KR101825957B1 - Saccharomyces cerevisiae Y297 with adaptability at low temperature and manufacturing method of fermented alchoholic beverage using the same - Google Patents

Abstract

The present invention relates to a new yeast Saccharomyces cerevisiae Y297 deposited with the deposit number KACC93244P and a fermentation liquor produced using the same. Specifically, among 500 fermentation strains, growth is carried out at a low temperature of 15 ° C Y297 was selected as an excellent yeast, Saccharomyces cerevisiae. The strain was found to have excellent low-temperature tolerance and alcohol-producing ability, and the activity of glucosidase and esterase of Y297 yeast was compared with that of yeast It was confirmed that the quality of the fermented wine was 2 to 5 times better than that of the commercial yeast. As a result of confirming the quality characteristics of the fermented wine, the acidity was low, the alcohol content was high, the organic acid content was high and the free amino acid content was high. Y297 yeast can be used to produce high quality fermented beverage with excellent flavor, sweetness and aroma.

Description

Y297 and a fermented juice prepared by using the fermented yeast Saccharomyces cerevisiae Y297 with adaptability at low temperature and manufacturing method of fermented alchoholic beverage using the same,

The present invention relates to a novel Saccharomyces cerevisiae strain Y297 deposited with the deposit number KACC93244P, a strain or a culture thereof, a fermentation liquor using the same and a method for producing the same.

A traditional fermented beverage in Korea is a type of fermented beverage that contains various components such as alcohol as a result of decomposition of carbohydrates by microorganisms. Examples of fermentation sources are cereals based on starch, fruits such as potatoes and sugar-based fruits, molasses do.

Yakju and Takju as fermented beans are traditionally made by using the cereals and yeast, and they are made from jade fermentation. After brewing, the sake is sieved and the appearance is opaque is called makgeolli or takju. .

Generally, after immersion, the raw material component is decomposed and fermented by an enzyme action by the microorganism in the yeast, so that the alcohol concentration becomes 12-15%, and water is added thereto to prepare Takju with an alcohol concentration of 6-8%. Takju contains nutrients such as lysine, leucine, glutamic acid, proline and glutathion which are essential amino acids including vitamin B group. Organic acid which is involved in acidity is effective in relieving thirst and facilitating metabolism.

In the process of manufacturing Yakju, yeast is important not only directly involved in the fermentation process that produces alcohol but also forming the quality of Yakju, such as producing various metabolites and volatile flavor components (Baek SY, et al. , Microbiol Biotechnol Lett, 2015, 43, 1-9, Lee SR., Et al., 1985, Ewha women's university press, p224-294, Shin KR, et al., 1999, J Korean Soc Food Sci Nutr, 28, 794-800 ). However, in the case of Saccharomyces cerevisiae , which is one of the major yeasts in the manufacture of Yakju, the properties such as protein transcription, cell membrane fluidity, and enzyme activity are also affected by low temperatures, It can also cause bad effects such as fermentation. Therefore, if fermentation can be performed using a yeast having excellent adaptability to low temperature, it will be possible to produce a better quality of the Yakju.

The temperature during the fermentation process is known to be an important factor in producing the volatile flavor components that influence the quality of the wine. In the case of wine production, low temperature fermentation (10 ~ 15 ℃) (Beltran G., et al., 2007, World J Microbiol Biotechnol, 23, 809-815, Chiva R., et al., 2012, FEMS Yeast Res, 831-843 , Torija MJ., Et al., 2003, Int J Food Microbiol, 80, 47-53). It is also known that fermentation at temperatures between 10 and 12 ° C increases ester production by more than 75% (Engan S., 1977, 16 ^th European brewery convention congress, Amsterdam, Netherlands, 591-607).

Ester, which is formed by the combination of organic acid and alcohol, is known as a flavor ingredient of various foods, and is used as a main index of flavor ingredient in fermented beverages. In the case of wines, the production of esters is achieved by the interaction of hydrolysis with esterase (EC 3.1.1.1) and synthetic reactions involving alcohol acetyltransferase (EC 2.3.1.84). Several studies have reported that the Saccharomyces sp. Strain has genes involved in ester synthesis and hydrolysis.

Korean Patent Laid-open Publication No. 10-2004-0117468 discloses a novel lactic acid bacterium and a method for producing a fermented liquor using the lactic acid bacterium as a prior art related to the manufacture of a traditional liquor, and 10-2010-0138957 discloses discovery of a yeast mutant having a low temperature tolerance , A yeast excellent in growth under a low temperature condition, and a fermentation product using the yeast are not known.

In order to solve the above problems, the present inventors selected Y297 as a yeast, Saccharomyces cerevisiae, which is excellent in growth at a low temperature of 15 ° C among 500 fermenting strains. The strain has excellent low-temperature tolerance and alcohol producing ability And the activity of glucosidase and esterase of Y297 yeast was found to be 2 to 5 times better than that of commercial yeast. The results of the confirmation of the quality characteristics of Y297 yeast , Low acidity, high alcohol content, organic acid content, and free amino acid content, compared with commercial yeast. Thus, the low temperature adaptable Y297 yeast of the present invention can be used for producing high quality fermented liquor with excellent flavor, sweetness and aroma The present invention has been completed.

An object of the present invention is a novel Saccharomyces cerevisiae Y297 strain deposited with Accession No. KACC93244P, a prophylactic composition comprising the strain or a culture thereof, a fermentation liquor using the same and a method for producing the same.

In order to achieve the above object,

A novel strain of Sacharomyces cerevisiae Y297 deposited with KACC93244P is provided.

The present invention also provides a probiotic composition comprising the strain or a culture thereof.

In addition,

1) Inoculating the cereal of claim 1 into cereals; And

2) fermenting the cereal in the step 1).

In addition, the present invention provides a fermented beverage produced by the method for producing a fermented beverage.

In the present invention, among the 500 fermenting strains, Y297 was selected as a yeast, Saccharomyces cerevisiae, which is excellent in growth at a low temperature of 15 ° C. The strain was found to have excellent low-temperature resistance and alcohol-producing ability, The activity of glucosidase and esterase of Y297 yeast was found to be 2 ~ 5 times better than that of commercial yeast. The results of the quality characteristics of Y297 yeast were found to be lower than that of commercial yeast , Alcohol content, organic acid content, and free amino acid content, the low temperature adaptable Y297 yeast of the present invention can be usefully used for producing high quality fermented liquor having excellent flavor, sweetness and aroma.

Fig. 1 is a view of Y297 yeast observed with a scanning electron microscope.
Fig. 2 is a diagram showing the identification and flow diagram of Y297 yeast.
FIG. 3 is a graph showing the enzyme activity of Y297 measured at low temperature.
FIG. 4 is a view showing a flavor pattern of a fermentation broth prepared from a low temperature adaptable Y297 yeast according to a fermentation temperature. FIG.
FIG. 5 is a chromatogram showing the aroma component of the fermented wine produced using commercial yeast or Y297 yeast.

Hereinafter, the present invention will be described in detail.

The present invention provides a novel strain of Sacharomyces cerevisiae Y297 deposited with KACC93244P.

Preferably, the strain has a nucleotide sequence of 26s rDNA of SEQ ID NO: 1 and is resistant to low-temperature growth at 10 to 20 ° C.

In a specific example of the present invention, 482 kinds of fermenting bacteria were cultured at a low temperature of 15 ° C to select Y297 yeast as a yeast having a higher viable cell count than the control (see Table 1) (See Fig. 2). The strain was deposited on Nov. 10, 2015 with the National Institute of Agricultural Science and Technology (National Academy of Agricultural Science) (Accession No .: KACC93244P ).

As a result of analyzing the characteristics of the above strains, it was confirmed that the low temperature resistance and alcohol producing ability were excellent (see Table 2), and the enzyme production ability related to the production of aroma components of yeast was compared to determine the glucosidase and esterase activities of Y297 yeast 2 to 5 times higher (see Fig. 3).

Therefore, it was confirmed that the strain of the present invention is a strain Y297 (KACC93244P) in a novel saccharomyces cerevisiae which is characterized by excellent low-temperature resistance and alcohol-producing ability.

In addition, the present invention provides a biocidal composition comprising the strain or a culture thereof.

The biocide composition may further comprise conventional pharmaceutical carriers and excipients. The composition may be freeze-dried or heat-dried according to a conventional method for producing a biocidal composition, and may be in an encapsulated form or in a culture suspension or a dry powder form. .

Further, according to the present invention,

1) Inoculating the cereal of claim 1 into cereals; And

2) fermenting the cereal in the step 1).

In the above method, the cereal in step 1) is preferably rice, barley, wheat, or sweet potato, but is not limited thereto.

In addition, the present invention provides a fermented beverage produced by the method for producing a fermented beverage.

In a specific example of the present invention, the quality characteristics of the fermentation broth fermented with the yeast Y297 yeast were examined. As a result, the pH was 0.3 to 0.5% higher and the acidity was lower by 0.05 to 0.15% than the fermented broth fermented with commercial yeast. It was confirmed that the alcohol content after the low temperature fermentation was 1 to 3% higher, and the fermentation stably occurred even at a low temperature (see Tables 3 to 5). In addition, it was confirmed that the content of essential amino acids and non-essential amino acids was high, and the contents of aspartic acid, glutamic acid and alanine, which are rich in the flavor and sweet taste, were high (see Table 6 and Table 7) ). In addition, the aroma pattern and aroma component of the fermented fermented product fermented with the low temperature adaptable yeast Y297 were different from those of the control (see Figs. 4, 5 and 8). As a result of the sensory evaluation, (See Table 9).

Therefore, it was confirmed that the fermentation broth fermented by the low temperature adaptable Y297 yeast of the present invention is a high quality fermentation broth having a different flavor, sweetness, and other aroma than the conventional fermentation broth.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

<Example 1> Selection of yeast having excellent growth and alcohol production ability at a low temperature (15 ° C)

482 strains isolated from 50 kinds of fermented foods such as meju and leek were collected from the whole country in order to select yeast with excellent growth and alcohol production ability at low temperature. Fermivin ( S. cerevisiae , Oenobrands, France; Con 1) and La parisienne ( S. cerevisiae , DB ingredients, UK; Con 2) were used as comparative strains. The yeasts used in the experiment were liquid medium (YPD) containing 1% of yeast extract, 2% of peptone, 2% of dextrose (Difico, Becton Dickinson and Company, After incubation, glycerol was added at 20% (v / v) and stored at -80 ° C for use in the experiment.

Then, the cells were plated on a YPD agar medium and cultured for 2 days, followed by pure separation. In order to select yeasts with good growth at low temperatures, the cells were incubated at 15 ° C for 2 days in a shaking incubator, and the absorbance was measured at 600 nm using a spectrophotometer. The number of viable cells at low temperature (15 ° C) As a result, as shown in Table 1, Y297 yeast showed higher viable cell count than the control, confirming excellent growth at low temperature (Table 1). As a result, it was confirmed that the strain Y297 Y297 yeast was finally selected.

Further, the degree of alcohol production after low-temperature (15 ° C) alcohol fermentation was measured. As a result, it was confirmed that Y297 yeast of the present invention had an alcohol-producing ability at the control level (Table 2).

Example 2 Identification of Y297 Yeast

To identify the isolated Y297 yeast, the D1 / D2 region of the gene was analyzed with 26S rDNA.

Specifically, the D1 / D2 region fragment of the gene was amplified with 26S rDNA using a primer of NL1 (5'-GCATATCAATAAGCGGAGGAAAAG-3 '; SEQ ID NO: 2) and NL4 (5'-GGTCCGTGTTTCAAGACGG-3' , And the nucleotide sequence was analyzed by ZENOTECH Co., Ltd. The homology of the genes was compared using the BLAST function of the National Center for Biotechnology Information (NCBI) website, and DNASTAR pro software was used to analyze the sequence. The gene analysis of the yeast was performed using the kimura2-parameter of MEGA6.0 method.

As a result, as shown in FIG. 1, the shape of Y297 yeast was confirmed using a scanning electron microscope (SEM), and it was confirmed that the size of the Y297 yeast was about 2 to 5 μm in an elliptical shape (FIG. 1). In order to identify the Y297 yeast by the nucleotide sequence analysis, the 26S rRNA (D1 / D2) region of Y297 yeast was analyzed by base sequence analysis and compared with the standard strain. As a result, the Y297 yeast was identified as Saccharomyces cerevisiae ( Saccharomyces cerevisiae) (Fig. 2).

<Experimental Example 1> Enzyme activity related to perfume ingredient production

To investigate the activity of β-glucosidase and esterase, enzymes affecting the production of aroma-related substances during yeast fermentation process at low temperature (15 ℃), yeast cytoplasmic proteins And the activity of the enzyme was confirmed.

Specifically, the protein was extracted using a yeast protein extraction buffer kit (GE healthcare, St Louis, USA) to confirm the yeast enzyme activity. The activity of β-glucosidase (EC 3.2.1.21.) was tested by the method of Greover et al. (Biochem Biophys Acta, 1997, 482, 98-108). 100 μl of 10 mM ρ-nitrophenyl-β-D-glucopyranoside (Sigma-Aldrich Co., St Louise, USA) was added and 890 μl of 100 mM citrate phosphate buffer (pH 4.0) was added. After incubation at 45 ° C, the reaction was stopped with 0.5 M sodium carbonate (Sigma-Aldrich Co), and the supernatant was centrifuged and its activity was confirmed to be 405 nm by a spectrophotometer.

In addition, the activity of esterase (EC 3.1.1.6) was modified by the method of Frank et al. (Appl Environ Microbiol, 2006, 72, 7140-7147) and a 4.5 M ρ-nitrophenyl acetate Sigma-Aldrich) and incubated at 35 ° C to confirm enzyme activity. The activity was measured at 405 nm in 0.5 M sodium phosphate buffer (pH 7.0) at 40 ° C using 5 M ρ-nitrophenyl-β-butyric acid as a substrate. Yeast cells were cultured in YPD (25% glucose) medium at 120 rpm for 5 days, and 1 μmol of ρ-nitrophenol produced per minute was defined as 1 unit.

As a result, as shown in FIG. 3, the activity of β-glucosidase of Y297 yeast was about 2.5-5.0 times higher than that of control (commercial yeast). The activity of esterase was found to be higher when ρ-nitrophenyl acetate was used as a substrate (Commercial yeast, Con 2). When ρ-nitrophenyl-β-butyric acid was used as a substrate, the activity was about 2-5 times higher than that of the control (commercial yeast) (Fig. 3).

<Experimental Example 2> Determination of quality characteristics of fermented fermented soybeans with low temperature adaptive Y297 yeast

<2-1> Preparation of Fermented Juice Using Y297 Yeast

Wash 1 Kg of rice with flowing water, immerse at 15 ~ 20 ℃ for 2 ~ 3 hours, drain for 1 hour, and cook with high rice. After cooling the heat of rice cakes, 2 g of saccharogenic enzyme is added to 1.2 L of water, Y297 cultured for 2 days is inoculated, and fermented at 15 to 25 ° C for 10 to 20 days to prepare a Yakju.

<2-2> Determination of the pH of the fermented fermented by low temperature adaptive Y297 yeast

To determine the pH of the fermented yeast fermented with Y297 yeast, 10 ml of the Yakju sample prepared with Y297 yeast was measured with a pH meter (FE20, Mettler Toledo, Switzerland).

The pH of the Y297 yeast fermented and fermented by using the control (commercial yeast) was 0.3 ~ 0.5% higher than that of the control (Table 3).

<2-3> Determination of total acidity of fermented soybean fermented with low temperature adaptive Y297 yeast

To determine the total acidity of the fermented yeast fermented with Y297 yeast, 1 ~ 2 drops of phenolphthalein solution (0.5%) was added to 10 ml of Yakju prepared using Y297 yeast, and then 0.1N NaOH solution in burette To the point where it is necessary. Calculate the total amount by calculating the conversion factor (acetic acid) to the appropriate amount consumed.

The total acidity of Y297 yeast fermented soybean was 0.05 ~ 0.15% lower than that of the control. The acidity range of general fermented soybean was 0.2 ~ 0.5%, which was lower than that of the control (Table 4) .

<2-4> Determination of Alcohol Content in Fermented Juice Fermented with Low Temperature Adaptive Y297 Yeast

To determine the alcohol content of Y297 fermented yeast, 100 ml of distilled water was added to 100 ml of Yakju prepared with Y297 yeast, transferred to a 500 ml Erlenmeyer flask, 0.5 ml of defoamer and liquefying enzyme were added, And then distilled. Fill the measuring cylinder with 70 ml of emulsion, add 30 ml of distilled water, and measure at 15 ° C using an umbrella.

The alcohol content of fermented Y297 fermented yeast was 1 ~ 3% higher than that of the control. Especially, alcohol content after low temperature fermentation showed higher alcohol content than that of the control, indicating that alcohol fermentation stably occurs at low temperature (Table 5).

<2-5> Analysis of Organic Acid Content of Fermented Soybean Fermented by Low Temperature Adaptive Y297 Yeast

In order to determine the content of organic acid in the fermented yeast fermented with Y297 yeast, 10 ml of Yakju prepared with Y297 yeast was centrifuged at 5,000 rpm and 2 ml of the supernatant was transferred to 0.45 um membrane filter (Millipore co., Bedford, MA, USA) and then discarded with Sep-pak C18 cartridge (Waters Oasis, Milford, Mass., USA). The samples were analyzed by HPLC (Chromaster 5000, Hitachi, Ltd., Tokyo, Japan) and analyzed using ODS-100W (4.6 × 250 mm) column. Standard curves were prepared using oxalic acid, citric acid, tartaric acid, malic acid, succinic acid, fumaric acid, lactic acid, formic acid and acetic acid.

Y297 fermented yeast fermented soybeans showed higher content of organic acids than the control (Table 6).

N.D: Measurement not possible.

<2-6> Free amino acid analysis of fermented soybean fermented with low temperature adaptive Y297 yeast

To determine the free amino acid content of Y297 fermented yeast, the supernatant was filtered through a 0.45 um membrane filter (Millipore co., Bedford, MA, USA) and centrifuged at 5,000 rpm. Sep-pak C18 cartridge (Waters Oasis, Milford, MA, USA). Add 1 ml of Ninhydrin Reaction Solution and 1 ml of Buffer (PF-1) to 100 μl of the sample, and heat at 100 ° C for 3 minutes. After the reaction, it was analyzed using an amino acid analyzer (L-8900 Amino Acid Analyzer, HITACHI, Ltd., Tokyo, Japan). 20 μl of the sample was analyzed using a HPLC packed column (4.6 × 60 mm) Ion exchange column (HITACHI, Ltd., Tokyo, Japan) and a UV-detector (570 nm, 440 nm).

As shown in Table 7, the content of essential amino acid and non-essential amino acid was higher than that of the control, and it is expected that this will affect the taste of the wine. In addition, it is expected that the content of aspartic acid, glutamic acid and alanine, which are rich in amino acids such as richness and sweetness, is high and sweetness and richness of fermented beverage are high.

<2-7> Analysis of aroma pattern of fermented fermented soybeans with low temperature adaptive Y297 yeast

The fermentation patterns of fermented soybeans prepared with low temperature adaptive Y297 yeast were different according to fermentation temperature (15, 20, 25 ℃). When fermented at low temperature of 15 and 20 ℃, (Fig. 4).

In addition, fragrance components of the fermented juice made from low-temperature adaptive Y297 yeast were analyzed by GC-TOF / MS, and octanoic acid was used as an internal standard for quantitative analysis of the samples.

As a result, it was confirmed that the fermented yeast prepared from Y297 yeast exhibited different fragrance components from the fermented fermented fermented soybean (FIG. 5, Table 8).

<Experimental Example 3> Sensory properties of fermented soybean fermented with low temperature adaptive Y297 yeast

1 kg of rice was steamed, then 2 g of hydrogen peroxide was added to 1.2 L of water, followed by inoculation with a selective yeast Y297 cultured for 2 days. The fermented soybean was fermented for 20 days, and then 13 strains were obtained from the National Institute of Agricultural Science and Technology The preference of low temperature adaptive Y297 yeast was investigated by sensory evaluation. As a result, although statistically significant results were not obtained, it was confirmed that Y297 yeast was best evaluated in taste, taste and color (Table 9).

National Academy of Agricultural Sciences KACC93244P 20151023

<110> Rural Development Administration <120> Saccharomyces cerevisiae Y297 with adaptability at low          temperature and manufacturing method of fermented alchoholic          beverage using the same <160> 3 <170> KoPatentin 3.0 <210> 1 <211> 1253 <212> DNA <213> Saccharomyces cerevisiae <400> 1 atcatcgaat ctttgaacgc acattgcgcc ccttggtatt ccagggggca tgcctgtttg 60 agcgtcattt ccttctcaaa cattctgttt ggtagtgagt gatactcttt ggagttaact 120 tgaaattgct ggccttttca ttggatgttt ttttttccaa agagaggttt ctctgcgtgc 180 ttgaggtata atgcaagtac ggtcgtttta ggttttacca actgcggcta atctttttta 240 tactgagcgt attggaacgt tatcgataag aagagagcgt ctaggcgaac aatgttctta 300 aagtttgacc tcaaatcagg taggagtacc cgctgaactt aagcatatca ataagcggag 360 gaaaagaaac caaccgggat tgccttagta acggcgagtg aagcggcaaa agctcaaatt 420 tgaaatctgg taccttcggt gcccgagttg taatttggag agggcaactt tggggccgtt 480 ccttgtctat gttccttgga acaggacgtc atagagggtg agaatcccgt gtggcgagga 540 gtgcggttct ttgtaaagtg ccttcgaaga gtcgagttgt ttgggaatgc agctctaagt 600 gggtggtaaa ttccatctaa agctaaatat tggcgagaga ccgatagcga acaagtacag 660 tgatggaaag atgaaaagaa ctttgaaaag agagtgaaaa agtacgtgaa attgttgaaa 720 gggaagggca tttgatcaga catggtgttt tgtgccctct gctccttgtg ggtaggggaa 780 tctcgcattt cactgggcca gcatcagttt tggtggcagg ataaatccat aggaatgtag 840 cttgcctcgg taagtattat agcctgtggg aatactgcca gctgggactg aggactgcga 900 cgtaagtcaa ggatgctggc ataatggtta tatgccgccc gtcttgaaac acggaccaag 960 gagtctaacg tctatgcgag tgtttgggtg taaaacccat acgcgtaatg aaagtgaacg 1020 taggttgggg cctcgcaaga ggtgcacaat cgaccgatcc tgatgtcttc ggatggattt 1080 gagtaagagc atagctgttg ggacccgaaa gatggtgaac tatgcctgaa tagggtgaag 1140 ccagaggaaa ctctggtgga ggctcgtagc ggttctgacg tgcaaatcga tcgtcgaatt 1200 tgggtatagg ggcgaaagac taatcgaacc atctagtagc tggttcctgc cga 1253 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 2 gcatatcaat aagcggagga aaag 24 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> Artificial sequence <400> 3 ggtccgtgtt tcaagacgg 19

Claims (7)

A novel strain of Sacharomyces cerevisiae Y297 having the nucleotide sequence of 26s rDNA of SEQ ID NO: 1 deposited with KACC93244P.
delete The novel strain of Saccharomyces cerevisiae Y297 according to claim 1, wherein the strain is resistant to low-temperature growth at 10 to 20 占 폚.
A prophylactic composition comprising the strain of claim 1 or a culture thereof.
1) Inoculating the cereal of claim 1 into cereals; And
2) fermenting the cereal of step 1) above.
6. The method of claim 5, wherein the cereal in step (1) is rice, barley, wheat, or sweet potato.
A fermented beverage produced by the method of claim 5.

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