JP7749558B2 - Beer-flavored alcoholic beverages - Google Patents

Description

The present invention relates to a beer-flavored alcoholic beverage.

With the recent diversification of consumer preferences, there is a demand for the development of beer-flavored alcoholic beverages with a variety of flavor and taste characteristics.

Patent document 1 discloses that a peptide of a specific molecular weight is added to improve the flavor of a beer-flavored alcoholic beverage.

Japanese Patent Application Laid-Open No. 2016-149975

Patent document 1 uses indicators such as beer-like taste, smooth flow of flavor, and roughness remaining in the mouth as evaluation indicators for beverages, and describes that by including a certain concentration of 10-20 kDa peptides, a beverage can be obtained that has a high sensory evaluation score based on these indicators.

Here, the taste felt immediately after drinking a beer-flavored beverage is a taste that cannot be expressed by the five basic tastes, namely, sweetness, saltiness, sourness, bitterness, and umami, and is sometimes preferred for its strength, breadth, depth, persistence, or balanced strength. Such characteristics are referred to as "fullness" in this specification. The beverage described in Patent Document 1 can be said to be one for which there is room for further improvement in terms of fullness, and a method for enhancing the fullness of beer-flavored alcoholic beverages has been desired.

The present invention aims to provide a beverage with enhanced swelling. The present invention aims to provide a beer-flavored alcoholic beverage with an enhanced swelling, in which the ratio of malt in the ingredients is 50% by weight or more. The present invention also aims to provide a beer-flavored alcoholic beverage with an enhanced swelling, in which the ratio of malt in the ingredients is less than 50% by weight. Beer-flavored alcoholic beverages with a malt ratio of less than 50% by weight tend to have poor swelling, so it is particularly desirable to enhance the swelling of these beverages.

That is, the present invention relates to the following beer-taste alcoholic beverages:
[1] A beer-flavored alcoholic beverage in which the ratio of malt in the raw materials is 50% by weight or more, and in which the concentration of 2'-deoxyadenosine is 11 ppm or more.
[2] The beer-taste alcoholic beverage according to [1] above, wherein the concentration of 2'-deoxyadenosine is 11 to 50 ppm.
[3] The beer-flavored alcoholic beverage according to [1] or [2] above, further comprising a protein having a molecular weight of 35 to 50 kDa, the protein having a concentration of 10 ppm or more.
[4] The beer-flavored alcoholic beverage according to [3], wherein the protein concentration is 200 ppm or less.
[5] A beer-flavored alcoholic beverage in which the proportion of malt in the raw materials is less than 50% by weight, and in which the concentration of 2'-deoxyadenosine is 9.2 ppm or more.
[6] The beer-taste alcoholic beverage according to [5] above, wherein the concentration of 2'-deoxyadenosine is 9.2 to 100 ppm.
[7] Further, the composition contains a protein having a molecular weight of 35 to 50 kDa.
The beer-taste alcoholic beverage according to [5] or [6] above, wherein the protein concentration is 1 ppm or more.
[8] The beer-flavored alcoholic beverage according to [7] above, wherein the protein concentration is 30 ppm or less.

According to the present invention, a beer-taste alcoholic beverage having an enhanced fullness and containing 50% by weight or more of malt as an ingredient can be provided.
Furthermore, according to the present invention, a beer-taste alcoholic beverage having an enhanced fullness can be provided in which the ratio of malt in the raw materials is less than 50% by weight.

In the beer-taste alcoholic beverage according to the first embodiment of the present invention, the proportion of malt in the raw materials is 50% by weight or more.
In the beer-taste alcoholic beverage of the first embodiment of the present invention, the proportion of malt is preferably 100% by weight.

In the beer-taste alcoholic beverage according to the second embodiment of the present invention, the proportion of malt in the raw materials is less than 50% by weight.
In the beer-taste alcoholic beverage of the second embodiment of the present invention, the malt content is preferably 30% by weight or more. Alternatively, the beer-taste alcoholic beverage of the second embodiment of the present invention may contain 0% malt in its ingredients.

Hereinafter, in this specification, explanations common to the first embodiment of the present invention, a beer-taste beverage in which the proportion of malt in the raw materials is 50% by weight or more, and the second embodiment of the present invention, a beer-taste alcoholic beverage in which the proportion of malt in the raw materials is less than 50% by weight, will be simply referred to as "beer-taste alcoholic beverage."

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 beer-taste alcoholic beverage of the present invention contains 2'-deoxyadenosine. 2'-Deoxyadenosine is a type of deoxyribonucleoside. In this specification, 2'-deoxyadenosine may be abbreviated as 2'DA.

In a beer-taste alcoholic beverage according to the first embodiment of the present invention, in which the proportion of malt in the raw materials is 50% by weight or more, the concentration of 2'-deoxyadenosine is 11 ppm or more.
In a beer-taste alcoholic beverage whose raw materials contain 50% or more by weight of malt, a 2'-deoxyadenosine concentration of 11 ppm or more can enhance the fullness of the beer-taste alcoholic beverage. The correlation between the inclusion of a certain concentration or more of 2'-deoxyadenosine and the fullness of a beer-taste alcoholic beverage whose raw materials contain 50% or more by weight of malt was previously unknown, and was a finding discovered by the present inventors. In a beer-taste alcoholic beverage whose raw materials contain 50% or more by weight of malt, the 2'-deoxyadenosine concentration is preferably 15 ppm or more.

In the beer-taste alcoholic beverage of the first embodiment of the present invention, the concentration of 2'-deoxyadenosine is preferably 50 ppm or less.
This is because if the concentration of 2'-deoxyadenosine is too high in a beer-flavored alcoholic beverage containing 50% by weight or more of malt as an ingredient, the aftertaste may be perceived as being strongly bitter and/or unpleasant.
In one embodiment, the concentration of 2'-deoxyadenosine in the beer-taste alcoholic beverage of the first embodiment of the present invention is preferably 11 to 50 ppm, and more preferably 15 to 50 ppm.

In a beer-taste alcoholic beverage according to the second embodiment of the present invention, in which the proportion of malt in the raw materials is less than 50% by weight, the concentration of 2'-deoxyadenosine is 9.2 ppm or more.
In beer-taste alcoholic beverages whose raw materials contain less than 50% malt, a 2'-deoxyadenosine concentration of 9.2 ppm or more can enhance the fullness of the beer-taste alcoholic beverage. The correlation between the inclusion of a certain concentration or more of 2'-deoxyadenosine and the fullness of the beer-taste alcoholic beverages whose raw materials contain less than 50% malt was previously unknown and was discovered by the present inventors. In beer-taste alcoholic beverages whose raw materials contain less than 50% malt, the 2'-deoxyadenosine concentration is preferably 9.5 ppm or more.

In a beer-taste alcoholic beverage according to the second embodiment of the present invention, in which the raw materials contain less than 50% by weight of malt, the concentration of 2'-deoxyadenosine is preferably 100 ppm or less. This is because, in a beer-taste alcoholic beverage in which the raw materials contain less than 50% by weight of malt, if the concentration of 2'-deoxyadenosine is too high, the aftertaste may be perceived as being strongly bitter. In a beer-taste alcoholic beverage in which the raw materials contain less than 50% by weight of malt, the concentration of 2'-deoxyadenosine is more preferably 50 ppm or less.
In one embodiment, the concentration of 2'-deoxyadenosine in a beer-taste alcoholic beverage in which the proportion of malt in the raw materials is less than 50 wt % is preferably 9.2 to 100 ppm, more preferably 9.5 to 100 ppm, and even more preferably 9.5 to 50 ppm.

The beer-taste beverage of the present invention preferably further contains a protein with a molecular weight of 35 to 50 kDa.
In the first embodiment of the present invention, a beer-taste alcoholic beverage containing 50% by weight or more of malt as an ingredient further contains a protein with a molecular weight of 35 to 50 kDa, and the concentration of the protein is preferably 10 ppm or more.

In the second form of the present invention, a beer-flavored alcoholic beverage in which the proportion of malt in the raw materials is less than 50% by weight, it is preferable that the beverage further contains a protein with a molecular weight of 35 to 50 kDa, and that the concentration of the protein is 1 ppm or more.

Proteins with a molecular weight of 35 to 50 kDa are proteins found in the molecular weight range of 35 to 50 kDa when a beer-taste alcoholic beverage is subjected to SDS-PAGE electrophoresis. Prior to subjecting the beer-taste alcoholic beverage to SDS-PAGE electrophoresis, the beer-taste alcoholic beverage may be subjected to ultrafiltration using a 30 kDa cutoff membrane as a pretreatment, for example.
The protein preferably has a molecular weight of 35 to 45 kDa, more preferably about 40 kDa. In this specification, a protein with a molecular weight of 35 to 50 kDa is also referred to as a 40 kDa protein.

The 40 kDa protein is preferably a cereal-derived protein.
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 protein derived from a known barley used in the production of beer-flavored alcoholic beverages. Examples of such barley include barley, wheat, rye, oats, oats, and oats, with barley being preferred. Furthermore, 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.

Furthermore, as the 40 kDa protein, Serpin Z4 (also known as BSZ4, HorvuZ4, Major Endosperm Albumin, or Protein Z) derived from barley (scientific name: Hordeum vulgare) and Serpin Z7 (also known as BSZ7 or HorvuZ7) derived from barley are preferred. Furthermore, the above proteins may have an amino acid sequence in which some amino acids have been deleted, substituted, inserted, and/or added.

By further including a 40 kDa protein in addition to 2'-deoxyadenosine, the fullness of a beer-flavored alcoholic beverage containing 50% by weight or more of malt as an ingredient can be further enhanced.
In a beer-taste beverage according to the first embodiment of the present invention, in which the proportion of malt in the raw materials is 50% by weight or more, the concentration of the 40 kDa protein is preferably 25 ppm or more, even more preferably 30 ppm or more, and is preferably 200 ppm or less, even more preferably 100 ppm or less. In one embodiment, in a beer-taste beverage according to the first embodiment of the present invention, the concentration of the 40 kDa protein is preferably 10 to 200 ppm, more preferably 25 to 200 ppm, even more preferably 30 to 200 ppm, and particularly preferably 30 to 100 ppm.

By further including a 40 kDa protein in addition to 2'-deoxyadenosine, the fullness of the beer-taste alcoholic beverage of the second embodiment of the present invention, in which the proportion of malt in the raw materials is less than 50 wt %, can be further enhanced.
In the beer-taste beverage of the second form of the present invention, the concentration of the 40 kDa protein is preferably 5 ppm or more, and more preferably 30 ppm or less. In one embodiment, in the beer-taste beverage of the second form of the present invention, the concentration of the 40 kDa protein is preferably 1 to 30 ppm, and more preferably 5 to 30 ppm.

When both 2'-deoxyadenosine and 40 kDa protein are contained, the synergistic effect of 2'-deoxyadenosine and 40 kDa protein can effectively enhance the fullness of a beer-flavored alcoholic beverage.

The beer-taste alcoholic beverage of the present invention is a beer-taste beverage that contains alcohol, and the alcohol concentration is preferably 1% (v/v) to 10% (v/v), but is not particularly limited. Furthermore, the origin of the alcohol contained in the beer-taste alcoholic beverage is not limited to fermented or non-fermented. Note that alcohol here refers to ethanol and does not include aliphatic alcohols, which will be described later. A beer-taste beverage is a carbonated beverage that has a beer-like flavor.

Below, a general process for producing a beer-taste alcoholic beverage common to the first and second embodiments of the present invention is shown.
First, enzymes such as amylase are added as needed to a mixture containing water and other raw materials, including malt and other barley, as well as other grains, starch, sugars, bittering agents, or coloring agents, to gelatinize and saccharify the mixture. The mixture is then filtered to produce a saccharified liquid. Hops, bittering agents, and other ingredients are added as needed to the saccharified liquid, which is then boiled and solids such as coagulated proteins are removed in a clarifying tank. As an alternative to this saccharified liquid, hops may be added to malt extract and warm water, and the mixture is 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 process, boiling process, solids removal process, and other processes. Known conditions may be used for the fermentation and storage processes. The resulting fermented liquid is filtered, and carbon dioxide gas is added to the resulting filtrate. The resulting saccharified liquid is then filled into containers and sterilized to produce the desired beer-flavored alcoholic beverage.

A second form of the present invention, a beer-flavored alcoholic beverage in which the proportion of malt in the raw materials is less than 50% by weight, may be produced by adding a barley-derived alcoholic beverage such as a barley-derived distilled liquor (e.g., spirits, shochu, etc.) to a beer-flavored alcoholic beverage produced by the method described above.

When producing a beer-taste alcoholic beverage produced without using malt as a raw material, which is an example of a beer-taste beverage according to the second embodiment of the present invention, 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. This liquid sugar solution is boiled. When hops are used as a raw material, hops may be mixed with the liquid sugar solution during boiling rather than before the start of boiling. As an alternative to this saccharified solution, hops may be added to an extract made from a raw material other than malt, to which warm water is added, and the mixture is then boiled. Hops may be added at any stage from the start of boiling to the end of boiling. Known conditions may be used for the fermentation and storage processes. The resulting fermented liquid is filtered, and carbon dioxide gas is added to the resulting filtrate. The beverage is then filled into containers and sterilized to obtain a beer-taste alcoholic beverage in which the malt content of the desired raw materials is less than 50% by weight.

Beer-flavored alcoholic beverages that are non-fermented and contain alcohol may have their alcohol content adjusted in the final product by adding raw material alcohol, etc. The raw material alcohol may be added at any stage from the saccharification process to the filling process.

The alcohol content (v/v%) of the beer-taste alcoholic beverage of the present invention refers to the alcohol content (v/v%) in the beverage and can be measured by any known method, for example, a vibration density meter. Specifically, a sample is prepared by removing carbon dioxide from the beverage using filtration or ultrasound, and the sample is then distilled over an open flame. 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). For low concentrations of less than 1.0% alcohol, a commercially available alcohol measuring device or gas chromatography may also be used.

An aliphatic alcohol may be added to the beer-taste alcoholic beverage according to 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 having 4 to 5 carbon atoms are preferred. In the present invention, preferred aliphatic alcohols having 4 carbon atoms include 2-methyl-1-propanol and 1-butanol, and preferred aliphatic alcohols having 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% by weight, more preferably 0.0003 to 0.0006% by weight. In this specification, the content of the aliphatic alcohol can be measured using headspace gas chromatography.

The carbohydrates contained in the beer-taste alcoholic beverage according to 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 remaining after excluding protein, lipids, dietary fiber, ash, alcohol, and water from the food. 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 by the nitrogen quantitative conversion method, the amount of lipids is measured by the ether extraction method, the chloroform-methanol mixed liquid extraction method, the Gerber method, the acid hydrolysis method or the Roese-Gottlieb method, the amount of dietary fiber is measured by high-performance liquid chromatography or the Prosky method, the amount of ash is measured by the magnesium acetate addition ashing method, the direct ashing method or the sulfuric acid addition ashing method, and the amount of moisture is measured by the Karl Fischer method, the drying aid method, the reduced-pressure superheat drying method, the normal pressure heat drying method or the plastic film method.

The beer-flavored alcoholic 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 beer-flavored alcoholic 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.

In the beer-taste alcoholic beverage according to the present invention, hops can be used as part of the ingredients.
When using hops, typical 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 beer-flavored alcoholic beverage of the present invention. The amount of hops added is not particularly limited, but is typically about 0.0001 to 1% by weight based on the total amount of the beverage.

The beer-taste alcoholic 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 and soybeans, 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.

The beer-taste alcoholic beverage of the present invention can be packaged in a container. There are no restrictions on the shape of the container, and the beverage can be filled into a sealed container such as a bottle, can, barrel, or plastic bottle to create a packaged beverage.

The method for producing the beer-taste alcoholic beverage of the present invention is not particularly limited, but may include, for example, a method in which a predetermined amount of 2'-deoxyadenosine is added to a beer-taste alcoholic beverage whose raw materials contain 50% or more by weight of malt, or a beer-taste alcoholic beverage whose raw materials contain less than 50% by weight of malt.
Furthermore, it is preferable to add a 40 kDa protein to a beer-taste alcoholic beverage in which the ratio of malt in the raw materials is 50% by weight or more according to the first embodiment of the present invention, or to a beer-taste alcoholic beverage in which the ratio of malt in the raw materials is less than 50% by weight according to the second embodiment of the present invention.

The 2'-deoxyadenosine and 40 kDa protein to be added can be prepared, for example, by the procedures described in the Examples below.
Furthermore, the contents of 2'-deoxyadenosine and 40 kDa protein may be increased by adjusting the conditions in the production process of the beer-taste alcoholic beverage.

The present invention will be explained in detail below using examples, but the present invention is not limited to the following examples.

(Purification of 2'-deoxyadenosine)
2'-deoxyadenosine (2'DA) was purified as follows.
(1) Fractionation of beer using HP20: 60 L of beer was fractionated using 10 L of Diaion (registered trademark) HP20 (manufactured by Mitsubishi Chemical Corporation). Before use, the HP20 was washed three times with ethanol, then three times with 50% ethanol. The washed HP20 was loaded into a large-scale fractionation column and purged with water. An equal amount of distilled water was mixed with 60 L of degassed beer and passed through the HP20 column using a medium-pressure pump. The solution that passed through the HP20 column was obtained as the flow-through fraction. 40 L of distilled water was passed through using a medium-pressure pump, and the eluate was obtained as the water-eluted fraction. Similarly, 40 L of aqueous ethanol (10% ethanol, 30% ethanol, and 70% ethanol) was passed through the column, and the eluates were obtained as the 10% ethanol-eluted fraction, the 30% ethanol-eluted fraction, and the 70% ethanol-eluted fraction, respectively. Each eluted fraction was dried using an evaporator and a freeze-dryer and stored in a refrigerator.

(2) LH-20 Fractionation of 30% Ethanol-Eluted Fraction The 30% ethanol-eluted fraction from the HP20 fraction was fractionated using 1.2 kg of Sephadex® LH-20. The LH-20 washed with ethanol was loaded onto a large-scale fractionation column and substituted with water. Of the 30% ethanol-eluted fraction (87.9 g) obtained by HP20 fractionation, 17.6 g was dissolved in distilled water and applied to the LH-20 column. 13.5 L of distilled water was passed through using a medium-pressure pump, and water-eluted fractions 1 to 6 were obtained. Next, 7 L of aqueous ethanol (35% ethanol, 70% ethanol, and 100% ethanol) was passed through each column, and the eluates were obtained as the 35% ethanol-eluted fraction, the 70% ethanol-eluted fraction, and the 100% ethanol-eluted fraction, respectively. Each eluted fraction was dried using an evaporator and a freeze-dryer and stored in a refrigerator.

(3) 86.4 mg of the water-eluted fraction-4 (0.56 g) obtained by LH-20 fractionation of 2'DA was eluted with 10% ethanol using HPLC (COSMOSIL 5C18-PAQ, 20 × 250 mm). The eluate from 10 to 13 minutes was concentrated and eluted with a gradient mixture of ethanol and water (5:95 → 15:85) using HPLC (COSMOSIL 5C18-PAQ, 20 × 250 mm) to obtain Compound (I) (0.5 mg, tR = 21 min).
Compound (I) was identified as 2'-deoxyadenosine by analysis of physical data from MS and NMR and comparison with authentic samples.
The analytical instruments used are as follows:
LC-MS: Q Exactive, manufactured by Thermo Fisher Scientific NMR: AVANCE 400, manufactured by Bruker

(Purification of 40 kDa protein)
A 40 kDa protein was purified from commercially available beer (1 L) as follows.

(1) Fractionation by Cation Exchange Resin: 50 mL of cation exchange resin SP Sepharose was placed in an empty column. Beer was adsorbed 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 the fractions were collected. The resulting fractions were evaluated by SDS-PAGE, and the fractions containing the 40 kDa protein were collected and used as the cation exchange resin-bound fraction.

(2) Ultrafiltration (buffer exchange)
10 mL of 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: 20 mM phosphate buffer (pH 7.0) + 2 M ammonium sulfate was placed in a beaker, and the concentrate obtained in (2) 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 40 kDa purified protein (Bradford quantification (bovine serum albumin (BSA) equivalent), 20.4 mg/mL, 2.21 mL). The purity of the obtained 40 kDa protein was confirmed by SDS-PAGE.

The 40 kDa protein was digested with an enzyme and then analyzed by LC-MS/MS to attempt to identify the protein.
A band around 40 kDa separated by SDS-PAGE was excised 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 a 10 ng/μL chymotrypsin solution (5 mM calcium chloride, 50 mM ammonium bicarbonate solution) containing 0.01% Protease Max and 15 μL of a 5 mM calcium chloride, 50 mM ammonium bicarbonate solution were added, followed by overnight incubation, after which the enzyme digestion solution was recovered. The recovered solution was evaporated to dryness under reduced pressure and redissolved in 0.1% formic acid solution.
This was used for LC-MS/MS analysis.

(Measurement by LC-MS/MS)
The LC-MS/MS measurement was carried out under the following conditions.
Equipment used: Direct flow nanoLC system Easy-nLC 1000™ (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: 300 nL/min
Gradient: 0-40% B/0-30 min, 40-60% B/30-35 min, 60-90% B/35-37 min, 90% B/37-45 min
Injection volume: 10μL
Ionization mode: ESI Positive
Measurement range: MS1 (m / z 350-1750)
Data Dependent Scan Mode

(4) Protein analysis Protein identification was carried out under the following conditions.
Search software: Proteome Discoverer 2.2.0.388 (ThermoFisher)
Biological species: Barley (Hordeum vulgare), hops (Humulus), yeast (Saccharomyces cerevisiae)
Search conditions: Digestive enzyme: Chymotrypsin
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

As a result, it was found that the 40 kDa protein was barley-derived Serpin Z4 (sequence coverage: 77.2%) and barley-derived Serpin Z7 (sequence coverage: 72.8%).

<I. Sensory evaluation of commercially available beer-flavored alcoholic beverages containing 50% or more by weight of malt as raw materials when 2'DA was added>
In Examples 1 and 2, 2'-deoxyadenosine (2'DA) was added to a commercially available beer-taste alcoholic beverage (A), and a sensory evaluation of fullness was performed.
The beer-taste alcoholic beverage (A) is a beer-taste alcoholic beverage containing 50% by weight or more of malt as raw materials, including malt and hops, and contains, as nutritional components, 5.5% alcohol, 0.4 to 0.6 g of protein, 3.6 g of carbohydrates, and approximately 12.5 mg of purines per 100 ml.

The criteria for the sensory evaluation are as follows:
Five expert panelists scored the items in increments of 0.05 points according to the following criteria, and the scores were averaged.
The swelling strength is based on the following criteria:
0 point: Not detectable at all 1 point: Slightly detectable 2 points: Clearly detectable 3 points: Very detectable As a benchmark, a commercially available beer-taste alcoholic beverage (B) different from the commercially available beer-taste alcoholic beverage (A) to be evaluated was used as the reference beer-taste alcoholic beverage (I) and its fullness was assigned a benchmark score of 0.7 points. In addition, a beer-taste alcoholic beverage identical to the commercially available beer-taste alcoholic beverage (A) to be evaluated was used as the reference beer-taste alcoholic beverage (II) and its fullness was assigned a benchmark score of 1.5 points.
A beer-taste alcoholic beverage (I) according to this standard is a beer-taste alcoholic beverage in which the proportion of malt in the raw materials is more than 0% by weight and less than 50% by weight.
The ingredients are happoshu, malt, hops, sugar, dietary fiber, and spirits (wheat), and the nutritional content per 100ml is 4% alcohol, 0-0.2g protein, 0.5-0.8g carbohydrates, and approximately 2.0mg purines.

The procedure for the sensory evaluation was as follows.
(1) Dispense the beer-flavored alcoholic beverage to be evaluated into a vial at 1/10 of the final volume (v/v). (2) Weigh out an arbitrary amount of 2'DA and add it. (3) Sonicate for 30 seconds. (4) Let stand at room temperature for 30 minutes. (5) Fill the beer-flavored alcoholic beverage to the final volume. (6) Dispense, swallow, and evaluate.

(Analysis of commercially available beer-flavored alcoholic beverages)
The concentrations of 2'DA contained in the commercially available beer-flavored alcoholic beverages used in the sensory evaluation were quantified by LC-MS according to the following procedure.
(1) Preparation of Standards and Preparation of Calibration Curve 2'DA was diluted to the following concentrations, passed through a 0.22 μm filter, and then subjected to measurement.
Final concentration: 0.001ppm, 0.025ppm, 0.050ppm, 0.100ppm, 0.200ppm, 0.300ppm, 0.500ppm, 0.750ppm, 1.000ppm
(1 ppm = 1 μg/mL)
The diluent used was a 5% (v/v) aqueous ethanol solution.
In addition, the measurement values used were those at a dilution ratio such that the measurement values fell within the range where the linearity of the calibration curve was maintained (R ² >0.99) in the analysis results of the standard samples.

The LC-MS measurement conditions are as follows.
LC-MS: X500R manufactured by AB Sciex
Separation column: Waters HSST3 1.8 μm, 2.1 x 150 mm
Eluent:
Solution A: 0.1% formic acid/water, Solution B: 0.1% formic acid/acetonitrile Gradient: Solution A: Solution B = 98:2 → 2:98 (27 min)
Injection volume: 5μL
Flow rate: 0.2mL/min
Column oven: 40°C
(MS)
Ionization mode: ESI Positive
Measurement range: MS1 (m / z 100-1000)
Data Independent Scan mode Ion source temperature: 350°C

(2) Preparation of measurement samples from commercially available beer-flavored alcoholic beverages. A commercially available beer-flavored alcoholic beverage was degassed by sonication, and after the bubbles had settled, the beverage was diluted appropriately and passed through a 0.22 μm filter before being used for measurement.
The diluent used was a 5% (v/v) aqueous ethanol solution.
The concentration of 2'DA contained in a commercially available beer-flavored alcoholic beverage (A) was designated as Control 1.

The concentration of the 40 kDa protein contained in the commercially available beer-flavored alcoholic beverages used in the sensory evaluation was measured using a LabChip™ GXII device (PerkinElmer) with an HT Protein Express chip according to the standard protocol (n=3).
The concentration of the 40 kDa protein contained in the commercially available beer-flavored alcoholic beverage (A) was 25 ppm.

(Example 1: Evaluation by adding 2'DA)
The concentration of 2'DA contained in the commercially available beer-flavored alcoholic beverage (A) (Control 1) was 8 ppm.
To this, 2'DA was added so that the 2'DA concentrations became 11 ppm, 15 ppm, 20 ppm, and 50 ppm, respectively, and a sensory evaluation was carried out.
The results of the sensory evaluation are shown in Table 1.

The results shown in Table 1 show that in beer-flavored alcoholic beverages with a malt ratio of 50% or more by weight in the raw materials, a 2'DA concentration of 11 ppm or more enhances fullness.

Example 2: Evaluation of synergistic effect of 2'DA and 40 kDa protein
A commercially available beer-flavored alcoholic beverage (A) containing 50% or more by weight of malt as a raw material was used as a reference beverage to which 2'DA was added to bring the 2'DA concentration to 11 ppm. The 40 kDa protein was then added to the beverage to evaluate the synergistic effect of 2'DA and 40 kDa protein. The 40 kDa protein was added so that the concentrations of the 40 kDa protein were 30 ppm, 35 ppm, and 45 ppm, respectively. The 40 kDa protein used was the one purified as described above.
For comparison, a commercially available beer-flavored alcoholic beverage (A) was also evaluated by adding 5 ppm of the 40 kDa protein alone (control sample 2-1).
The results of the sensory evaluation are shown in Table 2.

The results shown in Table 2 indicate that the volume of a commercially available beer-flavored alcoholic beverage (A) containing 50% or more by weight of malt as a raw material can be further enhanced by adding 2'DA and further adding 40 kDa protein.
The results for comparison sample 2-1 show that the addition of 40 kDa protein alone can enhance swelling. However, the increase in sensory evaluation value from the control for sample 2-2 (0.21) is greater than the additive effect (0.17) predicted when 2'DA and 40 kDa protein are added, calculated by adding the increase in sensory evaluation value from the control for sample 2-1 (0.11) and the increase in sensory evaluation value from the control for comparison sample 2-1 (0.06). It can be said that the combined use of 2'DA and 40 kDa protein exerts an unexpected synergistic effect in enhancing swelling.

Example 3: Evaluation of synergistic effect of 2'DA and 40 kDa protein
2'DA and 40 kDa protein were added to a commercially available beer-taste alcoholic beverage (referred to as beer-taste alcoholic beverage (C)), and a sensory evaluation was conducted. The beer-taste alcoholic beverage (C) evaluated in Example 3 is a beer-taste alcoholic beverage containing 50% or more by weight of malt as an ingredient. This beer-taste alcoholic beverage (C) is a different beer-taste alcoholic beverage from the commercially available beer-taste alcoholic beverage (A) used in Examples 1 and 2.
The ingredients of the beer-flavored alcoholic beverage (C) are malt, hops, rice, corn, and starch, and the nutritional components per 100 ml are 5% alcohol, 0.2 to 0.4 g of protein, 3.0 g of carbohydrates, and 5 to 6 mg of purines.

The concentrations of 2'DA and 40 kDa protein in the beer-taste alcoholic beverage (C) were analyzed using the methods described above. The concentration of 2'DA in the beer-taste alcoholic beverage (C) (Control 2) was 10 ppm, and the concentration of the 40 kDa protein was 6.5 ppm.
A sensory evaluation was conducted on this beer-taste alcoholic beverage (C) by adding 2'DA to a 2'DA concentration of 11 ppm (Sample 3-2). Another sensory evaluation was conducted on the beer-taste alcoholic beverage (C) by adding 2'DA to a 2'DA concentration of 11 ppm and then adding 40 kDa protein to a 40 kDa protein concentration of 10 ppm (Sample 3-3).

The criteria and procedures for the sensory evaluation were the same as those in Example 1.
The results of the sensory evaluation are shown in Table 3.

The results shown in Table 3 show that even when the 40 kDa protein concentration is as low as 6.5 ppm, adding 2'DA to a commercially available beer-flavored alcoholic beverage (C) containing 50% or more by weight of malt as an ingredient can enhance its volume. Furthermore, it can be seen that the volume of a beer-flavored alcoholic beverage can be more effectively enhanced by increasing the 2'DA concentration to 11 ppm and the 40 kDa protein concentration to 10 ppm.

<II. Sensory Evaluation of a Commercially Available Beer-Taste Alcoholic Beverage (B) in Which the Malt Ratio in the Raw Materials is Less than 50% by Weight When 2'DA Is Added>
2'-deoxyadenosine (2'DA) was added to a commercially available beer-flavored alcoholic beverage (B), and a sensory evaluation of the swelling was carried out.
The beer-taste alcoholic beverage (B) contains malt as an ingredient, and the proportion of malt in the ingredients is less than 50% by weight.
The ingredients are happoshu, malt, hops, sugar, dietary fiber, and spirits (wheat), and the nutritional content per 100ml is 4% alcohol, 0-0.2g protein, 0.5-0.8g carbohydrates, and approximately 2.0mg purines.

(Analysis of commercially available beer-flavored alcoholic beverages)
The concentration of 2'DA contained in the commercially available beer-flavored alcoholic beverage (B) used in the sensory evaluation was quantified by LC-MS using the same procedure as above. The concentration of 2'DA contained in the commercially available beer-flavored alcoholic beverage (B) was designated Control 3.

(Example 4: Evaluation by adding 2'DA)
The concentration of 2'DA contained in the commercially available beer-flavored alcoholic beverage (B) (Control 3) was 1.9 ppm.
To this, 2'DA was added so that the 2'DA concentrations became 9.2 ppm, 9.5 ppm, 15 ppm, 20 ppm, 50 ppm, and 100 ppm, respectively, and sensory evaluation was carried out.
The criteria and procedures for the sensory evaluation were the same as those in Example 1.
The results of the sensory evaluation are shown in Table 4.

The results shown in Table 4 show that in beer-flavored alcoholic beverages in which the proportion of malt in the raw materials is less than 50% by weight, a 2'DA concentration of 9.2 ppm or more enhances fullness.

Example 5: Evaluation of synergistic effect of 2'DA and 40 kDa protein
A commercially available beer-flavored alcoholic beverage (B) (Control 3) was supplemented with 2'DA to achieve a 2'DA concentration of 9.2 ppm, and the synergistic effect of 2'DA and 40 kDa protein was evaluated by further adding 40 kDa protein. The concentrations of the 40 kDa protein were 1 ppm, 5 ppm, 10 ppm, and 25 ppm. The 40 kDa protein used was the one purified as described above.
For comparison, a commercially available beer-flavored alcoholic beverage (B) to which only the 40 kDa protein was added was also evaluated.
The results of the sensory evaluation are shown in Table 5.

The results shown in Table 5 indicate that the volume of a commercially available beer-flavored alcoholic beverage (B), whose raw material contains less than 50% by weight of malt, can be further enhanced by adding 2'DA and further adding 40 kDa protein.
The results for comparison samples 5-1 to 5-3 demonstrate that the addition of 40 kDa protein alone can enhance swelling. However, the sensory evaluation increase (0.21) for sample 5-2 over the control is greater than the predicted additive effect (0.13) of combining 2'DA and 40 kDa protein, calculated by adding the sensory evaluation increase (0.10) for sample 5-1 and the sensory evaluation increase (0.03) for comparison sample 5-1. The sensory evaluation increase (0.35) for sample 5-3 over the control is also greater than the sum of the sensory evaluation increases (0.10 and 0.09, respectively) for sample 5-1 and comparison sample 5-2. The sensory evaluation increase (0.47) for sample 5-5 over the control is also greater than the sum of the sensory evaluation increases (0.10 and 0.20, respectively) for sample 5-1 and comparison sample 5-3. These results suggest that the combined use of 2'DA and the 40 kDa protein produces an unexpected synergistic effect in enhancing the fullness of beer-flavored beverages containing less than 50% by weight of malt as raw material.

According to the present invention, it is possible to provide a beer-taste alcoholic beverage with enhanced fullness, in which the malt content of the ingredients is 50% by weight or more. Also, according to the present invention, it is possible to provide a beer-taste alcoholic beverage with enhanced fullness, in which the malt content of the ingredients is less than 50% by weight.

Claims (2)

A beer-flavored alcoholic beverage in which the ratio of malt in the raw materials is less than 50% by weight, the beer-flavored alcoholic beverage having a 2'-deoxyadenosine concentration of 9.2 to 100 ppm, and a barley-derived protein concentration of 35 to 50 kDa molecular weight of 1 to 10 ppm. 2. The beer-taste alcoholic beverage according to claim 1 , wherein the concentration of 2'-deoxyadenosine is 20 to 100 ppm.