JP7081905B2 - Beer taste fermented alcoholic beverage and its manufacturing method - Google Patents

Description

The present invention relates to a beer-taste fermented alcoholic beverage using malt and / or ungerminated wheat as a raw material and a method for producing the same.

As a technique for imparting richness and thickness to a beer-taste fermented alcoholic beverage, a technique using a peptide containing carboxymethyl lysine as a richness-imparting substance (Patent Document 1) is known. However, there was still room for improvement in the realization of the flavor of beer-taste fermented alcoholic beverages. Further, as far as the inventors know, no example has been reported in which an attempt is made to improve the flavor of beer from the viewpoints of imparting a soft and smooth texture, achieving a long-lasting taste, and imparting a lingering finish.

Japanese Unexamined Patent Publication No. 2014-158502

Beer has a soft and smooth texture compared to low-malt beer and new genres, and has favorable taste characteristics such as a strong body feeling. Based on this difference, the present inventors considered that if technological development for further enhancing the characteristics of beer is realized, it will be possible to produce beer with unprecedented flavor characteristics.

An object of the present invention is to provide a beer-taste fermented alcoholic beverage having a new flavor, which has a soft and smooth texture like beer and has a long-lasting taste, and a method for producing the same. It is also an object of the present invention to provide a flavor improving agent and a flavor improving method for a beer-taste fermented alcoholic beverage.

The present inventors have found that in a beer-taste fermented alcoholic beverage, a peptide having a specific molecular weight and an α-glucan having a specific degree of polymerization contribute to imparting a soft and smooth texture like beer and sustaining the taste. The present inventors have also found that by keeping the ratio of peptides having a specific molecular weight and the content of α-glucan having a specific degree of polymerization within a specific range, a softer and smoother texture and taste persistence more like beer can be realized. rice field. The present inventors have further specifically identified peptides that contribute to improving the flavor of beer-taste fermented alcoholic beverages. The present invention is based on these findings.

According to the present invention, the following inventions are provided.
[1] A beer-taste fermented alcoholic beverage using malt and / or ungerminated wheat as a raw material, and the ratio of the amount of peptide having a molecular weight of 10 to 20 kDa (HPLC gel filtration method) to the total amount of protein (hereinafter). , Simply referred to as "peptide ratio") is greater than 4.8%, beer-taste fermented alcoholic beverage.
[2] The beer-taste fermented alcoholic beverage according to the above [1], wherein the concentration of α-glucan having a degree of polymerization of 5 to 10 is 7.1 mg / mL or more.
[3] The beer-taste fermented alcoholic beverage according to the above [1] or [2], wherein the malt usage ratio is 50% or more.
[4] A method for producing a beer-taste fermented alcoholic beverage, which comprises a step of blending one or more peptides having a molecular weight of 10 to 20 kDa (HPLC gel filtration method).
[5] The method for producing a beer-taste fermented alcoholic beverage according to the above [4], wherein the peptide is derived from a beer-taste fermented alcoholic beverage using malt and / or ungerminated wheat as a raw material.
[6] The method for producing a beer-taste fermented alcoholic beverage according to the above [4] or [5], further comprising a step of blending α-glucan having a degree of polymerization of 5 to 10.
[7] Any of the above [4] to [6], wherein the peptide having a molecular weight of 10 to 20 kDa (HPLC gel filtration method) is blended so as to have a ratio of more than 4.8% with respect to the total amount of protein in the beverage. A method for producing a beer-taste fermented alcoholic beverage according to.
[8] The method for producing a beer-taste fermented alcoholic beverage according to the above [6] or [7], wherein α-glucan having a degree of polymerization of 5 to 10 is blended at a concentration of 7.1 mg / mL or more.
[9] The method for producing a beer-taste fermented alcoholic beverage according to any one of [4] to [8] above, further comprising a step of preparing the peptide from a raw material containing the peptide.
[10] The method for producing a beer-taste fermented alcoholic beverage according to the above [9], wherein the peptide is prepared by an ultrafiltration method and an ammonium sulfate precipitation method.
[11] The method for producing a beer-taste fermented alcoholic beverage according to the above [9] or [10], wherein the raw material containing the peptide is malt and / or ungerminated wheat or a processed product thereof.
[12] The beer according to any one of [9] to [11] above, wherein the raw material containing the peptide is a beer-taste fermented alcoholic beverage containing at least a part of malt and / or ungerminated wheat as a raw material. Taste Fermented alcoholic beverage manufacturing method.
[13] Improvement of flavor of beer-taste fermented alcoholic beverages containing one or more peptides having a molecular weight of 10 to 20 kDa (HPLC gel filtration method) and / or α-glucan having a degree of polymerization of 5 to 10 as an active ingredient. Agent.
[14] The beer according to the above [13], wherein the peptide and / or α-glucan is derived from a beer-taste fermented alcoholic beverage using malt and / or ungerminated wheat as a raw material. Taste Fermented alcoholic beverage flavor improver.
[15] Flavor of a beer-taste fermented alcoholic beverage comprising the step of adding one or more peptides having a molecular weight of 10 to 20 kDa (HPLC gel filtration method) and / or α-glucan having a degree of polymerization of 5 to 10. How to improve.
[16] The beer according to [15] above, wherein the peptide and / or α-glucan is derived from a beer-taste fermented alcoholic beverage made from malt and / or ungerminated wheat as a raw material. How to improve the flavor of taste fermented alcoholic beverages.

According to the present invention, by blending a peptide having a molecular weight of 10 to 20 kDa or setting the ratio of the peptide within a predetermined value range, there is a soft and smooth texture like beer, and a beer taste in which the lasting taste is recognized. Fermented alcoholic beverages can be provided. In particular, beer with a high malt content has a soft and smooth texture that is typical of beer, and the sustainability of the taste is enhanced, making it possible to provide beer with new flavor characteristics, and it is possible to meet the diverse taste needs of consumers. It is advantageous.

It is a figure which showed the amount of the peptide added to the beer-based malt alcoholic beverage in Reference Example 1. FIG. It is a figure which showed the amount of α-glucan added to the beer-based malt alcoholic beverage in Reference Example 1. FIG. It is a figure which showed the sensory evaluation result of Reference Example 1. FIG. The sensory evaluation score was shown for each added fraction. The sensory evaluation score of the additive-free control was set to 2.5. It is a figure which showed the calibration curve prepared from the holding time of the HPLC gel filtration method carried out in Example 1 and the molecular weight of a known substance. It is a bubble graph which showed the sensory evaluation result (taste smoothness) of Examples 1 and 3. Each sample was plotted with the vertical axis representing the α-glucan concentration (mg / mL) having a degree of polymerization of 5 to 10 and the horizontal axis representing the 10 to 20 kDa peptide ratio (%). The horizontal stripe pattern is sample numbers 1 to 3 (commercially available), the rhombus pattern is sample numbers 4 to 8 (trial product), and the vertical stripe pattern is sample number 1 (commercially available) with peptides and glucan added. Numbers 9 to 13 (additives). The bubble size represents the score of "taste smoothness". It is a bubble graph which showed the sensory evaluation result (taste persistence) of Examples 1 and 3. Each sample was plotted with the vertical axis representing the α-glucan concentration (mg / mL) having a degree of polymerization of 5 to 10 and the horizontal axis representing the 10 to 20 kDa peptide ratio (%). The horizontal stripe pattern is sample numbers 1 to 3 (commercially available), the rhombus pattern is sample numbers 4 to 8 (trial product), and the vertical stripe pattern is sample number 1 (commercially available) with peptides and glucan added. Numbers 9 to 13 (additives). The bubble size represents the score of "taste persistence". It is a figure which evaluated the correlation between the α-glucan concentration of the degree of polymerization 5-10 and the peptide concentration of 10-20 kDa and a sensory evaluation result. FIG. 7A is a diagram plotting the sensory evaluation results with respect to the α-glucan concentration having a degree of polymerization of 5 to 10. FIG. 7B is a diagram plotting the sensory evaluation results for peptide concentrations of 10 to 20 kDa. FIG. 7C is a diagram plotting the sensory evaluation results against the value obtained by multiplying the α-glucan concentration having a degree of polymerization of 5 to 10 by the peptide concentration of 10 to 20 kDa. It is a figure which showed the result of 2D-PAGE electrophoresis about the test group 2 of the beverage produced in the reference example 1. FIG. The left side of the figure is a molecular weight marker (kDa). The bands indicated by the arrows correspond to the protein identification results in Table 14.

Specific description of the invention

In the present invention, the "beer taste" means the taste and aroma peculiar to beer obtained when beer is normally produced, that is, when beer is produced based on fermentation with yeast or the like.

In the present invention, the "beer-taste fermented alcoholic beverage" means a beverage fermented with yeast using carbon sources, nitrogen sources, water and the like as raw materials, and alcohol is used for beer, sparkling liquor and beer and sparkling liquor using malt as a raw material. (For example, a liqueur-based new genre beverage classified as "liqueur (foaming) (1)" under the Liquor Tax Law) can be mentioned.

The beer-taste fermented alcoholic beverage of the present invention can use at least malt as a raw material derived from wheat, in which case the malt use ratio can be, for example, 50% or more, preferably 60% or more. Is. Here, the "malt use ratio" means the ratio of the malt mass to the mass of all raw materials excluding brewing water.

In the beer-taste fermented alcoholic beverage of the present invention, the ratio of the molecular weight of 10 to 20 kDa to the total protein amount (peptide ratio) (preferably the peptide ratio of molecular weight of 10 to 20 kDa derived from the raw material of the beer-taste fermented alcoholic beverage). It is characterized by having a specific value or more. Here, in calculating the peptide ratio, the protein concentration (mg / mL) in the beverage is taken as the total protein amount, and the peptide concentration (mg / mL) having a molecular weight of 10 to 20 kDa in the beverage is taken as the peptide amount having a molecular weight of 10 to 20 kDa. can do. Further, in the present specification, the peptide amount which is the basis for calculating the "peptide ratio" is calculated by totaling the contents of one or more kinds of peptides having a molecular weight of 10 to 20 kDa. Further, in the present specification, the "molecular weight" of the peptide is measured by the HPLC gel filtration method, and specific examples of the measurement are as shown in Example 1 below. The quantification of peptides and proteins can be carried out by the Lowry method.

The beer-taste fermented alcoholic beverage of the present invention is characterized by having a new flavor, that is, the smoothness of the taste and the persistence of the taste are enhanced. Here, "smoothness of taste" means the degree of softness in the mouth when the taste is felt by the tongue. In addition, "taste persistence" means the degree of aftertaste of taste that is sustained after drinking.

In the beer-taste fermented alcoholic beverage of the present invention, the ratio of the peptide amount having a molecular weight of 10 to 20 kDa to the total protein amount can be set to a ratio larger than 4.8%, preferably 4.9% or more, more preferably 5. It is 1% or more, more preferably 5.2% or more, particularly preferably 5.4% or more, and most preferably 5.7% or more. The ratio can be set to an upper limit from the viewpoint of taste harmony, for example, 8.0% can be set as the upper limit.

The beer-taste fermented alcoholic beverage of the present invention is also characterized in that the concentration of α-glucan having a degree of polymerization of 5 to 10 is at least a specific value. In the present specification, the "α-glucan concentration" is calculated by summing up the contents of one or more α-glucans having a degree of polymerization of 5 to 10. Further, in the present specification, "α-glucan" is a linear or branched glucan (for example, α-1,4-glucoside) composed of a plurality of glucose molecules bonded by α-1,4-glucosidic bonds. It means a branched glucan composed of a bond and an α-1,6 glucosidic bond). Further, in the present specification, the "degree of polymerization" of α-glucan means the number of glucose residues constituting the glucan, and not only the number of glucose residues constituting the linear glucan but also the glucose constituting the branched structure. Includes the number of residues. The degree of polymerization and content of α-glucan can be measured by, for example, HPLC analysis using a corona CAD detector, and specific examples of the measurement are as shown in Example 1 below. In addition, the degree of polymerization may be simply expressed as "G" in this specification and drawings.

In the beer-taste fermented alcoholic beverage of the present invention, the α-glucan concentration having a degree of polymerization of 5 to 10 can be set to 7.1 mg / mL or more, preferably higher than 7.1 mg / mL, more preferably. It is 11.1 mg / mL or more. The α-glucan concentration can be set to an upper limit from the viewpoint of taste harmony, for example, 16.0 mg / mL can be set as the upper limit.

From the viewpoint of further enhancing the smoothness and sustainability of the taste in the beer-taste fermented alcoholic beverage of the present invention, the α-glucan concentration having a degree of polymerization of 5 to 10 is set as a predetermined value in addition to the peptide ratio having a molecular weight of 10 to 20 kDa. Is preferable. In the beer-taste fermented alcoholic beverage of the present invention (particularly, those having a malt use ratio of 50% or more), the peptide having a molecular weight of 10 to 20 kDa is set to a predetermined ratio, and the α-glucan concentration having a polymerization degree of 5 to 10 is set to a predetermined concentration. By adjusting, the smoothness of the taste and the sustainability of the taste are further enhanced. That is, in the beer-taste fermented alcoholic beverage of the present invention (particularly, the malt use ratio is 50% or more), the ratio of the peptide amount having a molecular weight of 10 to 20 kDa (HPLC gel filtration method) to the total protein amount is 4.8%. Along with a large ratio (preferably 4.9% or more, more preferably 5.1% or more, still more preferably 5.2% or more, particularly preferably 5.4% or more, most preferably 5.7% or more). , The concentration of α-glucan having a degree of polymerization of 5 to 10 can be set to a concentration of 7.1 mg / mL or more (preferably a concentration larger than 7.1 mg / mL, more preferably a concentration of 11.1 mg / mL or more). .. Both the peptide ratio and the α-glucan concentration can be set to the upper limit as described above.

The beer-taste fermented alcoholic beverage of the present invention can be used to produce a normal beer-taste fermented alcoholic beverage as long as the peptide ratio having a molecular weight of 10 to 20 kDa and / or the α-glucan concentration having a degree of polymerization of 5 to 10 is adjusted within a predetermined range. It can be manufactured according to the procedure. For example, a fermented malt beverage can be produced by adding fermenting beer yeast to malt prepared from brewing raw materials such as malt, hops, auxiliary raw materials, and brewing water to ferment and brew a fermented liquid. .. The obtained fermented alcoholic beverage with a beer taste can be stored at a low temperature, and then yeast can be removed by a filtration step. It goes without saying that among beer-taste fermented alcoholic beverages, all-malt beer can be produced from malt, hops, and water.

In the above production procedure, wort can be produced according to a conventional method. For example, a mixture of brewing raw materials and brewing water is saccharified and filtered to obtain wort, hops are added to the wort, and then the wort is boiled and the boiled wort is cooled to prepare the wort. Can be done. Wort can also be prepared by adding a commercially available enzyme preparation during the saccharification step. For example, a protease preparation for proteolysis, an α-amylase preparation, β-amylase preparation, glucoamylase preparation, plulanase preparation, etc. for glycolysis, β-glucanase preparation, fibrinolysis for fibrinolysis, etc. Enzyme preparations (for example, hemicellulase preparations) and the like can be used respectively, or mixed preparations thereof can also be used.

In the production of the beer-taste fermented alcoholic beverage of the present invention, in addition to malt, ungerminated wheat (for example, ungerminated barley (including extracted), ungerminated wheat (including extracted)); rice. Auxiliary raw materials specified by the Liquor Tax Law such as corn, corn, malt, starch, sugar (for example, liquid sugar); nitrogen sources such as proteolytic products and yeast extract; fragrances, pigments, foaming / foam retention improvers, water quality Other additives such as modifiers and fermentation aids can be used as brewing ingredients. In the beer-taste fermented alcoholic beverage of the present invention, the raw materials used other than the water for brewing can be at least malt and hops, and in some cases, sugars, rice, corn, starch and the like can be further used as raw materials.

In order to adjust the peptide ratio of the molecular weight of 10 to 20 kDa in the produced beverage in the method for producing a beer-taste fermented alcoholic beverage of the present invention within a predetermined value, for example, malt and / or ungerminated wheat as a raw material can be used. It can be adjusted by suppressing the protein decomposition in the preparation / saccharification process and the degree of protein decomposition in the malting process of the raw material malt. Examples of the proteolytic decomposition include proteases inherent in malt and ungerminated wheat, and protease preparations added from the outside. Degradation can be suppressed by reducing the amount of the protease preparation added, reducing the treatment time at the action temperature of protein decomposition, changing the action pH from the optimum conditions, and the like.

In the method for producing a beer-taste fermented alcoholic beverage of the present invention, in order to adjust the α-glucan concentration having a degree of polymerization of 5 to 10 in the produced beverage within a predetermined range, for example, the raw material malt and / or ungerminated Use liquid sugar that suppresses α-glucan decomposition in the wheat preparation and saccharification process, suppresses α-glucan decomposition in the wheat sprouting process of raw material, or suppresses the degree of α-glucan decomposition. It can be adjusted by such things. Examples of the α-glucan decomposition include α-amylase and β-amylase inherent in malt and ungerminated wheat, or α-amylase preparations, glucoamylase preparations, pullulanase preparations and the like added from the outside. The decomposition can be suppressed by reducing the addition amount of the α-amylase preparation, the glucoamylase preparation, the pullulanase preparation, etc., reducing the treatment time at the action temperature of α-glucan decomposition, changing the action pH from the optimum conditions, and the like. ..

In the present invention, a fraction containing the peptide and a fraction containing the α-glucan are prepared from a raw material containing a peptide having a molecular weight of 10 to 20 kDa and a raw material containing α-glucan having a degree of polymerization of 5 to 10. By adding the fraction to a beer-taste fermented alcoholic beverage, the beer-taste fermented alcohol in which the peptide ratio having a molecular weight of 10 to 20 kDa and the α-glucan concentration having a degree of polymerization of 5 to 10 are adjusted within the predetermined values, respectively. Beverages can be produced. Typically, a beer-taste fermented alcoholic beverage is produced, from which a fraction containing a peptide having a molecular weight of 10 to 20 kDa and an α-glucan having a degree of polymerization of 5 to 10 are contained according to the procedure described in Example 1. By preparing a fraction and adding the fraction to a beer-taste fermented alcoholic beverage, the peptide ratio having a molecular weight of 10 to 20 kDa and the α-glucan concentration having a degree of polymerization of 5 to 10 are adjusted within a predetermined range. It is possible to produce fermented alcoholic beverages with a beer taste.

That is, according to the present invention, it is derived from a beer-taste fermented alcoholic beverage using at least one or more peptides having a molecular weight of 10 to 20 kDa (preferably malt and / or ungerminated wheat as a raw material. A beer-taste fermented alcoholic beverage containing one or more peptides having a molecular weight of 10 to 20 kDa is provided, and the beverage is a part of the beer-taste fermented alcoholic beverage of the present invention. The beverage containing the peptide has an improved or improved flavor as a beer-taste fermented alcoholic beverage, and specifically, a beverage having enhanced smoothness and sustainability of taste.

The amount of the peptide to be blended in the beverage of the present invention can be blended so that the ratio of the peptide amount having a molecular weight of 10 to 20 kDa to the total protein amount is larger than 4.8%, preferably 4.9% or more. It is preferably 5.1 or more, more preferably 5.2% or more, particularly preferably 5.4% or more, and most preferably 5.7% or more. The ratio can be set to an upper limit from the viewpoint of taste harmony, for example, 8.0% can be set as the upper limit.

One or two peptides with a molecular weight of 10 to 20 kDa (particularly, one or 2 with a molecular weight of 10 to 20 kDa derived from a beer-taste fermented alcoholic beverage using at least a part of malt and / or ungerminated barley as a raw material. Examples of peptides) are α-amylase / trypsin inhibitor, trypsin inhibitor, α-amylase inhibitor (BDAI-1), non-specific lipid-transfer protein 1 and avenin. -Like a1 is mentioned, preferably these proteins and peptides are derived from barley, some of which are derived from wheat. When α-amylase / trypsin inhibitor, trypsin inhibitor, α-amylase inhibitor (BDAI-1), non-specific lipid transfer protein 1 or avenin-like a1 is added to a beer-taste fermented alcoholic beverage, these peptides or proteins. May be a peptide or protein other than those prepared from beer-taste fermented alcoholic beverages made from malt and / or ungerminated wheat as at least a portion of the raw material.

In the present invention, α-glucan having a degree of polymerization of 5 to 10 can be blended in addition to one or more peptides having a molecular weight of 10 to 20 kDa. The blending amount of the glucan can be blended so that the α-glucan concentration having a degree of polymerization of 5 to 10 in the beverage is 7.1 mg / mL or more, preferably larger than 7.1 mg / mL. Preferably, it can be blended so as to be 11.1 mg / mL or more. The α-glucan concentration can be set to an upper limit from the viewpoint of taste harmony, for example, 16.0 mg / mL can be set as the upper limit.

The glucan that can be blended in the beer-taste fermented alcoholic beverage of the present invention is α-with a degree of polymerization of 5 to 10 derived from a beer-taste fermented alcoholic beverage using malt and / or ungerminated wheat as a raw material. Glucans can be used, but are not limited to, and may be glucans prepared from raw materials other than beer-taste fermented alcoholic beverages using malt and / or ungerminated wheat as at least a part of the raw materials.

In the beer-taste fermented alcoholic beverage of the present invention, it is preferable to add α-glucan having a degree of polymerization of 5 to 10 in addition to the peptide having a molecular weight of 10 to 20 kDa from the viewpoint of further enhancing the smoothness of the taste and the sustainability of the taste. In the beer-taste fermented alcoholic beverage of the present invention (particularly, those having a malt use ratio of 50% or more), the peptide having a molecular weight of 10 to 20 kDa is set to a predetermined ratio, and the α-glucan concentration having a polymerization degree of 5 to 10 is set to a predetermined concentration. By adjusting, the smoothness of the taste and the sustainability of the taste are further enhanced. That is, in the beer-taste fermented alcoholic beverage of the present invention (particularly, the ratio of malt used is 50% or more), the ratio of the peptide amount having a molecular weight of 10 to 20 kDa (HPLC gel filtration method) to the total protein amount is 4.8%. It is said to be large (preferably 4.9% or more, more preferably 5.1% or more, still more preferably 5.2% or more, particularly preferably 5.4% or more, most preferably 5.7% or more). The peptide is blended and the glucan has a concentration of α-glucan having a degree of polymerization of 5 to 10 of 7.1 mg / mL or more (preferably larger than 7.1 mg / mL, more preferably 11.1 mg / mL or more). Can be blended. Both the peptide ratio and the α-glucan concentration can be set to the upper limit as described above.

One or more peptides having a molecular weight of 10 to 20 kDa and α-glucan having a degree of polymerization of 5 to 10 to be blended in a beer-taste fermented alcoholic beverage are prepared from a raw material containing the peptide or a raw material containing the α-glucan. can do. Raw materials containing one or more peptides having a molecular weight of 10 to 20 kDa include malt and / or ungerminated wheat, processed products thereof, and cereal protein degradation products (for example, soybean protein degradation products and corn protein degradation products). Can be mentioned. Processed products of malt and / or ungerminated wheat include malt and / or malt made from ungerminated wheat as a raw material, ungerminated wheat extract, malt and / or ungerminated wheat. Examples thereof include beer-taste fermented alcoholic beverages containing at least a part of the raw material, preferably a beer-taste fermented alcoholic beverage containing at least a part of malt and / or ungerminated wheat, more preferably malt. A beer-taste fermented alcoholic beverage that is part or all of the derived ingredients. Examples of means for preparing one or more kinds of peptides having a molecular weight of 10 to 20 kDa from a raw material include a combination of an ultrafiltration method and a sulfur precipitation method, and a combination of a gel filtration fraction and a solid-phase extraction column. From the viewpoint of specific production, a combination of the ultrafiltration method and the sulfurized cheap precipitation method is preferable. When preparing one or more peptides having a molecular weight of 10 to 20 kDa by a combination of the ultrafiltration method and the ammonium sulfate precipitation method, the ultrafiltration method can be carried out, and then the ammonium sulfate precipitation method can be carried out. .. Examples of the raw material containing α-glucan having a degree of polymerization of 5 to 10 include malt and / or ungerminated wheat and processed products thereof, rice, corn, potatoes, and sugars. Processed products of malt and / or ungerminated wheat include those mentioned above. Examples of means for preparing α-glucan having a degree of polymerization of 5 to 10 from raw materials include a combination of a gel filtration fraction and a solid-phase extraction column, and separation means such as ultrafiltration and chromatographic separation. It may be carried out in combination with an impurity removing means using an ion exchange resin or activated carbon.

One or more peptides having a molecular weight of 10 to 20 kDa and α-glucan having a degree of polymerization of 5 to 10 contained in a beer-taste fermented alcoholic beverage are preferably at least one of malt and / or ungerminated wheat as a raw material. It can be prepared from a beer-taste fermented alcoholic beverage (more preferably, a beer-taste fermented alcoholic beverage that uses at least malt as a raw material derived from wheat, particularly preferably all-malt beer). For example, a beer-taste fermented alcoholic beverage is produced, and a fraction containing a peptide having a molecular weight of 10 to 20 kDa and an α-glucan having a polymerization degree of 5 to 10 are contained from the beverage using a gel filtration fraction or a solid phase extraction column. Fractions can be prepared. Peptides with a molecular weight of 10 to 20 kDa and α-glucans having a degree of polymerization of 5 to 10 do not necessarily have to be isolated and purified, and peptide fractions and α-glucans obtained by fractionation treatment from beer-taste fermented alcoholic beverages. The fraction can be blended into a beer-taste fermented alcoholic beverage.

The blending of a peptide having a molecular weight of 10 to 20 kDa and an α-glucan having a degree of polymerization of 5 to 10 into a beer-taste fermented alcoholic beverage can be added to the pre-fermentation liquid before fermentation, the fermented liquid during fermentation, or the fermented liquid after fermentation. It can be carried out. For example, a beer-taste fermented alcoholic beverage is produced, a fraction containing a peptide having a molecular weight of 10 to 20 kDa and a fraction containing α-glucan having a degree of polymerization of 5 to 10 are prepared from the beverage, and the fraction is fermented. By adding to the beer-taste fermented alcoholic beverage, it is possible to produce a beer-taste fermented alcoholic beverage containing a peptide having a molecular weight of 10 to 20 kDa and α-glucan having a polymerization degree of 5 to 10.

The beer-taste fermented alcoholic beverage of the present invention is blended so that peptides having a molecular weight of 10 to 20 kDa are blended in a predetermined ratio, and in some cases, α-glucan concentration having a polymerization degree of 5 to 10 is further blended so as to be a predetermined value. Can be produced according to the procedure for producing a normal beer-taste fermented alcoholic beverage. For example, fermented malt beverages can be brewed by adding fermenting beer yeast to malt prepared from brewing raw materials such as malt, hops, auxiliary raw materials, and brewing water to ferment. The obtained fermented alcoholic beverage with a beer taste can be stored at a low temperature, and then yeast can be removed by a filtration step.

In the above production procedure, wort can be produced according to a conventional method. For example, a mixture of brewing raw materials and brewing water is saccharified and filtered to obtain wort, hops are added to the wort, and then the wort is boiled and the boiled wort is cooled to prepare the wort. Can be done.

In the production of the beer-taste fermented alcoholic beverage of the present invention, a raw material other than malt can be used, and specifically, a raw material other than malt can be used according to the description of the beer-taste fermented alcoholic beverage of the present invention.

According to another aspect of the present invention, beer comprising one or more peptides having a molecular weight of 10 to 20 kDa (HPLC gel filtration method) and / or α-glucan having a degree of polymerization of 5 to 10 as an active ingredient. A flavor improving agent for a taste-fermented alcoholic beverage is provided. Yet another aspect of the invention comprises the step of adding one or more peptides having a molecular weight of 10-20 kDa (HPLC gel filtration method) and / or α-glucan having a degree of polymerization of 5-10. , A method for improving the flavor of a fermented alcoholic beverage having a beer taste is provided. As used herein, "improving the flavor of a fermented alcoholic beverage with a beer taste" means that the smoothness of the taste and the sustainability of the taste are enhanced. In addition, one or more peptides having a molecular weight of 10 to 20 kDa (HPLC gel filtration method) and α-glucan having a degree of polymerization of 5 to 10 are preferably at least a part of malt and / or ungerminated wheat as a raw material. It is derived from the beer-taste fermented alcoholic beverage. The flavor improving agent and the flavor improving method of the present invention can be carried out according to the description of the beer-taste fermented alcoholic beverage of the present invention and the method for producing the beverage.

The present invention will be described in more detail based on the following examples, but the present invention is not limited to these examples. In the following examples, the ratio (%) represents mass% unless otherwise specified.

Reference Example 1: Specifying the peptide fraction and α-glucan fraction that impart a favorable flavor to beer-taste beverages (1) Production of beer-taste fermented alcoholic beverages Using barley malt, hops, and enzyme preparations, the infusion method is used. A beer-taste fermented alcoholic beverage was produced.

In Test Group 1, 100 g of barley malt was placed in 300 mL of hot water at 50 ° C., an enzyme preparation was added and held for 60 minutes, the temperature was raised to 65 ° C. and held for 60 minutes, and the temperature was further raised to 78 ° C. for 5 minutes. After holding, it was filtered to obtain wort. In the test group 2, wort was obtained in the same manner except that the 50 ° C. step was not performed and the enzyme preparation was not added. Subsequently, 0.8 g / L of hops was added and boiled at 100 ° C. for 90 minutes, and then filtered to obtain a pre-fermentation solution.

Then, fermentation was carried out with brewer's yeast according to a conventional method, and the flavor of the fermented liquid was confirmed. The panel of 8 people has a minimum of 1 point and a maximum of 5 points, with the index of "softness of taste", that is, the soft and smooth texture like beer, and the harmonious taste. The sensory evaluation was performed in five stages, and the average score was calculated. The results were as shown in Table 1.

As shown in Table 1, the sensory evaluation score (softness of taste) was better in Test Group 2 than in Test Group 1, and the impression of flavor was also favorable.

(2) Gel filtration fraction The fermented liquid obtained in (1) above was filtered with a 0.45 μm filter, the filtered fermented liquid was weighed, and freeze-dried. The dried product was dissolved in 100 mM NaCl solution to prepare a 5-fold concentrated solution, which was used as a sample for fractionation. The sample was subjected to gel filtration fraction under the following conditions.

Column: Hiload Superdex 30pg 26/600 (manufactured by GE Healthcare)
Sample injection volume: 5 mL
Eluent composition: 100 mM NaCl
Flow velocity: 2.5 mL / min (constant flow velocity)
Detection wavelength: 215 nm
Fraction: 19.1 mL (fraction 0) from 0.29 cv (column volume), then 5 mL increments from 0.35 cv (fractions 1-51)

According to the sensory evaluation of the fractions, the fractions were divided into 9 groups of pre-fractions, A1, A2, B, C, D, E, F and G as shown in Table 2 according to the difference in flavor characteristics.

(3) Purification of peptide fraction and α-glucan fraction Each fraction obtained in (2) above is a solid-phase extraction column (fractions A1, A2 and B are Bond Elute C18 EWP, fraction C, D, E, F, and G are adsorbed with Bond Elute C18 (manufactured by Agilent technologies), washed with desalted water, eluted with 50% aqueous ethanol solution, and concentrated to dryness. Was rehydrated with demineralized water to prepare a concentrated solution. This was used as a peptide fraction. The passing fraction of the solid-phase extraction column is further desalted with an anion / cation / ion exchange resin (Amberlite IR120H and Amberlite XE583, manufactured by Organo Corporation), deodorized with a small amount of activated carbon, and concentrated to dryness. After solidification, this was reconstituted with desalted water to prepare a concentrated solution. This was taken as the α-glucan fraction.

(4) Protein quantification by the Lowry method The protein quantification of the peptide fraction obtained by the gel filtration fraction of the above (3) was performed by the Lowry method using a commercially available kit (DC protein assay, manufactured by Bio-Rad). .. First, 50 μL of the above fractionated solution was taken, concentrated under reduced pressure and dried, and 10 μL of ultrapure water was added and redissolved to prepare 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 the color reaction was carried out at room temperature for 15 minutes, 350 μL was transferred to a 96-well plate and the absorbance at 750 nm was measured. The amount of peptide was calculated based on the obtained absorbance and the calibration curve prepared in advance. The calibration curve was prepared using BSA (bovine serum albumin).

(5) Quantification of α-glucan The quantification of α-glucan contained in the α-glucan fraction obtained in (3) above is glucoamylase and a buffer solution (final concentration 10 U / mL, 100 mM Na acetate pH 4.5). ) Was added and reacted at 40 ° C. overnight, and then measured as glucose with a commercially available glucose C-II test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). The chain length distribution of the contained α-glucan was evaluated by an MCI-gel CK02AS column (20 × 250 mm) and a corona CAD detector (corona charged particle detector) without glucoamylase treatment. Specifically, the degree of polymerization (chain length) was analyzed under the following conditions. G1-G7 maltooligosaccharides and amylose DP17 were used as standard mobility products.

Column: MCI-gel CK02AS column (20 x 250 mm, manufactured by Mitsubishi Chemical Corporation)
Mobile phase: MQ water flow rate: 1.0 mL / min Column temperature: 85 ° C
Detector: Corona CAD detector

(6) Sensory evaluation These fractionated / purified samples are added to a commercially available beer-based alcoholic beverage using barley and barley malt with a malt usage ratio of less than 49%, by adding 50% to each fraction contained in the beverage. (See FIGS. 1 and 2), and a panel of 5 people performed a sensory evaluation.

The index of sensory evaluation was evaluated on a five-point scale of 1 to 5 as a comprehensive evaluation of "umami, sweetness, thickness, body, and astringency / taste discord as an off-flavour". The additive-free control was given a score of 2.5. The average value of the sensory evaluation score is as shown in FIG. 3, and the sensory evaluation comment is as shown in Table 3.

The peptide fraction had a high sensory evaluation score (FIG. 3) in Test Group 2 from the pre-fraction to D, which was in agreement with the sensory evaluation result of the fermented liquid before fractionation. In addition, in the fractions A1 and A2 of the test group 2, the score was high, and the sensory evaluation comment was that the softness and the taste were reduced (Table 3). In the pre-fraction of test group 2, the score was equally high, but the sensory evaluation comment was that it was soft but the taste itself was low. The fractions B to D had the same high scores, but the sensory evaluation comments evaluated that the contributions of thickness, body, and umami were stronger. That is, in the pre-fractions, A1, A2, B, C, and D, the evaluation of the test group 2 is high, but the taste quality is different from each other. It turned out that there is an effect of.

It was found that in the α-glucan fraction, when the fractions B and C were added, the sensory evaluation score (Fig. 3) was high, and in the sensory evaluation comment (Table 3), it was expressed as sweetness, mellowness, and smoothness. .. The α-glucan derived from fractions A1 and A2 did not have a clear flavor.

The peptide fraction was analyzed on the Superdex 75 10/300 column described in Example 1, and the molecular weight was estimated. As a result, the peptide of the fraction A1 was distributed at a molecular weight of about 10 to 20 kDa, and SDS-PAGE electricity was performed. By electrophoresis, it was confirmed that peptides having a similar molecular weight of about 10 to 20 kDa were distributed in the fractions A1 to A2 (data not shown). Further, it was confirmed that the amount of the peptide was increased in the test group 2 which was excellent in flavor (FIG. 1).

Further, when the degree of polymerization distribution of the α-glucan fraction was confirmed, it was as shown in Table 4.

As shown in Table 4, it was confirmed that the main components of the fractions B and C, in which a clear flavor was felt, contained a degree of polymerization (DP) in the range of 2 to 10. In addition, it was confirmed that the amount of α-glucan in the fraction was large in Test Group 2, which was excellent in flavor. The α-glucans derived from the fractions A1 and A2 had a longer chain length of the main component (8 to about 40), and no clear flavor was felt.

From the above results, the fractionated and purified peptide fractions A1 and A2 having a molecular weight of about 10 to 20 kDa and the α-glucan fractions B and C having a glucose polymerization degree of 2 to 10 were harmonized with suppressed miscellaneous taste. It has become clear that beer-based beverages can be given a taste that is typical of beer.

Example 1: Production and sensory evaluation of beer-taste fermented alcoholic beverages
A: Production of beer-taste fermented alcoholic beverages In the production of beer-taste fermented alcoholic beverages, barley malt was used as the main raw material. For saccharification, an enzyme preparation was used, the temperature and time of saccharification were adjusted, and filtration was performed to obtain worts having different compositions. That is, the temperature range for saccharification was selected from 62 to 68 ° C, such as 62 ° C, 64 ° C, 65 ° C or 68 ° C. The saccharification time was adjusted between 5 and 40 minutes in each temperature step. The heat treatment temperature of the raw material was selected from 70 ° C. to 100 ° C. Specifically, wort was obtained as follows.

(A) Saccharification conditions of sample No. 4 After adding 40 parts by mass of barley malt and an enzyme preparation to 100 parts by mass of hot water at 65 ° C. and holding for 40 minutes, the temperature was raised to 78 ° C. and held for 5 minutes. It was filtered to obtain wort.

(B) Saccharification conditions of sample No. 5 33.4 parts by mass of barley malt and an enzyme preparation were added to 100 parts by mass of hot water at 65 ° C. and held for 30 minutes, then heated to 78 ° C and held for 5 minutes. After that, it was filtered to obtain wort.

(C) Saccharification conditions of sample numbers 6 to 8 To 100 parts by mass of hot water at 62 ° C, 40 parts by mass of barley malt and an enzyme preparation were added and held for 40 minutes, then heated to 78 ° C and held for 5 minutes. After that, it was filtered to obtain wort. While the temperature was maintained at 62 ° C., a part of the mash was transferred to another kettle, heat-treated, and then returned to the original kettle.

Following the above wort preparation steps (a) to (c), hops were added to each of the obtained worts and boiled at 100 ° C. for 90 minutes, and then the wort was allowed to stand to separate the trube. After that, it was cooled to obtain a pre-fermentation wort. Then, bottom fermentation yeast was added to the pre-fermentation liquid, and main fermentation and post-fermentation were carried out according to a conventional method. Subsequently, the fermented liquid after post-fermentation was stored at a lower temperature and filtered to obtain a clear beer-taste fermented alcoholic beverage (sample numbers 4 to 8).

B: Commercial beer Commercial beer (sample numbers 1 to 3) (commercially available) having a malt usage ratio of 67% or more was subjected to an analytical test.

C: Quantification of α-glucan amount in beer-taste fermented alcoholic beverages (a) Sample preparation for HPLC analysis Sample numbers 1 to 3 (commercially available) and sample numbers 4 to 8 (trial) are solid-phase extraction columns. The sample was separated using and a sample for fractionation was obtained. Specifically, the product liquid is adsorbed on a solid-phase extraction column (Bond Elute C18, manufactured by Agilent technologies) to recover the passing fraction, and the recovered liquid is further collected by Bond Elute C18 EWP (manufactured by Agilent technologies). And the passage fraction was collected. The recovered pass-through fractions were further treated with ion exchange resins Sep Pak QMA and Sep Pak CM (both from Waters) in order. The obtained liquid was centrifugally concentrated, and the dried product was condensed to prepare a sample for fractionation.

(A) Sugar analysis by HPLC The chain length distribution of α-glucan contained in the sample was evaluated using an MCI-gel CK02AS column (20 × 250 mm) and a corona CAD detector. Specifically, the degree of polymerization (chain length) was analyzed under the following conditions. The concentration was analyzed by a calibration curve using G5 to G10 maltooligosaccharide as a standard product. G5 is Maltopentaose, G6 is Maltohexaose, G7 is Maltoheptaose, G8 is Maltooctaose, G9 is Maltononaose, and G10 is Maltononaose. (Maltodecaose). Maltopentaos
e) For Maltohexaose and Maltoheptaose, purchase standard products from Tokyo Kaseisha, and for Maltooctaose, Maltononaose and Maltodecaose, purchase standard products. A standard product was purchased from Elicityl SA, and the conditions for the mass spectrometer were set.

<HPLC analysis conditions>
Column: MCI-gel CK02AS column (20 x 250 mm, manufactured by Mitsubishi Chemical Corporation)
Mobile phase: MQ water flow rate: 1.0 mL / min Column temperature: 85 ° C
Detector: Corona CAD detector

The results of the analysis of sample numbers 1 to 8 under the analysis conditions set in (a) above are as shown in Table 5.

D: Protein quantification (a) Sample preparation for gel filtration fractionation Sample numbers 1 to 3 (commercially available) and sample numbers 4 to 8 (test brew) were weighed and freeze-dried. The dried product was dissolved in 50 mM sodium phosphate buffer (containing pH 7.0 150 mM NaCl) to prepare a 2.5-fold concentrated solution, which was used as a sample for fractionation.

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

(C) Setting the fraction range In the column, eluent, flow velocity, and detection wavelength described in the HPLC gel filtration method conditions of (a) above, a peptide having a known molecular weight is appropriately dissolved in ultrapure water at 0.1 to 5 mg / mL. 50 μL of the above was injected and HPLC analysis was performed to confirm the retention time (Table 7). A calibration curve (FIG. 4) was prepared from the holding time and molecular weight, and the molecular weight range and the fractionation range were determined.

(D) Protein quantification of fractionated liquid by Lowry method Protein quantification was performed by the Lowry method described in Reference Example 1. A calibration curve was created from the obtained absorbance and BSA concentration, the peptide amount of the fraction (BSA equivalent) was calculated, and the product-equivalent peptide concentration (mg / mg /) of the fraction was calculated from the fraction amount and concentration ratio. mL) was calculated.

(E) Quantification of 10 to 20 kDa peptide The 10 to 20 kDa peptide concentration is the protein concentration contained in fraction 3 in the range of the molecular weight of 10 to 20 kDa determined from the calibration curve in the above HPLC gel filtration method, and is the peptide concentration equivalent to the product. It was calculated by converting it to (mg / mL). The ratio of the amount of 10 to 20 kDa peptide to the total amount of protein, that is, the peptide ratio of 10 to 20 kDa was calculated by the following formula.

E: Sensory evaluation Sample numbers 1 to 3 (commercially available) and sample numbers 4 to 8 (test brew) were subjected to sensory evaluation by 7 trained panelists. The index of the evaluation item was "smoothness of taste" and "sustainability of taste", and each was evaluated with a 9-point score of 1 (weak) to 9 (strong). The analysis results and sensory evaluation results of each sample are as shown in Table 8. In addition, it is shown in FIGS. 5 and 6 together with the results of Example 3 described later.

As shown in Table 8 and FIGS. 5 and 6, test brewing in which the peptide ratio of 10 to 20 kDa was higher than 4.8% and the amount of α-glucan having a degree of polymerization of 5 to 10 was higher than 7.1 mg / mL. In the product, "smoothness of taste" and "sustainability of taste" were improved.

The above result is the result when analyzed by the method described in Example 1, that is, the analysis value obtained by using the corona CAD detector (corona charged particle detector). The detector is independent of the chemical structure of the substance and is characterized by consistent substance weight-dependent responsiveness under the same analytical conditions (according to Thermofisher scientific).

Example 2: Evaluation of relationship between α-glucan and peptide and sensory evaluation For beer-taste effervescent alcoholic beverages such as commercially available beer, low-malt beer, and new genre beverages, α-glucan concentration with a degree of polymerization of 5 to 10 and 10 to 20 kDa The correlation coefficient (R2) between the peptide concentration and the sensory evaluation was evaluated. The method for analyzing the peptide concentration of 10 to 20 kDa was carried out according to the procedure described in Example 1. The value including both of the two components was calculated by multiplying the α-glucan concentration (mg / mL) having a degree of polymerization of 5 to 10 and the peptide concentration (mg / mL) having a degree of polymerization of 10 to 20 kDa. In the sensory evaluation, 10 trained panelists evaluate "taste harmony", that is, 10 that does not feel 0 at all in the range of 11 steps from 0 to 10 with the taste harmony related to beer-likeness as an index. It was evaluated as having never felt any more in the recognition of the person.

The α-glucan concentration having a degree of polymerization of 5 to 10 was analyzed as follows.
(A) Preparation of samples for saccharide analysis Weigh beer products and product samples manufactured in a test brewing facility, and quantify a product diluted 50-fold with a 30% (v / v) acetonitrile / 70% (v / v) aqueous solution. It was used as a sample.

(B) Quantification of sugars by LC / MS / MS HPLC conditions were as follows.
Equipment used: 1200 Series (manufactured by Agilent technologies)
Column: ACQUITY UPLC BEH Amide 2.1 mm x 50 mm (particle size 1.7 μm) (manufactured by Waters)
Mobile phase A: 95% (v / v) acetonitrile water + 0.1% (v / v) formic acid + 10 mM ammonium formate Mobile phase B: water + 0.1% (v / v) formic acid + 10 mM ammonium formate Injection amount: 5 μL
Column temperature: 55 ° C
Sample temperature: 20 ° C
Granant conditions / flow velocity: As shown in Table 9.

(C) Setting of mass spectrometer conditions Example 1. C. In the same manner as in (a), the concentration was analyzed by a calibration curve using G5 to G10 maltooligosaccharide as a standard product. The purchased standard product was adjusted to 100 ppm using 50% (v / v) acetonitrile / 50% (v / v) water / 0.1% (v / v) formic acid / 10 mM ammonium formate solution, and 3200Qtrap manufactured by ABSicex. Various parameters were optimized by injecting directly into the water by the introduction method.

Peptide concentration analysis of 10 to 20 kDa is carried out in Example 1. It was carried out according to the description of D.

The α-glucan concentration having a degree of polymerization of 5 to 10 and the peptide concentration of 10 to 20 kDa and the sensory evaluation results were as shown in Table 10. The relationship between the concentration and the sensory evaluation result was as shown in FIG.

As shown in Table 10 and FIG. 7, the correlation coefficient with the sensory evaluation result was obtained for each of the α-glucan concentration having a degree of polymerization of 5 to 10 and the peptide concentration of 10 to 20 kDa (R2 = 0, respectively). It was clarified that the correlation coefficient with the two components (value obtained by multiplying the concentrations of the two components) is higher than that of .29, 0.46) (R2 = 0.58). From this result, it was confirmed that it is effective to use two components as an index, not each of α-glucan having a degree of polymerization of 5 to 10 and a peptide having a degree of polymerization of 10 to 20 kDa alone.

Example 3: Addition of α-glucan and peptide to beer-taste fermented alcoholic beverages and sensory evaluation
A: Preparation of peptide fraction (A-1) Gel filtration fraction Sample No. 3 (commercially available) (all malt beer) was degassed and then freeze-dried. The dried product was dissolved in a 100 mM NaCl solution to prepare a 5-fold concentrated solution, and the sample used as a fractionation sample was subjected to gel filtration fractionation under the following conditions.

<Gel filtration fractionation conditions>
Column: Hiload Superdex 30pg 26/600 (GE Healthcare)
Sample injection volume: 5 mL
Eluent composition: 100 mM NaCl
Flow velocity: 2.5 mL / min (constant flow velocity)
Detection wavelength: 215 nm
Fraction: 19.1 mL (fraction 0) from 0.29 CV (column volume), then 5 mL increments from 0.35 CV (fractions 1-51)

By the sensory evaluation of the fractions, the fractions were divided into 9 groups of pre-fractions, A1, A2, B, C, D, E, F and G as shown in Table 2 above according to the difference in flavor characteristics.

(A-2) Purification of Peptide Fraction Of the fractions obtained in (A-1) above, the A1 fraction (fraction numbers: F1 to 12) was adsorbed on a solid-phase extraction column (Bond Elute C18 EWP). After the treatment and washing with demineralized water, the mixture was extracted with a 50% ethanol aqueous solution, concentrated to dryness, and reconstituted with demineralized water to prepare a concentrated solution. This was used as a peptide fraction.

(A-3) Protein quantification by Lowry method The protein quantification of the peptide fraction obtained by the gel filtration fraction of (A-2) above is performed by the Lowry method using a commercially available kit (DC protein assay, Bio-Rad). gone. First, the concentration of the peptide fraction solution was adjusted to be 10-fold and 40-fold diluted with respect to the product, 25 μL of solution A was added to 5 μL of the sample and stirred, and then 200 μL of solution B was added and stirred. .. After performing a color reaction at room temperature for 15 minutes, the absorbance at 750 nm was measured. The amount of peptide was calculated based on the obtained absorbance and the calibration curve prepared in advance. The calibration curve was prepared using BSA (bovine serum albumin).

B: Preparation of α-glucan fraction (B-1) Preparation of α-glucan fraction Low molecular weight sugar is removed from commercially available malto-oligosaccharide by dialysis so as to approach the sugar composition of sample number 1 (commercially available) (all malt beer). The α-glucan fraction was prepared. Float-A-Lyzer G2 (Spectrum Laboratories) was used as the dialysis membrane, and the oligosaccharides and the pore size of the dialysis membrane were as follows.

1 g of each maltooligosaccharide was dissolved in ion-exchanged water so as to be 10 mL, and dialysis was performed for 30 hours to obtain an α-glucan fraction.

(B-2) Quantification of α-glucan The quantification of α-glucan contained in the α-glucan fraction prepared in (B-1) above is described in Example 1. C. It was carried out by high performance liquid chromatography (HPLC) according to the method described in (a).

(B-3) Sugar composition analysis of α-glucan fraction Example 1. C. The sugar composition of the α-glucan fraction was analyzed by HPLC according to the method described in (a). The results are shown in Table 12 below.

C: Preparation of tasting sample To sample number 1 (commercially available) (all malt beer), the peptide fraction obtained in A above and the α-glucan fraction obtained in B above were added, and sample numbers 9 to 13 (sample numbers 9 to 13 (all malt beer) were added. Additives) were prepared. Specifically, the peptide fraction was added so that the peptide amount of 10 to 20 kDa was 0.25 mg / mL and 0.30 mg / mL. Similarly, the α-glucan fraction was added so that the amount of α-glucan having a degree of polymerization of 5 to 10 was 11.1 mg / mL and 13.1 mg / mL. The correspondence between the tasting sample and the amount and ratio of peptide and the amount of glucan is shown in Table 13.

D: Sensory evaluation No additive-free sample No. 1 (commercially available product) and sample numbers 9 to 13 (additive product) obtained in C were subjected to sensory evaluation by seven trained panelists. The index of the evaluation item is "smoothness of taste" and "sustainability of taste", and the evaluation is carried out in Example 1. It was carried out according to the method described in E. The sensory evaluation results of each sample were as shown in Table 13. In addition, the above-mentioned Example 1. Together with the results of E, they are illustrated in FIGS. 5 and 6.

As shown in Table 13, 5 and 6, by increasing the peptide ratio of 10-20 kDa above 4.8% and the amount of α-glucan at a degree of polymerization 5-10 above 7.1 mg / mL. , "Smoothness of taste" and "sustainability of taste" were all improved with respect to the commercial product 1.

Example 4: Identification of molecular species of protein fraction In this example, a 2D-PAGE and a quadrupole-time-of-flight mass spectrometer have a soft and smooth texture like beer, and a protein that contributes to a harmonious taste. I tried to identify it.

200 μL of an equal amount mixture of the gel filtration fraction obtained in Reference Example 1 and the peptide fractions A1 and A2 obtained by solid-phase extraction and purification was purified by TCA acetone precipitation, electrophoresed by 2D-PAGE, and then silver-stained. gone. There is not much difference in the amount of the thick band around the molecular weight of 40 kDa between the samples of the test group 1 and the test group 2 in which the flavor is different, and there is a quantitative difference in the band distributed in the range of 10 to 20 kDa. Was found (data not shown).

Next, the band of the arrow shown in FIG. 8 was cut out from the 2D-PAGE gel of Test Group 2, and the protein was digested with trypsin (ProteaseMAX ™ Surfactant, Trypsin Enhancer, manufactured by Promega). Obtain MS / MS spectra of the obtained trypsin-digested peptide solution using a quadrupole-time-of-flight mass spectrometer (manufactured by SCIEX), and identify proteins using the database registered in SWISS-PROT. gone.

The results of protein identification by a quadrupole-time-of-flight mass spectrometer are as shown in Table 14.

The proteins in the gel filtration fraction (fractions A1 to A2) include α-amylase / trypsin inhibitor CMa, α-amylase / trypsin inhibitor CMb, α-amylase / trypsin inhibitor CMd, trypsin inhibitor CMe, α-amylase inhibitor. (BDAI-1), non-specific lipid-transfer protein 1 and avenin-like a1 were identified. The identified proteins were identified as those derived from barley ( Hordeum vulgare ) and, in part, from wheat ( Triticum aestivum ). α-amylase / trypsin inhibitor CMa, α-amylase / trypsin inhibitor CMb, α-amylase / trypsin inhibitor CMd, trypsin inhibitor CMe are known as protease inhibitor proteins, and α-amylase inhibitor (BDAI-1) is α. Known as an amylase inhibitor protein. In addition, non-specific lipid-transfer protein 1 is known as a lipid transfer protein.

Example 5: Addition of peptides prepared by ultrafiltration and sulfur precipitation to beer-taste fermented alcoholic beverages and sensory evaluation In this example, the method for preparing peptides suitable for large-scale production and their effects on sensory evaluation were examined. ..

A: Preparation of peptide fraction (A-1) Fractionation by ultrafiltration After degassing sample number 1 (commercially available) (all malt beer), ultrafiltration membrane (molecular weight cut off; 6000, AIP- Peptides were concentrated and fractionated by 1013D, manufactured by Asahi Kasei Corporation). Specifically, sample No. 1 was concentrated about 4 times, the concentrated solution was collected, and freeze-dried. Then, the dried product was rehydrated with ultrapure water so as to have a concentration 6 times that of the commercially available product, and then dialyzed by a dialysis membrane Spectra / Por1 (manufactured by Spectrum Laboratories, the same applies hereinafter) having a molecular weight cut off of 6000 to 8000.

(A-2) Separation of Peptide by Ammonium Sulfate Precipitation A 0-40% saturated ammonium sulfate precipitate was obtained by performing a 40% saturated ammonium sulfate precipitation fraction on the solution obtained in (A-1) above. The precipitate was dissolved in ultrapure water and dialyzed with a dialysis membrane Spectra / Por1 to sufficiently remove the salt. Then, it was freeze-dried and dissolved in ultrapure water to obtain a peptide concentrate. It was confirmed by the stained image of SDS-PAGE electrophoresis that the concentrated solution was mainly composed of a peptide of 10 to 20 kDa (data not shown).

B: Preparation of α-glucan fraction Preparation of α-glucan fraction is carried out in Example 3. It was done according to the description of B.

C: Preparation of tasting sample The α-glucan fraction obtained in B above was added to sample number 1 to prepare sample number 14 (additive 6). In sample number 14, Example 3. A 10-20 kDa peptide fraction purified from sample number 1 was added using the methods described in (A-1) and (A-2) to prepare sample number 15 (additive 7). Further, the 10 to 20 kDa peptide fraction obtained in A above was added to sample number 14 to prepare sample number 16 (additive 8). Specifically, each peptide fraction was added so that the 10 to 20 kDa peptide ratio of the additives 7 and 8 was 5.6%. Further, the α-glucan fraction was added so that the amount of α-glucan having a degree of polymerization of 5 to 10 of the additives 6 to 8 was 8.5 mg / mL. The correspondence between the tasting sample and the amount and ratio of peptide and the amount of α-glucan is shown in Table 15.

D: Sensory evaluation For sample numbers 14 to 16 (additives) obtained in C above, Example 1. Sensory evaluation was performed according to the description of E. The sensory evaluation results of each sample were as shown in Table 15.

As shown in Table 15, compared with the beverage without 10 to 20 kDa peptide (Sample No. 14), the beverage with 10 to 20 kDa peptide obtained by combining ultrafiltration and sulfur precipitation (Sample No. 16). Then, it was confirmed that "smoothness of taste" and "sustainability of taste" were improved as in the case of the beverage (sample No. 15) to which the 10 to 20 kDa peptide obtained by the gel filtration fraction and the solid-phase extraction was added. Was done.

Example 6: Addition of peptides prepared by different purification methods to beer-taste fermented alcoholic beverages and sensory evaluation
A: Preparation of peptide fraction Example 3. Purified sample 1 (peptide A1 fraction) was prepared from sample number 1 (commercially available) (all malt beer) according to the methods described in (A-1) and (A-2). In addition, Example 3. The A1 fraction prepared according to the method described in (A-1) was used in Example 3. Instead of purifying by the method described in (A-2) (purification by a solid-phase extraction column), the precipitate is precipitated with 70% saturated ammonium sulfate and then dialyzed against the dialysis membrane Spectra / Por1 to sufficiently remove the salt. , Purified sample 2 was prepared.

In addition, sample number 1 (commercially available product) (all malt beer) was used in Example 5. Purified sample 3 (10 to 20 kDa peptide) by performing ultrafiltration and concentration by the same method as described in (A-1), then performing 40% saturated ammonium sulfate precipitation fractionation and dialysis to sufficiently remove the salt. Fraction) was prepared. Further, 40 to 70% saturated ammonium sulfate precipitate fraction and dialysis were performed from the supernatant fraction, and the salt was sufficiently removed to prepare a purified sample 4 (40 kDa peptide fraction). In addition, Example 5. Except for the 70% saturated ammonium sulfate precipitation fraction. Purified sample 5 (10-40 kDa peptide fraction) was prepared according to the same method described in A. For each purified sample (purified samples 1 to 5), it was confirmed by SDS-PAGE electrophoresis stained images that peptides of 10 to 20 kDa, 40 kDa or 10 to 40 kDa were the main components (data not shown). ..

B: Preparation of tasting sample Sample number 17 (commercially available product) (commercially available product) in which each purified sample (purified samples 1 to 5) obtained in A above is added by 25% to each fraction contained in sample number 1. Sample numbers 18 to 22 were prepared by increasing the peptide ratio by adding to a beer-taste fermented alcoholic beverage with less than 50% malt.

C: Sensory evaluation With sample number 17 (commercially available) as a control, sample numbers 18 to 22 (additives) prepared in B were subjected to sensory evaluation by trained panelists. Sample numbers 18 and 19 were evaluated by 12 panelists, and sample numbers 20 to 22 were evaluated by 13 panelists. The index of the evaluation items was "beer-like taste", "roughness remaining in the mouth", and "thickness, body feeling". Here, the "beer-like taste" means that the soft and smooth texture of beer is felt and is in harmony, and was evaluated on a scale of 1 (weak) to 9 (strong). "Roughness remaining in the mouth" refers to unpleasant tastes such as astringency and a rough texture remaining on the tongue, and was evaluated on a 9-point scale from 1 (weak) to 9 (strong). For the two items of "beer-like taste" and "roughness remaining in the mouth", the obtained sensory evaluation scores for each individual were standardized and shown as the average value of the values. The standardized score is a value obtained by subtracting the average value from each sensory evaluation score (evaluation score) using the average value and standard deviation of the sensory evaluation score (evaluation score) for each individual, and then dividing by the standard deviation. To say. The formula for the standardized score is as shown below.

"Thickness, body feeling" means the thickness and strength of the taste after swallowing, and the taste and afterglow remain, and the number of panels where the indication was seen is 0-1-2 people. Was evaluated as +, 3 to 4 people as ++, and 5 to 6 people as +++. In addition, since the smooth texture is evaluated in the "beer-like taste" as described above, the "taste smoothness" evaluated in Examples 3 and 5 is also evaluated in this example. In addition, since it is evaluated that the aftertaste of the taste remains in the "thickness and body feeling", the "sustainability of taste" evaluated in Examples 3 and 5 is also evaluated in this Example. The sensory evaluation results of each sample were as shown in Table 16.

As shown in Table 16, even when each purified fraction is added to a beer-taste fermented alcoholic beverage (commercially available) with less than 50% malt, by increasing the 10 to 20 kDa peptide ratio, "beer-like taste", The evaluations of "roughness remaining in the mouth" and "thickness and body feeling" were improved with respect to the commercially available product (sample number 17). Specifically, the fraction purified by the solid-phase extraction method in sample No. 19 to which the fraction further fractionated and purified by 70% saturated sulfate precipitation was added instead of the solid-phase extraction method after fractionation by gel filtration. The same effect as that of sample No. 18 to which was added was obtained. In addition, a beverage (sample number 20) to which a 10 to 20 kDa peptide fraction (purified sample 3) purified by ultrafiltration and a sulfur-precipitated fraction from all-malt beer (commercially available) was added, and a 10 to 40 kDa peptide fraction. In the beverage (sample No. 22) to which (fraction containing 10 to 20 kDa peptide) (purified sample 5) was added, the same effect as that of sample No. 18 was obtained. In addition, a beverage (sample No. 21) to which a 40 kDa peptide fraction (purified sample 4) was added was also weak but effective.

Claims (9)

A beer-taste fermented alcoholic beverage made from malt and / or ungerminated wheat as a raw material, the beverage having a molecular weight of 10 derived from a beer-taste fermented alcoholic beverage made from barley malt as a raw material. It contains a peptide fraction of up to 20 kDa (HPLC gel filtration method), and the ratio of the amount of peptide contained in the peptide fraction to the total amount of protein in the beverage is more than 4.8% and 8.0% or less . Moreover, a beer-taste fermented alcoholic beverage having a concentration of α-glucan having a degree of polymerization of 5 to 10 in the beverage of 7.1 mg / mL or more and 16.0 mg / mL or less . The beer-taste fermented alcoholic beverage according to claim 1, wherein the malt usage ratio is 50% or more. A step of blending a peptide fraction having a molecular weight of 10 to 20 kDa (HPLC gel filtration method) derived from a beer-taste fermented alcoholic beverage using barley malt as a raw material, and an α-glucan having a polymerization degree of 5 to 10 are blended. A method for producing a beer-taste fermented alcoholic beverage, which comprises the steps of: 4.8% or less and 8.0% or less of the peptide contained in the peptide fraction with respect to the total amount of protein in the beverage. A production method in which the α-glucan is blended so as to have a concentration of 7.1 mg / mL or more and 16.0 mg / mL or less in a beverage. The method for producing a beer-taste fermented alcoholic beverage according to claim 3, further comprising a step of preparing the peptide from a raw material containing the peptide. The method for producing a beer-taste fermented alcoholic beverage according to claim 4, wherein the peptide is prepared by an ultrafiltration method and an ammonium sulfate precipitation method. The method for producing a beer-taste fermented alcoholic beverage according to claim 4 or 5, wherein the raw material containing the peptide is barley malt or a processed product thereof. The method for producing a beer-taste fermented alcoholic beverage according to any one of claims 4 to 6, wherein the raw material containing the peptide is a beer-taste fermented alcoholic beverage containing at least a part of barley malt as a raw material. A flavor improving agent for a beer-taste fermented alcoholic beverage containing a peptide fraction having a molecular weight of 10 to 20 kDa (HPLC gel filtration method) and α-glucan having a degree of polymerization of 5 to 10 as active ingredients, which is used in the peptide fraction. The contained peptide was used so as to have a ratio of more than 4.8% and 8.0% or less with respect to the total amount of protein in the beverage, and the α-glucan was used at a concentration of 7.1 mg / mL or more in the beverage 16 A flavor-improving agent derived from a beer-taste fermented alcoholic beverage that is used so as to be 0.0 mg / mL or less and the peptide fraction and the α-glucan are at least a part of barley malt as a raw material. A method for improving the flavor of a beer-taste fermented alcoholic beverage, which comprises a step of adding a peptide fraction having a molecular weight of 10 to 20 kDa (HPLC gel filtration method) and α-glucan having a degree of polymerization of 5 to 10, and the peptide drawing . The peptide contained in the minute was added so as to have a ratio of more than 4.8% and 8.0% or less with respect to the total amount of protein in the beverage, and the α-glucan was added to the beverage at a concentration of 7.1 mg / mL. A method for improving flavor, which is derived from a beer-taste fermented alcoholic beverage in which the peptide fraction and the α-glucan are added so as to be 16.0 mg / mL or less and the barley malt is at least a part of the raw material.

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