Novel native fermented yeast Saccharomyces cerevisiae EXTRASMALL01 for using production of high-alcohol craft beer containing high esters with excellent flavor-related sensory

Abstract

The present invention relates to a novel indigenous fermentation yeast, *Saccharomyces cerevisiae* EXTRASMALL01, which can be utilized in the production of high-alcohol craft beer containing high esters and possessing excellent flavor-related sensory properties. The yeast of the present invention has the advantage of being resistant to high concentrations of sugar and acidic environments, enabling rapid growth and the production of high concentrations of alcohol, and can be used as a substitute for foreign brewing yeasts due to enhanced flavor compounds.

Description

Technology Field

[0001] The present invention relates to a novel indigenous fermentation yeast, Saccharomyces cerevisiae, which can be utilized in the production of high-alcohol craft beer containing high esters with excellent flavor-related sensory properties. Saccharomyces cerevisiae This is about EXTRASMALL01. Background Technology

[0003] Beer is a brewed alcoholic beverage made by fermenting malted barley with the addition of hops, a spice, and is known as the most popular and oldest alcoholic beverage in the world. The earliest traces of fermented alcohol made from grains as early as around 8000 BC have been discovered in Erich (present-day Jordan).

[0004] The fact that beer was brewed in Europe a long time ago and spread throughout the world is deeply connected to raw materials such as pure water, high-quality barley, and hops with delicate aromas and flavors. In addition, various types of beer are produced depending on brewing methods and regional traditions. It is estimated that modern beer was first introduced during the late 19th century in Korea, along with Western culture.

[0005] In Korea, the foundation for the craft beer market was laid with the approval of small-scale beer licenses in 2002 and the possibility of external distribution in 2014. Since 2014, as distribution channels such as convenience stores and large supermarkets have expanded, general consumers have been able to easily access craft beer.

[0006] Craft beer is produced using four main ingredients—malt, hops, yeast, and water—just like conventional beer, and is primarily made by individuals or small-scale breweries. Craft beer is mainly made according to self-developed recipes and is characterized by its ability to have a variety of flavors depending on the type of malt, malt content, type of yeast, fermentation method, fermentation temperature, aging period, and auxiliary ingredients.

[0007] The market experienced explosive growth during the 2020 pandemic as the culture of drinking alone spread, coinciding with the popularity of 'label beer.' Experts predict that in the future, companies will need to compete with a diverse range of beers based on quality and taste, rather than relying on the trend of mass-market label beer, in order to increase their market share.

[0008] However, since most yeast used domestically relies on imported yeast, there is a need to develop high-quality yeast that can replace it. Prior art literature

[0010] Korean Patent No. 10-1087760 (November 22, 2011) discloses a novel recombinant yeast strain and a method for the simultaneous production of ethanol and a target protein using the same. Korean Patent No. 10-1073145 (October 6, 2011) discloses Saccharomyces cerevisiae KK1, a high-yield ethanol-producing yeast, and an ethanol fermentation method using the same. The problem to be solved

[0011] The present invention relates to Saccharomyces cerevisiae of accession number KFCC 12061P ( Saccharomyces cerevisiae One of the purposes is to provide EXTRASMALL01.

[0012] The present invention relates to Saccharomyces cerevisiae of accession number KFCC 12061P ( Saccharomyces cerevisiae Another purpose is to provide an application use of EXTRASMALL01 as a brewing strain. means of solving the problem

[0014] The present invention relates to Saccharomyces cerevisiae of accession number KFCC 12061P ( Saccharomyces cerevisiae ) Provides EXTRASMALL01.

[0015] In the present invention, the above Saccharomyces cerevisiae ( Saccharomyces cerevisiae EXTRASMALL01 is a brewing strain for beer fermentation.

[0016] The present invention relates to the production of ethanol using yeast, wherein the yeast used is Saccharomyces cerevisiae of accession number KFCC 12061P ( Saccharomyces cerevisiae A method for producing ethanol characterized by using EXTRASMALL01 is provided.

[0017] The present invention relates to Saccharomyces cerevisiae of accession number KFCC 12061P ( Saccharomyces cerevisiae Provides beer brewed using EXTRASMALL01 as a brewing strain. Effects of the invention

[0019] The yeast of the present invention has the advantage of being resistant to high concentrations of sugar and acidic environments, enabling rapid growth and high-concentration alcohol production, and enhancing flavor compounds, allowing it to be used as a substitute for foreign brewing yeasts. Specific details for implementing the invention

[0021] In the following, redundant content has been omitted to prevent clutter. In other words, the content of the invention is not limited solely to the following, and should be interpreted in accordance with the overall context of the invention.

[0022] Currently, the yeast used for the production of craft beer relies on imported yeast. Accordingly, this invention sought to isolate a yeast from traditional fermented foods that grows more rapidly in high-concentration sugar and acidic environments and can produce high concentrations of ethanol.

[0023] In the present invention, the above-mentioned strain is 'Saccharomyces cerevisiae ( Saccharomyces cerevisiaeIt was named 'EXTRASMALL01' and deposited with the Korean Culture Collection of Microorganisms, receiving accession number KFCC 12061P on October 21, 2025.

[0024] Accordingly, the present invention relates to Saccharomyces cerevisiae of accession number KFCC 12061P ( Saccharomyces cerevisiae ) Provides EXTRASMALL01.

[0025] Saccharomyces cerevisiae of the present invention ( Saccharomyces cerevisiae EXTRASMALL01 has been confirmed to have improved acid resistance, high concentration ethanol production ability, and resistance to high concentration ethanol (4~7%) compared to the control yeast, so it can be used to replace foreign brewing yeast that was previously dependent on imports.

[0026] In addition, in one embodiment of the present invention, Saccharomyces cerevisiae ( Saccharomyces cerevisiae By using the EXTRASMALL01 strain, the content of ethyl acetate, ethyl decanoate, phenethyl acetate, isoamyl acetate, isobutyl acetate, isoamyl propionate, hexyl acetate, and ethyl palmitate was increased or newly identified compared to commercial yeast (USA). In addition, it was confirmed that ethyl octanoate and acetaldehyde accounted for the highest content in the beer. Based on the emission of these flavor components, it was confirmed that the present invention makes it possible to produce beer having fresh fruit aromas of peach, apricot, and green apple, as well as floral aromas of rose.

[0027] Accordingly, it was confirmed that using the yeast of the present invention in beer production enables faster beer production and the production of beer with excellent flavor containing a high concentration of alcohol. That is, the Saccharomyces cerevisiae of the present invention ( Saccharomyces cerevisiae ) EXTRASMALL01 is preferably used as a brewing strain for beer fermentation.

[0028] Meanwhile, the present invention relates to Saccharomyces cerevisiae of accession number KFCC 12061P ( Saccharomyces cerevisiae Provides beer brewed using EXTRASMALL01 as a brewing strain.

[0029] The beer of the present invention can preferably be produced through a method comprising: (a) adding crushed malt to water at 50 to 80°C and adding brewing salt; (b) raising the temperature to 60 to 90°C to extract sugars; (c) raising the temperature to 65 to 95°C to sparge; (d) sterilizing and cooling; and (e) inoculating with the yeast of the present invention.

[0030] At this time, the sugar extraction step must be set at a temperature higher than that of the malt additive to facilitate sugar extraction, and the sparging step must be set at a temperature higher than that of the sugar extraction step to be suitable for extracting residual sugar.

[0031] Meanwhile, in the above sterilization process, additional hop or yeast nutrients may be optionally added, which can impart flavor to the beer, inhibit bacterial growth, and promote yeast growth.

[0032] In addition, the above cooling refers to the process of cooling to a temperature of 15 to 45°C, and the temperature range is not limited as long as it is a temperature suitable for the growth temperature of yeast to be inoculated into beer.

[0033] In addition, commonly known techniques may be applied for the manufacture of beer, and other processes widely known to those in the industry may be utilized; therefore, specific descriptions thereof will be omitted.

[0034] Meanwhile, the present invention relates to the production of ethanol using yeast, wherein the yeast comprises Saccharomyces cerevisiae of accession number KFCC 12061P ( Saccharomyces cerevisiae A method for producing ethanol characterized by using EXTRASMALL01 is provided.

[0035] The yeast of the present invention has the advantage of being resistant to high concentrations of sugar and acidic environments, enabling rapid growth and high-concentration alcohol production, and enhancing flavor compounds, allowing it to be used as a substitute for foreign brewing yeasts.

[0037] The contents of the present invention will be explained in more detail below through the following examples or experimental examples. However, the scope of the present invention is not limited to the following examples or experimental examples, but includes variations of equivalent technical concepts.

[0039] [ Examples 1: Selection and Identification of the Yeast Strain of the Present Invention]

[0040] The yeast according to the present invention is a cherry tree in a park in Yeonsu-gu, Incheon ( Prunus yedoensis It was separated from the fruit (cherry).

[0041] Specifically, to isolate yeast, a YPD medium (Yeast extract 10 g / L, Peptone 20 g / L, Glucose 20 g / L) containing ampicillin (100 µg / mL) or kanamycin (100 µg / mL), respectively, was used.

[0042] After fermenting the sample in YPD medium supplemented with antibiotics at 25°C for 2 weeks, 10 -1 ~ 10 -7 The samples were diluted and plated, and then cultured statically at 30°C for 3 days. Afterward, to preserve single colonies that appeared to be yeast, the samples were prepared to contain 15–20% glycerol and stored in an ultra-low temperature freezer (-75°C to -80°C).

[0043] In addition, the selected strain was named 'EXTRASMALL01' and its identification was performed. To identify the strain, the 18S rRNA sequence was sequenced.

[0044] Specifically, first, PCR was performed on colonies of the selected strain using Dyne Direct PCR (containing UDG / UTP) (BN430, Dainbio) with NS1 (5'-GTAGTCATATGCTTGTCTC-3') and NS24 (5'-AAACCTTGTTACGACTTTTA-3') primers. Subsequently, the PCR products were purified using the LaboPass™ PCR Purification Kit (CMR0112, Cosmogenetec). Afterward, sequencing was commissioned to Macrogen using the ABI 3730XL System with the same primers. The sequencing analysis was performed by running a BLAST program to identify the nucleotide sequences, and the EXTRASMALL01 strain was identified as Saccharomyces cerevisiae ( Saccharomyces cerevisiae It was identified as ). In addition, the sequence of the 18S rRNA of the EXTRASMALL01 strain is shown in Sequence No. 1.

[0046] [ Experimental Example 1: Evaluation of the characteristics of the yeast strain of the present invention]

[0047] In the following, to compare the characteristics of the yeast strain according to the present invention, a commercial yeast known to be mainly used in the production of craft beer (White Labs Liquid Yeast WLP001 (White Labs California Ale Yeast) from the USA) was used as a control.

[0049] 1. Ethanol generation ability comparison

[0050] Commercial yeast, which served as the control strain, and the experimental strain were inoculated into 10 ml of 2% YPD medium and cultured in a shaking incubator at 30°C and 200 rpm for 24 hours. Subsequently, the strains were inoculated into 10 ml each of 2%, 10%, 20%, and 30% YPD medium to achieve an initial OD of 0.2, and cultured in a shaking incubator at 30°C and 200 rpm for 48 hours, during which samples were collected at 24 and 48 hours.

[0051] The ethanol production was determined by analyzing the collected samples using HPLC. The HPLC used for the analysis was an Agilent 1200 Series, the column was a Phenomenex Rezex ROA-Organic Acid H+(8%) Ion Exclusion HPLC Column, and the mobile phase was 5 mM sulfuric acid buffer. All samples were measured three times, and the data were analyzed. The results are shown in Table 1 below.

[0052] Ethanol production capacity (g / L) strain name hour YPD 2% YPD 10% YPD 20% YPD 30% WLP001 24h 5.28 25.88 24.17 17.52 48h 0.53 34.33 29.36 32.84 EXTRASMALL01 24h 8.02 42.35 44.83 27.05 48h 8.11 44.29 80.11 62.56

[0054] As a result of the experiment, the EXTRASMALL01 yeast strain exhibited a higher ethanol production than the control strain, commercial yeast (USA), under all medium conditions, confirming the superior ethanol production ability of the EXTRASMALL01 yeast strain.

[0056] 2. Internal party comparison

[0057] A single colony of the strain was inoculated into 10 ml of YPD (Yeast extract 10 g / L, Peptone 20 g / L, Glucose 20 g / L) and cultured in a shaking incubator at 30°C and 200 rpm for 24 hours.

[0058] Subsequently, the culture medium was inoculated into 10 ml each of YPD medium (Yeast extract 10 g / L, Peptone 20 g / L, Glucose 20–300 g / L) containing 2%, 10%, and 30% glucose, respectively, to achieve an initial OD of 0.2. Each inoculated culture medium was dispensed into 96-well plates in 180 µl doses in three repetitions. Afterward, the plates were incubated for 48 hours at 30°C and 180 rpm using a microplate reader (SPECTROstar Nano, BMG Labtech, Germany). Subsequently, absorbance was measured at 600 nm every 60 minutes and compared, and the results are shown in Tables 2 and 3 below.

[0059] Non-growth rate comparison (OD) 600 / h) strain name YPD 2% YPD 10% YPD 20% YPD 30% WLP001 0.111 0.084 0.061 0.036 EXTRASMALL01 0.135 0.049 0.037 0.035

[0060] Comparison of maximum absorbance (OD) 600 ) strain name YPD 2% YPD 10% YPD 20% YPD 30% WLP001 1.264 1.438 1.474 1.233 EXTRASMALL01 1.290 1.683 1.426 1.108

[0062] Experimental results showed that the EXTRASMALL01 strain possessed a high specific growth rate and maximum absorbance similar to that of commercial yeast (USA), confirming that the EXTRASMALL01 strain possesses excellent sugar tolerance.

[0064] 3. acid resistance comparison

[0065] 2% YPD with pH values ​​of 5.5, 4.5, 3.5, and 2.5 was prepared by adding HCl to YPD medium containing 2% glucose and used as the medium. Subsequently, a single colony was inoculated into 10 ml of YPD medium and cultured in a shaking incubator at 30°C and 200 rpm for 24 hours.

[0066] The culture medium was inoculated into 10 mL of each of 2% YPD medium at pH 5.5, 4.5, 3.5, and 2.5 to achieve an initial OD of 0.2. Each inoculated culture medium was dispensed into 96-well plates in 180 µL doses in three repetitions. Subsequently, the plates were incubated for 48 hours at 30°C and 180 rpm using a microplate reader (SPECTROstar Nano, BMG Labtech, Germany). Afterward, absorbance was measured at 600 nm every 60 minutes and compared, and the results are shown in Tables 4 and 5 below.

[0067] Non-growth rate comparison (OD) 600 / h) strain name YPD 2%, pH 5.5 YPD 2%, pH 4.5 YPD 2%, pH 3.5 YPD 2%, pH 2.5 WLP001 0.100 0.098 0.070 0.000 EXTRASMALL01 0.132 0.132 0.129 0.005

[0068] Comparison of maximum absorbance (OD) 600 ) strain name YPD 2%, pH 5.5 YPD 2%, pH 4.5 YPD 2%, pH 3.5 YPD 2%, pH 2.5 WLP001 1.124 1.096 1.163 0.191 EXTRASMALL01 1.180 1.164 1.321 0.597

[0070] Experimental results showed that the EXTRASMALL01 strain had a higher specific growth rate and maximum absorbance than commercial yeast (USA) in an acidic environment of pH 2.5 to 4.5, confirming that the EXTRASMALL01 strain possesses excellent acid resistance.

[0072] 4. Comparison of Ethanol Resistance

[0073] To compare the ethanol resistance of the EXTRASMALL01 strain, media containing 4%, 7%, 10%, 12%, and 15% ethanol in 2% YPD were prepared, respectively. The control strain, commercial yeast, and the experimental group strain were each inoculated into 10 ml of 2% YPD medium and cultured in a shaking incubator at 30°C and 200 rpm for 24 hours.

[0074] Subsequently, the culture medium was inoculated into 10 ml each of 2% YPD medium containing 4%, 7%, 10%, 12%, and 15% of pre-prepared ethanol, respectively, to achieve an initial OD of 0.5, and cultured in a shaking incubator at 30°C and 200 rpm for 48 hours. Afterward, the absorbance was measured at 600 nm, and the results are shown in Table 6 below.

[0075] Absorbance Comparison (OD) 600 ) strain name YPD 2%, EtOH 0% YPD 2%, EtOH 4% YPD 2%, EtOH 7% YPD 2%, EtOH 10% YPD 2%, EtOH 12% YPD 2%, EtOH 15% WLP001 6.400 1.050 0.720 0.720 0.640 0.610 EXTRASMALL01 5.994 4.675 3.157 0.474 0.386 0.273

[0077] As a result of the experiment, it was found that the EXTRASMALL01 strain had a higher 48-hour OD than commercial yeast (USA) under high ethanol concentration conditions of 4–7%, confirming that the EXTRASMALL01 strain possesses excellent ethanol resistance.

[0079] [ Examples 2: Preparation of beer using the yeast strain of the present invention]

[0080] 5 kg of 2-Row Pale Malt (Weyermann, German) was ground and placed in 23 L of water set to 67°C. Then, 5 g of brewing salt (calcium sulfate, calcium chloride) was added.

[0081] Sugars were extracted from the malt by maintaining the water temperature at 67°C for 1 hour, raising it to 78°C for 10 minutes, and maintaining it for 10 minutes. To extract residual sugars from the malt, a sparging process was performed by adding 10 L of water at 78°C. For sterilization, the mixture was boiled at 100°C for 60 minutes.

[0082] To impart bitterness and flavor to the beer while boiling, 20 g of Magnum hops (12.0% Alpha Acid, Yakima Valley Hops) were added.

[0083] After the pasteurization step was completed, cold water was circulated to cool the wort to the fermentation temperature of 20–22°C. 10 L of the cooled wort was filled into each of two 20 L fermentation tanks, and pre-cultured commercial yeast strains (White Labs California Ale Yeast WLP001, USA) and EXTRASMALL01 strain were inoculated into the wort at a concentration of 7 million cells / mL. After inoculation, the wort was fermented for 7 days while maintaining a temperature of 20–22°C to produce beer.

[0085] [ Experimental Example 2: Analysis of Beer Flavor Components]

[0086] Forty metabolites that significantly affect flavor during fermentation were secured, and quantitative and qualitative analyses were performed on the flavor components using SPME-GC / MS (PAL3 RSI autosampler, CTC Analytics AG, Zwingen, Switzerland / Agilent 7890A GC / Agilent 5973 MSD, Agilent Co., PalAlto, CA, USA).

[0087] All samples for analysis were prepared by placing 10 ml of beer sample and 3 g of NaCl into a 20 ml vial and sealing it. For sample analysis, the vials containing the samples were stirred and maintained at 60°C for 20 minutes, and then exposed to SPME fiber for 20 minutes to adsorb volatile components.

[0088] Afterwards, the adsorbed cellulose was placed at the front inlet of the GC and desorbed at 250°C for 3 minutes.

[0089] To perform chromatography, a DB-WAX capillary column (30 m * 0.25 mm, 0.25 μm) was used, and the oven temperature was set to 50°C for 2 minutes, then increased at a rate of 10°C / min to 250°C and maintained for 3 minutes. The temperature of the GC front inlet was maintained at 250°C, and the temperatures of the transfer line and MSD source were maintained at 280°C.

[0090] Helium was used as the carrier gas at a flow rate of 1.0 ml / min, and the analysis was performed with the MSD molecular weight screening range (m / z) set to 50–500.

[0091] The analysis results are shown in Table 7 below.

[0092] Fragrance component analysis results (GC peak, Area value) fragrance ingredients WLP001 EXTRASMALL01 Acetaldehyde 4023222 5743427 Ethyl acetate 171579 503685 1-Propanol Non-detectable Non-detectable Isobutyl acetate Non-detectable 74692 Ethyl butyrate 44544 83809 Ethyl Isovalerate Non-detectable Non-detectable Isoamyl formate Non-detectable Non-detectable Isobutanol 54237 52941 Isoamyl acetate 146376 1292174 2,3-Hexanedione Non-detectable Non-detectable Ethyl valerate Non-detectable Non-detectable Isoamyl propionate Non-detectable 18111 3-Methyl-1-butanol 1769942 2621831 Ethyl hexanoate 201964 292076 Pentanol Non-detectable Non-detectable Hexyl acetate Non-detectable 10239 Hexanol Non-detectable Non-detectable Ethyl Octanoate 3934935 6119571 Isoamyl hexanoate Non-detectable Non-detectable Ethyl nonanoate 63379 81537 Isoamyl lactate Non-detectable Non-detectable Nonanol Non-detectable Non-detectable Ethyl decanoate 469848 1741905 Isoamyl octanoate Non-detectable Non-detectable 3-Mercaptohexyl acetate 3401 Non-detectable Phenethyl acetate 510327 4454778 3-Mercapto-1-hexanol Non-detectable Non-detectable Ethyl dodecanoate 37368 349343 Isoamyl decanoate Non-detectable grain Phenylethyl Alcohol 2448692 4782255 Ethyl myristate Non-detectable Non-detectable 4-Ethylphenol Non-detectable Non-detectable 4-Vinylguaiacol Non-detectable Non-detectable Ethyl palmitate Non-detectable 186890

[0094] Experimental results showed that the yeast according to the present invention (EXTRASMALL01 strain) had increased content of acetaldehyde, ethyl acetate, isobutyl acetate, ethyl butyrate, isoamyl acetate, isoamyl propionate, 3-methyl-1-butanol, ethyl hexanoate, hexyl acetate, ethyl octanoate, ethyl nonanoate, ethyl decanoate, phenyl acetate, phenylethyl alcohol, and ethyl palmitate compared to commercial yeast (USA). I was able to confirm it.

[0095] Specifically, compared to the control group, the beer produced using the strain of the present invention showed that the content of ethyl acetate, which has the characteristics of apple and pear-type fruit aromas, increased by about 2.9 times, ethyl decanoate, which has the characteristics of apple and peach-type fruit aromas, increased by about 3.7 times, phenyl acetate, which has the characteristics of rose, floral, and honey aromas, increased by about 8.7 times, and isoamyl acetate, which has the characteristics of banana aromas, increased by about 8.8 times.

[0096] In addition, isobutyl acetate, which has apple and tropical fruit scents, isoamyl propionate, which has pineapple and pear scents, hexyl acetate, which has pear scents, and ethyl palmitate, which has wax scents, were not detected in the control group but were detected in the beer produced using the strain of the present invention.

[0097] In addition, it was confirmed that in beer produced using the strain of the present invention, the content of ethyl octanoate, which has the characteristics of peach and apricot fruit aromas, and acetaldehyde, which has the characteristics of grass and green apple aromas, was the highest among the aroma components.

[0098] Accordingly, it was confirmed that it is possible to finally produce a beer rich in fresh fruit aromas of peach, apricot, and green apple, and floral aromas of rose using the yeast (EXTRASMALL01 strain) according to the present invention.

[0100] Depository Name: Korean Culture Collection of Microorganisms (Domestic) Trustee Number: KFCC12061P Date of Deposit: 2025-10-21

Claims

Claim 1 Saccharomyces cerevisiae of accession number KFCC 12061P, which promotes the production of the aroma compounds ethyl octanoate and isoamyl acetate ( Saccharomyces cerevisiae ) EXTRASMALL01. Claim 2 delete Claim 3 delete Claim 4 Saccharomyces cerevisiae of accession number KFCC 12061P as described in Paragraph 1 ( Saccharomyces cerevisiae Beer brewed using EXTRASMALL01 as the brewing strain.