TW202338097A - Method for preparing γ-aminobutyric acid, and method for preparing energy drink containing γ-aminobutyric acid - Google Patents

Abstract

The disclosure provides a koji fungus, a yeast, a method for preparing γ-aminobutyric acid (GABA) by using the koji fungus, and a method for producing a GABA-containing functional beverage by combining the koji fungus fermented product with yeast. The method for preparing GABA by using koji fungus includes adding the koji fungus to the koji fungus basal medium for fermentation to obtain a koji fungus fermented product containing GABA. The method for producing a functional beverage containing GABA comprises adding koji fungus to a koji fungus basal medium for fermentation to obtain a koji fungus fermented product, mixing the koji fungus fermented product with a beverage medium, and adding yeast to obtain functional beverage medium, and to perform second-stage fermentation to obtain functional beverage containing GABA. Through the above-mentioned method, GABA and functional beverage having GABA can be produced.

Description

Methods for preparing gamma-aminobutyric acid, and methods for preparing functional drinks containing gamma-aminobutyric acid

The present invention relates to a method for preparing gamma-aminobutyric acid (GABA). Specifically, it relates to a method of preparing γ-aminobutyric acid and an energy drink containing γ-aminobutyric acid by using Kojima yeast compound.

γ-Aminobutyric Acid (GABA) is a non-protein natural amino acid that exists in plants, animals and microorganisms. It mainly catalyzes glutamine through glutamate decarboxylase (GAD). Acid decarboxylation is produced. The production methods of GABA include chemical synthesis, enzyme method, microbial fermentation, etc. The existence of glutamic acid decarboxylase has been discovered in various microorganisms. Therefore, using microbial fermentation to produce GABA is compared to chemical synthesis and plant enrichment. The GABA method has the advantages of low cost, high yield, and can be safely used in food. Microbial fermentation methods generally use glutamic acid, its sodium salt, or substances rich in glutamic acid as raw materials, and then ferment them using microorganisms such as yeast, fungi, or lactic acid bacteria. GABA has been proven to have a variety of physiological effects, including lowering blood pressure, calming nerves, enhancing memory, anti-anxiety, etc. Therefore, the development of functional foods containing GABA has great market potential.

With the rise of health awareness, the functional beer market is also gradually growing. Low-alcohol, low-purine, low-calorie and natural-flavored beers (no artificial flavors added) are favored by consumers, prompting the diversified development of craft beer, and new global beers. Developed towards unique flavors or health benefits, diversified products attract new consumer groups and expand the beer beverage market. At present, functional beer is mainly constructed from upstream bacterial strain mutation or genetic engineering to construct low-alcohol production yeast strains, and develops non-alcoholic and low-alcohol beer series products, or is produced through seasoning (fermentation without added yeast), or yeast After fermentation, other processes are used to remove alcohol, or fruits or spices are added as a source of flavor, or carbon dioxide is added to prepare a product with a beer-like taste.

In summary, a method that can simply prepare GABA, or a method that can directly contain GABA when preparing energy drinks is in demand by the market.

In order to achieve the above purpose, this case provides a method for preparing γ-aminobutyric acid (GABA), which includes: fermenting Monascus pilosus and a Monascus pilosus basic culture medium to obtain GABA-containing Monascus pilosus Fermentation product; wherein the koji strain is BCRC31527 or BCRC 31505.

Preferably, the koji basic culture medium includes a carbon source, a nitrogen source, an amino acid sodium salt and rice raw materials.

Preferably, the carbon source is glucose, sucrose or a combination thereof.

Preferably, the nitrogen source is protein derived from animal sources, protein derived from plants, or a combination thereof.

Preferably, the rice raw material is brown rice, white rice or germinated rice.

Preferably, based on the weight of the koji bacteria basic culture medium, the koji bacteria basic culture medium includes 0.1-5 wt% carbon source, 0.1-3 wt% nitrogen source, and 0.5-5.0 wt% amino acid salts. And 1.0~5.0 wt% of rice raw materials.

According to another object of the present invention, a method of manufacturing a beverage containing gamma-aminobutyric acid (GABA) is also provided, which includes: adding a koji fermentation product prepared by the aforementioned method to a beverage culture medium to obtain a beverage culture medium. The energy drink culture medium is added to the energy drink culture medium with a yeast cultured in a yeast basic culture medium to perform the second stage of fermentation.

Preferably, the yeast strain is Saccharomyces cerevisiae BCRC21377 or BCRC 21800.

Preferably, the combination of the koji and the yeast is a combination of BCRC31527 and BCRC 21377, a combination of BCRC31527 and BCRC 21800, or a combination of BCRC 31505 and BCRC 21377.

Preferably, the beverage culture medium contains wort and sucrose.

Preferably, the volume ratio of the koji fermentation product to the beverage culture medium is 2:8 to 4:6.

Preferably, the fermentation time is 7 to 14 days and the temperature is 25˚C.

Through the above technical features, the present invention at least provides the following advantages: (1) Provide a simple method for preparing GABA, which can increase GABA production and reduce production costs. (2) A method of manufacturing a GABA-containing beverage is provided, which can contain GABA when manufacturing the beverage, save additional addition costs, and reduce manufacturing steps.

The above and other objects, features and advantages of the present invention will become apparent with reference to the following detailed description and preferred embodiments and the accompanying drawings.

In the following detailed description, in order to explain the present invention, numerous specific details are provided to provide a thorough understanding of the disclosed embodiments. It will be apparent, however, that one or more embodiments may be practiced without the specific details recited. In other instances, well-known structures and processes are shown schematically in order to simplify the drawings.

Details of each specific embodiment of the present invention are described below. Other features of the present invention will be clearly presented through the detailed description and patent scope of each specific example below. Without further elaboration, it is believed that one with ordinary skill in the art to which the present invention belongs can utilize the present invention to the widest extent based on the foregoing description. Therefore, it can be understood that the following description is only for illustrative purposes and does not limit the remaining disclosure in any way. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In order to achieve the purpose of the present invention, a fermentation test of koji bacteria and rice was first performed to screen strains with GABA production potential, and the raw material composition formula and fermentation conditions were discussed, and then a method for preparing GABA was completed. Next, the koji fermentation product obtained by the koji fermentation is combined with yeast for a second stage of fermentation to produce an energy drink containing GABA. Specifically, this case provides a method for preparing GABA through koji bacteria with high GABA production; and a second-stage fermentation of the koji bacteria fermentation product containing GABA with an energy drink culture medium and yeast to obtain a functional product with high GABA content. Drink method.

First, refer to Figure 1, which illustrates a method for preparing GABA according to an embodiment of the present invention. In step S00, the koji bacteria with high GABA production potential are screened out and the rice used as the koji bacteria basic culture medium is fermented and cultured to obtain high-yield GABA. In this embodiment, the rice used in the koji basic culture medium can be brown rice, white rice, or germinated rice, preferably rice produced in Taiwan.

Referring to Figure 2, Figure 2 is a flow chart of a method for manufacturing a GABA-containing energy drink according to an embodiment of the present invention.

In step S10, rice is used as the koji bacteria basic culture medium, and koji bacteria are added to perform the first stage of fermentation culture. In this embodiment, the koji bacteria can be Aspergillus oryzae (BCRC 31163) or Monascus ruber (BCRC 31535) , Monascus pilosus (BCRC 31502, BCRC 31505, BCRC 31526, BCRC 31527) , Monascus purpureus (BCRC 31540, BCRC 31615)), the rice used in the koji bacteria basic culture medium can be brown rice, white rice, germinated rice, preferably rice produced in Taiwan. The screening methods for Koji bacteria will be explained in detail later. In step S20, the koji fermentation product obtained in step S10 is mixed with the beverage culture medium. In this embodiment, the beverage culture medium includes wort and sucrose. Specifically, the ratio of sucrose to wort can be 2~6 g/100 mL, preferably 3~5 g/100 mL, and more preferably It is 4~4.5 g/100mL. The present invention is not limited to this, and the sucrose content can be adjusted according to fermentation requirements, sweetness, etc.

In step S30, a yeast basic culture medium (Saccharomyces cerevisiae) containing yeast is added to the energy drink culture medium obtained by mixing the koji fermentation product and the beverage culture medium, and fermentation is performed. In this embodiment, the fermentation environment is at a temperature of 24˚C to 26˚C, preferably 25˚C, and the fermentation time is 7 to 14 days to obtain an energy drink containing GABA. According to a preferred embodiment of the present invention, the fermented fermentation product can be precipitated, filtered or centrifuged to remove bacterial cells, and the resulting liquid can be collected. The obtained functional drinks include functional beverage products (alcohol content <0.5%) or functional beer products (alcohol content >0.5%).

The methods for screening koji and yeast included in the present invention and the basis for judgment will be described in detail below through examples.

(1) GABA analysis: high performance liquid chromatography (HPLC method)

GABA content was analyzed by HPLC using GABA-OPA derivatization method. Prepare GABA solutions with different concentrations from 5 to 500 ppm. Mix the sample and OPA derivatization agent at a ratio of 1:5 (v/v). After reacting for 1 minute in the dark at room temperature, filter with a 0.22 μm filter and take 20 μL. The filtrate was analyzed by HPLC. Gradient conditions are shown in Table 1. HPLC column: RP-18 column (5μm, 120*4mm, Merck) HPLC mobile phase: A:Phosphate buffer B: Acetonitrile C: Deionized water (DIW) Detection wavelength: UV338nm

Table 1. GABA gradient conditions for HPLC analysis Time (min) Mobile phase A(%) Mobile phase B(%) Mobile phase C(%) 0 87 13 0 10 87 13 0 12 0 15 85 20 0 85 15 25 87 13 0 30 87 13 0

According to the chromatogram analyzed by HPLC, the retention time of GABA is about 17.5 minutes. The integrated area of GABA at different concentrations analyzed by HPLC is plotted against its content to obtain the calibration line of GABA. The GABA content in the sample can be obtained by incorporating the integrated area obtained from HPLC analysis of the fermentation sample into the formula.

(2) Screening of strains with GABA production potential

1. Strains

The strains selected in this case are "biological materials that are commercially available to the public" and "biological materials that have been deposited in a reputable depository institution before application and are freely transferable." According to Article 30 of the current Patent Law A proviso stipulates that deposit is not required.

According to the screening steps of the embodiments of the present invention, koji bacteria are screened from the following strains: (1) Aspergillus oryzae (BCRC 31163) and (2) Monascus ruber BCRC 31535, Monascus pilosus (BCRC 31502, BCRC 31505, BCRC 31526, BCRC 31527) and Monascus purpureus (BCRC 31540, BCRC 31615).

Yeast was screened from the following 7 strains of Saccharomyces cerevisiae : BCRC 21680 (NO.12), BCRC 21377 (NO.14), BCRC 21800 (NO.16), BCRC 20848 (NO.1), BCRC 21791 (NO.5), BCRC 21459 (NO.6), BCRC 20262 (NO.7).

2. Culture medium

According to an embodiment of the present invention, it includes (1) Kojima basic culture medium, (2) Yeast basic culture medium, (3) GABA screening medium, (4) beverage culture medium and (5) functional beverage culture medium. Detailed ingredients are as follows.

(1) Koji bacteria basic culture medium, including rice produced in Taiwan: brown rice, white rice, germinated rice, rice is finely ground into powder with a particle size of 10 mesh to 100 mesh, glucose (purchased from Merck), monosodium glutamate (sodium glutamate, MSG) , purchased from Weiwang Co., Ltd.) and Peptone (Peptone, purchased from Merck). The detailed proportions added per liter of solution are shown in Table 2.

Table 2 composition g/L rice flour 20 glucose 40 MSG 10 Protein 10

(2) Yeast basic culture medium, including yeast extract, malt extract, proteinaceous and glucose. The detailed proportions added per liter of solution are shown in Table 3.

table 3 Yeast basic medium ( purchased from Difco) g/L yeast extract 3.0 malt extract 3.0 Protein 5.0 glucose 10.0 Agar (Merck) 15.0

(3)GABA screening medium

Add 0.01% bromocresol green (Sigma-Aldrich), 0.2% glyoxylic acid (Sigma-Aldrich) and 0.2% succinic acid (Merck) to the aforementioned yeast basic culture medium.

(4) Beverage culture medium, the composition is shown in Table 4

Table 4 composition share Basic wort (7.3%)※ 210mL Sucrose (Taiwan Sugar Refined Special Sand) 9g

(5) Functional beverage culture medium, the composition is shown in Table 5

table 5 composition share Koji ferment 90mL Basic wort (7.3%)※ 210mL Sucrose (Taiwan Sugar Refined Special Sand) 9 g ※Basic wort: Black Rock Pale Ale concentrated malt (Black Rock Pale Ale), purchased from Beer King, contains hops (Green Bullet and Pacific Gem), add water to prepare 7.3% (w/w) wort.

A. Screening test for GABA production of Koji mold

Use koji bacteria basic culture medium (white rice), add koji bacteria (1 strain of koji bacteria, 7 strains of red koji bacteria) at 25˚C, 125rpm for fermentation, and screen the koji bacteria with the potential to produce GABA. The fermentation product was fermented for 7 days and 11 The appearance of the sky is shown in Figure 3. The results of analyzing the GABA content of Koji fermentation products are shown in Figure 4. There are 4 strains with GABA production higher than 200 ppm, namely BCRC 31502, BCRC 31505, BCRC 31527 and BCRC 31163.

B. Yeast GABA production analysis test

131 strains of Saccharomyces cerevisiae were activated with yeast basic culture medium (yeast plate), cultured at 25°C for 3 days, and then a single colony was picked using an inoculation ring to GABA selection medium, cultured at 25°C for 3 days, and observed The blue-green color of the colony represents the ability of the tested strain to produce GABA. As shown in Figure 5, the higher the GABA content, the darker the color of the colony.

Seven strains of Saccharomyces cerevisiae with GABA production potential were screened out from 131 strains of Saccharomyces cerevisiae: BCRC 21680 (NO.12), BCRC 21377 (NO.14), BCRC 21800 (NO.16), BCRC 20848 (NO.1), BCRC 21791 (NO.5), BCRC 21459 (NO.6), BCRC 20262 (NO.7).

7 strains of yeast were activated on the yeast plate at 25˚C for 2 days. Use sterilized RO water to wash off the cells from the yeast plate so that the OD ₆₀₀ of the bacterial suspension is 20~30. Use an inoculation amount of 0.5 % was inoculated into the beverage culture medium and fermented at 25˚C. The GABA content of the fermentation samples was analyzed on days 7 and 14, as shown in Figure 6. In addition, the sugar content (Brix%), alcohol content, observation of beverage bubbles and sensory evaluation results are shown in Table 6. This result was then compared with the data of low-alcohol Saccharomyces cerevisiae, and three Saccharomyces cerevisiae strains with low alcohol and GABA production potential were identified, namely BCRC 21680 (NO.12), BCRC 21377 (NO.14) and BCRC 21800(NO.16). Based on the total sensory evaluation score and preference ranking, to evaluate the palatability and acceptability of GABA-containing beverages, a total of 5 yeast strains were selected, namely NO.5 (BCRC 21791), NO.6 (BCRC 21459), and NO.12 (BCRC 21680), NO.14 (BCRC 21377) and NO.16 (BCRC 21800).

Table 6 Strain number NO. Brix% Amount of bubbles ( maximum 5 points) Alcohol (%) Ranking of taste ratings 1 6.4 3 3.5 6 5 6.6 4 3.0 5 6 4.9 2 5.0 3 7 5.4 5 4.5 7 12 5.1 1 4.5 4 14 9.2 1 1.0 1 16 6.7 5 3.0 2

Based on the above results, the koji bacteria BCRC 31505, BCRC 31527 and BCRC 31163 were selected, and the top five yeasts NO.5 (BCRC 21791), NO.6 (BCRC 21459), NO.12 (BCRC 21680) and NO.14 were selected. (BCRC 21377) and NO. 16 (BCRC 21800) conducted GABA functional beverage fermentation experiments in the specific examples described below.

(3) Specific embodiments

The effect of the method for preparing GABA provided in this case is illustrated below through Example 1 of fermentation test of the selected Koji bacteria.

Example 1

First, Example 1 of this case uses koji basic culture medium (germinated rice, brown rice, white rice), and adds koji bacteria (koji bacteria (Koji mold BCRC 31163, red koji bacteria BCRC 31502, BCRC 31505, BCRC 31527)) at 25˚C, 125rpm. Fermentation was carried out in a grooved shake flask. The appearance of the fermentation product at 7 days (a) and 14 days (b) is shown in Figure 7. The results of analyzing the GABA content of the Kojima fermentation product are shown in Figure 8 (7 days) and Figure 7. 9 (14 days) and Figure 10 (comprehensive average of different strains of the same raw material), where three kinds of raw materials (germinated rice, brown rice, white rice) were mixed with koji bacteria and fermented for 7 days, the koji of the combination of BCRC 31163 and brown rice The GABA content of the yeast fermentation products was higher than 75 ppm, and after 14 days of fermentation, most of the GABA content in the koji fermentation products showed a significant increase (Figure 9). In addition, after comparing the three raw materials after 14 days of fermentation, the GABA content produced by each strain was higher than 75 ppm on average, and there was no significant difference between different raw materials, as shown in Figure 10. In addition, as shown in Figure 7, the appearance of the koji fermentation product of BCRC 31527 was red. At this point, the GABA component can be obtained from the koji fermentation product obtained by koji fermentation.

As mentioned above, the method for preparing GABA provided by the present invention includes fermenting Koji bacteria and Koji bacteria basic culture medium; wherein the Koji bacteria can be Aspergillus oryzae (BCRC 31163) or Monascus ruber (BCRC 31502) , BCRC 31505, BCRC 31527).

Next, the method for manufacturing a GABA-containing energy drink provided in this case is explained by performing the first-stage fermentation with Koji and combining it with yeast for the second-stage fermentation to achieve the production of a GABA-containing energy drink in Example 2. Effect.

Example 2

Example 2 of this case uses the koji bacteria basic culture medium (white rice), adds koji bacteria (koji bacteria 31163, red koji bacteria 31505, 31527) at 25˚C, 125rpm and uses a grooved shake flask to ferment for 14 days. The koji bacteria fermentation After filtering, the material is mixed with the beverage culture medium in a ratio of 3:7 to obtain the functional beverage culture medium. This embodiment is not limited to this. The ratio of the koji fermentation product to the beverage culture medium may be 2:8, 2.5:7.5, 3:7, 3.5:6.5, 4:6, etc.

Then, activate the yeasts on the YM plate at 25˚C for 2 days, use sterilized RO water to wash the bacterial cells from the YM plate, so that the OD ₆₀₀ of the bacterial suspension is 20~30, and divide 5 strains with GABA production into Potential Saccharomyces cerevisiae (NO.5, NO.6, NO.12, NO.14, NO.16) was inoculated into the energy drink medium with an inoculation amount of 0.5%, and the energy drink was obtained after fermentation at 25˚C for 7 days. These functional drinks were analyzed for GABA content, sugar content, alcohol content and sensory evaluation. The results are shown in Figures 11 to 14 and Table 7. Figure 11 shows the appearance of using koji bacteria basic medium (white rice) and fermenting it in a grooved shake flask at 25˚C for 14 days. It can be seen that the fermentation product of BCRC 31527 is in the same state as Example 1 and still appears red. Figure 12 shows the GABA content of koji bacteria basic culture medium (white rice) fermented at 25˚C for 14 days using a grooved shake flask. As shown in the figure, it can be seen that BCRC 31527 has the highest GABA production. Figure 13 shows the appearance of 15 kinds of beverages obtained by fermenting different koji and yeast for 7 days according to the embodiment of the present invention. After fermentation by yeast, the fermentation product of BCRC 31527 still remains a red liquid. Figure 14 shows the GABA content measured in 15 kinds of beverages fermented by different koji and yeast for 7 days according to the embodiment of the present invention. In terms of GABA content, the yeast is Saccharomyces cerevisiae , preferably BCRC 21791 (NO. 5), BCRC 21800 (NO. 16 low alcohol potential strain) and BCRC 21377 (NO. 14 low alcohol potential strain). On the other hand, these energy drinks can also be sedimented, filtered or centrifuged to remove bacteria to improve their taste. The obtained functional beverage can include functional beverage products (alcohol content <0.5%) or functional beer products (alcohol content >0.5%) through formula adjustment.

Combining the results in Figures 11 to 14 and Table 7, it is possible to select a koji and yeast combination that has both good taste and high GABA content. The culture results showed that the process was reproduced well. The koji fermentation product containing BCRC 31527 in the energy drink culture medium still showed a red appearance. In addition, there were 6 beverages with GABA production higher than 100 ppm, namely 31505-5, 31505-14, and 31505- 16, 31527-5, 31527-14 and 31527-16, among which 31527-14, 31527-16 and 31505-14 ranked the top three in terms of consumer sensory evaluation. After consumer sensory evaluation and comprehensive GABA content consideration, the best combinations of koji and yeast are 31527-14, 31527-16 and 31505-14.

Table 7 Koji yeast Brix (%) Alcohol(%) Likeability ranking 31163 5 3.9 4.0 14 6 3.0 4.9 10 12 3.0 4.9 4 14 3.9 4.0 9 16 4.1 3.8 5 31505 5 4.2 3.6 12 6 3.1 4.7 8 12 3.1 4.7 15 14 6.3 1.5 3 16 3.3 4.5 13 31527 5 4.3 4.0 11 6 3.2 5.1 7 12 3.1 5.2 6 14 6.2 2.1 1 16 3.3 5.0 2

In summary, through the embodiments of the present invention, a method for preparing GABA can be provided, and GABA can be prepared in a simple manner. In addition, the method of preparing GABA by Koji mold can be combined with yeast to perform a second-stage fermentation to provide a method of manufacturing an energy drink containing GABA, which can achieve the effects of reducing manufacturing steps and saving costs. In addition, GABA-containing energy drinks with different appearances and different tastes can be provided by selecting a combination of different strains.

The above is merely illustrative and not restrictive. Any equivalent modifications or changes that do not depart from the spirit and scope of the present invention shall be included in the scope defined by the patent application.

S10~S50: steps

Figure 1 is a flow chart of a method for preparing GABA according to an embodiment of the present invention. Figure 2 is a flow chart of a method for manufacturing a GABA-containing energy drink according to an embodiment of the present invention. Figure 3 shows the appearance of the fermentation product on day 7 (left) and day 11 (right) of using koji bacteria basic culture medium and koji bacteria for fermentation to screen koji bacteria with GABA potential according to an embodiment of the present invention. Figure 4 shows the GABA content measured by the HPLC method on the 7th and 11th day of the koji fermentation product fermented with the koji basic culture medium according to the embodiment of the present invention. Figure 5 shows the appearance of yeast colonies with GABA production potential through plate rapid screening according to an embodiment of the present invention. Figure 6 shows the GABA content measured by HPLC method on the 7th day and the 14th day of the fermentation product of the yeast fermented with the beverage culture medium according to the embodiment of the present invention. Figure 7 shows the appearance of the koji yeast fermentation product on the 7th day (a) and the 14th day (b) according to the embodiment of the present invention, using the koji bacteria basic medium and adding koji bacteria for fermentation. Figure 8 shows the GABA content measured by the HPLC method on the 7th day of the koji fermentation product fermented with the koji basic culture medium according to the embodiment of the present invention. Figure 9 shows the GABA content measured by the HPLC method on the 14th day of the koji fermentation product fermented with the koji basic culture medium according to the embodiment of the present invention. Figure 10 shows the average GABA content measured in the koji fermentation products of three kinds of rice raw materials fermented by different koji bacteria for 14 days according to the embodiment of the present invention. Figure 11 shows the appearance of the koji fermentation product using koji bacteria basic culture medium (white rice) and fermented at 25˚C for 14 days in a grooved shake flask according to an embodiment of the present invention. Figure 12 shows the GABA content of fermentation at 25˚C for 14 days using koji bacteria basic culture medium (white rice) according to an embodiment of the present invention. Figure 13 shows the appearance of 15 kinds of beverages obtained by fermenting different koji and yeast for 7 days according to the embodiment of the present invention. Figure 14 shows the GABA content measured in 15 kinds of beverages fermented by different koji and yeast for 7 days according to the embodiment of the present invention.

S00: Steps

Claims (12)

A method for preparing gamma-aminobutyric acid (GABA), which includes: fermenting Monascus pilosus and a Monascus pilosus basic culture medium, wherein the Monascus pilosus strain is BCRC 31527 or BCRC 31505. The method of claim 1, wherein the koji bacteria basic culture medium includes a carbon source, a nitrogen source, an amino acid sodium salt and rice raw materials. The method according to claim 2, wherein the carbon source is glucose, sucrose or a combination thereof. The method of claim 2, wherein the nitrogen source is protein derived from animal sources, protein derived from plants, or a combination thereof. The method as described in claim 2, wherein the rice raw material is brown rice, white rice or germinated rice. The method as described in claim 1, wherein based on the weight of the koji bacteria basic culture medium, the koji bacteria basic culture medium includes 0.1-5 wt% carbon source, 0.1-3 wt% nitrogen source, 0.5-5.0 wt% of amino acid salts and 1.0 to 5.0 wt% of rice raw materials. A method of manufacturing an energy drink containing gamma-aminobutyric acid (GABA), which includes: adding a koji fermentation product prepared by the method described in any one of claims 1 to 6 to a beverage culture medium to An energy drink culture medium is obtained, a yeast ( Saccharomyces cerevisiae ) cultured in a yeast basic culture medium is added to the energy drink culture medium, and then the second stage of fermentation is performed. The method of claim 7, wherein the yeast strain is BCRC 21377 or BCRC 21800. The method of claim 7, wherein the combination of the koji and the yeast is a combination of BCRC31527 and BCRC 21377, a combination of BCRC31527 and BCRC 21800, or a combination of BCRC 31505 and BCRC 21377. The method of claim 7, wherein the beverage culture medium contains wort and sucrose. The method according to claim 7, wherein the volume ratio of the koji fermentation product and the beverage culture medium is 2:8 to 4:6. The method as described in claim 7, wherein the fermentation time of the second stage fermentation is 7 to 14 days and the temperature is 25˚C.