KR20220039942A - Saccharomyces cerevisiae FT4-4 strain having antioxidant activity and tyrosinase inhibitory activity isolated from blueberry and uses thereof - Google Patents

Abstract

The present invention relates to a strain of Saccharomyces cerevisiae FT4-4 and uses thereof. The Saccharomyces cerevisiae FT4-4 strain (Accession No: KCCM12522P) of the present invention is resistant to an alcohol, sugars and a sulfurous acid, and has alcohol production ability, β-glucosidase activity, tyrosinase inhibitory activity and antioxidant activity. In addition, the Saccharomyces cerevisiae FT4-4 strain does not produce biogenic amines and harmful enzymes, but produces skin physiologically active substances.

Description

Saccharomyces cerevisiae FT4-4 strain having antioxidant activity and tyrosinase inhibitory activity isolated from blueberry and uses thereof isolated from blueberry

The present invention relates to a Saccharomyces cerevisiae FT4-4 strain having tyrosinase inhibitory activity and antioxidant activity isolated from blueberries and uses thereof.

In Korea, fermentation technology and aging technology used in food production have been developed since ancient times, and traditional wine using fermentation technology has been continuously developed. Traditional liquor is largely divided into traditional distilled liquor and traditional fermented liquor according to the manufacturing method. Saccharomyces ( Saccharomyces ) genus, Aspergillus genus, Bacillus genus, Lactobacillus genus, etc. are used for fermentation of traditional liquor, and recently, the physiological activity of these fermented strains has been used. The health functional fermented wine emphasized is being produced.

Antioxidant means to prevent aging by inhibiting oxidation, and reactive oxygen species in the body attack normal cells and act as a cause of aging and various diseases. Therefore, it can be said that removing free radicals is a way to prevent cell oxidation, and antioxidant enzymes that change free radicals into harmless substances are important. However, it is known that it is good to consume antioxidants through food because the ability of the antioxidant enzymes produced in the body to remove active oxygen rapidly decreases with age.

Melanocytes in the basal layer, the lowest layer of the epidermis, make melanin by various stimulating factors of the skin and send it to the upper layer. Skin whitening is the process of quickly discharging unnecessary melanin and restoring the original skin's transparency. Substances registered as whitening ingredients by the Food and Drug Administration include mulberry extract, arbutin, ethyl ascorbyl ether, oil-soluble licorice extract, ascorbyl glucoside, niacinamide, alpha-bisabolol, and ascorbyl tetraisopalmitate, such substances Whitening cosmetics is to make lotions, liquids, creams, and mask packs using

On the other hand, alcoholic fermentation of berries such as wine is made by repeating complex chemical reactions step by step by the action of various microorganisms. In many foreign countries, Saccharomyces cerevisiae is used as a starter to improve the quality of wine, and efforts are being made to select yeast that has excellent alcohol fermentation ability and helps improve the quality of wine. .

On the other hand, Korean Patent No. 1743526 discloses 'Saccharomyces cerevisiae strain with excellent sugar tolerance isolated from domestic grapes', and Korean Patent No. 1703249 discloses 'New Saccharomyces cerevisiae Y28 Saccharomyces cerevisiae FT4-4 strain having tyrosinase inhibitory activity and antioxidant activity isolated from blueberries and its 'Saccharomyces cerevisiae FT4-4 strain and its There is no mention of 'use'.

The present invention was derived from the above needs, and the present inventors have resistance to alcohol, sugar and sulfurous acid from blueberries and blueberry extracts, alcohol production ability, β-glucosidase activity, tyrosinase (tyrosinase) Saccharomyces cerevisiae FT4-4 has inhibitory and antioxidant activity, does not produce biogenic amines histamine and tyramine, and urease, a harmful enzyme, and has a high content of β-glucan, a skin bioactive substance A strain (Accession No.: KCCM12522P) was isolated. By confirming that the Saccharomyces cerevisiae FT4-4 strain of the present invention can be used in a starter strain for wine production and a cosmetic composition for whitening, the present invention has been completed.

In order to solve the above problems, the present invention has resistance to alcohol, sugar and sulfurous acid, alcohol production ability, β-glucosidase activity, tyrosinase inhibitory activity, antioxidant activity, and biogenic It does not produce amines, harmful enzymes, and produces skin physiologically active substances, with an accession number of KCCM12522P Saccharomyces cerevisiae ) Provides a FT4-4 strain.

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

In addition, the present invention provides a cosmetic composition for skin whitening comprising the strain, its culture medium, a concentrate of the culture medium, or a dried product thereof as an active ingredient.

In addition, the present invention provides a starter composition for producing a fermented food comprising the strain, its culture medium, a concentrate of the culture medium, or a dried product thereof as an active ingredient.

Saccharomyces cerevisiae FT4-4 strain of the present invention has resistance to alcohol, sugar and sulfurous acid, alcohol production ability, β-glucosidase activity, tyrosinase inhibition It has active and antioxidant activity, does not produce biogenic amines and harmful enzymes, and has a high content of skin bioactive substances, so it can be very usefully used in related industrial fields such as starter strains for wine production and cosmetic compositions for skin whitening. there is.

1 shows a phylogenetic diagram of the Saccharomyces cerevisiae FT4-4 strain isolated in the present invention.
2 is a result of measuring the dry cell mass, the number of viable cells and the absorbance according to the culture time of the Saccharomyces cerevisiae FT4-4 strain isolated in the present invention. The average value of the experiment repeated three times is shown. △: dry cell mass (g/L), ■: viable cell count (log ₁₀ CFU/ml), ●: absorbance (660 nm)
3 is a result showing the alcohol (A), sugar (B) and sulfurous acid (C) resistance of the Saccharomyces cerevisiae FT4-4 strain isolated in the present invention. The average value of the experiment repeated three times is shown.

In order to achieve the object of the present invention, the present invention has resistance to alcohol, sugar and sulfurous acid, alcohol production ability, β-glucosidase activity, tyrosinase inhibitory activity, antioxidant activity, It provides a biogenic amine, Saccharomyces cerevisiae FT4-4 strain that does not produce a biogenic amine, a harmful enzyme, and produces a skin bioactive substance, with an accession number of KCCM12522P.

In the present invention, there is resistance to alcohol, sugar and sulfurous acid from blueberries and blueberry extracts grown and harvested in Sunchang-gun, Jeollabuk-do, alcohol-producing ability, β-glucosidase activity, tyrosinase inhibitory activity, Yeast that has antioxidant activity, does not produce biogenic amines and harmful enzymes, and produces skin bioactive substances was isolated, and as a result of identification through 18S rRNA sequencing of the finally selected strain, Saccharomyces cerevisiae Jiae ( Saccharomyces cerevisiae ) It was confirmed that the strain. The present inventor named the final selected strain Saccharomyces cerevisiae FT4-4 strain and deposited it with the Korea Center for Microorganisms Conservation (KCCM) on May 3, 2019 (Accession No.: KCCM12522P) .

In the Saccharomyces cerevisiae FT4-4 strain whose deposit number is KCCM12522P of the present invention, the biogenic amine is histamine and tyramine, and the harmful enzyme is urease, and the skin The physiologically active substance may be β-glucan, but is not limited thereto. In addition, the alcohol resistance is alcohol resistance to 8-10% of absolute ethanol, sugar resistance is sugar resistance to 30-50% glucose, and sulfurous acid resistance is sulfurous acid resistance to 100-300 ppm potassium metabisulfite am.

The present invention also provides a composition for producing alcohol comprising, as an active ingredient, the Saccharomyces cerevisiae FT4-4 strain, the culture solution thereof, the concentrate of the culture solution, or the dried product thereof, the accession number of which is KCCM12522P. .

In the composition according to an embodiment of the present invention, the alcohol may be alcohol obtained by fermenting fruits, and most preferably may be wine obtained by fermenting berries, but is not limited thereto.

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 particular method.

When using as an additive the concentrate of the strain or the culture of the strain or the culture solution of the strain obtained in the step of culturing the strain of the present invention, the concentrate of the strain or the culture of the strain or 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 active ingredient may be appropriately determined depending on the intended use thereof.

The present invention also provides a cosmetic composition for skin whitening comprising, as an active ingredient, the Saccharomyces cerevisiae FT4-4 strain whose accession number is KCCM12522P, a culture solution thereof, a concentrate of the culture solution, or a dried product thereof as an active ingredient do.

The cosmetic composition according to the present invention contains a Saccharomyces cerevisiae FT4-4 strain having an accession number of KCCM12522P having tyrosinase inhibitory activity, a culture solution thereof, a concentrate of the culture solution or a dried product thereof as an active ingredient and can be used for skin whitening.

In the cosmetic composition according to an embodiment of the present invention, the cosmetic composition for skin whitening is a cream, softening lotion, nutrient lotion, pack, essence, hair tonic, shampoo, conditioner, hair conditioner, hair treatment, gel, skin lotion , Skin Softener, Skin Toner, Astringent, Milk Lotion, Moisture Lotion, Nourishing Lotion, Massage Cream, Nourishing Cream, Moisture Cream, Hand Cream, Foundation, Nourishing Essence, Sunscreen, Soap, Cleansing Foam, Cleansing Lotion, Cleansing Cream, Body It may have any one formulation selected from the group consisting of lotions and body cleansers, but is not limited thereto. The cosmetic composition composed of each of these formulations may contain various bases and additives necessary and appropriate for the formulation of the formulation, and the types and amounts of these components can be easily selected by those skilled in the art.

The cosmetic composition of the present invention may further contain excipients including fluorescent substances, fungicides, hydrophobicity inducers, moisturizers, fragrances, fragrance carriers, proteins, solubilizers, sugar derivatives, sunscreens, vitamins, plant extracts, and the like. .

The present invention also provides a Saccharomyces cerevisiae FT4-4 strain whose accession number is KCCM12522P, a culture solution thereof, a concentrate of the culture solution or a dry product thereof as an active ingredient. Starter for manufacturing fermented food A composition is provided.

In the present invention, a starter for fermented food production means a preparation or composition containing microorganisms involved in fermentation for the production of fermented food. It is used to provide microorganisms that can grow in fermented foods or microorganisms that can grow as dominant species by adding them during the manufacture of fermented foods. In the case of manufacturing food using the starter for food fermentation, by the microorganisms included in the starter for food fermentation, the quality of food is constantly controlled, or for a specific purpose, for example, no off-flavor is generated in food or reduced. purpose can be achieved. In the present invention, by using the Saccharomyces cerevisiae FT4-4 strain or its culture medium as a starter strain, there is resistance to alcohol, sugar and sulfurous acid, alcohol production ability, β-glucosidase activity, tyrosina It has tyrosinase inhibitory activity and antioxidant activity, does not produce biogenic amines and harmful enzymes, and can produce fermented food with a high content of skin bioactive substances.

In the starter composition for manufacturing fermented food according to an embodiment of the present invention, the fermented food may be a fermented wine obtained by fermenting fruits, preferably wine obtained by fermenting berries, but is not limited thereto.

Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited thereto.

Materials and Methods

1. Strain isolation and culture conditions

Blueberries and blueberry extracts grown and harvested in Sunchang-gun, Jeollabuk-do were purchased and used in the experiment. For enrichment, blueberries and blueberry extracts were added to 250 ml of YPD (1% yeast extract, 2% peptone, 2% dextrose) liquid medium, and fermented at 30° C. for 48 hours, which was used to isolate strains. 10 g of blueberries and blueberry extract, enriched blueberries and blueberry extracts are homogenized, put in 90 ml of sterile saline (0.85% NaCl), diluted in decimal, and then 100 μg/ml of It was smeared on YPD solid medium containing chloramphenicol and incubated at 30°C for 2 days. Among them, strains producing alcohol were selected, mixed in YPD liquid medium containing 20% glycerol, and stored at 4°C. Thereafter, it was activated by culturing at 30° C. for 2 days in YPD liquid medium, and then used for the experiment.

2. Selection of alcohol-producing strains

For the primary screening of strains with excellent alcohol-producing ability, the gas-producing ability was confirmed through the Durham tube test. For culturing of the selected strain, it was cultured with stirring at 30° C. for 24 hours in YPD medium, and 2% of the culture medium was inoculated into 10 ml of YPD medium containing 24% glucose, and left at 30° C. for 48 hours without stirring. cultured. After that, the alcohol-producing strains were first selected by checking whether gas was produced, and the first selected strains were inoculated in a YPD liquid medium containing 24% glucose and cultured for 48 hours, then the culture solution was centrifuged and the supernatant was taken 0.45 After filtration using a ㎛ syringe filter, strains with excellent alcohol production ability were secondarily selected through HPLC analysis according to the conditions according to Table 1.

HPLC instrument analysis conditions for alcohol production capacity analysis Instrument Agilent 1200 series Column Aminex HPX 87H column, 300x7.8 mm Detector RID detector Eluant 5 mM sulfuric acid in H ₂ O flow rate 0.6 ml/min Temperature 65℃ Injection volume 10 μl

3. Biogenic amine analysis

To investigate the biogenic amine generation of the selected strains, each strain is inoculated in a YPD liquid medium, and after pre-culturing at 30° C. for 24 hours in a shaking incubator at 150 rpm, 1 ml of the culture medium is tyrosine and histidine, which are precursor amino acids of tyramine and histamine. It was inoculated into 9 ml of this 0.1% YPD liquid medium and incubated for 48 hours at 30° C. in a shaking incubator at 150 rpm. The culture solution was centrifuged at 13,000 rpm for 30 minutes to remove cells, and the supernatant from which the cells were removed was used as a sample for biogenic amine analysis. After taking 0.5 ml of each of the sample solution and the standard solution, 0.25 ml of 1,7-diaminoheptane (sigma, USA), 0.25 ml of saturated sodium carbonate solution, 1% acetone, and 0.4 ml of Dansyl chloride were mixed, followed by mixing at 45°C for 1 hour. derivatized. After adding 0.25 ml of 10% proline to the derivatized sample, removing the remaining amount of dansyl chloride, adding 2.5 ml of ether, shaking for 3 minutes, taking the separated supernatant, evaporating, and dissolving the remaining residue in 0.5 ml of acetonitrile After filtration through a 0.45 μm filter, HPLC analysis was performed. The analysis conditions are shown in Table 2 below.

HPLC instrument analysis conditions for biogenic amine analysis Instrument Agilent 1200 series Column CapcellPak C18 Column Detector DAD detector (254 nm) mobile phase A: 0.1% formic acid in H2O
B: 0.1% formic acid in ACN gradient condition A : B = 45 : 55, 0~10 min
A : B = 35 : 65, 10~15min
A : B = 20 : 80, 15~20 min
A : B = 10 : 90, 20~30 min
A : B = 10 : 90, 40 min over flow rate 1.0 ml/min Temperature 40℃ Injection volume 20 μl

4. Hazardous Enzyme Production Analysis

The generation of harmful enzymes in the selected strain was confirmed using the Urea rapid test kit (MB cell, Korea). Selected strains were inoculated into the kit, and vaseline oil was added, followed by incubation at 37° C. for 24 hours in a state in which oxygen was blocked, and color change was observed at 4 hour intervals to determine whether urease was generated.

5. β-Glucosidase Enzyme Activity Assay

To analyze the activity of the β-glucosidase enzyme, the selected strain was placed on a Lactobacillus MRS agar medium prepared by adding 1.0% esculin and 0.5% ferric ammonium citrate. After inoculation and incubation at 37° C. for 24 hours, the activity of the β-glucosidase enzyme was measured according to the presence or absence of a black transparent ring formed around the colony.

6. Measurement of tyrosinase inhibitory activity

In order to measure the tyrosinase inhibitory activity of the selected strain, it was inoculated into YPD broth and incubated at 30° C. at 150 rpm for 48 hours. The culture medium was centrifuged at 13,000 rpm for 10 minutes to recover the cells. The cells were suspended in 50 mM PBS buffer (pH 6.8). After disrupting the cells at 60° C. for 1 hour using an ultrasonic crusher, the supernatant was used by centrifugation at 13,000 rpm for 10 minutes. The substrate was used by completely dissolving 1 mM L-tyrosine solution in 50 mM PBS buffer (pH 6.8). 100 μl of L-tyrosine solution was mixed with 50 μl of sample, 50 μl of tyrosinase (50 U/ml) was added, and reacted at 37° C. for 30 minutes, absorbance was measured at 470 nm and tyrosinase was calculated using the following formula. Inhibitory activity values were calculated.

Tyrosinase inhibitory activity (%)=[1-(A/B)x100]

A: Absorbance of sample-added tyrosinase solution at 470 nm

B: Absorbance of sample-free tyrosinase solution at 470 nm

7. Antioxidant Activity Assay

For the analysis of the antioxidant activity of the selected strains, the supernatant was used as a sample by incubating in YPD liquid medium at 30° C., 150 rpm for 48 hours with shaking, followed by centrifugation at 4° C., 13,000 rpm for 10 minutes. Free radical scavenging ability using DPPH (2,2-diphenyl-1-picryl-hydrazyl) was measured by mixing 180 μl of 100 μM DPPH ethanol solution with 20 μl of sample and reacting in the dark for 30 minutes with UV/VIS spectrometer. After measuring the absorbance at 517 nm, it was calculated by substituting the formula below, and YPD liquid medium was used as a control.

DPPH free radical scavenging activity (%)=[1-(A/B)x100]

A: Absorbance of sample-added DPPH solution at 517 nm

B: Absorbance of sample-free DPPH solution at 517 nm

8. Identification and phylogenetic of selected strains

18S rRNA gene using ITS1 (5'-TCCGTAGGTGAACCTGCGG-3': SEQ ID NO: 2) and ITS4 primer (5'-TCCTCCCGCTTATTGATATGC-3': SEQ ID NO: 3) by extracting DNA from the cells to identify the finally selected strain was amplified, and the amplified product was identified by requesting Cosmogene tech. For the base sequence, the base sequence of the standard strain was obtained through the ExTaxone-e server (http://www.eztaxon.org), and the nucleotide sequence was compared using the MEGA 6.0 program and then a phylogenetic diagram was prepared. The phylogenetic tree was analyzed using the neighbor-joining algorithm, and the robustness of the phylogenetic tree was confirmed by bootstrapping with 1,000 repetitions.

9. β-glucan content analysis

In order to investigate the β-glucan content of the selected strain, the cells were cultured with shaking at 150 rpm for 48 hours in YPD liquid medium, and the cells separated by centrifugation at 3,000 rpm were washed 3 times with distilled water and lyophilized. was used for the analysis. The β-glucan content was investigated using a Yeast β-glucan kit (K-YBGL, Megazyme International Ireland Ltd.).

10. Cell Growth Analysis

In order to analyze the growth of the selected strains according to time, the selected strains were inoculated into the liquid medium and pre-cultured for 2 days in a shaking incubator at 30°C and 150 rpm. As a medium for cell growth investigation, a YPD liquid medium containing 2% glucose was used, and 2% of the pre-culture solution was inoculated into each liquid medium, and then cultured at 30° C. at 200 rpm for 36 hours. Samples were taken every 4 hours, and the dry cell mass, viable cell count, and absorbance were measured. To measure the dry cell mass, 10 ml of the culture solution was centrifuged at 13,000 rpm for 20 minutes. The separated cells were washed three times with distilled water, dried at 80° C. until constant weight, and then the weight was measured. In addition, 1 ml of the culture medium was recovered, diluted stepwise, spread on YPD solid medium, and cultured at 30° C. for 48 hours, and then the number of viable cells was measured. For absorbance measurement for comparison with the dry cell mass, 1 ml of the culture medium was recovered, centrifuged at 13,000 rpm for 20 minutes, washed three times in distilled water, resuspended in 1 ml of sterile distilled water, and then resuspended in 1 ml of sterile distilled water using an ultraviolet/visible light spectrophotometer ( Absorbance was measured and recorded using a UV/VIS spectrophotometer, SPECORD 200, Analytik Jena, Jena, Germany).

11. Analysis of alcohol tolerance, sugar tolerance, and sulfurous acid tolerance

Alcohol resistance of the selected strain was analyzed by inoculating the strain in YPD broth, immediately adding 0, 8, 14, and 20% absolute ethanol, and incubating at 30° C. for 72 hours by measuring the dry cell mass. Glucose tolerance was analyzed by measuring the dry cell mass after incubating the strain in YPD broth containing 3, 30, 40 and 50% glucose at 30° C. for 48 hours. Sulfurous acid resistance was analyzed by measuring the amount of dried cells after incubation for 48 hours at 30° C. in YPD liquid medium containing 100, 200 and 300 ppm potassium metabisulfite.

Example 1. Selection of alcohol-producing strains

About 150 strains were selected from blueberries and blueberry extracts, and a durham tube test was performed on them to first select 53 strains with excellent gas-generating ability. However, since there are strains with low gas-producing ability but high alcohol-producing ability, additionally, as a result of confirming whether or not alcohol is produced through HPLC analysis, a total of 60 strains were selected. was finally selected (Table 3).

Analysis of alcohol-producing ability of selected strains strain Alcohol production capacity (%) Commercial yeast (fruit wine) 10.28 ± 0.01 Commercial yeast (grain wine) 10.52 ± 0.05 FT4-4 13.37 ± 0.03 FT4-7 10.11 ± 0.05 FT4-32 11.01 ± 0.02 FT4-98 9.09 ± 0.03 FT4-140 7.08 ± 0.01

Example 2. Biogenic amine analysis

Biogenic amines exist in wine and fermented foods, and are produced by various yeasts and lactic acid bacteria during alcoholic fermentation. Ingestion of excess biogenic amines is known to cause symptoms such as nausea, vomiting, high blood pressure, and headache. , was examined whether the biogenic amines histamine and tyramine were produced in the five selected strains. As a control, a YPD liquid medium that was not inoculated with the strain was used. The results are shown in Table 4, and the strain that does not produce both biogenic amines among the five selected strains was identified as FT4-4, and in most strains, two types of biogenic amines were detected but not quantified. showed insignificant values. Therefore, it was confirmed that the most suitable yeast as an industrial strain was FT4-4 through the alcohol production ability and biogenic amine production results.

Analysis of biogenic amine-producing ability of selected strains biogenic amines selection strain FT4-4 FT4-7 FT4-32 FT4-98 FT4-140 tyramine N.D. ^a NQ ^b N.D. N.Q. 1.25±0.03 histamine N.D. N.Q. 2.22±0.03 N.Q. N.D.

Results were measured three times and expressed as standard deviation.

^a ND - not detected

^b NQ - not quantified

Example 3. Harmful enzyme production analysis

As a mechanism of cellular damage, Helicobacter pylori infection causes cell damage by ammonia produced by urease, vacuolization of epithelial cells by cytostatic toxin, and mediator of inflammation secreted by neutrophils Cells are also damaged by substances and reactive oxygen species. Damage to epithelial cells causes atrophic changes in cells, and cells that have actively proliferated due to a compensatory mechanism have a greater chance of being damaged by carcinogens. , As a result, it was confirmed that urease was not produced in the FT4-4, FT4-7 and FT4-140 strains (Table 5).

Analysis of urease production in selected strains strain Urease activity FT4-4 - FT4-7 - FT4-32 + FT4-98 + FT4-140 -

Example 4. β-Glucosidase Enzyme Activity Assay

As microorganisms that produce β-glucosidase, strains belonging to various phylogenetic groups such as mold, yeast, soil bacteria and hyperthermia archaea are known. Many genes are also known. In addition, β-glucosidase is known to be involved in the catalytic action of converting soybean isoflavone glycosides genistin and daidgin, which are mainly present in traditional fermented foods, into non-glycosides for body absorption. Accordingly, the present inventors analyzed the enzyme activity of β-glucosidase against the selected strain, and as a result, it was confirmed that there was activity in 4 types of yeast, and it was confirmed that the activity of β-glucosidase of the FT4-4 strain was the best. became (Table 6).

Analysis of β-glucosidase enzyme activity of selected strains strain β-glucosidase enzyme activity FT4-4 +++ T4-7 - T4-32 + T4-98 ++ T4-140 +

-; not detected, +; 1.0-1.4 cm, ++; 1.5-1.9 cm, +++; 2.0 cm or more

Example 5. Measurement of tyrosinase inhibitory activity

In skin cells, melanin is synthesized by the activity of tyrosinase, L-tyrosine is converted to L-dihydroxy phenylalanine (L-DOPA), and L-DOPA is converted back to DOPA quinone to finally produce melanin. do. The melanin produced in this way induces intracellular pigmentation to form spots and freckles, so the expression of melanin synthetase must be inhibited. Accordingly, the present inventors measured the tyrosinase inhibitory activity against the selected strain, and confirmed that the FT4-4 strain showed the highest activity (Table 7).

Analysis of tyrosinase inhibitory activity of selected strains strain Tyrosinase inhibitory activity (%) FT4-4 64.75 FT4-7 51.11 FT4-32 52.12 FT4-98 54.92 FT4-140 55.84

Example 6. Measurement of antioxidant activity

Free radicals generated by various reactions in the body cause oxidative damage to biological enzymes and proteins, causing gene damage and cancer. However, it is known that by removing free radicals through enzymatic or non-enzymatic methods using some microorganisms, antioxidant activity is enhanced and aging can be inhibited. Accordingly, the present inventors measured the DPPH activity against the selected strains, and as a result, all of the selected strains showed an antioxidant activity of 30% or more, and in particular, FT4-4 was confirmed as the strain with the highest antioxidant activity (Table 8).

Analysis of DPPH activity of selected strains strain DPPH activity (%) FT4-4 54.41 FT4-7 32.11 FT4-32 33.51 FT4-98 42.63 FT4-140 48.51

Example 7. Identification and phylogenetic of selected strains

As a result of analyzing the 18S rRNA gene base sequence (SEQ ID NO: 1) to identify the strain FT4-4 identified as the best strain through the above Examples, it was found to be Saccharomyces cerevisiae , and compared with a standard strain with high homology by the SeqMatch program As a result, it showed 100% similarity to Saccharomyces cerevisiae NRRL Y-12632 (FIG. 1). Finally, the strain selected in the present invention was named Saccharomyces cerevisiae FT4-4, and was named Saccharomyces cerevisiae FT4-4 at the Korean Culture Center of Microorganisms (KCCM) on 5, 2019. It was deposited on the 3rd of the month (Accession No.: KCCM12522P).

Example 8. β-glucan content analysis

β-glucan, one of the physiologically active substances for skin, is a homogeneous polysaccharide in which glucose is linked by a β-(1,3)-D-glycosidic bond, and occupies the largest amount among the components of the yeast cell wall. β-glucan is known to have various physiological activity promoting effects such as anticancer, anticholesterol, immune enhancement and skin regeneration. In particular, β-glucan obtained from yeast plays a role in protecting the skin and regenerating the skin in case of skin damage, and is known to play an important role in strengthening the immune system in the human body. Accordingly, the present inventors analyzed the content of β-glucan produced in the final selection strain FT4-4 using the Yeast β-glucan kit, and as a result, it was confirmed to be 28.31 mg per cell dry weight (g).

Example 9. Cell growth measurement of selected strains

Changes in the dry cell mass according to the incubation time of the final selected strain FT4-4 were investigated. The growth of the cells had a lag phase of 0-8 hours and a stationary phase of 12-16 hours. The cell mass recorded 3.1647 g/L at 16 hours, and then showed a log phase in which the cell mass gradually increased or decreased. It was confirmed that the cell mass and the number of viable cells for comparison appeared in the same pattern (FIG. 2).

Example 10. Analysis of alcohol tolerance, sugar tolerance and sulfurous acid tolerance of selected strains

Since high concentrations of alcohol, glucose and sulfurous acid are added to wine to enhance the storage period and alcoholic fermentation ability of grape wine, resistance to these is one of the essential elements as yeast for wine making. In order to confirm the potential of the FT4-4 strain finally selected in the present invention as an industrial yeast, an analysis for alcohol, sugar and sulfurous acid resistance was performed.

In order to investigate the alcohol resistance of FT4-4, 0, 8, 14, and 20% of absolute ethanol were added to YPD liquid medium, which is a general yeast culture medium, and the dry cell mass was measured. As a result, when 8% of alcohol was added, the dry cell mass was reduced to 3.81 g/L compared to the control (4.69 g/L), and it was confirmed that almost no cells were grown when high concentration of ethanol was added (FIG. 3A) . This is consistent with the previous report that Saccharomyces cerevisiae hardly grew in the initial low sugar concentration and high alcohol concentration.

The results of the sugar tolerance of FT4-4 are the same as in Figure 3B, and in the experimental group in which 30% glucose was added compared to 4.55 g/L of the YPD liquid medium as a control, the cell mass was increased by about 2 times to 10.51 g/L, In the experimental group to which 40% and 50% glucose was added, the cell mass was increased to 7.94 g/L and 5.69 g/L, respectively, compared to the control group, confirming that FT4-4 had excellent sugar tolerance.

Sulfurous acid is a substance added for the purpose of preserving food, beverages, and pharmaceuticals, and has effects such as anti-oxidation, antibacterial and browning prevention. Therefore, sulfurous acid is used to prevent oxidation and selectively inhibit harmful microorganisms in the production of wine, and yeast for wine production needs to have resistance to it. According to the current domestic food standards, sulfurous acid resistance of FT4-4 strain was investigated by adding 100, 200 and 300 ppm of sulfurous acid in accordance with the allowable addition of 350 mg/L. As a result, even when the concentration of added sulfurous acid increased, the cell mass of FT4-4 was not significantly affected, confirming that the yeast had excellent sulfurous acid resistance (FIG. 3C).

Korea Microorganism Center (Overseas) KCCM12522P 20190503

<110> Microbial Institute for Fermentation Industry <120> Saccharomyces cerevisiae FT4-4 strain having antioxidant activity and tyrosinase inhibitory activity isolated from blueberry and uses it <130> PN20179 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 1730 <212> DNA <213> Saccharomyces cerevisiae <400> 1 atgtctaagt ataagcaatt tatacagtga aactgcgaat ggctcattaa atcagttatc 60 gtttatttga tagttccttt actacatggt ataactgtgg taattctaga gctaatacat 120 gcttaaaatc tcgacccttt ggaagagatg tatttattag ataaaaaatc aatgtcttcg 180 gactctttga tgattcataa taacttttcg aatcgcatgg ccttgtgctg gcgatggttc 240 attcaaattt ctgccctatc aactttcgat ggtaggatag tggcctacca tggtttcaac 300 gggtaacggg gaataagggt tcgattccgg agagggagcc tgagaaacgg ctaccacatc 360 caaggaaggc agcaggcgcg caaattaccc aatcctaatt cagggaggta gtgacaataa 420 ataacgatac agggcccatt cgggtcttgt aattggaatg agtacaatgt aaatacctta 480 acgaggaaca attggagggc aagtctggtg ccagcagccg cggtaattcc agctccaata 540 gcgtatatta aagttgttgc agttaaaaag ctcgtagttg aactttgggc ccggttggcc 600 ggtccgattt tttcgtgtac tggatttcca acggggcctt tccttctggc taaccttgag 660 tccttgtggc tcttggcgaa ccaggacttt tactttgaaa aaattagagt gttcaaagca 720 ggcgtattgc tcgaatatat tagcatggaa taatagaata ggacgtttgg ttctattttg 780 ttggtttcta ggaccatcgt aatgattaat agggacggtc gggggcatca gtattcaatt 840 gtcagaggtg aaattcttgg atttattgaa gactaactac tgcgaaagca tttgccaagg 900 acgttttcat taatcaagaa cgaaagttag gggatcgaag atgatcagat accgtcgtag 960 tcttaaccat aaactatgcc gactagggat cgggtggtgt ttttttaatg acccactcgg 1020 caccttacga gaaatcaaag tctttgggtt ctggggggag tatggtcgca aggctgaaac 1080 ttaaaggaat tgacggaagg gcaccaccag gagtggagcc tgcggcttaa tttgactcaa 1140 cacggggaaa ctcaccaggt ccagacacaa taaggattga cagattgaga gctctttctt 1200 gattttgtgg gtggtggtgc atggccgttc ttagttggtg gagtgatttg tctgcttaat 1260 tgcgataacg aacgagacct taacctacta aatagtggtg ctagcatttg ctggttatcc 1320 acttcttaga gggactatcg gtttcaagcc gatggaagtt tgaggcaata acaggtctgt 1380 gatgccctta gacgttctgg gccgcacgcg cgctacactg acggagccag cgagtctaac 1440 cttggccgag aggtcttggt aatcttgtga aactccgtcg tgctggggat agagcattgt 1500 aattattgct cttcaacgag gaattcctag taagcgcaag tcatcagctt gcgttgatta 1560 cgtccctgcc ctttgtacac accgcccgtc gctagtaccg attgaatggc ttagtgaggc 1620 ctcaggatct gcttagagaa gggggcaact ccatctcaga gcggagaatt tggacaaact 1680 tggtcattta gaggaactaa aagtcgtaac aaggtttccg taggtgaacc 1730 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 tccgtaggtg aacctgcgg 19 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 tcctcccgct tattgatatg c 21

Claims (6)

It is resistant to alcohol, sugar and sulfurous acid, has alcohol production ability, β-glucosidase activity, tyrosinase inhibitory activity, antioxidant activity, does not produce biogenic amines, harmful enzymes, and skin Saccharomyces cerevisiae FT4-4 strain with an accession number of KCCM12522P to produce a physiologically active substance. The strain according to claim 1, wherein the biogenic amine is histamine and tyramine, the harmful enzyme is urease, and the skin bioactive material is β-glucan. . A composition for producing alcohol comprising the strain of claim 1 or 2, a culture solution thereof, a concentrate of the culture solution, or a dried product thereof as an active ingredient. The composition according to claim 3, wherein the alcohol is berry wine. A cosmetic composition for skin whitening comprising the strain of claim 1 or 2, a culture solution thereof, a concentrate of the culture solution, or a dried product thereof as an active ingredient. A starter composition for producing a fermented food comprising the strain of claim 1 or 2, a culture solution thereof, a concentrate of the culture solution, or a dried product thereof as an active ingredient.

Images