JP2012147680A - High-ester production yeast for distilled liquor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide yeast for producing distilled liquor which can be simply and easily used for producing distilled liquor, having resistance to high temperature and resistance to citric acid, for producing distilled liquor having high ethyl caproate forming capability, and having gorgeous and well-proportioned flavors.SOLUTION: High ester forming mutant refined sake yeast which forms much ethyl caproate selected and obtained in a cerulenin-containing medium, is crossed with yeast for distilled spirit to obtain mutant yeast for distilled liquor which forms much ethyl caproate and which belongs to Saccharomyces cerevisiae, and has resistance to high temperature and resistance to citric acid.

Description

  The present invention relates to a yeast for high-ester producing distilled liquor, a breeding method thereof, a method for producing distilled liquor using the yeast, and the like.

  There are many foods produced by brewing and fermentation using yeast, including sakes such as sake, beer, shochu, and wine. The quality of these foods is often influenced by the properties (characteristics) of the yeast used, and for the purpose of producing products of better quality, attempts to breed excellent yeasts having characteristics superior to those of existing yeasts. It is actively done.

  In recent years, the demand for diversification of alcoholic beverages has increased, and not only for sake, but also for authentic shochu, which is a traditional Japanese distilled spirit, consumer demand for flavorful products has become very high. .

  In order to respond to such needs, breeding of new excellent yeast with high fragrance is performed, for example, as one of the methods for obtaining mutant yeast that can produce fragrant liquor, as an antibiotic that inhibits fatty acid synthase A method for obtaining ethyl caproate high-producing yeast obtained from a known cerulenin resistant strain (Patent Document 1) is disclosed.

  However, the sake-producing yeast with high production of ethyl caproate according to this patent document produces a large amount of caproic acid which causes unpleasant odor at the same time as producing a high amount of ethyl caproate. The patent document also discloses that a cerulenin-resistant strain can be obtained from a shochu yeast mutant, but this method obtains a shochu yeast mutant that produces ethyl caproate at a high level from the characteristics of the shochu yeast itself. Is difficult.

  It should be noted that high-capacity ethyl caproate sake yeast cannot be used as it is in distilled liquor production, but in the production of shochu and awamori, fermentation at high temperature and high citric acid concentration is often required, Because sake yeast with high production of ethyl caproate does not have high temperature resistance or citric acid resistance, the production of distilled liquor using the sake yeast requires strict condition control or when ethyl caproate is not produced sufficiently high This is not a simple and practical method.

  Among such background technologies, the development of mutant yeast that has a trait of citric acid resistance and high temperature resistance and can produce ethyl caproate with high yield, which can be used simply and practically for the production of distilled spirits, It has been strongly desired by the distilled liquor manufacturing industry, etc.

Japanese Patent Laid-Open No. 8-23954

  The present invention produces a distilled liquor that has a high temperature resistance, citric acid resistance, and high caproic acid ethyl acetate production ability, and has a fragrant and well-flavored flavor that can be easily and easily used in the production of distilled spirits. It aims at providing the yeast for distilled liquor manufacture.

  In order to achieve the above-mentioned object, the present inventors conducted intensive research, crossed a high ester-producing sake yeast that produces a large amount of ethyl caproate and a yeast for shochu, produced a large amount of ethyl caproate, and was resistant to high temperatures. And by obtaining a mutant yeast belonging to Saccharomyces cerevisiae having citric acid resistance, it is possible to easily produce a distilled liquor having a high ethyl caproate content and a fragrant and well-flavored flavor. The headline, the present invention has been reached.

That is, the embodiment of the present invention is as follows.
(1) Mutating the sake yeast, selecting a high ester-producing sake yeast that produces a large amount of ethyl caproate from a strain that grows on a cerulenin-containing medium, crossing the high ester-producing sake yeast and the shochu yeast The obtained mutant yeast for distilled liquor belonging to Saccharomyces cerevisiae, which produces a large amount of ethyl caproate and has high-temperature resistance and citric acid resistance.
(2) Yeast as described in (1), which is obtained by crossing Kyoto 1601 yeast (K-1601) and Kyoto S-2 yeast.
(3) Saccharomyces cerevisiae NS-2-16 strain (NITE P-1021), which is the yeast according to (2).
(4) Ethyl caproate content characterized by performing brewing using the yeast according to any one of (1) to (3) and performing distillation (vacuum distillation, atmospheric distillation, etc.) Of distilled liquor with high fragrance and fragrance.
(5) A method for producing an alcoholic beverage (such as sake liqueur), wherein the distilled liquor produced by the method according to (4) is added to sake.
(6) Mutating the sake yeast, selecting a high ester-producing sake yeast that produces a large amount of ethyl caproate from a strain that grows on a cerulenin-containing medium, and crossing the high ester-producing sake yeast and the shochu yeast A method for breeding a mutant yeast for distilled liquor belonging to Saccharomyces cerevisiae, which produces a large amount of ethyl caproate and is resistant to high temperature and citric acid.
(7) The method according to (6), wherein the high-ester producing sake yeast is Kyoto 1601 yeast (K-1601), and the shochu yeast is Kyoto S-2 yeast.

  According to the present invention, a high ester-producing yeast that can be easily used in distilled liquor production, which is a production condition of high temperature and high citric acid concentration, can be obtained, so distilled liquor such as authentic shochu with a high ethyl caproate content Can be easily manufactured. Furthermore, since caproic acid, which is an unpleasant odor, is not concentrated in the distillation process, the content of ethyl caproate is high (for example, 8.5 mg / L or more in distilled liquor) and the fragrance is gorgeous and well-flavored (low unpleasant odor) ) Aromatic distilled liquor can be produced.

  In the present invention, in obtaining the target yeast for distilled sake, first, a high ester producing sake yeast that produces a large amount of ethyl caproate from a strain that grows on a cerulenin-containing medium by subjecting the yeast for sake use to mutation is selected. In addition, as a representative example of the high ester-producing sake yeast obtained by the method, there is Kyokai No. 1601 yeast (K-1601).

  Next, the high ester-producing sake yeast and shochu yeast are crossed. As the shochu yeast, any shochu yeast can be used as long as it has the high-temperature resistance and citric acid resistance necessary for the production of distilled liquor. Is mentioned. By this crossing, the yeast for high-ester-producing distilled liquor can be obtained more simply and efficiently than when the yeast for distilled liquor is mutated and selected with a selective medium.

  In addition, a known method can be used for the hybridization method, and examples thereof include a haploid (mating type) mating method, protoplast fusion, transformation, and mutation. In addition, the selection of a hybrid strain can be further facilitated by using a marker gene.

  The citric acid resistance of the resulting yeast for high-ester producing distilled liquor is only required to be resistant in a solution having a citric acid concentration of 1.0 g / L or more, for example, 1.0 to 3.0 g / L (fermented liquid ) Degree of citric acid resistance is exemplified. Moreover, high temperature tolerance should just be able to brew (fermentation) at 30 degreeC or more, for example, 30-35 degreeC. All of these are important components for easily producing distilled liquor.

  In the present invention, the objective mutant yeast for excellent distilled liquor can be easily obtained by the method as described above. As an example, we succeeded in obtaining an excellent mutant yeast by crossing Kyoto 1601 yeast and Kyoto S-2 yeast and named it NS-2-16 strain. December 17, 2010 (Heisei 22), this stock was incorporated in the National Institute of Technology and Evaluation Microorganisms Depositary Center (2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture, Japan 292-0818). At the same time, it was deposited as NITE P-1021.

Examples of the main bacteriological properties of Saccharomyces cerevisiae NS-2-16 strain obtained according to the present invention are as follows.
(A) Bacteria morphology when grown in YM liquid medium (1) Vegetative cell size: size 4-8 μm, width 4-6 μm
(2) Vegetative cell shape: oval and elliptical (3) Growth type: budding (b) Presence / absence of sporulation (c) Physiological and chemical taxonomic properties (1) Optimal growth conditions (pH ): 5.0
(2) Growth range (pH): 2.7 to 7.2
(3) Utilization of nitrate:-
(4) Fat breakdown:-
(5) Decomposition of urea:
(6) Liquefaction of gelatin:-
(7) Carotenoid production: −
(8) Formation of remarkable organic acid: −
(9) Production of starch-like substance:-
(10) Vitamin requirement: Calcium pantothenate is required adaptively (±), pyridoxine, inositol, biotin and thiamine are not required (11) Carbon source utilization: glucose, galactose, sucrose, maltose, raffinose , Fructose, glycerol and α-methyl glucoside are assimilated and lactose, starch, D-xylose, D-arabinose, D-sorbitol, melibiose, cellobiose and D-mannitol are not assimilated (12) Carbon source fermentability: glucose, Galactose, sucrose, maltose, raffinose and fructose are fermented and lactose, starch, D-xylose, D-arabinose, D-sorbitol, melibiose, cellobiose, D-mannitol, melezitose, trehalose Scan and α- methyl glucoside does not ferment

  And the content of ethyl caproate in the distilled liquor produced by the yeast for high-ester producing distilled liquor as described above is 8.5 mg / L or more (for example, 8.5 to 17.0 mg / L, preferably 10 0.01 to 17.0 mg / L, more preferably 15.0 to 17.0 mg / L). In the present invention, caproic acid (normal caproic acid) and aroma that emit an unpleasant odor by a distillation process such as 0.01 to 0.02 MPa, vacuum distillation at 40 to 50 ° C., and atmospheric distillation at 90 to 100 ° C. Since the components ethyl caproate and ethanol can be separated to some extent, caproic acid is not concentrated in the distillate and ethyl caproate is concentrated (the caproic acid content of the distillate is less than the ethyl caproate content) and aroma It is possible to obtain an aromatic distilled liquor that is gorgeous and well-flavored. Note that the present invention does not necessarily require an operation (continuous distillation) that repeats distillation that has been conventionally performed to remove unpleasant odors twice or more, and obtains an aromatic distilled liquor even by one distillation (single distillation). It is characteristic that

  Furthermore, by adding distilled liquor such as the above-mentioned aromatic shochu to refined sake, it is also possible to produce novel alcoholic beverages (for example, sake liqueur etc.) having a novel and characteristic aroma. The same applies when mixing with alcohol other than sake.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

(1) Isolation of haploid strain of Kyokai No. 1601 yeast (K-1601) and determination of mating type First, Kyokai No. 1601 yeast (high ester-producing sake yeast) was designated as Ballg. The cells were immersed in a 15 ° wort medium at 30 ° C. for 2 days. The yeast cells were washed with sterilized water, and then applied to an agar medium (spore-forming medium) containing 0.4% potassium acetate, and cultured at 30 ° C. for 2 days to form spores. The formed spores were washed with sterilized water, and a 2% glucose-1 / 5M phosphate buffer (pH 8.0) containing 0.05% cell wall lytic enzyme (Zymolase 100T Seikagaku Corporation) was used. The cell wall was lysed by acting at 30 ° C. for 30 to 60 minutes to release spores, and then heated at 58 ° C. for 20 minutes to sterilize the diploid strain. Then Ballg. The haploid strain was grown by coating on a 10 ° broth agar plate medium and culturing at 30 ° C. for 7 to 10 days.

  A colony of the grown haploid strain was randomly picked and Ballg. Saccharomyces cerevisiae standard strains Saccharomyces cerevisiae IFO 10175 (MATa) and Saccharomyces cerevisiae IFO 10176 (MATα) and Ballg. After cross streaking on a 10 ° wort agar medium and culturing at 30 ° C. for 3 days, a part of the crossing portion was picked and Ballg. After culturing at 10 ° wort medium at 30 ° C. for 2 days, spore formation is carried out in a spore formation medium as described above, and the haploid type of K-1601 yeast is determined by the presence or absence of zygote formation after crossing. did. The resulting haploid strain was designated 1601-Hp1 (joined type (MATa)).

(2) Crossing of haploid strain 1601-Hp1 (mating type (MATa)) of Kyoto 1601 yeast and shochu yeast S-2 yeast (S-2 strain) -2 strain) was immersed in a YPD medium at 30 ° C. for 24 hours, washed with sterilized water, and then 1/15 M phosphorus containing 0.05% cell wall lytic enzyme (Zymolase 100T Seikagaku). Using an acid buffer solution (containing 0.6 M KCl, pH 7.6), the cell wall was lysed and spores were released by acting at 30 ° C. for 30 to 60 minutes. Thereafter, the diploid strain was sterilized by heating at 55 ° C. for 10 minutes, and then the spore and the haploid strain 1601-Hp1 (mating type (MATa)) of the yeast No. 1601 in this reaction solution were added to the YPD medium. The yeast cells cultured at 30 ° C. for 24 hours at 50 ° C. were mixed with 50 ml of YAD medium, and mixed by stationary culture for 2 days. Crosses are confirmed by comparing the bacteriological properties of haploid strains (1601-Hp1), diploid strains (S-2 strains) and hybrid diploid strains, and finally NS The -2-16 strain was selected.

(3) Confirmation of breeding strain NS-2-16 strain was inoculated into 10 ml of YPD medium (yeast extract 1%, polypeptone 2%, glucose 2%), cultured at 30 ° C. for 2 days, and then cerulenin medium (Difco Yeast Carbon). Base 0.67%, ammonium sulfate 0.5%, cerulenin 3 ppm, agar 2.0%), citrate medium (Difco Yeast Nitrogen Base 0.67%, trisodium citrate dihydrate 1.56%, agar 2 0.0%, pH 4.0), citrate medium containing 0.5 to 2.0 ppm of cerulenin by combination of both media (Difco Yeast Nilogen Base 0.67%, trisodium citrate dihydrate 1.56%, cerulenin 0.5 to 2.0 ppm, 2.0% agar, pH 4.0) The cells were applied so that the number of cells was 200 to 300 per plate and cultured at 30 ° C. for 3 to 7 days. These results are shown in Table 1 below.

  The haploid strain 1601-Hp1 (mating type (MATa)) of Kyoto No. 1601 grows well in the cerulenin medium, whereas the S-2 strain has the property that it cannot grow at all. The S-2 strain grows well in a citrate medium, whereas 1601-Hp1 has the property that it cannot grow at all. On the other hand, the hybrid strain NS-2-16 grows well in both media and can be confirmed to have been crossed because it differs from the properties of 1601-Hp1 and S-2 strains. Furthermore, by using a citrate medium containing 0.5 to 2.0 ppm of cerulenin, which is a combination of both culture media, it is possible to confirm hybridization using a single petri dish. Thus, as a result of comparing the viability in each culture medium, it was confirmed that NS-2-16 was a hybrid of 1601-Hp1 and S-2. It was also confirmed that cerulenin resistance was inherited by the hybrid strain.

(1) Small preparation fermentation test with breeding strains (NS-2-16 strain) and S-2 strain (control) The small preparation fermentation test is the composition shown in Table 2 below. Using koji mold, the fermentation temperature was kept constant at 20 ° C. and 25 ° C. for both primary and secondary.

  Regarding the results of small preparation fermentation test of the breeding strain (NS-2-16 strain) and the haploid strain (1601-Hp1), the breeding strain has more ethyl caproate than the parent strains S-2 and K-1601. Generated. Moreover, it was a strain with a small amount of acid production and a very strong fermenting ability. The comparison results including the parent strains (K-1601, S-2 strain) are shown in Table 3 (Made in USA: 20 ° C) and Table 4 (Made in Rice: 25 ° C). In addition, a distilled liquor having a fragrant and well-flavored flavor with a reduced caproic acid ratio by distillation was obtained. The comparison results with this parent strain (S-2 strain) are shown in Table 5 (General analysis of fermentation broth: 20 ° C.) and Table 6 (General analysis of distillate: 20 ° C.).

(2) Distilled liquor production test by breeding strain (NS-2-16 strain) and S-2 strain (control) Breeding strain (NS-2-16 strain) and parent strain S-2 strain (control strain) As a result of distilled liquor production with the feed blend, the breeding strain produced more ethyl caproate than the control strain, as in the small feed fermentation test. Moreover, it was a strain with a small amount of acid production and a very strong fermenting ability. The comparison results are shown in Table 8. In addition, a distilled liquor having a fragrant and well-flavored flavor with a reduced caproic acid ratio by distillation was obtained. The comparison results are shown in Table 9 (General analysis of fermentation broth: 23 ° C.) and Table 10 (General analysis of distillate: 23 ° C.).

  Although the results are not shown, it was confirmed that the breeding strain (NS-2-16 strain) can be fermented similarly to the above results even at a temperature of 30 to 35 ° C. and can easily produce an aromatic shochu. It was.

  The present invention is summarized as follows.

  The present invention produces a distilled liquor that has a high temperature resistance, citric acid resistance, and high caproic acid ethyl acetate production ability, and has a fragrant and well-flavored flavor that can be easily and easily used in the production of distilled spirits. It aims at providing the yeast for distilled liquor manufacture.

  Then, a high ester-producing mutant sake yeast that produces a large amount of ethyl caproate obtained by selection in a cerulenin-containing medium is crossed with a yeast for shochu to produce a large amount of ethyl caproate, and is resistant to high temperature and citric acid. The above-mentioned problem can be solved by obtaining a mutant yeast for distilled liquor belonging to Saccharomyces cerevisiae having Saccharomyces cerevisiae.

The accession numbers of the microorganisms deposited in the present invention are shown below.
(1) Saccharomyces cerevisiae NS-2-16 strain (NITE P-1021).

Claims (7)

  It was obtained by mutating sake yeast and selecting high ester-producing sake yeast that produces a large amount of ethyl caproate from strains that grow on cerulenin-containing medium and crossing the high ester-producing sake yeast and shochu yeast. A mutant yeast for distilled liquor belonging to Saccharomyces cerevisiae, which produces a large amount of ethyl caproate and has high temperature resistance and citric acid resistance.   The yeast according to claim 1, wherein the yeast is obtained by crossing Kyoto 1601 yeast (K-1601) and Kyoto S-2 yeast.   Saccharomyces cerevisiae NS-2-16 strain (NITE P-1021), which is the yeast according to claim 2.   Brewing using the yeast according to any one of claims 1 to 3, and distillation, characterized by a high content of ethyl caproate and a fragrant and well-flavored flavored sake. Production method.   A method for producing an alcoholic beverage comprising adding the distilled liquor produced by the method according to claim 4 to sake.   It is characterized by selecting a high ester-producing sake yeast that produces a large amount of ethyl caproate from a strain that grows on a cerulenin-containing medium by mutating the sake yeast and crossing the high ester-producing sake yeast with the shochu yeast. A method for breeding a mutant yeast for distilled liquor belonging to Saccharomyces cerevisiae, which produces a large amount of ethyl caproate and has high temperature resistance and citric acid resistance.   The method according to claim 6, wherein the high ester-producing sake yeast is Kyoto No. 1601 yeast (K-1601) and the shochu yeast is No. S-2 yeast.