KR102474061B1 - Method for culturing Saccharomyces cerevisiae Y297 strain - Google Patents

Abstract

The present invention relates to a method for culturing Saccharomyces cerevisiae Y297 strain using a medium containing sucrose, soy peptone, yeast extract, MgSO ₄ 7H ₂ O, KH ₂ PO ₄ , CaCl ₂ and D-biotin, and the above It relates to a method for producing rice beer using the Y297 strain in cultured Saccharomyces cerevisiae. Using the method of culturing the Y297 strain in Saccharomyces cerevisiae provided by the present invention, yeast strains suitable for rice beer production can be effectively cultivated and used for production of rice beer, thereby producing better quality rice beer. It will be widely used.

Description

Method for culturing Saccharomyces cerevisiae Y297 strain {Method for culturing Saccharomyces cerevisiae Y297 strain}

The present invention relates to a method for culturing Saccharomyces cerevisiae Y297 strain, and more specifically, the present invention relates to sucrose, soy peptone, yeast extract, MgSO ₄ 7H ₂ O, KH ₂ PO ₄ , CaCl ₂ and D - It relates to a method for culturing Saccharomyces cerevisiae Y297 strain using a medium containing biotin and a method for producing rice beer using the cultured Saccharomyces cerevisiae Y297 strain.

Beer is a fermented liquor made from barley and hops, and is one of the popular alcoholic beverages. Rice, corn, starch, sugars, etc. are used as starch supplements depending on the situation or preference of the country or region. It contains less carbon dioxide and bitter taste components of hops, so it has the effect of promoting digestion and helping diuresis.

In recent years, the consumption of imported beer is rapidly increasing in Korea rather than domestic beer, and the demand for various and differentiated high-quality beers is significantly increasing, especially among young people. In addition, according to the trend of enjoying various and unique beer tastes, differentiated craft beer is developed. and craft beer markets are expanding.

In line with the expansion of the craft beer market and consumption trends, the government is continuously pushing for tax exemption and distribution regulation easing policies for craft beer to promote domestic rice consumption and vitalize the craft beer industry. In the case of craft beer that uses more than 20% of rice, tax reduction is provided to support the expansion and vitalization of the craft beer market. However, beer sold in the domestic craft beer market adheres to the general beer manufacturing method using only malt, so there is no differentiation in quality by company, and a problem of losing diversity and quality competitiveness compared to imported beer is emerging.

As a way to solve this problem, developing rice varieties suitable for rice beer production (Korean Patent Publication No. 10-2019-0053734), developing a rice processing method (Korean Patent Registration No. 10-1729733), or rice Developing a method for manufacturing malt (Korean Patent No. 10-1976565), developing fermentation strains (Korean Patent No. 10-1825957), or developing various additional ingredients (Korean Patent No. 10-1020626) Various studies are being conducted. However, in the case of the fermented strain, it exhibits excellent growth even at a low temperature of 15 ° C., but research on how to effectively cultivate it has not yet been conducted, and rice beer has not been commercially produced using it. .

Under this background, the present inventors have made intensive research efforts to develop a method for producing rice beer using Saccharomyces cerevisiae Y297 strain (Accession No. KACC93244P), which is known to exhibit excellent growth even at a low temperature of 15 ° C. Results , developed a culture method using a medium suitable for culturing the Y297 strain, and completed the present invention.

The main object of the present invention is to provide a method for culturing strain Y297 in Saccharomyces cerevisiae.

Another object of the present invention is to provide a method for producing rice beer using the Y297 strain in Saccharomyces cerevisiae cultured by the above method.

One embodiment of the present invention for achieving the above object is sucrose, soy peptone, yeast extract, MgSO ₄ 7H ₂ O, KH ₂ PO ₄ , CaCl ₂ and D- biotin in a medium containing Saccharomyces Provided is a method for culturing Saccharomyces cerevisiae Y297 strain (Accession No. KACC93244P), comprising the step of inoculating and culturing the Y297 strain (Accession No. KACC93244P) in Celebijie.

The term "Saccharomyces cerevisiae Y297 strain (accession number KACC93244P)" of the present invention means a yeast strain developed by the Rural Development Administration and deposited with the National Academy of Agricultural Sciences as deposit number KACC93244P on November 10, 2015 However (Korean Patent Registration No. 10-1825957), it is known to exhibit resistance to low-temperature growth of 10 to 20 ° C.

The content of each component included in the medium is not particularly limited thereto, but as an example, 10 to 60 g/L sucrose, 1 to 10 g/L soy peptone, 10 to 60 g/L yeast extract, 0.1 to 5 g/L MgSO ₄ .7H ₂ O, 1 to 10 g/L KH ₂ PO ₄ , 0.1 to 2.0 g/L CaCl ₂ and 1 to 5 g/L D-biotin; As another example, 20 to 40 g/L sucrose, 3 to 7 g/L soy peptone, 20 to 40 g/L yeast extract, 1 to 3 g/L MgSO ₄ 7H ₂ O, 2 to 6 g/L KH ₂ PO ₄ , 0.3 to 1.0 g/L CaCl ₂ and 1.5 to 3 g/L D-biotin; As another example, 30 g/L sucrose, 5 g/L soy peptone, 30 g/L yeast extract, 1.6 g/L MgSO ₄ 7H ₂ O, 4 g/L KH ₂ PO ₄ , 0.5 g/L CaCl ₂ and 2 g/L D-Biotin.

In order to prevent sedimentation and browning during wet sterilization, the sugar component and nitrogen source presented above are separated and sterilized, and the remaining components are also sterilized, and then a medium can be prepared by mixing them. At this time, the conditions for carrying out the sterilization are not particularly limited thereto, but as an example, it may be performed using a pressure sterilizer (autoclave) at 100 to 150 ° C. for 5 to 60 minutes, and as another example, 110 It may be carried out using an autoclave (autoclave) at 140 ° C for 10 to 30 minutes, and as another example, it may be performed using an autoclave (autoclave) at 121 ° C for 15 minutes.

The Saccharomyces cerevisiae strain Y297 can be cultured as follows: inoculate the strain into YPD solid medium, grow colonies obtained therefrom in YPD medium, and then inoculate the medium to shake culture method can be cultivated with At this time, the culture temperature is not particularly limited thereto, but as an example, it may be 20 to 40 ° C, as another example, it may be 25 to 35 ° C, and as another example, it may be 30 ° C. In addition, the shaking condition is not particularly limited thereto, but as an example, it may be 100 to 300 rpm, as another example, it may be 150 to 250 rpm, and as another example, it may be 200 to 220 rpm. Finally, the incubation time is also not particularly limited thereto, but as an example, it may be 10 to 30 hours, as another example, it may be 20 to 28 hours, and as another example, it may be 24 hours.

A pre-cultivation step may be further performed before culturing the Y297 strain in Saccharomyces cerevisiae. The pre-cultivation step may include culturing in YPD solid medium to obtain colonies; It may include the step of inoculating and culturing the obtained colony in YPD liquid medium.

At this time, the YPD medium used is not particularly limited thereto, but as an example, a medium containing 1 to 20 g/L yeast extract, 1 to 10 g/L peptone, and 1 to 20 g/L dextrose may be used ; As another example, a medium containing 5 to 15 g/L yeast extract, 3 to 7 g/L peptone, and 5 to 15 g/L dextrose may be used; As another example, a medium containing 10 g/L yeast extract, 5 g/L peptone and 10 g/L dextrose may be used.

The pre-cultivation may be performed under the same conditions as those of the Y297 strain in Saccharomyces cerevisiae described above, except that the medium used is different.

Another embodiment of the present invention provides a method for producing rice beer using Saccharomyces cerevisiae Y297 strain (Accession No. KACC93244P) cultured by the above method.

Specifically, the method for producing rice beer provided by the present invention includes the steps of (a) adding a rice flour-derived component to the saccharification solution of malt and heating it to obtain wort; (b) adding Y297 strain (Accession No. KACC93244P) to Saccharomyces cerevisiae cultured by the above method to the obtained wort and performing alcoholic fermentation to obtain a fermentation broth; and (c) preparing rice beer by aging the obtained fermentation broth.

The alcoholic fermentation in step (b) may be performed at 10 ° C to 30 ° C for 3 to 10 days; Aging in step (c) may be performed at 10 ° C to 30 ° C for 1 day to 10 days.

The term "rice beer" of the present invention means beer prepared using rice as a sub-material.

The rice beer has the advantage of having a low gluten content that causes indigestion because the malt content is low compared to conventional barley beer, and the content of rice and the type of beer material used to enhance the taste and flavor of beer , germination of beer materials, types of enzymes used, etc. can be appropriately selected within the range of ordinary ability of those skilled in the art.

The term “malt” of the present invention refers to germinated product that is poured into barley with water at an appropriate temperature and left for about 3 days to germinate. As the activity of amylase, an enzyme, increases during germination, it can be used for making amylase, starch syrup, and brewing beer. do. Undried malt is called green malt, and dried malt is called dry malt.

In the present invention, the rice flour-derived component used in the preparation of the rice beer is not particularly limited thereto, but as an example, powder obtained by milling polished rice, rice flour gelatinized liquid obtained by hydrolyzing and heating the powder, etc. It can be used, and as another example, it can be a rice flour gelatinized liquid obtained by hydrolyzing and heating the powder obtained by milling polished rice, and as another example, obtained by milling the hadeulmi or alchanmi It may be a gelatinization liquid obtained by hydrolyzing and heating the powder.

The term "Haedulmi" of the present invention refers to an early-ripening rice variety first harvested in 2019 developed together with the Icheon Agricultural Research and Extension Services and the Rural Development Administration as an alternative to Koshihikari, and is known to have an amylose content of about 18%.

The term "alchanmi" of the present invention is one of the rice varieties of the middle species, and is known to indicate an amylose content of about 18.6%.

In the step (a), the mixing ratio of rice flour and malt contained in the wort is not particularly limited thereto, but may be, for example, 5:5 to 1:9 (w/w); another example may be 4:6 to 1:9 (w/w); As another example, it may be 2:8 (w/w).

The term "wort" of the present invention, also known as wort juice, is a liquid obtained by pouring hot water into pulverized malt to saccharify it and filtering to remove substances insoluble in liquid, which is the main raw material of beer. After boiling wort with hops, the liquid from which hop dregs are removed is called Ga-hop wort, and cold wort is cooled to 10 ° C or lower to remove coagulated sediment.

The step of obtaining the wort is usually performed by a process known as saccharification. In the saccharification, the malt is put in water and heated so that the enzymes contained in the malt convert the starch and protein contained in the rice flour into sugar and nitrogen compounds, respectively. means the process of

In the present invention, the wort may be obtained by adding rice flour gelatinization solution to malt saccharification solution, and the saccharification process may be changed by the addition of the rice flour gelatinization solution, so that the conventional wort containing pure malt saccharification solution It is distinct from wort.

Heating conditions performed when obtaining the wort are not particularly limited thereto, but may be performed while increasing the temperature in three steps, for example, 1 to 20 minutes at 40 to 60 ℃, 10 to 60 minutes and at 90 to 110 °C in the order of 30 to 90 minutes; As another example, it may be performed in the order of 5 to 15 minutes at 45 to 55 ° C, 20 to 40 minutes at 65 to 75 ° C, and 50 to 80 minutes at 95 to 105 ° C; As another example, it may be performed in the order of 50 ℃ 10 minutes, 70 ℃ 30 minutes and 100 ℃ 60 minutes.

After performing step (a) and before performing step (b), a step of adding hops to the obtained wort and heating may be further included.

In the heating step, various functional ingredients such as ginseng, black raspberry, goji berry, etc. may be added in order to enhance the taste and flavor of beer and to add functionality. The time for boiling to concentrate the wort can be appropriately selected according to the characteristics of the additive material.

The term "alcohol fermentation" of the present invention refers to a process in which sugar in wort is decomposed by yeast to produce alcohol and carbon dioxide. Diacetyl, organic acids, alcohols, esters, sulfur compounds, aldehydes, phenols, etc. are produced as by-products of the fermentation, and these by-products affect the taste and flavor of beer.

The alcohol fermentation step must be performed in an anaerobic state, that is, in a state of being blocked from air. However, it is okay to temporarily introduce an aerobic state in order to proliferate the yeast in an appropriate number.

In the fermentation step of the present invention, it is preferable to determine the fermentation time and fermentation temperature in consideration of the degree of preference of beer, and those skilled in the art can use the type of beer material, whether or not the beer material has germinated, and ungerminated grains within the range of their ordinary ability. Depending on the type of enzyme used in the case, the preference of the finally obtained beer, etc., the optimal fermentation conditions may be determined based on known techniques or experimentally.

Before adding Y297 strain (Accession No. KACC93244P) to Saccharomyces cerevisiae to perform the alcohol fermentation, a step of pre-proliferating and activating the strain may be further included, and Saccharomyces Prior to adding strain Y297 (Accession No. KACC93244P) to Celebige, a step of adding oxygen may be further included.

The amount of Y297 strain (Accession No. KACC93244P) added to the Saccharomyces cerevisiae is not particularly limited thereto, but as an example, it may be 0.1 to 0.25% (w / v) relative to the volume of wort, and as another example, It may be 0.15 to 0.20% (w / v) by volume of wort, and as another example, it may be 0.18% (w / v) by volume of wort.

The term "maturation" of the present invention is a process of precipitating the suspended matter of the fermentation broth to clear the fermentation broth and carbon dioxide to be dissolved and saturated in the fermentation broth. Specifically, the aging may be performed at 10 ° C to 30 ° C for 1 to 10 days, but is not limited thereto.

In the present invention, the aging process is not particularly limited thereto, but as an example, it may be carried out in a manner of standing at 1 to 20 ° C. for 1 to 4 months; As another example, it may be carried out in a manner in which it is allowed to stand at 1 to 17° C. for 1 to 2 months; As another example, it may be carried out in a manner in which it is allowed to stand for 1.5 months at 1 to 10 °C.

Using the method of culturing the Y297 strain in Saccharomyces cerevisiae provided by the present invention, yeast strains suitable for rice beer production can be effectively cultivated and used for production of rice beer, thereby producing better quality rice beer. It will be widely used.

Figure 1 is a chart showing the results of performing RSM for each component of the G3 growth medium.
Figure 2 is a chart showing the ANOVA analysis results for the results of performing RSM for each component of the G3 growth medium.
Figure 3 is a diagram showing the second-order polynomial obtained using the optimal combination of G3 growth media.
Figure 4 is a three-dimensional graph for each component derived from the second-order polynomial obtained using the optimal combination of G3 growth medium.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited to these examples.

Example 1: Development of Saccharomyces cerevisiae Y297 strain (accession number KACC93244P) culture medium

Example 1-1: Preculture of strain Y297 in Saccharomyces cerevisiae

Saccharomyces cerevisiae Y297 strain (accession number KACC93244P) (hereinafter abbreviated as "Y297 strain") was obtained, and it was added to YPD solid medium (1% agarose, 10 g/L yeast extract, 5 g/L peptone and 10 g/L dextrose) and then cultured at 37° C. for 2 days to obtain a large number of colonies. Each colony obtained above was inoculated into 5 ml of YPD liquid medium (10 g/L yeast extract, 5 g/L peptone, and 10 g/L dextrose) and cultured with shaking at 37 ° C. for 1 day to form a primary culture was obtained. Each of the obtained primary cultures was inoculated into 30 ml of YPD liquid medium and cultured with shaking at 37 ° C. for 1 day to obtain secondary cultures, and then the highest growth rate among Y297 strains included in each culture Strains showing were selected.

Example 1-2: Primary selection of culture medium

Cell growth rate while inoculating and culturing the Y297 strain selected in Example 1-1 in each G1 growth medium (glucose, soy peptone, yeast extract, D-biotin and minor components) containing various components. By measuring, the medium with the best cell growth rate was selected. At this time, a combination of various components such as KH ₂ PO ₄ , K ₂ HPO ₄ , MgSO ₄ .7H ₂ O, and CaCl ₂ was used as minor components. As a result, a G3 growth medium capable of exhibiting the maximum growth rate of strain Y297 was selected (Table 1).

Composition of G3 growth medium ingredient Concentration (g/L) glucose
Soy Peptone
yeast extract
MgSO ₄ ·7H ₂ O
KH ₂ PO ₄
CaCl ₂
D-Biotin (mg/L) 20
5
25
2
2
0.5
2

Example 1-3: Secondary selection using RSM (response surface method)

RSM experiments were performed on each component of the G3 growth medium selected in Example 1-2 to determine the optimum content of each component.

The RSM is an experiment that informs the final concentration of each component by calculating an equation for productivity between medium components with a second-order polynomial, and is a method commonly used in the final stage of media optimization. The optimal concentration of each component is a second-order polynomial The point at which the value of is maximized can be analyzed using a 3-dimensional response surface diagram. At this time, the significance of the second-order polynomial model is determined by the P value given during ANOVA analysis. It is acknowledged.

Specifically, a total of 32 experiments were performed based on 4 variables and the central composite design method (FIG. 1).

Figure 1 is a chart showing the results of performing RSM for each component of the G3 growth medium.

As shown in Figure 1, cell growth was shown in a narrow range of about OD600 = 17 to 28, and the highest cell growth was confirmed in 16 combinations.

ANOVA analysis was performed on the RSM results of FIG. 1 using Design expert, a statistical program (FIG. 2).

Figure 2 is a chart showing the ANOVA analysis results for the results of performing RSM for each component of the G3 growth medium.

As shown in FIG. 2, the p-value was 0.0485, confirming that the model performed in the experiment was significant.

A second-order polynomial for two-dimensional response surface analysis was obtained using the combination of No. 16 obtained in FIG. 1 (FIG. 3).

Figure 3 is a diagram showing the second-order polynomial obtained using the optimal combination of G3 growth media.

The obtained second-order polynomial for the two-dimensional response surface analysis was applied to the response surface analysis method to obtain a three-dimensional graph, and analysis for each variable (concentration of each component) was performed (FIG. 4).

Figure 4 is a three-dimensional graph for each component derived from the second-order polynomial obtained using the optimal combination of G3 growth medium.

Finally, based on the results of the RSM performed, a single experiment was conducted to exchange glucose, an existing carbon source, with sucrose for the competitiveness of the yeast culture process and the stable supply of the medium for food additives. Finally, the composition of the GM growth medium was determined. (Table 2).

Composition of GM growth medium ingredient Concentration (g/L) sucrose
Soy Peptone
yeast extract
MgSO ₄ ·7H ₂ O
KH ₂ PO ₄
CaCl ₂
D-Biotin (mg/L) 30
5
30
1.6
4
0.5
2

As a result of culturing the Y297 strain using the determined GM growth medium, it was confirmed that the OD600 value was 30 to 33.

Example 2: Preparation of rice beer using the cultured Y297 strain

Y297 strain was cultured using the GM growth medium determined in Example 1-3, and rice beer was prepared using the cultured Y297 strain.

Example 2-1: Obtaining rice flour

Rice beer was prepared using two varieties of rice (Haedeulmi or Alchanmi) showing high amylose content.

As a result of analyzing the taste characteristics of the hadeulmi, it was confirmed that it contained an alkali disintegration of 6.0, a protein content of 6.1% and an amylose content of 18%. Disintegration was found to include a protein content of 5.6% and an amylose content of 18.6%.

The hadeulmi and alchanmi were polished to obtain white rice, and each of the obtained white rice was ground twice to obtain each rice flour.

Example 2-2: Preparation of rice beer

Each rice flour obtained in Example 2-1 was gelatinized for 80 minutes from 45 ° C to 100 ° C using a method of raising the temperature step by step with a time difference without adding an enzyme. In the last stage of gelatinization of rice, malt was separately saccharified after 10 minutes, and after 20 minutes, rice flour gelatinized liquid and saccharified malt were mixed to obtain a mixture. The obtained mixture was saccharified at 65° C. for 60 minutes, and final saccharification was completed at 78° C. to obtain wort.

Y297 strain was inoculated into the obtained wort, alcoholic fermentation was performed at 15 to 18 ° C for 6 days, and when the target specific gravity was reached, the temperature was raised by about 2 ° C and left for 1 day, and then cooled to 10 ° C or less. Post-fermentation was carried out for 7 days. After the post-fermentation was completed, rice beer was prepared by aging at 0 to 1 ° C. for 2 weeks, and its processability (pH, bitterness and alcohol content) was measured (Table 3).

Comparison of processing aptitude of rice beer rice variety pH bitterness Alcohol content (%) Hadeulmi
good taste 4.22±0.01
4.25±0.01 12.33±0.84
13.33±0.99 3.15±0.05
3.87±0.15

As shown in Table 3, it was confirmed that the rice beer prepared using Alchanmi showed a comparative superior quality than the rice beer prepared using Hadeulmi as a whole.

Example 2-3: Comparison of rice beer according to rice flour content

From the results of Example 2-2, it was confirmed that rice beer prepared using Alchanmi rather than Haedulmi showed a comparative advantage in quality, so the quality of beer according to the rice flour content of Alchanmi was analyzed.

Roughly, each rice beer was prepared by performing the method of Example 2-2 except for setting the content of rice flour to 0, 10, 20 or 40%, and the quality of each rice beer produced were compared (Table 4). At this time, commercially available beer was used as a control group.

Comparison of quality of rice beer according to rice flour content experimental group rice flour content Alcohol(%) Acidity (%) PH Brix amino acid(%) control group
experimental group 1
experimental group 2
experimental group 3
experimental group 4 -
0%
10%
20%
40% 4.5
4.6
4.2
4.25
4.43 0.27
0.21
0.21
0.23
0.2 4.45
4.53
4.27
4.42
4.28 5.2
6.21
6.17
6.17
6.99 0.01
0.07
0.02
0.03
0.03

As shown in Table 4, compared to the case where rice flour was not added, when rice flour was added, the overall alcohol content was lowered, but it was confirmed that it showed a tendency to increase in proportion to the added rice flour content.

It was confirmed that the sweetness and aroma components, which can affect the taste of beer, are determined by the content of soluble solids (Brix), and the content of soluble solids tends to increase in proportion to the increase in rice flour content. did

In addition, it was confirmed that the pH that maintains the stability of yeast changes according to the addition of rice flour, but is maintained within a certain range (pH 4.27 to 4.53).

On the other hand, acidity is changed by the content of rice flour, and when 20% rice flour is added, it shows a relatively high level, confirming that acidity can be added to the prepared rice beer.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without changing its technical spirit or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not limiting. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the claims to be described later and equivalent concepts rather than the detailed description above are included in the scope of the present invention.

Claims (10)

Saccharomyces cerevisiae Y297 strain (Accession No. KACC93244P) was inoculated and cultured in a medium containing sucrose, soy peptone, yeast extract, MgSO ₄ 7H ₂ O, KH ₂ PO ₄ , CaCl ₂ and D-biotin A method for culturing Saccharomyces cerevisiae Y297 strain (Accession No. KACC93244P) comprising the steps of:
According to claim 1,
The content of sucrose contained in the medium is 10 to 60 g/L, the content of soy peptone is 1 to 10 g/L, the content of yeast extract is 10 to 60 g/L, and MgSO ₄ 7H ₂ O The content of is 0.1 to 5 g/ℓ, the content of KH ₂ PO ₄ is 1 to 10 g/ℓ, the content of CaCl ₂ is 0.1 to 2.0 g/ℓ, and the content of D-biotin is 1 to 5 g/ℓ ℓ.
(a) adding a rice flour-derived component to the saccharification solution of malt and heating it to obtain wort;
(b) adding Y297 strain (Accession No. KACC93244P) to Saccharomyces cerevisiae cultured by the method of claim 1 or 2 to the wort obtained above and performing alcohol fermentation to obtain a fermentation broth; and
(c) A method for producing rice beer, comprising the step of aging the obtained fermentation broth to prepare rice beer.
According to claim 3,
In the step (a), the rice flour-derived component is a powder obtained by milling polished rice or a rice powder gelatinized liquid obtained by hydrolyzing and heating the powder.
According to claim 4,
The method, wherein the rice is hadeulmi or fruitful rice.
According to claim 3,
In step (a), the mixing ratio of rice flour and malt contained in the wort is 5:5 to 1:9 (w / w).
According to claim 6,
In step (a), the heating is performed in the order of 1 to 20 minutes at 40 to 60 ° C, 10 to 60 minutes at 60 to 80 ° C, and 30 to 90 minutes at 90 to 110 ° C.
According to claim 3,
The alcoholic fermentation of step (b) is carried out at 10 ° C to 30 ° C for 3 to 10 days.
According to claim 3,
The method of aging in step (c) is carried out at 10 ° C to 30 ° C for 1 to 10 days.
Rice beer prepared by the method of claim 3.

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