Kit for measuring lactose and method for measuring lactose

Abstract

To provide a kit enabling simple measurement of lactose in a sample that may simultaneously contain glucose, without influence from glucose.SOLUTION: A kit for measuring lactose in a sample, comprising a solution A and a solution B: solution (A) containing glucose oxidase, catalase, and mutarotase; solution (B) containing β-galactosidase and a catalase inactivator; where one of the solutions A and B contains a coupler compound, the other contains a new Trinder reagent, one of them further contains peroxidase, and the content of β-galactosidase in the solution B is within a range of 50 U / mL to 200 U / mL.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a lactose measurement kit and a lactose measurement method. [Background technology]

[0002] Lactose is a sugar found in mammalian milk, accounting for over 99% of its content. The main foods containing lactose are milk and various dairy products made from milk. While milk and dairy products are produced worldwide and highly valued as nutritious foods, two-thirds of the world's population is lactose intolerant, meaning they cannot digest lactose. To ensure these people do not miss out on the excellent nutrients, such as protein and calcium, that come from consuming dairy products, dairy products with reduced or no lactose have been developed, and lactose quantification is essential for managing the manufacturing process of these products.

[0003] There are methods such as high-performance liquid chromatography (ion exchange chromatography) for quantitative analysis of sugars such as lactose. However, the sample preparation method is complicated, and measurement requires specialized equipment and a certain level of skill.

[0004] On the other hand, several enzyme-based lactose measurement kits are also commercially available. Most of these involve breaking down lactose into glucose and galactose using β-galactosidase, and then measuring the amount of glucose or galactose produced in a 1:1 ratio with lactose. For example, the method for measuring lactose specified in German industrial standards follows the above method.

[0005] Patent Document 1 describes a trehalose analysis reagent that can be used in a method including a step of measuring the amount of glucose produced by enzymatic degradation, and that is a combination of a glucose elimination pretreatment reagent containing at least mutarotase, glucose oxidase, and catalase, and a produced glucose detection reagent containing at least trehalose phosphorylase, sodium azide, peroxidase, 4-aminoantipyrine, and a hydrogen donor. Patent Document 1 discloses that trehalose at concentrations of 1 to 10 g / L (approximately 2.6 to 26 mM) can be quantified based on the color development obtained by adding 50 μL of the glucose elimination pretreatment reagent to a test solution and incubating at 37°C, followed by adding the produced glucose detection reagent and incubating at 37°C. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Publication No. 10-99098 Summary of the Invention [Problem to be solved by the invention]

[0007] In the above-mentioned methods using commercially available kits, the amount of glucose or galactose originally contained in the sample must be measured and subtracted from the total amount of glucose or galactose to calculate the amount of lactose. Therefore, there are many reaction steps, and the process of calculating the lactose concentration from the measurement results is complicated. Furthermore, the method described in Patent Document 1, which measures trehalose, measures trehalose. An object of the present invention is to provide a novel kit for easily measuring lactose, particularly a kit for accurately and easily measuring lactose in a sample that may also contain glucose, without being affected by the coexisting glucose, by using a lactose solution as a standard solution. [Means for solving the problem]

[0008] The present inventors have conducted extensive research to solve the above problems and have completed the present invention.

[0009] Specifically, the present invention provides the following as representative embodiments. [1] A kit for measuring lactose containing solutions A and B: (Solution A) a solution containing glucose oxidase, catalase, and mutarotase; (Solution B) a solution containing β-galactosidase and a catalase inactivator; However, one of the solutions A and B contains a coupler compound, the other contains a new Trinder reagent, and at least one contains peroxidase; A kit in which the content of β-galactosidase in solution B is in the range of 50 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 the new Trinder 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 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 [1] to [5], further comprising a lactose standard solution. [7] The kit according to any one of [1] to [6], which includes a device having an LED light source for measuring the amount of the produced pigment. [8] A method for measuring lactose, comprising: Step 1 and Step 2: (Step 1) allowing a sample to coexist with glucose oxidase, mutarotase, and catalase in a container; (Step 2) Following Step 1, in the same vessel, a catalase inactivator is applied to inactivate the catalase, and glucose oxidase, mutarotase, peroxidase, a coupler compound, and a new Trinder reagent are applied to the glucose produced by the action of β-galactosidase on lactose to produce a dye. Including, A method in which step 2 is carried out in a solution having a β-galactosidase concentration of 17 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 a solution A containing glucose oxidase, mutarotase, and catalase to a sample; 7 to 20 minutes after adding solution A, solution B containing β-galactosidase and catalase inactivator is added to start 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 the solution B.

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

[11] , wherein in step 1, the sample is further allowed to coexist with ascorbic acid oxidase. [Effects of the Invention]

[0010] The present invention provides a lactose measurement kit using an enzyme, which allows for simple measurement. The kit of the present invention allows lactose to be measured in a few steps, even in samples that may also contain glucose, without the need for a separate glucose measurement. Furthermore, the kit of the present invention allows lactose to be used as a standard substance, making it possible to directly quantify lactose without performing calculations for concentration conversion after measurement. Furthermore, the kit of the present invention has good storage stability and is highly versatile. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a diagram showing the principle of lactose measurement using the kit of the present invention. [Figure 2]This shows the time course of a 100 mg / L lactose standard solution and water (blank) after adding solution B in measurements using solutions A and B. [Figure 3] The calibration curve for lactose is shown. [Figure 4] The time course of a 200 mg / L lactose solution with and without mutarotase in solution A is shown. [Figure 5] 1 is a graph showing the specificity of the kit of the present invention for disaccharides. [Figure 6] 1 is a graph showing the effect of glucose coexisting in a sample. [Figure 7] 1 is a graph showing the effect of ascorbic acid coexisting in a sample. [Figure 8] 1 is a graph showing the effect of glucose coexisting in a sample. [Figure 9] FIG. 1 is a graph comparing the time course of lactose solution after addition of solution B in measurements using solutions A and B before and after storage at 40° C. for one week. [Figure 10] This figure compares the time courses of measurements using solutions A and B before and after storage at 40°C for one week, and a blank sample (distilled water) after the addition of solution B. [Figure 11] The correlation between the kit of the present invention and existing kits is shown. [Figure 12] 1 shows the correlation between the kit of the present invention and HPLC. [Figure 13] This shows the time course of a 100 mg / L lactose standard solution when the amount of β-galactosidase in solution B is changed. [Figure 14] This figure compares the shelf life (change in absorbance due to addition of ascorbic acid to a sample) of kits containing ascorbic acid oxidase in solution A, using combinations of coupler compounds, new Trinder reagent, and peroxidase. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention provides a kit for measuring lactose in a sample, which contains the following solution A and solution B. In this specification, the term "measurement of lactose" refers to the detection of lactose, measurement of lactose content, etc.

[0013] The principle of lactose measurement using the kit of the present invention is shown in Figure 1. First, Solution A is added, and the reaction in Step 1 proceeds. In Step 1, glucose in the measurement sample is decomposed by glucose oxidase and then catalase, eliminating the influence of glucose originally present in the sample. If Solution A also contains ascorbic acid oxidase, the ascorbic acid originally present in the sample can be further decomposed and its influence eliminated. Next, Solution B is added to the sample to which Solution A has been added, and the reaction in Step 2 proceeds. Lactose is decomposed by β-galactosidase (lactase), and the resulting glucose is oxidized by glucose oxidase. The hydrogen peroxide produced by the oxidation of glucose is in a 1:1 ratio with the lactose contained in the sample. This hydrogen peroxide reacts with peroxidase, a coupler compound, and the new Trinder's reagent (4-AA and TOOS in Figure 1) under catalase inhibition to produce a dye. Lactose is quantified based on the amount of this dye.

[0014] The kit of the present invention was compared with existing lactose measurement kits in terms of the type of enzyme acting on glucose, whether or not mutarotase was used, whether or not heating was used during the reaction process, the type of standard solution (lactose solution or glucose solution), whether or not separate measurement of coexisting glucose was required, and whether or not the concentration could be roughly estimated by visual inspection of the resulting dye (Table 1). Unlike any of the existing lactose measurement kits listed in Table 1, the kit of the present invention does not require prior measurement of coexisting glucose in the sample and can be measured at room temperature without heating throughout the entire 20-30 minute reaction process. Furthermore, this kit uses a lactose solution as the standard solution and only two reagent solutions, allowing lactose to be quantified at the endpoint of the enzymatic reaction. Furthermore, while existing lactose measurement kits are intended for professionals with knowledge of biochemistry and analytical chemistry, the lactose measurement kit of the present invention can be easily performed at room temperature without the need for special equipment or skilled techniques, making it suitable for analysis in food processing facilities or educational institutions.

[0015] [Table 1]

[0016] (A liquid, B liquid) 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 β-galactosidase and a catalase inactivator. However, one of the solutions A and B contains a coupler compound, the other contains a new Trinder reagent, and at least one contains peroxidase.

[0017] In the kit of the present invention, solution A contains mutarotase, an enzyme that converts α-glucose to β-glucose as follows:

[0018] [ka]

[0019] By incorporating mutarotase into Solution A, the inventors have made it possible to measure lactose in a short time at room temperature using the kit of the present invention. Glucose oxidase is an enzyme that oxidizes β-D-glucose to D-glucono-1,5-lactone. The addition of mutarotase to Solution A is thought to rapidly convert α-glucose, which exists in a constant equilibrium in the sample, to β-glucose, thereby accelerating the glucose oxidase reaction. Furthermore, while β-galactosidase-mediated enzymatic reactions are typically performed at temperatures of 37°C or higher, the presence of mutarotase in the reaction rapidly decomposes the product glucose, accelerating the β-galactosidase-mediated reaction of lactose to glucose, making it possible to measure lactose at room temperature.

[0020] Furthermore, in the kit of the present invention, either Solution A or Solution B contains peroxidase. Peroxidase is an enzyme that acts in Step 2 in Figure 1, which is performed after the addition of Solution B, and therefore, from the perspective of the measurement principle, it is sufficient that it is contained in Solution B. However, the present inventors have found that regardless of whether Solution A or Solution B contains peroxidase, both Solutions A and B are stable, can be stored as a kit for a long period of time, and do not affect the measurement results.

[0021] In the kit of the present invention, one of solutions A and B contains a coupler compound, and the other contains a new Trinder reagent. By containing the coupler compound and the new Trinder reagent in separate solutions, and in particular by not containing the coupler compound, the new Trinder reagent, and peroxidase in the same solution, it is possible to prevent the reagents themselves from developing color. In a preferred embodiment, solution A contains the new Trinder reagent, and solution B contains the coupler compound. This improves the stability of both solutions A and B. In this specification, the coupler compound, the new Trinder reagent, or a combination thereof may be referred to as a color former.

[0022] The kit of the present invention can be used to measure lactose in a sample that may also contain glucose.

[0023] The kit of the present invention may further contain ascorbic acid oxidase in one or more of solutions A and B, preferably solution A. Ascorbic acid (vitamin C) is a component that may be contained in the measurement target, and the presence of ascorbic acid in the sample inhibits the color reaction. By including ascorbic acid oxidase, the kit of the present invention can be used to measure lactose in a sample that may contain ascorbic acid or erythorbic acid.

[0024] When solution A contains ascorbic acid oxidase, it is preferred, from the viewpoint of enzyme stability, that solution A contains peroxidase and a new Trinder reagent, and solution B contains a coupler compound.

[0025] (enzyme) In the present invention, known enzymes such as glucose oxidase, β-galactosidase, mutarotase, catalase, peroxidase, and ascorbic acid oxidase can be used.

[0026] Glucose oxidase is an enzyme that oxidizes β-D-glucose to D-glucono-1,5-lactone, and examples thereof include glucose oxidase derived from microorganisms such as Aspergillus niger.

[0027] β-galactosidase is an enzyme also known as lactase. Examples of β-galactosidases used in the present invention include β-galactosidases derived from microorganisms such as Aspergillus oryzae, Kluyveromyces lactis, and Escherichia coli.

[0028] Mutarotase may include mutarotase derived from pig kidney.

[0029] Examples of catalases include catalases derived from bovine liver, as well as catalases derived from microorganisms such as Aspergillus niger and Corynebacterium glutamicum.

[0030] An example of peroxidase is horseradish peroxidase.

[0031] Examples of ascorbate oxidase include ascorbate oxidase derived from cucumber or pumpkin.

[0032] (New Trinder Reagent) In the present invention, known new Trinder reagents can be used. Examples of new Trinder reagents that can be 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 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 ...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-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAOS), N-ethyl-N Examples of suitable aniline include N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxy-4-fluoroaniline (FDAOS), N-ethyl-N-sulfopropyl-3,5-dimethoxy-4-fluoroaniline (FDAPS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (ADOS), and N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (HDOS). It is preferable to use any of 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).

[0033] (Coupler Compounds) In the present invention, known coupler compounds can be used. Any compound that produces color in combination with the new Trinder reagent can be used. Examples include 4-aminoantipyrine (4-AA), vanillindiamine sulfonic acid, methylbenzthiazolinone hydrazone (MBTH), sulfonated methylbenzthiazolinone 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 a derivative thereof. 4-aminoantipyrine (4-AA) is preferred.

[0034] (Other ingredients) Solution A or B included in the kit of the present invention may further contain a preservative. Known preservatives can be used. Examples include sodium azide, Proclin 300, Proclin 950, and chloramphenicol.

[0035] Solutions A and B may contain a component having pH buffering ability, such as acetate, phosphate, citrate, borate, tartrate, Tris (tris(hydroxymethyl)aminomethane), and Hepes ([2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid]).

[0036] Both solutions A and B are stable even if they do not substantially contain bovine serum albumin, which is used as an enzyme stabilizer (for example, even if the amount of bovine serum albumin is less than 0.02 wt %, or 0.01 wt % or less), and as shown in the examples, the enzyme activity in the solution can be maintained for a long period of time even without containing bovine serum albumin.

[0037] (Content of ingredients) The glucose oxidase content of Solution A is preferably 20 to 500 U / mL, more preferably 50 to 200 U / mL, and particularly preferably 100 to 500 U / mL, more preferably 100 to 300 U / mL, to enable measurement in a short time and to increase the stability of Solution A. The catalase content in Solution A is preferably 500 to 2000 U / mL, and 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, and 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), and 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 concentration can be 1 to 30 U / mL, and preferably 2 to 10 U / mL.

[0038] The content of β-galactosidase in solution B may be in the range of 50 U / mL to 200 U / mL, and preferably in the range of 60 U / mL to 150 U / mL. The concentration is preferably in the range of 70 U / mL to 100 U / mL. The enzyme unit (U) of β-galactosidase in this case is the international unit determined by measuring the amount of o-nitrophenol produced per minute at 37°C at pH 7.3 using o-nitrophenyl-β-D-galactopyranoside as a substrate. By using β-galactosidase at a concentration of 10 U / mL to 200 U / mL, the enzymatic reaction can proceed rapidly at room temperature using the kit of the present invention. Furthermore, it has been demonstrated that even when Solution B contains β-galactosidase at a concentration of 50 U / mL to 200 U / mL, which is higher than the amount of enzyme typically used, it is stable enough to withstand use as a kit that can be stored for a certain period of time. To complete lactose measurement at room temperature in a shorter time, the β-galactosidase content in Solution B is preferably 70 U / mL or more. In the reaction in Step 2 using Solution B, lactose is decomposed by β-galactosidase to produce glucose, which is then oxidized by glucose oxidase. The resulting hydrogen peroxide is then reacted with peroxidase, a coupler compound, and the new Trinder reagent under catalase inhibition to produce a dye. By increasing the amount of β-galactosidase in Solution B, the lactose decomposition reaction proceeds rapidly, even at room temperature, and the resulting dye production reaction can be completed within a short time (e.g., within 10 minutes) after the addition of Solution B. This invention is a colorimetric method that uses endpoint measurement of the enzyme reaction and does not require strict time management. Therefore, it is a simpler and more novel method for quantifying lactose.

[0039] Solution B preferably contains an inorganic salt together with β-galactosidase. The inclusion of an inorganic salt can enhance enzymatic activity. Examples of inorganic salts include magnesium sulfate, manganese chloride, cobalt chloride, and any combination thereof, with magnesium sulfate being preferred. When Solution B contains an inorganic salt, its concentration may be 0.2 mM to 3.0 mM, and preferably 0.5 mM to 2.0 mM.

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

[0041] (Lactose standard solution) The kit of the present invention may contain a lactose standard solution in addition to the above-mentioned solutions A and B. The concentration of the lactose standard solution can be appropriately adjusted, specifically, for example, to 100 to 300 mg / L, and preferably 100 mg / L.

[0042] (Other components of the kit) The kit of the present invention may include a device with a light source for measuring the amount of the produced pigment. The kit of the present invention may also include a dropper for collecting solution A and a dropper for collecting solution B, and may further include a colorimeter (sometimes referred to as an "LED colorimeter" in this specification) for measuring the amount of the produced pigment using an LED as a light source, and a plastic cell for measurement. The LED colorimeter may be of a type that displays the measurement results in absorbance or in concentration (a value calculated from the absorbance of the color-developing solution of the measurement sample and the standard solution).

[0043] (Application) The kit of the present invention can be used to measure lactose. Specifically, it can be used to measure lactose in a measurement target. The measurement target is not particularly limited, and can be anything that is expected to contain lactose. The measurement target may also be something that may contain glucose. Specific examples include human or other mammalian breast milk, cow's milk, composition-adjusted milk, low-fat milk, non-fat milk, processed milk, dairy drinks, skim milk powder, modified milk powder, yogurt, ice cream, and various confectioneries. The measurement object is preferably provided for measurement as an aqueous solution. For the measurement object in the form of a solution containing insoluble matter, it is also preferable to prepare a sample by filtering or centrifuging.

[0044] Using the kit of the present invention, for example, when considering the case where solutions A and B are both used in an amount 10 times the volume of the sample, it is possible to accurately measure the concentration of a lactose solution of 10 to 800 mg / L, preferably 20 to 600 mg / L. The measurement target may be diluted with water to prepare a sample so as to fall within a concentration range that allows for more accurate measurement, depending on the mixing ratio of the reagents selected for the kit of the present invention. In this case, the water used for dilution is not limited to distilled water or deionized water; tap water can also be used to dilute the measurement target.

[0045] Furthermore, the kit of the present invention can be used to accurately measure the concentration of lactose in a sample that may also contain glucose, without measuring the glucose separately. For example, the lactose content can be accurately measured in a sample containing 20 to 600 mg / L of lactose, even if the sample contains up to 1000 mg / L of glucose.

[0046] When measuring with the kit of the present invention, the volume of Solution A is preferably 1 to 100 times, more preferably 5 to 20 times, and even more preferably 10 times, the volume of the sample, and the volume of Solution B is preferably 1 to 100 times, more preferably 5 to 20 times, and even more preferably 10 times the volume of the sample. The amount of Solution B used relative to the volume of Solution A can be determined based on the amounts of components in each solution and is not particularly limited. From the practical standpoint of preventing mistakes in the amounts added and making them easy to remember, it is preferable that the volumes of Solutions A and B used are equal (volume ratio). By using Solutions A and B at a volume of 1 or more relative to the sample and using approximately the same amount of Solution B relative to Solution A, the amount of β-galactosidase in the solution during the lactose hydrolysis reaction can be adjusted to a range of approximately 17 U / mL to 100 U / mL.

[0047] (Method for measuring lactose) The present invention also relates to a method for measuring lactose, which comprises the following steps 1 and 2. This method can be carried out using the kit of the present invention, and for details of the enzymes, reagents, samples, etc. used, please refer to the explanation of the kit above.

[0048] (Step 1) a step of allowing a sample, glucose oxidase, mutarotase, and catalase to coexist in a container X; (Step 2) Following Step 1, in a container X, a catalase inactivator is applied to inactivate catalase, and the glucose produced by the action of β-galactosidase on lactose is reacted with glucose oxidase, mutarotase, peroxidase, a coupler compound, and the new Trinder reagent to produce a dye.

[0049] Here, step 2 is carried out in a solution with a β-galactosidase concentration of 17 U / mL to 100 U / mL. By using the enzyme at this concentration, the enzymatic reaction can proceed rapidly, even at room temperature, for example. The β-galactosidase concentration is preferably 20 U / mL to 75 U / mL, and more preferably 23 U / mL to 50 U / mL.

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

[0051] In step 1, if the sample contains glucose, the glucose is decomposed using glucose oxidase and then catalase to eliminate the influence of glucose present in the sample. Specifically, the hydrogen peroxide produced by the oxidative decomposition of glucose by glucose oxidase is decomposed using catalase. Here, the coexistence of mutarotase promotes the conversion of glucose from α-form to β-form, and glucose is rapidly decomposed by glucose oxidase. In step 1, the sample may also be coexisted with ascorbic acid oxidase. In this case, ascorbic acid or erythorbic acid in a sample containing ascorbic acid or erythorbic acid is decomposed by ascorbic acid oxidase, thereby eliminating their influence.

[0052] In step 2, the catalase present in step 1 is inactivated by the action of a catalase inactivator. This is to prevent the decomposition of hydrogen peroxide by catalase in step 2. Also in step 2, lactose in the sample is decomposed by β-galactosidase, and the resulting glucose is oxidized by glucose oxidase. The coexistence of mutarotase derived from step 1 promotes the conversion of glucose from α- to β-form, and glucose is rapidly decomposed by glucose oxidase. The hydrogen peroxide produced by the oxidation of glucose reacts with peroxidase, a coupler compound, and the new Trinder reagent to produce a dye. For example, the concentration of lactose can be quantified by measuring the absorbance at the dye's absorption maximum (555 nm when using TOOS and 4-AA) using a spectrophotometer. Step 2 can be initiated, for example, by adding the above-mentioned solution B to the mixture of the sample and solution A after the step.

[0053] In step 1, if the sample does not contain glucose, the reaction with glucose oxidase and catalase is not carried out, and if the sample does not contain ascorbic acid or erythorbic acid, the decomposition of ascorbic acid or erythorbic acid by the action of ascorbic acid oxidase on the ascorbic acid or erythorbic acid is not carried out.

[0054] In the method of the present invention, after the reaction in step 1 (the reaction by solution A when using the kit of the present invention) has progressed sufficiently, step 2 is carried out (solution B is added when using the kit of the present invention). In step 2, only glucose produced by the reaction of lactose, the measurement target in the sample, is decomposed by glucose oxidase present since step 1 to produce hydrogen peroxide, which is then reacted with a color former and peroxidase to produce color. In step 2, catalase present since step 1 is inactivated by a catalase inactivator, so the color reaction is not inhibited by catalase.

[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 at a temperature range including room temperature, for example, in an environment of 15°C to 42°C. While enzymatic reactions using β-galactosidase are typically carried out at temperatures of 37°C or higher, the present inventors have discovered that in the method of the present invention, in which step 2 is carried out in the presence of mutarotase and with an increased amount of β-galactosidase enzyme, the reaction proceeds rapidly even at room temperature. Since step 1 can also be carried out at room temperature in the method of the present invention, all steps 1 and 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 interpreted as meaning "without cooling or heating." Furthermore, in this specification, room temperature refers to a temperature of 15°C to 35°C, preferably 20°C to 30°C. The pH conditions may be appropriately set based on the optimum pH of each enzyme used. Usually, steps 1 and 2 can be carried out at a pH of 6.0 to 8.0.

[0056] Steps 1 and 2 can each be performed for 5 minutes or more, preferably 7 minutes or more, more preferably 9 minutes or more, and preferably 30 minutes or less, more preferably 20 minutes or less. From the practical standpoint of being able to easily remember the time settings in a short time, steps 1 and 2 are each preferably performed for 10 minutes. Furthermore, in cases where a higher amount of coexisting glucose may be present, step 1 is also preferably performed for 20 minutes from the standpoint of eliminating its influence. For example, the instructions for the kit of the present invention may state that solution A is added, followed by solution B 10 or 20 minutes later, and the amount of pigment is measured 10 minutes after the addition of solution B.

[0057] The color generated in step 2 can be measured as absorbance at a specific wavelength in step 3 using a spectrophotometer with a light source such as a halogen lamp or xenon lamp. Alternatively, the degree of color generation can be measured as an electrical signal (e.g., voltage value) using a colorimeter with a phototransistor or other light-receiving element and an LED light source with a specific wavelength range. When using an LED as a light source, the wavelength of the light source for measuring color generation can be selected according to the type of coloring agent used. For example, when TOOS is used as the new Trinder reagent, the absorption maximum of the resulting dye is 555 nm, so a 555 nm pure green LED is suitable as a light source. Based on the absorbance measured in steps 1 and 2 for the coloring solution of the sample, the concentration of lactose in the sample can be calculated by comparing the absorbance measured with the absorbance of a previously measured coloring solution of a lactose solution of known concentration.

[0058] For example, the method of the present invention may further include the following steps 12, 13, and 14. (Step 12) a step of reacting β-galactosidase with lactose in a standard lactose solution in a container Y to produce glucose, and then reacting glucose oxidase, mutarotase, peroxidase, a coupler compound, and the new Trinder reagent with the glucose to produce a dye; (Step 13) A step of measuring the amount Y of the pigment produced in step 12 (Step 14) A step of calculating the lactose concentration in the sample from the pigment amount Y, the pigment amount X, and the lactose concentration of the lactose standard solution. Step 14 may be performed by a device equipped with an information processing unit that performs the above calculations. For example, a colorimeter may be equipped with an information processing unit.

[0059] In order to measure lactose with higher accuracy, the method of the present invention preferably further comprises the following step 11. (Step 11) A step of allowing glucose oxidase, mutarotase, and catalase to coexist in a lactose standard solution in a container Y. Following step 11, step 12 is carried out in container Y. The lactose concentration in the sample can be calculated from the dye amount Y, the dye amount X, and the lactose concentration of the lactose standard solution. Steps 11 to 13 are preferably performed before steps 1 and 2. For example, in an apparatus equipped with an information processing unit, calibration is performed using the color-developing solution of the lactose standard solution, and then the color-developing solution of the measurement sample is measured, whereby the lactose concentration of the measurement sample can be calculated from the absorbance. Calibration is preferably performed based on the results of measurements using the lactose standard solution as well as measurements using water (distilled water) instead of the lactose standard solution.

[0060] The method for measuring lactose of the present invention can be carried out either manually or using an automatic analyzer. [Example]

[0061] (Example 1) Solution A and Solution B Examples of the compositions of solutions A and B are shown in Table 2. These compositions were used in the following examples.

[0062] [Table 2] Mutarotase used was a mutarotase suspension (Buda kidney-derived) (Code No. 133-07501) manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. 0.5 vol% corresponds to 50 U / mL.

[0063] (Example 2) Time course 0.05 mL of a 100 mg / L lactose standard solution or water (blank) was placed in a disposable cell, 0.5 mL of Solution A was added, the cells were gently shaken, and the cells were left at room temperature for 10 minutes. 0.5 mL of Solution B was then added, the cells were gently shaken, and the change in absorbance at 555 nm at room temperature was measured over time (Figure 2). The reaction endpoint 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 lactose solution was placed in a disposable cell, 0.5 mL of solution A was added, the cells were gently shaken, and the cells were left at room temperature for 10 minutes. Next, 0.5 mL of solution B was added, the cells were gently shaken, and the cells were left at room temperature for 10 minutes. The absorbance at 555 nm was then measured using an LED colorimeter (Figure 3). The calibration curve was linear in the lactose range of 10 to 600 mg / L, demonstrating that quantitative measurement is possible within this range.

[0065] (Example 4) Effect of adding mutarotase Measurements were carried out using a 200 mg / L lactose solution as a sample according to the following procedure. 0.05 mL of lactose solution was placed in two 1 cm square disposable cells. 0.5 mL of Solution A was added to one cell, and 0.5 mL of Solution A without mutarotase was added to the other. The cells were then left to stand at room temperature for 10 minutes. 0.5 mL of Solution B was then added to each cell, and the change in absorbance at 555 nm over time at room temperature was measured. As shown in Figure 4, the reaction proceeded slowly when Solution A without mutarotase was used, whereas the endpoint was reached 10 minutes after the addition of Solution B when Solution A with mutarotase was used.

[0066] (Example 5) Specificity of the kit of the present invention 0.05 mL of 1 mM solutions of lactose and naturally occurring disaccharides (cellobiose, lactulose, maltose, isomaltulose, sucrose (cane sugar), and trehalose) were placed in disposable cells, 0.5 mL of solution A was added, the cells were gently shaken, and the cells were left at room temperature for 10 minutes. Next, 0.5 mL of solution B was added, the cells were gently shaken, and the cells were left at room temperature for 10 minutes. The absorbance of the color-developed solutions of each disaccharide was measured at 555 nm using an LED colorimeter. As shown in Figure 5, disaccharides other than lactose showed little color development. Therefore, it was demonstrated that the lactose measurement kit according to the method of the present invention can specifically measure only lactose.

[0067] (Example 6) Effect of coexisting substances Measurements were performed using a solution containing 100 mg / L lactose and 0 to 600 mg / L glucose or a solution containing 100 mg / L lactose and 0 to 400 mg / L ascorbic acid as a sample, and the effects of coexisting glucose and coexisting ascorbic acid were examined using the following procedure. 0.05 mL of sample was placed in a disposable cell, 0.5 mL of solution A was added, the mixture was gently shaken, and the mixture was left at room temperature for 10 minutes. Next, 0.5 mL of solution B was added, the mixture was gently shaken, and the mixture was left at room temperature for 10 minutes. The absorbance at 555 nm was then measured using an LED colorimeter. It was confirmed that the measurement was not affected by glucose (Figure 6) and ascorbic acid (Figure 7), which coexist in the sample.

[0068] Furthermore, measurements were carried out using a solution containing 100 mg / L of lactose and 0 to 1000 mg / L of glucose as a sample, and the influence of the coexisting glucose was examined using the following procedure. 0.05 mL of sample 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 20 minutes. 0.5 mL of solution B was then added, and the mixture was gently shaken and left at room temperature for 10 minutes, after which the absorbance at 555 nm was measured using an LED colorimeter. It was confirmed that the measurement was not affected by glucose (Figure 8) present in the sample. A similar study was performed with the lactose 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 Solutions A and B were stored in a 40°C incubator for 1 week. The time course of color development was measured using solutions A and B before storage and after 1 week of storage at 40°C, using lactose solution (100 mg / L) as a sample. Additionally, distilled water was used as a blank. Specifically, 0.05 mL of lactose 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, gently shaken, and left at room temperature for 10 minutes. 0.5 mL of solution B was added, gently shaken, and the change in absorbance at a wavelength of 555 nm over time was measured using an LED colorimeter. There was no significant difference in the time course of color development for solutions A and B before and after 1 week of storage at 40°C, and both reached their endpoints approximately 10 minutes after the addition of solution B (Figure 9). Furthermore, even after storing solutions A and B at 40°C for one week, no significant increase in the blank was observed (Figure 10). Therefore, the stability of the kit of the present invention containing solutions A and B was confirmed.

[0070] (Example 8) Correlation with existing kits Among existing lactose measurement methods, one example that uses an enzyme is the E-Kit Liquid from R-Biopharm. Using the kit of the present invention and the E-Kit Liquid, the lactose concentrations in dairy products were measured, and the correlation between the measured values ​​from both kits was evaluated. All samples were dissolved in water as appropriate, then deproteinized using an acetic acid solution. The clear supernatant obtained after centrifugation was used as the measurement sample. Table 3 shows the measurement results, and Figure 11 shows the correlation between the kit of the present invention and E-kit Liquid.

[0071] [Table 3]

[0072] As shown in FIG. 11, a strong correlation was observed between the measurements using the kit of the present invention and the measurements using E-kit Liquid.

[0073] (Example 9) Correlation with measurements from external analysis institute The lactose concentrations contained in the measurement targets shown in Table 4 were measured using the kit of the present invention. 0.05 mL of each sample was placed in a disposable cell, 0.5 mL of Solution A was added, and the cells were gently shaken and left to stand at room temperature for 20 minutes. Next, 0.5 mL of Solution B was added, and the cells were gently shaken and left to stand at room temperature for 10 minutes. The absorbance at 555 nm was measured using an LED colorimeter. The lactose concentration was determined based on the calibration curve in Figure 3. The lactose concentration of the same samples was also measured by HPLC at an external analytical institution (Tsukuba Food Evaluation Center, Inc.). All samples were dissolved in water as appropriate, then deproteinized using an acetic acid solution. The clear supernatant obtained after centrifugation was used as the measurement sample. Table 4 shows the measurement results, and Figure 12 shows the correlation between the kit of the present invention and HPLC.

[0074] [Table 4]

[0075] (Example 9) Examination of enzyme amount A 100 mg / L lactose standard solution was treated and the change in absorbance at 555 nm over time was measured in the same manner as in Example 2, except that the amount of β-galactosidase in Solution B in the example shown in Table 2 was changed from 100 U / mL to 70 U / mL, 35 U / mL, 14 U / mL, or 7.0 U / mL. The results are shown in Figure 13. The results in FIG. 13 show that when the β-galactosidase content in solution B is 50 U / mL or more, particularly 70 U / mL or more, the reaction endpoint is reached in a short time of around 10 minutes.

[0076] Example 10: Stability of kits containing ascorbic acid oxidase In the example solutions A and B (No. 1 in Table 5) shown in Table 2, the formulations of TOOS, 4-AA, and peroxidase (POD) were changed as shown in Table 5 below, and the stability was compared.

[0077] [Table 5]

[0078] Kits were prepared for Nos. 1 to 4 listed in Table 5, immediately after preparation and after one week of storage at 40°C. Using these kits, 100 mg / L lactose solutions containing ascorbic acid at concentrations of 0 to 400 mg / L were used as samples. 0.5 mL of Solution A was added to 0.05 mL of the sample, and the mixture was left for 20 minutes. 0.5 mL of Solution B was then added, and the mixture was left for 10 minutes, after which the absorbance at 555 nm was measured. Figure 14 shows the change in absorbance depending on the concentration of added ascorbic acid, with the absorbance in the absence of added ascorbic acid (0 mg / L) set at 100%.

[0079] The results shown in Figure 14 show that when reagents stored at 40°C for one week were used, No. 1 was hardly affected by the addition of ascorbic acid, but reagents No. 2 to No. 4 were affected by the addition of ascorbic acid, resulting in discoloration of the reaction solution. This suggests that ascorbic acid oxidase was inactivated in all combinations except for No. 1.

[0080] From the above results, it can be seen that in a lactose measurement kit containing ascorbic acid oxidase in solution A, the shelf life of the reagent can be further improved by combining solution A containing peroxidase and the new Trinder reagent and solution B containing a coupler compound.

Claims

1. A kit for measuring lactose, containing solution A and solution B: (Solution A) a solution containing glucose oxidase, catalase, and mutarotase; (Solution B) a solution containing β-galactosidase and a catalase inactivator; However, one of the solutions A and B contains a coupler compound, the other contains a new Trinder reagent, and at least one contains peroxidase; A kit, wherein the content of β-galactosidase in solution B is in the range of 50 U / mL to 200 U / mL.

2. 2. The kit of claim 1, wherein Solution A further comprises ascorbic acid oxidase.

3. 3. The kit of claim 2, wherein solution A comprises new Trinder's reagent and peroxidase.

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

5. 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 comprising a lactose standard solution.

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

8. 1. A method for measuring lactose, comprising: Step 1 and Step 2: (Step 1) allowing a sample to coexist with glucose oxidase, mutarotase, and catalase in a container; (Step 2) Following Step 1, in the same vessel, a catalase inactivator is applied to inactivate the catalase, and glucose oxidase, mutarotase, peroxidase, a coupler compound, and a new Trinder reagent are applied to glucose produced by the action of β-galactosidase on lactose to produce a dye. Including, A method in which step 2 is carried out in a solution having a β-galactosidase concentration of 17 U / mL to 100 U / mL.

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

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

11. Step 1 begins by adding Solution A containing glucose oxidase, mutarotase, and catalase to a sample; 7 to 20 minutes after the addition of Solution A, Solution B containing β-galactosidase and a catalase inactivator is added 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 solution B.

12. The method according to any one of claims 8 to 10, wherein in step 1, the sample is further made to coexist with ascorbic acid oxidase.

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