JP2023145804A - Beer taste beverage with reduced unfavorable taste - Google Patents

Abstract

To provide a beer taste beverage that includes malt and/or un-germinated wheat as a part of raw materials, the beverage having a reduced unfavorable taste derived from the raw materials.SOLUTION: There is provided a beer taste beverage that includes malt and/or un-germinated wheat as a part of raw materials. A percentage (molar ratio) of amino acids causing an unfavorable taste, to all amino acids constituting a peptide with a molecular weight of 90 to 2600 Da (HPLC gel filtration) in the beverage, is 9.7% or less. The amino acids causing the unfavorable taste include valine, leucine and isoleucine.SELECTED DRAWING: None

Description

The present invention relates to a beer-taste beverage that uses malt and/or ungerminated barley as part of its raw materials.

In fermented malt beverages that use malt as the main ingredient, malt, which is the main ingredient, greatly contributes to the flavor of beer and low-malt beer. However, in other sparkling fermented alcoholic beverages such as happoshu and beer-flavored fermented alcoholic beverages, the proportion of malt used is low, so simply saccharification will not produce amino acids through the decomposition of proteins derived from malt, which is the raw material. If there is too little, there will be a lack of nitrogen source, which will cause yeast proliferation and fermentability to worsen, leading to deterioration of flavor. To avoid such problems, when producing low-malt beer and sparkling fermented alcoholic beverages, it is possible to add proteins from barley, rice, corn, soybeans, etc. and proteolytic enzymes as nitrogen sources to generate amino acids. This is done to prevent deterioration of fermentation and deterioration of flavor.

On the other hand, in the production of low-malt beer and beer-flavored fermented alcoholic beverages, proteolytic enzymes are used to break down proteins other than malt. However, it is known that when hydrolyzed with proteolytic enzymes, undesirable flavors such as astringency, bitterness, and astringency are produced due to enzymatic decomposition. Such an unpleasant taste may impair the refreshing and refreshing drinking sensation.

Patent Document 1 discloses a method for producing a beer-like beverage with reduced astringency by contacting an intermediate liquid obtained during the production process with a cation exchange resin.

Japanese Patent Application Publication No. 2013-006154

If it is possible to reduce the unpleasant taste derived from raw materials in beer-taste beverages that use auxiliary raw materials other than malt, it is possible to provide beverages that are refreshing, refreshing, and easy to drink. Furthermore, if such unpleasant tastes are removed even in beer that does not use auxiliary raw materials, it is possible to provide a beer that is cleaner, more refreshing, and easier to drink.

That is, an object of the present invention is to provide a beer-taste beverage that uses malt and/or ungerminated barley as a part of the raw materials, and has reduced unpleasant tastes derived from the raw materials.

The present inventors recently focused on peptides contained in beer-taste beverages that use malt as a raw material, and investigated their relationship with the off-taste of beer-taste beverages. As a result, we found that peptides with a molecular weight of 90 to 2,600 Da contained in beer-taste beverages separated by preparative gel filtration were involved in off-taste, and that the proportion of amino acids involved in off-taste constituting the peptides was adjusted within a specified range. It has been found that by adjusting the amount, it is possible to effectively reduce the unpleasant taste of beer-taste beverages. The present invention is based on this knowledge.

According to the present invention, the following inventions are provided.
[1] A beer-taste beverage that uses malt and/or ungerminated barley as part of the raw materials, and has an unpleasant taste for all amino acids that make up the peptides with a molecular weight of 90 to 2,600 Da (HPLC gel filtration method) in the beverage. A beer-taste beverage in which the proportion (molar ratio) of amino acids involved is 9.7% or less, and the amino acids involved in the off-taste are valine, leucine, and isoleucine.
[2] At least one of valine, leucine and isoleucine has the following content ratio:
- The beer-taste beverage according to [1] above, which satisfies the following conditions: - valine content: less than 2.1% - leucine content: less than 3.3% - isoleucine content: less than 4.3%.
[3] The ratio (mole ratio) of amino acids involved in off-taste to all amino acids constituting peptides with a molecular weight of 90 to 2600 Da (HPLC gel filtration method) in the beverage is 17.2% or less,
The beer-taste beverage according to [1] or [2] above, wherein the amino acids involved in unpleasant taste are valine, leucine, isoleucine, phenylalanine, and arginine.
[4] At least one of valine, leucine, isoleucine, phenylalanine, and arginine has the following content ratio:
・Valine content: less than 2.1% ・Leucine content: less than 3.3% ・Isoleucine content: less than 4.3% ・Phenylalanine content: 3.0% or less ・Arginine content: The beer-taste beverage according to [3] above, which satisfies 4.5% or less.
[5] The beer-taste beverage according to any one of [1] to [4] above, which has reduced unpleasant taste.
[6] The beer-taste beverage according to any one of [1] to [5] above, which is a fermented alcoholic beverage.
[7] The beer-taste beverage according to [6] above, wherein the malt usage ratio is less than two-thirds.

According to the present invention, it is possible to provide a beer-taste beverage that uses malt and/or ungerminated barley as a part of the raw materials, with reduced unpleasant tastes derived from the raw materials.

FIG. 2 is a diagram schematically showing two methods of determining the molecular weight range from the retention time of the HPLC gel filtration method carried out in Example 2 (2). FIG. 2 is a diagram showing sensory evaluation results (evaluation scores) for a control product and a test product (using a proteolytic enzyme preparation). FIG. 3 is a diagram showing a calibration curve created from the retention time of the HPLC gel filtration method and the molecular weight of a known substance, which was carried out in Example 2 (2). FIG. 2 is a diagram showing sensory evaluation results (evaluation scores) for preparative gel filtration fractions 32 to 34 (F32, F33, F34) obtained from a control product and a test product (using a proteolytic enzyme preparation). . FIG. 3 is a diagram showing the analysis results of amino acid content ratios for preparative gel filtration fractions 32 to 34 obtained from a control product and a test product (using a proteolytic enzyme preparation). FIG. 2 is a diagram showing the analysis results of amino acid content ratios for preparative gel filtration fractions 32 to 34 obtained from commercially available beer, commercially available low-malt beer, control product, and test product (using proteolytic enzyme preparations). .

Specific description of the invention

In the present invention, "beer-taste beverage" refers to the case where beer is normally produced, that is,
Refers to beverages with the taste and aroma unique to beer obtained when beer is produced based on fermentation with yeast, etc., and includes "beer-taste alcoholic beverages" and "beer-taste non-alcoholic beverages."

"Beer-taste alcoholic beverages" also include "beer-taste fermented alcoholic beverages" that are fermented by yeast using carbon sources, nitrogen sources, water, etc. as raw materials. , Happoshu, beer-taste sparkling alcoholic beverages that do not use barley or malt as raw materials (e.g., new genre brewed beverages classified as "other brewed liquors (sparkling) (1)" under the Liquor Tax Act) and raw materials. Beverages made by adding alcohol to beer and low-malt beer that use malt (for example, a new genre of liqueur drinks classified as "liqueur (sparkling) (1)" under the Liquor Tax Act) are examples.

The beer-taste alcoholic beverage of the present invention uses malt and/or ungerminated barley as a part of the raw materials, and any malt usage ratio can be used, but for example, the malt usage ratio can be two-thirds. Above, it can be less than two-thirds or less than 50%. In the present invention, the "malt usage ratio" refers to the ratio of malt mass to the mass of all raw materials excluding brewing water.

"Beer-taste non-alcoholic beverage" refers to a non-alcoholic beverage that is unfermented and therefore does not contain alcoholic components derived from fermentation, yet has a beer-like flavor. Furthermore, in the present invention, the term "non-alcoholic beverage" refers to a beverage that does not contain alcohol at all, that is, the ethanol concentration is 0.00 v/v%, and is synonymous with "completely alcohol-free beverage."

The beer-taste beverage of the present invention is characterized in that the ratio of amino acids involved in off-taste to all amino acids constituting peptides in a specific molecular weight range in the beverage is below a predetermined value. Here, the amino acids involved in the off-flavor are amino acids with high side chain Δg values (KH Ney: Z. Lebensm
Unters. Forach., 147, 64 (1971)), for example, valine, leucine,
Examples include isoleucine, phenylalanine and arginine.

The peptides in drinks, which are the basis for calculating the content ratio of amino acids involved in off-taste, are 90-2.
It is a peptide with a molecular weight of 600 Da. The molecular weight of the peptide is determined by gel filtration method using high performance liquid chromatography (herein sometimes referred to as "HPLC gel filtration method").
It is measured by Specifically, fractions from a preparative gel filtration column (for example, Superdex 30pg) are analyzed by HPLC gel filtration, and the retention time from the beginning of the peak rising from the baseline to the end of the peak falling to the baseline (Fig. The molecular weight range corresponding to the arrow range A) shown in 1 is defined as the molecular weight of the fraction. Molecular weight can be calculated from retention time using a calibration curve of HPLC gel filtration method (for example, FIG. 3). In addition, when calculating the molecular weight based on the retention time from the first peak top to the last peak top included in the above range (arrow range B shown in Figure 1), the content ratio of amino acids involved in off-flavor can be calculated. The molecular weight of the peptide in the drink, which is the basis for calculation, is 330 to 1040 Da. A specific example of molecular weight measurement by HPLC gel filtration method is as shown in Example 2 below. In addition, peptide quantification is performed using Low
It can be carried out by the y method (Lowry method).

The "ratio of amino acids involved in the off-taste" in the beer-taste beverage of the present invention is 90 to 2.
This is the ratio of the content of amino acids involved in unpleasant taste to the content of amino acids constituting all peptides having a molecular weight of 600 Da, and the content ratio is calculated by molar ratio. 90～
All peptides with a molecular weight of 2600 Da can be obtained by preparative gel filtration according to the examples below. These peptides were decomposed into amino acids by the acid decomposition method described in Example 2, and the content ratio of amino acids constituting the peptides was calculated from the difference in the values of presence/absence of acid decomposition.

The amino acid content can also be measured by high performance liquid chromatography, for example by using a commercially available amino acid analyzer according to the method described in Example 2.

In the present invention, the content ratio of amino acids involved in off-taste can be calculated based on the total amount of valine, leucine, and isoleucine, and in this case, the content ratio (molar ratio) of amino acids involved in off-taste is 9. It can be 7% or less, preferably less than 3.5%.

In the present invention, the content ratio of amino acids involved in off-taste can also be calculated based on the total amount of valine, leucine, isoleucine, phenylalanine, and arginine, and in this case, the content ratio of amino acids involved in off-taste ( molar ratio) can be 17.2% or less, preferably less than 4.2%.

In the present invention, the content ratio of amino acids involved in off-taste can be specified by the content ratio of each amino acid involved in off-taste. For example, in beverages with a molecular weight of 90 to 2,600 Da (HP
The content ratio of each of valine, leucine, isoleucine, phenylalanine, and arginine to all amino acids constituting the peptide (LC gel filtration method) can be specified as follows.
・Valine content: less than 2.1% (preferably less than 1.1%)
- Leucine content: less than 3.3% (preferably less than 1.1%)
- Isoleucine content: less than 4.3% (preferably less than 1.3%)
- Phenylalanine content: 3.0% or less (preferably less than 0.7%)
- Arginine content: 4.5% or less (preferably 0.0%)

The beer-taste beverage of the present invention can be a beverage in which at least one or two of valine, leucine, and isoleucine satisfy the above content ratio, and preferably, all of valine, leucine, and isoleucine satisfy the above content ratio. The beer-taste beverage of the present invention can also be a beverage in which at least one, two, three or four of valine, leucine, isoleucine, phenylalanine and arginine satisfy the above content ratio, preferably valine, leucine, This is a beverage in which isoleucine, phenylalanine, and arginine all satisfy the above content ratios.

A preferred embodiment of the beer-taste beverage of the present invention includes a fermented malt beverage (especially low-malt beer) in which the malt usage ratio is less than two-thirds, and in this case, the following conditions (a-1) and/or (a -2).
(a-1) The ratio (molar ratio) of amino acids involved in off-taste to all amino acids constituting peptides with a molecular weight of 90 to 2600 Da (HPLC gel filtration method) in the beverage is 9.7% or less (however, The amino acids involved in taste are valine, leucine and isoleucine).
(a-2) The ratio (mole ratio) of valine, leucine, and isoleucine to all amino acids constituting the peptide with a molecular weight of 90 to 2,600 Da (HPLC gel filtration method) in the beverage satisfies at least one of the following conditions.
・Valine content: less than 2.1% ・Leucine content: less than 3.3% ・Isoleucine content: less than 4.3%

A preferred embodiment of the beer-taste beverage of the present invention also includes a fermented malt beverage (especially beer) in which the malt usage ratio is two-thirds or more; in this case, the following conditions (b-1) and (
It can be a beverage that satisfies b-2).
(b-1) The ratio (molar ratio) of amino acids involved in off-taste to all amino acids constituting peptides with a molecular weight of 90 to 2600 Da (HPLC gel filtration method) in the beverage is 17.2% or less (however, The amino acids involved in taste are valine, leucine, isoleucine, phenylalanine and arginine).
(b-2) The ratio (mole ratio) of valine, leucine, isoleucine, phenylalanine, and arginine to all amino acids constituting the peptide with a molecular weight of 90 to 2600 Da (HPLC gel filtration method) in the beverage satisfies all of the following.
・Valine content: less than 2.1% ・Leucine content: less than 3.3% ・Isoleucine content: less than 4.3% ・Phenylalanine content: 3.0% or less ・Arginine content: 4.5% or less

A more preferable embodiment of the beer-taste beverage of the present invention includes a fermented malt beverage (especially beer) in which the malt usage ratio is two-thirds or more, and in this case, the following conditions (c-1) and/or
Or it can be a beverage that satisfies (c-2).
(c-1) The ratio (mole ratio) of amino acids involved in off-taste to all amino acids constituting peptides with a molecular weight of 90 to 2600 Da (HPLC gel filtration method) in the beverage is less than 3.5% (however, The amino acids involved in taste are valine, leucine and isoleucine).
(c-2) The ratio (mole ratio) of valine, leucine, and isoleucine to all amino acids constituting the peptide with a molecular weight of 90 to 2,600 Da (HPLC gel filtration method) in the beverage satisfies at least one of the following conditions.
・Valine content: less than 1.1% ・Leucine content: less than 1.1% ・Isoleucine content: less than 1.3%

A more preferable embodiment of the beer-taste beverage of the present invention includes a fermented malt beverage (especially beer) in which the malt usage ratio is two-thirds or more; in this case, the following conditions (d-1) and/or ( It can be a beverage that satisfies d-2).
(d-1) The ratio (molar ratio) of amino acids involved in off-taste to all amino acids constituting peptides with a molecular weight of 90 to 2600 Da (HPLC gel filtration method) in the beverage is less than 4.2% (however, The amino acids involved in taste are valine, leucine, isoleucine, phenylalanine and arginine).
(d-2) The ratio (molar ratio) of valine, leucine, isoleucine, phenylalanine, and arginine to all amino acids constituting peptides with a molecular weight of 90 to 2,600 Da (HPLC gel filtration method) in the beverage satisfies at least three of the following: Fulfill.
・Valine content: less than 1.1% ・Leucine content: less than 1.1% ・Isoleucine content: less than 1.3% ・Phenylalanine content: less than 0.7% ・Arginine content: 0.0%

The beer-taste beverage of the present invention has reduced unpleasant taste compared to beverages outside the scope of the present invention. Therefore, the beer-taste beverage of the present invention can be provided as a refreshing, refreshing, and easy-to-drink beverage. The beer-taste beverage of the present invention is particularly advantageous in that it is a refreshing and easy-to-drink beverage because it has less unpleasant taste in the mouth. Here, the term "miscellaneous taste" is used to include astringent, bitter, and astringent tastes. Although not bound by the following theory, the unpleasant taste was reduced by reducing the amino acids involved in the unpleasant taste contained in peptides with a molecular weight of 90 to 2,600 Da (HPLC gel filtration method) from the beverage. Beverages will likely be provided.

In the beer-taste beverage of the present invention, the ratio (molar ratio) of amino acids involved in off-taste to all amino acids constituting peptides with a molecular weight of 90 to 2,600 Da (HPLC gel filtration method) in the beverage is adjusted within a predetermined range. It can be manufactured according to normal beer-taste beverage manufacturing procedures.

For example, beer-taste alcoholic beverages are produced by adding fermentation beer yeast to wort prepared from brewing raw materials such as malt, hops, auxiliary raw materials, and brewing water, and then fermenting to create a fermented liquid. I can do it. Moreover, various additives described below can be added as raw materials as necessary. It goes without saying that among beer-taste alcoholic beverages, all-malt beer can be produced from malt, hops, and water.

In the above manufacturing procedure, wort can be prepared according to a conventional method. For example, wort is prepared by saccharifying and filtering a mixture of brewing raw materials and brewing water to obtain wort, adding hops to the wort, boiling it, and cooling the boiled wort. I can do it. In addition, the wort is
It can also be produced by adding a commercially available enzyme preparation during the saccharification process. For example, protease preparations are used for proteolysis, α-amylase preparations, glucoamylase preparations, pullulanase preparations, etc. are used for carbohydrate decomposition, and β-glucanase preparations, fibrinolytic enzyme preparations, etc. are used for fibrinolysis. or a mixture of these can be used.

In a preferred embodiment of the production of the beer-taste beverage of the present invention, an enzyme preparation containing an endo-type protease and an exo-type protease can be used alone or in combination with other protease preparations for proteolysis in the saccharification step. By carrying out such a saccharification process, the molecular weight of 90 to 2600 Da (H
It is possible to reduce the content of amino acids that are involved in off-taste and constitute peptides (PLC gel filtration method), and it is therefore possible to produce a beer-taste beverage with reduced off-taste. That is, the present invention provides a method for producing a beer-taste beverage, which is characterized in that an enzyme preparation containing an endo-type protease and an exo-type protease is used for proteolysis in the saccharification step.

The activity characteristics of the enzyme preparation used for the above proteolysis can be specified based on the exo-type protease activity and the endo-type protease activity. For example, the total value of the exo-type protease activity and the endo-type protease activity is 100. ~10,000 (units/g), and the ratio (percentage) of exo-type protease activity (ratio (percentage) of exo-type protease activity to the total value of exo-type protease activity and endo-type protease activity) is 10 to 80. Enzyme preparations that are % can be used. More preferably, the total value of exo-type protease activity and endo-type protease activity is 1000 to 7000 (units/g), and the ratio (percentage) of the exo-type protease activity (exo-type protease activity and endo-type protease activity) Enzyme preparations can be used in which the ratio (percentage) of exo-protease activity to the total activity is between 30 and 70%. Exo-type protease activity can be expressed as the sum of acid carboxypeptidase (CP) activity, leucine-aminopeptidase (LAP) activity, and X-prolyl-dipeptidyl-aminopeptidase (DAP) activity, and the measurement of each activity is It can be carried out according to the procedure described in 1. Furthermore, endo-protease activity can be measured according to the procedure described in Example 1.

Alternatively, in the production of the beer-taste beverage of the present invention, a beer-taste beverage with a molecular weight of 90 to 2,600 Da (HPL
It is also possible to produce a beer-taste beverage with reduced unpleasant taste by reducing the content of peptides that are involved in the unpleasant taste (C gel filtration method).

In the production of the beer-taste alcoholic beverage of the present invention, in addition to malt, ungerminated barley (for example, ungerminated barley (including extract), ungerminated wheat (including extract)); rice, Auxiliary raw materials stipulated by the Liquor Tax Law such as corn, koryan, potato, starch, sugars (e.g. liquid sugar); Nitrogen sources such as protein decomposition products and yeast extract; fragrances, pigments, foaming/foam retention improvers, water quality adjustment Other additives such as fermentation agents, fermentation aids, etc. can be used as brewing raw materials. That is, the beer-taste alcoholic beverage of the present invention can use at least malt, ungerminated barley (preferably ungerminated barley), and hops as raw materials other than brewing water,
Depending on the case, sugars, rice, corn, starch, etc. may also be used as raw materials.

In addition, for beer-taste non-alcoholic beverages, malt, hops, and other raw materials (
For example, a preparation liquid is prepared from raw materials such as fragrances, pigments, dietary fibers, protein hydrolysates, foaming/foam retention improvers, water quality conditioners, etc.), water, etc., and solids are removed from the preparation liquid by standing. After that, by performing filtration, a beer-taste non-alcoholic beverage can be produced. The above-mentioned preparation liquid can be prepared according to a conventional method similar to the method for preparing the wort for the above-mentioned beer-taste alcoholic beverage. In the saccharification process, enzyme preparations containing endo- and exo-proteases can be used for proteolysis.

The beverage provided by the present invention may be subjected to a step of adding carbon dioxide gas, as the case may be, and may be provided as a packaged beverage through steps such as a filling step and a sterilization step. Sterilization may be performed before or after filling the container. Furthermore, if the pH of the beverage is adjusted to less than 4, the beverage can be packed into containers by directly performing the filling process without going through the sterilization process.

The containers used for the beverage according to the invention may be any containers commonly used for filling beverages, such as metal cans, barrels, plastic bottles (e.g. PET bottles, cups),
Examples include paper containers, bottles, pouch containers, etc., but preferred are metal cans/barrel containers, plastic bottles (for example, PET bottles), and bottles.

According to another aspect of the present invention, there is provided a method for reducing the unpleasant taste of a beer-taste beverage containing malt and/or ungerminated barley as a raw material, the method comprising:
LC gel filtration method) A method characterized by adjusting the proportion (molar ratio) of amino acids involved in off-flavor to all amino acids constituting the peptide to 9.7% or less (however, the amino acids involved in off-taste are valine, leucine and isoleucine). The method of reducing off-taste of the present invention can be carried out according to the description regarding the beer-taste beverage of the present invention. In particular, the ratio (molar ratio) of amino acids involved in off-taste to all amino acids constituting peptides with a molecular weight of 90 to 2,600 Da (HPLC gel filtration method) in the beverage can be determined according to the description regarding the beer-taste beverage of the present invention.

The present invention will be explained more specifically based on the following examples, but the present invention is not limited to these examples.

Example 1: Production and evaluation using a pilot plant (1) Production using a pilot plant Happoshu with a malt usage ratio of less than 25% was produced in a pilot plant. Specifically, malt, barley, corn, and liquid sugar are used as raw materials so that the malt usage ratio is less than 25%. In addition to the enzyme preparation used, an enzyme preparation for proteolysis (hereinafter sometimes referred to as "enzyme preparation for proteolysis") is used, and after being held at 50 °C, the temperature is raised to 65 °C, and then to 78 °C. Then, saccharification was carried out for a total of 270 minutes. The product using the proteolytic enzyme preparation was used as a test product, and the product not using the proteolytic enzyme preparation was used as a control product. The test product and the control product were manufactured under the same conditions except for the use of proteolytic enzyme preparations in the saccharification process.The brewing liquid was prepared according to the normal low-malt beer manufacturing method, fermented with yeast, filtered, and bottled. was manufactured.

(2) Regarding proteolytic enzyme preparations The proteolytic enzyme preparations used in (1) above are enzyme preparations containing endo-type proteases and exo-type proteases, and can be identified based on exo-type protease activity and endo-type protease activity. . Specifically, the enzyme preparation has a total value of exo-type protease activity and endo-type protease activity of 1029 units/g, and a ratio (percentage) of the exo-type protease activity (exo-type protease activity and endo-type protease activity). This is an enzyme preparation in which the ratio (percentage) of exo-type protease activity to the total value of type protease activity is 47.9%.

Exo-type protease activity The activity of exo-type protease is the sum of acidic carboxypeptidase (CP) activity, leucine-aminopeptidase (LAP) activity and X-prolyl-dipeptidyl-aminopeptidase (DAP) activity. Each activity of the proteolytic enzyme preparation was measured as follows.

[Measurement of acidic carboxypeptidase (CP) activity]
The enzyme preparation was appropriately diluted with 0.2M acetate buffer pH 4.0. 0.25mM H-Gl
0.2 mL of enzyme dilution solution was mixed with 1 mL of u-Tyr-Glu, reacted at 37° C. for 60 minutes, and then boiled for 5 minutes to stop the reaction. Thereafter, 3 mL of OPA (o-phthalaldehyde) reagent was added, and after 3 minutes, the absorbance at 340 nm was measured using an absorbance meter (manufactured by Shimadzu Corporation). As a result, the amount of Glu released due to the enzyme reaction was determined. In addition, after performing the same treatment as above on the enzyme diluted solution inactivated by boiling for 5 minutes, color was developed with OPA reagent, and this absorbance was subtracted from the absorbance of the enzyme reaction solution as a blank, and created with L-Glu standard solution. Enzyme activity was determined using the calibration curve obtained. Enzyme activity was defined as 1 unit, which is the amount of enzyme that releases 1 μmol of Glu per minute. The OPA reagent was first dissolved in ethanol to give 4% o-phthalaldehyde, and then the final concentrations were 0.08% o-phthalaldehyde, 50 mM sodium tetraborate, 1% SDS, and 0.2%, respectively. The mixture was mixed to make an aqueous solution of mercaptoethanol and prepared for use.

[Measurement of leucine-aminopeptidase (LAP) activity]
After dissolving 36 mg of L-leucine-p-nitroanilide HCl in 2.5 mL of dimethyl sulfoxide (DMSO), 100 m
Messed up to L. The enzyme preparation was appropriately diluted with ion-exchanged water. 80 μL of the substrate solution and 20 μL of the diluted enzyme solution were placed in an Eppendorf tube that had been cooled on ice, and the tube was capped and stirred, followed by reaction at 40° C. for 5 minutes. The reaction was stopped by adding 200 μL of 1.5M acetic acid solution and stirring. The absorbance of the supernatant at 405 nm was measured using an absorbance meter (manufactured by Molecular Devices). Enzyme activity was calculated based on a calibration curve prepared with pNA (p-nitroaniline). Enzyme activity is defined as the amount of enzyme that releases 1 μmol of pNA per minute.
It was defined as a unit.

[Measurement of X-prolyl-dipeptidyl-aminopeptidase (DAP) activity]
H-Gly-Pro-pNA-tosylate (manufactured by Bachem) was dissolved to 2mM in 100mM potassium phosphate buffer (pH 7.5) to prepare a substrate solution. The enzyme preparation was appropriately diluted with ion-exchanged water. 80 μL of the substrate solution and 80 μL of the diluted enzyme solution were placed in an Eppendorf tube that had been cooled on ice, and the tube was capped and stirred, followed by a reaction at 40° C. for 10 minutes. During the reaction, add 20 μL of 1M acetic acid solution to the microplate and add 10 μL of 1M acetic acid solution to the microplate.
After 10 minutes, 100 μL of the reaction solution was added and stirred to completely stop the reaction. Further, after mixing 50 μL of the substrate solution and 20 μL of 1M acetic acid solution, 50 μL of the enzyme dilution solution was added and mixed, which was used as an enzyme blank. For these solutions, use an absorbance meter (manufactured by Molecular Devices).
The absorbance at 405 nm was measured. Enzyme activity was calculated based on a calibration curve prepared with pNA (p-nitroaniline). Enzyme activity was defined as the amount of enzyme that releases 1 μmol of pNA per minute as 1 unit.

Endo-type protease activity The activity of endo-type protease was measured using a method in which an enzyme reaction was performed using milk casein (pH 6) as an enzyme reaction substrate, and amino acids released in the solution were measured. The specific measurement method is as follows.

A 13.3 mM sodium phosphate buffer (pH 6.0) containing 0.6% milk casein was used as a milk casein substrate. The enzyme preparation was appropriately diluted with ion-exchanged water. 1 mL of the diluted enzyme preparation solution was added to 5 mL of the milk casein substrate solution, and a reaction was performed at 30° C. for 10 minutes. After the reaction was completed, 5 mL of 0.44 M trichloroacetic acid solution was added, mixed well, and left at 30° C. for 30 minutes to stop the reaction and insolubilize the undecomposed substrate. No. The reaction solution was filtered through 6 filter paper. Add 2.5 mL of 0.55 M sodium carbonate solution to 1 mL of filtrate and dilute 3-fold phenol reagent 0.5
mL was added and left at 30°C for 30 minutes to allow sufficient color development. The absorbance at 660 nm was measured using an absorbance meter (manufactured by Shimadzu Corporation) to quantify the amino acid released from the substrate. Enzyme activity is 1
The amount of enzyme that produces an increase in Folin's reagent color substance equivalent to 1 μg of tyrosine per minute is
It was defined as a unit.

(3) Sensory evaluation of test products The test products and control products were subjected to sensory evaluation by five trained expert panelists. Specifically, the off-taste (astringency, bitterness, astringency) of the control product was set as 5, and the off-taste (astringency, astringency) of the test product was set as 5.
Bitterness and astringency) were evaluated on a nine-point scale from 0 to 8. The average value of the evaluation scores of each of the five panelists was calculated, and if it was 4 or less, it was judged that the off-taste (astringency, bitterness, astringency) was reduced.

The results of the sensory evaluation are shown in FIG. From the results shown in FIG. 2, it was confirmed that by using the proteolytic enzyme preparation, it was possible to reduce unpleasant tastes (astringency, bitterness, astringency) and produce low-malt beer with a refreshing aftertaste.

Example 2: Evaluation of preparative gel filtration fraction (1) Preparation of preparative gel filtration fraction The test product and control product obtained in Example 1 (1) were each weighed and freeze-dried. Each lyophilized product was dissolved in a 100 mM NaCl solution to prepare a 5-fold concentrated solution, and each was used as a sample for gel filtration fractionation. Gel filtration fractionation was performed on each sample under the following conditions.

Column: Hiload Superdex30pg 26/600 (manufactured by GE Healthcare)
Sample injection volume: 5mL
Eluent composition: 100mM NaCl
Flow rate: 2.5mL/min (constant flow rate)
Detection wavelength: 215nm
Fractionation: 19.1 mL (fraction 0) from 0.29 cv (column volume), then 5 mL fractions from 0.35 cv (fractions 1 to 51)

Protein quantification of the fraction was carried out by the Lowry method using a commercially available kit (DC Protein Assay, Bio-Rad). Specifically, first, 50 μL of the above fractionated solution was taken, centrifuged, concentrated to dryness under reduced pressure, and redissolved by adding 10 μL of ultrapure water to obtain an analysis sample. 50 μL of solution A was added thereto and stirred, and then 400 μL of solution B was added and stirred. After carrying out a color reaction for 15 minutes at room temperature, 350 μL of the mixture was transferred to a 96-well plate, and the absorbance at 750 nm was measured.
The peptide concentration (mg/mL) was calculated based on the obtained absorbance and a calibration curve prepared in advance. Note that the calibration curve was created using BSA (bovine serum albumin).

(2) Measurement of molecular weight by HPLC gel filtration method (a) Sample preparation for HPLC gel filtration The fraction obtained in (1) above was used as a sample.

(a) HPLC gel filtration method The conditions for the HPLC gel filtration method were as follows.
<HPLC gel filtration analysis conditions>
Column: Superdex 75 10/300 (manufactured by GE Healthcare)
Sample injection volume: 100μL
Eluent composition: 50mM sodium phosphate (pH 7.0), 20% (v/v) acetonitrile, 150mM NaCl
Flow rate: 0.5mL/min (constant flow rate)
Detection wavelength: 215nm

(C) Creation of molecular weight calibration curve Under the column, eluent, flow rate, and detection wavelength described in the HPLC gel filtration conditions above, inject 50 μL of a peptide with a known molecular weight dissolved in ultrapure water at 0.1 to 5 mg/mL as appropriate. Then H
PLC analysis was performed to confirm the retention time (Table 1). A calibration curve (Figure 3) was created from the retention time and molecular weight.

(d) Calculation of molecular weight For the peptides contained in fractions 32 to 34 of fractions 1 to 51, the molecular weight was calculated from the retention time according to the method in the range A in Figure 1. The molecular weight was 90 to 2.
It was in the range of 600 Da.

(3) Sensory evaluation (a) Determination of fractions with off-taste Each fraction (fractions 1 to 51) of the control product obtained in Example 1 (1) was subjected to sensory evaluation by five trained panelists. Specifically, evaluation was performed based on the presence or absence of off-taste (astringent, bitter, astringent).

As a result, among fractions 1 to 51 of the control product obtained in Example 1 (1), fraction 3
It was confirmed that fractions 2 to 34 had an unpleasant taste, and no unpleasant taste was felt in the other fractions (data not shown).

(B) Sensory evaluation of fractions with off-taste by five trained panelists on fractions 32 to 34 (fraction with off-taste) of the test product and control product obtained in Example 1 (1) Served. Specifically, the taste (astringency, bitterness, astringency) was evaluated on a five-point scale from 0 to 4. The average value of the evaluation scores of each panelist was calculated, and if the evaluation value of the test product was lower than that of the control product, it was determined that the unpleasant taste had been reduced.

The results of the sensory evaluation are shown in FIG. From the results shown in FIG. 4, it was confirmed that by using the proteolytic enzyme preparation, the off-taste can be reduced in the fraction having the off-taste.

(4) Amino Acid Analysis Fractions 32 to 34 (fraction with off-taste) of the test product and control product obtained in Example 1 (1) were hydrolyzed using the hydrolysis method of JASCO Corporation. 100 μL of the sample was dispensed into a hydrolysis tube and evaporated to dryness using a heated centrifugal evaporator. The tube was set in a hydrolysis vessel, 294 μL of 6M hydrochloric acid and 6 μL of 10% phenol were dropped into the vessel, the pressure was reduced for 1 to 2 minutes, and then hydrolysis was carried out at 110° C. for 24 hours under reduced pressure. After hydrolysis, residual hydrochloric acid was removed using a vacuum pump. Thereafter, the sample was suspended in sulfosalicylic acid so as to be diluted 4 times, and the filtrate obtained by filtration with a 0.45 μm filter was used as a sample solution for amino acid analysis. Amino acid analysis was performed on the sample using a high-speed amino acid analyzer L-8900 (manufactured by Hitachi High-Tech Science). The content ratio of amino acids constituting the peptide was calculated from the difference between the values with and without acid degradation.

The results of amino acid analysis are shown in Figure 5. From the results in Figure 5, it is clear that in the fraction with reduced off-taste, amino acids (Val, He, Leu, Tyr, Phe,
, Lys, Arg) was confirmed to have decreased.

Example 3: Comparison with commercial products (1) Preparation of preparative gel filtration fractions Gel filtration fractionation was performed on commercial products (commercially available beer and commercially available low-malt beer) in the same manner as in Example 2 (1).

(2) Sensory evaluation The fractions (fractions 1 to 51) of each commercial product obtained in (1) above were used in Example 2 (3).
It was subjected to sensory evaluation in the same manner as in (a). As a result, among fractions 1 to 51 of each commercially available product obtained in (1) above, fractions 32 to 34 were fractions with an unpleasant taste, similar to Example 2 (3) (a), and the other fractions It was confirmed that no unpleasant taste was detected in the test (data not shown).

(3) Amino acid analysis Fractions 32 to 34 (fraction with off-taste) of each commercially available product obtained in (1) above were subjected to amino acid analysis in the same manner as in Example 2 (4).

The results of amino acid analysis are shown in FIG. In addition, the data of Example 2 (4) was used for the data of the test product and the control product. From the results in Figure 6, it can be seen that in the fraction having an off-taste, the proportions of Val, He, Leu, Phe, and Arg among the amino acids involved in the off-taste are different between beer and low-malt beer. It was confirmed that the ratio of amino acids involved in ``low-malt beer'' was higher than that of beer. In addition, in the control product that did not use a proteolytic enzyme preparation, the amino acid ratio involved in the above-mentioned off-flavor was at the same level as that of low-malt beer, but in the test product that used a proteolytic enzyme preparation, the above-mentioned off-taste was present. It was revealed that the ratio of amino acids involved in beer was reduced to the same level as beer.

From the above, it is clear that the astringency and other miscellaneous tastes felt in beer-taste drinks such as low-malt beer are due to the presence of amino acids that are involved in specific off-flavors, and that the off-flavors can be reduced by reducing the amino acids. It became.

Claims (7)

A beer-taste beverage that uses malt and/or ungerminated barley as a raw material, amino acids that contribute to the off-taste relative to all amino acids that constitute peptides with a molecular weight of 90 to 2,600 Da (HPLC gel filtration method) in the beverage. A beer-taste beverage in which the proportion (molar ratio) of At least one of valine, leucine and isoleucine contains the following content:
The beer-taste beverage according to claim 1, which satisfies the following conditions: - valine content: less than 2.1% - leucine content: less than 3.3% - isoleucine content: less than 4.3%. The ratio (molar ratio) of amino acids involved in off-taste to all amino acids constituting peptides with a molecular weight of 90 to 2600 Da (HPLC gel filtration method) in the beverage is 17.2% or less, and the amino acids involved in off-taste are valine. , leucine, isoleucine, phenylalanine and arginine, the beer-taste beverage according to claim 1 or 2. At least one of valine, leucine, isoleucine, phenylalanine and arginine contains the following content:
・Valine content: less than 2.1% ・Leucine content: less than 3.3% ・Isoleucine content: less than 4.3% ・Phenylalanine content: 3.0% or less ・Arginine content: The beer-taste beverage according to claim 3, which satisfies 4.5% or less. The beer-taste beverage according to any one of claims 1 to 4, which has reduced unpleasant taste. The beer-taste beverage according to any one of claims 1 to 5, which is a fermented alcoholic beverage. The beer-taste beverage according to claim 6, wherein the malt usage ratio is less than two-thirds.

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