Sucrose measurement kit and method for measuring sucrose

Abstract

To provide a novel kit that enables simple measurement of sucrose in a sample that may simultaneously contain glucose, without influence from glucose.SOLUTION: A kit for measuring sucrose in a sample, comprises a solution A and a solution B: (solution A) a solution containing glucose oxidase, catalase, and mutarotase; (solution B) a solution containing invertase and a catalase inactivator; wherein one of the solution A and the solution B contains a coupler compound, the other contains a Neo-Trinder reagent, one of the solution A and the solution B contains peroxidase, and the content of invertase in the solution B is in a range of 10 U / mL to 200 U / mL.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a kit for measuring sucrose and a method for measuring sucrose.

Background Art

[0002] Sucrose is a major sweetening component, and some fruits and vegetables contain a large amount of sucrose. Also, sucrose is the main component of sugar, with approximately 100% of granulated sugar and 97.9% of refined sugar being sucrose. There is a need for simple quantitative analysis of sucrose in various scenarios, such as in the manufacturing processes and quality control of juices, soft drinks, confectionery, and processed foods, or in the variety improvement tests and cultivation management of agricultural products like vegetables and fruits, and even in the classification on the tariff schedule for imported foods.

[0003] For the measurement of sucrose, high-performance liquid chromatography (HPLC) can be selected. However, operations such as sample pretreatment and selection of column measurement conditions are complicated, and a certain degree of skilled technique is required for measurement. Also, since the analytical instruments for sucrose measurement by HPLC and biosensors capable of measuring sucrose are very expensive devices, the measurement of sucrose using these analytical instruments is mainly carried out in universities, specialized analytical institutions, public research institutions, or research institutes of large enterprises.

[0004] On the other hand, a plurality of kits for measuring sucrose using enzymes are also commercially available. Many of them use invertase, which decomposes sucrose into glucose and fructose, and measure the amount of glucose generated in a 1:1 relationship with sucrose. Therefore, in order to calculate the amount of sucrose, it is necessary to measure the amount of glucose initially contained in the sample and subtract it from the total amount of glucose.

[0005] Patent Document 1 discloses an enzyme electrode for measuring sucrose concentration, which has a first electrode for detecting hydrogen peroxide and a second electrode for electrolytically oxidizing a substance that interferes with the detection of hydrogen peroxide by the first electrode. The first electrode and the second electrode are each made of a porous membrane formed with a platinum layer. Glucose oxidase, mutarotase, and β-fructosidase are immobilized on the first electrode, and glucose oxidase and mutarotase are immobilized on the second electrode. The second electrode is arranged on the test solution side with respect to the first electrode. It is described that the influence of substances such as glucose can be easily removed by this electrode.

[0006] Patent Document 2 describes a reagent for trehalose analysis applicable to a method including a step of measuring the amount of glucose generated by enzymatic decomposition, which consists of a combination of a glucose elimination pretreatment reagent containing at least mutarotase, glucose oxidase, and catalase, and a generated glucose detection reagent containing at least trehalose phosphorylase, sodium azide, peroxidase, 4-aminoantipyrine, and a hydrogen donor. Patent Document 2 discloses that 50 μL of the glucose elimination pretreatment reagent is added to the test solution, incubated at 37°C, then the generated glucose detection reagent is added, incubated at 37°C, and based on the resulting color development, quantification of trehalose at a concentration of 1 to 10 g / L (about 2.6 to 26 mM) can be performed.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0008] The method described in Patent Document 1 includes current measurement using an immobilized enzyme electrode and is suitable for incorporation into an electrochemical biosensor, but it is difficult to apply in various scenarios. Further, the method described in Patent Document 2 measures trehalose. An object of the present invention is to provide a novel kit capable of easily measuring sucrose. In particular, an object is to provide a kit that can accurately and easily measure sucrose in a sample that may simultaneously contain glucose, using a sucrose solution as a standard solution, without being affected by the coexisting glucose.

Means for Solving the Problems

[0009] The present inventors repeatedly studied a kit applicable to the procedure of adding invertase after decomposing glucose in a sample before allowing the sucrose-degrading enzyme (invertase) to act on sucrose, and completed the present invention.

[0010] Specifically, the present invention provides the following. [1] A kit for measuring sucrose, containing Solution A and Solution B: (Solution A) A solution containing glucose oxidase, catalase, and mutarotase; (Solution B) A solution containing invertase and a catalase deactivator; However, in Solution A and Solution B, one of them contains a coupler compound, the other contains a new Trinder's reagent, and at least one of them contains peroxidase, The content of invertase in Solution B is in the range of 10 U / mL to 200 U / mL. [2] The kit according to [1], wherein Solution A further contains ascorbic acid oxidase. [3] The kit according to [1] or [2], wherein Solution A contains a new Trinder's reagent and peroxidase. [4] The kit according to any one of [1] to [3], wherein the coupler compound is 4-aminoantipyrine. [5] The kit according to any one of [1] to [4], wherein the novel Trinder's reagent is N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS) or N-ethyl-N-sulfopropyl aniline (ALPS). [6] The kit according to any one of [1] to [5], further comprising a sucrose standard solution. [7] The kit according to any one of [1] to [6], comprising an apparatus having an LED light source for measuring the amount of the produced dye. [8] A method for measuring sucrose, Steps 1 and 2: (Step 1) A step of co-existing a sample, glucose oxidase, mutarotase, and catalase in a container; (Step 2) Subsequently to Step 1, in the same container, a catalase inactivator is allowed to act to inactivate the catalase, and invertase is allowed to act on sucrose, and glucose oxidase, mutarotase, peroxidase, a coupler compound, and a novel Trinder's reagent are allowed to act on the produced glucose to produce a dye. comprising a method, wherein Step 2 is carried out in a solution having an invertase concentration of 3.3 U / mL to 100 U / mL. [9] The method according to [8], wherein Step 2 is carried out at room temperature.

[10] The method according to [8], wherein Steps 1 and 2 are carried out at room temperature.

[11] Step 1 is initiated by adding Solution A containing glucose oxidase, mutarotase, and catalase to the sample, after adding Solution A, Solution B containing invertase and a catalase inactivator is added 7 to 20 minutes later to initiate Step 2, the method according to any one of [8] to

[10] , wherein the color development by the dye is measured 5 to 10 minutes after adding Solution B.

[12] The method according to any one of [8] to

[11] , wherein in Step 1, ascorbic acid oxidase is further co-existed with the sample.

Advantages of the Invention

[0011] According to the present invention, there is provided a novel kit for measuring sucrose using an enzyme, which enables simple measurement. With the kit of the present invention, even a sample that may simultaneously contain glucose can measure sucrose in fewer steps without separately measuring the glucose. Further, in the kit of the present invention, it is possible to use sucrose as a standard substance, and it is possible to directly quantify sucrose without calculation for concentration conversion after measurement. Furthermore, the kit of the present invention has good storage stability and high versatility.

Brief Description of the Drawings

[0012]

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Mode for Carrying Out the Invention

[0013] The present invention provides a kit for measuring sucrose in a sample, which contains the following Solution A and Solution B. In this specification, when measuring sucrose, it means detecting sucrose, measuring the content of sucrose, etc.

[0014] The measurement principle of sucrose using the kit of the present invention is shown in FIG. 1. First, Solution A is added to proceed with the reaction in Step 1. In Step 1, glucose in the measurement sample is decomposed by glucose oxidase and then by catalase to remove the influence of glucose originally contained in the sample. When Solution A further contains ascorbic acid oxidase, ascorbic acid originally contained in the sample can be further decomposed to remove this influence. Next, Solution B is further added to the sample to which Solution A has been added above to proceed with the reaction in Step 2. Sucrose is decomposed by invertase, and the generated glucose is oxidized by glucose oxidase. Hydrogen peroxide generated along with the oxidation of glucose is in a 1:1 ratio with the sucrose originally contained in the sample. This hydrogen peroxide is reacted with peroxidase, a coupler compound, and a new Trinder's reagent (4-AA and TOOS in FIG. 1) under the inhibition of catalase to generate a dye. Based on the amount of this dye, sucrose is quantified.

[0015] The kit of the present invention was compared with existing sucrose measurement kits from the viewpoints of the type of enzyme acting on glucose, the presence or absence of mutarotase use, the presence or absence of heating in the reaction process, the type of standard solution (sucrose solution or glucose solution), and the necessity of separately measuring coexisting glucose (Table 1). Different from any of the existing sucrose measurement kits shown in Table 1, the kit of the present invention does not require pre-measurement of glucose coexisting in the sample and can be measured at room temperature without the need for heating in the entire reaction process of 20 to 30 minutes. Further, it is a kit of a method capable of quantifying sucrose at the end point of the enzyme reaction using a sucrose solution as the standard solution and only two types of reagent solutions. Furthermore, all existing sucrose measurement kits are for experts with knowledge of biochemistry and analytical chemistry, but the sucrose measurement kit of the present invention can be easily performed at room temperature without special equipment or skilled techniques, and thus can be used for analysis at sites related to agriculture and food processing or in educational institutions.

[0016]

Table 1

[0017] (Solution A, Solution B) In the kit of the present invention, Solution A and Solution B are as follows. (Solution A) A solution containing glucose oxidase, catalase, and mutarotase; (Solution B) A solution containing invertase and a catalase deactivator. However, in Solution A and Solution B, one of them contains a coupler compound, the other contains a new Trinder reagent, and at least one of them contains peroxidase.

[0018] In the kit of the present invention, Solution A contains mutarotase. Mutarotase is an enzyme that converts α-type glucose to the β-type as follows.

[0019]

Chemical formula

[0020] By including mutarotase in Solution A, the kit of the present invention enables the measurement of sucrose at room temperature in a short time. Glucose oxidase is an enzyme that oxidizes β-D-glucose to D-glucono-1,5-lactone. It is considered that the addition of mutarotase to Solution A allows the α-type glucose present in a certain equilibrium relationship in the sample to be quickly converted to the β-type, and as a result, the reaction of glucose oxidase can proceed quickly. Also, although the enzymatic reaction by invertase is usually carried out at a temperature of 37°C or higher, by allowing the reaction to proceed in the presence of mutarotase, the product glucose is quickly decomposed, and the production reaction of glucose from sucrose by invertase is promoted, making it possible to perform the measurement even at room temperature.

[0021] In addition, in the kit of the present invention, Solution A or Solution B contains peroxidase. Since peroxidase is an enzyme that acts in Step 2 of FIG. 1 after the addition of Solution B, from the perspective of the measurement principle, it suffices to be contained in Solution B. However, the inventors have found that whether Solution A or Solution B contains peroxidase, both Solution A and Solution B have good stability, can be stored for a long time as a kit, and have no influence on the measurement results.

[0022] In the kit of the present invention, one of Solution A and Solution B contains a coupler compound, and the other contains a new Trinder's reagent. By having the coupler compound and the new Trinder's reagent contained in separate solutions, in particular, by not having the coupler compound, the new Trinder's reagent, and peroxidase contained in the same solution, it is possible to prevent the reagent itself from developing color. In a preferred embodiment, Solution A contains the new Trinder's reagent, and Solution B contains the coupler compound. This improves the stability of both Solution A and Solution B. In the present specification, a coupler compound, a new Trinder's reagent, or a combination thereof may be referred to as a chromogenic agent.

[0023] The kit of the present invention can be used to measure sucrose in a sample that may contain glucose.

[0024] The kit of the present invention contains one or more of Liquid A and Liquid B. Preferably, Liquid A may contain ascorbic acid oxidase. Ascorbic acid (vitamin C) is a component contained in fruits (fruit juices) or juice products that can be the measurement target. However, if ascorbic acid is present in the sample, the color reaction will be inhibited. By containing ascorbic acid oxidase, the kit of the present invention can be used to measure sucrose in a sample that may contain ascorbic acid or erythorbic acid.

[0025] When Liquid A contains ascorbic acid oxidase, from the viewpoint of the stability of the enzyme, it is preferable that Liquid A contains peroxidase and a new Trinder reagent, and Liquid B contains a coupler compound.

[0026] (enzyme) In the present invention, as the enzyme, known glucose oxidase, invertase, mutarotase, catalase, peroxidase, and ascorbic acid oxidase can be used.

[0027] Glucose oxidase is an enzyme that oxidizes β-D-glucose to D-glucono-1,5-lactone, and examples of glucose oxidase derived from microorganisms such as Aspergillus niger can be mentioned.

[0028] Invertase is an enzyme also known as β-fructofuranosidase. Examples of invertase used in the present invention include invertase derived from microorganisms such as Saccharomyces cerevisiae.

[0029] Examples of mutarotase include mutarotase derived from pig kidneys.

[0030] Examples of catalase include catalase derived from bovine liver, as well as catalase derived from microorganisms such as Aspergillus niger and Corynebacterium glutamicum.

[0031] Examples of peroxidase include peroxidase derived from Japanese horseradish.

[0032] Examples of ascorbic acid oxidase include ascorbic acid oxidase derived from cucumber or pumpkin.

[0033] (New Trinder's reagent) In the present invention, those known as novel Trinder's reagents can be used. Examples of the novel Trinder's reagents used in the present invention include N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (ADOS), N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (HDAOS), N-ethyl-N-sulfopropyl-3-methoxyaniline (ADPS), N-ethyl-N-sulfopropylaniline (ALPS), N-ethyl-N-sulfopropyl-3-methylaniline (TOPS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAOS), N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline (DAPS), N-(2-carboxyethyl)-N-ethyl-3,5-dimethoxyaniline (CEDB), N-(2-carboxyethyl)-N-ethyl-3-methoxyaniline (CEMO), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxy-4-fluoroaniline (FDAOS), and N-ethyl-N-sulfopropyl-3,5-dimethoxy-4-fluoroaniline (FDAPS). It is preferable to use any one of N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (ADOS), N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (HDAOS), N-ethyl-N-sulfopropyl-3-methoxyaniline (ADPS), N-ethyl-N-sulfopropylaniline (ALPS), N-ethyl-N-sulfopropyl-3-methylaniline (TOPS), and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAOS), and it is more preferable to use N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS).

[0034] (Coupler compound) In the present invention, those known as coupler compounds can be used. In the present invention, any compound that produces color in combination with a new Trinder's reagent may be used. Examples include 4-aminoantipyrine (4-AA), vanillyldiaminesulfonic acid, methylbenzothiazolinone hydrazone (MBTH), sulfonated methylbenzothiazolinone hydrazone (SMBTH), aminodiphenylamine-1-(4-sulfophenyl)-2,3-dimethyl-4-amino-5-pyrazolone (CP2-4), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine or its derivatives. It is preferable to use 4-aminoantipyrine (4-AA).

[0035] (Other components) The solution A or solution B contained in the kit of the present invention may further contain a preservative. Known preservatives can be used. Examples include sodium azide, procaine 300, procaine 950, and chloramphenicol.

[0036] Solution A and solution B may contain components having pH buffering ability. Examples of such components include acetates, phosphates, citrates, borates, tartrates, Tris (tris(hydroxymethyl)aminomethane), and Hepes ([2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid]).

[0037] Neither solution A nor solution B substantially contains bovine serum albumin used as an enzyme stabilizer (for example, the amount of bovine serum albumin is less than 0.02% by weight, 0.01% by weight or less), and as shown in the examples, the enzyme activity in the solution can be maintained for a long time even without containing bovine serum albumin.

[0038] (Content of components) The content of glucose oxidase in Solution A is preferably 20 to 500 U / mL, more preferably 50 to 200 U / mL. In particular, for enabling measurement in a short time and making the stability of Solution A higher, 100 to 500 U / mL is preferable, and 100 to 300 U / mL is more preferable. The content of catalase in Solution A is preferably 500 to 2000 U / mL, more preferably 1000 to 1500 U / mL. When Solution A contains peroxidase, the content of peroxidase in Solution A is preferably 5 to 30 U / mL, more preferably 10 to 20 U / mL. The content of mutarotase in Solution A is preferably in the range of 0.1 to 1.5 vol% (10 to 150 U / mL), more preferably in the range of 0.3 to 0.8 vol% (30 to 80 U / mL). When Solution A contains ascorbic acid oxidase, the ascorbic acid oxidase can be 1 to 30 U / mL, and preferably 2 to 10 U / mL.

[0039] The content of invertase in Solution B may be in the range of 10 U / mL to 200 U / mL, preferably in the range of 50 U / mL to 100 U / mL. In this case, the enzyme unit (U) of invertase adopts the international unit determined by measuring the amount of glucose produced in 1 minute at 30 °C at pH 4.0 using sucrose as a substrate. By using invertase at a concentration of 10 U / mL to 200 U / mL, the enzyme reaction can proceed rapidly even at room temperature using the kit of the present invention. Further, in the present invention, it has been clarified that even when containing invertase at a concentration of 10 U / mL to 200 U / mL, which is higher than the usually used enzyme amount, Solution B has the stability to withstand use as a kit that is stored and used for a certain period of time. In addition, in order to complete the sucrose measurement in a shorter time at room temperature, the content of invertase in Solution B is preferably 50 U / mL or more. In the reaction of Step 2 with Solution B, the glucose produced by decomposing sucrose with invertase is oxidized by glucose oxidase, and the produced hydrogen peroxide is reacted with peroxidase, a coupler compound, and a new Trinder reagent under catalase inhibition to produce a dye. Here, by increasing the amount of invertase in Solution B, the decomposition reaction of sucrose can proceed rapidly even at room temperature, and as a result, the dye generation reaction can be completed in a short time (for example, within 10 minutes) after adding Solution B. The present invention is a colorimetric method by measuring the endpoint of a so-called enzyme reaction and does not require strict time management. Therefore, it is a simpler and novel method for quantifying sucrose.

[0040] The content of the catalase deactivator in Solution B is preferably in the range of 0.01 to 0.09% by mass, more preferably in the range of 0.05 to 0.09% by mass. When Solution B contains peroxidase, the content of peroxidase in Solution B is preferably 5 to 30 U / mL, more preferably 10 to 20 U / mL. Peroxidase is preferably 2 to 15 U / mL, more preferably 5 to 10 U / mL, in the solution obtained by mixing the sample, Solution A, and Solution B. The content of the new Trinder's reagent in Solution A or Solution B is preferably 0.1 to 1.0 μmol / mL, more preferably 0.2 to 0.5 μmol / mL. The content of the coupler compound in Solution A or Solution B is preferably in the range of 0.1 to 1.0 μmol / mL, more preferably in the range of 0.2 to 0.5 μmol / mL.

[0041] (Sucrose standard solution) In addition to the above Solution A and Solution B, the kit of the present invention may contain a sucrose standard solution. The concentration of the sucrose standard solution can be appropriately set. Specifically, for example, it can be 100 to 300 mg / L, and preferably 100 mg / L.

[0042] (Other components of the kit) The kit of the present invention may include an apparatus having a light source for measuring the amount of the generated dye. Further, the kit of the present invention may include a pipette for collecting Solution A and a pipette for collecting Solution B. Furthermore, it may include a colorimeter (sometimes referred to as an "LED colorimeter" in this specification) for measuring the amount of the generated dye using an LED as the light source, and a plastic cell for measurement. The LED colorimeter may be of a type in which the measurement result is displayed as absorbance or a type in which it is displayed as concentration (a calculated value from the absorbances of the coloring solutions of the measurement sample and the standard solution).

[0043] (Use) The kit of the present invention can be used for measuring sucrose. Specifically, it can be used for measuring sucrose in the measurement target. The measurement target is not particularly limited as long as it is expected to contain sucrose. The measurement target may possibly contain glucose at the same time. Specific examples include agricultural products, alcoholic beverages such as wine and beer, beverages such as juice, food raw materials, processed foods, and plant cells. The measurement target is preferably provided as an aqueous solution for measurement. For measurement targets in the form of solutions containing insolubles, it is also preferable to perform filtration or centrifugation in advance to obtain a sample.

[0044] When using the kit of the present invention, for example, considering the case where both Solution A and Solution B are used in an amount 10 times the volume of the sample, a sucrose solution concentration of 10 to 800 mg / L, preferably 20 to 600 mg / L, can be accurately measured. The measurement target can be diluted with water to form a sample so as to be within a concentration range that allows for more accurate measurement according to the mixing ratio of the reagents selected by the kit of the present invention. In this case, the water for dilution is not limited to distilled water or deionized water, and the measurement target can be diluted using tap water.

[0045] Also, when using the kit of the present invention, even in a sample that may simultaneously contain glucose, the concentration of the sucrose solution can be accurately measured without separately measuring the glucose. For example, in a sample containing 20 to 600 mg / L of sucrose, the sucrose content can be accurately measured even if glucose is contained up to 1000 mg / L.

[0046] When measuring using the kit of the present invention, Solution A is preferably used in an amount 1 to 100 times the volume of the sample, more preferably 5 to 20 times the volume, and even more preferably 10 times the volume. Solution B is preferably used in an amount 1 to 100 times the volume of the sample, more preferably 5 to 20 times the volume, and even more preferably 10 times the volume. Also, the usage amount of Solution B relative to the usage amount of Solution A may be determined according to the component amounts in each solution and is not particularly limited. From a practical perspective of being less likely to make mistakes in the addition amount and being easy to remember, it is preferable that the usage amounts of Solution A and Solution B are the same (volume ratio). By using Solution A and Solution B in an amount of 1 or more times the sample and the usage amount of Solution B relative to Solution A being about the same or more, the amount of invertase in the solution during the sucrose decomposition reaction can be in the range of about 3.3 U / mL to 100 U / mL.

[0047] (Method for measuring sucrose) The present invention also relates to a method for measuring sucrose including the following Step 1 and Step 2. This method can be carried out using the kit of the present invention. For details such as the enzymes, reagents, and samples to be used, reference can be made to the description of the above kit.

[0048] (Step 1) A step of allowing a sample, glucose oxidase, mutarotase, and catalase to coexist in container X; (Step 2) Following Step 1, in container X, a catalase inactivator is allowed to act to inactivate catalase, and glucose oxidase, mutarotase, peroxidase, a coupler compound, and a new Trinder reagent are allowed to act on the glucose produced by allowing invertase to act on sucrose to produce a dye.

[0049] Here, Step 2 is carried out in a solution having an invertase concentration of 3.3 U / mL to 100 U / mL. By allowing the enzyme to act at this concentration, the enzyme reaction can proceed rapidly even, for example, in an environment at room temperature. For measurement in a shorter time, the invertase concentration of the solution for carrying out Step 2 is preferably 4.8 U / mL or more, more preferably 6.0 U / mL or more, still more preferably 10 U / mL or more, and particularly preferably 17 U / mL or more. Further, the above invertase concentration is preferably 50 U / mL or less.

[0050] The method of the present invention preferably further includes the following Step 3. (Step 3) A step of measuring the amount of dye X of the dye produced in Step 2.

[0051] In Step 1, when the sample contains glucose, the glucose is decomposed by glucose oxidase and then by catalase to eliminate the influence of the glucose present in the sample. Specifically, the hydrogen peroxide generated by the oxidative decomposition of glucose by glucose oxidase is decomposed by catalase. Here, by co-existing with mutarotase, the conversion of glucose from the α-form to the β-form proceeds, and glucose is rapidly decomposed by glucose oxidase. In Step 1, the sample and ascorbic acid oxidase may further co-exist. At this time, ascorbic acid or erythorbic acid in the sample containing ascorbic acid or erythorbic acid is decomposed by ascorbic acid oxidase, and these influences can be removed.

[0052] In Step 2, a catalase inactivator is allowed to act on the catalase present in Step 1 to inactivate the catalase. This is to prevent the decomposition of hydrogen peroxide by catalase in Step 2. Also, in Step 2, the sucrose in the sample is decomposed by invertase, and the generated glucose is oxidized by glucose oxidase. Here, due to the co-existence of mutarotase derived from Step 1, the conversion of glucose from the α-form to the β-form proceeds, and glucose is rapidly decomposed by glucose oxidase. The hydrogen peroxide generated upon the oxidation of glucose is reacted with peroxidase, a coupler compound, and a new Trinder's reagent to generate a dye. For example, the concentration of sucrose can be quantified by measuring the absorbance at the absorption maximum of the dye (555 nm when TOOS and 4-AA are used) with a spectrophotometer. Step 2 can be initiated, for example, by adding the above-mentioned Solution B to the mixture of the sample after the step and Solution A.

[0053] Note that in Step 1, when the sample does not contain glucose, the reactions by glucose oxidase and catalase are not carried out, and when the sample does not contain ascorbic acid or erythorbic acid, ascorbic acid or erythorbic acid is not decomposed by allowing ascorbic acid oxidase to act on ascorbic acid or erythorbic acid.

[0054] In the method of the present invention, after the reaction in Step 1 (the reaction with Solution A when using the kit of the present invention) has proceeded sufficiently, Step 2 is carried out (when using the kit of the present invention, Solution B is added). In Step 2, only the glucose generated by the reaction of sucrose, which is the measurement target in the sample, is decomposed by the glucose oxidase present from Step 1 to generate hydrogen peroxide, and the color developer and peroxidase are reacted with this hydrogen peroxide to cause color development. In this Step 2, the catalase present from Step 1 is inactivated by the catalase inactivator in Step 2, so no inhibition of the color development reaction by catalase occurs.

[0055] In the method of the present invention, the reaction in Step 2 can be carried out at room temperature. That is, it can be carried out in a temperature range including room temperature, for example, in an environment of 15°C to 42°C. Even if the reaction in Step 2 is carried out at room temperature, the reaction ends (reaches the end point) in proportion to the content of sucrose contained in the sample. Although the enzymatic reaction by invertase is usually carried out at a temperature of 37°C or higher, in the method of the present invention in which Step 2 is carried out with an increased amount of invertase in the presence of mutarotase, the present inventors have found that the reaction proceeds rapidly even at room temperature. Since Step 1 in the method of the present invention can also be carried out at room temperature, the entire steps of Step 1 and Step 2 can be carried out in an environment of 15°C to 42°C, particularly at room temperature. Thus, the method of the present invention can be carried out rapidly even in an environment without heating equipment. Note that room temperature is generally understood to mean "without cooling or heating". Also, in this specification, when referring to room temperature, it may be 15°C to 35°C, but preferably 20°C to 30°C. The pH conditions may be appropriately set based on the optimum pH of the enzyme used. Step 1 can be carried out at pH 6.0 to pH 8.0, and Step 2 can be carried out at pH 5.5 to pH 7.0.

[0056] Further, Steps 1 and 2 can each be carried out if they are 5 minutes or longer, preferably 7 minutes or longer, and more preferably 9 minutes or longer. The upper limit is not particularly limited, but it may be 60 minutes or shorter, preferably 30 minutes or shorter, and more preferably 20 minutes or shorter. From a practical perspective that the time setting is easy to remember in a short time, Steps 1 and 2 are each preferably carried out for 10 minutes. Also, when more coexisting glucose may be contained, from the perspective of eliminating its influence, Step 1 is also preferably carried out for 20 minutes. For example, in the instruction manual of the kit of the present invention, it can be described that after adding Solution A, Solution B is added 10 minutes or 20 minutes later, and the amount of the dye is measured 10 minutes after adding Solution B.

[0057] The color development occurring in Step 2 can be measured as the absorbance at a specific wavelength in Step 3 by a general method, for example, a spectrophotometer using a light source such as a halogen lamp or a xenon lamp. Also, the degree of color development can be measured as an electrical signal (for example, a voltage value) by a colorimeter using an LED in a specific wavelength range as the light source and using a phototransistor or the like in the light receiving part. When an LED is used as the light source, the wavelength of the light source for measuring color development may be selected according to the type of the color former used. For example, when TOOS is used as a new Trinder reagent, since the absorption maximum of the resulting dye is 555 nm, a pure green LED of 555 nm is suitable as the light source. Based on the absorbance of the color-developed solution of the sample measured in Steps 1 and 2, the concentration of sucrose in the sample can be calculated by comparing it with the measured absorbance value of the color-developed solution of a sucrose solution with a known concentration measured in advance.

[0058] For example, the method of the present invention can further include the following Steps 12, 13, and 14. (Step 12) A step of reacting glucose generated by reacting invertase with sucrose in a sucrose standard solution in container Y with glucose oxidase, mutarotase, peroxidase, a coupler compound, and a new Trinder reagent to generate a dye. (Step 13) A step of measuring the amount of the dye Y of the dye generated in Step 12 (Step 14) Step of calculating the sucrose concentration in the sample from the amount of pigment Y, the amount of pigment X, and the sucrose concentration of the sucrose standard solution. Step 14 may be carried out by an apparatus provided with an information processing unit for performing the above calculation. For example, a colorimeter may be provided with an information processing unit.

[0059] In order to measure sucrose with higher accuracy, the method of the present invention preferably further includes the following Step 11. (Step 11) Step of causing glucose oxidase, mutarotase, and catalase to coexist in the sucrose standard solution in container Y. Subsequent to Step 11, Step 12 is carried out in container Y. The sucrose concentration in the sample can be calculated from the amount of pigment Y, the amount of pigment X, and the sucrose concentration of the sucrose standard solution. Steps 11 to 13 are preferably before Steps 1 and 2. For example, in an apparatus provided with an information processing unit, if calibration is performed with the color-developing solution of the sucrose standard solution and then the color-developing solution of the measurement sample is measured, the sucrose concentration of the measurement sample calculated from the absorbance can be obtained. The calibration is preferably performed based on the results of measurement using water (distilled water) instead of the sucrose standard solution together with the measurement using the sucrose standard solution.

[0060] The sucrose measurement method of the present invention can be carried out by either a manual method or a method using an automatic analyzer.

Example

[0061] (Example 1) Solution A and Solution B Examples of the compositions of Solution A and Solution B are shown in Table 2. This composition was used in the following Examples 1 to 9.

[0062]

Table 2

[0063] (Example 2) Time Course 0.05 mL of a 100 mg / L sucrose standard solution or water (blank) was placed in a disposable cell, 0.5 mL of Solution A was added, and the mixture was gently shaken and left at room temperature for 10 minutes. Subsequently, 0.5 mL of Solution B was added, gently shaken, and the change in absorbance at 555 nm over time at room temperature was measured (Figure 2). The end point of the reaction was reached 10 minutes after the addition of Solution B, and there was no significant increase in absorbance in the blank.

[0064] (Example 3) Calibration Curve 0.05 mL of each concentration of sucrose solution was placed in a disposable cell, 0.5 mL of Solution A was added, gently shaken, and left at room temperature for 10 minutes. Subsequently, 0.5 mL of Solution B was added, gently shaken, and after leaving at room temperature for 10 minutes, the absorbance at 555 nm was measured with an LED colorimeter (Figure 3). The calibration curve was linear in the range of 10 - 600 mg / L of sucrose, indicating that quantitative measurement can be performed within this range.

[0065] (Example 4) Effect of Adding Mutarotase Using a 200 mg / L sucrose solution as a sample, measurements were carried out according to the following procedure. 0.05 mL of the sucrose solution was placed in each of two 1 cm square disposable cells. 0.5 mL of Solution A was added to one, and 0.5 mL of a solution without mutarotase in Solution A was added to the other, and left at room temperature for 10 minutes. Then, 0.5 mL of Solution B was added to each, and the change in absorbance at 555 nm over time at room temperature was measured. As shown in Figure 4, when using Solution A without mutarotase, the progress of the reaction was slow, whereas when using Solution A with added mutarotase, the end point was reached 7 - 10 minutes after the addition of Solution B.

[0066] (Example 5) Specificity of the Kit of the Present Invention For sucrose and naturally occurring disaccharides, 0.05 mL of a 1 mM solution of each was placed in a disposable cell, 0.5 mL of Solution A was added, and the mixture was gently shaken and allowed to stand at room temperature for 10 minutes. Subsequently, 0.5 mL of Solution B was added, and the mixture was gently shaken. After allowing it to stand at room temperature for 10 minutes, the absorbance at 555 nm of the colored solution of each disaccharide was measured with an LED colorimeter. As shown in Fig. 5, almost no coloring occurred for disaccharides other than sucrose. Therefore, it was shown that the sucrose measurement kit according to the method of the present invention can specifically measure only sucrose.

[0067] (Example 6) Influence of coexisting substances Measurements were performed using as samples solutions containing 100 mg / L of sucrose and 0 to 600 mg / L of glucose or solutions containing 100 mg / L of sucrose and 0 to 400 mg / L of ascorbic acid, and the influence of coexisting glucose and the influence of coexisting ascorbic acid were examined by the following procedure. 0.05 mL of the sample was placed in a disposable cell, 0.5 mL of Solution A was added, and the mixture was gently shaken and allowed to stand at room temperature for 10 minutes. Subsequently, 0.5 mL of Solution B was added, and the mixture was gently shaken. After allowing it to stand at room temperature for 10 minutes, the absorbance at 555 nm was measured with an LED colorimeter. It was confirmed that the measurements were not affected by coexisting glucose (Fig. 6) and ascorbic acid (Fig. 7) in the measurement samples.

[0068] Furthermore, measurements were performed using as a sample a solution containing 100 mg / L of sucrose and 0 to 1000 mg / L of glucose, and the influence of coexisting glucose was examined by the following procedure. 0.05 mL of the sample was placed in a disposable cell, 0.5 mL of Solution A was added, and the mixture was gently shaken and allowed to stand at room temperature for 20 minutes. Subsequently, 0.5 mL of Solution B was added, and the mixture was gently shaken. After allowing it to stand at room temperature for 10 minutes, the absorbance at 555 nm was measured with an LED colorimeter. It was confirmed that the measurements were not affected by coexisting glucose (Fig. 8) in the measurement samples. Similar measurements were performed with the sucrose amount changed to 20 mg / L or 600 mg / L, and similar results were obtained.

[0069] (Example 7) Stability of Solution A and Solution B Solution A and Solution B were stored in an incubator at 28°C for 6 weeks. Using the Solutions A and B before the start of storage and the Solutions A and B after being stored at 28°C for 6 weeks, the time course of color development was measured with a sucrose solution (100 mg / L) as the sample. Also, using distilled water as the sample for the blank, the time course of color development was measured. Specifically, 0.05 mL of the sucrose solution (100 mg / L) or 0.05 mL of distilled water was placed in a 1 cm square disposable cell, 0.5 mL of Solution A was added, and after leaving it to stand at room temperature for 10 minutes, 0.5 mL of Solution B was added, and the time course of color development, that is, the change over time in the absorbance at 555 nm, was measured with an LED colorimeter. For Solutions A and B, there was no significant difference in the time course of color development before the start of storage at 28°C and after 6 weeks of storage, and in both cases, the endpoint was reached within 10 minutes after the addition of Solution B (Figure 9). Also, although not shown in the figure, no significant increase was observed in the blank even when Solutions A and B were stored at 28°C for 6 weeks. Therefore, the stability of the kit of the present invention comprising Solutions A and B was confirmed.

[0070] (Example 8) Correlation with an existing kit (F-kit) Among existing sucrose measurement methods, there is an F-kit manufactured by Roche as an example of a method using an enzyme. Using the kit of the present invention and the F-kit, the sucrose concentration contained in fruits and beverages was measured, and the correlation between the measured values by both kits was evaluated. Regarding fruits, the water extract of the edible part was used as the sample. In the method using the kit of the present invention, 0.05 mL of the sample was placed in a disposable cell, 0.5 mL of Solution A was added, gently shaken, and left to stand at room temperature for 20 minutes. Subsequently, 0.5 mL of Solution B was added, gently shaken, and after leaving it to stand at room temperature for 10 minutes, the absorbance at 555 nm was measured with an LED colorimeter. Based on the calibration curve in Figure 3, the sucrose concentration was determined. Table 3 shows the measurement results, and Figure 10 shows the correlation between the kit of the present invention and the F-kit.

[0071]

Table 3

[0072] A strong correlation was observed between the measured values obtained using the kit of the present invention and those obtained using the F-kit, and the correlation coefficient was 0.997.

[0073] (Example 9) Correlation with Measured Values of External Analytical Institutions The sucrose concentration included in the measurement target shown in Table 4 was measured using the kit of the present invention. 0.05 mL of the sample was placed in a disposable cell, 0.5 mL of Solution A was added, and it was gently shaken and left at room temperature for 20 minutes. Subsequently, 0.5 mL of Solution B was added, gently shaken, and after leaving it at room temperature for 10 minutes, the absorbance at 555 nm was measured with an LED colorimeter. Based on the calibration curve in Figure 3, the sucrose concentration was determined. Also, the sucrose concentration of the same sample was measured by HPLC method at an external analytical institution (Tsukuba Food Evaluation Center Co., Ltd.). For beverages, they were diluted with water as appropriate. For fruits, the filtrate of a 10-fold extract of the edible part with water was used as the sample. For dairy products, protein removal was performed using an acetic acid solution, and the clear supernatant after centrifugation was used as the measurement sample. The measurement results are shown in Table 4, and the correlation between the kit of the present invention and HPLC is shown in Figure 11.

[0074]

Table 4

[0075] (Example 10) Examination of Enzyme Amount A solution in which the amount of invertase in Solution B of the example shown in Table 2 was changed from 100 U / mL to 50 U / mL, 25 U / mL, 12.5 U / mL, 6.25 U / mL, or 3.12 U / mL was used as Solution B, and in the same manner as in Example 2, a 100 mg / L sucrose standard solution was treated, and the change over time in the absorbance at 555 nm was measured. The results are shown in Figure 12. From the results in Figure 12, it can be seen that when the content of invertase in Solution B is 10 U / mL or more, the reaction endpoint is reached in a short time of around 10 minutes.

[0076] (Example 11) Stability of the Kit Containing Ascorbic Acid Oxidase In the liquid A and liquid B of the example shown in Table 2 (No. 1 in Table 5), the formulations of TOOS, 4-AA, and peroxidase (POD) were changed as shown in Table 5 below to compare the stability.

[0077]

Table 5

[0078] For No. 1 to No. 4 described in Table 5, kits were prepared immediately after preparation and after storage at 40 °C for 1 week. Using these kits, a 100 mg / L sucrose solution containing ascorbic acid at a concentration of 0 to 400 mg / L was used as a sample. After adding 0.5 mL of liquid A to 0.05 mL of the sample and leaving it for 20 minutes, 0.5 mL of liquid B was added and left for 10 minutes, and then the absorbance at 555 nm was measured. Figure 13 shows the change in absorbance depending on the ascorbic acid addition concentration with the absorbance without ascorbic acid addition (0 mg / L) taken as 100%.

[0079] From the results shown in Figure 13, when using the reagent stored at 40 °C for 1 week, No. 1 is hardly affected by the addition of ascorbic acid, but for the reagents of No. 2 to No. 4, it can be seen that they are affected by the addition of ascorbic acid and the reaction solution has faded. That is, it was suggested that ascorbic acid oxidase was inactivated in cases other than the combination of No. 1.

[0080] From the above results, it can be seen that in a sucrose measurement kit containing ascorbic acid oxidase in liquid A, by making liquid A contain peroxidase and a new Trinder reagent and liquid B contain a coupler compound, the storage stability of the reagent can be further improved.

Claims

1. A kit for measuring sucrose, containing liquid A and liquid B: (Liquid A) A liquid containing glucose oxidase, catalase, and mutarotase; (Liquid B) A liquid containing invertase and a catalase deactivator; However, for liquid A and liquid B, one of them contains a coupler compound, the other contains a new Trinder reagent, and at least one of them contains peroxidase, The content of invertase in liquid B is in the range of 10 U / mL to 200 U / mL.

2. The kit according to claim 1, wherein liquid A further contains ascorbic acid oxidase.

3. The kit according to claim 2, wherein liquid A contains a new Trinder reagent and peroxidase.

4. The kit according to any one of claims 1 to 3, wherein the coupler compound is 4-aminoantipyrine.

5. The kit according to claim 4, wherein the new Trinder reagent is N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS) or N-ethyl-N-sulfopropylaniline (ALPS).

6. The kit according to any one of claims 1 to 3, further containing a sucrose standard solution.

7. The kit according to any one of claims 1 to 3, including a device having an LED light source for measuring the amount of the generated dye.

8. A method for measuring sucrose, comprising: Steps 1 and 2: (Step 1) A step of co-existing a sample, glucose oxidase, mutarotase, and catalase in a container; (Step 2) Following step 1, in the same container, a catalase deactivator is allowed to act to inactivate the catalase, and invertase is allowed to act on sucrose to generate glucose, and then glucose oxidase, mutarotase, peroxidase, a coupler compound, and a new Trinder reagent are allowed to act on the generated glucose to generate a dye is included, The method, wherein step 2 is carried out in a solution with an invertase concentration of 3.3 U / mL to 100 U / mL.

9. The method according to claim 8, wherein step 2 is carried out at room temperature.

10. The method according to claim 8, wherein steps 1 and 2 are carried out at room temperature.

11. Step 1 is started by adding liquid A containing glucose oxidase, mutarotase, and catalase to the sample, After adding liquid A, liquid B containing invertase and a catalase deactivator is added 7 to 20 minutes later to start step 2. The method according to any one of claims 8 to 10, wherein the color development by the dye is measured 5 to 10 minutes after the addition of liquid B.

12. The method according to any one of claims 8 to 10, wherein in step 1, the sample and ascorbate oxidase are further allowed to coexist.

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