KR100768566B1 - 3 Novel alcohol producing yeast Saccharomyces cerevisiae HA3 - Google Patents
3 Novel alcohol producing yeast Saccharomyces cerevisiae HA3 Download PDFInfo
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Abstract
Description
도 1은 ITS 1 프라이머를 이용하여 HA3로부터 ITS 염기서열을 증폭하여 블라스트 서치한 결과를 나타낸다.Figure 1 shows the result of blast search by amplifying the ITS sequence from HA3 using the
도 2는 ITS 4 프라이머를 이용하여 HA3로부터 ITS 염기서열을 증폭하여 블라스트 서치한 결과를 나타낸다.Figure 2 shows the result of blast search by amplifying the ITS sequence from HA3 using the ITS 4 primer.
본 발명은 신규한 주조용 효모 사카로마이세스 세레비지아에 HA3 균주에 관한 것으로 더욱 상세하게는 상기 균주의 분리동정 및 그 이용에 관한 것이다. The present invention relates to a novel cast yeast Saccharomyces cerevisiae HA3 strain, and more particularly, to isolate identification and use of the strain.
술은 지역, 민족, 기후 풍토 및 인간의 문화적 특성에 따라 다양한 형태의 술로 개발 전승되어 왔으며, 또 각 민족은 독특한 주조법을 개발하여 민족 고유의 전통주를 갖게 되었으며, 수많은 전통주들이 탄생하여 360종의 술이 개발되었다. 그러나 일제의 주세정책을 이어받은 정부의 통제행정에 의해 자가양조가 금지되어 일제시대를 거치면서 우리나라 주류는 약주, 탁주, 소주로 획일화되고 민속주의 전승이 단절되었다. Liquor has been developed and passed on in various forms according to the region, ethnicity, climate, and human cultural characteristics.Each nation has developed a unique casting method and has traditional liquor. This was developed. However, the self-brewing was prohibited by the government's control administration, which inherited the Japanese tax policy, and the Japanese liquor was uniformized into Yakju, Takju, and Soju, and the folklore tradition was cut off.
그러나 1982년 문공부에서 전통민속주의 지정·조사를 시작으로 하여, 국세청기술연구소와 우리나라 전래 민속주에 대한 문헌을 중심으로 그 종류와 주조법상의 주종으로 분류 고찰하였으며, 1994년도부터 과기처에서 주관하는 G-7 Project가 수행되면서 뒤늦게 전통누룩과 민속주의 품질향상과 산업화 기술에 대한 과학적인 제조방법의 연구가 진행되고 있다,However, in 1982, the Ministry of Education began to designate and investigate traditional folkism, and classified them into the types and types of casting methods mainly in the literature on the National Tax Agency's research institute and Korean folklore. As the project is carried out, studies on the scientific manufacturing method of the traditional yeast and the improvement of nationalism and the technology of industrialization are being conducted.
전통주에 대한 지금까지 연구들을 살펴 보면, 주로 재래식 약·탁주의 효율적인 제조기술개발, 원료 및 술덧 등의 각종 화학성분의 분석, 발효제 종균개발 및 전통주의 증류특성에 대한 분석, 누룩 및 술덧 중의 미생물과 효소의 분포, 저장성 연장 및 품질개선, 생리기능성 물질의 생산 등에 대해 보고되었다, 또한 전통주의 개량을 목적으로 18개 지역의 누룩으로부터 생전분 분해성이 우수한 균주 Rhizopous, Absidia, Aspergillus , Avtinomucor , Bortyotruchum , Cladosporum 속 균들을 분리 선발하여 보고하기도 하였으나 우리나라 전통주에 대한 연구는 대부분이 제조기술과 살균 및 저장성 증가 기술, 발효 또는 저장 중의 이화학적 변화에만 치중되어 있으며 이러한 기초연구를 바탕으로 산업화를 한 예는 매우 적다.So far, researches on traditional liquor have mainly focused on the development of efficient manufacturing techniques of conventional medicine and Takju, analysis of various chemical components such as raw materials and sake, development of fermenter seed and distillation characteristics of traditional liquor, microorganisms in yeast and sake. Enzyme distribution, extended shelf life and quality improvement, and production of physiologically functional substances have been reported. Also , strains of Rhizopous, Absidia, Aspergillus , Avtinomucor , Bortyotruchum , Cladosporum , which have high biodegradability from yeast in 18 regions for the purpose of improving traditional stocks . Although some isolates were selected and reported, most of the researches on Korean traditional liquor are focused on the manufacturing technology, the technology of increasing sterilization and shelf life, and the physicochemical changes during fermentation or storage, and there are very few examples of industrialization based on such basic research. .
1996년 대구무형문화재 제11호로 지정된 지역 전통주인 하향주가 민속주로써 현대까지 구전되어오고 있으며, 주요 식문헌에도 등장하는 전통명주를 보유하고 있으나 문화재관리국의 주도로 소략한 조사가 이루어진 이후 아직 체계적인 조명이 이루어지고 있지 않은 실정이다.Dangju, a local traditional liquor designated as Daegu Intangible Cultural Heritage No. 11 in 1996, has been handed down to modern times as a folk liquor, and it has traditional silk that appears in major food literature, but has been systematically illuminated after a brief investigation led by the Cultural Heritage Administration. It is not happening.
따라서, 본 발명자는 지역고유의 전통주인 하향주의 우수성을 규명하기 위한 기초자료로, 주조시 이용되는 전통 누룩으로부터 미생물자원인 효모를 분리하고, 분리된 효모들 중 하향주의 대량생산 및 산업화로 이용할 수 있는 효모를 선발, 동정하는데 성공하였다.Therefore, the present inventors can separate the yeast, which is a microbial resource, from the traditional yeast used in casting, and use it as a mass data and industrialization of the down yeast among the separated yeasts. Successfully selected and identified yeast.
본 발명은 주조시 이용되는 전통 누룩으로부터 효모를 분리하는 단계; 분리된 효모 중 하향주의 대량생산으로 이용성이 큰 효모를 선발 동정하는 단계와; 그 특성을 비교하는 단계로 구성된다.The present invention comprises the steps of separating the yeast from the traditional yeast used in casting; Selecting and identifying a yeast having high usability through mass production of the down yeast from the separated yeast; Comparing the characteristics.
삭제delete
본 발명을 실시예에 의해 상세히 설명하지만 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Although the present invention will be described in detail by way of examples, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.
실시예Example 1: 본 발명 1: present invention 신규한New 효모 균주의 분리 Isolation of Yeast Strains
대구 무형문화재 제11호인 하향주 제조용 누룩으로부터 효모를 분리하기 위하여 분쇄한 누룩 1g에 멸균증류수 10ml을 첨가하고 충분히 혼합(vortexing) 한 후 10배 희석한 희석액 100㎕를 yeast malt extract agar(agar 2%, glucose 1%, peptone 0.5%, yeast extract 3%, malt extract 3%, pH 6.0)에 도말하고 24시간동안 28℃에서 배양시킨 후 생성된 집락을 분리하였다.In order to separate yeast from the yeast for the production of downward liquor, Daegu Intangible Cultural Heritage No. 11, 10 ml of sterile distilled water was added to 1 g of crushed yeast, and thoroughly mixed (vortexed), and 100 µl of the diluted solution diluted 10-fold was added to yeast malt extract agar (agar 2%,
실험 결과, 상기 하향주 제조용 전통 누룩으로부터 29개 효모를 분리 하였으며, 분리된 효모는 Yeast Malt Extract agar(YM agar) 배지에 계대하여 4℃에서 보 관하면서 공시재료로 본 발명에 사용하였다.As a result of the experiment, 29 yeasts were isolated from the traditional yeast for the production of the down strain, and the separated yeasts were used in the present invention as a test material while being stored at 4 ° C. in passage in Yeast Malt Extract agar (YM agar) medium.
실시예Example 2: 분리된 효모의 당 내성 측정 2: Determination of Sugar Tolerance in Isolated Yeast
상기 실시예 1에서 분리한 효모의 당내성 측정은 glucose를 40%, 50% 첨가한 yeast malt extract broth(glucose 1%, peptone 0.5%, yeast extract 3%, malt extract 3%, pH 6.0)에 분리된 효모를 l loop 접종하고 28℃에서 180rpm으로 24시간 동안 진탕배양시킨 후 spectrophotometer를 이용하여 600nm에서 흡광도를 측정하여 균주의 성장을 비교하였다. 이때 control은 2% glucose를 첨가한 yeast malt extract broth(YM broth)에서 분리된 효모균주의 성장으로 하였다.Measurement of glucose tolerance of the yeast isolated in Example 1 was isolated on yeast malt extract broth (
분리된 효모 29개 균주를 대상으로 glucose 40%, 50%를 첨가한 YM broth에서의 균주의 생육과 control인 2% glucose를 첨가한 YM broth에서 균주의 생육을 비교한 결과. N25, HA1, HA2, HA3, HA4를 제외하고는 대체적으로 50% glucose가 첨가된 YM broth에서도 양호한 생육을 나타냈으며, 특히 N8의 경우는 50% glucose 첨가시에도 control에서 균주 생육의 80%를 나타내고 있어 표 1에서 보듯이 고농도의 당에서 충분히 생육 가능한 우수한 내당성 균주로 확인하였다.Comparison of the growth of strains in the YM broth with 40% and 50% glucose and the growth of the YM broth with 2% glucose control was performed on 29 isolated yeast strains. Except for N25, HA1, HA2, HA3, and HA4, YM broth showed good growth in 50% glucose. In particular, N8 showed 80% of strain growth in control even when 50% glucose was added. As shown in Table 1, it was confirmed as an excellent sugar-resistant strain capable of sufficiently growing in a high concentration of sugar.
본 실시예 및 이하에서, 세포 성장은 spectrophotometer(600nm)에서 흡광도를 측정하였다.In this example and below, cell growth was measured for absorbance on a spectrophotometer (600 nm).
실시예Example 3: 효모의 에탄올 내성 측정 3: Determination of ethanol tolerance of yeast
상기 분리된 각 효모의 ethanol 함유 배지에서의 생육정도를 측정을 하여 분리된 효모들의 에탄올에 대한 내성을 조사하고자 하였다. 즉 ethanol이 각각 0%, 5%, 10%, 15%의 농도로 함유된 YM broth 10ml에 전 배양한 효모 배양액을 0.1% 농도로 접종하고 28℃에서 5일간 정치 배양 후 600nm에서 흡광도를 측정하여 균의 생육 정도를 조사하였다. The growth of ethanol-containing medium of each yeast was measured to investigate the resistance of the yeast to ethanol. That is, inoculated with 10% YM broth 10ml of YM broth containing ethanol at concentrations of 0%, 5%, 10%, and 15%, respectively, and inoculated at 0.1% concentration. The degree of growth of the bacteria was examined.
에탄올 내성 효모를 선발하기 위하여 에탄올이 각각 0%, 5%, 10%, 15%의 농도로 함유된 YM broth에 분리된 효모를 접종하여 5일 후 에탄올 무첨가(0%)와 생육정도를 비교한 결과, 에탄올 5% 첨가시에는 control과 비교시 분리 효모들이 생육이 모두 양호하였으나 10% 에탄올 첨가시에 N22, HA2 HA3, HA4 균주를 제외하고는 생육이 급격히 저하되었으며, 15% 에탄올 첨가시에는 분리된 효모들의 생육은 거의 저해되었다(표 2).In order to select ethanol resistant yeasts, ethanol was inoculated with YM broth containing 0%, 5%, 10% and 15%, respectively, and 5 days later, ethanol-free (0%) and growth were compared. As a result, when the 5% ethanol was added, the isolated yeasts showed good growth compared to the control, but when the 10% ethanol was added, the growth decreased dramatically except for the N22, HA2 HA3, and HA4 strains. Growth of dried yeast was almost inhibited (Table 2).
실시예Example 4: 분리된 효모의 산 4: acid of isolated yeast 생성능Generation 측정 Measure
분리된 효모의 산 생성능 조사를 위하여 산 생성배지( glucose 5%, yeast extract 0.5%, agar 2%, pH 6.0 CaCO3 5%)를 제조하고 분리된 효모를 tooth pick 접종하여 28℃에서 2일간 배양 후 균체 주변에 생성된 clear zone을 측정하였다.Acid production medium (glucose 5%, yeast extract 0.5%, agar 2%, pH 6.0 CaCO3 5%) was prepared to investigate the acid production ability of the isolated yeast, and after incubating at 28 ° C. for 2 days by inoculating the tooth yeast with the isolated yeast Clear zones generated around the cells were measured.
분리된 효모를 tooth pick하여 접종하고 배양 후 균체 주변에 생성된 clear zone을 측정하여 균주의 산생성능을 조사한 결과, N2,N6, N17, HA3, HA4가 산생성능이 특히 우수하였으며, N4, N8, N10, N11, N12, N15, N19, N24도 산 생성능이 양호하였다(표 3).After inoculation by tooth picking the isolated yeast and measuring the clear zones generated around the cells after incubation, the acid production performance of the strain was examined, and N2, N6, N17, HA3 and HA4 were particularly excellent in acid production. N10, N11, N12, N15, N19, and N24 also had good acid generating ability (Table 3).
실시예Example 5: 분리된 효모의 에탄올 5: Ethanol from Isolated Yeast 발효능Fermentation ability 측정 Measure
분리된 효모의 에탄올 생성량은 25% glucose를 첨가한 100ml YB broth에 YM broth에 전 배양한 효모 배양액 100㎕를 접종하고 28℃에서 180rpm으로 48시간 동안 진탕배양시킨 후 10,000rpm에서 10분간 원심분리하여 배양 상등액을 회수하고 배양상등액에 생성된 에탄올을 함량을 측정하였다, 에탄올 함량은 비중법을 이용하여 배양상등액 100ml를 증류하여 70%를 메스실린더에 회수하고 다시 증류수를 이용하여 100ml로 정용하고 주정계를 이용하여 측정하였다,Ethanol production of isolated yeast was inoculated into 100ml YB broth to which 25% glucose was added, 100 μl of pre-cultured yeast culture in YM broth, shaken at 28 ° C for 180 hours at 180 rpm, and centrifuged at 10,000 rpm for 10 minutes. The culture supernatant was recovered and the content of ethanol produced in the culture supernatant was measured. The ethanol content was distilled from 100 ml of the culture supernatant using specific gravity method, and 70% was recovered to a measuring cylinder. Measured using
1차적으로 선발된 상기 13개 균주를 대상으로 25% glucose 첨가한 YB broth에서 각 균주의 에탄올 생성량을 주정계를 이용하여 측정한 결과, HA2, HA3, HA4가 각각 에탄올 생성량이 10.8%, 10.45%, 10%로 선발된 균주들 중 가장 우수하였다(표 4).As a result of measuring the ethanol production of each strain in a YB broth added with 25% glucose to the 13 selected strains using a ethanol, HA2, HA3 and HA4 produced 10.8%, 10.45%, It was the best among the strains selected at 10% (Table 4).
실시예Example 6: 분리된 효모 중 주조용 효모의 1차 선발 6: First Selection of Brewing Yeast from Isolated Yeast
분리된 효모 29개 균주를 대상으로 상기의 결과를 토대로 하여 내당성, 내알콜성, 산생성능이 우수한 N2, N3 N,5, N6, N8, N12, N17, N18, N22, HA1, HA2, HA3, HA4는 하향주의 대량생산을 위하여 산업용으로 이용할 수 있는 효모로 1차적으로 선발하였다. N2, N3, N, 5, N6, N8, N12, N17, N18, N22, HA1, HA2, HA3 with excellent glucose tolerance, alcohol resistance, and acid productivity, based on the above-mentioned results in 29 isolated yeast strains In particular, HA4 was selected primarily as an industrial yeast for mass production of downstream strains.
실시예Example 7: 선발된 효모의 동정 7: Identification of Selected Yeasts
선발된 에탄올 내성 효모의 동정은 현미경상 형태적 특성을 조사 한 후 Biolog사의 동정시스템(MicrologTM4.0)과 ITS의 영역을 증폭한 sequence를 blast search 하여 동정하였다.The ethanol-resistant yeast was identified by microscopic morphological examination, followed by blast search for sequences amplified from Biolog's identification system (Microlog ™ 4.0) and ITS.
1차적으로 선발된 주조용 13개 균주를 현미경상 형태적 특성을 조사 한 후 Biolog사의 동정시스템(MicrologTM4.0)과 ITS 1 primer(5'-TCCGTAGGTGAACCTGCGG-3'), ITS 4 primer(5'-TCCTCCGCTTATTGATATGC-3')를 이용하여 PCR 증폭하여 sequencing 후 blast search한 결과, N2, N3, N5, N6, N8, N12, N17, N18, N22는 Pichia anomala에 모두 99% 상동성을 나타내어 Pichia anomala로 동정 되었으며, HA1, HA2, HA3, HA4는 Saccharomyces cerevisiae로 각각 97%, 94%, 99%, 97% 상동성을 나타내어 Saccharomyces cerevisiae HA1, Saccharomyces cerevisiae HA2, Saccharomyces cerevisiaeHA3, Saccharomyces cerevisiaeHA4로 명명하였다(도 1 및 도 2). The microscopical morphological characteristics of the 13 selected casting strains were examined under the microscope, followed by Biolog's identification system (MicrologTM4.0), ITS 1 primer (5'-TCCGTAGGTGAACCTGCGG-3 '), and ITS 4 primer (5'- PCR amplification using TCCTCCGCTTATTGATATGC-3 ') and after sequencing blast search, N2, N3, N5, N6, N8, N12, N17, N18, N22 are Pichia 99% homology to both anomala Pichia anomala was identified and HA1, HA2, HA3 and HA4 were identified as Saccharomyces. cerevisiae with 97%, 94%, 99%, 97% homology, respectively, Saccharomyces cerevisiae HA1, Saccharomyces cerevisiae HA2, Saccharomyces cerevisiae HA3, Saccharomyces cerevisiae HA4 (Figs. 1 and 2).
실시예Example 8: 주조용 효모 균주 최종 선발 8: Final Selection of Casting Yeast Strains
동정된 효모들 중 Pichia anomala의 경우 에탄올 생성량도 미약하며 주정 발효시에 막을 형성하여 이상발효 및 주질의 품질을 저하시키는 산막효모이므로 산업용 효모로 이용가치가 없으므로 Saccharomyces cerevisiae HA1, Saccharomyces cerevisiae HA2, Saccharomyces cerevisiaeHA3, Saccharomyces cerevisiae HA4를 대상으로 내당성, 내알콜성, 산생성능을 조사한 상기결과를 다시 검토한 결과 이들은 내당성은 서로 큰 차이가 없었으나, 산생성능은 Saccharomyces cerevisiaeHA3, Saccharomyces cerevisiaeHA4가 우수하였고, 내알콜성 및 에탄올 발효능은 Saccharomyces cerevisiae HA2, Saccharomyces cerevisiaeHA3이 우수하였다. 따라서 하향주의 대량생산시 주조용 산업적 이용성이 가장 큰 효모로서 사카로마이세스 세레비지아에 HA3(Saccharomyces cerevisiae HA3) 균주를 최종 선발하고 농업생명공학연구원에 2006년 5월 12일자 KACC 93039P로 기탁하였다. Pichia among identified yeasts In the case of ethanol production anomala yeast is also weak, and because it lodges that degrade the quality of fermentation and jujil over the film during alcoholic fermentation because there is no use value to industrial yeast Saccharomyces cerevisiae HA1, Saccharomyces cerevisiae HA2, Saccharomyces cerevisiae HA3, Saccharomyces cerevisiae After reviewing the results of glucose tolerance, alcohol resistance, and acid production in HA4, they did not differ significantly from each other, but acid performance was not affected by Saccharomyces cerevisiae HA3, Saccharomyces. cerevisiae HA4 was excellent, and alcohol resistance and ethanol fermentation capacity were Saccharomyces cerevisiae HA2 and Saccharomyces cerevisiae HA3 were excellent. Therefore, HA3 ( Saccharomyces ) in Saccharomyces cerevisiae as the yeast with the highest industrial availability for casting in the mass production of downstream liquor. cerevisiae HA3) was finally selected and deposited with KACC 93039P dated May 12, 2006 to the Institute of Agricultural Biotechnology.
이상에서 명백한 바와 같이, 본 발명은 우리나라 하향주 대량샌산시 주조용 산업적 이용성이 가장 큰 신규한 효모 균주를 산업계에 제공할 수 있는 뛰어난 효과가 있다.As is apparent from the above, the present invention has an excellent effect that can provide the industry with a new yeast strain of the largest industrial usability for casting mass production in China downstream.
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| KR890016163A (en) * | 1988-04-12 | 1989-11-28 | 박용성 | Saccharomyces cerevisiae Doosan No. 1, a Highly Concentrated Alcohol-producing Yeast, and Alcohol Fermentation Method Using the Same |
| JPH08173147A (en) * | 1994-12-26 | 1996-07-09 | Takara Shuzo Co Ltd | New yeast and its use |
| JPH10262652A (en) | 1997-03-21 | 1998-10-06 | Oozeki Kk | New yeast strain resistant to alcohol, sugar and high temperature and production of liquor with the same |
| KR20010103304A (en) * | 2000-05-09 | 2001-11-23 | 진익렬 | Saccahromyces cerevisiae KNU5377 strains having multi-stress tolerance |
| KR20030040605A (en) * | 2001-11-15 | 2003-05-23 | 주식회사 두산 | An Ethanol-tolerant Strain, Saccharomyces cerevisiae SE211 and The Process For Manufacturing Rice Wine Using This Strain |
| JP2006000025A (en) | 2004-06-16 | 2006-01-05 | Akita Prefecture | New yeast and method for producing sake therewith |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR890016163A (en) * | 1988-04-12 | 1989-11-28 | 박용성 | Saccharomyces cerevisiae Doosan No. 1, a Highly Concentrated Alcohol-producing Yeast, and Alcohol Fermentation Method Using the Same |
| JPH08173147A (en) * | 1994-12-26 | 1996-07-09 | Takara Shuzo Co Ltd | New yeast and its use |
| JPH10262652A (en) | 1997-03-21 | 1998-10-06 | Oozeki Kk | New yeast strain resistant to alcohol, sugar and high temperature and production of liquor with the same |
| KR20010103304A (en) * | 2000-05-09 | 2001-11-23 | 진익렬 | Saccahromyces cerevisiae KNU5377 strains having multi-stress tolerance |
| KR20030040605A (en) * | 2001-11-15 | 2003-05-23 | 주식회사 두산 | An Ethanol-tolerant Strain, Saccharomyces cerevisiae SE211 and The Process For Manufacturing Rice Wine Using This Strain |
| JP2006000025A (en) | 2004-06-16 | 2006-01-05 | Akita Prefecture | New yeast and method for producing sake therewith |
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| KR101284263B1 (en) * | 2011-03-22 | 2013-07-08 | 인천대학교 산학협력단 | Saccharomyces cerevisiae NK28 for producing wine and method for producing wine using the same |
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