Microbiol. Biotechnol. Lett. (2015), 43(1), 56 64 pissn 1598-642X eissn 2234-7305 Microbiology and Biotechnology Letters 주류의풍미및품질향상을위한야생효모의분리및특성분석 백성열 1, 이유정 1,2, 김재현 1, 여수환 1 * 1 농촌진흥청국립농업과학원농식품자원부발효식품과 2 충북대학교농업생명과학대학식품생명공학과 Received: February 24, 2015 / Revised: March 6, 2015 / Accepted: March 9, 2015 Isolation and Characterization of Wild Yeasts for Improving Liquor Flavor and Quality Seong Yeol Baek 1, You Jung Lee 1,2, Jae Hyun Kim 1, and Soo-Hwan Yeo 1 * 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 producing harmful biogenic amines and hydrogen sulfide were excluded from further study, and eventually 12 yeast strains belonging 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 서 론 우리나라의전통주는주로찹쌀이나멥쌀을원료로하고누룩을발효제로사용하여만들어진다. 전통주는담금후, 누룩중의미생물에의한효소작용으로원료성분이분해되어당분, 아미노산, 유기산등이생성된다. 그리고효모나젖산균등의미생물에의한알코올발효로휘발성향미성분이생성되어색과함께품질의조화를이룬다 [22]. 전통주의담금과정중미생물학적변화에대한연구는지금까지많은연구자들에의하여진행되었으며, Seo 등 [21] 은탁 약주발효과정중의미생물균총변화를, Lee 등 [14, 15] 은효모종류를달리하여탁주술덧의품질특성과향기성분을, Kim [12] 은증자와무증자탁 약주의품질특성과발효미생물분석을연구하였다. 또한 Kwon 등 [13] 은 PCR- DGGE 법을이용한막걸리발효중의미생물다양성을조 *Corresponding author Tel: +82-63-238-3610, Fax: +82-63-238-3843 E-mail: yeobio@korea.kr 2015, The Korean Society for Microbiology and Biotechnology 사한결과, Pichia kudriavzevii, Saccharomyces cerevisiae, Asidia idahoensis, Kluyveromyces marxianus, Saccharomycopsis fibuligera 및 Torulaspora delbrueckii로 6종이주요효모로나타났으며, 우점효모는배양 2일까지 Pichia kudriavzevii 가이후에는 Saccharomyces cerevisiae로나타났다고보고하였다. 최근와인제조분야에서는 S. cerevisiae가아닌 non- Saccharomyces 효모에관한연구가활발히진행되고있다. non-saccharomyces 효모는포도밭에서기인한야생효모이며, 다양한효소를세포밖으로분비함으로써와인의향기성분증가에중요한영향을준다고알려져있다 [3]. 와인제조사및미생물학자들은 non-saccharomyces 효모가와인의관능적특성에관여하고있음을인지하고, 산업적응용에대한관심을가지고있다 [4]. non-saccharomyces 효모의일부종과 S. cerevisiae와의혼합발효를통해와인의품질을향상시킨연구결과도보고되었다 [4]. 발효능이낮은레몬형효모인 Hanseniaspora uvarum와이의무성세대인 Kloeckera apiculata는포도과피표면에가장많이존재하는미생물이며, 와인의알코올발효초기에우점종으로작용하는것으로
Characterization of Yeasts for Improving Liquor Quality 57 관찰되었다 [6]. 이러한효모들은와인의휘발성향기성분생성에중요한역할을하는것으로알려져있다 [4]. 국내연구진에의해 non-saccharomyces인 Pichia anomala Y197-13 으로막걸리품질향상을위한연구가수행되었다 [11]. 일본의대표술인사케의향기성분중 isoamyl acetate는과일향과달콤한향을내는주요성분이며, 이것은 alcohol acetyltransferase와 esterase의가수분해에의해 isoamyl alcohol과 acetyl coenzyme A의합성물질로알려져있다 [28]. Isoamyl alcohol은 leucine 생합성과정의전구물질로 α-ketoisocaproate로부터생성된다 [10]. Leucine 생합성과정에서 α-isopropyl malate 생합성효소 (α-ipm) 가 L- leucine의양이많을때피드백억제에의해조절되어 α- isopropyl malate에서 α-kotoisovalerate로의변환을촉진한다 [2]. 이러한경우 isoamyl alcohol 합성이감소된다. 효모중에서 α-ipm의피드백조절을상실되어 L-leucine이과생산되는균주는에스테르와고급알코올류의생산이증가하여 isoamyl alcohol과 isoamyl acetate 또한증가한다. 이러한균주의탐색법은 5,5,5-trifluor-DL-leucine (TFL) 즉 L- leucine 유사물질에미생물을노출시켜저항성균주를선발하는것으로보고되었다 [1]. 최근에 L-leucine 피드백조절이제거된 LEU4 유전자 (α-ipm) 돌연변이균주는 isoamyl alcohol 을고생산하는것으로나타난연구결과도보고되었다 [19]. 효모가내는또다른중요한향기성분은에스테르인 ethyl caproate이다 [28]. 이물질의축적과합성은전구체 (ethanol, caproic acid), alcohol acyl transferase와 esterase 효소의존재에달려있다 [25]. 지방산생합성은 cerulenin에의해억제되는지방산생합성효소에의해촉진되며, 지방산고생성균주의탐색은 cerulenin 저항성균주를탐색하는것으로알려져있다 [8]. 본연구의목적은발효식품에서분리된효모의특성연구를통해, 우리술의품질향상을위한우수한효모종균의다양성을확보하고자하였다. 따라서효모의세포외효소인 β- glucosidase, glucanase, protease, amino acid decarboxylase 등을조사하였으며, 에탄올내성, 황화수소생성능등효모의환경내성을탐색하였다. 또한효모의경우, 아미노산생합성및지방산생합성경로를통해고급알코올류, 향기성에스테르계열의물질을생산하는데이생합성경로저해제에대해내성을가지는균주를발굴함으로써, 향기가뛰어난것으로예상되는효모생물자원 12 균주를확보하였다. 재료및방법 주로시판효모인 Fermivin (Sacchromyces cerevisiae, Gist Brocades, Denmark), Frootzen (Pichia kluyvery, Chr. Hansen, Denmark) 를사용하였다. 배지및효소활성분석수집된발효식품 (10 g) 을 0.85% NaCl (90 ml) 용액에현탁하여 typd (1% yeast extract, 2% peptone, 2% glucose, 0.0018% tartaric acid) 한천배지 [16] 에 100 μl 도말하여 30 o C에서 48시간배양하였다. 배양된효모의집락모양, 크기, 색깔등형태적특징을관찰하여서로다른균주를 YPD (1% yeast extract, 2% peptone, 2% glucose) 한천배지를사용하여순수분리하였다. 순수분리된효모는 YPD 액체배지에배양후, glycerol를 20% 함유하도록첨가하여 80 o C 에서보관하여실험에사용하였다. 분리효모의 β-glucosidase (EC 3.2.1.21.), glucanase (EC 3.2.1.6.), protease (EC 3.4.), amino acid decarboxylase (EC 4.1.1.) 활성을확인하였다. 한천평판법으로실험하였고각효소의적합한한천평판을만든후분리효모를접종하여효소활성유무를확인하였다. β-glucosidase 활성은 esculin (Sigma-Aldrich Co., St. Louis, USA) 을 YPD 한천배지에농도가 0.5% (w/v) 가되도록첨가하고 ph 5.0으로조정후멸균한다음, 1% ferric ammonium citrate (Sigma-Aldrich Co., St. Louis, USA) 를농도가 2% (v/v) 되도록첨가하여배지를제조하였다. 이배지에효모를접종하고 30 o C에서 24시간배양후집락주변에검은색환의생성유무에따라평가하였다 [7]. Glucanase는 β-d-glucan (Sigma-Aldrich Co., St. Louis, USA) 을 YPD 한천배지전체농도의 0.2% (w/v) 로첨가하여제조후효모를접종하고 30 o C에서 48시간배양한다음 congo red (Sigma-Aldrich Co., St. Louis, USA) 를 0.1% 으로희석한용액으로염색후집락주변에형성된투명환의생성여부로활성을확인하였다 [23]. Protease는 YM (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1% glucose) 한천배지에 skim milk 농도가 1% 되도록첨가하여제조후효모를접종하고 30 o C에서 48시간배양한다음집락주변에형성된투명환으로활성을확인하였다. 그리고 amino acid decarboxylase는 YPD 한천배지에아미노산 1% (histidine, tyrosine, phenylalanine, tryptophane, lysine, leucine) (Sigma-Aldrich Co., St. Louis, USA), bromocresol purple (Sigma-Aldrich Co., St. Louis, USA) 을 0.006% 가되도록첨가하고 ph 5.3으로조정한다음효모를접종후집락주변보라색환의생성여부에따라평가하였다 [18]. 효모균주및사용시약본실험에사용한효모는전국에서수집한발효액등의발효식품 126 점에서 1,007 주를분리하여사용하였다. 비교균 에탄올, 아황산내성및황화수소생성능분석에탄올내성평가는 YPD 한천배지에 5%, 10%, 15% 의에탄올을첨가하여제조한후효모를접종하여집락의생성여 March 2015 Vol. 43 No. 1
58 Baek et al. 부에따라내성을평가하였다 [17]. 아황산내성평가는 YPD 한천배지에 tartaric acid (Sigma-Aldrich Co., St. Louis, USA) 을전체농도의 1.8% 가되도록첨가하고 1 M K 2 S 2 O 5 를 (Daejung Chemicals Co., Ltd, Korea) 첨가하여 SO 2 의농도가 20 mg으로조정한후효모를접종하여집락의생성여부에따라내성을평가하였다 [5]. 황화수소생성능을알아보기위해 Biggy 한천배지 (Becton Dickinson GmbH, Heidelberg, Germany) 에효모를접종하여 30 o C에서 48시간배양한다음, 집락색깔의진하기에따라황화수소생성능을평가하였다 [5]. Cerulenin 및 5,5,5-trifluor-DL-leucine 내성 Cerulenin과 5,5,5-trifluor-DL-leucine (TFL) (Sigma- Aldrich Co., St. Louis, USA) 의대한저항성은 0.67% YNB (yeast nitrogen base), 2% glucose, 2% agar 조성의한천배지에 cerulenin (25 μm) 과 TFL (1 mm) 을각각첨가하여제조후효모를접종하고 30 o C에서 48시간배양한다음집락생성여부에따라평가하였다 [26]. 효모의동정분리효모를동정하기위하여 26S rrna 유전자의 D1/D2 부위염기서열을분석하였다. 프라이머 [27] 로 NL1 (5'- GCATATCAATAAGCGGAGGAAAAG-3') 및 NL4 (5'- GGTCCGTGTTTCAAGACGG-3') 를사용하여 26S rrna D1/D2 부위단편을증폭후, ZEOTECH사에의뢰하여염기서열을분석하였다. 염기서열은 National Center for Biotechnology Information (NCBI, Bethesda, MD, USA) 의 BLAST를사용하여 26S rrna 유전자단편의염기서열상동성을비교하였으며, 염기서열은 DNASTAR pro software (SeqMan Pro, Ver 8.1.5., Lasergene) 를이용하여수행하였다. 효모의계통분석도 (phylogenetic tree) 작성은 GenBank 에보고된다른효모균주의 26S rrna 염기서열을비교분석하여, MEGA version 4.0 [24] 의 neighbor-joining method [20] 로작성하였다. 당및에탄올내성측정분리효모의포도당에대한내성측정은포도당농도를 20, 30, 40% 첨가한 YPD 액체배지 (yeast extract 1%, peptone 2%, glucose 1.5%, ph 6.5) 에효모를접종하고 30 o C에서 180 rpm으로 24시간동안진탕배양한후 600 nm에서흡광도를측정하여균주의성장을비교하였다. 이때대조구는포도당 1% 를첨가한 YPD 액체배지에서효모균주의성장으로하였다. 에탄올내성은에탄올이각각 0, 5, 10, 15% 의농도로함유된 YPD 액체배지 10 ml에전배양한효모의배양액을 0.1% 농도로접종하고 25 o C에서 5일간배양후, 600 nm에서흡광도를측정하여균주의성장을조사하였다. 에탄올발효능분석효모의에탄올생성량은전배양한효모를포도당 25% 가함유된 YPD 액체배지 100 ml 에 0.1% 의농도로접종하고 30 o C 에서 48 시간동안정치배양한후, 10,000 rpm 에서 10 분간원심분리하여상등액을회수하고생성된에탄올함량을측정하였다. 에탄올함량은비중법을이용하여배양상등액 100 ml 를증류한다음 70% (v/v) 를메스실린더에회수하고다시증류수를이용하여 100 ml 로정용한다음주정계를이용하여측정하였다 [9]. 결과및고찰 효모의효소활성효모의 β-glucosidase, glucanase, protease 및 amino acid decarboxylase 효소활성을확인한결과는 Table 1 에나타내었다. β-glucosidase 는분리균주 1,007 주중에서 566 균주가활성을보였고, glucanase 는 45 균주만이활성을보였다. Protease 활성은 401 균주에서나타났다. 바이오제닉아민 (biogenic amine, BAs) 은아미노산의탈탄산작용, 아미노기전이작용등의화학적작용에의해생성되는질소화합물이다. 바이오제닉아민류는단백질을함유한식품이미생물에의해분해되는과정에서생성되며, 알러지유발물질인히스타민 (histamine) 과티라민 (tyramine) 이대표적이다 [18]. Amino acid decarboxylase 활성을보유한효모는바이오제닉아민류생성가능성이높으며, 각아미노산기질을바이오제닉아민류로변화시킨균주를조사한결과, histidine 은 69 균주, tyrosine 은 306 균주, phenylanine 은 171 균주, 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 2 S production Cerulenin TFL 5% 10% 15% 563 610 934 792 307 500
Characterization of Yeasts for Improving Liquor Quality 59 Table 2. Production of extracellular enzymes of selected wild type yeasts and commercial yeasts. Strains β-glucosidase Glucanase Protease AA decarboxylation 1 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. tryptophane은 23 균주, lysine은 197 균주, leucine은 198 균주로나타났다. 또한 cerulenin, TFL, SO 2, 에탄올내성균주및황화수소생성능에대한결과는 Table 1에나타내었다. Cerulenin, TFL 내성균주는각각 563, 610 균주로나타났고, SO 2 내성은 22 균주로상대적으로적은수의효모에서나타났다. 에탄올내성의경우, 에탄올농도가높을수록내성을나타내는균주는현저히감소하였으며, 에탄올농도가 5%, 10%, 15% 에대하여각각 934, 792, 307 균주로나타났다. 황화수소를생성할것으로추정되는효모는 500 균주로분석되었다. 위결과를바탕으로 biogenic amine decarboxylase 활성미보유균주, 황화수소저생성균주중심으로 1차선별하였으며, 이후효소활성능, cerulenin, TFL, SO 2, 에탄올내성특성등을보유한 12 균주를선별하였다 (Table 2). β-glucosidase는와인제조시비휘발성향기성분으로중요한역할을한다고알려져있으며, 이는 non-saccharomyces 효모에서많이발견된다고보고되었다 [7, 23]. 특히, Hansenispora vinea와 Candida 종이생산한 glucosidase가와인의다양한향에영향을미친다고알려져있다 [23]. β-glucosidase 활성은시판효모인 S. cerevisiae, P. kluyvery보다분리효모인 N43-8, N77-4, SM2-7, Y685, BY30-1, M1-9, SD1-2 등 7 균주가더우수한것으로나타났다. Glucanase 활성은시판효모인 S. cerevisiae, P. kluyvery 에서나타나지않았으며, N43-8, Y685 2 균주만활성을나 타내었다. Glucanase 활성을보유한균주로는 Candida stellata, C. hellenica, C. pulcherrima, Kloeckera apiculata 효모들로보고된바있다 [23]. Protease 의경우, 약한활성을보인균주가 7 균주로나타났다. 또한, amino acid decarboxylase 활성은선발효모와시판효모모두음성으로나타난것으로보아바이오제닉아민류생성가능성이매우낮을것으로예상되었다. Cerulenin, TFL, SO 2, 알코올내성균주효모가생성하는고급알코올류와에스테르계열의성분들이주류의향을증진시킬수있음을감안하여, cerulenin 과 TFL 에내성을보유한효모를탐색하였다. Cerulenin 과 TFL 은 isoamyl alcohol 과 caproic acid 를생산할수있는균을선별하는데사용되어왔다 [8]. 선별된 12 주의효모중 cerulenin 과 TFL 에대하여내성을나타낸효모는각각 8, 7 균주로나타났다 (Table 3). N43-8, N56-10, N77-4, SM2-7, Y447, Y685 및 HP1-2 균주는 cerulenin 과 TFL 에대한내성을동시에가지며, CM4-5 균주는 cerulenin 내성만가지는것으로나타났다. A9-1, BY30-1, M1-9, SD1-2 효모와시판효모인 S. cerevisiae 는두가지항생물질에대해민감한것으로나타났다. non-saccharomyces 시판효모인 P. kluyvery 는 cerulenin 과 TFL 에내성을나타내어, 두항생물질에감수성을보인 S. cerevisiae 와대조적인결과를나타내었다. Cerulenin 과 TFL 에내성을나타낸 7 주의효모는고급알코 March 2015 Vol. 43 No. 1
60 Baek et al. Table 3. The cerulenin, TFL, SO 2 and ethanol tolerance and production of H 2 S of selected wild type yeasts and commercial yeasts. Ethanol con. H 2 S Strains Cerulenin TFL SO 2 5% 10% 15% production 1 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. 올류와에스테르계열의향기성분을생산할수있을것으로예상되며, 이에대한추가적인연구가필요할것으로판단된다. SO 2 에대하여내성을나타낸효모는 2 균주로 A9-1 과 HP1-2 로나타났다. 시판효모인 S. cerevisiae 는 SO 2 에대한내성이높은것으로나타났다. 알코올내성에대해서는대부분의효모가높은내성을보였으며, 그중 N56-10, Y447 균주는알코올내성이상대적으로낮은것으로나타났다. 황화수소는와인이나알코올음료에좋지않는냄새로미생물발효에의해생성될수있는데 [5], 본연구에사용한시판효모를포함하여대부분의효모는황화수소생성능이낮았으며, SM2-7, Y447, CM4-5, M1-9, SD1-2, HP1-2 균주는생성능이거의없는것으로보아와인이나전통주발효산업에사용가능할것으로기대된다. 효모동정선발된효모 12 균주의 26S rrna 유전자의 D1/D2 부위염기서열분석결과를 Fig. 1에나타내었다. 효모 12주는 C. tropicalis, H. opuntiae, H. uvarum, P. kudriavzevii, S. cerevisiae 및 Wickerhamomyces anomalus로각각동정되었다. SD1-2, M1-9, CM4-5, A9-2, BY30-1 균주는 S. cerevisiae로동정되었다. HP1-2 균주는 H. opuntiae, N56-10 균주는 H. uvarum, N43-8, SM2-7, Y685 균주는 W. anomalus, Y447 균주는 C. tropicalis, N77-4 균주는 P. kudriavzevii로각각동정되었다. 효모의내당성및에탄올내성주류제조에이용되는효모는고농도의당에서충분히생육을하면서알코올발효를진행할수있어야하므로선발된효모 12 균주를대상으로포도당이 20%, 30%, 40% 로첨가된 YM 액체배지에서생육과당내성을비교하였다 (Fig. 2). S. cerevisiae 효모중에서 BY30-1 균주를제외하고, 대체적으로포도당 40% 가첨가된 YM 액체배지에서양호한생육을나타내었다. non-saccharomyces 효모중에서는 H. opuntiae HP1-2, C. tropicalis Y447 균주를제외하고포도당 40% 가첨가된 YM 액체배지에서생육이양호한것으로보아내당성이우수하며주류제조에사용가능성이충분한것으로판단된다. 일반적으로일정수준이상의에탄올농도에서는효모생육이저해되어더이상알코올발효가일어나지않는것으로알려져있으며 [24], 주류등알코올발효음료제조용효모는에탄올에우수한내성을보유하는것이바람직하다. 효모 12 균주를대상으로에탄올을 5%, 10%, 15% 농도로첨가한 YM 액체배지에서효모생육과에탄올을첨가하지않은 YM 액체배지에서의생육을비교한결과 (Fig. 3), 배양 5 일뒤 S. cerevisiae 효모중에서 BY30-1 를제외하고는 10% 에탄올첨가한 YM 액체배지에서효모의생육은양호하였으나 15% 에탄올농도에서는모두생육이낮게나타났다. non-saccharomyces 효모중에서는균주마다다른특성이나타났다. 5% 에탄올농도의경우, H. uvarum N56-10, H.
Characterization of Yeasts for Improving Liquor Quality 61 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 o C for 24 h. Glucose tolerance was determined by absorbance of yeasts growth at 660 nm. The data were averages based on three trials. March 2015 Vol. 43 No. 1
62 Baek et al. 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 o C for 5 days. Ethanol tolerance was determined 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 o C for 48 h. Ethanol production was determined by alcoholometer. The data were averages based on three trials. *, significance between commercial yeasts (Fermivin and Frootzen) and selected strains, p <0.05. opuntiae HP1-2 균주를제외하고는양호한생육을보였다. 그러나 10% 에탄올첨가한 YM 액체배지에서효모의생육은 P. kudriavzevii N77-4 를제외한모든효모가저조하였으며, P. kudriavzevii N77-4 는에탄올농도 15% 에서생육이낮은것으로나타나에탄올내성이 non-saccharomyces 효모중에서상대적으로높은것으로나타났다. 효모의에탄올발효능효모 12 균주를대상으로포도당을 25% 첨가한 YM 액체배지에서 48시간배양한후각균주의에탄올생성량을측정한결과 (Fig. 4), S. cerevisiae SD1-2 균주의에탄올생성량이 9.5% 로가장우수하였으며, S. cerevisiae 속균주들은최소 7.3% 에서최고 9.5% 의에탄올생성량을나타내었다. non-saccharomyces 균주중에서는 W. anomalus N43-8, SM2-7 균주가 4.7% 으로높은에탄올생성량을보였으며, 최 소 2.2% 에서최고 4.7% 로나타났다. Kim 등 [11] 이 P. anomala Y197-13 효모로막걸리제조연구에서알코올함량이 11.1% 로나타나막걸리제조시좋은후보균주가될수있다고보고하였다. 또한 Pichia 속효모와 non-saccharomyces 속효모들은와인등의아로마형성에중요한역할을한다고알려져 [3, 23] 본연구에서선발한 7 주의 non-saccharomyces 속효모균주역시추가적인주류제조연구를진행하고있으며독특한향미를가진주류발효음료제조가능성이있을것으로판단된다. 요 약 non-saccharomyces 효모는야생효모로서, 다양한효소를세포밖으로분비하여와인의향기성분증가에중요한영향을준다고알려져있다. 본연구에서는한국의발효식품에
Characterization of Yeasts for Improving Liquor Quality 63 서분리된효모 1,007주의세포외효소활성을조사하였다. 그결과, β-glucosidase 566, glucanase 45, protease 401로나타났으며, AA decarboxylase 활성균주는각아미노산별로 His 69, Tyr 306, Phe 171, Trp 23, Lys 197, Leu 198 균주로나타났다. Cerulenin 과 TFL 내성균주는각각 563, 610주로, 15% 에탄올내성균주는 307주로나타났다. 황화수소생성균은 500주로조사되었다. AA decarboxylation와황화수소저생성균주중유용효모 12 균주를선발하여 26S rdna 염기서열을분석한결과, C. tropicalis, H. opuntiae, H. uvarum, P. kudriavzevii, S. cerevisiae, W. anomalus 로각각동정되었다. 12주의효모중당내성우수균주는 9주이며, 알코올내성우수균주는 5 균주로나타났다. 알코올발효능은 Saccharomyces 효모는 8% 이상으로나타났으며, non-saccharomyces 효모는 3% 내외로나타났다. Acknowledgments This work was carried out with the support of Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ00947701) Rural Development Administration, Republic of Korea. References 1. Bussey H, Umbarger HE. 1970. Biosynthesis of branchedchain amino acids in yeast: a trifluoroleucine-resistant mutant with altered regulation of leucine uptake. J. Bacteriol. 103: 286-294. 2. Casalone E, Fia G, Barberio C, Cavalieri D, Turbanti L, Polsinelli M. 1997. Genetic and biochemical characterization of Saccharomyces cerevisiae mutants resistant to trifluoroleucine. Res. Microbiol. 148: 613-623. 3. Charoenchai C, Fleet GH, Henschke PA, Todd BEN. 1997. Screening of non-saccharomyces wine yeasts for the presence of extracellular hydrolytic enzymes. Australian J. Grape Wine Res. 3: 2-8. 4. Ciani M, Beco L, Comitini F. 2006. Fermentation behavior and metabolic interactions of multistarter wine yeast fermentations. Int. J. Food Microbiol. 108: 239-245. 5. Comitini F, Gobbi M, Domizio P, Romani D, Lencioni L, Mannazzu I, et al. 2011. Selected non-saccharomyces wine yeasts in controlled multistarter fermentations with Saccharomyces cerevisiae. Food Microbiol. 28: 873-882. 6. Fleet GH. 1993. The microorganisms of winemaking-isolation, enumeration and identification. In: Fleet GH (ed) Wine microbiology and biotechnology. Harwood Academic Publishers, Switzerland. pp. 1-25. 7. Hernández LF, Espinosa JC, Ferna ndez-gonzález M, Briones A. 2003. β-glucosidase activity in a Saccharomyces cerevisiae wine strain. Int. J. Food Microbiol. 80: 171-176. 8. Ichikawa E, Hosokawa N, Hata Y, Abe Y, Suginami K, Imayasu S. 1991. Breeding of sake yeast with improved ethyl caproate productivity. Agric. Biol. Chem. 55: 2153-2154. 9. Jung HK, Park CD, Park HH, Lee GD, Lee IS, Hong JH. 2006. Manufacturing and characteristics of Korean traditional liquor, Hahyangju prepared by Saccharomyces cerevisiae HA3 isolated from traditional Nuruk. Korean J. Food Sci. Technol. 38: 659-667. 10. Kaosowski G, Czuprynski B. 2006. Kinetics of acetals and esters formation during alcoholic fermentation of various starchy raw materials with application of yeasts Saccharomyces cerevisiae. J. Food Eng. 72: 242-246. 11. Kim HR, Kim JH, Bai DH, Ahn BH. 2012. Feasibility of brewing Makgeolli using Pichia anomala Y197-13, a Non-Saccharomyces cerevisiae. J. Microbiol. Biotechnol. 22: 1749-1757. 12. Kim MJ, Kim BH, Han JK, Lee SY, Kim KS. 2011. Analysis of quality properties and fermentative microbial profiles of Takju and Yakju brewed with or without steaming process. J. Fd Hyg. Safety 26: 64-69. 13. Kwon SJ, Ahn TY, Sohn JH. 2012. Analysis of microbial diversity in Makgeolli fermentation using PCR-DGGE. J. Life Sci. 22: 232-238. 14. Lee HS, Lee TS, Noh BS. 2007. Volatile flavor components in the mashes of Takju prepared using different yeasts. Korean J. Food Sci. Technol. 39: 593-599. 15. Lee HS, Park CS, Choi JY. 2010. Quality characteristics of the mashes of Takju prepared using different yeasts. Korean J. Food Sci. Technol. 42: 56-62. 16. Lee MN. 2010. Isolation and characteristics of urease production yeasts from Korean traditional Nuruk. MS thesis, Kyungpook National University, Korea, pp. 5-6. 17. Lee YJ, Choi YR, Lee SY, Park JT, Shim JH, Park KH, et al. 2011. Screening wild yeast strains for alcohol fermentation from various fruits. Korean Soc. Mycol. 39: 33-39. 18. Nikolaoua E, Soufleros EH, Bouloumpasi E, Tzanetakis N. 2006. Selection of indigenous Saccharomyces cerevisiae strains according to their oenological characteristics and vinification results. Food Microbiol. 23: 205-211. 19. Oba T, Nomiyana S, Hirakawa H, Tashiro K, Kuhara S. 2005. Asp578 in Leu4p is one of the key residues for leucine feedback inhibition release in sake yeast. Biosci. Biotechnol. Biochem. 69: 1270-1273. 20. Saitou N, Nei M. 1987. The neighbor joining-methods: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406-425. 21. Seo MY, Lee JK, Ahn BH, Cha SK. 2005. The changes of microflora during the fermentation of Takju and Yakju. Korean J. Food Sci. Technol. 37: 61-66. 22. Shin KR, Kim BC, Yang JY, Kim YD. 1999. Characterization of Yakju prepared with yeasts from fruits. 1. Volatile components in Yakju during fermentation. J. Korean Soc. Food Sci. Nutr. 28: 794-800. 23. Strauss MLA, Jolly NP, Lambrechts MG, Van Rensburg P. March 2015 Vol. 43 No. 1
64 Baek et al. 2001. Screening for the production of extracellular hydrolytic enzymes by non-saccharomyces wine yeasts. J. Appl. Microbiol. 91: 182-190. 24. Tamura K, Dudley J, Nei M, Kumar S. 2007. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24: 1596-1599. 25. Verstrepen KJ, van Laere SDM, Vanderhaegen BMP, Derdelinckx G, Dufour JP, Pretorius IS, et al. 2003. Expression levels of the yeast alcohol acetyltransferase genes ATF1, Lg- ATF1, and ATF2 control the formation of a broad range of volatile esters. Appl. Environ. Microbiol. 69: 5228-5237. 26. Viana F, Gil JV, Genovés S, Vallés S, Manzanares P. 2008. Rational selection of non-saccharomyces wine yeasts for mixed starters based on ester formation and enological traits. Food Microbiol. 25: 778-785. 27. Yi SH, Kwon SJ, Ahn C, Yoo JY. 1997. Isolation, identification and cultural conditions of yeasts from traditional Meju. Korean J. Appl. Microbiol. Biotechnol. 25: 435-441. 28. Yoshikawa K. 1999. Sake: production and flavor. Food Rev. Int. 15: 83-107.