KR101599270B1 - Saccharomyces cerevisiae BA33 strain producing alcohol and nonproducing biogenic amine isolated from Mulberry and uses therof - Google Patents

Abstract

The present invention relates to a strain Saccharomyces cerevisiae BA33 (KCCM11508P) which produces an alcohol isolated from an ory and does not produce a biogenic amine, and a composition for producing an alcohol comprising the strain or a culture thereof as an active ingredient , It is possible to provide a safe oedewine that does not contain a biogenic amine by using the strain of the present invention.

Description

Saccharomyces cerevisiae BA33 strain which does not produce a biosgenic amine and produces alcohols separated from the mulberry and its use {Saccharomyces cerevisiae BA33 strain producing alcohol and nonproducing biogenic amine isolated from Mulberry and uses therof}

The present invention relates to a strain of Saccharomyces cerevisiae BA33 which produces alcohols isolated from an ory and does not produce a biogenic amine, and more particularly to a process for producing an alcohol separated from an ory and producing a biogenic amine Saccharomyces cerevisiae strain BA33 (KCCM11508P), which does not produce a biogenic amine, and a composition for producing an 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, an aim was to develop a fermentation strain suitable for producing an audi wine by using an audi, which has antioxidative effect and is attracting attention as a black food.

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 process for producing a fermented beverage using the same, but the strain Saccharomyces cerevisiae BA33 which does not produce a biogenic amine by producing the alcohol isolated from the present invention and its use has been disclosed There is no bar.

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 the production of an audi wine by separating a strain of Saccharomyces cerevisiae BA33 which 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 BA33 (KCCM11508P) which does not produce a biogenic amine by producing an alcohol separated from an audi.

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, it was confirmed that Saccharomyces cerevisiae strain BA33 isolated from an ory produces alcohol and does not produce a biogenic amine. It is expected that the use of the strain Saccharomyces cerevisiae BA33 according to the present invention can provide a safe oedewine free from biogenic amines and thus can be industrially useful.

Figure 1 shows the result of investigating the strain growth rate and alcohol production ability of the strain BA33 according to the incubation time. ●; Glucose concentration, O; The amount of yeast cells, ▲; Ethanol concentration.
Fig. 2 shows the results of examining resistance of BA33 strain to different concentrations of alcohol, sugar, and sulfurous acid. (A); Alcohol tolerance, (B); Sugar tolerance, (C); Sulfurous acid tolerance.

In order to accomplish the object of the present invention, the present invention provides Saccharomyces cerevisiae strain BA33 (KCCM11508P) which does not produce a biogenic amine by producing an alcohol separated from an oti.

The bacterium Saccharomyces cerevisiae BA33 according to the present invention does not produce the biogenic amine when the alcohol is produced from the strains isolated from the oti and the odi wines are produced. Therefore, the strain is selected as the most suitable strain for the fermentation for the production of the odd wines .

The selected BA33 strain was sequenced by the API 20 C AUX yeast identification kit and the 18S rRNA region. As a result, the BA33 strain of the present invention was identified as Saccharomyces cerevisiae S. cerevisiae ).

The strain Saccharomyces cerevisiae BA33 of the present invention was deposited at the Korean Microorganism Conservation Center on Jan. 22, 2014 (Accession No .: KCCM11508P).

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, it may be an audi fermented with an audi, but 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, 10 kinds of audi were purchased from Sunchang, Jeongeup and Gochang of Cholla province. 10 g of oats were weighed into 100 ml of sterile physiological saline and diluted by decidual method. To inhibit bacterial growth, YPD (yeast extract 1%, peptone 2%, glucose 3%) containing 100 쨉 g / ml of chloramphenicol 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). Tramine, 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 produce an Audi wine. 10 ml of ODW wine was added to 10 ml of 0.1 N hydrochloric acid solution, and 1 ml of this sample solution and standard solution were 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, the yeast BA33 suitable for Audi wine was selected from the screened strains. To identify the selected strains, the sugar availability was analyzed using the API 20 C AUX Yeast Identification Kit, and the 18S rRNA gene was analyzed by extracting DNA It was commissioned by Morgentech 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. 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 BA33, 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 measuring the amount of dried cells in a YPD liquid medium supplemented with 100, 200 and 300 ppm of potassium metabisulfite at 30 DEG C for 48 hours.

Example  One. From Audi  Isolation of yeast

For the isolation of yeast suitable for the fermentation of the wines, yeast strains were isolated from the seeds. The isolated strains were stored at 4 ℃ and used in the following study.

Example  2. Gas Production capacity  Selection of Yeast

Experiments were carried out using more than 300 strains isolated from oedi. The gas formation in Duraham test tubes was observed after culturing at 30 ° C for 48 hours. All culture supernatants were used to investigate the formation of gas, and 110 strains were 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, five yeasts producing more than 5% 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. Onal A. (2007, Food Chemistry, 103: 1475-1486) detected biogenic amines in wine, and we prepared 5 kinds of yeast from selected yeast to produce histidine, tyramine, And cadaverine were investigated. The results were as shown in Table 3. All strains did not produce cadaverine, and tyramine and putrescine were detected in all strains except BA33. However, as a control, . In particular, the BA33 strain showed the lowest histamine content of 1.55 mg / l, which was higher than that of Daeshel et al. (1998, Am. J. Enol. Vitic., 49 (3): 279-282) (1998, Am. J. Enol. Vitic., 49 (3): 266-278) showed a tolerable limit in stability at concentrations lower than 8 to 20 mg / l which have toxicity.

Biogenic Amine Content of Audi Wines Prepared with Selected Strains Strain Class name Biogenic amine concentration (mg / l) Tyramine Histamine Putrescine Cadaverine Control ^a - ND ^b 22.47 ± 0.02 NQ ^c ND ^b BA29 S. cerevisiae NQ ^c 2.3 ± 0.01 ND ^b ND ^b BA31 S. cerevisiae NQ ^c 2.45 ± 0.02 ND ^b ND ^b BA32 S. cerevisiae NQ ^c 1.8 ± 0.03 NQ ^c ND ^b BA33 S. cerevisiae ND ^b 1.55 ± 0.01 ND ^b ND ^b BA34 S. cerevisiae ND ^b 2.337 + 0.02 NQ ^c ND ^b

^a Oddi juice itself

^b Not detected

^c Not quantified

Example  4. Identification of yeast

The availability of the carbon source of the selective strain BA33 was examined by using API 20 C AUX, and the result of BA33 was analyzed by S. cerevisiae . The gene was amplified using the ITS1 primer (5'-TCCGTAGGTGAACCTGCGG-3 '; SEQ ID NO: 2) and the ITS4 primer (5'-TCCTCCGCTTATTGATATGC-3'; SEQ ID NO: 3) to examine the base sequence through molecular diagnosis 18S rRNA (SEQ ID NO: 1) was analyzed and identified as the same Saccharomyces cerevisiae as API 20C AUX.

Carbon source availability of BA33 strain using API 20 C AUX Carbon source Strain BA33 Carbon source Strain BA33 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, BA33 strain was cultured in YPD liquid medium supplemented with 24% glucose. As a result, 12.338% of alcohol was produced when incubated for 25 hours, and the maximum alcohol production capacity was 12.845 %. Glucose was completely consumed at 28 hours, and dry cell mass was about 30 hours (Fig. 1), about twice as much as that of culture (8.33 g / ℓ) in the primary growth medium (19.45 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 were investigated in order to confirm the possibility of yeast for the production of Odihin wine. To investigate the alcohol resistance of BA33 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 dry cell mass was examined to confirm resistance. The amount of dry cell mass was as shown in Fig. 2A. In the case of 8% added cells, the amount of dried cells was decreased to 0.64 g / ℓ compared with that of the control.

The sugar tolerance was as shown in Fig. 2B, and increased from 7.81 g / L to 16.91 g / L in 30% glucose supplemented group compared to that of YPD as a 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, it decreased to 5.73 g / ℓ and 0.12 g / ℓ in 40% and 50% glucose added groups. This was presumably due to the decrease in biomass production and the difficulty of circulating water in the culture medium due to the addition of high concentration of sugar, resulting in a decrease in dry cell mass.

Sulfurous acid is a substance to be added for the preservation of foods, beverages and medicines, and is a substance to be used because of the effects such as antioxidation, antibacterial effect and browning prevention. Therefore, it is used for antioxidant and selective harmful microorganism inhibitory ability in wine production. Therefore, it is necessary for yeast to have sulfurous acid resistance for wine production. The results of the resistance test by adding sulfurous acid of 100, 200 and 300 ppm in accordance with the addition standard of 350 ㎎ / ℓ in domestic food standard are as shown in Fig. 2C. According to the addition of sulfurous acid, BA33 strain is not greatly influenced, It was confirmed that it could be used as a yeast for manufacturing.

Korea Microorganism Conservation Center (overseas) KCCM11508P 20130122

<110> Sunchang Research Center for Fermentation Microbes (SRCM) <120> Saccharomyces cerevisiae BA33 strain producing alcohol and          nonproducing biogenic amine isolated from Mulberry and uses          therof <130> PN14030 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 811 <212> DNA <213> Saccharomyces cerevisiae <400> 1 aaggatcatt aaagaaattt aataattttg aaaatggatt ttttttgttt tggcaagagc 60 atgagagctt ttactgggca agaagacaag agatggagag tccagccggg cctgcgctta 120 agtgcgcggt cttgctaggc ttgtaagttt ctttcttgct attccaaacg gtgagagatt 180 tctgtgcttt tgttatagga caattaaaac cgtttcaata caacacactg tggagttttc 240 atatctttgc aactttttct ttgggcattc gagcaatcgg ggcccagagg taacaaacac 300 aaacaatttt atttattcat taaatttttg tcaaaaacaa gaattttcgt aactggaaat 360 tttaaaatat taaaaacttt caacaacgga tctcttggtt ctcgcatcga tgaagaacgc 420 agcgaaatgc gatacgtaat gtgaattgca gaattccgtg aatcatcgaa tctttgaacg 480 cacattgcgc cccttggtat tccagggggc atgcctgttt gagcgtcatt tccttctcaa 540 acattctgtt tggtagtgag tgatactctt tggagttaac ttgaaattgc tggccttttc 600 attggatgtt tttttttcca aagagaggtt tctctgcgtg cttgaggtat aatgcaagta 660 cggtcgtttt aggttttacc aactgcggct aatctttttt atactgagcg tattggaacg 720 ttatcgataa gaagagagcg tctaggcgaa caatgttctt aaagtttgac ctcaaatcag 780 gtaggagtac ccgctgaact taagcatatc a 811 <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 BA33 strain (KCCM11508P), which produces alcohol isolated from an oti 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 an Audi wine.

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