JPS6043959B2 - Inosine and/or hypoxanthine measurement method and test strip - 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 fish meat, livestock meat, etc. decrease in freshness, 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.

Measuring trimethylamine, PH) volatile basic nitrogen, etc. in fish meat has been proposed as an indicator to judge the freshness of fish meat, etc., but it is not always satisfactory in terms of knowing the so-called ``quality of life.'' It cannot be said that it is an appropriate indicator.

Adenosine Ξ, which is an energy source for animals, is a more effective indicator for determining the quality of life of fish meat, etc.
It is known to investigate the evolution of phosphoric acid (ATP) decomposition products.

Adenosine Ξ-lysoic acid decomposes as follows, and as freshness decreases, inosine and hypoxanthine are generated and accumulate in fish meat. Adenosine triphosphate (ATP) → adenosine diphosphate (, ADP) → adenosine-phosphate (AMP) → inosinic acid (IMP) → inosine (HxR) → hypoxanthine (1Ix) Therefore, in determining freshness, fish meat Inosine (hereinafter abbreviated as HxR) and hypoxanthine (
It is important to quantify Hx (hereinafter abbreviated as Hx).

Conventionally known methods for the simple determination of HxR (5Hx) include column methods and enzyme methods.

In the column method, the sample solution is subjected to protein removal treatment in advance, and then the above-mentioned ATP decomposition products are separated and fractionated using column chromatography using an ion exchange resin, and their concentrations are determined from the ultraviolet absorbance of the HxR and Hx fractions. It is a method of measurement. The enzymatic method uses nucleoside phosphorylase and xanthine oxidase to decompose HxR and Hx in the sample solution into uric acid, then measures ultraviolet absorbance to quantify uric acid, and calculates the HxR and Hx concentrations in the sample solution from this value. (For the column method and enzyme method, "Fisheries Biochemistry and Food Science Experiment Book" pages 17-28 (published by Seiseisha Kosekaku, 1936-10)
15th of the month). However, these conventional methods require complicated operations such as bringing the sample into the laboratory and performing protein removal treatment, separation and fractionation, or measuring ultraviolet absorption, so they cannot be carried out easily and quickly on site. This cannot be said to be a reliable measurement method. Therefore, an object of the present invention is to easily and quickly perform HxR. To provide a method for measuring 1IxR (5Hx); To provide a method for easily and quickly measuring 1IxR (5Hx), especially in the field, by measuring the absorbance or macroscopically examining the shade of color; Another object of the present invention is to provide a test paper that can be used to carry out the above measurement method more effectively.

In other words, the method for measuring HxR and removal of the present invention involves allowing nucleoside phosphorylase, xanthine oxidase, tetrazolium salt, and a deoxidizing material to act on a sample solution containing HxR and/or Hx, and determining the concentration of formazan dye in the sample solution based on the concentration of the formazan dye produced. The method is characterized by measuring HxR and/or Hx concentration. The nucleoside phosphorylase used in the present 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
・This is the enzyme No. 2, which oxidizes Hx and decomposes it 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, ATP may be decomposed into Hx.
There are some substances that only degrade up to xR, and if xanthine oxidase is used alone, it does not act on HxR and therefore the amount of HxR cannot be measured, but when used together with nucleoside phosphorylase, HxR is degraded to Hx, so this The total amount of Hx produced by decomposition and Hx originally contained in the sample is oxidatively decomposed by xanthine oxidase and converted into uric acid. In the conventional enzymatic method, the uric acid thus produced was directly measured by ultraviolet absorption, but the present invention differs from the conventional enzymatic method in that uric acid is not directly measured. That is, according to the present invention, a tetrazolium salt, which is a redox dye, is present in the enzyme reaction system as described above, and when xanthine oxidase oxidizes Hx to uric acid, the tetrazolium salt is simultaneously reduced to produce a red formazan dye. Let it be generated. The amount of formazan dye produced changes depending on the amount present in the reaction system; if the amount of lull is large, the amount of formazan dye produced increases and the color develops into a deep color, and if the amount of Hx is small, the amount of formazan dye produced is also decreases and the color becomes lighter. Therefore, by measuring the density of the formazan dye, it is possible to determine the amount of Hx in the reaction system.
The lull in the reaction system at this time is the HxR in the sample solution. 5Hx
It should be noted that this corresponds to the total amount of As the tetrazolium salt, neotetrazolium chloride, triphenyltetrazolium chloride, etc. can be preferably used.

The formazan dye production reaction by reduction of the tetrazolium salt described above does not proceed effectively if oxygen is present in the reaction system, so it must be carried out under deaerated conditions. However, in the present invention, sodium sulfite, etc. By having an oxygen scavenger present, it is possible to provide a deoxidized state even in the atmosphere, and the reduction reaction of the tetrazolium salt can be carried out reliably.

When carrying out the present invention, first, a solution is prepared in which the two types of enzymes described above, a tetrazolium salt, and an oxygen scavenger are dissolved.

In addition, in order to keep the activity of the enzyme stable in the solution, the pH is 7.
．． It is desirable to add 5 to 8 buffers into the solution.
Next, a sample solution prepared by homogenizing fish meat or the like is brought into contact with the solution and left for several minutes. During this time, decomposition of HXR(7)HX in the sample solution by the enzyme, oxidation of Hx to uric acid, and accompanying reduction of the tetrazolium salt occur, producing a red formazan dye having absorption in the visible region. 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 carbon tetrachloride, and then It is desirable to determine the shade of color by measuring visible absorption of the liquid.

In order to carry out the present invention easily and quickly in the field, it is preferable to use a test paper pre-absorbed with an enzyme, a tetrazolium salt, and an oxygen scavenger.

That is, two types of enzymes and a tetrazolium salt are made into an aqueous solution, which is absorbed into a filter paper or the like, and then dried using a freeze-vacuum dryer to prevent the enzymes from deactivating, thereby producing enzyme monochromatic paper. In this case as well, the pH is adjusted to keep the enzyme activity stable.
It is desirable to use a buffer solution or to add antioxidants and stabilizers to the aqueous solution to prevent enzyme deactivation. On the other hand, the oxygen absorber is made into an aqueous solution alone, absorbed into a filter paper or the like different from the above, and then dried in a vacuum to produce an oxygen absorber paper. The reason why the oxygen absorbing paper is produced separately from the enzyme-dyed paper is to prevent the enzyme from being deactivated by the oxygen absorber during production and storage. By superimposing the thus obtained enzyme-dyed paper and oxygen-absorbing paper, HxR and Hx
A test strip for measurement of .

When using this test paper, contact the sample solution made by homogenizing fish meat etc. from the oxygen-absorbing paper side of the test paper for 5 to 5 minutes.
1. After leaving it for a while, judge the shade of color on the enzyme-dyed paper side. The shade of color on the filter 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. The HxR and H weir degrees in the liquid 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, HxR and Hx concentration, which are indicators of the freshness of fish meat, etc. can be measured easily and quickly in the atmosphere, and the sample required in the conventional method can be measured easily and quickly. Complex operations such as protein removal treatment of the liquid, fractionation operation using a column, and measurement of ultraviolet absorption can also be made unnecessary.

In particular, according to the present invention that uses a test paper, the HxR and calm concentration in the sample can be immediately measured simply by bringing the sample liquid into contact with the test paper and visually determining the shade of color on the test paper. Using special analytical equipment, the freshness of fish meat can be confirmed very easily and quickly at retail stores and other locations. The present invention will be further explained below with reference to Examples.

Example 1 Phosphate buffer pH 7.8 4.5 ml 110 n 1 M triphenyltetrazolium chloride 0.5 ml 11 xanthine oxidase 0.1 enzyme unit, nucleoside phosphorylase 0. hydrogen unit, and sodium sulfite 0.1
mm01es was mixed.

HxR or Hx concentration in this mixed solution is 0.
A substrate solution was added to the solution at a concentration of about 0.02, 0.05, 0.1, and 0.2 rT1M, and the solution was left to stand to produce a color-developing insoluble formazan produced by reduction of the above-mentioned tetrazolium salt. This solution was then diluted with 1N HClO. 5 ml of acetone (0.5 ml) and carbon tetrachloride (5 ml) were added and shaken to extract color-developing insoluble formazan into the carbon tetrachloride phase, and the absorbance of this extract was measured at a wavelength of 485 nn1. FIG. 1 shows the relationship between HxR concentration and absorbance, and FIG. 2 shows the relationship between Hx concentration and absorbance. In addition, HxR and Hx at this time
The factors of the substrate solution are 0.903 and 0.903, respectively.
It was 873. As can be seen from the graphs in FIGS. 1 and 2, HxR or Hx
can be easily and accurately quantified under atmospheric conditions.

Example 2 Phosphate buffer 5 ml pH 7.8, triphenyltetrazolium chloride 0.1 mm 0 Ies 1 xanthine oxidase lance enzyme unit, nucleoside phosphorylase 2
Add water to Vf elementary unit, 1' sucrose (stabilizer), 0.2 ml of 1% mercaptoethanol (antioxidant) to make 15 m
1 and an enzyme-dye solution was prepared.

This solution was absorbed into a chromatographic filter paper, and then freeze-dried in vacuum to prepare an enzyme monochrome paper. Separately, a solution in which about 8 g of sodium sulfite was dissolved in 100 ml of water was absorbed into a chromatographic filter paper, and then vacuum dried to prepare an oxygen-absorbing paper.

Next, the enzyme-dyed paper obtained above and the oxygen-absorbing paper were laminated to produce a test paper of the present invention. HxR or Hx concentration is 0.05, 0.01, 0.15, 0.20 respectively
, 0.30, 0.40rT1M was added dropwise from the oxygen-absorbing paper side of the test paper of the present invention.
After cleaning, check the shading of the color on the enzyme-dye paper side using a color chart (JISZ-
8721). The results are shown in Table 1. As can be seen from Table 1, the test paper of the present invention has FIxR
Alternatively, the Hx concentration can be measured by the shade of color (visible red) on the test paper.

Example 3 A carp was 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 solution was dropped from the oxygen-absorbing paper side of the test paper of the present invention prepared in Example 2, and the shade of color on the enzyme-dyed paper was measured using a color chart in the same manner as in Example 2. From the reading, HxR. (The total concentration of 5Hx was determined. The results are shown in Table 2. In parallel with the measurement using the test paper of the present invention described above, 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 packed with ion exchange resin (strongly basic anion exchange resin). The total concentration of 11xR and Hx in the sample solution was determined by measuring the absorbance of 11xR and Hx. The results are also listed in Table 2. 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 4 11 samples each from raw yellowtail and macro yellowtail sold at retail stores (sample numbers NO.1 to NOll)
) were collected, and sample solutions were prepared from each sample in the same manner as in Example 3.

HxR. The total concentration of l5Hx is
Measurement was carried out in the same manner as in Example 3 using the method using the test paper of the present invention 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 the total A
'IT' related compounds (ATP, ADP, AMP, IMP
, HxR, Hx) is the percentage of HxR + Hx amount to the total amount, and is calculated by the following formula: (For example, refer to the above-mentioned "Fisheries Biochemistry and Food Science Experiment Book" 1st page). The K value according to the test paper method of the present invention was calculated by measuring the concentration of total ATP-related compounds in 2008.
Calculated as es/g, macro 7.5 μM Moles/V.

The results are summarized in Table 3 for yellowtail and Table 4 for macro.

As can be seen from these results, the values obtained by the test paper method of the present invention are substantially the same as those obtained by the column method, and from this, the test paper method of the present invention can be used to determine the freshness of fish meat extremely easily and quickly. It can be seen that it can be measured.

[Brief explanation of the drawing]

Figure 1 shows the color-forming insoluble formazan dye produced in the reaction solution by the method of the present invention, extracted with carbon tetrachloride. FIG. 2 is a graph showing the relationship between absorbance and HxR concentration of the extracted liquid at a wavelength of 485 r1m, and FIG. 2 is a graph showing the relationship between absorbance and Hx concentration measured in the same manner as in FIG.

Claims (1)

[Scope of Claims] 1. Nucleoside phosphorylase, xanthine oxidase, tetrazolium salt, and oxygen scavenger are allowed to act on a sample solution containing inosine and/or hypoxanthine in the atmosphere, and the sample is determined based on the intensity of the color of the formazan dye produced. A method for measuring inosine and/or hypoxanthine, which comprises measuring the concentration of inosine and/or hypoxanthine in a liquid. 2. The method according to claim 1, wherein a solution containing nucleoside phosphorylase, xanthine oxidase, a tetrazolium salt, and an oxygen scavenger is brought into contact with the sample solution, and the shade of color of the formed formazan dye is measured by absorbance. . 3. Hold nucleoside phosphorylase, xanthine oxidase, and tetrazolium salt in a first immobilization material to produce an enzyme-dye immobilization product, and hold a deoxidizing material in a second immobilization material to create a deoxidized material immobilization product. After superposing these enzyme-dye immobilized products and deoxidizing material immobilized products and immersing the sample solution from the deoxidizing material immobilized product side, the color density of the formazan dye produced on the enzyme-dye immobilized product side was determined. 2. The method according to claim 1, which comprises visually measuring . 4 An enzyme-dye immobilized product in which nucleoside phosphorylase, xanthine oxidase, and tetrazolium salt are held in a first immobilization material, and a deoxidizing material immobilized product in which a deoxidizing material is held in a second immobilization material. A test strip for measuring inosine and/or hypoxanthine.