Non-alcoholic beer-flavored beverage having enhanced richness

Abstract

Disclosed is a non-alcoholic beer-flavored beverage in which the total amount of free carboxymethyl lysine (CML), free carboxyethyl lysine (CEL), and free methylglyoxal-derived hydroimidazolone-1 (MG-H1) is 160 ppb or greater. The non-alcoholic beer-flavored beverage has enhanced richness.

Description

A non-alcoholic beer-flavored beverage with enhanced richness

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

[0002] In recent years, there has been an increasing demand for non-alcoholic beer-flavored beverages due to health concerns. However, non-alcoholic beer-flavored beverages have various flavor and balance issues due to their low alcohol content.

[0003] In the art, various efforts have been made to solve such problems, and for example, Patent Document 1 discloses a method of adding a freeze-dried product derived from beer to impart a beer-like flavor to a beer-like beverage. Also, Patent Document 2 discloses a malt beverage using dark malt and a coloring agent, a method for producing the same, and a method for masking unpleasant flavors.

[0004] 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.

[0005] JP 2013-201976 A JP 2013-188160 A

[0006] Food Chemistry, 99, 600-604

[0007] The present inventors have discovered the problem that non-alcoholic beer-flavored beverages lack body and the grain flavor characteristic of beer.

[0008] The present inventors have investigated the above-mentioned problems and found that some of the Maillard reaction products (MRPs) produced during the heating and aging process can enhance the body of non-alcohol beer-flavored beverages within a specific concentration range. The present invention is based on this finding.

[0009] Therefore, the present invention provides a non-alcohol beer-taste beverage with enhanced body and a method for producing the same.

[0010] The present invention provides the following: (1) A non-alcoholic beer-flavored 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 160 ppb or more. (2) A non-alcoholic beer-flavored beverage according to (1) above, in which the total content of free CML, free CEL, and free MG-H1 in the beverage is 250 to 800 ppb or more. (3) A non-alcoholic beer-flavored beverage according to (1) or (2) above, in which the total content of bound CML, bound CEL, and bound MG-H1 in the beverage is 35 ppb or more, and the bound CML, bound CEL, and bound MG-H1 are contained in a peptide fraction with a molecular weight of 400 to 3,000 Da. (4) The non-alcoholic beer-taste beverage according to (3) above, in which the total content of bound CML, bound CEL, and bound MG-H1 in the beverage is 35 to 300 ppb. (5) The non-alcoholic beer-taste beverage according to any one of (1) to (4) above, in which the alcohol (ethanol) concentration is less than 0.01 v / v%. (6) A method for producing a non-alcoholic beer-taste beverage, the method comprising the step of adjusting the total content of free CML, free CEL, and free MG-H1 in the non-alcoholic beer-taste beverage to 160 ppb or more. (7) The method according to (6) above, further comprising the step of adjusting the total content of bound CML, bound CEL, and bound MG-H1 in the beverage to 35 ppb or more, in which 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 method according to (6) or (7) above, wherein the alcohol (ethanol) concentration of the non-alcoholic beer-taste beverage is less than 0.01 v / v %. (9) A method for enhancing the body of a non-alcoholic beer-taste beverage, comprising the step of adjusting the total content of free CML, free CEL, and free MG-H1 in the non-alcoholic beer-taste beverage to 160 ppb or more.(10) The method according to (9) above, further comprising the step of adjusting the total content of bound CML, bound CEL, and bound MG-H1 in the beverage to 35 ppb or more, wherein 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. (11) A method for enhancing the beer-like graininess in a non-alcoholic beer-taste beverage, comprising the step of adjusting the total content of free CML, free CEL, and free MG-H1 in the non-alcoholic beer-taste beverage to 160 ppb or more. (12) The method according to (11) above, further comprising the step of adjusting the total content of bound CML, bound CEL, and bound MG-H1 in the beverage to 35 ppb or more, wherein 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. (13) The method according to any one of (9) to (12) above, wherein the alcohol (ethanol) concentration of the non-alcoholic beer-taste beverage is less than 0.01 v / v%.

[0011] According to the present invention, it is possible to enhance the richness of a non-alcohol beer-taste beverage, and it is also possible to enhance the beer-like grain flavor of a non-alcohol beer-taste beverage.

[0012] Fig. 1 is a plot diagram showing the relationship between MRP concentration and the evaluation results of "full body" (vertical axis: bound MRP concentration, horizontal axis: free MRP concentration, bubble size: full body). Fig. 2 is a plot diagram showing the relationship between MRP concentration and the evaluation results of "beer-like grain flavor" (vertical axis: bound MRP concentration, horizontal axis: free MRP concentration, bubble size: beer-like grain flavor). Specific Description of the Invention

[0013] As described above, the present invention makes it possible to enhance the body of a non-alcoholic beer-taste beverage and also to enhance the beer-like grain flavor. The present invention is particularly advantageous in that it makes it possible to enhance the body and beer-like grain flavor even in a non-alcoholic beer-taste beverage with an alcohol content of 0.00 v / v% obtained through a wort fermentation process. In particular, it has been confirmed that the present invention not only enhances the grain flavor, but also enhances the beer-like grain flavor, and this effect is included in the effects described in the examples below.

[0014] In the present invention, the term "beer-taste beverage" refers to a beverage that has a beer-like flavor. "Beer-like flavor" means that the beverage exhibits the unique taste and aroma of beer that can be obtained when beer is produced in the usual way, i.e., when beer is produced based on fermentation using yeast or the like.

[0015] In the present invention, the term "non-alcoholic beer-taste beverage" refers to a beer-taste beverage with an alcohol (ethanol) concentration of less than 1% (v / v). According to a preferred embodiment of the present invention, the alcohol concentration of the non-alcoholic beer-taste beverage of the present invention is less than 0.75% (v / v), more preferably less than 0.5% (v / v), even more preferably less than 0.05% (v / v), even more preferably less than 0.01% (v / v), even more preferably less than 0.009% (v / v), even more preferably less than 0.008% (v / v), even more preferably less than 0.007% (v / v), and even more preferably The alcohol content is preferably less than 0.006% (v / v), more preferably less than 0.005% (v / v), even more preferably less than 0.004% (v / v), even more preferably less than 0.003% (v / v), and even more preferably less than 0.002% (v / v), and is preferably 0.002% (v / v) or more, more preferably 0.003% (v / v) or more, even more preferably 0.003% (v / v) or more, and even more preferably 0.004% (v / v) or more. Alcohol (ethanol) is a component that contributes to the harmony of the beer-like flavor and aroma in alcoholic beer-flavored beverages, and the lower its concentration, the more likely flavor problems will arise.

[0016] The alcohol concentration in a beverage can be quantified by gas chromatography (GC) with an FID detector. To achieve more accurate concentration measurements, it is desirable to use a calibration curve based on the measurements of several control samples with known concentrations. It is preferable that these control samples have a concentration in the same range as the concentration to be measured. It is also preferable to use an internal standard, such as 2-propanol.

[0017] 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:

[0018] 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."

[0019] In this specification, the unit "ppm" is synonymous with "mg / L", and the unit "ppb" is synonymous with "μg / L".

[0020] The non-alcoholic beer-taste 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 non-alcoholic beer-taste beverage of the present invention also contains three bound active ingredients, the total content of which is within a predetermined range. Such a non-alcoholic beer-taste 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.

[0021] The total content of the three free active ingredients in the non-alcohol beer-taste beverage of the present invention is 160 ppb or more, and preferably 250 to 800 ppb. Furthermore, the total content of the three free active ingredients in the non-alcohol beer-taste beverage of the present invention may be 160 to 1000 ppb, 200 to 1000 ppb, 300 to 1000 ppb, 400 to 1000 ppb, 160 to 900 ppb, 200 to 900 ppb, 300 to 900 ppb, 400 to 900 ppb, or 160 to 80 The concentration may be 0 ppb, 200-800 ppb, 300-1000 ppb, 400-1000 ppb, 160-700 ppb, 200-700 ppb, 300-700 ppb, 400-700 ppb, 160-600 ppb, 200-600 ppb, 300-600 ppb, or 400-600 ppb. The free active ingredients may be derived from the raw material, added separately from the plant raw material, or produced by fermentation. The concentration of the free active ingredients can be controlled, for example, by controlling the composition of the raw material and the fermentation conditions.

[0022] According to a preferred embodiment of the present invention, the total content of the three bound active ingredients in the non-alcoholic beer-taste beverage of the present invention is 0 ppb or more, more preferably 35 ppb or more, and even more preferably 35 to 300 ppb. Furthermore, the total content of the three bound active ingredients in the non-alcoholic beer-taste beverage of the present invention may be 35 to 400 ppb, 50 to 400 ppb, 100 to 400 ppb, 160 to 400 ppb, 35 to 350 ppb, 50 to 350 ppb, 100 to 350 ppb, 160 to 350 ppb, or 35 to 300 ppb, 50 to 300 ppb, 100 to 300 ppb, or 160 to 300 ppb. The three bound active ingredients may be derived from the raw materials, added separately from the plant raw materials, or even produced by fermentation. The concentration of the combined active ingredients can be controlled by, for example, controlling the composition of the raw materials, the processing conditions of the raw materials, the fermentation conditions, and the like.

[0023] 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.

[0024] In a preferred embodiment of the present invention, the carbohydrate content of the non-alcoholic beer-taste beverage of the present invention is 0.5 g / 100 mL or more, preferably 1.0 g / 100 mL or more, and is less than 7.0 g / 100 mL, less than 6.95 g / 100 mL, less than 6.9 g / 100 mL, less than 6.85 g / 100 mL, less than 6.8 g / 100 mL, less than 6.75 g / 100 mL, or less than 6. . Less than 7g / 100mL, less than 6.65g / 100mL, less than 6.6g / 100mL, less than 6.55g / 100mL, less than 6.5g / 100mL, less than 6.4g / 100mL, less than 6.3g / 100mL, less than 6.2g / 100mL, less than 6.1g / 100mL, less than 6.0g / 100mL, less than 5.5g / 100mL, less than 5.0g / 100mL.

[0025] The carbohydrate concentration can be measured by known methods, and can be calculated by subtracting the amounts of water, protein, fat, ash, and dietary fiber from the mass of the sample (see the Nutrition Labeling Standards (Partially revised Consumer Affairs Agency Notification No. 9 of December 16, 2009)).

[0026] The true extract concentration can be 0.3 w / w% or more, preferably 0.5 w / w% or more, more preferably 1 w / w% or more, 1.5 w / w% or more, or 2 w / w% or more, and can be less than 10 w / w%, less than 9 w / w%, less than 8 w / w%, less than 7 w / w%, less than 6 w / w%, or less than 5 w / w%. The "true extract concentration" can be measured, for example, by the EBC method (BCOJ Beer Analysis Methods, 7.2 (2004), edited by the Brewers Association of Japan).

[0027] The non-alcoholic beer-taste beverage of the present invention may contain other ingredients as long as the effects of the present invention are not impaired. That is, the non-alcoholic beer-taste beverage of the present invention can contain, as ingredients, colorants (e.g., caramel color), sweeteners (e.g., high-intensity sweeteners), seasoning ingredients (e.g., amino acids), flavorings (e.g., commercially available beer flavors containing typical beer aroma components), acidulants (e.g., gluconic acid), bittering agents (e.g., hop extract), dietary fiber (e.g., indigestible dextrin), fruit juice, herbs, yeast extract, antioxidants, salts, and the like.

[0028] The pH (measured at 25°C) of the non-alcoholic beer-taste beverage of the present invention is preferably 3.5 or higher, more preferably 3.7 or higher, even more preferably 3.8 or higher, and preferably 4.6 or lower, more preferably 4.4 or lower, even more preferably less than 4.4, even more preferably 4.35 or lower, and even more preferably 4.3 or lower. The pH of the non-alcoholic beer-taste beverage of the present invention can be adjusted by using a pH adjuster, adjusting process (saccharification / fermentation) conditions, or adjusting the blend of raw materials. The pH of the non-alcoholic beer-taste beverage can be measured using a commercially available pH meter (e.g., a benchtop pH meter manufactured by Horiba, Ltd.).

[0029] The EBC color of the non-alcoholic beer-taste beverage of the present invention is preferably 8.5 EBC or less, more preferably 8.4 EBC or less, even more preferably 8.3 EBC or less, even more preferably 8.2 EBC or less, even more preferably 8.1 EBC or less, even more preferably 8.0 EBC or less, even more preferably 7.9 EBC or less, even more preferably 7.8 EBC or less, even more preferably 7.7 EBC or less, even more preferably 7.6 EBC or less, even more preferably 7.5 EBC or less, even more preferably 7.4 EBC or less, even more preferably 7.3 EBC or less, even more preferably 7.2 EBC or less, even more preferably 7.1 EBC or less, and even more preferably 7.0 EBC or less. The EBC color of the non-alcoholic beer-taste beverage of the present invention can be measured using "Revised BCOJ Beer Analysis Method 4.3.8, compiled by the International Technical Committee (Analysis Committee) of the Brewery Association of Japan, Brewery Society of Japan."

[0030] The bitterness value of the non-alcoholic beer-taste beverage of the present invention is preferably 20 or less, more preferably 19.9 or less, even more preferably 19.8 or less, even more preferably 19.7 or less, even more preferably 19.6 or less, even more preferably 19.5 or less, even more preferably 19.4 or less, even more preferably 19.3 or less, even more preferably 19.6 or less, even more preferably 19.5 or less, even more preferably 19.4 or less, even more preferably 19.3 or less, even more preferably 19.2 or less, even more preferably 19.1 or less, even more preferably 19 or less, even more preferably 18.9 or less, even more preferably 18.8 or less, even more preferably 18.7 or less, even more preferably 18.6 or less, even more preferably 18.5 or less, even more preferably 18.4 or less, even more preferably 18.3 or less, even more preferably 18.2 or less, even more preferably 18.1 or less, and even more preferably 18 or less. The bitterness value of the non-alcohol beer-taste beverage of the present invention can be measured using the method described in Section 8.15, Bitterness Value, of BCOJ Beer Analysis Methods (published in 1997).

[0031] The total nitrogen concentration of the non-alcoholic beer-taste beverage of the present invention is preferably 400 mg / L or more, more preferably 405 mg / L or more, even more preferably 410 mg / L or more, even more preferably 415 mg / L or more, even more preferably 420 mg / L or more, even more preferably 425 mg / L or more, even more preferably 430 mg / L or more, even more preferably 435 mg / L or more, even more preferably 440 mg / L or more, even more preferably 445 mg / L or more, even more preferably 450 mg / L or more, even more preferably 500 mg / L or more, even more preferably 550 mg / L or more, and even more preferably 600 mg / L or more. The total nitrogen concentration of the non-alcoholic beer-taste beverage of the present invention can be determined using an analytical method registered as the combustion method by the European Breweries Committee (EBC). Specifically, the "combustion method" involves burning a sample at high temperature, reducing the resulting nitrogen oxides to nitrogen, separating the resulting nitrogen gas in a column, and detecting it with a thermal conductivity detector (Analytica-EBC, 9.9.2 Dumas Combustion Method (1997)).

[0032] According to a preferred embodiment of the present invention, the non-alcoholic beer-taste beverage of the present invention is a non-alcoholic beer-taste beverage made from malt, more preferably a non-alcoholic beer-taste beverage made from barley malt. According to another preferred embodiment of the present invention, the non-alcoholic beer-taste beverage of the present invention is a non-alcoholic beer-taste beverage made from ungerminated barley. According to another preferred embodiment of the present invention, the non-alcoholic beer-taste beverage of the present invention is a non-alcoholic beer-taste beverage made from wort. According to another preferred embodiment of the present invention, the non-alcoholic beer-taste beverage of the present invention is a non-alcoholic beer-taste beverage made from hops. According to another preferred embodiment of the present invention, the non-alcoholic beer-taste beverage of the present invention is a non-alcoholic beer-taste beverage that is not made from malt extract. According to another preferred embodiment of the present invention, the non-alcoholic beer-taste beverage of the present invention is a non-alcoholic beer-taste beverage that is not made from starch. According to another preferred embodiment of the present invention, the non-alcoholic beer-taste beverage of the present invention is a non-alcoholic beer-taste beverage that is not made from corn. According to another preferred embodiment of the present invention, the non-alcoholic beer-taste beverage of the present invention is a non-alcoholic beer-taste beverage that is not made from rice. According to another preferred embodiment of the present invention, the non-alcoholic beer-taste beverage of the present invention is a non-alcoholic beer-taste beverage that is not made from flavorings. According to another preferred embodiment of the present invention, the non-alcoholic beer-taste beverage of the present invention is a non-alcoholic beer-taste beverage that is substantially free of propylene glycol. Here, "substantially free of propylene glycol" means that the beverage may contain a small amount of propylene glycol, which is sometimes contained in non-alcoholic beer-taste beverages produced via a fermentation process using malt, hops, various enzymes, and yeast, but does not contain propylene glycol in amounts exceeding this. For example, the non-alcoholic beer-taste beverage of the present invention may contain propylene glycol at or below the detection limit, or at 20 ppm or less.According to another preferred embodiment of the present invention, the non-alcoholic beer-taste beverage of the present invention is a beverage containing dissolved carbon dioxide, i.e., a non-alcoholic beer-taste carbonated beverage. According to another preferred embodiment of the present invention, the non-alcoholic beer-taste beverage of the present invention is a non-alcoholic beer-taste beverage obtained through a wort fermentation process.

[0033] According to a preferred embodiment of the present invention, the proportion of barley malt in the raw material malt can be, for example, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100%.

[0034] According to a preferred embodiment of the present invention, the non-alcoholic beer-taste beverage of the present invention can be a carbonated beverage. The carbon dioxide pressure (gas pressure at 20°C) can be, for example, 0.05 MPa or more, 0.06 MPa or more, 0.07 MPa or more, 0.08 MPa or more, 0.09 MPa or more, or 0.1 MPa or more, or 0.4 MPa or less, 0.39 MPa or less, 0.38 MPa or less, 0.37 MPa or less, 0.36 MPa or less, or 0.35 MPa or less.

[0035] The non-alcoholic beer-taste beverage of the present invention can be produced by adjusting the content of the three free active ingredients and / or the three bound active ingredients at any stage using a general method well known in the art. Such a general method includes, for example, a method comprising a wort preparation step and a filtration step. This method can be carried out, for example, by sequentially performing the following steps: (a) saccharifying a mixture of malt powder and water and filtering it to obtain wort; (b) adding hops to the obtained wort and then boiling it; (c) cooling the boiled wort; and (d) filtering the cooled wort. Another general method includes a method in which alcohol is removed from a fermented malt beverage such as beer after the beverage has been produced. Methods for removing alcohol from beverages include (i) distillation under reduced pressure or normal pressure to remove alcohol and low-boiling components; (ii) removal of alcohol and low-molecular-weight components using a reverse osmosis (RO) membrane; and (iii) removal of volatile components by adsorption with steam using centrifugal force.

[0036] The non-alcohol beer-taste beverage of the present invention can be produced by using an enzyme preparation at any stage of the fermentation process, such as a saccharifying enzyme such as α-amylase, glucoamylase, or pullulanase, or a protease.

[0037] According to another aspect of the present invention, there is provided a method for enhancing the body of a non-alcoholic beer-taste beverage, the method comprising the step of adjusting the total content of free CML, free CEL, and free MG-H1 in the non-alcoholic beer-taste beverage to 160 ppb or more.

[0038] According to yet another aspect of the present invention, there is provided a method for enhancing the beer-like graininess in a non-alcoholic beer-taste beverage, the method comprising the step of adjusting the total content of free CML, free CEL, and free MG-H1 in the non-alcoholic beer-taste beverage to 160 ppb or more.

[0039] The present invention will be specifically described based on the following examples, but the present invention is not limited to these examples.

[0040] 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.

[0041] Example 1: Preparation of Maillard Reaction Products (F-MRP and B-MRP) 1-1: Trial Production of Non-Alcoholic Beer-Taste Beverage A non-alcoholic beer-taste beverage was prepared by trial production as follows. A fermented malt beverage was produced using malt and hops, and then this beverage was subjected to an alcohol removal process. The resulting liquid was used as a base beverage sample. Specifically, ground malt, an enzyme agent, a water treatment agent, and warm water were added to a brewing tank, and saccharification was carried out at a temperature of 60°C to 78°C. The saccharified liquid was filtered and transferred to a boiling kettle, where hops were added and boiled for 70 minutes. After boiling, additional warm water was added to the resulting mixture to replace the evaporated amount. The heat trough was removed in a whirlpool tank, and the mixture was cooled to 10°C to obtain cold wort. Brewer's yeast was added to this wort, and the wort was fermented at around 10°C for 7 days, after which the brewer's yeast was removed. The resulting mixture was transferred to another tank and aged for 7 days, then cooled to around -1°C and stabilized for 14 days. The resulting mixture was then diluted with degassed water and filtered to obtain a fermented wort liquor (fermented malt beverage). The mixture was then sprayed into a degassing tank to remove carbon dioxide, and heated to approximately 50°C. The mixture was then contacted with steam heated to approximately 50°C in a vacuum column at approximately 60 mbar, allowing the volatile components to adsorb onto the steam. The alcohol and volatile components were then removed, yielding non-alcoholic beer-flavored beverage test samples A to C. Of these, sample C's various analytical values ​​were: alcohol concentration 0.001 v / v%, carbohydrate concentration less than 5.0 g / 100 ml, and true extract concentration 4.56 w / w%.

[0042] 1-2: Quantification of Free and Bound Maillard Reaction Products (MRPs) (1) Quantification of Free Maillard Reaction Products (F-MRPs) Quantification of the free Maillard reaction product fraction 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.

[0043]

[0044]

[0045] (2) Gel filtration fractionation The sample was filtered through a 0.45 μm filter, and the non-alcoholic beer-taste beverage test sample was weighed and freeze-dried. The dried product was dissolved in 100 mM NaCl solution to prepare a 5-fold concentrated solution. The resulting concentrate 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.

[0046]

[0047] (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.

[0048]

[0049] 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.

[0050]

[0051] (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.

[0052] 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.

[0053] 1-3: Purification of Maillard reaction product fractions From the sample (Sample C) described in 1-1, which had a sensory evaluation score of 3.8 or higher for "body intensity" (score when evaluated according to the following evaluation criteria), a fraction obtained by the method of 1-2(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 adsorbate 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 bound Maillard reaction product.

[0054] 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.

[0055] The sensory evaluation criteria for the "body strength" were as follows: First, test sample A, which had the weakest body strength in the preliminary tasting, was identified as the negative control. Next, a trained panel of six people evaluated the body strength (a comprehensive evaluation consisting of flavor strength, complexity, and persistence) of all samples on a nine-point scale from 1 point (weak) to 9 points (strong). At this time, the score of test sample A, which was the negative control, was fixed at 2.

[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 1-2, and used for the added tasting.

[0057] Example 2: Effect on flavor of adjusting the concentrations of free Maillard reaction products (F-MRP) and bound Maillard reaction products (B-MRP) in a non-alcoholic beer-taste beverage (1) Base beverage sample Of the non-alcoholic beer-taste beverage test products produced in Example 1-1, Sample A (alcohol concentration 0.0002 v / v%, carbohydrate concentration less than 5.0 g / 100 ml, true extract concentration 1.57 w / w%) was used as the base beverage sample (base beverage sample A). The concentrations of free and bound Maillard reaction products (MRP) contained in base beverage sample A were measured according to the method described in Example 1.

[0058] Additionally, free and bound Maillard reaction products (MRPs) were added to base beverage sample A at the concentrations shown in the table below.

[0059] (2) Tasting with the addition of purified MRP fractions To the base beverage sample A shown in (1) above, purified MRP was added to the concentrations shown in the table below to obtain each sample (sample numbers 2 to 11). The obtained tasting samples were subjected to a sensory evaluation by a trained panel of six people, with base beverage sample A (sample number 1) as a control. The evaluation items and evaluation criteria were as follows:

[0060] Evaluation item 1 was "full-body strength" (an overall evaluation consisting of flavor strength, complexity, and persistence), which was evaluated on a 17-point scale ranging from 1 point (weak) to 9 points (strong). Evaluation item 2 was "beer-like grain flavor" (a moderate grain flavor found in alcohol-containing beers), which was evaluated on a 17-point scale ranging from 1 point (weak) to 9 points (strong). For the sensory evaluation, base beverage sample A (sample number 1) was used as a control (full-body strength: fixed at 2.0, beer-like grain flavor: fixed at 2.0).

[0061] The results are shown in the table below.

[0062] The control sample No. 1 (base beverage sample A) had a very weak body, and no beer-like grain flavor was perceived. Samples with a score of 3.3 or higher for "body strength" and a score of 3.8 or higher for "beer-like grain flavor" had a body, and a beer-like grain flavor. Furthermore, samples with a score of 5.2 or higher for "body strength" and a score of 5.3 or higher for "beer-like grain flavor" had a strong body, and a more beer-like grain flavor. These samples were considered to have a favorable flavor as a beer-taste beverage. Therefore, it was considered that a "body strength" score of 3.3 or higher is preferable, with a score of 5.2 or higher being more preferable, and a "beer-like grain flavor" score of 3.8 or higher being more preferable, with a score of 5.3 or higher being more preferable, for the flavor of a beer-taste beverage.

[0063] 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 free MRP content in a non-alcoholic beer-taste beverage is preferably 160 ppb or more. Furthermore, although there is no need to limit the total bound MRP content, it was found that it is preferable for it to be 35 ppb or more.

[0064] A plot showing the relationship between MRP concentration and the evaluation results of "beer-like graininess" (vertical axis: bound MRP concentration, horizontal axis: free MRP concentration, bubble size: beer-like graininess) is shown in Figure 2. This figure shows that the total content of free MRP in a non-alcoholic beer-taste beverage does not need to be limited, but is preferably 160 ppb or more, and more preferably 250 to 800 ppb. It also shows that the total content of bound MRP does not need to be limited, but is preferably 35 ppb or more, and more preferably 35 to 300 ppb.

[0065] Example 3: Confirmation of Effects in Non-Alcoholic Beer-Taste Beverages with Different Malt Ratios Beverage samples D and E were produced by varying the malt ratio in the ingredients according to the method described in Example 1-1. The malt ratio was varied by using 25% rice, corn, and starch as ingredients other than malt. As in Example 2, beverage samples A to C, D, and E produced in Example 1-1 were subjected to sensory evaluation by a trained panel of six people, using base beverage sample A (sample number 1) as a control. The evaluation items and evaluation criteria were the same as in Example 2. The concentrations of free and bound Maillard reaction products (MRPs) contained in base beverage samples B to E were measured according to the method described in Example 1. Furthermore, various analytical values ​​for beverage samples B, D, and E all showed an alcohol concentration of less than 0.01 v / v%, a carbohydrate concentration of less than 5.0 g / 100 ml, and a true extract concentration of 2 w / w% or more.

[0066] The results are shown in the table below.

[0067] Since the same effects of enhancing richness and enhancing the beer-like grain flavor as the evaluation results described above were confirmed within the same numerical range as in Example 2, it was thought that similar effects could be expected for non-alcoholic beer-flavored beverages with different malt ratios.

Claims

1. A non-alcoholic beer-flavored 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 160 ppb or more.

2. A non-alcoholic beer-taste beverage according to claim 1, in which the total content of free CML, free CEL and free MG-H1 in the beverage is 250 to 800 ppb or more.

3. A non-alcoholic beer-taste beverage as described in claim 1, wherein the total content of bound CML, bound CEL, and bound MG-H1 in the beverage is 35 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.

4. The non-alcoholic beer-taste beverage according to claim 3, wherein the total content of bound CML, bound CEL, and bound MG-H1 in the beverage is 35 to 300 ppb.

5. A non-alcoholic beer-flavored beverage according to any one of claims 1 to 4, having an alcohol (ethanol) concentration of less than 0.01 v / v %.

6. A method for producing a non-alcoholic beer-taste beverage, comprising the step of adjusting the total content of free CML, free CEL and free MG-H1 in the non-alcoholic beer-taste beverage to 160 ppb or more.

7. The method according to claim 6, further comprising a step of adjusting the total content of bound CML, bound CEL and bound MG-H1 in the beverage to 35 ppb or more, wherein 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 method according to claim 6 or 7, wherein the alcohol (ethanol) concentration of the non-alcoholic beer-taste beverage is less than 0.01 v / v %.

9. A method for enhancing the richness of a non-alcoholic beer-taste beverage, the method comprising the step of adjusting the total content of free CML, free CEL and free MG-H1 in the non-alcoholic beer-taste beverage to 160 ppb or more.

10. The method according to claim 9, further comprising a step of adjusting the total content of bound CML, bound CEL and bound MG-H1 in the beverage to 35 ppb or more, wherein 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.

11. A method for enhancing the beer-like graininess in a non-alcoholic beer-taste beverage, the method comprising the step of adjusting the total content of free CML, free CEL and free MG-H1 in the non-alcoholic beer-taste beverage to 160 ppb or more.

12. The method according to claim 11, further comprising a step of adjusting the total content of bound CML, bound CEL and bound MG-H1 in the beverage to 35 ppb or more, wherein 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.

13. The method according to any one of claims 9 to 12, wherein the alcohol (ethanol) concentration of the non-alcoholic beer-taste beverage is less than 0.01 v / v %.

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