JPH08173147A - New yeast and its use - Google Patents
New yeast and its useInfo
- Publication number
- JPH08173147A JPH08173147A JP33664694A JP33664694A JPH08173147A JP H08173147 A JPH08173147 A JP H08173147A JP 33664694 A JP33664694 A JP 33664694A JP 33664694 A JP33664694 A JP 33664694A JP H08173147 A JPH08173147 A JP H08173147A
- Authority
- JP
- Japan
- Prior art keywords
- strain
- yeast
- saccharomyces cerevisiae
- sake
- ethyl caproate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Alcoholic Beverages (AREA)
Abstract
Description
【0001】[0001]
【産業上の利用分野】本発明は、カプロン酸エチル高生
産酵母の発酵性能及びアルコール耐性の改善された酵母
及び該酵母を用いた香気成分の豊かな酒類、食品の製造
方法に関する。更に詳細には、本発明は、カプロン酸感
受性で、香気成分のうちカプロン酸エチルを多く生成す
る酵母の中から、発酵性能及びアルコール耐性の改善さ
れた新菌株を容易に選択し、該酵母を用いて、香気成分
の豊かな酒類、食品を製造する方法に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a yeast having a high ethyl caproate-producing yeast having improved fermentation performance and alcohol tolerance, and a method for producing alcoholic beverages and foods rich in aroma components using the yeast. More specifically, the present invention easily selects a new strain having improved fermentation performance and alcohol resistance from yeasts that are caproic acid-sensitive and produce a large amount of ethyl caproate among aroma components, The present invention relates to a method for producing alcoholic beverages and foods rich in aroma components using the same.
【0002】[0002]
【従来の技術】酒類、特に清酒の製造において、フルー
ティな吟醸香成分の含量の高い清酒を製造するには、低
精白米を使用し、15℃前後で発酵させる普通酒仕込で
は困難である。従来、吟醸香に富んだ清酒を得るために
は高度精白米を使用し、低温発酵を行ってきた。しかし
ながら、この方法では時間とコストがかかる。現状で
は、低精白米の加圧蒸しやリパーゼ処理等が行われるよ
うになったが、低精白米の普通酒仕込から吟醸香に富ん
だ清酒を製造することは、困難であることより、酵母の
育種という観点からの技術開発に依存する方法が進めら
れ、これまでに、5′,5′,5′−トリフルオロ−
D,L−ロイシン耐性を用いる方法(特開昭62−66
69号)、セルレニン耐性を用いる方法(特開昭63−
309175号)、4−アザ−DL−ロイシン耐性を用
いる方法(特開平1−257423号)、及び固形色素
平板培地上で異なる色調を示す株を選択する方法(特開
平2−13368号)が公開されている。吟醸香は単一
の香気成分によって成り立っていないことは、多くの専
門家によって指摘されるところであり、したがって、吟
醸香を構成するより多くの成分を、官能的にバランスを
保った形で高生産する酵母の育種が持たれていた。先
に、本発明者らは、清酒用酵母の変異処理株又は交雑株
について、一定濃度のカプロン酸を含む最少培地での生
育試験を行い、当該培地で生育できないカプロン酸感受
性株を分離し、カプロン酸エチル高生産株を取得した
(特願平5−253630号)が、変異処理によりカプ
ロン酸エチルを多く生成する酵母ほど発酵性能及びアル
コール耐性が低下する傾向が認められ、カプロン酸エチ
ル及び酢酸イソアミルがバランスよく高生成され、かつ
発酵性能及びアルコール耐性の改善された酵母の提供が
望まれていた。また、清酒以外の酒類では、焼酎の製造
においても、香りの良い焼酎を製造するためには、同様
の問題点を有し、更には食品の製造においても、同様で
あった。2. Description of the Related Art In the production of alcoholic beverages, in particular sake, it is difficult to prepare low-polished rice and ferment it at about 15 ° C. to prepare ordinary sake which has a high content of fruity Ginjo aroma components. Traditionally, in order to obtain sake with a rich scent of Ginjo, low-temperature fermentation has been performed using highly polished rice. However, this method is time consuming and costly. At present, low-polished rice has been subjected to pressure steaming, lipase treatment, etc., but it is difficult to produce sake with rich ginjo aroma from low-polished rice ordinary liquor preparation. A method that depends on technological development from the viewpoint of breeding has been developed, and so far, 5 ', 5', 5'-trifluoro-
Method using D, L-leucine resistance (JP-A-62-66)
69), a method using resistance to cerulenin (JP-A-63-
No. 309175), a method using 4-aza-DL-leucine resistance (JP-A-1-257423), and a method for selecting a strain showing a different color tone on a solid dye plate medium (JP-A-2-13368). Has been done. It has been pointed out by many experts that Ginjo incense is not composed of a single aroma component, and therefore, more of the components that make up Ginjo incense are produced in a sensually balanced manner at high levels. It had the breeding of yeast. First, the present inventors, for mutant strains or crosses of sake yeast, a growth test in a minimal medium containing a constant concentration of caproic acid, to isolate a caproic acid-sensitive strain that cannot grow in the medium, A high-capacity ethyl caproate-producing strain was obtained (Japanese Patent Application No. 5-253630), but it was observed that yeasts that produce more ethyl caproate by mutation treatment tend to have lower fermentation performance and alcohol tolerance. It has been desired to provide yeast in which isoamyl is highly produced in a balanced manner and which has improved fermentation performance and alcohol tolerance. Further, with alcoholic beverages other than sake, even in the production of shochu, there was the same problem in producing shochu with a good fragrance, and also in the production of food.
【0003】[0003]
【発明が解決しようとする課題】香気成分の豊かな吟醸
酒の場合、原料コストが高く、また製造日数も長くかか
ることから、安定に大量の吟醸酒を製造することは困難
であった。また、低精白米を用いる普通酒仕込では、吟
醸香に富んだ清酒を製造することは更に困難であった。
本発明の目的は、前記の従来技術の問題点を解決すべ
く、普通酒仕込で吟醸香が高く、かつ生成エタノール濃
度の高い清酒の原酒を製造することにあり、そのために
カプロン酸エチル及び酢酸イソアミルがバランスよく高
生成し、発酵力の強い新規酵母菌を提供すること、及び
該酵母を用いて低精白米から、安定にかつ大量に、吟醸
香の豊かな酒類を製造する方法、更には該酵母を用いて
吟醸香の豊かな食品を製造する方法を提供することにあ
る。In the case of Ginjo liquor with a rich aroma component, it is difficult to stably produce a large amount of ginjo liquor because the raw material cost is high and the number of production days is long. In addition, it was more difficult to produce refined sake rich in ginjo aroma by using ordinary sake prepared with low-polished rice.
In order to solve the above-mentioned problems of the prior art, an object of the present invention is to produce a sake brewed with ordinary liquor, which has a high ginjo aroma and a high ethanol concentration, and therefore ethyl caproate and acetic acid are used. Isoamyl is produced in a well-balanced manner and provides a novel yeast having a strong fermenting power, and a method for producing a liquor rich in ginjo aroma in a stable and large amount from low-polished rice using the yeast, and further It is intended to provide a method for producing a food product rich in Ginjo fragrance using the yeast.
【0004】[0004]
【課題を解決するための手段】本発明を概説すれば、第
1の発明は、カプロン酸感受性で、カプロン酸エチル高
生産のサッカロミセス・セレビシエ(Saccharomyces ce
revisiae)に属する酵母における発酵性能及びアルコー
ル耐性の改善されたサッカロミセス・セレビシエに属す
る新規酵母に関し、第2の発明は、本発明の第1の発明
の当該酵母を用いることを特徴とする香気成分の豊かな
酒類、食品の製造方法に関する。SUMMARY OF THE INVENTION The first aspect of the present invention is summarized as follows. A first aspect of the present invention is Saccharomyces cereciens, which is caproic acid-sensitive and produces high ethyl caproate.
revisiae) relates to a novel yeast belonging to Saccharomyces cerevisiae which has improved fermentation performance and alcohol tolerance in yeast belonging to revisiae, and a second invention is the use of the yeast of the first invention of the present invention, wherein It relates to a method for producing rich alcoholic beverages and foods.
【0005】本発明者らは、先に清酒用酵母の変異処理
株又は交雑株を、一定濃度のカプロン酸を含む最少培地
で生育させ、当該培地で生育できないカプロン酸感受性
株を分離し、清酒小仕込試験を重ねて生成清酒の官能試
験、機器分析により、カプロン酸エチルの高生産株を選
別した。次に、前記カプロン酸エチルの高生産酵母菌株
の発酵性能及びアルコール耐性がカプロン酸エチルの生
成濃度に反比例する現象を見出し、カプロン酸エチル高
生産酵母から、(1)18v/v%エタノール中で長く
生存する株、(2)0.5M酢酸リチウムに対する耐性
度の高くなった株、(3)0.01w/v%ラウリル硫
酸ナトリウムに対する耐性度の高くなった株、(4)日
本醸造協会701号のハプロイド株と交雑させた株を
得、清酒小仕込試験によりそれらの中から選択すること
で、カプロン酸エチル高生産株の発酵性能及びアルコー
ル耐性が改善され、更にはカプロン酸エチルと酢酸イソ
アミルをバランスよく高生成するサッカロミセス・セレ
ビシエを得ることができることを見出し、本発明を完成
させた。The inventors of the present invention firstly grow mutant strains or hybrid strains of sake yeast in a minimal medium containing a constant concentration of caproic acid, isolate a caproic acid sensitive strain that cannot grow in the medium, and High-capacity ethyl caproate producing strains were selected by sensory test of produced sake and instrumental analysis after repeated small-scale preparation tests. Next, we found that the fermentation performance and alcohol tolerance of the ethyl caproate high-producing yeast strain were inversely proportional to the ethyl caproate production concentration, and from the ethyl caproate high-producing yeast, (1) in 18 v / v% ethanol A long-lived strain, (2) a strain highly resistant to 0.5 M lithium acetate, (3) a strain highly resistant to 0.01 w / v% sodium lauryl sulfate, (4) Japan Brewing Society 701 The strains that were hybridized with the haploid strain of No. No. 1 were obtained and selected from among them by a small-scale sake-brewing test, the fermentation performance and alcohol tolerance of the ethyl caproate high-producing strains were improved, and further ethyl caproate and isoamyl acetate were selected. It was found that Saccharomyces cerevisiae capable of producing a high yield of S. cerevisiae was obtained, and the present invention was completed.
【0006】本発明で得られるカプロン酸エチル高生産
株の発酵性能及びアルコール耐性が改善された酵母は、
カプロン酸感受性がそのまま保持されたものと保持され
ないものとがあるが、カプロン酸感受性がそのまま保持
されたものが性能上好ましい。[0006] A yeast having improved fermentation performance and alcohol tolerance of the ethyl caproate high-producing strain obtained by the present invention,
There are some that retain the caproic acid sensitivity as it is and those that do not retain it, but those that retain the caproic acid sensitivity as they are are preferable in terms of performance.
【0007】本発明において発酵性能の改善とは、醪末
期における生成エタノール濃度が高くなることや日本酒
度の切れがよくなる、すなわち発酵力が強くなることを
いい、また、アルコール耐性の改善とは、醪末期におけ
る高濃度のエタノール存在下で死滅しにくく、醪末期で
も速やかに発酵を行えるようになることをいう。In the present invention, the improvement of fermentation performance means that the concentration of ethanol produced at the end of the life is high and the sake level is well cut off, that is, the fermentation ability is strong, and the improvement of alcohol tolerance is It means that it is difficult to die in the presence of high-concentration ethanol at the end of life and that fermentation can be carried out quickly even at the end of life.
【0008】本発明におけるカプロン酸エチル高生産株
とは、その基質となるカプロン酸、又はカプロン酸及び
カプリル酸の高生産株も含む。更には、4−ヒドロキシ
酪酸エチルの生成が多くなった株をも含む。The ethyl caproate high-producing strain of the present invention also includes caproic acid or a caproic acid and caprylic acid high-producing strain as a substrate thereof. Furthermore, it also includes strains that have increased production of ethyl 4-hydroxybutyrate.
【0009】選択される酵母細胞の母集団は、変異処理
株、交雑株、馴養株、細胞融合株及びプラスミド等によ
る形質転換株等の人工変異株のほかに野生株を含む。The population of yeast cells selected includes wild-type strains as well as mutation-treated strains, hybrid strains, acclimatized strains, cell fusion strains and artificial mutant strains such as transformants with plasmids.
【0010】本発明における変異処理としては、酵母に
公知の変異誘導法、例えば、紫外線、放射線等を照射さ
せる方法、又はN−メチル−N′−ニトロ−N−ニトロ
ソグアニジン、エチルメタンスルホネート等の薬剤を接
触させる方法を適宜用いることにより行えばよい。有効
な変異処理としてはエチルメタンスルホネート処理が好
ましい。Examples of the mutagenesis treatment in the present invention include known mutagenesis methods for yeasts, for example, methods of irradiating ultraviolet rays, radiation, etc., or N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate and the like. It may be carried out by appropriately using the method of contacting the drug. Ethyl methanesulfonate treatment is preferable as an effective mutation treatment.
【0011】ここに得られるカプロン酸エチル高生産株
の発酵性能及びアルコール耐性の改善された酵母は、清
酒酵母、焼酎酵母、ワイン酵母、ビール酵母、ウイスキ
ー酵母、アルコール酵母、及びパン酵母のいずれでもよ
く、これらの酵母を用いて清酒、焼酎、ワイン、ビー
ル、ウイスキー等の酒類、又はパン等の食品を製造すれ
ば、香気の豊かな製品を製造することができる。更に、
これらの酵母を用いて芳香性の香気液を製造することも
可能である。清酒、焼酎、ワイン、ビール、ウイスキ
ー、香気液、パン等の製造方法は特に限定するものでは
なく、一般的な方法に従って製造することができる。The yeast having a high ethyl caproate-producing strain with improved fermentation performance and alcohol tolerance is any of sake yeast, shochu yeast, wine yeast, brewer's yeast, whiskey yeast, alcohol yeast, and baker's yeast. Of course, if these yeasts are used to produce alcoholic beverages such as sake, shochu, wine, beer and whiskey, or foods such as bread, it is possible to produce fragrant products. Furthermore,
It is also possible to produce an aromatic aroma liquid using these yeasts. The method for producing sake, shochu, wine, beer, whiskey, aroma liquid, bread and the like is not particularly limited and can be produced according to a general method.
【0012】以下に本発明の酵母の取得方法の1例を示
す。 (酵母の変異処理方法)清酒用泡なし酵母、日本醸造協
会901号(以下、K−901と略称する)株のカプロ
ン酸エチル高生産株を取得するために、以下の工程で変
異処理を行った。YPD液体培地(酵母エキス1w/v
%、ポリペプトン2w/v%、グルコース2w/v%)
5mlで30℃にて一夜振とう培養した。遠心分離で菌
体を集菌した後、0.2Mリン酸バッファー(pH8.
0)で菌体を洗浄した。洗浄菌体を4.6mlの0.2
Mリン酸バッファー(pH8.0)に懸濁し、40w/
v%グルコース溶液を0.25ml添加してよくかくは
んした後、0.15mlの3w/v%エチルメタンスル
ホネート(EMS)水溶液を添加し、30℃にて1時間
おだやかに振とうした。その0.2mlを9.8mlの
6w/v%チオ硫酸ナトリウム溶液に加えて、室温で1
0〜15分維持し、そのうちの0.2mlを19.8m
lの滅菌水に添加し、0.1mlずつ100枚のSD培
地〔イーストニトロゲンベース(アミノ酸不含)0.6
7w/v%、グルコース2w/v%〕の平板寒天培地に
塗布し、30℃で3日間培養した。An example of the method for obtaining yeast of the present invention is shown below. (Yeast mutation treatment method) In order to obtain a strain-free yeast for sake, a high-capacity ethyl caproate strain of Japan Brewing Association No. 901 (hereinafter abbreviated as K-901) strain, mutation treatment is performed in the following steps. It was YPD liquid medium (yeast extract 1 w / v
%, Polypeptone 2 w / v%, glucose 2 w / v%)
The cells were shake-cultured in 5 ml at 30 ° C. overnight. After collecting the cells by centrifugation, 0.2M phosphate buffer (pH 8.
The cells were washed in 0). Washed cells with 4.6 ml of 0.2
Suspended in M phosphate buffer (pH 8.0), 40 w /
After adding 0.25 ml of v% glucose solution and stirring well, 0.15 ml of 3 w / v% ethyl methanesulfonate (EMS) aqueous solution was added, and the mixture was gently shaken at 30 ° C. for 1 hour. 0.2 ml thereof was added to 9.8 ml of 6 w / v% sodium thiosulfate solution, and the mixture was mixed at room temperature for 1 hour.
Maintain for 0 to 15 minutes, 0.2 ml of which is 19.8 m
100 ml of SD medium [yeast nitrogen base (without amino acid) 0.6)
7 w / v%, glucose 2 w / v%] was applied to a plate agar medium and cultured at 30 ° C. for 3 days.
【0013】(カプロン酸エチル高生産株の選択方法)
シャーレ1枚の平板寒天培地に約200個のコロニーを
形成させたもの合計100枚について、0.03v/v
%のカプロン酸を含むSD培地(選択培地)の平板寒天
培地にレプリカし、30℃で5日間培養した。後者の選
択培地に生育できない変異株1株(13−e)をカプロ
ン酸感受性株として選択した。該菌株はSaccharomyces
cerevisiae 13−eと命名、表示され、工業技術院生
命工学工業技術研究所にFERM P−13843とし
て寄託されている。カプロン酸感受性の変異株(13−
e)について、表1に示す仕込配合で清酒の小仕込試験
を行った。(Method for selecting ethyl ethyl caproate high-producing strain)
About 200 colonies were formed on one plate agar medium on a Petri dish. About 100 in total, 0.03 v / v
A replica was carried out on a plate agar medium of SD medium (selection medium) containing% caproic acid, and the cells were cultured at 30 ° C. for 5 days. The mutant 1 strain (13-e) that cannot grow in the latter selective medium was selected as a caproic acid sensitive strain. The strain is Saccharomyces
It is named and displayed as cerevisiae 13-e, and has been deposited as FERM P-13843 at the Institute of Biotechnology, Institute of Biotechnology, AIST. Mutants sensitive to caproic acid (13-
Regarding e), a small preparation test of sake was conducted with the preparation composition shown in Table 1.
【0014】[0014]
【表1】 [Table 1]
【0015】掛米は精米歩合77%(w/w)のα米
〔セブンライス工業(株)製〕を使用した。麹は、精米
歩合72%(w/w)の白米を用いて製造した。酵母は
5ml中に1×109 個含むものを添加した。発酵温度
は15℃一定で行った。留後13日目の醪のろ液につい
て、ガスクロマトグラフィーでカプロン酸エチルの定量
を行った結果、変異株13−e株は親株(K−901)
の約10倍のカプロン酸エチルを生成することを認め
た。また、変異株13−e株の醪のろ液について、ポー
ラパック樹脂を用いた方法(坂本宏司ら:日本農芸化学
会誌、第67巻、第685〜692頁、1993年)に
従って、中高沸点香気成分を分析した結果、K−901
株に比べて、約4倍のカプリル酸エチル及び約2倍の4
−ヒドロキシ酪酸エチルを生成していることを認めた。
以上のカプロン酸エチル高生産、あるいはカプロン酸エ
チル、カプリル酸エチル及び4−ヒドロキシ酪酸エチル
高生産という形質は、当該変異株がK−901株とは異
なる新規酵母菌であることを示すものである。しかしな
がら、変異株13−e株は最終生成エタノール濃度がK
−901株に比べて低く、また上槽前の酵母細胞のメチ
レンブルー染色による死滅率が極めて高くなっていた。As the rice for polishing, α rice (manufactured by Seven Rice Industry Co., Ltd.) having a polishing rate of 77% (w / w) was used. The koji was produced by using 72% (w / w) polished rice. Yeast containing 1 × 10 9 cells in 5 ml was added. The fermentation temperature was constant at 15 ° C. As a result of quantitative determination of ethyl caproate by gas chromatography, the mutant 13-e strain was found to be the parent strain (K-901) on the 13th day after the distillation.
It was confirmed that about 10 times more ethyl caproate was produced. Further, regarding the filtrate of the mash of the mutant strain 13-e, medium- and high-boiling-point aromas are obtained according to the method using Polapack resin (Koji Sakamoto et al., Journal of Japan Society of Agricultural Chemistry, Vol. 67, pp. 685-692, 1993). As a result of analyzing the components, K-901
4 times as much ethyl caprylate and 2 times as much as the strain
-It was confirmed that ethyl hydroxybutyrate was produced.
The above trait of high production of ethyl caproate or high production of ethyl caproate, ethyl caprylate and ethyl 4-hydroxybutyrate indicates that the mutant strain is a novel yeast strain different from the K-901 strain. . However, the mutant 13-e strain had a final ethanol concentration of K
It was lower than that of the -901 strain, and the killing rate of the yeast cells before the upper tank by methylene blue staining was extremely high.
【0016】(カプロン酸エチル高生産酵母の発酵性能
及びアルコール耐性の改善方法) (1)エタノール馴養法 13−e株を用いて製造した清酒醪(最終アルコール濃
度17.4v/v%)にエタノールを添加して18v/
v%に調整した。この醪を15℃で2日間静置し、生存
する細胞をYPD寒天平板培地上でコロニーとして生育
させ、清酒の小仕込試験により選択し、そのうちの1株
を18Al株とした。 (2)酢酸リチウム耐性法 13−e株を前記の方法でEMS変異処理し、この菌懸
濁液を適当に希釈して0.5M酢酸リチウムを含むSD
培地の寒天平板培地に塗布し、コロニーとして生育さ
せ、清酒の小仕込試験により選択し、そのうちの1株を
13L3株とした。 (3)ラウリル硫酸ナトリウム耐性法 0.008w/v%のラウリル硫酸ナトリウム(以下、
SDSと略称する)を含むSD培地の液体培地に13−
e株を接種し、30℃で振とう培養した。十分生育した
後、SDS濃度を少し上げたSD培地に移植し、同様に
培養した。最終的に、0.01w/v%SDSを含む培
地で生育した細胞をYPD培地の寒天平板上でコロニー
として生育させ、清酒の小仕込試験により選択し、その
うちの2株を13S5株、13S12株とした。13S
5株は0.03v/v%のカプロン酸に対して耐性を示
し、親株の13−e株とは異なる菌学的性質を示した。 (4)交雑法 13−e株の一倍体株(接合型:α型)と日本醸造協会
701号(以下、K−701と略述する)の一倍体株
(接合型:a型)のYPD培地での培養液それぞれ0.
1mlを、1mlのYPD培地に加え、30℃で一夜静
置培養した。この菌液をYPD培地の寒天平板培地に接
種し、生育の早い二倍体コロニーを清酒の小仕込試験に
より選択し、そのうちの1株を10−143−2株とし
た。(Method for Improving Fermentation Performance and Alcohol Tolerance of Yeast Highly Producing Ethyl Caproate) (1) Ethanol Acclimation Method Ethanol was added to sake mash (final alcohol concentration: 17.4 v / v%) produced using 13-e strain. 18v /
Adjusted to v%. This mash was allowed to stand for 2 days at 15 ° C., and the surviving cells were grown as colonies on a YPD agar plate medium and selected by a small-brewing test of sake, one of which was the 18Al strain. (2) Lithium acetate resistance method 13-e strain was subjected to EMS mutation treatment by the above-mentioned method, and this bacterial suspension was appropriately diluted to obtain SD containing 0.5M lithium acetate.
It was applied to an agar plate medium as a medium, grown as a colony, and selected by a small-brewing test of sake, one of which was designated as 13L3 strain. (3) Sodium lauryl sulfate resistance method 0.008 w / v% sodium lauryl sulfate (hereinafter,
The liquid medium of SD medium containing 13-
The e strain was inoculated and shake cultured at 30 ° C. After sufficient growth, the cells were transplanted to an SD medium with a slightly increased SDS concentration and cultured in the same manner. Finally, cells grown in a medium containing 0.01 w / v% SDS were grown as colonies on an agar plate of YPD medium and selected by a small-brewing test of sake, and two of them were selected as 13S5 strain and 13S12 strain. And 13S
Five strains were resistant to 0.03 v / v% caproic acid, and showed different mycological properties from the parent strain 13-e. (4) Hybridization method Haploid strain of 13-e strain (zygous type: α type) and haploid strain of Japan Brewing Society No. 701 (hereinafter abbreviated as K-701) (zygous type: a type) Culture solution in YPD medium of 0.
1 ml was added to 1 ml of YPD medium, and static culture was carried out at 30 ° C. overnight. This bacterial solution was inoculated on an agar plate medium of YPD medium, and diploid colonies with fast growth were selected by a small-brewing test of sake, and one of them was designated as 10-143-2 strain.
【0017】上記のように、本発明による菌株{変異・
馴養株:18Al、13L3、13S5、13S12、
交雑株:10−143−2}は、K−901株の変異・
馴養株又はK−701株との交雑株であるが、それらの
菌学的性質を以下に示す。 (菌学的性質) 1.形態学的性質 YPD培地で30℃、2日間培養した後、顕微鏡で観察
した。 a)形:卵円形 b)大きさ:長さ4.7〜7.9μm、幅3.8〜5.
5μm 2.胞子形成:有り 胞子形成用培地(酢酸カリウム2w/v%、グリコース
0.05w/v%、寒天2w/v%)で30℃、5日間
培養し、顕微鏡で観察した。 3.増殖の形態:出芽 4.生化学的観察 a)糖の発酵性 ウイッカーハムの炭素化合物同化試験用培地(ディフコ
社製)をダーラム管入り試験管に分注して、当該4菌株
を接種し、30℃で7日間培養して、その炭酸ガス発生
の有無を観察した。 グルコース (+) ガラクトース(+) スクロース (+) マルトース (+) ラクトース (−) メリビオース(−) ラフィノース(+) b)糖の資化性 ウイッカーハムの炭素化合物同化試験用培地(ディフコ
社製)を用いて、オキザノグラフ法により、30℃、1
4日後の生育を観察した。 グルコース (+) ガラクトース(+) スクロース (+) マルトース (+) ラクトース (−) c)硝酸塩の同化性:(−) 硝酸塩は硝酸カリウムとし、ウイッカーハムの炭素化合
物同化試験用培地(ディフコ社製)を用いて、オキザノ
グラフ法により生育を観察した。 d)TTC染色性:赤 e)β−アラニン培地、35℃、3日間培養での生育:As described above, the strain according to the present invention {mutation /
Acclimatized strains: 18Al, 13L3, 13S5, 13S12,
Hybrid strain: 10-143-2} is a mutation of the K-901 strain.
The acclimatized strain or a hybrid strain with the K-701 strain, whose mycological properties are shown below. (Mycological properties) 1. Morphological Properties After culturing in YPD medium at 30 ° C. for 2 days, it was observed under a microscope. a) Shape: oval b) Size: length 4.7-7.9 μm, width 3.8-5.
5 μm 2. Sporulation: Yes Spore formation medium (potassium acetate 2 w / v%, glucose 0.05 w / v%, agar 2 w / v%) was cultured at 30 ° C for 5 days and observed under a microscope. 3. Form of growth: budding 4. Biochemical Observation a) Fermentability of Sugar Wicker Ham's carbon compound assimilation test medium (manufactured by Difco) was dispensed into a test tube containing a Durham tube, inoculated with the 4 strains, and cultured at 30 ° C. for 7 days. Then, the presence or absence of the generation of carbon dioxide was observed. Glucose (+) Galactose (+) Sucrose (+) Maltose (+) Lactose (-) Mellibiose (-) Raffinose (+) b) Sugar assimilation medium Wickerham carbon compound assimilation test medium (manufactured by Difco) By using the oxanograph method,
The growth after 4 days was observed. Glucose (+) Galactose (+) Sucrose (+) Maltose (+) Lactose (-) c) Nitrate assimilation: (-) Nitrate was potassium nitrate, and Wickerham's carbon compound assimilation test medium (manufactured by Difco) was used. Then, the growth was observed by an oxanograph method. d) TTC stainability: red e) Growth in β-alanine medium at 35 ° C. for 3 days in culture:
【0018】[0018]
【表2】 [Table 2]
【0019】5.高泡の形成 清酒の小仕込を行ったところ、高泡の形成は観察されな
かった。以上、形態学的、生化学的結果は、本発明酵母
5菌株がサッカロミセス・セレビシエに属する酵母菌で
あることを示すものである。また、清酒の小仕込試験に
おいて高泡の形成も認められないことから、当該4菌株
はK−901株の変異株あるいはK−701株の誘導体
であることを示すものである。 6.薬剤に対する耐性 それぞれの薬剤を含むSD培地を用いて、30℃で3日
間培養した。5. Formation of high bubbles When a small amount of sake was charged, formation of high bubbles was not observed. As described above, the morphological and biochemical results show that the five yeast strains of the present invention are yeast strains belonging to Saccharomyces cerevisiae. Moreover, since formation of high bubbles was not observed in the small-brewing test of sake, the 4 strains were shown to be mutant strains of K-901 strain or derivatives of K-701 strain. 6. Resistance to Drugs Using an SD medium containing each drug, the cells were cultured at 30 ° C. for 3 days.
【0020】[0020]
【表3】 [Table 3]
【0021】a)5′,5′,5′−トリフルオロ−
D,L−ロイシン耐性 b)セルレニン耐性 c)4−アザ−DL−ロイシン耐性 d)クロトリマゾール耐性 7.カプロン酸に対する感受性 カプロン酸を含むSD培地を用いて、30℃で3日間培
養した。A) 5 ', 5', 5'-trifluoro-
D, L-leucine resistance b) cerulenin resistance c) 4-aza-DL-leucine resistance d) clotrimazole resistance 7. Sensitivity to Caproic Acid Using an SD medium containing caproic acid, the cells were cultured at 30 ° C. for 3 days.
【0022】[0022]
【表4】 [Table 4]
【0023】かくして、本発明により、13−e株を変
異あるいは馴養させ、又はK−701株と交雑させるこ
とによって、カプロン酸エチル及び酢酸イソアミルをバ
ランスよく高生成する、13−e株よりも発酵性能及び
アルコール耐性の改善された、普通酒仕込に使用可能な
香気高生産酵母が提供された。代表的な菌株である18
Al株、13L3株、13S12株、及び10−143
−2株は、それぞれSaccharomyces cerevisiae 18A
l、Saccharomyces cerevisiae 13L3、Saccharomy
ces cerevisiae 13S12、及びSaccharomyces cere
visiae 10−143−2と表示し、工業技術院生命工
学工業技術研究所に、各々FERM P−14606、
FERM P−14612、FERM P−1462
6、FERM P−14605として寄託してある。Thus, according to the present invention, the 13-e strain is fermented more than the 13-e strain, which produces a well-balanced high production of ethyl caproate and isoamyl acetate by mutating or acclimatizing the 13-e strain or crossing with the K-701 strain. An aroma-rich yeast that can be used for the preparation of ordinary sake with improved performance and alcohol tolerance is provided. 18 representative strains
Al strain, 13L3 strain, 13S12 strain, and 10-143.
-2 strains are Saccharomyces cerevisiae 18A, respectively
1, Saccharomyces cerevisiae 13L3, Saccharomy
ces cerevisiae 13S12, and Saccharomyces cere
Visiae 10-143-2 is displayed and FERM P-14606 and
FERM P-14612, FERM P-1462
6, deposited as FERM P-14605.
【0024】本発明の清酒、焼酎及びその他の酒類の製
造方法は、これらの酵母菌株を用いることを特徴とし、
醸造方法は特に限定するものではない。また、食品の製
造方法においても同様に製造方法は特に限定されるもの
ではない。The method for producing sake, shochu and other alcoholic beverages of the present invention is characterized by using these yeast strains,
The brewing method is not particularly limited. Moreover, the manufacturing method is not particularly limited in the manufacturing method of the food as well.
【0025】[0025]
【実施例】次に、本発明の菌株を用いた酒類製造の具体
例を挙げて、本発明を更に具体的に説明するが、本発明
はこれらの実施例に限定されない。EXAMPLES Next, the present invention will be described in more detail with reference to specific examples of alcohol production using the strain of the present invention, but the present invention is not limited to these examples.
【0026】実施例1 変異・馴養株4株(18Al、13L3、13S5、1
3S12株)及び交雑株1株(10−143−2株)に
ついて、表1に示す仕込配合で清酒の製造を行った。掛
米は精米歩合77%(w/w)のα米〔セブンライス工
業(株)製〕を使用した。麹は、精米歩合72%(w/
w)の白米を用いて製造した。酵母は5ml中に1×1
09 個含むものを添加した。発酵温度は15℃一定で行
った。対照株として親株の13−e株及びK−901株
を用いた。上槽液の分析結果を表5に示す。Example 1 Four mutant / acclimated strains (18Al, 13L3, 13S5, 1
3S12 strain) and one hybrid strain (10-143-2 strain) were produced with the blending ratio shown in Table 1. The rice used was α rice (manufactured by Seven Rice Industry Co., Ltd.) with a polishing rate of 77% (w / w). Koji has a 72% rice polishing rate (w /
It was produced using the white rice of w). 1 x 1 in 5 ml of yeast
One containing 0 9 was added. The fermentation temperature was constant at 15 ° C. The parent strain 13-e and K-901 were used as control strains. Table 5 shows the analysis results of the upper tank liquid.
【0027】[0027]
【表5】 [Table 5]
【0028】官能検査は3点法(1:良、2:普通、
3:悪)で行い、パネラー10名の平均値で表した。こ
の結果、18Al株、13L3株、13S5株、13S
12株及び10−143−2株の5株は親株の13−e
株に比べて発酵性能及びアルコール耐性が向上し、かつ
カプロン酸エチル生産能もK−901株の3倍から9倍
を示した。また、18Al株及び10−143−2株は
K−901株の3倍から5倍の酢酸イソアミルを生成し
た。この酢酸イソアミル高生産能は13−e株にはなか
った性能である。得られた5株は官能的にも華やかな香
りが認められ、特にカプロン酸エチルの生成量が13−
e株より増加した13L3株で製造した清酒は、リンゴ
様のフルーティな香りが強く、また酢酸イソアミル生成
量も増加した18Al株及び10−143−2株で製造
した清酒は、軽快で香りのバランスの良い優良な酒質と
なった。The sensory test is a three-point method (1: good, 2: normal,
3: Bad) and expressed as the average value of 10 panelists. As a result, 18Al strain, 13L3 strain, 13S5 strain, 13S strain
5 of 12 and 10-143-2 are 13-e of parent strain
Fermentation performance and alcohol tolerance were improved as compared with the strain, and ethyl caproate-producing ability was 3 to 9 times that of the K-901 strain. Further, the 18Al strain and the 10-143-2 strain produced 3 to 5 times as much isoamyl acetate as the K-901 strain. This high productivity of isoamyl acetate is a performance that the 13-e strain did not have. The 5 strains obtained had a sensually brilliant scent, and especially the amount of ethyl caproate produced was 13-.
Sake produced with the 13L3 strain, which was increased compared to the e strain, had a strong apple-like fruity scent, and the sake produced with the 18Al strain and the 10-143-2 strain, which also increased the amount of isoamyl acetate produced, had a light and aroma balance. The quality of sake was good.
【0029】実施例2 変異株1株(13L3株)及び交雑株1株(10−14
3−2株)について、表6に示す仕込配合で焼酎の製造
を行った。対照株として米焼酎用によく用いられるK−
701株を使用した。Example 2 One mutant strain (13L3 strain) and one hybrid strain (10-14)
3-2 strain), shochu was produced with the mixing composition shown in Table 6. K- which is often used as a control strain for rice shochu
The 701 strain was used.
【0030】[0030]
【表6】 [Table 6]
【0031】掛米は精米歩合70%(w/w)の低品位
米を使用した。麹は、精米歩合72%(w/w)の白米
を用いて製造した。酵母は5ml中に2×108 個含む
ものを添加した。酵素剤はスピターゼM〔ナガセ生化学
工業(株)製〕を使用した。発酵温度は20℃一定で行
った。留後14日目の醪を減圧度−700mmHgで減
圧蒸留し、留出アルコール20v/v%までの垂口をア
ルコール25.0v/v%に割水したものを分析した。
その結果を表7に示す。Low-grade rice with a polishing rate of 70% (w / w) was used as the rice for polishing. The koji was produced by using 72% (w / w) polished rice. Yeast containing 2 × 10 8 cells in 5 ml was added. As the enzyme agent, spitase M (manufactured by Nagase Seikagaku Corporation) was used. The fermentation temperature was constant at 20 ° C. On the 14th day after the distillation, the mash was vacuum-distilled at a vacuum degree of -700 mmHg, and the distillate alcohol up to 20 v / v% was divided into 25.0 v / v% alcohol for analysis.
The results are shown in Table 7.
【0032】[0032]
【表7】 [Table 7]
【0033】官能検査は3点法(1:良、2:普通、
3:悪)で行い、パネラー10名の平均値で表した。こ
の結果、13L3株を用いて製造した米焼酎は、吟醸香
であるカプロン酸エチルの含量が対照のK−701株に
比べて極めて多く、官能的にもリンゴ様のフルーティな
香りが強く認められた新しいタイプの焼酎となった。ま
た、10−143−2株を用いて製造した米焼酎は、酢
酸イソアミルの生成量がK−701株の2.5倍、カプ
ロン酸エチルの生成量が約4倍に増加しており、官能的
にも華やかな軽快な香りが認められた。The sensory test is a three-point method (1: good, 2: normal,
3: Bad) and expressed as the average value of 10 panelists. As a result, rice shochu produced using the 13L3 strain had a much higher content of ethyl caproate, a ginjo scent, than the control K-701 strain, and a strong apple-like fruity aroma was strongly recognized. It became a new type of shochu. In addition, rice shochu produced using the 10-143-2 strain increased the production amount of isoamyl acetate by 2.5 times that of the K-701 strain and the production amount of ethyl caproate by about 4 times. A gorgeous and light fragrance was recognized.
【0034】以上の結果は、18Al株、13L3株、
13S5株、13S12株、及び10−143−2株が
K−901株にない性質をもつ、すなわち香気エステル
を多く生成し、更には13−e株に比べて発酵性能及び
アルコール耐性の改善された新規酵母菌であることを示
すものである。The above results show that the 18Al strain, 13L3 strain,
The 13S5 strain, the 13S12 strain, and the 10-143-2 strain have a property that the K-901 strain does not have, that is, a large amount of aroma ester is produced, and further fermentation performance and alcohol tolerance are improved as compared with the 13-e strain. This shows that the yeast is a novel yeast.
【0035】[0035]
【発明の効果】本発明による酵母の選択法を用いて得ら
れるカプロン酸エチル高生産酵母の発酵性能及びアルコ
ール耐性の改善された新規酵母菌を使用することによ
り、高精白米、低温長期仕込の吟醸仕込を行わなくと
も、普通酒仕込あるいは低価格米による焼酎仕込におい
てカプロン酸エチル及び酢酸イソアミルを主とする香気
エステルの豊かでバランスの良い、芳香性に富んだ清
酒、焼酎及びその他の酒類の製造が、低コストで、しか
も短期間で安定して行うことが可能となる。また、食品
の製造においても同様に、本発明による新規酵母菌を用
いることにより、香気成分の豊かな食品を製造すること
が可能となる。EFFECT OF THE INVENTION By using a novel yeast having improved fermentation performance and alcohol tolerance of the yeast with high production capacity of ethyl caproate obtained by using the yeast selection method of the present invention, highly polished rice and low temperature long-term Even if ginjo is not charged, when sake is charged with normal sake or shochu with low-price rice, it is rich and well-balanced with aroma ester mainly composed of ethyl caproate and isoamyl acetate, aromatic sake, shochu and other alcoholic beverages. Manufacturing can be performed at low cost and stably in a short period of time. Further, also in the production of foods, by using the novel yeast according to the present invention, it becomes possible to produce foods rich in aroma components.
───────────────────────────────────────────────────── フロントページの続き (72)発明者 平岡 信次 滋賀県大津市瀬田3丁目4番1号 寳酒造 株式会社中央研究所内 (72)発明者 茂野 忠樹 滋賀県大津市瀬田3丁目4番1号 寳酒造 株式会社中央研究所内 (72)発明者 矢野 忠▲徳▼ 大阪府堺市竹城台3丁19−3 ─────────────────────────────────────────────────── --- Continuation of the front page (72) Inventor Shinji Hiraoka 3-4-1 Seta, Otsu City, Shiga Prefecture, Central Research Institute, Mina Shuzo Co., Ltd. (72) Inventor Tadashi Moshino 3--4 Seta, Otsu City, Shiga Prefecture No. 1 Takara Shuzo Co., Ltd. Central Research Institute (72) Inventor Tadashi Yano ▲ Toku ▼ 3-19-3 Takeshirodai, Sakai City, Osaka Prefecture
Claims (7)
高生産のサッカロミセス・セレビシエに属する酵母にお
ける発酵性能及びアルコール耐性の改善されたサッカロ
ミセス・セレビシエに属する新規酵母。1. A novel yeast belonging to Saccharomyces cerevisiae, which is improved in fermentation performance and alcohol tolerance in a yeast belonging to Saccharomyces cerevisiae that is caproic acid-sensitive and highly produces ethyl caproate.
酸感受性であるサッカロミセス・セレビシエに属する新
規酵母。2. The novel yeast according to claim 1, which belongs to Saccharomyces cerevisiae, which is sensitive to caproic acid.
v%エタノールに対する耐性を示すサッカロミセス・セ
レビシエに属する新規酵母。3. The novel yeast according to claim 1, which is 18 v /
A novel yeast belonging to Saccharomyces cerevisiae that exhibits resistance to v% ethanol.
酢酸リチウムに対する耐性を示すサッカロミセス・セレ
ビシエに属する新規酵母。4. The novel yeast according to claim 1 is 0.5M
A novel yeast belonging to Saccharomyces cerevisiae that exhibits resistance to lithium acetate.
w/v%のラウリル硫酸ナトリウムに対する耐性を示す
サッカロミセス・セレビシエに属する新規酵母。5. The novel yeast according to claim 1, which is 0.01
A novel yeast belonging to Saccharomyces cerevisiae that exhibits resistance to w / v% sodium lauryl sulfate.
myces cerevisiae13−e(FERM P−1384
3)と日本醸造協会701号との交雑株であるサッカロ
ミセス・セレビシエに属する新規酵母。6. The novel yeast according to claim 1, which is Saccharo.
myces cerevisiae 13-e (FERM P-1384
3) A new yeast belonging to Saccharomyces cerevisiae, which is a hybrid strain of 3) and Japan Brewing Society No. 701.
ビシエを用いることを特徴とする酒類、食品の製造方
法。7. A method for producing alcoholic beverages and foods, which comprises using the Saccharomyces cerevisiae according to claim 1.
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JP33664694A JP3506280B2 (en) | 1994-12-26 | 1994-12-26 | New yeast and its use |
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---|---|---|---|
JP33664694A JP3506280B2 (en) | 1994-12-26 | 1994-12-26 | New yeast and its use |
Publications (2)
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JP3506280B2 JP3506280B2 (en) | 2004-03-15 |
Family
ID=18301324
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JP33664694A Expired - Lifetime JP3506280B2 (en) | 1994-12-26 | 1994-12-26 | New yeast and its use |
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Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998055592A1 (en) * | 1997-06-04 | 1998-12-10 | Asahi Kasei Kogyo Kabushiki Kaisha | Low-alcohol sakes |
KR100768566B1 (en) * | 2006-05-26 | 2007-10-19 | 박환희 | 3 Novel alcohol producing yeast Saccharomyces cerevisiae HA3 |
CN102827783A (en) * | 2012-09-25 | 2012-12-19 | 江南大学 | Method for breeding fast fermentation yellow wine Saccharomyces cerevisiae |
JP6119881B1 (en) * | 2016-01-07 | 2017-04-26 | 菊正宗酒造株式会社 | Yeast having high ability to produce ethyl caproate and method for producing fermented product using the yeast |
JP2017086047A (en) * | 2015-11-17 | 2017-05-25 | 秋田県 | Caproic acid low production yeast |
JP2017121251A (en) * | 2017-03-10 | 2017-07-13 | 菊正宗酒造株式会社 | Yeast with high production capacity of ethyl caproate, and method of producing fermentation product using that yeast |
JP2017169471A (en) * | 2016-03-22 | 2017-09-28 | 独立行政法人酒類総合研究所 | Method for producing yeast strain with improved brewing characteristics |
JP7128561B1 (en) * | 2022-02-17 | 2022-08-31 | 合同会社ねっか | Refined sake manufacturing method and refined sake |
-
1994
- 1994-12-26 JP JP33664694A patent/JP3506280B2/en not_active Expired - Lifetime
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998055592A1 (en) * | 1997-06-04 | 1998-12-10 | Asahi Kasei Kogyo Kabushiki Kaisha | Low-alcohol sakes |
KR100768566B1 (en) * | 2006-05-26 | 2007-10-19 | 박환희 | 3 Novel alcohol producing yeast Saccharomyces cerevisiae HA3 |
CN102827783A (en) * | 2012-09-25 | 2012-12-19 | 江南大学 | Method for breeding fast fermentation yellow wine Saccharomyces cerevisiae |
JP2017086047A (en) * | 2015-11-17 | 2017-05-25 | 秋田県 | Caproic acid low production yeast |
JP6119881B1 (en) * | 2016-01-07 | 2017-04-26 | 菊正宗酒造株式会社 | Yeast having high ability to produce ethyl caproate and method for producing fermented product using the yeast |
JP2017121204A (en) * | 2016-01-07 | 2017-07-13 | 菊正宗酒造株式会社 | Yeast with high production capacity of ethyl caproate, and method of producing fermentation product using that yeast |
JP2017169471A (en) * | 2016-03-22 | 2017-09-28 | 独立行政法人酒類総合研究所 | Method for producing yeast strain with improved brewing characteristics |
JP2017121251A (en) * | 2017-03-10 | 2017-07-13 | 菊正宗酒造株式会社 | Yeast with high production capacity of ethyl caproate, and method of producing fermentation product using that yeast |
JP7128561B1 (en) * | 2022-02-17 | 2022-08-31 | 合同会社ねっか | Refined sake manufacturing method and refined sake |
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