JP7709972B2 - Beer-flavored alcoholic beverages - Google Patents

Description

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

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

JP 2016-149975 A

Patent document 1 uses indicators such as a beer-like taste, a smooth flow of flavor, and a roughness remaining in the mouth as evaluation indices for beverages, and describes how by containing a specified concentration of a 10-20 kDa peptide, a beverage can be obtained with a high sensory evaluation score based on these indices.

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

The present invention aims to provide a beverage with enhanced swelling. In particular, the present invention aims to provide a beer-flavored alcoholic beverage with enhanced swelling and a purine concentration of 5 mg/100 mL or more.

Specifically, the present invention relates to the following beer-taste alcoholic beverages.
[1] A beer-flavored alcoholic beverage having a purine concentration of 5 mg/100 mL or more and an adenosine concentration of 50 ppm or more.
[2] The beer-taste alcoholic beverage according to [1] above, wherein the adenosine concentration is 50 to 150 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 concentration of the protein being 10 ppm or more.
[4] The beer-flavored alcoholic beverage according to [3] above, wherein the protein concentration is 200 ppm or less.
[5] A beer-flavored alcoholic beverage containing adenosine and a protein having a molecular weight of 35 to 50 kDa, wherein the adenosine concentration is 45 ppm or more, the protein concentration is 30 ppm or more, and the purine concentration is 5 mg/100 mL or more.

According to the present invention, a beer-flavored alcoholic beverage with enhanced fullness can be provided.

The beer-taste alcoholic beverage of the present invention has a purine concentration of 5 mg/100 mL or more. In this specification, the term "purine" is not particularly limited as long as it is a compound having a purine nucleus structure. Therefore, examples of "purine" include purine bases (adenine, guanine, xanthine, hypoxanthine), purine nucleosides (adenosine, guanosine, inosine), purine nucleotides (adenylic acid, guanylic acid, inosinic acid), and low- or high-molecular-weight nucleic acids (oligonucleotides, polynucleotides).
In this specification, the term "purine concentration" refers to the total amount of four purine bases, adenine, guanine, xanthine, and hypoxanthine. Purines can be measured by known methods, for example, a method of detecting them using LC-MS/MS (liquid chromatography mass spectrometry) after hydrolysis with perchloric acid (see "Guide to Microanalysis of Purines in Alcoholic Beverages", Japan Food Research Center, General Incorporated Foundation, URL: http://www.jfrl.or.jp/item/nutrition/post-31.html).

The purine concentration in the beer-flavored alcoholic beverage of the present invention is preferably 5.5 mg/100 mL or more. The purine concentration in the beer-flavored alcoholic beverage of the present invention is preferably 15 mg/100 mL or less, and more preferably 13 mg/100 mL or less.

The beer-taste alcoholic beverage of the present invention contains adenosine, which is a type of nucleoside composed of adenine and ribose.
The above adenosine does not include derivatives of adenosine such as 5'-deoxyadenosine.

The beer-taste alcoholic beverage of the present invention has an adenosine concentration of 50 ppm or more.
An adenosine concentration of 50 ppm or more can effectively enhance the swelling of a beer-flavored alcoholic beverage having a purine concentration of 5 mg/100 mL or more. It was not previously known that the inclusion of adenosine at a certain concentration or higher is related to the swelling of a beer-flavored alcoholic beverage having a purine concentration of 5 mg/100 mL or more, and this is a finding discovered by the present inventors.

Furthermore, the proportion of malt in the raw materials of the beer-taste alcoholic beverage of the present invention is preferably at least 50% by weight, and most preferably 100% by weight.
Here, the "malt ratio" refers to the weight ratio of malt to the ingredients other than water and hops, such as malt, rice, corn, sorghum, potato, starch, non-malted barley, and sugars, etc. However, ingredients that can be added in small amounts, such as acidulants, sweeteners, bittering agents, seasonings, and flavorings, are not included in the calculation of the above ratio.

In the beer-taste alcoholic beverage of the present invention, the adenosine concentration is preferably 150 ppm or less.
If the adenosine concentration becomes too high, the taste may seem unbalanced.
In one embodiment, the adenosine concentration in the beer-taste alcoholic beverage is preferably 50 to 150 ppm.

Furthermore, it is preferable that the beer-taste alcoholic beverage of the present invention further contains a protein having a molecular weight of 35 to 50 kDa, and that the concentration of the protein is 10 ppm or more. This is because a concentration of 10 ppm or more of a protein having a molecular weight of 35 to 50 kDa can more effectively enhance the swelling of a beer-taste alcoholic beverage having a purine concentration of 5 mg/100 mL or more.

Proteins with a molecular weight of 35 to 50 kDa are proteins that are found in the molecular weight range of 35 to 50 kDa when a beer-taste alcoholic beverage is subjected to electrophoresis by SDS-PAGE. Prior to subjecting the beer-taste alcoholic beverage to electrophoresis by SDS-PAGE, for example, the beer-taste alcoholic beverage may be subjected to ultrafiltration using a 30 kDa cutoff membrane as a pretreatment.
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 above-mentioned 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-taste alcoholic beverages. Such barley may include barley, wheat, rye, oats, oats, and the like, 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.

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

By further containing a 40 kDa protein in addition to adenosine, the fullness of a beer-flavored alcoholic beverage can be effectively increased.
Moreover, the concentration of the 40 kDa protein is more preferably 25 ppm or more, further preferably 30 ppm or more, and is preferably 200 ppm or less.

In addition, when both adenosine and the 40 kDa protein are contained, the synergistic effect of adenosine and the 40 kDa protein can enhance the swelling of a beer-flavored alcoholic beverage with a purine concentration of 5 mg/100 mL or more. Therefore, the lower limit of the adenosine concentration required to enhance swelling may be low, and the lower limit of the 40 kDa protein concentration may be low.

In other words, another form of the beer-flavored alcoholic beverage of the present invention is a beer-flavored alcoholic beverage that contains adenosine and a protein with a molecular weight of 35 to 50 kDa, in which the concentration of the adenosine is 45 ppm or more, the concentration of the protein is 30 ppm or more, and the concentration of purines is 5 mg/100 mL or more.

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 thereto. 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.

The manufacturing process for a typical beer-flavored alcoholic beverage is shown below.
First, enzymes such as amylase are added as necessary to a mixture containing raw materials such as malt and other wheat, other grains, starch, sugars, bittering agents, or coloring agents as necessary, and water, to cause gelatinization and saccharification, and the mixture is filtered to obtain a saccharified liquid. Hops, bittering agents, etc. are added as necessary 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 then boiled. Hops may be mixed at any stage from the start of boiling to the end of boiling. Known conditions may be used for the saccharification process, boiling process, solids removal process, etc. Known conditions may be used for the fermentation and storage processes, etc. The obtained fermented liquid is filtered, and carbon dioxide gas is added to the obtained filtrate. The product is then filled into a container and sterilized to obtain the desired beer-flavored alcoholic beverage.
The beer-taste alcoholic beverage may be produced by adding a barley-derived alcoholic beverage such as a barley-derived distilled liquor (e.g., spirits or shochu) to a beer-taste alcoholic beverage produced by the method described above.

A beer-flavored alcoholic beverage produced without using malt as a raw material is prepared by mixing liquid sugar containing a carbon source, a nitrogen source as an amino acid-containing material other than barley or malt, hops, a coloring, etc. with warm water to produce a liquid sugar solution. The liquid sugar solution is boiled. When hops are used as a raw material, hops may be mixed into the liquid sugar solution during boiling, not before the start of boiling. As an alternative to this saccharified liquid, hops may be added to an extract made from a raw material other than malt, and the resulting mixture is boiled. Hops may be mixed 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 resulting mixture is then filled into containers and sterilized to produce the desired beer-flavored alcoholic beverage.

Beer-flavored alcoholic beverages that are non-fermented and contain alcohol may be those in which the alcohol content of the final product has been adjusted by adding raw material alcohol or the like. The raw material alcohol may be added at any stage from the saccharification process to the filling process.

The alcohol content of the beer-taste alcoholic beverage according to the present invention means 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 a direct flame. The density of the resulting distillate is measured at 15°C, and the alcohol content is calculated by conversion using "Table 2: Conversion Table of Alcohol Content, Density (15°C) and Specific Gravity (15/15°C)," which is an appendix to the National Tax Agency Specified Analysis Method (National Tax Agency Instruction No. 6 of 2007, revised June 22, 2007). For low concentrations of less than 1.0%, a commercially available alcohol measuring device or gas chromatography may be used.

From the viewpoint of imparting an alcoholic sensation to the beer-taste alcoholic beverage according to the present invention, an aliphatic alcohol may be added. There are no particular limitations on the aliphatic alcohol as long as it is a known alcohol, but an aliphatic alcohol having 4 to 5 carbon atoms is preferred. In the present invention, preferred aliphatic alcohols include 2-methyl-1-propanol, 1-butanol, etc. as those having 4 carbon atoms, and 3-methyl-1-butanol, 1-pentanol, 2-pentanol, etc. as those having 5 carbon atoms. These may be used alone or in combination of two or more kinds.
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 by headspace gas chromatography.

The carbohydrates contained in the beer-taste alcoholic 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 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 by the nitrogen quantitative conversion method, the amount of lipids is measured by the ether extraction method, the chloroform-methanol mixed extraction method, the Gerber method, the acid decomposition 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 heating 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 for low carbohydrates. Thus, 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. In addition, although there is no particular lower limit, it is usually about 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 a 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 according to the desired flavor. Also, processed hop products such as isomerized hops and reduced hops may be used. The hops used in the beer-flavored alcoholic beverage of the present invention include these. 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 coloring, plant-extracted saponin substances such as soybean saponin and quillaja saponin, plant protein and peptide-containing substances such as corn and soybeans, protein 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 limitations 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 produce a packaged beverage.

The method for producing the beer-taste alcoholic beverage of the present invention is not particularly limited, but an example is a method in which a predetermined amount of adenosine is added to a beer-taste alcoholic beverage having a purine concentration of 5 mg/100 mL or more.
It is also preferable to add a 40 kDa protein to a beer-flavored alcoholic beverage having a purine concentration of 5 mg/100 mL or more.
Adenosine and the 40 kDa protein to be added can be prepared, for example, by the procedure described in the Examples below.
Furthermore, the contents of adenosine and the 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 described in detail below with reference to examples, but the present invention is not limited to the following examples.

(Adenosine purification)
Adenosine was purified as follows.
(1) Fractionation of beer with HP20 60L of beer was fractionated using 10L of Diaion (registered trademark) HP20 (manufactured by Mitsubishi Chemical Corporation). Before use, HP20 was washed three times with ethanol and then three times with 50% ethanol. The washed HP20 was packed into a column for large-scale fractionation and replaced with water. An equal amount of distilled water was mixed with 60L 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 a pass-through fraction. 40L of distilled water was passed through using a medium pressure pump, and the eluate was obtained as a water elution fraction. Similarly, 40L of hydrous ethanol (10% ethanol, 30% ethanol, and 70% ethanol) was passed through each column, and the eluate was obtained as a 10% ethanol elution fraction, a 30% ethanol elution fraction, and a 70% ethanol elution fraction, respectively. Each eluted fraction was dried using an evaporator and a freeze-dryer and stored in a refrigerator.

(2) LH-20 fractionation of 10% ethanol eluted fraction Among the HP20 fractions, the 10% ethanol eluted fraction was fractionated using Sephadex (registered trademark) LH-20 (column capacity: 500 mL). LH-20 washed with ethanol was packed into a column for large-scale fractionation and replaced with water. The 10% ethanol eluted fraction (2.57 g) obtained by HP20 fractionation was dissolved in distilled water and applied to the LH-20 column. 1 L of distilled water was passed through to obtain water eluted fractions-1 to -5. Next, 1 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) Separation of Adenosine Water-eluted fraction-3 (82.4 mg) obtained by LH-20 fractionation was eluted with 10% ethanol using HPLC (COSMOSIL 5C18-PAQ, 20×250 mm). The eluate from 5 min to 12 min was concentrated and eluted with a mixed solution of ethanol-water (5:95→15:85) with a concentration gradient using HPLC (COSMOSIL 5C18-PAQ, 20×250 mm) to obtain compound (I) (tR=18.5 min).
Compound (I) was identified as adenosine 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: AVANCE400, 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). Next, elution was performed with 20 mM sodium acetate (pH 4.5) + 0.5 M-NaCl, and fractions were collected. The obtained fractions were evaluated by SDS-PAGE, and fractions containing a 40 kDa protein were collected and used as cation exchange resin-bound fractions.

(2) Ultrafiltration (buffer exchange)
10 mL of the cation exchange resin-bound fraction obtained in (1) was added to an ultrafiltration unit (Merck Amicon Ultra-15 30K) that had been washed with water, and the unit was centrifuged at 3,500 rpm for ultrafiltration to obtain a concentrate.

(3) Ammonium Sulfate Fraction 20 mM phosphate buffer (pH 7.0) + 2M ammonium sulfate was placed in a beaker, and the concentrated solution 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 concentrated solution was added to 20 mM sodium acetate (pH 4.5) and centrifuged (2330 g, 10 minutes, room temperature) to concentrate, and a 40 kDa protein purified product (Bradford quantification (bovine serum albumin (BSA) equivalent), 20.4 mg/mL, 2.21 mL) was obtained. 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 reduced with dithiothreitol (56°C, 1 hour) and carbamidomethylated with iodoacetamide (under light shielding, at room temperature, for 45 minutes). Then, 15 μL of 10 ng/μL chymotrypsin solution (5 mM calcium chloride, 50 mM ammonium bicarbonate solution) containing 0.01% ProteaseMax and 15 μL of 5 mM calcium chloride and 50 mM ammonium bicarbonate solution were added and incubated overnight, after which the enzyme digestion solution was collected. The collected solution was dried 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 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: 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 misscleavage: 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%).

(Sensory evaluation of commercially available beer-flavored alcoholic beverage 1 with adenosine added)
Adenosine was added to a commercially available beer-taste alcoholic beverage 1, and a sensory evaluation of fullness was performed.
The beer-flavored alcoholic beverage is a beer-flavored alcoholic beverage in which the ratio of malt in the raw materials is 50% by weight or more.
The raw materials for this beer-flavored alcoholic beverage are malt and hops, and its nutritional components per 100 ml include 5.5% alcohol, 0.4 to 0.6 g of protein, 3.6 g of carbohydrates, and approximately 12.5 mg of purines.

The standard points for the sensory evaluation are as follows:
A panel of five experts 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 standard score, the fullness of a reference beer-taste alcoholic beverage (I) was set to 1.5 points using the same beer-taste alcoholic beverage as the commercially available beer-taste alcoholic beverage 1 to be evaluated. In addition, the fullness of a beverage obtained by adding 25 ppm of 40 kDa protein to the same beer-taste alcoholic beverage as the commercially available beer-taste alcoholic beverage 1 to make the 40 kDa protein concentration 50 ppm was set to 2 points.

The procedure for the sensory evaluation was as follows.
(1) Dispense the beer-flavored alcoholic beverage into a vial at 1/10 of the final volume (v/v). (2) Weigh out an arbitrary amount of adenosine and add it. (3) Sonicate for 30 seconds. (4) Allow to stand at room temperature for 30 minutes. (5) Fill up 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 adenosine 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 Adenosine was diluted to the following concentrations and passed through a 0.22 μm filter before being used for 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 measured values of the dilution ratios were adopted so that the measured 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 measurement conditions for LC-MS are as follows.
LC-MS: AB Sciex X500R
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-taste alcoholic beverages. A commercially available beer-taste alcoholic beverage was degassed by sonication, and after the air bubbles had settled, it 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 adenosine contained in commercially available beer-flavored alcoholic beverage 1 was designated as Control 1.

The concentration of the 40 kDa protein contained in the commercially available beer-taste alcoholic beverage 1 used in the sensory evaluation was measured using an HT Protein Express chip on a LabChip™ GXII device (PerkinElmer) according to the standard protocol (n=3).
The concentration of the 40 kDa protein contained in the commercially available beer-taste alcoholic beverage 1 was 25 ppm.

(Example 1: Evaluation by adding adenosine)
The concentration of adenosine contained in commercially available beer-taste alcoholic beverage 1 (Control 1) was 41.9 ppm.
Adenosine was added to each of the samples so that the adenosine concentrations became 50 ppm, 100 ppm, and 150 ppm, respectively, and sensory evaluation was performed (samples 2, 4, and 5).
The results of the sensory evaluation are shown in Table 1.

Example 2: Evaluation of synergistic effect of adenosine and 40 kDa protein
A 40 kDa protein and adenosine were added to a commercially available beer-flavored alcoholic beverage 1 (Control 1) so that the 40 kDa protein concentration was 30 ppm and the adenosine concentrations were 45 ppm and 50 ppm, respectively, and a sensory evaluation test was performed (Sample 1 and Sample 3).
The 40 kDa protein used was the one purified as described above.
For comparison, 5 ppm of the 40 kDa protein alone was added to commercially available beer-flavored alcoholic beverage 1, and an evaluation was also performed when the 40 kDa protein concentration was 30 ppm (control sample 1).
The results of the sensory evaluation are shown in Table 1.
The purine concentration of each sample is shown in Table 1. As described above, the "purine concentration" in this specification refers to the total amount of four purine bases, adenine, guanine, xanthine, and hypoxanthine, and can be detected, for example, by using LC-MS/MS (liquid chromatography mass spectrometry) after hydrolysis with perchloric acid.

The results for Samples 2, 4, and 5 in Table 1 show that for beer-flavored alcoholic beverages with a purine concentration of 5 mg/100 mL or more, when the adenosine concentration was 50 ppm or more, the average sensory evaluation score was 0.1 points higher than the control (1.5), effectively enhancing swelling. This suggests that by setting the adenosine concentration in a beer-flavored alcoholic beverage with a purine concentration of 5 mg/100 mL or more to a specified level or higher, swelling can be effectively enhanced compared to the control.
Furthermore, the results of Comparative Sample 1 show that simply adding 40 kDa protein to a beer-taste alcoholic beverage increases the score by 0.08 points compared to the control, and enhances the swelling. However, the sensory evaluation of Sample 1, which uses adenosine and 40 kDa protein in combination, shows an increase of 0.1 points from the control, indicating that even when the adenosine concentration is less than 50 ppm, the combination with 40 kDa protein effectively enhances swelling. This shows that the combination of adenosine and 40 kDa protein exerts an unexpected synergistic effect in enhancing swelling.

(Example 3: Evaluation by adding adenosine)
The adenosine concentration in the commercially available beer-taste alcoholic beverage 2 (Control 2) was 17.6 ppm, and the 40 kDa protein concentration was 6.5 ppm.
A sensory evaluation was conducted by adding adenosine to this commercially available beer-taste alcoholic beverage 2 to bring the adenosine concentration to 50 ppm (Sample 6). In addition, adenosine was added to commercially available beer-taste alcoholic beverage 2 to bring the adenosine concentration to 50 ppm, and 40 kDa protein was further added to bring the 40 kDa protein concentration to 10 ppm (Sample 7). The procedure for the sensory evaluation was the same as that in Example 1, and the reference points for the sensory evaluation were as follows:
The results of the sensory evaluation are shown in Table 2. The purine concentration of each sample is also shown in Table 2. As described above, the "purine concentration" in this specification refers to the total amount of four purine bases, adenine, guanine, xanthine, and hypoxanthine, and can be detected, for example, by using LC-MS/MS (liquid chromatography mass spectrometry) after hydrolysis with perchloric acid.

The beer-taste alcoholic beverage 2 evaluated in Example 3 is a beer-taste alcoholic beverage in which the ratio of malt in the ingredients is 50% by weight or more. This beer-taste alcoholic beverage 2 is a different beer-taste alcoholic beverage from the commercially available beer-taste alcoholic beverage 1 used in Examples 1 and 2. The ingredients of beer-taste alcoholic beverage 2 are malt, hops, rice, corn, and starch, and the nutritional components per 100 ml are 5% alcohol, 0.2-0.4 g of protein, 3.0 g of carbohydrates, and 5-6 mg of purines.

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 2.

The results for Sample 6 in Table 2 show that for beer-taste alcoholic beverage 2, which has a purine concentration of 5 mg/100 mL or more, when the adenosine concentration is 50 ppm or more, the average sensory evaluation score is 0.07 points higher than Control 2 (1.0), and swelling is enhanced, even when the 40 kDa protein concentration is as low as 6.5 ppm. This shows that by setting the adenosine concentration to a predetermined level or higher, swelling can be enhanced compared to Control 2, even when the 40 kDa protein concentration is low.
Furthermore, the increase in the sensory evaluation value from control 2 for sample 7, which has an adenosine concentration of 50 ppm and a 40 kDa protein concentration of 10 ppm, is 0.1 points or more, indicating that the adenosine concentration is equal to or higher than a predetermined concentration and the 40 kDa protein concentration is equal to or higher than a predetermined concentration, thereby effectively enhancing swelling. This indicates that an unexpected synergistic effect is exerted in enhancing swelling by having an adenosine concentration of 50 ppm or more and a 40 kDa protein concentration of 10 ppm or more.

According to the present invention, a beer-flavored alcoholic beverage having a purine concentration of 5 mg/100 mL or more can be provided, which has enhanced volume.

Claims (1)

A beer-flavored alcoholic beverage having a purine concentration of 6.98 to 17.97 mg/100 mL, an adenosine concentration of 50 to 150 ppm, and a total concentration of barley-derived Serpin Z4 and barley-derived Serpin Z7 of 25 to 30 ppm.