Method for controlling quality of alcoholic beverage

Abstract

Provided is a method for controlling the quality of an alcoholic beverage, the method comprising: (1) a step for preparing a plurality of probe solutions having different probes, ion intensities, or pH, the probes containing (a) a cationic polymer and (b) an environment-responsive fluorophore, the fluorophore being covalently bonded to a part of the primary amino group in the cationic polymer; (2) a step for mixing the plurality of probe solutions and an analysis sample that contains an alcoholic beverage; (3) a step for measuring the fluorescence intensity of the mixed liquid prepared in step (2); and (4) a step for comparing the fluorescence intensity pattern obtained in step (3) with a fluorescence intensity pattern obtained from a reference sample.

Description

Alcoholic beverage quality control methods

[0001] The present invention relates to a method for controlling the quality of alcoholic beverages, and in particular to a method for evaluating differences in quality due to origin, manufacturer, production method or storage condition of alcoholic beverages.

[0002] Alcoholic beverages are a general term for beverages containing ethanol, and most are produced through a fermentation process involving microorganisms such as yeast and Aspergillus oryzae. The fermentation process is influenced by a complex array of biological and chemical factors, including the type and activity of microorganisms, the type and characteristics of raw materials, and environmental factors such as pH, temperature, and oxygen concentration. Therefore, supplying alcoholic beverages of consistent quality and developing alcoholic beverages with new flavors is extremely challenging. Traditional brewing methods are particularly challenging, as they rely on the skill, experience, and intuition of artisans. These challenges are particularly pronounced in the production of sake. Sake is brewed using a uniquely Japanese method, and the production process is extremely complex.

[0003] Sake contains a wide variety of components, making it difficult to assess its quality. To standardize the quality of alcoholic beverages, the National Tax Agency's analytical methods specify the analysis items of alcohol content, amino acid content, acidity, and sake meter value, and specify the methods for measuring these. However, sake's taste can vary significantly depending on factors other than these, and the evaluation indices based on these measurements (sweet / dry, rich / light) do not necessarily coincide with sensory evaluation. Furthermore, sake quality can vary significantly depending on storage conditions. However, due to the complexity of its composition, it is largely unknown which components in sake affect its taste and how.

[0004] Attempts have been made to develop analytical methods for objectively evaluating sake quality. For example, models for predicting sake sensory evaluation based on metabolomics analysis and GC-MS analysis have been reported (Non-Patent Documents 1 and 2). Furthermore, a machine learning-based sake quality evaluation model has been reported that combines expert sensory evaluation with analysis of physicochemical properties (Non-Patent Document 3). However, building these evaluation models requires a huge amount of analytical data, and the analysis is technically difficult, requiring a great deal of time and effort.

[0005] On the other hand, an optical sensor array using a conjugated polymer has been reported as a simple method for analyzing sake (Non-Patent Document 4). However, this method can only quantify glucose and pyruvate, and does not provide sufficient information for evaluating the quality of sake.

[0006] J. Agric. Food Chem., 2010; 58:374-383J. Biosci. Bioeng., 2014; 118:406-414J. Am. Soc. Brew Chem., 2022; 80:146-154 Polym. J., 2021; 53:1287-1291

[0007] The present invention has been made with the objective of solving the problems of the prior art and providing an easy, rapid and consistent quality control technology for alcoholic beverages that does not rely on sensory evaluation.

[0008] The present inventors have successfully used a cross-reactive sensing method to distinguish samples containing a wide variety of proteins and samples containing microflora (WO 2018 / 088510, WO 2020 / 262413). Based on the above method, the present inventors have established a method for determining the quality of alcoholic beverages with high accuracy.

[0009] That is, according to one embodiment, the present invention provides a method for controlling the quality of alcoholic beverages, comprising: (1) preparing a plurality of probe solutions having different probes, ionic strengths, or pHs, wherein the probes comprise: (a) a cationic polymer having at least five primary amino groups per molecule and a weight-average molecular weight of 1,000 to 500,000; and (b) an environmentally responsive fluorophore, the fluorophore being covalently bonded to some of the primary amino groups in the cationic polymer; (2) mixing the plurality of probe solutions with an analytical sample containing alcoholic beverages; (3) measuring the fluorescence intensity of the mixture prepared in step (2); and (4) comparing the fluorescence intensity pattern obtained in step (3) with the fluorescence intensity pattern obtained for a reference sample.

[0010] The environmentally responsive fluorophore is preferably selected from the group consisting of a fluorophore having a naphthalenesulfonic acid skeleton, a fluorophore having a benzofurazan skeleton, a fluorophore having a xanthene skeleton, a fluorophore having a pyrene skeleton, and an aggregation-induced emission fluorophore.

[0011] The cationic polymer is preferably a linear or branched polyamino acid, polyallylamine, polyamidoamine or polyalkyleneimine.

[0012] It is preferable that the environmentally responsive fluorescent group be covalently bonded to 1 to 50% of the primary amino groups in the cationic polymer.

[0013] It is preferable that at least a portion of the primary amino groups of the cationic polymer to which the environmentally responsive fluorophore is not covalently bonded be modified with an acyl group or an amino acid.

[0014] In the step (2), an analysis sample containing an alcoholic beverage may be added to each of the plurality of probe solutions.

[0015] Alternatively, in step (2), the plurality of probe solutions may be added simultaneously to the analysis sample containing alcoholic beverages.

[0016] The measurement of the fluorescence intensity in (3) above is preferably carried out at a plurality of excitation wavelengths and fluorescence wavelengths.

[0017] Step (4) allows for evaluation of differences in quality due to the origin, manufacturer, production method, or storage conditions of the alcoholic beverage.

[0018] According to the method of the present invention, the quality of alcoholic beverages can be determined easily, accurately, and consistently by using only one or a few probes and obtaining a fluorescence intensity pattern that reflects the sum of nonspecific interactions between all components and the probes, without requiring a comprehensive analysis of the components in alcoholic beverages or sensory evaluation by an expert. Therefore, according to the method of the present invention, it is possible to perform quality control of alcoholic beverages, for example, by using alcoholic beverages of guaranteed quality as a reference sample and simply comparing the fluorescence intensity pattern.

[0019] FIG. 1 shows the structural formulas of the probes (probes 1 to 24) used in Example A. The values ​​in parentheses indicate the ClogP values ​​of the sites where functional groups were introduced. FIG. 2 shows the change in fluorescence intensity when various concentrations of sake (S3 or S11) were added to 750 nM of probe 1 (TPE-None) and probe 8 (TPE-Phe) / purified water. FIG. 3 shows the change in fluorescence intensity (I-I) obtained by measuring 12 sakes (12 brands) x 1 solvent condition (purified water (no buffer)) x 12 probes (probes 1 to 12) x 2 wavelength sets x 10 times. ０ 4 shows the change in fluorescence intensity (I-I) obtained from 12 sakes (12 brands) × 2 solvent conditions (acetic acid (pH 5.0) or EPPS (pH 8.0)) × 6 probes (probes 1, 3, 6, 7, 8, and 12) × 2 wavelength sets × 10 measurements. ０ ) is a heat map. Figure 5 is a diagram plotting the second discriminant scores obtained by analyzing the data for six probes (probes 1, 3, 6, 7, 8, and 12) from the data in Figure 3 using supervised linear discriminant analysis. Figure 6 is a diagram plotting the second discriminant scores obtained by analyzing the data for one solvent condition (acetic acid (pH 5.0)) from the data in Figure 4 using supervised linear discriminant analysis. Figure 7 is a diagram plotting the second discriminant scores obtained by analyzing the data for one solvent condition (EPPS (pH 8.0)) from the data in Figure 4 using supervised linear discriminant analysis. Figure 8 is a graph showing the results of quantifying peptide components contained in 12 brands of sake using the Bradford method. Figure 9 is a diagram plotting the first principal component (top row) and the second principal component (bottom row) obtained by analyzing the data for six probes (probes 1, 3, 6, 7, 8, and 12) selected from Figure 3 and the data in Figure 4 using unsupervised principal component analysis against the concentration of the peptide components. FIG. 10 shows the change in fluorescence intensity (I-I) obtained by measuring sake (4 specific name classifications) × 1 solvent condition (purified water) × 6 probes (probes 1, 3, 6, 7, 8, and 12) × 2 wavelength sets × 16 times. ０) is a heat map. Figure 11 is a diagram plotting the first and second discriminant scores obtained by analyzing the data in Figure 10 by supervised linear discriminant analysis. Figure 12 is a graph plotting the change in fluorescence intensity (I-I) obtained by measuring 12 sets of sake (2 brands x 4 storage periods) x 1 solvent condition (purified water) x 6 probes (probes 1, 3, 6, 7, 8, and 12) x 2 wavelength sets. ０ ) is a heat map. Figure 13 is a diagram plotting the first and second discriminant scores obtained by analyzing the data for two storage periods (1 day and 13 days) of the data in Figure 12 using supervised linear discriminant analysis. Figure 14 is a diagram plotting the first and second principal components obtained by analyzing the data in Figure 12 using unsupervised principal component analysis. Figure 15 is a diagram plotting the first and second principal components (PC1) and (PC2) obtained by analyzing the data in Figure 12 using unsupervised principal component analysis against storage period. Figure 16 is a diagram showing changes in fluorescence intensity when various concentrations of liqueur (F2 or F4) were added to 750 nM probe 1 (TPE-None) and probe 10 (TPE-Suc) / 20 mM MOPS (pH 7.0). Figure 17 is a diagram showing changes in fluorescence intensity (I-I) obtained by 6 liqueurs x 2 solvent conditions x 6 probes (probes 1, 3, 6, 7, 8, and 12) x 2 wavelength sets x 6 measurements. ０ ) is a heat map. Figure 18 is a diagram plotting the second discriminant scores obtained by analyzing the data for five probes (probes 1, 3, 6, 8, and 12) from the data in Figure 17 using supervised linear discriminant analysis. Figure 19 is a diagram showing the change in fluorescence intensity when various concentrations of awamori (A1 or A2) are added to 750 nM probe 1 (TPE-None) and probe 10 (TPE-Suc) / purified water. Figure 20 shows the change in fluorescence intensity (I-I) obtained from 6 awamori samples x 2 solvent conditions x 6 probes (1, 6, 8, and 10-12) x 2 wavelength sets x 6 measurements. ０) is a heat map. Figure 21 is a diagram plotting the results up to the second discriminant score obtained by analyzing the data of Figure 20 by supervised linear discriminant analysis. Figure 22 is a diagram plotting the results up to the second discriminant score obtained by analyzing a portion of the data of Figure 20 ([6 probes x 1 solvent condition (purified water) x 2 wavelength sets] + [2 probes (TPE-Nle and TPE-Phe) x 1 solvent condition (20 mM EPPS (pH 8.0)) x 2 wavelength sets]) by supervised linear discriminant analysis. Figure 23 is a diagram showing changes in fluorescence intensity when various concentrations of red wine (W1) are added to 300 nM probe 13 (FITC-None) and probe 22 (FITC-Suc) / purified water. Figure 24 is a diagram showing the change in fluorescence intensity (I-I) obtained by 6 red wines x 2 solvent conditions x 6 probes (probes 13, 18, 20, and 22-24) x 1 wavelength set x 6 measurements. ０ ) is a heat map. Figure 25 is a diagram plotting the first and second discriminant scores obtained by analyzing the data for one solvent condition (purified water) from the data in Figure 24 by supervised linear discriminant analysis. Figure 26 is a diagram plotting the change in fluorescence intensity (I-I) obtained by red wine (1 brand x 7 storage periods) x 2 solvent conditions x 6 probes (probes 13, 18, 20, and 22 to 24) x 1 wavelength set x 6 measurements. ０ ) is a heat map of the data. Figure 27 is a diagram plotting the first and second discriminant scores (left) and the first and third discriminant scores (right) obtained by analyzing a portion of the data in Figure 26 ([6 probes × 1 solvent condition (purified water) × 1 wavelength set] + [2 probes (FITC-None and FITC-Phe) × 1 solvent condition (20 mM MOPS (pH 7.0)) × 1 wavelength set]) by supervised linear discriminant analysis. Figure 28 is a diagram showing the structural formulas of the probes (probes 8, 25, and 26) used in Example B. Figure 29 is a diagram showing the change in fluorescence intensity when mixtures of probe 8 (TPE-Phe) / purified water, Dnc-PAMAM (probe 25) / purified water, and DBD-PLL (probe 26) / purified water were added to sake (S3 or S11) at various concentrations. FIG. 30 shows the change in fluorescence intensity (I-I) obtained by measuring 10 sakes (10 brands) x 3 types of probe mixtures x 6 wavelength sets x 6 times.０ 31 is a diagram plotting the first and second discriminant scores (left) and the first and third discriminant scores (right) obtained by analyzing the data in FIG. 30 by supervised linear discriminant analysis.

[0020] The present invention will be described in detail below, but the present invention is not limited to the embodiments described in this specification.

[0021] According to a first embodiment, the present invention provides a method for controlling the quality of alcoholic beverages, comprising: (1) preparing a plurality of probe solutions having different probes, ionic strengths, or pHs, wherein the probes comprise: (a) a cationic polymer having at least five primary amino groups per molecule and a weight-average molecular weight of 1,000 to 500,000; and (b) an environmentally responsive fluorophore, the fluorophore being covalently bonded to some of the primary amino groups in the cationic polymer; (2) mixing the plurality of probe solutions with an analytical sample containing alcoholic beverages; (3) measuring the fluorescence intensity of the mixture prepared in step (2); and (4) comparing the fluorescence intensity pattern obtained in step (3) with the fluorescence intensity pattern obtained for a reference sample.

[0022] First, the probe used in the method of this embodiment will be described. The probe used in the method of this embodiment contains (a) a cationic polymer having at least five primary amino groups per molecule and a weight-average molecular weight of 1,000 to 500,000, and (b) an environmentally responsive fluorophore, with the fluorophore covalently bonded to some of the primary amino groups in the cationic polymer.

[0023] The cationic polymer (a) of the probe in this embodiment may be any polymer as long as it has a weight-average molecular weight of 1,000 to 500,000 and at least five primary amino groups per polymer molecule. Here, "polymer" refers to a compound formed by polymerizing two or more monomers, which may be the same or different, and therefore may be a homopolymer or a copolymer. The degree of polymerization of the polymer is not particularly limited, and therefore "polymer" also includes oligomers formed by polymerizing several (e.g., 3 to 20) monomers.

[0024] The weight average molecular weight of the cationic polymer of the probe in this embodiment is 1,000 to 500,000, preferably 1,500 to 200,000, and particularly preferably 2,000 to 100,000.

[0025] The cationic polymer of the probe in this embodiment has at least 5, preferably 7 or more, and particularly preferably 10 or more primary amino groups in one molecule of the polymer.

[0026] In this embodiment, the cationic polymer that can be used for the probe is preferably a linear or branched polyamino acid, polyallylamine, polyamidoamine, or polyalkyleneimine. Furthermore, these cationic polymers may be copolymerized with polyethylene glycol.

[0027] In this embodiment, the polyamino acid that can be used for the probe may be a polymer of the same type of amino acid residues, or may be a polymer of different types of amino acid residues. Furthermore, the amino acid residues that make up the polyamino acid may be either L- or D-isomers. Examples of polyamino acids include polylysine, polyornithine, random copolymers of lysine and phenylalanine, and random copolymers of lysine and tyrosine. A preferred polyamino acid in this embodiment is polylysine or polyornithine.

[0028] In this embodiment, examples of polyalkyleneimine that can be used for the probe include polyethyleneimine, polypropyleneimine, polybutyleneimine, etc. A preferred polyalkyleneimine in this embodiment is polyethyleneimine.

[0029] The environmentally responsive fluorophore (b) of the probe in this embodiment may be any fluorophore whose fluorescence properties change depending on the environment around the fluorescent molecule. Examples of such fluorophores include, but are not limited to, fluorophores whose fluorescence properties change depending on the polarity around the fluorescent molecule, fluorophores whose fluorescence properties change depending on the pH around the fluorescent molecule, and fluorophores whose fluorescence properties change depending on the degree of crowding around the fluorescent molecule.

[0030] Examples of fluorophores whose fluorescent properties change depending on the polarity around the fluorescent molecule include fluorophores having a naphthalenesulfonic acid skeleton, such as 5-dimethylaminonaphthalene-1-sulfonyl (dansyl), 1-anilinonaphthalene-8-sulfonic acid (ANS), N-methyl-2-anilinonaphthalene-6-sulfonic acid (MANS), and 2-p-toluidinylnaphthalene-6-sulfonic acid (TNS); fluorophores having a benzofurazan skeleton, such as 4-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole (DBD), 7-nitro-2,1,3-benzoxadiazole (NBD), 4-(aminosulfonyl)-2,1,3-benzoxadiazole (ABD), and ammonium 2,1,3-benzoxadiazole-4-sulfonate (SBD); and fluorescent derivatives thereof.

[0031] Examples of fluorophores whose fluorescence properties change depending on the pH around the fluorescent molecule include fluorophores having a xanthene skeleton, such as fluorescein, fluorescein isothiocyanate (FITC), 5(6)-carboxyfluorescein (5(6)-FAM), 2'-7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), and seminaphthalodafluorescein (SNARF); fluorophores having a pyrene skeleton, such as 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HTPS); and fluorescent derivatives thereof.

[0032] Examples of fluorophores whose fluorescence properties change depending on the degree of crowding around the fluorescent molecule include aggregation-induced emission (AIE) fluorophores such as tetraphenylethylene (TPE), 10,10',11,11'-tetrahydro-5,5'-bisbenzo[a,d][7]annulenylidene (THBA), and 1,1,2,3,4,5-hexaphenylsilole (HPS), as well as fluorescent derivatives thereof.

[0033] Preferred environmentally responsive fluorophores in this embodiment may be dansyl, NBD, DBD, FITC or TPE or fluorescent derivatives thereof.

[0034] The probe in this embodiment comprises a cationic polymer (a) having an environmentally responsive fluorophore (b) covalently bonded to a portion of the primary amino groups in the cationic polymer (a). Here, "a portion" preferably refers to 1 to 50%, and particularly preferably 5 to 20%, of the primary amino groups in the cationic polymer molecule.

[0035] The probe in this embodiment can be synthesized by a conventionally known chemical synthesis method, or the chemical synthesis method described in the examples of WO 2018 / 088510 or WO 2020 / 262413, or a chemical synthesis method equivalent thereto. Specifically, for example, the probe can be prepared by labeling the amino group of the cationic polymer (a) with an environmentally responsive fluorophore (b) activated by an active ester group such as an N-hydroxysuccinimide (NHS) ester group, a pentafluorophenyl (PFP) ester group, or an O-acylisourea group, an isothiocyanate group, or a halogenated alkyl group.

[0036] In the probe of this embodiment, at least a part of the primary amino groups to which an environmentally responsive fluorophore has not been introduced may be modified with a modifying group. In other words, the primary amino groups to which an environmentally responsive fluorophore has not been introduced (-NH ２ In at least a portion of the probes, a hydrogen atom (H) may be substituted with a monovalent modifying group (X) (-NHX). The modifying group may have either a negative or positive charge and may preferably be selected from the group consisting of a guanidino group, an acyl group, and an amino acid. Examples of amino acids include leucine, valine, isoleucine, tyrosine, tryptophan, phenylalanine, serine, asparagine, glutamine, or derivatives thereof. The probe in this embodiment may be modified with one or more modifying groups selected from the above.

[0037] Modification of an amino group with a modifying group can be carried out by a conventionally known chemical synthesis method. Modification of an amino group with a guanidino group can be carried out by guanylation of the amino group using, for example, 1H-pyrazole-1-carboxamidine hydrochloride. Modification of an amino group with an acyl group can be carried out by, for example, a nucleophilic acyl substitution reaction using a carboxylic acid anhydride such as acetic anhydride, phthalic anhydride, or naphthalenedicarboxylic acid anhydride. Modification of an amino group with an amino acid can be carried out by, for example, dehydration condensation of the carboxyl group of the amino acid with the amino group.

[0038] The probe in this embodiment can interact nonspecifically with any molecule contained in alcoholic beverages. In addition to the main component ethanol, alcoholic beverages contain proteins, peptides, amino acids, polysaccharides, monosaccharides, nucleic acids, nucleotides, fatty acids, organic acids, esters, polyphenols, terpenes, minerals, and the like, and the probe in this embodiment can interact with any of these. Furthermore, the molecules targeted by the probe in this embodiment can include not only known molecules but also unknown molecules.

[0039] The method of this embodiment uses multiple probe solutions that differ in one or more of the following: probe, ionic strength, and pH. The multiple probe solutions in this embodiment may be prepared, for example, by dissolving one type of probe in multiple solvents with different ionic strengths and / or pHs, or by dissolving multiple types of probes in the same solvent. For example, solvents with two or more different ionic strengths and / or pH conditions can be used, preferably three or more, and particularly preferably six or more different ionic strengths and / or pH conditions can be used.

[0040] In the method of this embodiment, one type of probe may be used, but it is more preferable to use multiple types of probes, for example, two or more types, preferably three or more types, and particularly preferably six or more types of probes. For example, by using two types of solvents and three types of probes, six types of probe solutions can be prepared, resulting in six-dimensional data for one analytical sample. For example, by using five types of solvents and three types of probes, fifteen types of probe solutions can be prepared, resulting in fifteen-dimensional data for one analytical sample. In this way, by increasing the number of solvents and probes, more multidimensional data can be obtained.

[0041] The solvent for dissolving the probe may be an aqueous solvent containing any buffer and / or salt. Examples of the buffer include MES, MOPS, EPPS, HEPES, Tris, phosphoric acid, acetic acid, citric acid, boric acid, glycine, etc. Examples of the salt include NaCl, KCl, MgCl. ２ , Na ２ SO ４ , K. ２ SO ４ , MgSO ４ , NaI, NaSCN, etc. In this embodiment, the pH of the solvent is preferably 4.0 to 10.0, and particularly preferably 5.0 to 7.0. In this embodiment, the ionic strength of the solvent is preferably 10 to 500 mM. In addition, the concentration of the probe in the probe solution is preferably 0.1 to 100 μg / mL.

[0042] Next, the multiple probe solutions are mixed with an analytical sample containing alcoholic beverages. In this step, the probes interact nonspecifically with any molecules contained in the alcoholic beverages.

[0043] The "alcoholic beverages" targeted by the method of this embodiment may be any beverage containing ethanol and may be produced from any raw materials, and the production method is not particularly limited. Furthermore, the characteristics of the alcoholic beverages (e.g., color, clarity, sparkling quality) are not particularly limited. Therefore, the alcoholic beverages targeted by the method of this embodiment may be brewed alcoholic beverages, distilled alcoholic beverages, or mixed alcoholic beverages. Examples of brewed alcoholic beverages include sake (Japanese rice wine), wine, beer, Shaoxing wine, and makgeolli. Examples of distilled alcoholic beverages include shochu, awamori, whiskey, brandy, vodka, and tequila. Examples of mixed alcoholic beverages include, but are not limited to, liqueurs, vermouth, sherry, and mirin. The country of origin / region of the alcoholic beverages is also not particularly limited. A wide variety of alcoholic beverages produced in various countries / regions are commercially available, and the method of this embodiment can analyze any of them.

[0044] The analytical sample in this embodiment may contain a single alcoholic beverage selected from any of the above alcoholic beverages, or a mixture of two or more alcoholic beverages, and the ratio of alcoholic beverages in the mixture may be arbitrary. Furthermore, the analytical sample in this embodiment may consist solely of alcoholic beverages, or may be a beverage containing alcoholic beverages. The beverage containing alcoholic beverages may be, for example, a cocktail made by mixing alcoholic beverages with non-ethanolic beverages such as fruit juice, water, or carbonated water. The final concentration of alcoholic beverages in the mixture of the analytical sample and the probe solution may be, for example, 0.001 to 99.9 v / v%, preferably 0.01 to 95 v / v%, and more preferably 0.1 to 90 v / v%. If the concentration of alcoholic beverage components in the analytical sample is unknown, the sample may be serially diluted as appropriate and added to the probe solution.

[0045] In the method of this embodiment, an analytical sample containing alcoholic beverages may be added to each of multiple probe solutions, or multiple probe solutions may be added simultaneously to an analytical sample containing alcoholic beverages.

[0046] Next, the fluorescence intensity of the mixture of the probe solution and the analytical sample is measured. In the method of this embodiment, fluorescence intensity can be measured at an excitation wavelength of 300 to 500 nm and a fluorescence wavelength of 400 to 700 nm. Furthermore, in the method of this embodiment, it is preferable to measure the fluorescence intensity for each measurement target at multiple excitation wavelength / fluorescence wavelength sets (e.g., excitation wavelength (nm) / fluorescence wavelength (nm): 340 / 520, 330 / 480, 345 / 505, 360 / 530, etc.), and the fluorescence intensity can be measured using, for example, two, three, or four sets of excitation wavelength / fluorescence wavelength.

[0047] The fluorescence intensity of the probe solution varies depending on the type, amount, and state of the alcoholic beverage components, as well as the conditions of the solvent the probe is dissolved in. Therefore, this step allows us to obtain a fluorescence intensity pattern (fluorescence fingerprint) specific to the sample.

[0048] The fluorescence intensity pattern obtained for the analytical sample is then compared with that obtained for the reference sample. The fluorescence intensity pattern for the reference sample may be obtained by measuring the fluorescence intensity in parallel with the analytical sample, or may be a predetermined fluorescence intensity pattern prepared in advance. The comparison of the fluorescence intensity patterns is preferably performed by multivariate analysis such as principal component analysis, linear discriminant analysis, or hierarchical cluster analysis to reduce the number of dimensions and compress the differences between the fluorescent fingerprints into two or three dimensions for comparison.

[0049] The method of this embodiment can determine the quality of alcoholic beverages based solely on differences in fluorescence intensity patterns. In this embodiment, the "quality" of alcoholic beverages refers to the overall palatability of alcoholic beverages, including flavors such as sweetness, sourness, saltiness, bitterness, umami, astringency, and aroma, as well as richness and crispness. The quality of alcoholic beverages varies significantly depending on numerous factors, including the place of origin, manufacturer, raw materials, and production method, as well as storage temperature and duration. However, it is extremely difficult and impractical to individually identify which components in alcoholic beverages change to what extent and in what way to change their quality. In contrast, the method of this embodiment can detect differences in the overall quality of alcoholic beverages by acquiring a fluorescence intensity pattern (fluorescence fingerprint) that reflects the sum of nonspecific interactions between a wide variety of alcoholic beverage components and probes, and comparing it with the fluorescence intensity pattern of a reference sample.

[0050] Therefore, in certain embodiments, for example, the fluorescent fingerprints obtained for different brands of sake A, B, or C are compared with the fluorescent fingerprint obtained for the analytical sample. If the fluorescent fingerprint obtained for the analytical sample is probabilistically closest to the distribution of the fluorescent fingerprint obtained for sake A, it can be estimated or identified that the analytical sample is sake A.

[0051] In another specific embodiment, for example, the fluorescent fingerprints obtained for Junmai sake A1, Ginjo sake A2, and Honjozo sake A3, which are the same brand but have different specific name classifications (i.e., production methods), are compared with the fluorescent fingerprint obtained for the analytical sample. If the fluorescent fingerprint obtained for the analytical sample is probabilistically closest to the distribution of the fluorescent fingerprint obtained for Junmai sake A1, it can be estimated or identified that the analytical sample is Junmai sake A1.

[0052] In another specific embodiment, for example, a fluorescent fingerprint obtained for sake X (reference sample) immediately after purchase is compared with fluorescent fingerprints obtained for sakes X1, X2, and X3 (analytical samples) of the same brand / specific name classification but stored under different conditions (e.g., storage temperature and / or duration). If the fluorescent fingerprint obtained for sake X1 is probabilistically closest to the distribution of the fluorescent fingerprints obtained for the reference sample, it can be estimated or determined that the quality of sake X1 has not deteriorated.

[0053] According to the method of this embodiment, it is possible to easily, quickly, objectively and consistently control the quality of alcoholic beverages without relying on the intuition of a craftsman or sensory tests by professional tasters.

[0054] The present invention will be further described below with reference to examples, which should not be construed as limiting the scope of the present invention.

[0055] [A: Construction of an analysis system using multiple wells] In this example, the following nonspecific fluorescent probe synthesized in the examples of WO 2020 / 262413 was used. The structural formula of the probe is shown in Figure 1. (i) PEG-b-PLL probes into which TPE groups and functional groups have been introduced (probes 1 to 12) Probe 1: A probe in which tetraphenylethylene (TPE) has been introduced into polyethylene glycol-block-poly-L-lysine trifluoroacetate (PEG-b-PLL, Mw: 17200) [number of ethylene glycol repeating units: 104, number of L-lysine trifluoroacetate repeating units: 52] (hereinafter referred to as "TPE-None") Probe 2: A probe obtained by guanylation of the amino group in TPE-None (probe 1) with 1H-pyrazole-1-carboxamidine hydrochloride (hereinafter referred to as "TPE-hA") Probe 3: A probe obtained by dehydration condensation reaction of the amino group in TPE-None (probe 1) with 2,3-diaminopropionic acid (hereinafter referred to as "TPE-Dap") Probe 4: Probe obtained by dehydration condensation reaction of the amino group in TPE-None (probe 1) with proline (hereinafter referred to as "TPE-Pro") Probe 5: Probe obtained by dehydration condensation reaction of the amino group in TPE-None (probe 1) with leucine (hereinafter referred to as "TPE-Leu") Probe 6: Probe obtained by dehydration condensation reaction of the amino group in TPE-None (probe 1) with norleucine (hereinafter referred to as "TPE-Nle") Probe 7: Probe obtained by dehydration condensation reaction of the amino group in TPE-None (probe 1) with glycylglycylglycine (hereinafter referred to as "TPE-Gly") ３") Probe 8: A probe obtained by subjecting the amino group in TPE-None (probe 1) to a dehydration condensation reaction with phenylalanine (hereinafter referred to as "TPE-Phe") Probe 9: A probe obtained by subjecting the amino group in TPE-None (probe 1) to a dehydration condensation reaction with 4-pyridylalanine (hereinafter referred to as "TPE-Pyri") Probe 10: A probe obtained by subjecting the amino group in TPE-None (probe 1) to a nucleophilic acyl substitution reaction with succinic anhydride (hereinafter referred to as "TPE-Suc") Probe 11: A probe obtained by subjecting the amino group in TPE-None (probe 1) to a nucleophilic acyl substitution reaction with phthalic anhydride (hereinafter referred to as "TPE-Pht") Probe 12: A probe obtained by subjecting the amino group in TPE-None (probe 1) to a nucleophilic acyl substitution reaction with 2,3-pyrazinedicarboxylic anhydride (Hereinafter referred to as "TPE-Pyr")

[0056] (ii) PEG-b-PLL probes into which an FITC group and a functional group have been introduced (probes 13 to 24) Probe 13: A probe obtained by introducing fluorescein isothiocyanate (FITC) into polyethylene glycol-block-poly-L-lysine trifluoroacetate (PEG-b-PLL, Mw: 17200) [number of ethylene glycol repeating units: 104, number of L-lysine trifluoroacetate repeating units: 52] (hereinafter referred to as "FITC-None"). Probe 14: A probe obtained by guanylation of the amino group in FITC-None (probe 13) with 1H-pyrazole-1-carboxamidine hydrochloride (hereinafter referred to as "FITC-hA"). Probe 15: A probe obtained by dehydration condensation reaction of the amino group in FITC-None (probe 13) with 2,3-diaminopropionic acid. (hereinafter referred to as "FITC-Dap") Probe 16: A probe obtained by subjecting the amino group in FITC-None (probe 13) to a dehydration condensation reaction with proline (hereinafter referred to as "FITC-Pro") Probe 17: A probe obtained by subjecting the amino group in FITC-None (probe 13) to a dehydration condensation reaction with leucine (hereinafter referred to as "FITC-Leu") Probe 18: A probe obtained by subjecting the amino group in FITC-None (probe 13) to a dehydration condensation reaction with norleucine (hereinafter referred to as "FITC-Nle") Probe 19: A probe obtained by subjecting the amino group in FITC-None (probe 13) to a dehydration condensation reaction with glycylglycylglycine (hereinafter referred to as "FITC-Gly") ３") Probe 20: A probe obtained by subjecting the amino group in FITC-None (probe 13) to a dehydration condensation reaction with phenylalanine (hereinafter referred to as "FITC-Phe") Probe 21: A probe obtained by subjecting the amino group in FITC-None (probe 13) to a dehydration condensation reaction with 4-pyridylalanine (hereinafter referred to as "FITC-Pyri") Probe 22: A probe obtained by subjecting the amino group in FITC-None (probe 13) to a nucleophilic acyl substitution reaction with succinic anhydride (hereinafter referred to as "FITC-Suc") Probe 23: A probe obtained by subjecting the amino group in FITC-None (probe 13) to a nucleophilic acyl substitution reaction with phthalic anhydride (hereinafter referred to as "FITC-Pht") Probe 24: A probe obtained by subjecting the amino group in FITC-None (probe 13) to a nucleophilic acyl substitution reaction with 2,3-pyrazinedicarboxylic acid anhydride (hereinafter referred to as "FITC-Pyr")

[0057] 1. Analysis of Sake Information on the sake analyzed in this example is shown in Table 1. S1 to S12 are from different breweries and brands.

[0058] Table 1. Sake (S1-12)

[0059] (1-1) Changes in Probe Fluorescence Due to Sake Probe 1 (TPE-None) and probe 8 (TPE-Phe) were dissolved in purified water to prepare two types of probe solutions (7500 nM). Sake (S3 or S11) was diluted to various concentrations with purified water and added to a 96-well half-well microplate (Corning, 3993) at 108 μL / well using an automatic dispenser (Andrew+, Andrew Alliance). After incubation at 35°C for 10 minutes, the fluorescence intensity (I) at an excitation wavelength of 330 nm was measured using a microplate reader (Cytation5, BioTek). ０The fluorescence intensity (I) was measured (fluorescence wavelength: 460 nm). Then, the probe solution was added (12 μL / well) using an automatic dispenser, and after incubation at 35°C for 10 minutes, the fluorescence intensity (I) was measured under the same conditions (final concentrations: 750 nM probe, 0-10% v / v sake).

[0060] The results are shown in Figure 2. The Y-axis (ΔF.I.) represents the change in fluorescence intensity (I-I) before and after adding the probe. ０ ) are shown. The fluorescence intensity of probe 1 (TPE-None) monotonically increased in a sake concentration-dependent manner. The fluorescence intensity of probe 8 (TPE-Phe) decreased in a sake concentration-dependent manner at or below 0.5 v / v% and increased above 0.5 v / v%. These results suggest that nonspecific fluorescent probes exhibit diverse responses to sake.

[0061] (1-2) Discrimination of different sake Probes 1 to 12 (TPE-None, TPE-hA, TPE-Dap, TPE-Pro, TPE-Leu, TPE-Nle, TPE-Gly) ３ , TPE-Phe, TPE-Pyri, TPE-Suc, TPE-Pht, and TPE-Pyr) were dissolved in purified water to prepare 12 probe solutions (7500 nM). Sake samples (S1 to S12) were diluted (8.8 v / v% each) with purified water, 22.2 mM acetate buffer (pH 5.0), or 22.2 mM EPPS buffer (pH 8.0), and added to a 96-well half-well microplate (Corning, 3993) at 108 μL / well using an automated dispenser (Andrew+, Andrew Alliance). After incubation at 35°C for 10 minutes, the fluorescence intensity (I ) was measured using a microplate reader (Cytation5, BioTek) at the following two sets of excitation wavelength (nm) / emission wavelength (nm): ０ The fluorescence intensity (I) was measured: (Ch1) 330 / 480, (Ch2) 360 / 530. Then, probe solution was added (12 μL / well) using an automated pipetting device, and after incubation at 35°C for 10 minutes, the fluorescence intensity (I) was measured under the same conditions (final concentrations: 750 nM probe, 8 v / v% sake, 0 or 20 mM buffer). Each measurement (12 sake samples x 3 solvent conditions x 12 probes x 2 wavelength sets) was performed 10 times.

[0062] The change in fluorescence intensity before and after adding the probe (I-I ０ Heat maps of the fluorescence intensity of each probe are shown in Figures 3 and 4. Figure 3 shows the results of measurements using sake diluted with purified water and probes 1 to 12 (12 sakes × 12 probes × 2 wavelength sets × 10 measurements), while Figure 4 shows the results of measurements using sake diluted with 22.2 mM acetate buffer (pH 5.0) or 22.2 mM EPPS buffer (pH 8.0) and probes 1, 3, 6, 7, 8, and 12 (12 sakes × 2 solvent conditions × 6 probes × 2 wavelength sets × 10 measurements). The fluorescence intensity of each probe varied depending on the type of sake and the solvent conditions, resulting in unique fluorescence patterns for each sake.

[0063] Measurement data from probes 1, 3, 6, 7, 8, and 12 in solvent conditions of purified water (no buffer), acetate buffer (pH 5.0), or EPPS buffer (pH 8.0) were analyzed by supervised linear discriminant analysis, and the results up to the second discriminant score were plotted and shown in Figures 5 to 7. The clusters of each sake were distributed without overlapping, with some exceptions. Furthermore, when these results were analyzed by the jackknife method, it was confirmed that each sake could be identified with 100% accuracy.

[0064] In a previous application, the inventors developed the probe used in this example to interact primarily with proteins and their peptide fragments. Therefore, the peptide components in sake were quantified using Bradford reagent (Sigma-Aldrich). The results are shown in Figure 8. Furthermore, unsupervised principal component analysis was performed on the measurement data obtained with probes 1, 3, 6, 7, 8, and 12 in purified water (no buffer), acetate buffer (pH 5.0), or EPPS buffer (pH 8.0). The first and second principal components were plotted against the peptide component concentrations, as shown in Figure 9. For the measurement data in purified water, there was little correlation between the first principal component and the peptide component concentrations (r = 0.08), whereas there was a strong correlation between the second principal component and the peptide component concentrations (r = -0.75). For the measurement data in buffer, there was a strong correlation between the first principal component and the peptide component concentrations (r = -0.81 (pH 5.0) and r = 0.71 (pH 8.0)). These results suggested that in purified water, the probe interacted mainly with components other than the peptide.

[0065] Furthermore, the correlation between the first or second principal component and the sweetness / dryness or strength of sake is shown in Table 2. Neither the first nor the second principal component showed much correlation with sweetness / dryness or strength. These results confirmed that the fluorescence patterns obtained by the above measurements reflect information different from sweetness / dryness or strength, suggesting that complex information can be obtained by the interaction of nonspecific fluorescent probes with various components.

[0066] Table 2. Correlation coefficients between the results of principal component analysis and the sweetness / dryness or strength of sake

[0067] (1-3) Identification of sake with different specific name classifications Sake from the same brewery and brand with different specific name classifications (Junmai Daiginjo, Daiginjo, Tokubetsu Honjozo, and Futsushu: S13 to 16 (Table 3)) were analyzed using purified water as the solvent using the same procedures and conditions as in (1-2) above.

[0068] Table 3. Sake (S13-16)

[0069] A heat map of the measurement results (4 specific name classifications x 1 solvent condition x 6 probes x 2 wavelength sets x 16 measurements) is shown in Figure 10. Unique fluorescence patterns were obtained for each specific name classification, even for the same brand. Furthermore, the fluorescence patterns were analyzed using supervised linear discriminant analysis, and the resulting first and second discriminant scores were plotted. The clusters were distributed without overlapping. Furthermore, when these results were analyzed using the jackknife method, it was confirmed that each specific name could be identified with 100% accuracy.

[0070] (1-4) Identification of sakes with different storage periods Sake (S3 or S11) was dispensed into microtubes and stored in a sealed, light-shielded state at 37°C (storage period: 0, 5, 8, or 12 days). After that, purified water was used as the solvent and the analysis was carried out using the same procedures and conditions as in (1-2) above.

[0071] A heat map of the measurement results (4 storage periods × 1 solvent condition × 6 probes × 2 wavelength sets × 12 measurements) is shown in Figure 12. Unique fluorescence patterns were obtained for each different storage period. Furthermore, the fluorescence patterns for samples on days 1 and 13 (i.e., storage periods of 0 and 12 days) were analyzed using supervised linear discriminant analysis, and the resulting first and second discriminant scores were plotted. The clusters were distributed without overlapping. Furthermore, when these results were analyzed using the jackknife algorithm, it was confirmed that each sample could be distinguished with 98% accuracy.

[0072] The same data was analyzed using unsupervised principal component analysis, and the results up to the second principal component obtained for each sample were plotted as shown in Figure 14. It was confirmed that the clusters for all sake samples shifted in a certain direction over time. Figure 15 also shows the results of plotting the first and second principal components against the number of days elapsed. For both S3 and S11, the first principal component changed in proportion to time. These results indicate that changes in sake due to storage period are reflected in the fluorescence pattern, suggesting the possibility of evaluating sake quality using a nonspecific fluorescent probe.

[0073] 2. Analysis of Liqueurs Information on the liqueurs analyzed in this example is shown in Table 4. F4 to F6 are plum liqueurs from the same manufacturer.

[0074] Table 4. Liqueurs (F1 to F6)

[0075] (2-1) Changes in Probe Fluorescence Due to Liqueur Probe 1 (TPE-None) and probe 10 (TPE-Suc) were dissolved in purified water to prepare two types of probe solutions (7500 nM). Liqueurs (F2 and F4) were diluted to various concentrations with 20 mM MOPS buffer (pH 7.0) and added to a 384-well microplate (Corning, 3575) at 54 μL / well using an automatic dispenser (Andrew+, Andrew Alliance). After incubation at 35°C for 10 minutes, the fluorescence intensity (I ) at an excitation wavelength of 330 nm was measured using a microplate reader (Cytation 5, BioTek). ０ ) was measured (fluorescence wavelength: 480 nm). Then, a probe solution was added (6 μL / well) using an automatic dispenser, and the mixture was incubated at 35°C for 10 minutes, after which the fluorescence intensity (I) was measured under the same conditions (final concentration: 750 nM probe, 0 to 6.4 v / v% liqueur).

[0076] The results are shown in Figure 16. The Y-axis (ΔF.I.) represents the change in fluorescence intensity (I - I ０ ) are shown. The fluorescence intensity of probe 1 (TPE-None) increased in a concentration-dependent manner when the liqueur concentration was below 0.4 v / v%, but began to decrease when the liqueur concentration exceeded 0.4 v / v%. The fluorescence intensity of probe 10 (TPE-Suc) increased monotonically in a liqueur concentration-dependent manner, and the response of TPE-None increased significantly at 6.4% F4. These results suggest that nonspecific fluorescent probes exhibit diverse responses to liqueurs.

[0077] (2-2) Discrimination of different liqueurs Probes 1, 3, 6, 7, 8, and 12 (TPE-None, TPE-Dap, TPE-Nle, TPE-Gly) ３, TPE-Phe, TPE-Pyr) were dissolved in purified water to prepare six types of probe solutions (7500 nM). Liqueurs (F1 to F6) were diluted (3.3 v / v% each) with purified water or 22.2 mM EPPS buffer (pH 8.0) and added to a 384-well microplate (Corning, 3575) at 54 μL / well using an automatic dispenser (Andrew+, Andrew Alliance). After incubation at 35°C for 10 minutes, the fluorescence intensity (I ) was measured using a microplate reader (Cytation5, BioTek) at the following two sets of excitation wavelength (nm) / emission wavelength (nm): ０ The fluorescence intensity was measured: (Ch1) 330 / 480, (Ch2) 360 / 530. Then, probe solution was added (6 μL / well) using an automated pipetting device, and after incubation at 35°C for 10 minutes, the fluorescence intensity was measured under the same conditions (final concentrations: 750 nM probe, 3% v / v liqueur, 0 or 20 mM EPPS buffer (pH 8.0)). Each measurement (6 liqueurs x 2 solvent conditions x 6 probes x 2 wavelength sets) was performed six times (I).

[0078] The change in fluorescence intensity before and after adding the probe (I-I ０ A heat map of the results is shown in Figure 17. The fluorescence intensity of each probe varied depending on the fruit type and solvent conditions, resulting in unique fluorescence patterns for each liqueur. Of these data, data from five probes (TPE-None, TPE-Dap, TPE-Nle, TPE-Phe, TPE-Pyr) × 2 solvent conditions × 1 wavelength set (Ch1) was analyzed using supervised linear discriminant analysis, and the results up to the second discriminant score were plotted in Figure 18. The clusters derived from each liqueur were distributed without overlapping. Furthermore, when these results were analyzed using the jackknife algorithm, it was confirmed that each liqueur could be identified with 100% accuracy.

[0079] The fruit used as the raw material for F1 (lychee) belongs to the Sapindaceae family, while the fruits used as the raw materials for F2 to F6 (apricot, apple, and plum) all belong to the Rosaceae family. More specifically, apricot and plum belong to the genus Prunus in the Rosaceae family, while apple belongs to the genus Malus in the Rosaceae family. In Figure 18, the clusters of F2 and F4 to F6 were located close to each other, whereas the cluster of F1 was far away from the clusters of F2 to F6. These results indicated that the response of the nonspecific fluorescent probe can reflect the phylogenetic classification of the fruits used as the raw materials for liqueurs.

[0080] 3. Analysis of Awamori Information on the Awamori analyzed in this example is shown in Table 5. A3 and A5 are different brands of Awamori from the same brewery, and the other Awamori are from different breweries / brands.

[0081] Table 5. Awamori (A1 to A6)

[0082] (3-1) Probe Fluorescence Changes Due to Awamori Probe 1 (TPE-None) and probe 10 (TPE-Suc) were dissolved in purified water to prepare two types of probe solutions (7500 nM). Awamori (A1 and A2) were diluted to various concentrations with 20 mM MOPS buffer (pH 7.0) and added to a 384-well microplate (Corning, 3575) at 54 μL / well using an automatic dispenser (Andrew+, Andrew Alliance). After incubation at 35°C for 10 minutes, the fluorescence intensity at an excitation wavelength of 330 nm (fluorescence wavelength 480 nm) was measured using a microplate reader (Cytation5, BioTek) (I ０ Then, the probe solution was added (6 μL / well) using an automatic dispenser, and after incubation at 35° C. for 10 minutes, the fluorescence intensity was measured under the same conditions (final concentration: 750 nM probe, 0 to 64 v / v% awamori) (I).

[0083] The results are shown in Figure 19. The Y-axis (ΔF.I.) represents the change in fluorescence intensity (I - I ０) are shown. The fluorescence intensity of probe 1 (TPE-None) tended to decrease depending on the awamori concentration. The fluorescence intensity of probe 10 (TPE-Suc) decreased at 1 v / v% awamori, but increased at concentrations exceeding 1%, and decreased again at even higher concentrations. Furthermore, at awamori concentrations exceeding 2 v / v%, probe 10 (TPE-Suc) consistently responded more strongly to A2 than to A1. These results suggest that nonspecific fluorescent probes exhibit diverse responses to awamori.

[0084] (3-2) Discrimination of Different Awamori Probes 1, 6, 8, and 10-12 (TPE-None, TPE-Nle, TPE-Phe, TPE-Suc, TPE-Pht, TPE-Pyr) were dissolved in purified water to prepare six probe solutions (7500 nM). Awamori (A1-A6) was diluted (22.2 v / v% each) with purified water or 22.2 mM EPPS buffer (pH 8.0) and added to a 384-well microplate (Corning, 3575) at 54 μL / well using an automated dispenser (Andrew+, Andrew Alliance). After incubation at 35°C for 10 minutes, the fluorescence intensity (I ) was measured using a microplate reader (Cytation5, BioTek) at the following two sets of excitation wavelength (nm) / emission wavelength (nm): ０ ) were measured: (Ch1) 330 / 480, (Ch2) 360 / 530. Then, probe solution was added (6 μL / well) using an automated pipetting device, and after incubation at 35°C for 10 minutes, fluorescence intensity was measured under the same conditions (final concentrations: 750 nM probe, 20 v / v% awamori, 0 or 20 mM EPPS (pH 8.0)) (I). Each measurement (6 awamori × 2 solvent conditions × 6 probe × 2 wavelength sets) was performed six times.

[0085] The change in fluorescence intensity before and after adding the probe (I-I ０) is shown in Figure 20. The fluorescence intensity of each probe varied depending on the awamori brewery / brand and solvent conditions, resulting in a unique fluorescence pattern for each distinct awamori. The above data was analyzed using supervised linear discriminant analysis, and the results obtained up to the second discriminant score are plotted in Figure 21. The clusters derived from each awamori were distributed without overlapping, with some exceptions. Furthermore, when these results were analyzed using the jackknife algorithm, it was confirmed that each awamori could be distinguished with 94% accuracy. In addition, a portion of the above data was selected ([6 probes x 1 solvent condition (purified water) x 2 wavelength sets] + [2 probes (TPE-Nle and TPE-Phe) x 1 solvent condition (20 mM EPPS buffer (pH 8.0)) x 2 wavelength sets]) and analyzed using supervised linear discriminant analysis. The results up to the second discriminant score are shown in Figure 22. The clusters derived from each awamori were distributed without overlapping. Furthermore, when these results were analyzed using the jackknife algorithm, it was confirmed that each awamori could be distinguished with 100% accuracy.

[0086] In both analyses, the cluster A1 (25% alcohol by volume) was significantly different from the clusters of other awamori (30% alcohol by volume), indicating that the alcohol content is strongly reflected in the response of the non-specific fluorescent probe. However, the clusters A2 to A6 were also diversely distributed, suggesting that the non-specific fluorescent probe responded to components other than alcohol.

[0087] 4. Analysis of Red Wines Information on the red wines analyzed in this example is shown in Table 6. W1 to W6 are from different wineries and brands.

[0088] Table 6. Red wine (W1-W6)

[0089] (4-1) Probe Fluorescence Changes Induced by Red Wine Probe 13 (FITC-None) and probe 22 (FITC-Suc) were dissolved in purified water to prepare two probe solutions (3000 nM). Red wine (W1) was diluted to various concentrations with 20 mM MOPS buffer (pH 7.0) and added to a 384-well microplate (Corning, 3575) at 54 μL / well using an automatic dispenser (Andrew+, Andrew Alliance). After incubation at 35°C for 10 minutes, the fluorescence intensity at an excitation wavelength of 460 nm (emission wavelength 520 nm) (I ０ Then, the probe solution was added (6 μL / well) using an automatic dispenser, and after incubation at 35°C for 10 minutes, the fluorescence intensity was measured under the same conditions (final concentration: 300 nM probe, 0 to 6.4 v / v% red wine) (I).

[0090] The results are shown in Figure 23. The Y-axis (ΔF.I.) represents the change in fluorescence intensity (I - I ０ The fluorescence intensities of both probe 13 (FITC-None) and probe 22 (FITC-Suc) decreased monotonically in a red wine concentration-dependent manner, but the fluorescence intensity of FITC-None decreased at lower concentrations. These results suggest that nonspecific fluorescent probes exhibit diverse responses to red wine.

[0091] (4-2) Discrimination of Different Red Wines. Probes 13, 18, 20, and 22-24 (FITC-None, FITC-Nle, FITC-Phe, FITC-Suc, FITC-Pht, and TPE-Pyr) were dissolved in purified water to prepare six probe solutions (3000 nM). Red wines (W1-W6) were diluted (2.2% v / v each) with purified water or 22.2 mM MOPS buffer (pH 7.0) and added to a 384-well microplate (Corning, 3575) at 54 μL / well using an automated pipetting device (Andrew+, Andrew Alliance). After incubation at 35°C for 10 minutes, the fluorescence intensity (I) was measured using a microplate reader (Cytation 5, BioTek) at an excitation wavelength of 460 nm and an emission wavelength of 520 nm. ０ ) was measured. Then, probe solution was added (6 μL / well) using an automatic pipetting device, and after incubation at 35°C for 10 minutes, fluorescence intensity was measured under the same conditions (final concentrations: 300 nM probe, 2 v / v% red wine, 0 or 20 mM MOPS buffer (pH 7.0)) (I). Each measurement (6 red wines × 2 solvent conditions × 6 probes × 1 wavelength set) was performed six times.

[0092] The change in fluorescence intensity before and after adding the probe (I-I ０ ) is shown in Figure 24. The fluorescence intensity of each probe varied depending on the brand of red wine and the solvent conditions, resulting in a unique fluorescence pattern for each different red wine. Data from purified water was analyzed using supervised linear discriminant analysis, and the results up to the second discriminant score were plotted in Figure 25. The clusters derived from each red wine were distributed without overlapping. Furthermore, when these results were analyzed using the jackknife method, it was confirmed that each red wine could be identified with 100% accuracy.

[0093] Furthermore, in Figure 25, French red wines (W1, W3, W5) and Italian red wines (W2, W4, W6) each formed a cluster, indicating that the response of the nonspecific fluorescent probe can reflect the origin of the red wine.

[0094] (4-3) Identification of Red Wines with Different Storage Periods Red wine (W1) was dispensed into microtubes and stored in a sealed, light-shielded state at 37°C (storage period: 0 days, 7 days, 14 days, 21 days, 28 days, 35 days, or 42 days). After that, it was analyzed using the same procedures and conditions as in (4-2) above.

[0095] A heat map of the measurement results (7 storage periods × 2 solvent conditions × 6 probes × 1 wavelength set × 6 measurements) is shown in Figure 26. Unique fluorescence patterns were obtained for each different storage period. A portion of the data was selected ([6 probes × 1 solvent condition (purified water) × 1 wavelength set] + [2 probes (FITC-None and FITC-Phe) × 1 solvent condition (20 mM MOPS (pH 7.0)) × 1 wavelength set]) and analyzed using supervised linear discriminant analysis. The results of plotting the first and second discriminant scores and the first and third discriminant scores are shown in Figure 27. On the plane where the first and second discriminant scores are plotted, the clusters from day 29 (storage period of 28 days) and day 36 (storage period of 35 days) overlap, but the other clusters are distributed without overlap. On a plane plotting the first and third discriminant scores, the clusters from day 29 (38 days of storage) and day 36 (35 days of storage) were distributed at separate locations without overlapping. Furthermore, analysis of these results using the jackknife algorithm confirmed that each sample could be distinguished with 97% accuracy. These results indicate that changes in red wine due to storage time are reflected in the fluorescence pattern, suggesting the possibility of evaluating the quality of red wine using a nonspecific fluorescent probe.

[0096] [B: Construction of an analytical system using a single well] In the following examples, in addition to TPE-Phe (probe 8), a nonspecific fluorescent probe synthesized in an example of WO 2018 / 088510 was used. The structural formulas of the probes are shown in Figure 28. Probe 25: A probe in which dansyl (Dnc) has been introduced into a PAMAM dendrimer (ethylenediamine core, generation 4.0) (hereinafter referred to as "Dnc-PAMAM") Probe 26: A probe in which dimethylaminosulfonylbenzoxadiazole (DBD) has been introduced into poly-L-lysine trifluoroacetate (PLL, Mw: 12000) [number of L-lysine trifluoroacetate repeat units: 55] (hereinafter referred to as "DBD-PLL")

[0097] <5. Analysis of Sake (Single Well)> (5-1) Changes in Probe Fluorescence Due to Sake Three types of probe solutions (40 μg / mL) were prepared by dissolving TPE-Phe (probe 8), Dnc-PAMAM (probe 25), and DBD-PLL (probe 26) in purified water. Sake (S3 or S11) was diluted to various concentrations with purified water and added to a 384-well microplate (Corning, 3575) at 54 μL / well using an automatic dispenser (Andrew+, Andrew Alliance). After incubation at 35°C for 10 minutes, the fluorescence intensity (I ) was measured using a microplate reader (Cytation5, BioTek) at the following two sets of excitation wavelength (nm) / emission wavelength (nm): ０ ) were measured: (Ch1) 340 / 460, (Ch2) 340 / 520, (Ch3) 440 / 560. Then, a mixture of the three probes was added (6 μL / well) using an automatic pipetting device, and after incubation at 35°C for 10 minutes, the fluorescence intensity was measured under the same conditions (final concentration: 4 μg / mL of each probe, 0-90% v / v sake) (I).

[0098] The results are shown in Figure 29. The Y-axis (ΔF.I.) represents the change in fluorescence intensity (I - I ０ The fluorescence intensity of the three probes varied in a complex concentration-dependent manner under all measurement conditions (wavelength sets), confirming that each probe responded differently to sake.

[0099] (5-2) Discrimination of Different Sakes TPE-Phe (probe 8), Dnc-PAMAM (probe 25), and DBD-PLL (probe 26) were dissolved in purified water to prepare three probe solutions (40 μg / mL). Undiluted sake (S1, S3 to S6, and S8 to S12) was added to a 384-well microplate (Corning, 3575) at 54 μL / well using an automated pipetting device (Andrew+, Andrew Alliance). After incubation at 35°C for 10 minutes, the fluorescence intensity (I ) was measured using a microplate reader (Cytation5, BioTek) at the following six sets of excitation wavelength (nm) / emission wavelength (nm). ０ The following were measured: (Ch1) 340 / 460, (Ch2) 340 / 420, (Ch3) 440 / 560, (Ch4) 390 / 510, (Ch5) 340 / 580, (Ch6) 440 / 620. Then, a mixture of the three probes was added (6 μL / well) using an automated pipetting device, and after incubation at 35°C for 10 minutes, the fluorescence intensity was measured under the same conditions (final concentration: 4 μg / mL of each probe, 90 v / v% sake) (I). Each measurement (12 sake × 3 probe mixture × 6 wavelength sets) was performed six times.

[0100] The change in fluorescence intensity before and after adding the probe (I-I ０ A heat map of the results is shown in Figure 30. The fluorescence intensity of each probe varied depending on the type of sake, resulting in a unique fluorescence pattern for each sake. The above data was analyzed using supervised linear discriminant analysis, and the plots of the first and second discriminant scores and the first and third discriminant scores are shown in Figure 31. The clusters derived from each sake were distributed with almost no overlap. Furthermore, when these results were analyzed using the jackknife algorithm, it was confirmed that each sake could be distinguished with 98% accuracy. These results demonstrate that the quality of sake can be determined simply by adding multiple nonspecific fluorescent probes directly to sake and measuring the fluorescence response, without adjusting the pH or ionic strength using a buffer solution.

Claims

1. A method for controlling the quality of alcoholic beverages, comprising: (1) preparing a plurality of probe solutions having different probes, ionic strengths or pHs, wherein the probes comprise: (a) a cationic polymer having at least five primary amino groups per molecule and a weight-average molecular weight of 1,000 to 500,000; and (b) an environmentally responsive fluorophore, the fluorophore being covalently bonded to a portion of the primary amino groups in the cationic polymer; (2) mixing the plurality of probe solutions with an analytical sample containing alcoholic beverages; (3) measuring the fluorescence intensity of the mixture prepared in step (2); and (4) comparing the fluorescence intensity pattern obtained in step (3) with the fluorescence intensity pattern obtained for a reference sample.

2. The method according to claim 1, wherein the environmentally responsive fluorophore is selected from the group consisting of a fluorophore having a naphthalenesulfonic acid skeleton, a fluorophore having a benzofurazan skeleton, a fluorophore having a xanthene skeleton, a fluorophore having a pyrene skeleton, and an aggregation-induced emission fluorophore.

3. The method according to claim 1 or 2, wherein the cationic polymer is a linear or branched polyamino acid, a polyallylamine, a polyamidoamine or a polyalkyleneimine.

4. The method according to any one of claims 1 to 3, wherein the environmentally responsive fluorophore is covalently attached to 1 to 50% of the primary amino groups in the cationic polymer.

5. The method according to any one of claims 1 to 4, wherein at least a portion of the primary amino groups of the cationic polymer to which the environmentally responsive fluorescent group is not covalently bound is modified with an acyl group or an amino acid.

6. The method according to any one of claims 1 to 5, wherein in step (2), an analytical sample containing an alcoholic beverage is added to each of the plurality of probe solutions.

7. The method according to any one of claims 1 to 5, wherein in step (2), the plurality of probe solutions are added simultaneously to an analytical sample containing alcoholic beverages.

8. The method according to any one of claims 1 to 7, wherein the measurement of fluorescence intensity in step (3) is performed for a plurality of excitation wavelengths and fluorescence wavelengths.

9. The method according to any one of claims 1 to 8, wherein the step (4) evaluates differences in quality due to the place of origin, manufacturer, production method, or storage condition of the alcoholic beverage.

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