KR101862487B1 - Saccharomycopsis fibuligera SSU2601-09 strain for fermentation of Korean traditional alcohol and the method for preparing makgeolli using the same - Google Patents

Saccharomycopsis fibuligera SSU2601-09 strain for fermentation of Korean traditional alcohol and the method for preparing makgeolli using the same Download PDF

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KR101862487B1
KR101862487B1 KR1020160162590A KR20160162590A KR101862487B1 KR 101862487 B1 KR101862487 B1 KR 101862487B1 KR 1020160162590 A KR1020160162590 A KR 1020160162590A KR 20160162590 A KR20160162590 A KR 20160162590A KR 101862487 B1 KR101862487 B1 KR 101862487B1
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서정아
캐롤 에밀리
김민주
김민수
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    • C12G3/02—Preparation of other alcoholic beverages by fermentation
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    • C12G3/022—Preparation of other alcoholic beverages by fermentation of botanical family Poaceae, e.g. wheat, millet, sorghum, barley, rye, or corn of botanical genus Oryza, e.g. rice
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Abstract

본 발명은 전통주 발효 효모 사카로마이콥시스 피브리게라(Saccharomycopsis fibuligera) SSU2601-09 균주(KCTC13034BP) 및 이를 이용한 막걸리 제조방법에 관한 것으로서, 상기 균주는 당화, 발효능 및 독특한 풍미를 가지고 있다. 특히, 상기 균주는 단일 균주로 당화 및 발효를 동시에 할 수 있으며 제품 개선을 위한 기초적인 자료로 활용될 수 있으며, 주류 산업에 유용한 정보를 제공할 것이다. 상기 균주와 같은 우수한 발효능과 기능성을 가진 종균 개발과, 전통주의 표준화 및 고급화 공정에 활용할 수 있는 기반 기술 개발은 바이오산업에서의 국제 경쟁력 제고에 크게 기여할 것으로 예상된다.The present invention relates to a strain of Saccharomycopsis fibuligera SSU2601-09 (KCTC13034BP) and a method for producing a makgeolol using the same, wherein the strain has saccharification, efficacy and unique flavor. In particular, the strain can be saccharified and fermented simultaneously as a single strain, and can be used as basic data for improving the product, and will provide useful information to the mainstream industry. It is expected that the development of seeds having excellent efficacy and functionality such as the above strains and the development of a base technology that can be utilized in the standardization and upgrading process of traditional strains will greatly contribute to enhancement of international competitiveness in the biotechnology industry.

Description

전통주 발효 효모 사카로마이콥시스 피브리게라 SSU2601-09 균주 및 이를 이용한 막걸리 제조방법{Saccharomycopsis fibuligera SSU2601-09 strain for fermentation of Korean traditional alcohol and the method for preparing makgeolli using the same}(Saccharomycopsis fibuligera strain SSU2601-09 strain for fermentation of Korean traditional alcohol and the method for preparing makgeolli using the same)

본 발명은 전통주 발효 효모 사카로마이콥시스 피브리게라(Saccharomycopsis fibuligera) SSU2601-09 균주 및 이를 이용한 막걸리 제조방법에 관한 것이다. The present invention relates to my Cobb cis blood debris galley (Saccharomycopsis fibuligera) SSU2601-09 rice wine strains and production method using the same in traditionalism fermenting yeast Saccharomyces.

우리나라는 오래전부터 김치, 술, 간장, 된장과 같이 전통발효 방법을 사용하여 다양한 발효식품을 제조해온 나라임에도 불구하고 국내 전통식품 관련 발효 미생물의 유전체 정보를 확보하고 이를 기반으로 하는 종균 개발 및 유용자원 활용 기술 개발 연구는 외국에 비해 상대적으로 미진하다. 국내 전통주 산업은 대표적인 2∼3개 업체를 제외하면 매우 영세한 규모로 운영되어 기본적인 품질관리조차 제대로 이루어지지 않는 업체가 대부분이다. 특히 아직 우수한 종균 발명이 미비한 관계로 매년 고액의 로열티가 해외 생물자원의 사용 대가로 지불되고 있다.Although Korea has been manufacturing fermented foods using traditional fermentation methods such as kimchi, liquor, soy sauce, and soybean paste for a long time, it has acquired genome information of fermented microorganisms related to traditional foods in Korea, Utilization technology development research is relatively small compared to foreign countries. Except for the typical two to three companies, the traditional Korean traditional industry operates on a very small scale, and most of the companies do not even perform basic quality control properly. Especially, since there are not enough excellent seeds invented yet, a large amount of royalties are paid every year for the use of overseas biological resources.

외국의 전통주와 달리 한국 전통주는 복합 누룩 균주에 의해 제조되고 있는데, 한국 전통주의 표준화, 과학화, 세계화를 위해서 핵심 종균들에 대한 유전체, 대사체 정보의 확보가 필수적이다. 국내 전통주에서 분리한 효모균들을 대상으로 우수한 발효능과 기능성을 가진 종균 개발에 필요한 유용 유전자원을 확보하고, 전통주의 표준화 및 고급화 공정에 활용할 수 있는 기반 기술 개발은 바이오산업에서의 국제 경쟁력 제고에 크게 기여할 것이다. Unlike the traditional Korean traditional alcoholic beverages, Korean traditional alcoholic beverages are manufactured by the complex Nuruk strains. For the standardization, scientific and globalization of Korean traditional alcoholic beverages, it is essential to secure the genome and metabolic information for the key insecticides. The development of the basic technology that can be used for the standardization and the upgrading process of the traditional wine is secured for the enhancement of the international competitiveness in the bio industry by securing the useful genetic resources necessary for the development of the seed culture having excellent efficacy and functionality, Will contribute.

유전체 해독 및 대사체 프로파일링을 통해 유용 형질과 관련된 유전자들을 발굴하고 관련 대사경로를 이해할 수 있으며 이를 기반으로 종 다양성 분석 연구를 수행하여 야생 균주 및 우량 형질 보유 균주들을 체계적으로 선발 및 확보하고, 더 나아가 우수 목표 형질을 가진 균주로의 개량이 필요하다. Genetic analysis and metabolism profiling can be used to identify genes associated with useful traits and to understand relevant metabolic pathways. Based on this analysis, species diversity analysis studies are conducted to systematically screen and secure wild and well trait strains. Furthermore, it is necessary to improve the strain to have excellent target traits.

국내에서 일부 모델 생물체로 활용되는 곰팡이 혹은 효모 대상의 오믹스 분석 연구는 진행된 바 있으나, 전통 주류 진핵 미생물에 관한 유전체 기반 오믹스 분석 연구는 미비한 상태이므로, 효모 및 곰팡이를 이용한 전통 주류의 유전체 기능에 대한 총체적인 연구 및 풍미, 품질관리에 대한 미생물 유전체와 발효조건의 상관관계에 대한 연구가 필요한 실정이다. Studies on the analysis of omics for fungi or yeast, which are used as some model organisms in Korea, have been carried out, but studies on the genome-based omics of traditional mainstream eukaryotic microorganisms have not been conducted so that the genome functions of traditional liquor using yeast and fungi And the relationship between microbial genome and fermentation conditions for flavor and quality control is needed.

전통 주류산업에서 효모 균주의 생화학적 작용에 의해 다양한 대사체들이 최종적으로 생성되며, 이들에 의해 전통 발효주의 기능성, 영양학적 특성 및 품질 특성이 결정된다. 특히, 제품의 기호도와 밀접한 연관이 있는 풍미와 관련한 미생물 대상의 유전체/대사체에 대한 총체적인 연구는 제품 개선을 위한 기초적인 자료로 활용될 수 있으며, 산업적으로도 품질 관리에 유용한 정보를 제공할 것이다.A variety of metabolites are finally produced by the biochemical action of yeast strains in traditional liquor industry, which determine the functional, nutritional and quality characteristics of traditional fermented beverages. In particular, a comprehensive study of the genome / metabolism of microorganisms in relation to the flavor closely related to the preference of the product can be used as basic data for product improvement, and it will provide useful information for quality control in industry .

한국공개특허 제10-2014-0055917호(2014.05.09 공개)Korean Patent Publication No. 10-2014-0055917 (published May 2014, 2014)

본 발명의 목적은 전통주 발효 효모 사카로마이콥시스 피브리게라(Saccharomycopsis fibuligera) SSU2601-09 균주 및 이를 이용한 막걸리 제조방법을 제공하는 데에 있다.It is an object of the present invention to provide a strain of Saccharomycopsis fibuligera SSU2601-09 , a traditional strain fermenting yeast, and a process for producing makgeolli using the strain.

상기 목적을 달성하기 위하여, 본 발명은 수탁번호 KCTC13034BP로 기탁된, 당화 및 발효능이 우수한 전통주 발효 효모 사카로마이콥시스 피브리게라(Saccharomycopsis fibuligera) SSU2601-09 균주를 제공한다. In order to achieve the above object, the present invention provides Saccharomycopsis fibuligera strain SSU2601-09, which has been deposited with Accession No. KCTC13034BP and has excellent saccharification and fermentation efficiency.

또한, 본 발명은 상기 균주를 사용하여, 곡류 원료로 막걸리를 제조하는 방법을 제공한다.Further, the present invention provides a method for producing makgeolli using the strain as a raw material for grain.

본 발명은 전통주 발효 효모 사카로마이콥시스 피브리게라(Saccharomycopsis fibuligera) SSU2601-09 균주(KCTC13034BP) 및 이를 이용한 막걸리 제조방법에 관한 것으로서, 상기 균주는 당화, 발효능 및 독특한 풍미를 가지고 있다. 특히, 상기 균주는 단일 균주로 당화 및 발효를 동시에 할 수 있으며 제품 개선을 위한 기초적인 자료로 활용될 수 있으며, 주류 산업에 유용한 정보를 제공할 것이다. 상기 균주와 같은 우수한 발효능과 기능성을 가진 종균 개발과, 전통주의 표준화 및 고급화 공정에 활용할 수 있는 기반 기술 개발은 바이오산업에서의 국제 경쟁력 제고에 크게 기여할 것으로 예상된다.The present invention relates to a strain of Saccharomycopsis fibuligera SSU2601-09 (KCTC13034BP) and a method for producing a makgeolol using the same, wherein the strain has saccharification, efficacy and unique flavor. In particular, the strain can be saccharified and fermented simultaneously as a single strain, and can be used as basic data for improving the product, and will provide useful information to the mainstream industry. It is expected that the development of seeds having excellent efficacy and functionality such as the above strains and the development of a base technology that can be utilized in the standardization and upgrading process of traditional strains will contribute to enhancement of international competitiveness in the bio industry.

도 1은 본 발명에 따른 사카로마이콥시스 피브리게라(Saccharomycopsis fibuligera) SSU2601-09 (KCTC13034BP)를 현미경으로 관찰한 결과이다.
도 2는 분리 및 동정된 다양한 균주에서 있어서, 밀기울 코지의 효소활성을 나타낸다.
도 3은 본 발명에 따른 사카로마이콥시스 피브리게라(Saccharomycopsis fibuligera) SSU2601-09 (KCTC13034BP)로 빚은 막걸리의 관능 평가 결과이다.1 is a microscopic observation of Saccharomycopsis fibuligera SSU2601-09 (KCTC13034BP) according to the present invention.
Figure 2 shows the enzymatic activity of bran koji in various strains isolated and identified.
FIG. 3 shows the sensory evaluation results of rice wine with Saccharomycopsis fibuligera SSU2601-09 (KCTC13034BP) according to the present invention.

본 발명은 수탁번호 KCTC13034BP로 기탁된, 당화 및 발효능이 우수한 전통주 발효 효모 사카로마이콥시스 피브리게라(Saccharomycopsis fibuligera) SSU2601-09 균주를 제공한다.The invention in the, superior efficacy to saccharification and fermentation of yeast Saccharomyces traditionalism deposited as accession number KCTC13034BP Mai Cobb cis blood debris galley (Saccharomycopsis fibuligera ) strain SSU2601-09.

상세하게는, 상기 균주는 누룩으로부터 분리되었으며, 알파-아밀라아제(Alpha-Amylase), 글루코아밀라아제(Glucoamylase) 또는 프로테아제(Protease) 활성이 우수한 것을 특징으로 한다.Specifically, the strain is isolated from yeast and is characterized by excellent activity of Alpha-Amylase, Glucoamylase or Protease.

상세하게는, 상기 균주는 서열번호 3으로 표시되는 내부 전사 영역(Internal transcribed spacer; ITS)으로 이루어질 수 있으나, 이에 한정되는 것은 아니다.In detail, the strain may be an internal transcribed spacer (ITS) represented by SEQ ID NO: 3, but is not limited thereto.

또한, 본 발명은 상기 균주를 사용하여, 곡류 원료로 막걸리를 제조하는 방법을 제공한다.Further, the present invention provides a method for producing makgeolli using the strain as a raw material for grain.

상세하게는, 상기 곡류 원료는 막걸리를 제조할 수 있는 찹쌀, 멥쌀, 보리쌀, 현미, 옥수수, 고구마, 밀 등의 곡류 원료일 수 있으며, 보다 상세하게는 쌀 또는 밀기울일 수 있으나, 이에 한정되는 것은 아니다.Specifically, the cereal raw materials may be cereal raw materials such as glutinous rice, rice, barley, brown rice, corn, sweet potato, and wheat which can produce rice wine, and more specifically rice or wheat. no.

상세하게는, 상기 균주를 사용하여 제조된 막걸리는 풍미가 우수한 것을 특징으로 한다.Specifically, the rice wine produced using the strain is characterized by excellent flavor.

본 발명에 있어서, '막걸리'는 막걸리는 찹쌀, 멥쌀, 보리쌀, 현미, 옥수수, 고구마, 밀 등의 전분질을 원료로 하고 당화, 발효제로서 진핵미생물 군집의 총합체인 누룩을 첨가하여 발효시킨 술덧을 혼탁하게 제성한 우리나라 고유의 전통주로, 단맛, 신맛, 쓴맛, 매운맛과 청량감이 있고 알코올 함량이 2-8%인 술이다. 다른 주류와 달리 막걸리는 각종 영양원이 풍부하게 함유되어 있는데, 인체 신진대사에 관여하는 비타민 B군을 비롯한 라이신, 류신, 아르기닌 등의 필수 아미노산, 풍미물질인 에틸아세테이트, 아밀아세테이트, 에틸카르로에이트 등의 에스테르, 새콤한 맛을 내어 갈증을 해소케 하는 유기산, 그리고 간 기능을 도와주는 아세틸콜린 등이 함유되어 있다. 이와 같이 막걸리는 영양학적 및 기능적 가치가 높을 뿐만 아니라 생효모가 함유되어 있기 때문에 다른 주류와 비교할 수 없는 독특한 맛을 지니고 있다.In the present invention, "makgeolli" means that makgeolli is prepared by using starch such as glutinous rice, rice, barley, brown rice, corn, sweet potato and wheat as a raw material and adding yeast, which is a total of eukaryotic microbial communities as a saccharification and fermentation agent, Is a Korean traditional alcoholic beverage which has a sweet, sour, bitter, hot and refreshing taste and alcohol content of 2-8%. Unlike other alcoholic beverages, Makgeolli is rich in various nutrients. It contains vitamins B, which are involved in human metabolism, essential amino acids such as lysine, leucine and arginine, flavors such as ethyl acetate, amyl acetate, ethyl caroate , An organic acid to relieve thirst with a sour taste, and acetylcholine to help liver function. In addition to high nutritional and functional value, makkolli has a unique flavor that can not be compared with other alcoholic beverages because it contains raw yeast.

이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

<< 실시예Example 1> 전통주 발효 효모 사카로마이콥시스 피브리게라( 1> Traditional fermented yeast Saccharomyces cispifbrigera ( Saccharomycopsis fibuligeraSaccharomycopsis fibuligera ) SSU2601-09 균주의 분리) Isolation of SSU2601-09 strain

1. 균주의 분리1. Isolation of strain

본 발명자들은 당화, 발효 및 풍미를 가진 균주를 발굴하기 위하여 2013년 7월 제주지역의 전통방식으로 만든 누룩을 수집하여 4℃에서 보관하고 고르게 분쇄한 누룩 10 g을 90 ml의 0.1% 펩톤(peptone)에 현탁한 후 단계적으로 10-2~10-7배 희석하였다. 희석한 현탁액은 100 ㎕씩 분리용 평판배지에 도말하여 25℃에 배양하였다. 이때 사용되는 분리배지는 Dichloran-glycerol agar (DG18: Peptone 0.5%, Glucose 1%, KH2PO4 0.1%, MgSO4·7H2O 0.05%, 0.2% Dichloran 1.0 ml, Chloramphenicol 0.01%, Agar 1.5%) 배지와 Dichloran rose bengal chloramphenicol agar (DRBC: Peptone 0.5%, Glucose 1%, KH2PO4 0.1%, MgSO4·7H2O 0.05%, 0.2% Dichloran 1.0 ml, Rose bengal 0.0025%, Chloramphenicol 0.01%, Agar 1.5%) 배지이며, 이후 분리된 균주는 Malt extract agar (MEA: Malt extract 3%, Mycological peptone 0.5%, Agar 1.5%) 배지에 접종하고 보존하였다.In order to identify strains with saccharification, fermentation, and flavor, we collected the yeast in traditional Jeju area in July 2013, stored at 4 ℃, weighed 10 g of uniformly crushed koji in 90 ml of 0.1% peptone ) And then diluted with 10 -2 to 10 -7 times in a stepwise manner. 100 [mu] L of the diluted suspension was plated on a separate plate culture medium and cultured at 25 [deg.] C. The separation medium used was Dichloran-glycerol agar (DG18: Peptone 0.5%, Glucose 1%, KH 2 PO 4 0.1%, MgSO 4揃 7H 2 O 0.05%, 0.2% Dichloran 1.0 ml, Chloramphenicol 0.01%, Agar 1.5% ) Medium and Dichloran rose bengal chloramphenicol agar (DRBC: Peptone 0.5%, Glucose 1%, KH 2 PO 4 0.1%, MgSO 4揃 7H 2 O 0.05%, 0.2% Dichloran 1.0 ml, Rose bengal 0.0025%, Chloramphenicol 0.01% Agar 1.5%). The isolated strains were inoculated and stored in Malt extract agar (MEA: Malt extract 3%, Mycological peptone 0.5%, Agar 1.5%) medium.

2. 선별된 균주의 동정2. Identification of selected strains

(1) 형태학적 동정(1) Morphological identification

상기에서 선별된 균주를 대상으로 현미경을 통해 균주의 형태학적 모양을 관찰하였다. 25℃에서 Potato dextrose agar에서 5일간 배양한 균주를 관찰 결과, 동정된 균주는 5 ~ 10 μm 크기의 단일세포와 긴 hypae를 형성하는 모양을 갖는 것을 관찰할 수 있었다(도 1). The morphological shape of the strain was observed through a microscope. The strains cultivated in Potato dextrose agar at 25 ° C for 5 days showed that the strain had a shape of 5-10 μm single cells and long hypae (Fig. 1).

(2) 분자생물학적 동정(2) Molecular biology identification

상기에서 선별한 균주의 분자생물학적 동정을 위해 CTAB 방법을 이용하여 DNA를 추출하였고, ITS (Internal transcribed spacer)를 PCR (polymerase chanin reaction) 증폭하기 위하여 배양체로부터 비드 비팅(bead beating) 방법을 이용하여 DNA를 추출하였고, 내부 전사되는 ITS(Internal transcribed spacer)를 PCR (polymerase chain reaction) 증폭하기 위하여 ITS1 (TCCGTAGGTGAACCTGCGG; 서열번호 1) / ITS4 (TCCTCCGCTTATTGATATGC; 서열번호 2)의 프라이머 세트를 각각 이용하여 증폭한 후 정제를 통해 (주)Macrogen에 시퀀싱을 의뢰하여 염기서열을 결정하였다. 분석된 ITS 부위의 염기서열은 서열번호 3에 나타냈다. DNA was extracted using the CTAB method for the molecular identification of the strains selected above, and DNA was extracted from the cultured beads by a bead beating method to amplify the internal transcribed spacer (PCR) (polymerase chain reaction) (TCCGTAGGTGAACCTGCGG; SEQ ID NO: 1) / ITS4 (TCCTCCGCTTATTGATATGC; SEQ ID NO: 2) in order to amplify an internal transcribed spacer (ITS) Sequencing was commissioned by Macrogen Inc. to determine the nucleotide sequence. The nucleotide sequence of the analyzed ITS region is shown in SEQ ID NO: 3.

(3) 균학적 동정(3) Mycological identification

상기 동정한 전통주 발효 효모 사카로마이콥시스 피브리게라(Saccharomycopsis fibuligera) SSU2601-09 균주의 균학적 특징을 조사하였는데, 즉 다양한 온도 조건, 다양한 배지 및 질소원 하에서 최적의 배양 조건을 조사하였다. The mycological characteristics of Saccharomycopsis fibuligera strain SSU2601-09 were investigated. The optimal culture conditions were investigated under various temperature conditions, various media and nitrogen source.

조사 결과, 표 1에서 나타난 바와 같이 생육 온도는 25 ~ 42℃의 온도에서 성장하는 특징이 있는 것을 나타났으며, 특히 25 ~ 37℃에서 성장이 우수한 것으로 나타났다(표 1).As shown in Table 1, the growth temperature was 25 to 42 ° C, and growth was particularly good at 25 to 37 ° C (Table 1).

배지badge 25℃25 ℃ 37℃37 ℃ 42℃42 ° C 45℃45 ° C CMCM ++++++ ++++++ ++ -- PDAPDA ++++++ ++++++ ++ -- YPDYPD ++++++ ++++++ ++ -- MMMM -- -- -- -- MM (NaNO3)MM (NaNO 3 ) -- -- -- -- MM (NH4Cl)MM (NH 4 Cl) -- -- -- --

<< 실시예Example 2> 전통주 발효 효모 사카로마이콥시스 피브리게라( 2> Traditional yeast fermentation yeast Saccharomyces cispifbrigera ( Saccharomycopsis fibuligeraSaccharomycopsis fibuligera ) SSU2601-09 균주의 효소 역가 특성) Enzyme activity characteristics of strain SSU2601-09

본 발명에서 동정한 균주 및 여러 비교 균주로 만든 쌀코지와 밀기울 코지의 알파-아밀라아제, 글루코아밀라아제, 프로테아제(곰팡이성)의 역가를 확인하였다(표 2). 또한, 분리 및 동정된 다양한 균주에서 있어서, 밀기울 코지의 효소활성을 도 2에 나타냈다. 분석방법은 식품의약품안전처의 식품첨가물 공전에 따라 분석하였다.The activity of alpha-amylase, glucoamylase, and protease (fungal) of rice koji and bran koji made from strains identified in the present invention and various comparative strains was confirmed (Table 2). In addition, for various strains isolated and identified, the enzyme activity of bran koji is shown in Fig. Analysis methods were analyzed according to the Food Additives Code of the Food and Drug Administration.

알파-아밀라아제
(Alpha-Amylase)Alpha-amylase
(Alpha-Amylase) 글루코아밀라아제
(Glucoamylase)Glucoamylase
(Glucoamylase) 프로테아제
(Protease) Protease
(Protease) 사카로마이콥시스 피브리게라 SSU2601-09Sakaromai Cobsis Fibriegera SSU2601-09 쌀코지
(Rice Koji)Rice koji
(Rice Koji) 11.8711.87 552.97552.97 291.99291.99 밀기울 코지
(Wheat Koji)Bran koji
(Wheat Koji) 12.2212.22 977.91977.91 573.73573.73 사카로마이콥시스 피브리게라 LNJJ81Sakaromi Cobisis Fibriegera LNJJ81 쌀코지
(Rice Koji)Rice koji
(Rice Koji) 2.822.82 00 00 밀기울 코지
(Wheat Koji)Bran koji
(Wheat Koji) 7.867.86 131.55131.55 53.2753.27 사카로마이콥시스 피브리게라 LNPH12Sakaromycops Cispifbrigera LNPH12 쌀코지
(Rice Koji)Rice koji
(Rice Koji) 4.404.40 31.0431.04 425.25425.25 밀기울 코지
(Wheat Koji)Bran koji
(Wheat Koji) 1.971.97 68.0668.06 537.06537.06 아스퍼질러스
오리재
CN2601-05
Aspergillus
Duck
CN2601-05
쌀코지
(Rice Koji)Rice koji
(Rice Koji) 5.365.36 100.32100.32 444.02444.02 밀기울 코지
(Wheat Koji)Bran koji
(Wheat Koji) 42.3642.36 205.52205.52 1197.781197.78 리케테이미아
라모사
LNPH11Lycetemia
Ramosa
LNPH11 쌀코지
(Rice Koji)Rice koji
(Rice Koji) 0.610.61 233.12233.12 1286.301286.30 밀기울 코지
(Wheat Koji)Bran koji
(Wheat Koji) 1.641.64 83.7183.71 7852.907852.90 리케테이미아
라모사
LNJJ15-06Lycetemia
Ramosa
LNJJ15-06 쌀코지
(Rice Koji)Rice koji
(Rice Koji) 3.163.16 43.0943.09 2763.572763.57 밀기울 코지
(Wheat Koji)Bran koji
(Wheat Koji) 3.863.86 397.48397.48 11264.3611264.36

Alpha-amylase: the amount of ceralpha units per gram of koji. Alpha-amylase: the amount of ceralpha units per gram of koji.

The glucose activity: the units of glucose released per gram of koji.The glucose activity: the units of glucose released per gram of koji.

One unit of protease: the amount of enzyme required to release 1μg of tyrosine/hour.One unit of protease: the amount of enzyme required to release 1 μg of tyrosine / hour.

<< 실시예Example 3> 전통주 발효 효모 사카로마이콥시스 피브리게라( 3> Traditional yeast fermentation yeast Saccharomyces cispifbrigera ( Saccharomycopsis fibuligeraSaccharomycopsis fibuligera ) SSU2601-09 균주로 빚은 막걸리의 특성Characteristics of rice wine made with strain SSU2601-09

1. 쌀 코지1. rice koji

쌀을 세미 후 2시간 동안 침지한 다음 30분간 물 빼기를 한 뒤 증미기를 이용하여 1시간 30분 증자한다. 증자된 쌀을 실온에서 냉각한 뒤 쌀 중량의 0.1%의 종균을 접종하여 항온항습배양기에서 30℃, 상대습도 70% 조건에서 72시간 배양하였다. The rice is immersed for 2 hours after the semi-baking, and then the rice is drained for 30 minutes. Then, the rice is baked for 1 hour and 30 minutes by using a steamer. After the rice was cooled at room temperature, 0.1% of rice seed weight was inoculated and cultured in a constant temperature and humidity incubator at 30 ° C and 70% relative humidity for 72 hours.

2. 밀기울 코지2. Bread Cozy

밀기울 50g에 증류수 30mL를 첨가해 반죽한 뒤 상온에서 10분간 방치한 뒤 밀기울 반죽 30g씩 500mL 삼각 플라스크에 넣고 121℃, 20분간 가압 살균한다. 균주별로 배양시킨 사면배지에 증류수 10mL를 첨가해 균 현탁액을 제조하여 실온에서 냉각한 밀기울에 접종한다. 접종 완료 후 항온항습배양기에서 30℃, 상대습도 70% 조건에서 72시간 배양하였다.30 g of distilled water is added to 50 g of wheat bran, kneaded, and left at room temperature for 10 minutes. Put 30 g of bran dough in 500 mL Erlenmeyer flask and sterilize by pressurizing at 121 ° C for 20 minutes. 10 ml of distilled water is added to the slurry medium cultivated for each strain to prepare a suspension, which is then inoculated into bran cooled at room temperature. After inoculation, the cells were cultured in a constant temperature and constant-humidity incubator at 30 ° C and 70% relative humidity for 72 hours.

3. 막걸리 만들기3. Make rice wine

1.4L의 물에 4×108cells/g의 균주를 넣고 코지 200g을 넣은 다음 800g의 고두밥을 넣고 90% Lactic acid 5.5mL를 넣는다. 25℃에서 7일간 배양 후 120 mesh 필터로 거른 다음, 21~50세의 12명 남녀에게 관능평가(향, 단맛, 신맛, 알콜향, 탄산의 세기, 바디감)를 분석하고, 유기산 및 알콜 함량을 분석하기 위하여 -70℃에 보관하였다(표 3). 제조된 막걸리의 분석결과는 표 4에 나타냈으며, 관능평가 결과는 표 5 및 도 3에 나타냈다. Add 4 × 10 8 cells / g of the strain to 1.4 L of water, add 200 g of kojie, add 800 g of high-brow seed, and add 5.5 mL of 90% Lactic acid. After incubation at 25 ° C for 7 days, the mixture was filtered through a 120 mesh filter and analyzed by sensory evaluation (aroma, sweetness, acidity, alcoholic strength, acidity and body sensation) And stored at -70 ° C for analysis (Table 3). The analytical results of the makgeolli produced are shown in Table 4, and the sensory evaluation results are shown in Table 5 and Fig.

compoundscompounds R.T.R.T. Relative peak area
(mean±SD)Relative peak area
(mean ± SD) Odor description *Odor description * Methyl propanedioic acidMethyl propanedioic acid 07:14.307: 14.3 N.D.N.D. 　 2-(methoxyimino)-Propanoic acid2- (methoxyimino) -propanoic acid 07:15.107: 15.1 N.D.N.D. 　 Lactic acidLactic acid 07:28.307: 28.3 0.489±0.0440.489 + 0.044 Weak,sour,buttermilkodorandtaste Weak, sour, buttermilkodorandtaste Hexanoic acidHexanoic acid 07:47.007: 47.0 N.D.N.D. Heavy, fatty, cheesey-sweaty odor and tasteHeavy, fatty, cheesey-sweaty odor and taste Acetic acidAcetic acid 07:48.307: 48.3 0.028±0.0040.028 ± 0.004 Pungent, sour, vinegar odor with sour, acid tastePungent, sour, vinegar odor with sour, acid taste 2-Ethylhexanoic acid2-Ethylhexanoic acid 07:59.807: 59.8 N.D.N.D. Mild, fatty-cheese-fruity, sour rancid, herbaceous musty notesMild, fatty-cheese-fruity, sour rancid, herbaceous musty notes Octanoic acidOctanoic acid 08:15.908: 15.9 0.016±0.0030.016 0.003 Fatty, rancid, animallic, sweaty odor; cheese like taste in dilutionFatty, rancid, animallic, sweaty odor; cheese like taste in dilution Oxalic acidOxalic acid 08:56.408: 56.4 0.018±0.0030.018 ± 0.003 　 Propanoic acidPropanoic acid 09:08.809: 08.8 N.D.N.D. Pungent, sour milk odor; sour milk and cheese tastePungent, sour milk odor; sour milk and cheese taste Butanoic acidButanoic acid 09:25.809: 25.8 0.006±0.0010.006 ± 0.001 Strong, cheese, butter-like, sour-rancid odor and tasteStrong, cheese, butter-like, sour-rancid odor and taste 3-methylbutanoic acid3-methylbutanoic acid 09:28.809: 28.8 0.011±0.0010.011 ± 0.001 Very sour,"sweaty", cheesy, odor; fruity on dilutionVery sour, "sweaty", cheesy, odor; fruity on dilution Carbamic acidCarbamic acid 10:48.310: 48.3 N.D.N.D. 　 Succinic acidSuccinic acid 11:59.711: 59.7 1.715±0.0331.715 + 0.033 Odorless, sour acid taste; flavor, umami & saltiness enhancerOdorless, sour acid taste; flavor, umami & saltiness enhancer Pyruvic acidPyruvic acid 12:30.412: 30.4 0.002±0.0000.002 ± 0.000 Sour burnt acetic odor; mild sour, burnt, caramel, maple tasteSour burnt acetic odor; mild sour, burnt, caramel, maple taste Fumaric acidFumaric acid 12:31.612: 31.6 N.D.N.D. Odorless with a clean, tart, acidic-sour tasteOdorless with a clean, tart, acidic-sour taste Nonanoic acidNonanoic acid 12:41.012: 41.0 N.D.N.D. Mild, fatty, dairy-cheese, nut-like odor; fatty-waxy-cheese & nutty tasteMild, fatty, dairy-cheese, nut-like odor; fatty-waxy-cheese & nutty taste 3-methylbenzoic acid3-methylbenzoic acid 12:45.412: 45.4 0.037±0.0040.037 ± 0.004 　 Pentanoic acidPentanoic acid 12:55.512: 55.5 N.D.N.D. Powerful, "sweaty" cheese-like odor; fruity, cheese, dairy taste dilutedPowerful, "sweaty" cheese-like odor; fruity, cheese, dairy taste diluted Dipropylacetic acidDipropylacetic acid 13:12.413: 12.4 N.D.N.D. 　 2-methylmalic acid2-methylmalic acid 14:10.014: 10.0 0.044±0.0130.044 0.013 　 Malic acidMalic acid 14:23.314: 23.3 0.019±0.0030.019 ± 0.003 Almost odorless with a sharp tart acidic tasteAlmost odorless with a sharp tart acidic taste Mevalonic acidMevalonic acid 15:16.615: 16.6 N.D.N.D. 　 Pentanedioic acidPentanedioic acid 15:30.515: 30.5 0.004±0.0010.004 ± 0.001 Sour taste; flavor modifier & citral stabilizerSour taste; flavor modifier & citral stabilizer Benzenepropanoic acidBenzenepropanoic acid 15:38.715: 38.7 N.D.N.D. Weak, sweet, balsamic odor; sweet vanilla-tonka tasteWeak, sweet, balsamic odor; sweet vanilla-tonka taste Dodecanoic acidDodecanoic acid 16:43.316: 43.3 N.D.N.D. Weak, refreshing, fatty, waxy odor; fatty-waxy-soapy tasteWeak, refreshing, fatty, waxy odor; fatty-waxy-soapy taste Citric acidCitric acid 20:16.320: 16.3 0.03±0.0020.03 0.002 Odorless with a clean, acid, tart tasteOdorless with a clean, acid, tart taste 1,2-Benzenedicarboxylic acid1,2-Benzenedicarboxylic acid 32:58.432: 58.4 N.D.N.D. Aromatic, weak benzoic odor with a sour bitter tasteAromatic, weak benzoic odor with a sour bitter taste

* Flavor-base 프로그램 이용 odor description* Using the Flavor-base program odor description

균주명Strain name 막걸리 결과Makgeolli result pHpH BrixBrix Reducing SugarsReducing Sugars LiquidLiquid
VolumeVolume
(ml)(ml) RiceRice
WeightWeight
(g)(g) 사카로마이콥시스Sakaromycops 피브리게라Friburgera SSU2601SSU2601 -09-09 4.44.4 1313 22.2922.29 600600 3161.683161.68 사카로마이콥시스Sakaromycops 피브리게라Friburgera LNJJ81LNJJ81 4.24.2 1313 24.5024.50 10001000 3118.773118.77 사카로마이콥시스Sakaromycops 피브리게라Friburgera LNPH12LNPH12 4.14.1 17.517.5 20.1620.16 700700 3179.163179.16 아스퍼질러스Aspergillus 오리재 CN2601-05 Duck reed CN2601-05 4.44.4 17.617.6 138.03138.03 13001300 3174.93174.9 리케테이미아Lycetemia 라모사 Ramosa LNPH11LNPH11 4.64.6 19.719.7 146.64146.64 12801280 3160.13160.1 리케테이미아Lycetemia 라모사  Ramosa LNJJ15LNJJ15 -06-06 4.64.6 18.318.3 160.23160.23 13001300 3139.983139.98

관능평가 결과Sensory evaluation result ScentScent 3.63.6 SweetSweet 1.81.8 SourSour 3.63.6 Alcohol/BitternessAlcohol / Bitterness 3.63.6 탄산의 세기Strength of carbonic acid 2.82.8 BodyBody 5.85.8 Overall AcceptabilityOverall Acceptability 33

4. 막걸리의 향기성분 조성 분석4. Analysis of aroma composition of makgeolli

제조된 막걸리를 vial에 4g씩 담아, 50℃에서 25분간 평형(equilibrium)시킨 뒤, 휘발성 향기성분을 CAR/PDMS/DVB fiber에 25분간 흡착(adsorption)시켰다. 그 후, GC 주입구(250℃)에서 5분간 탈착(desorption)시켰다. 분석에 사용된 GC-MS 조건은 다음과 같으며, 분석 결과는 표 6에 나타냈다.The prepared makgeolli was equilibrated at 50 ° C for 25 minutes, and volatile fragrance components were adsorbed on CAR / PDMS / DVB fiber for 25 minutes. Then, it was desorbed at a GC injection port (250 ° C) for 5 minutes. The GC-MS conditions used in the analysis are as follows, and the results of the analysis are shown in Table 6.

* 6890N GC system, 5975 mass detector 이용* Using 6890N GC system, 5975 mass detector

* 칼럼: DB-Wax (30m length x 0.25mm i.d. x 0.25μm film thickness, J&W Scientific)* Column: DB-Wax (30 m length x 0.25 mm i.d. x 0.25 μm film thickness, J & W Scientific)

* 오븐온도: 40℃에서 5분간 유지한 뒤, 150℃까지 2℃/min의 속도로 올리고, 이를 다시 6℃/min의 속도로 220℃까지 올려 5분간 유지* Oven temperature: maintained at 40 ° C for 5 minutes, then raised to 150 ° C at a rate of 2 ° C / min, raised to 220 ° C at a rate of 6 ° C / min and held for 5 minutes

* 이동상기체: 헬륨(He) 0.8mL/min* Mobile phase gas: helium (He) 0.8 mL / min

* mass scan range: 35-350m/z* mass scan range: 35-350m / z

CompoundsCompounds R.T.R.T. 사카로마이콥시스 피브리게라 SSU2601-09Sakaromai Cobsis Fibriegera SSU2601-09 Odor descriptionOdor description Ethyl AcetateEthyl Acetate 4.614.61 3.247±0.6543.247 + 0.654 Ethereal, sharp, wine-brandy like odorEthereal, sharp, wine-brandy like odor Isobutyl acetateIsobutyl acetate 8.3528.352 0.184±0.0420.184 + 0.042 Fruity, banana-apple-pear-pineapple notes Fruity, banana-apple-pear-pineapple notes 1-Propanol1-Propanol 9.6259.625 0.129±0.0580.129 ± 0.058 Alcoholic, fermented sweet odor; bland fermented, fusel fruity flavor Alcoholic, fermented sweet odor; bland fermented, fusel fruity flavor IsobutanolIsobutanol 12.76712.767 2.438±0.7462.438 + 0.746 Breathtaking, sweet, sweaty-chemical; fermented, whiskey-like in dilution Breathtaking, sweet, sweaty-chemical; fermented, whiskey-like in dilution Isoamyl acetateIsoamyl acetate 13.33713.337 2.824±1.2662.824 ± 1.266 Sweet, fruity, banana, pear odor & tasteSweet, fruity, banana, pear odor & taste 1-Butanol1-Butanol 16.6716.67 N.D.N.D. Breathtaking, winey, fusel oil-like Breathtaking, winey, fusel oil-like Butyl butyrateButyl butyrate 18.8918.89 N.D.N.D. Strong, sweet, fruity, banana-pear-pineapple-apple notes Strong, sweet, fruity, banana-pear-pineapple-apple notes Ethyl hexanoate Ethyl hexanoate
(Ethyl caproate)(Ethyl caproate) 19.42319.423 N.D.N.D. Strong, fruity, pineapple, banana with strawberry, pear & tropical notes Strong, fruity, pineapple, banana with strawberry, pear & tropical notes 2-Methyl-1-butanol2-Methyl-1-butanol 19.52119.521 3.522±1.5463.522 + 1.546 Ethereal, fusel oil, fermented winey notes Ethereal, fusel oil, fermented winey notes Isoamyl alcoholIsoamyl alcohol 19.71419.714 11.819±2.30211.819 + - 2.302 Breathtaking, alcoholic odor; in dilution a winey-brandy taste Breathtaking, alcoholic odor; in dilution a winey-brandy taste Ethyl octanoateEthyl octanoate
(Ethyl caprylate)(Ethyl caprylate) 32.57232.572 3.633±0.6093.633 ± 0.609 Fruity, winey, sweet odor; cognac-apricot taste Fruity, winey, sweet odor; cognac-apricot taste Ethyl decanoate Ethyl decanoate
(ethyl caprate)(ethyl caprate) 44.65744.657 3.219±0.2263.219 + 0.226 Sweet, fatty, nut-like, winey-cognac odor Sweet, fatty, nut-like, winey-cognac odor Ethyl (E)-4-decenoateEthyl (E) -4-decenoate 46.03146.031 0.091±0.010.091 ± 0.01 Fatty, waxy, green, pineapple and pear-apple notes Fatty, waxy, green, pineapple and pear-apple notes Methyl salicylateMethyl salicylate 51.38251.382 2.8±0.2352.8 ± 0.235 Warm, sweet, wintergreen odor and taste Warm, sweet, wintergreen odor and taste 2-Phenethyl acetate2-Phenethyl acetate 53.88153.881 1.635±0.2861.635 + - 0.286 Sweet, rose, fruity, honey like odor; floral-honey taste Sweet, rose, fruity, honey like odor; floral-honey taste 2-Phenylethanol2-Phenylethanol 58.96158.961 48.659±5.18148.659 ± 5.181 Ethyl hexadecanoate
(Ethyl palmitate)Ethyl hexadecanoate
(Ethyl palmitate) 69.08769.087 1.61±0.2041.61 + - 0.204 Faint, waxy, sweet odor; nearly tasteless; creamy mouthfeel Faint, waxy, sweet odor; nearly tasteless; creamy mouthfeel

한국생명공학연구원Korea Biotechnology Research Institute KCTC13034BPKCTC13034BP 2016060720160607

<110> Soongsil University Research Consortium techno-PARK <120> Saccharomycopsis fibuligera SSU2601-09 strain for fermentation of Korean traditional alcohol and the method for preparing makgeolli using the same <130> ADP-2016-0598 <150> KR 16/0082215 <151> 2016-06-30 <160> 3 <170> KopatentIn 2.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 tccgtaggtg aacctgcgg 19 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 tcctccgctt attgatatgc 20 <210> 3 <211> 576 <212> DNA <213> Saccharomycopsis fibuligera <400> 3 cgcggtttaa taaactctta tacacagtgt ttttgtttgc gaatttggtt tagtttgttg 60 gttttcattc gaaaggatga agattgattg ctaaatctta ttcagctttt taaactcaga 120 tctcttttta agagaaatgt atttttttaa ttacaactag tcgattttac aaactaaaag 180 tttaaaactt tcagcaacgg atctcttggt tctcgcatcg atgaagaacg cagcgaattg 240 cgataagtaa tgtgaattgc agattttcgt gaatcatcga atctttgaac gcatattgcg 300 ctctatagta ttctatagag catgcctgtt tgagcgtcat ttctctctta aacctttggg 360 tttagtattg aaggttgtgt tagcttctgc taactccttt gaaatgactt ggcaattgat 420 tgagttttcc atatatttgc ttaaggattt aatattaggt tctaccaact tattaaatac 480 ccttttgcga aggacttact cgtgtatcaa ggccttataa ctttgtcatt aattttgacc 540 tcaaatcagg taaggatacc cgctgaactt aagcat 576 <110> Soongsil University Research Consortium techno-PARK <120> Saccharomycopsis fibuligera SSU2601-09 strain for fermentation of          Korean traditional alcohol and the method for preparing makgeolli          using the same <130> ADP-2016-0598 <150> KR 16/0082215 <151> 2016-06-30 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 tccgtaggtg aacctgcgg 19 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 tcctccgctt attgatatgc 20 <210> 3 <211> 576 <212> DNA <213> Saccharomycopsis fibuligera <400> 3 cgcggtttaa taaactctta tacacagtgt ttttgtttgc gaatttggtt tagtttgttg 60 gttttcattc gaaaggatga agattgattg ctaaatctta ttcagctttt taaactcaga 120 tctcttttta agagaaatgt atttttttaa ttacaactag tcgattttac aaactaaaag 180 tttaaaactt tcagcaacgg atctcttggt tctcgcatcg atgaagaacg cagcgaattg 240 cgataagtaa tgtgaattgc agattttcgt gaatcatcga atctttgaac gcatattgcg 300 ctctatagta ttctatagag catgcctgtt tgagcgtcat ttctctctta aacctttggg 360 tttagtattg aaggttgtgt tagcttctgc taactccttt gaaatgactt ggcaattgat 420 tgagttttcc atatatttgc ttaaggattt aatattaggt tctaccaact tattaaatac 480 ccttttgcga aggacttact cgtgtatcaa ggccttataa ctttgtcatt aattttgacc 540 tcaaatcagg taaggatacc cgctgaactt aagcat 576

Claims (7)

수탁번호 KCTC13034BP로 기탁된, 당화 및 발효능이 우수한 전통주 발효 효모 사카로마이콥시스 피브리게라(Saccharomycopsis fibuligera) SSU2601-09 균주로서, 상기 균주의 내부 전사 영역(Internal transcribed spacer; ITS)은 서열번호 3으로 표시되는 것을 특징으로 하는 사카로마이콥시스 피브리게라(Saccharomycopsis fibuligera) SSU2601-09 균주.The Saccharomycopsis fibuligera strain SSU2601-09 , which is a sugar-fermenting yeast having excellent glycation and efficacy, deposited under accession number KCTC13034BP, wherein the internal transcribed spacer (ITS) 3. A strain of Saccharomycopsis fibuligera SSU2601-09 , characterized in that it is represented by SEQ ID NO : 3. 제1항에 있어서, 상기 균주는 누룩으로부터 분리된 것을 특징으로 하는 사카로마이콥시스 피브리게라(Saccharomycopsis fibuligera) SSU2601-09 균주.The strain of Saccharomycopsis fibuligera SSU2601-09 according to claim 1, wherein the strain is isolated from the yeast. 제1항에 있어서, 상기 균주는 알파-아밀라아제(Alpha-Amylase), 글루코아밀라아제(Glucoamylase) 또는 프로테아제(Protease) 활성이 우수한 것을 특징으로 하는 사카로마이콥시스 피브리게라(Saccharomycopsis fibuligera) SSU2601-09 균주.The strain according to claim 1, wherein the strain is Saccharomycopsis fibuligera SSU2601-09 ( SEQ ID NO: 1 ), which is characterized by excellent activity of Alpha-Amylase, Glucoamylase or Protease. Strain. 삭제delete 제1항 내지 제3항 중 어느 한 항의 균주를 사용하여, 곡류 원료로 막걸리를 제조하는 방법.A method for producing makgeolli using a strain of any one of claims 1 to 3 as a raw material for grain. 제5항에 있어서, 상기 곡류 원료는 쌀 또는 밀기울인 것을 특징으로 하는 막걸리를 제조하는 방법.[6] The method of claim 5, wherein the cereal raw material is rice or wheat bran. 제5항에 있어서, 상기 막걸리는 풍미가 우수한 것을 특징으로 하는 막걸리를 제조하는 방법.[6] The method according to claim 5, wherein the rice wine is excellent in flavor.
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