JPS5934120B2 - Food freshness determination method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for quickly and simply determining the freshness of fresh foods, especially fish.

Among fresh foods, fish are consumed in large quantities in Japan, and their quality is closely related to freshness.

Freshness is an important factor that affects the quality of each product, not only when it is used as fresh fish, but also when using batter products and frozen fish, and several methods have been known to date to determine this freshness. In particular, as shown below, it is effective to measure the concentration of substances at each stage in which ATP-related substances in fish meat are decomposed into hypoxanthine by the action of enzymes as the freshness of fish decreases, and use this as a guideline for determining freshness. It is said that there is.

Here, these symbols represent the following, respectively.

For example, the literature (Bull. Jap-SOc-Fish-
24,749C59)) describes a method of determining freshness from the K value as shown below.

This is based on the total amount of ATP-related compounds (]]XRl+
[Hx] is expressed as a percentage, and it has already been applied to a considerable range of fish species and has been shown to be effective in determining freshness. In particular, since the ratio of substance concentrations is taken, there is an advantage that the analytical value remains constant regardless of the dilution of the sample.
Japanese Unexamined Patent Publication No. 50-98892 describes a freshness determination device that determines this K value.

This takes advantage of the property of the anion exchange resin to allow only HxR and Hx among the above ATP-related compounds to pass through, and the sample liquid (containing only HxR and Hx) that has passed through the anion exchange resin column (or bed) The absorbance ratio of the sample liquid (including all ATP-related substances) that is not allowed to pass through is determined spectroscopically, and this is used as a guideline for the K value. Furthermore, a literature document (Nissui Shishi, 32,716 (66)) describes a method for spectroscopically determining the total amount of HxR and Hx using enzymes that specifically act on these.

This uses nucleoside phosphorylase and xanthine oxidase, with the former converting HxR to Hx,
The latter converts Hx into uric acid and measures the absorbance of the produced uric acid in the ultraviolet region. Each of the above methods has its own problems.

In the method of determining the K value using an anion exchange resin, the sample liquid is passed through a column or bed filled with an anion exchange resin, so solid matter tends to clog in these. For this reason, pretreatment such as centrifugation and filtration of the sample is required. Furthermore, complicated operations are involved, such as recycling and reusing the resin. On the other hand, the above method of spectroscopically determining the total amount of HxR and Hx (molecular portion of the K value) using an enzyme is advantageous in that it does not require separation operations using columns or beds. However, since it is a spectroscopic method, transparency of the analysis sample is still required, and the sample must be pretreated for this purpose. Furthermore, since only the molecular part of the K value is determined, analysis errors are likely to occur when diluting the sample. The present invention relates to a new method that improves these conventional freshness determination methods. The literature listed above (Nissuishi,
32,716 ('66)), HxR + Hx is IMP
It has been reported that the increase is almost equivalent to the decrease in the The freshness measuring method of the present invention is based on these HxR, Hx
, MP, and calculate the ratio L, M of these substances expressed by the following formula with respect to the conventional K value. Furthermore, in order to determine the L value or M value, an enzyme that specifically acts on each substance that makes up these ratios is immobilized on or near the current collector, and these substances are subjected to an enzymatic reaction using an electrode. It is characterized by electrochemically detecting the change in substance during this enzymatic reaction.

As an enzyme that specifically acts on inosinic acid (IMP),
MP dehydrogenase or 5'-nucleotide phosphohydrolase (5'-nucleotidase) can be used. The former is nicotinamide adenine dinucleotide (
It catalyzes the following reaction using NAD) as a coenzyme. The latter catalyzes the following reactions. Examples of enzymes that specifically act on inosine (HxR) include leosidase), inosine ribohydrolase (inosinase), and purine nucleoside phosphorylase (nucleoside phosphorylase).

The three types of enzymes mentioned above each catalyze the following reactions. Between nucleosidase and inosinase, nucleosidase has a slightly broader specificity and can also act on other substances.

As enzymes that specifically act on hypoxanthine (Hx), xanthine oxidase and xanthine dehydrogenase can be used.

Each of these catalyzes the following reactions. Above (1)~
The L value or M value can be determined quickly and easily by combining the multiple types of enzymatic reactions shown in equation (7) with an electrochemical measurement method.

In other words, for the molecular part of the L value or M value (total amount of HxR and Hx), one of reactions (3), (4), and (5) is combined with one of reactions (6) and (7), and For the denominator part of the L value (total amount of IMP, HxR, Hx), this combination is further combined with reaction (2),
Furthermore, regarding the denominator part of the M value (amount of IMP), the reaction (
1), electrochemically active substances (02, uric acid, H2O2, reduced N
It can be determined by electrochemically measuring the concentration of AD). That is, a composite enzyme consisting of at least one enzyme selected from the group consisting of nucleosidase, inosinase, and nucleoside phosphorylase and at least one enzyme selected from the group consisting of xanthine oxidase and xanthine dehydrogenase is current-collected. Two types of electrodes were used: an electrode fixed on or near the body, and an electrode fixed on or near the current collector along with this complex enzyme. The above L value can be determined by allowing an enzyme to act on an extract from a food and electrochemically detecting changes in substance concentration during this enzymatic reaction. That is, at one electrode, HxR is converted to Hx, and both the Hx generated by this reaction and the originally existing Hx are oxidized to uric acid, and various substances accompanying this oxidation reaction (
The total amount of HxR and Hx can be determined by electrochemically measuring changes in the concentrations of 02, uric acid, H2O2, NAD). In addition, at the other electrode, by using a ternary complex enzyme system in which a complex enzyme is further combined with a nucleotidease, and reaction (2) is also utilized, MP, HxR
, Hx. Regarding the M value, a complex consisting of at least one enzyme selected from the group consisting of nucleosidase, inosinase, and nucleoside phosphorylase, and at least one enzyme selected from the group consisting of xanthine oxidase and xanthine dehydrogenase Two types of electrodes are used: an electrode in which an enzyme is immobilized on or in the vicinity of a current collector, and an electrode in which at least inosinate dehydrogenase is immobilized on or in the vicinity of a current collector, and the enzyme is immobilized on these electrodes. The L value can be determined in the same manner as the above-mentioned L value by allowing the enzyme to act on an extract from a food and electrochemically detecting the change in substance concentration during this enzymatic reaction.

Below, based on the present invention, L associated with the preservation of actual fish meat is shown.
An example is shown in which a change in value or M value is determined.

Example 1 First, the following 2 electrodes were used as electrochemical measurement electrodes.
constitute a species.

(,A) An electrode in which inosinase and xanthine oxidase are immobilized in a thin film on the surface of a disc-shaped graphite current collector.

Enzyme immobilization can be carried out by applying a mixture of the enzymes to the surface of a graphite plate and then treating it with a bifunctional crosslinking reagent such as glutaraldehyde or imidoester.

(B) An electrode in which 5'-nucleotidase, inosinase, and xanthine oxidase are immobilized in the form of a thin film on the surface of a disc-shaped graphite current collector. The immobilization method is the same as that for electrode A.

These two types of electrodes are incorporated into the measurement system shown in FIG.

1 and 1' are the enzyme electrodes A and B described above, 2 is a reference electrode made of a saturated calomel electrode (SCE), and 3 is a counter electrode made of platinum.

These electrodes are connected to a potentiostat 4. Electrodes 1 and V are each at a constant potential (0.4) with respect to SCB.
and measure the anode current flowing between the electrode and the counter electrode. A buffer solution is fed into the cell 5 in which the electrodes 1, V, and 2 are assembled at a constant flow rate by a liquid pump 6 through a thin tube 7 made of Teflon.

The enzyme-immobilized surface of electrode 1,1/3 is directed into the cell and comes into contact with the inflowing buffer. 8 is a storage tank for buffer solution, and 9 is a storage tank for waste liquid flowing out through the cell.

A sample injector 10 is provided on the way from the liquid pump to the cell. Through this injector, a sample solution is pumped into the buffer stream by a suitable syringe 11, such as a microsyringe, and reaches the cell, where an enzyme reaction is carried out. FIG. 2 is an example of a change curve of an anode current curve measured by a potentiostat due to sample injection.

When a sample is injected at the arrow in the figure, the current increases rapidly, reaches a peak, then decreases and returns to the position before sample injection. In practice, the peak heights of the currents flowing through the electrodes 1 and V are read, respectively, and the ratio of the heights of these current values is calculated. Reference numeral 12 in FIG. 1 is a circuit section that processes the anode current signal obtained from the potentiostat and calculates the peak height ratio. Next, a method for preparing samples used in the method of the present invention will be described.

Frozen macro fillets were thawed and stored, and the stored fillets were sequentially sampled, cut into pieces, and then squeezed, and the extract was used as an injection sample. Although the extract contains solids, no particular treatment such as centroid separation was performed. Table 1 shows the change in the height ratio of the response peaks of electrodes A and B of the injected sample as the number of days of storage changed. The peak ratio (corresponding to the above-mentioned L value) increases with the passage of storage days, indicating that this value can be used as a measure of freshness. Note that the response current in this case is expressed by the reaction equation (6
) is produced based on the oxidation current of uric acid.Example 2 The following two types of electrodes for electrochemical measurement are constructed.

(An electrode in which O-nutaleoside phosphorylase and xanthine oxidase are immobilized on a cellophane membrane and brought into contact with a platinum plate current collector.

Both enzymes can be immobilized on the cellophane membrane using a crosslinking reagent in the same manner as in Example 1.

(1) An electrode in which IMP dehydrogenase and NAD were immobilized on the surface of a graphite current collector similar to that in Example 1. As in Example 1, these enzymes and coenzymes can also be immobilized by treating a mixture of the two with a crosslinking reagent. These electrodes C and D were incorporated into the same measurement system as in Example 1, and the potential was set using a potentiostat (0.
4V vs SCE), and the peak ratio of the anode current accompanying sample injection was determined.

Table 2 shows the change in the peak ratio with storage when the same sample as in Example 1 was used. The peak ratio (corresponding to the above-mentioned M value) increases with the passage of storage days, and this value can be used as a measure of freshness.

Note that the anode current flowing through the electrode C in this case is an oxidation current of uric acid and H2O2 generated based on reaction formula (6). Further, the anode current flowing through the electrode D is an oxidation current of reduced NAD generated based on reaction formula (1). In the above examples, the peak ratios (each L
(corresponding to the M value) can be used as a measure of freshness.

Compared to the K value, we have not determined the concentrations of the three substances ATP, ADP, and AMP, but in reality, ATP
Among the decomposition pathways from to Hx, the change to IMP is said to be very rapid and almost complete before rigor mortis. Therefore, determining freshness based on the changes in the three types of substances MP, HxR, and Hx is a convenient but effective method. Moreover, compared to conventional spectroscopic measurement methods, the method of the present invention, which combines the specificity of an enzyme reaction and the simplicity of electrochemical measurement, allows measurements regardless of coloration, cloudiness, etc. of the sample liquid, and The complexity of processing can be greatly reduced, and there is no need for separation processing using columns or the like. Furthermore, since this method is based on the concentration ratio of substances (determined from the response current ratio), a constant value can be obtained regardless of sample dilution or sample injection amount. In reality, if the amount of sample injection is too small, the original response current will decrease, which can easily cause errors; if it is too large, it will take a long time to return to the original steady value after the current value reaches its peak. Although there is a problem that it takes time, a sample injection amount of 20 to 50 P1 was suitable for the measurement system according to the present invention. With this sample injection volume,
Measured values can be obtained with good reproducibility, and the time required for measurement is 1
It was only about a minute. In Examples 1 and 2, the ratio was directly determined from the peak value of the response current obtained from the thorn electrode, and L
However, in Example 1, a value (corresponding to the MP concentration) obtained by subtracting the peak value obtained by electrode A from the peak value obtained by electrode B is obtained, and this is used to calculate the M value. It is also possible to find the value corresponding to . In the actual electrode,
Due to variations in the amount of immobilized enzyme, the response peaks will not necessarily be the same even if the electrodes are of the same type. I was able to solve the problem by multiplying by

As mentioned above, there are various enzymes that can be used in the present invention other than those described in the examples, and enzymes that are selective for inosinic acid, inosine, and hypoxanthine can be used.

In addition, grapheye and platinum were used as electrode current collectors, but
Conductive metal oxides such as tin oxide can also be used.
Furthermore, enzymes were immobilized directly on electrodes (Example 1) and indirectly through cellophane membranes (Example 2). It can also be used as In this case, a so-called Clark oxygen concentration meter is applied to measure the decrease in oxygen concentration based on reaction (6). Furthermore, in the present invention, coenzyme (N
AD) does not necessarily need to be immobilized, and can also be used after being dissolved in a buffer.

Furthermore, the enzyme electrode according to the present invention can of course be used for the purpose of measuring the concentration of a substance such as inosinic acid, and is not limited to use only in the method of the present invention. The electrodes produced by combining various enzymes and current collectors used in the present invention have not been previously known, and the present invention is also novel in this respect. As described above, using the method and electrode of the present invention, the freshness of foods, especially fish meat, can be measured simply and quickly.

[Brief explanation of drawings]

FIG. 1 is a schematic configuration diagram of a measurement system used in an example of the present invention, and FIG. 2 shows a response current curve due to sample injection into an electrode.

Claims (1)

[Scope of Claims] 1. At least one enzyme selected from the group consisting of N-ribosylpurine ribohydrolase, inosine ribohydrolase, and purine nucleoside phosphorylase, and at least one enzyme selected from the group consisting of xanthine oxidase and xanthine dehydrogenase. An electrode in which a composite enzyme consisting of an enzyme of A food product characterized in that the M value expressed by the following formula is determined by applying a converted enzyme to an extract from the food and electrochemically detecting the change in substance concentration during this enzymatic reaction. Freshness determination method. M={[HxR]+[Hx]}/{[IMP]} (where, [IMP] represents the concentration of inosinic acid, [HxR] represents the concentration of inosine, and [Hx] represents the concentration of hypoxanthine.
)2 Consists of at least one enzyme selected from the group consisting of N-ribosylpurine ribohydrolase, inosine ribohydrolase, and purine nucleoside phosphorylase, and at least one enzyme selected from the group consisting of xanthine oxidase and xanthine dehydrogenase. an electrode in which a complex enzyme is immobilized on or near a current collector;
Using an electrode in which 5'-nucleotide phosphohydrolase is immobilized on or near a current collector together with the complex enzyme, the enzyme immobilized on these electrodes is allowed to act on the extract from the food, and during this enzymatic reaction. A method for determining the freshness of food, characterized by determining an L value expressed by the following formula by electrochemically detecting changes in substance concentration. L={[HxR]+[Hx]}/{[IMP]+[Hx
R] + [Hx]} (where, [IMP] represents the concentration of inosinic acid, [HxR] represents the concentration of inosine, and [Hx] represents the concentration of hypoxanthine.)