WO2022004715A1

WO2022004715A1 - Beer-taste alcoholic beverage

Info

Publication number: WO2022004715A1
Application number: PCT/JP2021/024544
Authority: WO
Inventors: 理沙高木; 菜子首藤; 恵子岩佐; 正晃小沢; 直人神田; 嘉英松尾
Original assignee: サントリーホールディングス株式会社
Priority date: 2020-07-01
Filing date: 2021-06-29
Publication date: 2022-01-06
Also published as: JPWO2022004715A1; TW202206586A

Abstract

The purpose of the present invention is to provide: a beer-taste alcoholic beverage in which the proportion of malt in starting materials is more than 0 wt% and less than 50 wt% and which has an enriched bulging taste; and a beer-taste alcoholic beverage in which the proportion of malt in starting materials is 50 wt% or more and which has an enriched bulging taste. The present invention pertains to: a beer-taste alcoholic beverage in which the proportion of malt in starting materials is more than 0 wt% and less than 50 wt% and which contains at least one kind of diketopiperazine selected from the group consisting of cycloleucyl proline, cyclovalyl proline and cycloisoleucyl proline, wherein the concentration of the diketopiperazine contained in the largest amount, among these diketopiperazines, is 0.8 ppm or more; and a beer-taste alcoholic beverage in which the proportion of malt in starting materials is 50 wt% or more and which contains at least one kind of diketopiperazine selected from the group consisting of cycloleucyl proline, cyclovalyl proline and cycloisoleucyl proline, wherein the concentration of the diketopiperazine contained in the largest amount, among these diketopiperazines, is 2.2 ppm or more.

Description

Beer taste alcoholic beverage

The present invention relates to beer-taste alcoholic beverages.

With the diversification of consumer tastes in recent years, it is desired to develop beer-taste alcoholic beverages having various flavor characteristics.

Patent Document 1 discloses that a peptide having a specific molecular weight is contained in order to improve the flavor of a beer-taste alcoholic beverage.

Japanese Unexamined Patent Publication No. 2016-149975

In Patent Document 1, indexes such as beer-like taste, smooth taste flow, and roughness remaining in the mouth are used as evaluation indexes for beverages, and by containing a peptide of 10-20 kDa at a predetermined concentration, these indexes can be used. It is stated that a beverage with a high sensory evaluation score based on it can be obtained.

Here, as the taste that can be felt immediately after drinking a beer-taste beverage, the strength, spread, thickness, and taste of the five basic tastes, that is, the tastes that cannot be expressed by sweetness, saltiness, acidity, bitterness, and umami, are maintained. Alternatively, the characteristic that the strength of taste is well-balanced may be preferred. Such a feature is referred to as "bulge" in the present specification.
It can be said that the beverage described in Patent Document 1 is a beverage that has room for further improvement from the viewpoint of swelling, and a method for enhancing swelling has been desired.

An object of the present invention is to provide a beverage having an enhanced bulge.
An object of the present invention is to provide a beer-taste alcoholic beverage in which the proportion of malt in the raw material exceeds 0% by weight and is less than 50% by weight, and the swelling is enhanced. Beer-taste alcoholic beverages in which the proportion of malt exceeds 0% by weight and is less than 50% by weight are particularly desired to enhance swelling because they tend to have poor swelling.
Another object of the present invention is to provide a beverage with enhanced swelling, particularly in a beer-taste alcoholic beverage in which the ratio of malt in the raw material is 50% by weight or more.

That is, the present invention relates to the following beer-taste alcoholic beverages.
[1] A beer-taste alcoholic beverage in which the proportion of malt in the raw material exceeds 0% by weight and is less than 50% by weight, and is selected from the group consisting of cycloleuicylproline, cyclovalylproline and cycloisoleucylproline. A beer-taste alcoholic beverage containing at least one diketopiperazine and having a concentration of the most abundant diketopiperazine among the above-mentioned diketopiperazines of 0.8 ppm or more.
[2] The beer-taste alcoholic beverage according to [1] above, wherein the concentration of diketopiperazine contained most in the above-mentioned diketopiperazine is 10 ppm or less.
[3] The beer-taste alcoholic beverage according to the above [1] or [2], further comprising a protein having a molecular weight of 35 to 50 kDa and having a protein concentration of 1 ppm or more.
[4] The beer-taste alcoholic beverage according to the above [3], wherein the concentration of the protein is 30 ppm or less.
[5] A beer-taste alcoholic beverage in which the ratio of malt in the raw material is 50% by weight or more, and at least one didi selected from the group consisting of cycloleuicylproline, cyclovalylproline and cycloisoleucilproline. A beer-taste alcoholic beverage containing ketopiperazine and having a concentration of diketopiperazine, which is the most abundant among the diketopiperazines, of 2.2 ppm or more.
[6] The beer-taste alcoholic beverage according to [5] above, wherein the concentration of diketopiperazine contained most in the above-mentioned diketopiperazine is 10 ppm or less.
[7] The beer-taste alcoholic beverage according to the above [5] or [6], further comprising a protein having a molecular weight of 35 to 50 kDa and having a protein concentration of 10 ppm or more.
[8] The beer-taste alcoholic beverage according to any one of the above [5] to [7], further comprising a protein having a molecular weight of 35 to 50 kDa and having a protein concentration of 30 ppm or more.
[9] The beer-taste alcoholic beverage according to the above [7] or [8], wherein the concentration of the protein is 200 ppm or less.

According to the present invention, it is possible to provide a beverage with enhanced swelling in a beer-taste alcoholic beverage in which the ratio of malt in the raw material exceeds 0% by weight and is less than 50% by weight. Further, according to the present invention, it is possible to provide a beverage with enhanced swelling in a beer-taste alcoholic beverage in which the ratio of malt in the raw material is 50% by weight or more.

In the beer-taste alcoholic beverage according to the first aspect of the present invention, the proportion of malt in the raw material exceeds 0% by weight and is less than 50% by weight.
The malt ratio is preferably 30% by weight or more.

The beer-taste alcoholic beverage according to the second aspect of the present invention has a malt content of 50% by weight or more in the raw material.
The malt ratio is preferably 100% by weight.

Hereinafter, in the present specification, in the beer-taste beverage in which the ratio of malt in the raw material according to the first embodiment of the present invention is more than 0% by weight and less than 50% by weight, and in the raw material according to the second embodiment of the present invention. The description common to beer-taste alcoholic beverages having a malt ratio of 50% by weight or more is simply described as "beer-taste alcoholic beverage".

Here, the "malt ratio" refers to the ratio of the weight of malt to water and raw materials other than hops, such as malt, rice, corn, kouryan, potato, starch, wheat other than malt, and sugars. However, ingredients that can be added in trace amounts, such as acidulants, sweeteners, bitterness agents, seasonings, and flavors, are not included in the above ratio calculation.

The beer-taste alcoholic beverage of the present invention contains at least one diketopiperazine selected from the group consisting of cycloleucylproline, cyclovalylproline and cycloisoroycilproline.
Diketopiperazine is a cyclic dipeptide in which two amino acids are linked.
Cycloleucylproline is a combination of leucine and proline, and is sometimes referred to as Cyclo (Leu-Pro) in the present specification.
Cyclovalylproline is a combination of valine and proline, and is sometimes referred to as Cyclo (Val-Pro) in the present specification.
Cycloisoleucine proline is a combination of isoleucine and proline, and is sometimes referred to as Cyclo (Ile-Pro) in the present specification.

In the beer-taste alcoholic beverage in which the ratio of malt in the raw material according to the first embodiment of the present invention exceeds 0% by weight and is less than 50% by weight, the concentration of diketopiperazine contained most in the above-mentioned diketopiperazine is 0. It is 8.8 ppm or more.
When the concentration of these diketopiperazines is 0.8 ppm or more, the swelling in the beer-taste alcoholic beverage in which the ratio of malt in the raw material exceeds 0% by weight and is less than 50% by weight can be enhanced. It has been known so far that the content of a specific diketopiperazine above a predetermined concentration is related to the swelling in beer-taste alcoholic beverages in which the proportion of malt in the raw material exceeds 0% by weight and is less than 50% by weight. This is the finding found by the present inventors.

In the beer-taste alcoholic beverage in which the ratio of malt in the raw material, which is the second embodiment of the present invention, is 50% by weight or more, the concentration of diketopiperazine contained most in the above-mentioned diketopiperazine is 2.2 ppm or more.
When the concentration of these diketopiperazines is 2.2 ppm or more, the swelling in the beer-taste alcoholic beverage in which the ratio of malt in the raw material is 50% by weight or more can be enhanced. It has not been known so far that the content of a specific diketopiperazine in a predetermined concentration or more is related to the swelling in a beer-taste alcoholic beverage in which the ratio of malt in the raw material is 50% by weight or more. Is the finding found by.

In the beer-taste alcoholic beverage of the present invention, the concentration of diketopiperazine, which is contained most in the diketopiperazine, is preferably 10 ppm or less.
This is because if the concentration of diketopiperazine becomes too high, the taste such as bitterness and astringency becomes stronger.

Further, the beer-taste alcoholic beverage in which the ratio of malt in the raw material, which is the first embodiment of the present invention, exceeds 0% by weight and is less than 50% by weight, further contains a protein having a molecular weight of 35 to 50 kDa, and the concentration of the above protein. Is preferably 1 ppm or more.

Further, the beer-taste alcoholic beverage in which the ratio of malt in the raw material, which is the second embodiment of the present invention, is 50% by weight or more, further contains a protein having a molecular weight of 35 to 50 kDa, and the concentration of the protein is 10 ppm or more. Is preferable. Moreover, it is preferable that the concentration of the above protein is 30 ppm or more.

A protein having a molecular weight of 35 to 50 kDa is a protein found in a region having a molecular weight of 35 to 50 kDa when a beer-taste alcoholic beverage is subjected to electrophoresis by SDS-PAGE. Before subjecting the beer-taste alcoholic beverage to electrophoresis by SDS-PAGE, for example, the beer-taste alcoholic beverage may be subjected to extrafiltration by using a 30 kDa cut-off membrane as a pretreatment.
The protein is preferably a protein having a molecular weight of 35 to 45 kDa, more preferably a protein having a molecular weight of about 40 kDa. In the present specification, a protein having 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.
When the cereal is wheat, it can contain a known protein derived from wheat used in the production of beer-taste alcoholic beverages. Examples of such wheat include barley, wheat, rye, oats, oats, and barley, and barley is preferable. Further, either germinated wheat or ungerminated wheat may be used, but germinated malt is preferable. These may be contained alone or in combination of two or more kinds.

Further, as the 40 kDa protein, Serpin Z4 (also known as BSZ4, HorvuZ4, Major endosperm albumin or Protein Z) derived from barley (scientific name: Hordeum bulgare) and Serpin Z7 (also known as BSZ7 or HorvZ) derived from barley are preferable. Further, in the above protein, a protein having an amino acid sequence in which a part of the amino acids in the amino acid sequence is deleted, substituted, inserted and / or added may be used.

By further containing 40 kDa protein in addition to diketopiperazine, the proportion of malt in the raw material according to the first embodiment of the present invention is further increased by more than 0% by weight and less than 50% by weight in the beer-taste alcoholic beverage. Can be enhanced.
In a beer-taste alcoholic beverage in which the ratio of malt in the raw material according to the first aspect of the present invention exceeds 0% by weight and is less than 50% by weight, the concentration of 40 kDa protein is preferably 30 ppm or less.

By further containing 40 kDa protein in addition to diketopiperazine, the swelling in a beer-taste alcoholic beverage in which the ratio of malt in the raw material according to the second aspect of the present invention is 50% by weight or more can be further enhanced.
In a beer-taste alcoholic beverage in which the ratio of malt in the raw material, which is the second embodiment of the present invention, is 50% by weight or more, the concentration of 40 kDa protein is preferably 200 ppm or less.

When both diketopiperazine and 40 kDa protein are contained, the swelling of the beer-taste alcoholic beverage can be effectively enhanced by the synergistic effect of diketopiperazine and 40 kDa protein.

The beer-taste alcoholic beverage of the present invention is a beer-taste beverage containing 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 content contained in the beer-taste alcoholic beverage is not limited to fermented and non-fermented. The alcohol here refers to ethanol and does not include the aliphatic alcohol described later. A beer-taste beverage is a carbonated beverage with a beer-like flavor.

The following shows the manufacturing process of a general beer-taste alcoholic beverage common to the first and second embodiments of the present invention.
First, in addition to wheat such as malt, if necessary, enzymes such as amylase are added to a mixture containing raw materials such as other grains, starch, saccharides, bitterness agents, or coloring agents and water, and glue is added. It is saccharified and saccharified, and filtered to obtain a saccharified solution. If necessary, add hops and bitterness to the saccharified solution and boil, and remove solids such as coagulated protein in a clarification tank. As an alternative to this saccharified solution, hops may be added to a malt extract mixed with warm water and boiled. Hops may be mixed at any stage from the start of boiling to the end of boiling. As the conditions in the saccharification step, the boiling step, the solid content removing step and the like, known conditions may be used. As the conditions in the fermentation / liquor storage process and the like, known conditions may be used. The obtained fermentation broth is filtered, and carbon dioxide gas is added to the obtained filtrate. After that, it is filled in a container and subjected to a sterilization step to obtain a desired beer-taste alcoholic beverage.

The beer-taste alcoholic beverage in which the ratio of malt in the raw material, which is the first embodiment of the present invention, exceeds 0% by weight and is less than 50% by weight, is derived from wheat in the beer-taste alcoholic beverage produced by the above method. It may be produced by adding a wheat-derived alcoholic beverage such as distilled liquor (for example, spirits or shochu).

The non-fermented and alcohol-containing beer-taste alcoholic beverage may be one in which the alcohol content of the final product is adjusted by adding alcohol for raw materials or the like. The alcohol for raw materials may be added in any step from the saccharification step to the filling step.

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. It can be measured by a meter. Specifically, a sample from which carbon dioxide gas has been removed from the beverage by filtration or ultrasonic waves is prepared, and the sample is directly distilled to measure the density of the obtained distillate at 15 ° C. Converted using the "Table 2 Alcohol Content and Density (15 ° C) and Specific Weight (15/15 ° C) Conversion Table" attached to (Heisei 19 National Tax Agency Royal Instruction No. 6, revised on June 22, 2007). Can be asked. When the alcohol content is a low concentration of less than 1.0%, a commercially available alcohol measuring device or gas chromatography may be used.

Fatty alcohol may be added to the beer-taste alcoholic beverage according to the present invention from the viewpoint of imparting a feeling of alcohol. The aliphatic alcohol is not particularly limited as long as it is known, but an aliphatic alcohol having 4 to 5 carbon atoms is preferable. In the present invention, preferred aliphatic alcohols include 2-methyl-1-propanol and 1-butanol as those having 4 carbon atoms and 3-methyl-1-butanol and 1-pen as those having 5 carbon atoms. Examples thereof include tanol and 2-pentanol. These can be used in one type or a combination of two or more types.
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 by using a headspace gas chromatograph method.

The sugar contained in the beer-taste alcoholic beverage according to the present invention means a sugar based on the nutrition labeling standard for foods (Ministry of Health, Labor and Welfare Notification No. 176, 2003). Specifically, carbohydrate refers to food obtained by removing proteins, lipids, dietary fiber, ash, alcohol and water. The amount of sugar in a food is calculated by subtracting the amount of protein, lipid, dietary fiber, ash and water from the weight of the food. In this case, the amounts of protein, lipid, dietary fiber, ash and water are measured by the methods listed in the nutrition labeling standards. Specifically, the amount of protein is measured by the nitrogen quantitative conversion method, the amount of lipid is measured by the ether extraction method, the chloroform / methanol mixed solution extraction method, the gelbell method, the acid decomposition method or the Reesegotleave method, and the amount of dietary fiber is measured. Is measured by high-speed liquid chromatograph method or Proski method, the amount of ash is measured by magnesium acetate-added ashing method, direct ashing method or sulfuric acid-added ashing method, and the amount of water is measured by Karl Fisher method and drying aid. Measure by the method, vacuum overheating drying method, normal pressure heating drying method or plastic film method.

The beer-taste alcoholic beverage according to the present invention may be low-carbohydrate in accordance with the recent taste for low-carbohydrate. Therefore, the sugar content of the beer-taste alcoholic beverage according to the present invention may be less than 2.5 g / 100 mL or less than 0.5 g / 100 mL. Although the lower limit is not particularly set, 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 raw material.
When hops are used, ordinary pellet 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. Further, processed hop products such as isometric hops and reduced hops may be used. These are included in the hops used in the beer-taste alcoholic beverage according to 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 according to the present invention may use other raw materials as needed, as long as the effects of the present invention are not impaired. For example, sweeteners (including high-sweetness sweeteners), bitterness agents, flavors, yeast extracts, colorants such as caramel pigments, plant-extracted saponin-based substances such as soybean saponin and kiraya saponin, plant proteins such as corn and soybean, and Peptide-containing substances, protein-based substances such as bovine serum albumin, seasonings such as dietary fiber and amino acids, and antioxidants such as ascorbic acid can be used as needed as long as they do not interfere with the effects of the present invention.

The beer-taste alcoholic beverage according to the present invention can be packed in a container. The form of the container is not limited in any way, and it can be filled in a sealed container such as a bottle, a can, a barrel, or a PET bottle to make a beverage in a container.

The method for producing a beer-taste alcoholic beverage of the present invention is not particularly limited. For example, a beer-taste alcoholic beverage in which the ratio of malt in the raw material exceeds 0% by weight and less than 50% by weight, or a beer-taste alcoholic beverage in which the ratio of malt in the raw material is 50% by weight or more, the diketopiperazine. Is exemplified by a method of adding a predetermined amount of.
Further, the ratio of malt in the raw material according to the first embodiment of the present invention is more than 0% by weight and less than 50% by weight, or the ratio of malt in the raw material according to the second form of the present invention is 50. It is preferable to add 40 kDa protein to a beer-taste alcoholic beverage having a weight of% by weight or more.
The diketopiperazine and 40 kDa protein to be added can be prepared, for example, by the procedure described in Examples described later.
Further, regarding diketopiperazine and 40 kDa protein, the content thereof may be increased by adjusting various conditions in the manufacturing process of the beer-taste alcoholic beverage.

Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to the following Examples.

(Purification of diketopiperazine)
Diketopiperazine was purified according to the following.
(1) Fractionation of beer by HP20 60 L of beer was fractionated using 10 L of Diaion (registered trademark) HP20 (manufactured by Mitsubishi Chemical Corporation). HP20 was washed 3 times with ethanol and then 3 times with 50% ethanol before use. The washed HP20 was filled in a mass fractionation column and replaced with water. The same amount of distilled water was mixed with 60 L of degassed beer and flowed to the HP20 column using a medium pressure pump. A solution that passed through the HP20 column was obtained as a passing fraction. 40 L of distilled water was flowed using a medium pressure pump to obtain an eluate as a water-eluting fraction. Similarly, 40 L of hydrous ethanol (10% ethanol, 30% ethanol, and 70% ethanol) was poured, and the eluate was used as a 10% ethanol elution fraction, a 30% ethanol elution fraction, and a 70% ethanol elution fraction, respectively. Obtained. Each eluted fraction was refrigerated as a dried product using an evaporator and a lyophilizer.

(2) LH-20 Fraction of 30% Ethanol Elution Fraction Of the HP20 fractions, the 30% ethanol-eluting fraction was fractionated using 1.2 kg of Sephadex® LH-20. LH-20 washed with ethanol was filled in a mass fractionation column and replaced with water. Of the 30% ethanol-eluted fraction (87.9 g) obtained by the HP 20 fraction, 17.6 g was dissolved in distilled water and applied to the LH-20 column. 13.5 L of distilled water was flowed using a medium pressure pump to obtain water elution fractions -1 to 6. Then, 7 L of hydrous ethanol (35% ethanol, 70% ethanol, and 100% ethanol) was poured, and the eluate was obtained as a 35% ethanol elution fraction, a 70% ethanol elution fraction, and a 100% ethanol elution fraction, respectively. rice field. Each eluted fraction was refrigerated as a dried product using an evaporator and a lyophilizer.

(3) Separation of Diketopiperazine 86.7 mg of the water-eluted fraction-3 (1.04 g) obtained by the LH-20 fraction was used by HPLC (COSMOSIL 5C18-PAQ, 20 × 250 mm). It was eluted with 10% ethanol. Then, the eluate from 15 min to 20 min was concentrated and eluted with a mixed solution of ethanol-water (5:95 → 20:80) using HPLC (COSMOSIL 5C18-PAQ, 20 × 250 mm) to compound 1. (6.7 mg, tR = 25 min) was obtained. Similarly, the eluate from 27.5 min to 40 min is concentrated and eluted with a mixed solution of ethanol-water (5:95 → 20:80) using HPLC (COSMOSIL 5C18-PAQ, 20 × 250 mm). , Compound 2 (2.2 mg, tR = 43 min) and Compound 3 (2.6 mg, tR = 46 min) were obtained.
Compound 1 was identified as Cyclo (Pro-Val) by analysis of physical data of MS and NMR and literature values. Compound 2 was identified as Cyclo (Ile-Pro) by analysis of physical data of MS and NMR and literature values. Compound 3 was identified as Cyclo (Leu-Pro) by analysis of physical data of MS and NMR and literature values.
The references referred to are J. Mol. Agric. Food Chem. 2007,55,75-79. Is.
The analytical instruments used are as follows.
LC-MS; Q Active, Thermo Fisher Scientific NMR; AVANCE400, Bruker

(Purification of 40 kDa protein)
40 kDa protein was purified from 1 L of commercially available beer according to the following.

(1) Fractionation by cation exchange resin 50 mL of cation exchange resin SP Sepharose was placed in an empty column. Beer was adsorbed on the resin. Then, the resin was transferred to a column and washed with 20 mM sodium acetate buffer (pH 4.5). Then, it was eluted with 20 mM sodium acetate (pH 4.5) + 0.5M-NaCl and fractions were collected. The obtained fraction was evaluated by SDS-PAGE, and the fraction containing a protein of 40 kDa was collected and used as a cation exchange resin bound fraction.

(2) Extrafiltration (buffer exchange)
10 mL each of the cation exchange resin bonded fraction obtained in (1) was added to a water-washed ultrafiltration unit (Amicon Ultra-15 30K manufactured by Merck), and the mixture was centrifuged at 3500 rpm to obtain a concentrated solution. ..

(3) Ammonium sulfate fraction 20 mM phosphate buffer (pH 7.0) + 2M 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 is added to 20 mM sodium acetate (pH 4.5), centrifuged (2330 g, 10 minutes, room temperature), concentrated, and 40 kDa protein assay (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.

An attempt was made to identify the protein by digesting the 40 kDa protein with an enzyme and then analyzing it by LC-MS / MS.
Bands around 40 kDa separated by SDS-PAGE were excised, reduced with dithiothreitol (56 ° C., 1 hour), and carbamid methylated with iodoacetamide (light-shielded, room temperature, 45 minutes). Then, 15 μL of 10 ng / μL chymotrypsin solution (5 mM calcium chloride, 50 mM ammonium hydrogencarbonate solution) containing 0.01% ProteinMax, 15 μL of 5 mM calcium chloride, 50 mM ammonium hydrogencarbonate solution was added, and the mixture was incubated overnight, and then the enzyme digestive juice was recovered. .. The recovered solution was dried under reduced pressure and redissolved in a 0.1% formic acid solution.
This was used for LC-MS / MS analysis.

(Measurement by LC-MS / MS)
The LC-MS / MS measurement was performed under the following conditions.
Equipment used: Direct flow nanoLC system Easy-nLC 1000TM (Thermo Scientific)
Trap column: Acclim PepMap® (Thermo Scientific)
Analytical column: NANO HPLC CAPILLARY COLUMN (Nikkyo Technos Co., Ltd.)
Liquid Chromatograph Mass Spectrometer Q Active Plus (Thermo Scientific)
Mobile phase: Liquid A: 0.1% formic acid / water, liquid B: 0.1% formic acid / acetonitrile Flow velocity: 300 nL / min
Radiant: 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
Measurement range: MS1 (m / z 350-1750)
Data Dependent Scan mode

(4) Protein analysis Protein identification was performed under the following conditions.
Search software: Proteome Discoverer 2.2.0.3888 (manufactured by Thermo Fisher)
Species: Barley (Hordeum vulgare), Hops (Humulus), Yeast (Saccharomyces cerevisiae)
Search condition: Digestive enzyme: Chymotrypsin
Precursor ion mass error range: Monoisotopic, ± 10 ppm
Product ion mass error range: ± 0.02 Da
Maximum number of missed cribes: 5
Confidence level (Percolator): High (the most probable level of the three levels of certainty)
Database: SwissProt

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

(Sensory evaluation when diketopiperazine is added to beer-taste alcoholic beverage C in which the ratio of malt in commercially available raw materials exceeds 0% by weight and is less than 50% by weight)
Diketopiperazine was added to the beer-taste alcoholic beverage C in which the ratio of malt in the commercially available raw material was more than 0% by weight and less than 50% by weight, and the sensory evaluation of the swelling was performed.
The beer-taste alcoholic beverage is a beer-taste alcoholic beverage in which the proportion of malt in the raw material exceeds 0% by weight and is less than 50% by weight.
The raw materials are low-malt beer, malt, hops, sugars, dietary fiber, and spirits (wheat). As nutritional components, alcohol content is 4%, protein is 0 to 0.2 g, sugar is 0.5 to 0.8 g, and purine is used. Contains about 2.0 mg.

The reference points for sensory evaluation are as follows.
Five expert panels scored in increments of 0.05 points according to the following criteria, and the score values were averaged.
The bulge strength is based on the following criteria.
0 points: Not felt at all 1 point: Slightly felt 2 points: Clearly felt 3 points: As a very felt reference point, the ratio of malt in the above-mentioned commercially available raw materials to be evaluated exceeds 0% by weight, 50 points The above-mentioned commercially available beer-taste alcoholic beverage (I), which is the same as the beer-taste alcoholic beverage C having a weight% of less than C, was used as the standard beer-taste alcoholic beverage (I), and its bulge was set to 0.7 points. Further, the beer-taste alcoholic beverage A in which the ratio of malt in other commercially available raw materials was 50% by weight or more was set as the standard beer-taste alcoholic beverage (II), and the bulge was set to 1.5 points as the reference point.
The standard beer-taste alcoholic beverage (II) is a beer-taste alcoholic beverage in which the ratio of malt in the raw material is 50% by weight or more.
The raw materials of the beer-taste alcoholic beverage A are malt and hops, and contain 5.5% alcohol, 0.4 to 0.6 g of protein, 3.6 g of sugar, and about 12.5 mg of purine as nutritional components per 100 ml.

The procedure for sensory evaluation is as follows.
(1) Dispense the beer-taste alcoholic beverage to be evaluated into 1/10 volume (v / v) vials of the final volume (2) Weigh and add diketopiperazin to any weight (3) 30 seconds Sonication (4) Let stand at room temperature for 30 minutes (5) Fill up the beer-taste alcoholic beverage to the final volume (6) Dispense and evaluate by swallowing

(Analysis of commercial beer-taste alcoholic beverages)
The concentration of diketopiperazine contained in the commercially available beer-taste alcoholic beverage used for the sensory evaluation was quantified by LC-MS by the following procedure.
(1) Preparation of standard and preparation of calibration curve Diketopiperazine was diluted to the following concentrations, passed through a 0.22 μm filter, and then subjected to measurement.
Final concentration: 0.001 ppm, 0.025 ppm, 0.050 ppm, 0.100 ppm, 0.200 ppm, 0.300 ppm, 0.500 ppm, 0.750 ppm, 1.000 ppm
(1 ppm = 1 μg / mL)
A 5% (v / v) aqueous ethanol solution was used as the diluent.
Note that in the analysis of a preparation was employed measurements of the dilution ratio as the measured value is in the range (R ^2> 0.99) the linearity of the calibration curve is maintained.

The measurement conditions for LC-MS are as follows.
LC-MS: X500R manufactured by AB SIX Co., Ltd.
Separation column: Waters HSST3 1.8 μm, 2.1x150 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: 5 μL
Flow rate: 0.2 mL / 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 sample from commercially available beer-taste alcoholic beverages Commercially available beer-taste alcoholic beverages are degassed by sonication, diluted appropriately after the bubbles have settled, passed through a 0.22 μm filter, and then measured. Served.
A 5% (v / v) aqueous ethanol solution was used as the diluent.
The concentration of each diketopiperazine contained in the commercially available beer-taste alcoholic beverage C was set as control 1.

(Example 1: Evaluation by addition of Cyclo (Leu-Pro))
The concentration of Cyclo (Leu-Pro) contained in the beer-taste alcoholic beverage C in which the ratio of malt in the commercially available raw material was more than 0% by weight and less than 50% by weight was 0.504 ppm.
On the other hand, sensory evaluation was performed by adding Cyclo (Leu-Pro) so that the concentration of Cyclo (Leu-Pro) was 0.8 ppm, 1 ppm, 5 ppm, 10 ppm, 15 ppm, and 20 ppm, respectively.
The results of the sensory evaluation are shown in Table 1.

From the results shown in Table 1, for a beer-taste alcoholic beverage C in which the proportion of malt in the raw material exceeds 0% by weight and is less than 50% by weight, swelling occurs when the degree of Cyclo (Leu-Pro) is 0.8 ppm or more. It can be seen that it is enhanced.
In addition, in the cases where the Cyclo (Leu-Pro) concentration exceeds 10 ppm (15 ppm, 20 ppm), it has been evaluated that bitterness and harshness are felt from a viewpoint different from that of swelling. From this, it is considered more preferable that the Cyclo (Leu-Pro) concentration is 10 ppm or less.

(Example 2: Evaluation of synergistic effect between Cyclo (Leu-Pro) and 40 kDa protein)
Cyclo (Leu-Pro) is added to the beer-taste alcoholic beverage C in which the ratio of malt in the commercially available raw material exceeds 0% by weight and is less than 50% by weight, and the concentration of Cyclo (Leu-Pro) is 0.8 ppm. The synergistic effect of Cyclo (Leu-Pro) and 40 kDa protein was evaluated by further adding 40 kDa protein based on the beverage. The concentration of 40 kDa protein was 1 ppm, 5 ppm, 10 ppm, and 25 ppm. The 40 kDa protein used was the one purified above.
Further, for comparison, evaluation was performed by adding only 40 kDa protein to the beer-taste alcoholic beverage C in which the ratio of malt in the commercially available raw material exceeds 0% by weight and is less than 50% by weight.
The results of the sensory evaluation are shown in Table 2.

From the results shown in Table 2, Cyclo (Leu-Pro) is added to the beer-taste alcoholic beverage C in which the proportion of malt in the commercially available raw material exceeds 0% by weight and is less than 50% by weight, and further 40 kDa protein is added. It can be seen that the bulge can be further increased.
From the results of the comparison sample 1, it can be seen that the swelling can be enhanced only by adding the 40 kDa protein. However, the diketopiperazine and 40 kDa protein obtained by the sum of the increase value (0.16) from the sensory evaluation control in the sample 1 and the increase value (0.09) from the sensory evaluation control in the contrast sample 1 can be obtained. Since the increase value (0.27) from the control of the sensory evaluation in sample 8 is larger than the additive effect (0.25) when used in combination, it cannot be predicted by using diketopiperazine and 40 kDa protein in combination. It can be said that the synergistic effect is exhibited.

(Example 3: Evaluation by addition of Cyclo (Val-Pro))
The concentration of Cyclo (Val-Pro) contained in the beer-taste alcoholic beverage C in which the ratio of malt in the commercially available raw material was more than 0% by weight and less than 50% by weight was 0.396 ppm.
On the other hand, sensory evaluation was performed by adding Cyclo (Val-Pro) so that the concentration of Cyclo (Val-Pro) was 0.8 ppm. Further, 5 ppm of 40 kDa protein was added and sensory evaluation was performed.
The results of the sensory evaluation are shown in Table 3.

From the results shown in Table 3, it is possible to enhance the swelling by adding Cyclo (Val-Pro) to the beer-taste alcoholic beverage C in which the ratio of malt in the commercially available raw material exceeds 0% by weight and is less than 50% by weight. I understand.
Further, it can be seen that the swelling can be further enhanced by further adding 40 kDa protein.
From the results of the comparison sample 1 (see Table 2), it can be seen that the swelling can be enhanced only by adding the 40 kDa protein. However, diketopiperazine, which is obtained by the sum of the increase value (0.09) from the sensory evaluation control in the sample 11 and the increase value (0.09) from the sensory evaluation control in the comparison sample 1 shown in Table 2. Since the increase value (0.21) from the control of the sensory evaluation in sample 12 is larger than the additive effect (0.18) when the 40 kDa protein is used in combination with diketopiperazine, the 40 kDa protein should be used in combination. Therefore, it can be said that an unexpected synergistic effect is exhibited.

(Example 4: Evaluation by addition of Cyclo (Ile-Pro))
The concentration of Cyclo (Ile-Pro) contained in the beer-taste alcoholic beverage C in which the ratio of malt in the commercially available raw material was more than 0% by weight and less than 50% by weight was 0.265 ppm.
On the other hand, sensory evaluation was performed by adding Cyclo (Ile-Pro) so that the concentration of Cyclo (Ile-Pro) was 0.8 ppm. Further, 5 ppm of 40 kDa protein was added and sensory evaluation was performed.
The results of the sensory evaluation are shown in Table 4.

From the results shown in Table 4, it is possible to enhance the swelling by adding Cyclo (Ile-Pro) to the beer-taste alcoholic beverage C in which the ratio of malt in the commercially available raw material exceeds 0% by weight and is less than 50% by weight. I understand.
Further, it can be seen that the swelling can be further enhanced by further adding 40 kDa protein.
From the results of the comparison sample 1 (see Table 2), it can be seen that the swelling can be enhanced only by adding the 40 kDa protein. However, diketopiperazine, which is obtained by the sum of the increase value (0.10) from the sensory evaluation control in the sample 13 and the increase value (0.09) from the sensory evaluation control in the comparison sample 1 shown in Table 2. Since the increase value (0.23) from the control of the sensory evaluation in the sample 14 is larger than the additive effect (0.19) when the 40 kDa protein is used in combination with diketopiperazine, the 40 kDa protein should be used in combination. Therefore, it can be said that an unexpected synergistic effect is exhibited.

(Sensory evaluation when diketopiperazine is added to a commercially available beer-taste alcoholic beverage)
Diketopiperazin is added to a beer-taste alcoholic beverage A in which the ratio of malt in a commercially available raw material is 50% by weight or more, or a beer-taste alcoholic beverage B in which the ratio of malt in a commercially available raw material is 50% by weight or more. Then, the sensory evaluation of the bulge was performed.
The beer-taste alcoholic beverages A and B are beer-taste alcoholic beverages in which the ratio of malt in the raw material is 50% by weight or more.
The raw materials of the beer-taste alcoholic beverage A are malt and hops, and contain 5.5% alcohol, 0.4 to 0.6 g of protein, 3.6 g of sugar, and about 12.5 mg of purine as nutritional components per 100 ml.
The raw materials for beer-taste alcoholic beverage B are malt, hops, rice, corn, and starch. As nutritional components, alcohol content is 5%, protein is 0.2 to 0.4 g, sugar is 3.0 g, and purine is 5 to 6 mg. including.

The reference point and procedure for sensory evaluation are the same as the reference point and procedure in Example 1. The concentrations of each diketopiperazine contained in the commercially available beer-taste alcoholic beverages A and B were set to controls 2 and 3, respectively.

(Example 5: Evaluation by addition of Cyclo (Leu-Pro) to a commercially available beer-taste alcoholic beverage A)
The concentration of Cyclo (Leu-Pro) contained in the beer-taste alcoholic beverage A in which the ratio of malt in the commercially available raw material was 50% by weight or more was 0.518 ppm.
On the other hand, sensory evaluation was performed by adding Cyclo (Leu-Pro) so that the concentration of Cyclo (Leu-Pro) was 2.2 ppm, 3 ppm, 5 ppm and 10 ppm, respectively.
The results of the sensory evaluation are shown in Table 5.

From the results shown in Table 5, it can be seen that the swelling is enhanced when the Cyclo (Leu-Pro) concentration is 2.2 ppm or more for the beer-taste alcoholic beverage A in which the ratio of malt in the raw material is 50% by weight or more. ..

(Example 6: Evaluation of synergistic effect of Cyclo (Leu-Pro) and 40 kDa protein on a commercially available beer-taste alcoholic beverage A)
Based on a beverage with a concentration of Cyclo (Leu-Pro) of 2.2 ppm by adding Cyclo (Leu-Pro) to a beer-taste alcoholic beverage A in which the ratio of malt in commercially available raw materials is 50% by weight or more. Further, the synergistic effect of Cyclo (Leu-Pro) and 40 kDa protein was evaluated by adding 40 kDa protein. The concentration of 40 kDa protein was 30 ppm, 35 ppm, and 45 ppm. The 40 kDa protein used was the one purified above.
For comparison, only 40 kDa protein was added to the beer-taste alcoholic beverage A in which the ratio of malt in the commercially available raw material was 50% by weight or more, and the concentration of 40 kDa protein was evaluated as 30 ppm (contrast sample 2). ).
The results of the sensory evaluation are shown in Table 6.

From the results shown in Table 6, the swelling was further enhanced by adding Cyclo (Leu-Pro) to the beer-taste alcoholic beverage A in which the ratio of malt in the commercially available raw material was 50% by weight or more, and further adding 40 kDa protein. I know I can do it.
From the results of the comparison sample 2, it can be seen that the swelling can be enhanced only by adding the 40 kDa protein. However, the diketopiperazine and 40 kDa protein obtained by the sum of the increase value (0.18) from the sensory evaluation control in the sample 15 and the increase value (0.09) from the sensory evaluation control in the contrast sample 2 are obtained. Since the increase value (0.36) from the control of the sensory evaluation in sample 19 is larger than the additive effect (0.27) when used in combination, it cannot be predicted by using diketopiperazine and 40 kDa protein in combination. It can be said that the synergistic effect is exhibited.

(Example 7: Evaluation by addition of Cyclo (Val-Pro) to a commercially available beer-taste alcoholic beverage A)
The concentration of Cyclo (Val-Pro) contained in the beer-taste alcoholic beverage A in which the ratio of malt in the commercially available raw material was 50% by weight or more was 0.167 ppm.
On the other hand, sensory evaluation was performed by adding Cyclo (Val-Pro) so that the concentration of Cyclo (Val-Pro) was 2.2 ppm. Further, the evaluation was performed with the concentration of 40 kDa protein set to 30 ppm.
The results of the sensory evaluation are shown in Table 7.

From the results shown in Table 7, it can be seen that the swelling can be enhanced by adding Cyclo (Val-Pro) to the beer-taste alcoholic beverage A in which the ratio of malt in the commercially available raw material is 50% by weight or more.
Further, it can be seen that the swelling can be further enhanced by further adding 40 kDa protein.
From the results of the comparison sample 2 (see Table 6), it can be seen that the swelling can be enhanced only by adding the 40 kDa protein. However, diketopiperazine, which is obtained by the sum of the increase value (0.09) from the sensory evaluation control in the sample 22 and the increase value (0.09) from the sensory evaluation control in the comparison sample 2 shown in Table 6. Since the increase value (0.25) from the control of the sensory evaluation in the sample 23 is larger than the additive effect (0.18) when the 40 kDa protein is used in combination with diketopiperazine, the 40 kDa protein should be used in combination. Therefore, it can be said that an unexpected synergistic effect is exhibited.

(Example 8: Evaluation by addition of Cyclo (Ile-Pro) to a commercially available beer-taste alcoholic beverage A)
The concentration of Cyclo (Ile-Pro) contained in the beer-taste alcoholic beverage A in which the ratio of malt in the commercially available raw material was 50% by weight or more was 0.260 ppm.
On the other hand, sensory evaluation was performed by adding Cyclo (Ile-Pro) so that the concentration of Cyclo (Ile-Pro) was 2.2 ppm. Further, the evaluation was performed with the concentration of 40 kDa protein set to 30 ppm.
The results of the sensory evaluation are shown in Table 8.

From the results shown in Table 8, it can be seen that the swelling can be enhanced by adding Cyclo (Ile-Pro) to the beer-taste alcoholic beverage A in which the ratio of malt in the commercially available raw material is 50% by weight or more.
Further, it can be seen that the swelling can be further enhanced by further adding 40 kDa protein.
From the results of the comparison sample 2 (see Table 6), it can be seen that the swelling can be enhanced only by adding the 40 kDa protein. However, diketopiperazine, which is obtained by the sum of the increase value (0.10) from the sensory evaluation control in the sample 24 and the increase value (0.09) from the sensory evaluation control in the comparison sample 2 shown in Table 6. Since the increase value (0.29) from the control of the sensory evaluation in the sample 25 is larger than the additive effect (0.19) when the 40 kDa protein is used in combination with diketopiperazine, the 40 kDa protein should be used in combination. Therefore, it can be said that an unexpected synergistic effect is exhibited.

(Example 9: Evaluation by addition of Cyclo (Leu-Pro) to a commercially available beer-taste alcoholic beverage B)
The concentration of Cyclo (Leu-Pro) contained in the beer-taste alcoholic beverage B in which the ratio of malt in the commercially available raw material was 50% by weight or more was 0.365 ppm.
On the other hand, sensory evaluation was performed by adding Cyclo (Leu-Pro) so that the concentration of Cyclo (Leu-Pro) was 2.2 ppm, 3 ppm, and 5 ppm, respectively.
The results of the sensory evaluation are shown in Table 9.

From the results shown in Table 9, it can be seen that the swelling is enhanced when the Cyclo (Leu-Pro) concentration is 2.2 ppm or more for the beer-taste alcoholic beverage B in which the ratio of malt in the raw material is 50% by weight or more. ..

(Example 10: Evaluation of synergistic effect of Cyclo (Leu-Pro) and 40 kDa protein on a commercially available beer-taste alcoholic beverage B)
Based on a beverage in which the concentration of Cyclo (Leu-Pro) is 2.2 ppm by adding Cyclo (Leu-Pro) to the beer-taste alcoholic beverage B in which the ratio of malt in the commercially available raw material is 50% by weight or more. Further, the synergistic effect of Cyclo (Leu-Pro) and 40 kDa protein was evaluated by adding 40 kDa protein. The concentration of the 40 kDa protein was 10 ppm, 15 ppm, and 20 ppm.
For comparison, only 40 kDa protein was added to the beer-taste alcoholic beverage B in which the ratio of malt in the commercially available raw material was 50% by weight or more, and the concentration of 40 kDa protein was evaluated as 10 ppm (contrast sample 3). ).
The results of the sensory evaluation are shown in Table 10.

From the results shown in Table 10, the swelling was further enhanced by adding Cyclo (Leu-Pro) to the beer-taste alcoholic beverage B in which the ratio of malt in the commercially available raw material was 50% by weight or more, and further adding 40 kDa protein. I know I can do it.
From the results of the comparison sample 3, it can be seen that the swelling can be enhanced only by adding the 40 kDa protein. However, the diketopiperazine and 40 kDa protein obtained by the sum of the increase value (0.12) from the sensory evaluation control in the sample 26 and the increase value (0.08) from the sensory evaluation control in the contrast sample 3 are obtained. Since the increase value (0.21) from the control of the sensory evaluation in the sample 29 is larger than the additive effect (0.20) when used in combination, it cannot be predicted by using diketopiperazine and 40 kDa protein in combination. It can be said that the synergistic effect is exhibited.

Claims

A beer-taste alcoholic beverage in which the proportion of malt in the raw material is greater than 0% by weight and less than 50% by weight, at least one selected from the group consisting of cycloleucylproline, cyclovalylproline and cycloisoleucylproline. A beer-taste alcoholic beverage containing the seed diketopiperazine and having a concentration of diketopiperazine, which is the most abundant among the diketopiperazines, of 0.8 ppm or more.
The beer-taste alcoholic beverage according to claim 1, wherein the concentration of diketopiperazine contained most in the diketopiperazine is 10 ppm or less.
In addition, it contains a protein with a molecular weight of 35-50 kDa,
The beer-taste alcoholic beverage according to claim 1 or 2, wherein the protein concentration is 1 ppm or more.
The beer-taste alcoholic beverage according to claim 3, wherein the protein concentration is 30 ppm or less.
A beer-taste alcoholic beverage in which the proportion of malt in the raw material is 50% by weight or more, and at least one diketopiperazine selected from the group consisting of cycloleuicylproline, cyclovalylproline and cycloisoleucilproline. A beer-taste alcoholic beverage containing, and the concentration of diketopiperazine contained most in the above-mentioned diketopiperazine is 2.2 ppm or more.
The beer-taste alcoholic beverage according to claim 5, wherein the concentration of diketopiperazine contained most in the diketopiperazine is 10 ppm or less.
In addition, it contains a protein with a molecular weight of 35-50 kDa,
The beer-taste alcoholic beverage according to claim 5 or 6, wherein the protein concentration is 10 ppm or more.
In addition, it contains a protein with a molecular weight of 35-50 kDa,
The beer-taste alcoholic beverage according to any one of claims 5 to 7, wherein the protein concentration is 30 ppm or more.
The beer-taste alcoholic beverage according to claim 7 or 8, wherein the protein concentration is 200 ppm or less.