WO2014148583A1

WO2014148583A1 - Quality evaluation device

Info

Publication number: WO2014148583A1
Application number: PCT/JP2014/057623
Authority: WO
Inventors: 慶一野間; 宮崎　秀樹; 裕子村田; 昌一村田; メイコ木村; 博章鈴木
Original assignee: フジデノロ株式会社
Priority date: 2013-03-22
Filing date: 2014-03-19
Publication date: 2014-09-25
Also published as: JP2014185934A; JP6192092B2

Abstract

Provided is a quality evaluation device for effortless and rapid measurement of a value relating to the quality of a measured item, including fish, shellfish, edible meats, red meat, and processed products using these. The device is characterized by having: an extraction receptacle (12) for extracting a nucleic acid-related substance which is contained in measured item (8) including fish, shellfish, edible meats, red meat, and processed products using these, and which serves as an indicator of freshness and quality; an electrochemical sensor (14) having at least a working electrode (32) and a counter electrode (34) selected from among three different types of electrode, namely, a working electrode (32), a counter electrode (34), and a reference electrode (36), the surface of the working electrode (32) including an enzyme immobilizing portion on which is immobilized an enzyme that reacts with the nucleic acid-related substance, and which is adapted to be presented with the nucleic acid-related substance extracted by extraction receptacle (12) as well as to apply a voltage across the working electrode (32) and the counter electrode (34); and a measuring device (16) for measuring the concentration of the nucleic acid-related substance on the basis of the output of the electrochemical sensor (14), and calculating a value relating to the quality.

Description

Quality evaluation device

The present invention relates to an apparatus for measuring indices for managing the freshness and quality of fish meat, seafood, meat, livestock meat, and processed products using these.

As an index for managing the freshness and quality of fish meat, seafood, meat, livestock, and processed products using these (hereinafter referred to as “fish meat”), numerical values based on the content of nucleic acid-related substances are used. It is being used. For example, the K value is an example. For example, in fish muscle, adenosine diphosphate (ADP), adenosine monophosphate (AMP), inosinic acid (IMP), inosine (HxR), hypoxanthine accompanying the degradation of adenosine triphosphate (ATP) after death (Hx) is sequentially produced, and the percentage of the weight ratio or the amount (mole) ratio of inosine and hypoxanthine to all these substances is the K value. This K value has been widely used as one of the indexes representing freshness.

By the way, conventionally, as a method for measuring the concentration and mass of nucleic acid-related substances such as ATP, ADP, AMP, IMP, HxR, Hx, etc., for example, it was obtained through repeated mixing, centrifugation, etc. with a homogenizer. It has been carried out by a complicated procedure and a method requiring a large equipment, such as detecting the supernatant using chromatography. In particular, since fish and the like decompose ATP quickly, there may be a problem that even if measurement is performed over time, the obtained value is already different from that of actual seafood. As described above, the conventional method for measuring the K value may be difficult to use in the field of distribution, and a means for measuring the K value by a simpler method has been expected.

Furthermore, as described above, the K value is the ratio of HxR and Hx to all substances of ATP, ADP, AMP, IMP, HxR, and Hx. In the hours after the death of seafood, ATP is often decomposed. In fish such as horse mackerel and red sea bream, IMP decomposition, that is, generation of HxR and Hx is not often performed over time. As a result, depending on the type of fish, although ATP decreases several hours after the death of seafood, this is not reflected in the K value, and it was difficult to evaluate the freshness using the K value. Therefore, in such a situation, an index that changes to a K value for evaluating the freshness of seafood and the like has been demanded. FIG. 14 is a diagram showing the ratio of each nucleic acid-related substance, the K value, and the ratio of ATP with the passage of time when the maji meat as a detection target is stored in a refrigerator. For example, comparing immediately after death (0h) and after 6 hours (6h), HxR and Hx are hardly generated, so there is no change in the K value, but the ATP concentration is greatly reduced. It can be seen that the freshness is changing.

JP 2012-047606 A

Patent Document 1 proposes a method for measuring the amount of nucleic acid substances in fish proposed under such circumstances. According to the method of Patent Document 1, the amount of nucleic acid-related substance contained in the fish meat can be measured by electrochemically measuring the body fluid of the fish meat. However, in such a method, it is necessary to take out body fluid from fish meat, and a method for taking out body fluid more efficiently is still desired.

The present invention has been made against the background of the above circumstances, and its object is to provide a quality measuring device capable of measuring the freshness and quality of fish by collecting body fluid more efficiently from fish. Is to provide.

The invention according to claim 1 for achieving the object includes (a) fish, fish and shellfish, meat, livestock meat, and an object to be measured comprising these, and an indicator of freshness and quality; An extraction container for extracting a nucleic acid-related substance, and (b) having at least a working electrode and a counter electrode among the three types of electrodes, a working electrode, a counter electrode, and a reference electrode, and reacting with the nucleic acid-related substance on the surface of the working electrode An electrochemical sensor that includes an enzyme immobilization part on which an enzyme is immobilized, the nucleic acid-related substance extracted by the extraction container is provided, and a voltage is applied between the working electrode and the counter electrode; and (c) the electrochemical And a measuring device that measures the concentration of the nucleic acid-related substance based on the output of the sensor and calculates the value related to the quality.

According to the first aspect of the present invention, the extraction liquid can be easily taken out from the measurement object by the extraction container. By providing the extract to the electrochemical sensor, the enzyme in the enzyme fixing part provided on the working electrode is allowed to react with the nucleic acid-related substance in the extract. A voltage is applied between at least the working electrode and the counter electrode of the electrochemical sensor. Further, based on the output of the electrochemical sensor at that time, the concentration of the nucleic acid-related substance in the extract is measured by the measuring device, and a value related to the quality of the measurement object is calculated based on the measured value. Therefore, a value relating to the quality of the measurement object can be obtained easily and quickly by a simple method.

In addition, a feature of the second invention is that the electrochemical sensor has a plurality of working electrodes, and an enzyme is immobilized on each of the enzyme fixing portions corresponding to the plurality of working electrodes. It is in. In this way, the concentration of each of a plurality of nucleic acid-related substances that react with a plurality of enzymes can be measured by a single measurement, so that more efficient measurement is possible.

Further, the third invention is characterized in that the extraction container includes a crushing part for crushing the measurement object, a pusher for pressing the measurement object against the crushing part, and the crushing part. And a filtration member that filters the liquid extracted from the measurement object crushed by the above, and the crushing part is composed of a plurality of granular crushed particles. In this way, since the measurement object can be efficiently crushed in the extraction container, an extract of the measurement object can be obtained more quickly, and the filtration unit can remove the extraction object from the extract. It becomes possible to remove substances other than nucleic acid-related substances to be measured in the electrochemical sensor.

According to a fourth aspect of the invention, the nucleic acid-related substance is ATP (adenosine triphosphate), ADP (adenosine diphosphate), AMP (adenosine monophosphate), IMP (inosinic acid), HxR. (Inosine) and Hx (hypoxanthine). In this way, it is possible to measure the concentration of ATP and the nucleic acid-related substances produced by the degradation of ATP. Therefore, fish, seafood, meat, livestock meat containing ATP, and processed products using these can be obtained. A quality-related value can be measured.

Further, a feature of the fifth invention is that the filtration member selectively absorbs oil and fat components from the liquid extracted from the measurement object. If it does in this way, it will prevent that the fats and oils component contained in the extract of the said measuring object becomes a disorder | damage | failure which a nucleic acid related substance and an enzyme react in the said enzyme fixing | fixed part, and a more appropriate measurement will be attained.

Further, the sixth invention is characterized in that an index relating to the freshness of the measurement object is calculated based on the concentration of the nucleic acid-related substance, particularly ATP, in the liquid extracted from the measurement object. . In this way, when the measurement target is fish meat, seafood, meat, livestock meat, etc., the ATP decreases after the death, and the freshness of the measurement target is appropriately measured by measuring the concentration of ATP. Can be obtained.

Further, the seventh invention is characterized in that an index relating to the quality of the measurement object is calculated based on the concentration of the nucleic acid-related substance, particularly IMP, in the liquid extracted from the measurement object. . In this way, since IMP corresponds to a so-called umami component, an index relating to the quality of the measurement object can be obtained based on the concentration of IMP. Moreover, even if the measurement object is already processed at the time of processing such as processed food, an index relating to the quality can be obtained.

Further, the eighth invention is characterized in that the K value or Ki value of the measurement object is calculated based on the concentration of the nucleic acid-related substance in the liquid extracted from the measurement object. . In this way, it is possible to easily obtain the K value or Ki value that has been conventionally used as one of the indexes representing freshness for the measurement object.

It is a figure which shows the outline | summary of the quality measurement apparatus in one Example of this invention. It is a figure explaining the outline | summary of the extraction container in the quality measuring device of FIG. It is a figure explaining an example of the crushed grain in the extraction container of FIG. It is a figure which compares the ratio of the extract with respect to a measuring object with the extraction container of a present Example, and a comparative example. It is a figure explaining an example of a structure of the electrochemical sensor in the quality measuring device of FIG. It is a figure explaining an example of reaction of the nucleic acid related substance and enzyme in the working electrode of an electrochemical sensor, (a) is ATP, (b) is IMP, (c) is each reaction of HxR and Hx FIG. It is a figure which shows the time change of the electric current value detected by a measuring device, when detecting ATP with an electrochemical sensor. It is a figure which shows the correlation with the value of the electric current detected with a measuring device, and the density | concentration of ATP of an actual measurement object. It is a figure which shows the correlation with the value of the electric current detected with a measuring device, and the density | concentration of IMP of an actual measurement object. It is a figure which shows the correlation with the value of the electric current detected with a measuring device, and the density | concentration of HxR of an actual measurement object. It is a figure which shows the correlation with the value of the electric current detected with a measuring device, and the density | concentration of ATP in the ATP solution as an actual measuring object. It is a figure explaining the structure of the electrochemical sensor in another Example of this invention. It is a figure explaining the structure of the electrochemical sensor in another Example of this invention, Comprising: It is a figure corresponding to FIG. It is the figure shown with progress of time about each of the ratio of each nucleic acid related substance, K value, and the ratio of ATP at the time of storing chilled meat as a detection target object refrigerated.

Hereinafter, an embodiment of the present invention will be described in detail with reference to the drawings.

FIG. 1 is a diagram for explaining an outline of a quality measuring apparatus according to an embodiment of the present invention. In FIG. 1, a quality measuring apparatus 10 is an electrical signal indicating the degree of reaction between a nucleic acid-related substance and an enzyme contained in an extraction container 12 for extracting an extraction liquid from a measurement object, and the extraction liquid extracted from the extraction container 12. And a measurement device 16 that measures the concentration of the nucleic acid-related substance based on the output signal from the electrochemical sensor 14 and calculates a value related to quality.

FIG. 2 shows an example of the extraction container 12. The extraction container 12 is configured to include, for example, a cylindrical tubular portion 20 and a pusher 22 that are open at one end and closed at the other end. A discharge portion 20 a whose inner diameter is smaller than the inner diameter of the cylindrical portion 20 is continuously provided on the distal end side of the cylindrical portion 20, and is extracted from the measurement object 8 placed inside the cylindrical portion 20. The extracted liquid is discharged. The pusher 22 has a push plate 22a at its tip, and these are moved integrally. The pusher 22 is inserted into the inside of the cylindrical portion 20 from the opening end so that the push plate 22a becomes the tip, and the measuring object 8 having the push plate 22a placed inside the cylindrical portion 20 is inserted into the cylindrical portion 20. The tip is pressed toward the tip 20a side. Here, the push plate 22a is provided so that the size thereof is smaller than the inner diameter of the cylindrical portion 20 so that the inside of the cylindrical portion 20 can be moved. The space between the push plate 22a and the push plate 22a may or may not be airtight.

The filtration member 24 and the crushing part 26 are provided in the end part by the side of the discharge part 20a of the cylindrical part 20 from the front-end | tip part. Among these, the filtration member 24 is made into the same shape as the cross-sectional shape of the cylindrical part 20, and the extract liquid which the measurement object 8 is crushed and extracted by the crushing part 26 mentioned later reaches from the cylindrical part 20 to the discharge part 20a. In the process, filtration, adsorption or absorption is performed. The filtration member 24 is capable of filtering, adsorbing, or absorbing substances other than nucleic acid-related substances to be measured, particularly substances that interfere with measurement in an electrochemical sensor described later, in the extract. The material is selected. In this embodiment, a nonwoven fabric is used for the purpose of removing oils and fats from the extract. The thickness of the filter member 24 is appropriately set according to the amount of substances that can be filtered or adsorbed by the filter member 24 and the exchange frequency. Furthermore, in the filtration member 24, when the measuring object 8 is crushed in the crushing part 26 mentioned later, the solid substance contained in an extract is filtered.

The crushing unit 26 crushes the measurement object 8 and extracts the extract. The crushing part 26 is composed of a plurality of crushed grains 28. The plurality of crushed grains 28 may be inserted into the cylindrical portion 20 after being preliminarily bonded to form a predetermined shape, or may be inserted into the cylindrical portion 20 as individual grains. The size of the crushing portion 26, in other words, the amount of the crushing particles 28 constituting the crushing portion 26 is set according to the size of the cylindrical portion 20, the size of the measurement object 8 to be extracted, and the like. Specifically, for example, when the pusher 22 is pushed into the cylindrical portion 20 and the push plate 22a presses the measurement target 8, the measurement target 8 is crushed, and the push plate 22a approaches or contacts the crushing portion 26. The crushing part 26 is provided to such an extent that it can be performed. In addition, the shape of the crushing part 26 is not particularly limited, but preferably the measurement object 8 is sandwiched between the push plate 22a and the crushing part 26 so that the measurement object 8 is crushed without unevenness. Both shapes can be designed.

FIG. 3 is a diagram showing an example of the shape of the crushed grains 28. In the present embodiment, the crushed grains 28 have a spherical shape, a so-called bead shape. When the measuring object 8 is pressed against the crushed grains 28 having such a shape by the push plate 22a, the measuring object 8 is broken and pressed, and the extract is extracted. In the crushing portion 26, the directions of the plurality of crushed grains 28 do not need to be aligned. The size, specifically the outer diameter, the inner diameter, the length, and the like of the crushed particles 28 are determined according to the measurement object 8 to be crushed. Specifically, for example, when the measurement object 8 is a piece of meat such as tuna, horse mackerel, or tie according to the viscosity or hardness of the measurement object 8, the measurement object has a diameter of 0.5 to 2 mm. What is necessary is just to determine experimentally so that the amount of extraction may increase according to the kind of the thing 8. FIG. The material of the crushed particles 28 may be determined according to the viscosity or hardness of the measurement object or according to the magnitude of the force that presses the pusher 22. Specifically, in this embodiment, the crushed grains 28 are made of glass. In addition, although the crushing particle | grains 28 shown in FIG. 3 are chamfered, this is not necessarily essential.

FIG. 4 is a diagram comparing the extraction amount of the extraction liquid from the measurement object 8 by the extraction container 12 of this example with other methods. In FIG. 4, the particle diameter Φ0.5 mm, the particle diameter Φ1 mm, and the particle diameter Φ2 mm correspond to the present embodiment, and the outer diameters of the crushed particles 28 are 0.5 mm, 1 mm, and 2 mm, respectively. It corresponds to the case. Further, both the manual push and the screw type are provided with a disk-shaped plate instead of the crushing portion 26 of the present embodiment, and are provided on the disk-shaped plate and the pusher 22. The extraction liquid is extracted by sandwiching and crushing the measuring object 8 with the pressing plate 22a. The extraction liquid extracted by crushing the measurement object 8 is discharged from the discharge port 20a through the filter member 24. Both the manual push and the screw type are different in the manner in which the pusher 22 is pushed. The hand push is a push by hand, and the screw type pushes the pusher by a propulsion mechanism using a screw (not shown). . Further, for each of the particle diameters Φ0.5 mm, particle diameter Φ1 mm, and particle diameter Φ2 mm, the pusher is pushed by hand in the same manner as the above-described hand pushing. In addition, pushing with a hand is the state in which force was applied to such an extent that the extract was obtained from the measuring object 8.

The fish meat was put in the extraction container 12 as the measurement object 8, and the extraction liquid was extracted several times under the above five conditions. The value (mass) of the extract (body fluid) obtained when the extraction time is 1 minute, the value (mass) of the fish meat as the measurement object 8 placed in the extraction container 12, and the ratio thereof are shown in the figure. As shown in FIG. As shown in FIG. 4, in the case of the particle diameter Φ0.5 mm, the particle diameter Φ1 mm, and the particle diameter Φ2 mm, that is, when the crushing portion 26 of the present embodiment is applied, the same force is applied. In addition to the case, the ratio of the amount of the obtained extract to the mass of the measurement object 8 is large even in comparison with the screw type in which a larger force is applied, and it is short and efficient. It can be seen that the extraction liquid is being extracted. Further, when the respective cases of the particle diameter Φ0.5 mm, the particle diameter Φ1 mm, and the particle diameter Φ2 mm are compared, the ratio of the amount of the obtained extract to the mass of the measurement object 8 is different. As described above, the size, material, and the like of the crushed particles can be selected according to the measurement object 8. In the example of FIG. 4, the most efficient is achieved by selecting the diameter Φ of the crushed particles 28 as 1 mm. It will be good.

It should be noted that the crushing portion 26 and the filtering member 24 in the extraction container can be replaced every time extraction is performed.

FIG. 5 is a diagram for explaining an example of the configuration of the electrochemical sensor 14. As shown in FIG. 5, the electrochemical sensor 14 of this embodiment includes a working electrode 32, a counter electrode 34, and three electrodes provided on a substrate 30 made of, for example, glass epoxy resin by vapor deposition or coating. A reference electrode 36 is provided. In FIG. 5, the working electrode 32, the counter electrode 34, and the reference electrode 36 are exposed on the substrate 30 in the terminal portion 42 on the left side of the horizontally long substrate 30, and are connected to the measuring device 16 described later. It functions as a terminal. In the right sensor unit 46, the working electrode 32, the counter electrode 34, and the reference electrode 36 are exposed on the substrate 30, and the extraction liquid of the measurement object 8 extracted by the extraction container 12 is dropped. For example, the working electrode 32, the counter electrode 34, and the reference electrode 36 can be attached so as to cover them. On the other hand, in the intermediate portion 44 between the terminal portion 42 and the sensor portion 46, the three electrodes of the substrate and the working electrode 32, the counter electrode 34, and the reference electrode 36 are covered with the insulating portion 40. The insulating part 40 is made of, for example, an insulating film or an insulating resist, so that these three electrodes are not electrically connected by an extract or the like at a place other than the sensor part 46.

Further, on the sensor unit 46 side, the working electrode 32 and the counter electrode 34 are preferably made of platinum, gold, glassy carbon, or carbon paste, and the reference electrode 36 is made of silver / silver chloride or the like.

On the surface of the working electrode 32, acetyl cellulose, collagen, polyvinyl chloride, polyvinyl alcohol, polyvinyl glutamic acid, polyvinyl butyral, polyvinyl acrylamide, an enzyme that reacts with a nucleic acid-related substance to be detected by the electrochemical sensor 14 including the working electrode. An enzyme immobilization section 38 is provided that is immobilized using an immobilization material such as nitrocellulose, carboxymethylcellulose, or a photocurable resin. Specifically, when the nucleic acid-related substance to be detected is ATP, enzymes immobilized on the enzyme immobilization unit 38 are, for example, glycerol kinase (GK) and glycerol 3-phosphate oxidase (G3PO). In addition, when the nucleic acid-related substance to be detected is IMP, the enzyme immobilized on the enzyme immobilization unit 38 is 5′-nucleotidase (NT) or alkaline phosphatase (AP), nucleotide phosphorylase (NP), xanthine oxidase. (XOD) or the like, and when the nucleic acid-related substance to be detected is HxR or Hx, the enzyme immobilized on the enzyme immobilization unit 38 is nucleotide phosphorylase (NP), xanthine oxidase (XOD), or the like. Further, interfering substances contained in the measurement object, specifically, for example, ascorbic acid (vitamin C), acetaminophen, uric acid, etc., cause a potential difference between the working electrode 32 and the counter electrode 34 in the electrochemical sensor described later. When a voltage is applied to occur, it can decompose below that voltage. In order to reduce the influence of these interfering substances, an ion exchange membrane such as acetyl cellulose or Nafion (registered trademark), polyvinyl alcohol, or the like may be provided on the substrate side of the enzyme fixing part 38 of the working electrode 32. In this way, it is possible to prevent or reduce these interference substances from reaching the surface of the working electrode 32 and being decomposed.

FIG. 6 is a diagram for explaining an example of the reaction between the nucleic acid-related substance and the enzyme at the working electrode 32 of the electrochemical sensor 14. The principle will be described with reference to FIG. FIG. 6 shows an example in which the electrochemical sensor 14 detects ATP. ATP contained in the extract adhering to the enzyme fixing part 38 of the working electrode 32 is decomposed into ADP by GK, which is an enzyme fixed to the enzyme fixing part 38, and glycerol triphosphate is generated. This glycerol triphosphate is also decomposed by G3PO, which is an enzyme fixed to the enzyme fixing part 38, to generate hydrogen peroxide. The reference electrode 36 is provided to refer to a reference voltage when measuring the potential of the working electrode 32 and the counter electrode 34, and a relative potential difference between the working electrode 32 and the counter electrode 34 can be obtained. Is not necessarily required.

Returning to FIG. 1, the measuring device 16 measures the concentration of the nucleic acid-related substance that the electrochemical sensor 14 attempts to detect based on the output of the electrochemical sensor 14. Further, a value related to the quality of the measurement object 8 is calculated based on the measured concentration of the nucleic acid-related substance. In the present embodiment, the measurement device 16 has a display unit 50 including, for example, a display device, and the measured concentration of the nucleic acid-related substance, the value related to the calculated quality, and the like are displayed on the display unit 50.

The measuring device 16 and each

electrode

32, 34, 36 of the electrochemical sensor 14 are connected by a cable 18, and a voltage is applied between the electrodes of the electrochemical sensor 14, or a current flowing between the electrodes is applied. It can be measured. In the present embodiment, when the electrochemical sensor 14 detects ATP as described above with reference to FIGS. 5 and 6, the voltage of the working electrode 32 is higher than the voltage of the counter electrode 34 by, for example, 0.7 V. Thus, when a voltage is applied to generate a potential difference, hydrogen peroxide is oxidized and electrons are transferred to the working electrode 32 as shown in FIG. That is, a current flows from the working electrode 32 to the counter electrode 34. This 0.7 V corresponds to the decomposition voltage of hydrogen peroxide, and may be larger or smaller as long as hydrogen peroxide can be decomposed. The measuring device 16 detects the magnitude of this current. Thus, the measuring device 16 has a function of applying a voltage having a value set for each of the

electrodes

32, 34, and 36 of the electrochemical sensor 14, and the working electrode 32 of the electrochemical sensor 14 It has a function of measuring the current flowing between the counter electrode 34.

FIG. 7 shows the time change of the current value detected by the measuring device 16 when ATP is the detection target in the electrochemical sensor 14 of FIG. Specifically, a solution prepared so that the ATP concentration is 2.5, 1.0, 0.75, 0.5, 0.25, 0.1 mM (mmol / L, volume molar concentration) in advance. In addition, when each of the liquids (Blank) not containing ATP is the measurement object 8, the time change of the current detected by the measuring device 16 of the present embodiment is shown. As shown in FIG. 7, when a certain period of time elapses from the start of detection for each concentration, the change in the ATP concentration in the measurement object 8 is settled, and the gradient of the detected current is stable.

On the other hand, FIG. 8 is a diagram showing the correlation between the ATP concentration and the current value measured as shown in FIG. 7 for each of the measurement objects 8 that are ATP measurement objects in FIG. Here, as described with reference to FIG. 7, since a certain time elapses from the start of detection and the gradient of the detected current is stabilized, the current value is specifically shown in FIG. 7 after the gradient of the current is stabilized. In the example, an average of current values measured a plurality of times is used from 10 seconds to 60 seconds after the start of detection. The ATP concentration is calculated based on the value of the current flowing between the working electrode 32 and the counter electrode 34 measured by the measuring device 16 by approximating the correlation obtained in this way by, for example, the least square method. Is possible. That is, the measuring device 16 has a relationship between the value of the current flowing between the working electrode 32 and the counter electrode 34 illustrated in FIG. 8 and the concentration of the nucleic acid-related substance of the measurement target 8 for each nucleic acid-related substance. A function of outputting the value of the nucleic acid-related substance with respect to the value of the generated current. The above relationship may be a diagram as shown in FIG. 8, but is not limited to this, and may be in the form of a formula or a data table.

FIGS. 9 to 10 are diagrams showing the correlation between the IMP concentration and the current value and the correlation between the HxR concentration and the current value, respectively, obtained by the same method as in FIG.

That is, the measuring device 16 has a relationship between the value of the current flowing between the working electrode 32 and the counter electrode 34 illustrated in FIG. 8 and the concentration of the nucleic acid-related substance of the measurement target 8 for each nucleic acid-related substance. A function of outputting the value of the nucleic acid-related substance with respect to the value of the generated current. The above relationship may be a diagram as shown in FIG. 8, but is not limited to this, and may be in the form of a formula or a data table.

Furthermore, the measuring device 16 calculates a value relating to the quality based on the concentration of the nucleic acid-related substance obtained as described above. Specifically, when the electrochemical sensor 14 detects ATP as described above, the measurement device 16 has the maximum ATP concentration obtained for the measurement object 8, and the maximum of the measurement object 8. That is, the ratio of the ratio to the ATP concentration immediately after death expressed as a percentage is calculated as a value relating to freshness. That is,
(Value relating to freshness) = (ATP concentration of measurement object 8) / (ATP concentration immediately after death of measurement object 8) × 100 (1)
It is. Here, the maximum ATP concentration of the measurement object 8 is a value set for each measurement object 8, that is, for each type of fish, or even for the same fish, for each part or production area. In general, in the measurement object 8 of the quality measuring device of the present invention such as fish meat, seafood, meat, livestock meat, etc., the concentration of ATP becomes the maximum value immediately after death. Or just get it statistically. This value may be stored in advance in a storage device (memory or the like) (not shown) in the measurement device 16 or may be input by the operator every time measurement using the measurement device 16 is performed.

While the ATP concentration in the measurement object 8 becomes maximum immediately after death as described above, and thereafter decreases with the passage of time, the value relating to freshness obtained as in the above equation (1) is effective as an index representing freshness. It is. In addition, it is possible to evaluate the freshness regardless of fish species, production area, and the like.

On the other hand, when the electrochemical sensor 14 uses IMP as a detection target, the measuring device 16 expresses the ratio of the IMP concentration obtained for the measurement object 8 to the maximum ATP concentration of the measurement object 8 as a percentage. Things are calculated as quality values. That is,
(Value related to quality) = (IMP concentration of measurement object 8) / (maximum ATP concentration of measurement object 8) × 100 (2)
It is. Here, the maximum ATP concentration of the measurement object 8 is the same as that described for the above-described value relating to freshness, and is a value obtained experimentally or statistically in advance.

When the measurement object 8 is a fish, the IMP increases with the decrease in ATP in the fish meat that is the measurement object 8. That is, IMP increases by the amount of ATP decomposition. Therefore, it can be said that the higher the quality-related value obtained by Equation (2) is, the more ATP is decomposed. Further, since IMP corresponds to a so-called umami component, it can be determined that the umami is increased when the value related to the quality obtained by equation (2) is high.

When the electrochemical sensor 14 cannot directly detect the concentration of IMP, for example, an electrochemical sensor capable of detecting the total concentration of IMP, HxR, and Hx and the total concentration of HxR and Hx, respectively. It is also possible to obtain the IMP concentration by subtracting the sum of the HxR and Hx concentrations from the sum of the IMP, HxR and Hx concentrations.

When the electrochemical sensor 14 uses IMP as a detection target, the measuring device 16 displays the ratio of the IMP concentration obtained for the measurement target 8 to the reference value of the IMP concentration of the measurement target 8 as a percentage. It can also be calculated as a quality value. That is,
(Value relating to quality) = (IMP concentration of measurement object 8) / (Reference value of IMP concentration of measurement object 8) × 100 (3)
It is. Here, the reference value of the IMP concentration of the measurement object 8 is a value set in advance for each type of the measurement object 8, and for example, the maximum value of the IMP concentration is used.

Thus, since the value regarding the quality obtained by the above formula (3) is defined without using the ATP concentration, when the measurement object 8 is a processed fish product, that is, before or at the processing stage. Even when ATP has already been decomposed, the quality can be evaluated using the value relating to the quality obtained by the equation (3).

The result of the experiment conducted for verifying the effect of the quality measuring apparatus 10 of the present embodiment is shown below. Fish (Aji) fish meat that had passed a sufficient amount of time to decompose almost the entire amount of ATP was put into the extraction container 12 of this example, and the extract was extracted. The extracted extract was mixed with an ATP standard solution to produce a liquid having an ATP concentration close to the actual body fluid of fish (hereinafter referred to as an ATP solution) and used as a measurement object. For each of the ATP solutions having a plurality of ATP concentrations, the current value when a predetermined voltage was applied in the electrochemical sensor 14 of the measuring device 16 was measured. At this time, the enzyme for decomposing ATP described above is fixed to the enzyme fixing portion 38 of the electrochemical sensor 14 by the fixing material. FIG. 11 shows the results. As shown in FIG. 11, this result shows that the ATP concentration and the measured current value have a certain linear relationship. Therefore, it can be seen that, based on this relationship, the measuring device 16 can calculate the ATP concentration from the value of the current when the electrochemical sensor 14 measures the current to be measured 8.

Also for IMP, HxR, and Hx, as in ATP, it has been confirmed that the concentration and the measured current value have a certain linear relationship. Therefore, when the current is measured by the electrochemical sensor 14 for a certain measurement object 8 based on this relationship, the measuring device 16 can calculate the concentration from the value of the current.

According to the quality measuring apparatus 10 of the present embodiment, (a) a nucleic acid that is included in the measurement object 8 including fish, seafood, meat, livestock meat, and processed products using these, and serves as an index of freshness and quality. An extraction container 12 for extracting a related substance; and (b) a working electrode 32, a counter electrode 34, and a reference electrode 36, and at least the working electrode 32 and the counter electrode 34. An electrochemical sensor 14 that includes an enzyme immobilization unit 38 that immobilizes an enzyme that reacts with a substance, and that provides a nucleic acid-related substance extracted by the extraction container 12 and a voltage is applied between the working electrode 32 and the counter electrode 34; (C) a measurement device 16 that measures the concentration of the nucleic acid-related substance based on the output of the electrochemical sensor 14 and calculates a value related to the quality. Thereby, the extraction liquid can be easily taken out from the measurement object 8 by the extraction container 12, and the concentration of the nucleic acid-related substance in the extraction liquid is measured by the electrochemical sensor 14 and the measuring device 16. Since the value related to the quality of the measurement object 8 is calculated based on the measured value, the value related to the quality of the measurement object can be obtained easily and quickly by a simple method.

Further, the extraction container 12 of the present embodiment includes a crushing portion 26 for crushing the measurement object 8, a pusher 22 and a push plate 22 a for pressing the measurement object 8 against the crushing portion 26, and a crushing portion 26. And a filtering member 24 for filtering the liquid extracted from the crushed measurement object 8, and the crushing portion 26 is composed of a plurality of granular crushed particles 28. Thereby, since the measuring object 8 can be efficiently crushed in the extraction container 12, the extract of the measuring object 8 can be obtained more quickly, and the electrochemical sensor 14 can be obtained from the extract by the filtering member 24. It is possible to remove substances other than nucleic acid-related substances to be measured in step (b).

In addition, the nucleic acid-related substances to be measured by the quality measuring apparatus 10 of this embodiment are ATP (adenosine triphosphate), ADP (adenosine diphosphate), AMP (adenosine monophosphate), and IMP (inosinic acid). , HxR (inosine), and Hx (hypoxanthine), it is possible to measure the concentration of ATP and nucleic acid-related substances produced by the decomposition thereof, so that fish, seafood, meat, and livestock meat containing ATP can be measured. , And a value related to the quality of a processed product using these can be measured.

Moreover, since the filtration member 24 of a present Example selectively absorbs fat and oil components from the liquid extracted from the measuring object 8, the fat and oil components contained in the extract of the measuring object 8 are enzyme-immobilized. It is possible to prevent the nucleic acid-related substance and the enzyme from becoming obstacles in the unit 38, and more appropriate measurement is possible.

Moreover, since the quality measuring apparatus 10 of the present embodiment calculates an index related to the freshness of the measurement target 8 based on the concentration of ATP in the liquid extracted from the measurement target 8, the measurement target is fish meat or seafood. When ATP decreases after death, as in the case of meat, meat, livestock, etc., an index relating to the freshness of the measurement object can be obtained appropriately by measuring the concentration of ATP.

Moreover, since the quality measuring apparatus 10 of the present embodiment calculates an index relating to the quality of the measurement object 8 based on the concentration of the IMP in the liquid extracted from the measurement object 8, the IMP corresponds to a so-called umami component. Therefore, an index relating to the quality of the measurement object can be obtained based on the concentration of IMP. Moreover, even if the measurement object is already processed at the time of processing such as processed food, an index relating to the quality can be obtained.

Subsequently, another embodiment of the present invention will be described. In the following description, portions common to the embodiments are denoted by the same reference numerals and description thereof is omitted.

FIG. 12 is a view for explaining the configuration of the electrochemical sensor 114 in the embodiment of the quality measuring apparatus 10 of the present invention, and corresponds to FIG. 5 in the first embodiment. Also in the electrochemical sensor 114 of FIG. 12, as with the electrochemical sensor 14 in the above-described first embodiment, for example, three types of electrodes provided by vapor deposition or coating on the substrate 30 made of glass epoxy resin or the like. As a working electrode 32, a counter electrode 34, and a reference electrode 36. However, the electrochemical sensor 114 of this embodiment is different from the electrochemical sensor of Embodiment 1 in that it has a plurality of types of working electrodes, that is, three working

electrodes

32a, 32b, and 32c in the example of FIG.

The three working

electrodes

32a, 32b, and 32c are provided with

enzyme fixing portions

38a, 38b, and 38c, respectively, as in the first embodiment. Here, preferably, in order for the electrochemical sensor 114 to detect a plurality of types of nucleic acid-related substances in one measurement, the enzyme immobilized on each of the plurality of

enzyme immobilization units

38a, 38b, 38c is Different types of nucleic acid-related substances are used. Specifically, for example, the working electrode 32a is for detecting ATP, and glycerol kinase (GK) and glycerol 3-phosphate oxidase (G3PO) are fixed to the fixing part 38a provided on the working electrode 32a. The working electrode 32b is for detecting IMP, HxR and Hx, and the fixing part 38b provided on the working electrode 32b has an alkaline phosphatase (AP), 5'-nucleotidase (NT), nucleotide phosphoric acid. Rase (NP) and xanthine oxidase (XOD) are immobilized. Further, the working electrode 32c is for detecting HxR and Hx, and nucleotide phosphorylase (NP) and xanthine oxidase (XOD) are fixed to the fixing part 38c provided on the working electrode 32c.

FIG. 13 is a diagram for explaining another embodiment of the electrochemical sensor 114 in the present embodiment, and corresponds to FIG. In the electrochemical sensor 114 shown in FIG. 13, the arrangement and shape of the

electrodes

32, 34, and 36 are different from those in FIG. As shown in FIGS. 12 and 13, in the case where a plurality of working electrodes 32 are provided, the arrangement is limited to these arrangements as long as the distances between the working electrodes 32 and the counter electrode 34 can be equal. Not.

Further, in FIG. 13, each of the

electrodes

32, 34, and 36 is such that only a portion in contact with the extraction liquid in the sensor portion 46 is exposed, while the other portions are covered with the insulating portion 40. This is different in the example of FIG. If it does in this way, the part which is not used for a measurement among each

electrode

32,34,36 can be protected, and the improvement of durability of the electrochemical sensor 114 can be aimed at. In the

electrochemical sensors

14 and 114 shown in FIG. 5 and FIG. 12, the insulating portion 40 may similarly cover the

electrodes

32, 34, and 36 except for the portions in contact with the extract.

The electrochemical sensor 114 according to Example 2 described above has a plurality of working

electrodes

32a, 32b, and 32c, and the

enzyme fixing portions

38a, 38b, and 38c corresponding to the plurality of working

electrodes

32a, 32b, and 32c, respectively. Since different enzymes are immobilized, the concentration of each of multiple types of nucleic acid-related substances that react with multiple types of enzymes can be measured with a single measurement, enabling more efficient measurement. .

As mentioned above, although the Example of this invention was described in detail based on drawing, this invention is applied also in another aspect.

For example, in the above-described embodiment, the measuring device 16 calculates the values related to freshness or the values related to quality expressed by the above formulas (1) to (3), but instead, You may make it calculate the value of the natural logarithm of what was shown by Formula (1) thru | or Formula (3) as a value regarding freshness, or a value regarding quality. In this way, even if the value changes sharply, it can be an easily understood index. Further, according to the type of the measurement object 8, specifically, the calculation method may be different for each fish type.

In the above-described embodiment, the measuring device 16 calculates the values related to freshness or the values related to quality expressed by the above formulas (1) to (3), but instead, You may make it display the parameter | index set for every predetermined numerical range defined beforehand. Specifically, for example, when the value relating to the freshness calculated by the expression (1) is 0 or more and 20 or less, it is class 1 or more and 20 or more and 40 or less. In some cases, it can be displayed as class 5 if it exceeds

class

3, 60 and 80 or less, and class 4 if it exceeds 80 and 100 or less. This classification may be defined differently depending on the type of measurement object 8, specifically for each fish type.

In the above-described embodiment, the measuring device 16 indicates a value related to freshness or a value related to quality. However, when both are measured for the same measurement object 8, both are taken into consideration. Evaluation may be performed and displayed. Specifically, for example, when the value related to the freshness is high and the value related to the quality is low, that is, when the content of ATP is high and the content of IMP is low, the freshness is evaluated to be good. If the value is low and the quality value is high, the quality is evaluated as being high or eaten, and if both the freshness value and the quality value are low, the quality is evaluated as poor. Can do.

In the above-described embodiment, the value related to freshness is defined by the equation (1), but is not limited thereto. For example, the ratio of the ATP concentration obtained for the measurement object 8 to the total concentration of all nucleic acid-related substances of the measurement object 8 expressed as a percentage can be calculated as a value related to freshness. That is,
(Value relating to freshness) = (ATP concentration of measurement object 8) / (total of ATP, IMP, HxR, Hx concentration of measurement object 8) × 100 (1 ′)
It can also be. Similarly, in the above-described embodiment, the value related to quality is defined by the equation (2), but is not limited thereto. For example, the ratio of the IMP concentration obtained for the measurement object 8 to the total concentration of all nucleic acid-related substances of the measurement object 8 expressed as a percentage can be calculated as the value related to freshness. That is,
(Value relating to quality) = (IMP concentration of measurement object 8) / (Total of concentrations of ATP, IMP, HxR, Hx of measurement object 8) × 100 (2 ′)
It can also be. The total nucleic acid-related substance means, for example, when the measurement object 8 is fish meat, each nucleic acid-related substance ATP (adenosine triphosphate), ADP (adenosine diphosphate), AMP (adenosine monophosphate) contained therein. Phosphoric acid), IMP (inosinic acid), HxR (inosine), and Hx (hypoxanthine), but ADP and AMP, which are substances that rapidly decrease with degradation, can be considered to have a concentration of 0 .

In Example 2 described above, three working

electrodes

32a, 32b, and 32c are provided on the substrate 30, and different enzymes are fixed to the three working

electrodes

32a, 32b, and 32c, respectively. However, it is not limited to such an embodiment. Specifically, for example, the same nucleic acid-related substance can be measured three times at the same time by immobilizing the same enzyme on these three working

electrodes

32a, 32b, and 32c. And the tolerance of a measured value can be made small by making the average of the measured value for 3 times measured in this way into a measured value. Furthermore, while the same enzyme is fixed to two of the three working

electrodes

32a, 32b, and 32c, different enzymes can be fixed to the remaining one working electrode. In this way, both the case where different enzymes are fixed to each of the three working

electrodes

32a, 32b and 32c and the case where the same enzyme is fixed to each of the three working

electrodes

32a, 32b and 32c. The effect can be obtained at the same time. Further, the number of working electrodes 32 provided on the substrate 30 is not limited to three, and the same effect can be obtained if the number of working electrodes 32 is plural.

In the above-described embodiment, the measurement device 16 indicates a value related to freshness or a value related to quality. However, in the measurement device 16, the total concentration of HxR and Hx, and ATP, ADP, AMP, When the sum of the concentrations of IMP, HxR, and Hx is respectively obtained, the K value, which is the ratio of the sum of these concentrations, is used instead of or in addition to the value relating to freshness or the value relating to quality (3) It can be calculated and displayed as an expression.
(K value) = (total concentration of HxR and Hx of the measurement object 8) / (total concentration of ATP, ADP, AMP, IMP, HxR, and Hx of the measurement object 8) × 100 ( 3)
Thereby, it becomes possible to evaluate the measuring object 8 with the K value widely used conventionally. Instead of the K value, an approximate K value represented by the following equation (4) may be calculated as the K value.
(Approximate pseudo K value) = (sum of concentrations of HxR and Hx of the measuring object 8) / (sum of concentrations of ATP, IMP, HxR and Hx of the measuring object 8) × 100 (4)
This is because the ADP and AMP concentrations in the measurement object 8 do not occupy a large proportion over time, so even if it is a value calculated as in equation (4), the measurement object is in accordance with the K value. It is because it is possible to evaluate. Furthermore, instead of the K value or the approximate K value, a Ki value represented by the following equation (5) may be calculated.
(Ki value) = (total of HxR and Hx concentrations of measurement object 8) / (total of IMP, HxR and Hx concentrations of measurement object 8) × 100 (5)
This Ki value can be used as one of effective indexes that can replace the K value in a state in which ATP, ADP, and AMP in the measurement object 8 are almost completely decomposed.
In this way, the K value conventionally used by the quality evaluation apparatus of the present invention or an approximate K value or Ki value equivalent thereto can be easily obtained.

In the above-described embodiment, the crushed particles 28 are spherical. However, the present invention is not limited to this, and when a large number of crushed particles 28 are provided as the crushing portion 26, the shape of the crushed particles 28 and the crushed particles. The shape may be any shape as long as the liquid can be extracted from the measurement object 8 by the 28 gaps between them. For example, the shape may be a chamfered polyhedron.

In the above-described embodiment, the crushed particles 28 are made of glass. However, the present invention is not limited to this. For example, the extract is extracted by pressing the measurement object 8 such as resin, metal, ceramic, activated carbon, or the like. Anything is possible.

In the above-described embodiment, the substrate 30 of the electrochemical sensor 14 is made of glass epoxy resin, but is not limited to this. For example, acrylic resin (PMMA), polystyrene (PS), polyethylene terephthalate (PET). Polycarbonate (PC) or the like may be used.

8: Measurement object 10: Quality measuring device 12: Extraction container 14: Electrochemical sensor 16: Measuring device 22: Pusher 24: Filtration member 26: Crushing part 28: Crushing particle 32: Working electrode 34: Counter electrode 36: Reference electrode 38: Enzyme fixing part

Claims

An extraction container for extracting nucleic acid-related substances, which are included in measurement objects including fish, seafood, meat, livestock meat and processed products using these, and serve as indicators of freshness and quality;
The working electrode, the counter electrode, and the reference electrode have at least a working electrode and a counter electrode. The surface of the working electrode includes an enzyme immobilization part on which an enzyme that reacts with the nucleic acid-related substance is immobilized, and the extraction An electrochemical sensor in which the nucleic acid-related substance extracted by the container is provided and a voltage is applied between the working electrode and the counter electrode;
A measurement device that measures the concentration of the nucleic acid-related substance based on the output of the electrochemical sensor, and calculates values relating to the freshness and quality;
A quality measuring device characterized by comprising:
The electrochemical sensor has a plurality of working electrodes, and each of the plurality of working electrodes has an enzyme immobilized on an enzyme immobilization portion corresponding to each of the working electrodes;
The quality measuring device according to claim 1, wherein:
The extraction container includes a crushing part for crushing the measurement object, a pusher for pressing the measurement object against the crushing part, and a liquid extracted from the measurement object crushed by the crushing part. A filtering member for filtering,
The crushing part is composed of a plurality of granular crushed particles;
The quality measuring apparatus according to claim 1 or 2, wherein
The nucleic acid-related substance is ATP (adenosine triphosphate), ADP (adenosine diphosphate), AMP (adenosine monophosphate), IMP (inosinic acid), HxR (inosine), and Hx (hypoxanthine). The quality measuring device according to any one of claims 1 to 3, wherein
The filtration member selectively absorbs fat and oil components from the liquid extracted from the measurement object;
The quality measuring apparatus according to claim 2, wherein:
6. The quality according to claim 1, wherein an index relating to the freshness of the measurement object is calculated based on the concentration of the nucleic acid-related substance in the liquid extracted from the measurement object. Measuring device.
6. The quality according to claim 1, wherein an index relating to the quality of the measurement target is calculated based on the concentration of the nucleic acid-related substance in the liquid extracted from the measurement target. Measuring device.
6. The K value or Ki value of the measurement object is calculated based on the concentration of the nucleic acid-related substance in the liquid extracted from the measurement object. 6. Quality measuring device.