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Microbiol. Biotechnol. Lett. (2015), 43(3), 227 235 pissn 1598-642X eissn 2234-7305 Microbiology and Biotechnology Letters 야생효모종류에따른알코올발효특성 백성열 1,2, 이유정 1, 김명동 3, 이재형 4, 문지영 1, 여수환 1 * 1 농촌진흥청국립농업과학원농식품자원부발효식품과 2 경북대학교농업생명과학대학식품공학과 3 강원대학교식품생명공학과 4 강원도농업기술원농식품연구소 Received: July 15, 2015 / Revised: August 5, 2015 / Accepted: August 5, 2015 Characterization of Ethanol Fermentation with Wild Type Yeast Strains Seong Yeol Baek 1,2, You Jung Lee 1, Myoung-Dong Kim 3, Jae-Hyoung Yi 4, Ji-Young Mun 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 Technology, Kyungpook National University, Daegu 702-701, Republic of Korea 3 Department of Food Science and Biotechnology, Kangwon National University, Chuncheon 200-701, Republic of Korea 4 Agro-food Research Institute, Gangwondo Agricultural Research and Extension Services, Chuncheon 200-822, Republic of Korea The objective of this study was to improve the quality of Korean rice wine with wild type yeast strains isolated from various traditional Korean fermented foods. Herein the fermentation and sensory characterization of wild yeast, for the purposes of brewing Korean rice wine, was investigated. 12 yeast strains were examined for their ethanol and glucose tolerance. In addition, the ph, soluble solids, acidity, amino acidity, ethanol content, organic acids, and volatile compounds were also studied for the alcoholic beverages made with the wild yeasts. Almost all Saccharomyces genera yeasts were showed to have a tolerance at 10% ethanol, but non-saccharomyces genera yeasts displayed a low tolerance. The alcoholic beverages fermented by non-saccharomyces yeasts demonstrated higher levels of soluble solids, titratable acidity, amino acids, and lower ethanol content, when compared with the alcoholic beverages fermented by Saccharomyces genera yeasts. The organic acid content, such as malic acid, acetic acid, and succinic acid, was seen to also be higher. The electronic nose was analyzed, and discriminant function analysis (DFA) was used for discriminating wild yeast strains. The DFA plots indicated a significant separation of Saccharomyces genera and non-saccharomyces yeast strains. For volatile compounds, ethyl acetate from non-saccharomyces yeasts, and ethanol from Saccharomyces genera yeast, a high area ratio was observed. Keywords: Korean rice wine, fermentation, volatile compound, non-saccharomyces, electronic nose 서 론 발효는오래전부터자연발생적으로미생물에의해생겨난현상으로미생물이밝혀지기전부터이를이용해왔다. 과학이발달하면서미생물의존재와그기능이밝혀지게되었고, 사람들은미생물을이용하여다양한발효식품을만들어왔다 [20]. 최근대부분의발효는자연계에서분리된미생물을배양하여종균 (starter) 형태로개발된균을사용하고있다. 이 *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 러한목적으로다양한종류의효모가분리 동정되었으며그중일부는빵과술을만드는 Saccharomyces cerevisiae 로알려져있다 [15]. 우리나라의경우도전통주를빚기전에주모를제조한다. 주모란밑술또는술밑으로효모를순수하게배양해놓은일종의스타터이다. 주모로사용하는대표적인미생물은 S. cerevisiae 이며, 효모중에서알코올생산에관여하는미생물이다 [9, 10]. 우리고유의전통주인탁주는자연상태의곰팡이, 효모및세균에의해발효된누룩을이용하여전분질원료를병행복발효방식으로알코올발효한것이다. 병행복발효주는전분질을미생물이생성한효소로당화시켜효모에의해알코올발효한것으로탁주, 약주및청주등이있다. 효모에의한발효는탁주의맛을좌우하는

228 Baek et al. 여러요인중하나이며효모의종류에의해고유의향미를생성한다고알려져있다 [8]. 수입주류와경쟁력을갖춘우리술을개발하기위해서는주류제조의원천기반기술인우수한토착발효미생물의확보가필수적이다. 예로부터우리나라는발효공학적으로가장특징적인누룩을이용한양조기술이발전되었지만, 일제강점기를거쳐주류제조법의현대화과정에서주미생물원인누룩의가치가제대로평가받지못하면서전통양조기술이위축되었다 [11]. 그러나최근전통주에대한소비자의관심증대와소비가늘어남에따라전통주의품질향상을위해, 전국의전통누룩으로부터분리한야생효모의향미증가와양조특성에관한연구가수행되고있다. 유럽의와인산업계에서는 Saccharomyces 속효모가아닌야생효모에대한관심과연구가증가하고있다. 특히, non-saccharomyces 효모는포도밭에서분리되는야생효모로다양한효소를생산함으로써와인의향미에중요한역할을한다고보고되었다 [2]. 와인제조사및미생물학자들은 non-saccharomyces 효모가와인의관능적특성에관여하고있음을인지하고, 산업적응용에대해관심을가지고있다. 그리고 non-saccharomyces 효모의일부종과 S. cerevisiae 와의혼합발효를통해와인의풍미와품질을향상시킨연구결과도보고되었다 [3]. 발효능이낮은레몬형효모인 Hanseniaspora uvarum과이의무성세대인 Kloeckera apiculata는포도과피표면에가장많이존재하는미생물로써와인의알코올발효초기에우점종으로작용하는야생효모로보고되었다 [5]. 이러한효모들은와인의휘발성향기성분생성에중요한역할을하는것으로알려져있다 [3]. 전통주에관한 non-saccharomyces 효모연구는 Kim 등 [12] 에 의해 Pichia anomala를이용한막걸리의품질향상연구가수행되었다. 우리술의주질개선연구중야생효모에관한연구로는 Kim 등 [11] 의청주제조, Lee 등 [16] 의탁주향기성분, Kim 등 [13] 의포도주제조, Lee 등 [17] 의오미자발효주의제조등으로대부분 S. cerevisiae에관한연구이며, non-saccharomyces 효모에관한연구는 Kim 등 [12] 의 P. anomala 효모에관한막걸리연구가처음보고되었다. 본연구는전보 [1] 에서선발된야생효모 12 균주에대한알코올과당내성을조사하였으며, 낱알누룩의당화액을야생효모로알코올발효시켜이들의양조적성을분석하여우리술의품질향상과다양성확보를위한야생효모의활용가능성을검토하였다. 재료및방법 효모균주본실험에사용한효모는전보 [1] 에서선발한효모 12균주를사용하였다 (Table 1). 비교균주로시판효모인 Fermivin (Saccharomyces cerevisiae, Oenobrands, France), Frootzen (Pichia kluyvery, Chr. Hansen, Denmark) 을사용하였다. 실험효모는 YPD 액체배지에배양후, 글리세롤을 20% 함유하도록첨가하여초저온냉동고 (-80 o C) 에보관하여실험에사용하였다. 당화액및알코올발효액제조본연구에서는효모에의한알코올발효특성을조사하기위해, Kim 등 [11] 의방법을응용하여낱알누룩을이용한당 Table 1. List of isolated wild type yeasts from different kind of fermented foods and commercial yeasts. Species Strain Source Wickerhamomyces anomalus N43-8 Nuruk, Jeonju-si, Jeollabuk-do Wickerhamomyces anomalus SM2-7 Yakju (Sinju), Bucheon-si, Gyeonggi-do Wickerhamomyces anomalus Y685 Meju, Jeongseon-gun, Gangwon-do Pichia kudriavzevii N77-4 Nuruk, Seoul Hanseniaspora opuntiae HP1-2 Peach, Suwon-si, Gyeonggi-do Hanseniaspora uvarum N56-10 Nuruk, Seogwipo-si, Jeju-do Candida tropicalis Y447 Meju, Gunsan-si, Jeollabuk-do Saccharomyces cerevisiae BY30-1 Fermented Liquid, Namyangju-si, Gyeonggi-do Saccharomyces cerevisiae A9-2 Apple wine, Gongju-si, Chungcheongnam-do Saccharomyces cerevisiae CM4-5 Yakju (Chukbae), Bucheon-si, Gyeonggi-do Saccharomyces cerevisiae M1-9 Nuruk, Bucheon-si, Gyeonggi-do Saccharomyces cerevisiae SD1-2 Yakju (Sinju), Bucheon-si, Gyeonggi-do Saccharomyces cerevisiae Fermivin Strain n 8906 Champagne, France Pichia kluyvery Frootzen Wine, New Zealand

Characterization of Fermentation with Wild Yeasts 229 화액을제조하였다. 당화액제조는황국균 (Aseprgillus oryzae, Choongmoo Fermentation Co. Ltd., Ulju, Korea) 으로낱알누룩을제조하여, 낱알누룩 250 g을물 2 L에혼합후, 고두밥 1 kg을넣고 60 o C에서 110 rpm으로 24시간당화하였다. 당화액을원심분리 (8,000 rpm, 10 min) 하여상등액만회수하여당도를 16 brix로조정하였다. 또한젖산 (lactic acid) 을첨가하여 ph 3.7로조정하고 65 o C에서 30분간저온살균하였다. 발효를위한효모접종은글리세롤보존된효모를 YPD broth 5 ml에 500 µl 넣어접종후 30 o C 에서 48시간배양하였다. 배양한효모를 200 ml YPD broth 에 1 ml씩접종하여 30 o C에서 48시간배양한후, 배양액을 1/200 희석하여, 세포계수기 (hematocytometer) 에 10 µl씩분주하여세포수를측정하였다. 각효모를당화액에 1 10 6 cell/ml이되도록접종하고 25 o C에서 5일간정치배양한후, 8,000 rpm에서 10분원심분리하여효모를제거하고, 상등액을분석하였다. 알코올내성및내당성알코올내성은 YM (yeast extract 3%, malt extract 3%, peptone 0.5%, glucose 1%) 액체배지에무수에탄올농도를 5, 10, 15% (v/v) 가되도록각각첨가하였다. 효모를접종하고 30 o C에서 5일간배양한후 660 nm에서흡광도를측정하여비교하였다. 내당성은 YM 액체배지에 glucose가 20, 30, 40% (w/v) 가되도록첨가한후 30 o C에서 2일간배양한후 660 nm에서흡광도를측정하여비교하였다 [10]. 후, 0.45 µm membrane filter (Millipore Co., Bedford, MA, USA) 로여과한다음 Sep-pak C18 cartridge (Waters Oasis, Milfort, MA, USA) 로색소를제거하고 high performance liquid chromatography (HPLC, Chromaster 5000, Hitachi, Ltd, Tokyo, Japan) 로분석하였다. 분석컬럼은 ODS-100W (4.6 mm 250.0 mm) 를사용하였다. 이동상은 3 mm perchloric acid를사용하였으며, flow rate는 0.6 ml/min, column oven 온도는 63 o C로하였다. 컬럼을통과한분리물은반응용액 (0.2 mm bromothymol blue, 15 mm Na 2 HPO 4, 2 mm NaOH) 과반응한후, UV 440 nm에서검출하였다. 이때반응용액의 flow rate는 0.9 ml/min로하였다. 전자코분석 Metal oxide sensor (MOS) 가장착된전자코시스템 (FOX- 2000, Alpha MOS, Toulouse, France) 을이용하여야생효모에의한알코올발효액의향기양상을측정하였다. 시료 1ml 를 10 ml 헤드스페이스용기에담아밀봉하고, 실온에서 24 시간방치후측정하였다. 시료는 80 o C 에서 20 분간 250 rpm 으로교반하고, 주입구온도는 130 o C 상태에서주입하였다. 이때사용한가스는공기이며분당 150 ml 의유속으로흘려분석하였다. 각시료는 3 회반복을실시하였다. 통계처리는 Alpha MOS 에서제공된소프트웨어를사용하였으며, 판별함수분석 (Discriminant Function Analysis; DFA) 으로각시료간의휘발성분의차이를전체적인패턴으로나타내었다 [14]. 이화학적분석 ph는시료 10 ml를취하여 ph meter (FE20, Mettler Toledo, Switzerland) 를사용하여측정하였으며, 고형분함량은당도계 (ATAGO Pocket PAL-1, ATAGO Co., Tokyo, Japan) 를사용하여측정하였다. 적정산도는시료 10 ml에 0.5% phenolphthalein 2 3방울을떨어뜨린다음, 0.1 N NaOH로중화적정하였으며소비된용액의양을 acetic acid (%) 로환산하였다. 아미노산도는시료 10 ml를 0.1 N NaOH로중화시킨후, 중성포르말린용액 5 ml를가하여혼합하고여기에 0.1 N NaOH 용액을적정하여 ph 8.3이될때까지소요된 0.1 N NaOH의 ml 수로표시하였다 [6]. 알코올함량은시료 100 ml를가열하여증류액을 80 ml까지받은후, 100 ml까지물로정용한후, 온도계와주정계를사용하여그표시도를읽어 Gay-Lussac 표로서 15 o C로보정하여나타내었다 [19]. 유기산분석야생효모에의한알코올발효액의유기산은다음과같이분석하였다. 시료 2 ml 을 17,000 rpm 에서 15 분간원심분리 휘발성화합물분석야생효모에의한알코올발효액의향기성분은 GC-MS로분석하였다. 시료를 8,000 rpm에서 10분간원심분리하여 Oasis TM HLB Plus LP 추출카트리지 (Waters Co., MA, USA) 에시료를흡착시켰다. 펜탄 (pentane) 과에테르 (ether) 를 1:1로혼합한용액 1 ml을카트리지에넣어흡착된성분을튜브에받은후, Na 2 SO 4 를넣어용매성분을제거하여전용용기에담아분석하였다. 분석은가스크로마토그래피 GCMS-QP2010 PLUS (Shimadzu Co., Kyoto, Japan) 기기를사용하였다. 컬럼은 HP-Innowax fused silica capillary column (60 m 0.25 mm, 0.25 µm film thickness) 을사용하였고, 컬럼의온도는 40 o C에서 5분, 230 o C에서 30분, 주입구와검출기의온도는 250 o C로하였다. flow rate는 1.0 ml/min, 시료의양은 1 µl 를주입하였다. 결과및고찰 야생효모의알코올내성과당내성야생효모의알코올내성 (alcohol 5, 10, 15% (v/v)) 과당

230 Baek et al. Table 2. Effect of alcohol and glucose tolerance about different wild yeasts and commercial yeasts. Yeast Alcohol tolerance (OD 600 ) 1 Glucose tolerance (OD 600 ) 2 strains 0 5 10 15 1 20 30 40 Non-Sacchromyces yeasts N43-8 0.92 ± 0.09 d 0.78 ± 0.05 g,h 0.01 ± 0.01 a 0.00 ± 0.00 0.96 ± 0.05 b,c,d,e 1.24 ± 0.03 c 1.07 ± 0.04 d,e 0.56 ± 0.00 d SM2-7 0.89 ± 0.02 d 0.79 ± 0.01 g,h 0.01 ± 0.01 a 0.00 ± 0.00 0.96 ± 0.02 b,c,d,e 1.31 ± 0.01 c,d 1.13 ± 0.01 e 0.57 ± 0.02 d Y685 0.76 ± 0.01 c 0.85 ± 0.07 h,i 0.01 ± 0.01 a 0.00 ± 0.00 1.02 ± 0.08 d,e,f 1.39 ± 0.02 d,e 1.21 ± 0.02 f 0.66 ± 0.04 d,e N77-4 0.56 ± 0.09 b 0.66 ± 0.05 f 0.24 ± 0.01 b 0.00 ± 0.00 1.04 ± 0.00 e,f 1.25 ± 0.03 c 1.01 ± 0.01 d 0.34 ± 0.01 b HP1-2 0.37 ± 0.00 a 0.09 ± 0.01 b 0.00 ± 0.00 a 0.00 ± 0.00 0.87 ± 0.07 a,b 1.04 ± 0.06 a 0.43 ± 0.08 a 0.01 ± 0.00 a N56-10 0.39 ± 0.01 a 0.01 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 0.84 ± 0.04 a 1.07 ± 0.02 a 0.90 ± 0.00 c 0.44 ± 0.01 c Y447 0.83 ± 0.01 c,d 0.92 ± 0.06 i 0.00 ± 0.00 a 0.00 ± 0.00 1.08 ± 0.03 f 1.07 ± 0.05 a 0.37 ± 0.07 a 0.04 ± 0.05 a Frootzen 0.91 ± 0.02 d 0.72 ± 0.03 f,g 0.01 ± 0.01 a 0.00 ± 0.00 1.11 ± 0.00 f 1.15 ± 0.01 b 0.83 ± 0.00 b,c 0.25 ± 0.01 b S. cerevisiae A9-2 0.90 ± 0.02 d 0.78 ± 0.00 g,h 0.50 ± 0.05 c 0.00 ± 0.00 0.92 ± 0.03 a,b,c 1.40 ± 0.00 e 1.30 ± 0.05 g,h 0.72 ± 0.02 e,f CM4-5 0.79 ± 0.01 c 0.82 ± 0.00 h 0.57 ± 0.04 c 0.00 ± 0.00 1.01 ± 0.01 c,d,e,f 1.43 ± 0.02 e 1.23 ± 0.04 f,g 0.79 ± 0.01 f,g SD1-2 0.78 ± 0.00 c 0.48 ± 0.03 d 0.50 ± 0.00 c 0.00 ± 0.00 1.10 ± 0.07 f 1.44 ± 0.08 e 1.22 ± 0.01 f,g 0.81 ± 0.08 f,g M1-9 0.86 ± 0.05 c,d 0.57 ± 0.00 e 0.77 ± 0.00 d 0.00 ± 0.00 1.06 ± 0.01 e,f 1.59 ± 0.05 f 1.34 ± 0.00 h 0.89 ± 0.11 g BY30-1 0.91 ± 0.03 d 0.34 ± 0.01 c 0.21 ± 0.09 b 0.00 ± 0.00 0.93 ± 0.03 a,b,c,d 1.39 ± 0.03 d,e 0.79 ± 0.04 b 0.31 ± 0.07 b Fermivin 0.84 ± 0.02 c,d 0.67 ± 0.06 f 0.52 ± 0.04 c 0.00 ± 0.00 0.92 ± 0.04 a,b,c,d 1.42 ± 0.00 e 1.27 ± 0.01 f,g,h 0.76 ± 0.04 e,f 1 Yeast strains were cultured at 30 o C for 5 days in YM broth containing 0%, 5%, 10% or 15% ethanol. 2 Yeast strains were cultured at 30 o C for 24 hours in YM broth containing 1%, 20%, 30% or 40% glucose. 내성 (glucose 20, 30, 40% (w/v)) 분석결과를 Table 2 에나타내었다. 알코올 10% 에서효모 M1-9 의흡광도값은 0.77 이며대조군 (Fermivin) 은 0.52 로 M1-9 가알코올내성이높았으며, BY30-1 을제외한나머지 S. cerevisiae 효모는대조군과유사한내성을나타내었다. Non-Saccharomyces 효모중알코올 10% 에서내성을나타내는효모는 N77-4 뿐이었으며, S. cerevisiae 보다전반적으로알코올내성이낮은것으로나타났다. 그리고알코올 15% 에서내성을보인효모는없었다. 당 40% 에서내성은 M1-9 가가장높았으며, 대조군의흡광도값은 0.76 이며 M1-9 은 0.89 로당내성이높았다. 당 40% 에서 non-saccharomyces 효모보다 S. cerevisiae 효모들이내성이높았으며, HP1-2, Y447 을제외한효모들은 glucose 40% 에서내성을가지는것으로나타났다. Kim 등 [11] 이보고한알코올내성효모와비교해본결과, 본연구에사용한효모는알코올 15% 에서내성이매우낮았으며, 당에대한내성도비슷한것으로나타났다. 알코올발효액의이화학적특성당화액에야생효모를 5 일간발효한발효액의 ph, 고형분, 적정산도, 아미노산도및알코올함량을분석한결과는 Table 3 과같다. Saccharomyces 속효모의 ph 는 3.28 3.49 로나타났으며, A9-2 효모가가장낮았고 BY30-1 가가장높게나타났다. non-saccharomyces 속효모의 ph 는 3.01 3.61 로나 타났으며, N77-4 가가장낮았으며, Y447 효모가 ph 3.61 로가장높게나타났다. Saccharomyces 속효모의고형분함량은 8.73 9.27 Brix 로나타났으며, M1-9가가장낮았고 SD1-2가가장높았다. non-saccharomyces 속효모의고형분함량은 8.90 13.10 Brix로, N77-4가가장낮았고 Y447가가장높게나타났다. Saccharomyces 속효모의고형분함량은 non-saccharomyces 속효모보다낮은함량을나타내었고, 이는 Saccharomyces 속효모가당을이용하여알코올발효가더진행된것으로보인다. Saccharomyces 속효모의적정산도는 0.25 0.28% 로나타났으며, M1-9가가장낮았고 SD1-2가가장높았다. non- Saccharomyces 속효모는 0.28 0.33% 로나타났으며, Y447 가가장낮았고 Y685가가장높았다. Non-Saccharomyces 속효모의적정산도가 Saccharomyces 속효모보다높은경향을보였다. Saccharomyces 속효모의아미노산도는 1.61 2.02 g/100 ml로나타났으며, CD4-5가가장낮았고 SD1-2가가장높았다. non-saccharomyces 속효모는 1.91 2.61 g/100 ml로나타났으며, N77-4가가장낮았고 N56-10가가장높았다. 야생효모의알코올생성능력을비교하기위하여초기당화액의당도를 16 Brix로하여발효하였다. 5일발효후의알코올함량은 A9-2가 8.5% 로가장높았으나대조군을포

Characterization of Fermentation with Wild Yeasts 231 Table 3. Chemical content of alcohol fermentation with different wild yeasts and commercial yeasts. Yeast strains ph Soluble solid ( Brix) Titratable acidity (%) Amino acid contents (g/100 ml) Alcohol (%) Non-Sacchromyces yeasts N43-8 W. anomalus 3.40 ± 0.005 b,c 10.05 ± 0.150 c,d 0.32 ± 0.025 c 2.12 ± 0.057 e 3.80 ± 1.13 c SM2-7 W. anomalus 3.40 ± 0.000 b,c 09.65 ± 0.250 b,c 0.32 ± 0.006 c 2.10 ± 0.021 e 3.65 ± 0.21 c Y685 W. anomalus 3.41 ± 0.025 b,c 10.45 ± 0.650 d,e 0.33 ± 0.008 c 2.11 ± 0.028 e 3.10 ± 0.14 a,b,c N77-4 P. kudriavzevii 3.01 ± 0.020 a 08.90 ± 0.100 a.b 0.29 ± 0.006 b,c 1.91 ± 0.014 c 2.25 ± 0.07 a HP1-2 H. opuntiae 3.47 ± 0.157 c 11.70 ± 0.200 f 0.31 ± 0.026 c 2.26 ± 0.007 f 2.50 ± 0.42 a,b N56-10 H. uvarum 3.51 ± 0.030 c,d 11.45 ± 0.050 e,f 0.29 ± 0.003 b,c 2.61 ± 0.021 g 3.60 ± 0.85 b,c Y447 C. tropicalis 3.61 ± 0.049 d 13.10 ± 1.131 g 0.28 ± 0.055 b,c 2.21 ± 0.007 f 3.00 ± 0.28 a,b,c Frootzen P. kluyveri 3.05 ± 0.025 a 12.15 ± 0.150 f 0.31 ± 0.011 c 1.75 ± 0.014 b 3.10 ± 0.14 a,b,c S. cerevisiae A9-2 S. cerevisiae 3.28 ± 0.078 b 09.13 ± 0.577 a,b 0.26 ± 0.042 a,b 1.72 ± 0.021 b 8.50 ± 0.42 d CM4-5 S. cerevisiae 3.31 ± 0.098 b 08.83 ± 0.493 a 0.26 ± 0.015 a,b 1.61 ± 0.014 a 7.50 ± 0.14 d SD1-2 S. cerevisiae 3.39 ± 0.074 b,c 09.27 ± 0.643 a,b 0.28 ± 0.021 a,b,c 2.02 ± 0.021 d 8.40 ± 0.28 d M1-9 S. cerevisiae 3.30 ± 0.081 b 08.73 ± 0.651 a 0.25 ± 0.026 a,b 1.88 ± 0.028 c 7.80 ± 0.00 d BY30-1 S. cerevisiae 3.49 ± 0.080 c,d 08.90 ± 0.000 a,b 0.26 ± 0.011 a,b 1.62 ± 0.021 a 7.40 ± 0.71 d Fermivin S. cerevisiae 3.29 ± 0.091 b 09.00 ± 0.624 a 0.24 ± 0.038 a 1.75 ± 0.007 b 8.20 ± 0.00 d Data are means ± standard deviations of two independent experiments. Values displaying different superscript letter ( a,b,c,d,e,f,g ) according to the Duncan test (0.05%). Table 4. Concentrations of organic acid of alcohol fermentation with different wild yeasts and commercial yeasts. (Unit: g/l) Yeast strains Oxalic acid Malic acid Lactic acid Acetic acid Citric acid Succinic acid Non-Sacchromyces yeasts N43-8 W. anomalus 0.27 ± 0.001 e,f,g 0.21 ± 0.033 a,b 1.66 ± 0.031 a 1.56 ± 0.032 d 0.10 ± 0.036 a,b 0.20 ± 0.048 b,c SM2-7 W. anomalus 0.29 ± 0.008 f,g 0.26 ± 0.025 b 1.68 ± 0.033 a 1.72 ± 0.220 d 0.12 ± 0.042 a,b 0.22 ± 0.068 b,c Y685 W. anomalus 0.29 ± 0.001 f,g 0.26 ± 0.090 b 1.75 ± 0.154 a,b 1.58 ± 0.180 d 0.15 ± 0.002 b 0.17 ± 0.067 a,b,c N77-4 P. kudriavzevii 0.24 ± 0.018 c,d,e,f 0.60 ± 0.197 c 1.86 ± 0.162 a,b 0.29 ± 0.018 a,b 0.09 ± 0.048 a,b 0.27 ± 0.071 c HP1-2 H. opuntiae 0.28 ± 0.007 f,g 0.29 ± 0.069 b 1.94 ± 0.164 a,b 0.41 ± 0.071 b 0.08 ± 0.022 a 0.18 ± 0.077 b,c N56-10 H. uvarum 0.30 ± 0.003 g 0.32 ± 0.110 b 1.82 ± 0.079 a,b 0.77 ± 0.275 c 0.10 ± 0.025 a,b 0.21 ± 0.140 b,c Y447 C. tropicalis 0.23 ± 0.027 c,d,e 0.24 ± 0.034 a,b 1.94 ± 0.258 a,b 0.30 ± 0.085 a,b 0.08 ± 0.023 a 0.22 ± 0.038 b,c Frootzen P. kluyveri 0.29 ± 0.004 f,g 0.61 ± 0.175 c 1.96 ± 0.026 a,b 0.49 ± 0.007 b 0.10 ± 0.020 a,b 0.14 ± 0.039 a,b,c S. cerevisiae A9-2 S. cerevisiae 0.16 ± 0.035 a 0.26 ± 0.033 b 1.95 ± 0.103 a,b 0.31 ± 0.079 a,b 0.06 ± 0.016 a 0.10 ± 0.011 a,b CM4-5 S. cerevisiae 0.18 ± 0.033 a,b 0.25 ± 0.056 a,b 1.92 ± 0.146 a,b 0.36 ± 0.062 a,b 0.08 ± 0.004 a 0.12 ± 0.092 a,b,c SD1-2 S. cerevisiae 0.21 ± 0.020 b,c,d 0.28 ± 0.003 b 1.91 ± 0.082 a,b 0.49 ± 0.071 b 0.08 ± 0.006 a 0.22 ± 0.005 b,c M1-9 S. cerevisiae 0.18 ± 0.030 a,b 0.28 ± 0.047 b 1.89 ± 0.145 a,b 0.43 ± 0.099 b 0.06 ± 0.004 a 0.13 ± 0.060 a,b,c BY30-1 S. cerevisiae 0.22 ± 0.000 b,c,d 0.21 ± 0.019 a,b 2.06 ± 0.298 b 0.35 ± 0.026 a,b 0.08 ± 0.029 a 0.13 ± 0.060 a,b Fermivin S. cerevisiae 0.19 ± 0.020 a,b,c 0.19 ± 0.052 a,b 2.00 ± 0.033 a,b 0.25 ± 0.152 a,b 0.08 ± 0.020 a 0.16 ± 0.017 b,c Medium 0.31 ± 0.040 g 0.05 ± 0.039 a 1.89 ± 0.132 a,b 0.09 ± 0.041 a 0.09 ± 0.021 a nd a Data are means ± standard deviations of two independent experiments. Values displaying different superscript letter ( a,b,c,d,e,f,g ) according to the Duncan test (0.05%). nd : Not detected.

232 Baek et al. 함한 S. cerevisiae 6 종류는 7.4 8.5% 로유의적인차이는없었다. 대조군을포함한 non-saccharomyces 속 8 종류는최소 2.25% 에서최대 3.8% 까지알코올생성능력을보였으며, N43-8, SM2-7 이 non-saccharomyces 속에서가장높은알코올함량을나타내었다. 최근, Kim 등 [11] 이보고한알코올함량은유사하거나높은편으로나타났다. 야생효모에따른알코올발효액의유기산분석당화액에효모를 5일간발효한다음유기산함량을분석한결과는 Table 4와같다. 유기산중사과산 (malic acid) 함량은 Saccharomyces 속효모의경우, 0.21 0.28 g/l로나타났으며, BY30-1가가장낮았으며 SD1-2과 M1-9가가장높은함량을나타내었다. non-saccharomyces 속효모의사과산함량은 0.21 0.60 g/l로나타났으며, N77-4은 N43-8보다약 3배가량높았다. P. kudriavzevii N77-4 효모로발효한당화액의사과산함량이높았으며이는시판효모 (Frootzen) 와유사한결과를나타내었다. Kim 등 [12] 의연구에서도 S. cerevisiae 보다 P. anomala가생성한사과산이더높았으며, 본연구의결과와유사하였다. 초산 (acetic acid) 함량은 Saccharomyces 속효모의경우 0.31 0.49 g/l로나타났으며, A9-2가가장함량이낮았으며 SD1-2가가장높은함량을나타내었다. Non-Saccharomyces 속효모의초산함량은 0.29 1.72 g/l로나타났으며, P. kudriavzevii N77-4가가장낮았으며, W. anomalus SM2-7가가장높았다. 주류제조중품질에나쁜영향을미치는초산함량은전반적으로 non- Saccharomyces 속효모에서함량이높은것으로보아이들야생효모는 Saccharomyces 속효모보다초산을더생산하는것으로추측된다. 하지만 N77-4의초산함량은 Saccharomyces 속효모와유사하거나낮은결과를나타내었다. 호박산 (succinic acid) 함량은 Saccharomyces 속효모의경우 0.10 0.22 g/l 로나타났으며, A9-2 가가장낮았고 SD1-2 가가장높게나타났다. Non-Saccharomyces 속효모는 0.17 0.27 g/l 로나타났으며, W. anomalus Y685 가가장낮았으며, P. kudriavzevii N77-4 가가장높았다. 야생효모에따른유기산분석결과사과산, 초산, 호박산등이초기당화액에비해증가하였으며, 그중 P. kudriavzevii N77-4 는낮은초산함량과사과산, 호박산의높은함량을보여유기산에의한관능적특성이뛰어난것을알수있었다. 야생효모에따른알코올발효액의전자코분석당화액에효모를접종하여 5 일간발효한다음, 각효모의향기패턴을조사하기위해, 전자코를사용하여시료간의판별함수분석결과를 Fig. 1 에나타내었다. Fig. 1 에서와같이당화액과효모에의한알코올발효액을대상으로분석한결과, 당화액이오른쪽에위치하였고효모 12 균주의발효액시료가 DF1 값의왼쪽에위치하는것으로보아 DF1 값이오른쪽방향에서왼쪽으로이동할수록휘발성분이감지되는것으로분석되었다. 그결과, 가로축에해당하는 DF1 의 F 값이 98.45%, 세로축에해당하는 DF2 의값이 1.54% 로시료간의차별성이 x 축과 y 축이 60:1 의비율로영향을받아구분되었다. 당화액시료는 DF1 의오른쪽범주에속했으며, 효모에의한알코올발효액은 DF1 의왼쪽범주에속하여발효에따른뚜렷한차이를보였다. 또한 S. cerevisiae 와비교하였을때 non-saccharomyces 속의일부효모는 DF2 의아래쪽범주에속하여효모발효에의한향기패턴의차이가나타났다. Cordero-Bueso 등 [4] 이보고한포도주발효를위 Fig. 1. Discriminant function analysis (DFA) of the obtained data from electronic nose date for the flavor patterns of alcohol fermentation with different wild yeasts and commercial yeasts. ; Medium, ; Non-Saccharomyces yeasts, ; S. cerevisiae.

Characterization of Fermentation with Wild Yeasts 233 한 non-saccharomyces 연구에의하면효모의종에따라생산하는주요향기성분의차이를주성분분석 (PCA, principal component analysis) 으로나타내었으며, S. cerevisiase, P. kudiavzevii, W. anomalus, C. stellate 등이서로다른군집을형성하는것으로보고되어본연구의결과와유사하였다. 효모종마다생산하는주요향기성분과기질에따른변화등추가적인연구가필요한것으로사료된다. 야생효모에따른알코올발효액의휘발성향기성분당화액에효모종류를달리하여 5 일간발효한알코올발효액의휘발성향기성분을 GC-MS 로분석한결과를 Table 5, 6 에나타내었다. Non-Saccharomyces 속효모의주요휘발성향기성분은 ethyl acetate (11.71 91.88%) 와 ethanol (2.16 52.26%) 로나타났다. 반면 S. cerevisiase 효모의주요휘발성향기성분은 ethanol (37.67 62.89%) 과 ethyl acetate (1.32 11.97%) 로분석되었다. 알코올류에대해시험구별로 살펴보면 S. cerevisiase와 non-saccharomyces 속효모로크게구별되었는데, S. cerevisiase는 ethanol 외에도 3-methyl-abutanol, 2-methyl-1-propanol, 2-methyl-1-butanol 등다양한종류의알코올들이검출되었다. 반면 non-saccharomyces 속효모는에탄올및알코올류등의휘발성향기성분들이낮은비율을나타내었다. 국내생산되는대부분의막걸리향기성분은원료, 누룩, 주모및담금후술덧에생육하는각종미생물의발효작용에의해생성되며, 고급알코올류에속하는향기성분들은 isoamyl alcohol과 2-methyl-1-propanol로보고되었다 [16]. 본연구에서 2-methyl-1-propanol은모든 S. cerevisiase 시험구에서검출되었으며 (0.68 3.12%), non- Saccharomyces 속효모는 C. tropicalis Y447에서 4.41% 로가장높게나타났다. Non-Saccharomyces 속알코올발효액의주요휘발성향기성분은 ethyl acetate로나타났다. W. anomalus 효모 (N43-8, SM 2-7, Y685) 의 ethyl acetate의휘발성향기성분 Table 5. Volatile compound content of alcohol fermentation with different non-saccharomyces yeasts and commercial yeast. (Unit : peak area %) Volatile compounds N43-8 SM2-7 Y685 N77-4 HP1-2 N56-10 Y447 Frootzen Medium Acetic acid 0.69 nd 0.6 nd 0.89 1.81 nd nd nd Tetrahydro-2-methyl-furan nd nd nd nd 0.33 23.64 nd 0.08 0.61 Ethyl acetate 91.88 90.82 78.97 74.72 34.2 25.03 11.71 60.81 5.41 Isoamyl acetoacetate 0.49 0.125 nd 0.715 1.09 1.025 4.49 nd nd Ethanol 2.3 2.16 2.91 8.89 41.16 20.17 52.26 17.47 nd Heptyl alcohol nd nd nd nd nd 0.63 nd 0.11 1.29 2-Butanol nd nd nd nd 0.12 nd nd nd nd 1-Heptanol nd nd nd nd 0.31 nd nd nd nd 2-Methyl-1-pentanol nd nd nd nd nd nd nd nd 1.58 3-Methyl-1-butanol 0.04 nd 0.07 nd nd 0.58 nd nd 1.23 2-Methyl-1-propanol 0.15 0.08 nd nd 0.93 nd 4.41 nd nd 2-Methyl-1-butanol 0.15 nd 0.25 nd 0.8 1.03 1.42 nd nd 2-Phenyl ethanol nd nd nd nd 0.37 nd nd nd nd 3,3-dimethyl-4-[(1-methylethyl)amino]-2-Butanone 1.25 2.94 1.61 6.23 nd 5.05 7.42 13.59 19.23 2-Methylheptane nd nd nd nd nd nd nd nd 11.19 Benzene nd nd 7.18 nd nd nd nd nd nd 1,2-Benzenedicarboxylic acid, nd nd 2.38 nd nd nd nd nd nd bis(2-methoxyethyl) ester 2,4-dimethyl hexane 1.15 0.89 0.9 4.34 7.18 6.79 11.18 7.31 6.59 Decane 1.25 0.56 0.72 2.76 6.57 7.70 7.55 1.50 5.21 Dodecane nd nd nd 1.32 nd nd nd 5.42 10.43 Propadiene 0.08 nd nd nd 0.4 1.03 nd nd nd 1-Decene 0.08 nd nd nd 0.40 1.03 nd nd 1.2 nd : Not detected.

234 Baek et al. Table 6. Volatile compound content of alcohol fermentation with different wild-type S. cereviaise yeasts and commercial yeast. (Unit : peak area %) Volatile compounds A9-2 CM4-5 SD1-2 M1-9 BY30-1 Fermivin Medium Acetic acid 0.39 nd 0.43 nd nd nd nd Tetrahydro-2-methyl-furan 0.24 nd 0.14 nd 0.2 nd 0.61 Ethyl acetate 4.01 8.29 2.49 3.44 5.02 5.35 5.41 Isoamyl acetoacetate 4.00 9.15 1.32 3.80 5.46 11.97 nd Ethanol 62.89 41.71 51.96 37.67 40.19 37.12 nd Heptyl alcohol 0.54 nd 0.18 nd 0.47 nd 1.29 2-Butanol nd nd nd nd nd nd nd 1-Heptanol 0.10 nd nd nd nd nd nd 2-Methyl-1-pentanol 0.57 nd nd nd nd nd 1.58 3-Methyl-1-butanol 0.52 0.43 0.17 0.37 nd 0.24 1.23 2-Methyl-1-propanol 2.27 3.12 0.68 2.31 1.22 2.58 nd 2-Methyl-1-butanol 2.12 3.24 0.63 2.57 0.79 3.04 nd 3,3-dimethyl-4-[(1-methylethyl)amino]-2-Butanone nd nd nd nd nd nd nd 2-Methylheptane 7.71 9.16 nd 12.76 13.36 6.39 19.23 Benzene nd nd nd nd nd nd nd 1,2-Benzenedicarboxylic acid, nd nd nd nd nd nd nd bis(2-methoxyethyl) ester 2,4-dimethyl hexane 3.74 nd nd 3.68 15.69 8.68 11.19 Decane 6.45 11.06 9.26 12.34 13.19 9.32 6.59 Dodecane 1.88 10.77 0.94 11.19 11.84 10.24 5.21 Propadiene nd nd 47.84 3.11 nd nd 10.43 1-Decene 0.48 nd 0.2 0.4 nd 0.53 nd nd : Not detected. 이높게나타났으며, 그중 W. anomalus N43-8 효모가 91.88% 로써시판효모인 Frotzen과 Fermivin보다 1.5배에서 17배이상높게나타났다. 그리고 P. kudriavzevii N77-4 효모도 74.72% 로높게나타났다. 특히, ethyl acetate 화합물은시럽의향료로사용되며배향, 바나나향, 사과향등의향을내는에스테르중의하나로알려져있다 [21]. 휘발성향기성분인 ethyl acetate는우리나라전통주, 맥주뿐만아니라일본증류식소주의대표적인에스테르성분이지만농도가높으면오히려쓴맛의원인물질로알려져있고 [7], 방향성향기성분으로서알코올류보다향미기여도가높다고보고되었다 [16]. Acetic acid는주류의발효과정중세균과효모의발효작용으로생성되는산화생성물로서자극취를나타내는산미성분이며 [15], non-saccharomyces 속효모중 H. uvarum N56-10, H. opuntiae HP1-2, W. anomalus N43-8, Y685에서소량 (1.81%, 0.89%, 0.69%, 0.6%) 생성되었다. S. cerevisiae A9-2와 SD1-2의초산은각각 0.39%, 0.43% 로나타나 non-saccharomyces 속효모에비해초산함량이낮거 나거의생성되지않았다. 본연구의결과는 Lee 등 [18] 의누룩종류를달리한탁주술덧과효모종류를달리한탁주술덧 [16] 보다향기성분의종류는적었지만검출된휘발성향기성분중 ethanol, ethyl acetate, 2-methyl-1-propanol, acetic acid 등 5 종은 Lee 등 [16, 18] 의결과와공통된향기성분을가지고있었다. 그러나 tetrahydro-2-methyl-furan, isoamyl acetoacetate, decane, dodecane 등 17 종은본연구에서만검출된것으로보아효모종류에따라면적비율이나주성분등에차이가있는것을알수있었다. 요 약 발효식품에서분리한야생효모 12 균주의주류제조가능성을조사하기위해, 알코올발효에관여하는특성을분석하였다. 알코올및당내성을조사한후, 당화액을제조하여 ph, 고형분함량, 적정산도, 아미노산도, 알코올, 유기산및

Characterization of Fermentation with Wild Yeasts 235 향기성분등을분석하였다. S. cerevisiae 효모의알코올내성은 10% 에서보였으나 non-saccharomyces 효모는 P. kudriavzevii N77-4 를제외하고내성이낮았다. 당내성은 H. opuntiae HP1-2, C. tropicalis Y447 을제외한효모에서모두우수하였다. 야생효모를이용한알코올발효액의 ph 는 3.01 3.61, 고형분함량은 8.73 13.10 Brix 로 S. cerevisiae 보다 non-saccharomyces 가높았다. 적정산도는 0.25 0.33% 로, 아미노산도는 1.61 2.61 g/100 ml 으로 non-saccharomyces 효모가높은값을나타내었다. 알코올함량은 2.25 8.5% 로 S. cerevisiae 에비해 non-saccharomyces 효모가매우낮았다. 야생효모로알코올발효시유기산은사과산, 초산, 호박산등이증가하였고, 그중 P. kudriavzevii N77-4 는낮은초산함량과높은사과산및호박산함량을나타내었다. 당화액을전자코로분석한결과, DF1 의오른쪽범주에속했으며, 효모발효액은 DF1 의왼쪽범주에속하여발효에따른뚜렷한차이를보였다. 휘발성향기성분은 non-saccharomyces 효모에서 ethyl acetate 가, S. cerevisiae 효모는 ethanol 화합물이높은향기성분을나타내었다. 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. Baek SY, Lee YJ, Kim JH, Yeo SH. 2015. Isolation and characterization of wild yeasts for improving liquor flavor and quality. Korean J. Microbiol. Biotechnol. 43: 56 64. 2. Charoenchai C, Fleet GH, Henschke PA, Todd BEN. 1997. Screening of non-saccharomyces wine yeasts for the present of extracellular hydrolytic enzymes. Australian J. Grape Wine Res. 3: 2 8. 3. Ciani M, Beco L, Comitini F. 2006. Fermentation behavior and metabolic interactions of multistarter wine yeast fermentations. Int. J. Food Microbiol. 108: 239 245. 4. Cordero-Bueso G, Esteve-Zarzoso B, Cabellos JM, Gil-Diaz M, Arroyo T. 2013. Biotechnological potential of non-saccharomyces yeasts isolated during spontaneous fermentations of Malvar (Vitis vinifera cv.l.). Eur. Food Res. Technol. 236: 193 207. 5. 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. 6. Im SY, Baek CH, Baek SY, Park HY, Choi HS, Choi JH, et al. 2014. Quality characteristics of Takju according to different rice varieties and mixing ratio of Nuruk. Korean J. Food Preserv. 21: 892 902. 7. In HY, Lee TS, Lee DS, Noh BS. 1995. Volatile components and fusel oils of sojues and mashes brewed by Korean traditional method. Korean J. Food Sci. Technol. 27: 235 240. 8. Jeon HJ, Yu JC, Kim GW, Kong HS. 2014. Quality characteristics of Takju by yeast strain type. Korean J. Food Nutr. 27: 971 978. 9. Jin TY, Kim ES, Eun JB, Wang SJ, Wang MH. 2007. Changes in physicochemical and sensory characteristics of rice wine, yakju prepared with different amount of red yeast rice. Korean J. Food Sci. Technol. 39: 309 314. 10. 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. 11. Kim HR, Baek SH, Seo MJ, Ahn BH. 2006. Feasibility of Cheongju Brewing with wild type yeast strains from Nuruks. Korean J. Microbiol. Biotechnol. 34: 244 249. 12. Kim HR, Kim JH, Bai DH, Ahn BH. 2012. Feasibility of brewing Makgeolli using Pichia anomala Y197-13, a Non-Saccharomyces cerevisiae. Korean J. Microbiol. Biotechnol. 22: 1749 1757. 13. Kim JI, Lee NK, Hahm YT. 2007. Isolation and Identification of wild Yeast and its use for the production of grapewine. Korean J. Microbiol. 43: 217 221. 14. Kim KH, Dong HM, Han HJ, Lee YH, Moon JY, Bang KH, Noh BS. 2013. Analysis of geographical origin of red ginseng extract using mass spectrometer-based electronic nose. Korean J. Food Sci. Technol. 45: 652 656. 15. Kurtzman CP, Fell JW. 1998. The Yeasts, A Taxonomic Study (fourth edition). Elsevier, Amsterdam, Netherlands. pp. 358 371. 16. 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. 17. Lee SH, Park HK, Kim MH. 2010. Isolation and identification of wild yeasts from Schizandra (Schizandra chinesis) for wine production and its characterization for physicochemical and sensory evaluations. J. Korean Soc. Food Sci. Nutr. 39: 1860 1866. 18. Lee TS, Choi JY. 2005. Volatile flavor components in mash of takju prepared by using Aspergillus kawachii nuruks. Korean J. Food Sci. Technol. 37: 944 950. 19. Min YK, Lee MK, Jeong HS. 1997. Fermentation characteristics of jujube alcoholic beverage from different additional level of jujube fruit. J. Korean Agric. Chem. Soc. 40: 433 437. 20. Montville TJ, Matthews KR. 2005. Food microbiol. : an introduction. ASM Press, Washington, D.C., USA. pp. 223 239. 21. Nishiaya T. 1997. Composition of soju. J. Jpn. Soc. Brew. 72: 415-432.