Highly water-absorbent polymer for colorimetric analysis and analysis method using the same

Abstract

To provide a highly visible method for measuring food or biological components such as L-glutamic acid, glucose, lactic acid, and pyruvic acid without the need for expensive analytical devices, and to provide materials therefor.SOLUTION: Provided are a colorimetric analysis material to be used after immersion in a sample solution, absorption, and coloration, in which hydrogen peroxide-generating oxidase, peroxidase, a new Trinder's reagent, and a coupler compound are held within a highly water-absorbent polymer; and a method for analyzing a component that is a substrate for hydrogen peroxide-generating oxidase, comprising: immersing the material in a sample solution, allowing it to swell, and activating the hydrogen peroxide-generating oxidase, peroxidase, coupler compound, and new Trinder's reagent on the components inside the swollen material to form a pigment, thereby obtaining a colored sample material (sample material); and calculating the concentration of the component in the sample solution by comparing the color of the sample material with a color sample or a material (standard material) that is immersed in a standard solution, absorbs water, and becomes colored.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a highly water-absorbent polymer for colorimetric analysis and a method for analyzing ingredients using the same. The present invention is useful in fields such as food production. [Background technology]

[0002] L-glutamic acid is known as one of the umami taste components, and also functions as an excitatory neurotransmitter that transmits nerve signals via glutamate receptors in the body of animals. Therefore, detection and quantification of L-glutamic acid is very important in the fields of food and biochemistry. A method using L-glutamic acid oxidase (Non-Patent Document 1) has been developed to measure L-glutamic acid in a sample. For example, Patent Document 1 describes a kit containing reaction solution I containing ascorbic acid oxidase, peroxidase, and a new Trinder reagent, and reaction solution II containing L-glutamic acid oxidase, a coupler compound, and a catalase deactivator. It is explained that this kit solves problems associated with freeze-dried products, such as foaming of the solution, by dividing the reaction solution into two, and can be stored in a stable state for a long period of time. can be done.

[0003] On the other hand, highly water-absorbent polymers, which have a water absorption capacity of several hundred to several thousand times their own weight, are used as sanitary goods, soil water retention agents, food drip absorbents, cooling materials, etc. by utilizing their high water absorption and water retention capacity, and are also used as gel fragrances and insect repellents for clothing by retaining ingredients in gel. For example, Patent Document 2 describes an insect repellent and mite repellent for clothing, which is characterized in that an insect repellent and mite repellent composition for clothing containing 3-methyl-5-phenyl-1-pentanol is retained in aqueous gel beads. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2017-012169 A (Patent No. 6218894) [Patent Document 2] JP 2020-40991 A [Non-patent literature]

[0005] [Non-Patent Document 1] Agric. Biol. Chem., 47(6);1323,1983 Summary of the Invention [Problem to be solved by the invention]

[0006] Measurement of umami components such as L-glutamic acid and sweet components such as glucose is important for objectively evaluating the deliciousness of food. Measurement of lactic acid and pyruvic acid is also important for the production process management of fermented foods such as sake and pickles. However, in order to spread the measurement of chemical components in the production process management and quality control of fermented foods in small and medium-sized enterprises and micro-enterprises, it is desirable to have a highly visible measurement method that does not require expensive analytical equipment and can be easily performed by anyone on-site. [Means for solving the problem]

[0007] The present invention provides the following: [1] A colorimetric analytical material comprising a hydrogen peroxide-generating oxidase, a peroxidase, a new Trinder reagent, and a coupler compound supported on a highly water-absorbent polymer, which is to be immersed in a sample solution, absorbed, and developed color. [2] 2. The material according to 1, wherein the hydrogen peroxide generating oxidase is any one selected from the group consisting of L-glutamate oxidase, L-lysine oxidase, L-arginine oxidase, L-histidine oxidase, glycine oxidase, L-tryptophan oxidase, L-phenylalanine oxidase, L-aspartate oxidase, glucose oxidase, lactate oxidase, pyruvate oxidase, hexose oxidase, cholesterol oxidase, galactose oxidase, pyranose oxidase, alcohol oxidase, nucleoside oxidase, D-mannitol oxidase, xylitol oxidase, oxalate oxidase, D-aspartate oxidase, L-amino acid oxidase, D-amino acid oxidase, amine oxidase, D-glutamate oxidase, putrescine oxidase, sarcosine oxidase, urate oxidase, glutathione oxidase, and xanthine oxidase. [3] The material described in 1, further comprising any one selected from a catalase inactivator, a preservative, a stabilizer, and a pH buffering agent retained in the highly absorbent polymer. [4] The material according to 1, wherein the highly absorbent polymer is a polyacrylate-based polymer. [5] The material according to 1, wherein the superabsorbent polymer is in the form of beads. [6] A material according to any one of 1 to 5 for quantifying food components. [7] A kit for analyzing a component that is a substrate for hydrogen peroxide generating oxidase, comprising the material described in any one of 1 to 5. [8] The kit according to 7, further comprising a color sample or a standard solution of the component. [9] A method for producing the material described in 1, comprising a step of drying bead-shaped superabsorbent polymer impregnated with an enzyme solution containing hydrogen peroxide-generating oxidase, peroxidase, new Trinder's reagent, and a coupler compound at 25°C to 55°C.

[10] The method according to 9, wherein the enzyme solution is a solution of 0.02 to 300 U / mL of hydrogen peroxide generating oxidase, 0.5 to 50 U / mL of peroxidase, 0.05 to 4 mM of new Trinder's reagent, 0.05 to 4 mM of a coupler compound, and a pH buffer.

[11] A method for analyzing a component contained in a sample, the component being a substrate for hydrogen peroxide generating oxidase, comprising the steps of: A step of immersing the material according to any one of claims 1 to 5 in a sample solution, swelling the material, and obtaining a colored material (sample material), in which the hydrogen peroxide-generating oxidase, the peroxidase, the coupler compound, and the new Trinder's reagent act on the components inside the swollen material to form a dye; and A process in which the concentration of the components in the sample solution is calculated by comparing the color of the sample material with a color sample or a material (standard material) that has been immersed in a standard solution, allowed to absorb water, and allowed to develop color.

[12] The method according to claim 11, wherein the step of comparing colors and calculating includes photographing the sample material with an imaging means and calculating the concentration of the components in the sample solution using the obtained image data and previously prepared calibration curve data.

[13] The method according to claim 12, wherein the photographing means is a camera mounted on a smartphone.

[14] The method according to claim 11, wherein the sample solution is diluted with tap water. Effect of the Invention

[0008] When analyzing the target component, highly visible analysis can be performed without requiring expensive analytical equipment. [Brief description of the drawings]

[0009] [Figure 1] Photographs of beads absorbed with enzyme solutions for L-glutamic acid colorimetric measurement, lactate colorimetric measurement, glucose colorimetric measurement, and pyruvic acid colorimetric measurement (top), and photographs of beads obtained by drying them: L-glutamic acid colorimetric measurement beads (GUCB), lactate colorimetric measurement beads (LACB), glucose colorimetric measurement beads (GCCB), and pyruvic acid colorimetric measurement beads (PVCB) (bottom) [Diagram 2] Photographs (top) of GUCB colored with L-glutamic acid standard solutions of various concentrations (taken out of the standard solution 1 hour after the beads were added) and the calibration curve (bottom) [Diagram 3] Photographs of LACB (removed from the standard solution 1 hour after the beads were added) colored with lactic acid standard solutions of various concentrations (top) and the calibration curve (bottom) [Figure 4] Photographs of GCCB (removed from the standard solution 1 hour after the beads were added) colored with glucose standard solutions of various concentrations (top), and calibration curve (bottom) [Diagram 5] Photographs of PVCB (removed from the standard solution 1 hour after adding the beads) colored with pyruvic acid standard solutions of various concentrations (top), and the calibration curve (bottom) [Figure 6] Only the inside of the GUCB develops color, and the external liquid (sample liquid) does not develop color. [Figure 7] Correlation graph between glutamate measurements using glutamate colorimetric measurement beads for 12 types of sake (quantified by comparison with color samples 30 minutes after the beads are added) and measurements using a glutamate measurement kit manufactured by Enzyme Sensor Co., Ltd. [Figure 8] A correlation graph between lactate measurements using lactate colorimetric measurement beads for 12 types of sake (quantified by comparison with color samples 30 minutes after the beads are added) and measurements using a lactate measurement kit manufactured by Enzyme Sensor Co., Ltd. [Figure 9] A correlation graph between glucose measurement values ​​(quantified by comparison with a color sample 30 minutes after the beads are added) using glucose colorimetric measurement beads for 12 types of sake and the measurements using a glucose measurement kit manufactured by Enzyme Sensor Co., Ltd. [Figure 10] Correlation graph between pyruvate measurements using pyruvate colorimetric measurement beads for 12 types of sake (quantified by comparison with color samples 30 minutes after the beads are added) and measurements using a pyruvate measurement kit manufactured by Enzyme Sensor Co., Ltd. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] [Colorimetric analysis materials, kits] The present invention relates to a colorimetric analysis material comprising a hydrogen peroxide-generating oxidase, a peroxidase, a new Trinder reagent, and a coupler compound supported on a highly water-absorbent polymer, which is used by immersing the colorimetric analysis material in a sample solution, allowing it to absorb water and develop color.

[0011] (Super absorbent polymer) In the present invention, the term "superabsorbent polymer (SAP)" refers to a polymer that absorbs 300 times or more of its own weight in pure water, unless otherwise specified. Examples of superabsorbent polymers used in the material include sodium polyacrylate crosslinked body, polyvinyl alcohol crosslinked polymer, sodium acrylate-vinyl alcohol copolymer, starch-acrylonitrile graft polymer hydrolyzate, starch-acrylic acid graft polymer, starch-styrene sulfonic acid graft polymer, starch-vinyl sulfonic acid graft polymer, starch-acrylamide graft polymer, cellulose-acrylonitrile graft polymer, cellulose-styrene sulfonic acid graft polymer, carboxymethylcellulose crosslinked body, hyaluronic acid, agarose, etc., and in one embodiment, one or more of these can be used in combination.

[0012] The colorimetric analysis material composed of a highly water-absorbent polymer can be in various forms such as powder, fine powder, pearl, bead, flake, block, etc. The material is preferably in the form of beads (small spheres). When in the form of beads, the particle size is not particularly limited, but from the viewpoints of the amount of sample solution used in the analysis, ease of observation when swollen, ease of handling, etc., the diameter of the beads in a non-swollen state is about 0.5 to 5 mm, preferably 0.8 mm to 3 mm, and more preferably 1 to 2 mm.

[0013] In the colorimetric material of the present invention, a hydrogen peroxide-generating oxidase, a peroxidase, a new Trinder's reagent, and a coupler compound are held in a highly water-absorbent polymer.

[0014] (Hydrogen peroxide generating oxidase) In the present invention, a hydrogen peroxide-generating oxidase is used. In the present invention, the hydrogen peroxide-generating oxidase refers to an oxidoreductase (denoted by EC number EC 1.XX), which is an enzyme that generates hydrogen peroxide by a reaction. Examples of preferred hydrogen peroxide-generating oxidases are any one selected from L-glutamate oxidase, L-lysine oxidase, glucose oxidase, lactate oxidase, and pyruvate oxidase. All of these enzymes are commercially available, and commercially available products can be used. For example, L-glutamate oxidase (manufactured by Yamasa Shoyu Co., Ltd.), L-lysine oxidase (manufactured by Yamasa Shoyu Co., Ltd.), glucose oxidase (manufactured by Toyobo Co., Ltd.), lactate oxidase (manufactured by Toyobo Co., Ltd.), and pyruvate oxidase (manufactured by Toyobo Co., Ltd.) are commercially available. Also usable are known oxidases that generate hydrogen peroxide, such as glycine oxidase, L-histidine oxidase, L-arginine oxidase, L-tryptophan oxidase, and L-phenylalanine oxidase, as well as enzymes classified as oxidoreductases and that act on other sugars or organic acids to generate hydrogen peroxide. For example, any one selected from hexose oxidase, cholesterol oxidase, galactose oxidase, pyranose oxidase, alcohol oxidase, nucleoside oxidase, D-mannitol oxidase, xylitol oxidase, oxalate oxidase, D-aspartate oxidase, L-amino acid oxidase, D-amino acid oxidase, amine oxidase, D-glutamate oxidase, putrescine oxidase, sarcosine oxidase, urate oxidase, glutathione oxidase, and xanthine oxidase may be used as the hydrogen peroxide generating oxidase in the present invention.

[0015] When L-glutamate oxidase is used, it is not particularly limited as long as it is an enzyme that can act on L-glutamic acid to generate hydrogen peroxide and catalyzes the oxidative deamination of L-glutamic acid to generate hydrogen peroxide, but an enzyme with high substrate specificity is preferred. Examples include L-glutamate oxidase derived from microorganisms such as Streptomyces sp. X-119-6, Streptomyces violascens, and Streptomyces endus. Commercially available L-glutamate oxidase derived from microorganisms may be used.

[0016] When lactate oxidase is used, it is preferable that the enzyme is capable of producing pyruvic acid and hydrogen peroxide by acting on lactic acid and has high substrate specificity. Examples of lactate oxidase include lactate oxidase derived from microorganisms such as Pediococcus sp. and Aerococcus viridans. Commercially available lactate oxidase derived from microorganisms may be used.

[0017] When glucose oxidase is used, it is preferable that the enzyme is capable of producing gluconolactone and hydrogen peroxide by acting on glucose and has high substrate specificity. Examples of glucose oxidase include glucose oxidase derived from microorganisms such as Aspergillus niger and Penicillium amagasakiense. Commercially available glucose oxidase derived from microorganisms may be used.

[0018] When pyruvate oxidase is used, it is preferable that the enzyme is capable of producing acetyl phosphate and hydrogen peroxide by acting on pyruvic acid and has high substrate specificity. Examples of pyruvate oxidase include pyruvate oxidase derived from microorganisms such as Aerococcus sp. and Lactobacillus plantarum. Commercially available pyruvate oxidase derived from microorganisms may be used.

[0019] (Peroxidase) In the present invention, the peroxidase used has hydrogen peroxide as a substrate. Examples of peroxidase include peroxidase derived from plants such as horseradish, and peroxidase derived from microorganisms such as bacteria and mold. Commercially available products such as peroxidase from horseradish (manufactured by Sigma-Aldrich), peroxidase derived from horseradish (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and peroxidase (POD (Horseradish roots)) (manufactured by Oriental Yeast Co., Ltd.) can be used.

[0020] (New Trinder Reagent) In the present invention, known new Trinder reagents can be used. Examples of the new Trinder reagents that can be used include N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt (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 ...-methoxyaniline (CEMO), 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-methoxyaniline (CEMO), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAOS), N-ethyl-N-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 sodium salt (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), or N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAOS) is preferably used, and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt (TOOS) is more preferably used.

[0021] (Coupler Compounds) In the present invention, known coupler compounds can be used. In the present invention, a coupler compound (sometimes called a color-forming reagent) that produces color in combination with the new Trinder reagent may be used, and examples thereof include 4-aminoantipyrine (4-AA), vanillin diamine sulfonic acid, methyl benzthiazolinone hydrazone (MBTH), sulfonated methyl benzthiazolinone 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).

[0022] (Other ingredients) In the colorimetric analytical material of the present invention, in addition to the above-mentioned enzymes, any one selected from a catalase deactivator, a preservative, a stabilizer, and a pH buffer may be further retained by the highly water-absorbent polymer.

[0023] The catalase deactivator is not particularly limited as long as it is an agent that can deactivate the catalytic action of catalase, an enzyme that catalyzes the reaction of converting hydrogen peroxide into oxygen and water, without interfering with the measurement of the target component when catalase is present in the sample. Examples include metal azides such as sodium azide, barium azide, and lithium azide, and 3-amino-1,2,4-triazole. One suitable example is sodium azide.

[0024] Known preservatives can be used, such as chloramphenicol, and Proclin (registered trademark) series such as Proclin 150, 200, 300, and 950 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), which contain isothiazolinones (5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one) as active ingredients.

[0025] The activity of the substrate-specific oxidase can be stably maintained during storage by the stabilizer. Examples of the stabilizer used in the present invention include disaccharides, specifically, trehalose, lactose, sucrose, lactulose, maltose, cellobiose, kojibiose, nigerose, isomaltose, isotrehalose, neotrehalose, sophorose, laminaribiose, gentiobiose, turanose, maltulose, palatinose, gentiobiose, mannobiose, melibiose, melibiose, neolactose, galactosucrose, scillabiose, rutinose, rutinulose, vicianose, xylobiose, and primeverose. Among the above disaccharides, those that do not affect the enzyme reaction and have excellent solubility at relatively low temperatures and at a pH at which the hydrogen peroxide generating oxidase is stable, such as trehalose, lactose, sucrose, maltose, cellobiose, and isomaltose, are preferred, with trehalose, lactose, and maltose being more preferred, and trehalose being even more preferred. A mixture of multiple disaccharides may also be used as the disaccharide.

[0026] Other examples of stabilizers that can be used in the present invention include cyclodextrin, dextran, sugar alcohols, surfactants such as Tween 20, polyglutamic acid, polylysine, and polyethylene glycol, which have excellent solubility at a pH level at which the hydrogen peroxide-generating oxidase is stable.

[0027] The pH buffer can maintain the pH of the system during the enzyme reaction at a level at which the enzyme can effectively act. Examples of pH buffers that can be used include acetate, phosphate, citrate, borate, tartrate, Tris (tris(hydroxymethyl)aminoethane), and Hepes ([2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid]).

[0028] In the colorimetric analysis material of the present invention, the superabsorbent polymer may further retain ascorbic acid oxidase. When ascorbic acid, which is known to inhibit color-developing reactions in food analysis or biological sample component analysis due to its strong reducing power, is present in a sample, ascorbic acid oxidase is used to eliminate the influence of the ascorbic acid without inhibiting the measurement of L-glutamic acid. Ascorbic acid oxidase may be derived from plants isolated from pumpkin, cucumber, or chayote (JP Patent Publication No. 56-88793), or may be derived from microorganisms such as Aspergillus or Penicillium (JP Patent Publication No. 58-51891). Examples of the ascorbic acid oxidase include enzymes that catalyze the oxidation reaction of ascorbic acid, and commercially available products may also be used.

[0029] (Application) Specifically, the colorimetric analysis material of the present invention is used by immersing it in a sample solution prepared by diluting a sample or the like, allowing it to absorb water and develop color.

[0030] The colorimetric analytical material of the present invention can be used to analyze a component that is a substrate for hydrogen peroxide generating oxidase in a sample that is expected to contain the component. Analysis includes detection of the component and quantification (measurement) of the component.

[0031] The sample is not particularly limited. Specific examples include alcoholic beverages such as sake and beer, beverages, food, agricultural products, food ingredients, processed foods, aqueous solutions prepared from these food samples, culture media for cells or microorganisms, culture fluid during or after culture, biological samples such as blood, serum, plasma, urine, sweat, saliva, amniotic fluid, tissues, cells, and organs, and aqueous solutions prepared from these biological samples.

[0032] (Other components of the kit) The colorimetric material of the present invention can be a component of a kit for analyzing a component that is a substrate for hydrogen peroxide generating oxidase. Such a kit may contain a color sample or a standard solution of the component in addition to the colorimetric material. For example, the color sample may be a color sample obtained by immersing the colorimetric material of the present invention in standard solutions of 1.25 mg / L, 2.5 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, 30 mg / L, and 40 mg / mL of the component that is a substrate for hydrogen peroxide generating oxidase, allowing it to absorb water, and printing a color equivalent to the color developed.

[0033] In addition, when the kit includes a standard solution, the concentration of the standard solution can be appropriately set, but from the viewpoint of serial dilution to prepare a calibration curve during measurement, the concentration is preferably around the upper measurement limit of the kit. Specifically, the concentration can be, for example, 20 mg / L or 40 mg / mL, or may be 30 mg / mL.

[0034] The kit may further include a dropper for collecting a sample solution, a transparent container for use in immersing the colorimetric material in the sample solution, etc. When diluting a sample to be used in the colorimetric material of the present invention, tap water can be used, and therefore a special liquid for diluting the sample does not need to be included in the kit.

[0035] The kit may also include a package insert, such as instructions on how to detect the component of interest, instructions for each component, such as an enzyme, contained in the colorimetric material, etc.

[0036] [Manufacturing method of colorimetric analysis material] The present invention also provides a method for producing the above-mentioned colorimetric analysis material. The colorimetric analysis material may include a step of impregnating a highly water-absorbent polymer with an enzyme solution containing a hydrogen peroxide generating oxidase, a peroxidase, a new Trinder's reagent, and a coupler compound, and drying the swollen highly water-absorbent polymer at a temperature at which the enzyme is not inactivated, for example, at 25°C to 55°C. The drying time may be appropriately set. For example, it may be 12 hours to several days, or may be 1 to 5 days.

[0037] The concentrations of each component in the enzyme solution for producing the colorimetric analysis material can be, for example, 0.02 to 300 U / mL of hydrogen peroxide generating oxidase, 0.5 to 50 U / mL of peroxidase, 0.05 to 4 mM of new Trinder's reagent, and 0.05 to 4 mM of coupler compound.

[0038] More specifically, the content of each oxidase is preferably in the range of 0.1 to 2 U / mL for L-glutamate oxidase, 10 to 300 U / mL for glucose oxidase, 1 to 10 U / mL for lactate oxidase, 0.02 to 1 U / mL for L-lysine oxidase, and 0.1 to 3 U / mL for pyruvate oxidase. The contents of other enzymes and reagents are preferably in the range of 5 to 30 U / mL for peroxidase, 2 to 20 U / mL for ascorbic acid oxidase, 0.1 to 2 μmol / mL for new Trinder's reagent, and 0.1 to 2 μmol / mL for coupler compound.

[0039] Various buffer solutions can be used to dissolve the enzymes and reagents, including, for example, acetic acid, phosphoric acid, citric acid, boric acid, trisaminomethane, HEPES, MES, Bis-tris, ADA, ACES, PIPES, MOPSO, MOPS, BES, TES, DIPSO, TAPSO, TAPS, CHES, CAPSO, CAPS, and salts thereof.

[0040] When producing a colorimetric analytical material in which, in addition to the above-mentioned enzymes, any one of a catalase deactivator, a preservative, and a stabilizer is further retained in the superabsorbent polymer, the enzyme solution to be impregnated into the superabsorbent polymer may contain the necessary member selected from the catalase deactivator, the preservative, and the stabilizer.

[0041] When the enzyme solution contains a catalase deactivator, its concentration can be 0.01% to 3.0%, and preferably 0.02% to 0.1%.

[0042] When a preservative is contained in the enzyme solution, its concentration can be 0.01% to 0.1%, and preferably 0.02% to 0.05%.

[0043] When a stabilizer such as trehalose is contained in the enzyme solution, the concentration thereof can be 0.01% to 5% (0.1 to 50 mg / mL), preferably 0.02% to 3% (0.2 to 30 mg / mL), and more preferably 0.04% to 2% (0.4 to 20 mg / mL).

[0044] When the enzyme solution contains ascorbic acid oxidase, the concentration thereof can be 1 U / mL to 100 U / mL, and preferably 2 U / mL to 20 U / mL.

[0045] The colorimetric analysis material obtained by impregnating it with an enzyme solution, swelling it, and drying it may be promptly sealed together with silica gel in an aluminum-evaporated bag, or otherwise treated for long-term storage.

[0046] [Analysis method] The present invention also provides a method for analyzing a component contained in a sample, which is a substrate for hydrogen peroxide generating oxidase, using a colorimetric agent, comprising the steps of: A colorimetric material is immersed in a sample solution, causing it to swell and develop color (sample material). Inside the swollen colorimetric material, hydrogen peroxide-generating oxidase, peroxidase, coupler compounds, and the new Trinder's reagent act on the components to form pigments; and the sample material is compared with a color sample or a material (standard material) that has been immersed in a standard solution, allowed to absorb water, and developed color to determine the concentration of the components in the sample solution.

[0047] When carrying out a method for detecting and / or quantifying a component contained in a sample that is a substrate for hydrogen peroxide generating oxidase, a colorimetric analysis material is first immersed in a standard solution of a known concentration of the component that is a substrate for hydrogen peroxide generating oxidase to cause color development, and a color sample is prepared in which the degree of color development corresponds to the concentration. The concentration of the component in the sample solution can then be determined by visually comparing the sample material with the color sample.

[0048] The detection limit concentration of a component that is a substrate for the hydrogen peroxide generating oxidase in the present invention can be 1.25 mg / L as the component concentration in the sample to be subjected to the colorimetric analysis material of the present invention. If the component concentration in the test sample is determined to be higher than the highest concentration set in the above color sample, i.e., 40 mg / L, a test can be performed using a solution further diluted with tap water or deionized water, and the concentration determined by the color sample can be multiplied by the dilution factor to determine the concentration of the component in the actual test sample.

[0049] The colorimetric and calculation step may include photographing the sample material and the color sample or standard material together with an image capturing means, and calculating the concentration of the component of the sample solution from the obtained image data with a calculation means. The image capturing means may be a digital camera, but may also be a camera mounted on a terminal such as a smartphone, tablet terminal, or personal computer that is located at the user's hand.

[0050] The calculation means may store data obtained by previously analyzing the correlation between the degree of color development of the colorimetric analysis material using a standard solution and the concentration of the component as a programmed application, and may calculate the concentration of the component based on that program.

[0051] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples. EXAMPLES

[0052] [Example 1: Production of beads for colorimetric measurement of L-glutamic acid using highly water-absorbent sodium polyacrylate beads] Approximately 80 highly water-absorbent sodium polyacrylate beads with a diameter of approximately 1.5 mm (trade name "Tenshi no Beads", Life Taste Co., Ltd., Japan. In the following examples, the same beads were used unless otherwise specified) were placed in a plastic tray with a shallow bottom, and 20 mL of an enzyme solution for L-glutamic acid measurement with the composition shown in the table below was added, and the beads were impregnated with the enzyme solution in a refrigerator (4°C to 8°C) until the enzyme solution was almost completely used up. The beads that were sufficiently swollen were transferred to another container and dried in a 40°C air-blowing incubator for 24 hours. These dried beads (diameter approximately 2 mm) were used as highly water-absorbent polymer beads for colorimetric measurement of L-glutamic acid (hereinafter referred to as "GUCB") (Figure 1).

[0053] [Table 1]

[0054] [Example 2: Production of beads for measuring lactate colorimetrically using highly water-absorbent sodium polyacrylate beads] Highly water-absorbent sodium polyacrylate beads (approximately 80 beads) with a diameter of approximately 1.5 mm were placed in a shallow plastic tray, and 20 mL of the enzyme solution for lactic acid measurement with the composition shown in the table below was added. The beads were impregnated with the enzyme solution in a refrigerator (4°C to 8°C) until almost all of the enzyme solution was gone. The beads that had fully swollen were transferred to another container and dried in a 40°C air-blowing incubator for 24 hours. These dried beads (approximately 2 mm in diameter) were used as highly water-absorbent polymer beads for lactic acid colorimetric measurement (hereinafter referred to as "LACB") (Figure 1).

[0055] [Table 2]

[0056] [Example 3: Production of beads for glucose colorimetric measurement using highly water-absorbent sodium polyacrylate beads] Highly water-absorbent sodium polyacrylate beads (approximately 80 beads) with a diameter of approximately 1.5 mm were placed in a shallow plastic tray, and 20 mL of the enzyme solution for glucose measurement with the composition shown in the table below was added. The beads were impregnated with the enzyme solution in a refrigerator (4°C to 8°C) until almost all of the enzyme solution was gone. The beads that had fully swollen were transferred to another container and placed in a 40°C air-blowing incubator to dry for 72 hours. These dried beads (approximately 2 mm in diameter) were used as highly water-absorbent polymer beads for glucose colorimetric measurement (hereinafter referred to as "GCCB") (Figure 1).

[0057] [Table 3]

[0058] [Example 4: Production of beads for colorimetric measurement of pyruvic acid using highly water-absorbent sodium polyacrylate beads] Highly water-absorbent sodium polyacrylate beads (approximately 80 beads) with a diameter of approximately 1.5 mm were placed in a shallow plastic tray, and 20 mL of enzyme solution for pyruvic acid measurement with the composition shown in the table below was added. The beads were impregnated with the enzyme solution in a refrigerator (4°C to 8°C) until almost all of the enzyme solution was gone. The beads that had fully swollen were transferred to another container and placed in a 40°C air-blowing incubator to dry for 24 hours. These dried beads (approximately 2 mm in diameter) were used as highly water-absorbent polymer beads for pyruvic acid colorimetric measurement (hereinafter referred to as "PVCB") (Figure 1).

[0059] [Table 4]

[0060] [Example 5: Relationship between color development of beads for measuring L-glutamic acid and L-glutamic acid concentration (substitute calibration curve)] (1) The highly water-absorbent polymer beads (GUCB) for colorimetric measurement of L-glutamic acid produced in Example 1 were placed one by one into a small transparent plastic bottle (diameter 1.2 cm, height 4.5 cm). (2) Approximately 2.5 mL of water or L-glutamic acid standard solution (0 mg / L to 20 mg / L) was added to each small bottle containing one GUCB, and the bottle was left at room temperature to initiate the color reaction. Coloration began within a few minutes, and a proportional relationship between the color development and the L-glutamic acid concentration was observed within about 10 minutes. (3) After 60 minutes had passed since the L-glutamic acid standard solution (0 mg / L to 20 mg / L) was added and color development began, the GUCB that had turned reddish purple and swelled to a diameter of about 7 mm was removed, drained, and placed in a small test tube, to which 1 mL of water was added and left overnight. Due to leakage of the dye, the color of the inside of the GUCB became the same as the color of the external solution (water). (4) By measuring the absorbance of the external solution at 555 nm, a calibration curve (substitute calibration curve) was created from the relationship between the absorbance of the external solution, which reflects the color intensity inside the GUCB, and the concentration of the L-glutamic acid standard solution (Figure 2). The surrogate calibration curve in this case reflects the correlation between the amount of pigment trapped inside the beads and the concentration of the standard solution, but is not used in the process of determining the concentration of the components in the sample solution by visually comparing the sample material with a color sample, so it is called a surrogate calibration curve.

[0061] [Example 6: Relationship between color development and lactic acid concentration of highly absorbent polymer beads for colorimetric measurement of lactic acid (substitute calibration curve)] (1) The highly absorbent polymer beads (LACB) for colorimetric measurement of lactic acid produced in Example 2 were placed one by one into a small transparent plastic bottle (diameter 1.2 cm, height 4.5 cm). (2) Approximately 2.5 mL of water or lactic acid standard solution (0 mg / L to 20 mg / L) was added to a small bottle containing one bead to initiate the color reaction. Coloring began within a few minutes, and a proportional relationship between the degree of color development and the lactic acid concentration was observed between 10 and 60 minutes. (3) After 60 minutes had passed since the lactic acid standard solution (0 mg / L - 20 mg / L) was added and color development began, the LACB that had turned blue-green and swelled to a diameter of about 7 mm was removed and placed in a small test tube, 1 mL of water was added, and the tube was left overnight. Due to leakage of the dye, the color of the inside of the LACB became the same as the color of the external solution (water). (4) By measuring the absorbance of the external solution at 630 nm, a surrogate calibration curve was created from the relationship between the absorbance of the external solution, which reflects the color intensity inside the LACB, and the concentration of the lactic acid standard solution (Figure 3).

[0062] [Example 7: Relationship between color development and glucose concentration of highly absorbent polymer beads for glucose colorimetric measurement (substitute calibration curve)] (1) One GCCB produced in Example 3 was placed in each small transparent plastic bottle (diameter 1.2 cm, height 4.5 cm). (2) After 60 minutes had passed since the glucose standard solution (0 mg / L - 20 mg / L) was added and color development began, the GCCB that had turned reddish purple and swelled to a diameter of about 7 mm was removed and placed in a small test tube, 1 mL of water was added, and the tube was left overnight. Due to leakage of the dye, the color of the inside of the GCCB became the same as the color of the external solution (water). (3) By measuring the absorbance of the external solution at 555 nm, a surrogate calibration curve was created from the relationship between the absorbance of the external solution, which reflects the color intensity inside the GCCB, and the concentration of the glucose standard solution (Figure 4).

[0063] [Example 8: Relationship between color development and pyruvic acid concentration of highly absorbent polymer beads for colorimetric measurement of pyruvic acid (substitute calibration curve)] (1) One piece of the PVCB produced in Example 4 was placed in each small transparent plastic bottle (diameter 1.2 cm, height 4.5 cm). (2) After 60 minutes had passed since the color development began after adding pyruvic acid standard solution (0 mg / L - 20 mg / L), the PVCB that had developed a reddish purple color and swelled to a diameter of about 7 mm was removed and placed in a small test tube, 1 mL of water was added, and the tube was left overnight. Due to leakage of the dye, the color of the inside of the PVCB became the same as the color of the external solution (water). (3) By measuring the absorbance of the external solution at 555 nm, a surrogate calibration curve was created from the relationship between the absorbance of the external solution, which reflects the color intensity inside the PVCB, and the concentration of the pyruvic acid standard solution (Figure 5). [Example 9: Relationship between the degree of color development inside the beads and the degree of color development in the external liquid (test sample liquid) during color development of highly water-absorbent polymer beads for colorimetric measurement of L-glutamic acid] (1) The L-glutamic acid colorimetric measurement beads (GUCB) produced in Example 1 were placed one by one into a small transparent plastic bottle (diameter 1.2 cm, height 4.5 cm). (2) Approximately 2.5 mL of L-glutamic acid standard solution (30 mg / L) was added to each small vial containing one GUCB, and the color reaction was initiated. (3) The time when the L-glutamic acid standard solution (30 mg / L) was added was set as zero time, and the swollen beads were taken out at 10, 20, 30, and 40 minutes, and the absorbance (555 nm) of the external solution was measured. Next, the swollen beads were weighed, and the diameter of the beads was measured to calculate the volume of the beads, and then the beads were placed in a small test tube. 2 mL of water was added to this test tube and the tube was left overnight. Due to the leakage of the dye from inside the beads, the color of the beads in the small test tube became the same as the color of the external solution (water). (4) The absorbance of the external solution at 555 nm was measured. The absorbance value reflecting the color intensity inside the beads at each time point was calculated by dividing the absorbance of the external solution at 555 nm by the volume of the beads (Figure 6). It was confirmed by visual inspection as well as by the measured absorbance values ​​that only the inside of the beads initially developed color, and that the external solution (containing the target compound) did not develop color within a short period of time (approximately 1 hour).

[0064] [Example 10: Simple visual measurement of glutamic acid concentration in commercially available sake using highly absorbent polymer beads for colorimetric measurement of L-glutamic acid] For 12 types of commercially available sake, the color development of GUCB (produced in Example 1) soaked in a 10-fold water dilution of sake for 30 minutes was visually compared with a color sample created based on GUCB colored with an L-glutamic acid standard solution to perform a simplified numerical measurement of L-glutamic acid concentration (n=5). Separately, the L-glutamic acid concentrations of these 12 types of sake were measured using an L-glutamic acid measurement kit manufactured by Enzyme Sensor Co., Ltd. The correlation coefficient of the L-glutamic acid concentration measurements using both methods was 0.936 (Figure 7).

[0065] [Example 11: Simple visual measurement of lactic acid concentration in commercially available sake using highly absorbent polymer beads for lactic acid colorimetric measurement (hereinafter referred to as "LACB")] For 12 types of commercially available sake, the color development of LACB after soaking in a 10-fold diluted solution of sake for 30 minutes was visually compared with a color sample created based on LACB colored with a lactic acid standard solution to perform a simplified numerical measurement of the lactic acid concentration (n=5). Separately, the lactic acid concentration of these 12 types of sake was measured using a lactic acid measurement kit manufactured by Enzyme Sensor Co., Ltd. The correlation coefficient of the lactic acid concentration measured by both methods was 0.920 (Figure 8).

[0066] [Example 12: Simple visual measurement of glucose concentration in commercially available sake using highly absorbent polymer beads for glucose colorimetric measurement] For 12 types of commercially available sake, the color development of GCCB (produced in Example 3) soaked in a 1000-fold diluted solution of sake for 30 minutes was visually compared with a color sample created based on GCCB colored with a glucose standard solution to perform a simplified numerical measurement of glucose concentration (n=5). Separately, the glucose concentrations of these 12 types of sake were measured using a glucose measurement kit manufactured by Enzyme Sensor Co., Ltd. The correlation coefficient between the glucose concentration measurements obtained by both methods was 0.981 (Figure 9).

[0067] [Example 13: Simple visual measurement of pyruvic acid concentration in commercially available sake using highly absorbent polymer beads for colorimetric pyruvic acid measurement] For 12 types of commercially available sake, the color development of PVCB (produced in Example 4) soaked in a 10-fold water dilution of sake for 30 minutes was visually compared with a color sample created based on PVCB colored with a pyruvic acid standard solution to perform a simplified numerical measurement of pyruvic acid concentration (n=5). Separately, the pyruvic acid concentration of these 12 types of sake was measured using a pyruvic acid measurement kit manufactured by Enzyme Sensor Co., Ltd. The correlation coefficient of pyruvic acid concentration measured by both methods was 0.987 (Figure 10).

Claims

1. A colorimetric analysis material, which comprises a hydrogen peroxide generating oxidase, a peroxidase, a new Trinder reagent, and a coupler compound supported on a highly water-absorbent polymer, is used by immersing it in a sample solution, allowing it to absorb water and develop color.

2. The material according to claim 1, wherein the hydrogen peroxide generating oxidase is any one selected from the group consisting of L-glutamate oxidase, L-lysine oxidase, L-arginine oxidase, L-histidine oxidase, glycine oxidase, L-tryptophan oxidase, L-phenylalanine oxidase, L-aspartate oxidase, glucose oxidase, lactate oxidase, pyruvate oxidase, hexose oxidase, cholesterol oxidase, galactose oxidase, pyranose oxidase, alcohol oxidase, nucleoside oxidase, D-mannitol oxidase, xylitol oxidase, oxalate oxidase, D-aspartate oxidase, L-amino acid oxidase, D-amino acid oxidase, amine oxidase, D-glutamate oxidase, putrescine oxidase, sarcosine oxidase, uric acid oxidase, glutathione oxidase, and xanthine oxidase.

3. The material according to claim 1, further comprising any one selected from the group consisting of a catalase inactivator, a preservative, a stabilizer, and a pH buffering agent retained in the highly water-absorbent polymer.

4. 2. The material of claim 1, wherein the superabsorbent polymer is a polyacrylate-based polymer.

5. 10. The material of claim 1, wherein the superabsorbent polymer is in the form of beads.

6. A material according to any one of claims 1 to 5 for quantifying ingredients in food products.

7. A kit for the analysis of a component which is a substrate for a substrate-specific oxidase, comprising a material according to any one of claims 1 to 5.

8. 8. The kit of claim 7, further comprising a color sample or a standard solution of the component.

9. 2. A method for producing the material according to claim 1, comprising the step of drying beads of a highly absorbent polymer impregnated with an enzyme solution containing a hydrogen peroxide generating oxidase, a peroxidase, a new Trinder reagent, and a coupler compound at 25°C to 55°C.

10. 10. The method according to claim 9, wherein the enzyme solution is a solution of 0.02 to 300 U / mL of hydrogen peroxide generating oxidase, 0.5 to 50 U / mL of peroxidase, 0.05 to 4 mM of new Trinder's reagent, 0.05 to 4 mM of a coupler compound, and a pH buffer.

11. A method for analyzing a component contained in a sample and which is a substrate for hydrogen peroxide generating oxidase, comprising the steps of: A process for obtaining a colored material (sample material) by immersing the material according to any one of claims 1 to 5 in a sample solution, causing it to swell, and forming a dye inside the swollen material, in which hydrogen peroxide-generating oxidase, peroxidase, coupler compound, and new Trinder's reagent act on the components inside the swollen material; and A process in which the concentration of the components in the sample solution is calculated by comparing the color of the sample material with a color sample or a material (standard material) that has been immersed in a standard solution, allowed to absorb water, and allowed to develop color.

12. The method according to claim 11, wherein the step of comparing colors and calculating includes photographing the sample material with an imaging means, and calculating the concentration of the component of the sample solution using the obtained image data and calibration curve data prepared in advance.

13. The method according to claim 12, wherein the photographing means is a camera mounted on a smartphone.

14. 12. The method of claim 11, wherein the sample solution is diluted with tap water.

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