KR0185018B1 - Saccharomyces cerevisiae ctb 14 used for the preparation of sake - Google Patents
Saccharomyces cerevisiae ctb 14 used for the preparation of sake Download PDFInfo
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Abstract
본 발명은 청주의 양조에 사용되는 사카로마이세스 세레비제(Saccharomyces cerevisiae) YBB 효모를 에틸메탄설포네이트(E.M.S. Ethyl Methanesulfonate)변이 처리한 후, 선별배지에서 분리 개량시킨 청주의 주요 향기 성분들을 고농도로 생산하는 미생물 및 이를 이용한 청주의 제조방법에 곤한 것으로, 더욱 상세히는 사카로마이세스 세세비제 YBB 효모를 변이 유기제인 EMS로 처리한 후, 트라이플루오로류신의 최종 농도가 50∼500μM, 셀률레닌의 최종농도가 3∼50μMC 첨가한 최소재비에서 선발된 내성균주를 다시 효모용 선별배지에 접종하여 호흡장애가 있는 균주를 제거하고, 국즙배지에서 배양후, 가스크로마토그라피로 향성분을 분석하여 고향기 효모를 분리 개량하여 얻어진청주에 좋은 풍미를 부여하는 향기성분을 다량 생산하는 효모균주 사카로마이세스 세레비제 CTB14(KFCC-10936호)와 이를 증미배지에서 발효시킴으로서 청주를 제조하는 방법에 관한 것이다.The present invention, Saccharomyces cerevisiae YBB yeast used in the brewing of Cheongju after treatment of the mutant of Ethyl Methanesulfonate (EMS Ethyl Methanesulfonate), the main flavor components of Cheongju separated and improved in a selective medium at high concentration The microorganism to be produced and the preparation method of the sake using the same, more specifically, Saccharomyces sebase YBB yeast is treated with EMS as a variant organic agent, the final concentration of trifluoroleucine is 50 ~ 500μM, celenolenin Resistant strains selected at the minimum concentration of 3 ~ 50μMC added to the final concentration inoculated again in the yeast screening medium to remove strains with respiratory distress, cultured in broth broth, and then analyzed by fragrance by gas chromatography in homegrown yeast Saccharomyces cerevis yeast strain producing a large amount of fragrance ingredients that give good flavor to sake CTB14 (KFCC-10936 Lake) and to a method for producing a fermented rice wine sikimeuroseo it in jeungmi medium.
Description
본 발명은 청주의 양조에 사용되는 사카로마이세스 세리베제(Saccharomyces cerevisiae) YBB 효모를 에틸메탄설포네이트(E.M.S., Ethyl Methanesulfonate)변이 처리한 후, 선별배지에서 분리 개량시킨 청주의 주요 향기 성분들을 고농도로 생산하는 미생물 및 이를 이용한 청주의 제조방법에 관한 것이다.The present invention is the main fragrance components of Saccharomyces cerevisiae (Saccharomyces cerevisiae) YBB yeast used in the brewing of Cheongju after mutated ethylmethanesulfonate (EMS, Ethyl Methanesulfonate), isolated from the selection medium It relates to a microorganism produced at a high concentration and a method of manufacturing cheongju using the same.
최근 몇 년 사이 소비자들의 다양한 취향에 부응하여 기존의 대표적 술이었던 맥주, 소주에 비해 청주의 소비량이 비약적으로 증가되었다. 청주는 쌀을 주원료로 제조되며, 제조 특성상 복합적인 맛과 향을 갖고 있다. 청주의 맛과 향에는 많은 향기성분들이 관여하지만 직접적으로 영향을 미치는 것으로는 아세트알데히드(Aectaldehyde), 다이아세틸(Diacethyl), 초산에틸(Ethyl acetate), 이소초산 아밀(Isoamyl acetate), 이소아밀알코올(Isoamyl alcohol), 페닐에틸알코올(phenylethyl alcohol), 초산페닐에틸(Phenylethyl acetate), 카프로산 에틸(Ethyl caproate)등이 있다.In recent years, in order to meet the various tastes of consumers, the consumption of sake has increased dramatically compared to beer and shochu, which were the representative alcohols. Cheongju is made from rice as the main ingredient and has a complex taste and aroma due to its manufacturing characteristics. Many flavors are involved in the taste and aroma of Cheongju, but those that directly affect acetaldehyde, diacetyl, ethyl acetate, isoamyl acetate and isoamyl alcohol Isoamyl alcohol, phenylethyl alcohol, phenylethyl acetate, and ethyl caproate.
맛과 향이 우수한 청주 효모 균주를 얻긱위한 기존의 방법들은 뚜렷한 선발방법이 없기 때문에 많은 균주를 인위적인 혹은 자연적인 돌연변이에 의해 분리된 균주를 일일이 청주를 제조하여서 향성분을 분석해야 했다. 따라서 많은 시간과 인력을 필요로 하며 설사 이러한 방법에 의해 균주가 선발된 후에라도 선발된 균주들이 흔히 원래의 모균주가 갖고 있었던 우수한 발효적 특성들을 유실하는 경우가 많았다. 따라서 좋은 향기성분을 다량으로 생산하는 균주지만 청주발효적 특성이 좋지 않기 때문에 실제로 이용되기에는 적합하지 않는 경우가 대부분이었다.Since existing methods for obtaining sake yeast strains with excellent taste and aroma do not have a clear selection method, many strains were isolated from artificial or natural mutations to prepare sake and then analyzed the fragrance components. Therefore, it takes a lot of time and manpower, and even after the strain was selected by this method, the selected strains often lost the excellent fermentation properties of the original parent strain. Therefore, the strain that produces a large amount of good fragrance components, but because the fermentation properties of the cheongju is not good in most cases was not suitable for practical use.
트라이플루오로류신(T.F.L., Trifluoroleucine)은 아미노산이 류신(Leucine)의 유도체로서 이 물질이 첨가된 배지에서 생존이 가능한 효모균주들은 이소초산아밀과 이소아밀알코올의 생합성 경로에서 중요한 역할을 수행하는 효소인 알파아이피엠 신테이즈(α-IPM synthase)의 특성이 변하여 류신에 의한 저해를 극복함으로써 다량의 이소초산아밀과 이소아밀알코올을 생산하게 된다. 한편, 세룰레닌(Cerulenin)은 곰팡이에서 유래한 것으로 지방산이 합성에 관여하는 효소들을 저해함으로써 항진균성, 항세균성을 띠는 물질이다. 그런데 세룰레닌이 첨가된 배지에 생장한 효모균주들은 기존 균주에 비해 청주의 중요한 향성분중 하나인 카프로산 에틸(Ethyl caproate)을 다량으로 생산함이 보고되었다.Trifluoroleucine (TFL, Trifluoroleucine) is a derivative of leucine (amino acid). Yeast strains that can survive in medium containing this substance are enzymes that play an important role in the biosynthetic pathway of isoacetate and isoamyl alcohol. The properties of alpha-IPM synthase are changed to overcome the inhibition by leucine to produce a large amount of isoamyl acetate and isoamyl alcohol. On the other hand, cerulenin is derived from the fungus, fatty acids inhibit the enzymes involved in the synthesis of the antifungal, antibacterial substances. However, it was reported that yeast strains grown in the medium containing cerulein produced a large amount of ethyl caproate, one of the important flavor components of saengju, compared to the existing strains.
따라서 본 발명자들은 트라이플루오로류신과 세룰레닌의 이러한 특성에 착안하여, 두 물질을 배지에 동시에 첨가함으로써 이소초산아밀, 이소아밀알코올 및 카프로산 에틸을이미 다량으로 분비하는 우수 청주 효모 균주를 선발하였으며, 그 결과 본래 갖고 있었던 발효적 특성을 유지하면서 다량의 향기성분을 분비하는 변이주 CTB 14를 선발하였다.Therefore, the inventors have focused on these properties of trifluoroleucine and cerulenin, and have selected excellent sake yeast strains that already secrete large amounts of isoacetate, isoamyl alcohol and ethyl caproate by adding both substances to the medium at the same time. As a result, the mutant strain CTB 14, which secretes a large amount of fragrance components, was selected while maintaining the fermentation properties originally possessed.
따라서 본 발명은 사카로마이세스 세레비제(Saccharomyces cerevisiae) YBB 효모를 변이 유기제인 EMS로 처리한 후, 트라이플루오로류신의 최종 농도가 50∼500μM 첨가한 최소배지에서 선발된 내성 균주를 다시 효모용 선별배지에 접종하여 호흡장애가 있는 균주를 제거하고, 국즙배지에서 배양후, 가스크로마토그라피로 향성분을 분석하여 고향기 효모를 분리 개량하여 얻어진 청주에 좋은 풍미를 부여하는 향기성분은 다량 생산하는 효모균주 사카로마이세스 세레비제 CTB14(KFCC-10936호)와 이를 증미배지에서 발효시킴으로서 청주를 제조하는 방법에 관한 것이다.Therefore, the present invention, after treating Saccharomyces cerevisiae YBB yeast with EMS as a variant organic agent, the resistant strain selected from the minimum medium added 50 ~ 500μM final concentration of trifluoroleucine again for yeast After inoculation into the selective medium to remove strains with respiratory disorders, cultured in a broth medium, and analyzed the fragrance component by gas chromatography to separate the home yeast, the flavor component to give a good flavor to the sake obtained by producing a large amount of yeast bacteria The present invention relates to Saccharomyces cerevisiae CTB14 (KFCC-10936) and a method for producing sake by fermenting it in a thickening medium.
이때 최소배지는 15∼25g/L의 포도당, 12∼18g/L의 한천, 4∼6g/L의 황산 암모늄, 0.8∼1.2g/L의 인산제일칼륨, 0.4∼0.6g/L의 황산마그네슘 및 미량의 비타민 및 무기염류를 포함하는 배지를 사용하고, 효모용 선별배지는 40∼60g/L의 포도당, 3∼5g/L의 효모엑기스, 4∼6g/L의 카시톤 및 미량의 무기염류를 포함하는 배지를 사용한다.The minimum medium is 15-25 g / L glucose, 12-18 g / L agar, 4-6 g / L ammonium sulfate, 0.8-1.2 g / L potassium phosphate, 0.4-0.6 g / L magnesium sulfate and A medium containing a small amount of vitamins and inorganic salts is used, and the selective screening medium for yeast contains 40 to 60 g / L of glucose, 3 to 5 g / L of yeast extract, 4 to 6 g / L of carton and a small amount of inorganic salts. Use medium containing.
본 발명의 변이주를 유도한 방법은 다음과 같다.The method of inducing the mutant strain of the present invention is as follows.
청주 발효 효모인 사카로마이세스 세레비제 YBB에 변이 유기제인 이엠에스를 처리하여 트라이플루오로류신의 최종농도가 50∼500μM, 세룰레닌의 최종농도가 3∼50μM이 되도록 첨가한 최소배지(포도당 20g/l, 황산암노늄 5g/L, 비오틴 2㎍/L, 판토덴산 칼슘 400㎍/L, 엽산 2㎍/L, 이노시톨 2000㎍/L, 나이아신 400㎍/L, 파라아미노안식향산 200㎍/L, 피리독신 염산염 400㎍/L, 리보플라빈 200㎍/L, 티아민 염산염 400㎍/L, 붕산 500㎍/L, 황산동 40㎍/L, 요화칼륨 100㎍/L, 염화제이철 200㎍/L, 황산망간 400㎍/L, 몰리브덴산 소오다 020㎍/L, 황산아연 400㎍/L, 인산제일칼륨 1g/L, 황산마그네슘 0.5g/L, 염화나트륨 0.1g/L, 염화칼슘 0.1g/L, 한천 15g/L)에 도말하고 23∼27℃에서 7∼8일간 배양하여 트라이플루오로류신-세룰레닌 동시 내성 균주를 얻었다.Saccharomyces fermented yeast Saccharomyces cerevisiae YBB was treated with a variant organic agent EMS to add a minimum concentration of trifluoro leucine 50 ~ 500μM, the final concentration of cerulenin 3 ~ 50μM (20g per glucose / l, ammonium sulfate 5g / L, biotin 2µg / L, calcium pantothenate 400µg / L, folic acid 2µg / L, inositol 2000µg / L, niacin 400µg / L, paraaminobenzoic acid 200µg / L, Pyridoxine hydrochloride 400 µg / L, riboflavin 200 µg / L, thiamine hydrochloride 400 µg / L, boric acid 500 µg / L, copper sulfate 40 µg / L, potassium iodide 100 µg / L, ferric chloride 200 µg / L, manganese sulfate 400 µg / L, sodium molybdate 020µg / L, zinc sulfate 400µg / L, potassium phosphate 1g / L, magnesium sulfate 0.5g / L, sodium chloride 0.1g / L, calcium chloride 0.1g / L, agar 15g / L) Plated and cultured at 23 to 27 ° C. for 7 to 8 days to obtain a trifluoroleucine-serulenin co-resistant strain.
트라이플루오로류신-세룰레닌 동시 내성 균주들을 다시 효모용 선별배지(효모엑기스 4g/L, 카시톤 5g/L, 포도당 50g/L, 인산제일칼륨 0.55g/L, 염화칼륨 0.425g/L, 염화칼슘 0.125g/L, 황산마그네슘 0.125g/L, 염화제이철 0.0025g/L, 황산망간 0.0025g/L, 한천 15g/L, 브로모크레졸그린 0.022g/L)에 스트리킹(Streaking) 하여 호흡에 장애가 있는 균주들을 배제한 단일 콜로니(Colony)를 분리하였다.Trifluoroleucine-serulenin co-resistant strains were again screened for yeast (4 g / L yeast extract, 5 g / L cachetium, 50 g / L glucose, 0.55 g / L potassium phosphate, 0.425 g / L potassium chloride, 0.125 calcium chloride) g / L, magnesium sulfate 0.125g / L, ferric chloride 0.0025g / L, manganese sulfate 0.0025g / L, agar 15g / L, Streaking in bromocresol green 0.022g / L) Single colonies (Colony) excluding these were separated.
위에서 얻은 변이주들을 20㎖의 국즙배지(그늘에서 자연건조시킨 국(麴) 250g에 물을 첨가하여 1.5L로 부피를 맞춘 후 65℃에서 8시간 당화를 시킨 다음 원심분리하여 상등액만을 분리하고, 이 상등액을 다시 와트만(Whatman) 4번 여과지로 거른 후 121℃에서 15분간 멸균한 것)에 접종한 후 10∼14℃에서 15∼19일간 정치배양한 다음 배양액을 가스크로마토그래피로 정량분석하여 향기성분을 다량으로 생성하는 효모를 선발하였으며, 이를 사카로마이세스 세레비제 ctb14로 명명하였다. 이 균주는 1996년 11월 27일 한국종균협회의 한국미생물보존센터에 기탁하여 KFCC-10936호로 기탁번호를 부여받았다.The mutants obtained above were added to 20 ml of broth (250 g of naturally dried broth) in water, adjusted to 1.5 L, and then glycosylated at 65 ° C. for 8 hours, followed by centrifugation to separate only the supernatant. The supernatant was again inoculated with Whatman No. 4 filter paper and sterilized at 121 ° C. for 15 minutes), and then cultured at 10-14 ° C. for 15-19 days, and the culture solution was quantitatively analyzed by gas chromatography. Yeast producing a large amount of ingredients was selected and named as Saccharomyces cerevisiae ctb14. This strain was deposited on November 27, 1996 with the Korea Microbiological Conservation Center of the Korean spawn association and was given accession number to KFCC-10936.
상기의 방법에 따라 분리된 개량균주를 이용하여 증미를 주성분으로 하고 누룩과 물 및 소량의 당화효소를 첨가한 배지에서 통상의 방법으로 발효시켜 청주를 제조한다.Cheongju is prepared by fermenting in a conventional manner in a medium containing jeungmi as a main component and adding yeast, water, and a small amount of glycosylating enzyme using the improved strain isolated according to the above method.
[실시예 1]Example 1
사용균주 : 모균주 YBB와 본 발명의 변이주 CTB14(KFCC-10936호)Use strain: Mother strain YBB and mutant strain CTB14 of the present invention (KFCC-10936)
종배지 : 포도당 20g/L, 펩톤 20g/L, 효모엑기스 10g/L, 맥아엑기스 10g/LSpecies medium: glucose 20g / L, peptone 20g / L, yeast extract 10g / L, malt extract 10g / L
발효배지 : 국즙배지Fermentation medium: broth
발효방법 : 상기 종배지 2㎖를 4㎖ 바이알에 분주하고 121℃에서 15분간 멸균한 후 사용균주를 접종하고 30℃에서 24시간 정치배양하여 종배양액으로 사용하였다. 발효배지 19㎖를 100㎖ 시험관에 분주하고 121℃에서 15분간 멸균한 후, 종배양액 1㎖를 식균한 다음 12℃에서 17일간 정치배양하였다. 상기 발효액을 회수하여 향기성분을 측정한 결과는 다음 표 1과 같다.Fermentation method: 2 ml of the seed medium was dispensed into 4 ml vials, sterilized at 121 ° C. for 15 minutes, inoculated with the used strain, and incubated at 30 ° C. for 24 hours to use as a seed culture solution. 19 ml of the fermentation broth were dispensed into 100 ml test tubes, sterilized for 15 minutes at 121 ° C., 1 ml of the seed culture solution was inoculated, and then cultured at 12 ° C. for 17 days. The results of measuring the fragrance components by recovering the fermentation broth are shown in Table 1 below.
본 발명에 따라 얻어진 사카로마이세스 세레비제 CTB14를 사용하여 청주를 제조함으로서 향기성분인 이소초산아밀, 이소아밀알코올 및 카프로산 에틸을 다량 함유한 풍미가 좋은 청주를 쉽게 제조할 수 있다.By preparing sake using Saccharomyces cerevisiae CTB14 obtained according to the present invention, savory sake containing a large amount of ammonium isoacetate, isoamyl alcohol and ethyl caproate can be easily produced.
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