JP2857607B2 - Method and apparatus for measuring freshness of seafood - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to relates to a method and apparatus for measuring fish freshness from the concentration ratio of ATP-related compounds contained in fish such, To be more specific, K _I by FIA method (flow analysis) The present invention relates to a method and an apparatus capable of quickly calculating a value and determining freshness of fish and shellfish in a short time.

[0002]

2. Description of the Related Art People's interest in food quality is increasing year by year, and there is a need for delicious, fresh and safe foods. In order to meet these demands in the food industry, it is necessary to inspect and control the quality of raw materials, intermediate products and final products in the manufacturing process from various aspects. In this case, since a considerable number of man-hours and a skilled panel (inspector) are required to objectively judge the quality of the food by the sensory test, a scientific food quality judgment method using a sensor or the like is required. ing.

[0003] Among the materials handled in the food industry, regarding the freshness of marine products, (a) changes within a very short time, (b) changes in freshness follow various courses depending on fish species, (c) Because of the difficulty of objective judgment, many methods have been considered. Among them, a method of using the ratio (K value) of the amount of the ATP-related compound contained in fish meat as an index of freshness has been established at present.

[0004] That is, after the death of the fish, ATP in muscle is
ATP (adenosine 5'-triphosphate) → ADP (adenosine 5'-diphosphate) → AMP (adenosine 5'-phosphate) → IMP (inosine 5'-phosphate) → H _X R (inosine) → H _X is metabolized (hypoxanthine), but is further decomposed to uric acid and hydrogen peroxide, where H _X R and H _X is accumulated. Therefore, a value obtained by taking the ratio of the sum of the total value and the concentration of all components in the concentration of H _X R and H _X is K value, represented by the following formula (unit:%). [ATP] and the like are the concentrations of the respective components. K value _{= {([H X R]} + [H X]) / ([ATP] + [ADP] + [AMP] + [IMP] + [H
_X R] + [H _X ])} × 100

[0005] Also, 10 to 20 hours after the death of the fish, AT
Components up to P, ADP, and AMP are reduced to almost negligible levels. Therefore, for the seafood that is normally distributed, it is known that expressed the freshness of K values in a simplified below K _I value (unit:%). K _I value = {([H _X R] + [H _X ]) / ([IMP] + [H _X R] + [H _X ])} × 100

[0006] Conventionally, as an index K _I value described above, FIA
As a system for judging the freshness of fish and shellfish by the method, the one having the configuration shown in FIG. 9 has been proposed. In the FIA system of FIG. 9, A is the first carrier liquid, B is the pump,
C is injector, D is uricase immobilized reactor, E
Is a reactor immobilized with alkaline phosphatase (AP),
F is a delay coil, G is a blank reactor, H is a second carrier liquid, I is a pump, J is a mixing coil, K is a purine nucleoside phosphorylase (PNP) / xanthine oxidase (XOD) simultaneous immobilization reactor, L
Indicates a glassy carbon electrode for measuring uric acid, M indicates a potentiostat, and N indicates a recorder.

In the system of FIG. 9, the first carrier liquid A
The sample is injected from the injector C, and H _X R is H _X in the reactor K is in the carrier solution A passing through the flow channel P, H _X is converted into hydrogen peroxide and uric acid, detected the uric acid in the electrode L Is done. Further, the carrier liquid A passing through the passage Q then IMP is converted to H _X R in reactor E, H _X R is the H _X in the reactor K, H _X is converted into hydrogen peroxide and uric acid, This uric acid is detected by the electrode L. At this time, since in the present system it is provided with a delay coil F, H _X a first peak corresponding to the total concentration of R and H _X, IMP, a second corresponding to the total concentration of H _X R and H _X because there is a peak obtained it can be determined K _I values according to the equation based on these peak heights.

In this case, the enzymatic reaction by AP in reactor E is inhibited by phosphate ions,
Since the enzymatic reaction of PNP by phosphorylation is phosphorolysis, a phosphate ion is required. Therefore, in the present system, the first carrier liquid A for introducing the sample into the reactor E is used.
Is used without phosphate ions.
As the carrier liquid H, the one containing phosphate ions is used, and the second carrier liquid H containing phosphate ions is mixed with the first carrier liquid A before the reactor K.

The reason why the uricase-immobilized reactor D is arranged in the apparatus shown in FIG. 9 is that uric acid originally coexisting in the sample gives a positive error, so that the uric acid is decomposed and removed. In the same FIA system, instead of the measurement of uric acid by the glassy carbon electrode L, a reagent that reacts with hydrogen peroxide and emits light is added to the reacted carrier liquid, and the amount of light emitted at this time is measured by a photoresponsive element. in by measuring, it has also been proposed to obtain the K _I values obtained two peaks of similar to the above.

[0010]

The conventional F shown in FIG.
The IA system, enzyme reaction of AP is inhibited by phosphate ions, since enzymatic reaction with PNP is in need of phosphate ions, necessary IMP, the H _X R and H _X to convert into hydrogen peroxide and uric acid Are immobilized by dividing them into two reactors, and the AP immobilized reactor E
The carrier liquid that does not contain phosphate ions is used as the carrier liquid for introducing the sample into the sample, and the phosphate ions are added to the carrier solution when the sample is introduced into the subsequent PNP / XOD simultaneous immobilization reactor K.

[0011] However, AP, conventional FIA systems immobilized by dividing the PNP and XOD two reactors may take about 5 minutes to measure the K _I values of one sample, poor rapidity of measurement Had the disadvantage that That is,
Converts IMP to H _X R, and converts the H _X R to H _X, successive enzymatic reactions like converting a H _X to hydrogen peroxide and uric acid, all required for continuous reaction in a single reactor It is desirable from the viewpoint of reaction efficiency that the enzyme is immobilized in order to increase the reaction rate. However, in the conventional FIA system, the enzyme required for the continuous reaction is divided into two reactors, so that the reaction is difficult. The measurement was time-consuming due to low efficiency and low reaction rate.

[0012] The present invention has been made in view of the above circumstances, and uses the enzyme reaction of AP, PNP and XOD to provide FI.
In the case of obtaining the K _I values of seafood by the A method, 1
The K _I value of the sample can be calculated in a short time of about 1 minute, thus an object to provide a very rapid freshness measuring method and apparatus for fish which can perform freshness of seafood .

[0013]

Means for Solving the Problems The present inventors have conducted intensive studies in order to achieve the above object, and have obtained the following findings. (1) The enzymatic reaction by AP has been considered to be inhibited by phosphate ions so far, and in fact, when the concentration of phosphate ions is high, the reaction is significantly inhibited.
However, the present inventors have set the phosphate ion concentration in the carrier liquid to be at least twice the total concentration of inosine 5′-phosphate and inosine in the sample and not more than 5 mM (unit M is mol / l in the present specification). when the low concentration range are shown), the enzymatic reaction by the AP advanced without being most inhibited by phosphate ions, it is satisfactorily converted to H _X R, also enzymatic reaction by the PNP, phosphoric acid ion concentration of the carrier liquid also proceeds favorably in the range below 5mM at least twice the total concentration of IMP and H _X R in a sample,
H _X R has knowledge that it is converted to H _X. Therefore,
Using those following 5mM at least twice the total concentration of IMP and H _X R of phosphoric acid ion concentration in the sample as a carrier liquid, AP, AP / PNP with immobilized PNP and XOD in one reactor / XOD simultaneous immobilization reactor enables continuous enzyme reaction of IMP → H _X R → H _X → uric acid / hydrogen peroxide with high reaction efficiency and reaction rate,
It was found that the total concentration of IMP, H _X R and H _{X in} the sample could be quickly detected.

[0014] (2) The present inventors have found that phosphate ion concentration as described above by using the carrier liquid in the range below 5mM at least twice the total concentration of IMP and H _X R AP / PNP /
When the measurement was performed using the XOD simultaneous immobilization reactor,
The concentration of the components of H _X or the like to be detected in total from 0.06 to 30
It was found that the calibration curve for each component was linear at a low concentration of 0 μM. Therefore, AP / PNP
/ XOD IMP carrier liquid for introducing a sample into simultaneous immobilization reactors, the total concentration of H _X R and H _X from 0.06 to 3
By introducing a sample into the carrier liquid such that the 00MyuM, it has been found that it is possible to determine the K _I values correctly. However, when detecting such a low-concentration component, a detector having high sensitivity is required.

(3) In the conventional FIA system shown in FIG. 9, since the sample solution having a high concentration is used for the measurement, the amount of the immobilized enzyme necessary for sufficiently decomposing the components in the sample solution is reduced. As a result, the size of the reactor is increased, and it is considered that the reaction takes time in this respect as well. That is, the conventional has been using fish plant extract samples, such preparation conditions such that the concentration of the components, such as H _X is about 5mM in total, so that the inner diameter 3mm as column reactor, those having a length of about 50mm Is needed. In contrast, (2) a total concentration of components such as H _X as described in 0.06 to 30
When the measurement is performed using a fish meat extract sample prepared under such a condition that the concentration is as low as 0 μM, the amount of the immobilized enzyme required to sufficiently decompose the components in the sample may be small,
Since the reaction time can be shortened by reducing the size of the reactor, the measurement can be speeded up.

(4) To determine the K _I value, AP / P
Using the NP / XOD simultaneous immobilization reactor, the total concentration of IMP, H _X R and H _X in the sample is detected as described in (1), and the same sample is detected using the PNP / XOD simultaneous immobilization reactor. What is necessary is just to detect the total concentration of H _X R and H _{X in} the mixture. In this case, in order to determine the K _I values correctly, it is necessary not to decomposition of IMP occurs in the flow path arranged PNP / XOD simultaneous immobilization reactor. In this regard, the present inventors used a carrier solution having a phosphate ion concentration of 5 mM or more as a carrier solution for introducing a sample into the PNP / XOD simultaneous immobilization reactor, thereby achieving a low concentration as described in (2). It has been found that the decomposition of IMP can be suppressed.

(5) Using two reactors, the AP / PNP / XOD simultaneous immobilization reactor and the PNP / XOD simultaneous immobilization reactor, the peak corresponding to the total concentration of IMP, H _X R and H _X and H _X R If and peaks corresponding to the total concentration of H _X to obtain simultaneously, compared with the case where two peaks are detected at different times by using the delay coil as in the system of FIG. 9, to shorten the measurement time be able to.

The present invention has been made based on the above findings (1) to (5), and provides the following method and apparatus. The first method in which the phosphate ion concentration is at least twice the total concentration of inosine 5′-phosphate and inosine in the sample but not more than 5 mM.
A sample obtained from a fish and shellfish is introduced into a carrier liquid, and the first carrier liquid is passed through a reactor for simultaneous immobilization of alkaline phosphatase / purine nucleoside phosphorylase / xanthine oxidase, and the inosine 5′-phosphate in the sample, After converting inosine and hypoxanthine to hydrogen peroxide and uric acid, the concentration of hydrogen peroxide or uric acid in the first carrier liquid is detected to detect inosine 5′- in the sample.
Obtaining a first physical quantity corresponding to the total concentration of phosphoric acid, inosine and hypoxanthine, introducing the sample into a second carrier solution containing phosphate ions, and purifying the second carrier solution with purine nucleoside phosphorylase / xanthine oxidase After the inosine and hypoxanthine in the sample are converted into hydrogen peroxide and uric acid through the co-immobilization reactor, the inosine and hypoxanthine in the sample are detected by detecting the concentration of hydrogen peroxide or uric acid in the second carrier liquid. second physical quantity obtained, said first physical quantity and the fish freshness method for measuring and calculating the K _I value represented by the following formula from the second physical quantity corresponding to the total concentration of. K _I values _{= {([H X R]} + [H X]) / ([IMP] + [H X R] + [H X])} × 100 [IMP]: Inosine 5'-phosphate concentration [H _X R]: Inosine concentration [H _X ]: Hypoxanthine concentration

[0019] provided with a device alkali phosphatase / purine nucleoside phosphorylase / xanthine oxidase simultaneous immobilization reactors, inosine phosphate ion concentration in the sample 5 '
-Adding a first carrier liquid, which is at least twice the total concentration of phosphoric acid and inosine and 5 mM or less, to the first carrier liquid in the first carrier liquid;
Total of syn 5'-phosphate, inosine and hypoxanthine
A sample obtained from fish and shellfish is introduced so as to have a concentration of 0.06 to 300 μM, and the first carrier liquid is passed through the reactor to inosine 5′-phosphate in the sample,
After converting inosine and hypoxanthine into hydrogen peroxide and uric acid, the total concentration of inosine 5'-phosphate, inosine and hypoxanthine in the sample is determined by detecting the concentration of hydrogen peroxide or uric acid in the first carrier liquid. A first channel for obtaining a first physical quantity corresponding to the above, a purine nucleoside phosphorylase / xanthine oxidase simultaneous immobilization reactor, and the sample is introduced into a second carrier liquid having a phosphate ion concentration of 5 mM or more. In addition, the second carrier liquid is passed through the reactor to convert inosine and hypoxanthine in the sample to hydrogen peroxide and uric acid, and then the concentration of hydrogen peroxide or uric acid in the second carrier liquid is detected to thereby obtain a sample. A second channel for obtaining a second physical quantity corresponding to a total concentration of inosine and hypoxanthine in the first physical quantity; Beauty freshness measuring apparatus seafood, characterized by comprising an arithmetic unit for calculating the K _I value represented by the following formula from the second physical quantity.

Hereinafter, the present invention will be described in more detail. Enzyme alkaline phosphatase (AP) is derived from bovine intestinal mucosa, purine nucleoside phosphorylase (PNP) is derived from calf spleen, thermophilic microorganism, and xanthine oxidase (XOD) is derived from milk And PODs derived from thermophilic microorganisms. Known PNPs and XODs are preferably those derived from thermophilic microorganisms because of their high stability and high specific activity.

[0021] The reactor AP / PNP / XOD simultaneous immobilization reactor is intended to AP, the PNP and XOD were immobilized in a single reactor, PNP / XOD simultaneous immobilization reactor fixed the PNP and XOD in a single reactor It is a thing. In this case, each reactor can be prepared by filling a column with a carrier on which an enzyme is immobilized, and a carrier on which a single enzyme is immobilized is placed in the column in the order of AP / PNP / XOD or POD.
It is preferable to use a method in which AP, PNP and XOD or a substance in which PNP and XOD are immobilized on a single carrier is packed in a column, rather than a method in which NP / XOD is arranged in the order of NP / XOD, since the reaction efficiency is higher. Incidentally, as the carrier, any one such as porous glass modified with an aminopropyl group can be used.

Examples of the carrier liquid first carrier liquid, the phosphate ion concentration is less 5mM at more than twice the total concentration of IMP and H _X R in a sample,
Preferably, 1 mM to 2 mM is used. Decomposition of H _X R of the phosphate ion concentration is less than twice the total concentration of IMP and H _X R in the sample in the sample not sufficiently, IM
The total concentration of P, H _X R and H _X cannot be obtained correctly. On the other hand, if it is more than 5 mM, the decomposition of IMP in the sample does not sufficiently occur, and similarly, the total concentration of IMP, H _X R and H _X cannot be obtained correctly. The pH of the first carrier liquid is 7.0-9.
0, especially 7.5 to 8.5. More specifically, as the first carrier liquid, 0.1 pH of about 8 is used.
M Tris-HCl buffer solution to which 1-2 mM phosphate ions are added can be used. If necessary, magnesium ions can be added to the first carrier liquid for the purpose of activating the enzymatic reaction by the AP.

The second carrier liquid contains a phosphate ion concentration, and preferably has a phosphate ion concentration of 5 m.
M or more, especially 5 mM to 1 M, more preferably 50 to 20 M
Use 0 mM one. If the phosphate ion concentration is less than 5 mM, several percent decompose and H _X
R is generated, and the total concentration of H _X R and H _X may not be determined correctly. If it is more than 200 mM, it may not be possible to adjust the pH to a value suitable for chemiluminescence when performing detection by a chemiluminescence method. The pH of the second carrier liquid is 7.0.
-9.0, especially 7.5-7.8.
More specifically, a 0.1 M phosphate buffer solution having a pH of about 7.8 can be used as the second carrier liquid.

Sample As the sample, a sample obtained by cutting off a part of fish meat and removing the biological component of the fish meat by means of homogenization, grinding or the like can be used. At this time, protein removal may be performed by treating fish meat with a strong acid such as perchloric acid or Tris-hydrochloric acid.

[0025] When introducing the sample into the first carrier liquid introduction of the sample into the carrier liquid, IMP of the first carrier liquid, the total concentration of H _X R and H _X .06 to 30
It is appropriate to introduce so that the concentration is 0 μM, preferably 1 to 100 μM. In this case, the calibration curve for each component becomes linear, and the K _I value can be calculated correctly. Further, the reaction time can be shortened by reducing the size of the AP / PNP / XOD simultaneous immobilization reactor.

[0026] When introducing the sample into the second carrier liquid, the total concentration of H _X R and H _X of the second carrier liquid is 0.06
It is appropriate to introduce so that the concentration is 300 μM, preferably 1 to 100 μM. In this case, PNP
/ XOD simultaneous immobilization reactor can be miniaturized to shorten the reaction time.

Detecting means The detecting means for detecting the concentration of hydrogen peroxide or uric acid in the carrier liquid after the reaction to obtain the first physical quantity and the second physical quantity include, for example, the following means, but are not limited thereto. It is not something to be done.

A reagent that reacts with hydrogen peroxide and emits light is introduced into the carrier liquid after the reaction, and the amount of light emitted at this time is measured by a photoresponsive element. As the luminescent reagent, for example, luminols, peroxalates, and the like can be used. As the photoresponsive element, for example, a photodiode, a phototransistor, a PIN photodiode, an avalanche photodiode, a photoconductive cell, a photomultiplier tube Etc. can be used. The concentration of hydrogen peroxide in the carrier liquid after the reaction is measured using an electrode such as a hydrogen peroxide electrode. The uric acid concentration in the carrier liquid after the reaction is measured using an electrode such as a glassy carbon electrode.

When the concentration of hydrogen peroxide is detected by the light emission method or the electrode method as described above, since a peroxide coexisting in the sample may respond and cause a positive error, It is preferable to dispose a peroxidase-immobilized column or catalase-immobilized column before the reactor to remove peroxide from the sample. Further, when detecting the uric acid concentration as in the above, since uric acid coexisting in the sample becomes an interfering substance, it is preferable to remove the uric acid from the sample by disposing a uricase-immobilized column in front of the reactor. . In these cases, the column used for removing the interfering substance may be a small column, and thus does not affect the speed of the measurement.

[0030]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG. 1 is a flow chart showing an example of an apparatus for measuring the freshness of fish and shellfish according to the present invention. In FIG.
2 the first carrier liquid below 5mM at least twice the total concentration of IMP and H _X R in a sample concentration of phosphate ions, the second carrier liquid ion concentration phosphoric acid is more than 5mM 4, 6,
8 is a luminescent reagent solution containing a luminescent reagent that emits light by reacting with hydrogen peroxide, 10 is a sample solution obtained from fish and shellfish, 12 is a peristaltic pump, 14 is a first carrier liquid flow path, and 16 is a second carrier liquid flow. Passages, 18 and 20, luminescence reagent liquid flow paths, 22 a sample liquid flow path, 24 an injector for introducing the sample liquid 10 into the first carrier liquid, 26 an AP / AP interposed in the first carrier liquid flow path 14. A PNP / XOD simultaneous immobilization reactor (inner diameter 2 mm, length 30 mm), 28 detects the luminescence reagent solution 6 introduced into the first carrier liquid exiting the reactor 26 and measures the amount of luminescence at this time with a photoresponsive element. , 30 is an injector for introducing the sample liquid 10 into the second carrier liquid, 32 is a second
A PNP / XOD simultaneous immobilization reactor (inner diameter 2 mm, length 30 mm) interposed in the carrier liquid flow path 16, and the luminescence reagent liquid 8 is introduced into the second carrier liquid exiting the reactor 32 to emit light. A detection unit 36 for measuring the amount with the light-responsive element, and a calculation unit (personal computer) for calculating a K _I value from the light emission amount (first physical amount and second physical amount) measured by the detection units 28 and 34.

In this apparatus, the first carrier liquid flow path 14, the luminescent reagent liquid flow path 18, the injector 24, the AP / PNP /
The XOD simultaneous immobilization reactor 26 and the detection unit 28 constitute a first channel 38, and the second carrier liquid flow path 1
6. Luminescent reagent liquid channel 20, injector 30, PNP /
The XOD simultaneous immobilization reactor 32 and the detection unit 34 constitute a second channel 40.

In the present apparatus, the first carrier liquid flow path 1
The sample liquid 10 is introduced from the injector 24 into the first carrier liquid flowing through the first carrier liquid 4, and the first carrier liquid is supplied to the first carrier liquid AP / PNP /
IM in sample through XOD simultaneous immobilization reactor 26
After converting P, H _X R and H _X to hydrogen peroxide and uric acid, respectively, the IMP, H _X R and H _{X in the} sample are detected by detecting the concentration of hydrogen peroxide in the first carrier liquid by the detection unit 28.
To obtain a first physical quantity (light emission quantity) corresponding to the total concentration of Further, the sample liquid 10 is introduced from the injector 30 into the second carrier liquid flowing through the second carrier liquid flow path 16, and the second carrier liquid is passed through the PNP / XOD simultaneous immobilization reactor 32 so that the H _X R and the After converting H _X into hydrogen peroxide and uric acid, respectively, the detection unit 34 detects the concentration of hydrogen peroxide in the second carrier liquid, thereby obtaining H _X R and H _{X in the} sample.
To obtain a second physical quantity (light emission quantity) corresponding to the total concentration of Then, the arithmetic unit 36, the first physical quantity _{([IMP] + [H X} R] +
And it calculates the K _I values by the following formulas corresponding to [H _X]) and a second physical quantity (corresponding to _{[H X R] + [H} X]). K _I value = (second physical quantity / first physical quantity) × 100

[0033]

The following experiment was carried out using the apparatus shown in FIG.
The effect of the present invention was confirmed. In this case, the detection units 28 and 34
A silicon photodiode was used as the photoresponsive element, and a voltage signal corresponding to light detected by the silicon photodiode was transferred from the detection units 28 and 34 to the calculation unit 36. The voltage value was recorded by the calculating unit 36 and used for calculating the peak height.

Experimental Example 1 Influence of Phosphate Ions in the First Carrier Solution The first carrier solution was treated with a 0.1 M Tris-HCl buffer solution (pH
8.0, containing 10 mM magnesium ion), and sodium phosphate was added thereto to reduce the phosphate ion concentration to 1
It was varied in the range from mM to 100 mM. AP / PNP /
As the XOD simultaneous immobilization reactor 26, a reactor in which an enzyme-immobilized support was packed in a column having an inner diameter of 2 mm and a length of 5 mm was used. Under these conditions, 100 μM of IMP, H _X R and H _X were each measured as a 0.02 mM bis-Tris-HCl buffer solution (20 μl, containing 1 mM phosphate ion), and IMP / H _X R and H _X R were measured. / H _X peak height ratios were compared. The results are shown in FIG. From FIG. 2, the ratio of IMP / H _X R corresponding to the activity of AP decreases linearly in this range.
It turned out to be about 0.9. Further, the ratio of H _X R / H _X corresponding to the activity of PNP is determined by the phosphate ion concentration of 1-2.
It was found to be around 0.8 in mM. Therefore, the phosphate ion concentration in the first carrier liquid is set to 1 to 2 m
M, the enzymatic reaction by AP proceeds without being substantially inhibited by phosphate ions, and PN
It was confirmed that the enzymatic reaction with P also proceeded favorably.
At the time when IMP was added to the first carrier liquid, H
Although the breakdown into _X R and H _X is likely to occur (described later), so ultimately measured as _{[IMP] + [H X R} ] + [H X], is believed not to affect the measurement results .

Experimental Example 2 : Low concentration I in the second carrier liquid
MP Stability In the device of the present invention, the IMP
Although but it is necessary not to decompose, IMP is unstable in alkaline and acidic, there is a partially decomposed to H _X R or H _X. Therefore, a 1 μM low-concentration IMP solution was prepared using various buffer solutions, and the amount of decomposition into H _X R and H _X was measured by high performance liquid chromatography. as a result,
0.1 M Tris-HCl buffer solution (pH 7.8 and pH 8.
Although in 0,1mM phosphate ion-containing) it was observed degradation of about 8% to H _X R, 0.1 M Tris-HCl buffer solution (pH7.8, 5,10,30mM containing phosphate ions) and 0 In a 1M phosphate buffer solution (pH 7.8), almost no decomposition to H _X R or H _X was observed (1%
Less than). Accordingly, those phosphate ion concentration is 5~100mM as the second carrier liquid 4, for example, the use of 0.1M phosphate buffer solution of about pH 7.8, even IMP low concentration of about 1 [mu] M, H _X R and H It has been found that the measurement can be performed with little decomposition into _X.

Experimental Example 3 : Effect of pH in Simultaneous AP / PNP / XOD Immobilization Reactor The pH of the first carrier liquid 2 was examined in the same manner as in Experimental Example 1. The same study as in Experimental Example 1 was performed using a 0.1 M Tris-HCl buffer solution (containing 1 mM phosphate ion and 10 mM magnesium ion) as the first carrier solution and changing the pH between 7.0 and 8.7. Was done. The results are shown in FIG. As a result, the first carrier liquid has a pH of 7.0.
Those having a range of about 5 to 8.7 can be used.
It turned out that the vicinity of H8 was appropriate.

Experimental Example 4 : Effect of pH in PNP / XOD Simultaneous Immobilization Reactor The pH of the second carrier liquid 2 was examined in the same manner as in Experimental Example 3. The same study as in Experimental Example 3 was performed using a 0.1 M phosphate buffer solution as the second carrier solution and changing the pH between 7.5 and 8.7. FIG. 4 shows the results. As a result, the pH of the second carrier liquid was 7.5 to 7.5.
A range of about 8.7 can be used.
A value around 7.8 was found to be appropriate.

Experimental Example 5 : Effect of Reactor Length The effect of the length of the AP / PNP / XOD simultaneous immobilized reactor 26 was examined. The first carrier solution is 0.1 M Tris-HCl buffer solution (pH 8.0, 1 or 2 mM phosphate ion, 10 M
mM magnesium ion). FIG. 5 shows the results. As a result, when the reactor length was 30 mm, the peak height was almost the same for all three components, and the ratio was about 0.99. Therefore, the simultaneous immobilization reactor of AP / PNP / XOD requires 3
That allows the conversion of sufficiently IMP → H _X R → H _X observed in 0mm length. In addition, the same examination was performed for the PNP / XOD simultaneous immobilization reactor 32, and as a result, AP
30mm same as / PNP / XOD simultaneous immobilization reactor
Sufficiently conversion of H _X R → H _X it is found that enables the length of the.

[0039] Experimental Example 6 was performed IMP, H _X R, IMP in calibration following measurement conditions for H _X, H _X R, the creation of a calibration curve for H _X. (1) Luminescent reagent (the same for both the first and second channels): 0.8 M sodium carbonate buffer solution containing 35 µM luminol and 25 µg ml ^-1 peroxidase (pH 10.
0) (2) First carrier solution: 0.1 M Tris-HCl buffer solution (p
H8.0, 1 mM phosphate ion, 10 mM magnesium ion) (3) Second carrier solution: 0.1 M phosphate buffer solution (pH
7.8) (4) Flow velocity: 0.6 m for flow channels 14, 16, 18, and 20
l / min (5) Reactor: Both the reactors 26 and 32 have an inner diameter of 2 mm,
Length 30mm (6) Temperature: 25 ± 0.5 ° C (7) Sample volume: 20μl

The obtained calibration curves are shown in FIGS. FIG. 6 is a calibration curve by the AP / PNP / XOD simultaneous immobilization reactor 26, and FIG. 7 is a calibration curve by the PNP / XOD simultaneous immobilization reactor 32. As a result, a linear calibration curve was obtained in the range of 0.06 to 300 μM, and it was confirmed that the components of the concentration in this range could be measured according to the present invention.

[0041] Experimental Example 7: IMP, measured IMP20μM samples containing H _X R and H _X, simulated samples were prepared containing H _X R2μM and H _X 2 [mu] M, a and a measurement of the sample in the same manner as in Experimental Example 6 went. FIG. 8 shows the response of the detection unit. In this case, the first
The K _I value is calculated from the ratio between the peak height of the output X of the channel and the peak height of the output Y of the second channel. According to FIG. 8, according to the present invention, the K _I value is shortened to about one minute.
It was confirmed that _I value could be calculated.

[0042]

According to the present invention, AP / PNP / XOD
By using the simultaneous immobilization reactor, IMP, H _X
Since the R and H _X can be performed at high speed with high reaction efficiency of the enzyme reaction to be converted to hydrogen peroxide and uric acid, it is possible to calculate the K _I values of one sample in a short time of about 1 minute, seafood Can be determined very quickly.
Further, in the present invention, since a low-concentration sample may be used, the amount of immobilized enzyme necessary for sufficiently decomposing the components in the sample may be small, and the reaction time may be shortened by downsizing the reactor. Therefore, the measurement can be speeded up also in this respect.

[Brief description of the drawings]

FIG. 1 is a flowchart showing an example of a fish and shellfish freshness measuring apparatus according to the present invention.

FIG. 2 shows phosphate ion concentration and IMP in a first carrier liquid.
/ H _X R, is a graph showing the relationship between the ratio of the peak heights of H _X R / H _X.

FIG. 3 shows the pH and IMP / H _X R and H _X R of the first carrier liquid.
/ Peak of H _X is a graph showing the relationship between the height ratio.

FIG. 4 is a graph showing the relationship between the pH of the second carrier liquid and the ratio of the peak height of H _X R / H _X.

5 is a graph showing the relationship between the AP / PNP / XOD simultaneous immobilization reactor length and _{IMP / H X R, H X} R / H X peak height ratio of a.

FIG. 6 is a graph showing the relationship between the concentration of IMP, H _X R and H _X and the peak height when a reaction is carried out by an AP / PNP / XOD simultaneous immobilization reactor.

FIG. 7 is a graph showing the relationship between the concentration of H _X R and H _X and the peak height when a reaction is carried out by a PNP / XOD simultaneous immobilization reactor.

8 is a waveform chart showing an example of a response when a sample containing IMP, H _X R, and H _X is measured using the apparatus of FIG.

FIG. 9 is a flowchart showing an example of a conventional seafood freshness measuring apparatus.

[Explanation of symbols]

 2 First carrier liquid 4 Second carrier liquid 6 Luminescent reagent liquid 8 Luminescent reagent liquid 10 Sample liquid 12 Peristaltic pump 14 First carrier liquid flow path 16 Second carrier liquid flow path 18 Luminescent reagent liquid flow path 20 Luminescent reagent liquid flow path 22 Sample liquid flow path 24 Injector 26 AP / PNP / XOD simultaneous immobilization reactor 28 Detector 30 Injector 32 PNP / XOD simultaneous immobilization reactor 34 Detector 36 Calculation unit 38 First channel 40 Second channel

──────────────────────────────────────────────────続 き Continuation of the front page (72) Inventor Masao Kube 1-3-16 Higashi-Arima, Miyamae-ku, Kawasaki City, Kanagawa Prefecture (56) References Talanta, Vol. 43, No. 2 (Feb. 1996) p. 283-289 Anal. Chim. Acta, Vol. 260, no. 1 (1992) p. 93-98 (58) Field surveyed (Int. Cl. ⁶ , DB name) C12Q 1/42 BIOSIS (DIALOG) WPI (DIALOG)

Claims (4)

(57) [Claims] 1. The method according to claim 1, wherein the phosphate ion concentration is at least twice the total concentration of inosine 5′-phosphate and inosine in the sample and 5 m
A sample obtained from fish and shellfish is introduced into a first carrier liquid having a molecular weight of not more than M, and the first carrier liquid is passed through a reactor for simultaneous immobilization of alkaline phosphatase / purine nucleoside phosphorylase / xanthine oxidase, and the inosine in the sample is removed. After converting 5'-phosphate, inosine and hypoxanthine into hydrogen peroxide and uric acid, the concentration of hydrogen peroxide or uric acid in the first carrier liquid is detected to detect inosine 5'-phosphate, inosine in the sample. And obtaining the first physical quantity corresponding to the total concentration of hypoxanthine, introducing the sample into a second carrier liquid containing phosphate ions, and purifying the second carrier liquid with a purine nucleoside phosphorylase / xanthine oxidase simultaneous immobilization reactor To convert inosine and hypoxanthine in samples to hydrogen peroxide and uric acid By detecting the concentration of hydrogen peroxide or uric acid in the second carrier liquid, a second physical quantity corresponding to the total concentration of inosine and hypoxanthine in the sample is obtained. From the first physical quantity and the second physical quantity, seafood method of freshness measurements and calculates the K _I values shown. K _I values _{= [([H X R]} + [H X]) / ([IMP] + [H X R] + [H X])] × 100 [IMP]: Inosine 5'-phosphate concentration [H _X R]: Inosine concentration [H _X ]: Hypoxanthine concentration 2. The total concentration of inosine 5'-phosphate, inosine and hypoxanthine in the first carrier liquid is 0.06.
The method according to claim 1, wherein the sample is introduced into the first carrier liquid so as to have a concentration of ~ 300 µM. 3. The method according to claim 1, wherein the second carrier liquid has a phosphate ion concentration of 5 mM or more. 4. It is provided with a reactor for simultaneous immobilization of alkaline phosphatase / purine nucleoside phosphorylase / xanthine oxidase, and has a phosphate ion concentration of at least 2 times the total concentration of inosine 5'-phosphate and inosine in the sample, and 5 mM. the first carrier liquid is less than, the first calibration
Inosine 5'-phosphate of the rear liquid, Lee Noshin and Hipoki
So that the total concentration of santin is 0.06-300 μM
A sample obtained from fish and shellfish is introduced into the sample, and the first carrier liquid is passed through the reactor to convert inosine 5′-phosphate, inosine and hypoxanthine in the sample into hydrogen peroxide and uric acid. A first channel for obtaining a first physical quantity corresponding to the total concentration of inosine 5′-phosphate, inosine and hypoxanthine in the sample by detecting the concentration of hydrogen peroxide or uric acid in one carrier liquid; and a purine nucleoside phosphonate. comprising a Foriraze / xanthine oxidase simultaneous immobilization reactors, phosphate ion conc
The sample is introduced into a second carrier liquid having a concentration of 5 mM or more, and the second carrier liquid is passed through the reactor to convert inosine and hypoxanthine in the sample to hydrogen peroxide and uric acid. A second channel that obtains a second physical quantity corresponding to the total concentration of inosine and hypoxanthine in the sample by detecting the concentration of hydrogen peroxide or uric acid in the sample, and is represented by the following equation from the first physical quantity and the second physical quantity. K
_An apparatus for measuring the freshness of fish and shellfish, comprising: a calculation unit for calculating an _I value. K _I values _{= [([H X R]} + [H X]) / ([IMP] + [H X R] + [H X])] × 100 [IMP]: Inosine 5'-phosphate concentration [H _X R]: Inosine concentration [H _X ]: Hypoxanthine concentration