KR102529249B1 - Novel native fermented yeast Saccharomyces cerevisiae BC-2500 for using production of craft beer containing high esters with excellent flavor-related sensory - Google Patents

Abstract

The present invention relates to Saccharomyces cerevisiae BC-2500, a native fermentation yeast which can be used in production of craft beer containing high esters with excellent flavor-related functionality, wherein the yeast of the present invention is resistant to high concentrations of sugar and an acidic environment and is capable of rapid growth and high concentration of alcohol production, and can used as a substitute for a foreign brewing yeast as flavor substances are enhanced.

Description

Novel native fermented yeast Saccharomyces cerevisiae BC-2500 for using production of craft beer containing high esters with excellent flavor-related sensory}

The present invention is a native fermentation yeast Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) about BC-2500.

Beer is the world's oldest fermented alcoholic beverage and one of the most popular alcoholic beverages. The reason why beer was brewed in Europe a long time ago and spread all over the world is deeply related to the raw materials such as pure water, high-quality barley, and delicate aroma and taste of hops. In addition, various beers are produced according to manufacturing methods and local traditions. Modern beer is first estimated to have been introduced with Western culture at the end of the 19th century.

On the other hand, the first beers produced on the Korean Peninsula were Joseon Beer and Sohwa Kirin Beer, which were established by Japanese capital in 1933. The two companies were managed by the U.S. military government after liberation on August 15, but were sold to the private sector, and Chosun Beer became Crown Beer and Sohwa Kirin Beer became Oriental Beer. This leads to Hite Beer and OB Beer, respectively.

According to the Liquor Tax Act of Korea, beer is defined as "fermented with one or more of germinated barley, hops (including those extracted from hops), rice, barley, corn, sorghum, potato, starch, sugar, caramel, and water as raw materials. It is defined as "purified by filtering or filtering."

A commonly used beer manufacturing method includes a malt grinding step of grinding malt in a grain grinder; A malt juice preparation step of putting the pulverized malt into a saccharification tank containing water and heating it to extract malt juice; A wort filtration step of transferring and filtering the extracted wort into a filtration tank; A wort boiling step of mixing and heating the filtered wort and hops in a boiling tank (boiling tank); A whirlpool step of removing malt and hop residues by putting the wort after the boiling step into a whirlpool and allowing it to stand; A cooling and transfer step of lowering the temperature of the malt juice after the whirlpool step through a heat exchanger and transferring it to a fermenter, injecting yeast in the middle of the transfer and supplying oxygen; A fermentation step of fermenting the wort transferred to the fermentation tank for 2 to 7 days to produce young beer; A maturation step of putting the immature beer into a maturation tank and aging it by lowering the temperature; and filtering the aged beer. Therefore, beer has four main ingredients: malt, hops, yeast, and water.

On the other hand, craft beer (craft beer) is manufactured using the same four main ingredients as malt, hops, yeast, and water, as in conventional beer, and this is mainly done by individuals or small breweries. Homemade beer is mainly made according to a manufacturing method developed by itself, and is characterized in that it can have various tastes depending on the type of malt, the content of malt, the type of yeast, fermentation method, fermentation temperature, aging period, and auxiliary ingredients.

The size of the Korean craft beer (craft beer) market in 2020 was KRW 118 billion, a 50% increase compared to the previous year. The Korea Craft Beer Association predicts that the size of the Korean craft beer market will grow to 230 billion won by 2023.

However, most of the yeast used in Korea relies on imported yeast. Accordingly, it is necessary to develop high-quality yeast that can replace imported yeast.

Korean Patent Registration No. 10-1087760 (November 22, 2011) discloses a novel recombinant yeast strain and a method for simultaneous production of ethanol and a target protein using the same. Korean Patent Registration No. 10-1073145 (2011.10.06) discloses a high-yield ethanol producing yeast, Saccharomyces cerevisiae KK1, and an ethanol fermentation method using the same.

An object of the present invention is to provide domestic yeast isolated from domestic traditional fermented foods as a substitute for imported brewing yeast.

Another object of the present invention is to provide a method for producing ethanol using domestic yeast.

Another object of the present invention is to provide a method for producing beer using domestic yeast.

The present invention is Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) BC-2500 (accession number: KFCC11937P).

At this time, the Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) BC-2500 is preferably a brewing strain for beer fermentation.

In addition, in the present invention, in producing ethanol using yeast, the yeast, Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) BC-2500 (accession number: KFCC11937P) is provided.

In addition, the present invention is Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) BC-2500 (accession number: KFCC11937P) as a brewing strain.

The yeast of the present invention has the advantage of being resistant to high concentrations of sugar and acidic environments, enabling rapid growth and production of high concentrations of alcohol, and being able to be used as a substitute for foreign brewing yeast due to enhanced flavor substances.

Yeast used for the production of craft beer is currently dependent on imported yeast. Therefore, in the present invention, yeast capable of rapidly growing in a high concentration of sugar and acidic environment and producing high concentration of ethanol was searched for and isolated from traditional fermented foods.

In the present invention, the above strain was named ' Saccharomyces cerevisiae BC-2500', and was deposited with the Korea Microorganism Conservation Center and was given accession number KFCC11937P on July 27, 2022.

Saccharomyces cerevisiae BC-2500 (accession number: KFCC11937P) of the present invention has sugar tolerance, acid resistance and ethanol production ability comparable to control yeast, which is a foreign brewing yeast, and ethanol resistance compared to control yeast As it has been confirmed that this has been improved, it can be used as a substitute for foreign brewing yeast that has been dependent on existing imports.

In addition, in one embodiment of the present invention, Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) By using the BC-2500 strain, the productivity of isoamyl acetate is enhanced, and the flavor components having the fresh fruit flavor, banana flavor, and orientation are promoted It was confirmed that the production of beer was possible.

Accordingly, it was confirmed that the yeast of the present invention can be used to produce beer faster and to produce beer with excellent flavor containing high concentration of alcohol. That is, Saccharomyces cerevisiae of the present invention ( Saccharomyces cerevisiae ) BC-2500 (accession number: KFCC11937P) is preferably used as a brewing strain for beer fermentation.

On the other hand, the present invention Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) BC-410 (accession number: KFCC11937P).

The beer of the present invention preferably comprises the steps of (a) adding pulverized malt to water at 50 to 80 ° C and adding salt for brewing; (b) extracting sugar by raising the temperature to 60-90°C; (c) sparging by raising the temperature to 65-95 ° C; (d) sterilizing and cooling; And (e) inoculating the yeast of the present invention; it can be prepared through a method comprising the.

At this time, in the step of extracting the sugar, a higher temperature should be set than the temperature of the malt additives to facilitate sugar extraction, and in the sparging step, a higher temperature should be set than in the step of extracting the sugar to extract residual sugar. Suitable.

Meanwhile, in the sterilization process, hops or yeast nutrients may be optionally further added, and through the process, flavor may be imparted to beer, growth of bacteria may be inhibited, and growth of yeast may be promoted.

In addition, the cooling refers to a process of cooling to a temperature of 15 to 45 ° C., and the temperature range is not limited (limited) as long as the temperature is suitable for the growth temperature of yeast for inoculating beer.

In addition, conventionally known techniques can be further applied for the production of beer, and processes widely known to those skilled in the art can be used, and detailed description thereof will be omitted.

On the other hand, in the present invention, in producing ethanol using yeast, the yeast, Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) BC-2500 (accession number: KFCC11937P) is provided.

The yeast of the present invention has the advantage of being resistant to high concentrations of sugar and acidic environments, enabling rapid growth and production of high concentrations of alcohol, and being able to be used as a substitute for foreign brewing yeast due to enhanced flavor substances.

Hereinafter, the contents of the present invention will be described in more detail through the following examples or experimental examples. However, the scope of the present invention is not limited only to the following examples or experimental examples, and includes modifications of equivalent technical ideas.

[ Example 1: Screening and identification of yeast strains of the present invention]

1. Selection of yeast strains

1,000 pieces of yeast were separated from Nuruk to select strains with excellent sugar tolerance, acid resistance, and ethanol production ability.

YPDA (Yeast extract 10g/L, Peptone 20g/L, Glucose 20g/L, Agar 15-20g/L) medium containing ampicillin (100μg/ml) or kanamycin (100μg/ml) respectively to isolate yeast was used.

After washing the sample three times with sterilized distilled water, it was diluted with 10 ^-2 to 10 ^-8 and spread on the YPDA medium prepared above, followed by stationary culture at 30 °C for 3 days. Thereafter, in order to preserve single colonies that appear to be yeast, they were made to contain 15 to 20% glycerol and stored in a cryogenic freezer (-75 ° C to -80 ° C).

In addition, in order to compare the efficacy of the yeast strain according to the present invention, commercial yeast (USA) known to have excellent ethanol production ability, sugar tolerance, acid resistance and ethanol resistance was used as a control strain.

2. Ethanol ability to produce comparison

In order to select strains with excellent ethanol-producing ability, ethanol-producing ability experiments were conducted using the strains selected above. Specifically, the control strain commercial yeast (USA) and the experimental group strains were inoculated in 10 ml of 2% YPD medium and incubated for 24 hours at 30 ° C. and 200 rpm in a shaking incubator. Then, the strain was inoculated to an initial OD of 0.1 in 10 ml each of 2%, 10% and 20% YPD medium, cultured for 24 hours at 30 ° C. and 200 rpm in a shaking incubator, and samples were taken immediately after the culture was completed.

The collected samples were analyzed by HPLC to confirm the ethanol production. The HPLC used in the analysis was Agilent 1100 Series, the column was Aminex's HPX-87H, and the mobile phase was 5 mM sulfuric acid buffer. All samples were measured three times to analyze the data, and the results are shown in Table 1 below.

strain/condition 2% YPD 10% YPD 20% YPD commercial yeast 5.83g/L 22.53g/L 19.13g/L BC-2500 5.12 g/L 22.04g/L 17.49 g/L

As a result of the experiment, the BC-2500 yeast strain shows ethanol production comparable to that of commercial yeast (USA). By showing the production, it was confirmed that the BC-2500 strain had excellent ethanol production ability.

3. glucose tolerance comparison

In order to compare the sugar tolerance of the selected strains, a single colony was inoculated into 10 ml of YPD (Yeast extract 10g/L, Peptone 20g/L, Glucose 20g/L) and cultured for 24 hours at 30℃ and 200rpm in a shaking incubator. .

Thereafter, the culture medium was added to YPD (Yeast extract 10g/L, Peptone 20g/L, Glucose 20-300g/L) medium containing 2%, 10%, 20%, and 30% glucose, respectively, with an initial OD of 0.1 in 10 ml each. Inoculated as much as possible. Each of the inoculated cultures was dispensed twice in an amount of 180 µl into a 96-well plate. Thereafter, the cells were incubated for 48 hours at 30° C. and 180 rpm in a Microplate reader (Synergy HT, BioTek instrument, Winooski, VT, USA). Then, the absorbance at 600 nm was measured every 30 minutes and compared, and the results are shown in Tables 2 and 3 below, with the BC-2500 yeast strain showing the best sugar tolerance as a representative.

Comparison of specific growth rates strain/condition 2% YPD 10% YPD 20% YPD 30% YPD commercial yeast 0.105 0.051 0.031 0.029 BC-2500 0.092 0.045 0.031 0.027

Comparison of maximum absorbance strain/condition 2% YPD 10% YPD 20% YPD 30% YPD commercial yeast 1.295 1.575 1.455 1.351 BC-2500 1.269 1.573 1.514 1.298

As a result of the experiment, the BC-2500 strain was found to have a specific growth rate and maximum absorbance comparable to commercial yeast (USA), and it was confirmed that the BC-2500 strain also had excellent sugar tolerance.

4. acid resistance comparison

Lactic acid was added to YPD medium containing 2% glucose to prepare 2% YPD at pH 5.5, 4.5, 3.5 and 2.5, respectively, and used as the medium. Thereafter, a single colony was inoculated into 10 ml of YPD medium and cultured for 24 hours at 30° C. and 200 rpm in a shaking incubator.

The culture medium was inoculated to an initial OD of 0.1 in 10 ml each of 2% YPD medium at pH 5.5, 4.5, 3.5 and 2.5. Each of the inoculated cultures was dispensed twice in an amount of 180 µl into a 96-well plate. Thereafter, the cells were incubated for 48 hours at 30° C. and 180 rpm in a Microplate reader (Synergy HT, BioTek instrument, Winooski, VT, USA). Afterwards, the absorbance at 600 nm was measured every 30 minutes and compared, and the results are shown in Tables 4 and 5 with BC-2500 yeast strain showing the best acid resistance as a representative.

Comparison of specific growth rate strain/condition 2% YPD , pH3 .5 2% YPD , pH4 .5 2% YPD , pH5 .5 commercial yeast 0.046 0.095 0.087 BC-2500 0.042 0.084 0.089

Comparison of maximum absorbance strain/condition 2% YPD , pH3 .5 2% YPD , pH4 .5 2% YPD , pH5 .5 commercial yeast 1.317 1.233 1.264 BC-2500 1.338 1.169 1.233

As a result of the experiment, both the control and experimental strains did not grow in YPD 2%, pH 2.5 medium. However, in other acidic conditions, the BC-2500 strain was found to have a high specific growth rate and maximum absorbance comparable to commercial yeast (USA), confirming that the BC-2500 strain had excellent acid resistance.

5. Ethanol Resistance Comparison

Through the above experiments, the ethanol resistance of the BC-2500 strain, which was identified as a strain with excellent sugar tolerance, acid resistance, and ethanol production ability, was confirmed. Specifically, a medium containing 3%, 5%, 7%, and 10% ethanol in 2% YPD was prepared.

Control strains, commercial yeast (USA) and BC-2500 strain, were each inoculated into 10 ml of 2% YPD medium and incubated for 24 hours at 30° C. and 200 rpm in a shaking incubator. Then, the culture medium was inoculated to an initial OD of 0.1 in each of 10 ml of YPD 2% medium containing 3, 5, 7, and 10% of ethanol prepared in advance, and cultured for 24 hours at 30 ° C. and 200 rpm in a shaking incubator. As a result, are shown in Table 6 below.

Absorbance comparison strain/condition 2% YPD , EtOH 3% 2% YPD , EtOH 5% 2% YPD , EtOH 7% 2% YPD , EtOH 10% commercial yeast 7.87 6.67 3.92 0.15 BC-2500 5.66 4.19 2.83 0.19

As a result of the experiment, the BC-2500 strain showed a higher 24-hour OD than the commercial yeast (USA) at 10% ethanol concentration compared to the control strain, confirming that the BC-2500 strain had excellent ethanol resistance.

6. Identification of the BC-2500 strain

As a result of the synthesis of the above experiments, 'BC-2500', a strain showing excellent sugar tolerance, acid resistance, ethanol production ability and ethanol resistance, was selected as the strain of the present invention and identified. For identification of the strain, 18S rRNA nucleotide sequence was sequenced.

Specifically, Dyne Direct PCR (containing UDG/UTP) (BN430, Dynbio) was first performed on the colonies of the selected strain using NS1 (5'-GTAGTCATATGCTTGTCTC-3') and NS24 (5'-AAACCTTGTTACGACTTTTA-3') primers. PCR was performed using Then, the PCR product was purified using LaboPass™ PCR Purification Kit (CMR0112, Cosmogenetech). Thereafter, sequencing was requested to Macrogen using the ABI 3730XL System with the same primers. A BLAST program was run to identify the sequencing-analyzed nucleotide sequence, and it was confirmed that the strain had 99% homology with Saccharomyces cerevisiae. In addition, the sequence of 18S rRNA of strain BC-2500 is shown in SEQ ID NO: 1.

[ Example 2: Production of beer using the yeast strain of the present invention]

After grinding 5 kg of 2-Row Pale Malt (Weyermann, German) malt, it was put into 28 L of water set at 66°C. Then, 15 g of brewing salt (Calcium sulfate, Calcium Chloride) was added.

The temperature of the water was maintained at 66 ° C for 1 hour, the temperature was raised to 78 ° C for 10 minutes, and then maintained for 10 minutes to extract sugar from malt. In order to extract residual sugar from malt, a sparging process was performed. At this time, 5 L of water at 80 ° C. was added to extract residual sugar. For sterilization, boiling was performed at 100 ° C. for 60 minutes.

After the sterilization step, it was cooled by circulating cold water to the fermentation temperature of 21 ~ 23 ℃. Two 20L fermenters were filled with 10L each of the cooled wort, and commercial yeast (USA) and BC-2500 strain, each of which had been cultured in advance, were inoculated into the wort to be 6 million cells/ml. After inoculation, beer was prepared by fermentation for 7 days while maintaining a temperature of 21 to 23 ° C.

[ Experimental example 1: Analysis of flavor components]

Fragrance components were analyzed using SPME-GC/MS (PAL3 RSI autosampler, CTC Analytics AG, Zwingen, Switzerland/Agilent 7890A GC/Agilent 5973 MSD, Agilent Co., Pal Alto, CA, USA). For the extraction of volatile components, SPME fiber (80 μm, DVB/Carbon WR/PDMS, CTC Analytics AG, Zwingen, Switzerland) was used.

The beer sample was prepared by putting 10 ml of the beer sample and 3 g of NaCl in a 20 ml headspace vial and sealing it with a teflon cap.

For sample analysis, the vial containing the sample was stirred at 60° C. for 20 minutes, and then the SPME fiber was exposed for 20 minutes to adsorb volatile components to the fiber. Thereafter, the cellulose on which material adsorption was completed was placed in the front inlet of the GC and desorbed at 250° C. for 3 minutes.

A DB-WAX column (60 m length/0.25 mm film thickness/0.25 μm inner diameter, Agilent Co., Pal Alto, CA, USA) was used as a GC column for chromatography, and the oven temperature was 2 at 50 °C. After leaving for a minute, the temperature was raised at a rate of 10°C/min and maintained for 3 minutes after reaching 250°C.

The temperature of the front inlet of the GC was maintained at 250 ° C, the transfer line and MSD source temperatures were maintained at 280 ° C, and helium was used as a carrier gas at 1.0 mL / min. was used at a flow rate of The molecular weight screening range (m/z) of MSD was analyzed by setting it to 50 to 500, and the results are shown in Table 7 below.

Fragrance component analysis result (GC peak, area value) fragrance ingredient commercial yeast BC-2500 Ethyl butyrate 436245 543598 Ethyl hexanoate 2160383 2212918 Ethyl Nonanoates 517443 503851 Isoamyl acetate 16191802 22796137 Phenylethylacetate 5811229 10727649

As a result of the experiment, ethyl butyrate with apple flavor, pineapple flavor and orange juice flavor, ethyl hexanoate with anise seed flavor and fennel flavor, ethyl nonanoate with apple, pear and cognac flavor characteristics, sun fruit flavor and banana It was confirmed that substances such as isoamyl acetate having characteristics of fragrance and orientation and phenylethyl acetate having characteristics of producing fragrance such as honey were detected.

In particular, among the fragrance components, the productivity of isoamyl acetate was found to be significantly higher in the BC-2500 strain than in commercial yeast (USA), and thus, the flavor components with fresh fruit, banana, and pear flavors were enhanced through the BC-2500 strain. It was confirmed that the production of beer was possible.

Korea Microbial Conservation Center (Korea) KFCC11937P 20220727

Claims (4)

Saccharomyces strains for beer fermentation that can grow under the condition of ethanol concentration of 3-7% (v/v) and promote the production of isoamyl acetate and phenylethyl acetate, which are flavor components. Levisiae ( Saccharomyces cerevisiae ) BC-2500 (accession number: KFCC11937P).
delete delete Saccharomyces strains for beer fermentation that can grow under the condition of ethanol concentration of 3-7% (v/v) and promote the production of isoamyl acetate and phenylethyl acetate, which are flavor components. Beer prepared using Saccharomyces cerevisiae BC-2500 (accession number: KFCC11937P) as a brewing strain.