KR20140103590A - Eurotium cristatum sp. strain, golden flower fungi isolated from Chinese Fuzhuan brick-tea - Google Patents

Abstract

The present invention relates to an Eurotium cristatum sp. strain (KACC93171P) which is a golden flower fungus isolated from Chinese Fuzhuan brick-tea and, more specifically, to a novel Eurotium cristatum sp. strain (KACC93171P) isolated from Fuzhuan brick-tea and used as a starter of fermented tea. The golden flower fungus which is a kind of microorganism zymogens is isolated as a single clone from the Fuzhuan brick-tea, and base sequences are analyzed by performing PCR amplification on genes from genomic DNA, so that it is confirmed that the golden flower fungus has a novel gene sequence. Using the same as a starter, golden flower tea which is microorganism fermented tea can be developed under adjusted temperature and humidity.

Description

A gold coin isolated from a Chinese fermentation car, Eurotium cristatum sp. strain, golden flower fungi isolated from Chinese Fuzhuan brick-tea}

The present invention relates to a method for producing a Eurotium < RTI ID = 0.0 > cristatum sp.) strain (KACC93171P).

After the microorganisms such as fermented tea (post-fermented tea) it is in mainly Aspergillus that the auto-oxidation and non-enzymatic oxidation caused by the microorganism (Aspergillus), passage tium in (Eurotium), debari Oh, my process in (Debaryomyces) It is known to be involved. The tea that fermented by these microorganisms is called dark green tea, and there are Pu'er tea, Raw dark green tea, and Fuzhuan brick-tea. Among the beneficial yellow mold fungi, which are naturally occurring in the high-grade bochahas and tributaries, which are one of the black tea among Chinese fermented tea, they are called "golden flora" This includes the Eurotium . These lesions have been used as anticholinergic drugs in men and have been reported to be effective against diarrhea / diarrhea and have excellent antimicrobial activity.

The jade process of the electric car was made from the raw dark green tea, steamed and softened, and then deposited at 80 ° C. overnight to form a brick. The microbial fermentation was continued for 15-17 days and then dried for 6 months It is a process of aging. Most of the high-grade microbial fermentation tea in China grows naturally, so it is important to make fermentation conditions. However, in Korea's climate, temperature and humidity, there is a limit to making high-grade microbial fermentation tea naturally.

On the other hand, Korean Patent Application No. 10-2012-7009778 discloses that aspergillus sp. (PK-1) fungi [Aspergillus sp. (PK-1), Aspergillus sp. (AO-1) bacteria [Aspergillus oryzae (NBRS 4214) sp. (AO-1)], Aspergillus sp. (SK-1) fungi [Aspergillus awamori (NBRS 4122) sp. (SK-1)], or the europium sp. (KA-1) bacteria [Eurotium sp. (KA-1)] is subjected to an extraction treatment to obtain a functional microbial fermentation tea extract containing various extracts and a novel polyphenol derivative, and further, a novel polyphenol derivative-hatching functional fermentation But there is no mention of gold coins which the present inventors have isolated from Chinese fermentation tanks.

Therefore, the present inventors have confirmed that a gold coccus, which is a kind of microbial fermenting bacteria, is isolated from a complex carotid artery by a single clone, and the gene is PCR amplified from the genomic DNA and subjected to base sequencing analysis. The aim of this study was to develop golden flower tea which is a microorganism fermentation tea under controlled temperature and humidity.

It is an object of the present invention to provide a new strain of Eurotium < RTI ID = 0.0 > cristatum sp.) strain (KACC93171P).

In order to achieve the above object, the present invention provides a novel strain, Eurotium, which is separated from a zygotic body and used as a seed of a fermentation tea, cristatum sp.) strain (KACC93171P). Specifically, the fermentation tea is a gold tea, and the fermented tea produced using the strain is characterized in that the content of catechin is increased.

"Catechin" is a kind of polyphenol, commonly known as a bitter taste component of green tea. Catechin has various effects such as inhibition of carcinogenesis, arteriosclerosis, inhibition of blood pressure increase, prevention of thrombosis, antiviral, anti-obesity, antidiabetic, antibacterial, detoxifying action, anti-inflammatory action, prevention of tooth decay, prevention of browning, normalization of intestinal flora. Catechin's antioxidant and antioxidant activity, which is a fundamental element of aging, has a stronger effect on the removal of free radicals than vitamin C and vitamin E, and its antioxidant activity has the effect of protecting against cardiovascular diseases such as low density lipoprotein oxidation Are reported.

The Eurotium < RTI ID = 0.0 > cristatum sp.) strain (KACC93171P) was isolated from a Chinese hybrid electric vehicle and screened and identified according to the method of the following example. The identification of the microorganisms was based on his morphological, physiological and biochemical characteristics.

(KACC93171P), which is a gold coin isolated from a Chinese embankment. The gold microorganism, which is a kind of microbial fermenting bacteria, is isolated from a blastocyst by a single clone and a gene is isolated from a genomic DNA by PCR It was confirmed that the strain was a gold-bearing strain having a novel gene sequence by amplification and sequencing, and golden flower tea which is a microbial fermentation tea under the control of temperature and humidity can be developed as a seed. The gold tea can be used as a substitute for a folk medicine to stop diarrhea and diarrhea and further develop a functional food having anti-diabetic or anti-cancer effect.

Fig. 1 shows an enlarged photograph of a petri dish (petridish) cultivated for 48 hours (B) and 72 hours (C) from a Chinese hybrid electric kettle (A) and a gold coin isolated from a gold fermentor. In addition, as scanning electron microscope (SEM) photograph (D) at 200 times magnification, it is possible to observe the cleistothecium and hyphae.
2 is a photograph (A) of a genomic DNA of a preliminary gold-plating microorganism isolated from two petri dishes and a PCR-amplified β-tubulin gene and a polymerase ) ≪ RTI ID = 0.0 > II < / RTI >
3 shows the β-tubulin gene (A) and the RNA polymerase II gene (B) obtained through sequencing. The forward and reverse primers used for each gene were boxed.
FIG. 4 is a graph showing the results obtained by using the β-tubulin gene sequence of the isolated gold cocci that has been subjected to the nucleotide sequence analysis and the already registered nucleotide sequence of the β-tubulin gene in Eurotium It shows the phylogenetic tree created.
FIG. 5 shows the results of comparison of caffeine and catechin contents of the gold coins produced in ppm. Total catechin content was expressed as the sum of EGC (epigallocatechin), EC (epicatechin), EGCG (epigallocatechin gallate) and ECG (epicatechin gallate).

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.

< Example  1> China From a tram  Isolation and culture

In this study, we investigated the effect of homogenization on the potato dextrose agar (PDA) plate culture medium used for the cultivation of fungi and yeast by mixing a part with 0.85% physiological saline, 1 mL of each of the solutions is inoculated into each well and inoculated on 10 plate culture media and cultured at 25 ° C for 24 hours. After 72 hours from inoculation, yellow or yellow spores were formed on the plate medium and judged to be E. cristatum (Fig. 1). At this time, one platinum is collected from the spores judged to be gold coins, streaked on 10 PDA plate media, and cultured at 25 ° C. This method is repeated at intervals of 72 hours, and 1-2 pieces of yellow or yellow spore-forming medium, which is judged to be gold coins, are separated and refrigerated.

< Example  2> Identification of isolated strains PCR  Amplification and sequencing

2 g of gold-bearing bacteria grown on a plate medium is scraped off with a glass rod and the gold microorganism DNA is separated by the following method. Genomic DNA of the isolated gold leaf fungus was made into a 1% agarose gel and subjected to electrophoresis at 80 V for 1 hour to confirm DNA isolation (FIG. 2A). Using 50 ng of isolated gold coin genomic DNA as a template, the known europium crystals ( Eurotium PCR is carried out by synthesizing primers using the well-conserved β-tubulin gene and the RNA polymerase II gene of the cristatum NRRL4222 strain (Table 1).

Genes and primers used for gold coin gene amplification β- Tubulin (β- Tubulin )
( E. cristatum NRRL4222 strain) RNA Polymerase (Polymerase) II
( E. cristatum NRRL 4222 strain) Forward Primer
(5 '- &gt;3') 5'-TGGTATGTCTGCATTATAATCAGA-3 '
(SEQ ID NO: 1) 5'-ACCCGTGTCACCCGTGATCTTCAA-3 '
(SEQ ID NO: 3) Reverse Primer
(5 '- &gt;3') 5'-CCAGTTGTTCCAGGCACCGGACTG-3 '
(SEQ ID NO: 2) 5'-CTCACGGATATCCCGAACCAAACT-3 '
(SEQ ID NO: 4)

Table 2 shows the execution conditions of the PCR after synthesizing the primers. PCR was performed using 20ul premix kit (Accu PowerTM Bioneer) and Takara® PCR Thermal cycler Dice instrument.

PCR conditions used for amplification division Temperature and time conditions Number of cycles
( cycle number ) Denaturation 94 ° C 4 min One Annealing 94 ° C 1 min
48 ° C 1 min
72 ° C 1 min 40 Extension 72 ° C 10 min One

The DNA amplified by PCR was confirmed by electrophoresis (FIG. 2B). The identified PCR DNA fragment was cloned into a T / A cloning vector (pGEMTeasy) to analyze the DNA base sequence (Fig. 3). The sizes of the β-tubulin gene and the polymerase II gene were 400 base pairs (bp) and 600 bp, respectively. Each represented by SEQ ID NO: 5 and SEQ ID NO: 6.

Blast analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was performed on the gene data base to determine the similarity of the above genes. As a result, the nucleotide sequence of Eurotium cristatum It was found that the most similarity was high. In the case of β-tubulin, one nucleotide sequence (1 bp) was different from Eurotium cristatum . In the case of RNA polymerase II, three nucleotide sequences (3 bp) I could see this difference.

In order to investigate how the β-tubulin gene sequence of the gold coins analyzed in this study differs from the known β-tubulin gene of Eurotium , the Sequence Alignment Multigroup Sequence Alignment) was analyzed using Clustalw (http://www.genome.jp/tools/clustalw/) program, and then the UPGMA (Unweighted Pair Group Method using Arithmetic Algorithm) Genetic system. The result was a gold bacteria isolated from this invention is the similar to the particular flow path in tium (Eurotium) Euro tium Christa term (Eurotium cristatum), sub-species of the path tium Christa term (Eurotium cristatum) (subspecies, E. cristatum sp.) Respectively.

Therefore, the present inventors deposited the above strain with the National Institute of Agricultural Science and Technology, National Institute of Agricultural Science and Technology (Accession No. KACC93171P).

< Example  3> using isolate Gold tea  Produce

Green tea, yellow tea, fermented tea and the like are used as the green tea used in the present invention in order to produce gold tea which is a fermented microorganism. Firstly, inoculation of Eurotium cristatum sp. Separated in a liquid or solid medium and cultivation for more than 72 hours, golden flower tea which is a microbial fermentation tea treated with tea leaves was prepared Respectively. The catechin-caffeine content of the gold tea thus produced was analyzed by HPLC chromatogram. First, 0.1 g of the tea sample is diluted with 50 ml of distilled water, extracted with hot water for 1 hour in a reflux-cooled extractor fixed at 80 ° C, and then filtered through Whatman® No.2 filter paper. Add 50 ml of ethyl acetate (EtOAc) to the filtrate, mix with a separatory funnel, and collect the supernatant. After concentrating the supernatant under reduced pressure, the concentrate is dissolved in 5 ml of methyl alcohol (MeOH), filtered through a 0.45 μm syringe and analyzed by injecting 10 μl into HPLC. Instrumental analysis conditions are as follows (Table 3).

HPLC conditions for comparison of caffeine and catechin content of gold coins HPLC operating conditions Item Condition Column TOSHO ODS-80Tm (4.6 x 150 mm) Wave length 280 nm Flow rate 1 mL / min Injection volume 10 μL 오빈 온도 40 ℃ Mobile phase Solvent A: 0.2% H ₃ PO ₄
Solvent B: 100% Acetonitrile Time (min) Solvent A (%) Solvent B (%) 0 85 15 7 85 15 15 0 100 15.05 85 15 20 85 15

Analysis of the catechin-caffeine content of the golden flower tea by HPLC chromatogram revealed that the content of catechin in the tea leaves was higher than that of the untreated tea in the tea leaves of 200 ~ 300ppm and total catechin content was about 4,900ppm. It is considered that the strain specifically increases the content of catechin by about 8% than that of the common car (Table 4 and FIG. 5).

(Unit: ppm) EGC EC EGCG ECG Caffeine Total Catechin Gold coin control ¹ 6103.42 3381.39 27093.68 8334.60 6943.99 51857.08 Gold coins ² 7074.18 3695.33 30818.89 8612.67 6606.96 56808.04 1. Tea not treated with gold coins, 2. Tea treated with gold coins

EGC (epigallocatechin), EC (epicatechin), EGCG (epigallocatechin gallate), ECG (epicatechin gallate)

Agricultural Biotechnology Research Institute KACC93171P 20130118

<110> Institute of Hadong Green Tea <120> Eurotium cristatum sp. strain, golden flower fungi isolated from          Chinese Fuzhuan brick-tea <130> DP-2013-0004 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> beta-tubulin forward primer <400> 1 tggtatgtct gcattataat caga 24 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> beta-tubulin reverse primer <400> 2 ccagttgttc caggcaccgg actg 24 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> RNA polymerase II forward primer <400> 3 acccgtgtca cccgtgatct tcaa 24 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> RNA polymerase II reverse primer <400> 4 ctcacggata tcccgaacca aact 24 <210> 5 <211> 412 <212> DNA <213> Eurotium cristatum sp. <400> 5 tggtatgtct gcattataat cagagtcgga tttagtgata tactaacagt atcacaggca 60 gactatctcc ggcgagcacg gtctcgacgg ctctggtgtg taagtacagt cgggtctccg 120 ggatggatgc gtatcggata tggatatcta aatggattgc agctacaatg gctcctccga 180 cctccaattg gagcgtatga acgtctactt caacgaggtt tgcttaattc attcgtgttt 240 atgcggaaaa cagttctgac agtgacaggc ctccaacaac aaatatgtcc cccgtgccgt 300 cctcgtcgac cttgagcccg gtaccatgga cgccgtccgt gacggtccct tcggtcagct 360 cttccttccc gacaacttcg tcttcggtca gtccggtgcc tggaacaact gg 412 <210> 6 <211> 599 <212> DNA <213> Eurotium cristatum sp. <400> 6 acccgtgtca cccgtgatct tcaacggtac gtgcaaagat gcgtggaaac aaaccgtgag 60 atctacttga acattggtat caaggccagc accttgaccg aggtttgaaa tatgctttgg 120 ccacgggtaa ctggggtgaa cagaagaagg cggctagtgc aaaggcaggt gtgtcgcagg 180 tgctgagtcg atacacatat gcttttacct tgtcgcatct tggtcggaca aacaccccaa 240 ttggtcgtga cggaaagatc gccaaaccgc gacaacttca caacacccac tggggtttgg 300 tgtgtccagc agaaactcca gaaggtcaag cttgtggtct ggtcaagaac ctggcactta 360 tgtgttacat taccgttggt acgcctagcg agcctattat cgacttcatg attcagcgga 420 acatggaagt tctcgaagag ttcgaacctc aagttacgcc taatgccacc aaggtctttg 480 tcaacggtgt ctgggttggt attcaccggg actctgcgca tctggtgaac accatgcttg 540 ccttgcgtcg gcgcaacatg atttcgcacg aagttagttt ggttcgggac atccgtgag 599

Claims (3)

A new strain, Eurotium, was isolated from the blastocyst and used as a seed for fermentation tea. cristatum sp.) strain (KACC93171P). According to claim 1 wherein the fermented tea novel strain flow path, characterized in that gold car tium Christa term (Eurotium cristatum sp.) strain (KACC93171P). The fermented tea according to claim 1, wherein the fermented tea has a catechin content enhanced, a new strain of Eurotium &lt; RTI ID = 0.0 &gt; cristatum sp.) strain (KACC93171P).

Images