KR101350796B1 - Kazachstania exigua CCSY27 strain having probiotics properties and preparation of makgeolli by using the strain - Google Patents

Kazachstania exigua CCSY27 strain having probiotics properties and preparation of makgeolli by using the strain Download PDF

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KR101350796B1
KR101350796B1 KR1020120016343A KR20120016343A KR101350796B1 KR 101350796 B1 KR101350796 B1 KR 101350796B1 KR 1020120016343 A KR1020120016343 A KR 1020120016343A KR 20120016343 A KR20120016343 A KR 20120016343A KR 101350796 B1 KR101350796 B1 KR 101350796B1
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조현국
조계만
서원택
이주영
이동철
김동현
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경남과학기술대학교 산학협력단
영농조합법인 오름주가
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Abstract

본 발명에서는 내산성, 위액 내성 내담즙성, 내당성, 내알코올성 및 알코올 생성능이 우수한 카자크스타니아 엑시구아 CCSY27 균주 및 이를 이용한 막걸리 제조방법이 제공된다. In the present invention, there is provided a Kazakstania exigua CCSY27 strain excellent in acid resistance, gastric juice resistance bile resistance, sugar resistance, alcohol resistance, and alcohol producing ability, and a method for producing makgeolli using the same.

Description

생균제제 특성을 갖는 카자크스타니아 엑시구아 ＣＣＳＹ27 균주 및 이를 이용한 막걸리 제조 {Kazachstania exigua CCSY27 strain having probiotics properties and preparation of makgeolli by using the strain}Kazakstania exigua CCSY27 strain having probiotics properties and preparation of makgeolli by using the strain}

본 발명은 생균제제(probiotics) 특성 및 알코올 생성능을 갖는 카자크스타니아 엑시구아 (Kazachstania exigua) CCSY27 균주 및 이를 이용한 막걸리 제조방법에 관한 것으로, 더 상세히는 내산성, 위액 내성 내담즙성, 내당성, 내알코올성 및 알코올 생성능이 우수한 카자크스타니아 엑시구아 CCSY27 균주 및 이를 이용한 막걸리 제조방법에 관한 것이다.
The present invention relates to a Kazachstania exigua CCSY27 strain having probiotics and alcohol-producing ability, and a method for producing makgeolli using the same. More particularly, the present invention relates to acid resistance, gastric juice resistance, bile resistance, glucose tolerance, and resistance. The present invention relates to a Kazakstania exigua CCSY27 strain having excellent alcoholicity and alcohol producing ability, and a method for producing makgeolli using the same.

생균제제 (프로바이오틱스; probiotics)는 장내 미생물 균형에 도움을 주는 미생물, 항균 활성과 효소 활성을 가진 미생물, 및 그들이 생산해 내는 생산물을 말한다(Fuller, R. J Appl Bacteriol. 66(5):365-378, 1989). 아울러 생균제제는 사람이나 동물에 건조된 세포형태나 발효산물 형태로 공급되어 사람이나 동물 숙주의 장내 균총을 개선함으로서 좋은 영향을 주는 단일 또는 복합 균주 형태의 생균으로 정의되고 있다. 생균제제가 갖추어야 할 특성은 인간의 장내를 서식지로 하고, 비병원성, 무독성, 장으로 가는 동안 살아남아야 한다. 더 나아가서 전달 식품 안에서 소비되기 전에 생존율과 활성을 유지 및 감염 예방으로 사용되는 항생제에 대해 민감해야 하며, 항생제 내성을 갖는 플라스미드를 보유하지 않아야 한다. 또한 장내 환경에서 산, 효소, 담즙에 대한 내성을 갖추어야 한다(Mishra, C. et al., Asia Pacific J Clin Nutr. 5:20-24, 1996). Probiotics are microorganisms that help intestinal microbial balance, microorganisms with antimicrobial and enzymatic activity, and the products they produce (Fuller, R. J Appl Bacteriol. 66 (5): 365-378 , 1989). In addition, the probiotic is defined as a single or complex strain of live bacteria which is supplied to humans or animals in the form of dried cells or fermented products to improve the intestinal flora of human or animal hosts. Probiotics should have the human gut as their habitat, and must survive on their path to non-pathogenicity, nontoxicity and intestines. Furthermore, they should be sensitive to antibiotics used to maintain viability and activity and prevent infection before they are consumed in the delivered food, and should not have antibiotic resistant plasmids. It must also be resistant to acids, enzymes and bile in the intestinal environment (Mishra, C. et al., Asia Pacific J Clin Nutr. 5: 20-24, 1996).

최근 웰빙 및 로하스 트렌드 확산으로 우리민족 고유의 전통주에 관한 관심이 높아지면서 특유의 청량미와 낮은 알코올 함량의 혼탁한 술인 막걸리는 새로운 전환기를 맞이하고 있다. Recently, with the spread of well-being and LOHAS trends, interest in Korean traditional liquor has increased, and rice wine, a muddy liquor with a unique refreshing taste and low alcohol content, is entering a new turning point.

막걸리는 찹쌀, 멥쌀, 보리쌀, 현미, 옥수수, 고구마, 밀 등의 전분질을 원료로 하고 발효제로서 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. Makgeolli is a Korean traditional liquor made from glutinous rice, non-glutinous rice, barley rice, brown rice, corn, sweet potato, wheat, and other ingredients. It is a refreshing drink with 2-8% alcohol content. 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. Makgeolli is not only high in nutritional and functional value, but also contains fresh yeast and has a unique taste that cannot be compared with other alcoholic beverages.

현재 통상적으로 막걸리 제조는 2단 혹은 3단 담금법에 의하는데, 누룩, 알코올 발효 효모 및 용수를 이용하여 발효 (1단 담금)하여 밑술을 제조한 후, 밑술에 곡류 원료, 누룩 및 용수를 추가하여 발효시켜 (2단 담금) 막걸리를 완성한다. 이때 사용되는 알코올 발효 효모로는 주로 사카로마이세스 세레비아제 (Saccharomyces cereviase) 종이었다. Currently, makgeolli is usually prepared by two-stage or three-stage immersion method, and fermentation (single-stage immersion) using yeast, alcoholic fermentation yeast and water to prepare the base liquor. Fermentation (two steps immersion) completes rice wine. The alcoholic fermentation yeast used at this time was mainly Saccharomyces cereviase .

이에 사카로마이세스 세레비아제 균주 이외에 전통 발효식품의 토착 미생물 발굴과 막걸리 주조성을 가지면서도 그 외 기능성을 갖는 대체 균주 개발과 더불어 그것을 이용한 막걸리의 제조방법의 개발이 요구되어 왔다.
Therefore, in addition to Saccharomyces cerevisiae strain, it has been required to develop indigenous microorganisms of traditional fermented foods and to develop alternative strains having other functions while also producing rice wine.

종래 기술에서의 요구에 부응하기 위해 예의 연구한 결과, 본 발명자들은 전통 발효식품 유래 막걸리 주조성 및 생균제제 특성을 갖는 카자크스타니아 엑시구아 (Kazachstania exigua) CCSY27 균주를 발견/동정하고 이 균주는 사카로마이세스 세레비아제 균주를 대체하여 우수한 특성의 막걸리를 제조할 수 있게 한다는 사실을 확인하고 본 발명을 완성하기에 이르렀다. As a result of diligent research to meet the demands of the prior art, the present inventors have discovered / identified the Kazachstania exigua CCSY27 strain having traditional fermented food-derived makgeolli castability and probiotic properties, which strain The present invention was completed by confirming that it is possible to manufacture makgeolli with superior properties by replacing strains of Loyces serevivia.

본 발명의 목적은 막걸리 제조를 위한 효모 균주로 사용될 수 있는 카자크스타니아 엑시구아 CCSY27 균주를 제공하는 것이다. It is an object of the present invention to provide a Kazakstania exigua CCSY27 strain which can be used as a yeast strain for the production of makgeolli.

본 발명의 또 다른 목적은 카자크스타니아 엑시구아 CCSY27 균주를 이용하여 막걸리를 제조하는 방법을 제공하는 것이다.
Still another object of the present invention is to provide a method for preparing makgeolli using a strain of Kazakstania exigua CCSY27.

상기 목적을 달성하기 위하여, 본 발명은 막걸리 제조를 위한 효모 균주로 사용될 수 있는 카자크스타니아 엑시구아 CCSY27 균주를 제공한다. In order to achieve the above object, the present invention provides a strain of Kazakstania exigua CCSY27 that can be used as a yeast strain for manufacturing rice wine.

본 발명자들은 식초로부터 생균제제 특성 (내산성, 위액 내성, 내답즙성) 및 막걸리 주조성 (내당성, 내알코올성 및 알코올 발효능) 등 우수한 특성을 지닌 균주를 확인/동정하였고, 이 균주를 카자크스타니아 엑시구아 CCSY27 로 명명한 후 국립농업과학원 농업유전자원센터에 기탁하여 2011년 4월 11일에 수탁번호 KACC93119P를 부여받았다 (실시예 1).The present inventors identified / identified strains having excellent properties such as probiotic properties (acid resistance, gastric juice resistance, juice resistance) and rice wine casting (sugar resistance, alcohol resistance, and alcohol fermentation ability) from vinegar, and this strain was identified as Kazakstania. It was named exigua CCSY27 and deposited with the National Institute of Agricultural Science, Agricultural Genetic Resource Center, and received accession number KACC93119P on April 11, 2011 (Example 1).

카자크스타니아 엑시구아 CCSY27 균주의 26S rDNA 염기서열 (도 1)을 결정하였고 이를 기초로 하여 BLAST 네트워크 서비스와 DNA 서열 데이터베이스를 제공하는 미국국립생물정보센터(NCBI)에서 얻은 다른 균종의 26S rDNA 염기서열과 정렬하여 카자크스타니아 속에 속함을 알 수 있었다 (도 2).The 26S rDNA sequence of the strain of Kazakstania exigua CCSY27 (FIG. 1) was determined and based on this, the 26S rDNA sequence of another species obtained from the National Institute of Biological Information (NCBI), which provides BLAST network service and DNA sequence database. It can be seen that belonging to the genus Kazakstania in alignment with (Fig. 2).

카자크스타니아 엑시구아 CCSY27 균주는 pH 3.0 조건에서 3 시간 배양후 60% 이상의 생존율을 보이고, 인공위액 조건에서 3 시간 배양후 50% 이상의 생존율을 보이고 담즙 1~4% 조건에서 생육가능하였고, 당 농도 30%에서 OD600nm 4 이상의 생육성, 알코올 농도 10%에서 OD600nm 3.00 이상의 생육성을 나타냈고, 10%(v/v) 의 알코올 발효능을 가졌다 (표 1~ 표 5, 표 7). The Kazakstania exigua CCSY27 strain showed a survival rate of 60% or more after 3 hours incubation at pH 3.0, 50% or more survival after 3 hours in artificial gastric juice, and was viable in 1-4% of bile. At 30% growth of OD 600nm 4 or higher, alcohol concentration of 10% showed growth of OD 600nm 3.00 or higher, and had an alcohol fermentation capacity of 10% (v / v) (Tables 1 to 5 and Table 7).

본 발명의 또 다른 목적에 따라서, 카자크스타니아 엑시구아 CCSY27 균주를 알코올 발효 균주로 사용하는 것을 특징으로 하는 막걸리 제조방법이 제공된다. According to still another object of the present invention, there is provided a method for producing makgeolli, characterized by using the Kazakstania exigua CCSY27 strain as an alcoholic fermentation strain.

본 발명의 막걸리 제조에 사용되는 곡류 원료로는 쌀, 도정밀을 단독으로 또는 두 가지 이상 혼합하여 사용할 수 있다. 곡류 원료는 물로 세척 후, 물을 부어 실온에서 충분히 침지한 후 물기를 제거하고 95 ~ 100℃에서 30 ~ 60분 동안 증자하여 냉각한 후 사용한다. 특히 쌀과 도정밀을 5 : 5 또는 4 : 6의 중량비로 혼합하여 사용하는 것이 가장 바람직하다. 또한 쌀과 도정밀은 가루로 만들어 사용할 수도 있다. As the grain raw material used in the production of makgeolli of the present invention, rice and fine grains may be used alone or in combination of two or more. Grain raw materials are washed with water, pour water, immersed sufficiently at room temperature, then drained and cooled by steaming for 30 to 60 minutes at 95 ~ 100 ℃. In particular, it is most preferable to mix rice and fine grains in a weight ratio of 5: 5 or 4: 6. Rice and fine grains can also be used as a powder.

막걸리 제조는 통상의 방법에 의한다. 예를들면 다음과 같이 2단 단금법으로 제조할 수 있다. 누룩 : 용수를 1 : 1.5의 중량비로 혼합한 후 본 발명의 카자크스타니아 엑시구아 CCSY27 균주 배양물를 첨가하여 25℃에서 20 ~ 28 시간 발효시켜 밑술을 제조한다 (1단 담금). 이때 본 발명의 카자크스타니아 엑시구아 CCSY27 균주 배양물의 첨가량은 2 ~ 5 %(v/v)가 바람직하다. 제조된 밑술 18 ~ 20 중량%에 곡류 원료 25 ~ 27 중량% , 누룩 2 ~ 3 중량% 및 용수 52 ~ 53 중량%를 혼합하여 25℃에서 5 ~ 10일 동안 발효한다 (2단 담금).Makgeolli production is by a conventional method. For example, it can manufacture by the two stage forging method as follows. Yeast: Water is mixed at a weight ratio of 1: 1.5, and then the Kazakstania exigua CCSY27 strain culture of the present invention is added and fermented at 25 ° C. for 20 to 28 hours to prepare a base liquor (single immersion). At this time, the addition amount of the Kazakstania exigua CCSY27 strain culture of the present invention is preferably 2 to 5% (v / v). 18 to 20% by weight of the prepared base material, 25 to 27% by weight of grain raw material, 2 to 3% by weight of yeast and 52 to 53% by weight of water are mixed and fermented at 25 ° C. for 5 to 10 days (two-stage immersion).

본 발명의 제조방법에 따라 제조된 막걸리는 통상의 알코올 발효 효모인 사카로마이세스 세레비아제 균주로 제조한 막걸리에 필적하는 pH, 산도, 당도 및 알코올 함량을 갖는다 (실시예 3). 또한 본 발명의 제조방법에 따라 제조된 막걸리는 기호성도 우수하였다 (실시예 3 및 4). 또한 본 발명의 제조방법에 따라 제조된 막걸리는 생균제제를 제공하는 기능성 식품으로서의 역활도 한다.
Makgeolli prepared according to the production method of the present invention has a pH, acidity, sugar content and alcohol content comparable to that of Makgeolli prepared with Saccharomyces cerevisiae strain which is a conventional alcohol fermenting yeast (Example 3). In addition, the rice wine prepared according to the production method of the present invention was also excellent in palatability (Examples 3 and 4). In addition, makgeolli prepared according to the production method of the present invention also serves as a functional food providing a probiotic agent.

본 발명의 카자크스타니아 엑시구아 CCSY27 균주는 막걸리 주조성 및 생균제제 특성을 지녀서 종래의 막걸리 제조용 효모 균주인 사카로마이세스 세레비아제 균주를 대체하여 우수한 특성의 막걸리를 제조할 수 있게 한다.The Kazakstania exigua CCSY27 strain of the present invention has the characteristics of castability and probiotic, so that it is possible to prepare the rice wine with excellent properties by replacing the strain of Saccharomyces cerevia, a yeast strain for manufacturing conventional rice wine.

본 발명에 따른 막걸리는 생균제제를 제공하는 기능성 식품으로서의 역활도 한다.Makgeolli according to the present invention also serves as a functional food providing a probiotic.

본 발명의 막걸리 제조방법은 막걸리 주조성이 우수하여 우리밀과 우리쌀의 활용성을 극대화할 수 있다.
Makgeolli manufacturing method of the present invention can maximize the usability of rice mill and our rice due to the excellent rice wine castability.

도 1은 카자크스타니아 엑시구아 CCSY27 균주의 26S 라이보좀 DNA 염기서열이다.
도 2는 카자크스타니아 엑시구아 CCSY27 균주의 계통발생학적 유연관계도이다.
도 3은 카자크스타니아 엑시구아 CCSY27 균주를 이용한 막걸리의 제조의 일례를 보여주는 공정도이다.Figure 1 is a 26S ribosomal DNA sequence of the Kazakstania exigua CCSY27 strain.
FIG. 2 is a phylogenetic relationship diagram of strains of Kazakstania exigua CCSY27.
Figure 3 is a process chart showing an example of the production of makgeolli using the Kazakstania exigua CCSY27 strain.

다음의 실시예들에 의해 본 발명이 더 상세히 설명된다. 이들 실시예는 본 발명을 예시하기 위한 것이며, 본 발명의 범위가 이들에 의해 제한되어서는 안된다.
The present invention will be explained in more detail by the following examples. These examples are for illustrating the present invention, and the scope of the present invention should not be limited by them.

실시예Example

실시예Example 1: 균주 분리 및 특성 확인 1: Isolate and characterize the strain

막걸리(진주 탁주 및 고성 하이 막걸리), 식초(감식초 및 포도식초), 와인(포도 와인 및 다래 와인)으로부터 효모 균주를 다음과 같이 순수 분리했다: 각각의 시료를 1 ml 취하여 9 ml의 멸균증류수에 순차적으로 희석하여 10,000배로 희석하고 이를 0.1 ml 취하여 감자한천배지(PDA; potato dextrose agar, Becton & Dicknson사, USA)에 도말하였다. 도말한 배지를 30℃에서 48시간 배양한 후 크고 탁한 전형적인 효모 형태의 클론을 선택하여 다시 PDA에서 순수 분리하여 30℃에서 48시간 배양하였다.Yeast strains were isolated purely from makgeolli (pearl takju and Goseong high makgeolli), vinegar (persimmon vinegar and grape vinegar), and wine (grape wine and daerae wine) as follows: 1 ml of each sample was taken in 9 ml of sterile distilled water. Diluted sequentially, diluted 10,000-fold and taken 0.1 ml of this was plated on potato agar medium (PDA; potato dextrose agar, Becton & Dicknson, USA). After culturing the plated medium at 30 ° C. for 48 hours, a large, cloudy, typical yeast form of clone was selected, purely separated from PDA, and incubated at 30 ° C. for 48 hours.

순수 분리된 21개 균주에 대해 CCSY01 ~ CCSY20 및 CCSY27로 칭하여 분류한 후, 내산성, 인공위액 내성, 담즙산 내성, 당 내성, 알코올 내성에 대해 시험하였다. 양성 대조구로서 통상의 막걸리 제조에 사용되어 오던 효모 균주인 사카로마이세스 세레비아제 TJY01 균주 (진주탁주협회에서 분양)를 사용하여 동일하게 시험하였다. Twenty-one purely isolated strains were classified as CCSY01 to CCSY20 and CCSY27, and then tested for acid resistance, artificial gastric juice resistance, bile acid resistance, sugar resistance, and alcohol resistance. As a positive control, the same test was carried out using a strain of TJY01 made by Saccharomyces cerevisiae (prepared by the Pearl Takju Association), a yeast strain that has been used for the production of conventional rice wine.

내산성 측정은 각각의 균주를 PDB (potato dextrose broth, Becton & Dicknson사, USA) 액체배지에서 30℃에서 48시간 배양하고 3M 염산으로 pH 3으로 조정된 새로운 PDB 액체배지로 106 CFU/ml로 희석했다. 희석된 균체들을 37℃에서 3시간 및 6시간 배양 후, 배지 산성도를 중화시키기 위해서 포스페이트 버퍼 (0.1 M, pH 6.2)에서 균체를 연속적으로 희석시키고 30℃에서 48시간 배양한 후에 PDA 평판배지 상에서 생균수를 측정했다. 생존율은 초기 균의 농도와 PDA 평판배지 상에 형성된 효모의 콜로니 비율을 비교하여 계산했고, 그 결과를 표 1에 나타냈다. Acid resistance was determined by incubating each strain for 48 hours at 30 ° C. in PDB (potato dextrose broth, Becton & Dicknson, USA) liquid medium and diluting to 10 6 CFU / ml with fresh PDB liquid medium adjusted to pH 3 with 3M hydrochloric acid. did. After incubating the diluted cells for 3 hours and 6 hours at 37 ° C, the cells were serially diluted in phosphate buffer (0.1 M, pH 6.2) to neutralize medium acidity and incubated for 48 hours at 30 ° C. The number was measured. Survival was calculated by comparing the concentration of the initial bacteria and the colony ratio of yeast formed on the PDA plate medium, the results are shown in Table 1.

균주Strain 분리원Separation source 생존률Survival rate (%)(%) 균주Strain 분리원Separation source 생존률Survival rate (%)(%) 배양시간Incubation time 배양시간Incubation time 33 66 33 66 TJY01TJY01 막걸리Makgeolli 16.616.6 0.60.6 CCSY11CCSY11 막걸리Makgeolli 18.118.1 0.60.6 CCSY01CCSY01 식초vinegar 73.873.8 35.035.0 CCSY12CCSY12 막걸리Makgeolli 26.126.1 5.45.4 CCSY02CCSY02 식초vinegar 69.069.0 29.529.5 CCSY13CCSY13 와인wine 67.367.3 32.732.7 CCSY03CCSY03 식초vinegar 48.648.6 19.319.3 CCSY14CCSY14 와인wine 25.325.3 1.41.4 CCSY04CCSY04 식초vinegar 37.537.5 14.014.0 CCSY15CCSY15 와인wine 12.112.1 00 CCSY05CCSY05 식초vinegar 44.444.4 22.222.2 CCSY16CCSY16 식초vinegar 52.052.0 33.633.6 CCSY06CCSY06 식초vinegar 12.912.9 1.81.8 CCSY17CCSY17 식초vinegar 48.648.6 23.823.8 CCSY07CCSY07 막걸리Makgeolli 37.837.8 6.36.3 CCSY18CCSY18 식초vinegar 66.666.6 28.328.3 CCSY08CCSY08 막걸리Makgeolli 14.014.0 0.10.1 CCSY19CCSY19 식초vinegar 45.745.7 22.422.4 CCSY09CCSY09 막걸리Makgeolli 27.527.5 5.95.9 CCSY20CCSY20 식초vinegar 45.545.5 18.118.1 CCSY10CCSY10 막걸리Makgeolli 15.015.0 0.30.3 CCSY27CCSY27 식초vinegar 60.860.8 29.329.3 PD 액체배지에서 3반복 수행하였음.Three repetitions were performed in PD liquid medium.

<균주의 산(pH 3.0)에서 생존율>Survival rate in acid (pH 3.0) of strains

인공위액 내성 측정은 PDB 액체배지에 1% 펩신을 첨가하여 pH 3으로 맞춰 인공위액을 만든 후, 그 외 과정은 위에서 기재된 내산성 측정과 동일하게 실시했고, 그 결과를 표 2에 나타냈다:The gastric juice resistance measurement was performed by adding 1% pepsin to the PDB liquid medium to make the gastric juice adjusted to pH 3, and the other procedures were performed in the same manner as the acid resistance measurement described above, and the results are shown in Table 2:

균주Strain 분리원Separation source 생존률Survival rate (%)(%) 균주Strain 분리원Separation source 생존률Survival rate (%)(%) 배양시간Incubation time 배양시간Incubation time 33 66 33 66 TJY01TJY01 막걸리Makgeolli 7.17.1 0.20.2 CCSY11CCSY11 막걸리Makgeolli 4.74.7 00 CCSY01CCSY01 식초vinegar 58.758.7 25.325.3 CCSY12CCSY12 막걸리Makgeolli 13.413.4 2.32.3 CCSY02CCSY02 식초vinegar 55.955.9 20.120.1 CCSY13CCSY13 와인wine 51.751.7 26.526.5 CCSY03CCSY03 식초vinegar 40.440.4 22.922.9 CCSY14CCSY14 와인wine 12.112.1 00 CCSY04CCSY04 식초vinegar 23.423.4 3.63.6 CCSY15CCSY15 와인wine 1.41.4 00 CCSY05CCSY05 식초vinegar 41.041.0 18.718.7 CCSY16CCSY16 식초vinegar 43.043.0 23.623.6 CCSY06CCSY06 식초vinegar 32.032.0 9.79.7 CCSY17CCSY17 식초vinegar 36.736.7 2.12.1 CCSY07CCSY07 막걸리Makgeolli 15.115.1 0.40.4 CCSY18CCSY18 식초vinegar 50.350.3 24.724.7 CCSY08CCSY08 막걸리Makgeolli 5.25.2 00 CCSY19CCSY19 식초vinegar 25.525.5 2.12.1 CCSY09CCSY09 막걸리Makgeolli 17.617.6 3.23.2 CCSY20CCSY20 식초vinegar 31.431.4 15.315.3 CCSY10CCSY10 막걸리Makgeolli 9.09.0 00 CCSY27CCSY27 식초vinegar 52.952.9 28.628.6 PD 액체배지에서 3반복 수행하였음.Three repetitions were performed in PD liquid medium.

<균주의 인공위액 산(pH 3.0) 조건에서 생존율>Survival under artificial gastric acid (pH 3.0) conditions

담즙산 내성은 30℃에서 48시간동안 PDB 액체배지에서 배양한 균체 15㎕(106 CFU/ml과 동일)를 다른 농도(1, 2, 4% w/v)의 황소담즙(oxgall bile)을 함유한 각각의 PDA 평판배지 상에 스폿팅(spotting)한 후, 30℃에서 5일 동안 배양했다. 스폿을 육안 검사하여 균주 생장 여부를 판단하여 그 결과를 표 3에 나타냈다: Bile acid resistance contains 15 µl (same as 10 6 CFU / ml) cultured in PDB liquid medium at 30 ° C for 48 hours and contains oxgall bile at different concentrations (1, 2, 4% w / v). After spotting on each PDA plate, the cells were incubated at 30 ° C. for 5 days. Visual inspection of the spots to determine strain growth and the results are shown in Table 3:

분리균주Isolated strain 분리원Separation source 생육정도Degree of growth 분리균주Isolated strain 분리원Separation source 생육정도Degree of growth 담즙산Bile acid 농도(%) density(%) 담즙산Bile acid 농도(%) density(%) 1One 22 33 44 1One 22 33 44 TJY01TJY01 막걸리Makgeolli ++ ++ ++ ++ CCSY11CCSY11 막걸리Makgeolli ++ ++ ++ ++ CCSY01CCSY01 식초vinegar ++ ++ ++ ++ CCSY12CCSY12 막걸리Makgeolli ++ ++ ++ ++ CCSY02CCSY02 식초vinegar WW WW WW WW CCSY13CCSY13 와인wine ++ ++ ++ ++ CCSY03CCSY03 식초vinegar ++ ++ ++ ++ CCSY14CCSY14 와인wine ++ ++ ++ ++ CCSY04CCSY04 식초vinegar ++ ++ ++ ++ CCSY15CCSY15 와인wine ++ ++ ++ ++ CCSY05CCSY05 식초vinegar ++ ++ ++ ++ CCSY16CCSY16 식초vinegar ++ ++ ++ ++ CCSY06CCSY06 식초vinegar ++ ++ ++ ++ CCSY17CCSY17 식초vinegar ++ ++ ++ ++ CCSY07CCSY07 막걸리Makgeolli ++ ++ ++ ++ CCSY18CCSY18 식초vinegar ++ ++ ++ ++ CCSY08CCSY08 막걸리Makgeolli ++ ++ ++ ++ CCSY19CCSY19 식초vinegar ++ ++ ++ ++ CCSY09CCSY09 막걸리Makgeolli ++ ++ ++ ++ CCSY20CCSY20 식초vinegar ++ ++ ++ ++ CCSY10CCSY10 막걸리Makgeolli ++ ++ ++ ++ CCSY27CCSY27 식초vinegar ++ ++ ++ ++ PD 고체배지에서 3반복 수행하였음.Three repetitions were performed in PD solid medium.

<균주의 담즙 농도별 생육여부; w(week): 생약정도가 약함> <Growth of bile concentrations of strains; w (week): weak herbal level>

당 내성 측정은 PDB 액체배지에 0 ~ 30% 설탕을 첨가하고 각각의 균주를 5.0%(v/v)로 첨가한 후 37℃에서 48시간 액체배양한 후 흡광도(600nm)를 측정하여 균주 생육정도를 결정하였고 그 결과를 표 4에 나타냈다: Sugar resistance was measured by adding 0-30% sugar to PDB liquid medium, adding 5.0% (v / v) of each strain, and incubating the liquid at 37 ° C for 48 hours, and then measuring the absorbance (600nm). Was determined and the results are shown in Table 4:

균주Strain 분리원Separation source 생육정도Degree of growth ( ( ODOD 600600 nmnm )) 균주Strain 분리원Separation source 생육정도Degree of growth ( ( ODOD 600600 nmnm )) 설탕 농도(%)Sugar concentration (%) 설탕 농도(%)Sugar concentration (%) 00 1010 2020 3030 00 1010 2020 3030 TJY01TJY01 막걸리Makgeolli 7.157.15 5.445.44 4.324.32 3.213.21 CCSY11CCSY11 막걸리Makgeolli 8.198.19 7.707.70 6.576.57 5.155.15 CCSY01CCSY01 식초vinegar 6.106.10 5.085.08 4.214.21 3.083.08 CCSY12CCSY12 막걸리Makgeolli 7.817.81 6.136.13 5.095.09 4.234.23 CCSY02CCSY02 식초vinegar 5.225.22 4.114.11 3.223.22 2.262.26 CCSY13CCSY13 와인wine 7.697.69 5.905.90 4.884.88 3.463.46 CCSY03CCSY03 식초vinegar 8.818.81 6.516.51 5.655.65 4.384.38 CCSY14CCSY14 와인wine 3.843.84 4.254.25 2.532.53 2.132.13 CCSY04CCSY04 식초vinegar 9.759.75 8.188.18 6.186.18 3.513.51 CCSY15CCSY15 와인wine 8.378.37 5.115.11 5.855.85 4.754.75 CCSY05CCSY05 식초vinegar 8.448.44 7.027.02 5.745.74 5.045.04 CCSY16CCSY16 식초vinegar 7.857.85 6.586.58 5.695.69 5.235.23 CCSY06CCSY06 식초vinegar 6.786.78 6.036.03 5.255.25 4.714.71 CCSY17CCSY17 식초vinegar 7.477.47 6.536.53 5.575.57 4.724.72 CCSY07CCSY07 막걸리Makgeolli 7.117.11 5.025.02 4.374.37 3.693.69 CCSY18CCSY18 식초vinegar 7.797.79 6.416.41 5.505.50 5.465.46 CCSY08CCSY08 막걸리Makgeolli 7.227.22 5.655.65 4.314.31 3.303.30 CCSY19CCSY19 식초vinegar 8.948.94 6.556.55 5.295.29 4.244.24 CCSY09CCSY09 막걸리Makgeolli 7.507.50 6.416.41 4.834.83 3.813.81 CCSY20CCSY20 식초vinegar 7.607.60 6.246.24 4.524.52 4.034.03 CCSY10CCSY10 막걸리Makgeolli 7.087.08 4.394.39 3.533.53 2.162.16 CCSY27CCSY27 식초vinegar 8.698.69 6.076.07 5.005.00 4.444.44 PD 고체배지에서 3반복 수행하였음.Three repetitions were performed in PD solid medium.

<균주의 설탕 농도별 생육여부><Growth status by sugar concentration>

알코올 내성 측정은 PDB 액체배지에 0 ~ 15% 알코올을 첨가하고 각각의 균주를 5.0%(v/v) 첨가한 후 37℃에서 48시간 액체배양한 후 흡광도(600 nm)에서 측정하여 균주 생육정도를 결정하였고 그 결과를 표 5에 나타냈다: Alcohol resistance was measured by adding 0-15% alcohol to PDB liquid medium, adding 5.0% (v / v) of each strain, and incubating the liquid at 37 ° C for 48 hours, and then measuring the absorbance (600 nm). Was determined and the results are shown in Table 5:

균주Strain 분리원Separation source 생육정도Degree of growth ( ( ODOD 600600 nmnm )) 균주Strain 분리원Separation source 생육정도Degree of growth ( ( ODOD 600600 nmnm )) 알코올 농도(%)Alcohol concentration (%) 알코올 농도(%)Alcohol concentration (%) 00 55 1010 1515 00 55 1010 1515 TJY01TJY01 막걸리Makgeolli 7.217.21 5.185.18 1.361.36 0.220.22 CCSY11CCSY11 막걸리Makgeolli 7.407.40 5.875.87 2.632.63 0.450.45 CCSY01CCSY01 식초vinegar 6.466.46 2.832.83 0.260.26 0.200.20 CCSY12CCSY12 막걸리Makgeolli 6.646.64 4.84.8 2.82.8 0.330.33 CCSY02CCSY02 식초vinegar 4.624.62 4.214.21 0.240.24 0.140.14 CCSY13CCSY13 와인wine 6.986.98 4.274.27 1.081.08 0.180.18 CCSY03CCSY03 식초vinegar 8.298.29 4.454.45 2.792.79 0.290.29 CCSY14CCSY14 와인wine 3.113.11 2.372.37 0.130.13 0.070.07 CCSY04CCSY04 식초vinegar 9.119.11 8.738.73 5.335.33 0.300.30 CCSY15CCSY15 와인wine 7.497.49 5.195.19 3.413.41 0.310.31 CCSY05CCSY05 식초vinegar 7.567.56 6.796.79 0.260.26 0.110.11 CCSY16CCSY16 식초vinegar 7.907.90 4.824.82 2.252.25 0.290.29 CCSY06CCSY06 식초vinegar 6.076.07 4.124.12 2.052.05 0.230.23 CCSY17CCSY17 식초vinegar 6.86.8 3.333.33 1.731.73 0.290.29 CCSY07CCSY07 막걸리Makgeolli 7.137.13 4.634.63 3.653.65 0.300.30 CCSY18CCSY18 식초vinegar 7.087.08 6.136.13 1.841.84 0.320.32 CCSY08CCSY08 막걸리Makgeolli 7.287.28 5.345.34 1.301.30 0.200.20 CCSY19CCSY19 식초vinegar 9.279.27 5.345.34 3.463.46 0.570.57 CCSY09CCSY09 막걸리Makgeolli 8.188.18 5.325.32 2.982.98 0.400.40 CCSY20CCSY20 식초vinegar 6.716.71 4.814.81 2.352.35 0.380.38 CCSY10CCSY10 막걸리Makgeolli 7.907.90 6.446.44 2.892.89 0.350.35 CCSY27CCSY27 식초vinegar 7.657.65 4.864.86 3.343.34 0.390.39 PD 고체배지에서 3반복 수행하였음.Three repetitions were performed in PD solid medium.

<균주의 알코올 농도별 생육여부><Growth status by alcohol concentration>

위와 같은 내산성, 위액 내성, 담즙산 내성, 당 내성 및 알코올 내성의 특성을 모두 고려하여 분리된 균주들 중에서 CCSY27을 선별하게 되었다.
CCSY27 was selected from the isolated strains in consideration of the characteristics of acid resistance, gastric juice resistance, bile acid resistance, sugar resistance and alcohol resistance as described above.

실시예 2: 카자크스타니아 엑시구아 CCSY27 균주 동정 및 기탁Example 2: Identification and Deposit of Kazakstania Exigua CCSY27 Strains

선별된 CCSY27 균주를 PDB 액체배지 5 ml에 2.5%(0.125 ml, v/v) 접종하고 30℃에서 48시간 배양한 후 1.5 ml 소형 원심분리 튜브를 사용하여(3회) 약 5 ml 정도 취한 후 13,000 rpm에서 5분간 원심분리한 후, 균체를 모집하고 DNAzol kit(Invtrogen사, USA)를 이용하여 게놈 DNA를 분리했다. 분리된 게놈 DNA를 주형으로 하여 94℃에서 1분간 변성, 52℃에서 30초간 풀림, 72℃에서 30초 신장의 사이클을 30 회 수행하여 26S rDNA (0.6 kb 단편)을 증폭했다. 증폭된 26S rDNA PCR 산물을 1% 아가로스에 전기영동하고 0.6 kb 단편을 회수 및 정제한 후 pGEM-T Easy (Promega, Madison, USA)를 사용하여 클로닝하고 대장균(Escherichia coli) DH5α에 형질전환 후, 형질전환체를 무작위로 선정하여 순수 분리하고, 순수 분리된 형질전환체를 50 μg의 앰피실린 함유된 LB (Luria-Bertani broth, Becton & Dicknson사, USA) 액체배지에 접종하여 37 ℃에서 16시간 배양한 후 균체를 모집하고 플라스미드 정제 Kit (Intron, Suwon, Korea)에 기술된 대로 플라스미드를 분리 정제했다. 분리된 플라스미드를 서열 결정 주형가닥으로 사용하였다. 핵산 염기서열은 PRISM Ready Reaction Dye terminator/primer cycle sequencing kit를 사용한 디데옥시 사슬 종결법을 이용하여 분석하여 그 염기서열 639 bp를 결정하였다 (도 1). 이 26S rDNA 염기서열은 BLAST network service와 DNA sequence database를 제공하는 미국 국립생물정보센터(NCBI)에서 얻은 다른 균들의 것과 상동성을 분석하여 최종 동정했다 (도 2). The selected CCSY27 strain was inoculated with 2.5% (0.125 ml, v / v) of 5 ml of PDB liquid medium and incubated at 30 ° C. for 48 hours, followed by taking about 5 ml using a 1.5 ml small centrifuge tube (3 times). After centrifugation at 13,000 rpm for 5 minutes, cells were recruited and genomic DNA was isolated using DNAzol kit (Invtrogen, USA). Using the isolated genomic DNA as a template, 26S rDNA (0.6 kb fragment) was amplified by performing 30 cycles of denaturation at 94 DEG C for 1 minute, 52 DEG C for 30 seconds, and 72 DEG C for 30 seconds. The amplified 26S rDNA PCR product was electrophoresed in 1% agarose, 0.6 kb fragment was recovered and purified, cloned using pGEM-T Easy (Promega, Madison, USA) and Escherichia coli ) After transforming to DH5α, transformants were randomly selected and purified, and the isolated isolates were inoculated into LB (Luria-Bertani broth, Becton & Dicknson, USA) liquid medium containing 50 μg of ampicillin. After culturing at 37 ° C. for 16 hours, cells were recruited and the plasmids were separated and purified as described in the plasmid purification kit (Intron, Suwon, Korea). The isolated plasmid was used as a sequencing template strand. The nucleotide sequence was analyzed by the dideoxy chain termination method using the PRISM Ready Reaction Dye terminator / primer cycle sequencing kit to determine the base sequence 639 bp (FIG. 1). This 26S rDNA sequence was finally identified by analysis of homology with other microbes obtained from the National Center for Biotechnology Information (NCBI), which provides BLAST network service and DNA sequence database (Fig. 2).

이와 같이 동정된 균주를 카자크스타니아 엑시구아 CCSY27로 명명하고, 국립농업과학원 농업유전자원센터에 특허균주를 기탁하여 2011년 4월 11일자로 수탁번호 KACC93119P를 부여받았다.
The identified strain was named as Kazakstania exigua CCSY27, and a patent strain was deposited at the National Institute of Agricultural Science, Agricultural Genetic Resource Center, and was assigned accession number KACC93119P on April 11, 2011.

실시예 3: 카자크스타니아 엑시구아 CCSY27 균주를 사용하여 막걸리 제조Example 3: Manufacture of Makgeolli Using Kazakstania Exigua CCSY27 Strain

실시예 2에서 선별된 카자크스타니아 엑시구아 CCSY27 균주를 밀 당화액(10% 밀당화액, 설탕 2%, 펩톤 1%, 효모 추출물 1%) 50 ml에 2.5%(1.25 ml) 접종하여 30℃ 48시간 배양한 후 그 배양액을 누룩 ((주)오름주가)과 정제수를 다음 표 6과 같은 조성으로 1.2 리터 유리 발효조에 넣고 25℃에서 1일 동안 발효하여 밑술 제조(1단 담금)한 후, 증자된 쌀, 누룩 및 정제수를 표 6와 같이 첨가한 후, 25℃에서 7일 동안 발효 (2단 담금)하여 막걸리를 완성하였다. 양성대조구로서 사카로마이세스 세레비아제 TJY01 균주 (진주탁주협회에서 분양받음)를 사용하여 동일하게 막걸리를 제조하였다. Kazakstania exigua CCSY27 strain selected in Example 2 was inoculated with 2.5% (1.25 ml) of 50% wheat glycosylated solution (10% wheat glycosylated solution, sugar 2%, peptone 1%, yeast extract 1%) at 30 ° C After incubating for 48 hours, the culture solution was put into a 1.2 liter glass fermenter with Nuruk (Oreum Co., Ltd.) and purified water as shown in Table 6, followed by fermentation at 25 ° C. for 1 day to prepare a base liquor (single immersion). Steamed rice, koji and purified water were added as shown in Table 6, followed by fermentation (single-stage two) at 25 ° C. for 7 days to complete makgeolli. Makgeolli was prepared in the same manner using the TJY01 strain (available from the Pearl Takju Association) made by Saccharomyces cerevisiae as a positive control.

1담금1 soaking 효모
누룩
정제수 leaven
yeast
Purified water 4 mL
56 g
80 mL4 mL
56 g
80 mL 0.52%
7.27%
10.39%0.52%
7.27%
10.39% 2담금2 soaking 증자 쌀
누룩
정제수 Steamed rice
yeast
Purified water 200 g
20 g
410 mL200 g
20 g
410 mL 25.97%
2.60%
53.25%25.97%
2.60%
53.25% 합계Sum 770 mL(g)    770 mL (g) 100.00%    100.00%

<막걸리 담금 비율><Makgeolli immersion ratio>

제조된 막걸리의 pH, 산도, 당도 및 알코올 함량을 측정하여 그 결과를 표 7에 나타냈다. The pH, acidity, sugar and alcohol content of the prepared makgeolli were measured and the results are shown in Table 7.

하기 실시예에서 제조된 막걸리에 대해서는 pH 및 산도, 브릭스 당도, 알코올 함량, 갈변도, 탁도를 분석하였고 관능평가를 수행하였다. 분석을 위해 막걸리 를 4겹의 거즈로 여과한 시료를 사용하였다.For the makgeolli prepared in the following examples, pH and acidity, brix sugar, alcohol content, browning, and turbidity were analyzed and sensory evaluation was performed. Samples of makgeolli filtered with four layers of gauze were used for analysis.

pH는 여과한 시료 50 mL을 pH 미터 (model 3510, Jenway, UK)를 사용하여 측정하였고, 산도는 여과한 시료 10 mL를 0.1N-NaOH 용액으로 pH 8.3±0.1까지 중화시키데 소요된 0.1N-NaOH의 소비 mL 수를 구한 후 초산(acetic acid)으로 환산하였고, 당도는 여과한 시료를 원심분리기(Hanil micro-12, Korea)로 원심분리한 후 상등액을 취하여 굴절당도계(N-1α, Atago Co., Tokyo, Japan)를 이용하여 측정하였고, 알코올 함량은 여과한 시료 100 mL에 동량의 증류수를 가한 후 증류한 다음 주정계를 이용하여 측정하였으며 Gay Luccac Table을 이용하여 15℃로 보정하였다. The pH was measured using a pH meter (model 3510, Jenway, UK) for 50 mL of the filtered sample, and the acidity was 0.1 N required to neutralize 10 mL of the filtered sample to pH 8.3 ± 0.1 with 0.1 N-NaOH solution. -Calculate the number of consumed mL of NaOH and convert it into acetic acid, and the sugar content was centrifuged with a centrifuge (Hanil micro-12, Korea), and the supernatant was taken. Co., Tokyo, Japan), alcohol content was measured by distillation after adding the same amount of distilled water to 100 mL of the filtered sample and calibrated to 15 ℃ using the Gay Luccac Table.

균주Strain 분리원Separation source 분석항목Analysis item pHpH 산도Acidity
(%, 젖산)(% Lactic acid) 브릭스Brix
당도(°)Sugar content (°) 알코올Alcohol
(%, v/v)(%, v / v) TJY01TJY01 막걸리Makgeolli 3.983.98 1.611.61 8.28.2 10.010.0 CCSY27CCSY27 식초vinegar 4.044.04 1.801.80 8.08.0 10.010.0 각각의 균주를 5.0%(v/v) 접종하여 25에서 7일간 막걸리를 발효하였음. Each strain was inoculated with 5.0% (v / v) to ferment makgeolli for 25 to 7 days.

<막걸리의 이화학적 특성>Physicochemical Properties of Makgeolli

상기 표로부터, 본 발명의 카자크스타니아 엑시구아 CCSY27 균주로 제조한 막걸리는 사카로마이세스 세레비아제 TJY01 균주로 제조한 막걸리에 필적하는 pH, 산도, 당도를 가지며, 알코올 함량 또한 10%로 높았다.
From the above table, Makgeolli prepared with the strain of Kazakstania exigua CCSY27 of the present invention had a pH, acidity, and sugar content comparable to Makgeolli prepared with the strain TJY01 made by Saccharomyces cerevisiae, and the alcohol content was also high as 10%. .

실시예 4: 카자크스타니아 엑시구아 CCSY27를 이용한 막걸리 제조시 우리밀-쌀 최적 혼합비 시험Example 4: Uri wheat-rice optimum mixing ratio test for manufacturing rice wine using Kazakstania exigua CCSY27

카자크스타니아 엑시구아 CCSY27를 이용하여 쌀과 밀의 혼합비를 달리하여 막걸리를 제조하여 최적 혼합비를 구하였다. Makazuli was prepared by varying the mixing ratio of rice and wheat using the Kazakstania exigua CCSY27 to obtain the optimum mixing ratio.

쌀을 1 kg 달아 물로 세척한 다음 충분한 물을 부은 후 실온(15-20℃)에서 12시간 침지하였고, 도정밀 1 kg 달아 물로 세척한 다음 충분한 물을 부은 후 실온에서 4시간 침지한 후 30분간 물기를 제거했다. 물기가 제거된 각각의 시료를 100℃에서 1시간 동안 증자하여 냉각했다. 증자된 쌀과 증자된 도정밀을 10 : 0, 8 : 2, 6 : 4, 5 : 5, 4 : 6, 2 : 8 및 0 : 10 중량비로 혼합하여, 표 8과 같은 담금 비율하는 것을 제외하고는 상기 실시예 3에서와 동일한 과정으로 막걸리를 제조하였다 (도 3). 1 kg of rice was washed with water and poured with sufficient water, and then immersed at room temperature (15-20 ° C) for 12 hours. Drained off. Each sample from which water was removed was cooled by steaming at 100 ° C. for 1 hour. The cooked rice and the cooked fine grain were mixed in a 10: 0, 8: 2, 6: 4, 5: 5, 4: 6, 2: 8 and 0: 10 weight ratio, except that the immersion ratios as shown in Table 8 And makgeolli was prepared in the same process as in Example 3 (Fig. 3).

1담금1 soaking 효모  leaven
누룩  yeast
정제수Purified water 40       40 mLmL
560 g     560 g
800      800 mLmL 0.52%      0.52%
7.27%      7.27%
10.39%     10.39% 2담금2 soaking 쌀 : 밀 = 4 : 6
누룩
정제수 Rice: wheat = 4: 6
yeast
Purified water 2000 g
200 g
4100 mL2000 g
200 g
4100 mL 25.97%
2.60%
53.25%25.97%
2.60%
53.25% 합계Sum 7700     7700 mLmL (g)(g) 100.00%    100.00%

제조된 막걸리의 이화학적 특징, 균수, 갈변도, 탁도, 관능평가를 수행하였다. Physicochemical characteristics, bacterial count, browning degree, turbidity, and sensory evaluation of the prepared rice wine were performed.

이화학적 특징은 상기 실시예 3에 기재된 바와 같이 측정하였고, 그 결과를 표 9에 나타냈다.Physicochemical characteristics were measured as described in Example 3 above, and the results are shown in Table 9.

비율ratio
(쌀 : 밀)(Rice: wheat) 분석항목Analysis item pHpH 산도Acidity
(%, 젖산)(% Lactic acid) 브릭스Brix 당도(°) Sugar content (°) 알코올Alcohol
(%, v/v)(%, v / v) 10 : 010: 0 3.863.86 1.141.14 7.87.8 12.012.0 8 : 28: 2 3.863.86 1.481.48 7.87.8 11.011.0 6 : 46: 4 3.853.85 1.501.50 7.67.6 11.011.0 5 : 55: 5 3.883.88 1.521.52 7.47.4 11.011.0 4 : 64: 6 3.873.87 1.541.54 7.27.2 10.010.0 2 : 82: 8 3.853.85 1.721.72 7.27.2 9.09.0 0 : 100: 10 3.883.88 1.741.74 7.27.2 9.09.0

<밀과 쌀의 혼합비에 따른 막걸리의 이화학적 특징>Physicochemical Characteristics of Makgeolli by Mixing Ratio of Wheat and Rice

젖산균수는 여과한 시료를 멸균생리수로 적당히 희석하여 브로모크레솔 퍼플(bromocresol purple; BCP) 0.02%를 함유한 MRS (lactobacilli MRS broth, Becton & Dicknson사, USA) 평판배지에 도말하고 30℃ 배양기에서 48시간을 배양 후 황색 집락을 계수하여 총 젖산균으로 계산하였고, 효모균수 역시 여과한 시료를 멸균생리수로 적당히 희석하여 크로람페니콜(1.5 mg/mL) 함유된 PDA 배지에 도말한 후 30℃에서 48시간을 배양 후 생성된 집락을 계측하였다. 각 실험은 3회 반복하여 수행하여 평균값으로 나타내었으며 시료 mL당 콜로니 형성 단위(CFU/mL)로 표시하였다. 그 결과를 표 10에 나타냈다. The lactic acid bacterium was diluted with sterile physiological water and plated on MRS (lactobacilli MRS broth, Becton & Dicknson, USA) plate medium containing 0.02% bromocresol purple (BCP). After 48 hours of incubation in the incubator, yellow colonies were counted and counted as total lactic acid bacteria, and yeast bacteria were also diluted with sterile physiological water and plated in PDA medium containing chromamphenicol (1.5 mg / mL). Colonies generated after incubation at 30 ° C. for 48 hours were counted. Each experiment was performed three times and expressed as an average value and expressed as colony forming units (CFU / mL) per mL of sample. The results are shown in Table 10.

비율ratio
(쌀 : 밀)(Rice: wheat) 생균수(Viable cell count ( cfucfu /Of mlml )) 총균수Total number of bacteria 젖산균수Number of lactic acid bacteria 효모균수Yeast count 10 : 010: 0 3.6×107 3.6 × 10 7 1.6×106 1.6 × 10 6 2.3×106 2.3 × 10 6 8 : 28: 2 4.0×107 4.0 × 10 7 2.3×106 2.3 × 10 6 2.7×106 2.7 × 10 6 6 : 46: 4 4.1×107 4.1 × 10 7 2.1×106 2.1 × 10 6 2.5×106 2.5 × 10 6 5 : 55: 5 4.4×107 4.4 × 10 7 2.9×106 2.9 × 10 6 2.6×106 2.6 x 10 6 4 : 64: 6 4.3×107 4.3 × 10 7 3.1×106 3.1 × 10 6 2.4×106 2.4 × 10 6 2 : 82: 8 4.1×107 4.1 × 10 7 2.5×106 2.5 × 10 6 2.5×106 2.5 × 10 6 0 : 100: 10 4.2×107 4.2 × 10 7 2.9×106 2.9 × 10 6 2.2×106 2.2 × 10 6

<밀과 쌀의 혼합비에 따른 막걸리의 총균수 및 효모균수, 젖산균수><Total Number of Rice Wine, Yeast, and Lactic Acid Bacteria by Mixing Ratio of Wheat and Rice>

갈변도는 여과한 시료를 원심분리기(Hanil micro-12, Korea)로 원심분리한 후 상등액을 취하여 분광광도계(Spectronic 2D, Thermo Electron Co., Califonia, USA)를 이용하여 흡광도 (420 nm)를 측정하였고, 탁도는 여과한 시료를 분광광도계(Spectronic 2D)를 이용하여 흡광도 (600 nm)를 측정하여 그 결과를 표 11에 나타냈다. Browning degree was measured by centrifugation of the filtered sample in a centrifuge (Hanil micro-12, Korea), and then the supernatant was taken to measure the absorbance (420 nm) using a spectrophotometer (Spectronic 2D, Thermo Electron Co., Califonia, USA). Turbidity was measured by measuring the absorbance (600 nm) of the filtered sample using a spectrophotometer (Spectronic 2D) and the results are shown in Table 11.

비율ratio
(쌀 : 밀)(Rice: wheat) 갈변도(Browning degree ( OD420OD420 nmnm )) 탁도(Turbidity ( OD600OD600 nmnm )) 10 : 010: 0 0.1270.127 2.4252.425 8 : 28: 2 0.1670.167 2.4302.430 6 : 46: 4 0.1690.169 2.4322.432 5 : 55: 5 0.1900.190 2.4312.431 4 : 64: 6 0.2090.209 2.4472.447 2 : 82: 8 0.2370.237 2.4292.429 0 : 100: 10 0.3000.300 2.4412.441

<쌀과 밀 혼합비에 따른 막걸리의 갈변도와 탁도>Browning and Turbidity of Makgeolli According to Rice and Wheat Mixing Ratio

관능평가는 성인 30명 (20 ~ 30대)을 대상으로 제조된 막걸리의 향, 맛, 색 및 전체적인 기호도에 대해 '매우 좋다 5점, 좋다 4점, 보통이다 3점, 나쁘다 2점, 매우 나쁘다 1점'으로 하는 5점 척도법으로 관능성을 평가하였고 그 결과를 표 12에 나타냈다. The sensory evaluation was based on the flavor, taste, color and overall taste of makgeolli made for 30 adults (ages 20 to 30). 'Very good 5 points, good 4 points, normal 3 points, bad 2 points, very bad Sensory evaluation was carried out by the 5-point scale method of '1 point' and the results are shown in Table 12.

비율ratio
(쌀 : 밀)(Rice: wheat) 향incense 맛flavor 색color 전체적인Overall
기호도Likelihood 10 : 010: 0 3.90 3.90 4.55 4.55 4.25 4.25 4.15 4.15 8 : 28: 2 3.85 3.85 4.50 4.50 3.80 3.80 4.15 4.15 6 : 46: 4 3.95 3.95 4.55 4.55 3.80 3.80 4.20 4.20 5 : 55: 5 4.25 4.25 4.50 4.50 4.20 4.20 4.50 4.50 4 : 64: 6 4.30 4.30 4.70 4.70 4.10 4.10 4.55 4.55 2 : 82: 8 4.15 4.15 3.95 3.95 4.15 4.15 4.10 4.10 0 : 100: 10 3.75 3.75 3.80 3.80 4.00 4.00 4.05 4.05

<쌀과 밀 혼합비에 따른 막걸리의 관능평가><Sensory Evaluation of Makgeolli According to Rice and Wheat Mixing Ratio>

상기 결과에 의하면, 본 발명에 따른 제조 방법에서 곡류 원료로서, 쌀과 밀을 단독으로 사용하는 것보다 혼합하여 사용하는 것이 바람직하며, 가장 바람직하게는 쌀과 밀을 5:5 또는 4:6의 중량비로 혼합하여 사용하는 것임을 알 수 있다.
According to the above results, in the production method according to the present invention, it is preferable to use a mixture of rice and wheat as a raw material of grains, rather than to use alone, most preferably rice and wheat of 5: 5 or 4: 6 It can be seen that the mixture is used in a weight ratio.

국립농업과학원농업유전자원센터National Institute of Agricultural Science, Genetic Resources Center KACC93119KACC93119 2011032520110325

Claims (6)

막걸리 제조를 위한 효모 균주용 카자크스타니아 엑시구아 (Kazachstania exigua) CCSY27 (수탁번호 KACC93119P) 균주. Kazachstania exigua CCSY27 (Accession No. KACC93119P) strain for yeast strains for the production of makgeolli. 제 1항에 있어서, 상기 균주는 pH 3.0 조건에서 3 시간 배양시 60.8%의 생존율, 인공위액 조건에서 3 시간 배양후 52.9%의 생존율, 담즙 1~4% 조건에서 생육성, 당 농도 30%에서 OD600nm 4.44의 생육성, 알코올 농도 10%에서 OD600nm 3.34의 생육성, 및 10%(v/v)의 알코올 발효능을 갖는 것을 특징으로 하는 균주. According to claim 1, wherein the strain has a survival rate of 60.8% when cultured for 3 hours at pH 3.0 conditions, 52.9% survival rate after 3 hours in artificial gastric juice conditions, viable growth at 1-4% bile conditions, at a sugar concentration of 30% A strain characterized by having a viability of OD 600nm 4.44, a viability of OD 600nm 3.34 at an alcohol concentration of 10%, and an alcohol fermentation capacity of 10% (v / v). 곡류 원료로 막걸리를 제조하는 방법으로,
카자크스타니아 엑시구아 (Kazachstania exigua) CCSY27 (수탁번호 KACC93119P) 균주를 사용하는 것을 특징으로 하는 방법. As a method of manufacturing makgeolli from raw grains,
Kazachstania exigua CCSY27 (Accession No. KACC93119P) strain. 제 3항에 있어서, 곡류 원료는 쌀과 밀을 혼합한 것인 막걸리를 제조하는 방법. 4. The method of claim 3, wherein the grain raw material is a mixture of rice and wheat. 제 4항에 있어서, 쌀과 밀은 5: 5의 중량비로 혼합하는 것인 막걸리를 제조하는 방법. The method of claim 4, wherein the rice and wheat are mixed in a weight ratio of 5: 5. 제 4항에 있어서, 쌀과 밀은 4: 6의 중량비로 혼합하는 것인 막걸리를 제조하는 방법.The method of claim 4, wherein the rice and wheat are mixed in a weight ratio of 4: 6.
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