KR102251303B1 - Saccharomyces cerevisiae SRCM101756 strain for increasing rutin content in fermented mulberry and uses thereof - Google Patents

Abstract

The present invention relates to method for producing berry wine with enhanced rutin contents using a Saccharomyces cerevisiae SRCM101756 strain, and to berry wine with enhanced rutin contents produced by the method. The Saccharomyces cerevisiae SRCM101756 strain of the present invention is resistant to alcohol, sugar and sulfurous acid, has the ability to produce alcohol, and does not produce biogenic amines. Since it is possible to increase the rutin contents in a fermented product during fermentation of berries, the Saccharomyces cerevisiae SRCM101756 strain may be usefully utilized as brewing yeast for the production of fruit wine with enhanced flavor and functional ingredients useful for the human body.

Description

{Saccharomyces cerevisiae SRCM101756 strain for increasing rutin content in fermented mulberry and uses thereof}

The present invention relates to a strain of Saccharomyces cerevisiae SRCM101756 for increasing rutin content in a fermented mulberry and its use.

As the standard of living improves with economic growth, wine is becoming more popular and consumption is increasing further. In addition, as the social atmosphere that enjoys lightly drinking and the increasing number of consumers who value health are increasing, the preference of alcoholic beverages with lower alcohol content is increasing than alcoholic beverages with higher alcohol content.

Wine, as a fermented liquor, is an alcohol fermentation of various fruits or juices as well as grapes, and the flavor increases as it goes through the fermentation process, and physiological activity increases as the functional polymer substances possessed by the fruit are converted into low molecular weight substances. In such fermented fruit wines, the characteristics of raw materials have the greatest impact on the quality, so product development is being actively carried out as the marketability of wines manufactured using functional fruits such as mulberry, bokbunja, blueberries and plums is attracting attention.

Enhancement of wine's unique flavor and palatability is achieved through fermentation, and fermented liquor using mulberry ( Morus alba ), grape (Vitis coignetiae ), blueberry ( Vaccinium corymbosum ) and bokbunja ( Rubus coreanus ), etc. It is manufactured mainly using Saccharomyces cerevisiae , which is known to produce. In order for fruit fermented liquor to be competitive as described above, research related to improving the quality of fermented liquor is required by providing various functional ingredients and flavors to fermented liquor through the development of new yeast as well as diversification of raw materials.

Mulberry is a fruit of a mulberry tree, an arboreal deciduous tree belonging to the Moraceae genus Morus. It has black or magenta color, is mainly distributed in temperate and tropical regions, and is classified as a berry with the oldest cultivation history in Korea along with bokbun. . Audi is attracting attention as a new functional material that has an effect on aging, cholesterol suppression, antioxidant, anti-inflammatory and vision improvement, diabetes, etc., but it is not only easy to be damaged or fragile due to its weak pulp, but also has a high moisture content, so it is distributed in a frozen state. It is processed into beverages, jams, jelly, and sake, and is widely distributed. In particular, although the sugar content of mulberry is somewhat insufficient in the production of fermented liquor, it can be very useful as a fruit for the production of fermented liquor because the strong color and scent of mulberry is not diluted by the addition of sugar.

In addition, as interest in health has increased in recent years, interest in functional fermented liquor that emphasizes the efficacy and characteristics of useful ingredients by increasing the functional ingredients in the fermentation process or by producing by-products in the fermentation process along with fermented ingredients has increased have. Accordingly, the present inventors isolated Saccharomyces cerevisiae SRCM101756 strain (accession number: KCCM12776P) from traditional fermented foods such as makgeolli and yeast, and when the mulberry juice is fermented with the Saccharomyces cerevisiae SRCM101756 strain, it is fermented with mulberry. It was confirmed that the rutin content in water was increased.

On the other hand, Korean Patent Application Publication No. 2015-0125381 discloses'a method of manufacturing a berry wine using GBY3 and the yeast in the yeast Saccharomyces cerevisiae isolated from domestic mulberry'. Biogenic amine-free Saccharomyces cerevisiae BA34 strain and its use for the production of berry wines are disclosed, but the Saccharomyces cerevisiae SRCM101756 strain which increases the rutin content in the mulberry fermented product of the present invention and There is no description of its use.

The present invention was derived from the above requirements, and the present inventors isolated various yeast strains of the genus Saccharomyces from makgeolli and yeast, which are traditional fermented foods, and using the isolated strains of the genus Saccharomyces, the present inventors Saccharomyces cerevisiae SRCM101756 strain (accession number: KCCM12776P), which can be fermented to increase the rutin content in the mulberry fermentation product to the highest level, was isolated. The Saccharomyces cerevisiae SRCM101756 strain of the present invention not only increases the rutin content in the mulberry fermented product when the mulberry is fermented, but also has alcohol, sugar and sulfurous acid resistance, has alcohol-producing ability, and has histamine, tyramine ( tyramine), putrescine, and cadaverine, such as biogenic amines, are not produced, and thus the present invention has been completed by confirming that it can be used in fermented food-related industries using berries.

In order to solve the above problems, the present invention provides a Saccharomyces cerevisiae (Saccharomyces cerevisiae) SRCM101756 strain having an accession number of KCCM12776P, characterized in that increasing the rutin content in the fermented product by fermenting berries.

In addition, the present invention provides a composition for alcohol production comprising the strain, a culture solution thereof, a concentrate of the culture solution, or a dried product thereof as an active ingredient.

In addition, the present invention provides a composition for producing vinegar comprising the strain, a culture solution thereof, a concentrate of the culture solution, or a dried product thereof and acetic acid bacteria as active ingredients.

In addition, the present invention provides a method for producing a berry wine with increased rutin content, comprising the step of inoculating and fermenting the Saccharomyces cerevisiae SRCM101756 strain having an accession number of KCCM12776P on the berries.

In addition, the present invention provides a berry wine with an enhanced rutin content produced by the above method.

The Saccharomyces cerevisiae SRCM101756 strain of the present invention is resistant to alcohol, sugar and sulfurous acid, has alcohol-producing ability, does not produce biogenic amines, and can increase the rutin content in the fermented product during fermentation of berries. Therefore, it may be usefully used as a brewing yeast for the production of fruit wine with increased flavor and functional ingredients useful for the human body.

Figure 1 is a rutin standard product (routin high rate, rutin hydrate; A), non-fermented mulberry juice (B) and Saccharomyces cerevisiae (Saccharomyces cerevisiae) with an accession number of KCCM12776P, mulberry juice fermented with the SRCM101756 strain (C) This is a chromatogram displayed by performing high-performance liquid chromatography (HPLC) on the target.
Figure 2 shows a schematic diagram of the Saccharomyces cerevisiae SRCM101756 strain isolated in the present invention.

In order to solve the above problems, the present invention provides a strain of Saccharomyces cerevisiae (Saccharomyces cerevisiae) SRCM101756 having an accession number of KCCM12776P, characterized in that increasing the rutin content in the fermented product by fermenting berries.

In the present invention, it is resistant to alcohol, sugar and sulfurous acid from makgeolli and yeast, which are traditional fermented foods, has alcohol-producing ability, and is biogenic such as histamine, tyramine, putrescine, and cadaverine. A yeast strain of Saccharomyces genus that can increase the content of rutin in the fermented product when fermenting berries without producing amine (biogenic amine) was isolated. Among the isolated yeast strains, rutin in the fermentation product when fermenting berries fruit The yeast strain that can increase the content the most is finally selected and As a result of identification through the nucleotide sequence (SEQ ID NO: 1) of the 18S rRNA gene, it was confirmed that it was a Saccharomyces cerevisiae strain. The present inventors named the final selected strain as Saccharomyces cerevisiae SRCM101756 strain, and deposited with the Korea Microbial Conservation Center (KCCM) on August 5, 2020 (accession number: KCCM12776P).

The present invention also provides a composition for alcohol production comprising the Saccharomyces cerevisiae SRCM101756 strain of which the accession number is KCCM12776P, a culture solution thereof, a concentrate of the culture solution, or a dried product thereof as an active ingredient.

The method of culturing the strain of the present invention may be cultured according to a method commonly used in the art, and is not limited to a specific method.

When the strain obtained in the step of culturing the strain of the present invention or the culture product of the strain or the concentrate of the culture solution of the strain is used as an additive, the strain or the culture product of the strain or the concentrate of the culture solution of the strain is added as it is, or Other additives may be used together, and may be appropriately used according to a conventional method. The mixing amount of the ingredients may be suitably determined depending on the purpose of use thereof.

In the composition of the present invention, the alcohol may be wine fermented from berries, preferably mulberry ( Morus alba ), bokbunja ( Rubus coreanus ), and blueberry ( Vaccinium corymbosum ) any one selected from the fermented wine It may, and most preferably, may be a wine fermented mulberry, but is not limited thereto.

In the composition of the present invention, the berry fruit may be used as the fruit itself, or a juice, pulverized product, concentrate, or dried product of the fruit, but is not particularly limited thereto.

The present invention also provides a composition for producing vinegar comprising the strain of the present invention, a culture solution thereof, a concentrate of the culture solution or a dried product thereof and acetic acid bacteria as active ingredients. Since the strain of the present invention is a strain capable of producing alcohol, after producing alcohol using the strain of the present invention, vinegar can be produced by inoculating and fermenting the produced alcohol with acetic acid bacteria such as Acetobacter aceti. have.

The present invention also provides a method for producing a berry wine with enhanced rutin content, comprising the step of inoculating and fermenting the Saccharomyces cerevisiae SRCM101756 strain having an accession number of KCCM12776P to the berries.

In the method according to an exemplary embodiment of the present invention, the berries may be any one selected from mulberry, bokbunja, and blueberry, and preferably may be mulberry, but the present invention is not limited thereto. In addition, the berries may be the fruit itself, or the juice, pulverized product, concentrate, or dried product of the fruit, but is not particularly limited thereto.

In the method according to an embodiment of the present invention, the Saccharomyces cerevisiae SRCM101756 strain can increase the rutin content in the fermented product during fermentation of berries fruits, has alcohol, sugar, and sulfurous acid resistance, and alcohol-producing ability. In addition, since there is a characteristic that it does not produce biogenic amines, when the above method is used, the content of rutin, a functional ingredient that has an effect on antioxidant, arteriosclerosis, high blood pressure, cerebral hemorrhage, diabetes, obesity, etc., is increased, thereby improving the quality of functional berries wine. Can be manufactured.

In the method according to an embodiment of the present invention, the berry wine is supplemented with 21 to 23 ° brix for berry fruit juice, and the Saccharomyces cerevisiae SRCM101756 strain having an accession number of KCCM12776P is 0.5 to 1.5% (v /v) may be fermented for 3 to 5 days at 140 to 160 rpm at 26 to 28°C, preferably 22° brix for berries fruit juice, and the deposit number is KCCM12776P. Ses cerevisiae SRCM101756 strain may be inoculated with 1% (v/v) and then fermented at 27° C. at 150 rpm for 4 days, but is not limited thereto.

The present invention also provides a berry wine with an enhanced rutin content produced by the above method.

Hereinafter, it will be described in detail by examples of the present invention. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

Materials and methods

1. Yeast strain selection for mulberry fermentation

1-1. Collection of fermented foods for separation of useful yeast

In order to separate the yeast for brewing from traditional fermented foods, yeast strains were isolated from commercially available makgeolli and yeast. Specifically, 1 ml of makgeolli or 1 g of koji was suspended in 9 ml of sterile physiological saline, and then ^{diluted to 10 6} by a decimal method. YM (yeast mold) solid medium containing penicillin and streptomycin was used as a separation medium, and 100 µl of the diluted solution was smeared on the medium and cultured at 29°C for 48 hours, and then independent colonies were selected. I did. After that, it was subcultured in YM medium, mixed in the same amount with 30% glycerol, and stored at 80°C for use.

1-2. Confirmation of growth of yeast strains using Lysine medium

My process in a saccharide to target a variety of yeast strains isolated from rice wine or yeast (Saccharomyces sp.) And Caro Ibiza My process in (non- Saccharomyces sp.) To separate the secret history Caro My process selective medium lysine (lysine agar medium ) Was used to confirm the growth of the strain. In order to check the growth, YPD medium and lysine medium were prepared in the composition of Table 1 below, and then autoclaved at 121°C for 15 minutes, and the liquid medium for preparing yeast preculture was 21 g/L of YM medium. After dissolving into the composition, it was used by autoclaving at 121°C for 15 minutes. The isolated yeast strains were inoculated in YM liquid medium at 1% (v/v) and cultured with shaking at 29°C and 240 rpm for 48 hours, and then one loop of the culture medium was taken and SCI (single colony isolation) in YPD medium and lysine medium, respectively. It was carried out and cultured at 29°C for 48 hours, and then growth was confirmed.

Composition of the medium of the present invention YPD medium Lysine badge Yeast extract 10 g/L - Peptone 20 g/L - Glucose 20 g/L 10 g/L Lysine - 5.6 g/L KH ₂ PO ₄ - 0.85 g/L MgSO ₄ 7H ₂ 0 - 1.02 g/L Agar 20 g/L 20 g/L

2. Evaluation of fermentation suitability of yeast strains isolated from fermented foods

2-1. Confirmation of alcohol production ability of yeast strain

In order to confirm the alcohol fermentation ability of the isolated yeast strain, YM liquid medium (21 g/L) added with 30% (w/v) glucose was autoclaved at 121°C for 15 minutes and 1% of the pre-culture solution of the yeast strain (v/v) was inoculated and cultured with shaking at 29° C. and 180 rpm for 48 hours, followed by further cultivation at 29° C. for 48 hours to induce alcohol fermentation.

To measure the produced alcohol content, the culture solution was centrifuged at 4,000 rpm at 4°C for 10 minutes (GYROZEN, 1580MGR, Korea) to obtain a supernatant, and then 10% (v/v) of the same amount as an internal standard. Isopropyl alcohol was mixed and distilled, and the distillate was filtered through a 0.45 μm membrane filter and analyzed by a gas chromatograph-flame ionization detector (GC-FID). For GC (HP 6890 system) analysis of the produced alcohol, the column is DB-5 (30 m×0.25 mm id, 0.25 μm film thickness), the detector is FID (flame ionization detector), the mobile phase is helium gas, and the column flow rate is 1.3. Ml/min, the split ratio was 50:1, the detector temperature was 250°C, the oven temperature was maintained at 70°C for 2 minutes, and then set to increase to 150°C by 20°C per minute, and 10 μl of the sample was injected. The alcohol concentration of the sample was calculated based on the standard curve prepared using ethanol and isopropyl alcohol for each concentration as standard substances for quantitative analysis.

2-2. Confirmation of film formation of yeast strain

The isolated yeast strain was inoculated with 1% (v/v) in YM medium and cultured with shaking at 29° C. and 240 rpm for 48 hours. To confirm the formation of a film, 1% (v/v) of the yeast preculture was inoculated into the YM liquid medium prepared by adding glucose 12% (w/v), followed by stationary culture at 29°C for 72 hours, and then 24, 48 And it was confirmed the degree of formation of the film formed on the surface of the liquid medium at intervals of 72 hours.

2-3. Check whether yeast strains produce gas

In order to check whether gas is generated, a Durham tube is added to the YM medium prepared by adding glucose 12% (w/v), sterilized at 121° C. for 15 minutes, and then the yeast pre-culture solution 1 in the sterilized YM medium. % (v/v) was inoculated and cultured at 29° C. for 72 hours, and it was confirmed whether bubbles were generated on the surface of the durum tube and the liquid medium at intervals of 24, 48, and 72 hours.

2-4. Confirmation of alcohol, sugar and sulfurous acid resistance of yeast strains

500 µl of YM medium was dispensed into 48-well plates, and 1% (5 µl, v/v) of the yeast preculture was inoculated into the medium, and the 48-well plate was wound with parafilm for 48 hours at 29°C and 240 rpm. It was pre-cultured by shaking culture during the period.

In the case of sugar tolerance evaluation, glucose was added to the YM medium at a concentration of 1, 40, 50 or 60% (w/v) to prepare a sugar-containing medium, and in the case of sulfite tolerance evaluation, potassium metabisulfite (potassium disulfite) was used. Sulfurous acid medium added at 0, 250 or 500 ppm (mg/L, w/v) was prepared, and in the case of ethanol tolerance evaluation, an alcohol medium added with ethanol at a concentration of 0, 10, 12.5 or 15% (v/v) Was prepared and then autoclaved at 121° C. for 15 minutes.

500 µl of YM liquid medium added with sugar, sulfurous acid or alcohol for each concentration was dispensed into a 48-well plate, and 1% (5 µl, v/v) of the yeast preculture was inoculated into the medium, and then a 48-well plate was prepared. It was wound with parafilm and cultured with shaking at 29° C. and 240 rpm for 72 hours, and absorbance was measured at 600 nm at intervals of 24, 48 and 72 hours to confirm the growth degree of the strain.

2-5. The ability of yeast strains to produce biogenic amines

In order to evaluate the ability of the isolated yeast strain to produce biogenic amine, a medium to which an amino acid precursor was added was prepared. Specifically, after dissolving 2.1 g of YM medium in 50 ml of distilled water, histidine, tyrosine, which are amino acid precursors of biogenic amines histamine, tyramine, putrescine and cadaverine (tyrosine), ornithine and lysine were added to the liquid medium at 0.1% (w/v), cresol red 0.006% (w/v) and glycerol 0.25% (v/v) ) Were mixed. After adjusting the pH to 5.0 with 1N HCl and quantifying the medium to 100 mL, the medium was prepared by autoclaving at 121°C for 15 minutes, and then 500 µl of each was dispensed into a 48-well plate, and 1% (5 µl) of the yeast pre-culture solution was added to the medium. , v/v) after inoculation, the 48-well plate was wound with parafilm and cultured with shaking at 29°C and 240 rpm for 48 hours, and the color change was observed by measuring the absorbance at 540 nm after the culture was completed. When a basic biogenic amine is generated by precursor metabolism, the color of the biogenic amine changes from yellow to reddich purple, and the presence or absence of the biogenic amine was determined by absorbance.

2-6. Identification of yeast strains

Molecular phylogenetic identification of the finally selected yeast strains was analyzed by PCR for amplification of 28S rDNA and 5.8S rRNA regions. After denatured at 95° C. for 15 minutes, colony PCR was performed with the obtained template DNA, and the nucleotide sequence of the 18S rDNA gene of the isolated strain was analyzed and identified.

3. Fermentation of mulberry using enzyme strain

3-1. Preparation of medium and yeast culture

In order to check the optimal fermentation conditions for mulberry fermentation using yeast strains, Saccharomyces cerevisiae S1 held by the Fermentation Microorganism Industry Promotion Agency was used in YM (yeast mold) medium at 27℃, 150 rpm for 72 hours. It was stirred for a while, activated while pre-cultured, and used as a fermentation strain.

3-2. Establishment of fermentation conditions

After culturing by adjusting the amount of bacteria inoculation (0%, 1%, 5%), stirring speed (0rpm, 80rpm, 150rpm), culture temperature (27℃, 35℃) and sugar content (stock solution 2.4 °brix or 22 °brix) Saccharomyces cerevisiae by measuring pH, acidity, sugar content, alcohol content and viable cell count When the mulberry was fermented with the strain, it was attempted to confirm the optimal fermentation conditions (Table 2).

(A) Changes according to the amount of bacteria inoculated

A stock solution (2.4 ° brix) or a culture broth of the strain pre-cultured in YM medium with a sugar content of 22 ° brix was inoculated with 0%, 1% or 5% each, and stirred at 27° C. for 96 hours at 150 rpm, followed by fermentation. PH, acidity, sugar content, alcohol concentration, and viable cell count were measured. The strain inoculation amount (%) refers to the addition of 10 ml of the strain culture solution based on 1 L of mulberry juice, and the strain concentration of the strain culture solution is 1×10 ⁶ CFU/ml.

(B) Change according to the stirring speed

Inoculate 1% of the culture solution of the strain pre-cultured in a stock solution (2.4 ° brix) or YM medium with a sugar content of 22 ° brix, and stir at 27° C. for 96 hours at different speeds of 0 rpm, 80 rpm, and 150 rpm. After the fermentation broth, pH, acidity, sugar content, alcohol concentration, and viable cell count were measured.

(C) Change according to culture temperature

Inoculate 1% of the culture solution of the strain pre-cultured in a stock solution (2.4 ° brix) or YM medium with a sugar content of 22 ° brix, and the culture temperature was varied between 27 °C and 30 °C and stirred at 150 rpm for 96 hours. After the fermentation broth was measured for pH, acidity, sugar content, alcohol concentration, and viable cell count.

(D) Changes depending on whether sugar content is adjusted or not

Inoculate 1% of the culture solution of the pre-cultured strain in a stock solution (2.4 ° brix) or YM medium with a sugar content of 22 ° brix, and agitate at 150 rpm for 96 hours by varying the culture temperature between 27 °C and 30 °C. The fermentation broth was measured for pH, acidity, sugar content, alcohol concentration, and viable cell count.

(E) Change according to the concentration of Audi

Inoculate 1% of the culture solution of the pre-cultured strain in a stock solution (2.4 ° brix) or YM medium with a sugar content of 22 ° brix, and vary the concentration of the Audi juice to 100%, 70% or 50% at 27°C. After stirring at 150 rpm for 96 hours, the pH, acidity, sugar content and number of viable cells of the fermentation broth were measured.

4. Analysis of fermentation characteristics and rutin content in fermentation products

Based on the optimal fermentation conditions identified in the experiment for each condition, the isolated yeast strains were different to confirm the fermentation characteristics of mulberry and the rutin content in the fermented product for each yeast strain.

4-1. Fermentation characteristics

The Audi juice (concentration 100%) was sterilized with the sugar content adjusted to 22 ° brix, and 1% of the yeast strain was inoculated into the Audi juice, followed by stirring at 27° C. for 96 hours at 150 rpm to obtain the pH and acidity of the fermentation broth. (acidity), sugar content, alcohol content, and viable cell count were measured.

4-2. Analysis of rutin content in fermentation

(A) Reagents and standards

Reagents were tertiary DW, methanol (Duksan, HPLC grade), acetonitrile (JT Baker, HPLC grade), formic acid (Samjeon, GP), and rutin hydrate (Sigma, 94%) as a rutin standard. Was used.

(B) Equipment and facilities

As the analyzer, an HPLC-DAD (Diode Array Detector) Agilent 1200 series HPLC system (Palo Alto, USA) and an ultrasonic extraction device (Lab companion UC-20 JEIO TECH, Korea) were used.

(C) Standard solution analysis process

Rutin standard was prepared at 1 mg/ml, the solution was sequentially diluted to 5 concentrations of 50, 25, 10, 5 and 2.5 ppm, filtered, and injected into HPLC. HPLC analysis conditions are shown in Table 3.

(D) Sample preparation

After the sample was lyophilized, 0.1 g was precisely taken, dissolved in 5 ml of methanol, and sonicated for 30 minutes. The sonicated extract was centrifuged to take a supernatant, and then filtered through a 0.45 μm syringe filter and used as an analysis sample.

(E) Analysis and calculation

The content was calculated by calculating the concentration of rutin in the test solution using a calibration curve obtained using the peak area of the standard solution.

Example 1. Isolation of yeast strain from fermented food

In order to isolate useful yeast strains from traditional fermented foods (makgeolli, yeast) collected nationwide, a number of single colonies were purely isolated from YM medium supplemented with antibiotics. Biochemical and molecular biology methods such as film formation, gas production, alcohol resistance, sugar resistance, sulfite resistance, biogenic amine resolution, alcohol production ability, and strain identification are used to select yeast for brewing among the isolated yeast strains. Then, the useful yeast strain was evaluated.

Saccharomyces sp. and non- Saccharomyces sp. were cultivated using a non-Saccharomyces selective lysine medium to differentiate strains isolated into makgeolli and yeast on a medium to differentiate between Saccharomyces sp. and non-Saccharomyces sp. By doing this, it was confirmed whether the strain was grown.

As a result, most strains showed a pattern of inhibition of growth by lysine added to the medium, and yeast strains isolated from makgeolli and yeast were expected to be strains of the genus Saccharomyces (Table 4).

The result of culturing the isolated strain in lysine medium Strain name Growth in Lysine medium A1 - A2 - A3 - SRCM 101756 - A4 - A5 - A6 - A7 - A8 - A9 - A10 - A11 - A12 -

Example 2. Results of fermentation suitability evaluation of yeast strains isolated from fermented foods

To evaluate the fermentation suitability of the yeast strain of Saccharomyces isolated from rice wine and yeast, the amount of alcohol produced, gas production, film formation, alcohol tolerance, sugar tolerance and sulfurous acid tolerance were measured, and the results are shown in Table 5 below. . Yeast strains that produced alcohol in an amount of 10.5% or more, formed a mountain film (film), produced gas, and were resistant to alcohol, sugar, and sulfurous acid were selected. Alcohol, sugar and sulfurous acid tolerance concentrations for the determination of fermentation suitability of the isolated yeast were set as standards that can be used during brewing.

In addition, as a result of confirming the production of biogenic amines in yeast strains isolated from makgeolli and yeast, it was confirmed that the production of biogenic amines such as histamine, tyramine, putrescine and cadaverine was not detected in all yeast strains ( Table 6). Therefore, the yeast isolated from makgeolli and malt in the present invention is a useful yeast for wine production, including alcoholic beverages for vinegar production, and does not produce biogenic amines harmful to the body, so it was expected to be a strain suitable for application of fermented liquor.

Whether the isolated strain produces biogenic amines division Biogenic amine producing strain Histamine None Tyramine None Putrescine None Cadaverine None

Example 3. Confirmation of Optimal Fermentation Conditions of Mulberry

In order to confirm the optimal fermentation conditions of mulberry using yeast strains isolated from makgeolli and yeast, fermentation characteristics were analyzed according to the conditions of Table 2 using Saccharomyces cerevisiae S1 strain possessed by the Fermentation Microorganism Industry Promotion Agency. I did.

As a result, there was no significant difference in the inoculation amount of the strain in 1~5%, and when the culture temperature was 27°C, the stirring speed was 150 rpm, and the sugar content was 22° brix, it was confirmed that the alcohol-producing ability and the number of viable cells were the highest. .

Based on the above conditions, as a result of fermenting mulberry juice by varying only the concentration of mulberry juice (50, 70, 100%) at a sugar content of 22 ° brix, bacteria inoculation amount 1%, culture temperature 27 ℃, stirring speed 150 rpm, mulberry juice concentration There was no significant difference in fermentation characteristics according to, and in consideration of sensory characteristics, 100% concentration of mulberry juice was used in subsequent experiments (Table 7).

Example 4. Analysis of fermentation characteristics and rutin content according to mulberry fermentation by strain

After fermenting the mulberry juice by only different strains under the optimal fermentation conditions (sugarity 22 ° brix, bacterial inoculum 1%, culture temperature 27°C, stirring speed 150 rpm, mulberry juice 100%), the quality characteristics of the mulberry fermentation product were determined. The confirmed results are shown in Table 8 below. Most all fermented mulberry products produced a high concentration (12% or more) of alcohol after 4 days of fermentation, and through this result, the yeast strain isolated from makgeolli or yeast in the present invention is suitable as a fermentation strain for mulberry fermentation. Confirmed.

In addition, as a result of measuring the content of rutin in the fermented mulberry fermented with each of the isolated strains, it was confirmed that the content of rutin was increased in the fermented mulberry fermented with yeast strains isolated from makgeolli or yeast compared to the control (non-fermented mulberry). In particular, it was confirmed that the rutin content was significantly increased in the mulberry fermented product fermented with the SRCM101756 strain (Table 9 and FIG. 1). Through this, the SRCM101756 strain having the most increased rutin content in the mulberry fermentation product was finally selected as a yeast strain for the mulberry fermentation.

Example 5. Identification of selected strains

As a result of analyzing the 18S rRNA gene sequence (SEQ ID NO: 1) of the finally selected SRCM101756 strain , it was found to be Saccharomyces cerevisiae , and homology after creating a phylogenetic tree based on the standard strain and 18S rRNA sequence in MEGA-X. Analysis showed high similarity to Saccharomyces cerevisiae NRRL Y-12632 (FIG. 2). Finally, on to the August 05 date of 2020 as Saccharomyces cerevisiae SRCM101756 on was named as the invention Celebi jiae My access the selected strains as Saccharomyces (Saccharomyces cerevisiae) SRCM101756 in, Korea Culture Center of Microorganisms (KCCM, Korean Culture Center of Microorganisms ) Deposited (accession number: KCCM12776P).

Korea Microorganism Conservation Center (overseas) KCCM12776P 20200805

<110> Microbial Institute for Fermentation Industyry <120> Saccharomyces cerevisiae SRCM101756 strain for increasing rutin content in fermented mulberry and uses thereof <130> PN20348 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1708 <212> DNA <213> Saccharomyces cerevisiae <400> 1 cttaactctt cctgtcacgt aaagcaattt atacagtgaa actgcgaatg gctcattaaa 60 tcagttatcg tttatttgat agttccttta ctacatggta taactgtggt aattctagag 120 ctaatacatg cttaaaatct cgaccctttg gaagagatgt atttattaga taaaaaatca 180 atgtcttcgg actctttgat gattcataat aacttttcga atcgcatggc cttgtgctgg 240 cgatggttca ttcaaatttc tgccctatca actttcgatg gtaggatagt ggcctaccat 300 ggtttcaacg ggtaacgggg aataagggtt cgattccgga gagggagcct gagaaacggc 360 taccacatcc aaggaaggca gcaggcgcgc aaattaccca atcctaattc agggaggtag 420 tgacaataaa taacgataca gggcccattc gggtcttgta attggaatga gtacaatgta 480 aataccttaa cgaggaacaa ttggagggca agtctggtgc cagcagccgc ggtaattcca 540 gctccaatag cgtatattaa agttgttgca gttaaaaagc tcgtagttga actttgggcc 600 cggttggccg gtccgatttt ttcgtgtact ggatttccaa cggggccttt ccttctggct 660 aaccttgagt ccttgtggct cttggcgaac caggactttt actttgaaaa aattagagtg 720 ttcaaagcag gcgtattgct cgaatatatt agcatggaat aatagaatag gacgtttggt 780 tctattttgt tggtttctag gaccatcgta atgattaata gggacggtcg ggggcatcag 840 tattcaattg tcagaggtga aattcttgga tttattgaag actaactact gcgaaagcat 900 ttgccaagga cgttttcatt aatcaagaac gaaagttagg ggatcgaaga tgatcagata 960 ccgtcgtagt cttaaccata aactatgccg actagggatc gggtggtgtt tttttaatga 1020 cccactcggc accttacgag aaatcaaagt ctttgggttc tggggggagt atggtcgcaa 1080 ggctgaaact taaaggaatt gacggaaggg caccaccagg agtggagcct gcggcttaat 1140 ttgactcaac acggggaaac tcaccaggtc cagacacaat aaggattgac agattgagag 1200 ctctttcttg attttgtggg tggtggtgca tggccgttct tagttggtgg agtgatttgt 1260 ctgcttaatt gcgataacga acgagacctt aacctactaa atagtggtgc tagcatttgc 1320 tggttatcca cttcttagag ggactatcgg tttcaagccg atggaagttt gaggcaataa 1380 caggtctgtg atgcccttag acgttctggg ccgcacgcgc gctacactga cggagccagc 1440 gagtctaacc ttggccgaga ggtcttggta atcttgtgaa actccgtcgt gctggggata 1500 gagcattgta attattgctc ttcaacgagg aattcctagt aagcgcaagt catcagcttg 1560 cgttgattac gtccctgccc tttgtacaca ccgcccgtcg ctagtaccga ttgaatggct 1620 tagtgaggcc tcaggatctg cttagagaag ggggcaactc catctcagag cggagaattt 1680 ggacgatctg tctgatactc tgatgacg 1708

Claims (8)

It is resistant to alcohol, sugar and sulfurous acid, has alcohol-producing ability, does not produce biogenic amines of histamine, tyramine, putrescine and cadaverine. , Saccharomyces cerevisiae (Saccharomyces cerevisiae) SRCM101756 strain, which has an accession number of KCCM12776P, characterized in that to increase the rutin content in the fermentation by fermenting berries. delete A composition for producing alcohol comprising the strain of claim 1, a culture solution thereof, a concentrate of the culture solution, or a dried product thereof as an active ingredient. A composition for producing vinegar comprising the strain of claim 1, a culture solution thereof, a concentrate of the culture solution, or a dried product thereof and acetic acid bacteria as active ingredients. A method for producing a berry wine with enhanced rutin content, comprising the step of inoculating and fermenting the Saccharomyces cerevisiae SRCM101756 strain of the accession number KCCM12776P on the berries. The method of claim 5, wherein the berry wine is stored at 21 to 23 ° brix for berry fruit juice, and after inoculation of the Saccharomyces cerevisiae SRCM101756 strain having an accession number of KCCM12776P, 140 to 160 rpm at 26 to 28 °C. Method characterized in that the fermentation for 3 to 5 days. The method of claim 5, wherein the berries are at least one selected from mulberry (Morus alba ), bokbunja ( Rubus coreanus ), and blueberry ( Vaccinium corymbosum). Berries wine with increased rutin content prepared by the method of any one of claims 5 to 7.

Images