Novel native fermented yeast Saccharomyces cerevisiae BC25 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* BC25, 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 concerns ) 25 BC. Background Technology

[0003] Beer is an alcoholic beverage made by sprouting barley to create malt, filtering the resulting wort, adding hops, and fermenting it with yeast. There are two theories regarding the etymology of the word "beer." One theory suggests it originated from the Latin word "bibere," meaning "to drink," and the other from the Germanic word "bior," meaning "grain." Beer is known as the oldest fermented beverage in the world. Depending on the circumstances and preferences of each country and region, rice, corn, starch, and sugars are used as starchy supplements. However, it has a relatively low alcohol content and contains carbon dioxide and the bitter components of hops, which have the effect of promoting digestion and aiding in diuretic action.

[0004] 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.

[0005] 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.

[0006] Recently, the consumption of imported beer in Korea has been surging compared to that of domestic beer, and there has been a significant increase in demand for diverse, differentiated, high-quality beers, particularly among the younger generation. Furthermore, driven by the trend of enjoying diverse and unique beer flavors, the development of distinctive craft beers and the expansion of the craft beer market are underway.

[0007] In response to the expansion of the craft beer market and consumption trends, the government has been continuously pursuing policies to reduce taxes on craft beer and ease distribution regulations to promote domestic rice consumption and revitalize the craft beer industry. Additionally, since April 2018, the National Tax Service has been providing policy support for the expansion and revitalization of the craft rice beer market by reducing taxes on craft beers that use 20% or more of rice.

[0008] 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.

[0009] However, beers sold in the domestic craft beer market adhere to standard brewing methods using only malt, resulting in a lack of differentiation in quality among manufacturers and a loss of diversity and quality competitiveness compared to imported beers. Consequently, there is a need to develop high-quality yeast capable of replacing imported yeast. Prior art literature

[0011] 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

[0012] The present invention relates to Saccharomyces cerevisiae of accession number KFCC 12060P ( Saccharomyces cerevisiae One of the purposes is to provide BC25.

[0013] The present invention relates to Saccharomyces cerevisiae of accession number KFCC 12060P ( Saccharomyces cerevisiae Another purpose is to provide an application use of BC25 as a brewing strain. means of solving the problem

[0015] The present invention relates to Saccharomyces cerevisiae of accession number KFCC 12060P ( Saccharomyces cerevisiae ) Provides BC25.

[0016] In the present invention, the above Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) BC25 is a brewing strain for beer fermentation.

[0017] The present invention relates to the production of ethanol using yeast, wherein the yeast used is Saccharomyces cerevisiae of accession number KFCC 12060P ( Saccharomyces cerevisiae ) Provides a method for producing ethanol characterized by using BC25.

[0018] The present invention relates to Saccharomyces cerevisiae of accession number KFCC 12060P ( Saccharomyces cerevisiae Provides beer brewed using BC25 as a brewing strain. Effects of the invention

[0020] 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

[0022] 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.

[0023] 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.

[0024] In the present invention, the above-mentioned strain is 'Saccharomyces cerevisiae ( Saccharomyces cerevisiae It was named 'BC25' and deposited with the Korean Culture Collection of Microorganisms, receiving accession number KFCC 12060P on October 21, 2025.

[0025] Accordingly, the present invention relates to Saccharomyces cerevisiae of accession number KFCC 12060P ( Saccharomyces cerevisiae ) Provides BC25.

[0026] Saccharomyces cerevisiae of the present invention ( Saccharomyces cerevisiae It has been confirmed that BC25 has 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.

[0027] In addition, in one embodiment of the present invention, Saccharomyces cerevisiae ( Saccharomyces cerevisiaeBy using the EXTRASMALL01 strain, the content of phenylethyl alcohol, isoamyl acetate, phenethyl 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 acetaldehyde and phenethyl acetate accounted for the highest content in the beer. Based on the release of these flavor components, it was confirmed that the present invention enables the production of beer with a rich aroma of deeper rose, honey, and green apple.

[0028] 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 BC25 is preferably used as a brewing strain for beer fermentation.

[0029] Meanwhile, the present invention relates to Saccharomyces cerevisiae of accession number KFCC 12060P ( Saccharomyces cerevisiae Provides beer brewed using BC25 as a brewing strain.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] Meanwhile, the present invention relates to the production of ethanol using yeast, wherein the yeast comprises Saccharomyces cerevisiae of accession number KFCC 12060P ( Saccharomyces cerevisiae ) Provides a method for producing ethanol characterized by using BC25.

[0036] 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.

[0038] 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.

[0040] [ Examples 1: Selection and Identification of the Yeast Strain of the Present Invention]

[0041] The yeast according to the present invention is blackberries from North Korea ( Rubus fruticosus It was separated from the fruit.

[0042] 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.

[0043] 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).

[0044] In addition, the selected strain was named 'BC25' and its identification was performed. To identify the strain, the 18S rRNA sequence was sequenced.

[0045] Specifically, PCR was first 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, Cosmogenetech). 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 BC25 was identified as Saccharomyces cerevisiae ( Saccharomyces cerevisiae It was identified as ). In addition, the sequence of the 18S rRNA of the BC25 strain is shown in Sequence No. 1.

[0047] [ Experimental example 1: Evaluation of the characteristics of the yeast strain of the present invention]

[0048] 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.

[0050] 1. Ethanol generation ability comparison

[0051] 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.

[0052] 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.

[0053] 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 BC25 24h 8.43 36.05 26.33 12.26 48h 8.42 43.41 67.83 40.26

[0055] As a result of the experiment, the BC25 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 BC25 yeast strain.

[0057] 2. Internal party comparison

[0058] 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.

[0059] 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.

[0060] 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 BC25 0.135 0.049 0.037 0.035

[0061] 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 BC25 1.290 1.683 1.426 1.108

[0063] Experimental results showed that the BC25 strain possessed a high specific growth rate and maximum absorbance similar to that of commercial yeast (USA), confirming that the BC25 strain has excellent sugar tolerance.

[0065] 3. acid resistance comparison

[0066] 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.

[0067] 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.

[0068] 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 BC25 0.132 0.132 0.129 0.005

[0069] 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 BC25 1.180 1.164 1.321 0.597

[0071] Experimental results showed that the BC25 strain had a higher specific growth rate and maximum absorbance than commercial yeast (USA) in an acidic environment of pH 2.5 to 5.5, confirming that the BC25 strain possesses excellent acid resistance.

[0073] 4. Comparison of Ethanol Resistance

[0074] To compare the ethanol resistance of the BC25 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.

[0075] 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.

[0076] 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 BC25 5.994 4.675 3.157 0.474 0.386 0.273

[0078] As a result of the experiment, it was found that the BC25 strain had a higher 48-hour OD than commercial yeast (USA) under high ethanol concentration conditions of 4–7%, confirming that the BC25 strain possesses excellent ethanol resistance.

[0080] [ Examples 2: Preparation of beer using the yeast strain of the present invention]

[0081] 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.

[0082] 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.

[0083] To impart bitterness and flavor to the beer while boiling, 20 g of Magnum hops (12.0% Alpha Acid, Yakima Valley Hops) were added.

[0084] 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 (White Labs California Ale Yeast WLP001, USA) and BC25 strains were inoculated into the wort to 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.

[0086] [ Experimental example 2: Analysis of Beer Flavor Components]

[0087] 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).

[0088] 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.

[0089] Afterwards, the adsorbed cellulose was placed at the front inlet of the GC and desorbed at 250°C for 3 minutes.

[0090] 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.

[0091] 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.

[0092] The analysis results are shown in Table 7 below.

[0093] Fragrance component analysis results (GC peak, Area value) fragrance ingredients WLP001 BC25 Acetaldehyde 4023222 8389428 Ethyl acetate 171579 572794 1-Propanol Non-detectable Non-detectable Isobutyl acetate Non-detectable 248293 Ethyl butyrate 44544 53945 Ethyl Isovalerate Non-detectable Non-detectable Isoamyl formate Non-detectable Non-detectable Isobutanol 54237 117808 Isoamyl acetate 146376 1936623 2,3-Hexanedione Non-detectable Non-detectable Ethyl valerate Non-detectable Non-detectable Isoamyl propionate Non-detectable 23827 3-Methyl-1-butanol 1769942 3637336 Ethyl hexanoate 201964 172063 Pentanol Non-detectable Non-detectable Hexyl acetate Non-detectable 13007 Hexanol Non-detectable Non-detectable Ethyl Octanoate 3934935 4654868 Isoamyl hexanoate Non-detectable Non-detectable Ethyl nonanoate 63379 69548 Isoamyl lactate Non-detectable Non-detectable Nonanol Non-detectable Non-detectable Ethyl decanoate 469848 1010255 Isoamyl octanoate Non-detectable Non-detectable 3-Mercaptohexyl acetate 3401 Non-detectable Phenethyl acetate 510327 8115839 3-Mercapto-1-hexanol Non-detectable Non-detectable Ethyl dodecanoate 37368 389211 Isoamyl decanoate Non-detectable grain Phenylethyl Alcohol 2448692 7509079 Ethyl myristate Non-detectable Non-detectable 4-Ethylphenol Non-detectable Non-detectable 4-Vinylguaiacol Non-detectable Non-detectable Ethyl palmitate Non-detectable 148652

[0095] As a result of the experiment, the yeast according to the present invention (BC25 strain) compared to commercial yeast (USA) showed acetaldehyde, ethyl acetate, isobutyl acetate, ethyl butyrate, isobutanol, isoamyl acetate, isoamyl propionate, 3-methyl-1-butanol, hexyl acetate, ethyl octanoate, ethyl nonanoate, ethyl decanoate, phenethyl acetate, ethyl dodecanoate, phenylethyl alcohol, and ethyl palmitate. It was confirmed that the content of palmitate was increased.

[0096] Specifically, the beer produced using the strain of the present invention showed that, compared to the control group, phenylethyl alcohol, which has a rose-like floral scent, increased by about 3.0 times, isoamyl acetate, which has a banana scent, increased by about 13.2 times, and phenyl acetate, which has rose and honey scents, increased by about 15.9 times.

[0097] 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.

[0098] In addition, it was confirmed that in beer produced using the strain of the present invention, the content of acetaldehyde, which has characteristics of grass and green apple scents, and phenethyl acetate, which has characteristics of rose and honey scents, was the highest among the aroma components.

[0099] Accordingly, it was confirmed that it is possible to produce beer with a deeper rose scent, honey scent, and green apple scent through the yeast (BC25 strain) according to the present invention.

[0101] Depository Name: Korean Culture Collection of Microorganisms (Domestic) Trustee Number: KFCC12060P Date of Deposit: 2025-10-21

Claims

Claim 1 Saccharomyces cerevisiae of accession number KFCC 12060P, which promotes the production of the aroma compound acetaldehyde ( Saccharomyces cerevisiae ) BC25. Claim 2 delete Claim 3 delete Claim 4 Saccharomyces cerevisiae of accession number KFCC 12060P as described in Paragraph 1 ( Saccharomyces cerevisiae Beer brewed using BC25 as the brewing strain.