Isolation and Characterization of Wild Yeasts for Improving Liquor Flavor and Quality

주류의 풍미 및 품질 향상을 위한 야생 효모의 분리 및 특성분석

백성열¹, 이유정^1,2, 김재현¹, 여수환¹*

1농촌진흥청국립농업과학원농식품자원부발효식품과

2충북대학교농업생명과학대학식품생명공학과

Received: February 24, 2015 / Revised: March 6, 2015 / Accepted: March 9, 2015

서 론

우리나라의전통주는주로찹쌀이나멥쌀을원료로하고 누룩을발효제로사용하여만들어진다

^.

^,

^,

^,

^.

생성되어색과함께품질의조화를이룬다

^[22].

전통주의담금과정중미생물학적변화에대한연구는지 금까지많은연구자들에의하여진행되었으며

^{, Seo}

^[21]

^·

^{, Lee}

^{[14, 15]}

은효모종류를달리하여탁주술덧의품질특성과향기성분 을

^{, Kim [12]}

^·

^.

^Kwon

^[13]

^PCR-

DGGE

사한결과

, Pichia kudriavzevii, Saccharomyces cerevisiae, Asidia idahoensis, Kluyveromyces marxianus, Saccharomycopsis fibuligera

Torulaspora delbrueckii

⁶

^,

²

Pichia kudriavzevii

Saccharomyces cerevisiae

^.

최근 와인 제조 분야에서는

S. cerevisiae

non- Saccharomyces

^.

non-Saccharomyces

며

^,

분증가에중요한영향을준다고알려져있다

^[3].

사및미생물학자들은

non-Saccharomyces

^,

[4]. non-Saccharomyces

S. cerevisiae

^[4].

Hanseniaspora uvarum

Kloeckera

apiculata

며

^,

Isolation and Characterization of Wild Yeasts for Improving Liquor Flavor and Quality

Seong Yeol Baek

, You Jung Lee

, Jae Hyun Kim

, and Soo-Hwan Yeo

*

1

Fermented Food Division, Department of Agro-food Resource, NAAS, RDA, Jeollabuk-do 565-851, Republic of Korea

2

Department of Food Science and Biotechnology, Chungbuk National University, Cheongju 362-763, Republic of Korea It has been known for some time to the wine industry that non-Saccharomyces yeasts play an important role in increasing volatile components through the secretion of extracellular enzymes. The objective of this study was to investigate what types of enzymes are produced by 1,007 non-Saccharomyces yeast strains isolated from Korean fermented foods. Among 1,007 yeast strains, the 566, 45 and 401 strains displayed β-glucosidase, glucanase and protease activity, respectively. In addition, the 563 and 610 strains possessed tolerances against cerulenin and TFL, and the 307 strain was tolerant to 15% ethanol. Yeasts pro- ducing harmful biogenic amines and hydrogen sulfide were excluded from further study, and eventually 12 yeast strains belong- ing to the genera Wickerhamomyces, Hanseniaspora, Pichia, Saccharomyces were identified, based on the 26S rRNA gene sequences. Among the 12 strains, the 9 and 5 strains possessed glucose and ethanol tolerance, respectively. Yeasts belonging to the genus Saccharomyces produced more than 8% alcohol, but non-Saccharomyces yeasts produced only 3% alcohol.

Keywords: Yeast, non-Saccharomyces, extracellular enzyme, fermentation, liquor

*Corresponding author

Tel: +82-63-238-3610, Fax: +82-63-238-3843 E-mail: [email protected]

© 2015, The Korean Society for Microbiology and Biotechnology

관찰되었다

^[6].

성에중요한역할을하는것으로알려져있다

^[4].

진에의해

non-Saccharomyces

Pichia anomala Y197-13

^[11].

일본의대표술인사케의향기성분중

isoamyl acetate

^,

^alcohol acetyltransferase

^esterase

^isoamyl alcohol

acetyl coenzyme A

[28]. Isoamyl alcohol

^leucine

-ketoisocaproate

[10]. Leucine

-isopropyl malate

⁽

^-IPM)

^L-

leucine

^-

isopropyl malate

-kotoisovalerate

^[2].

isoamyl alcohol

^.

^-IPM

^L-leucine

isoamyl alcohol

isoamyl acetate

^.

5,5,5-trifluor-DL-leucine (TFL)

^L-

leucine

는것으로보고되었다

^[1].

L-leucine

^LEU4

⁽

^-IPM)

isoamyl alcohol

^[19].

효모가내는또다른중요한향기성분은에스테르인

^ethyl caproate

^[28].

^(ethanol, caproic acid), alcohol acyl transferase

^esterase

^[25].

^cerulenin

^,

균주의탐색은

cerulenin

려져있다

^[8].

본연구의목적은발효식품에서분리된효모의특성연구 를통해

^,

^.

^- glucosidase, glucanase, protease, amino acid decarboxylase

^,

^,

^.

^,

^,

^,

¹²

^.

재료 및 방법

효모 균주 및 사용 시약

본실험에사용한효모는전국에서수집한발효액등의발 효식품

¹²⁶

^1,007

^.

주로시판효모인

Fermivin (Sacchromyces cerevisiae, Gist Brocades, Denmark), Frootzen (Pichia kluyvery, Chr.

Hansen, Denmark)

^.

수집된발효식품

^{(10 g)}

0.85% NaCl (90 ml)

tYPD (1% yeast extract, 2% peptone, 2% glucose, 0.0018% tartaric acid)

^[16]

¹⁰⁰

^l

30

C

48

.

,

^,

^YPD (1% yeast extract, 2% peptone, 2% glucose)

사용하여순수분리하였다

^.

^YPD

배지에배양후

^{, glycerol}

^20%

⁸⁰

^C

^.

분리효모의β

-glucosidase (EC 3.2.1.21.), glucanase (EC 3.2.1.6.), protease (EC 3.4.), amino acid decarboxylase (EC

4.1.1.)

^.

효소의적합한한천평판을만든후분리효모를접종하여효 소활성 유무를 확인하였다

^.

-Glucosidase

^esculin (Sigma-Aldrich Co., St. Louis, USA)

^YPD

0.5% (w/v)

^{pH 5.0}

, 1% ferric ammonium citrate (Sigma-Aldrich Co., St. Louis, USA)

^{2% (v/v)}

^.

³⁰

^C

²⁴

[7]. Glucanase

-D-glucan (Sigma-Aldrich Co., St.

Louis, USA)

^YPD

0.2% (w/v)

³⁰

^C

⁴⁸

congo red (Sigma-Aldrich Co., St. Louis, USA)

^0.1%

으로희석한용액으로염색후집락주변에형성된투명환 의 생성 여부로 활성을 확인하였다

[23]. Protease

^YM (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1%

glucose)

skim milk

^1%

³⁰

^C

⁴⁸

^.

amino acid decarboxylase

^YPD

1% (histidine, tyrosine, phenylalanine, tryptophane, lysine, leucine) (Sigma-Aldrich Co., St. Louis, USA), bromocresol purple (Sigma-Aldrich Co., St. Louis, USA)

^0.006%

도록첨가하고

^{pH 5.3}

주변보라색환의생성여부에따라평가하였다

^[18].

에탄올, 아황산 내성 및 황화수소 생성능 분석

에탄올내성평가는

^YPD

5%, 10%, 15%

부에따라내성을평가하였다

^[17].

^YPD

tartaric acid (Sigma-Aldrich Co., St. Louis, USA)

^1.8%

^{1 M K}

S

O

(Daejung Chemicals Co., Ltd, Korea)

^SO

농도가

^{20 mg}

여부에따라내성을평가하였다

^[5].

^Biggy

(Becton Dickinson GmbH, Heidelberg, Germany)

³⁰

^C

⁴⁸

^,

^[5].

Cerulenin 및 5,5,5-trifluor-DL-leucine 내성

Cerulenin

5,5,5-trifluor-DL-leucine (TFL) (Sigma- Aldrich Co., St. Louis, USA)

^{0.67% YNB} (yeast nitrogen base), 2% glucose, 2% agar

cerulenin (25

^M)

TFL (1 mM)

³⁰

^C

⁴⁸

^[26].

효모의 동정

분리효모를동정하기위하여

^{26S rRNA}

^D1/D2

^.

^[27]

^{NL1 (5'-} GCATATCAATAAGCGGAGGAAAAG-3')

^{NL4 (5'-} GGTCCGTGTTTCAAGACGG-3')

^{26S rRNA} D1/D2

^,

^ZEOTECH

^.

National Center for Biotechnology Information (NCBI, Bethesda, MD, USA)

^BLAST

^{26S rRNA}

^,

DNASTAR pro software (SeqMan Pro, Ver 8.1.5., Lasergene)

^.

(phylogenetic tree)

^GenBank

에보고된다른효모균주의

^{26S rRNA}

석하여

, MEGA version 4.0 [24]

neighbor-joining method [20]

^.

당 및 에탄올 내성 측정

분리효모의포도당에대한내성측정은포도당농도를

^20,

30, 40%

^YPD

(yeast extract 1%, peptone 2%, glucose 1.5%, pH 6.5)

³⁰

^C

180 rpm

²⁴

^{600 nm}

^.

포도당

^1%

^YPD

으로하였다

^.

0, 5, 10, 15%

농도로함유된

^YPD

^{10 ml}

배양액을

^0.1%

²⁵

^C

⁵

^,

600 nm

^.

에탄올 발효능 분석

효모의에탄올생성량은전배양한효모를포도당

^25%

^YPD

^{100 ml}

^0.1%

30

C

⁴⁸

, 10,000 rpm

10

^.

^{100 ml}

^{70% (v/v)}

하고다시증류수를이용하여

^{100 ml}

를이용하여측정하였다

^[9].

결과 및 고찰

효모의 효소활성

효모의 β

-glucosidase, glucanase, protease

^amino acid decarboxylase

^{Table 1}

^.

-Glucosidase

^1,007

⁵⁶⁶

, glucanase

⁴⁵

. Protease

⁴⁰¹

^.

(biogenic amine, BAs)

^,

^.

^,

(histamine)

^(tyramine)

^[18].

Amino acid decarboxylase

^,

, histidine

69

^{, tyrosine}

³⁰⁶

, phenylanine

¹⁷¹

^,

Table 1. Yeast isolates used in this study.

β

-Glucosidase Glucanase Protease AA decarboxylation

His Tyr Phe Trp Lys Leu

566 45 401 69 306 171 23 197 198

Tolerance Ethanol tolerance H

S production

Cerulenin TFL 5% 10% 15%

563 610 934 792 307 500

tryptophane

²³

^{, lysine}

¹⁹⁷

^{, leucine}

¹⁹⁸

^.

cerulenin, TFL, SO

,

주및황화수소생성능에대한결과는

^{Table 1}

다

. Cerulenin, TFL

^{563, 610}

^{, SO}

²²

^.

^,

^,

5%, 10%, 15%

934, 792, 307

^.

⁵⁰⁰

^.

위결과를바탕으로

biogenic amine decarboxylase

^,

¹

^,

, cerulenin, TFL, SO

,

¹²

(Table 2).

β

-Glucosidase

요한역할을한다고알려져있으며

^,

non-Saccharomyces

^{[7, 23].}

, Hansenispora vinea

^Candida

glucosidase

^[23].

-Glucosidase

S. cerevisiae, P. kluyvery

N43-8, N77-4, SM2-7, Y685, BY30-1, M1-9, SD1-2

⁷

^.

Glucanase

S. cerevisiae, P. kluyvery

, N43-8, Y685 2

타내었다

. Glucanase

^Candida stellata, C. hellenica, C. pulcherrima, Kloeckera apiculata

[23]. Protease

^,

⁷

^.

, amino acid decarboxylase

^.

Cerulenin, TFL, SO₂, 알코올 내성 균주

효모가생성하는고급알코올류와에스테르계열의성분

들이주류의향을증진시킬수있음을감안하여

, cerulenin

과

^TFL

. Cerulenin

TFL

isoamyl alcohol

caproic acid

^[8].

¹²

cerulenin

^TFL

^{8, 7}

(Table 3). N43-8, N56-10, N77-4, SM2-7, Y447, Y685

^HP1-2

^cerulenin

^TFL

^{, CM4-5}

cerulenin

. A9-1, BY30-1, M1-9, SD1-2

S. cerevisiae

. non-Saccharomyces

P. kluyvery

^cerulenin

^TFL

^,

S. cerevisiae

^. Cerulenin

^TFL

⁷

Table 2. Production of extracellular enzymes of selected wild type yeasts and commercial yeasts.

Strains

-Glucosidase Glucanase Protease AA decarboxylation

His Tyr Phe Trp Lys Leu

N43-8 +++ + - - - - - - -

N56-10 - - + - - - - - -

N77-4 +++ - + - - - - - -

SM2-7 +++ - + - - - - - -

Y447 - - - - - - - - -

Y685 +++ + - - - - - - -

A9-1 - - + - - - - - -

BY30-1 ++ - - - - - - - -

CM4-5 + - + - - - - - -

M1-9 ++ - + - - - - - -

SD1-2 ++ - + - - - - - -

HP1-2 + - - - - - - - -

Frootzen ++ - - - - - - - -

Fermivin + - + - - - - - -

Positive result clear or fluorescence zone of more than 1 mm margin from edge of colony: +, 1 mm; ++, 2 mm; +++, 3 mm; –, negative result.

1

Amino acid decarboxylase activity means biogenic amines production.

올류와에스테르계열의향기성분을생산할수있을것으로 예상되며

^,

^{. SO}

²

^A9-1

HP1-2

^.

S. cerevisiae

^SO

^.

^,

N56-10, Y447

^.

효에의해생성될수있는데

^[5],

를포함하여대부분의효모는황화수소생성능이낮았으며

^, SM2-7, Y447, CM4-5, M1-9, SD1-2, HP1-2

^.

효모 동정

선발된효모

¹²

^{26S rRNA}

^D1/D2

^{Fig. 1}

^.

¹²

^C.

tropicalis, H. opuntiae, H. uvarum, P. kudriavzevii, S.

cerevisiae

Wickerhamomyces anomalus

. SD1-2, M1-9, CM4-5, A9-2, BY30-1

^S.

cerevisiae

^{. HP1-2}

H. opuntiae, N56- 10

H. uvarum, N43-8, SM2-7, Y685

^W.

anomalus, Y447

C. tropicalis, N77-4

^P.

kudriavzevii

^.

효모의 내당성 및 에탄올 내성

주류제조에이용되는효모는고농도의당에서충분히생 육을하면서알코올발효를진행할수있어야하므로선발 된효모

¹²

20%, 30%, 40%

^YM

^(Fig.

2). S. cerevisiae

^BY30-1

^,

^40%

^YM

. non-Saccharomyces

^H.

opuntiae HP1-2, C. tropicalis Y447

^40%

^YM

^.

일반적으로일정수준이상의에탄올농도에서는효모생 육이저해되어더이상알코올발효가일어나지않는것으

로알려져있으며

^[24],

모는에탄올에우수한내성을보유하는것이바람직하다

^.

¹²

5%, 10%, 15%

^YM

^YM

^{(Fig. 3),}

5

S. cerevisiae

^BY30-1

^10%

에탄올첨가한

^YM

으나

^15%

^.

non-Saccharomyces

^{. 5%}

, H. uvarum N56-10, H.

Table 3. The cerulenin, TFL, SO

and ethanol tolerance and production of H

S of selected wild type yeasts and commercial yeasts.

Strains Cerulenin TFL SO

Ethanol con. H

S

production

5% 10% 15%

N43-8 + + - + + + +

N56-10 + + - + - - +

N77-4 + + - + + + +

SM2-7 + ++ - + + + -

Y447 + + - + - - -

Y685 + + - + + + +

A9-1 - - + + + + +

BY30-1 - - - + + - +

CM4-5 + - - + + + -

M1-9 - - - + + + -

SD1-2 - - - + + + -

HP1-2 + + + + + - -

Frootzen + + + + + - ++

Fermivin - - ++ + + + +

Tolerance symbols ; + growth, - no growth.

1

-, no color; +, light brown; ++, dark brown.

Fig. 1. Phylogenetic tree based on 26S rDNA sequences showing the relative genetic position of the selected 12 yeast strains isolated from Korean fermented food.

Fig. 2. Glucose tolerance of selected wild yeast strains and commercial yeasts. The selected wild yeast strains were grown shaking

on 100 ml of YPD broth medium containing 1, 20, 30, and 40% glucose in 250 ml flask at 30

C for 24 h. Glucose tolerance was determined

by absorbance of yeasts growth at 660 nm. The data were averages based on three trials.

opuntiae HP1-2

^.

그러나

^10%

^YM

은

P. kudriavzevii N77-4

, P. kudriavzevii N77-4

^15%

non-Saccharomyces

^.

효모의 에탄올 발효능

효모

¹²

^25%

^YM

⁴⁸

(Fig. 4), S. cerevisiae SD1-2

^9.5%

, S. cerevisiae

^7.3%

^9.5%

^. non-Saccharomyces

W. anomalus N43-8, SM2-7

^4.7%

^,

소

^2.2%

^4.7%

^{. Kim}

^[11]

^P.

anomala Y197-13

량이

^11.1%

있다고보고하였다

^.

^Pichia

non-Saccharomyces

^{[3, 23]}

⁷

non-Saccharomyces

^.

요 약

non-Saccharomyces

^,

^.

Fig. 3. Ethanol tolerance of selected wild yeast strains and commercial yeasts. The selected wild yeast strains were grown static culture on 10 ml of YPD broth medium containing 0, 5, 10, and 15% ethanol in test tube at 25

C for 5 days. Ethanol tolerance was deter- mined by absorbance of yeasts growth at 660 nm. The data were averages based on three trials.

Fig. 4. Ethanol production by selected wild yeast strains and commercial yeasts. The selected wild yeast strains were grown static

culture on 100 ml of YPD broth containing 25% glucose in 500 ml flask at 30

C for 48 h. Ethanol production was determined by alco-

holometer. The data were averages based on three trials. *, significance between commercial yeasts (Fermivin and Frootzen) and selected

strains, p < 0.05.

서분리된효모

^1,007

^.

^,

-glucosidase 566, glucanase 45, protease 401

, AA decarboxylase

His 69, Tyr 306, Phe 171, Trp 23, Lys 197, Leu 198

. Cerulenin

^TFL

^563, 610

^{, 15%}

³⁰⁷

^.

⁵⁰⁰

. AA decarboxylation

¹²

^26S rDNA

, C. tropicalis, H. opuntiae, H. uvarum, P. kudriavzevii, S. cerevisiae, W. anomalus

^{. 12}

9

^,

⁵

^.

Saccharomyces

^8%

, non-Saccharomyces

^3%

^.

Acknowledgments

This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Develop- ment (Project No. PJ00947701)” Rural Development Administra- tion, Republic of Korea.

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