TW202212556A - Beer-taste alcoholic beverage - Google Patents

Abstract

The purpose of the present invention is to provide: a beer-taste alcoholic beverage in which the ratio of malt in the raw material is less than 50 wt%, said beverage having a purine concentration of less than 5 mg/100 mL, wherein the beer-taste alcoholic beverage has enhanced swelling; and a beer-taste alcoholic beverage in which the ratio of malt in the raw material is less than 50 wt%, said beverage having a purine concentration of less than 5 mg/100 mL, wherein the beverage has enhanced swelling and beer-like flavor. The present invention relates to, inter alia, a beer-taste alcoholic beverage in which the ratio of malt in the raw material is less than 50 wt%, wherein the beer-taste alcoholic beverage has a purine concentration of less than 5 mg/100 mL and an adenosine concentration of 5-10 ppm.

Description

beer-flavored alcoholic beverages

The present invention relates to beer-flavored alcoholic beverages.

With the diversification of consumers' tastes in recent years, the development of a beer-flavored alcoholic beverage having various flavor characteristics is desired.

Patent Document 1 discloses a peptide containing a specific molecular weight in order to improve the flavor of a beer-flavored alcoholic beverage.

In addition, as the health orientation increases, the demand for low-calorie, low-carbohydrate, and low-purine beverages also increases. However, in such low-calorie, low-carbohydrate and low-purine beverages, the aroma characteristics of these ingredients will disappear, and the malt of the raw material is also insufficient, so the aroma characteristics derived from malt may disappear. question. Patent Document 2 discloses a method for increasing the taste of beer while reducing the purine concentration. [Prior Art Literature] [Patent Literature]

[Patent Document 1] Japanese Patent Laid-Open No. 2016-149975 [Patent Document 2] International Publication No. 2019/130458

[Problems to be Solved by the Invention]

Patent Document 1 describes a beverage, which is used as an evaluation index for the beverage, using such indicators as beer-like flavor, smooth taste flow, and gritty feeling remaining in the mouth. , the sensory evaluation score based on these indicators is higher. In addition, Patent Document 2 describes, as an evaluation index for beverages, a comprehensive evaluation using the mouthfeel of beer as a beer-flavored beverage.

Here, as a taste immediately after drinking a beer-flavored beverage, the five basic tastes, namely, sweet, salty, sour, bitter, and umami, cannot be expressed as the intensity, diffusion, and richness of the taste. Characteristics of a balance of intensity, flavor persistence or flavor intensity are preferred. Such a feature is called "swelling degree" in this specification. The beverages described in Patent Document 1 and Patent Document 2 are beverages with room for improvement from the viewpoint of swelling degree, and a method for enhancing the swelling degree of beer-flavored alcoholic beverages is desired. Also, a beverage with enhanced flavors in addition to swelling is also desired.

The purpose of the present invention is to provide a beverage with enhanced swelling. It is also an object of the present invention to provide a beverage with enhanced swelling and beer-like bitterness. In particular, the purpose of the present invention is to provide a beverage with enhanced swelling in a beer-flavored alcoholic beverage in which the proportion of malt in the raw material is less than 50% by weight and the concentration of purines is less than 5 mg/100 mL. Furthermore, in a beer-flavored alcoholic beverage in which the ratio of malt in a raw material is less than 50% by weight and the concentration of purines is less than 5 mg/100mL, the swelling degree and the beer-like bitterness of the beer-flavored alcoholic beverage are also enhanced. One of the objectives of the present invention. Beer-flavored alcoholic beverages with a malt ratio of less than 50% by weight tend to lack swelling or beer-like bitterness. Further, if the purine concentration is less than 5 mg/100mL, swelling or beer-like bitterness tends to occur. A tendency to lack even more. Therefore, such a beverage is particularly desired to be a beverage that enhances swelling, or in addition to swelling, also enhances beer-like bitterness. Here, in the present invention, the beer-like bitterness means a refreshing and high-quality bitterness that is felt when drinking a beer-flavored alcoholic beverage, and a bitterness that does not leave bad staying power. [Means of Solving Problems]

That is, the present invention relates to one of the following beer-flavored alcoholic beverages. [1] A beer-flavored alcoholic beverage, which is a beer-flavored alcoholic beverage in which the proportion of malt in the raw material is less than 50% by weight, the concentration of purine is less than 5 mg/100 mL, and the concentration of adenosine is 5-10 ppm. [2] A beer-flavored alcoholic beverage, which is a beer-flavored alcoholic beverage in which the proportion of malt in the raw material is less than 50% by weight, the concentration of purine is less than 5 mg/100 mL, and the concentration of adenosine is 12-18 ppm. [3] The beer-flavored alcoholic beverage according to [1] or [2], further comprising a protein having a molecular weight of 35 to 50 kDa, and the concentration of the protein is 1 ppm or more. [4] The beer-flavored alcoholic beverage according to [3], wherein the protein concentration is 30 ppm or less. [Effect of invention]

According to the present invention, it is possible to provide a beverage which is a beer-flavored alcoholic beverage in which the ratio of malt in the raw material is less than 50% by weight, and the purine concentration is less than 5 mg/100 mL, and the swelling degree is enhanced. And, by the present invention, it is possible to provide a beverage, which is a beer-flavored alcoholic beverage in which the ratio of malt in the raw material is less than 50% by weight, and in the above-mentioned beverage in which the concentration of purine is less than 5 mg/100mL, swelling degree and similar. The bitterness of the beer is enhanced.

The proportion of malt in the beer-flavored alcoholic beverage of the present invention is less than 50% by weight. The ratio of malt is preferably 30% by weight or more. Moreover, the ratio of the malt in the beer-flavored alcoholic beverage system raw material of this invention may be 0%. The "ratio of malt" here means the ratio of the weight of malt, rice, corn, sorghum, potato, starch, wheat other than malt and sugar to water and malt other than hops. However, ingredients that can be added in trace amounts, such as sour, sweet, bitter, seasoning, and spice, are not included in the calculation of the above ratio.

The beer-flavored alcoholic beverage of the present invention has a purine concentration of less than 5 mg/100 mL. In this specification, "purine body" means a compound as long as it has a purine core structure, and is not particularly limited. Therefore, examples of "purinosomes" include purine bases (adenine, guanine, xanthine, hypoxanthine), purine nucleosides (adenosine, guanosine, inosine), and purine nucleotides (adenosine). Nucleotides, bird nucleotides, inosinic acid) and low-molecular or high-molecular nucleic acids (oligonucleotides, polynucleotides), etc. In addition, in the present specification, the "purinosome concentration" means the total amount of the four purinosome bases of adenine, guanine, xanthine, and hypoxanthine. The determination of purine bodies can be carried out by a known method, for example, by a method that can be measured by using LC-MS/MS (liquid chromatography mass spectrometry) after hydrolysis with perchloric acid (“Purine bodies in wines”). "Introduction to Microanalysis", Japan Food Analysis Center, URL: http://www.jfrl.or.jp/item/nutrition/post-31.html) to measure.

The concentration of the purine body in the beer-flavored alcoholic beverage of the present invention is preferably 4.5 mg/100 mL or less, more preferably 4.2 mg/100 mL or less.

Adenosine is contained in the beer-flavored alcoholic beverage of the present invention. Adenosine is a type of nucleosides containing adenine and ribose. In addition, the above-mentioned adenosine is not a derivative of adenosine including 5'deoxyadenosine. The present invention relates to a beer-flavored alcoholic beverage in which the proportion of malt in the raw materials is less than 50% by weight, the concentration of purine is less than 5 mg/100 mL, and the concentration of adenosine is 5-10 ppm, or a A beer-flavored alcoholic beverage is a beer-flavored alcoholic beverage in which the proportion of malt in the raw material is less than 50% by weight, the concentration of purine is less than 5 mg/100 mL, and the concentration of adenosine is 12-18 ppm. In this specification, the beer-flavored alcoholic beverage of the present invention with adenosine concentration of 5-10 ppm is described as the first aspect of the present invention, and the beer-flavored alcoholic beverage of the present invention with adenosine concentration of 12-18 ppm is described as the present invention The second form is explained. In addition, the description common to the 1st aspect and the 2nd aspect of this invention is simply described as "this invention" or "the beer-flavored alcoholic beverage of this invention".

In the beer-flavored alcoholic beverage of the first aspect of the present invention, the concentration of the adenosine is 5 ppm or more. If the concentration of adenosine is 5 ppm or more, the ratio of malt in the raw material is less than 50% by weight, and the swelling degree of beer-flavored alcoholic beverages whose purine concentration is reduced to less than 5 mg/100 mL can be enhanced. The fact that the present inventors discovered that adenosine containing a specific concentration or more of adenosine in a beer-flavored alcoholic beverage with a purine concentration of less than 5 mg/100 mL and the swelling degree of the beer-flavored alcoholic beverage has not yet been known.

In the beer-flavored alcoholic beverage of the first aspect of the present invention, the concentration of adenosine is 10 ppm or less. This is because when the concentration of adenosine becomes too high, in addition to an increase in swelling degree, a taste such as spiciness may become stronger. Therefore, in the beer-flavored alcoholic beverage of the present invention, by setting the concentration of adenosine to 5 to 10 ppm, the degree of swelling can be enhanced without causing an increase in spiciness. In the beer-flavored alcoholic beverage of the first aspect of the present invention, the concentration of adenosine is preferably 5-8 ppm.

In the beer-flavored alcoholic beverage of the second aspect of the present invention, the concentration of the adenosine is 12 ppm or more. If the concentration of adenosine is 12 ppm or more, it can enhance the swelling degree of the beer-flavored alcoholic beverage in which the ratio of malt in the raw material is less than 50% by weight and the purine concentration is reduced to less than 5 mg/100mL, and, It can also increase the beer-like bitterness in the taste. The fact that the present inventors discovered that 12 ppm or more of adenosine in a beer-flavored alcoholic beverage with a purine concentration of less than 5 mg/100 mL is related to the swelling degree and beer-like bitterness of the beer-flavored alcoholic beverage has not yet been known.

In the beer-flavored alcoholic beverage of the second aspect of the present invention, the concentration of adenosine is 18 ppm or less. When the concentration of adenosine becomes too high, in addition to the enhancement of swelling degree and beer-like bitterness, bitterness or astringent taste of stamina may be strongly felt. Therefore, in the beer-flavored alcoholic beverage of the present invention, by setting the concentration of adenosine to 12-18 ppm, the swelling degree and the beer-like bitterness can be enhanced without increasing the bitterness or astringency of the stamina.

In addition, the beer-flavored alcoholic beverage of the present invention further contains a protein with a molecular weight of 35 to 50 kDa, and the concentration of the protein is preferably 1 ppm or more.

A protein with a molecular weight of 35 to 50 kDa is a protein whose molecular weight appears in the region of 35 to 50 kDa when a beer-flavored alcoholic beverage is subjected to SDS-PAGE electrophoresis. Before supplying the beer-flavored alcoholic beverage to the electrophoresis of SDS-PAGE, for example, the beer-flavored alcoholic beverage may be subjected to extra-limited filtration using a 30 kDa cut off membrane as a pretreatment. The above-mentioned protein is preferably a protein with a molecular weight of 35-45 kDa, more preferably a protein of about 40 kDa. In this specification, proteins with a molecular weight of 35 to 50 kDa are also referred to as 40 kDa proteins.

The 40kDa protein is preferably a protein derived from cereals. It is preferable that the above-mentioned cereals are at least one selected from the group consisting of barley, wheat, corn, rice, and soybeans. Moreover, when a cereal grain is wheat, the protein derived from the well-known wheat used for manufacture of a beer-flavored alcoholic beverage can be contained. Examples of such wheat include barley, wheat, rye, black wheat, barley, oats, and the like, and barley is preferred. In addition, it may be either malted wheat or unmalted wheat, but it is preferably malted wheat malt. These may be contained individually or in combination of 2 or more types.

Further, as the 40 kDa protein, Serpin Z4 (alias: BSZ4, HorvuZ4, Major endosperm albumin or Protein Z) derived from barley (scientific name: Hordeum vulgare) and Serpin Z7 (alias: BSZ7 or HorvuZ7) derived from barley are preferable. In addition, among the above-mentioned proteins, amino acids which are part of the amino acid sequence may also have amino acid sequences deleted, substituted, inserted and/or added.

In addition to adenosine, the swelling degree of a beer-flavored alcoholic beverage can be further enhanced by further containing a 40 kDa protein. Furthermore, the concentration of the 40kDa protein is preferably 5 ppm or more, and more preferably 30 ppm or less.

In addition, when adenosine and 40kDa protein are contained at the same time, the swelling degree of the beer-flavored alcoholic beverage can be effectively enhanced by the synergistic effect of adenosine and 40kDa protein. Moreover, when the beer-flavored alcoholic beverage of the second aspect of the present invention contains both adenosine and 40kDa protein, the synergistic effect of adenosine and 40kDa protein can effectively enhance the swelling degree and similarity of the beer-flavored alcoholic beverage. Bitterness of beer.

The beer-flavored alcoholic beverage of the present invention is a beer-flavored beverage containing alcohol, and the alcohol concentration is preferably 1% (v/v) to 10% (v/v), but is not particularly limited. Furthermore, the source of the alcohol component contained in the beer-flavored alcoholic beverage is not limited to fermentation and non-fermentation. In addition, the alcohol here means ethanol, and is not included in the aliphatic alcohol mentioned later. The beer-flavored beverage means a carbonated beverage having a beer-like flavor.

The manufacturing steps of a general beer-flavored alcoholic beverage common to the first aspect and the second aspect of the present invention are shown below. First of all, in addition to the wheat including malt, other raw materials such as grains, starches, sugars, bitters or colorants and water as necessary, enzymes such as amylase are added as necessary to perform gelatinization, Saccharified and filtered as saccharified liquid. If necessary, add hops, bitters, etc. to the saccharification solution, boil it, and remove solids such as coagulated proteins in a clarification tank. As an alternative to this saccharification solution, hops may be added to the malt extract with warm water added and boiled. Hops can also be blended at any stage from the start of boiling until the end of boiling. The conditions in the saccharification step, boiling step, solid content removal step, etc. may be well-known conditions. For conditions such as fermentation and wine storage steps, well-known conditions can be used. The obtained fermentation broth was filtered, and carbon dioxide gas was added to the obtained filtrate. Then, it is filled in a container, and it goes through a sterilization process to obtain the desired beer-flavored alcoholic beverage. The beer-flavored alcoholic beverage may be produced by adding wheat-derived alcoholic beverages such as barley-derived distilled spirits (eg, distilled spirits, shochu, etc.) to the beer-flavored alcoholic beverages produced by the above-described method.

Beer-flavored alcoholic beverages manufactured without using malt as a raw material are liquid sugar containing carbon sources, nitrogen sources other than malt or malt as amino acid-containing materials, hops, pigments, etc. are mixed with warm water, as Liquid sugar solution. The liquid sugar solution is boiled. When using hops as a raw material, the hops may be mixed with the liquid sugar solution during the boiling, not before the start of boiling. As an alternative to this saccharified liquid, it is also possible to add hops to an extract using a raw material other than malt and to add warm water, and then boil it. Hops can also be blended at any stage from the start of boiling until the end of boiling. For conditions such as fermentation and wine storage steps, well-known conditions can be used. The obtained fermentation broth was filtered, and carbon dioxide gas was added to the obtained filtrate. After that, it was filled in a container and subjected to a sterilization step to obtain a beer-flavored alcoholic beverage in which the ratio of malt in the target raw material was less than 50% by weight.

For non-fermented beer-flavored alcoholic beverages containing alcohol, the alcohol content of the final product can also be adjusted by adding alcohol for raw materials. The addition of the alcohol for raw materials can also be carried out in any step from the saccharification step to the filling step.

The alcohol content of the beer-flavored alcoholic beverage according to the present invention means the content (v/v%) of the alcohol component in the beverage, and can be measured by any known method, but can be measured by, for example, a vibrating density meter. Specifically, it is possible to prepare a sample for removing carbonic acid gas from beverages by filtration or ultrasonic waves, and subject the sample to direct-fire distillation to measure the density of the obtained distillate at 15°C, using the analytical method specified by the National Taxation Bureau (flat). 19. Order No. 6 of the National Taxation Bureau (revised on June 22, 2019) in the attached table "Table 2 Alcohol Composition and Density (15°C) and Specific Gravity (15/15°C) Conversion Table" to convert and calculate. When the alcohol content is less than 1.0%, a commercially available alcohol measuring device or gas chromatography can also be used.

In the beer-flavored alcoholic beverage according to the present invention, an aliphatic alcohol may be added from the viewpoint of imparting a sense of alcohol. The aliphatic alcohol is not particularly limited as long as it is a known one, but an aliphatic alcohol having 4 to 5 carbon atoms is preferable. In the present invention, as preferable aliphatic alcohols, those having 4 carbon atoms include 2-methyl-1-propanol, 1-butanol, and the like, and those having 5 carbon atoms include 3-methyl- 1-butanol, 1-pentanol, 2-pentanol, etc. These can 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 the present specification, the content of the aliphatic alcohol can be measured using headspace gas chromatography.

The saccharide contained in the beer-flavored alcoholic beverage according to the present invention means the saccharide based on the nutrition representation standard for foods (Ministry of Health, Labour and Welfare Notice No. 176 in 2005). Specifically, a carbohydrate means a protein, lipid, dietary fiber, ash, alcohol content, and water that are removed from food. Also, the amount of sugar in a food can be calculated by deducting the amounts of protein, lipid, dietary fiber, ash, and moisture from the weight of the food. At this time, the amounts of protein, lipid, dietary fiber, ash, and moisture were measured by the methods disclosed in the Nutritional Standard. Specifically, the amount of protein was measured by nitrogen quantitative conversion method, the amount of lipid was measured by ether extraction method, chloroform-methanol mixed liquid extraction method, Gerber method, acid decomposition method or Reese-Gottlieb method, dietary fiber The amount of ash is determined by high-speed liquid chromatography or Prosky method, the amount of ash is determined by magnesium acetate adding ashing method, direct ashing method or sulfuric acid adding ashing method, and the amount of moisture is determined by Karl-Fischer method, drying aid method. Dosage method, reduced pressure superheat drying method, atmospheric pressure heating drying method or plastic film method to measure.

The beer-flavored alcoholic beverage related to the present invention caters to the preference for low sugar content in recent years, and can also be low sugar. Therefore, the sugar content of the beer-flavored alcoholic beverage related to the present invention may be less than 2.5 g/100mL or less than 0.5 g/100mL. In addition, the lower limit is not particularly set, but is usually about 0.1 g/100 mL, for example, 0.15 g/100 mL or more or 0.2 g/100 mL or more.

In the beer-flavored alcoholic beverage according to the present invention, hops can be used as part of the raw material. When hops are used, general flake hops, powdered hops, and hop extracts used in the production of beer and the like can be appropriately selected and used according to the desired flavor. In addition, processed hops such as dissimilated hops and reduced hops can also be used. These are included in the hops used in the beer-flavored alcoholic beverage related to the present invention. In addition, the addition amount of hops is not particularly limited, but is typically about 0.0001 to 1% by weight with respect to the total amount of the beverage.

In the beer-flavored alcoholic beverage related to the present invention, other raw materials may be used as necessary within the range that does not hinder the effects of the present invention. Coloring materials such as sweeteners (including high-intensity sweeteners), bitters, flavors, yeast extracts, caramel colors, and soybean saponins can be used as necessary within the range that does not hinder the effects of the present invention. Or plant extract saponins such as saponins, plant proteins such as corn or soybeans and substances containing peptides, bovine serum albumin and other protein-based substances, dietary fibers or amino acids and other seasonings, ascorbic acid, etc. of antioxidants.

The beer-flavored alcoholic beverage related to the present invention can be packaged as a container. There is no restriction on the form of the container, and it can be filled in sealed containers such as bottles, cans, barrels, or pet bottles, and can be used as beverages contained in containers.

The method for producing the beer-flavored alcoholic beverage of the present invention is not particularly limited, but is exemplified in the beer-flavored alcoholic beverage in which the ratio of malt in the raw material is less than 50% by weight and the concentration of purines is less than 5 mg/100 mL. The method of adding adenosine so that the purinosome concentration does not exceed 5mg/100mL. Furthermore, it is preferable to add 40 kDa protein to the beer-flavored alcoholic beverage in which the ratio of malt in the raw material is less than 50% by weight and the concentration of purines is less than 5 mg/100 mL. The preparation of the added adenosine and the 40 kDa protein can be carried out, for example, in the order described in the following Examples. Moreover, regarding adenosine and 40kDa protein, the content of these can also be increased by adjusting various conditions in the production process of the beer-flavored alcoholic beverage. [Example]

The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples.

(purification of adenosine) Purification of adenosine was carried out as follows. (1) Fractionation of HP20 of beer 60 L of beer was fractionated using 10 L of Diaion (registered trademark) HP20 (manufactured by Mitsubishi Chemical Co., Ltd.). The HP20 was washed three times with ethanol and then three times with 50% ethanol before use. The washed HP20 was packed in a column for a large number of fractionation and replaced with water. Mix the degassed 60L beer with the same amount of distilled water, use a medium pressure pump, and flow into the HP20 column. A solution that passed through the HP20 column was obtained as the pass fraction. Using a medium-pressure pump, 40 L of distilled water was poured into the solution to obtain an elution solution, which was used as a water elution fraction. Similarly, 40 L of water-containing ethanol (10% ethanol, 30% ethanol, and 70% ethanol) were each poured to obtain eluates, which were used as 10% ethanol eluted fractions, 30% ethanol eluted fractions, and 70% ethanol eluted fractions. The respective eluted fractions were refrigerated and stored as dried products using an evaporator and a freeze dryer.

(2) LH-20 Fractionation of 10% Ethanol Dissolution Fraction Among the HP20 fractions, the 10% ethanol eluted fraction was fractionated using Sephadex (registered trademark) LH-20 (column amount: 500 mL). The LH-20 washed with ethanol was packed into a column for fractionation in large quantities, and replaced with water. The 10% ethanol-eluted fraction (2.57 g) obtained by fractional distillation with HP20 was dissolved in distilled water and used for LH-20 column. Flow into 1L of distilled water to obtain water-dissolved fractions -1~5. Then, 1 L of water-containing ethanol (35% ethanol, 70% ethanol and 100% ethanol) was respectively poured into the solution to obtain eluates, which were respectively used as 35% ethanol eluted fraction, 70% ethanol eluted fraction and 100% ethanol eluted fraction. The respective eluted fractions were refrigerated and stored as dried products using an evaporator and a freeze dryer.

(3) Separation of adenosine The water-eluted fraction-3 (82.4 mg) obtained by fractional distillation of LH-20 was eluted with 10% ethanol using HPLC (COSMOSIL 5C18-PAQ, 20×250 mm). Next, the eluate was concentrated from 5 minutes to 12 minutes, and eluted by a mixture of a concentration gradient of ethanol-water (5:95→15:85) using HPLC (COSMOSIL 5C18-PAQ, 20×250 mm), Compound (I) was obtained (tR=18.5 min). Compound (I) was identified as adenosine by analysis of the physical data of MS and NMR and comparison with the standard substance. The analytical machine used is as described below. LC-MS; Q Exactive, manufactured by Thermo Fisher Scientific NMR; AVANCE400, manufactured by Bruker

(purification of 40kDa protein) Purification of the 40 kDa protein was performed from a commercial beer (1 L) as described below.

(1) Fractionation of cation exchange resin The cation exchange resin SP Sepharose 50mL was put into the empty column. Make the beer adsorb the resin. After that, the resin was converted into a column and washed with 20 mM sodium acetate buffer (pH 4.5). Next, it was eluted 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 contained in the 40 kDa protein were collected as cation exchange resin bound fractions.

(2) Filter outside the limit (buffer exchange) 10 mL of each of the cation exchange resin-bound fractions obtained in (1) was added to an extra-limit filtration unit (Amicon Ultra-15 30K manufactured by Merck) after washing with water, and centrifuged at 3500 rpm to obtain an extra-limit filtration concentrate.

(3) Ammonium sulfate fractionation 20 mM phosphate buffer (pH 7.0) + 2 M ammonium sulfate was added to the beaker, and the concentrated solution obtained in (2) was added dropwise and stirred. The suspension was then centrifuged (2330 g, 10 min, room temperature). Collect the supernatant in another container. The collected solution was concentrated using an out-of-limit filter unit. The concentrate was added to 20 mM sodium acetate (pH 4.5), centrifuged (2330 g, 10 minutes, room temperature), and concentrated to obtain a 40 kDa protein purified product (Bradford quantification (bovine serum albumin (BSA) conversion), 20.4 mg/mL, 2.21 mL). The purity of the obtained 40 kDa protein was confirmed by SDS-PAGE.

After the 40kDa protein was digested with enzymes, the identification of the protein was attempted by analysis by LC-MS/MS. Fragments around 40kDa separated by SDS-PAGE were excised and subjected to reduction of dithiothreitol (56°C, 1 hour) and methylation of carbamoylamine with iodoacetamide (under light-shielding, 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% ProteaseMax, 15 μL of 5 mM calcium chloride, 50 mM ammonium bicarbonate solution was added, and after overnight incubation, the enzyme digestion was recovered. liquid. The recovered solution was dried under reduced pressure, and then dissolved in 0.1% formic acid solution. This was used in LC-MS/MS analysis.

(Measurement by LC-MS/MS) The measurement by LC-MS/MS was carried out under the following conditions. Equipment used: direct flow nanoLC system Easy-nLC 1000TM (Thermo Scientific) Capture column: Acclaim PepMap (registered trademark) (Thermo Scientific) Analytical column: NANO HPLC CAPILLARY COLUMN (Nikyo-tec Co., Ltd.) Liquid chromatography mass analyzer 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 Gradients: 0-40%B/0-30min, 40-60%B/30-35min, 60-90%B/35-37min, 90%B/37-45min Injection volume: 10 μL Ionization Mode: ESI Positive Measuring range: MS1 (m/z 350-1750) Data Dependent Scan Mode

(4) Analysis of protein Protein identification was performed under the following conditions. Search software: Proteome Discoverer 2.2.0.388 (manufactured by ThermoFisher) Biological species: barley (Hordeum vulgare), hops (Humulus), yeast (Saccharomyces cerevisiae) Search terms: Digestive enzyme: chymotrypsin Precursor ion mass error range: monoisotope, ±10ppm Product ion mass error range: ±0.02Da Maximum number of missed cleavages: 5 Percolator: High (the highest degree of accuracy in the three stages of accuracy) Database: SwissProt

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

(Sensory evaluation of adding adenosine to commercially available beer-flavored alcoholic beverages) Adenosine was added to a commercially available beer-flavored alcoholic beverage, and sensory evaluation of swelling degree was performed. The beer-flavored alcoholic beverage is a beer-flavored alcoholic beverage in which the raw material contains malt, the proportion of the malt in the raw material is less than 50% by weight, and the concentration of purine is less than 5 mg/100 mL. The raw materials are sparkling wine, malt, hops, sugar, dietary fiber, and distilled liquor (wheat). As nutrients, each 100ml contains 4% alcohol, 0~0.2g protein, 0.5~0.8g carbohydrate, and purine. About 2.0mg.

The standard score of the sensory evaluation of swelling degree is as follows. Five professional evaluators were assigned points on a scale of 0.05 points according to the following criteria, and the score values were averaged. The swelling degree strength is based on the following criteria. 0 points: no feeling at all 1 point: Slightly felt 2 points: clearly felt 3 points: very feeling As a reference point, the swelling degree of the beer-flavored alcoholic beverage (I) which is the same as the above-mentioned commercially available beer-flavored alcoholic beverage as the evaluation object was set as a reference was made 0.7 point. In addition, other commercially available beer-flavored alcoholic beverages were used as the standard beer-flavored alcoholic beverages (II), and the swelling degree was set to 1.5 points of the standard. The standard beer-flavored alcoholic beverage (II) is a beer-flavored alcoholic beverage in which the proportion of malt in the raw material is 50% by weight or more. The raw materials are malt and hops. As nutrients, each 100ml contains 5.5% alcohol content, 0.4~0.6g protein, 3.6g carbohydrate, and about 12.5mg purine.

In addition, the standard score of the sensory evaluation of the bitterness like beer is as follows. With four professional evaluators, the scale was 0.05 points, and it was scored according to the following criteria, and the scores were averaged. The bitterness intensity of beer-like is the following standard. 0 points: no feeling at all 1 point: Slightly felt 2 points: clearly felt 3 points: very feeling As a reference point, the beer-flavored alcoholic beverage (I) which is the same as the above-mentioned commercially available beer-flavored alcoholic beverage as the evaluation object was set as a reference, and its beer-like bitterness was set as 1.0 point. In addition, regarding the beer-flavored alcoholic beverage (II) of the other commercially available beer-flavored alcoholic beverages described above, the beer-like bitterness was set to 2.0 points of the standard.

The order of sensory evaluation is as follows. (1) Pour beer-flavored alcoholic beverages into small glass bottles of 1/10 of the final capacity (v/v) (2) Weigh any weight of adenosine and add (3) Vibration treatment for 30 seconds (4) Stand at room temperature for 30 minutes (5) Fill the beer-flavored alcoholic beverage to the final capacity (6) Drinking evaluation after injection

(Analysis of Commercially Available Beer-Flavored Alcoholic Beverage) The concentration of adenosine contained in the commercially available beer-flavored alcoholic beverage used in the sensory evaluation was quantified by LC-MS by the following procedure. (1) Preparation of standard substance and preparation of calibration curve Regarding adenosine, each of the adenosine was diluted to the following concentrations, passed through a 0.22 μm filter, and then supplied for measurement. Final concentration: 0.001ppm, 0.025ppm, 0.050ppm, 0.100ppm, 0.200ppm, 0.300ppm, 0.500ppm, 0.750ppm, 1.000 ppm (1ppm=1μg/mL) Diluent use 5% (v/v) ethanol in water. And, in the analysis result of the standard product, the measured value of the dilution ratio within the range of maintaining the linearity of the calibration curve (R ² >0.99) was used.

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

(2) Preparation of test samples from commercially available beer-flavored alcoholic beverages Commercially available beer-flavored alcoholic beverages were degassed by vibrating, and after the bubbles were relieved, they were appropriately diluted and passed through a 0.22 μm filter before being supplied to the measurement. A 5% (v/v) ethanol aqueous solution was used as the diluent. The concentration of each adenosine contained in a commercially available beer-flavored alcoholic beverage was taken as a control group.

(Example 1: Evaluation of the swelling degree of adenosine and/or 40kDa protein added in the first form of the present invention) The concentration of adenosine contained in the commercially available beer-flavored alcoholic beverage (control group) was 4.25 ppm. On the other hand, adenosine was added so that the concentration of adenosine was 5 ppm and 8 ppm, respectively, and sensory evaluation was performed (Sample 1 and Sample 2). Also, the concentration of the 40 kDa protein contained in the commercially available beer-flavored alcoholic beverage (control group) was 0 ppm. On the other hand, adenosine and a 40 kDa protein were added so that the adenosine concentration was 5 ppm and the 40 kDa protein concentration was 5 ppm and 9 ppm, respectively, and functional evaluation was performed (samples 3 and 4). Then, 40kDa protein was added so that the concentration of 40kDa protein contained in a commercially available beer-flavored alcoholic beverage (control group) was 5 ppm, 9 ppm, and 25 ppm, and sensory evaluations were performed (Comparative Sample 1, Comparative Sample 2, and Comparative Sample 3). Among the 40kDa proteins, those purified above were used. The results of the sensory evaluation are shown in Table 1. In addition, the purine body concentration of each sample is shown in Table 1 below. The "purinomer concentration" in the present specification is the total amount of the four purinomer bases of adenine, guanine, xanthine, and hypoxanthine, as described above, and can be used, for example, after hydrolysis with perchloric acid using LC- MS/MS (liquid chromatography mass spectrometry) to detect.

From the results shown in Table 1, it can be known that the ratio of malt in the raw material is less than 50% by weight, and the concentration of purine is less than 5mg/100mL. 1 (5ppm), sample 2 (8ppm)), the degree of swelling will increase. In addition, it was found that the swelling degree can be further enhanced by adding adenosine and 40 kDa protein to a commercially available beer-flavored alcoholic beverage (Sample 3 and Sample 4). From the results of Comparative Sample 1, Comparative Sample 2, and Comparative Sample 3, it can be seen that only adding 40 kDa protein can enhance the degree of swelling. However, the sum of the increase in the control group (0.03) from the sensory evaluation of Sample 1 above and the increase in the control group (0.05) from the sensory evaluation of Comparative Sample 1 can be found, compared to the combination of adenosine (5 ppm). ) and 40kDa protein (5ppm) (0.08), the increase value (0.13) of the control group derived from the functional evaluation of sample 3 is larger, so by using adenosine in combination with 40kDa protein, it is possible to exert unexpected effects. Multiplier effect. Moreover, the sum of the increase value (0.03) of the control group from the sensory evaluation of the above-mentioned sample 1 and the increase value (0.08) of the control group from the sensory evaluation of the comparative sample 2 can be obtained, compared with the combined use of adenosine (5ppm). ) and 40kDa protein (9ppm), the increase value (0.17) of the control group from the functional evaluation of sample 4 was larger, so by using adenosine in combination with 40kDa protein, it was possible to exert unexpected effects. Multiplier effect.

(Example 2: Evaluation of the swelling degree of adenosine added in the second aspect of the present invention) The concentration of adenosine contained in the commercially available beer-flavored alcoholic beverage (control group) was 4.25 ppm. On the other hand, adenosine was added so that the concentration of adenosine was 8 ppm, 12 ppm, 18 ppm, and 23 ppm, respectively, and the sensory evaluation of the degree of swelling was performed (Comparative Sample 4, Sample 5, Sample 6, and Comparative Sample 5). The results of the sensory evaluation are shown in Table 2. In addition, the purine body concentration of each sample is shown in Table 2 below. The "purinomer concentration" in the present specification is the total amount of the four purinomer bases of adenine, guanine, xanthine, and hypoxanthine, as described above, and can be used, for example, after hydrolysis with perchloric acid using LC- MS/MS (liquid chromatography mass spectrometry) to detect.

From the results shown in Table 2, it can be known that in the beer-flavored alcoholic beverage in which the ratio of malt in the raw material is less than 50% by weight and the concentration of purine is less than 5mg/100mL, if the concentration of adenosine is 12ppm. Above (Sample 5, Sample 6, Comparative Sample 5), the evaluation of the degree of swelling of the control group will increase by 0.1 points or more, and the degree of swelling will be effectively enhanced. On the other hand, when the sample 6 and the comparative sample 5 in the above-mentioned Table 2 are compared, it can be confirmed that even if the adenosine concentration exceeds 18 ppm, the degree of swelling does not increase effectively as the concentration increases. Therefore, in the beer-flavored alcoholic beverage in which the ratio of malt in the raw material is less than 50% by weight and the concentration of purine body is less than 5 mg/100mL, the adenosine concentration is in the range of 12 to 18 ppm, and the swelling degree will be effective. enhanced.

(Example 3: Evaluation of swelling degree of adenosine and 40kDa protein added in the second form of the present invention) The concentration of adenosine contained in the above commercially available beer-flavored alcoholic beverage was 4.25 ppm and the concentration of 40 kDa protein was 0 ppm. On the other hand, adenosine was added to make the adenosine concentrations 8 ppm, 12 ppm, and 18 ppm, respectively, and 40 kDa protein was added to make the 40 kDa protein concentration 5 ppm, and the functional evaluation of the degree of swelling was performed (Comparative Sample 8, Sample 7, and Sample 8). ). Among the 40kDa proteins, those purified above were used. Furthermore, for comparison, only 40kDa protein was added to the above commercially available beer-flavored alcoholic beverages, and the 40kDa protein concentration was 5 ppm and 25 ppm, and sensory evaluation of swelling degree was performed (Comparative Sample 6 and Comparative Sample 7). The results of the sensory evaluation are shown in Table 3. In addition, the purine body concentration of each sample is shown in Table 3 below.

From the results in Table 3 above, it can be seen that the swelling degree can be further enhanced by adding adenosine and 40 kDa protein (5 ppm) to the commercially available beer-flavored alcoholic beverages (Comparative Sample 8, Sample 7, and Sample 8). In addition, it was found that the swelling degree can be enhanced even by adding only 40 kDa protein (Comparative Sample 6 and Comparative Sample 7). However, the sum of the increase in the control group from the sensory evaluation of Sample 5 (0.11) and the increase in the control group from the sensory evaluation of Comparative Sample 6 (0.06) can be obtained, compared to the combination of adenosine (12 ppm) and the 40kDa protein (5ppm), the additive effect (0.17) was greater in the control group (0.23) derived from the functional evaluation of sample 7. Therefore, by using adenosine in combination with the 40kDa protein, it was possible to exert an ineffective effect. The expected additive effect. The above-mentioned additive effect can be confirmed, and the sum of the increase value (0.14) of the control group from the sensory evaluation of the sample 6 and the increase value (0.06) of the control group from the sensory evaluation of the comparative sample 6 can be obtained. The additive effect (0.2) of adenosine (18 ppm) and 40 kDa protein (5 ppm) was greater for the control group from the functional evaluation of Sample 8 (0.25).

(Example 4: Evaluation of beer-like bitterness after the addition of adenosine and/or 40kDa protein in the second form of the present invention) The concentration of adenosine contained in the commercial beer-flavored alcoholic beverage (control group) was 4.25 ppm and the concentration of 40 kDa protein was 0 ppm. On the other hand, adenosine was added so that the concentration of adenosine was 8 ppm, 12 ppm, 18 ppm, and 23 ppm, respectively, and the sensory evaluation of beer-like bitterness was performed (Comparative Sample 4, Sample 5, Sample 6, and Comparative Sample 5). In addition, adenosine and 40 kDa protein were added to commercial beer-flavored alcoholic beverages so that the concentration of adenosine was 12 ppm, 18 ppm, and the concentration of 40 kDa protein was 5 ppm, respectively, and the sensory evaluation of beer-like bitterness was performed. (Sample 7, Sample 8). Among the 40kDa proteins, those purified above were used. And, for comparison, the sensory evaluation of the bitterness of beer-like in which only the 40 kDa protein contained in the commercially available beer-flavored alcoholic beverage was added (Comparative Sample 6) was performed. The results of the sensory evaluation are shown in Table 4. In addition, the purine body concentration of each sample is shown in Table 4 below.

From the results shown in Table 4, it can be known that in the beer-flavored alcoholic beverages in which the ratio of malt in the raw material is less than 50% by weight and the concentration of purine bodies is less than 5mg/100mL, if the adenosine concentration increases , the bitterness like beer will increase. In addition, it can be seen that if the adenosine concentration is 12 ppm or more (sample 5, sample 6, and comparative sample 5), the evaluation of beer-like bitterness will increase by 0.1 points or more compared to the control group, and the beer-like bitterness is effectively enhanced. . In addition, it was found that by adding adenosine and 40 kDa protein (5 ppm) to a commercially available beer-flavored alcoholic beverage, the bitterness like beer can be further enhanced (Sample 7 and Sample 8). In addition, it was found that the addition of only 40 kDa protein can enhance the bitterness like beer (Comparative Sample 6). However, the sum of the increase in the control group from the sensory evaluation of Sample 5 (0.19) and the increase in the control group from the sensory evaluation of Comparative Sample 6 (0.04) can be obtained, compared to the combined use of adenosine (12 ppm). ) and the 40kDa protein (5ppm), the additive effect (0.23) was greater in the control group (0.4) derived from the functional evaluation of sample 7. Therefore, by using adenosine in combination with the 40kDa protein, unexpected effects can be exhibited. Multiplier effect. The above-mentioned additive effect can be obtained from the sum of the increase value (0.34) of the control group from the sensory evaluation of the sample 6 and the increase value (0.04) of the control group from the sensory evaluation of the control sample 6. The additive effect (0.38) of nucleosides (18 ppm) and 40 kDa protein (5 ppm) was greater for the control group from the functional evaluation of Sample 8 (0.58). In addition, in beer-flavored alcoholic beverages in which the ratio of malt in the raw material is less than 50% by weight and the concentration of purine bodies is less than 5mg/100mL, if the concentration of adenosine exceeds 18ppm, there will be a feeling of stamina. A professional evaluator of bitterness or astringency, therefore believes that in the above-mentioned beer-flavored alcoholic beverages, it is not necessary to increase the bitterness or astringency of the stamina, and when the swelling degree and the bitterness like beer are enhanced, the concentration of adenosine is 12~18ppm. good.

From the above results, it can be confirmed that the ratio of malt in the raw material is less than 50% by weight, and the concentration of purine body is less than 5mg/100mL in beer-flavored alcoholic beverages. Enhance swelling and beer-like bitterness. Furthermore, by using adenosine in combination with a 40 kDa protein, an unexpected synergistic effect can be exhibited in the enhancement of swelling degree and beer-like bitterness. [industrial availability]

According to the present invention, it is possible to provide a beverage with enhanced swelling in a beer-flavored alcoholic beverage in which the proportion of malt in the raw material is less than 50% by weight. Furthermore, according to the present invention, it is possible to provide a beer-flavored alcoholic beverage in which the proportion of malt in the raw material is less than 50% by weight and the concentration of purines is less than 5 mg/100 mL, and the swelling degree and beer-like bitterness are enhanced.

Claims (4)

A beer-flavored alcoholic beverage, which is a beer-flavored alcoholic beverage in which the proportion of malt in the raw material is less than 50% by weight, the concentration of purine is less than 5mg/100mL, and the concentration of adenosine is 5-10ppm. A beer-flavored alcoholic beverage, which is a beer-flavored alcoholic beverage in which the proportion of malt in the raw material is less than 50% by weight, the concentration of purine is less than 5mg/100mL, and the concentration of adenosine is 12-18ppm. The beer-flavored alcoholic beverage according to claim 1 or 2, further comprising a protein with a molecular weight of 35 to 50 kDa, and the concentration of the protein is 1 ppm or more. The beer-flavored alcoholic beverage according to claim 3, wherein the concentration of the protein is 30 ppm or less.