, etc. decreases, and a test paper used in this method. In recent years, scientifically determining the freshness of fish and meat has become an important issue in the distribution of fresh foods from the viewpoint of quality control and price optimization. Traditionally, it has been used as an indicator to judge the freshness of fish meat, etc.
It has been proposed to measure trimethylamine, PH, volatile basic nitrogen, etc. in fish meat, but this is not necessarily a satisfactory indicator for understanding the so-called "quality of life." It is known that a more effective indicator for determining the viability of fish meat, etc. is to examine the evolution of decomposition products of adenosine triphosphate (ATP), which is an energy source for animals. Adenosine triphosphate decomposes as follows, and as freshness decreases, inosine and hypoxanthine are generated and accumulate in fish meat. Adenosine triphosphate (ATP) → adenosine diphosphate (ADP) → adenosine monophosphate (AMP)
→ Inosinic acid (IMP) → Inosine (HxR) → Hypoxanthine (Hx) Therefore, inosine in fish meat (hereinafter abbreviated as HxR) is used to judge freshness.
It is important to quantify and hypoxanthine (hereinafter abbreviated as Hx). As one of the simple methods for quantifying HxR and Hx, the following method using an enzyme has been proposed by the same applicant as the subject, and a patent application has already been filed. Patent application 1986-225491). That is, this prior invention allows nucleoside phosphorylase, xanthine oxidase, tetrazolium salt, and oxygen scavenger to act on a sample solution containing HxR and/or Hx, and determines the HxR in the sample solution from the density of the formazan dye produced.
and/or measuring Hx concentration. The nucleoside phosphorylase used in this prior invention is an enzyme listed in Enzyme Classification Tables 2, 4, 2, and 1, and phosphorolyzes HxR to Hx. On the other hand, xanthine oxidase is enzyme classification table 1, 2, 3, 2
It is an enzyme that oxidizes Hx and breaks it down into uric acid.
The reason for using these two types of enzymes together is as follows. In other words, depending on the species of fish, there are those that degrade ATP to Hx and those that only degrade it to HxR. When xanthine oxidase is used alone, it does not affect HxR and therefore the amount of HxR cannot be measured, but If phosphorylase is used in combination, HxR will be decomposed into Hx, so the Hx generated by this decomposition and the Hx originally contained in the sample will be
The total amount of uric acid undergoes oxidative decomposition by xanthine oxidase and is converted into uric acid. Since a tetrazolium salt, which is a redox dye, is present in the oxygen reaction system as described above, when xanthine oxidase oxidizes Hx to uric acid, it simultaneously cyclizes the tetrazolium salt to generate a formazan dye.
The amount of formazan dye produced depends on the amount of formazan dye present in the reaction system.
It changes depending on the amount of Hx; if the amount of Hx is large, the amount of formazan dye produced increases, resulting in a dark color, and if the amount of Hx is small, the amount of formazan dye produced is decreased, resulting in a pale color. Therefore, by measuring the density of the formazan dye, it is possible to determine the amount of Hx in the reaction system. In the reaction system at this time
The amount of Hx corresponds to the total amount of HxR and Hx in the sample solution. In this prior invention, neotetrazolium chloride or triphenyltetrazolium chloride is used as the tetrazolium salt, but the formazan dye production reaction by reduction of these tetrazolium salts does not proceed effectively if oxygen is present in the reaction system. In order to be able to carry out this measurement method in the atmosphere, it is necessary to have an oxygen scavenger such as sodium sulfite present in the reaction system. However, in order to carry out the method of the above-mentioned prior invention even more easily and quickly, when using a test paper in which an enzyme, a tetrazolium salt, and an oxygen scavenger have been absorbed in advance into an immobilizing material such as a filter paper, 2. An enzyme-dye paper that has absorbed various enzymes and a tetrazolium salt, and an oxygen-absorbing paper that has absorbed an oxygen scavenger must be prepared separately. This is because, if absorbed in the same fixative, the enzyme will be deactivated by the oxygen scavenger during the preparation and storage of the test strip. Also,
When using test paper, overlap the enzyme-dye paper and oxygen-absorbing paper, and make sure to contact the sample solution made by homogenizing fish meat etc. from the oxygen-absorbing paper side of the test paper, and then wait until the color develops on the enzyme-dye paper side. It is necessary to determine the shading. This is because if the enzyme is contacted from the dye paper side, the formazan dye production reaction by reduction of the tetrazolium salt in a deoxidized state will not occur reliably. When carrying out this test, the use of an oxygen scavenger is essential, which is complicated in the preparation and use of test strips. Therefore, the present invention has been improved so that the reduction of the tetrazolium salt and the reaction for producing the formazan dye can be carried out reliably and effectively without the use of an oxygen scavenger, which was essential in the above-mentioned earlier invention.
The purpose of this study was to provide a method for measuring HxR and Hx, as well as test strips for the measurement. Among the tetrazolium salts, the present inventors have particularly identified iodonitrotetrazolium chloride (formal name: 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl-2H-tetrazolium chloride) and nitroblue-tetrazolium chloride (formal name: 3,3'-(3,3'-dimethoxy-4,4'-biphenylylene)-bis(2-(p-
They found that when nitrophenyl-5-phenyl-2H-tetrazolium chloride) was used, it was reduced even in the presence of oxygen to easily form a formazan dye, and the present invention was completed. That is, the present invention allows nucleoside phosphorylase, xanthine oxidase, and a tetrazolium salt selected from iodonitrotetrazolium chloride or nitroblue tetrazolium chloride to act on a sample solution containing HxR and/or Hx, and determines the concentration of formazan dye in the sample based on the density of the produced formazan dye. This is a method for measuring HxR and/or Hx, which is characterized by measuring the HxR and/or Hx concentration of. This makes it possible to effectively measure HxR and Hx in the presence of oxygen without using an oxygen scavenger. When carrying out the present invention, first the above-mentioned two
A solution is prepared by dissolving various types of enzymes and tetrazolium salts. In addition, in order to keep the activity of the enzyme stable in the solution, it is desirable to add a phosphate buffer with a pH of 7.5 to 8 to the solution. Next, a sample solution obtained by homogenizing fish meat or the like is brought into contact with the solution and left for several minutes. During this time, the enzyme decomposes HxR in the sample solution into Hx, oxidizes Hx to uric acid,
The accompanying reduction of the tetrazolium salt occurs and a formazan dye having absorption in the visible region is produced, as in the prior invention described above. It goes without saying that the above coloring reaction can also be carried out in a vacuum if necessary. It is possible to visually observe the density of the formed formazan dye, but for more accurate measurement, extract the insoluble formazan dye from the reaction solution using an extraction solvent such as ethyl acetate, and then extract the insoluble formazan dye from the reaction solution. It is desirable to determine the shade of color by measuring the absorption in the visible region. The present invention can also be carried out more simply and quickly by using a test strip that has been preabsorbed with an enzyme and a tetrazolium salt, as in the prior invention. . That is, two types of enzymes and a tetrazolium salt are made into an aqueous solution, absorbed into a filter paper, etc., and then dried using a freeze-vacuum drier to prevent deactivation of the enzymes to prepare a test paper. In this case as well, it is desirable to use a phosphate buffer to keep the activity of the enzyme stable, and to add an antioxidant and a stabilizer to the aqueous solution to prevent deactivation of the enzyme. When using the test paper thus obtained, a sample solution prepared by homogenizing fish meat or the like is brought into contact with the test paper and left for 5 to 10 minutes, after which the intensity of color development on the test paper is determined. The shade of color on the test paper can be easily determined visually using a color chart, etc., and if the relationship between the color chart reading and the concentration of HxR and Hx is determined in advance, the sample can be determined from the color chart reading. HxR in liquid, Hx
The concentration can be quantified. In addition to filter paper, gelatin, collagen, etc. can be used as a fixing material for preparing test strips, if necessary. As can be seen from the above explanation, according to the method of the present invention, since iodonitrotetrazolium chloride or nitroblue-tetrazolium chloride, which can be reduced even in the presence of oxygen to form a formazan dye, is used as a tetrazolium salt, no oxygen scavenger is used. Both can effectively measure HxR and Hx. In particular, when carrying out the present invention using test paper, it is necessary to separately prepare enzyme-dye paper and oxygen-absorbing paper, as in the case of the prior invention using an oxygen scavenger, or to overlap them to remove oxygen. There is no need to contact the sample from the paper side, and there is an advantage that a single test paper in which the enzyme and the tetrazolium salt are absorbed in the same immobilizing material can be prepared and used. The present invention will be further explained below with reference to Examples. Example 1 10mM Iodonitrotetrazolium chloride
0.1 ml, 0.1 enzyme unit of xanthine oxidase, and 0.4 enzyme unit of nucleoside phosphorylase were mixed, and the mixture was made up to a constant volume with phosphate buffer at pH 7.8. A substrate solution is added so that the HxR or Hx concentration in this mixed solution is approximately 2, 5, 10, 20, 40, and 60 μM, respectively, and the mixture is left to stand to produce color-forming insoluble formazan produced by reduction of the tetrazolium salt. Ta. Next, add the same amount of ethyl acetate to this solution and shake it to extract the color-developing insoluble formazan into the ethyl acetate phase, and calculate the absorbance of this extract.
Measurements were made at a wavelength of 505 nm. FIG. 1 shows the relationship between Hx concentration and absorbance, and FIG. 2 shows the relationship between HxR concentration and absorbance. As can be seen from the graphs in FIGS. 1 and 2, by the measuring method of the present invention, it is possible to accurately quantify HxR or Hx by color development in the visible region and in the atmosphere. Example 2 10mM Nitroblue Tetrazolium Chloride
0.1 ml, 0.1 enzyme unit of xanthine oxidase, and 0.4 enzyme unit of nucleoside phosphorylase were mixed, and the mixture was made up to a constant volume with phosphate buffer at pH 7.8. A substrate solution is added so that the HxR or Hx concentration in this mixed solution is approximately 2, 5, 10, 20, 40, and 60 μM, respectively, and the mixture is left to stand to produce color-forming insoluble formazan produced by reduction of the tetrazolium salt. Ta. Next, add the same amount of ethyl acetate to this solution and shake it to extract the color-developing insoluble formazan into the ethyl acetate phase, and calculate the absorbance of this extract.
Measurements were made at a wavelength of 550 nm. The relationship between HxR concentration and absorbance is shown in FIG. 3, and the relationship between Hx concentration and absorbance is shown in FIG. 4. As can be seen from the graphs in FIGS. 3 and 4, by the measuring method of the present invention, it is possible to accurately quantify HxR or Hx by color development in the visible region and in the atmosphere. Example 3 5 ml of phosphate buffer at pH 7.8, 0.1 mmoles of iodonitrotetrazolium chloride, 10 enzyme units of xanthine oxidase, 20 enzyme units of nucleoside phosphorylase, 1 g of sucrose (stabilizer), 0.2 % mercaptoethanol (antioxidant) An enzyme-dye solution was prepared by adding water to make the volume 15 ml. This solution was absorbed into a chromatographic filter paper, and then freeze-dried under vacuum to produce a test paper of the present invention. HxR or Hx concentration is 0.02, 0.05, respectively
A substrate solution prepared to be 0.10, 0.15, 0.20, 0.30, 0.40mM was dropped onto the test paper of the present invention, and
After 10 minutes, check the color density of the test paper using a color chart (JIS Z-
8721). The results are shown in Table 1. As can be seen from Table 1, according to the test paper of the present invention, HxR or Hx concentration can be measured based on the shade of color (reddish-purple in the visible region) of the test paper.

[Table] Example 4 Carp were immediately killed and stored on ice, and samples were taken at intervals of a certain period of time, and 9 times the volume of water was added to homogenize to prepare a sample solution. This sample liquid was dropped onto the test paper of the present invention prepared in Example 3, and the shade of color on the test paper was measured using a color chart in the same manner as in Example 3. From the reading of the color chart, HxR in the sample solution was measured. and
The total concentration of Hx was determined. The results are shown in Table 2. In parallel with the measurement using the above-mentioned test paper of the present invention,
The concentrations of HxR and Hx were measured using a conventional column method. In measurements using this column method, the sample solution prepared above is treated with perchloric acid to remove protein, and then separated and fractionated using a column filled with ion exchange resin (strongly basic anion exchange resin). in the sample solution by measuring the absorbance of
The total concentration of HxR and Hx was determined. The results are also listed in Table 2.

[Table] As can be seen from Table 2, the method using the test paper of the present invention is less accurate than the column method, but it can be performed extremely easily and quickly in the field.
This can be effectively used to judge the freshness of fish meat during the distribution process. Example 5 Pacific saury, sardines, and sardines sold at retail stores
6 types of raw mackerel, salmon, tuna, and yellowtail
Muscle parts were collected from 14 samples (sample numbers No. 1 to No. 14), and respective sample solutions were prepared in the same manner as in Example 4. The total concentration of HxR and Hx in these sample solutions is
Measurement was carried out in the same manner as in Example 4 using the method using the test paper of the present invention obtained in Example 3 and the conventional column method. Furthermore, the K value (freshness constant) was determined using the test paper method of the present invention and the conventional column method. The K value is especially used as a measure of the freshness of fish, and it measures the total ATP-related compounds (ATP, ADP, AMP, IMP, HxR, Hx).
It is the percentage of HxR + Hx amount to the total amount, and is calculated by the following formula: K (%) = HxR + Hx / ATP + ADP + AMP + IMP +
HxR + Hx x 100 In addition, the K value by the column method is the total ATP
Although the K value was calculated by measuring the concentration of related compounds, in order to calculate the K value using the test paper method of the present invention, the total ATP related compound concentration was measured in advance for samples of each fish species other than those used in this example, and the average value was used as the denominator in the above equation. The results are shown in Table 3. As can be seen from these results, the values obtained by the test paper method of the present invention substantially match those obtained by the column method, and from this, the total concentration of HxR and Hx in fish meat and It can be seen that the K value can be measured extremely easily and quickly.

【table】

【table】 [Brief explanation of the drawing]

FIG. 1 is a graph showing the relationship between the absorbance at a wavelength of 505 nm and the Hx concentration of an extract obtained by extracting a color-forming insoluble formazan dye produced in a reaction solution by the method of the present invention using iodonitrotetrazolium chloride with ethyl acetate. , FIG. 2 is a graph showing the relationship between absorbance and HxR concentration measured in the same manner as in FIG. 1. Figure 3 shows the first step using nitro blue tetrazolium chloride.
This is a graph showing the relationship between the absorbance at a wavelength of 550 nm and the HxR concentration of the extract extracted in the same manner as in the figure, and Figure 4 is a graph showing the relationship between the absorbance and Hx concentration measured in the same manner as in Figure 3. It is.

Claims (1)

[Scope of Claims] 1. Formazan dye produced by allowing nucleoside phosphorylase, xanthine oxidase, and a tetrazolium salt selected from iodonitrotetrazolium chloride or nitroblue-tetrazolium chloride to act on a sample solution containing inosine and/or hypoxanthine. A method for measuring inosine and/or hypoxanthine, which comprises measuring the concentration of inosine and/or hypoxanthine in the sample liquid based on the shade of color. 2. The method according to claim 1, wherein a solution containing nucleoside phosphorylase, xanthine oxidase, and the tetrazolium salt is brought into contact with the sample solution, and the color density of the produced formazan dye is measured by absorbance. 3. Prepare an enzyme-dye immobilized product by holding nucleoside phosphorylase, xanthine oxidase, and the above-mentioned tetrazolium salt in an immobilization material. After immersing the sample solution in this oxygen-dye immobilized material, 2. The method according to claim 1, wherein the shade of color of the produced formazan dye is visually measured. 4 Inosine and/or hypoxanthine measurement characterized by comprising an enzyme-dye immobilized product in which a fixing material holds nucleoside phosphorylase, xanthine oxidase, and a tetrazolium salt selected from iodonitrotetrazolium chloride or nitroblue tetrazolium chloride test paper.