KR102226310B1 - Saccharomyces cerevisiae FT4-1 strain producing beta-glucan and not producing biogenic amine isolated from blueberry and uses therof - Google Patents

Abstract

The present invention relates to a Saccharomyces cerevisiae FT4-1 strain which is resistant to alcohol, sugar, and sulfurous acid; has beta-glucan producing ability, alcohol producing ability, and beta-glucosidase secretory ability; and does not produce biogenic amine and urease. The Saccharomyces cerevisiae FT4-1 strain is expected to be very usefully used in fermented food-related industries, such as a starter strain for wine production.

Description

Saccharomyces cerevisiae FT4-1 strain producing beta-glucan and not producing biogenic amine isolated from blueberry and uses therof}

The present invention relates to a Saccharomyces cerevisiae FT4-1 strain that produces beta-glucan isolated from blueberries and does not produce biogenic amines, and uses thereof.

Since ancient times, fermentation technology and aging technology used in food production have been developed in Korea, and accordingly, traditional liquor using fermentation technology is continuously being developed. Traditional liquor is largely divided into traditional distilled liquor and traditional fermented liquor according to the manufacturing method. In particular, traditional fermented liquor has a unique taste and aroma depending on the ingredients added, and each contains unique functionality.

There fermentation of traditionalism include saccharide as MY access (Saccharomyces) genus Aspergillus (Aspergillus) genus Bacillus (Bacillus) genus Lactobacillus bacteria (Lactobacillus) in such strains have been used, in recent years, with fermented material such fermentation strain Healthy functional fermented liquor is being produced that emphasizes the efficacy and characteristics of by-products produced during the fermentation process.

Gamma-amino butyric acid (GABA), one of the products produced by fermentation microorganisms during fermentation, is a type of non-proteinaceous amino acid distributed in nature and has a major inhibitory effect on the central nervous system such as the brain and spinal cord. It is known as an inhibitory neurotransmitter. Due to various pharmacological activities such as activating blood flow to the brain and increasing oxygen supply to the brain to promote metabolic function of brain cells, reducing triglycerides in the blood and improving liver function, headaches and ringing in the ears caused by cerebral artery dystrophy , It has been applied to treatments such as decreased motivation. In addition, it has been found to be effective in preventing and improving adult diseases such as hypertension, and interest as a functional food material is increasing, and recently, beverages containing a large amount of gamma-aminobutyric acid have been released.

On the other hand, biogenic amines are responsible for physiological functions such as growth control, inflammation control, and neurotransmission in the human body, but harmful symptoms appear when biogenic amines that exceed the human body's decomposition limit are consumed through food. Therefore, in fermentation, it is very important to select a fermentation strain that does not produce biogenic amines during the fermentation process.

Various microorganisms are involved in alcohol fermentation of berries such as wine, and it is achieved through various actions between microorganisms while repeating complex chemical steps. Many foreign countries use Saccharomyces cerevisiae as a starter strain for wine production to improve the quality of wine. I'm putting a lot of effort into it.

On the other hand, Korean Patent Registration No. 1599271 discloses'Saccharomyces cerevisiae BA42 strain that produces alcohol isolated from berries and does not produce biogenic amines and uses thereof', and Korean Patent No. 1599270 In the'Saccharomyces cerevisiae BA33 strain that produces alcohol isolated from Audi and does not produce biogenic amines and uses thereof' is disclosed, but'beta-glucan isolated from blueberries is produced and , Saccharomyces cerevisiae FT4-1 strain that does not produce biogenic amine and its use has not been disclosed at all.

The present invention was derived from the above requirements, and in the present invention, it has resistance to alcohol, sugar and sulfurous acid from blueberries and its extract, and β-glucan production ability, alcohol production ability and β-glucosida Saccharomyces cerevisiae (Saccharomyces cerevisiae) FT4-1 strain that has the ability to secrete β-glucosidase and does not produce biogenic amines and ureases was isolated. The present invention was completed by confirming that the Saccharomyces cerevisiae FT4-1 strain of the present invention can be used in a variety of industries related to fermented foods such as a starter strain for wine production.

In order to solve the above problems, the present invention has resistance to alcohol, sugar and sulfurous acid, has β-glucan production ability, alcohol production ability and β-glucosidase secretion ability, and is biogenic. It provides a Saccharomyces cerevisae FT4-1 strain (KCCM12519P) that does not produce amines and ureases.

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 starter composition for producing fermented food comprising the strain, a culture solution thereof, a concentrate of the culture solution, or a dried product thereof as an active ingredient.

Saccharomyces cerevisiae FT4-1 strain isolated from the berries of the present invention is resistant to alcohol, sugar and sulfurous acid, and β-glucan production ability, alcohol production ability and β- Since it has the ability to secrete glucosidase (β-glucosidase) and does not produce biogenic amines and ureases, it is expected to be very useful in fermented food related industries such as starter strains for wine production.

Figure 1 shows the nucleotide sequence of the 18S rRNA of the Saccharomyces cerevisae (Saccharomyces cerevisae) FT4-1 strain (KCCM12519P) isolated in the present invention.
Figure 2 shows the schematic diagram of the Saccharomyces cerevisae (Saccharomyces cerevisae) FT4-1 strain (KCCM12519P) isolated in the present invention.
3 is a result of measuring the amount of ethanol produced, the amount of dry cells, and the absorbance according to the culture time of the Saccharomyces cerevisae FT4-1 strain (KCCM12519P) isolated in the present invention. A: As a result of culturing on YPD liquid medium containing 2% glucose, B: As a result of culturing on YPD liquid medium containing 24% glucose, ●: absorbance (660 nm), ■: ethanol content, ○: dry cell weight.
Figure 4 is a result of analysis of ethanol, sugar and sulfurous acid resistance of the Saccharomyces cerevisae (Saccharomyces cerevisae) FT4-1 strain (KCCM12519P) isolated in the present invention. After incubation for 35 hours at 30°C and 200 rpm in YPD medium containing various concentrations of ethanol, sugar and sulfurous acid, the ethanol, sugar and sulfurous acid resistance of Saccharomyces cerevisiae was tested by dry cell weight (g/L) , B and C graphs.

In order to achieve the object of the present invention, the present invention is resistant to alcohol, sugar and sulfurous acid, has β-glucan production ability, alcohol production ability and β-glucosidase secretion ability, It provides a Saccharomyces cerevisae FT4-1 strain (KCCM12519P) that does not produce biogenic amines and ureases.

In the present invention, Saccharomyces cerevisiae strains were isolated from blueberries cultivated and harvested in Sunchang-gun, Jeollabuk-do, and extracts thereof, among which are resistant to alcohol, sugar and sulfurous acid, and β-glucan (β -glucan) production ability, alcohol production ability and β-glucosidase secretion ability, and the Saccharomyces cerevisiae strain, which does not produce biogenic amines and ureases, was isolated and identified. It was named as Cerevisiae FT4-1 strain and deposited with the Korea Microbial Conservation Center on May 03, 2019 (Accession No.: KCCM12519P).

In the strain according to an embodiment of the present invention, the biogenic amine may be histamine or tyramine, but is not limited thereto. The alcohol resistance is resistance to 8-20% ethanol, sugar resistance is resistance to 3-50% glucose, and sulfurous acid resistance is resistance to sulfurous acid of 100 to 300 ppm.

In addition, the present invention provides a composition for alcohol production comprising the strain or a culture medium thereof as an active ingredient.

In the composition according to an embodiment of the present invention, the alcohol may be an alcohol fermented fruits, most preferably wine fermented 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 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 active ingredient may be suitably determined according to the purpose of use thereof.

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

In the present invention, the 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 as dominant species by adding them in the manufacture of fermented foods. In the case of manufacturing food using the food fermentation starter, by the microorganisms included in the food fermentation starter, the quality of the food is constantly controlled, or for a specific purpose, for example, to prevent or reduce off-flavor in food. You can achieve your purpose. In the present invention, by using Saccharomyces cerevisiae FT4-1 strain or a culture solution thereof as a starter strain, β-glucan, alcohol and β-glucosidase are included, and biogenic amines and ureases are Fermented foods that do not contain can be manufactured.

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

Hereinafter, the present invention will be described in detail by examples. 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

Yeast separation and culture conditions

In order to separate various yeasts, blueberries grown and harvested in Sunchang-gun, Jeollabuk-do, and extracts thereof were purchased and used. For enrichment, 5% of blueberry or blueberry extract was added to 250 ml of YPD (yeast extract 1%, peptone 2%, glucose 2%) liquid medium, and fermented at 30° C. for 2 days and used for separation. 10 ml of the enriched blueberry and blueberry extract ferment were homogenized, and then diluted with a decimal method in 90 ml of sterile physiological saline, and YPD (yeast extract 1) containing 100 µg/ml of chloramphenicol to inhibit the growth of bacteria. %, peptone 2%, glucose 3%) were plated on a solid medium and incubated for 2 days at 30°C. In order to separate pure yeast colonies, single colonies were isolated and incubated for 2 days at 30°C in YPD liquid medium. Yeast suitable for fermentation was selected by producing alcohol among yeast strains, mixed with YPD liquid medium containing 20% glycerol, and stored at -80°C. Activation of the strain was used after culturing and activating at 30° C. for 2 days in YPD liquid medium.

Isolation of alcohol fermentable yeast

The gas generation ability was confirmed through the Duram test. For cultivation of the selected strain, the yeast was cultured by stirring at 30° C. for 24 hours in YPD liquid medium. 2% of the culture was inoculated into a Duram tube containing 10 ml of YPD liquid medium containing 20% glucose. Afterwards, all Duram test tubes were incubated at 30°C for 48 hours without agitation, and after 48 hours, the first screening was performed by checking whether gas was produced, and the first screening yeasts were inoculated into YPD liquid medium containing 24% glucose and cultured for 48 hours. After the culture was centrifuged, the culture supernatant was taken, filtered using a 0.45 μm syringe filter, and the alcohol production ability was analyzed using HPLC according to the conditions in Table 1 below.

HPLC analysis conditions used for detection of biogenic amines and ethanol HPLC analysis conditions used for detection of biogenic amines Instrument Agilent 1200 series
(Agilent Technologies, Santa Clara, CA, USA) Column CapcellPak C18 Column Detector DAD detector (254 nm) Mobile phase A: 0.1% formic acid in H ₂ O B: 0.1% formic acid in ACN Gradient condition A:B = 45:55, 0~10 min A:B = 35:65, 10~15 min 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 HPLC analysis conditions used for ethanol detection Instrument Agilent 1200 series
(Agilent Technologies, Santa Clara, CA, USA) Column Aminex HPX 87H column, 300 Х 7.8 mm
(Bio-Rad, Hercules, CA, USA) Detector RID detector Eluant 5 mM Sulfuric acid in H ₂ O Flow rate 0.6 ml/min Temperature 65℃ Injection volume 10 μL

Check whether biogenic amines are produced

To investigate the production of biogenic amines of the selected isolates, after inoculating each isolate in YPD liquid medium, shake culture at 30°C and 150 rpm for 24 hours before incubation. Inoculated into 9 ml of YPD liquid medium containing 0.1%, and cultured with shaking at 30° C. and 150 rpm for 48 hours, followed by main culture. The main culture solution was centrifuged at 13,000 rpm for 30 minutes to remove the cells, and the culture supernatant from which the cells were removed was used as a sample for biogenic amine analysis. After taking 0.5 ml each of the sample solution and the standard solution, 0.25 ml 1,7-diaminoheptane, 0.25 ml saturated Na ₂ CO ₃ solution, 1% acetone and 0.4 ml dansyl chloride were mixed and derivatized at 45° C. for 1 hour. I did. After adding 0.25 ml of 10% proline (Sigma-aldrich) to the derivatized sample, removing the remaining amount of dansyl chloride, adding 2.5 ml of ethyl ether and shaking for 3 minutes, the separated supernatant was taken and evaporated, and the remaining residue was 0.5 ml. It was used for analysis by filtration through a 0.45 μm syringe filter (Sartorius). Equipment analysis conditions for the analysis of the biogenic amine are shown in Table 1 above.

Confirmation of urease production

The production of urease, a harmful enzyme, was confirmed using a urea rapid test kit (MB cell, Korea). The selected strain was inoculated into the urea rapid test kit, and vaseline oil was added to incubate at 37° C. for 24 hours in an oxygen-blocked state, and color changes were observed at 4 hour intervals to confirm whether or not urease was produced.

β-glucosidase enzyme activity

In order to analyze β-glucosidase enzyme activity, the isolate was inoculated into YPD solid medium prepared by adding 1.0% esculin and 0.5% ferric ammonium citrate to 37°C. After incubation for 24 hours, β-glucosidase enzyme activity was investigated according to the presence or absence of black transparent rings around the colonies.

Identification of selected yeast and preparation of phylogenetic tree

Finally, the yeast FT4-1 suitable for fermentation of berries wine was selected from among the selected strains, and DNA was extracted to identify the selected strain, and the 18S rRNA gene was analyzed and identified by requesting Macrogen Co., Ltd. As for the base sequence, the base sequence of the standard strain was secured through the ExTaxone-e server (http://www.eztaxon.org), and a systematic diagram was prepared after mutual comparison between base sequences using the MEGA 6.0 program. For the tree, the neighbor-joining algorithm was used, and the robustness of the generated tree was confirmed by bootstrapping it with 1,000 iterations.

Analysis of β-glucan content in selected yeast

In order to investigate the β-glucan content of the finally selected FT4-1 strain, the cells were cultured with shaking at 30° C. and 150 rpm for 48 hours in YPD liquid medium, and centrifuged at 3,000 rpm with distilled water. Washed three times and freeze-dried samples were used for analysis. The β-glucan content was measured using a yeast β-glucan kit (K-YBGL, Megazyme International Ireland Ltd.).

Cell growth and alcohol production ability of selected yeast

In order to analyze the cell and alcohol production ability according to the culture time of the finally selected FT4-1 strain, the selected yeast was inoculated in a liquid medium and cultured with shaking at 30°C and 150 rpm for 2 days to conduct pre-culture for investigation of alcohol production and cell growth. Implemented. YPD liquid medium containing 24% glucose was used as the medium for the investigation of alcohol production ability, YPD liquid medium containing 2% glucose was used as the medium for cell growth investigation, and 2% of the pre-culture solution was inoculated into each liquid medium. Then, it was incubated at 30° C. and 200 rpm for 36 hours. In order to investigate the cell growth and alcohol production ability according to time, samples were taken every 2 hours and the dry cell mass, alcohol production ability, and absorbance were measured. In order to measure the amount of dried cells, 10 ml of the culture solution was centrifuged at 3,000 rpm for 20 minutes, and the separated cells were washed three times with distilled water, dried at 80° C. to a constant weight, and then the weight was measured. To measure the absorbance for comparison with the amount of dry cells, collect 1 ml of the culture solution, centrifuge at 3,000 rpm for 20 minutes, wash three times in distilled water, resuspend in 1 ml of sterile distilled water, /VIS spectrophotometer, SPECORD 200, Analytik Jena, Germany) was used to measure and record the absorbance. Alcohol production was measured through HPLC analysis under the conditions according to Table 1, and the average value was used for all experimental groups through repeated experiments three times.

Alcohol tolerance, sugar tolerance, sulfurous acid tolerance investigation

In order to investigate the alcohol tolerance of the finally selected FT4-1 strain, 0, 8, 14, and 20% of anhydrous ethanol were added immediately after inoculating the yeast in the YPD liquid medium. The dry cell mass was investigated by incubating at 30° C. for 72 hours. Sugar tolerance was investigated by culturing the yeast for 48 hours at 30° C. in YPD liquid medium containing 3, 30, 40 and 50% glucose, and then measuring the amount of dry cells. Sulfurous acid resistance was incubated at 30° C. for 48 hours in a YPD liquid medium to which 100, 200, and 300 ppm of potassium metabisulfite was added, and the amount of dried cells was measured.

Example 1. Isolation of alcohol fermentable yeast from blueberries and blueberry extract

Various microorganisms are involved in alcohol fermentation of berries such as wine, and it is achieved through various actions between microorganisms while repeating complex chemical steps. In many foreign countries, S. cerevisiae is used as a starter for wine production to improve the quality of wine. A lot of effort is being put into the selection of yeast. Therefore, about 300 kinds of yeast isolates were selected from blueberries and blueberry extracts for the isolation of yeast strains, and the selected isolates were stored at -80°C for the next study.

For the first screening of yeast having excellent alcohol fermentation ability for the selected isolates, gas-generating ability was investigated through a Durham tube test, and strains forming gas were first selected. However, according to reports, most of the yeasts play a role in producing alcohol without any identifiable gas-forming ability, so additionally, HPLC analysis was conducted to determine whether alcohol was produced, and as a result, five yeasts with an alcohol-producing ability of 5% or more were finally selected. (Table 2).

Ethanol production ability of the selected strain Strain Ethanol production ability (%) Commercial yeast (fruit wine) 10.28 ± 0.01 Commercial yeast (grain wine) 10.52 ± 0.05 FT4-1 11.99 ± 0.04 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. Selection of non-biogenic amine-producing yeast

The production of biogenic amines, histamine and tyramine, was investigated for the previously selected five yeasts. As a control, a YPD liquid medium without inoculation of the strain was used, and the results were identified as FT4-1, the strain that did not produce both histamine and tyramine among the final five selected strains, as disclosed in Table 3 below. In the strain of, two kinds of biogenic amines were detected, but were not quantified. Therefore, the FT4-1 strain was selected as a yeast suitable for wine production using berries based on the above results of alcohol fermentation ability and biogenic amine production.

Analysis of biogenic amine production ability of selected strains Biogenic amine Strain FT4-1 FT4-7 FT4-32 FT4-98 FT4-140 0.1% precursor
Containing YPD medium Tyramine N.D. ^a N.Q. N.D. N.Q. 1.25±0.03 Histamine N.D. N.Q. 2.22±0.03 NQ ^b N.D.

The results were measured 3 times and expressed as standard deviation.

^a ND-not detected.

^b NQ-not quantified.

Example 3. Confirmation of urease production

Cell damage is caused by ammonia produced by urease during Helicobacter pylori infection as a mechanism of cell damage, and epithelial cells are vacuolated by vacuolating cytotoxin gene A (Vac A), a secreted toxin of Helicobacter pylori. Upset occurs, and cells are also damaged by inflammatory mediators and free radicals secreted from neutrophils. Damage to epithelial cells causes atrophic change of cells, increases cell proliferation as a compensatory mechanism for cell damage, and cells that proliferate actively have a greater chance of damaging genes by carcinogens. Therefore, an experiment was conducted to determine whether urease was produced, and as a result of the measurement, it was confirmed that urease was not produced in the FT4-1, FT4-7, and FT4-140 strains (Table 4).

Comparison of urease activity for selected yeast strains Strain Urease activity FT4-1 - FT4-7 - FT4-32 + FT4-98 + FT4-140 -

Example 4. β-glucosidase enzyme activity

β-glucosidase is known to act as a catalyst for the formation of non-glycosides and hydrolysis of isoflavone glycosides. Microorganisms that produce β-glucosidase Fungi such as Aspergillus niger , Trichoderma reesei , Fusarium oxysporum and Saccharomyces cerevisiae , Schizocaromyces Pombe ( Schizosaccharoyces pombe ) In yeast and soil bacteria such as Streptomyces retina Cooley (Streptomyces reticuli), Bacillus standing subtilis (Bacillus subtilis), Bacillus poly miksa (Bacillus polymyxa), Bacillus circulator lance (Bacillu circulans), Agrobacterium tyumeo Pacific Enschede (Agrobacterium tumefaciens), and cellulose Pseudomonas species (Cellulomonas sp.) CS1-1 and high temperature property of archaea Pyrococcus Puri's sewage (Pyrococcus furiosus), etc. are known strains belonging to different clade, they produce β- glucosidase for which A number of enzymatic properties and genes were also revealed. As a result of confirming the activity of β-glucosidase, which has the potential to be utilized in the bioconversion process of converting genistine and daidgin, which are mainly present in traditional fermented foods to non-glycoside form for absorption in the body, FT4-1 strain It was confirmed that the activity of β-glucosidase was the most excellent (Table 5).

Comparison of β-glucosidase activity against selected yeast strains Strain β-glucosidase activity FT4-1 +++ FT4-7 - FT4-32 + FT4-98 ++ FT4-140 +

Example 5. Identification of yeast and preparation of phylogenetic tree

For the identification of the selected strain FT4-1, the genes were amplified using NS1 and NS8 primers, and the sequence of the 18S rRNA gene was analyzed. Based on the analysis result, the BLAST search result in GenBank was found to be S. cerevisiae , and the nucleotide sequence is shown in SEQ ID NO: 1 (FIG. 1). The nucleotide sequence was used to perform cross-comparison with the standard strain having high homology in the SeqMatch program. As a result of analyzing the phylogenetic tree by creating a phylogenetic tree based on the 18S rRNA gene sequence, it was found to be the closest closely related to Saccharomyces cerevisiae NRRL Y-12632 (FIG. 2). The finally selected strain was named Saccharomyces cerevisiae FT4-1 strain, and was deposited with the Korea Microbial Conservation Center (KCCM) on May 03, 2019, and was given the deposit number KCCM12519P.

Example 6. β-glucan content analysis

One of the skin physiologically active substances, β-glucan, is a homogeneous polysaccharide in which glucose is linked by β-(1-3)-D-glycoside bonds, and yeast is the largest component of the yeast cell wall. It is extracted from the cell wall. β-glucan has been reported to have various physiological activities promoting effects such as anti-cancer, anti-cholesterol, immunity enhancement and skin regeneration, and thus its use as a health food additive is increasing. In particular, β-glucan obtained from yeast protects the skin and plays a role in regenerating the skin in case of damage, and is known to play an important role in strengthening the immune system in the human body. In addition, β-glucan having a molecular structure of β-(1,3)-/β(1-6)-bonds than that of β-(1,3)- or β-(1,6)- bonds It is known to be excellent in promoting physiological activity. As a result of measuring the β-glucan content of yeast cells produced in Saccharomyces cerevisiae FT4-1 strain, it was found to be 21.76 mg per dry cell weight (g) (Table 6).

Analysis of β-glucan content for selected yeast strains Strain β-glucan content (mg/g of dry cell weight) FT4-1 21.76

Example 7. Cell growth and alcohol fermentation ability of the selected strain

When the Saccharomyces cerevisiae FT4-1 strain was cultured in YPD liquid medium containing 2% glucose, changes in ethanol production amount, absorbance, and dried cell weight according to culture time were investigated. As a result, as disclosed in Fig. 3A, the log phase of the cell growth phase was 4 to 12 hours and the stationary phase was 16 to 20 hours. During the incubation period, the cell mass increased to 3.9183 g/L at the maximum at 24 hours, and thereafter, the cell mass showed a death phase in the form of gradually increasing or decreasing. In the case of absorbance, the same form as the cell mass result was also shown. The alcohol content during the incubation period showed the highest alcohol production amount (0.8324%) in 20 hours, and the alcohol production amount basically depends on the amount of alcohol produced by the carbon source during the sugar metabolism process, so it contains at least 2% glucose. It is judged that the medium did not show high levels. Therefore, in the case of alcohol fermentation ability, it was investigated using YPD liquid medium added with 24% glucose, which was reported to produce 11 mg/mL of alcohol. As a result, alcohol produced at 32 hours during the incubation period was 11.4424%, showing the maximum alcohol production, and then gradually decreased. The amount of cells exhibited a cell weight of 11.9956 g/L, which was about 3 times higher than when cultured in the basic growth medium (YPD medium containing 2% glucose) (3.9183 g/L) (Fig. 3B).

Example 8. Alcohol resistance, sugar resistance and sulfurous acid resistance of selected strains

The seed culture of the Saccharomyces cerevisiae strain is a representative alcohol fermentation strain generally used due to the production of aromatic fragrance components and increased tolerance to sulfurous acid and alcohol. Resistance to alcohol, sugar, and sulfurous acid is an essential factor as a yeast for wine production, as high concentrations of glucose, alcohol, and sulfurous acid are added during the production of grape wine. Therefore, in order to confirm the possibility as a yeast for wine production using berries, the alcohol, sugar and sulfurous acid resistance of the selected strains were investigated. In order to investigate the alcohol tolerance of the FT4-1 strain, 0, 8, 14 and 20% of absolute ethanol were added to YPD liquid medium, which is a general yeast culture medium, respectively, and the amount of dry cells was investigated to confirm resistance. As a result, as disclosed in FIG. 4A, it was confirmed that the amount of dry cells was reduced to 0.51 g/L compared to the control (control, 4.96 g/L) in the experimental group to which 8% of ethanol was added, but it was viable. Almost did not grow. This confirmed a result similar to the results of previous studies that the final biomass concentration was low when the initial glucose and alcohol concentration was high. However, even when compared with other literature on alcohol tolerance, it did not grow well at concentrations above 8%, so there was no significant difference from other studies.

As a result of glucose tolerance, in the experimental group with 30% glucose added to the control group (control, 4.34 g/L), the cell mass increased approximately 2 times to 10.37 g/L, and the experimental group with 40% and 50% glucose added. Also, it was confirmed that the FT4-1 strain was excellent in glucose tolerance by showing an increase of 8.40 g/L and 5.94 g/L compared to the control (FIG. 4B).

Sulfuric acid is a substance added for preservation of foods, beverages and medicines, and exhibits antioxidant, antibacterial and browning effects. Therefore, since it is used for preventing oxidation and inhibiting selective harmful microorganisms in wine production, there is a need for yeast to have sulfurous acid resistance for wine production. Sulfurous acid resistance of FT4-1 strain was investigated by adding 100, 200 and 300 ppm of sulfurous acid according to the current domestic food standard of 350mg/L. As a result, it was confirmed that the FT4-1 strain was not significantly affected by the addition of sulfurous acid, thereby confirming that it was a yeast having excellent resistance to sulfurous acid (FIG. 4C).

Korea Microorganism Conservation Center (overseas) KCCM12519P 20190503

<110> Microbial Institute for Fermentation Industyry <120> Saccharomyces cerevisiae FT4-1 strain producing beta-glucan and not producing biogenic amine isolated from blueberry and uses therof <130> PN19263 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1688 <212> DNA <213> Saccharomyces cerevisiae <400> 1 tgtataagca atttatacag tgaaactgcg aatggctcat taaatcagtt atcgtttatt 60 tgatagttcc tttactacat ggtataactg tggtaattct agagctaata catgcttaaa 120 atctcgaccc tttggaagag atgtatttat tagataaaaa atcaatgtct tcggactctt 180 tgatgattca taataacttt tcgaatcgca tggccttgtg ctggcgatgg ttcattcaaa 240 tttctgccct atcaactttc gatggtagga tagtggccta ccatggtttc aacgggtaac 300 ggggaataag ggttcgattc cggagaggga gcctgagaaa cggctaccac atccaaggaa 360 ggcagcaggc gcgcaaatta cccaatccta attcagggag gtagtgacaa taaataacga 420 tacagggccc attcgggtct tgtaattgga atgagtacaa tgtaaatacc ttaacgagga 480 acaattggag ggcaagtctg gtgccagcag ccgcggtaat tccagctcca atagcgtata 540 ttaaagttgt tgcagttaaa aagctcgtag ttgaactttg ggcccggttg gccggtccga 600 ttttttcgtg tactggattt ccaacggggc ctttccttct ggctaacctt gagtccttgt 660 ggctcttggc gaaccaggac ttttactttg aaaaaattag agtgttcaaa gcaggcgtat 720 tgctcgaata tattagcatg gaataataga ataggacgtt tggttctatt ttgttggttt 780 ctaggaccat cgtaatgatt aatagggacg gtcgggggca tcagtattca attgtcagag 840 gtgaaattct tggatttatt gaagactaac tactgcgaaa gcatttgcca aggacgtttt 900 cattaatcaa gaacgaaagt taggggatcg aagatgatca gataccgtcg tagtcttaac 960 cataaactat gccgactagg gatcgggtgg tgttttttta atgacccact cggcacctta 1020 cgagaaatca aagtctttgg gttctggggg gagtatggtc gcaaggctga aacttaaagg 1080 aattgacgga agggcaccac caggagtgga gcctgcggct taatttgact caacacgggg 1140 aaactcacca ggtccagaca caataaggat tgacagattg agagctcttt cttgattttg 1200 tgggtggtgg tgcatggccg ttcttagttg gtggagtgat ttgtctgctt aattgcgata 1260 acgaacgaga ccttaaccta ctaaatagtg gtgctagcat ttgctggtta tccacttctt 1320 agagggacta tcggtttcaa gccgatggaa gtttgaggca ataacaggtc tgtgatgccc 1380 ttagacgttc tgggccgcac gcgcgctaca ctgacggagc cagcgagtct aaccttggcc 1440 gagaggtctt ggtaatcttg tgaaactccg tcgtgctggg gatagagcat tgtaattatt 1500 gctcttcaac gaggaattcc tagtaagcgc aagtcatcag cttgcgttga ttacgtccct 1560 gccctttgta cacaccgccc gtcgctagta ccgattgaat ggcttagtga ggcctcagga 1620 tctgcttaga gaagggggca actccatctc agagcggaga attggacgtc ccaacccata 1680 aggattga 1688

Claims (5)

It is resistant to alcohol, sugar and sulfurous acid, has β-glucan production ability, alcohol production ability and β-glucosidase secretion ability, and is biogenic of histamine and tyramine. Saccharomyces cerevisae FT4-1 strain (KCCM12519P) that does not produce amines and ureases. 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. The composition of claim 3, wherein the alcohol is a berry wine. A starter composition for producing fermented food 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.

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