JPH03219900A - Method for measuring freshness of fishes or shellfishes - Google Patents

Abstract

PURPOSE:To enable to rapidly, simply, accurately and inexpensively measure the freshness of a fish or shellfish by treating a system containing a sample solution for the measurement of the freshness thereof, a buffer solution and a color-generating reagent with a specific enzyme in the pressure of ribose-1- phosphoric acid. CONSTITUTION:>=0.04 Units of nucleotide phosphorylase and a specimen such as fish meal extract are added to and reacted with a solution containing a 10-100mM tris buffer solution not containing phosphoric acid and having a pH of approximately 7.5, etc., ribose-1-phosphoric acid and a color-generating reagent such as 4-aminoantipyrine to provide an absorbance E0 of the reaction solution (A). The component A is mixed and made to react with >=0.1 units of nuclotide oxidase to provide the absorbance E1 of the reaction solution (B). The component B is mixed and reacted with >=20 units of crude alkali phosphatase at 20-50 deg.C for 3-15min and the absorbance E2 of the reaction C is measured at a wavelength of 500nm, etc. K value is calculated from the absorbances by an equation. The freshness of a fish or shellfish is measured from a small value on the K value as a standard.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a method for measuring the freshness of seafood, and more particularly to a new method for easily and quickly measuring the freshness of seafood by using a combination of various enzyme reactions.

Traditionally, the freshness of seafood has been judged sensually, but with recent advances in freezing technology, packaging formats, and changes in distribution systems, the freshness of seafood has gradually been judged based on its appearance. This is becoming increasingly difficult, and instead of this, a scientific and objective freshness measurement technique and an index therefor are required.

Currently, the value calculated by the following formula (1) is known as the most reliable freshness index that meets the above requirements.

(1) (In the formula, ATP represents adenosine triphosphate, ADP represents adenosine diphosphate, AMP represents adenylic acid, IMP represents inosinic acid, HxR represents inosine, Hx represents hypoxanthine, and the same applies hereinafter). ) Also, the above ATP
Since XADP and AMP are rapidly decomposed in fish meat, even the Kl value calculated by the following formula (2), which omits this from the above formula (1), can represent freshness with sufficient accuracy for practical use. (For example, Japanese Patent Application Laid-Open No. 59-1072
Publication No. 56, 1, Agric, Food Che
m,, vol, 32. pp18-22 (19
84) etc.).

However, the value and Kl value of the above freshness index have traditionally been as follows (1
) to (4).
Each of the second-class methods has its own problems as described below, and none of them can be said to be sufficient at present.

That is, (1) fractionating each component using an ion exchange resin,
The method of measuring each of these parameters and calculating the values (simple chromatography method) requires 2 to 3 hours of operation time even for an expert to calculate the above indexes, and the operation is of course too complicated. However, there is a disadvantage that regeneration, equilibration, etc. of the ion exchange resin used are also required.

(2) The method of fractionating each component using a reversed-phase column, gel permeation chromatography column, etc., and determining the value by quantifying the components (HPLC method) does not require expensive HPLC equipment or chromatography. Accessories such as packs are required to determine the peak area of each component, measurement requires skilled techniques, standard solutions of each component must be prepared, and column equilibration is required for analysis. It also has the disadvantage of requiring cleaning and cleaning.

(3) The UV method using xanthine oxidase and nucleoside phosphorylase first extracts nucleic acid components from fish meat using perchloric acid, which is a powerful drug, and then
The total amount of nucleic acid-related compounds (A) is determined by measuring the absorption at nm, and then the above extract is treated with xanthine oxidase and nucleoside phosphorylase to extract HxR and HxR.
Convert x to uric acid, determine the amount of uric acid produced from the absorption at 293 nm, further multiply this by a conversion factor that differs depending on the species of fish to calculate (B), and from each value above A / B X10
This method calculates 0 and then obtains the value. However, as mentioned above, when extracting nucleic acid-related substances from fish meat, this method
This method requires the use of perchloric acid, which is a powerful chemical, since it does not interfere with UV absorption, which is dangerous, and has disadvantages in that it is complicated to operate and requires a UV spectrophotometer. Furthermore, this method
The amount of nucleic acid-related substances is determined by V absorption,
There is a possibility that there are substances with UV absorption in addition to the nucleic acid-related substances mentioned above in fish meat samples, and if this point is taken into consideration,
There is a problem with accuracy.

In addition, (4) the method of indirectly quantifying ATP-related compounds by measuring the amount of oxygen consumed in the enzymatic reaction using an oxygen electrode is to first divide the fish meat sample into two parts, and then divide the fish meat sample into two parts and add xanthine oxidase and nucleoside phosphorus to one part. HXR and HX are oxidized to uric acid by the action of urase, and the amount of enzyme consumed in the solution is measured with an oxygen electrode.
While determining the amount of r, a preliminary reaction is performed by treating the other sample with crude alkaline phosphatase, and ATP, A
DP This method calculates the value based on the ratio of the two measured values.

This method has the disadvantage that it requires expensive measuring equipment, and that it requires dividing the sample into two parts and separately determining the oxygen consumption for each part using an oxygen electrode.
In addition, a preliminary reaction with alkaline phosphatase is required, which is time consuming, and each enzymatic reaction is influenced by catalase, which is usually contaminated in the enzyme preparation used, which has the disadvantage of reducing accuracy.

As mentioned above, conventional techniques for measuring the freshness of seafood using value and 1 value as indicators have been found to be complicated and inaccurate, although the indicators themselves are said to be reliable. However, there is a current demand in the industry for the development of a new freshness measurement technique that can more quickly, simply, and accurately determine the above-mentioned index.

Problems to be Solved by the Invention The purpose of the present invention is to provide a quick, simple, and accurate freshness measuring method that meets the above-mentioned needs of the industry, and also to provide a method for measuring freshness that is quick, simple, and accurate, and that also uses HPLC equipment and oxygen electrodes found in conventional methods. The object of the present invention is to provide the above-mentioned method that does not require expensive equipment such as a measuring device or an ultraviolet spectrophotometer and can be carried out using an inexpensive analyzer.

Means to solve problems The above object is achieved by the following method.

That is, according to the present invention, in a system containing a sample solution for freshness measurement, a phosphate-free buffer solution, and a coloring reagent that develops color by oxidation, (1) nucleoside phosphorylase action is applied in the presence of ribose-1-phosphate. (2) allowing nucleoside oxidase to act to oxidize the nucleoside and developing a coloring reagent using the laccase-like activity of the nucleoside oxidase that is expressed in accordance with the amount of the nucleoside; 3) a step of acting with enzymes that convert adenosine triphosphate, adenosine diphosphate, adenylic acid, and inosinic acid into nucleosides; and (4) a step of measuring the degree of color development of the reaction solution after each of the above steps. A method for measuring the freshness of seafood is provided.

In more detail, the method of the present invention comprises (1) carrying out steps (1) to (3) in this order, and in step (3) using an enzyme selected from crude alkaline phosphatase, crude acid phosphatase, and crude apyrase. The measured values after step (2) are converted into step (3) using
) A method in which a value is obtained by dividing by the subsequent measured value and this is used as a seafood freshness index. (2) In the above, steps (1) to (3) are carried out in this order, and step (3) Using purified alkaline phosphatase and/or 5'-nucleotidase, divide the measured value after step (2) by the measured value after step (3) to calculate K.
(3) In the above, steps (1) to (3) are replaced by steps (
2), (1) and (3) in the order and step (3)
purified alkaline phosphatase and/or 5'-
Using nucleotidase, the value obtained by subtracting the measured value after step (2) from the measured value after step (1) of (a) is calculated in step (3).
) to determine the Hx ratio by dividing by the subsequent measurement value, (b) step (2)
The HxR ratio is obtained by dividing the measured value after step (3) by the measured value after step (3), and the HxR ratio is calculated from the measured value after step (3).
This includes a method in which the IMP ratio is obtained by subtracting the subsequent measured value and dividing the value obtained by subtracting the measured value after step (3), and using any of the obtained values as a seafood freshness index.

Any of the methods of the present invention described in (1) to (3) above can quickly, easily, and accurately measure the freshness of seafood, and is similar to conventional methods (HPLC equipment, measuring equipment using oxygen electrodes, It has the advantage that it does not require expensive equipment such as an ultraviolet spectrophotometer and can be carried out using an inexpensive analyzer.

To explain in detail the nucleoside oxidase used in the method of the present invention, this enzyme was previously developed by the present inventors (Japanese Patent Application No. 196632/1982) and was named [nucleoside oxidase YT-IJ]. It is an enzyme that catalyzes the reaction of the nucleoside with molecular oxygen to produce nucleoside-5'-aldehyde and then nucleoside-5'-carboxylic acid, but has the characteristic that hydrogen peroxide is not produced as a by-product. The nucleoside oxidation reaction catalyzed by this enzyme is shown by the following formulas (1) and (2), and is clearly distinguished from the oxidation reactions of other known nucleoside oxidases.

HOH2CBa5e OHCBa5eH
OH OHOH (1) OHOH01 (OH (2)) This enzyme also has the unique property of exhibiting laccase-like activity depending on the amount of the nucleoside at the same time as the oxidation reaction of the nucleoside represented by the above formula. and
The method of the present invention is characterized in that it utilizes the above-mentioned laccase-like activity specific to this enzyme.

The present enzyme YT-1 is produced using the enzyme-producing bacterium belonging to the genus Pseudomonas. Examples of the enzyme-producing bacteria used here include a strain newly isolated by the present inventors and having the following properties.

■, Mycological properties (A) Morphological properties ■ The cells are bacilli, and the size is 0.5X1.5~1.
It is 7 μm.

■ It is motile and the flagella are extremely flagella.

■ Spores are not formed.

■ Negative in Durham staining.

(B) Culture findings■ Growth on broth agar plate is moderate, colonies are circular, and the surface is smooth and yellow.

■ Meat juice agar slant culture The growth is moderate and the surface is smooth and yellow.

■ Meat juice liquid culture It grows on the surface and its growth is moderate.

Gravy gelatin puncture culture grows on the surface part and liquefies gelatin (Sfrati
form).

(c) Physiological properties■ Nitrate reduction Negative denitrification reaction Negative indole generation Negative hydrogen sulfide generation Negative starch hydrolysis Negative nitrate utilization Negative ammonium salt utilization Positive pigment generation Fluorescent pigments, biocyanin, and xanthomonadin are all Not generated. Broth agar may produce a brown, diffuse, pigment.

Urease Negative Oxidase Negative to weakly positive Catalase
It does not grow in the positive growth range of pH 4 and 5. Does not grow at 4°C and 41°C. The appropriate growth pH is between 5 and 8, and the temperature is 35°C.
is optimal.

Attitude towards oxygen 0-F test (Hugh) Production of acid from sugars - glucose, positive D-fructose, positive D-maltose, positive D-galactose, positive D-xylose, positive D-mannitol, negative sucrose, negative lactose, positive Aesculin decomposition arginine hydrase lysine decarboxylase ornithine decarboxylase phenylalanine deaminase egg yolk reaction Tween 20 decomposition aerobic Leifson method) Oxidative positive negative positive negative negative negative positive Requires auxotrophic methionine or cystine.

Based on the above properties, we conducted a search for this strain using Berji [Ber].
ge7's Manual of Systemati
According to C Bacteriology (1984)), this strain is: ■ Gram-negative rod and does not form spores; ■ It is catalase positive; ■ It moves by ultraflagella; and ■ It does not oxidize glucose. The Pseudomonas genus (P.
It was recognized that it belongs to the genus seudomonas). Additionally, ■ requires cystine or methionine for growth, ■
Negative for arginine dihydrolase; ■Does not accumulate polybata hydroxybutyrate; ■Colonies are yellow; ■Does not produce xanthomonadin;
■ Pseudomonas maltophilia (Pseudomonas maltophilia)
tophilia).

Based on the above results, the present inventors identified this strain as a strain of Pseudomonas maltophilia, and it is known as Pseudomonas maltophilia LB-86 (Pseudomonas maltophilia).
As maltophilia LB-g5).

The strain was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, with the above indication, and was approved by the Ministry of International Trade and Industry as a certified microorganism strain No. 9813 (FERM P).
-9813).

The present enzyme nucleoside oxidase YT-1 is derived from the above-mentioned LB
It can be produced by culturing microorganisms belonging to the genus Pseudomonas, including the -86 strain and its mutants, and having the ability to produce the above-mentioned nucleoside oxidase YT-1, and this culturing can be carried out in an appropriate nutrient-containing medium. As a medium,
Any of synthetic media, semi-synthetic media, and natural media containing various carbon sources, nitrogen sources, inorganic substances, etc. that are commonly used for culturing microorganisms belonging to the genus Pseudomonas can be used.

Specifically, as the carbon source, various carbon compounds that can be assimilated by microorganisms, such as higher fatty acids, starch decomposition products (dextrin), maltose, lactose, glucose, molasses, and fructose, are used singly or in combination of two or more. . Examples of the nitrogen source include peptone, meat extract, yeast extract, soybean flour, cottonseed flour, and corn steep liquor. As the inorganic substance, for example, in addition to common salt, inorganic salts such as phosphates and sulfates of inorganic metals such as potassium, sodium, magnesium, calcium, zinc, and iron can be used as appropriate.

The above-mentioned microorganisms can be cultured either in liquid culture or solid culture, and is preferably carried out aerobically under aeration and agitation conditions. Culture conditions such as pH 1 of the medium, culture temperature, and culture time are preferably selected from conditions that are suitable for the growth of the microorganism to be cultured and that produce as much of the desired enzyme as possible.

For example, the culture medium pH is in the range of about 6 to 8, and the culture temperature is in the range of about 20 to 8.
Preferably, the temperature is selected from the range of 35°C. The culture time may be selected so that the production and accumulation of the desired enzyme reaches its maximum, and is usually selected from a range of about 12 to 48 hours. Of course, each culture condition, culture method, etc. described above can be changed as appropriate depending on the type of microorganism used, external conditions for culture, etc.

In the culture thus obtained, the desired enzyme is usually accumulated in the bacterial cell compartment. The collection and purification of this enzyme can be carried out in accordance with various conventional methods conventionally employed in the production of enzymes and the like using microorganisms.

Examples of the above-mentioned purification methods include methods that utilize solubility in solvents, methods that utilize differences in ionic bonding strength, methods that utilize differences in molecular weight, methods that utilize differences in isoelectric points, and methods that utilize differences in hydrophobicity. Each of the means and the like can be employed alone or in an appropriate combination.

In more detail, the target enzyme can be collected from the above bacterial cell sections by suspending wet bacterial cells obtained by collecting them using a conventional method such as centrifugation in a phosphate buffer, Tris buffer, etc., and subjecting them to ultrasonic treatment. Various bacterial cell treatment methods such as French press and lysozyme treatment are employed in appropriate combinations to obtain a crude enzyme-containing liquid. The above-mentioned crude enzyme-containing solution can be further purified by a conventional method to obtain a purified enzyme preparation. As a purification method, for example, a precipitate is first collected from the crude enzyme-containing solution by a fractional precipitation method using an organic solvent such as ethanol or acetone, or a salting-out method using ammonium sulfate, aluminum sulfate, etc., and then the precipitate is purified by diethylaminoethyl This can be carried out by subjecting it to chromatography using an ion exchanger such as dextran or a gel filtration agent such as polyacrylamide gel, and the purified enzyme preparation obtained in this way can also be made into a purified powder preparation by further operations such as freeze-drying. can.

The present enzyme obtained as described above has the formula (1) described above.
In addition to being characterized by having an enzymatic action to catalyze the oxidation reaction shown in (2), it is also characterized by having the following physicochemical properties.

(1) Substrate specificity - Acts on various nucleosides such as inosine. It does not act on bases such as hypoxanthine, nucleotides such as inosinic acid, ribose, etc.

(2) Temperature and pH stability: pH 6.0, 60'C, 95% or more remained after 15 minutes, 7
It is inactivated at 0°C for 115 minutes.

In addition, when treated at 37°C for 160 minutes, the pH was 5.0 to 6.0.
More than 95% remains.

(3) Optimum pH: pH5.0-6. It is 0.

(4) Molecular weight: approximately 130,000.

(by gel filtration method) (5) Isoelectric point: pH 5.3.

(6) Effect of inhibitors Inhibited by potassium dianate and sodium azide.

(7) Visible absorption There is no absorption maximum at 450 nm or above in a neutral buffer such as diphosphate buffer. Oxidized enzymes that have not been reduced by the substrate, dinitzion (sodium hydrosulfide), etc., do not have an absorption maximum at 450 nm or higher.

(8) Metal content: Contains iron.

In the method of the present invention, inosine,
Utilizing the unique laccase-like activity of this enzyme, which oxidizes various nucleosides such as adenine using molecular oxygen as an electron acceptor and at the same time exhibits a laccase reaction depending on the amount of nucleosides in the presence of the nucleosides, hydroquinone, etc. By oxidizing typical laccase substrates such as 4-aminoantipyrine and phenol, aniline-based substances, etc., dyes such as quinoneimine can be produced in proportion to the amount of the nucleoside, and thus The amount of nucleoside can be quantified by measuring the amount of pigment produced.

As the coloring reagent that develops color by oxidation used in the method of the present invention, any substance that develops color by being oxidized by the laccase-like activity of the above-mentioned nucleoside oxidase can be used. The substances include reagents that are oxidized with a single compound and exhibit absorption in the visible region, and reagents that are oxidized and condensed with a combination of two or more compounds and exhibit absorption in the visible region.

Examples of the above single compounds include various laccase substrates,
For example, o-tolidine, o-toluidine, 0-dianisidine, 1O-N-methylcarbamoyl-3°7-dimethylamino-1O-H-phenothiazine, bis[3-bis(
Examples include aniline-based substances such as 4-chlorophenyl)-methyl-4-dimethylaminophenyl]amine. In addition, as a combination of two or more of the above-mentioned compounds, a combination of a so-called coupler and a Trinder reagent (hydrogen donor), as typified by 4-aminoantipyrine and phenol, which have been widely used as clinical diagnostic agents, may be used. I can give an example. Specific examples of the above couplers include 4-aminoantipyrine (4-AA), 2゜6-dibromoaminophenol, 3-methyl-benzothiazolinone hydrazone, etc., and specific examples of the Trindda reagent (hydrogen donor). Examples include phenol, β-chlorophenol, 2.4-
Dichlorophenol, 2,6-dichlorophenol, N
, N-dimethylaniline (DMA), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m
-Anisidine (ADO8), N-ethyl N-(2-hydroxy-3-sulfopropyl)aniline (ALO3)
, N-ethyl-N-(2hydroxy-3-sulfopropyl)-m-toluidine (TOO8), N-ethyl-N-sulfopropyl)-m-anisidine (ADPS
), N-ethyl-N-sulfopropylaniline (AL
PS), N-ethyl-N-sulfopropyl 3.5
-dimethoxyaniline sodium salt (DAPS),
N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt (
DAO8), N-sulfopropyl-3,5-dimethoxyaniline (HDAPS), N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (HDAO8), N-ethyl-N-( 2-Hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAO3), N-ethyl-N-sulfopropyl-3,5-dimethylaniline (MAPS), N-ethyl-N-sulfopropyl Room m-Truigi, N (TOP
S), N2-ethylN2- (3-methylphenyl)-
Examples include N'-acetylethylenediamine (EMAE).

In addition, in the method of the present invention, other reagents other than the above-mentioned nucleoside oxidase and coloring reagent, namely, a sample solution for freshness measurement, a phosphate-free buffer solution, ribose-1-phosphate,
Nucleoside phosphorylase and ATP, ADT
As the enzymes (typically alkaline phosphatase, etc.) that convert AMP and IMP into nucleosides (lxR), any enzymes similar to those used in conventional seed freshness measurement techniques are used. and can be prepared in a similar manner.

For example, as a sample solution for freshness measurement, an alkali-neutralized supernatant obtained by adding a seafood extract obtained according to a conventional method, such as homogenization with a 10% TCA solution and centrifugation, can be used.

Tris-HCl buffer, HEPES buffer, etc. can be used as a phosphate-free buffer.

ATP such as ribose-1-phosphate, nucleoside phosphorylase and alkaline phosphatase.

ADT, AMP, and IMP were combined with nucleosides (Hx
As the enzymes to be converted into R), any of various commercially available products can be used. Especially the above ATP, ADT, AM
Enzymes that convert P and IMP into nucleosides (HXR) are those selected from crude alkaline phosphatase, crude acid alkaline phosphatase, and crude apyrase, and purified enzymes according to the methods of the present invention described in (1) to (3) above. It is necessary to use alkaline phosphatase and/or 5'-nucleotidase properly.

The measurement method according to the method of the present invention will be explained in detail below.

According to the first method of the present invention, the freshness of seafood can be measured using the K value as an index.

In this method, the measurement items required to calculate the K value are ATP (adenosine triphosphate), At)P (adenosine diphosphate), AMP (adenylic acid), IMP (inosinic acid), )IXR (inosine), and HI (hypoxanthine), each of which is determined according to the present invention as follows.

That is, (1) Hx is converted to HxR by step (1) of allowing nucleoside phosphorylase to act in the presence of ribose-1-phosphate. However, the above nucleoside phosphorylase is originally an enzyme that phosphorolyzes nucleosides such as HIR to generate bases such as l(x) and ribose-1-phosphate. The reverse reaction of the reaction occurs, thus) I! More HxR is generated. Therefore, this reverse reaction is the subsequent step (2)
Accordingly, the present inventor has confirmed that by allowing the generated HXH to act with nucleoside oxidase to oxidize and remove it from the reaction meter, the reaction proceeds substantially 100%.

(2) ) (I R is directly oxidized in step (2) in which nucleoside oxidase is applied. Also, in step (2), the coloring reagent is also oxidized by a laccase-like reaction at the same time as the oxidation reaction of HxR described above, producing a colored substance. is produced and color development is observed.

(3) ATP + ADP + AMP + IMP is dephosphorylated in the step (3) of allowing alkaline phosphatase selected from crude alkaline phosphatase, crude acid alkaline phosphatase, and crude apyrase to act, and ATP is converted into ATP.

ADP and AMP become AdR (adenosine), and IM
P is converted to t(xR, respectively, and these are oxidized by the step of allowing nucleoside oxidase to act in the same manner as in step (2) above.

Therefore, the first method of the present invention includes steps (1) to (3).
are carried out in this order, and the value (EI Eo) obtained by subtracting the measured value before step (2) (denoted as Eo) from the measured value after step (2) (denoted as E+) is calculated as the value (EI Eo) after step (3). Measured value (E
By subtracting the measured value (Eo) before step (2) from (2) and dividing by the value (E2 EO), the value can be determined by the following formula.

K value-(E+Eo)/(E2-Eo)X
100(1') Particularly, this first method performs step (3) in a phosphate-free buffer solution and operates at a pH of about 7.5, thereby converting ATP, ADP, AMP, and I
1m from MP? The conversion from HX to IxR and the oxidation of HxR can be performed at the same pH, thus having the advantage that all items necessary for value measurement can be performed in the same system using a single sample. .

To explain in detail the operations in each step of implementing the first method of the present invention, in this method, first, in a solution in which ribose-1-phosphate and a coloring reagent are dissolved in a phosphate-free buffer solution,
Nucleoside phosphorylase and a sample for freshness measurement (for example, fish meat extract) are added and reacted (step (1)), and the absorbance Eo is measured.

Note that this can be replaced by adjusting the O of the absorbance measuring device. Next, nucleoside oxidase is added to the reaction solution and reacted (step (2)), and the absorbance E1 is measured. By the above operation, the reactions of each enzyme in step (1) and step (2) proceed simultaneously, and thus obtained E, -
The E value (or the El value when O adjustment is performed) indicates the total amount of Hx and HXH in the sample.

Next, crude alkaline phosphatase or the like is added to the reaction system to perform a dephosphorylation reaction (step (3)), and the absorbance is E2.
By measuring E2-EO (or E2 if O adjustment is performed), ATP.

ADP, AMP, IMP, HxR and FIZ
The total amount of can be found.

In the case of using alkaline phosphatase in the above, the pH in the system is preferably about 6.5 to 8.0, preferably around 7.0 from the action pH of each enzyme, and in the case of using acid phosphatase and apyrase, , pT (approximately 5.0-7
．． 0, especially around 6.0. As a buffer solution that does not contain phosphate, depending on the above pH conditions, for example, if the pH is around 7.5, a HEPES buffer or a Tris buffer can be used, and if the pH is around 6.0, a MES buffer can be used. can. The concentration of the secondary buffer is not particularly limited as long as it exhibits a sufficient buffering effect, but it is usually about 10 to 100 mM.

The amount of enzyme used in each of the above reactions may be appropriately determined depending on the type of the enzyme, and for nucleoside oxidase, it is approximately 0.
1 unit or more, preferably about 0.3 to 5 units, 0.04 units or more for nucleoside phosphorylase, preferably about 0.2 to 0.4 units, 20 units or more for alkaline phosphatase, preferably about 70 to 140 units, For acid phosphatase, it is appropriate to use 20 units or more, preferably around 100 units, and for apyrase (manufactured by Sigma), it is appropriate to use 20 g units or more, preferably in the range of about 60 to 100 units.

In addition, the reaction time of each of the above enzymatic reactions is appropriately determined depending on the type and amount of enzyme used, and is not particularly limited, but it is usually in the range of about 3 to 15 minutes, and the reaction temperature is Generally about 20-50℃, preferably about 25℃
The temperature may be about 40°C.

Furthermore, the measurement wavelength for absorbance measurement is appropriately determined according to the type of coloring reagent used. For example, when using 4-aminoantipyrine and phenol as coloring reagents, 500n
m, 555 nm for TOO8, 6 for MAO3
30 nm can be used, and 593 nm can be used for DAO8.

According to the second method of the present invention, the freshness of seafood can be measured using the Ki value as an index.

According to this method, steps (1) to (3) are performed in this order, purified alkaline phosphatase and 5'-nucleotidase are used in step (3), and step (2) is performed in this order.
The measured value before step (2) (Eo
) is subtracted from the measured value (E3) after step (3) to the measured value before step (2) (E
The Kl value can be determined according to the formula below by dividing by the value obtained by subtracting (E3 EO).

Kl value = (E+ -Eo) / (E3-Eo)
X100(2') That is, by using the purified alkaline phosphatase and 5'-nucleotidase, only IMF is converted to HXR, and thereby the value can be determined in the same manner as in the first method.

Further, according to the third method of the present invention, steps (1) to (3) are converted into step (2) based on the same principle as the second method.
, (1) and (3) in the order and using purified alkaline phosphatase and 5'-nucleotidase in step (3), (a) from the measured value after step (1) (referred to as At) to step ( 2) The value (At A2) after subtracting the measured value after step (3) is calculated as the measured value after step (3) (referred to as A2).
A3) to find the Hx ratio, (b) step (2)
The HXR ratio is obtained by dividing the measured value (A2) after step (3) by the measured value (A3) after step (3), and (c) the measured value (A3) after step (3) is divided by the measured value (A3) after step (1). The value (A3-At) obtained by subtracting the measured value (A1) is calculated as the measured value after step (3) (A
3) to determine the IMP ratio, and using at least one of these values as a seafood freshness index, the freshness of seafood can be measured. Each second-class index is calculated by the following formula (3) ~
It is shown in (5).

t(x ratio = (At - A2)/A3
(3) tlxR ratio - A2/A3 (4
)IMP ratio-(A3-At)/A3(5
) For details of the operation of this third method, for example, first, nucleoside oxidase is applied to a sample for freshness measurement, and the absorbance A2 (corresponding to the amount of HI'R) at that time is measured. Next, nucleoside phosphorylase is added to the reaction system, the reaction is caused, and the absorbance A1 is measured. This A2 is 1(X
Corresponds to the total amount of R and Hx.

Furthermore, purified alkaline phosphatase or 5
'-nucleotidase is applied and the absorbance A3 is measured. This A3 corresponds to the total amount of [M[', HXR and HK. From each absorbance determined above, fIX ratio, HXR ratio and I
MP ratio can be calculated.

Thus, according to the method of the present invention, the freshness of seafood can be accurately measured with a very simple operation.

EXAMPLES Reference examples, test examples, and examples are given below to explain the present invention in more detail.

In addition, nucleoside oxidase activity was measured by the following method.

<Nucleoside oxidase activity measurement method> 100mM phosphate buffer containing 7mM inosine (pH 6,0) 1
7A' is placed in an enzyme electrode cell, 5 μl of the enzyme preparation is added, and the initial rate of enzyme consumption is measured at 25°C. That is, the decrease in the amount of dissolved oxygen from 250 .mu.M in terms of saturated dissolved oxygen at 25.degree. C. is measured over time using an oxygen electrode, and 1 unit (U) is defined as the consumption of 1 .mu.mol of oxygen per minute.

Reference example 1 Pseudomonas maltophilia LB-86 (Pseu
+lomonas maNophilia LB-86
, Microtechnical Research Institute No. 9813), bouillon medium 100
500zI for yl! Inoculate into a volumetric flask and culture with shaking at 25°C for 12 hours (110 rpm).
to obtain a seed culture.

The obtained seed culture was placed in a 500 zl Sakalo flask with 100 yl of sterilized yeast extract/glucose medium (yeast extract 2.5%, glucose 3%, KCA'0.
1%, KH2PO40.1%, M g S Oa ・7
Inoculate (inoculation amount = 2%) into a container containing H200.05%, pH 7.2).

Cultivate for 24 hours at a culture temperature of 25°C, stirring at 110 rpm, and after the cultivation is complete, collect the bacteria and collect 3 microorganisms per flask.
The bacterial cells were suspended in 011 phosphate buffer (pH 7.0) and subjected to ultrasonication at 20 KHz and 200 W for 15 minutes. In this way, a bacterial cell extract is obtained.

The nucleoside oxidase activity of this product is 7, OU
/zA'.

After adding 20% streptomycin sulfate 6011 to the above-obtained bacterial cell extract 272yA' and removing the resulting precipitate by centrifugation, ammonium sulfate is added to 35% saturation. After removing the formed precipitate, ammonium sulfate is further added to 60% saturation. After leaving it in a cold place overnight to form a precipitate, this was collected and added to 20mM phosphate buffer (pH 6,0
). This was placed in a dialysis tube and dialyzed for - days, then applied to a DEAE-Toyopearl 650M column (manufactured by Toso Corporation) equilibrated with 20mM phosphate buffer (pH 6,0), washed thoroughly with the same buffer, and then 0 ~0.
Elute with a 5M linear gradient.

The active fraction was concentrated using an Amicon membrane (manufactured by Amicon) with a molecular weight cutoff of 20,000, and then added to a 20 mM phosphate buffer (
Sephacryl S-200 equilibrated at pH 6,0)
Charge the column (manufactured by Pharmacia) and allow the gel to pass through. The eluted active fractions were collected, concentrated again using an Amicon membrane with a molecular weight cutoff of 20,000, and separated with Sephacryl S-
200 ml of gel was washed repeatedly.

The active fractions eluted above are collected and freeze-dried to obtain purified nucleoside oxidase YT-1.

This purified product was disk electrophoretically single and had the properties described above.

The above purification results are summarized in Table 1 below.

Test Example 1 ATP, ADP, AMP according to the measurement method of the present invention
, IMP.

1 (In order to confirm that XR and HX can be quantified, standard solutions of various concentrations were prepared for each substance, and the relationship between concentration and absorbance was investigated.

■Measurement method (1) Measuring reagent (a) Coloring solution 23.4 mg of 4-aminoantipyrine, 5% phenol solution 2. Oyl, D-ribose-1-phosphate (manufactured by Boehringer Mannheim, product number +09240) 11.6
mg was dissolved in 50 mM HEPES buffer (pH 7,5) to make a total volume of 100 zl.

(b) Nucleoside phosphorylase manufactured by Boehringer Mannheim, product number 107956 (
Approximately 20 units/11) were used.

(c) Nucleoside oxidase Pse+++Iomonas malt obtained in Reference Example 1
Extracted from Ophilia LB-86 strain and purified (DE
AE-) Yopal eluate) was used (35 units/1 liter).

(d) Crude alkaline phosphatase manufactured by Boehringer Mannheim, Grade ■ (Grade
II), product number +011154 (approximately 1400 units/11) was used.

(2) Measurement procedure Add coloring solution 2.8111 nucleoside phosphorase 10 μl to a spectrophotometric cell, and 100 μl of standard solutions of various concentrations.
1 was added, the spectrophotometer was set, and zero adjustment was performed. Next, add 10 μl of nucleoside oxidase,
After standing for 5 minutes, absorbance E1 at 500 nm was measured. Next, 50 μl of crude alkaline phosphatase was added to this,
After standing for 5 minutes, absorbance E2 at 500 nm was measured. HX
The values of El were used for R and tb, and the values of E2 were used for the others. Furthermore, the measurements were performed at room temperature.

(3) Measurement results The measurement results for HXR and HX are shown in FIG.

In Figure 1, the horizontal axis represents inosine (
HXR) and hypoxanthine (Flx) addition amount (n
mol ), and the vertical axis shows absorbance (500 nm), in which (1) shows HXR and (2) shows HX.

From Figure 1, 1 (for both XR and HX, a good linear relationship between concentration and absorbance was obtained, and HxR and HX were plotted on the same straight line. From this, HXR and HX at the same concentration are the same. In the measurement method of the present invention,
It was confirmed that l(X was completely converted to HIH.

Furthermore, the measurement results for ATP, ADP, AMP and IMP are shown in FIG.

In the figure, the horizontal axis represents HXR added to the reaction system.

The amount (n mol) of each of ATP, ADP, XAMP, and IMP added, and the vertical axis is the absorbance (500 nm
), in the figure (1) represents HXR, (2) represents ATP,
(3) represents ADP, (4) represents AMP, and (5) represents I.
MF is shown respectively.

From Figure 2, ATP XADP , , AMP and IMP
It can be seen that a good linear relationship is also obtained for , and these are also plotted on the same straight line.

From FIGS. 1 and 2 above, it can be confirmed that all the components necessary for K value measurement can be satisfactorily measured by the measuring method of the present invention.

Test Example 2 In Test Example 1, instead of crude alkaline phosphatase,
Crude acid phosphatase (manufactured by Boehringer Mannheim,
Grade ■) and crude apyrase (manufactured by Sigma, No.
A6132) and HEPES buffer (
The same test was repeated using MES buffer (pH 6.0) instead of pH 7.5). The results obtained are shown in Table 2 below.

Table 2 From Table 2, crude acid phosphatase and crude apyrase (manufactured by Sigma, No.
, A6132), the same level of color development was similarly obtained, confirming that these enzyme preparations can also be used in the present invention.

Test Example 3 In this test, the method of the present invention was actually implemented using a fish meat extract, and its reproducibility and accuracy were examined.

■Preparation of sample Using yellowtail purchased at a fresh fish store in Otsu city, 4 g
10% TCA solution 82A' was added to homogenize. Next, a 10NKOH solution was added to the centrifuged supernatant to neutralize it, and the mixture was diluted to 1011 and used as a fish meat extract for freshness measurement.

■Freshness measurement method (1) Measuring reagent (a) Coloring solution 23.4n+g of 4-aminoantipyrine, 34.1mg of DAO8 (manufactured by Dojindo Chemical Co., Ltd.) and D-ribose 1-phosphate (manufactured by Boehringer Mannheim, product number 109240)
) 11. 6mg in 50mM HEPES buffer (
pH 7.5) to make the total amount 10011.

(b) Nucleoside phosphorylase Boehringer Mannheim product number 107956 (
Approximately 20 units/11) were used.

(c) Pseudomonas maltop obtained in Nucleoside Oxidase Reference Example 1
extracted from hilia L886 strain and purified (DEAE-
1 Yopal eluate) was used (35 units/11
).

(d) Crude alkaline phosphatase manufactured by Boehringer Mannheim, Grade ■ (Grade
II), product number 108154 (approximately 1400 units/
11) was used.

(2) Measurement procedure Spectrophotometric cell, coloring solution 2.8zA', nucleoside phosphorase 10μ! and 100 μl of fish meat extract were added, and the spectrophotometric system was set and zero adjustment was performed. Next, 10 μl of nucleoside oxidase was added, and after being left for 5 minutes, the absorbance E at 593 nm was measured. Next, 50 μl of crude alkaline phosphatase was added thereto, and after being left for 5 minutes, the absorbance E2 at 593 nm was measured. K value is E
It was determined from the formula +/E2X100.

Note that all measurement operations were performed at 30°C.

(3) Measurement results The same sample was measured 10 times, and an additional 10 measurements were performed the next day.
Measurements were made twice.

The average value, standard deviation, and variation coefficient of the obtained values were calculated.

The results are shown in Table 3 below.

Table 3 From the above table, it can be seen that the method of the present invention has sufficient accuracy and reproducibility for practical use.

Example 1 Burrs purchased at a fresh fish store were divided into three groups, and values were measured for each sample immediately after purchase, after being left in the refrigerator for 1 day, and after being left in the refrigerator for 2 days.

The method for preparing the sample and the method for measuring each value measurement item were the same as in Test Example 3.

The results obtained are shown in Table 4 below.

Table 4 From Table 4 above, it can be seen that the measurement method of the present invention increases the value of the test sample, squid, as time passes after purchase. It is clear that freshness measurements can be carried out.

Example 2 Yellowfin tuna, yellowtail, horse mackerel, sardine, smelt, chicken fillet, and yellowtail purchased at a fresh fish store were each divided into three groups and used as samples immediately after purchase, after being left at room temperature, and after being left at 30°C. The method of the present invention was carried out in the same manner, and the K value was determined.

In addition, the freshness value of the same sample as above was measured by HPLC method under the following conditions according to the conventional freshness measurement method. <HPLC method> Column: Cosmo Seal 5C18 (manufactured by Nacalai Tesque) Eluent: 10mM KH2P 04/methanol-88/3 Detection: UV254nm Flow rate: 1. The results of the values determined for each sample are shown in FIG.

In the figure, the horizontal axis shows the value (%) of each sample measured by the conventional HPLC method, and the vertical axis shows the value (%) of each sample determined according to the method of the present invention.

From Fig. 3 above, the value obtained by the method of the present invention and the conventional HP
A high correlation was observed between the values determined by the LC method,
The correlation coefficient was as follows.

γ=0.996 Regression equation y=1. ．． 04x+0.98 Example 3 Using yellowfin tuna and yellowtail purchased at a fresh fish store, samples were prepared in the same manner as in Test Example 3, and the It x R ratio, HX ratio, and IMP ratio were determined as follows for each of them. I asked for

That is, the same coloring reagent 2.8z/ as in Test Example 3 and Sample 4
Pour 0 μl into a spectrophotometer cell and perform zero adjustment, then add 10 μl (3
5 units/11) was added, and absorbance A1 was measured 5 minutes later.

Next, add nucleoside phosphorylase 10 to the above reaction solution.
μl was added and left for 5 minutes, and then the absorbance A2 was measured.

Furthermore, purified alkaline phosphatase (manufactured by Boehringer Mannheim, grade ■, product number 405) was added to the above reaction solution.
612) 50 μl was added, reacted for 5 minutes, and measured absorbance A3.

The measurement results are shown in Table 5 below.

In addition, A1 is the concentration of HXH, and A2 is Hl and HXR.
and A3 is the total concentration of IMP XHxR and HX
These values correspond to the total amount of , and from these, the lx ratio [
AI /A3 X 100], HXR ratio [(A2AI
)/A3 X100] and IMP ratio [(A3A2)
/A3 x 100] and the results of calculating the K1 value in the same manner are shown in Table 5.

Table 5 is a graph showing the relationship between the concentration and the concentration, and FIG. 3 shows the correlation between the values determined by the method of the present invention carried out in accordance with Example 2 of the present invention and the values determined according to the conventional HPLC method. This is a graph showing.

(Above) From Table 5 above, according to the method of the present invention, the HX ratio, HXR ratio, IMP ratio and values as freshness indicators of seafood can be calculated very easily. It can be seen that it is highly accurate and can be used to measure the freshness of seafood.

[Brief explanation of drawings]

Figures 1 and 2 show the concentration and absorption of each substance measured by the method of the present invention according to Test Example 1 of the present invention. Houtoki Aonokuchi (nmol) 00

Claims (1)

[Scope of Claims] [1] In a system containing a sample solution for freshness measurement, a phosphate-free buffer solution, and a coloring reagent that develops color by oxidation, (1) nucleoside phosphorylase is added in the presence of ribose-1-phosphate. (2) a step of causing a nucleoside oxidase to act to oxidize the nucleoside and developing a coloring reagent using the laccase-like activity of the nucleoside oxidase that is expressed in accordance with the amount of the nucleoside; (3) a step of acting with enzymes that convert adenosine triphosphate, adenosine diphosphate, adenylic acid, and inosinic acid into nucleosides; and (4) a step of measuring the degree of color development of the reaction solution after each of the above steps. A method for measuring the freshness of seafood. [2] In claim [1], steps (1) to (3)
are carried out in this order, and in step (3), enzymes selected from crude alkaline phosphatase, crude acid phosphatase, and crude apyrase are used, and the measured value after step (2) is divided by the measured value after step (3). A method of measuring the freshness of seafood by determining the K value and using this as an index of seafood freshness. [3] In claim [1], steps (1) to (3)
are carried out in this order, and purified alkaline phosphatase and/or 5'-nucleotidase are used in step (3), and the K_1 value is calculated by dividing the measured value after step (2) by the measured value after step (3). A seafood freshness measurement method that uses this as a seafood freshness index. [4] In claim [1], steps (1) to (3)
are carried out in the order of steps (2), (1) and (3), and using purified alkaline phosphatase and/or 5'-nucleotidase in step (3), (a) from the measured value after step (1) The value obtained by subtracting the measured value after step (2) is divided by the measured value after step (3), and Hx
(b) divide the measured value after step (2) by the measured value after step (3) to obtain the HxR ratio; and (c) calculate the HxR ratio from the measured value after step (3). The value after subtracting the measured value after step (3) is divided by the measured value after step (3).
A seafood freshness measurement method in which the P ratio is determined and at least one of the obtained values is used as a seafood freshness index.