KR102481206B1 - Novel native fermented yeast Saccharomyces cerevisiae BC-4100 for using production of craft beer containing high esters with excellent flavor-related sensory - Google Patents

Abstract

The present invention relates to novel native fermentation yeast Saccharomyces cerevisiae BC-4100 capable of being applied for manufacturing craft beer with high ester content and excellent flavor-related organoleptic properties. The yeast of the present invention has an advantage of having resistance to the high concentration of sugar and the acidic environment, thereby 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.

Description

Novel native fermented yeast Saccharomyces cerevisiae BC-4100 for using production of craft beer containing high esters with excellent flavor-related sensory}

The present invention relates to native fermented yeast Saccharomyces cerevisiae BC-4100 that can be used for producing high ester-containing craft beer having excellent flavor-related functionality.

Beer is known as the most popular and oldest alcoholic beverage in the world as a fermented alcoholic beverage made by adding hops, a spice, to malt produced from barley as the main ingredient. Already around 8,000 BC, the first traces of making fermented wine from grains have been found in Eriko (now Jordan).

Beer was brewed in Europe a long time ago and spread all over the world because of its raw materials, such as pure water, high-quality barley, and hops with a delicate aroma and taste. In addition, various beers are produced according to the manufacturing method and local tradition. Modern beer is first estimated to have been introduced with Western culture at the end of the 19th century during the Joseon Dynasty.

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.

Craft beer (craft beer) is made using the same four main ingredients as malt, hops, yeast, and water, as in conventional beer, but 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.

Meanwhile, this patent has the task number '2021-JB-RD-0118-01-101 (2021.10.08.~2022.04.07.)', the research project name 'Support for customized growth of research institute companies', and the research project name 'Fermentation Engineering-Based Advanced Technology' It was carried out with the help of 'development of native brewing yeast using strain development technology'.

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 provides Saccharomyces cerevisiae BC-4100 (Accession Number: KFCC11926P).

At this time, the Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) BC-4100 (accession number: KFCC11926P) 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-4100 (accession number: KFCC11926P) is provided.

In addition, the present invention provides beer prepared using Saccharomyces cerevisiae BC-4100 (Accession Number: KFCC11926P) 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.

1 is a result of confirming the ethanol production ability of the BC-4100 yeast strain of the present invention.

Yeast used for the production of craft beer is currently dependent on imported yeast. Therefore, in the present invention, yeast that can grow faster in a high concentration of sugar and acidic environment from traditional fermented foods and produce high concentration of ethanol was searched for and isolated.

In the present invention, the above strain was named ' Saccharomyces cerevisiae BC-4100', and was deposited with the Korea Microorganism Conservation Center and received accession number KFCC11926P on March 24, 2022.

Saccharomyces cerevisiae BC-4100 (accession number: KFCC11926P) of the present invention has improved sugar tolerance, acid resistance, ethanol production ability and ethanol resistance compared to the control yeast, depending on existing imports It can be used as a substitute for foreign brewing yeast.

In addition, in one embodiment of the present invention, by using the Saccharomyces cerevisiae BC-4100 strain, the productivity of ethyl nonanoate is enhanced, thereby enhancing the flavor component of apple, pear and cognac beer It was confirmed that the production of

Accordingly, it was confirmed that the yeast of the present invention can be used to produce beer faster and to produce beer containing a high concentration of alcohol and having excellent flavor. That is, Saccharomyces cerevisiae of the present invention ( Saccharomyces cerevisiae ) BC-4100 (accession number: KFCC11926P) is preferably used as a brewing strain for beer fermentation.

Meanwhile, the present invention provides beer prepared using Saccharomyces cerevisiae BC-4100 (Accession Number: KFCC11926P).

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-4100 (accession number: KFCC11926P) 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: Selection 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 to -80 ° C).

2. Comparison of ethanol production ability

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 and the data was analyzed (FIG. 1). 1 shows the result of confirming the ethanol producing ability of the commercial yeast (USA) as a control strain and the BC-4100 yeast strain showing the best ethanol producing ability among the experimental strains.

As a result of the experiment, it was confirmed that the BC-4100 yeast strain showed a higher yield of 63.8 g/L than the ethanol yield of 35.1 g/L of the control strain, commercial yeast (USA), in 20% YPD medium conditions, thereby confirming its excellent ethanol producing ability. could

3. Comparison of glucose tolerance

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). Thereafter, the absorbance at 600 nm was measured every 30 minutes and compared, and the results are shown in Tables 1 and 2 below, with the BC-4100 yeast strain showing the best sugar tolerance as a representative.

Comparison of specific growth rate strain/condition 2% YPD 10% YPD 20% YPD 30% YPD Commercial yeast (USA) 0.110 0.086 0.056 0.037 BC-4100 0.137 0.115 0.074 0.038

Comparison of maximum absorbance strain/condition 2% YPD 10% YPD 20% YPD 30% YPD Commercial yeast (USA) 1.332 1.817 1.883 1.852 BC-4100 1.401 1.890 1.976 1.870

As a result of the experiment, the BC-4100 strain was found to have a higher specific growth rate and maximum absorbance than commercial yeast (USA), confirming that the BC-4100 strain had excellent sugar tolerance.

4. Acid resistance comparison

Lactate 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 3 and 4 below, with the BC-4100 yeast strain showing the best acid resistance as a representative.

Comparison of specific growth rate strain/condition 2% YPD, pH 3.5 2% YPD, pH 4.5 2% YPD, pH 5.5 Commercial yeast (USA) 0.056 0.092 0.105 BC-4100 0.099 0.136 0.130

Comparison of maximum absorbance strain/condition 2% YPD, pH 3.5 2% YPD, pH 4.5 2% YPD, pH 5.5 Commercial yeast (USA) 1.274 1.233 1.286 BC-4100 1.517 1.438 1.460

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-4100 strain was found to have a higher specific growth rate and maximum absorbance than commercial yeast (USA), confirming that the BC-4100 strain had excellent acid resistance.

5. Ethanol Resistance Comparison

Through the above experiments, the ethanol resistance of the BC-4100 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-4100 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 5 below.

Absorbance comparison strain/condition 2% YPD, 3% EtOH 2% YPD, 5% EtOH 2% YPD, 7% EtOH Commercial yeast (USA) 4.59 0.37 0.29 BC-4100 4.76 3.07 2.17

As a result of the experiment, both the control strain and the BC-4100 strain did not grow in a medium containing 2% YPD and 10% ethanol. However, under other ethanol concentration conditions, the 24-hour OD of the BC-4100 strain was higher than that of commercial yeast (USA), confirming that the BC-4100 strain had excellent ethanol resistance.

6. Identification of the BC-4100 strain

As a result of the synthesis of the above experiments, 'BC-4100', 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.

[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-4100 strain, each of which had been previously cultured, 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: Flavor Component Analysis]

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. After this, the 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 6 below.

Fragrance component analysis result (GC peak, area value) fragrance ingredient Commercial yeast (USA) BC-4100 Ethyl hexanoate 30147202.33 17743683.33 Ethyl nonanoate 597130.67 1813042.67 Hexyl acetate 917705.33 578989.33 Isoamyl acetate 190219137.33 181319813.33 Ethyl butyrate 1310177.00 950518.00

As a result of the experiment, ethyl hexanoate, which has the characteristics of apple, anise, and fennel, and ethyl nonanoate, which has the characteristics of apple, pear, and cognac, and hexyl acetate, which has the characteristics of sweet, apple, and banana flavors, fresh fruit, and banana flavor , It was confirmed that substances such as isoamyl acetate having characteristics of orientation and ethyl butyrate having characteristics of pineapple flavor and orange juice flavor were detected.

In particular, among the flavor components, the productivity of ethyl nonanonate was found to be higher in the BC-4100 strain than in the commercial yeast (USA), and as a result, beer with enhanced flavor components with apple, pear, and cognac flavors through the BC-4100 strain. It was confirmed that the production of

Korea Microbial Conservation Center (Korea) KFCC11926P 20220324

<110> biocraft <120> Novel native fermented yeast Saccharomyces cerevisiae BC-4100 for using production of craft beer containing high esters with excellent flavor-related sensory <130> YP-22-046 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 970 <212> DNA <213> Saccharomyces cerevisiae <220> <221> rRNA <222> (1)..(970) <223> This is 18S rRNA of Saccharomyces cerevisiae BC-4100 <400> 1 agtgaaactg cgaatggctc attaaatcag ttatcgttta tttgatagtt cctttactac 60 atggtataac tgtggtaatt ctagagctaa tacatgctta aaatctcgac cctttggaag 120 agatgtattt attagataaa aaatcaatgt cttcggactc tttgatgatt cataataact 180 tttcgaatcg catggccttg tgctggcgat ggttcattca aatttctgcc ctatcaactt 240 tcgatggtag gatagtggcc taccatggtt tcaacgggta acggggaata agggttcgat 300 tccggagagg gagcctgaga aacggctacc acatccaagg aaggcagcag gcgcgcaaat 360 tacccaatcc taattcaggg aggtagtgac aataaataac gatacagggc ccattcgggt 420 cttgtaattg gaatgagtac aatgtaaata ccttaacgag gaacaattgg agggcaagtc 480 tggtgccagc agccgcggta attccagctc caatagcgta tattaaagtt gttgcagtta 540 aaaagctcgt agttgaactt tgggcccggt tggccggtcc gattttttcg tgtactggat 600 ttccaacggg gcctttcctt ctggctaacc ttgagtcctt gtggctcttg gcgaaccagg 660 acttttactt tgaaaaaatt agagtgttca aagcaggcgt attgctcgaa tatattagca 720 tggaataata gaataggacg tttggttcta tttttgttgg tttctaggac catcgtaatg 780 attaataggg acggtcgggg gcatcagtat tcaattgtca gaggtgaaat tcttggattt 840 attgaagact aactactgcg aaagcattgc ccaaggacgt tttcattaat caagaacgaa 900 agttagggga tcgaagatga tcagataccg tcgtagtctt aaccataaac tatgccgact 960 agggatccgg 970

Claims (4)

Saccharomyces cerevisiae BC-4100 (accession number: KFCC11926P) for beer fermentation that promotes the production of ethyl nonanoate, an aromatic component. delete delete Beer prepared using Saccharomyces cerevisiae BC-4100 (accession number: KFCC11926P) as a brewing strain for beer fermentation that promotes the production of ethyl nonanoate, an aromatic component.

Images