JP7313277B2 - Beer-taste alcoholic beverage, body-taste enhancer for beer-taste-alcoholic beverage, and method for enhancing body-taste of beer-taste-alcoholic beverage - Google Patents

Description

TECHNICAL FIELD The present invention relates to a beer-taste alcoholic beverage, a body taste enhancer for beer-taste alcoholic beverages, and a method for enhancing body taste of beer-taste alcoholic beverages.

With the diversification of consumer tastes in recent years, the development of beer-taste alcoholic beverages with various flavor characteristics is desired.

For example, in Patent Document 1, alcohol components,
It is disclosed that a sweetening component, an aldehyde component and the like are used as flavor improvers for beer-like beverages.

JP 2016-214262 A

However, flavor improvers such as those disclosed in Patent Document 1 affect flavor and are not suitable for use in some cases.

As raw materials for beer-taste alcoholic beverages, grain-derived raw materials such as barley and corn are commonly used. Since the grain-derived raw material is a raw material commonly used for beer-taste alcoholic beverages, manufacturers and consumers of beverages are less reluctant to use the grain-derived raw material as a material for enhancing the full-bodied taste. Therefore, there has been a demand for a method of enhancing richness by using cereal-derived raw materials.

An object of the present invention is to provide a beer-taste alcoholic beverage with a strong body using cereal-derived raw materials. Another object of the present invention is to provide a body taste enhancer for beer-taste alcoholic beverages, which is a cereal-derived raw material. Another object of the present invention is to provide a method for enhancing the richness of beer-taste alcoholic beverages using cereal-derived raw materials.

That is, the present invention relates to the following beer-taste alcoholic beverages and the like.
[1] A beer-taste alcoholic beverage containing less than 50% by weight of malt in the raw material, containing a sugar-modified protein having a molecular weight of 35 to 50 kDa in which the amino group of lysine constituting the cereal-derived protein is glycated, wherein the glycated lysine content of the sugar-modified protein is 30.0 nmol/mL or more.
[2] The beer-taste alcoholic beverage according to [1] above, wherein the glycated lysine content of the sugar-modified protein is 35.3 nmol/mL or more.
[3] The beer-taste alcoholic beverage of [1] or [2] above, wherein the content of glycated lysine contained in the sugar-modified protein is 30.0 to 1000 nmol/mL.
[4] The beer-taste alcoholic beverage according to any one of [1] to [3] above, wherein the sugar glycated to the amino group of the glycated lysine is a monosaccharide or a disaccharide.
[5] The beer-taste alcoholic beverage according to any one of [1] to [4] above, wherein the grain is at least one selected from the group consisting of barley, wheat, corn, rice and soybeans.
[6] The beer-taste alcoholic beverage according to any one of [1] to [5], which does not contain wort.
[7] A rich taste enhancer for beer-taste alcoholic beverages containing a sugar-modified protein with a molecular weight of 35 to 50 kDa in which the amino group of lysine constituting a cereal-derived protein is glycated, and the ratio of malt in the raw material is less than 50% by weight.
[8] A method for enhancing the body taste of a beer-taste alcoholic beverage, which comprises adding a sugar-modified protein having a molecular weight of 35 to 50 kDa in which the amino group of lysine constituting the cereal-derived protein is glycated to a beer-taste alcoholic beverage containing less than 50% by weight of malt in the raw material, thereby enhancing the body taste of the beer-taste alcoholic beverage.

According to the present invention, it is possible to provide a beer-taste alcoholic beverage with a strong body using grain-derived raw materials. In addition, it is possible to provide a rich taste enhancer for beer-taste alcoholic beverages, which is a cereal-derived raw material. Furthermore, it is possible to provide a method for enhancing body taste of beer-taste alcoholic beverages using cereal-derived raw materials.

The beer-taste alcoholic beverage of the present invention contains less than 50% by weight of malt in the ingredients.
The proportion of malt is preferably at least 30% by weight.
Also, the ratio of malt in the raw material may be 0%.
Here, the "ratio of malt" refers to the mass ratio of malt in raw materials other than water and hops, such as malt, rice, corn, sorghum, potato, starch, barley other than malt, and sugars. However, ingredients that can be added in small amounts, such as acidulants, sweeteners, bittering agents, seasonings, and flavoring agents, are not included in the calculation of the above ratio.
The beer-taste alcoholic beverage of the present invention may not contain wort.

The beer-taste alcoholic beverage of the present invention contains sugar-modified proteins.
Glycosylated proteins have a molecular weight of 35-50 kDa.

A protein with a molecular weight of 35 to 50 kDa is a protein found in the region of 35 to 50 kDa, which is measured by ultrafiltration using a 30 kDa cut-off membrane for a beer-taste alcoholic beverage raw material solution, followed by SDS-PAGE electrophoresis.
A protein of about 40 kDa is preferred, and the protein of about 40 kDa is also referred to herein as a 40 kDa protein.

Glycosylated proteins are grain-derived proteins.
The grains are preferably at least one selected from the group consisting of barley, wheat, corn, rice and soybeans.
Moreover, when the cereal is wheat, it can contain proteins derived from known wheat used for producing beer-taste alcoholic beverages. Examples of such barley include barley, wheat, rye, oats, oats, and oats, with barley being preferred. In addition, it may be either germinated barley or ungerminated barley, but preferably germinated barley malt. These may be contained independently and may be contained in combination of 2 or more types.

Glycosylated proteins are proteins in which the amino groups of lysines constituting cereal-derived proteins are glycated.
In the present invention, "the amino group of lysine is glycated" means that sugar is bound to the amino group of lysine.
Of the amino groups possessed by lysine, it is preferred that sugars are glycated to side chain amino groups.
Glycation of the amino group of lysine can be confirmed by colorimetric determination by the NBT method (Nitroblue tetrazolium method).
The sugar glycated to the amino group of lysine may be a monosaccharide, a disaccharide, or a polysaccharide of trisaccharide or higher, but is preferably a monosaccharide or a disaccharide.
Monosaccharides include all sugars including glucose, fructose, galactose and mannose.
Disaccharides include maltose-type disaccharides such as maltose, lactose and arabinose.
Trisaccharides include maltotriose, sertriose, fucosyllactose, sialyllactose and the like.

In the beer-taste alcoholic beverage of the present invention, the content of glycated lysine is 30.0 nmol/mL or more.
The content of glycated lysine is preferably 35.3 nmol/mL or more, preferably 50.0 nmol/mL or more, preferably 80.0 nmol/mL or more, and preferably 100 nmol/mL or more.
The glycated lysine content is preferably 30.0 to 1000 nmol/mL, more preferably 50.0 to 1000 nmol/mL, more preferably 80.0 to 1000 nmol/mL, and more preferably 100 to 1000 nmol/mL.
The content of glycated lysine can be quantified by colorimetric determination by the NBT method.

Since the beer-taste alcoholic beverage of the present invention contains the sugar-modified protein as described above, it can be a beer-taste alcoholic beverage with a strong body.
In addition, since the sugar-modified protein is a cereal-derived raw material, it is prevented from exerting an unintended influence on the flavor unlike the flavor improving agent described in Patent Document 1. In addition, since it is preferable that raw materials for beer-taste alcoholic beverages are only cereals and hops, it is a desirable characteristic for raw materials for beer-taste alcoholic beverages that the sugar-modified protein is a cereal-derived raw material.

In the present invention, “kokumi” is a taste that cannot be expressed by five basic tastes, that is, sweetness, saltiness, sourness, bitterness and umami, and is expressed by strength of taste (drinking response), spread of taste, thickness of taste, change over time of taste (lingering or persistence), etc. In the present invention, enhancing the richness includes, for example, imparting richness to a food or drink that does not have a richness, and enhancing the richness of a food or drink that has a richness. The presence or absence and degree of richness can be evaluated by a sensory evaluation by a specialized panel.

The beer-taste alcoholic beverage of the present invention preferably has a purine concentration of 0.01 to 10 mg/100 mL.
For beer-taste alcoholic beverages having a purine concentration within the above range, the addition of a sugar-modified protein can particularly enhance the richness.

The beer-taste alcoholic beverage of the present invention is an alcohol-containing beer-taste beverage. Alcohol refers to ethanol, and the ethanol content is preferably 1% to 10% by volume, but is not particularly limited. Furthermore, the origin of alcohol contained in the beer-taste alcoholic beverage is not limited to fermentation or non-fermentation.

Below, the production process of a general beer-taste alcoholic beverage is shown.
First, an enzyme such as amylase is optionally added to a mixture containing barley such as malt, raw materials such as other grains, starch, sugars, bittering agents, or coloring agents, and water as necessary, gelatinization, saccharification, and filtration to obtain a saccharified solution. If necessary, hops, bittering agents, etc. are added to the saccharified solution and boiled, and solids such as coagulated proteins are removed in a clarifying tank. As an alternative to this saccharified solution, hops may be added to malt extract and warm water, and the mixture may be boiled. Hops may be mixed at any stage from the beginning of boiling to the end of boiling. Known conditions may be used for the conditions in the saccharification step, the boiling step, the solid content removal step, and the like. Known conditions may be used as the conditions in the fermentation/storage process. The resulting fermented liquid is filtered, and carbon dioxide gas is added to the obtained filtrate. After that, it is filled into a container and goes through a sterilization step to obtain the intended beer-taste alcoholic beverage. In each of the steps described above, the addition of the kokumi enhancer may be performed in any step up to filling.

A beer-taste alcoholic beverage produced without using malt as a raw material is a liquid sugar solution by mixing liquid sugar containing a carbon source, nitrogen sources as amino acid-containing materials other than wheat or malt, hops, pigments, etc. with warm water. The liquid sugar solution is boiled. When hops are used as a raw material, the hops may be mixed into the liquid sugar solution during boiling rather than before the start of boiling. As an alternative to this saccharified solution, hops may be added to an extract obtained by adding raw materials other than malt to warm water, and the mixture may be boiled. Hops may be mixed at any stage from the beginning of boiling to the end of boiling. Known conditions may be used as the conditions in the fermentation/storage process. The resulting fermented liquid is filtered, and carbon dioxide gas is added to the obtained filtrate. After that, it is filled into a container and goes through a sterilization step to obtain the intended beer-taste alcoholic beverage. In each of the steps described above, the addition of the kokumi enhancer may be performed in any step up to filling.

The non-fermented alcohol-containing beer-taste alcoholic beverage may be one in which the alcohol content of the final product is adjusted by adding raw material alcohol or the like. The raw material alcohol 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. For example, it can be measured by a vibrating density meter. Specifically, a sample is prepared by removing carbon dioxide gas from the beverage by filtration or ultrasonic waves, and the sample is subjected to direct-fire distillation, and the density of the resulting distillate is measured at 15 ° C., and the density can be obtained by converting using the "Table 2 Alcohol content and density (15 ° C.) and specific gravity (15/15 ° C.) conversion table" attached to the National Tax Agency prescribed analysis method (2007 National Tax Agency Instruction No. 6, revised on June 22, 2007). If the alcohol content is as low as less than 1.0%, a commercially available alcohol measuring device or gas chromatography may be used.

An aliphatic 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 aliphatic alcohols having 4 to 5 carbon atoms are preferred. In the present invention, preferred aliphatic alcohols include those with 4 carbon atoms such as 2-methyl-1-propanol and 1-butanol, and those with 5 carbon atoms such as 3-methyl-1-butanol, 1-pentanol and 2-pentanol. These can be used singly or in combination of two or more.
The content of the aliphatic alcohol having 4 to 5 carbon atoms is preferably 0.0002-0.0007% by mass, more preferably 0.0003-0.0006% by mass. As used herein, the content of fatty alcohols can be measured using a headspace gas chromatographic method.

The carbohydrates contained in the beer-taste alcoholic beverage according to the present invention refer to carbohydrates based on the Nutrition Labeling Standards for Foods (Ministry of Health, Labor and Welfare Notification No. 176, 2003). Specifically, carbohydrates refer to foods from which proteins, lipids, dietary fibers, ash, alcohol and water have been removed. Also, 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, fat, dietary fiber, ash and moisture shall be measured by the methods listed in the Nutrition Labeling Standards. Specifically, the amount of protein was measured by the nitrogen quantitative conversion method, the amount of lipid was measured by the ether extraction method, the chloroform/methanol mixture extraction method, the Guerbel method, the acid decomposition method, or the Roese-Gottlieb method, the amount of dietary fiber was measured by the high-performance liquid chromatography method or the Prosky method, the amount of ash was measured by the magnesium acetate addition ashing method, the direct ashing method, or the sulfuric acid addition ashing method, and the water content was measured by the Karl Fischer method, the drying aid method, the vacuum superheat drying method, and the normal pressure heat drying method. or plastic film method.

The beer-taste alcoholic beverage according to the present invention may be low-sugar in line with recent low-sugar preferences. 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 part of the ingredients.
When using hops, 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. Also, processed hop products such as isoformed hops and reduced hops may be used. These hops 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 typically about 0.0001 to 1% by weight relative to the total amount of the beverage.

The beer-taste alcoholic beverage according to the present invention may use other raw materials, if necessary, as long as the effects of the present invention are not impaired. For example, sweeteners (including high-intensity sweeteners), bittering agents, flavors, yeast extracts, colorants such as caramel pigments, plant-extracted saponin-based substances such as soybean saponin and quillaja saponin, plant proteins and peptide-containing substances such as corn and soybeans, protein-based substances such as bovine serum albumin, seasonings such as dietary fiber and amino acids, and antioxidants such as ascorbic acid can be used as necessary 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 shape of the container is not limited at all, and the beverage can be packaged in a sealed container such as a bottle, a can, a barrel, or a PET bottle.

The body taste enhancer of the present invention contains a sugar-modified protein with a molecular weight of 35 to 50 kDa in which the amino group of lysine constituting the cereal-derived protein is glycated, and the ratio of malt in the raw material is less than 50% by weight. It is a beer taste enhancer for alcoholic beverages.

The body taste enhancer of the present invention is a sugar-modified protein itself or a composition containing a sugar-modified protein, which is used to enhance the body taste of beer-taste alcoholic beverages.
The sugar-modified protein is a protein with a molecular weight of 35 to 50 kDa in which the amino group of lysine constituting the cereal-derived protein is glycated, and the details are as described above.
When the body taste enhancer is a composition containing a sugar-modified protein, it may optionally contain known additives that can be added to beverages as long as the effects of the present invention are not impaired.

The content of glycated lysine in the sugar-modified protein contained in the body taste enhancer of the present invention is preferably 0.04 nmol/µg or more, more preferably 0.10 nmol/µg or more, as the content (nmol) of glycated lysine per 1 µg of sugar-modified protein.
Since such a sugar-modified protein has a large content of glycated lysine in the sugar-modified protein, it can be used as a body taste enhancer capable of enhancing the body taste of beer-taste alcoholic beverages when used in a small amount.

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

The method for producing the beer-taste alcoholic beverage of the present invention is not particularly limited, but is exemplified by a production method comprising the step of reducing the ratio of malt in the raw material to less than 50% by weight and increasing the content of a sugar-modified protein with a molecular weight of 35 to 50 kDa.
More specifically, to a beer-taste alcoholic beverage containing less than 50% by weight of malt in the raw material, a sugar-modified protein having a molecular weight of 35 to 50 kDa in which the amino group of lysine constituting the cereal-derived protein is glycated is added to obtain a beer-taste alcoholic beverage with enhanced body taste.
The method for producing a beer-taste alcoholic beverage by this method is also the method for enhancing the body taste of the beer-taste alcoholic beverage of the present invention.

When adding a body taste enhancer to a beer-taste alcoholic beverage, the amount of the body taste enhancer added is preferably such that the glycated lysine content of the sugar-modified protein is 30.0 nmol/mL or more.
In addition, the glycated lysine content of the sugar-modified protein is preferably 50.0 nmol/mL or more, preferably 80.0 nmol/mL or more, and preferably 100 nmol/mL or more.
In this case, the amount of the body taste enhancer to be added is preferably 7 μg/mL or more as the amount of sugar-modified protein.

(Purification of sugar-modified protein derived from beer)
North American malt was pulverized to an appropriate particle size and mixed with hot water after being placed in a brewing tank. A temperature suitable for malt enzymes was maintained for a sufficient time for saccharification, and starch was converted into sugar by the action of malt enzymes to produce a saccharified solution. After that, the saccharified liquid was filtered, hops were added, and it was boiled to produce hot wort, ready for fermentation. The hot wort was cooled and brewer's yeast was added to it. Over the course of a few days, the yeast worked to break down the sugar in the wort into alcohol and carbon dioxide, producing young beer. Young beer was transferred to storage tanks and stored at low temperatures for several tens of days. During this time, the beer was aged to harmonize the taste and aroma of the beer. After aging, the beer was filtered and bottled.
Sugar-modified proteins were purified from beer brewed from North American malt by the method described above.
(1) Fractionation by cation exchange resin 50 mL of cation exchange resin SP Sepharose was placed in an empty column. Beer brewed from North American malt was adsorbed onto the resin. The resin was then 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) + 0.5 M-NaCl and fractions were collected. The obtained fractions were evaluated by SDS-PAGE, and the fractions containing the 40 kDa sugar-modified protein were collected and used as the cation exchange resin-bound fraction.
(2) Ultrafiltration (buffer exchange)
The cation exchange resin-bound fraction obtained in (1) was centrifuged at 3500 rpm in a water-washed ultrafiltration unit (Amicon Ultra-15 30K, manufactured by Merck) and ultrafiltered to obtain a concentrate.
(3) Ammonium sulfate fractionation The concentrate obtained in (2) was added dropwise to 20 mM phosphate buffer (pH 7.0) + 2M ammonium sulfate and stirred. The suspension was then centrifuged (2330 g, 10 min). The supernatant was collected in another container. The collected solutions were concentrated using an ultrafiltration unit. 20 mM sodium acetate (pH 4.5) was added to the concentrate and centrifuged (2330 g, 10 minutes) for concentration to obtain a sugar-modified protein purified product (Bradford assay (bovine serum albumin (BSA) equivalent), 20.4 mg/mL, 2.21 mL). The purity of the obtained glycosylated protein purified product was confirmed by SDS-PAGE.

(Purification of sugar-modified protein derived from barley)
A glycosylated protein was purified from North American barley according to the following procedure.
(1) Threshing of barley 451.88 g of North American barley was threshed with a rice mill (SATAKE Gabamil RSKM 3D) to obtain 394.02 g of polished barley.
(2) Polished barley Freeze-ground 394.02 g of polished barley was placed in a ball mill, cooled with liquid nitrogen, and then ground (twice at 1100 rpm for 10 minutes) to obtain ground barley.
(3) Preparation of barley extract The pulverized barley was added to an Erlenmeyer flask containing 5 L of water and stirred for 1 hour. Thereafter, the ground barley suspension was placed in a centrifugal tube and centrifuged at 8000 rpm at 4° C. for 10 minutes to obtain a centrifugal supernatant as a barley extract.
(4) Ammonium sulfate fractionation (4-1) Put the 40% saturated ammonium sulfate-treated barley extract in a beaker, add 243 g of ammonium sulfate per 1 L while stirring with a stirrer bar, stir for 1 hour, then place the contents of the beaker in a centrifugal tube and centrifuge at 8000 rpm for 10 minutes at 4°C to collect 4.9 L of the centrifugal supernatant to obtain a 40% saturated ammonium sulfate-treated fraction.
(4-2) 40%-60% Saturated Ammonium Sulfate Treatment Put the whole amount of the 40% saturated ammonium sulfate treated fraction in a beaker, add 123 g of ammonium sulfate per 1 L while stirring with a stirrer bar, stir for 1 hour, then put the contents of the beaker into a centrifugal tube, centrifuge at 4° C. for 10 minutes at 8000 rpm, collect the precipitated fraction, and obtain the ammonium sulfate fraction of the barley extract.
(4-3) Buffer Exchange of Ammonium Sulfate Fraction To the ammonium sulfate fraction of malt extract, 100 mL of 20 mM sodium phosphate buffer (pH 6.5) was added. Thereafter, ultrafiltration was performed using an ultrafiltration unit (Merck Amicon Ultra-15 30K), and buffer exchange was performed using 20 mM phosphate buffer (pH 6.5).
(5) Anion resin fractionation Anion resin Q Sepharose 300 mL was packed in an econocolumn.
300 mL of Q Sepharose was added to the ammonium sulfate fraction of the barley extract, and batch adsorption was performed.
The contents of the beaker were then packed into a column and the flow-through collected (450 mL).
Then 3000 mL of 20 mM phosphate buffer (pH 6.5) was loaded.
Then, 900 mL of 20 mM phosphate buffer (pH 6.5) containing 0.1 M NaCl, 900 mL of 20 mM phosphate buffer (pH 6.5) containing 0.2 M NaCl, and 900 mL of 20 mM phosphate buffer (pH 6.5) containing 0.3 M NaCl were loaded. Fractions were collected every 300 mL and analyzed by SDS-PAGE, and fractions containing 40 kDa were collected and used as anion exchange resin-bound fractions.
Further, the concentrate was suspended in the smallest possible amount of 20 mM sodium acetate buffer (pH 4.5), and 20 mM acetate buffer (pH 4.5) was added until turbidity was removed.
Then, using an ultrafiltration unit (Merck Amicon Ultra-15 30K), buffer exchange was performed using 20 mM sodium acetate buffer (pH 4.5) by ultrafiltration to obtain a barley-derived sugar-modified protein (1.14 mg/mL, 16.1 mL).

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

(2) Enzymatic Digestion of Protein The excised gel piece was cut into small pieces, reduced with dithiothreitol (56° C., 1 hour), and carbamidomethylated with iodoacetamide (under light shielding, room temperature, 45 minutes). Then, 15 μL of 10 ng/μL chymotrypsin solution (5 mM calcium chloride, 50 mM ammonium hydrogen carbonate solution) containing 0.01% ProteaseMax and 15 μL of 5 mM calcium chloride, 50 mM ammonium hydrogen carbonate solution were added and incubated overnight, and then the enzymatic digestion fluid was recovered. The recovered solution was dried under reduced pressure and redissolved in a 0.1% formic acid solution.

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

(4) Protein and Modification Analysis Protein identification and modification search were performed under the following conditions.
Search software: Proteome Discoverer 2.2.0.388 (manufactured by ThermoFisher)
Species: barley (Hordeum vulgare), hops (Humulus), yeast (Saccharomyces cerevisiae)
Search condition: Digestive enzyme: Chymotrypsin
Modification (Static): Carbamidomethyl (Cysteine)
Modification (Dynamic): Oxidation (Methionine), Hex (K, R, Protein N-term), Hex (2) (K, R, Protein N-term), Hex (3) (K, R, Protein N-term), Acetyl (Protein N-term)
Precursor ion mass error range: Monoisotopic, ±10 ppm
Product ion mass error range: ±0.02 Da
Maximum number of missed revenge: 5
Confidence level (Percolator): High (the level with the highest probability among the three levels of certainty)
Database: SwissProt

As a result of modification analysis, it was confirmed that the lysine amino group of the sugar-modified protein was glycated.

(Evaluation of degree of sugar modification of sugar-modified protein)
The degree of sugar modification of the purified sugar-modified protein was colorimetrically determined by the NBT method using a Fructosamine Assay kit (abcam's product, ab228558).
The degree of glycosylation of barley-derived purified glycosylated protein and beer-derived purified glycosylated protein was determined.
The number of tests was set to n=2 for each protein, and the average value was adopted.
Table 3 shows the degree of sugar modification per weight of each purified sugar-modified protein.

(Measurement of concentration of glycated lysine in commercial beer-taste alcoholic beverage)
The concentration of glycated lysine in commercial beer-taste alcoholic beverages A and B, both of which had a malt content of less than 50% by weight, was measured as follows.
(1) Purification of sugar-modified proteins from commercial beer-taste alcoholic beverages A and B Purification of sugar-modified proteins from commercial beer-taste alcoholic beverages A and B was performed in the same manner as purification of sugar-modified proteins from beer brewed from North American malt.
A sugar-modified protein solution was obtained from a commercially available beer-taste alcoholic beverage A. A sugar-modified protein solution was obtained from a commercially available beer-taste alcoholic beverage B.
(2) Evaluation of the degree of sugar modification of sugar-modified proteins purified from commercially available beer-taste alcoholic beverages A and B The degree of sugar modification of sugar-modified proteins purified from commercially available beer-taste alcoholic beverages A and B was measured by the NBT method in the same manner as the method for measuring the degree of sugar modification of sugar-modified proteins purified from beer brewed from North American barley and North American malt.
(3) Concentration measurement of sugar-modified proteins in commercial beer-taste alcoholic beverages The concentration of sugar-modified proteins in beer of commercial beer-taste alcoholic beverages A and B was measured using a LabChip™ GXII device (manufactured by PerkinElmer) and an HT Protein Express chip according to a standard protocol (n = 2).
(4) Calculation of glycated lysine concentration in commercially available beer-taste alcoholic beverages The concentration of glycated lysine in commercially available beer-taste alcoholic beverages A and B was determined according to the following formula.
(concentration of glycated lysine per protein weight of sugar-modified protein) x (concentration of sugar-modified protein in beer-taste alcoholic beverage) = concentration of glycated lysine in beer-taste alcoholic beverage The concentration of glycated lysine in commercial beer-taste alcoholic beverage A was 28.8 nmol/mL, and the concentration of glycated lysine in commercial beer-taste alcoholic beverage B was 11.6 nmol/mL.

(Body taste evaluation when sugar-modified protein is added to commercially available beer-taste alcoholic beverage A)
A beer-derived refined sugar-modified protein was added to a commercially available beer-taste alcoholic beverage A, and a sensory evaluation of the richness was performed.
Purine body concentration of commercially available beer-taste alcoholic beverage A is 4.06 mg/100 mL. The total purine concentration was measured by the decomposition method at the Japan Food Research Laboratories (the same applies hereinafter).
For the sensory evaluation, the body taste of the commercially available beer-taste alcoholic beverage A to which no sugar-modified protein was added was set at 0.9 as a reference point, and the body taste obtained when 25 µg/mL of beer-derived sugar-modified protein was added to the commercially available beer-taste alcoholic beverage A was set at 1.3 points as a reference point, and scored in increments of 0.1 point by four expert panels.
Evaluation was carried out by varying the concentration of the glycosylated protein added, and the results are shown in Table 1.
The following criteria were used for the full-bodied taste.
0 points: Not felt at all 1 point: Slightly felt 2 points: Clearly felt 3 points: Very strongly felt Table 1 shows the glycated lysine content (nmol/mL) of sugar-modified proteins in beer-taste alcoholic beverages.

From the results shown in Table 1, by adding a sugar-modified protein to the commercial beer-taste alcoholic beverage A with a malt ratio of less than 50% by weight to increase the content of glycated lysine, it can be seen that the richness of the beer-taste alcoholic beverage can be enhanced.

(Evaluation of body taste when sugar-modified protein is added to commercially available beer-taste alcoholic beverage B)
A beer-derived sugar-modified protein was added to a commercially available beer-taste alcoholic beverage B, and sensory evaluation of the richness was performed.
The commercially available beer-taste alcoholic beverage B is a beer-taste alcoholic beverage that is said to have less body than the commercially available beer-taste alcoholic beverage A.
The purine body concentration of commercially available beer-taste alcoholic beverage B is 4.2 mg/100 mL.
The sensory evaluation was carried out in the same manner as the full-bodied taste evaluation when the beer-derived sugar-modified protein was added to the commercially available beer-taste alcoholic beverage A.
The body taste of the commercially available beer-taste alcoholic beverage A to which no glycosylated protein is added is set at 0.9 points as a reference point, and the body taste obtained when 25 μg/mL of beer-derived sugar-modified protein is added to the commercially available beer-taste alcoholic beverage A is set at 1.3 points as a reference point.
Table 2 shows the glycated lysine content (nmol/mL) of sugar-modified proteins in beer-taste alcoholic beverages.

From the results shown in Table 2, it can be seen that even beer-taste alcoholic beverages, which are originally considered to have a low body taste, can be enhanced in body taste by adding sugar-modified proteins and increasing the content of glycated lysine.

(Effect of adding sugar-modified proteins with different degrees of sugar modification to beer)
A barley-derived sugar-modified protein and a beer-derived sugar-modified protein were each added to a commercially available beer-taste alcoholic beverage A, and the effect on body taste was sensory evaluated.
The degree of sugar modification of each purified protein was as shown in Table 3.

表４に示す通り、糖修飾タンパク質の添加量が同じであっても、糖修飾タンパク質の糖修飾度によってコク強度が異なり、糖修飾度に応じてコクが向上することが明らかになった。 The sensory evaluation was carried out in the same manner as the full-bodied taste evaluation when the beer-derived sugar-modified protein was added to the commercially available beer-taste alcoholic beverage A.
Each sugar-modified protein was added to the commercially available beer-taste alcoholic beverage A for evaluation, and the results are shown in Table 4.
The amount of glycosylated protein added was 25 μg/mL.
Table 4 shows the glycated lysine content (nmol/mL) of sugar-modified proteins in beer-taste alcoholic beverages.

As shown in Table 4, even if the amount of sugar-modified protein added was the same, the richness intensity varied depending on the degree of sugar modification of the sugar-modified protein, and it became clear that the richness improved according to the degree of sugar modification.

According to the present invention, it is possible to provide a beer-taste alcoholic beverage with a strong body using grain-derived raw materials.

Claims (8)

A beer-taste alcoholic beverage in which the ratio of malt in the raw material is less than 50% by weight,
A sugar-modified protein with a molecular weight of 35 to 50 kDa in which the amino group of lysine constituting the cereal-derived protein is glycated,
A beer-taste alcoholic beverage, wherein the glycated lysine content of the sugar-modified protein is 30.0 nmol/mL or more. 2. The beer-taste alcoholic beverage according to claim 1, wherein the sugar-modified protein has a content of glycated lysine of 35.3 nmol/mL or more. 3. The beer-taste alcoholic beverage according to claim 1, wherein the glycated lysine content of said sugar-modified protein is 30.0 to 1000 nmol/mL. The beer-taste alcoholic beverage according to any one of claims 1 to 3, wherein the sugar glycated to the amino group of the glycated lysine is a monosaccharide or a disaccharide. The beer-taste alcoholic beverage according to any one of claims 1 to 4, wherein the grain is at least one selected from the group consisting of barley, wheat, corn, rice and soybeans. The beer-taste alcoholic beverage according to any one of claims 1 to 5, which does not contain wort. A body-taste enhancer for a beer-taste alcoholic beverage containing less than 50% by weight of malt in the raw material, comprising a sugar-modified protein having a molecular weight of 35 to 50 kDa, in which the amino group of lysine constituting a cereal-derived protein is glycated. A method for enhancing the body taste of a beer-taste alcoholic beverage, comprising adding a sugar-modified protein having a molecular weight of 35 to 50 kDa, in which the amino group of lysine constituting the cereal-derived protein is glycated, to the beer-taste alcoholic beverage containing less than 50% by weight of malt in the raw material, thereby enhancing the body taste of the beer-taste alcoholic beverage ,
A method for enhancing body taste of a beer-taste alcoholic beverage, wherein the sugar-modified protein is added so that the sugar-modified protein has a content of glycated lysine of 30.0 nmol/mL or more.