KR20170137237A - Saccharomycopsis fibuligera KJJ81 strain with high saccharification and fermentation capability and the method for preparing makgeolli - Google Patents

Abstract

The present invention relates to a Saccharomycopsis fibuligera kjj81 strain (KCTC18466P) with a high saccharification and fermentation capability and a rice wine manufacturing method using the same. Genetic resources for developing a starter having a fermentation capability and functionality through yeast fungi separated from traditional Korean liquor are secured and the basic technology for the standardization and gentrification of traditional liquor is developed, and thus, the present invention is capable of making a large contribution to international competitiveness in bio industries.

Description

(Saccharomycopsis fibuligera KJJ81 strain with high saccharification and fermentation capability and the method for preparing makgeolli) and a method for producing rice wine using the Saccharomycops fibrillaria KJJ81 strain

The present invention relates to superior efficacy to saccharification and fermentation of yeast Saccharomyces traditionalism as MY Cobb cis blood debris galley (Saccharomycopsis fibuligera) KJJ81 strain (KCTC18466P) and rice wine production method using the same. In particular, Saccharomycopsis ( Saccharomycopsis), which has excellent glycosylation and pharmacological activity but has not yet been genetically detoxified fibuligera ), and genome analysis.

Although Korea has been manufacturing fermented foods using traditional fermentation methods such as kimchi, liquor, soy sauce, and soybean paste for a long time, it has acquired genome information of fermented microorganisms related to traditional foods in Korea, Utilization technology development research is relatively small compared to foreign countries. Except for the typical two to three companies, the traditional Korean traditional industry operates on a very small scale, and most of the companies do not even perform basic quality control properly. Especially, since there are not enough excellent seeds invented yet, a large amount of royalties are paid every year for the use of overseas biological resources.

Unlike traditional Korean native rice, Korean traditional rice is produced by a complex Nuruk strain. It is essential to acquire reference genomic information for key species in order to standardize, scientificize, and globalize Korean traditional rice. The development of the basic technology that can be used for the standardization and the upgrading process of the traditional wine is secured for the enhancement of the international competitiveness in the bio industry by securing the useful genetic resources necessary for the development of the seed culture having excellent efficacy and functionality, Will contribute.

Genetic analysis can be used to identify genes related to useful traits and to understand related metabolic pathways. Based on this analysis, species diversity analysis studies will be conducted to systematically screen and secure wild strains and superior trait-bearing strains, To the strains of the strains.

Studies on the analysis of omics for fungi or yeast, which are used as some model organisms in Korea, have been carried out, but studies on the genome-based omics of traditional mainstream eukaryotic microorganisms have not been conducted so that the genome functions of traditional liquor using yeast and fungi And the relationship between microbial genome and fermentation conditions for flavor and quality control is needed.

A variety of metabolites are finally produced by the biochemical action of yeast strains in traditional liquor industry, which determine the functional, nutritional and quality characteristics of traditional fermented beverages. In particular, the overall study of transcripts / metabolites of microorganisms related to flavor closely related to product preference can be used as basic data for product improvement, and also provides useful information for quality control in industry will be.

Korean Patent Publication No. 10-2014-0055917 (2014.05.09)

It is an object of the present invention to provide a sugar-fermenting yeast Saccharomycopsis fibuligera ) KJJ81 strain and a method for producing rice wine using the same.

In order to achieve the above object, the present invention provides a system MY Cobb blood debris galley (Saccharomycopsis fibuligera) KJJ81 strain in the superior, efficacy to glycosylated and deposited as accession No. KCTC18466P traditionalism fermenting yeast Saccharomyces.

Further, the present invention provides a method for producing makgeolli using the strain as a raw material for grain.

The present invention relates to a glycosylated and rice wine production process in a superior to efficacy traditionalism fermenting yeast Saccharomyces Mai Cobb cis blood using debris galley (Saccharomycopsis fibuligera) KJJ81 strain (KCTC18466P) and this, superior to efficacy with the yeasts isolated from domestic traditionalism It is expected that the development of basic technologies that can be used for the standardization and upgrading process of traditional wines will contribute to the enhancement of international competitiveness in the bio industry.

Figure 1 shows the phylogenetic relationship of Saccharomycopsis fibuligera KJJ81 (KCTC18466P) according to the present invention through a whole genome comparison.
2 is a microscopic observation of Saccharomycopsis fibuligera KJJ81 (KCTC18466P) according to the present invention.
Fig. 3 shows the sensory evaluation results of rice wine made with Saccharomycopsis fibuligera KJJ81 (KCTC18466P) according to the present invention.

The invention in the, superior efficacy to saccharification and fermentation of yeast Saccharomyces traditionalism deposited as accession No. KCTC18466P Mai Cobb cis blood debris galley (Saccharomycopsis fibuligera ) KJJ81 strain.

Specifically, the strain is isolated from yeast and is characterized by excellent activity of Alpha-Amylase, Glucoamylase or Protease.

In detail, the strain may be an internal transcribed spacer (ITS) represented by SEQ ID NO: 3, but is not limited thereto.

Further, the present invention provides a method for producing makgeolli using the strain as a raw material for grain.

Specifically, the cereal raw materials may be cereal raw materials such as glutinous rice, rice, barley, brown rice, corn, sweet potato, and wheat which can produce rice wine, and more specifically rice or wheat. no.

In the present invention, 'makgeolli' is a Korean traditional rice liquor made of starch such as glutinous rice, rice, barley, brown rice, corn, sweet potato, and wheat as a raw material and fermented with fermented yeast as an fermentation agent , Sweet, sour, bitter, hot and refreshing, alcohol content of 2-8%. Unlike other alcoholic beverages, Makgeolli is rich in various nutrients. It contains vitamins B, which are involved in human metabolism, essential amino acids such as lysine, leucine and arginine, flavors such as ethyl acetate, amyl acetate, ethyl caroate , An organic acid to relieve thirst with a sour taste, and acetylcholine to help liver function. In addition to high nutritional and functional value, makkolli has a unique flavor that can not be compared with other alcoholic beverages because it contains raw yeast.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example  1> A new strain Sakaromycops Friburgera ( Saccharomycopsis  fibuligera ) Isolation of KJJ81

1. Isolation of strain

In order to discover new strains with glycosylation, fermentation and flavor, we collected koji in traditional Jeju area in July 2013, stored at 4 ℃, weighed 10 g of uniformly crushed koji in 90 ml of 0.1% peptone peptone) and then diluted ^10-2 to 10 ^-7 times in a stepwise manner. 100 [mu] L of the diluted suspension was plated on a separate plate culture medium and cultured at 25 [deg.] C. The separation medium used was Dichloran-glycerol agar (DG18: Peptone 0.5%, Glucose 1%, KH ₂ PO ₄ 0.1%, MgSO ₄揃 7H ₂ O 0.05%, 0.2% Dichloran 1.0 ml, Chloramphenicol 0.01%, Agar 1.5% ) Medium and Dichloran rose bengal chloramphenicol agar (DRBC: Peptone 0.5%, Glucose 1%, KH ₂ PO ₄ 0.1%, MgSO ₄揃 7H ₂ O 0.05%, 0.2% Dichloran 1.0 ml, Rose bengal 0.0025%, Chloramphenicol 0.01% Agar 1.5%). The isolated strains were inoculated and stored in Malt extract agar (MEA: Malt extract 3%, Mycological peptone 0.5%, Agar 1.5%) medium.

2. Identification of selected strains

(1) Morphological identification

The morphological shape of the strain was observed through a microscope. As a result of the observation, it was observed that a single cell of 5 to 10 μm in size forms a multipolar hypae, and each node forms a long hypae which is elongated to 20 to 40 μm 2).

(2) Molecular biology identification

DNA was extracted using the CTAB method for the molecular identification of the strains selected above, and DNA was extracted from the cultured beads by a bead beating method to amplify the internal transcribed spacer (PCR) (polymerase chain reaction) (TCCGTAGGTGAACCTGCGG; SEQ ID NO: 1) / ITS4 (TCCTCCGCTTATTGATATGC; SEQ ID NO: 2) in order to amplify an internal transcribed spacer (ITS) Sequencing was commissioned by Macrogen Inc. to determine the nucleotide sequence. The nucleotide sequence of the analyzed ITS region is shown in SEQ ID NO: 3.

(3) Mycological identification

The mycological characteristics of the identified strain, Saccharomycopsis fibuligera KJJ81, were investigated. The optimal culture conditions were investigated under various temperature conditions, various media and nitrogen source.

As shown in Table 1, the growth temperature was 25 to 42 ° C, and growth was particularly good at 25 to 37 ° C (Table 1).

badge 25 ℃ 37 ℃ 42 ° C 45 ° C CM +++ +++ + - PDA +++ +++ + - YPD +++ +++ + - MM - - - - MM (NaNO ₃ ) - - - - MM (NH ₄ Cl) - - - -

< Example  2> A new strain Sakaromycops Friburgera ( Saccharomycopsis  fibuligera ) Enzyme activity characteristics of KJJ81

The new strain Saccharomycopsis fibuligera KJJ81 identified in the present invention and the rice koji prepared with Saccharomycopsis fibuligera strain KPH12, which is a comparative strain, and bran koji alpha-amylase , Glucoamylase, and protease (fungal) (Table 2). Analysis methods were analyzed according to the Food Additives Code of the Food and Drug Administration.

Alpha-amylase
(Alpha-Amylase) Glucoamylase
(Glucoamylase) Protease
(Protease) Sakaromai Cobsis Pribragera KJJ81 Rice koji
(Rice Koji) 2.821 0 0 Bran koji
(Wheat Koji) 7.863 131.55 53.27 Sakaromycops cis fibrillera KPH12 Rice koji
(Rice Koji) 4.398 31.04 425.25 Bran koji
(Wheat Koji) 1.967 68.06 537.06

Alpha-amylase: the amount of ceralpha units per gram of koji.

The glucose activity: the units of glucose released per gram of koji.

One unit of protease: the amount of enzyme required to release 1 μg of tyrosine / hour.

< Example  3> A new strain Sakaromycops Friburgera ( Saccharomycopsis  fibuligera Characteristics of makgeolli made with KJJ81

1. rice koji

The rice is immersed for 2 hours after the semi-baking, and then the rice is drained for 30 minutes. Then, the rice is baked for 1 hour and 30 minutes by using a steamer. After the rice was cooled at room temperature, 0.1% of rice seed weight was inoculated and cultured in a constant temperature and humidity incubator at 30 ° C and 70% relative humidity for 72 hours.

2. Bread Cozy

30 g of distilled water is added to 50 g of wheat bran, kneaded, and left at room temperature for 10 minutes. Put 30 g of bran dough in 500 mL Erlenmeyer flask and sterilize by pressurizing at 121 ° C for 20 minutes. 10 ml of distilled water is added to the slurry medium cultivated for each strain to prepare a suspension, which is then inoculated into bran cooled at room temperature. After inoculation, the cells were cultured in a constant temperature and constant-humidity incubator at 30 ° C and 70% relative humidity for 72 hours.

3. Danyang  Make rice wine

Add 4 × 10 ⁸ cells / g of each strain to 1.4 L of water, add 200 g of coagulum, add 800 g of high-brow seed, and add 5.5 mL of 90% Lactic acid. After incubation at 25 ℃ for 7 days, the cells were filtered with a 120 mesh filter. The sensory evaluation (aroma, sweetness, freshness, alcohol, carbonic acid and body sensation) And stored at -70 ° C for analysis (Table 3). The analytical results of the makgeolli produced are shown in Table 4, and the sensory evaluation results are shown in Table 5 and Fig.

Organic acids Relative peak area
(mean ± SD) 2- (Methoxyimino) propanoic acid 1.298 + 0.178 Acetic acid 0.275 + 0.027 Oxalic acid 0.047 ± 0.01 Succinic acid 14.415 + 0.895 2-Butenedioic acid 0.129 + 0.014 2-Methylbenzoic acid 0.612 + 0.038 2- (Lactoyloxy) propanoic acid (lactic acid dimer) 7.966 + 0.183 Methyl valerate 0.022 0.005 2-Hydroxy-2-methylsuccinic acid 0.261 + 0.013 Malic acid 5.808 ± 0.108 Ethyl 4-hydroxybenzoate 0.887 ± 0.008 2,3,4-Trihydroxybutanoic acid 0.093 ± 0.007 Glutaric acid 4.264 ± 0.051 3-Phenylpropanoic acid 0.287 + 0.013 Phenylacetic acid 0.016 ± 0.004 Citric acid 0.789 + 0.039

Makgeolli result Sakaromycops Cispifbrigera
KJJ81 Sakaromycops Cispifbrigera
KPH12 pH 4.2 4.1 Brix 13 17.5 Reducing Sugars 24.50 20.16 Liquid Volume (ml) 1000 700 Rice Weight (g) 3118.77 3179.16

Sensory evaluation result Sakaromycops Cispifbrigera
KJJ81 Sakaromycops Cispifbrigera
KPH12 Scent 3.6 2.4 Sweet 3.4 2.6 Sour 6.2 7.4 Alcohol / Bitterness 6.2 5 Strength of carbonic acid 4 3.2 Body 3.8 3.4 Overall Acceptability 3.6 1.8

< Example  4> A new strain Sakaromycops Friburgera ( Saccharomycopsis  fibuligera ) Reference genomic sequencing and genomic analysis of KJJ81 strain

We performed long-reads, short reads, and long-mate pair reads sequencing for KJJ81 and KPH12 genome sequencing. Long-reads sequencing of a total of 94x was performed at an average size of 9kb using the Pacific Biosciences single molecule real time sequencing (SMRT) technique. Sequencing of 23X and 10X sequences was performed in the KPH12 and KJJ81 strains using the TruSeq Synthetic Long Reads (TSLR) sequencing library (Illumina). We also performed 100-bp paired-end sequencing using the Illumina HiSeq2500 sequencing technique with 86X or more short insert reads (a 500-bp library) and 87 X long-mated pair reads (a 15-kb library). The long-SMRT sequencing reads produced HGAP3, 15-kb mate-pair and 500-bp short insert reads with the generated sequencing data using SSPACE software. Gaps were corrected using GapCloser software using short read data. (Ref. #). The quality of the genome assembly was compared with the results of TSLR sequencing performed using QUAST software. BioNano physical mapping was performed with the KPH12 strain to perform repetitive sequence analysis and genomic scaffolds at the chromosome level The number of rRNA clusters was analyzed. KPH12 strain had 7 chromosomes and KJJ81 strain was 7 strains. The genomes of the two genomes of KPH12 and KJJ81 were analyzed to be about 19.7 and 38.6 Mb, respectively.

Korea Biotechnology Research Institute KCTC18466P 20160503

<110> Soongsil University Research Consortium techno-PARK          Chung-Ang University Industry-Academy Cooperation Foundation <120> Saccharomycopsis fibuligera KJJ81 strain with high          saccharification and fermentation capability and the method for          preparing makgeolli <130> ADP-2016-0156 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 tccgtaggtg aacctgcgg 19 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 tcctccgctt attgatatgc 20 <210> 3 <211> 558 <212> DNA <213> Saccharomycopsis fibuligera <400> 3 ctcttataca cagtgttttt gtttgcgaat ttggtttagt ttgttggttt tcattcgaaa 60 ggatgaagat tgattgctaa atcttattca gctttttaaa ctcagatctc tttttaagag 120 aaatgtattt ttttaattac aactagtcga ttttacaaac taaaagttta aaactttcag 180 caacggatct cttggttctc gcatcgatga agaacgcagc gaattgcgat aagtaatgtg 240 aattgcagat tttcgtgaat catcgaatct ttgaacgcat attgcgctct atagtattct 300 atagagcatg cctgtttgag cgtcatttct ctcttaaacc tttgggttta gtattgaagg 360 ttgtgttagc ttctgctaac tcctttgaaa tgacttggca attgattgag ttttccatat 420 atttgcttaa ggatttaata ttaggttcta ccaacttatt aaataccctt ttgcgaagga 480 cttactcgtg tatcaaggcc ttataacttt gtcattaatt ttgacctcga atcaggtaag 540 gatacccgct gaacttaa 558

Claims (6)

Saccharomycopsis fibuligera KJJ81 strain, a sugar-fermenting yeast strain excellent in saccharification and efficacy, deposited under accession number KCTC18466P. The method according to claim 1, wherein the strain is isolated from yeast. Saccharomycycopsis fibuligera ) KJJ81 strain. The method according to claim 1, wherein the strain is Saccharomycopsis fibuligera KJJ81, which is characterized by excellent activity of Alpha-Amylase, Glucoamylase or Protease. Strain. 2. The strain of Saccharomycopsis fibuligera KJJ81 according to claim 1, wherein the strain comprises an internal transcribed region (ITS) represented by SEQ ID NO: 3. A method for producing makgeolli using a strain of any one of claims 1 to 4 as a raw material for grain. [6] The method of claim 5, wherein the cereal raw material is rice or wheat bran.

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