KR102256699B1 - New Saccharomyces cerevisiae strain having high flocculation and fermentation ability and alcoholic beverages comprising the same - Google Patents

Abstract

In the present specification, FLO1, FLO5, and FLO9 genes are included as aggregation genes, and the aggregation (%) by the ASBC method is 20% or more, and Saccharomyces cerevisiae using α-methyl-D-glucoside as a carbon source ( Saccharomyces cerevisiae ) strain, the use of fermentation of the strain, a composition containing the strain, and a method of fermenting using the strain are disclosed, and when the strain is used, the fermentation ends and the yeast is settled through natural agglomeration through yeast reuse. This becomes possible and can reduce the cost of the filtration process.

Description

{New Saccharomyces cerevisiae strain having high flocculation and fermentation ability and alcoholic beverages comprising the same}

In the present specification, a novel Saccharomyces cerevisiae new strain having excellent cohesiveness and alcohol fermentation ability and a fermentation method using the same are disclosed.

Yeast flocculation is the property that when fermentable sugars are converted into ethanol and carbon dioxide through fermentation, yeast cells gather together with neighboring cells and settle to the bottom of the fermentation tank. Such cohesiveness is one of the characteristics of yeast that is important in the liquor industry, and when alcohol is prepared using yeast having cohesiveness, yeast cells can be separated and reused from the finished liquor, thereby reducing the cost of the manufacturing process and being environmentally friendly. . Natural flocculation can also reduce the cost of the filtration process.

Also, one of the most important characteristics of brewing yeast is its ability to ferment alcohol. Yeast is a microorganism having alcohol fermentation ability to convert to ethanol using a carbon source as a substrate, and the availability of various carbon sources and the ability to produce alcohol are important.

Until now, it has been very difficult to find a yeast strain having excellent cohesiveness and alcohol-producing ability as described above.

Korean Patent Publication No. 10-2019-0000970 A

E.V. Soares, Flocculation in Saccharomyces cerevisiae: a review, Journal of Applied Microbiology ISSN 1364-5072

In one aspect. An object of the present invention is to provide a reusable yeast strain for producing alcoholic beverages.

In one aspect, the present invention aims to provide a novel yeast strain for fermenting alcohol or alcohol, such as beer.

In one aspect, an object of the present invention is to provide a novel Saccharomyces cerevisiae strain having excellent cohesiveness.

In one aspect, an object of the present invention is to provide a novel Saccharomyces cerevisiae strain having both excellent cohesiveness and fermentation ability.

In one aspect, the present invention is Saccharomyces cerevisiae (Saccharomyces cerevisiae) strain, including FLO1, FLO5 and FLO9 genes as aggregation genes, and the aggregation (%) by the American Society of Brewing Chemists (ASBC) method It is 20% or more, and is a Saccharomyces cerevisiae strain using α-methyl-D-glucoside as a carbon source.

In one embodiment, the present invention is a yeast composition for fermentation of alcohol or alcohol, such as beer, comprising the above strain as an active ingredient.

In one embodiment, the present invention may be food, alcohol, alcohol, or beer including the above strain.

In one embodiment, the present invention is an alcohol fermentation method comprising fermenting by inoculating the above strain.

In one aspect, the present invention can significantly simplify the process and cost by providing a reusable yeast strain for producing alcoholic beverages.

In one aspect, the present invention enables mainstream fermentation with excellent fermentation efficiency even at low cost and simple process.

In one aspect, the present invention enables the production of alcoholic beverages with excellent fermentation efficiency while being environmentally friendly.

In one aspect, the present invention can provide a novel Saccharomyces cerevisiae strain having excellent cohesiveness.

In one aspect, the present invention can provide a novel Saccharomyces cerevisiae strain having both excellent cohesiveness and fermentation ability.

1 is a result of confirming the FLO gene family related to flocculation of yeast and positive control yeast according to an embodiment of the present invention through PCR.
2 is a result of measuring flocculation of yeast and positive control yeast according to an embodiment of the present invention by the American Society of Brewing Chemists (ASBC) method.
3 is a result of comparing the sedimentation properties of yeast for 6 hours after culturing the yeast and the positive control yeast according to an embodiment of the present invention in YPD broth at 25° C. for 48 hours, respectively.
4 is a scanning electron microscope observation photograph of yeast and positive control yeast according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail.

In one aspect, the present invention is a Saccharomyces cerevisiae (Saccharomyces cerevisiae) strain, including FLO1, FLO5 and FLO9 genes as aggregation genes, and the aggregation (%) by the ASBC method is 20% or more, and as a carbon source Saccharomyces cerevisiae using α-methyl-D-glucoside (Saccharomyces cerevisiae ) strain.

In one embodiment, the strain may be Saccharomyces cerevisiae AFY-7 strain.

In one embodiment, the strain is a strain deposited with the accession number KACC 93330P, Saccharomyces cerevisiae (Saccharomyces cerevisiae ) strain.

In one embodiment, the strain is having the 18S rRNA of SEQ ID NO: 9 Saccharomyces cerevisiae (Saccharomyces cerevisiae ) strain.

In one embodiment, the strain may have an alcohol fermentation ability. For example, the strain may be a beer fermentation strain.

In one embodiment, the strain has a cohesiveness (%) of 20.0% or more, 21.0% or more, 22.0% or more, 23.0% or more, 23. 5% or more, 23.6% or more, 23.8% or more, 24.0% or more according to the ASBC method. It may be greater than or equal to 24.2%, greater than or equal to 24.4%, greater than or equal to 24.6%, greater than or equal to 24.8%, greater than or equal to 25.0%, or greater than or equal to 25.1%.

There are several hypotheses about the mechanism of yeast aggregation, but they have not been accurately elucidated. Genes involved in aggregation have been reported to be FLO1, FLO5, FLO8, FLO9, FLO10, and FLO11.

The ASBC method presents a method for evaluating the cohesiveness of yeast (Spectroquant Prove -Analysis methods for the brewery industry (2018), Flocculation, absorbance method-yeast (ABSC method). 41-43).

[Equation 1]

Cohesiveness (%) = (A-B)/A × 100 (%)

In the above formula, A is the sample A value, and the sample A value refers to the absorbance value measured at 600 nm after resuspending and precipitation after adding EDTA to the pellet. B is the sample B value, which is the absorbance value measured at 600 nm after adding the washing solution to the pellet and then resuspending the obtained pellet with the suspension solution and resuspending it. For example, after shaking culture (200 rpm) at 30°C for 48 hours using YPD broth, OD (600 nm) was measured to equalize the absorbance of all strains, and then divided into samples A and B, respectively. , For Sample A, 100 mL of the strain was centrifuged at 2,500 rpm for 3 minutes to remove the supernatant, and then 9.9 mL of distilled water and 0.1 mL of 0.5M EDTA solution were added to the cell pellet, followed by 15 seconds using a vortex. Suspend for a while. Sample B 100 mL of the strain was centrifuged at 2,500 rpm for 3 minutes to remove the supernatant, and then _{10 mL of a washing solution (4.73 mM Ca 2} SO ₄ ) was added to the cell pellet and suspended for 15 seconds with a vortex. Then, it was centrifuged for 3 minutes at 2,500 rpm. After the supernatant was removed, a suspension solution (4.73 mM Ca ₂ SO ₄ , pH 4.5) was added and suspended by vortexing for 15 seconds, and then the solution was slowly mixed up and down for 15 seconds. After standing at room temperature for 6 minutes, 1 mL of the supernatant was obtained, added to 9 mL of sterile distilled water, and suspended in a vortex. Then, the absorbance is measured at 600 nm.

In one embodiment, the strain has an alcohol content of 5.07% or more, or 5.1% or more, and an apparent fermentation degree (%) of 77.06% or more, or 77.5% or more during beer fermentation, Saccharomyces cerevisiae ) strain. The alcohol frequency and specific gravity may be measured using, for example, an alcohol analyzer (Alex 500, Anton Paar, Austria). The specific gravity is measured using a hydrometer. The apparent fermentation degree can be calculated by measuring the initial gravity (OG) and final gravity (FG) and substituting them into a calculation formula. For example, the calculation formula is as follows:

[Equation 2]

Apparent attenuation = (OG-FG)/(OG-1) × 100 (%)

In one embodiment, the alcohol content and apparent fermentation degree were measured by adding 10 g of yeast to the wort, followed by fermentation at 20° C. for 1-2 weeks, bottled each into 500 mL of pressure-resistant PET bottles, and added 5 to 8 g of sugar. , It may be measured after fermentation at 20° C. for 7 days to generate carbonic acid and aging at 0 to 5° C. for 1 week. Specifically, after 10 g of yeast was added to wort, it was fermented at 20°C for 1.5 weeks, bottled into 500 mL of pressure-resistant PET bottles, and then 6.5 g of sugar was added and fermented at 20°C for 7 days to generate carbonic acid and at 2.5°C. It may be measured after aging for 1 week. More specifically, in 20-24 L of water (e.g., 22L) at 67°C, 3.40 kg of Pilsner malt, 0.80 kg of Carahell malt, 0.70 kg of Munich II malt are used in a grinder. After being pulverized and put into water, saccharification was performed at 67°C for 1 hour. Take the wort, put it in the grain layer and repeat until it becomes clear. Add 10 to 15 L of pre-warmed water (eg, 12.5 L) without breaking the filter layer, sparging for 10 minutes, and adding hops to boil at 100° C. for 1 hour. Citra 10 g 60 minutes, Citra 28 g 20 minutes, Mosaic was administered in the last 0 minutes, the wort was cooled to 26°C, and dried powdered yeast was each added for 10 minutes. After adding g. Fermented at 20°C for 1 to 2 weeks (eg, 1.5 weeks), bottled each into 500 mL of pressure-resistant PET bottles, and added 5 to 8 g (eg, 6.5 g) of sugar, then fermented at 20°C for 7 days to generate carbonic acid. It may be measured after aging for 1 week at 0-5°C (eg, 2.5°C).

In one embodiment, the strain of the present invention may have an alcohol content of 5.07% or more during beer fermentation. For example, 5.07% or more, 5.08% or more, 5.10% or more, 5.12% or more, 5.14% or more, 5.16% or more, 5.18% or more, 5.20% or more, 5.22% or more, 5.24% or more, 5.26% or more, 5.28% or more, Or 5.30% or more. The upper limit is set by the possible alcohol content of the beer, which is obvious to a person skilled in the art. For example, it may be 7.00% or less, 6.50% or less, or 6.00% or less. In one embodiment, the strain of the present invention may have an apparent fermentation degree of 77.06% or more during beer fermentation. For example, 77.10% or more, 77.20% or more, 77.30% or more, 77.40% or more, 77.50% or more, 77.60% or more, 77.70% or more, 77.80% or more, 77.90% or more, 78.00% or more, 78.10% or more, 78.20% or more, 78.30% or more, 78.40% or more, 78.50% or more, 78.60% or more, 78.70% or more, 78.80% or more, 78.90% or more, 79.00% or more, 79.10% or more, 79.20% or more, 79.30% or more, 79.40% or more, 79.50% Or more, 79.60% or more, 79.70% or more, 79.80% or more, 79.90% or more, 80.00% or more, 80.10% or more, 80.20% or more, 80.30% or more, 80.40% or more, 80.50% or more, 80.60% or more, 80.70% or more, It may be 80.72% or more, 80.74% or more, 80.76% or more, or 80.77% or more. The upper limit is set on the possible apparent fermentation degree of the beer, which is obvious to the person skilled in the art. For example, it may be 100% or less, 95% or less, or 90% or less.

In one embodiment, the strain has a ratio (%) of maltose and glucose content after fermentation to the maltose and glucose content in wort before fermentation is 1.80% or less and 4.00% or less, respectively, Saccharomyces cerevisiae ) strain. Specifically, the ratio (%) of maltose content after fermentation to maltose content in wort before fermentation is 1.80% or less, 1.78% or less, 1.76% or less, 1.74% or less, 1.72% or less, 1.70% or less, 1.68% or less, 1.66 % Or less, 1.64% or less, 1.62% or less, 1.60% or less, 1.59% or less, or 1.58% or less. In addition, in one embodiment, the ratio (%) of the glucose content after fermentation to the glucose content in wort before fermentation is 4.00% or less, 3.50% or less, 3.00% or less, 2.50% or less, 2.40% or less, 2.30% or less, 2.20 % Or less, 2.10% or less, 2.00% or less, 1.98% or less, 1.96% or less, 1.95% or less, or 1.94% or less. The fermentation may be the same as the fermentation in the measurement of the alcohol content and apparent fermentation degree.

In one embodiment, the present invention is a yeast composition for fermenting alcohol, liquor or beer comprising the above strain as an active ingredient.

In one embodiment, the yeast composition for fermentation of beer may be a yeast composition for fermentation of beer for use in fermenting beer with an alcohol content of 5.07% or more and an apparent fermentation degree (%) of 77.06% or more.

In one embodiment, the present invention may be food, alcohol, or beer including the above-described strain.

In one embodiment, the beer may have an alcohol content of 5.07% or more and an apparent fermentation degree (%) of 77.06% or more.

In one embodiment, the present invention is an alcohol fermentation method comprising fermenting by inoculating the above strain.

In one embodiment, the method may be for beer fermentation.

In one embodiment, the method comprises the steps of adding yeast to wort and first fermenting at 15 to 25° C. for 1 to 2 weeks; And

It may include the step of adding the first fermented fermented product to the bottle, sweetening it, and performing secondary fermentation at 15 to 25°C for 5 to 9 days.

In one embodiment, the method may further include aging the second fermented fermented product at 0 to 5° C. for 5 to 9 days.

All documents cited in this specification are incorporated by reference in their entirety as part of this specification.

Hereinafter, the present invention will be described in more detail through examples, but this is only an example and the scope of the rights is not limited thereto.

< Example 1> Isolation of new yeast

Add 10 g or 10 mL of yeast purchased at Saembat Marketplace, Sinbuk-eup, Chuncheon-si, Gangwon-do, Korea to 90 mL of distilled water, stir for 1 hour to extract, and then inoculate the extract into YPD broth (Yeast extract 1%, peptone 2%, dextrose 2%) And cultured at 25° C. for 2 hours. After incubating for 48 hours at 25°C in YPD agar (Yeast extract 1%, peptone 2%, dextrose 2%, agar 1.5%) medium containing 0.35% sodium propionate to inhibit the growth of fungi , A single colony was obtained, transferred to a new YPD agar, and cultured under the same conditions to isolate the AFY-7 strain.

< Example 2> AFY -7 yeast identification

One. Molecular biological identification

DNA was extracted using a yeast DNA prep kit, and an 18S rRNA site using primers NS1 (5'-GTAGTCATATGCTTGTCTC-3') (SEQ ID NO: 7), NS8 (5'-TCCGCAGGTTCACCTACGGA-3') (SEQ ID NO: 8) Was amplified by PCR. After the amplified site was purified, the base sequence was determined by requesting a base sequence analysis from Macrogen. AFY-7 strain is Saccharomyces cerevisiae (Saccharomyces cerevisiae ) strain was 99% identical, and if the identity was usually 97% or more, AFY-7 strain was Saccharomyces. cerevisiae . The strain Saccharomyces cerevisiae AFY-7 was deposited with the National Academy of Agricultural Sciences on August 22, 2019 (accession number: KACC 93330P). The nucleotide sequence of the analyzed 18S rRNA site is as follows (SEQ ID NO: 9).

Saccharomyces cerevisiae AFY-7 18S rRNA

AAGGGTTCGATTCCGGAGAGGGAGCCTGAGAAACGGCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCAATCCTAATTCAGGGAGGTAGTGACAATAAATAACGATACAGGGCCCATTCGGGTCTTGTAATTGGAATGAGTACAATGTAAATACCTTAACGAGGAACAATTGGAGGGCAAGTCTGGTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGTTGCAGTTAAAAAGCTCGTAGTTGAACTTTGGGCCCGGTTGGCCGGTCCGATTTTTTCGTGTACTGGATTTCCAACGGGGCCTTTCCTTCTGGCTAACCTTGAGTCCTTGTGGCTCTTGGCGAACCAGGACTTTTACTTTGAAAAAATTAGAGTGTTCAAAGCAGGCGTATTGCTCGAATATATTAGCATGGAATAATAGAATAGGACGTTTGGTTCTATTTTGTTGGTTTCTAGGACCATCGTAATGATTAATAGGGACGGTCGGGGGCATCAGTATTCAATTGTCAGAGGTGAAATTCTTGGATTTATTGAAGACTAACTACTGCGAAAGCATTTGCCAAGGACGTTTTCATTAATCAAGAACGAAAGTTAGGGGATCGAAGATGATCAGATACCGTCGTAGTCTTAACCATAAACTATGCCGACTAGGGATCGGGTGGTGTTTTTTTAATGACCCACTCGGCACCTTACGAGAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCAGGAGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGGAAACTCACCAGGTCCAGACACAATAAGGATTGACAGATTGAGAGCTCTTTCTTGATTTTGTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGCTTAATTGCGATAACGAACGAGACCTTAACCTACTAAATAGTGGTGCTAGCATTTGCTGG TTATCCACTTCTTAGAGGGACTATCGGTTTCAAGCCGATGGAAGTTTGAGGCAATAACAGGTCTGTGATGCCCTTAGACGTTCTGGGCCGCACGCGCGCTACACTGACGGAGCCAGCGAGTCTAACCTTGGCCG

2. Biochemical identification

The identified Saccharomyces cerevisiae AFY-7 strain was shown in Table 1 by evaluating the carbon source availability using the API 20C AUX kit to perform biochemical identification. Both the commercial yeast and the test strain were the same Saccharomyces cerevisiae, but the carbon source availability was different and it was confirmed that they are different strains. A saccharide as a commercial yeast with commercially available beer yeast as a control my process three Levy jiae (Saccharomyces cerevisiae ) strains, C1 (US-05, Fermentis, France), C2 (M44, Mangrove JACK'S, New Zealand) were used. Commercial yeast C1 and isolated yeast AFY-7 had the same carbon source utilization. It is the same as the carbon source utilization, but the phenotypes such as cohesiveness and fermentation capacity described below are not regarded as the same strain because they exhibit different aspects.

API 20 AUX Results division Commercial yeast Isolate yeast C1 C2 AFY-7 Glucose + + + Glycerol - - - 2-Keto-D-glucose - - - L-Arabinose - - - D-Xylose - - - Adonitol - - - Xylitol - - - D-Galactose + + + Inositol - - - D-Sorbitol - - - α-Methyl-D-Glucoside + - + N-Acetyl-D-Glucosamine - - - D-Celiobiose - - - D-Lactose - - - D-Maltose + + + D-Saccharose + + + D-Trehalose + + + D-Melezitose + + + D-Raffinose + + +

< Example 3> Cohesiveness evaluation

One. Coherence related genes PCR

The FLO gene family related to yeast flocculation was confirmed through PCR. Primer sequences for gene amplification are shown in Table 2, and PCR conditions are as follows: After initial denaturation at 94°C for 2 minutes, repeat the amplification process 28 times at 94°C for 1 minute, 57.5°C for 5 minutes, and 72°C for 2 minutes, and at 72°C for 7 minutes The last amplification was performed, and the results are shown in FIG. 1. At this time, as a positive control, three commercial yeasts sold as brewer's yeast are Saccharomyces cerevisiae (Saccharomyces cerevisiae ) strains, C1 (US-05, Fermentis, France), C2 (M44, Mangrove JACK'S, New Zealand) and C3 (WB-06, Fermentis, France) were used.

Primer name Base sequence (5'-> 3') Sequence number FLO1-F AAGTGTGCGTCACTTTTCCTACGGT One FLO1-R AGCGATGAGGCATTGTCATTT 2 FLO5-F GCAATAAACCACATGGCTACC 3 FLO5-R AGTGGTGCTAATCAATTTAAAGAA 4 FLO9-F AAGTTTACATTCATACCATTCTTCCTTGA 5 FLO9-R CGTTTCGACAGCCACTGCTA 6

As a result, it was found that the strains C1, C2, C3, and AFY-7 possess all three genes FLO1, FLO5, and FLO9 that regulate aggregation. The C3 strain is a commercial yeast known to have low cohesiveness, and as compared to C3, the band of the AFY-7 strain is darker, so it is expected that the cohesiveness will be higher.

2. Cohesiveness evaluation

Aggregation (%) for commercial yeast C1, C2 and AFY-7 strains was evaluated. Aggregation experiment was performed by shaking culture (200 rpm) at 30° C. for 48 hours using YPD broth, and then measuring OD (600 nm) to equalize the absorbance of all strains. Thereafter, the treatment was divided into samples A and B, respectively. In Sample A, 100 mL of the strain was centrifuged at 2,500 rpm for 3 minutes to remove the supernatant, and then 9.9 mL of distilled water and 0.1 mL of 0.5M EDTA solution were added to the cell pellet, and suspended for 15 seconds using a vortex. For sample B, 100 mL of the strain was centrifuged at 2,500 rpm for 3 minutes to remove the supernatant, and then _{10 mL of a washing solution (4.73 mM Ca 2} SO ₄ ) was added to the cell pellet for 15 seconds with a vortex. After being suspended, it was centrifuged at 2,500 rpm for 3 minutes. After removing the supernatant, a suspension solution (4.73 mM Ca ₂ SO ₄ , pH 4.5) was added and suspended by vortexing for 15 seconds, and then the solution was slowly mixed up and down for 15 seconds. After standing at room temperature for 6 minutes, 1 mL of the supernatant was obtained, added to 9 mL of sterilized distilled water, suspended with vortex, and absorbance was measured at 600 nm. Cohesiveness (%) = (AB) / AX was calculated as 100. The results are shown in FIG. 2.

As shown in FIG. 2, C1 was 17.7%, C2 was 19.3%, and AFY-7 was 26.1%, confirming that the AFY-7 strain had higher cohesion than commercial yeasts C1 and C2, respectively, 1.4 times and 1.5 times higher than that of commercial yeast C1 and C2. I did.

3. Sedimentation evaluation

In addition, after culturing the strains at 25° C. for 48 hours in YPD broth, respectively, the sedimentation properties of the yeast were compared for 6 hours. Strain No. 34 is a non-aggregating yeast owned by the Agricultural Food Research Institute of Gangwon-do Agricultural Technology Institute. In the AFY-7 strain, sedimentation began to appear after 1 hour, and most of the yeast was found to settle to the bottom in 4 hours, showing the fastest sedimentation among the five strains.

4. Morphological confirmation

After cell down the strain using a centrifuge, the supernatant was discarded and the cell pellet was washed twice with 0.1M phosphate buffer (pH 7.2). Be sure to wear gloves and put 4% glutaraldehyde 10 times the cell pellet in the hood with a pipette around the tube wall, taking care not to touch the cell pellet. After giving, the cells were fixed by refrigerating for 2 hours. After removing the supernatant by centrifugation at 13,000 rpm and 1 min condition, the sample was washed twice with 0.1 M phosphate buffer (pH 7.2) for 15 to 30 minutes. After washing once for 10 to 30 minutes using 30%, 50%, 70% 90% ethanol again, it was washed twice with 100% ethanol for 30 minutes. In order to remove the remaining ethanol, it was dried for 3 to 4 hours with a CPD (Critical Point Dryer). Pt coating for coating the sample took 30 minutes to 1 hour. The instrument was observed by magnifying 1,000 times and 5,000 times with a high resolution scanning electron microscope (UHR-SEM; Ultra High Resolution Scanning Electron Microscope, Hitachi S-4800, Hitachi, Japan) (FIG. 4).

The AFY-7 strain adhered a lot to neighboring yeast cells compared to strain No. 34 (non-aggregating yeast) owned by the Agricultural Food Research Institute of Gangwon-do Agricultural Technology Institute, and thus cohesiveness was confirmed.

< Example 4> AFY Brewing test using -7 strains

One. Beer manufacturing

3.40 kg of Pilsner malt, 0.80 kg of Karahell malt, and 0.70 kg of Munich II malt were pulverized with a grinder in 20 to 24 L of water at 67°C and put into water, and saccharification temperature Was carried out at 67° C. for 1 hour. Take the wort, put it in the grain layer, and repeat until it becomes clear. 10 to 15 L of pre-warmed water was added so as not to break the filter layer, followed by sparging for 10 minutes. The hops were added and boiled at 100°C for 1 hour, and the administration time was different for each type of hops. Citra 10 g 60 minutes, Citra 28 g 20 minutes, Mosaic was administered at the last 0 minutes. The wort was cooled to 26° C., and then 10 g of imported commercially available yeast and yeast isolated in a dry powder state were added. Fermentation was performed at 20° C. for 1 to 2 weeks, and after bottling each into 500 mL of a pressure-resistant PET bottle, 5 to 8 g of sugar was added, and then fermented at 20° C. for 7 days to generate carbonic acid. The beer after fermentation was aged for 1 week at 0~5℃.

2. Evaluation of fermentation properties

In order to evaluate the fermentation ability of commercial yeast and isolated yeast, the free sugar, alcohol content, and fermentation degree of beer prepared by strain were compared and analyzed.

2-1. Free sugar analysis

The free sugar content was analyzed using (Waters 2690, Milford, MA, USA) and ELSD (Waters, Milford, MA, USA). Mobile phase acetonitrile: d-water = 75:25 (v/v) using Prevail carbohydrate ES (4.6*250 mm, 5 μm, Alltech, USA), a column dedicated to sugar analysis, for 20 minutes at a flow rate of 1 mL/min. The sample injection amount was 20 μL, and the column temperature was 35°C. ELSD (Evaporative Light Scattering Detector) analysis conditions were 100 gain, 70°C drift, 50 psi gas pressure, and analyzed with nebulizer cooling. 10 mL of distilled water was added to 2 mL of the sample, passed through a 0.45 μm membrane filter, filtered, and used as an analysis sample. Maltose, glucose, and sucrose (Sigma Chemical Co., St, Louis, MO, USA) were used as standards to prepare a quantitative curve and the free sugar content in the sample was quantified.

As a result of free sugar analysis, it was confirmed that maltose and glucose content in wort was the lowest in beer fermented with AFY-7 strain compared to commercial yeast (Table 3), and it was confirmed that maltose and glucose utilization were high.

(Unit: g/L) division Wort C1 C2 AFY-7 Maltose 44.29 ± 1.15 0.84 ± 0.07 1.27 ± 0.08 0.70 ± 0.05 Glucose 12.83 ± 0.73 1.58 ± 0.09 0.53 ± 0.04 0.25 ±0.04 Sucrose 2.04 ± 0.09 - - -

2-2. Alcohol content and apparent fermentation

As described above, the alcohol content and apparent attenuation of beer (pale ale) fermented under the same conditions with imported commercial yeast and isolated yeast were measured. The alcohol content was measured using an Annar alcohol analyzer, and the apparent fermentation degree was calculated by measuring the original gravity and final gravity and substituting them into the calculation formula. It was confirmed that the alcohol content and fermentation degree showed higher fermentation ability in AFY-7 strain than commercial yeast (Table 4). This is converted into alcohol using a carbon source (maltose, etc.) in wort. The apparent attenuation was calculated as (OG-FG)/(OG-1) X 100.

division Commercial yeast AFY-7 C1 C2 Alcohol content (%) 5.06 4.35 5.30 Apparent fermentation (%)
(Apparent attenuation) 77.05 66.27 80.77

National Academy of Agricultural Sciences KACC93330P 20190822

<110> KANGWON PROVINCE <120> New Saccharomyces cerevisiae strain having high flocculation and fermentation ability and a method for fermenting using the same <130> 19p410/ind <160> 9 <170> KoPatentIn 3.0 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> FLO1-F <400> 1 aagtgtgcgt cacttttcct acggt 25 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> FLO1-R <400> 2 agcgatgagg cattgtcatt t 21 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> FLO5-F <400> 3 gcaataaacc acatggctac c 21 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> FLO5-R <400> 4 agtggtgcta atcaatttaa agaa 24 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> FLO9-F <400> 5 aagtttacat tcataccatt cttccttga 29 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FLO9-R <400> 6 cgtttcgaca gccactgcta 20 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> NS1 primer <400> 7 gtagtcatat gcttgtctc 19 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NS8 primer <400> 8 tccgcaggtt cacctacgga 20 <210> 9 <211> 1134 <212> DNA <213> Saccharomyces cerevisiae <400> 9 aagggttcga ttccggagag ggagcctgag aaacggctac cacatccaag gaaggcagca 60 ggcgcgcaaa ttacccaatc ctaattcagg gaggtagtga caataaataa cgatacaggg 120 cccattcggg tcttgtaatt ggaatgagta caatgtaaat accttaacga ggaacaattg 180 gagggcaagt ctggtgccag cagccgcggt aattccagct ccaatagcgt atattaaagt 240 tgttgcagtt aaaaagctcg tagttgaact ttgggcccgg ttggccggtc cgattttttc 300 gtgtactgga tttccaacgg ggcctttcct tctggctaac cttgagtcct tgtggctctt 360 ggcgaaccag gacttttact ttgaaaaaat tagagtgttc aaagcaggcg tattgctcga 420 atatattagc atggaataat agaataggac gtttggttct attttgttgg tttctaggac 480 catcgtaatg attaataggg acggtcgggg gcatcagtat tcaattgtca gaggtgaaat 540 tcttggattt attgaagact aactactgcg aaagcatttg ccaaggacgt tttcattaat 600 caagaacgaa agttagggga tcgaagatga tcagataccg tcgtagtctt aaccataaac 660 tatgccgact agggatcggg tggtgttttt ttaatgaccc actcggcacc ttacgagaaa 720 tcaaagtctt tgggttctgg ggggagtatg gtcgcaaggc tgaaacttaa aggaattgac 780 ggaagggcac caccaggagt ggagcctgcg gcttaatttg actcaacacg gggaaactca 840 ccaggtccag acacaataag gattgacaga ttgagagctc tttcttgatt ttgtgggtgg 900 tggtgcatgg ccgttcttag ttggtggagt gatttgtctg cttaattgcg ataacgaacg 960 agaccttaac ctactaaata gtggtgctag catttgctgg ttatccactt cttagaggga 1020 ctatcggttt caagccgatg gaagtttgag gcaataacag gtctgtgatg cccttagacg 1080 ttctgggccg cacgcgcgct acactgacgg agccagcgag tctaaccttg gccg 1134

Claims (17)

As a Saccharomyces cerevisiae strain,
The strain is,
Includes FLO1, FLO5 and FLO9 genes as aggregation genes,
The cohesiveness (%) by the ASBC method is 20% or more,
Using α-methyl-D-glucoside as a carbon source,
Saccharomyces cerevisiae AFY-7 (Saccharomyces cerevisiae) strain, which is a strain deposited with accession number KACC 93330P. delete delete The method of claim 1, wherein the strain has 18S rRNA of SEQ ID NO: 9 Saccharomyces cerevisiae (Saccharomyces cerevisiae ) strain. The method of claim 1, wherein the strain has alcohol fermentation ability, Saccharomyces cerevisiae (Saccharomyces cerevisiae ) strain. According to claim 5, wherein the strain is for fermentation of beer, Saccharomyces cerevisiae (Saccharomyces cerevisiae) strain. According to claim 5, The strain is beer fermentation, alcohol content of 5.07% or more, apparent fermentation (%) of 77.06% or more, Saccharomyces cerevisiae (Saccharomyces cerevisiae) strain. The method of claim 1, wherein the strain has a ratio (%) of maltose and glucose content after fermentation relative to the maltose and glucose content in wort before fermentation is 1.80% or less and 4.00% or less, respectively, Saccharomyces cerevisiae ) strain. An alcohol fermentation yeast composition comprising the strain according to any one of claims 1, 4 to 8 as an active ingredient. The yeast composition for alcohol fermentation according to claim 9, wherein the yeast composition for alcohol fermentation is for fermentation of beer, which is for fermentation into beer having an alcohol content of 5.07% or more and an apparent fermentation degree (%) of 77.06% or more. Composition. An alcoholic beverage comprising the strain according to any one of claims 1 and 4 to 8. The alcoholic beverage of claim 11, wherein the alcoholic beverage is beer. The alcoholic beverage according to claim 12, wherein the beer has an alcohol content of 5.07% or more and an apparent fermentation degree (%) of 77.06% or more. An alcohol fermentation method comprising inoculating and fermenting the strain according to any one of claims 1 and 4 to 8. 15. The alcohol fermentation method according to claim 14, wherein the method is for beer fermentation. The method of claim 15, wherein the method comprises:
Adding yeast to wort and first fermenting at 15 to 25° C. for 1 to 2 weeks; And
Comprising the step of bottling the first fermented fermented product, sweetening and secondary fermenting at 15 to 25°C for 5 to 9 days. The method of claim 16,
Further comprising the step of aging the secondary fermented fermented product at 0 to 5 ℃ for 5 to 9 days, alcohol fermentation method.

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