A Comparison of the Microbial Diversity in Korean and Chinese Post-fermented Teas

한국과 중국 미생물 발효차의 미생물 군집분석 및 비교

김병혁¹, 장종옥¹, 좌재호¹, 김진아¹, 송승엽¹, 임찬규¹, 김천환¹, 정영빈¹, 성기철¹, 김희식², 문두경¹*

1농촌진흥청국립원예특작과학원온난화대응농업연구소

2한국생명공학연구원세포공장연구센터

Received: February 28, 2017 / Revised: March 22, 2017 / Accepted: March 23, 2017

서 론

차는세계적으로넓게음용되는음료로서거의모든나라 에서소비되고있으며

^,

^,

^,

^.

(Camellia sinesis L.)

^,

^,

^,

^,

^,

^,

^,

⁽

⁾

^[1].

^,

⁽

^,

^,

⁾

^[2

^5].

차는제조방법에따라녹차

^,

(semi-oxidation tea),

(oxidation tea),

(post-fermented tea)

^,

^,

^,

^,

^[6].

^.

gallic acid, methoxy phenolic compound, poly-flavonoids

^,

^,

^,

이 증가되고 있다

^[7

^10].

Yunnan pu-erh tea, Hunan Fu-zhuan tea, Hubei Qing- zhuan tea, Sichuan Bian-xiao tea, Guangxi Liu-bio tea

^,

Yunnan pu-erh tea

^.

⁽

⁾

⁽

⁾

^,

[7].

후발효차는미생물의발효정도에따라서차의맛과향이

A Comparison of the Microbial Diversity in Korean and Chinese Post-fermented Teas

Byung-Hyuk Kim

, Jong-Ok Jang

, Jae-Ho Joa

, Jin-Ah Kim

, Seung-Yeob Song

, Chan Kyu Lim

, Chun Hwan Kim

, Young Bin Jung

, Ki-Cheol Seong

, Hee-Sik Kim

, and Doo-Gyung Moon

*

1

Agricultural Research Institute for Climate Change, National Institute of Horticultural and Herbal Science, RDA, Jeju 63240, Republic of Korea

2

Cell Factory Research Center, KRIBB, Daejeon 34141, Republic of Korea

Tea is the most popular beverage in the world. The three main types are green, black, and post-fermented.

Post-fermented teas are produced by the microbial fermentation of sun-dried green tea leaves (Camellia sinensis). In this study, the composition of the bacterial communities involved in the production of traditional oriental post-fermented teas (Korean algacha, dancha, and Chinese pu-erh) were investigated using 16S rRNA gene analysis. The dominant microorganisms present in the post-fermented teas included the α-proteobacteria Rhodobacteraceae and Sphingomonas, and the γ-proteobacteria Pantoea. Cluster analysis confirmed that the microbial populations present in both Korean and Chinese post-fermented teas grouped into the same class. Interestingly, the dominant microorganism present in the Korean post- fermented teas was a bacterium, while for the Chinese post-fermented tea, it was a fungus.

Keywords: Camellia sinensis, double gradient-DGGE, microbial community, post-fermented tea, tea

*Corresponding author

Tel: +82-64-741-2564, Fax: +82-64-749-2066 E-mail: [email protected]

© 2017, The Korean Society for Microbiology and Biotechnology

결정되기때문에

^,

^.

^,

^,

^[11

14].

^,

여미생물에관한연구는전무한상태이며

^,

^.

^,

^,

^.

하게여겨지고있다

^[15

^17].

물발효차에있어숙성미생물군집구조를규명하는연구는 후발효차의특성과품질을파악하는데매우중요한사항이 라판단된다

.

^1%

^.

^{16S rRNA}

^{[16, 18].}

한국전통후발효차

⁽

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⁾

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재료 및 방법

시료

한국전통후발효차인알가차와단차는

²⁰¹⁵

^,

²⁰¹⁵

^O

⁽

¹⁾

^T

⁽

²⁾

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DNA 추출

Total DNA

^{0.5 g}

^{, Fast DNA} Spin kit for Soil (MP bio, Korea)

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^DNA

NanoDrop ND-2000 (Thermo Scientific, USA)

^.

미생물 군집분석을 위한 PCR

미생물다양성을확인하기위해시료로부터얻은

^DNA

¹

^PCR

nested-PCR

^[19,

20]. 1

^PCR

27F (5'-AGA GTT TGA TCM TGG CTC AG-3')

1541R (5'-AAG GAG GTG ATC CAN CCR CA- 3')

⁵⁰

^l

1× PCR buffer, 20 mM MgCl

, 40 mM dNTP mixture,

primer (1

M), template DNA

0.5 U Taq polymerase

^{. PCR}

⁹⁴

^{5 min}

^DNA

pre-denaturation

^{, 94}

45 sec denaturation, 56

^{45 sec} annealing, 72

45 sec extension

^{25 cycles}

⁷²

^{7 min}

final extension

^. PCR

ethidium bromide

1% agarose gel

. Double Gradient-Denaturing Gradient Gel Electrophoresis (DG-DGGE)

^{, 1}

^PCR

²

^PCR

^,

^16S rDNA

^primer

⁴⁰

^GC- clamp

341F-GC (5'-CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG GCC TAC GGG AGG CAG CAG-3')

786R (5'-CTA CCA GGG TAT CTA ATC-3')

^[21

^{23]. 2}

^PCR

PCR

¹

^PCR

^,

PCR

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^{5 min}

^DNA

^pre- denaturation

^{, 94}

45 sec denaturation, 60

45 sec annealing, 72

45 sec extension

⁷²

^{7 min}

final extension

. Annealing

⁶⁰

^{1 cycle}

^0.5

20 cycles

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⁵⁰

^{15 cycles}

touch-down PCR

^.

²

^PCR

^DG-DGGE

^[20].

DG-DGGE 운전조건

PCR

^Dcode

System (Bio-Rad)

^DG- DGGE

[20, 24]. DG-denaturing gradient gel

⁸

12% polyacrylamide (37.5:1=acrylamide:bisacrylamide)

^urea

formamide

⁴⁰

^70%

^,

DG-DGGE gel

^PCR

³⁰

^l

^loading

1× TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA, pH 8.0)

⁶⁰

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²¹

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^DG- DGGE gel

ethidium bromide (1:10,000)

^, UV

^[16].

16S rRNA gene clone library

16S rRNA gene clone library

^PCR

^27F

(5'-AGA GTT TGA TCM TGG CTC AG-3')

1541R (5'-

AAG GAG GTG ATC CAN CCR CA-3')

[23].

^primer

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^{10× PCR}

buffer, 20 mM MgCl

, 40 mM dNTP mixture,

^primer (1

M), template DNA

0.5 U Taq polymerase

^PCR

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⁹⁴

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^{5 min}

DNA

pre-denaturation

^{, 94}

^C

1 min denaturation, 60

1 min annealing, 72

1 min 30 sec extension

⁷²

^{5 min}

final extension

^. 16S rRNA gene clone library

^PCR

1% agarose gel

^loading

^{1,500 bp}

^band

, LaboPass Gel Extraction kit (CosmoGen)

, All-in-one vector (Biofact)

^ligation

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plasmid

E. coli DH5

(RBC Korea)

^{LB +} kanamycin (50

^g/ml)

16S rRNA

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염기서열분석

DG-DGGE gel

^DNA

들을회수하기위하여각각의

^band

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3

^{DW 50}

^l

⁴

^{. Band}

^DNA

^nested PCR

^primer

^{, PCR}

agarose gel

DNA recovery kit (Biofact)

^cloning

^{. Cloning}

All-in-one vector (Biofact)

^,

^,

^{16S rRNA}

3730xl DNA Analyzer (ABI)

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NCBI (www.ncbi.nlm.nih.gov)

^GenBank database

BLAST search

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또한

, Phylogenetic tree

^ClusterX

^{, MEGA 5.0}

neighbor-joining

method

^[25].

미생물 군집 비교를 위한 정량 Real time PCR

각시료별미생물의군집의크기를정량적으로평가하기

위해

^qPCR

^.

27F

518R (5'-ATT ACC GCG GCT GCT GG-3') primer

[26], E. coil DH5

^{total DNA}

PCR

^{491 bp}

^PCR

^.

^,

SR-4F (5'-AG CCG CGG TAA TTC CAG CT-3')

SR-7R (5'-TCC TTG GGC AAA TGC TTT CGC-3')

, Aspergillus total DNA

^{388 bp}

^PCR

^[27].

^PCR

All-in-one vector (Biofact, Korea)

^cloning

^{, plasmid} DNA

LaboPass Mini Kit (CosmoGen, Korea)

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M13-20F and M13-20R

(All-in-one Vector Systems manual)

, BLAST search

^.

plasmid DNA

real-time PCR

^melting curve (Bio-Rad, USA)

(artificial standard clone)

^.

정량

^PCR

CFX96 Touch

Real-Time PCR Detection System (Bio-Rad)

^iTaq

^SYBR

^Green Supermix with ROX (Bio-Rad)

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^16S rRNA

^PCR

^primer

⁹⁵

^{15 min}

pre-denaturation

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30 sec denaturation, 67

30 sec annealing, 72

30 sec extension

fluorescence

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final extension

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5 min

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fluorescence

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팡이군집의정량을위해서앞서사용된

PCR program

용하였으며

, annealing

⁶⁹

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serial dilutions

^Real time-PCR

^{, DNA}

NanoDrop ND- 2000 (Thermo Scientific, USA)

^{1 ng/}

^l

^{[16, 28].}

결과 및 고찰

미생물후발효차는동아시아에서제한적으로생산되며

^,

^,

^,

^,

^.

^·

⁽²

⁾

⁽²

⁾

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DG-DGGE를 통한 미생물 군집분석

한국의알가차와단차그리고중국의보이차는전통미생 물발효차로서연구에사용한시료는

²⁰¹⁵

¹

^.

^DG- DGGE

^.

^DGGE

homo-duplexs

를분리하지못하는데비해

^DG-DGGE

하기때문에보다정확한염기서열간의차이를구분할수있 다는장점을가지고있다

^[29

^31].

^{total DNA}

^,

universal primer

^341F-GC

786R primer

^PCR

^,

^DG-DGGE

^{(Fig. 1).}

^,

해

DG-DGGE gel

⁷

³

, NCBI (www.ncbi.nlm.nih.gov)

GenBank database

BLAST search (blastn)

(Table 1). DG-DGGE

^genus

⁶

^, Pseudomonas (band 1, 2, 3) Burkholderia (band 4), Shigella sonnei (band 5), Ralstonia solanacearum (band

6, 7, 9), Sphingomonas (band 8)

Enterobacter (band 10)

^{(Table 1).}

미생물군집구조분석을수행한결과

^DG-DGGE

^,

^{. 9}

^,

Uncultured Ralstonia (LN624415)

(Fig. 1, Table 1). DG-DGGE

^,

^,

^,

^.

^.

^,

^.

16S rRNA

16S rRNA gene clone library

^.

universal primer

^27F

^1542R

^PCR

^, cloning

^{16S rRNA}

^, NCBI

BLAST search

^.

^{16S rRNA}

Rhodobacteraceae (27.6%), Pantoea ananatis (13.8%), Roseomonas (13.8%)

Uncultured bacterium (13.8%)

^,

Rhodobacteraceae (27.1%), Acidovorax (23.7%), Pantoea ananatis (10.2%)

Pseudomonas (10.2%)

^.

¹

Pantoea allii (19.4%), Gemmobacter caeni (16.1%), Sphingomonas (16.1%)

Reyranella (12.9%)

^,

²

Sphingomonas (17.7%), Exiguobacterium (16.1%), Pantoea allii (16.1%)

Klebsiella (12.9%)

Table 1. Identity of the bands obtained from 16S rRNA gene DG-DGGE profile of the oriental traditional post-fermented tea.

Band no. Accession no. Description Similarity

1 NR113600 Pseudomonas azotoformans strain NBRC 12693 100%

2 KX769816 Pseudomonas sp. strain PDSM-TXL-1 99%

3 KF147044 Pseudomonas azotoformans strain R2SpM3P1C3 99%

4 KR154612 Burkholderia sp. BL9 I2R2 96%

5 NR104826 Shigella sonnei strain CECT 4887 99%

6 JQ655458 Ralstonia solanacearum strain in4ss52 100%

7 JQ655458 Ralstonia solanacearum strain in4ss52 100%

8 KU199717 Sphingomonas sp. SG124 98%

9 LN624415 Uncultured Ralstonia sp. OH 55 99%

10 KR189894 Enterobacter sp. UIWRF0151 99%

Fig. 1. DG-DGGE patterns of 16S rDNA fragments of oriental

post-fermented tea bacteria. (A) Algacha, (B) Dancha, (C) Pu-

erh tea 1, and (D) Pu-erh tea 2.

으로우점하는것을확인하였다

(Fig. 2, Table 2).

한국후발효차인알가차와단차에가장우점하는미생물 은

Rhodobacteraceae (

-proteobacteria,

^{; 27.6%,}

^{; 27.1%)}

^,

²

Pantoea allii (

-proteobacteria,

^{1; 19.4%,}

^{2; 16.1%)}

Sphingomonas (

-proteobacteria,

1; 16.1%,

^{2; 17.7%)}

^.

-proteobacteria (Rhodobacteraceae, Roseomonas, Gemmobacter, Reyranella, Sphingomonas, Thioclava)

-proteobacteria (Acidovorax, Aeromonas, Klebsiella, Pantoea, Pseudomonas

⁾

^,

-proteobacteria

^- proteobacteria

^79%

(Fig. 3, Table 2). Pantoea

등에서분리가되었으며

, meta-genome

서우점하며

^,

Sphingomonas

^Bacillus

^{[32, 33].}

, Polyphenol tannic acid

gallic acid

gallic acid decarboxylase

Pantoea agglomerans T71

, gallic acid

tannic acid

^[34].

, Pantoea dispersa Y08

carotenoids

[35]. Sphingomonas

와

Rhodobacteraceae

^,

^[36

^41].

gallic acid, carotenoides

^Pantoea

Sphingomonas

^.

16S rRNA gene clone library

Rhodobacteraceae

²

, Pantoea vegans (

-proteobacteria)

^.

^,

Pantoea allii

Sphingomonas

, Gemmobacter, Klebsiella, Reyranella, Shigella, Thioclava

^.

^,

SPSS program (SPSS V18, USA)

^[42].

기반하여분석한결과는

^{Fig. 4}

중국의후발효차가뚜렷하게구분되었다

^.

^.

^. Fig. 2. Dominant bacterial operational taxonomic units detected in the post-fermented tea 16S rRNA gene clone libraries.

(A) Algacha, (B) Dancha, (C) Pu-erh tea 1, and (D) Pu-erh tea 2.

후발효차 우점 미생물의 군집 크기 비교

본연구는후발효차의군집분석을수행하여우점미생물 를분석하고

^,

^.

Real-time PCR

^{(Fig. 5).}

^{1.46 × 10}

^{1.32 × 10}

copy number,

^{1.69 × 10}

^{6.34 × 10}

copy number,

¹

6.00 × 10

^{4.11 × 10}

copy number,

²

^{2.63 × 10}

5.95 × 10

copy number

^.

^{1.46 × 10}

copy number

^,

^{6.00 × 10}

^copy

number

차미생물군집에비해약

^2.5

^.

곰팡이의총균수분석결과는알가차

5.61 × 10

^{2.63 ×} 10

copy number,

^{2.49 × 10}

^{3.09 × 10}

copy number,

¹

^{1.47 × 10}

^{1.75 × 10}

copy number,

²

^{1.86 × 10}

^{1.68 × 10}

copy number

^.

^{1.47 × 10}

copy number

^,

^{5.61 × 10}

copy number

^2.6

^.

^,

^.

Aspergillus

Penicillium

Table 2. Analysis of microbial community structure of the oriental traditional post-fermented tea based on 16S rRNA gene clone library analysis.

Operational taxonomic unit (OTU)

Algacha Dancha Pu-erh tea 1 Pu-erh tea 2

Clone number

Composi- tions

Clone number

Composi- tions

Clone number

Composi- tions

Clone number

Composi- tions

Acidovorax delafieldii 14 23.7%

Acinetobacter 4 6.9%

Aeromonas hydrophila 2 3.4%

Aquimonas voraii 1 1.6%

Burkholderia gladioli 2 3.4%

Exiguobacterium aurantiacum 4 6.9% 4 6.8% 6 9.7% 10 16.1%

Gemmobacter caeni 10 16.1% 3 4.8%

Klebsiella oxytoca 4 6.5% 8 12.9%

Methylobacterium radiotolerans 4 6.5%

Pantoea allii 12 19.4% 10 16.1%

Pantoea ananatis 8 13.8% 6 10.2% 4 6.5%

Pantoea vagans 6 10.3% 4 6.8%

Pseudomonas chengduensis 4 6.9% 6 10.2% 2 3.2%

Reyranella massiliensis 8 12.9% 4 6.5%

Roseomonas 8 13.8% 0.0%

Shigella flexneri strain 2 3.2% 1 1.6%

Sphingomonas oryziterrae 10 16.1% 11 17.7%

Thioclava atlantica 5 8.1% 6 9.7%

Uncultured bacterium clone

HH_aai33d01 8 13.8% 3 4.8%

Uncultured bacterium clone MACA-MR13 3 5.1%

Uncultured Rhodobacteraceae bacterium clone 3D6

16 27.6% 16 27.1%

Uncultured Rhodospirillales bacterium clone blastula 6

2 3.4%

58 100.0% 59 100.0% 62 100.0% 62 100.0%

Fig. 3. Phylogenetic tree based on 16S rRNA gene sequence using the neighbor-joining methods showing the oriental tradi-

tional post-fermented tea. *16S rRNA gene clone library, **DG-DGGE.

되었으며

^,

[13, 43, 44].

^,

^.

본연구는한국후발효차와중국후발효차의미생물군집 을분자생물학적기법으로분석하였다

^.

^,

Rhodobacteraceae

^,

차에는

^Pantoea

Sphingomonas

^.

^,

⁽

⁾

^,

^,

^.

^.

^,

^.

요 약

차는세계적으로인기있는음료로서

^,

(

^),

⁽

^),

⁽

^),

^.

(Camellia sinensis)

^.

⁽

^,

⁾

⁽

²

⁾

^{16S rRNA}

^.

발효차에 우점하는 미생물은 α

-proteobacteria

Rhodobacteraceae

Sphingomonas,

-proteobacteria

^Pantoea

^.

^cluster

^,

^.

^,

^.

Acknowledgments

This study was supported by 2016 the RDA Fellowship Program (PJ011964) of National Institute of Horticultural and Herbal Science, Rural Development Administraion, Republic of Korea.

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