Beer-flavored alcoholic beverage and method for producing same

Abstract

Disclosed is a low-carbohydrate beer-flavored alcoholic beverage in which the total content of free carboxymethyl lysine (CML), free carboxyethyl lysine (CEL), free methylglyoxal-derived hydroimidazolone-1 (MG-H1) is 434 ppb or more. In the low-carbohydrate beer-flavored alcoholic beverage, the richness is enhanced.

Description

Beer-flavored alcoholic beverage and its manufacturing method

[0001] The present invention relates to a beer-taste alcoholic beverage and a method for producing the same.

[0002] In recent years, growing health consciousness has led to an increasing demand for reduced-sugar beer-flavored beverages. However, reduced-sugar beer-flavored alcoholic beverages have the problem of losing flavor balance due to a reduced sugar content, resulting in a loss of body and an unpleasant sourness.

[0003] On the other hand, it has been reported that peptides with molecular weights of 1000 to 5000 Da among the products obtained by the Maillard reaction contribute to enhancing the richness of beer (Non-Patent Document 1), but the specific contributing components have not been clarified, and their effect on the flavor balance in beer is unknown.

[0004] Food Chemistry, 99, 600-604

[0005] The present inventors have found that in low-sugar beer-taste alcoholic beverages, some of the Maillard reaction products (MRPs) produced during the heating and aging process can enhance the body of the beer-taste alcoholic beverage within a specific concentration range. The present invention is based on this finding.

[0006] Therefore, the present invention provides a low-sugar, beer-flavored alcoholic beverage with enhanced body and a method for producing the same.

[0007] The present invention encompasses the following inventions: (1) A low-sugar, beer-flavored alcoholic beverage in which the total content of free carboxymethyllysine (CML), free carboxyethyllysine (CEL), and free methylglyoxal-derived hydroimidazolone-1 (MG-H1) in the beverage is 434 ppb or more. (2) A low-sugar, beer-flavored alcoholic beverage according to (1) above, in which the total content of bound CML, bound CEL, and bound MG-H1 in the beverage is 65 ppb or more, and the bound CML, bound CEL, and bound MG-H1 are contained in a peptide fraction having a molecular weight of 400 to 3,000 Da. (3) A low-sugar, beer-flavored alcoholic beverage according to (2) above, in which the total content of bound CML, bound CEL, and bound MG-H1 in the beverage is 72 ppb or more. (4) The low-sugar beer-flavored alcoholic beverage according to (1), wherein the total content of free CML, free CEL, free MG-H1, bound CML, bound CEL, and bound MG-H1 in the beverage is 499 ppb or more, and the bound CML, bound CEL, and bound MG-H1 are contained in a peptide fraction having a molecular weight of 400 to 3,000 Da. (5) The low-sugar beer-flavored alcoholic beverage according to (4), wherein the total content of free CML, free CEL, free MG-H1, bound CML, bound CEL, and bound MG-H1 in the beverage is 510 ppb or more. (6) The low-sugar, beer-flavored alcoholic beverage according to (4), in which the total content of free CML, free CEL, free MG-H1, bound CML, bound CEL, and bound MG-H1 in the beverage is 1,042 ppb or more. (7) The low-sugar, beer-flavored alcoholic beverage according to (1), in which the ratio (F / B) of the total content of free CML, free CEL, and free MG-H1 (F ppb) to the total content of bound CML, bound CEL, and bound MG-H1 (B ppb) in the beverage is 0.53 or more, and the bound CML, bound CEL, and bound MG-H1 are contained in a peptide fraction having a molecular weight of 400 to 3,000 Da. (8) The low-sugar, beer-flavored alcoholic beverage according to (7), in which F / B is 0.53 to 3.63.(9) The low-sugar beer-flavored alcoholic beverage according to (7), having an F / B ratio of 6.66 or higher. (10) The low-sugar beer-flavored alcoholic beverage according to any one of (1) to (9), having a purine concentration of 5.0 mg / 100 mL or lower. (11) A method for producing a low-sugar beer-flavored alcoholic beverage, wherein the total content of free carboxymethyllysine (CML), free carboxyethyllysine (CEL), and free methylglyoxal-derived hydroimidazolone-1 (MG-H1) in the beverage is adjusted to 434 ppb or higher. (12) A method for enhancing the richness of a low-sugar beer-flavored alcoholic beverage, wherein the total content of free carboxymethyllysine (CML), free carboxyethyllysine (CEL), and free methylglyoxal-derived hydroimidazolone-1 (MG-H1) in the beverage is adjusted to 434 ppb or higher. (13) A method for reducing unpleasant sourness in a low-sugar, beer-flavored alcoholic beverage, the method comprising adjusting the total content of free carboxymethyllysine (CML), free carboxyethyllysine (CEL), and free methylglyoxal-derived hydroimidazolone-1 (MG-H1) in the beverage to 434 ppb or more. (14) A method for reducing alcohol harshness in a low-sugar, beer-flavored alcoholic beverage, the method comprising adjusting the total content of free carboxymethyllysine (CML), free carboxyethyllysine (CEL), and free methylglyoxal-derived hydroimidazolone-1 (MG-H1) in the beverage to 434 ppb or more.

[0008] According to the present invention, it is possible to enhance the richness of a low-sugar, beer-flavored alcoholic beverage. Furthermore, according to the present invention, it is also possible to reduce unpleasant sourness in a low-sugar, beer-flavored alcoholic beverage. Furthermore, according to the present invention, it is also possible to reduce the harshness of alcohol in a low-sugar, beer-flavored alcoholic beverage.

[0009] FIG. 1 is a plot diagram showing the relationship between MRP concentration and the results of the "richness" evaluation (vertical axis: bound MRP concentration, horizontal axis: free MRP concentration, bubble size: richness). FIG. 2 is a plot diagram showing the relationship between MRP concentration and the results of the "richness" evaluation (vertical axis: bound MRP concentration, horizontal axis: free MRP concentration, bubble size: richness, diagonal auxiliary line: boundary line of the total amount of F-MRP and B-MRP). FIG. 3 is a plot diagram showing the relationship between MRP concentration and the results of the "richness" evaluation (vertical axis: bound MRP concentration, horizontal axis: free MRP concentration, bubble size: richness, diagonal auxiliary line: boundary line of the F-MRP / B-MRP ratio). Specific Description of the Invention

[0010] The three substances considered to be active ingredients in the present invention, carboxymethyllysine (CML), carboxyethyllysine (CEL), and methylglyoxal-derived hydroimidazolone-1 (MG-H1), have the following chemical structures:

[0011] The three free substances exist in beverages either as the above structures themselves or in a partially ionized state. The three bound substances are contained in the peptide fraction of a beverage having a molecular weight of 400 to 3,000 Da, and exist as residues in which the side chains of lysine or arginine are glycosylated. The amounts and concentrations of the above substances shown in the present invention are the masses and concentrations of the compounds having the above structures for both the free and bound forms. In this specification, the three free substances are sometimes referred to as the "three free active ingredients," and the three bound substances are sometimes referred to as the "three bound active ingredients."

[0012] In the present invention, the term "beer-taste alcoholic beverage" refers to beer (an alcoholic beverage obtained by fermenting malt and hops with brewer's yeast) or an alcoholic beverage having a flavor similar to beer. In the present invention, the term "alcoholic beverage" refers to a beverage with an alcohol (ethanol) concentration of 1% v / v or more. The beer-taste alcoholic beverage of the present invention is preferably a fermented beverage to which hops are used as an ingredient to impart a hop aroma. The beer-taste alcoholic beverage of the present invention is preferably a fermented malt beverage, i.e., a beverage using at least malt as an ingredient. Examples of such fermented malt beverages include beer and happoshu. The beer-taste alcoholic beverage of the present invention is preferably a fermented malt beverage with a malt usage ratio (also referred to as "malt ratio") of 0% to 100% by mass, more preferably 25% to 100% by mass, and even more preferably 50% to 100% by mass. The "malt usage ratio" refers to a value calculated in accordance with the Liquor Tax Act and the Interpretation Notice of Liquor Administration-Related Laws and Regulations, etc., which came into effect on April 1, 2018.

[0013] The beer-taste alcoholic beverage of the present invention is a low-sugar beer-taste alcoholic beverage, i.e., a beer-taste alcoholic beverage with a lower sugar content than usual. "Lower sugar content than usual" here means that the beer-taste alcoholic beverage has been produced using techniques to reduce the sugar content. The specific carbohydrate concentration in such a low-sugar beer-flavored alcoholic beverage is not particularly limited, but can be, for example, 1.5 g / 100 mL or less, preferably less than 1.5 g / 100 mL, more preferably 1.4 g / 100 mL or less, even more preferably 1.3 g / 100 mL or less, even more preferably 1.2 g / 100 mL or less, even more preferably 1.1 g / 100 mL or less, even more preferably 1.0 g / 100 mL or less, even more preferably less than 1.0 g / 100 mL, even more preferably 0.9 g / 100 mL or less, even more preferably 0.8 g / 100 mL or less, even more preferably 0.7 g / 100 mL or less, even more preferably 0.6 g / 100 mL or less, even more preferably 0.5 g / 100 mL or less, and even more preferably less than 0.5 g / 100 mL. In one embodiment, the carbohydrate concentration in the low-sugar beer-flavored alcoholic beverage is 0.4 g / 100 mL or less.

[0014] The carbohydrate concentration can be measured by known methods, and can be calculated by subtracting the amount of water, protein, fat, ash, and dietary fiber from the mass of the sample (see the Food Labeling Standards (March 30, 2015, Food Labeling Table No. 139), Appendix, Analytical Methods for Nutritional Components, etc.).

[0015] In the present invention, "richness" refers to a flavor determined by a comprehensive evaluation of the strength, complexity, and persistence of the flavor.

[0016] In the present invention, the term "unpleasant sourness" refers to an unpleasant sourness felt as an aftertaste or the like.

[0017] In the present invention, the term "alcoholic hotness" refers to the hotness accompanied by a tingling sensation felt when drinking an alcoholic beverage.

[0018] In the present invention, the unit "ppm" is synonymous with "mg / L", and the unit "ppb" is synonymous with "μg / L".

[0019] The beer-taste alcoholic beverage of the present invention has a total content of three free active ingredients within a predetermined range. Furthermore, according to a preferred embodiment of the present invention, the beer-taste alcoholic beverage of the present invention also contains three bound active ingredients, the total content of which is within a predetermined range. Such a beer-taste alcoholic beverage can be obtained by adjusting the total content of the three free active ingredients or the total content of the three bound active ingredients during production. Specific means for adjusting the concentrations of the free active ingredients and the three bound active ingredients are not particularly limited, and examples include adding the free active ingredients or the bound active ingredients, increasing or decreasing the amount of raw materials containing the free active ingredients or the bound active ingredients, increasing or decreasing the amount of raw materials that produce the free active ingredients or the bound active ingredients in the final product, and adjusting the concentration of substances that are converted to the free active ingredients or the bound active ingredients by fermentation with yeast.

[0020] The total content of the three free active ingredients in the beer-taste alcoholic beverage of the present invention is, for example, 434 ppb or more, preferably 438 ppb or more, and more preferably 700 ppb or more. Since beer-taste alcoholic beverages with a high total content of the three free active ingredients exhibit significant body-enhancing effects, reduced unpleasant sourness, and reduced alcohol harshness, the upper limit is not particularly limited. However, if one were to set an upper limit, it could be, for example, 3000 ppb, preferably 2000 ppb, more preferably 1800 ppb, even more preferably 1300 ppb, and even more preferably 1100 ppb. The three free active ingredients may be derived from raw materials, added separately from plant raw materials, or even produced by fermentation. The concentration of the three free active ingredients can be controlled, for example, by controlling the composition of the raw materials, raw material processing conditions, brewing conditions, fermentation conditions, etc.

[0021] According to a preferred embodiment of the present invention, the total content of the three bound active ingredients in the beer-taste alcoholic beverage of the present invention is 0 ppb or more, more preferably 65 ppb or more, even more preferably 72 ppb or more, even more preferably 100 ppb or more, and even more preferably 117 ppb or more. Since beer-taste alcoholic beverages with a high total content of the three bound active ingredients exhibit significant effects of enhancing richness, reducing unpleasant sourness, and reducing alcohol harshness, the upper limit of the content is not particularly limited. However, if one were to set an upper limit, it could be, for example, 2000 ppb, preferably 1600 ppb, more preferably 1000 ppb, and even more preferably 832 ppb. The three bound active ingredients may be derived from raw materials, added separately from plant raw materials, or produced by fermentation. The concentration of the three bound active ingredients can be controlled, for example, by controlling the composition of the raw materials, raw material processing conditions, mashing conditions, and fermentation conditions. According to another preferred embodiment of the present invention, the total content of the three bound active ingredients in the beer-taste alcoholic beverage of the present invention is preferably 65 ppb or more, from the viewpoint of reducing the harshness of alcohol.

[0022] According to one embodiment of the present invention, the total content of the three free active ingredients in the beer-taste alcoholic beverage of the present invention is, for example, 434 ppb or more, preferably 438 ppb or more, and more preferably 700 ppb or more, and the total content of the three bound active ingredients is 0 ppb or more, preferably 65 ppb or more, more preferably 72 ppb or more, even more preferably 100 ppb or more, and even more preferably 117 ppb or more. According to another embodiment of the present invention, the total content of the three free active ingredients in the beer-taste alcoholic beverage of the present invention is 1003 ppb or more, and the total content of the three bound active ingredients is 0 ppb or more, preferably 65 ppb or more, more preferably 72 ppb or more, and even more preferably 117 ppb or more.

[0023] According to another embodiment of the present invention, the total content of the three free active ingredients in the beer-taste alcoholic beverage of the present invention is 434 to 1,800 ppb, and preferably 438 to 1,100 ppb, and the total content of the three bound active ingredients is 0 to 832 ppb, preferably 65 to 832 ppb, more preferably 72 to 832 ppb, and even more preferably 117 to 832 ppb.

[0024] According to a preferred embodiment of the present invention, the total content of six components, including three free active ingredients and three bound active ingredients, in the beer-taste alcoholic beverage of the present invention is 499 ppb or more, more preferably 510 ppb or more, even more preferably 550 ppb or more, even more preferably 672 ppb or more, even more preferably 1042 ppb or more, and even more preferably 1203 ppb or more. Since beer-taste alcoholic beverages with high total contents of these six components exhibit significant effects of enhancing richness, reducing unpleasant sourness, and reducing alcohol harshness, there is no particular upper limit to this content. However, if one were to set an upper limit, it could be, for example, 5000 ppb, preferably 4000 ppb, more preferably 2500 ppb, even more preferably 2350 ppb, or even more preferably 1932 ppb. The preferred ranges for the total content of the three free active ingredients and the total content of the three bound active ingredients in this embodiment are as described in the above paragraphs.

[0025] According to a preferred embodiment of the present invention, in a beer-taste alcoholic beverage of the present invention, the ratio (F / B) of the total content of the three free active ingredients (F ppb) to the total content of the three bound active ingredients (B ppb) is 0.53 or greater. According to a further preferred embodiment of the present invention, F / B is 0.53 to 5.05, more preferably 0.53 to 3.62. According to another further preferred embodiment of the present invention, F / B is 6.66 or greater, more preferably 8.38 or greater. Since beer-taste alcoholic beverages with a high total content of the three free active ingredients exhibit a significant effect of enhancing body and reducing unpleasant sourness, the upper limit of F / B is not particularly limited. However, if one were to be set, it could be, for example, 50, preferably 30, more preferably 20, even more preferably 18, or even more preferably 15.34. The preferred ranges for the total content of the three free active ingredients and the total content of the three bound active ingredients in this embodiment are as described in the above paragraphs.

[0026] The amounts of the three free active ingredients and the three bound active ingredients in a beer-flavored alcoholic beverage can be determined by LC-MS / MS analysis as described in the Examples below. Furthermore, for more accurate concentration measurements, it is desirable to use a calibration curve prepared based on the measured values ​​of several samples spiked with standards of known concentrations.

[0027] The purine concentration in the beer-taste alcoholic beverage of the present invention is not particularly limited, but is preferably reduced compared to that of typical pale beers. Specifically, the purine concentration of the beer-flavored alcoholic beverage of the present invention is, for example, 5.0 mg / 100 mL or less, and is preferably set to a lower concentration (e.g., 4.5 mg / 100 mL or less, 4.0 mg / 100 mL or less, 3.5 mg / 100 mL or less, 3.0 mg / 100 mL or less, 2.5 mg / 100 mL or less, 2.0 mg / 100 mL or less, 1.5 mg / 100 mL or less, 1.4 mg / 100 mL or less, 1.3 mg / 100 mL or less, 1.2 mg / 100 mL or less, 1.1 mg / 100 mL or less, 1.0 mg / 100 mL or less, 0.9 mg / 100 mL or less, 0.8 mg / 100 mL or less, 0.7 mg / 100 mL or less, 0.6 mg / 100 mL or less, 0.5 mg / 100 mL or less, or less than 0.5 mg / 100 mL). A beverage with a purine concentration of less than 0.5 mg / 100 mL corresponds to a “zero-purine beverage.” There is no particular lower limit for the purine concentration of the beer-taste alcoholic beverage of the present invention, but if one were to be set, it could be, for example, 0.001 mg / 100 mL or more, preferably 0.01 mg / 100 mL or more.

[0028] Purines can be measured by known methods, for example, by a method in which purines are hydrolyzed with perchloric acid and then detected using LC-MS / MS (liquid chromatography mass spectrometry) (see Journal of Food Hygiene 55(2):110-116(2014)). In this specification, the term "purine concentration" refers to the total amount of four purine bases, i.e., adenine, xanthine, guanine, and hypoxanthine.

[0029] The purine concentration in the beer-flavored alcoholic beverage of the present invention can be reduced by known methods. Non-limiting examples of methods for reducing the purine concentration in a beer-flavored alcoholic beverage include a method in which a pre-fermentation solution or a fermentation solution is contacted with an adsorbent such as activated carbon or zeolite to reduce the purine concentration in the beverage (see JP 2003-169658 A, JP 2004-290071 A, JP 2004-290072 A, JP 2015-112090 A, etc.), and a method in which the purine concentration in a beverage is reduced using raw materials other than barley malt that contain less purine (e.g., wheat malt, soy protein, corn grits) (see JP 2014-117204 A, JP 2014-117205 A, JP 2022-140798 A, etc.).

[0030] The alcohol concentration in the beer-taste alcoholic beverage of the present invention is not particularly limited, but is preferably greater than 1% by volume (v / v%), more preferably 2% by volume (v / v%) or more, even more preferably 3% by volume (v / v%) or more, even more preferably 3.5% by volume or more, and even more preferably 4% by volume or more. The upper limit of the alcohol concentration in the beer-taste alcoholic beverage is not particularly limited as long as the effects of the present invention are achieved, but is, for example, 20% by volume, preferably 10% by volume, and more preferably 8% by volume. According to one embodiment of the present invention, the alcohol concentration in the beer-taste alcoholic beverage of the present invention is preferably 2 to 10% by volume, more preferably 3 to 10% by volume, and even more preferably 3 to 8% by volume.

[0031] The beer-taste alcoholic beverage of the present invention can be a carbonated beverage. The carbon dioxide pressure can be adjusted as desired, for example, within the range of 0.05 to 0.4 MPa (gas pressure at 20°C).

[0032] The pH of the beer-taste alcoholic beverage of the present invention can be adjusted to, for example, 2.0 to 5.0, preferably 2.3 to 4.9, and more preferably 2.9 to 4.9. The pH of the beverage can be easily measured using a commercially available pH meter (e.g., a tabletop pH meter manufactured by Horiba, Ltd.).

[0033] The beer-taste alcoholic beverage of the present invention is preferably provided as a packaged beverage. The container used for the beer-taste alcoholic beverage of the present invention may be any container commonly used for filling beverages, such as a metal can, a keg, a plastic bottle (e.g., a PET bottle or cup), a paper container, a bottle, or a pouch, with metal cans, keg containers, plastic bottles (e.g., a PET bottle), or bottles being preferred.

[0034] According to one embodiment of the present invention, the beer-taste alcoholic beverage of the present invention can be produced according to a typical method for producing a low-sugar beer-taste alcoholic beverage, except for adjusting the concentrations of the three free active ingredients and the three bound active ingredients in the beverage. Typical production methods include, for example, fermenting a pre-fermentation solution containing at least water and malt, i.e., adding fermenting beer yeast to a wort (pre-fermentation solution) prepared from brewing raw materials such as malt, fermenting the wort, and optionally storing the fermented solution at low temperature followed by filtration to remove the yeast. Furthermore, hydrolysis treatment using carbohydrate-degrading enzymes, cellulolytic enzymes, etc., may be performed at any time before the completion of the fermentation process (e.g., during brewing, fermentation, or both). Examples of carbohydrate-degrading enzymes include enzymes derived from brewing raw materials such as malt, and commercially available enzymes (e.g., α-amylase, β-amylase, glucoamylase, α-glucosidase, pullulanase, isoamylase, etc.). Examples of cellulolytic enzymes include commercially available enzymes (e.g., β-glucanase, xylanase, hemicellulase, etc.).

[0035] Hops (including processed hop products) can be added at any step in the production process of the beer-taste alcoholic beverage of the present invention. The amount of hops added can typically be adjusted to 0.1 to 5 g / L, preferably 0.1 to 2 g / L, and more preferably 0.2 to 1.5 g / L, calculated as dried hop cones relative to the volume of the pre-fermentation solution in the fermentation step.

[0036] In the present invention, in addition to malt, hops, and water, other brewing raw materials can be used, such as rice, corn, koryan (sorghum), potato, starch, sugars (e.g., liquid sugar), fruit, and coriander, as defined by the Liquor Tax Act, as well as other additives such as protein hydrolysates, nitrogen sources such as yeast extract, colorants, foaming and foam retention improvers, water quality conditioners, and fermentation aids. Ungerminated barley (e.g., ungerminated barley (including extracts) and ungerminated wheat (including extracts)) can also be used as brewing raw materials.

[0037] According to another aspect of the present invention, there is provided a method for enhancing the richness of a low-sugar, beer-taste alcoholic beverage, in which the total content of free carboxymethyllysine (CML), free carboxyethyllysine (CEL), and free methylglyoxal-derived hydroimidazolone-1 (MG-H1) in the beverage is adjusted to 434 ppb or more, preferably 438 ppb or more.

[0038] According to another aspect of the present invention, there is provided a method for reducing unpleasant sourness in a low-sugar, beer-taste alcoholic beverage, in which the total content of free carboxymethyllysine (CML), free carboxyethyllysine (CEL), and free methylglyoxal-derived hydroimidazolone-1 (MG-H1) in the beverage is adjusted to 434 ppb or more, preferably 438 ppb or more.

[0039] According to another aspect of the present invention, there is provided a method for reducing the alcoholic harshness of a low-sugar, beer-taste alcoholic beverage, in which the total content of free carboxymethyllysine (CML), free carboxyethyllysine (CEL), and free methylglyoxal-derived hydroimidazolone-1 (MG-H1) in the beverage is adjusted to 434 ppb or more, preferably 438 ppb or more.

[0040] The present invention will be specifically described based on the following examples, but the present invention is not limited to these examples.

[0041] In the following examples, the concentration of free Maillard reaction products (F-MRP) is the sum of the concentrations of free CML, free CEL, and free MG-H1. The concentration of bound Maillard reaction products (B-MRP) is the sum of the concentrations of bound CML, bound CEL, and bound MG-H1.

[0042] Example 1: Test brewing of beer-flavored beverages Low-sugar (including zero-sugar) beer-flavored beverages were prepared (also referred to as production) by test brewing as follows. Milled barley malt was used in a blending ratio of 50% by mass or more of malt, and saccharification was carried out using a carbohydrate-degrading enzyme and a cellulose-degrading enzyme. After being poured into a brewing tank containing warm water maintained at 50-60°C, the temperature was gradually increased, and a saccharified solution was obtained by adjusting the temperature and time conditions. The saccharified solution was then filtered to remove malt grains, and wort was obtained. Hops were added to the obtained wort, which was then boiled, subjected to solid-liquid separation, and cooled to obtain a clear wort. By adding yeast, adjusting the fermentation temperature and time, and filtering the resulting fermented liquid, test samples A to K (sample numbers 1 to 3, 9 to 11, 16, 19, 23 to 24, and 27) and test samples L to O, P to R, and T to X (sample numbers 30 to 33, 34, 49, 50, and 52 to 56) were prepared. Test sample S (sample number 51) was prepared in the same manner as test samples A to R and T to X, except that part of the barley malt was replaced with wheat malt. All of the obtained samples had an alcohol concentration of 4.6% and a pH of 3.8 to 4.1. The sugar content of test samples A to K and P to V was 0.3 to 0.4 g / 100 mL, and the sugar content of test samples L to O, W, and X was 0.6 to 0.9 g / 100 mL.

[0043] Example 2: Quantification of free and bound Maillard reaction products (MRPs) (1) Quantification of free Maillard reaction products (F-MRPs) Quantification of free Maillard reaction products fractions was performed by LC-MS / MS. Liquid samples or lyophilized samples were redissolved in ultrapure water. An equal volume of 6N sulfosalicylic acid was added and stirred. Centrifuged at 13,000 rpm for 5 minutes. The resulting supernatant was collected and mixed with one-third of the volume of 20% (v / v) methanol. The analytical sample was loaded onto a solid-phase extraction column (Bond Elut C18, Agilent Technologies), followed by loading an equal volume of 10% methanol. The resulting eluates were combined and stirred. The samples were then mixed 1:1 with separately prepared standard solutions (CEL, CML: 0, 50, 100, 200 ppb; MG-H1: 0, 100, 200, 400 ppb) and subjected to LC-MS / MS under the following conditions. The peak areas of the two ions obtained were used to calculate the Maillard reaction product concentrations in the samples by the standard addition method.

[0044]

[0045]

[0046] (2) Gel filtration fractionation The sample was filtered through a 0.45 μm filter, and the filtered fermentation broth was weighed and freeze-dried. The dried product was dissolved in 100 mM NaCl solution to prepare a 5-fold concentrated solution. The resulting concentrated solution was subjected to gel filtration fractionation under the following conditions, and a fraction between 0.66 CV (column volume) and 0.86 CV was collected and subjected to molecular weight analysis and quantification of bound Maillard reaction products.

[0047]

[0048] (3) Molecular weight analysis by HPLC gel filtration The fractions collected in (2) were dissolved in 50 mM sodium phosphate buffer (pH 7.0 containing 150 mM NaCl) and used as samples for molecular weight analysis, whereupon HPLC gel filtration was carried out as follows.

[0049]

[0050] To calculate molecular weights from the obtained chromatograms, 50 μL of peptides with known molecular weights dissolved in ultrapure water at concentrations ranging from 0.1 to 5 mg / mL were injected and subjected to HPLC gel filtration analysis under the same conditions to confirm retention times (Table 5). A calibration curve was created from the retention times and molecular weights, and the molecular weight range was determined based on the retention times from the start to the end of the main peak in the chromatogram of the fractions collected in (2). As a result, the molecular weights of the fractions were found to be between 400 and 3,000 Da.

[0051]

[0052] (4) Quantification of bound Maillard reaction products (B-MRP) To quantify bound Maillard reaction products, peptides and proteins in the sample were enzymatically hydrolyzed, and the free Maillard reaction products of the enzyme blank were analyzed by LC-MS / MS. The difference between the enzyme blank and the free Maillard reaction products was taken as the amount of bound Maillard reaction products. Sample treatment was performed as follows.

[0053] The fraction isolated in (2) was dialyzed for 24 hours using a 1 kDa dialysis membrane, and the resulting solution was lyophilized. The sample was then dissolved in 0.02 M hydrochloric acid containing pepsin and incubated at 37°C for 24 hours. Subsequently, a Tris buffer solution (pH 8.2) containing pronase E was added, mixed, and incubated at 37°C for 24 hours. Aminopeptidase M and prolidase were then added, mixed, and incubated at 37°C for 24 hours to cleave peptide bonds. This enzymatic reaction was repeated three times for 24 hours at 37°C, with a blank containing no enzyme buffer. The sample was lyophilized and redissolved in ultrapure water. An equal volume of 6 N sulfosalicylic acid was added, stirred, and centrifuged at 13,000 rpm for 5 minutes. The resulting supernatant was then mixed with 1 / 3 of the volume of 20% (v / v) methanol. The analytical sample was loaded onto a solid-phase extraction column (Bond Elut C18), followed by loading an equal volume of 10% methanol. The resulting eluates were combined and stirred. Then, the sample was mixed 1:1 with a separately prepared standard solution (CEL, CML: 0, 50, 100, 200 ppb; MG-H1: 0, 100, 200, 400 ppb). The sample was subjected to LC-MS / MS under the same conditions as in (1), and the concentration of bound Maillard reaction products in the sample was calculated by the standard addition method.

[0054] Example 3: Tasting with the addition of purified MRP fraction (1) Purification of Maillard reaction product fraction From samples whose "full-bodiedness" sensory evaluation score described in Example 1 was 4.2 or higher (score when evaluated according to the following evaluation criteria), the fraction obtained by the method of Example 2(2) was adsorbed onto a C18 solid-phase extraction column (Bond Elut C18). After washing with pure water, the obtained flow-through fraction and washings were further adsorbed onto a Diaion HP20 (Mitsubishi Chemical) column and washed with pure water. The adsorbed material from the C18 solid-phase extraction column was eluted with a 50% (v / v) aqueous ethanol solution. The eluate from the C18 solid-phase extraction column was concentrated to dryness and reconstituted with pure water to obtain a purified product of bound Maillard reaction products.

[0055] The fraction that passed through the Diaion HP20 column was freeze-dried and concentrated, and then dialyzed for about 10 hours using a dialysis membrane with a molecular weight of 100 to 500 Da until the electrical conductivity due to NaCl decreased. The external dialysis solution was replaced and dialyzed for another 20 hours. The external dialysis solution after removing NaCl was freeze-dried and then reconstituted with pure water to obtain a concentrate. This was used as a purified product of free Maillard reaction products.

[0056] The bound Maillard reaction products (B-MRP) and free Maillard reaction products (F-MRP) contained in each purified product were quantified by the method described in Example 2 and used for the added tasting.

[0057] (2) Tasting with the addition of purified MRP fractions To the brewed product B prepared in Example 1 and the brewed products C to K (sample numbers 3, 11, 16, 19, 24, and 27), purified MRP was added to the concentrations shown in the table below to obtain each sample (sample numbers 4 to 8, 12 to 15, 17 to 18, 20 to 22, 25 to 26, and 28 to 29). The obtained tasting samples were subjected to a sensory evaluation by a trained panel of five people, using the brewed product A (sample number 1) as a control. The evaluation items and evaluation criteria were as follows:

[0058] As evaluation item 1, the strength of body (an overall evaluation consisting of flavor strength, complexity, and persistence) was rated on a 9-point scale from 1 point (weak) to 9 points (strong). As evaluation item 2, "unpleasant sourness" (unpleasant sourness felt in the aftertaste, etc.) was rated on a 9-point scale from 1 point (weak) to 9 points (strong). For the sensory evaluation, the sample A prepared in Example 1 was used as a control (defined as body strength: 2.4, unpleasant sourness: 5.0).

[0059] The evaluation results are shown in the table below.

[0060] The control samples, Sample A and Sample B, had a score of less than 3 for "full body" and a score of 5.0 or more for "unpleasant sourness," indicating a very weak full body and an unpleasant sourness. Samples with a score of 4.2 or more for "full body" and a score of 4.8 or less for "unpleasant sourness" had a strong full body and a weak unpleasant sourness. Furthermore, samples with a score of 5.4 or more for "full body" and a score of 4.0 or less for "unpleasant sourness" had a strong full body and a weak unpleasant sourness. These samples were considered to have a favorable flavor and aroma for a beer-taste beverage. Therefore, a score of 4.2 or more for "full body" was evaluated as being preferable for the flavor and aroma of a beer-taste beverage, with a score of 5.4 or more being more preferable.

[0061] A plot showing the relationship between MRP concentration and the evaluation results of "fullness" (vertical axis: bound MRP concentration, horizontal axis: free MRP concentration, bubble size: fullness) is shown in Figure 1. This figure shows that the total content of free MRP in a beer-taste alcoholic beverage is preferably 438 ppb or more. Furthermore, although there is no need to limit the total content of bound MRP, it was found that it is preferable that it be 72 ppb or more, and even more preferable that it be 117 ppb or more.

[0062] Next, the above evaluation results are sorted and expressed by the total amount of F-MRP and B-MRP, and are shown in the table below.

[0063] A plot showing the relationship between MRP concentration and the results of the "full-body" evaluation (vertical axis: bound MRP concentration, horizontal axis: free MRP concentration, bubble size: full-body, diagonal auxiliary line: boundary between the total amount of F-MRP and B-MRP) is shown in Figure 2. From this figure, when the total amount of F-MRP and B-MRP in a beer-taste alcoholic beverage is 510 ppb or more within the F-MRP concentration range of 438 ppb or more, the "full-body" score is 4.2 or more and the "unpleasant sourness" score is 4.8 or less, indicating a full-body flavor with a weak unpleasant sourness, and considered to be a favorable flavor for a beer-taste beverage. Furthermore, when the total amount of F-MRP and B-MRP is 550 ppb or more, the "full-body" score is 4.2 or more and the "unpleasant sourness" score is 4.6 or less, indicating a full-body flavor with a weak unpleasant sourness, considered to be even more favorable. Furthermore, when the total amount of F-MRP and B-MRP was 672 ppb or more, the score for "full body" was 4.4 or more and the score for "unpleasant sourness" was 4.2 or less, indicating a rich flavor and a weak unpleasant sourness, which were considered even more preferable. Furthermore, when the total amount of F-MRP and B-MRP was 1,042 ppb or more, the score for "full body" was 5.2 or more and the score for "unpleasant sourness" was 3.6 or less, indicating a strong rich flavor and a weak unpleasant sourness, which were considered even more preferable. Furthermore, when the total amount of F-MRP and B-MRP was 1,203 ppb or more, the score for "full body" was 6.4 or more and the score for "unpleasant sourness" was 3.6 or less, indicating a strong rich flavor and a weak unpleasant sourness, which were considered even more preferable.

[0064] Next, the above evaluation results are sorted and expressed by the ratio of F-MRP to B-MRP (F-MRP / B-MRP), and the results are shown in the table below.

[0065] A plot showing the relationship between MRP concentration and the evaluation results of "fullness" (vertical axis: bound MRP concentration, horizontal axis: free MRP concentration, bubble size: fullness, diagonal auxiliary line: boundary line of F-MRP / B-MRP ratio) is shown in Figure 3. From this figure, it can be seen that when the F-MRP / B-MRP ratio in a beer-taste alcoholic beverage was 5.06 to 6.65 within the F-MRP range of 438 ppb or more, the "fullness" score was 4.2 to 4.8 and the "unpleasant sourness" score was 4.8 to 4.0, indicating a rich flavor and a weakly unpleasant sourness, which were considered to be favorable for a beer-taste beverage. Furthermore, when the F-MRP / B-MRP ratio was 6.66 to 8.37, the "full body" score was 4.2 to 5.8 and the "unpleasant sourness" score was 4.6 to 3.6, indicating a rich flavor and a weaker unpleasant sourness, which were considered even more preferable. Furthermore, when the F-MRP / B-MRP ratio was 3.63 to 5.05, the "full body" score was 6.0 to 6.4 and the "unpleasant sourness" score was 3.6 to 3.2, indicating a rich flavor and a weaker unpleasant sourness, which were considered even more preferable. Furthermore, when the F-MRP / B-MRP ratio was 0.53 to 3.62, the "full body" score was 5.4 to 8.2 and the "unpleasant sourness" score was 4.0 to 2.6, indicating a strong rich flavor and a weaker unpleasant sourness, which were considered even more preferable. Furthermore, when the F-MRP / B-MRP ratio was 8.38 or higher, the score for "richness" was 5.0 to 6.4 and the score for "unpleasant sourness" was 3.8 to 3.0, indicating a stronger richness and a weaker unpleasant sourness, which was considered even more preferable.

[0066] (3) Sensory Evaluation of Beer-Taste Beverages with Carbohydrate Content of 1 g / 100 mL or Less A trained panel of five members conducted a sensory evaluation of beer-taste beverage samples L to O (sample numbers 30 to 33), each with a carbohydrate content of 1 g / 100 mL or less, using sample A prepared in Example 1 as a control. The evaluation items and criteria were as described above.

[0067] The evaluation results are shown in the table below.

[0068] Within the same numerical range as (2) above, the same enhancement of richness and suppression of unpleasant sourness as the evaluation results described above were confirmed. Therefore, it is believed that a similar effect can be expected for beer-flavored beverages with a carbohydrate content of 1 g / 100 mL or less.

[0069] (4) Evaluation of samples with the same sum of B-MRP concentrations but different compositions of individual components The combinations of samples with approximately the same sum of F-MRP or B-MRP concentrations but different compositions of individual components and the results of the sensory evaluation are shown in the table below.

[0070]

[0071] Compared with sample 10, sample 9 contained less bound CEL and MG-H1 and more CML, but the total amount of the three components, the richness of the flavor, and the unpleasant sourness were all comparable.

[0072] From the above results, it was considered that the three active ingredients would have the same flavor effect if the total amount was the same, regardless of the composition of the individual components.

[0073] (5) Measurement of Purine Concentration The purine concentrations of the test products E and J prepared in Example 1 were measured. Specifically, with reference to Journal of Food Hygiene 55(2):110-116 (2014), the samples were decomposed with perchloric acid and the purine concentrations (mg / 100 mL) were measured by LC-MS / MS. LC-MS / MS was performed with reference to Measurement Method II in the above literature. The measurement results are shown in the table below.

[0074]

[0075] The purine concentrations of Sample 10 and Sample 24 were similar.

[0076] Example 4: Tasting with added purified MRP fraction (2) (1) Purification of Maillard reaction product fraction The purified bound Maillard reaction products and the purified free Maillard reaction products used were the purified products described in Example 3(1).

[0077] (2) Tasting with the addition of purified MRP fractions To the brewed product P (sample 34) prepared in Example 1, purified MRP was added to the concentrations shown in the table below to obtain each sample (samples 35 to 48). Note that for samples 39 and 40, free MRP, which has a different composition of individual components, was added to obtain the same free MRP concentration. The obtained tasting samples were subjected to a sensory evaluation by a trained panel of six people, using brewed product P (sample 34) as a control. The evaluation items and evaluation criteria were as follows:

[0078] As evaluation item 1, similar to Example 3(2), the strength of body (an overall evaluation consisting of flavor strength, complexity, and persistence) was evaluated on a nine-point scale from 1 point (weak) to 9 points (strong). As evaluation item 2, similar to Example 3(2), the "unpleasant sourness" (unpleasant sourness felt in the aftertaste, etc.) was evaluated on a nine-point scale from 1 point (weak) to 9 points (strong). As evaluation item 3, the "alcoholic bitterness" (spiciness accompanied by a tingling sensation) was evaluated on a nine-point scale from 1 point (almost no alcoholic bitterness) to 9 points (strong alcoholic bitterness). For the sensory evaluation, the brewed product P (Sample 34) prepared in Example 1 was used as a control (defined as body strength: 2.5, unpleasant sourness: 5.2, and alcoholic bitterness: 5.2).

[0079] The evaluation results are shown in the table below.

[0080]

[0081] The control, brewed product P (Sample 34), had a score of less than 3 for "full body", a score of 5.0 or more for "unpleasant sourness", and a score of 5.0 or more for "alcohol harshness", indicating a very weak full body, an unpleasant sourness, and an alcohol harshness. Samples with a score of 4.0 or more for "full body" and a score of 4.8 or less for "unpleasant sourness" had a rich body and a weak unpleasant sourness. Furthermore, samples with a score of 4.0 or less for "alcohol harshness" were perceived as having a weak alcohol harshness. These samples were considered to have a good flavor and aroma as a beer-taste beverage. Therefore, a beer-taste beverage with a score of 4.0 or more for "full body" can also be evaluated as having a favorable flavor and aroma.

[0082] (3) Evaluation of samples with the same free MRP (F-MRP) concentration but different compositions of individual components Samples 39 and 40 in Example 4(2) have the same F-MRP concentration but different compositions of individual components, and their compositions are shown in the table below.

[0083]

[0084] Sample 39 contained less free CEL and CML and more MG-H1 than Sample 40. On the other hand, as shown in Table 12, the total amount of the three components, the richness, unpleasant sourness, and alcoholic bitterness were similar.

[0085] From the above results, it was considered that the three active ingredients would have the same flavor effect if the total amount was the same, regardless of the composition of the individual components.

[0086] Example 5: Tasting of beer-taste beverage sample Q with different MRP concentrations (1) Tasting of beer-taste beverage Sample Q prepared in Example 1 was used as a tasting sample and subjected to a sensory evaluation in the same manner as in Example 4. The evaluation items and evaluation criteria were the same as in Example 4.

[0087] The evaluation results are shown in the table below.

[0088] Within the same numerical range as in Example 4 (2) above, the same enhancement of richness, suppression of unpleasant sourness, and suppression of alcohol harshness as the evaluation results described above were confirmed. Therefore, it was thought that similar effects could be expected for tasting samples of test products with different MRP concentrations.

[0089] Example 6: Tasting of beer-flavored beverage samples R to Y with different purine concentrations (1) Measurement of purine concentration The purine concentrations of the beer-flavored beverage samples R to X prepared in Example 1 were measured in the same manner as in the measurement of purine concentration described in Example 3(5). The measurement results are shown in the table below.

[0090] (2) Tasting of Beer-Taste Beverages with Purine Concentrations of 1.5 mg / 100 mL or Less A sensory evaluation was conducted in the same manner as in Example 4, using beer-taste beverage samples R to X (sample numbers 50 to 56) with purine concentrations of 1.5 mg / 100 mL or less as tasting samples and sample P (sample 34) prepared in Example 1 as a control. The evaluation items and evaluation criteria were the same as in Example 4. The evaluation results are shown in the table below.

[0091] As described in Example 4(2), the control brewed product P (Sample 34) had a score of less than 3 for "full body," a score of 5.0 or more for "unpleasant sourness," and a score of 5.0 or more for "alcohol harshness," indicating a very weak full body, an unpleasant sourness, and an alcohol harshness. On the other hand, Samples 50 to 56 had scores of 5.3 or more for "full body," and scores of 4.0 or less for "unpleasant sourness," indicating a strong full body and a weak unpleasant sourness. Furthermore, samples with a score of 4.0 or less for "alcohol harshness" were perceived as having a weak alcohol harshness. Therefore, it was considered to have a favorable flavor and taste for a beer-flavored beverage, and it was thought that similar effects could be expected for beer-flavored beverages with a purine concentration of 1.5 mg / 100 mL or less. Furthermore, Samples 50 to 56 had F-MRP levels of 438 ppb or higher in the beer-taste alcoholic beverage, F-MRP / B-MRP ratios of 1.95 to 2.07, scores of 5.3 to 5.7 for "full body" and scores of 3.3 to 3.9 for "unpleasant sourness," indicating a strong full body and a weak unpleasant sourness, and similar to Example 3(2), these were considered to be even more desirable as beer-taste beverages. Therefore, a beer-taste beverage with a score of 5.3 or higher for "full body" can also be evaluated as even more desirable in terms of flavor.

Claims

1. A low-sugar, beer-flavored alcoholic beverage in which the total content of free carboxymethyllysine (CML), free carboxyethyllysine (CEL), and free methylglyoxal-derived hydroimidazolone-1 (MG-H1) in the beverage is 434 ppb or more.

2. A low-sugar beer-flavored alcoholic beverage as described in claim 1, wherein the total content of bound CML, bound CEL, and bound MG-H1 in the beverage is 65 ppb or more, and the bound CML, bound CEL, and bound MG-H1 are contained in a peptide fraction having a molecular weight of 400 to 3,000 Da.

3. A low-sugar beer-flavored alcoholic beverage according to claim 2, in which the total content of bound CML, bound CEL and bound MG-H1 in the beverage is 72 ppb or more.

4. A low-sugar beer-flavored alcoholic beverage as described in claim 1, in which the total content of free CML, free CEL, free MG-H1, bound CML, bound CEL and bound MG-H1 in the beverage is 499 ppb or more, and the bound CML, bound CEL and bound MG-H1 are contained in a peptide fraction having a molecular weight of 400 to 3,000 Da.

5. A low-sugar beer-flavored alcoholic beverage according to claim 4, in which the total content of free CML, free CEL, free MG-H1, bound CML, bound CEL and bound MG-H1 in the beverage is 510 ppb or more.

6. A low-sugar beer-flavored alcoholic beverage according to claim 4, in which the total content of free CML, free CEL, free MG-H1, bound CML, bound CEL and bound MG-H1 in the beverage is 1042 ppb or more.

7. A low-sugar beer-flavored alcoholic beverage according to claim 1, wherein the ratio (F / B) of the total content (F ppb) of free CML, free CEL and free MG-H1 in the beverage to the total content (B ppb) of bound CML, bound CEL and bound MG-H1 in the beverage is 0.53 or more, and the bound CML, bound CEL and bound MG-H1 are contained in a peptide fraction having a molecular weight of 400 to 3,000 Da.

8. The low-sugar beer-flavored alcoholic beverage according to claim 7, wherein F / B is 0.53 to 3.

63.

9. The low-sugar beer-flavored alcoholic beverage according to claim 7, wherein F / B is 6.66 or more.

10. A low-sugar beer-flavored alcoholic beverage according to claim 1 or 2, having a purine concentration of 5.0 mg / 100 mL or less.

11. A method for producing a low-sugar beer-taste alcoholic beverage, wherein the total content of free carboxymethyllysine (CML), free carboxyethyllysine (CEL) and free methylglyoxal-derived hydroimidazolone-1 (MG-H1) in the beverage is adjusted to 434 ppb or more.

12. A method for enhancing richness in a low-sugar beer-taste alcoholic beverage, comprising adjusting the total content of free carboxymethyllysine (CML), free carboxyethyllysine (CEL) and free methylglyoxal-derived hydroimidazolone-1 (MG-H1) in the beverage to 434 ppb or more.

13. A method for reducing unpleasant sourness in a low-sugar beer-taste alcoholic beverage, comprising adjusting the total content of free carboxymethyllysine (CML), free carboxyethyllysine (CEL) and free methylglyoxal-derived hydroimidazolone-1 (MG-H1) in the beverage to 434 ppb or more.

14. A method for reducing the alcohol harshness in a low-sugar beer-flavored alcoholic beverage, the method comprising adjusting the total content of free carboxymethyllysine (CML), free carboxyethyllysine (CEL) and free methylglyoxal-derived hydroimidazolone-1 (MG-H1) in the beverage to 434 ppb or more.

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