Non-alcoholic beer-flavored beverage, agent for enhancing body flavor and agent for reducing acidity of non-alcoholic beer-flavored beverage, and method for enhancing body flavor and method for reducing acidity of non-alcoholic beer-flavored beverage

Abstract

The purpose of the present invention is to provide a non-alcoholic beer-flavored beverage produced using a grain-derived raw material and having a rich taste and reduced sourness. The present invention is a non-alcoholic beer-flavored beverage having an alcohol content of less than 1.0% and containing a sugar-modified protein having a molecular weight of 35-50 kDa, with the amino group of the lysin in the grain-derived protein being glycated.

Description

[Technical Field]

[0001] The present invention relates to a non-alcohol beer-taste beverage, a body flavor enhancer and an acidity reducer for a non-alcohol beer-taste beverage, and a method for enhancing body flavor and reducing acidity in a non-alcohol beer-taste beverage. [Background technology]

[0002] With the recent diversification of consumer tastes, there is a demand for the development of non-alcoholic beer-flavored beverages with various flavor characteristics.

[0003] For example, Patent Document 1 discloses that sweet components, aldehyde components, etc. are used as flavor improvers for beer-like beverages in order to enhance the richness, etc., of beer-like beverages. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 2016-214262 Summary of the Invention [Problem to be solved by the invention]

[0005] However, the flavor improving agent as described in Patent Document 1 affects the flavor and is therefore not suitable for use in some cases.

[0006] Furthermore, grain-derived ingredients such as barley and corn are commonly used as raw materials for non-alcoholic beer-flavored beverages. Because grain-derived ingredients are commonly used in non-alcoholic beer-flavored beverages, manufacturers and beverage consumers have little resistance to using grain-derived ingredients as ingredients for enhancing full-bodied flavor. Therefore, a method for enhancing full-bodied flavor using grain-derived ingredients has been desired.

[0007] In addition, acids such as citric acid are sometimes added to non-alcoholic beer-flavored beverages to lower the pH and prevent spoilage. However, adding acids makes the beverage taste sour, so a method to reduce the sourness while maintaining the pH has been desired.

[0008] An object of the present invention is to provide a non-alcoholic beer-taste beverage that uses a grain-derived ingredient and has a strong body and reduced acidity. Another object of the present invention is to provide a body-taste enhancer and an acidity reducer for a non-alcoholic beer-taste beverage that uses a grain-derived ingredient. A further object of the present invention is to provide a method for enhancing body-taste and a method for reducing acidity in a non-alcoholic beer-taste beverage that uses a grain-derived ingredient. [Means for solving the problem]

[0009] That is, the present invention relates to the following non-alcohol beer-flavored beverages, etc. [1] A non-alcoholic beer-flavored beverage with an alcohol content of less than 1.0%, which contains a glycosylated protein with a molecular weight of 35 to 50 kDa, in which the amino group of the lysine constituting a grain-derived protein is glycated. [2] The non-alcoholic beer-flavored beverage according to [1] above, wherein the glycosylated protein has a glycated lysine content of 4.7 nmol / mL or more. [3] The non-alcoholic beer-flavored beverage according to [1] or [2] above, wherein the glycosylated protein has a glycated lysine content of 9.4 nmol / mL or more. [4] The non-alcoholic beer-flavored beverage according to any one of [1] to [3] above, wherein the sugar glycated to the amino group of the glycated lysine is a monosaccharide or a disaccharide. [5] The non-alcoholic beer-taste beverage according to any one of [1] to [4] above, wherein the cereal is at least one selected from the group consisting of barley, wheat, corn, rice, and soybeans. [6] A non-alcoholic beer-flavored beverage according to any one of [1] to [5] above, which does not contain wort. [7] A flavor enhancer for non-alcoholic beer-flavored beverages with an alcohol content of less than 1.0%, which contains a glycosylated protein with a molecular weight of 35 to 50 kDa, in which the amino group of lysine constituting a grain-derived protein is glycated. [8] A method for enhancing the full-bodied taste of a non-alcoholic beer-flavored beverage, which comprises adding a glycosylated protein having a molecular weight of 35 to 50 kDa, in which the amino group of the lysine constituting a grain-derived protein is glycated, to the non-alcoholic beer-flavored beverage having an alcohol content of less than 1.0%, thereby enhancing the full-bodied taste of the non-alcoholic beer-flavored beverage. [9] An acidity reducer for non-alcoholic beer-flavored beverages with an alcohol content of less than 1.0%, which contains a glycosylated protein with a molecular weight of 35 to 50 kDa, in which the amino group of lysine constituting a grain-derived protein is glycated.

[10] A method for reducing the sourness of a non-alcoholic beer-flavored beverage, which comprises adding a glycosylated protein having a molecular weight of 35 to 50 kDa, in which the amino group of lysine constituting a grain-derived protein is glycated, to the non-alcoholic beer-flavored beverage having an alcohol content of less than 1.0%, thereby reducing the sourness of the non-alcoholic beer-flavored beverage. [Effects of the Invention]

[0010] According to the present invention, it is possible to provide a non-alcoholic beer-taste beverage that uses a grain-derived ingredient and has a strong body and reduced acidity. It is also possible to provide a body-taste enhancer and an acidity reducer for a non-alcoholic beer-taste beverage that uses a grain-derived ingredient. It is also possible to provide a method for enhancing the body-taste and a method for reducing the acidity of a non-alcoholic beer-taste beverage that uses a grain-derived ingredient. DETAILED DESCRIPTION OF THE INVENTION

[0011] The non-alcohol beer-taste beverage of the present invention contains a glycosylated protein. The molecular weight of the glycosylated protein is 35 to 50 kDa.

[0012] Proteins with a molecular weight of 35 to 50 kDa are proteins found in the 35 to 50 kDa range when the raw material liquid of a non-alcoholic beer-flavored beverage is ultrafiltered using a 30 kDa cutoff membrane and then electrophoresed by SDS-PAGE. A protein of about 40 kDa is preferred, and a protein of about 40 kDa is also referred to as a 40 kDa protein in this specification.

[0013] The glycosylated protein is a protein derived from a grain. The cereal is preferably at least one selected from the group consisting of barley, wheat, corn, rice, and soybean. Furthermore, when the grain is barley, it may contain a known barley-derived protein used in the production of non-alcoholic beer-taste beverages. Examples of such barley include barley, wheat, rye, oats, oats, and oats, with barley being preferred. Either germinated or ungerminated barley may be used, with germinated barley malt being preferred. These may be contained alone or in combination of two or more.

[0014] Glycosylated proteins are proteins in which the amino groups of lysines constituting grain-derived proteins are glycated. In the present invention, "the amino group of lysine is glycated" means that a sugar is bound to the amino group of lysine. Of the amino groups of lysine, it is preferred that a sugar be glycated to the amino group in the side chain. Glycation of the amino group of lysine can be confirmed by colorimetric determination using the NBT (Nitroblue tetrazolium) method. The sugar glycated to the amino group of lysine may be any of monosaccharides, disaccharides, and polysaccharides of trisaccharides or more, but is preferably a monosaccharide or disaccharide. Monosaccharides include all sugars including glucose, fructose, galactose, mannose, and arabinose. Examples of disaccharides include maltose-type disaccharides such as maltose and lactose. Examples of trisaccharides include maltotriose, certriose, fucosyllactose, and sialyllactose.

[0015] Because the non-alcohol beer-taste beverage of the present invention contains the glycosylated protein described above, it can be a non-alcohol beer-taste beverage with a strong body and reduced acidity. Furthermore, because glycosylated proteins are cereal-derived raw materials, they are prevented from unintentionally affecting flavor, as is the case with the flavor improving agent described in Patent Document 1. Furthermore, because cereal-derived raw materials are commonly used raw materials for non-alcohol beer-flavored beverages, manufacturers and beverage consumers will have little resistance to using cereal-derived raw materials as materials for enhancing fullness or reducing sourness.

[0016] In the present invention, "kokumi" refers to a taste that cannot be expressed by the five basic tastes, i.e., sweetness, saltiness, sourness, bitterness, and umami, and is expressed by taste intensity (drinkability), flavor breadth, flavor depth, and changes in flavor over time (aftertaste or persistence), etc. In the present invention, enhancing kokumi includes, for example, imparting kokumi to a food or drink that does not have kokumi, and enhancing the kokumi of a food or drink that does have kokumi. The presence or absence and degree of kokumi can be evaluated by sensory evaluation by a specialist panel. Furthermore, the presence or absence and degree of sourness can also be evaluated by sensory evaluation by a panel of experts.

[0017] In the non-alcohol beer-taste beverage of the present invention, the content of glycated lysine is preferably 4.7 nmol / mL or more, from the perspective of reducing the sourness of the non-alcohol beer-taste beverage. Furthermore, from the perspective of enhancing the full-bodied taste of a non-alcohol beer-flavored beverage, the content of glycated lysine is preferably 9.4 nmol / mL or more. Furthermore, the content of glycated lysine is preferably 50.0 nmol / mL or more, more preferably 80.0 nmol / mL or more, and even more preferably 100 nmol / mL or more. Furthermore, the content of glycated lysine is preferably 50.0 to 1000 nmol / mL, more preferably 80.0 to 1000 nmol / mL, and even more preferably 100 to 1000 nmol / mL. The content of glycated lysine can be determined by colorimetric determination using the NBT method.

[0018] The extract content of the non-alcohol beer-taste beverage of the present invention is not particularly limited, but is preferably 0.01 to 20.0 wt %. The non-alcohol beer-taste beverage of the present invention may use malt as a raw material. The proportion of malt in the raw material may be 0% by weight. Here, "malt ratio" refers to the weight ratio of malt to the ingredients other than water and hops, including malt, rice, corn, sorghum, potatoes, starch, non-malted barley, and sugars. However, ingredients that may be added in trace amounts, such as acidulants, sweeteners, bittering agents, seasonings, and flavorings, are not included in the calculation of the above ratio. The non-alcohol beer-taste beverage of the present invention does not necessarily contain wort.

[0019] The non-alcohol beer-taste beverage of the present invention preferably has a purine concentration of less than 5.0 mg / 100 mL, and also preferably has a purine concentration of 0 mg / 100 mL. For non-alcohol beer-flavored beverages with a purine concentration within the above range, the addition of a glycosylated protein can particularly enhance the full-bodied taste and reduce the sourness.

[0020] A non-alcoholic beer-flavored beverage is a beer-flavored beverage with an alcohol content of less than 1%, preferably containing substantially no alcohol, or may have an alcohol content of 0%. Beer-flavored drinks are carbonated drinks with a beer flavor. Here, beverages that are substantially alcohol-free do not exclude beverages that contain trace amounts of alcohol that are undetectable. Beverages with an alcohol content that is rounded to 0.0%, especially beverages with an alcohol content that is rounded to 0.00%, are included in the category of non-alcoholic beer-flavored beverages. Types of non-alcoholic beer-flavored beverages of the present invention include, for example, non-alcoholic beer-flavored beverages and beer-flavored soft drinks. Note that "alcohol content" here refers to the ethanol content and does not include aliphatic alcohols.

[0021] The alcohol content (v / v%) of the non-alcohol beer-taste beverage of the present invention refers to the alcohol content (v / v%) in the beverage and can be measured by any known method, such as a vibration density meter. Specifically, the beverage is decarbonated by filtration or ultrasonication to prepare a sample, which is then subjected to direct flame distillation. The density of the resulting distillate is measured at 15°C and converted using "Table 2: Conversion Table of Alcohol Content, Density (15°C), and Specific Gravity (15 / 15°C)," an appendix to the National Tax Agency's Prescribed Analysis Methods (National Tax Agency Ordinance No. 6 of 2007, revised June 22, 2007). When the alcohol content is less than 1.0%, a commercially available alcohol measuring device or gas chromatography may also be used.

[0022] The manufacturing process for a typical non-alcoholic beer-flavored beverage is shown below. By eliminating the need for a yeast fermentation process, non-alcoholic beer-flavored beverages can be easily produced. Typical non-fermented non-alcoholic beer-flavored beverages include those that use malt as an ingredient and those that do not, and can be produced as follows.

[0023] Non-alcoholic beer-flavored beverages produced using malt as a raw material are first prepared by adding enzymes such as amylase to a mixture containing malt and other barley, as well as other grains, starch, sugars, bittering agents, or coloring agents, as needed, and water, followed by gelatinization and saccharification, followed by filtration to obtain a saccharified solution. Hops, bittering agents, and other ingredients are added to the saccharified solution as needed, and the mixture is boiled, with solids such as coagulated proteins removed in a clarifying tank. As an alternative to this saccharified solution, hops may be added to malt extract and warm water, and then boiled. Hops may be added at any stage from the start of boiling to before the end of boiling. Known conditions may be used for the saccharification, boiling, and solids removal processes. After boiling, the resulting wort is filtered, and carbon dioxide gas is added to the filtrate. The resulting mixture is then filled into containers and sterilized to obtain the desired non-alcoholic beer-flavored beverage. If glycosylated proteins are added in any of the above processes, they may be added at any stage up to the filling stage.

[0024] When producing a non-alcoholic beer-flavored beverage that does not use malt as a raw material, first, a liquid sugar containing a carbon source, a nitrogen source as an amino acid-containing material other than barley or malt, hops, a color, etc. are mixed with warm water to form a liquid sugar solution. The liquid sugar solution is then boiled. When hops are used as a raw material, the hops may be mixed with the liquid sugar solution during boiling rather than before the start of boiling. Carbon dioxide gas is added to the boiled liquid sugar solution. The liquid sugar solution is then filled into containers and sterilized to obtain the desired non-alcoholic beer-flavored beverage. If a glycosylated protein is added in any of the above steps, it may be added at any step up to filling.

[0025] An aliphatic alcohol may be added to the non-alcohol beer-taste beverage of the present invention in order to impart a boozy flavor. There are no particular limitations on the aliphatic alcohol as long as it is a known alcohol, but aliphatic alcohols with 4 to 5 carbon atoms are preferred. In the present invention, preferred aliphatic alcohols with 4 carbon atoms include 2-methyl-1-propanol and 1-butanol, and those with 5 carbon atoms include 3-methyl-1-butanol, 1-pentanol, and 2-pentanol. These may be used alone or in combination of two or more. The content of the aliphatic alcohol having 4 to 5 carbon atoms is preferably 0.0002 to 0.0007 mass%, more preferably 0.0003 to 0.0006 mass%. In this specification, the content of the aliphatic alcohol can be measured using headspace gas chromatography.

[0026] The non-alcohol beer-taste beverage of the present invention is desirably low in calories in line with recent trends toward low-calorie beverages. Therefore, the calorie count of the non-alcohol beer-taste beverage of the present invention is preferably less than 5 kcal / 100 mL, more preferably less than 4 kcal / 100 mL, and even more preferably less than 3 kcal / 100 mL.

[0027] The number of calories contained in the non-alcoholic beer-taste beverage of the present invention is calculated essentially in accordance with the "Methods of Analysis of Nutritional Components, etc. in the Nutrition Labeling Standards" published in conjunction with the Health Promotion Act. In other words, in principle, the calorie content can be calculated as the sum of the amounts of each quantified nutrient component multiplied by the energy conversion coefficient for each component (protein: 4 kcal / g, fat: 9 kcal / g, carbohydrates: 4 kcal / g, dietary fiber: 2 kcal / g, alcohol: 7 kcal / g, organic acids: 3 kcal / g). For details, please refer to the "Methods of Analysis of Nutritional Components, etc. in the Nutrition Labeling Standards."

[0028] The specific method for measuring the amount of each nutrient contained in the non-alcohol beer-taste beverage of the present invention can be according to the various analytical methods described in the "Analytical Methods for Nutritional Components, etc. in the Nutrition Labeling Standards" of the Health Promotion Act. Alternatively, the calorie content and / or amount of each nutrient can be determined by requesting the Japan Food Research Laboratories Foundation.

[0029] The carbohydrates contained in the non-alcoholic beer-taste beverage of the present invention refer to the carbohydrates based on the Nutrition Labeling Standards for Foods (Ministry of Health, Labour and Welfare Notification No. 176 of 2003). Specifically, carbohydrates refer to the amount of food excluding protein, lipids, dietary fiber, ash, alcohol, and water. The amount of carbohydrates in a food is calculated by subtracting the amounts of protein, lipids, dietary fiber, ash, and water from the weight of the food. In this case, the amounts of protein, lipids, dietary fiber, ash, and water are measured using the methods set forth in the Nutrition Labeling Standards. Specifically, the amount of protein is measured using the nitrogen quantitative conversion method, the amount of lipids is measured using the ether extraction method, chloroform-methanol mixed liquid extraction method, Gerber method, acid hydrolysis method or Roese-Gottlieb method, the amount of dietary fiber is measured using high-performance liquid chromatography or the Prosky method, the amount of ash is measured using the magnesium acetate ashing method, direct ashing method or sulfuric acid ashing method, and the amount of moisture is measured using the Karl Fischer method, drying aid method, reduced-pressure superheat drying method, normal pressure heat drying method or plastic film method.

[0030] The non-alcohol beer-taste beverage of the present invention may be low in carbohydrates in line with the recent trend toward low carbohydrate foods. Therefore, the carbohydrate content of the non-alcohol beer-taste beverage of the present invention may be less than 2.5 g / 100 mL or less than 0.5 g / 100 mL. While there is no specific lower limit, it is typically around 0.1 g / 100 mL, and may be, for example, 0.15 g / 100 mL or more, or 0.2 g / 100 mL or more.

[0031] The non-alcoholic beer-taste beverage of the present invention may contain an acidulant. The acidulant preferably includes one or more acids selected from the group consisting of citric acid, lactic acid, phosphoric acid, and malic acid. In addition to the above acids, succinic acid, tartaric acid, fumaric acid, glacial acetic acid, and the like can also be used in the present invention. These acids may be used without limitation as long as they are approved for addition to foods. In the present invention, it is preferable to use a combination of lactic acid, which provides a mellow sourness, and phosphoric acid, which provides a slightly pungent sourness.

[0032] The content of the acidulant in the non-alcoholic beer-taste beverage of the present invention, calculated as citric acid, is preferably 200 ppm or more, more preferably 550 ppm or more, and even more preferably 700 ppm or more, from the viewpoint of imparting a beer-taste sensation, and is preferably 15,000 ppm or less, more preferably 5,500 ppm or less, and even more preferably 2,000 ppm or less, from the viewpoint of sourness. Therefore, in the present invention, the content of the acidulant, calculated as citric acid, is preferably in a suitable range of 200 ppm to 15,000 ppm, more preferably 550 ppm to 5,500 ppm, and even more preferably 700 ppm to 2,000 ppm. In this specification, the citric acid equivalent amount refers to the amount calculated from the acidity of each acidulant based on the acidity of citric acid. For example, the citric acid equivalent amount corresponding to 100 ppm of lactic acid is 120 ppm, the citric acid equivalent amount corresponding to 100 ppm of phosphoric acid is 200 ppm, and the citric acid equivalent amount corresponding to 100 ppm of malic acid is 125 ppm.

[0033] The content of acidulants in non-alcoholic beer-flavored beverages is calculated by analysis using high-performance liquid chromatography (HPLC) or other methods.

[0034] In the non-alcohol beer-taste beverage of the present invention, hops can be used as part of the ingredients. When using hops, conventional pelleted hops, powdered hops, or hop extracts used in the production of beer and the like can be appropriately selected and used depending on the desired flavor. Also, processed hop products such as isomerized hops and reduced hops may be used. These products are included in the hops used in the non-alcoholic beer-flavored beverage of the present invention. The amount of hops added is not particularly limited, but is typically about 0.0001 to 1% by weight of the total amount of the beverage.

[0035] The non-alcoholic beer-taste beverage of the present invention may contain other ingredients as needed, provided that the effects of the present invention are not impaired. For example, sweeteners (including high-intensity sweeteners), bittering agents, flavorings, yeast extracts, coloring agents such as caramel color, plant-extracted saponin substances such as soybean saponin and quillaja saponin, plant protein and peptide-containing substances from corn, soybeans, and the like, protein-based substances such as bovine serum albumin, seasonings such as dietary fiber and amino acids, and antioxidants such as ascorbic acid may be used as needed, provided that the effects of the present invention are not impaired.

[0036] The pH of the non-alcohol beer-taste beverage according to the present invention is preferably 3.0 to 5.0, more preferably 3.5 to 4.5, and even more preferably 3.5 to 4.0, from the viewpoint of improving the flavor of the beverage.

[0037] The non-alcohol beer-taste beverage of the present invention can be packaged in a container. The type of container is not particularly limited, and the beverage can be filled into a sealed container such as a bottle, can, barrel, or PET bottle to form a packaged beverage.

[0038] The body flavor enhancer of the present invention is a body flavor enhancer for non-alcoholic beer-flavored beverages with an alcohol content of less than 1.0%, which contains a glycosylated protein with a molecular weight of 35 to 50 kDa, in which the amino group of lysine constituting a cereal-derived protein is glycated. In addition, the acidity enhancer of the present invention is an acidity reducer for non-alcoholic beer-flavored beverages with an alcohol content of less than 1.0%, which contains a glycosylated protein with a molecular weight of 35 to 50 kDa, in which the amino group of lysine constituting a grain-derived protein is glycated. An agent containing a glycosylated protein with a molecular weight of 35 to 50 kDa, in which the amino group of the lysine constituting the grain-derived protein is glycated, can be used as an agent that simultaneously functions as a body-flavor enhancer and a sourness reducer for non-alcoholic beer-flavored beverages. Hereinafter, the body flavor enhancer and acidity reducer for non-alcohol beer-taste beverages will be described together.

[0039] The body flavor enhancer and acidity reducer of the present invention are glycosylated proteins themselves or compositions containing glycosylated proteins, which are used to enhance the body flavor and / or reduce the acidity of non-alcoholic beer-flavored beverages. Glycosylated proteins are proteins with a molecular weight of 35 to 50 kDa in which the amino groups of lysines constituting grain-derived proteins are glycated, and details thereof are as described above. When the body taste enhancer and acidity reducer are compositions containing glycosylated proteins, they may optionally contain known additives that can be added to beverages, as long as they do not inhibit the effects of the present invention.

[0040] The content of glycated lysine in the glycosylated protein contained in the body taste enhancer and sourness reducer of the present invention is preferably 0.04 nmol / μg or more, and more preferably 0.4 nmol / μg or more, in terms of the content (nmol) of glycated lysine per 1 μg of glycosylated protein. Such glycosylated proteins have a high content of glycated lysine, and therefore can be used as a body flavor enhancer that can enhance the body flavor of non-alcoholic beer-flavored beverages with a small amount of use, or as a sourness reducer that can reduce the sourness.

[0041] The body flavor enhancer and sourness reducer of the present invention can be obtained by purifying beer brewed from North American malt, which contains a large amount of sugar-modified proteins with a molecular weight of 35 to 50 kDa, in which the amino group of lysine constituting grain-derived proteins is glycated. Alternatively, it can be obtained by purifying cereal-derived proteins from malt or barley containing sugar-modified proteins with molecular weights of 35 to 50 kDa, in which the amino group of lysine constituting the cereal-derived protein is glycated. Furthermore, proteins may be extracted by mashing so as to contain a large amount of sugar-modified proteins with molecular weights of 35 to 50 kDa, in which the amino groups of lysines are glycated.

[0042] The method for producing the non-alcohol beer-taste beverage of the present invention is not particularly limited, but an example is a production method comprising the step of adding a glycosylated protein having a molecular weight of 35 to 50 kDa to a non-alcohol beer-taste beverage having an alcohol content of less than 1.0%. More specifically, a method for obtaining a non-alcoholic beer-flavored beverage with enhanced body and / or reduced acidity is exemplified by adding a sugar-modified protein having a molecular weight of 35 to 50 kDa, in which the amino group of the lysine constituting a grain-derived protein is glycated, to a non-alcoholic beer-flavored beverage with an alcohol content of less than 1.0%. The method for producing a non-alcohol beer-taste beverage using this method also serves as a method for enhancing the fullness of a non-alcohol beer-taste beverage and a method for reducing the sourness of a non-alcohol beer-taste beverage of the present invention.

[0043] When a body flavor enhancer or acidity reducer is added to a non-alcoholic beer-flavored beverage, the amount of body flavor enhancer or acidity reducer added is preferably such that the content of glycated lysine in the glycosylated protein is 9.4 nmol / mL or more from the perspective of enhancing body flavor, and preferably such that the content of glycated lysine in the glycosylated protein is 4.7 nmol / mL or more from the perspective of reducing acidity. It is also preferable that the content of glycated lysine in the glycosylated protein is 50.0 nmol / mL or more, preferably 80.0 nmol / mL or more, and preferably 100 nmol / mL or more. In this case, the amount of the flavor enhancer and acidity reducer added is preferably 5 μg / mL or more in terms of the amount of glycosylated protein from the viewpoint of enhancing the flavor, and preferably 10 μg / mL or more in terms of the amount of glycosylated protein from the viewpoint of reducing the acidity. [Example]

[0044] (Purification of glycosylated proteins derived from beer) North American malt was crushed to an appropriate particle size and placed in a mash tank, where it was mixed with warm water. The mash was then held at a temperature suitable for the malt enzymes for a sufficient time for saccharification, during which the malt enzymes converted the starch into sugar, producing a mash. The mash was then filtered, hops were added, and the mash was boiled to produce hot wort, which was then prepared for fermentation. The hot wort was cooled, and brewer's yeast was added to it. Over the course of several days, the yeast broke down the sugars in the wort into alcohol and carbon dioxide, producing green beer. The green beer was then transferred to a storage tank and stored at low temperatures for several dozen days. During this time, the beer was aged, allowing the flavor and aroma to harmonize. After aging was complete, the beer was filtered and bottled. Glycosylated proteins were purified from beer brewed using North American malt by the above method as follows. (1) Fractionation by cation exchange resin 50 mL of cation exchange resin SP Sepharose was placed in an empty column. Beer brewed from North American malt was allowed to adsorb onto the resin. The resin was then transferred to a column and washed with 20 mM sodium acetate buffer (pH 4.5). Elution was then performed with 20 mM sodium acetate (pH 4.5) + 0.5 M NaCl, and fractions were collected. The resulting fractions were evaluated by SDS-PAGE, and fractions containing the 40 kDa glycosylated protein were collected and used as the cation exchange resin-bound fraction. (2) Ultrafiltration (buffer exchange) The cation exchange resin-bound fraction obtained in (1) was added to a water-washed ultrafiltration unit (Merck Amicon Ultra-15 30K), centrifuged at 3500 rpm, and ultrafiltered to obtain a concentrate. (3) Ammonium sulfate fraction The concentrate obtained in (2) was placed in a beaker containing 20 mM phosphate buffer (pH 7.0) and 2 M ammonium sulfate, and the concentrate was added dropwise and stirred. The suspension was then centrifuged (2330 g, 10 minutes, room temperature). The supernatant was collected in a separate container. The collected solution was concentrated using an ultrafiltration unit. The concentrate was added to 20 mM sodium acetate (pH 4.5), and the mixture was centrifuged (2330 g, 10 minutes, room temperature) to concentrate, yielding a purified glycosylated protein (Bradford quantitation (bovine serum albumin (BSA) equivalent), 20.4 mg / mL, 2.21 mL). The purity of the purified glycosylated protein was confirmed by SDS-PAGE.

[0045] (Modification analysis of glycosylated proteins) (1) Preparation of gel pieces 10 μL of the purified glycosylated protein solution (concentration 1 mg / mL, 20 mM sodium acetate buffer (pH 4.5)) was electrophoresed on SDS-PAGE and stained with CBB (Coomassie Brilliant Blue), after which the gel of glycosylated proteins around 40 kDa was excised.

[0046] (2) Enzymatic digestion of proteins The excised gel pieces were cut into strips and subjected to reduction with dithiothreitol (56°C, 1 hour) and carbamidomethylation with iodoacetamide (protected from light, room temperature, 45 minutes). Next, 15 μL of 10 ng / μL chymotrypsin solution (5 mM calcium chloride, 50 mM ammonium bicarbonate solution) containing 0.01% Protease Max and 15 μL of 5 mM calcium chloride, 50 mM ammonium bicarbonate solution were added and incubated overnight. The enzyme digestion solution was then recovered. The recovered solution was evaporated to dryness under reduced pressure and redissolved in 0.1% formic acid solution.

[0047] (3) Measurement by LC-MS / MS The LC-MS / MS measurements were carried out under the following conditions. Equipment used: Direct flow nanoLC system Easy-nLC 1000TM (Thermo Scientific) Trap column: Acclaim PepMap® (Thermo Scientific) Analytical column: NANO HPLC CAPILLARY COLUMN (Nikkyo Technos Co., Ltd.) Liquid chromatograph mass spectrometer: Q Exactive Plus (Thermo Scientific) Mobile phase: Solution A: 0.1% formic acid / water, Solution B: 0.1% formic acid / acetonitrile Flow rate: 300nL / min Gradient: 0-40%B / 0-30min, 40-60%B / 30-35min, 60-90%B / 35-37min, 90%B / 37-45min Injection volume: 10μL Ionization mode: ESI Positive Measurement range: MS1 (m / z 350-1750) Data Dependent Scan Mode

[0048] (4) Protein and modification analysis Protein identification and modification search were performed under the following conditions. Search software: Proteome Discoverer 2.2.0.388 (ThermoFisher) Biological species: Barley (Hordeum vulgare), hops (Humulus), yeast (Saccharomyces cerevisiae) Search terms:Digestive enzymes:Chymotrypsin Modification (Static): Carbamidomethyl (Cysteine) Modification (Dynamic): Oxidation (Methionine), Hex (K, R, Protein N-term), Hex (2) (K, R, Protein N-term), Hex (3) (K, R, Protein N-term), Acetyl (Protein N-term) Precursor ion mass error range: Monoisotopic, ±10 ppm Product ion mass error range: ±0.02 Da Maximum number of misscleavages: 5 Confidence Level (Percolator): High (the highest probability level among the three levels of certainty) Database: SwissProt

[0049] Modification analysis confirmed that the amino groups of lysines in glycosylated proteins were glycated.

[0050] (Evaluation of glycosylation degree of glycosylated proteins) The degree of glycosylation of the purified glycosylated protein was determined colorimetrically by the NBT method using a Fructosamine Assay kit (Abcam, ab228558). The degree of glycosylation of purified glycosylated proteins derived from beer was determined. The number of tests for each protein was n=2, and the average value was used. The amount of glycated lysine per μg of glycosylated protein was 0.94 nmol / μg (glycosylated protein).

[0051] (Measurement of glycosylated protein concentrations in commercially available non-alcoholic beer-flavored beverages) The concentrations of glycosylated proteins in commercially available non-alcoholic beer-flavored beverages A and B were measured using LabChip. TM The analysis was performed using a GXII instrument (PerkinElmer) with HT Protein Express chips according to the standard protocol (n=2). As a result, the concentrations of glycosylated proteins in commercially available non-alcoholic beer-flavored beverages A and B were not detectable.

[0052] (Evaluation of sourness and body when glycosylated protein is added to commercially available non-alcoholic beer-flavored beverage A) A beer-derived purified glycosylated protein was added to a commercially available non-alcoholic beer-flavored beverage A, and a sensory evaluation of the sourness and richness was performed. The alcohol content of commercially available non-alcoholic beer-flavored beverage A is 0.00%. The purine concentration of commercially available non-alcoholic beer-flavored beverage A is less than 5.0 mg / 100 mL. The total purine concentration was measured by the Japan Food Research Laboratories using the degradation method (same below). For the sensory evaluation, commercially available non-alcoholic beer-flavored beverage A, which did not contain added glycosylated proteins, was scored in increments of 0.1 points by a panel of five experts, with the sourness being a reference score of 3.0 points and the fullness being a reference score of 0.5 points. The evaluation was carried out by varying the concentration of glycosylated protein added, and the results are shown in Table 1. The acidity and richness were determined based on the following criteria. 0 points: Not felt at all 1 point: Slightly felt 2 points: Clear feeling 3 points: Very strong feeling Table 1 shows the content (nmol / mL) of glycated lysine contained in glycomodified proteins in non-alcoholic beer-flavored beverages.

[0053] [Table 1]

[0054] The results shown in Table 1 indicate that by adding glycosylated proteins to a non-alcoholic beer-flavored beverage and increasing the content of glycated lysine, the sourness of the non-alcoholic beer-flavored beverage can be reduced and the full-bodied flavor can be enhanced.

[0055] (Evaluation of sourness and body when alcohol is added to commercially available non-alcoholic beer-flavored beverage A and glycosylated protein is also added) Ethanol was added to commercially available non-alcoholic beer-flavored beverage A to adjust the alcohol content to 0.999%. Purified glycosylated protein derived from beer was then added, and sensory evaluations of sourness and richness were performed. In this case too, the alcohol content is less than 1.0%, so it is a non-alcoholic beer-flavored beverage. The sensory evaluation was performed in the same manner as when a beer-derived glycosylated protein was added to commercially available non-alcoholic beer-flavored beverage A. Table 2 shows the content (nmol / mL) of glycated lysine contained in glycomodified proteins in non-alcoholic beer-flavored beverages.

[0056] [Table 2]

[0057] The results shown in Table 2 indicate that by adding glycosylated proteins to a non-alcoholic beer-flavored beverage with an alcohol content of 0.999% and increasing the content of glycated lysine, the sourness of the non-alcoholic beer-flavored beverage can be reduced and the full-bodied flavor can be enhanced.

[0058] (Evaluation of sourness and body when glycosylated protein is added to commercially available non-alcoholic beer-flavored beverage B) A beer-derived purified glycosylated protein was added to commercially available non-alcoholic beer-flavored beverage B, and sensory evaluations of sourness and richness were performed. The alcohol content of commercially available non-alcoholic beer-flavored beverage B is 0.00%. The purine concentration of commercially available non-alcoholic beer-flavored beverage B is less than 5.0 mg / 100 mL. For the sensory evaluation, commercially available non-alcoholic beer-flavored beverage A, which did not contain added glycosylated proteins, was scored in increments of 0.1 points by a panel of five experts, with the sourness being a reference score of 3.0 points and the fullness being a reference score of 0.5 points. The evaluation was carried out by varying the concentration of glycosylated protein added, and the results are shown in Table 3. The acidity and richness were determined based on the following criteria. 0 points: Not felt at all 1 point: Slightly felt 2 points: Clear feeling 3 points: Very strong feeling 4 points: Stronger than commercially available non-alcoholic beer-flavored beverage A Table 3 shows the content (nmol / mL) of glycated lysine contained in glycomodified proteins in non-alcoholic beer-flavored beverages.

[0059] [Table 3]

[0060] The results shown in Table 3 indicate that the acidity of different types of non-alcoholic beer-flavored beverages can be reduced and the full-bodied flavor enhanced by adding glycosylated proteins and increasing the content of glycated lysine. [Industrial Applicability]

[0061] According to the present invention, a non-alcohol beer-flavored beverage having a strong body and reduced acidity can be provided using a cereal-derived ingredient.

Claims

1. A non-alcoholic beer-flavored beverage having an alcohol content of less than 1.0%, The protein contains a glycosylated protein having a molecular weight of 35 to 50 kDa, in which the amino group of the lysine constituting the barley-derived protein is glycated, The content of glycated lysine is 4.7 nmol / mL or more; A non-alcoholic beer-flavored beverage having a purine concentration of less than 5.0 mg / 100 mL.

2. A non-alcoholic beer-flavored beverage as described in claim 1, having a glycated lysine content of 9.4 nmol / mL or more.

3. 3. The non-alcoholic beer-taste beverage according to claim 1 or 2, wherein the sugar glycated to the amino group of the glycated lysine is a monosaccharide or a disaccharide.

4. The non-alcohol beer-taste beverage according to any one of claims 1 to 3, which does not contain wort.

5. The acidity reducer for non-alcoholic beer-flavored beverages having an alcohol content of less than 1.0% contains a glycosylated protein having a molecular weight of 35 to 50 kDa, in which the amino group of lysine constituting a barley-derived protein is glycated.

6. A method for reducing the sourness of a non-alcoholic beer-flavored beverage comprising adding a glycosylated protein having a molecular weight of 35 to 50 kDa, in which the amino group of lysine constituting a barley-derived protein is glycated, to the non-alcoholic beer-flavored beverage having an alcohol content of less than 1.0%, thereby reducing the sourness of the non-alcoholic beer-flavored beverage.

7. 7. The method according to claim 6, wherein the glycosylated protein is added so that the content of glycated lysine contained in the glycosylated protein in the non-alcoholic beer-taste beverage is 4.7 nmol / mL or more.

8. 8. The method according to claim 6 or 7, wherein the glycosylated protein is added so that the content of glycated lysine contained in the glycosylated protein in the non-alcoholic beer-taste beverage is 9.4 nmol / mL or more.

9. The method according to claim 8, wherein the full body taste of the non-alcoholic beer-taste beverage is enhanced.

10. The method according to any one of claims 6 to 9, wherein the sugar glycated to the amino group of the glycated lysine is a monosaccharide or a disaccharide.

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