KR102611318B1 - Novel Saccharomyces cerevisiae W153 and use thereof - Google Patents

Abstract

The present invention relates to a novel Saccharomyces cerevisiae W153 strain, and more specifically, to the Saccharomyces cerevisiae W153 strain and a method for producing herbal medicine using the same. The Saccharomyces cerevisiae W153 strain according to the present invention has excellent low-temperature growth and fermentation ability and excellent resistance to fatty acid biosynthesis inhibitors. In addition, it was confirmed that herbal liquor prepared from the above strain had excellent sensory properties. Therefore, the Saccharomyces cerevisiae W153 strain of the present invention can be used in a variety of fields in the field of fermented foods, including fermented liquor.

Description

Novel Saccharomyces cerevisiae W153 strain and use thereof {Novel Saccharomyces cerevisiae W153 and use thereof}

The present invention relates to a novel Saccharomyces cerevisiae W153 strain, and more specifically, to the Saccharomyces cerevisiae W153 strain and a method for producing herbal medicine using the same.

Consumption of traditional liquor is currently decreasing compared to the increasing consumption of alcoholic beverages. The causes of stagnation and decline since 2014 include low accessibility to traditional liquor, price competitiveness, and lack of technological development. On the other hand, consumption of premium traditional liquor is showing an increase, but the consumption amount is still small compared to other alcoholic beverages.

Yakju is a type of traditional liquor, made by filtering the liquor made from starch-containing ingredients such as rice and wheat, soup, and water. According to Korea's alcohol classification, if the amount of yeast used compared to the weight of rice is more than 1%, it is classified as Takju and Yakju, and if it is less than 1%, it is classified as Cheongju (Enforcement Decree of the Liquor Tax Act, 2020). Unlike Takju, in which even the sake ants are squeezed out with a cotton cloth, the solid content is removed, making it clean and showing off its unique flavor.

Most yakju and takju are fermented at room temperature (20-25℃), and are characterized by the rapid growth of yeast that allows brewing in a relatively short period of time. However, fermentation at low temperatures lowers the pH, which can suppress harmful bacteria that spoil alcohol, and also shows differences from room temperature fermentation in the case of organic acids and volatile aroma components. Low-temperature fermentation is known to prevent the loss of primary aroma and increase the synthesis of secondary aroma (ethyl, acetate esters, etc.). In addition, it has been reported that in a low-temperature fermentation environment, yeast has a significant impact on the growth of yeast, including not only aroma component-related reactions, but also protein transcription, cell membrane fluidity, RNA structure stability, and enzyme activity. Therefore, by performing low-temperature fermentation using strains that are resistant to low temperatures, it is possible to prevent spoilage and produce sensory-characteristic herbal liquor.

The microorganisms involved in fermentation of herbal liquor are as follows. For saccharification of rice, molds such as Aspergillus , Rhizopus , and Mucor produce amylase to break down starch into small saccharides. Afterwards, alcohol and numerous organic substances are produced through the action of Saccharomyces cerevisiae and other microorganisms. Especially in alcoholic beverages, the results vary greatly depending on which yeast is involved in the fermentation. By using this and fermenting a mixture of S. cerevisiae and non-Saccharomyces strains specialized for alcohol fermentation, medicinal liquor with various aromatic components can be made. Fragrance components are also known to have a significant impact on sensuality and therefore overall preference.

In the case of aroma components, most of them are generated from alcohol and ester. Among them, there are ethyl caproate, which can be said to be the representative flavor of traditional liquor, 2-phenethyl alcohol with a rose scent, ethyl acetate with a pineapple scent, and isoamyl acetate with a banana scent. There are terpenes that produce aromatic scents.

Accordingly, the present inventors completed the present invention by isolating yeast from persimmons, plums, grapes, aronia, and yeast that grow naturally in Korea and confirming their characteristics.

Therefore, the purpose of the present invention is to provide a Saccharomyces cerevisiae W153 strain deposited under accession number KACC93338P and a method for producing medicinal liquor with excellent sensory properties using the same.

In order to achieve the above object, the present invention provides Saccharomyces cerevisiae W153 strain deposited under accession number KACC93338P.

In addition, the present invention provides a method for producing herbal medicine with excellent sensory properties, comprising culturing fermentation strains, raw rice, and fermentation agents.

The Saccharomyces cerevisiae W153 strain according to the present invention has excellent low-temperature growth and fermentation ability and excellent resistance to fatty acid biosynthesis inhibitors. In addition, it was confirmed that herbal liquor prepared from the above strain had excellent sensory properties. Therefore, the Saccharomyces cerevisiae W153 strain of the present invention can be used in a variety of fields in the field of fermented foods, including fermented liquor.

Figure 1 is a diagram showing the manufacturing process of Yakju.
Figure 2 is a diagram showing the results of PCR-RFLP pattern analysis of 79 initially selected strains.
Figure 3 is a diagram showing the results of phylogenetic analysis of the final selected strains W153, N373, G818, and A159.
Figure 4 shows the results of analyzing the enzyme activity of the final selected strains W153, N373, G818, and A159.
Figure 5 is a diagram showing the results of analyzing the volatile aroma components of herbal liquor prepared singly or in combination with the strains according to the present invention.

Hereinafter, the present invention will be described in detail.

According to an aspect of the present invention, the present invention provides Saccharomyces cerevisiae W153 strain deposited under accession number KACC93338P.

In an embodiment of the present invention, the Saccharomyces cerevisiae W153 strain is preferably isolated from wild grape.

Phylogenetic analysis was performed based on the nucleotide sequence of the ITS I-5.8S rDNA-ITS II region of the Saccharomyces cerevisiae W153 strain according to the present invention, and as a result, known strains of the same species and strains already used in industrialization was confirmed to be another new strain. It was named Saccharomyces cerevisiae W153 strain and deposited at the Agricultural Genetic Resources Center of the National Institute of Agricultural Sciences on September 17, 2020, and was given accession number KACC93338P.

In an embodiment of the present invention, the Saccharomyces cerevisiae W153 strain preferably includes the base sequence of SEQ ID NO: 1. The base sequence of SEQ ID NO: 1 is the base sequence of the ITS I-5.8S rDNA-ITS II region of the Saccharomyces cerevisiae W153 strain, and is the sequence used to identify the strain.

In an embodiment of the present invention, the Saccharomyces cerevisiae W153 strain preferably has resistance to low temperature. The tolerance to low temperature means excellent growth and fermentation ability at low temperature.

In an embodiment of the present invention, the Saccharomyces cerevisiae W153 strain preferably has resistance to cerulenin, a fatty acid biosynthesis inhibitor. When strains with high resistance to fatty acid biosynthesis inhibitors are used in brewing, large amounts of high-quality alcohol and aroma components are produced. In particular, the strain with strong cerurenin resistance produces up to 5 times more ethyl carproate, a representative aroma component of traditional Korean liquor, and the strain with strong resistance to fluorophenylalanine produces phenethyl alcohol and phenethyl acetate, which produce a rose scent. It is known to increase.

In the present invention, the Saccharomyces cerevisiae W153 strain itself or its culture medium, deposited under the accession number KACC93338P, can be used as long as the purpose of the present invention can be achieved.

In addition, it is clear to those skilled in the art that the method required to obtain the culture medium of the strain may use any method known in the art, and is not limited thereto.

The Saccharomyces cerevisiae W153 strain according to the present invention has excellent low-temperature growth and fermentation ability and excellent resistance to fatty acid biosynthesis inhibitors, so it can be used in a variety of fermented foods, including fermented liquor. .

According to another aspect of the present invention, the present invention provides a method for producing herbal liquor with excellent sensory properties, comprising culturing a fermentation strain, starch, and a fermentation agent. The fermentation strain may be Saccharomyces cerevisiae W153 strain deposited under accession number KACC93338P.

In an embodiment of the present invention, the fermentation strain is Saccharomyces cerevisiae W153, along with Pichia kudriabjevi N373 strain, deposited under accession number KACC93340P; Hansenia spora biniae G818, deposited under accession number KACC93341P strain; And Wickerhamomyces anomalus A159 strain deposited under accession number KACC93339P; it may further include one strain selected from the group consisting of.

In a preferred embodiment of the present invention, the fermentation strain is (a) Saccharomyces cerevisiae W153 strain; and (b) one strain selected from the group consisting of Pichia cudriabjevi N373 strain, Hansenia spora biniae G818 strain, and Wickerhamomyces anomalus A159 strain; at a weight ratio of 1:1 to 20. It is preferable that they are mixed, more preferably a weight ratio of 1:5 to 15, and most preferably a weight ratio of 1:9, but is not limited thereto.

In an embodiment of the present invention, the starch may be any starch-containing main raw material as long as it can be used to produce base liquor, and is preferably non-glutinous rice, glutinous rice, brown rice, white rice, black rice, barley, corn, waxy corn, and potatoes. , sweet potatoes, barley, glutinous barley, soybeans, wheat, glutinous wheat, and mung beans, and more preferably non-glutinous rice, but is not limited thereto.

In an embodiment of the present invention, the fermentation agent may be any fermentation agent as long as it can be used to produce base liquor, and preferably may be at least one selected from the group consisting of yeast, yeast, crude enzyme, and purified enzyme, More preferably, it is entry, most preferably rice entry, but it is not limited thereto.

In the present invention, the entry means that a mold containing an enzyme that produces alcohol is propagated on grains. The above entry must contain sufficient saccharification enzymes necessary for manufacturing Cheongju, Soju, Gamju, Mirim Soybean Paste, etc. The above-mentioned entry can be prepared according to a conventional method by inoculating grains such as steamed rice or barley with Baek chrysanthemum.

For example, rice can be manufactured by inoculating steamed rice with white chrysanthemum and propagating the chrysanthemum for 48 hours while maintaining the temperature and humidity at 33°C and 95% in a thermohygrostat.

In an embodiment of the present invention, the medicinal liquor prepared by the above method has an increased content of one or more selected from the group consisting of citric acid, succinic acid, lactic acid, and acetic acid. In herbal liquor, citric acid has a soft and refreshing sour taste, succinic acid has a soft and refreshing sour taste, and acetic acid has a strong irritant effect and is an organic acid that has a great influence on the sensory properties of herbal liquor.

In an embodiment of the present invention, the medicinal liquor prepared by the above method contains 1-propanol, iso-amyl alcohol, 2-phenethyl alcohol, and ethyl acetate. acetate, iso butyl acetate, ethyl hexanoate, ethyl nonanoate, ethyl decanoate, 2-phenethyl acetate It is preferable that the content of one or more selected from the group consisting of ethyl hexadecanoate is increased. The volatile aroma components of which the content of the herbal liquor is increased are aroma components with high sensory properties, and give the herbal liquor a fruity or floral aroma.

The method of manufacturing herbal liquor using Saccharomyces cerevisiae W153 of the present invention can significantly improve the flavor of the manufactured herbal liquor, and is expected to greatly contribute to increasing the consumption of traditional liquor.

Hereinafter, the present invention will be described in more detail through experimental examples and examples. These experimental examples and examples are only for illustrating the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be construed as limited by these experimental examples and examples. will be.

[Experimental example]

Experimental Example 1. Experimental materials and strains used

1-1. experiment material

The white rice used in this experiment was produced in 2018, and 100% non-glutinous rice (Shindongjin variety, Iksan, domestic production) was purchased and used at the Iksan Integrated Rice Processing Plant of Nonghyup Grain Co., Ltd., Jeollabuk-do on July 11, 2019. For entry, rice entry ( Aspergillus luchuensis , sp 60) product was purchased from Choeun-goksik Co. Ltd, Hwaseong, Korea. Additionally, the water used for brewing was commercially available bottled water (Icis, lottechilsung, Cheongju, Korea).

1-2. Culture of used strains and bacteria

The strains used in this experiment were about 500 strains of yeast isolated from persimmons, wild grapes, grapes, aronia, yeast, and plums owned by the Microbial Engineering Laboratory of the Department of Food Science and Technology at Kyungpook National University, and the commercial wine yeast Saccharomyces cerevisiae W-3 was used as a control strain. used. All strains were subcultured twice at 30°C and 150 rpm in YPD (1% yeast, 2% peptone, 2% dextrose) broth to yield 5% of the raw material, and centrifuged at the point when yeast activity was highest. SUPRA 22k PLUS, Hanil co., Daejeon, Korea) and then inoculated to proceed with fermentation.

Experimental Example 2. Analysis of low temperature tolerance and fermentation ability of strains

The strain was inoculated into YPD broth and cultured (15°C, 48 h), and absorbance was measured at 600 nm using a spectrophotometer (UV-1601, Shimazdu Co., Kyoto, Japan) to select strains with excellent growth. Afterwards, they were cultured in rice saccharification solution (15°C, 96 h) and strains with excellent fermentation ability were initially selected through gas production and sniffing tests.

Experimental Example 3. Ability to produce volatile aroma components

3-1. β-glucosidase enzyme activity

Measurement of β-glucosidase activity was performed by modifying Swangkeaw’s method (Swangkeaw et al., 2011). The first selected strain was cultured in YPD broth for 48 hours, then 1 mL was taken and centrifuged at 10000 rpm for 3 minutes. 0.2 mL of supernatant, 0.2 mL of 20 mM pNPG (ρ-nitrophenyl-β-d-glucopyranoside), and 0.4 mL of 0.1 M citrate phosphate buffer (pH 5.0) were mixed and reacted in a constant temperature water bath (40°C) for 30 minutes. Finally, 0.8 mL of 2 M sodium carbonate was added to terminate the reaction, and the absorbance was measured at 405 nm using a spectrophotometer. 1 unit (U) of β-glucosidase enzyme activity was defined as the amount of enzyme that hydrolyzes 1 μmol of pNPG per minute.

3-2. Determination of resistance to fatty acid and amino acid biosynthesis inhibitors

Strains resistant to cerulenin and FPA (ρ-Fluorophenylalanine) were selected. A single colony of the primary selection strain was inoculated into the medium shown in Table 1, cultured at 30°C for 48 hours, and resistance was confirmed by colony formation (Akita et al., 1990).

Medium Composition Concentration(%, w/v) Cerulenin Agar Yeast nitrogen base 0.67 10mM Cerulenin 0.5 Glucose 2.0 Agar 2.0 FPA Agar Yeast nitrogen base 0.67 1M FPA 0.4 Glucose 2.0 Agar 2.0

Experimental Example 4. PCR-RFLP (polymerase chain reaction-Restricted Fragment Length Polymorphism)

4-1. DNA extraction

The DNA of the experimental strain was isolated using a modified method of Looke et al. (2011). After culturing in YPD liquid medium at 30°C and 150 rpm for 24 hours, 100 μL of the culture medium was taken. The culture was centrifuged (10000 rpm, 1 min), the supernatant was removed, and 100 μL of 200 mM LiOAc 1% SDS solution was added to suspend it. The suspension was reacted in a constant temperature water bath (70°C) for 15 minutes and then resuspended by adding 300 μL of 95% ethanol. The suspension was centrifuged (12000 rpm, 3 min) and the supernatant was removed to obtain a pellet. The obtained pellet was washed with 500 μL of 70% ethanol. The washed pellet was dissolved in 50 μL of ultrapure water, and centrifuged (12,000 rpm, 15 sec) to obtain a supernatant. The finally obtained supernatant was used in the next experiment.

4-2. PCR(Polymerase chain reaction)

Primers used in PCR were purchased from Bioneer (Daejeon, Korea), and the yeast ITS (internal transcribed spacer) region was synthesized in both directions using universal primers ITS1 and ITS4. The primer base sequences used are as shown in Table 2, and the PCR device used was Tpersnal 48 (Biometra Co., Gottingen, Germany). The amount of reaction solution used for PCR was adjusted to a total of 50 μL. The composition and reaction conditions of the reaction solution are shown in Tables 3 and 4, respectively.

Primer Sequence ITS1 5’-TCC GTA GGT GAA CCT GCG G-3’ ITS4 5’-TCC TCC GCT TAT TGA TAT GC-3’

Composition Amount(μL) 10X Taq reaction buffer 5.00 Template DNA 2.00 Forward primer (10 pmol/ μL) 2.00 Reverse primer (10 pmol/ μL) 2.00 dNTP mixture (10mM) 1.00 Taq polymerase (5 U/μL) 0.25 Distilled water 37.75 Total volume 50.00

Cycles Denaturation Annealing Extension Frequency One 95℃/3min 50℃/1min 72℃/1min One 2 95℃/30s 50℃/1min 72℃/1min 32 3 72℃/10min One

4-3. Restriction enzyme treatment

For PCR-RFLP pattern analysis, restriction enzymes HinfⅠ, HaeⅢ, and HpaⅠ (Enzynomics Co., Daejeon, Korea) were used, and the reaction was performed at 37°C for 1 hour with the composition shown in Table 5. DNA fragments were confirmed through electrophoresis.

Composition Amount(μL) PCR product 5.0 Enzyme ( Hinf Ⅰ, Hae Ⅲ, Hpa Ⅰ) 0.5 Enzyme buffer (×10) 1.5 Distilled water 8.0 Total volume 15

4-4. DNA electrophoresis

DNA electrophoresis was performed at a voltage of 100 V, and 1.5% agarose gel (Molecular Biology grade, Korea) and 0.5× TBE buffer were used. When preparing the 1.5% agarose gel, 0.01% of DNA staining reagent (EcoDyeTM, Solgent Co., Daejeon, Korea) was added. The sample was loaded with DNA on a 1.5% agarose gel, electrophoresed for 35 minutes, and the electrophoresis image was observed using a UV transilluminator (TFX-20M, Vilber Lourmat. Paris, France). A 100 bp Plus DNA ladder (Biofact, Daejeon, Korea) was used as a marker to measure the size of DNA fragments for each strain.

Experimental Example 5. Identification of yeast

Identification of the isolated strain was performed through phylogenetic analysis based on the base sequence of the ITS I-5.8S rDNA-ITS II region. The DNA base sequence was determined using the sequencing service of Solgent (Daejeon, Korea), and the homology test of the base sequence was performed using BLAST from the National Center for Biotechnology Information (NCBI).

Experimental Example 6. Preparation of Yakju

In the manufacture of medicinal liquor, the material immersion ratio of each sample is shown in Table 6, and the specific manufacturing process is shown in Figure 1.

As shown in Figure 1, to make base liquor, 250 g of rice was washed, soaked in water for 2 hours, drained for 30 minutes, and then steamed in a steamer for 30 minutes to make godubap. I took out the godubap and spread it well to cool. In a fermentation vessel, cooled godubap, 250 g of white guk (rice malt), and 1 L of bottled water were mixed, and 2.5% of the total volume of yeast was cultured in YPD medium, collected, and then inoculated, and one-stage immersion was performed at 15°C. The yeast used to produce herbal liquor was either a single strain or a mixed strain as shown in Table 7. On the 5th day of alcohol fermentation, a second stage immersion (main immersion) was performed. For the second stage soaking, 1.1 kg of rice was washed, soaked, and steamed in the same way, and 1.3 L of mineral water was added. After completion of soaking, the fermented liquor was fermented at 15°C for 30 days.

ingredient Mix amount White Chrysanthemum 250g non-glutinous rice 1,350 g bottled water 2,300mL leaven 100mL

strain ratio Control ( S. cerevisiae W-3) Single strain W153 Single strain N373 Single strain G818 Single strain W153:N376 1:9 W153:G818 1:9 W153:A159 1:9

Experimental Example 7. Analysis of fermentation characteristics of Yakju

7-1. Sugar content

Sugar content was measured by centrifuging the sample at 8000 rpm for 10 minutes and measuring the supernatant using a refractive sugar meter (Ra250, ATAGO Co., Ltd., Kyoto, Japan).

7-2. reducing sugar

Reducing sugars were measured using the DNS (dinitrosalicylic acid) method (Ahmed, 2004). Specifically, 1 mL of DNS reagent was added to 0.3 mL of the sample, boiled at 95°C for 5 minutes, and then left to cool at room temperature. After adding 7 mL of distilled water, the absorbance was measured at 550 nm using a spectrophotometer (Shimazdu Co., UV-1601, Kyoto, Japan). The results were expressed by converting the measured absorbance value into glucose. Blank was used in the same manner as above, using 0.3 mL of distilled water instead of the sample.

7-3. pH and total acidity

10 mL of supernatant centrifuged at 8000 rpm for 10 minutes was measured using an automatic titrator (DL22 Food & Beverage Analyzer, Mettler-Toledo AG Analytical, Schwerzenbach, Switzerland), titrated with 0.1 N NaOH, and the amount consumed was converted to acetic acid. did.

7-4. Alcohol

Alcohol content was analyzed according to the alcohol analysis regulations of the National Tax Service Technology Research Institute. The sample centrifuged (8,000 rpm, 10 min) was taken into a 100 mL volumetric flask, transferred to a distillation flask, washed twice with 15 mL of ultrapure water, and then added to the sample in the distillation flask. The distillate was collected in a volumetric flask, and distillation was stopped when the distillate reached 70 mL. Distilled water was added to fill the volume up to the 100 mL line and mixed well to homogenize. It was adjusted to 15°C, the specific gravity was measured with a hydrometer, and the alcohol content was measured using the Gay-Lussac alcohol conversion table.

Experimental Example 8. Analysis of physicochemical properties of Yakju

8-1. Organic Acid Analysis

The organic acid content of the fermented Yakju was determined by activating the centrifuged supernatant with methanol and distilled water, using Sep-pak (Sep-pak plus C18 catridge, Waters Co., USA) and a membrane filter (Millex-HV, 0.45 ㎛, Millipore Co.). , Bedford, USA) and analyzed using a High Perfomanse Liquid Chromatograph (Model 600E, Waters Co., USA). HPLC (high performance liquid chromatography) conditions are shown in Table 9.

Items Conditions Instrument Waters Co. 600E Model Column PL Hi-Plex H (*?*7.7 mm × 300 mm) Column temp 65℃ Detector Refractive Index Detector (RI, Model 410) Mobile phase 0.005 M Sulfuric acid Flow rate 0.6mL/min Injection volume 20 μL

8-2. Volatile fragrance ingredient

Volatile aroma components were measured using a gas chromatography mass spectrometer (7890A GC-MS; Agilent, Santa Clara, USA) equipped with a flame ionization detector (FID). SPME fiber (50/30 ㎛ DVB/CAR/PDMS, Supelco, Bellefonte, PA, USA) was used to collect scent components using head-space analysis. Before adsorption, SPME fiber was preheated at 240°C for 1 hour. 5 mL of the sample was placed in a headspace vial (20 mm, PTFE/silicon septum, magnetic cap), then 25% (1.25 g) of NaCl was added and sealed. For SPME collection, the prepared sample was stirred in a water bath at 35°C for 20 minutes to equilibrate the volatile components of the sample and the headspace, and then SPME fiber was injected to collect the sample at 35°C for 40 minutes. After collection, it was injected into GC-MS to analyze its composition. The GC-MS analysis conditions are shown in Table 10, and the library used was Wiley9Nist 0.8 (Wiley9ist 0.8 library, mass spectral search program, version 5.0, USA) (Torrens et al., 2004).

Item Condition Instruments Agilent 7890A with Agilent 5975C inert XL MSD Column DB-WAX (60 m × 250 ㎛ × 0.25 mm, Waters) Column temp. 40℃ -(2℃/min)-> 220℃ -(20℃/min)-> 240℃(5min) Carrier gas He(1 mL/min) Inlet temp. 240℃ Split ratio 20:1

Experimental Example 9. Principal component analysis (PCA)

Principal component analysis was performed to derive the correlation between volatile aroma components and main components according to the strain used in manufacturing herbal liquor. Specifically, the principal component analysis used volatile aroma components as measurement variables, and samples according to the strain (single strain or mixed strain) used in herbal liquor were designated as observation targets. Principal component analysis was performed using SAS software, and the correlation coefficients of volatile compounds identified by SPME/GC-MS were analyzed using Pearson's correlation analysis.

Experimental Example 10. Analysis of enzyme activity of yeast

To test the enzyme activity of the isolated and selected yeast, the API ZYM kit (Biomerieux, France) was used. The API ZYM kit consists of 20 capsules in a strip, and each capsule contains 19 substrates excluding the negative control, so the color changes when microorganisms solubilize the substrate and the metabolites produced during the incubation period react with the added indicator. Enzyme activity is measured using the principle of observation. First, each yeast was grown on YPD agar medium for one day, then colonies were taken, dissolved in 2 mL of sterilized water, 65 μL was dispensed into each capsule, and cultured at 30°C for 4 hours. One drop each of ZYM A and ZYM B reagents were added to the incubated strip, reacted for 5 minutes, and the results were read. The reading result was expressed as 0 to 5 points (0: negative, 5: maximum intensity) depending on the degree of color change, and a score of 3 or more was judged positive.

Experimental Example 11. Color difference analysis

The color difference was measured by placing 5 mL of the sample in a transparent petri dish (35 , Japan) were used to measure L (lightness), a (redness), and b (yellowness) values. Each experiment was repeated 10 times.

L: Degree of lightness (light+100 ↔ 0 dark)

a : Degree of redness(red+100 ↔ 0 ↔ - 80 green)

b: Degree of yellowness (yellow+70 ↔ 0 ↔ - 80 blue)

Experimental Example 13. Statistical processing

All experiments were repeated three times, and the results were expressed as mean and standard deviation (Mean ± SD), and analysis of variance (ANOVA) was performed using SAS statistical processing (Statstical Analysis System, SAS Institute Inc., Cary, NC, USA). ) and Duncan's multiple range test (p<0.05) to verify significance.

[Example]

Example 1. Selection of brewing strains with excellent low-temperature fermentation ability

1-1. Selection of strains with excellent low temperature tolerance and fermentation ability

79 strains with excellent low-temperature growth and fermentation ability were initially selected from over 500 strains of yeast isolated from wild grapes, grapes, aronia, and yeast held by the Microbial Engineering Laboratory of the Department of Food Science and Technology at Kyungpook National University, and are shown in Table 11.

Strain Growth & fermentation activity Strain Growth & fermentation activity Strain Growth & fermentation activity A124 ++ ¹⁾ G439 + N266 ++ A126 + G441 ++ N275 + A142 + G442 + N289 + A144 + G444 ++ N311 + A159 ++ G463 + N341 + A173 ++ G471 + N342 + A182 + G472 + N344 + A245 + G473 ++ N358 + A251 ++ G475 + N362 + A255 + G478 ++ N368 + A262 + G479 + N369 + A266 + G483 + N371 + A271 + G557 ++ N372 + A275 + G571 + N373 ++ A279 ++ G573 + N374 + A291 + G576 + N376 ++ A293 + G578 + N377 ++ A313 + G581 ++ N381 ++ A322 ++ G583 + N383 + A328 + G818 ++ N384 + G119 + G836 + N393 + G122 + G838 + W141 ++ G123 + N247 + W149 ++ G139 + N249 ++ W153 ++ G167 ++ N253 + W256 + G195 ++ N254 ++ G244 ++ N264 + 1)++, Low-temperature resistance and producing a lot of gas; +, low-temperature resistance and producing gas moderately.

1-2. Classification and identification of selected strains

To identify the 79 first selected strains, the ITS I-5.8S rDNA-ITS II region was amplified, and the yeasts were classified through PCR-RFLP pattern analysis. After analyzing the base sequences of the ITS I-5.8S rDNA-ITS II region for each strain that showed the same pattern in PCR-RFLP analysis, a homology search was conducted using NCBI's BLAST. The results of PCR-RFLP pattern analysis are shown in Figure 2, and the strain identification results are shown in Table 12.

Strains Number of strains depending on the origin Isolates Grape Wild grapes Aronia Nuruk Saccharomyces cerevisiae 17 4 One G(119, 122, 123, 139, 167, 195, 439, 441, 442, 444, 473, 479, 483, 557, 573, 581, 583), N358, W(141, 149, 153, 256) Hanseniaspora vineae 11 One A271, G(244, 463, 471, 472, 475, 478, 571, 576, 578, 818, 836) Wickerhamomyces anomalus One 19 20 A(124, 126, 142, 144, 159, 173, 182, 245, 251, 255, 262, 266, 275, 279, 291, 293, 313, 322, 328), G838, N(247, 249, 253) , 254, 264, 266, 275, 289, 311, 341, 342, 344, 368, 369, 371, 372, 374, 377, 383, 384) Torulaspora delbrueckii 3 N(362, 381, 393) Pichia kudriavzevii 2 N(373, 376) Total 29 4 20 26 79

1-3. Evaluation of the ability of isolated strains to produce volatile aroma components

1-3-1. β-glucosidase enzyme activity assay

Since β-glucosidase cleaves the β-1,4 bond of non-volatile glycoside to produce various volatile aroma components, strains with higher β-glucosidase activity are known to be advantageous in producing high flavor during alcohol fermentation (Sarry and Gunata, 2004; Liu et al., 2017).

A β-glucosidase activity test was conducted on the 79 strains selected initially. In the β-glucosidase activity test, the higher the enzyme activity, the more pNPG is decomposed into pNP (ρ-nitrophenol) and glucose, and the higher the absorbance is due to the fluorescent pNP. The β-glucosidase activity test results are shown in Table 13.

Strain β-glucosidase activity (U/mL) Strain β-glucosidase activity (U/mL) Strain β-glucosidase activity (U/mL) A124 0.910 G439 0.511 N266 0.735 A126 0.756 G441 0.553 N275 0.655 A142 0.766 G442 0.540 N289 0.636 A144 0.793 G444 0.538 N311 0.617 A159 0.922 G463 0.435 N341 0.636 A173 0.792 G471 0.467 N342 0.768 A182 0.790 G472 0.429 N344 0.598 A245 0.813 G473 0.518 N358 0.623 A251 0.667 G475 0.481 N362 0.630 A255 0.685 G478 0.490 N368 0.687 A262 0.837 G479 0.505 N369 0.781 A266 0.551 G483 0.672 N371 0.739 A271 0.650 G557 0.619 N372 0.708 A275 0.688 G571 0.467 N373 1.332 A279 0.633 G573 0.612 N374 0.633 A291 0.878 G576 0.497 N376 1.408 A293 0.853 G578 0.469 N377 0.541 A313 0.850 G581 0.570 N381 1.151 A322 0.852 G583 0.661 N383 0.777 A328 0.706 G818 0.567 N384 0.568 G119 0.513 G836 0.488 N393 0.568 G122 0.490 G838 0.460 W141 0.586 G123 0.508 N247 0.828 W149 0.661 G139 0.500 N249 0.716 W153 0.679 G167 0.502 N253 0.669 W256 0.596 G195 0.467 N254 0.629 G244 0.314 N264 0.615 One unit(U) of enzyme activity was defined as the amount of enzyme that released 1 μmol of pNPG per minute under the experimental conditions

As shown in Table 13, it was confirmed that 37 strains showed higher absorbance than the control group. In particular, strains A159, N373, N376, and N381 had absorbance values higher than 0.4, indicating that the activity of β-glucosidase was significantly high.

1-3-2. Determination of resistance to fatty acid and amino acid biosynthesis inhibitors

Strains with high resistance to fatty acid and amino acid biosynthesis inhibitors produce large amounts of higher alcohols and aroma components. Strains with strong resistance to cerulenin can produce up to five times more ethyl carproate, a representative aroma component of Korean traditional liquor (Choi et al., 2013), and strains with high resistance to FPA (p-fluorophenylalanine) produce a rose scent. It is known to increase the production of phenethyl alcohol and phenethyl acetate (Osamu et al., 1990).

The results of confirming resistance to Cerulenin (50 μM) and FPA (4 mM) are shown in Tables 14 and 15, respectively.

Strain Cerulenin
resistance Strain Cerulenin
resistance Strain Cerulenin
resistance A124 + ¹⁾ G439 - N266 + A126 + G441 - N275 + A142 + G442 - N289 + A144 + G444 - N311 + A159 + G463 - N341 + A173 + G471 - N342 + A182 + G472 - N344 + A245 + G473 - N358 - A251 + G475 - N362 + A255 + G478 - N368 + A262 + G479 - N369 + A266 + G483 - N371 + A271 + G557 - N372 + A275 + G571 - N373 + A279 + G573 - N374 + A291 + G576 - N376 + A293 + G578 - N377 + A313 + G581 - N381 + A322 + G583 - N383 + A328 + G818 - N384 + G119 - G836 - N393 + G122 - G838 - W141 - G123 - N247 + W149 - G139 - N249 + W153 + G167 - N253 + W256 - G195 - N254 + G244 - N264 + ¹⁾ +, cerulenin resistance positive; -, cerulenin resistance negativie

Strain FPA resistance Strain FPA resistance Strain FPA resistance A124 - ^One) G439 - N266 - A126 - G441 - N275 - A142 - G442 - N289 - A144 - G444 - N311 - A159 - G463 + N341 - A173 - G471 + N342 - A182 - G472 + N344 - A245 - G473 - N358 - A251 - G475 + N362 - A255 - G478 + N368 - A262 - G479 - N369 - A266 - G483 - N371 - A271 - G557 - N372 - A275 - G571 + N373 + A279 - G573 - N374 - A291 - G576 + N376 + A293 - G578 + N377 - A313 - G581 - N381 + A322 - G583 + N383 - A328 - G818 + N384 - G119 - G836 + N393 - G122 - G838 + W141 - G123 - N247 - W149 + G139 - N249 - W153 - G167 - N253 - W256 - G195 - N254 - G244 + N264 - ¹⁾ +, ρ-fluoro phenylalanin (FPA) resistance positive; -, ρ-fluoro phenylalanin (FPA) resistance negativie

As shown in Table 14, 46 strains, including A159, N373, and W153, were confirmed to be Cerulenin resistant.

As shown in Table 15, 17 strains, including G818 and N373 strains, were confirmed to be FPA resistant.

1-4. Final selection of strains suitable for brewing among isolated strains

Among the first selection strains that completed PCR-RFLP analysis, strains with high results in volatile scent production experiments for each species were finally selected. Phylogenetic analysis was performed using the nucleotide sequence of the ITSⅠ-5.8S-ITSⅡ region of the final selected strain.

Information on the final selected strains is shown in Table 16, and the phylogenetic analysis results are shown in Figure 3.

Isolate
code Species Accession
No. Origin Accession number W153 Saccharomyces cerevisiae NR_111007 Wild grapes KACC93338P N373 Pichia kudriavzevii NR_131315 Nuruk KACC93340P G818 Hanseniaspora vineae NR_165975 Grape KACC93341P A159 Wickerhamomyces anomalus NR_111210 Aronia KACC93339P

As shown in Table 16 and Figure 3, the final selected strains W153, N373, G818, and A159 were all confirmed to be new strains. In addition, the strains W153, N373, G818 and A159 are characterized by containing the ITS I-5.8S rDNA-ITS II region represented by the base sequences of SEQ ID NOs: 1 to 4, respectively. The final selected strain was entrusted to the Agricultural Genetic Resources Center of the National Academy of Agricultural Sciences on September 17, 2020, and was given an accession number.

1-5. Enzyme activity analysis using API-ZYM kit

Using the API-ZYM kit, the enzyme activities of the strains W153, N373, G818, and A159 finally selected in Examples 1-4 were analyzed, and the results are shown in Figure 4.

As shown in Figure 4, it was confirmed that the strains W153, N373, G818, and A159 selected in Examples 1-4 had different enzyme activity patterns.

Example 2. Analysis of fermentation characteristics of herbal liquor produced using brewing strains with excellent low-temperature fermentation ability

Yakju was prepared using the brewing strains W153, N373, G818, and A159 with excellent low-temperature fermentation ability finally selected in Example 1. Herbal liquor was prepared by the process shown in Figure 1, using (i) single strains (W153, N373, G818, A159) or (ii) mixed strains (W153+N373, W153+G818, W153+A159). To analyze the fermentation characteristics of the prepared Yakju, the sugar content, reducing sugar, pH, total acid content, and alcohol content were measured, and the results are shown in Table 17.

Yeast strain Soluble solid
(˚Brix) Reducing sugar
(mg/ml) Ethanol
(%) pH Total acid
(%) control 6.93±0.06 ^d1)2) 0.13± ^0.00d 12.87± ^0.13ab 3.83± ^0.02a 0.64± ^0.02e W153 6.83±0.06 ^de 0.09± ^0.00d 13.12± ^0.43a 3.80± ^0.03a 0.62±0.01 ^e N373 8.63± ^0.06c 1.81± ^0.06c 9.53± ^0.06e 3.60± ^0.02c 0.90± ^0.03a G818 10.07± ^0.12b 3.93± ^0.08a 10.55± ^0.31d 3.82± ^0.04a 0.82± ^0.00b A159 10.37± ^0.06a 3.00± ^0.07b 10.53± ^0.41d 3.75± ^0.03b 0.80± ^0.01b W153+N373 6.67± ^0.06f 0.12± ^0.00d 13.02± ^0.33a 3.33± ^0.01d 0.72± ^0.01d W153+G818 6.73± ^0.06ef 0.11± ^0.00d 12.42±0.03 ^b.c. 3.32± ^0.02d 0.74±0.02 ^cd W153+A159 6.93± ^0.06d 0.12± ^0.00d 12.37± ^0.01c 3.35± ^0.01d 0.75± ^0.02c 1)All the data were expressed as mean ± SD (n=3).
2)Different letters within the same column indicate significant difference(p<0.05)

As shown in Table 17, Yakju produced with single strains N373, G818, and A159 had higher sugar content and reducing sugar content than the control group, and ethanol fermentation using sugar progressed less than the control group, so the alcohol content was lower than the control group. It was confirmed that the herbal liquor prepared from single strain W153 and mixed strains had similar levels of sugar and reducing sugar content as the control group, but the alcohol content of the produced herbal liquor was higher.

Herbal liquor prepared from single or mixed strains has a pH similar to that of the control group, but herbal liquor prepared from single strains N373, G818, and A159; and herbal liquor prepared from mixed strains tended to have a higher total acid content compared to the control group. This can be said to be because the type and content of organic acids produced by each strain are different. In addition, the fact that the total acid content increases but the pH does not decrease can be seen as a buffering effect due to the increase in amino acids as fermentation progresses.

Example 3. Analysis of physicochemical properties of herbal liquor prepared using brewing strains with excellent low-temperature fermentation ability

The physicochemical properties of herbal medicine prepared using single or mixed strains were analyzed. Specifically, organic acid content, volatile aroma components, color difference, and sensory properties, which directly affect the quality of herbal liquor, were analyzed.

3-1. Organic acid content analysis

The results of analyzing the organic acid content of herbal liquor prepared using single or mixed strains are shown in Table 18.

Yeast strain Citric acid Malic acid Succinic acid Lactic acid Acetic acid Control 1.41±0.01 ^f1)2) ND ³⁾ 1.05±0.02 ^b.c. 0.12±0.01 ^b.c. 0.12± ^0.01f W153 1.53±0.02 ^de N.D. 1.08± ^0.01b 0.10±0.01 ^b.c. 0.17± ^0.02e N373 1.56± ^0.01d N.D. 0.99± ^0.01d 0.41± ^0.02a 0.39± ^0.02b G818 2.06± ^0.02a N.D. 0.90± ^0.02f N.D. 0.29± ^0.02d A159 1.89± ^0.01b N.D. 0.96±0.02 ^de 0.04± ^0.01e 0.85± ^0.04a W153+N373 1.51± ^0.02e N.D. 0.95± ^0.02e 0.11±0.02 ^b.c. 0.36±0.03 ^b.c. W153+G818 1.63± ^0.03c N.D. 1.17± ^0.03a 0.07± ^0.01d 0.16± ^0.01ef W153+A159 1.61± ^0.02c N.D. 1.04± ^0.01c 0.09±0.02 ^cd 0.34± ^0.02c 1)All the data were expressed as mean ± SD (n=3).
2)Different letters within the same column indicate significant difference(p<0.05)
3)Not detected.

As shown in Table 18, a large amount of citric acid was produced in all herbal liquors, which appears to be the effect of rice malt used in the manufacture of herbal liquor. Lactic acid, which has a mild sour taste, was highest in the N373-only fermentation group, and highly irritating acetic acid was lowest in the W153-only fermentation group. Succinic acid was lower than the control group in all single or mixed treatments. In particular, the W153 single fermentation group had high contents of succinic acid and lactic acid. In addition, the W153 and N373 mixed fermentations had high contents of lactic acid and acetic acid, the W153 and G818 mixed fermentations had high contents of citric acid and succinic acid, and the W153 and A159 mixed fermentations had high contents of citric acid and acetic acid.

3-2. Volatile aroma component analysis

Volatile aroma components give each liquor different characteristics and contribute greatly to its taste. The results of analyzing the volatile aroma components of medicinal liquor prepared using single or mixed strains are shown in Table 19, and the results of principal component analysis are shown in FIG. 5.

Volatile compound Control W153 N373 G818 A159 W153+N373 W153+G818 W153+A159 Higher alcohols (mg/L) 1-propanol 30.40±0.33 ^b1)2) 6.82±0.19 ^g 10.01± ^0.62f 21.41± ^0.31d 6.78± ^0.45g 28.38± ^1.13c 48.46± ^0.86a 17.12± ^0.36e Iso-butanol 241.22± ^10.90a 144.52± ^4.81c 83.94± ^0.64e 39.25±2.84 ^f 25.09± ^0.98g 181.01±7.40 ^b 146.87± ^6.87c 114.08± ^3.78d Iso-amyl alcohol 158.89± ^8.89d 245.08±5.25 ^b 200.81± ^8.18c 196.14± ^9.31c 110.76± ^6.46e 204.56± ^5.63c 262.37± ^6.86a 48.93±5.12 ^f Methionol 2.89± ^0.77d 4.19± ^0.29c 10.92± ^0.39a 3.03± ^0.52d 8.26± ^0.86b 4.69± ^0.93c 4.43± ^0.27c 7.67± ^0.69b 2-Phenethyl alcohol 85.44±8.63 ^cd 95.92± ^6.22ab 76.80± ^4.08d 27.21±2.18 ^f 42.16± ^1.60e 87.86±6.16 ^b.c. 80.54±3.58 ^cd 104.26± ^6.24a 2,3-butandiol 36.75± ^1.79a 11.35± ^0.95ef 14.34± ^1.63d 10.13± ^0.62f 12.58±0.28 ^de 13.55± ^0.77d 18.95± ^0.96c 25.72±1.59 ^b Esters (μg/L) Ethyl acetate 26.08± ^2.69e 28.29±3.19 ^ef 237.70± ^28.80a 115.95±7.31 ^b 71.74±8.14 ^cd 85.72±7.17 ^c 56.62± ^5.13d 61.76± ^3.74d Isobutyl acetate 8.41± ^0.67a 9.17± ^0.77a 2.88± ^0.35c 1.06± ^0.16d 2.06±0.30 ^cd 9.07± ^1.32a 5.45± ^0.54b 7.97± ^1.22a Ethyl butyrate 0.42± ^0.03a 0.40± ^0.02ab 0.17± ^0.02d 0.09± ^0.01e 0.21± ^0.02d 0.42± ^0.02a 0.36±0.01 ^b.c. 0.33± ^0.06c Iso amyl acetate 10.62± ^1.56a 9.74± ^0.43ab 7.20± ^2.03c 4.04± ^0.36d 0.85± ^0.17e 8.12±1.27 ^b.c. 4.60± ^0.57d 5.02± ^0.16d Ethyl hexanoate 0.50± ^0.02b 0.54± ^0.01a 0.10±0.03 ^e 0.07± ^0.01ef 0.05± ^0.01f 0.49± ^0.02b 0.30± ^0.01d 0.35± ^0.03c Ethyl octanoate 1.36± ^0.22a 1.39± ^0.08a 0.16± ^0.13d 0.17± ^0.04d 0.06± ^0.02d 1.27± ^0.11a 0.81± ^0.12c 1.05± ^0.09b Ethyl nonanoate 5.84± ^0.32e 3.81± ^0.33f 7.17± ^0.41e 11.54± ^1.12c 15.88± ^0.35a 8.75± ^1.12d 10.60± ^1.36c 12.98± ^0.81b Ethyl decanoate 26.05± ^1.02ab 22.73± ^1.31c 4.04± ^0.47e 19.37± ^1.39d 1.80± ^0.08f 23.53± ^1.35c 28.08± ^1.98a 23.99±1.19 ^b.c. Diethyl succinate 2.59± ^0.36ab 2.26±0.15 ^b.c. 0.40± ^0.06d 1.93± ^0.27c 0.18± ^0.05d 2.34± ^0.24b 2.80± ^0.09a 2.39± ^0.08b Ethyl dodecanoate 2.63± ^0.15d 1.74±0.09 ^ef 1.60± ^0.11f 2.08± ^0.09e 1.49± ^0.08f 3.17± ^0.18c 4.41± ^0.27a 3.74± ^0.48b Methyl salicylate 1.43± ^0.18c 1.48± ^0.07c 1.65±0.16 ^b.c. 2.08± ^0.17a 2.05± ^0.24a 1.40± ^0.16c 1.71±0.19 ^b.c. 1.83± ^0.15ab 2-phenethyl acetate 3.18±0.28 ^de 5.77± ^0.35c 1.59±0.26 ^ef 33.76± ^1.80a 0.26± ^0.02f 4.08±0.94 ^cd 28.38±1.97 ^b 0.75± ^0.09f Ethyl tetradecanoate 2.66± ^0.13fg 2.38± ^0.33g 3.39± ^0.27e 8.44± ^0.45b 10.51± ^0.51a 3.07±0.28 ^ef 5.61± ^0.07c 4.66± ^0.38d Ethyl hexadecanoate 10.39±1.26 ^g 14.78± ^0.96f 17.78±2.47 ^f 38.00± ^2.33d 95.88± ^1.07a 29.40± ^2.39e 43.76± ^1.48c 50.65±1.93 ^b 1)All the data were expressed as mean ± SD (n=3).
2)Different letters within the same column indicate significant difference(p<0.05)

As shown in Table 19 and Figure 5, terpenes were not detected among the volatile aroma components in all test sections, and higher alcohol and ester were detected. Looking at the volatile components of herbal liquor for each test group, it was confirmed that herbal liquor prepared from single or mixed strains had a higher content of aromatic components with high preference compared to the control group. Fragrance components with high preference include fruity and floral scents, examples of which include Iso-amyl alcohol, 2-Phenetyl alcohol, Ethyl acetate, Iso amyl acetate, Ethyl hexanoate, Ethyl octanoate, Ethyl nonanoate, Ethyl decanoate, and 2-phenethyl acetate. and Ethyl hexadecanoate.

More specifically, in the N373 strain, ethyl acetate was detected about 9 times higher than the control group, and in the G818 strain, 2-phenethyl acetate was detected about 10 times higher than the control group. Additionally, among MCFA (Medium Chain Fatty Acid) esters, ethyl nonanoate was detected at high levels in the A159 strain, and ethyl hexanoate and ethyl nonanoate were detected at high levels in the W153 strain. Ethyl tetradecanoate and ethyl hexadecanoate were produced in the highest amount in the A159 strain, and in particular, ethyl hexadecanoate was confirmed to be about 9 times higher than the control group.

In addition, the single fermentation and mixed fermentation using the W153 strain had higher contents of the ingredients listed in Table 20 below than the control group and other test groups.

test zone High content of volatile fragrance ingredients W153 single fermentation port iso-amyl alcohol, 2-Phenethyl alcohol, Iso butyl acetate, Ethyl hexanoate W153 and N373 mixed fermentation pot Iso-amyl alcohol, 2-Phenethyl alcohol, Ethyl acetate, Iso butyl acetate, Ethyl hexanoate, Ethyl nonanoate, Ethyl dodecanoate, Ethyl hexadecanoate W153 and G818 mixed fermentation pot 1-propanol, Iso-amyl alcohol, 2-Phenethyl alcohol, Ethyl nonanoate, Ethyl decanoate, Ethyl dodecanoate, 2-phenethyl acetate, Ethyl hexadecanoate W153 and A159 mixed fermentation pot 2-Phenethyl alcohol, Ethyl nonanoate, Ethyl dodecanoate, 2-phenethyl acetate, Ethyl hexadecanoate

3-3. Color difference analysis

Sensuality is also greatly influenced by color. In particular, it is known that color in herbal liquor has a significant impact on the sensory properties as well as the aroma components. Yakju produced with strain W153; and herbal liquor prepared from mixed strains; the results of analyzing the color difference are shown in Table 21.

Sample L a b Control 62.38±0.01 ^d1)2) 0.14± ^0.00b -2.54± ^0.00e W153 62.47± ^0.02c 0.13± ^0.00c -2.18±0.00 ^d W153+N373 62.65± ^0.01b 0.10± ^0.01d -1.84±0.00 ^b W153+G818 62.27± ^0.00e 0.15± ^0.00a -1.68±0.00 ^a W153+A159 62.71± ^0.01a 0.07±0.00 ^e -1.95±0.01 ^{c 1)} All the data were expressed as mean ± SD (n=3).
²⁾ Different letters within the same column indicate significant difference (p<0.05)

As shown in Table 21, in the case of whiteness (L), the W153 and A159 mixed fermentation group showed the highest value at 62.71, and the W153 and N373 mixed fermentation group showed the second highest value at 62.65. Redness (a) tended to decrease as the L value increased. The L value was lowest at 0.07 in the W153 and A153 mixed fermentation plots, which had the highest, followed by the W153 and N373 mixed fermentation plots. Yellowness (b) showed negative values in all test groups, meaning that the control group ( S. cereevisiae only fermentation group) showed a lower value compared to the mixed fermentation group.

As a result of the physicochemical characteristic analysis, herbal liquor prepared from strains W153, N373, G818, and A159 each have various volatile aroma components, which means that unique low-water herbal liquor can be produced. It also suggests that it is possible to produce herbal liquor with more diverse aroma components and high sensory properties through mixed fermentation of the W153 strain and other strains (N373, G818, A159).

Overall, the present inventors isolated new monarchs W153, N373 G818, and A159 from wild grapes, grapes, aronia, and yeast. The strains have excellent low-temperature growth and fermentation ability, high β-glucosidase enzyme activity, and excellent resistance to fatty acid and amino acid biosynthesis inhibitors. Yakju prepared from the above strains W153, N373 G818 and A159; and herbal liquor prepared by mixing them showed excellent results in terms of scent production and sensory properties. Therefore, the strains W153, N373, G818, and A159 of the present invention can be used in a variety of fields in the field of fermented foods, including fermented liquor.

As above, specific parts of the present invention have been described in detail, and it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

National Institute of Agricultural Sciences Agricultural Genetic Resources Center KACC93338P 20200917 National Institute of Agricultural Sciences Agricultural Genetic Resources Center KACC93339P 20200917 National Institute of Agricultural Sciences Agricultural Genetic Resources Center KACC93340P 20200917 National Institute of Agricultural Sciences Agricultural Genetic Resources Center KACC93341P 20200917

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Novel Saccharomyces cerevisiae W153 and use thereof <130> 1-329P <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 808 <212> DNA <213> Saccharomyces cerevisiae <400> 1 gcggaaggat cattaaagaa atttaataat tttgaaaatg gatttttttg ttttggcaag 60 agcatgagag cttttactgg gcaagaagac aagagatgga gagtccagcc gggcctgcgc 120 ttaagtgcgc ggtcttgcta ggcttgtaag tttctttctt gctattccaa acggtgagag 180 atttctgtgc ttttgttata ggacaattaa aaccgtttca atacaacaca ctgtggagtt 240 ttcatatctt tgcaactttt tctttgggca ttcgagcaat cggggcccag aggtaacaaa 300 cacaaacaat tttatctatt cattaaattt ttgtcaaaaa caagaatttt cgtaactgga 360 aattttaaaa tattaaaaac tttcaacaac ggatctcttg gttctcgcat cgatgaagaa 420 cgcagcgaaa tgcgatacgt aatgtgaatt gcagaattcc gtgaatcatc gaatctttga 480 acgcacattg cgccccttgg tattccaggg ggcatgcctg tttgagcgtc atttccttct 540 caaacattct gtttggtagt gagtgatact ctttggagtt aacttgaaat tgctggcctt 600 ttcattggat gttttttttc caaagagagg tttctctgcg tgcttgaggt ataatgcaag 660 tacggtcgtt ttaggtttta ccaactgcgg ctaatctttt tttatactga gcgtattgga 720 acgttatcga taagaagaga gcgtctaggc gaacaatgtt cttaaagttt gacctcaaat 780 caggtaggag tacccgctga acttaagc 808 <210> 2 <211> 413 <212> DNA <213> Unknown <220> <223> Pichia kudriavzevii <400> 2 tgcgtgagcg gacgaaaacaa maacacctaa atgtggaata tagcatatag tcgacaagag 60 aaatctacga aaaacaaaca aaactttcaa caacggatct cttggttctc gcatcgatga 120 agagcgcagc gaaatgcgat acctagtgtg aattgcagcc atcgtgaatc atcgagttct 180 tgaacgcaca ttgcgcccct cggcattccg gggggcatgc ctgtttgagc gtcgtttcca 240 tcttgcgcgt gcgcagagtt gggggagcgg agcggacgac gtgtaaagag cgtcggagct 300 gcgactcgcc tgaaagggag cgaagctggc cgagcgaact agactttttt tcagggacgc 360 ttggcggccg agagcgagtg ttgcgagaca acaaaaagct cgacctcaaa tca 413 <210> 3 <211> 702 <212> DNA <213> Hanseniaspora vineae <400> 3 gagggatcat ttaagaaaat tactgaattt tcgagctgcc tgtgtggctg cagacagaga 60 gctaagcctg tgcgcctgcg cttaattgcg cggctgcggg tggcgttctt gctattggct 120 gtagtttgcg cggtggtttt gatttcattt cacactgtga agattttttc atactttact 180 tctttgggct gttttttcag cccaaaggtt ataaacacaa acaacttttt tttttattac 240 agacaatcaa gaaatttcta ttgaaataaa atattttaaa actttcaaca acggatctct 300 tggttctcgc atcgatgaag aacgtagcga attgcgataa gtaatgtgaa ttgcagattc 360 tcgtgaatca ttgaattttt gaacgcacat tgcgccctct ggtattccag agggcatgcc 420 tgtttgagcg tcatttcctt ctcaaaaacc cagtttttgg ttgtgagtga tactctgtta 480 cagggttaac ttgaaaatgc tatgcccatt tggctgcccc ttctctgagg ggactgcgcg 540 tctgtgcagg atgtaaccaa tgtatttagg tattcatacc aactttcatt gtgcgcgtct 600 tatgcagttg cagtccaccc aacctcggac acactgggct ggctgggcca acagtattca 660 taaagttgac ctcaaaatcag gtargaatac ccgctgaact ta 702 <210> 4 <211> 715 <212> DNA <213> Unknown <220> <223> Wickerhamomyces anomalus <400> 4 ctgcggaagg atcattatag tattctattg ccagcgctta attgcgcggc gataaacctt 60 acacacattg tctagttttt ttgaactttg ctttgggtgg tgggtgawcc tgcggaagga 120 tcattcttgt gatttattat tccatggygt gaccggaasg aaaccataac cacwtaaaat 180 gtggaaaata gcaattkttg gccaagagaa ttttrggaaa aacaaacaaa actttcaaca 240 acggatctct tggttctcgc atcgatgaag agcgcagcga aatgcgatac ctagtgtgaa 300 ttgcagccat cgtgaatcat cgagttcttg aacgcacatt gcgcccctcg gcattccggg 360 gggcatgcct gtttgagcgt cgtttccatc ttgcgcgtgc gcagagttgg gggagcggag 420 cggacgacgt gtaaagagcg tcggagctgc gactcgcctg aaagggagcg aaactggccg 480 agcgaactag actttttttc agggacgctt ggcggccgaa agcgagtgtt gcgcagacaa 540 caaaaagctc gacctcaaat cmtggagggaa tacccgctga acttaakcct attcaaaacg 600 gargaaagca ggtttagaag tattttaggc tcggcttaac aacaataaac taaaagtttg 660 acctcaaatc aggtaggact acccgctgaa cttaagcata tcaataagcg gagga 715

Claims (12)

Saccharomyces cerevisiae W153 strain, which contains the base sequence of SEQ ID NO: 1 and is resistant to low temperatures of 15-20°C, accession number KACC93338P. According to paragraph 1,
The strain is a Saccharomyces cerevisiae W153 strain isolated from wild grapes. delete delete According to paragraph 1,
The strain is a Saccharomyces cerevisiae W153 strain having resistance to cerulenin. Comprising: cultivating fermentation strain, starch, and fermentation agent,
The fermentation strain is a Saccharomyces cerevisiae W153 strain, which includes the base sequence of SEQ ID NO: 1 and is resistant to low temperatures of 15-20°C, accession number KACC93338P.
Method for manufacturing herbal liquor with excellent sensory properties. According to clause 6,
The fermentation strain is
Pichia Kudriabjevi N373 strain, accessioned with accession number KACC93340P;
Hanseniaspora biniae G818 strain, accessioned with accession number KACC93341P; and
Wickerhamyces anomalus A159 strain deposited under accession number KACC93339P; A method for producing medicinal liquor, further comprising one strain selected from the group consisting of. In clause 7,
The fermentation strain is
(a) Saccharomyces cerevisiae W153 strain; and
(b) one strain selected from the group consisting of Pichia cudriabjevi N373 strain, Hansenia spora biniae G818 strain, and Wickerhamomyces anomalus A159 strain; mixed at a weight ratio of 1: 1 to 20 The manufacturing method of herbal liquor. According to clause 6,
The starch is one or more selected from the group consisting of rice, glutinous rice, brown rice, white rice, black rice, barley, corn, waxy corn, potatoes, sweet potatoes, barley, waxy barley, soybeans, wheat, waxy wheat, and mung beans. According to clause 6,
The fermentation agent is at least one selected from the group consisting of yeast, yeast, crude enzyme, and purified enzyme. According to clause 6,
A method of manufacturing herbal liquor, wherein the herbal liquor prepared by the above method has an increased content of one or more selected from the group consisting of citric acid, succinic acid, lactic acid, and acetic acid. According to clause 6,
The medicinal liquor prepared by the above method includes 1-propanol, iso-amyl alcohol, 2-phenethyl alcohol, ethyl acetate, and isobutyl acetate ( iso butyl acetate, ethyl hexanoate, ethyl nonanoate, ethyl decanoate, 2-phenethyl acetate and ethyl hexadecanoate ( A method for manufacturing herbal liquor, wherein the content of one or more selected from the group consisting of ethyl hexadecanoate is increased.