KR101599271B1 - Saccharomyces cerevisiae BA42 strain producing alcohol and nonproducing biogenic amine isolated from berry and uses therof - Google Patents

Abstract

The present invention relates to a strain Saccharomyces cerevisiae BA42 (KCCM11509P) which produces an alcohol isolated from berries and does not produce a biogenic amine, and a strain for producing alcohol containing the strain or a culture thereof as an active ingredient With respect to the composition, it is possible to provide a safe bergermillant wine that does not contain a biogenic amine by using the strain of the present invention.

Description

Saccharomyces cerevisiae BA42 strain, which produces alcohol isolated from berries and does not produce biogenic amines, and uses thereof for producing bacterium from Saccharomyces cerevisiae BA42 strain and nonproducing biogenic amines from berry and uses therof,

The present invention relates to a strain Saccharomyces cerevisiae BA42 which produces alcohols isolated from berries and does not produce biogenic amines, and more particularly to a method for producing alcohols isolated from berries, Saccharomyces cerevisiae strain BA42 (KCCM11509P) which does not produce a biogenic amine and a composition for producing alcohol containing the strain or a culture solution thereof as an active ingredient.

Since ancient times, Korea has developed fermentation technology and aging technology which are used for the production of food, and accordingly, traditional fermentation technology is being continuously developed. Traditional berries are divided into traditional spirits and traditional fermented wines depending on the method of production. In particular, traditional fermented wines have unique flavors and aromas depending on the ingredients added, and they contain unique functionalities.

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 A health-functional fermented beverage has been produced which emphasizes the efficacy and characteristics of by-products produced during the fermentation process.

Biogenic amine (BA), one of the products produced by fermentation microorganisms during fermentation, is responsible for the physiological functions of growth regulation, inflammation regulation and neurotransmission in the human body. However, biogenic amines Adverse effects occur when the amine is ingested through food. Therefore, in fermentation, selection of fermentation strains that do not produce biogenic amines during the fermentation process is very important.

Accordingly, in the present invention, a fermentation strain suitable for producing wine using berries such as blueberry, audi, and bokbunja as a material was developed.

Korean Patent No. 1166489 discloses a fermented yeast Saccharomyces cerevisiae 183-2 and a fermented yeast prepared using the same. Korean Patent Publication No. 2009-0074855 discloses a fermented yeast Saccharomyces cerevisiae 98 -2-A 1122 and a method for producing a fermented liquor using the same. However, the strain Saccharomyces cerevisiae BA42 which does not produce a biogenic amine by producing an alcohol separated from the berries of the present invention and its use There is no description.

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above-mentioned needs, and it is an object of the present invention to provide a fermentation strain suitable for wine production by separating Saccharomyces cerevisiae BA42 strain, which produces alcohol from berries fruit and does not produce a biogenic amine The purpose is to provide.

In order to solve the above problems, the present invention provides Saccharomyces cerevisiae strain BA42 (KCCM11509P) which produces alcohol isolated from berries and does not produce biogenic amines.

The present invention also provides a composition for producing an alcohol comprising the strain or a culture solution thereof as an active ingredient.

In the present invention, three Levy jiae My process as a separate saccharide from fruit berries (Saccharomyces cerevisiae strain BA42 produced alcohol and did not produce biogenic amine. It is expected that the present invention can be used industrially because it is possible to provide a safe wine free from biogenic amines using the Saccharomyces cerevisiae BA42 strain of the present invention.

FIG. 1 shows the result of investigating the strain growth rate and alcohol production ability according to the incubation time of strain BA42. ▲; Cell mass of yeast,?; Ethanol concentration, O; Glucose concentration, ●; Absorbance.
Fig. 2 shows the results of investigating resistance of BA42 strain to different concentrations of alcohol, sugar, and sulfurous acid. A; Alcohol tolerance, B; Sugar tolerance, C; Sulfurous acid tolerance.

In order to achieve the object of the present invention, there is provided Saccharomyces cerevisiae strain BA42 (KCCM11509P) which produces alcohol isolated from berries and does not produce biogenic amines.

The bacterium Saccharomyces cerevisiae BA42 according to the present invention produced alcohols from strains isolated from about 20 kinds of bacterium, blueberry and olive fruit, and produced berry wine, blueberry wine and blueberry wine, Since it does not produce amines, it has been selected as the most suitable strain as a fermentation strain for producing berries.

The selected strain BA42 was subjected to sequencing analysis of the API 20 C AUX yeast identification kit and the 18S rRNA region. As a result, the strain BA42 of the present invention was identified as Saccharomyces cerevisiae S. cerevisiae ).

The strain Saccharomyces cerevisiae BA42 of the present invention was deposited at the Korean Microorganism Conservation Center on Jan. 22, 2014 (Accession No .: KCCM11509P).

In a strain according to an embodiment of the present invention, the biogenic amine is selected from the group consisting of histamine, putrescine, cadaverine, spermidine, spermine, tryptamine, , Hiseamine, 2-phenylethylamine, tyramine, serotonin, L-norepinephrine or dopamine, preferably histamine , Tyramine, putrescine or cadaverine, but is not limited thereto.

The present invention also provides a composition for producing an alcohol comprising the strain or a culture solution thereof as an active ingredient.

In the composition according to an embodiment of the present invention, the alcohol may be an alcohol fermented with cereal or fruit, and most preferably, fermented berries may be used, but the present invention is not limited thereto.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and methods

Yeast isolation and culture conditions

In order to isolate various yeast, 20 kinds of bokbunja, blueberry, and oodi fruit were purchased from Sunchang, Jeongeup and Gochang in Cholla Province. 10 g of fruit was put into 100 ml of sterilized physiological saline and diluted by deciduous method. Then, YPD (yeast extract 1%, peptone 2%, glucose 3%) containing 100 쨉 g / ml of chloramphenicol was dissolved in a solid medium And cultured at 30 DEG C for 2 days.

gas Production capacity  Isolation of yeast having

Gas production capacity was confirmed by Duraham test. Yeasts were cultured in YPD liquid medium at 30 ℃ for 24 hours for culture of selected strains. 2% of the culture was inoculated into a duramm tube containing 5 ml of YPD liquid medium containing 20% glucose. All Duraham test tubes were then incubated for 48 hours at 30 ° C without agitation, and after 48 hours gas production was confirmed. The supernatant was collected by centrifugation and the supernatant was analyzed for its ability to produce alcohol by HPLC according to the conditions shown in Table 1.

HPLC analysis conditions for sugar and alcohol analysis Instrument Agilent 1200 series
(Agilent Technologies, Santa Clara, CA, USA) Column Aminex HPX 87H column, 300 x 7.8 mm
(Bio-Rad, Hercules, Calif., USA) Detector RID detector Eluant 5mM sulfuric acid in H ₂ O Flow rate 0.6 ml / min Temperature 65 ℃ Injection volume 10 μl

Biogenic  Check for amine production

Tramine, histamine, putrescine, and cadaverine were analyzed by HPLC. Sample preparation and extraction for the assay were performed according to the method of Moret and Conte (1995, J. of Chromatography A, 718: 309-317) and analyzed for tyramine, histamine, putrescine, cadaverine, dansyl chloride and 1,7-diaminoheptane was purchased from Sigma-Aldrich, and proline, ether and hydrochloric acid were used. Acetonitrile and water were purchased from J.T Baker. Standard samples were prepared by dissolving in 0.1 N hydrochloric acid to be about 1 g / l, and then diluted with four standard solutions at a concentration of 0.1 to 100 mg / l to prepare calibration curves. For the analysis of biogenic amines, the selected strains were inoculated into a YPD liquid medium containing 100 ml of 24% glucose and cultured at 30 ° C for 5 days to prepare Audi wines, bokbunja wines and blueberry wines. 10 ml of 0.1 N hydrochloric acid solution was added to 10 ml of the wine sample, and 1 ml of each of the sample solution and the standard solution was taken in a test tube and 0.5 ml of 1,7-diaminoheptane (0.1 g / l) was added thereto. 0.5 ml of a saturated sodium carbonate solution and 1 ml of 1% dansyl chloride acetone solution were added and mixed, and the mixture was terminated at 45 ° C for 1 hour. After derivatization, 1 ml of 10% proline solution was added to remove excess dynechyl chloride, 5 ml of ether was added to the test tube and shaken for 3 minutes, and the supernatant was taken. The residue was completely dissolved in 2 ml of acetonitrile, filtered through a 0.45 쨉 l filter, and analyzed by HPLC under the same conditions as in Table 2.

HPLC analysis conditions for biogenic amine analysis 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 to 10 minutes
A: B = 35:65, 10-15 minutes
A: B = 20:80, 15-20 minutes
A: B = 10: 90, 20 to 30 minutes
A: B = 10: 90, more than 40 minutes Flow rate 1.0 ml / min Temperature 40 ℃ Injection volume 20 쨉 l

Identification of yeast

Finally, yeast BA42 strain suitable for berries wine was selected from among the selected strains. To identify the selected strains, the sugar availability was analyzed using API 20 C AUX yeast confirmation kit, and 18S rRNA gene was analyzed by DNA extraction It was commissioned to Cosmojin Tech and identified.

Cell growth and alcohols Production capacity  Research

In order to investigate alcohol production ability, 2% strain culture medium was inoculated into a YPD liquid medium containing 24% glucose and cultured at 30 ° C and 200 rpm for 24 hours. In order to investigate the alcohol production ability over time, samples were taken every 2 hours to investigate dry cell weight, alcohol production and sugar consumption. All experiments were repeated three times and the average value was used.

For the measurement of dry cell mass, 10 ml of the culture was centrifuged at 13,000 rpm for 20 minutes, washed three times in distilled water, dried at 80 ° C until it reached constant weight, and then weighed. 1 ml of the culture broth was recovered, centrifuged at 13,000 rpm for 20 minutes, washed three times in distilled water, resuspended in 1 ml of sterilized distilled water, and analyzed by ultraviolet / visible spectrophotometer (UV / VIS spectrophotometer) SPECORD 200, Analytic jeca). For the investigation of alcohol and sugar consumption, the supernatant was analyzed using HPLC under the conditions shown in Table 1. All the reagents necessary for the analysis were purchased from Sigma-Aldrich.

Alcohol tolerance, sugar tolerance, sulfurous acid tolerance

To investigate the alcohol tolerance of S. cerevisiae strain BA42, 0, 8, 14, and 20% anhydrous ethanol was added to the YPD broth immediately after inoculation with yeast. Dry cell mass was cultured at 30 ° C for 72 hours. The sugar tolerance was investigated by measuring the amount of dried cells after culturing the yeast in YPD liquid medium containing 3, 30, 40, and 50% glucose at 30 ° C for 48 hours. The sulfurous acid tolerance was determined by culturing the cells in a YPD liquid medium supplemented with 100, 200 and 300 ppm potassium metabisulfite at 30 DEG C for 48 hours.

Example  One. Berry  Isolation of Yeast from Fruit

For isolation of yeast suitable for fermentation of berries, yeast strains were isolated from berries. The isolated strains were stored at 4 ℃ for subsequent study.

Example  2. Gas Production capacity  Selection of Yeast

Experiments were carried out using selective strains. The gas formation in the Durham test tube was observed after incubation at 30 ° C for 48 hours. All culture supernatants were used to investigate the formation of gas, and the strains forming the gas were firstly selected. However, according to the report of JP Van Dijken et al. (1986, Yeast, 2: 123-127) that yeast produces alcohol without recognizable gas-forming ability, all culture supernatants were further investigated for alcohol production using HPLC. Finally, six yeasts that produce alcohol were selected.

Example  3. Biogenic  Amine Non-production  Selection of Yeast

Biogenic amines exist in beverages such as wine and beer, fermented foods, and also in grapes and grape juice itself. It may also be formed by yeast during alcohol fermentation. Studies have shown that when bioenergic amines are present in foods, symptoms such as nausea, vomiting, hypertension, and headaches can occur in the body of the food. Onan A. (2007, Food Chemistry, 103: 1475-1486) detected biogenic amines in wine. Based on the detection of six types of yeast, we selected the odin wines, bokbunja wines and blueberry wines And histamine, tyramine, putrescine and cadaverine were investigated. The results are shown in Table 3, and all BA42 strains were selected as strains suitable for the production of berries, because no biogenic amines were detected in all wines.

Biogenic Amine Content of Wines Made from Selected Strains wine
Kinds Strain Class name Biogenic amine concentration (mg / l) Tyramine Histamine Putrescine Cadaverine Audi
wine Control ^a - ND ^d 22.47 ± 0.02 NQ ^e ND ^d BA29 S. cerevisiae NQ ^e 2.3 ± 0.01 ND ^d ND ^d BA31 S. cerevisiae NQ ^e 2.45 ± 0.02 ND ^d ND ^d BA32 S. cerevisiae NQ ^e 1.8 ± 0.03 NQ ^e ND ^d BA33 S. cerevisiae ND ^d 1.55 ± 0.01 ND ^d ND ^d BA34 S. cerevisiae ND ^d 2.337 + 0.02 NQ ^e ND ^d BA42 S. cerevisiae ND ^d ND ^d ND ^d ND ^d Bokbunja
wine Control ^b - ND ^d 4.20 ± 0.01 ND ^d NQ ^e BA29 S. cerevisiae ND ^d NQ ^e ND ^d ND ^d BA31 S. cerevisiae ND ^d ND ^d ND ^d 1.04 + 0.11 BA32 S. cerevisiae NQ ^e ND ^d ND ^d NQ ^e BA33 S. cerevisiae NQ ^e NQ ^e ND ^d ND ^d BA34 S. cerevisiae ND ^d NQ ^e ND ^d ND ^d BA42 S. cerevisiae ND ^d ND ^d ND ^d ND ^d blue
Berry
wine Control ^c - ND ^d ND ^d 6.92 + 0.03 ND ^d BA29 S. cerevisiae ND ^d ND ^d ND ^d ND ^d BA31 S. cerevisiae ND ^d ND ^d ND ^d ND ^d BA32 S. cerevisiae ND ^d 1.25 + 0.02 ND ^d ND ^d BA33 S. cerevisiae ND ^d 1.21 ± 0.03 ND ^d ND ^d BA34 S. cerevisiae ND ^d 1.13 + 0.02 ND ^d ND ^d BA42 S. cerevisiae ND ^d ND ^d ND ^d ND ^d

^a Oddi juice itself

^b Bokbunja juice liquid itself

^c Blueberry juice itself

^{d not} detected

^e Not quantified

Example  4. Identification of yeast

The availability of the carbon source of the screening strain BA42 was examined using API 20 C AUX, and the results of analysis of the BA42 strain using S. cerevisiae were shown in Table 4. The gene was amplified using ITS1 (5'-TCCGTAGGTGAACCTGCGG-3 '; SEQ ID NO: 2) and ITS4 primer (5'-TCCTCCGCTTATTGATATGC-3'; SEQ ID NO: 3) rRNA (SEQ ID NO: 1) was analyzed and identified as the same Saccharomyces cerevisiae as API 20 C AUX.

Carbon availability of BA42 strain using API 20 C AUX Carbon source Strain BA42 Carbon source Strain BA42 Control - Methyl-aD-glucopyranoside - D-Glucose + N-Acetyl-glucosamine - Glycerol - D-Cellobiose - Calcium 2-keto-gluconate - D-Lactose (bovine origin) - L-Arabinose - D-Maltose + D-Xylose - Sucrose + Adonitol - D-Trehalose + Xylitol - D-Melezitose - D-Galactose + D-Raffinose + Inositol - Pseudohyphae - D-Sorbitol -

Example  5. Cell growth and alcohol Efficacy

In order to investigate alcohol efficacy and cell growth, BA42 strain was cultured in YPD liquid medium supplemented with 24% glucose. As a result, when the culture was incubated for 29 hours, 13.511% of maximum alcohol was produced and then gradually decreased. Glucose was completely consumed in 29 hours, and dry cell mass was increased to 24.1 g / ℓ in 33 hours (Fig. 1), which was about three times higher than that in the basic growth medium (8.23 g / ℓ).

Example  6. Alcohol tolerance, sugar tolerance and sulfurous acid resistance

Seed culture of S. cerevisiae strains is commonly used due to the production of aromatic fragrance components and an increase in sulfurous acid and alcohol tolerance. Therefore, alcohol, sugar, and sulfurous acidity are essential ingredients for wine making by adding high concentration of glucose, alcohol, and sulfite in the production of grape wines. Therefore, alcohol, sugar and sulfurous acid tolerance of selected strains were investigated to confirm the possibility of yeast for the production of berries. To investigate the alcohol resistance of BA42 strain, 0, 8, 14, and 20% of absolute ethanol was added to YPD liquid culture medium, which is a general yeast culture medium, and the amount of dried cells was examined to confirm tolerance. The dry cell mass was the same as in Fig. 2A, and it did not grow in all alcoholic beverages. The results indicate that the final biomass concentration is low when the initial concentrations of glucose and alcohol reported by B. Ortiz-Muniz et al. (2010, J. of Chemical Technology and Biotechnology, 85 (10): 1361-1367) Similar results were obtained. Compared with other literature on alcohol tolerance, however, it did not show a significant difference from other studies because it did not grow well at concentrations above 8%.

The sugar tolerance was as shown in FIG. 2B, and it was greatly increased to 17.2533 g / L in the experiment where 30% glucose was added compared with the amount of YPD (8.6033 g / L) in the yeast culture medium. (4) or more of sugars as proposed by Calado et al. (2003, J. Biosci. Bioeng., 96: 141-148) and Kim et al. (2006, J. Biotechnol. Bioeng., 21 (6): 479-483) The results were different from the results of low cell size and low alcohol production. On the other hand, in the experimental group added with 40% and 50% glucose, it was decreased to 6.69 g / ℓ and 0.11 g / ℓ, but it was confirmed that the growth was not significantly affected even in the case of 40% glucose added. However, the decrease of cell growth rate with addition of more than 50% glucose was analyzed because the decrease of the biomass production and the decrease of the dry cell mass due to the difficulty of water circulation in the culture medium due to the addition of high concentration of sugar.

Sulfurous acid is added to the preservation of foods, beverages and medicines. It is used for antioxidant, antibacterial and browning prevention and is used for prevention of oxidation and selective harmful microorganisms in wine production. Therefore, it is necessary for yeast to have sulfurous acid resistance for wine production. The tolerance was determined by adding sulfurous acid at 100, 200 and 300 ppm according to the standard of addition of 350 ㎎ / ℓ in the domestic food standard, and the result was as shown in FIG. 2C. According to the addition of sulfurous acid, the BA42 strain was 8.6033 g / As a result, it can be confirmed that it can be used as a yeast for the production of berries, since it is not influenced by 6.5733 g / l, 8.23 g / l, and 7.8267 g / l.

Korea Microorganism Conservation Center (overseas) KCCM11509P 20140122

<110> Sunchang Research Center for Fermentation Microbes (SRCM) <120> Saccharomyces cerevisiae BA42 strain producing alcohol and          nonproducing biogenic amine isolated from berry and uses therof <130> PN14031 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 815 <212> DNA <213> Saccharomyces cerevisiae <400> 1 cggaaggatc attaaagaaa tttaataatt ttgaaaatgg attttttttg ttttggcaag 60 agcatgagag cttttactgg gcaagaagac aagagatgga gagtccagcc gggcctgcgc 120 ttaagtgcgc ggtcttgcta ggcttgtaag tttctttctt gctattccaa acggtgagag 180 atttctgtgc ttttgttata ggacaattaa aaccgtttca atacaacaca ctgtggagtt 240 ttcatatctt tgcaactttt tctttgggca ttcgagcaat cggggcccag aggtaacaaa 300 cacaaacaat tttatttatt cattaaattt ttgtcaaaaa caagaatttt cgtaactgga 360 aattttaaaa tattaaaaac tttcaacaac ggatctcttg gttctcgcat cgatgaagaa 420 cgcagcgaaa tgcgatacgt aatgtgaatt gcagaattcc gtgaatcatc gaatctttga 480 acgcacattg cgccccttgg tattccaggg ggcatgcctg tttgagcgtc atttccttct 540 caaacattct gtttggtagt gagtgatact ctttggagtt aacttgaaat tgctggcctt 600 ttcattggat gttttttttt ccaaagagag gtttctctgc gtgcttgagg tataatgcaa 660 gtacggtcgt tttaggtttt accaactgcg gctaatcttt tttatactga gcgtattgga 720 acgttatcga taagaagaga gcgtctaggc gaacaatgtt cttaaagttt gacctcaaat 780 caggtaggag tacccgctga acttaagcat atcaa 815 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 tccgtaggtg aacctgcgg 19 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 tcctccgctt attgatatgc 20

Claims (4)

Saccharomyces cerevisiae strain BA42 (KCCM11509P), which produces alcohol isolated from berries and does not produce histamine or putrescine. delete A composition for producing an alcohol comprising the strain of claim 1 or a culture thereof as an active ingredient. 4. The composition of claim 3, wherein the alcohol is a Berry wine.

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