JPH0364120B2 - - Google Patents

Description

[Detailed description of the invention]

<Industrial Application Field> The present invention relates to a reagent composition that can easily and quickly measure inosine and hypoxanthine, which are produced as the freshness of fish meat, meat, etc. decreases. <Prior Art> 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. It has long been known that an effective indicator for determining the quality of life of fish meat, etc. is to examine the changes in the decomposition products of adenosine triphosphate (ATP), which is an energy source for animals. Adenosine triphosphate decomposes as freshness decreases, producing inosine and hypoxanthine, which accumulate in fish meat. Therefore, in determining freshness, it is important to quantify inosine (hereinafter abbreviated as HxR) and hypoxanthine (hereinafter abbreviated as Hx) in fish meat. 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 present application, and a patent application has already been filed (Japanese Patent Application Laid-Open No. 118098/1983). 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 decompose ATP to Hx and others that only decompose it to HxR, and when only xanthine oxidase is used, it does not affect HxR and therefore the amount of HxR cannot be measured. When used together with nucleoxide phosphorylase, HxR is decomposed into Hx, so the Hx generated by this decomposition and the Hx originally contained in the sample are
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 enzyme reaction system as described above, when xanthine oxidase oxidizes Hx to uric acid, it simultaneously reduces the tetrazolium salt and generates a formazan dye. The amount of formazan dye produced changes depending on the amount of Hx present in the reaction system; 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 also decreases and the color becomes lighter. Therefore, by measuring the density of the formazan dye, we can determine the Hx in the reaction system.
It is possible to find the quantity. The amount of Hx in the reaction system at this time corresponds to the total amount of HxR and Hx in the sample solution. In the above-mentioned prior invention, since the formazan dye production reaction by reduction of the tetrazolium salt does not proceed effectively in the presence of oxygen, it is essential to have an oxygen scavenger such as sodium sulfite present in the reaction system. Ta. On the other hand, among the tetrazolium salts, especially iodonitrotetrazolium chloride (official name: 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl-2H-tetrazolium chloride) and nitroblue-tetrazolium chloride (official name: 3 ,3'-(3,3'-dimethoxy-4,4'-biphenylylene)-bis(2-(p-
It was discovered that when nitrophenyl)-5-phenyl-2H-tetrazolium chloride) was used, it was reduced to reliably produce a formazan dye even in the presence of oxygen in the reaction system, and a patent application has already been filed for this. (Unexamined Japanese Patent Publication 1986-
49800). <Problems to be solved by the invention> However, in the above-mentioned prior invention,
When using an acid-extracted sample solution prepared by homogenizing fish meat and removing protein with perchloric acid or trichloroacetic acid, turbidity will occur in the reaction solution, and furthermore, in the case of reaction with high concentrations of HxR or Hx There is a phenomenon in which the formazan dye produced becomes insolubilized in the reaction solution. In this case, in order to determine the intensity of the color development of the formazan dye in the reaction solution, the absorbance cannot be measured accurately even if the reaction solution is directly measured using a colorimeter. The insoluble formazan dye in the reaction solution was extracted using a colorimeter, and the absorbance of the extract was measured using a colorimeter. Therefore, according to the present invention, even when an acid-extracted sample solution that has been subjected to protein removal treatment is used, the reaction solution does not become cloudy, and even when reacted with a high concentration of HxR or Hx, the formazan dye does not become insolubilized. The purpose of this invention is to provide a reagent composition for measuring HxR and Hx, which can reliably produce a transparent coloring solution that can be directly measured for absorbance using a colorimeter. <Means for Solving the Problems> The present inventors particularly found that 3-(4,5-dimethyl-2
-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide, no turbidity occurs in the reaction solution even when using an acid-extracted sample solution that has been subjected to protein removal treatment, and furthermore, high concentrations of HxR or Hx
The inventors completed this invention by discovering that a reddish-purple transparent coloring solution could be obtained even after reacting a sample solution to form a formazan dye. That is, HxR according to the present invention and/or
The reagent composition for Hx measurement consists of nucleoside phosphorylase, xanthine oxidase and 3-(4,5
-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide and phosphoric acid
It is characterized by consisting of a buffer with a pH of 7 to 9. When the reagent composition of this invention is brought into contact with the acid-extracted sample solution of fish meat, etc. that has been homogenized and subjected to protein removal treatment, and left for several minutes, the enzyme decomposes HxR in the sample solution to Hx, and oxidizes Hx to uric acid. This is the same as in the prior invention, in which the reduction of the tetrazolium salt occurs and a red to reddish-purple formazan dye having absorption in the visible region is produced. In this invention, the formazan dye produced does not become insolubilized, and therefore a transparent coloring solution can be obtained. The decomposition of HxR in the sample solution to Hx is achieved by phosphorolyzing HxR using the nucleoxide phosphorylase in the reagent composition of the present invention. Therefore, it is necessary to include phosphoric acid as a substrate in the reagent composition during this phosphorolysis. This phosphoric acid can be included in the reagent composition as a phosphate salt, but when it is made into a solution, the phosphoric acid should be present at a molar concentration equal to or higher than that of HxR in the sample solution. The buffer used in this invention is necessary to maintain the pH of the reaction solution at 7 to 9 and keep the enzyme activity stable when the reagent composition of this invention is brought into contact with a sample solution and reacted. For example, phosphate buffer, Tris buffer, borate buffer, glycine buffer, etc. can be used. In particular, it is preferable to use a phosphate buffer because it can also function as a phosphoric acid component in the reagent composition of the present invention. In other words, by using a phosphate buffer with a pH of 7 to 9 that contains phosphoric acid at the same molar concentration or higher than HxR in the sample solution, it can serve as both the phosphoric acid and buffer components in the reagent composition. . The reagent composition of the present invention can be provided in the form of a solution in which the components are mixed and dissolved, but it can also be provided as a powder mixture in which the components in powder form are mixed as they are. Those in powder form may be dissolved in water before use. It is known that albumin and surfactants are used as dye solubilizers, but
According to the reagent composition of the present invention, the produced formazan dye can be solubilized without using such a dye solubilizing agent. However, if desired, these known dye solubilizers can also be used with the reagent compositions of this invention. <Examples> The present invention will be further explained below with reference to Examples. Example 1 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide, xanthine oxidase, and nucleoside phosphorylase were added to 0.1 M phosphate buffer at pH 8.0 at final concentrations. 1mM, 0.033units/
ml and 0.066 units/ml to prepare reagent composition 1 of the present invention. Furthermore, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H in reagent composition 1
Instead of tetrazolium bromide, 3,3′-
(3,3'-dimethoxy-4,4'-biphenylylene)
-bis[2-(p-nitrophenyl)-5-phenyl-2H tetrazolium chloride] or 3
-(p-iodophenyl)-2-(p-nitrophenyl)-5-phenyl-2H Reagent composition 2 and reagent composition 3 were prepared, respectively, to which tetrazolium chloride was added. Then, chicken meat was deproteinized with perchloric acid (PCA), acid extracted sample solutions adjusted to pH 6.4 with potassium hydroxide solution were added to each of the above three reagent compositions, and the presence or absence of turbidity was examined. Also, the final concentration is
HxR was added as a substrate to each of the above three reagent compositions to a thickness of 50 μm, and the presence or absence of insolubilization of the formazan produced after the reaction was examined. The above test results are shown in Table 1. From Table 1, reagent composition 1 of the present invention does not produce turbidity even after addition of the acid extraction sample solution, and
It became clear that the coloring solution remained transparent even after reacting with HxR.

[Table] Example 2 An acid extraction sample solution (equivalent to 0.1 g of muscle per ml) was prepared from yellowfin tuna, sardine, scallop, Japanese squid, and Taisho shrimp in the same manner as in Example 1. In addition, HxR was added to each of these acid-extracted sample solutions so that the final concentration of HxR was 900 μm.
A HxR-added acid extraction sample solution was prepared. Each sample solution prepared above was added to Reagent Composition 1 of the present invention used in Example 1. The amount of each sample solution added was 1/30 volume of the reagent composition. After the addition, the solution was allowed to stand at room temperature for 20 minutes to generate formazan, and a clear colored solution was obtained in each case. These coloring solutions were applied directly to a colorimeter and the absorbance was measured at a wavelength of 565 nm to conduct an addition and recovery test. Note that the concentration was calculated using a calibration curve method. The results are shown in Table 2. As can be seen from Table 2, even when the coloring solution obtained using the reagent composition of the present invention was directly applied to a colorimeter, a high recovery rate was obtained.

[Table] <Effects of the Invention> As explained above, according to the reagent composition of the present invention, a transparent coloring solution can be obtained even after the formazan dye is generated in the reaction solution. By measuring the absorbance of the protein-removed acid-extracted sample solution,
Easily and quickly measure HxR and/or Hx concentration.
It is possible to measure with high precision.

Claims (1)

[Scope of Claims] 1 Consists of nucleoside phosphorylase, xanthine oxidase, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide, phosphoric acid, and a buffer with a pH of 7 to 9. A reagent composition for measuring inosine and/or hypoxanthine, which provides a transparent coloring solution when applied to a sample solution containing inosine and/or hypoxanthine. 2. Claim 1, characterized in that the phosphoric acid and the buffer are present in the form of a phosphate buffer.
The reagent composition described in Section.