JP2879351B2 - Method for measuring freshness of seafood - Google Patents

Description

Description: FIELD OF THE INVENTION The present invention relates to a method for measuring the freshness of fish and shellfish, and more particularly to a new method for measuring the freshness of fish and shellfish more easily and quickly by using various enzyme reactions in combination.

2. Description of the Related Art Conventionally, the freshness (lifeliness) of fish and shellfish has been sensually determined. However, with the recent progress in refrigeration technology, changes in packaging and distribution mechanisms, etc., the freshness of fish and shellfish by the appearance of the fish and shellfish has been determined. Judgment is becoming increasingly difficult, and a chemical and objective freshness measurement technique and an index therefor are required instead. At present, the K value calculated by the following equation (1) is known as the most reliable freshness index that meets the above demand.

(Where ATP is adenosine triphosphate and ADP is adenosine 2
Phosphoric acid, AMP is adenylic acid, IMP is inosinic acid, Hx
R represents inosine, Hx represents hypoxanthine, respectively,
The same applies hereinafter. Also, since ATP, ADP and AMP are rapidly decomposed in fish meat, even with the K ₁ value calculated by the following equation (2) omitting the above equation from the above equation (1), the K ₁ value is sufficiently accurate for practical use. It has been clarified that freshness can be represented (for example, JP-A-59-107256, J. Agric. Food Chem., Vol. 3).
2, pp 18-22 (1984)).

Thus, the freshness indicators K values and K ₁ value, conventionally the following (1) to (4) are obtained by such various methods, this such respective law specific to them as described below issues There are some points, and none of them are sufficient.

That is, (1) a method in which each component is fractionated using an ion-exchange resin and each of them is measured to calculate a K value (simple chromatography method) is a method in which even an expert can calculate the above-mentioned index by 2 to 2. The operation time is as long as 3 hours, which is disadvantageous in that the operation is too complicated, and the regeneration and equilibration of the ion exchange resin used are required.

(2) A method of separating each component using a reversed-phase column, a column for gel permeation chromatography, and the like, and quantifying the components to obtain a K value (HPLC method) is performed by using an expensive HPLC apparatus, a chromatopack, or the like. It is necessary to have accessories to determine the peak area of each component, and it requires skillful techniques for measurement.Moreover, it is necessary to prepare standard solutions of each component. There is also a drawback that requires cleaning.

(3) In the UV method using xanthine oxidase and nucleoside phosphorylase, a nucleic acid component is first extracted from fish meat using perchloric acid, a powerful drug, and the absorbance of the extract at 250 nm is measured to determine the total amount of nucleic acid-related compounds. (A) is determined, and then the above extract is treated with xanthine oxidase and nucleoside phosphorylase to convert HxR and Hx into uric acid, and the amount of uric acid generated is determined from the absorption at 293 nm.
Further, this is multiplied by a conversion factor different depending on the fish (B)
Is calculated, and A / B × 100 is calculated from the above values to obtain a K value. However, this method is dangerous because it requires the use of perchloric acid, a powerful drug, as it does not interfere with UV absorption when extracting nucleic acid-related substances from fish meat, as described above. There is a disadvantage that a total is required. Furthermore, this method is to determine the amount of nucleic acid-related substances by UV absorption, but there may be substances having UV absorption other than the nucleic acid-related substances in fish meat samples,
Considering this point, there is a problem in accuracy.

(4) A method of measuring the amount of oxygen consumed in an enzymatic reaction using an oxygen electrode and indirectly quantifying an ATP-related compound is as follows. First, a fish meat sample is divided into two, and one of them is xanthine oxidase and nucleoside phosphorylation. HxR and Hx are oxidized to uric acid by the action of an enzyme, and the amount of enzyme in the solution consumed at this time is measured with an oxygen electrode to determine the amount of HxR and Hx, while the other sample is allowed to act on crude alkaline phosphatase. ATP, ADP, AMP and IMP
Is converted to HxR, xanthine oxidase and nucleoside phosphorylase are allowed to act on this, and the amount of the enzyme consumed at this time is similarly measured with an oxygen electrode to obtain the total amount of ATP-related compounds, and the obtained measured values of both are obtained. Is a method of calculating the K value from the ratio of. This method has the disadvantage that the sample must be divided into two parts and the oxygen consumption must be determined separately using an oxygen electrode for each of these, with the disadvantage that an expensive measuring instrument is required. Preliminary reactions are also necessary and time consuming, and each enzymatic reaction is affected by catalase normally incorporated in the enzyme preparation used, which has the disadvantage of reducing accuracy.

As described above, seafood freshness measurement techniques as an index K value and K ₁ values from the prior art, despite the index itself is reliable, or a complicated measurement technology itself, accuracy However, the development of a new freshness measurement technique which can obtain the above-mentioned index more quickly, easily and accurately is demanded in this industry.

SUMMARY OF THE INVENTION The object of the present invention is to provide a method for measuring freshness quickly, easily and accurately, which meets the needs of the above-mentioned industry, and uses an HPLC apparatus and an oxygen electrode which are found in conventional methods. measuring device,
An object of the present invention is to provide the above method which can be performed by an inexpensive analyzer without requiring an expensive device such as an ultraviolet spectrophotometer.

Means for Solving the Problems The above object is achieved by the following method.

That is, according to the present invention, (1) nucleoside phosphorylase action in the presence of (1) ribose-1-phosphate in a system containing a freshness measurement sample solution, a phosphate-free buffer solution, and a coloring reagent that develops color by oxidation. (2) activating nucleoside oxidase to oxidize the nucleoside, and coloring the coloring reagent by the laccase-like activity of the nucleoside oxidase expressed corresponding to the amount of the nucleoside; 3) a step of reacting 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 freshness of fish and shellfish is provided.

More specifically, the method of the present invention comprises: (1) an enzyme selected from crude alkaline phosphatase, crude acid phosphatase and crude apyrase in the above step (1) to step (3) in this order; A method of dividing the measured value after the step (2) by the measured value after the step (3) to obtain a K value and using the K value as an index of freshness of fish and shellfish. Steps (3) to (3) are performed in this order, and the measured value after step (2) is divided by the measured value after step (3) using purified alkaline phosphatase and / or 5'-nucleotidase in step (3). And K ₁
And (3) performing the steps (1) to (3) in the order of steps (2), (1) and (3), and (3) Using purified alkaline phosphatase and / or 5'-nucleotidase in (3), (a) subtracting the measured value after step (2) from the measured value after step (1), (B) Divide the measured value after step (2) by the measured value after step (3) to obtain HxR.
(C) The value obtained by subtracting the measured value after step (1) from the measured value after step (3) is divided by the measured value after step (3) to obtain the IMP ratio. It also includes a method of using any of the above values as freshness indicators for fish and shellfish.

The method of the present invention of any of the above (1) to (3) is rapid,
It enables simple and accurate freshness measurement of fish and shellfish, and does not require expensive equipment such as HPLC equipment, oxygen electrode measurement equipment, ultraviolet spectrophotometer, etc. as in the conventional method, and is performed with an inexpensive analyzer. It has the advantages that it can.

The nucleoside oxidase used in the method of the present invention will be described in detail. This enzyme was first developed by the present inventors (Japanese Patent Application No. 63-196632), and is described in "Nucleoside Oxidase YT".
-1 "is an enzyme that catalyzes the oxidation reaction of a nucleoside, which reacts with the molecular oxygen to form a nucleoside through a nucleoside-5'-aldehyde.
It produces 5'-carboxylic acid, but does not produce hydrogen peroxide as a by-product. The nucleoside oxidation reaction catalyzed by the present enzyme is represented by the following formulas (1) and (2), and is clearly distinguished from other known nucleoside oxidase oxidation reactions.

In addition, the present enzyme also has a property unique to the enzyme that exhibits laccase-like activity according to the amount of the nucleoside simultaneously with the oxidation reaction of the nucleoside represented by the above formula. It is characterized by utilizing the above-mentioned specific laccase-like activity.

This enzyme YT-1 is produced using the enzyme-producing bacterium belonging to the genus Pseudomonas. Examples of the above-mentioned enzyme-producing bacteria used herein include strains having the following properties newly isolated by the present inventors.

I. Mycological properties (A) Morphological properties The cells are bacilli and have a size of 0.5 × 1.5-1.7 μm.

 It has motility and the flagella are extreme flagella.

 No spores are formed.

 It is negative in Gram staining.

(B) Culture findings Gravy agar plate culture Growth is moderate, colonies are round, and the surface is smooth and yellow.

 Gravy agar slope culture Growth is moderate, the surface is smooth and yellow.

 Broth liquid culture Growing on the surface, growth is moderate.

Broth gelatin stab culture Grow on the surface and liquefy gelatin (Stratifor
m) (C) Physiological properties Nitrate reduction Negative denitrification reaction Negative Indole formation Negative hydrogen sulfide formation Negative starch hydrolysis Negative nitrate use Negative use of ammonium salts Positive dye formation Fluorescent dye, pyocyanin and xanthomonadine Also does not generate. The broth agar medium may produce a brown diffusible pigment.

Urease negative Oxidase negative to weak positive Catalase positive Growth range Does not grow at pH 4.5. Does not grow at 4 ° C and 41 ° C. The growth pH is suitably in the range of 5 to 8 and the temperature is optimally 35 ° C.

 Attitude to oxygen Aerobic OF test (Hugh Leifson method) Oxidative generation of acid from saccharides D-glucose positive D-fructose positive D-maltose positive D-galactose positive D-xylose positive D-mannitol negative sucrose Negative Lactose Positive Esculin Degradation Positive Arginine Dihydrolase Negative Lysine Decarboxylase Positive Ornithine Decarboxylase Negative Phenylalanine Deaminase Negative Yolk Reaction Negative Tween 20 Degradation Positive Tween 80 Degradation Positive Polybeta-hydroxybutyrate Accumulation Negative auxotrophic methionine or cystine Request.

Based on the above properties, the search for this strain was carried out using barge [Berg
ey's Manual of Systematic Bacteriology (1984)]
This strain is a gram-negative bacillus, does not form spores, is catalase-positive, exercises on flagella, oxidatively degrades glucose, pH 7.0 normal agar. It was recognized as belonging to the genus Pseudomonas due to its characteristics such as growth on a medium. In addition, it requires cystine or methionine for growth, that it is arginine dihydrolase negative, that it does not accumulate polybeta-hydroxybutyric acid, that its colonies are yellow, that it does not produce xanthomonadine,
This was in good agreement with the characteristics of Pseudomonas maltophilia, such as the weak oxidase reaction.

From the above results, the present inventors identified this strain as a strain of Pseudomonas maltophilia, and identified this strain as Pseudomonas maltophilia LB-86 (Pseudomonas maltophiria).
lia LB-86). The strain was obtained from the Institute of Industrial Science and Technology of the Ministry of International Trade and Industry, Microbial Industrial Technology Research Institute,
No. 13 (FERM P-9813).

The present enzyme nucleoside oxidase TY-1 is obtained from the LB-
It can be produced by culturing a microorganism belonging to the genus Pseudomonas including 86 strains and mutants thereof and having the ability to produce nucleoside oxidase TY-1, and this culture can be carried out in a suitable nutrient-containing medium. As the medium, any of a synthetic medium, a semi-synthetic medium and a natural medium containing various carbon sources, nitrogen sources, inorganic substances and the like usually used for culturing microorganisms belonging to the genus Pseudomonas can be used. Specific examples of the carbon source include various carbon compounds that can be assimilated by microorganisms such as higher fatty acids, starch hydrolyzate (dextrin), maltose, lactose, glucose, molasses, fructose, etc., alone or in combination of two or more. Can be Examples of the nitrogen source include, for example, peptone, meat extract, yeast extract, soybean powder, cottonseed powder, and constellite liquor. As the inorganic substance, for example, in addition to salt, inorganic salts such as phosphates and sulfates of inorganic metals such as potassium, sodium, magnesium, calcium, zinc, and iron can be appropriately used as needed.

The cultivation of the microorganism can be carried out in either liquid culture or solid culture, and is preferably performed aerobically under aeration and stirring conditions. Culture conditions such as culture medium pH, culture temperature, and culture time are suitable for the growth of the microorganism to be cultured, and are preferably selected from conditions that maximize the production of the target enzyme. For example, the medium pH is preferably selected from the range of about 6 to 8, and the culture temperature is preferably selected from the range of about 20 to 35 ° C.
The cultivation time may be selected so long as the production and accumulation amount of the target enzyme reaches the maximum, and is usually selected from the range of about 12 to 48 hours. Of course, the conditions and culture method of the above culture can be appropriately changed according to the type of microorganism used, external conditions for culture, and the like.

In the culture thus obtained, the desired enzyme is usually accumulated in the cell body fraction. The collection and purification of the enzyme can be performed according to various ordinary methods conventionally employed in a method for producing an enzyme or the like using a microorganism. As the above-mentioned purification means, for example, a means utilizing solubility in a solvent, a means utilizing a difference in ionic bonding force, a means utilizing a difference in molecular weight, a means utilizing a difference in isoelectric point, and utilizing a difference in hydrophobicity The means and the like can be employed alone or in appropriate combination.

More specifically, the collection of the target enzyme from the bacterial cell compartment is described below.
For example, wet cells obtained by collection according to a conventional method such as centrifugation are suspended in a phosphate buffer or a Tris buffer or the like, and various cell processing means such as ultrasonic treatment, French press, and lysozyme treatment are appropriately combined. In this manner, a crude enzyme-containing solution is obtained. The crude enzyme content can be further purified by a usual method to obtain a purified enzyme preparation. As a purification means, for example, a precipitate is firstly recovered from the above crude enzyme-containing solution by a fractional precipitation method using an organic solvent such as ethanol or acetone, a salting-out method using ammonium sulfate, aluminum sulfate or the like, and then the precipitate is subjected to diethylaminoethyl. It can be carried out by performing a chromatography operation using an ion exchanger such as dextran or a gel permeating agent such as polyacrylamide gel, and the purified enzyme preparation thus obtained can be further purified to a purified powder preparation by an operation such as freeze-drying. it can.

The enzyme obtained as described above is characterized in that it has an enzymatic action of catalyzing the oxidation reaction represented by the above formulas (1) and (2), and has the following physicochemical properties and the like. It is also characterized in terms of points.

(1) Substrate specificity: It acts on various nucleosides such as inosine. It does not act on bases such as hypoxanthine, nucleotides such as inosinic acid, or ribose.

(2) Temperature and pH stability: 95% or more remains at pH 6.0, 60 ° C, 15 minutes, and deactivates at 70 ° C, 15 minutes.

Further, 95% or more remains at pH 5.0 to 6.0 in the treatment at 37 ° C. for 60 minutes.

(3) Optimum pH: pH 5.0 to 6.0.

(4) Molecular weight: about 130,000.

(5) Isoelectric point: pH 5.3.

(6) Influence of inhibitor: Inhibited by potassium cyanate and sodium azide.

(7) Visible absorption: In a neutral buffer such as a phosphate buffer, there is no absorption maximum at 450 nm or more. Oxidized enzymes that have not been reduced by the substrate, nitrite (sodium hydrosulfite) or the like have no absorption maximum at 450 nm or more.

(8) Metal content: contains iron.

In the method of the present invention, the present enzyme nucleoside oxidase YT-1 having the above properties is used to produce inosine,
Various nucleosides such as adenine are oxidized by using a molecular enzyme as an electron acceptor, and at the same time, a laccase reaction according to the amount of nucleoside is performed in the presence of the nucleoside, utilizing a laccase-like activity specific to the enzyme, such as hydroquinone. Oxidation of a representative laccase substrate such as, and oxidative coupling of 4-aminoantipyrine and phenol or an aniline-based substance or the like can produce a dye such as quinone imine in proportion to the amount of the nucleoside, and thus The amount of nucleoside can be quantified by measuring the amount of the dye formed.

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 nucleoside oxidase can be used. The substance includes a reagent that is oxidized by a single compound and absorbs in the visible region, and a reagent that is oxidized and condensed by a combination of two or more compounds and absorbs in the visible region. Examples of the single compound include various laccase substrates such as o-tolidine, o-toluidine, o-dianisidine, 10-N-methylcarbamoyl-3,7-dimethylamino-10-H-phenothiazine, bis [3- Screw (4
-Chlorophenyl) -methyl-4-dimethylaminophenyl] amine and the like. The combination of the two or more compounds may be a combination of a so-called coupler and a Trinder reagent (hydrogen donor), as represented by 4-aminoantipyrine and phenol, which have been widely used as clinical diagnostic agents. Can be illustrated. Specific examples of the above-mentioned couplers include, in addition to 4-aminoantipyrine (4-AA), 2,6-dibromoaminophenol, 3-methyl-benzothiazolinone hydrazone and the like, and specific examples of a Trinder 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 (AD
OS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -aniline (ALOS), N-ethyl-N-
(2-hydroxy-3-sulfopropyl) -m-triidine (TOOS), N-ethyl-N-sulfopropyl)
-M-anisidine (ADPS), N-ethyl-N-sulfopropylaniline (ALPS), N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline sodium salt (D
APS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline sodium salt (DAOS), N-sulfopropyl-3,5-dimethoxyaniline (HDAPS), N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (HDAO
S), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethylaniline (MAOS), N-
Ethyl-N-sulfopropyl-3,5-dimethylaniline (MAPS), N-ethyl-N-sulfopropyl-m-
Toluidine (TOPS), N ² - ethyl -N ² - (3- methylphenyl) can be exemplified -N'- acetylethylenediamine (EMAE) or the like.

Further, in the method of the present invention, other reagents other than the nucleoside oxidase and the coloring reagent, namely, a freshness measurement sample solution, a phosphate-free buffer, ribose-1-phosphate, nucleoside phosphorylase, and ATP, ADT, AMP
And enzymes that convert IMP to nucleosides (HxR) (eg, typically alkaline phosphatase, etc.) include:
Any of the same ones used in this kind of conventional freshness measuring technique can be used, and can be prepared in the same manner. For example, as the sample liquid for freshness measurement, an extract of fish and shellfish obtained according to an ordinary method, for example, an alkali neutralized product of a supernatant obtained by adding a 10% TCA solution, homogenizing and then centrifuging can be used.

As a phosphate-free buffer, a Tris-HCl buffer, a HEPES buffer, or the like can be used.

ATP, ADT, AMP and IMP such as ribose-1-phosphate, nucleoside phosphorylase and alkaline phosphatase
Enzymes that convert to nucleosides (HxR) include:
Any of various commercial products can be used. In particular, ATP, ADT,
Enzymes that convert AMP and IMP to nucleosides (HxR) are selected from crude alkaline phosphatase, crude acidic alkaline phosphatase and crude apyrase according to the method of the present invention described in (1) to (3) above. It is necessary to use alkaline phosphatase and / or 5'-nucleotidase properly.

 Hereinafter, the measuring method according to the method of the present invention will be described in detail.

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

In this method, ATP (adenosine triphosphate), ADT (adenosine diphosphate), AMP (adenylic acid), IMP (inosinic acid), HxR
(Inosine) and Hx (hypoxanthine), which are measured according to the present invention, respectively, as follows.

That is, (1) Hx is converted to HxR by the step (1) of allowing nucleoside phosphorylase to act in the presence of ribose-1-phosphate. Thus, the nucleoside phosphorylase is originally an enzyme that phosphorylates a nucleoside such as HxR to generate a base such as Hx and ribose-1-phosphate. A reverse reaction occurs, thus producing HxR from Hx. The inventor has confirmed that the reverse reaction substantially proceeds 100% according to the subsequent step (2) by reacting the produced HxR with nucleoside oxidase and oxidizing the HxR to remove it from the reactor. ing.

(2) HxR is directly oxidized by the step (2) of reacting nucleoside oxidase. In this step (2), the chromogenic reagent is also oxidized by the laccase-like reaction at the same time as the oxidation reaction of HxR to produce a colored substance, and color development is recognized.

(3) ATP + ADP + AMP + IMP is dephosphorylated in the step (3) of reacting alkaline phosphatase selected from crude alkaline phosphatase, crude acidic alkaline phosphatase and crude apyrase, and ATP, ADP and AMP are converted to AdR (adenosine), and IMP is converted to HxR. Are oxidized by the step of reacting nucleoside oxidase in the same manner as in the above step (2).

Therefore, in the first method of the present invention, the steps (1) to (3) are performed in this order, and the measured value (E ₁ ) after the step (2) is obtained.
The value (E ₁ −E ₀ ) obtained by subtracting the measurement value (E ₀ ) before the step (2) from the measurement value (E ₁ −E ₀ ) is calculated from the measurement value (E ₂ ) after the step (3). By dividing the previous measurement value (E ₀ ) by the subtracted value (E ₂ −E ₀ ), the K value can be obtained by the following equation.

K value = (E ₁ −E ₀ ) / (E ₂ −E ₀ ) × 100 (1 ′) In particular, in the first method, the step (3) is carried out in a phosphate-free buffer. ATP, ADP, AMP and IMP are converted to HxR by acting at about pH 7.5, H
The conversion of x to HxR and the oxidation of HxR can be performed at the same pH, and thus there is an advantage that all items necessary for K value measurement can be performed in the same system using a single sample.

The operation of each step in carrying out the first method of the present invention will be described in detail. In this method, riboside phosphorylase is first added to a solution in which ribose-1-phosphate and a coloring reagent are dissolved in a phosphate-free buffer. And a freshness measurement sample (for example, fish meat extract) are added and reacted (step (1)).
Measuring the absorbance E _0. This can be replaced with zero adjustment of the absorbance measuring device. Next, are carried out with the addition of nucleoside oxidase to the reaction solution (step (2)) to measure the absorbance E _1. By the above operation, the reaction of each enzyme in the step (1) and the step (2) proceeds simultaneously, and the thus obtained E ₁ -E ₀ value (or the E ₁ value in the case of performing the 0 adjustment) is the value in the sample. It shows the total amount of Hx and HxR.

The crude alkaline phosphatase or the like was added to the reaction system performs dephosphorylation reaction (step (3)), by measuring the absorbance E _2, when subjected to E ₂ -E ₀ (or 0 Adjustment As E ₂ ), the total amount of ATP, ADP, AMP, IMP, HxR and Hx can be determined. In the case of using alkaline phosphatase in the above, the pH in the system is about 6.5 to 8.0 from the working pH of each enzyme,
The pH is preferably about 7.0, and when acid phosphatase and apyrase are used, the pH is preferably about 5.0 to 7.0, particularly about 6.0. Depending on the above pH conditions, the phosphate-free buffer may be, for example, HEPES buffer or Tris buffer at about pH 7.5, or at pH 6.0 at about pH 6.0.
A MES buffer or the like can be used. The concentration of the buffer is not particularly limited as long as it exhibits a sufficient buffering action, but is usually preferably about 10 to 100 mM.

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

The reaction time of each of the above enzyme reactions is appropriately determined according to the type and amount of the enzyme to be used, and is not particularly limited, but may be usually in the range of about 3 to 15 minutes. Generally about 20-50 ° C, preferably about 25-40 ° C
It should be about the degree.

Further, the measurement wavelength for the absorbance measurement is appropriately determined according to the type of the coloring reagent to be used. For example, when 4-aminoantipyrine and phenol are used as a coloring reagent, 500 nm
555nm for TOOS, 630nm for MAOS, DAOS
593nm can be adopted respectively.

According to the second method of the present invention, it is possible to measure the seafood freshness K ₁ value as an index.

According to this method, the steps (1) to (3) are performed in this order, and the measured value (E ₁ ) after the step (2) is obtained by using the purified alkaline phosphatase and the 5′-nucleotidase in the step (3). ) Is subtracted from the measured value (E ₀ ) after the step (2), and the value (E ₁ −E ₀ ) is calculated from the measured value (E ₃ ) of the step (3) to the measured value (E ₃ ) before the step (2). By dividing E ₀ ) by the subtracted value (E ₃ −E ₀ ), the K ₁ value can be obtained according to the following equation.

K ₁ value = (E ₁ −E ₀ ) / (E ₃ −E ₀ ) × 100 (2 ′) That is, according to the use of the purified alkaline phosphatase and 5′-nucleotidase, only IMP is converted to HxR. , thereby may determine the K ₁ value in the same manner as in the first method.

According to the third method of the present invention, the steps (1) to (3) are performed in the order of the steps (2), (1) and (3) according to the same principle as the second method. And in step (3) purified alkaline phosphatase and 5'-
Using nucleotidase, step (a) (1) Measurement value after (A ₁ to) from step (2) Measurement value after (A ₂ to) the subtracted value (A ₁ -A _2), Measured value after step (3) (A ₃
Seeking Hx ratio divided by that), HxR and divided by the step (b) (2) Measurement value after (A ₂₎ a step (3) Measurement value after (A ₃₎
Determine the specific and step (c) (3) Measurement value after (A ₃₎ from step (1) Measurement value after (A ₁₎ obtained by subtracting the value (A ₃ -A ₁₎
The step (3) Measurement value after (A ₃₎ dividing seeking IMP ratio, this or the like can be performed freshness measurement seafood as seafood freshness indicator at least one of the values. These indices are represented by the following equations (3) to (5).

Hx ratio = (A ₁ −A ₂ ) / A ₃ (3) HxR ratio = A ₂ −A ₃ (4) IMP ratio = (A ₃ −A ₁ ) / A ₃ (5) Operation of this third method For details, for example, first, nucleoside oxidase is allowed to act on a freshness measurement sample, and the specific absorbance A ₂ (corresponding to the amount of HxR) is measured. Next, reacted by adding nucleoside phosphorylase measuring absorbance A ₁ in the reaction system. The A ₂ corresponds to the total amount of HxR and Hx. Further measuring the absorbance A ₃ by the action of purified alkaline phosphatase or nucleotidase in the reaction system. The A ₃ is IMP, corresponds to the total amount of HxR and Hx.
From the respective absorbances determined above, the above formulas (3) to (3) are respectively used.
According to (5), the Hx ratio, the HxR ratio, and the IMP ratio can be calculated.

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

Examples Hereinafter, reference examples, test examples and examples will be given in order to explain the present invention in further detail.

The nucleoside oxidase activity was measured by the following method.

<Method for measuring nucleoside oxidase activity> 1 ml of 100 mM phosphate buffer (pH 6.0) containing 7 mM inosine was placed in the cell of the enzyme electrode, and 5 µ of the enzyme preparation was added thereto.
The initial rate of enzyme consumption is measured in. That is, the decrease in the amount of dissolved oxygen from a saturated dissolved oxygen amount of about 250 μM at 25 ° C. is measured over time using an oxygen electrode, and the case where 1 μmol of oxygen is consumed per minute is defined as 1 unit (U).

Reference Example 1 Pseudomonas maltophilia LB-86 (Pseudomona
s maltophilia LB-86, No. 9813) is inoculated into a 500 ml Sakaguchi flask containing 100 ml of bouillon medium and shake-cultured (110 rpm) at 25 ° C. for 12 hours. Get.

The obtained seed culture was sterilized in a 500-ml Sakaguchi flask in advance with 100 ml of yeast extract / glucose medium (yeast extract 2.5%, glucose 3%, Kcl 0.1%, KH ₂ PO ₄ 0.1
%, MgSO ₄ .7H ₂ O 0.05%, pH 7.2) is inoculated (inoculation amount: 2%). The cells were cultured for 24 hours at a culture temperature of 25 ° C. and stirring at 110 rpm. After the completion of the culture, the cells were collected, and the cells were suspended in 30 ml of a phosphate buffer (pH 7.0) per flask. Sonicate for 15 minutes under conditions.
Thus, a sample extract is obtained.

The nucleoside oxidase activity of this is 7.0 U / m
l.

After adding 60 ml of 20% streptomycin sulfate to 272 ml of the bacterial cell extract obtained above, the precipitate formed is centrifugally removed, and ammonium sulfate is added so as to be 35% saturated. After removing the resulting precipitate, more ammonium sulfate is added until 60% saturation. After standing overnight in a cool place to form a precipitate, this is collected and dissolved in 20 mM phosphate buffer (pH 6.0). This was placed in a dialysis tube, dialyzed for one day, applied to a DEAE-Toyopearl 650M column (manufactured by Tosoh Corporation) previously equilibrated with a 20 mM phosphate buffer (pH 6.0), and sufficiently washed with the same buffer.
The salt is eluted with a linear gradient of 0 to 0.5M.

The active fraction was concentrated using an Amicon membrane having a molecular weight cutoff of 20,000 (manufactured by Amicon), and further concentrated in a 20 mM phosphate buffer (pH 6.
The gel is charged by charging a Sephacryl S-200 column (manufactured by Pharmacia) equilibrated in step (0). The eluted active fractions are collected, concentrated again with an Amicon membrane having a molecular weight cut off of 20,000, and gel filtration with Sephacryl S-200 is repeated.

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

This refined product was single by disc electrophoresis and had the properties described above.

 The above purification results are summarized in Table 1 below.

Test Example 1 ATP, ADP, AMP, IMP, HxR and H according to the measurement method of the present invention
To confirm that x can be quantified, standard solutions of various concentrations were prepared for each substance, and the relationship between the concentration and the absorbance was examined.

Measurement method (1) Measurement reagent (a) Coloring solution 4-aminoantipyrine 23.4 mg, 5% phenol solution
2.0 ml and 11.6 mg of D-ribose-1-phosphate (manufactured by Boehringer Mannheim, product number 109240) were dissolved in 50 mM HEPES buffer (pH 7.5) to make the total amount 100 ml.

(B) Nucleoside phosphorylase manufactured by Boehringer Mannheim, product number 107956 (about 20
Unit / ml) was used.

(C) Nucleoside oxidase Extracted from Pseudomonas maltophilia strain LB-86 obtained in Reference Example 1 and purified (DEAE-Toyopearl eluate) was used (35 units / ml).

(D) Crude alkaline phosphatase Boehringer Mannheim Grade II
I), product number 108154 (about 1400 units / ml) was used.

(2) Measurement operation 2.8 ml of a coloring solution, 10 μm of nucleoside phosphorase, and 100 μm of various concentrations of standard were added to a spectrophotometer cell, and the mixture was set in a spectrophotometer and zero-adjusted. next,
Nucleoside oxidase 10μ was added and left for 5 minutes, absorbance was measured E ₁ of 500 nm. Next, 50 μl of crude alkaline phosphatase was added thereto, and after leaving for 5 minutes, 500 nm
The absorbance E ₂ was measured. HxR and Hx is the value of E _1, the other is E ₂
Was used. The measurement was performed at room temperature.

(3) Measurement results The measurement results for HxR and Hx are shown in FIG.

In FIG. 1, the abscissa indicates the amount (n mol) of inosine (HxR) and hypoxanthine (Hx) added to the reaction system.
And the vertical axis indicates absorbance (500 nm), and (1) in the figure indicates HxR
And (2) shows Hx.

FIG. 1 shows that both HxR and Hx showed a good linear relationship between concentration and absorbance, and that HxR and Hx were plotted on the same line. From this, HxR and Hx at the same concentration show the same coloration ratio, and in the measurement method of the present invention, Hx is completely Hx
It was confirmed that it was converted to R.

FIG. 2 shows the measurement results for ATP, ADP, AMP and IMP.

In the figure, the horizontal axis is HxR added to the reaction system, ATP,
The vertical axis indicates the absorbance (500 nm) of each addition amount (n mol) of ADP, AMP, and IMP, and (1) indicates HxR, and (2)
Is ATP, (3) is ADP, (4) is AMP, and (5)
Indicates IMP, respectively.

From FIG. 2, it can be seen that good linear relationships are also obtained for ATP, ADP, AMP and IMP, which are also plotted on the same straight line.

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

Test Example 2 Instead of crude alkaline phosphatase in Test Example 1, crude acid phosphatase (Boehringer Mannheim, grade II) and crude apyrase (Sigma, No. A
6132), and using the MES buffer (pH 6.0) instead of the HEPES buffer (pH 7.5). The results obtained are shown in Table 2 below.

According to Table 2, crude crude phosphatase and crude apyrase (Sigma, N
o.A6132), the same level of color development was similarly obtained, and it was confirmed that the enzyme preparations of the present invention can be used in the present invention.

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

Sample preparation Using yellowtail purchased at a fresh fish store in Otsu City, 4g of its back meat
Was homogenized by adding 8 ml of a 10% TCA solution. Next, 10 NKOH solution was added to the centrifuged supernatant to neutralize it, and then the mixture was made up to 10 ml and used as a fish meat extract for freshness measurement.

Freshness measurement method (1) Measuring reagent (a) Coloring solution 4-aminoantipyrine 23.4 mg, DAOS (manufactured by Dojindo) 34.1 mg and D-ribose-1-phosphate (manufactured by Boehringer Mannheim, product number 109240) 11.6 mg 50mM HE
It was dissolved in PES buffer (pH 7.5) to make the total volume 100 ml.

(B) Nucleoside phosphorylase manufactured by Boehringer Mannheim, product number 107956 (about 20
Unit / ml) was used.

(C) Nucleoside oxidase Extracted from Pseudomonas maltophilia strain LB-86 obtained in Reference Example 1 and purified (DEAE-Toyopearl eluate) was used (35 units / ml).

(D) Crude alkaline phosphatase Boehringer Mannheim Grade II
I), product number 108154 (about 1400 units / ml) was used.

(2) Measurement operation 2.8 ml of a coloring solution, 10 µ of nucleoside phosphorase and 100 µ of fish meat extract were added to a spectrophotometer cell, and the mixture was set in a spectrophotometer and zero-adjusted. Next, 10 μ of nucleoside oxidase was added, and the mixture was left for 5 minutes.
And the absorbance was measured E ₁ of 3nm. Then added the crude alkaline phosphatase 50μ thereto, after standing 5 minutes, the absorbance was measured E ₂ of 593 nm. The K value was obtained from the equation of E ₁ / E ₂ × 100.

 All the measurement operations were performed at 30 ° C.

(3) Measurement results Ten measurements were performed for the same sample, and another ten measurements were performed the next day.

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

 The results are shown in Table 3 below.

From the above table, it can be seen that the method of the present invention has practically sufficient accuracy and reproducibility.

Example 1 Squid purchased at a fresh fish store was divided into three groups, and K values were measured as samples immediately after purchase, after standing in a refrigerator for one day, and after standing in a refrigerator for two days.

The preparation method of the sample and the measurement method of each of the K value measurement items were the same as in Test Example 3.

 The results obtained are shown in Table 4 below.

From Table 4 above, it can be seen that the K value of the test sample, squid, increases with the lapse of time after purchase according to the measurement method of the present invention. It is clear that can be done.

Example 2 Each of yellowfin tuna, yellowtail, horse mackerel, horse mackerel, sardine, smelt, squirrel and yellowtail purchased at a fresh fish store was 3
The sample was divided into groups, immediately after purchase, after standing at room temperature and after standing at 30 ° C., and the K value was determined by carrying out the method of the present invention in the same manner as in Test Example 3 above.

The K value of the same sample was measured by the HPLC method under the following conditions according to a conventional freshness measurement method. <HPLC method> Column: Cosmoseal 5C ₁₈ (manufactured by Nacalai Tesque) Eluent: 10 mM KH ₂ PO ₄ / methanol = 88/3 Detection: UV 254 nm Flow rate: 1.0 ml / min K value obtained for each sample The results are shown in FIG.

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

From FIG. 3, the K value obtained by the method of the present invention and the conventional PHLC
A high correlation was observed between the measured values and the K value, and the correlation coefficients were 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, a sample was prepared in the same manner as in Test Example 3, and the HxR ratio for each of them was as follows. , Hx ratio and IMP ratio were determined.

That is, 2.8 ml of the same coloring reagent as in Test Example 3 and 40 μl of the sample were used.
Into the cell of the spectrophotometer, and after zero adjustment,
Nucleoside oxidase 10μ (35 units / m
was added l), the absorbance was measured A ₁ after 5 minutes.

Next, nucleoside phosphorylase 10 was added to the above reaction solution.
adding μ left for 5 minutes, and then measuring the absorbance A _2.

Furthermore, purified alkaline phosphatase (manufactured by Boehringer Mannheim, grade I, product number 4056) was added to the above reaction solution.
12) was added and 50.mu., the absorbance was measured A ₃ is reacted for 5 minutes.

 The measurement results are shown in Table 5 below.

Also, the A ₁ is the concentration of HxR, A ₂ is the total concentration of the Hx and HxR, also A ₃ is a value corresponding respectively IMP, the total amount of HxR and Hx, Hx ratio than this such as [A ₁ / A ₃ × 100], HxR ratio [(A
With _{2 -A 1) / A 3 ×} 100] and IMP ratio _{[(A 3 -A 2) /} A 3 × results 100] were calculated, the results obtained in the same manner as the K ₁ value, Table 5 It is described together.

From the Table 5, Hx ratio as a freshness indicator of fish according to the method of the present invention, HxR ratio, also the IMP ratio and K ₁ value, very easy to calculate, yet this such indicators seafood It turns out that it is a high-precision thing which can be used sufficiently for the measurement of freshness.

BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1 and 2 are graphs showing the relationship between the concentration of each substance and the absorbance measured by the method of the present invention in accordance with Test Example 1 of the present invention, and FIG. 5 is a graph showing the correlation between the K value determined by the method of the present invention performed according to Inventive Example 2 and the K value determined by the conventional HPLC method.

──────────────────────────────────────────────────続 き Continued on front page (51) Int.Cl. ⁶ Identification code FI G01N 33/12 G01N 33/12

Claims (4)

(57) [Claims] 1. A system comprising a sample solution for freshness measurement, a buffer solution containing no phosphate and a coloring reagent which develops color by oxidation, is treated with (1) nucleoside phosphorylase in the presence of ribose-1-phosphate to form a hypopoxin. A step of converting xanthine to a nucleoside, (2) a step of oxidizing the nucleoside by the action of a nucleoside oxidase, and a step of causing the laccase-like activity of the nucleoside oxidase expressed corresponding to the amount of the nucleoside to develop a color-forming reagent, (3) adenosine Reacting enzymes that convert triphosphate, adenosine diphosphate, adenylic acid, and inosinic acid into nucleosides; and (4) measuring the degree of color development of the reaction solution after each of the above steps. Method for measuring the freshness of seafood. 2. The method according to claim 1, wherein the steps (1) to (3) are performed in this order, and the step (3) uses an enzyme selected from crude alkaline phosphatase, crude acid phosphatase and crude apyrase. 2) A method for measuring the freshness of seafood by dividing the measured value obtained after step (3) by the measured value obtained after step (3) to obtain a K value and using the K value as an index of freshness of seafood. 3. The method according to claim 1, wherein the steps (1) to (3) are performed in this order, and the purified alkaline phosphatase and / or 5'-nucleotidase is used in the step (3). obtains a K ₁ value by dividing the measured value by the measured value after step (3), the freshness measurement of fish to fish freshness indicator this. 4. The method according to claim 1, wherein the steps (1) to (3) are carried out in the order of steps (2), (1) and (3) and in step (3) purified alkaline phosphatase and / or 5 ' Using nucleotidase, (a) dividing the measured value after step (2) from the measured value after step (1) by the measured value after step (3) to obtain Hx
(B) dividing the measured value after step (2) by the measured value after step (3) to determine the HxR ratio; and (c) determining the HxR ratio from the measured value after step (3). The value obtained by subtracting the measurement value obtained after the process is divided by the measurement value obtained after the step (3) to obtain
A method for measuring the freshness of fish and shellfishes, in which the P ratio is determined and at least one of the obtained values is used as a seafood freshness index.