Method for producing wort

Abstract

To provide an enzyme preparation for increasing wort yield, by focusing not merely on cellulase activity as a whole, but rather on individual cellulase activities, such as CMC-degrading activity and filter paper-degrading activity, achieving increased filtrate volume and fermentable sugars in wort.SOLUTION: Provided is a method for producing wort, comprising adding, in a saccharification step, a cellulase preparation having filter paper-degrading activity and CMC-degrading activity, with the ratio between the respective activities being from 1:5 to 1:500. The cellulase may further have β-glucosidase activity and xylanase activity.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a method for producing wort with improved yield. [Background technology]

[0002] Beer is produced through the processes of malting, brewing, fermentation, and aging. During the brewing process, the starch contained in the malt is broken down into sugar by enzymes, and then the wort is produced through a filtration process. The first filtered wort is called the "first wort," and the remaining residue is poured with hot water to obtain the "second wort." Most beers are also produced using the second wort. Therefore, the more wort that is recovered, the higher the yield. In addition, the sugars broken down during the brewing process are assimilated by yeast during the fermentation process after the brewing process. The sugars that are assimilated are fermentable sugars (sugars with three or fewer sugars), while non-fermentable sugars remain in the beer. Therefore, if the rate at which starch is broken down into fermentable sugars during the brewing process increases, it will lead to a reduction in the sugar content of beer. In addition, the change in assimilation will lead to improved flavor.

[0003] Patent Document 1 discloses a method for producing beer wort having increased fermentable sugars by using two or more glucoamylases as a method for producing low-calorie beer.

[0004] Patent Document 2 discloses a technology for producing an alcoholic beverage with a high content of β-glucan, which retains as much β-glucan as possible in the fermentation raw material while achieving high wort filterability, by using a fiber-degrading enzyme with relatively high hemicellulase activity.

[0005] Patent Document 3 describes an improved mashing method for producing beer wort with low sugar content of 4 or more sugars and for producing beer, and describes the use of pullulanase.

[0006] Patent Document 4 describes a method for producing wort, which includes forming a mash containing 5% to 100% barley malt, and adding a protease and a cellulase before, during or after forming the mash, accomplished within 15 minutes of forming the mash. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2019 / 067287 [Patent Document 2] JP 2011-083239 A [Patent Document 3] U.S. Patent No. 8,765,199 [Patent Document 4] International Publication No. 2004 / 011591 Summary of the Invention [Problem to be solved by the invention]

[0008] Patent Document 4 describes the addition of cellulase to wort production, but does not describe activity values ​​or activity ratios of subdivided cellulase activities such as CMC decomposition activity and filter paper decomposition activity, etc. In the present invention, by focusing on subdivided cellulase activities such as CMC decomposition activity and filter paper decomposition activity, rather than on simple cellulase activity, it has been surprisingly found that the wort filtrate volume and fermentable sugars increase. [Means for solving the problem]

[0009] The present inventors discovered that when cellulase having filter paper decomposition activity and CMC decomposition activity with an activity ratio of 1:5 to 1:500 was added to the saccharification process, the amount of filtrate in the filtration process increased and the amount of fermentable sugars increased, and thus completed the present invention.

[0010] That is, the present invention provides the following [1] to

[10] . [1] An enzyme preparation for increasing wort volume, in which cellulase has filter paper decomposition activity and CMC decomposition activity and has an activity ratio of 1:5 to 1:500. [2] The enzyme preparation described in [1], wherein the cellulase further has β-glucosidase activity, and the ratio of β-glucosidase activity:CMC decomposition activity is 1:1 to 1:100. [3] The enzyme preparation according to [1] or [2], wherein the cellulase further has xylanase activity, and the ratio of xylanase activity:CMC decomposition activity is 1:0.01 to 1:10. [4] The enzyme preparation according to any one of [1] to [3], wherein the cellulase is a cellulase produced by Trichoderma reesei. [5] The enzyme preparation according to any one of [1] to [4], wherein the CMC decomposition activity is 100 to 15,000 U / g and the filter paper decomposition activity is 20 to 150 U / g. [6] A method for producing wort with improved yield, comprising adding the cellulase according to any one of [1] to [5] in a saccharification step. [7] The method for producing wort according to [6], wherein the malt used for producing the wort is pilsner malt. [8] The method for producing wort according to [6] or [7], wherein the reaction temperature of the cellulase is within the range of 20°C to 90°C. [9] The method for producing wort according to any one of [6] to [8], wherein the reaction time of the cellulase is within a range of 0.25 hours to 5 hours.

[10] A method for producing beer using any one of the enzyme preparations of [1] to [5] or the wort production methods of [6] to [9]. Effect of the Invention

[0011] According to the present invention, it is possible to obtain wort with an increased fermentable sugar content and improved yield. This technology leads to improved yield in beer production, shortened production time, and production of products with reduced sugar content. [Brief description of the drawings]

[0012] [Figure 1] FIG. 2 is a diagram showing temperature conditions during wort production. [Diagram 2] FIG. 2 is a graph showing the amount of wort filtrate in Production Example 1. [Diagram 3] FIG. 1 is a graph showing the amount of wort filtrate in Production Example 2. [Figure 4] FIG. 2 is a graph showing the turbidity of wort in Production Example 1. [Diagram 5] FIG. 1 is a graph showing the turbidity of wort in Production Example 2. [Figure 6] FIG. 2 is a graph showing the sugar concentration (total of maltotriose, maltose, glucose and sugar) of the wort in Production Example 1. [Figure 7] FIG. 2 is a graph showing the sugar concentration (total of maltotriose, maltose, glucose and sugar) of the wort in Production Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Beer production is well known in the art and generally comprises a first step (malting) of germinating ungerminated barley to produce malt, a second step (brewing) of producing wort containing sugars and amino acids using the malt, hops, and other auxiliary ingredients that are the raw materials for beer, a third step (fermentation) of fermenting the wort produced in the second step with yeast, and a fourth step (aging) of maturing the beer after fermentation. The second step of producing wort further comprises a grinding / hydration step, a saccharification step, a filtration step, and a boiling step. The method for producing wort according to the present invention includes a step of adding cellulase to the saccharification step in the second step to decompose cellulose contained in malt. Below, (1) raw materials for wort and (2) the amounts added, (3) the method for producing wort, and (4) uses will be described in this order.

[0014] <Raw materials for wort> <Cellulase> Cellulase is an enzyme that degrades the glycosidic bonds of cellulose, and in the present specification, it is sufficient that the cellulase has filter paper decomposition activity and CMC decomposition activity, and the activity ratio is 1:5 to 1:500. The filter paper decomposition activity:CMC decomposition activity ratio is preferably 1:8 to 1:100, more preferably 1:9 to 1:80, and particularly preferably 1:10 to 1:60. A higher filter paper decomposition activity / CMC decomposition activity ratio is more suitable for decomposing malt cellulose. The cellulase may further have β-glucosidase activity, and the ratio of β-glucosidase activity:CMC decomposition activity is preferably 1:1 to 1:100, more preferably 1:2 to 1:70, and particularly preferably 1:3 to 1:50. The cellulase may further have a xylanase activity, and the ratio of xylanase activity:CMC decomposition activity is preferably 1:0.01 to 1:10, more preferably 1:0.03 to 1:5, and particularly preferably 1:0.05 to 1:2.

[0015] When the cellulase preparation is used, the optimum (optimum) pH is preferably in the range of 3 to 7, more preferably 4 to 6, and even more preferably 4 to 5.5.

[0016] The optimum temperature when using the cellulase preparation is preferably in the range of 45°C to 70°C, and more preferably in the range of 55°C to 60°C.

[0017] The filter paper decomposition activity and CMC decomposition activity contained in the cellulase may be present in an active state in the saccharification process. In the saccharification process, the β-glucosidase activity contained in the cellulase may be present in an active state, and the xylanase activity contained in the cellulase may be present in an active state. It is preferable to inactivate the cellulase present in the wort by heating. It may be inactivated in the boiling process, or may be inactivated by heat before the boiling process. The heating conditions in the treatment to inactivate the enzyme are preferably high temperature and short time heating, for example, heating at 60 to 100°C for 1 to 30 minutes.

[0018] (Origin of cellulase) The type and origin of cellulase are not particularly limited. Examples of microorganisms include the genera Tricoderma, Aspergillus, Penicillium, Talaromyces, and Streptomyces. Specific examples include Tricoderma reesei, Tricoderma longibrachiatu, Aspergillus niger, and Talaromyces emersonii. As the cellulase, commercially available products can be used, and preferred examples of commercially available products include GODO Cellulase F from Godo Shusei Co., Ltd., Sumiteam A from Shin Nippon Chemical Industry Co., Ltd., and Cellulase SS from Nagase ChemteX Corporation. Commercially available cellulase preparations may be mixed and used.

[0019] (Cellulase manufacturing method) The cellulase used in the present invention can be one that has been recovered from a microorganism and purified by a conventional method. Therefore, the cellulase can be produced by genetic recombination or non-genetic recombination. For example, the cellulase can be obtained as a culture supernatant by culturing Tricoderma riisei in a medium or the like and filtering or centrifuging the culture. The culture supernatant can also be appropriately purified by column chromatography or ammonium sulfate fractionation before use. The enzyme solution obtained can be in the form of a liquid or a powder by spray drying or freeze drying. In this case, a stabilizer or excipient that is commonly used may be added.

[0020] (Amount of cellulase added) Cellulase is preferably added in the range of 0.01 to 500 U / g of CMC degrading activity and 0.005 to 5 U / g of filter paper degrading activity per gram of malt liquid (malted barley crushed and immersed in water to form a liquid). More preferably, it can be added in the range of 0.05 to 250 U / g of CMC degrading activity and 0.01 to 4 U / g of filter paper degrading activity. The β-glucosidase activity per gram of malt liquid is preferably in the range of 0.005 to 5 U / g, and more preferably in the range of 0.01 to 3 U / g. The xylanase activity per gram of malt liquid is preferably in the range of 0.1 to 200 U / g, and more preferably in the range of 0.5 to 150 U / g.

[0021] (Definition and Method of Cellulase Activity) Generally, cellulase preparations contain multiple activities. The addition amount (enzyme amount) of the above cellulase can be calculated as the enzyme activity per unit weight of the enzyme based on the CMC degrading activity, filter paper degrading activity, β-glucosidase activity, and xylan degrading activity defined by the following method.

[0022] <CMC Degrading Activity> Dissolve sodium carboxymethyl cellulose (product name: sodium carboxymethyl cellulose, company name: Junsei Chemical Co., Ltd.) in 0.05 M citrate buffer (pH 4.8) to a concentration of 1% (w / v). Measure the amount of reducing sugar produced by adding the enzyme solution under the conditions of 50 °C for 30 minutes by the dinitrosalicylic acid method. The enzyme amount that produces reducing sugar equivalent to 1 μmol of glucose per minute is defined as 1 U (Unit).

[0023] <Filter Paper Degrading Activity> Cellulose filter paper (product name: Cytiva (Whitman) qualitative filter paper No. 1) cut to a weight of 50 mg ± 1.5 mg (approximately 1 cm x 6 cm) was mixed with 1 mL of 0.05 M citrate buffer (pH 4.8). With the filter paper piled up at the bottom of the reaction vessel, the amount of reducing sugar generated by adding the enzyme solution was measured at 50°C for 60 minutes using the dinitrosalicylic acid method. The amount of enzyme that generates reducing sugar equivalent to 1 μmole of glucose per minute is defined as 1 U (Unit).

[0024] <β-glucosidase activity> Dissolve p-nitrophenyl-β-glucopyranoside in distilled water to a concentration of 5 mM (substrate solution). Mix 1.0 mL of 0.2 M acetate buffer (pH 5.0) with 0.5 mL of substrate solution. Measure the amount of p-nitrophenol produced by adding the enzyme solution at 50°C for 20 minutes based on the absorbance at 400 nm. The amount of enzyme that produces 1 μmole of p-nitrophenol per minute is defined as 1 U (Unit).

[0025] <Xylan decomposition activity> Xylan (product name: Sigma-Aldrich Xylan from Beechewood, sieved through 200 mesh before use) is dispersed in 0.05M citrate buffer (pH 5.5) to a concentration of 1.25% (w / v). The amount of reducing sugar produced by adding the enzyme solution is measured by the dinitrosalicylic acid method at 50°C for 30 minutes. The amount of enzyme that produces reducing sugar equivalent to 1 μmole of xylose per minute is defined as 1 U (Unit).

[0026] <malt> The malt used in the wort production of the present invention is not particularly limited as long as it is a common malt used in beer production, and means any malted grain, especially barley. For example, pale malt, pilsner malt, wheat malt, rye, kiln malt, Munich malt, Vienna malt, Carapils, Maris Otter, caramel malt, crystal malt, chocolate malt, black malt, roasted barley, etc. may be used. One type of malt may be used, or a mixture of multiple malts may be used. In the present invention, it is preferable to use base malt, especially pilsner malt. Commercially available malt may be used, or ungerminated barley may be germinated to produce the malt described above.

[0027] <Other enzymes> The wort produced by the production method of the present invention may contain other enzymes, as necessary, within the range that does not impair the effects of the present invention. Examples of such enzymes include pullulanase, isoamylase, glucoamylase, inulinase, and protease.

[0028] <Wort production method> The method for producing wort of the present invention includes a step of crushing malt and adding water, and a step of adding cellulase to decompose starch contained in the malt into sugar. The state of the malt used for the production of wort is preferably pulverized from the viewpoint of increasing the reactivity of the enzyme. The means for pulverizing the malt may be any generally known means, and is not particularly limited, such as crushing, grinding, or shredding. Means for pulverizing grains may also be used. The milled malt is steeped in water to form a liquid (malt liquid). The malt liquid herein may contain starch and may include milled malted barley, milled unmalted grains, other starch-containing materials, or combinations thereof.

[0029] The enzymes that break down the starch contained in malt into sugar are produced by the action of endogenous enzymes contained in the malt (amylase, hemicellulase, protease, etc.) and exogenous enzymes added. The sugar composition contained in the wort varies depending on the malt used, but generally consists of maltotriose, maltose, glucose, dextrin, etc., with 40-50% of the total sugar being maltose and 20-30% being dextrin.

[0030] The timing of adding cellulase is not particularly limited, and it may be added simultaneously with the water addition step or after that.

[0031] The cellulase treatment of the malt liquid is preferably carried out at a reaction temperature of 20°C to 90°C for 0.25 to 5 hours. The cellulase treatment of the malt liquid may be carried out as an independent step or may be carried out simultaneously with the saccharification step. The reaction temperature is preferably 20°C to 75°C, more preferably 40°C to 70°C, and even more preferably 50°C to 68°C. When the reaction temperature is 50°C to 68°C, the cellulase treatment of the malt liquid and the sugar processing step can be carried out simultaneously. In addition to improving the production efficiency, the effect of increasing the sugar concentration contained in the wort can be obtained. The reaction time is preferably 0.25 to 5 hours, more preferably 0.5 to 4 hours, and particularly preferably 1 to 3 hours. Before the malt liquid is treated with cellulase, the pH of the malt liquid may be adjusted to preferably be within the range of 3.0 to 8.0, more preferably be within the range of 3.5 to 7.5, even more preferably be within the range of 4.0 to 7.0, and particularly preferably be within the range of 5.0 to 6.0.

[0032] After the cellulase treatment of the malt liquid or after the saccharification process, filtration is repeated until the turbidity is removed. Any commonly known filtration method may be used, such as the Reuter method using a Reuter plate and the filter press method using a filter cloth. The solid residue remaining after filtration may be filtered by pouring hot water over it, and the wort may be recovered again. In the filtration process in the examples described below, filtration using filter paper was performed as a simple test, and filtration was performed twice.

[0033] <Uses of wort> The wort obtained by the present invention may be fermented with yeast to produce beer. Since the amount of sugars with 3 or less sugars that can be assimilated by yeast increases, it is expected to contribute to the production of low-sugar beer. In addition, it may be used as a beverage ingredient containing wort as a non-alcoholic beverage. EXAMPLES

[0034] The present invention will now be described in more detail with reference to examples, but the present invention is not limited thereto in any way.

[0035] <Preparation of cellulase> Table 1 shows the activities (FPase (filter paper decomposition activity), CMCase activity, β-glucosidase activity (β-GDase), xylanase activity) and their activity ratios of cellulase preparation I and cellulase preparation II used in this example.

[0036] [Table 1]

[0037] <Production of wort> <Production Example 1> (material) Malt: Pilsner (ground) (Weyermann Specialty Malts) (Manufacturing method) To 5 g of malt, four times the amount of water (20 mL, previously kept at 50°C) and cellulase preparation I shown in Table 1 were added to prepare a malt liquid. The activity values ​​(final concentrations) of cellulases in the malt liquid at this time are shown in Table 2. After heating at 50°C for 1 hour, the temperature was raised stepwise to 68°C and heated for a total of 2 hours under the conditions shown in Figure 1. This process combines enzyme reaction, protein rest, and saccharification. After heating, a No. 2 filter paper was placed on a PP φ90 mm spiral ribbed funnel, and the sample after the reaction was filtered. After 30 minutes from the start of filtration, the filtrate was collected, and the entire amount of the obtained filtrate was filtered again for 30 minutes on top of the malt residue to obtain the wort of Production Example 1.

[0038] [Table 2]

[0039] <Production Example 2> The wort of Production Example 2 was obtained in the same manner as in Production Example 1, except that cellulase preparation II shown in Table 1 was added. The activity values ​​(final concentrations) of each cellulase in the malt liquid are shown in Table 3.

[0040] <Comparative Example 1> The wort of Comparative Example 1 was obtained in the same manner as in Production Example 1, except that cellulase was not used.

[0041] [Table 3]

[0042] Evaluation <Test Example 1: Measurement of filtrate volume> The weights of the wort obtained in Production Examples 1 and 2 and Comparative Example 1 were measured. As shown in Figures 2 and 3, it can be seen that the filtrate significantly increased by adding cellulase. The rate of increase was greater in Production Example 1 than in Production Example 2. It is believed that the filterability improved due to the decomposition of cellulose or hemicellulose in the wort.

[0043] <Test Example 2: Measurement of turbidity of filtrate volume> The turbidity (OD 660 nm) of the wort obtained in Production Examples 1 and 2 and Comparative Example 1 was measured. As shown in FIG. 4, in Production Example 1, the turbidity decreased due to the addition of cellulase. It is believed that the turbidity was reduced due to the decomposition of cellulose in the filtrate. As shown in FIG. 5, in Production Example 2, the turbidity increased when cellulase was added at an activity ratio of C. It is believed that the improved filterability resulted in an increase in the components that migrated into the filtrate, which increased the turbidity.

[0044] <Test Example 3: Sugar analysis of filtrate> The worts obtained in Production Examples 1 and 2 and Comparative Example 1 were subjected to sugar analysis by high performance liquid chromatography (Allliance 295 Separation Module, manufactured by Nihon Waters K.K.). As shown in FIG. 6, in Production Example 1, when cellulase was added at an activity ratio of C, the amount of maltose increased, and the total amount of monosaccharides, disaccharides, and trisaccharides also increased accordingly. As shown in FIG. 7, in Production Example 2, when cellulase was added at an activity ratio of C, the amount of maltose decreased and glucose increased. No significant change was observed in the total amount of monosaccharides, disaccharides, and trisaccharides, but the total amount tended to increase.

Claims

1. An enzyme preparation for increasing wort volume, in which cellulase has filter paper decomposition activity and CMC decomposition activity, and the activity ratio is 1:5 to 1:

500.

2. The enzyme preparation according to claim 1, wherein the cellulase further has β-glucosidase activity, and the ratio of β-glucosidase activity:CMC decomposition activity is 1:1 to 1:

100.

3. The enzyme preparation according to claim 1 or 2, wherein the cellulase further has xylanase activity, and the ratio of xylanase activity:CMC decomposition activity is 1:0.01 to 1:

10.

4. The enzyme preparation according to any one of claims 1 to 3, wherein the cellulase is a cellulase produced by Trichoderma reesei.

5. The enzyme preparation according to any one of claims 1 to 4, wherein the CMC decomposition activity is 100 to 15,000 U / g and the filter paper decomposition activity is 20 to 150 U / g.

6. A method for producing wort with improved yield, comprising adding the cellulase according to any one of claims 1 to 5 in a saccharification process.

7. 7. The method for producing wort according to claim 6, wherein the malt used for producing the wort is pilsner malt.

8. The method for producing wort according to claim 6 or 7, wherein the reaction temperature of the cellulase is within the range of 20°C to 90°C.

9. The method for producing wort according to any one of claims 6 to 8, wherein the reaction time of the cellulase is within a range of 0.25 hours to 5 hours.

10. A method for producing beer, using the enzyme preparation according to any one of claims 1 to 5 or the method for producing wort according to any one of claims 6 to 9.

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