JPH0714335B2 - Mutant yeast culture method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing an alcoholic beverage or the like having rich aromaticity.

More specifically, the present invention relates to a method for producing various alcoholic beverages, foods, and further flavors by using a mutant yeast that has produced a large amount of aroma components such as isoamyl alcohol and isoamyl acetate by mutation. It is a thing.

In general, the aroma is an important factor for determining the quality of alcoholic beverages and foods, and the aroma ester isoamyl acetate is one of the highly preferable aroma components similar to the aroma of fruits. It has already been reported that isoamyl acetate is produced from isoamyl alcohol and acetyl CoA by the action of yeast acetyl CoA: alcohol acetyltransferase. (Yoshizawa et al., 1979, Annual Meeting of the 54th Annual Meeting of the Japanese Society of Agricultural Chemistry, P408) In addition, according to the research by the present inventor, acetyl-CoA is sufficiently present in yeast in this reaction, and therefore, in order to produce a large amount of isoamyl acetate. Found that isoamyl alcohol was the rate-determining factor. (Kuriyama et al .: Showa 57
Annual Meeting of the Japanese Society of Fermentation Engineering, P193)
Isoamyl alcohol is produced in the middle of the pathway of yeast biosynthesis of leucine, but when leucine accumulates, leucine inhibits the leucine biosynthetic pathway.
The biosynthesis of isoamyl alcohol will also be suppressed. Therefore, the inventors of the present invention have conducted intensive studies for a mutant strain in which the leucine biosynthetic pathway is not inhibited even if leucine accumulates, and found a method for obtaining a mutant yeast that produces a large amount of these aroma components. Furthermore, the present inventors have developed a method for producing alcoholic beverages with rich aromaticity using this mutant yeast.

In the present invention, any mutation method may be used to obtain mutant yeast. Examples of physical methods of mutation include ultraviolet irradiation and irradiation, and chemical methods include mutagens such as ethyl methanesulfonate, nitrosguanidine, 4-nitrosoquinoline, bromouracil, sulfurous acid, acridine compound, urethane. There is a mutation treatment to suspend in a solution such as.

In the present invention, these mutation methods can be used as appropriate,
It has a characteristic as a selection medium for mutant yeast. That is,
The selection medium must contain 5'-5'-5'-trifluoro-D, L-leucine.

In general, any leucine analog may be used to obtain a leucine analog resistant strain, but in the present invention, 5'-5'-5'-trifluoro- It was found that only D, L-leucine was effective, and only the resistant strain produced a significant amount of isoamyl alcohol and isoamyl acetate.

It is sufficient to add about 1 mM of 5'-5'-5'-trifluoro-D, L-leucine to a solid medium such as YNB agar medium.

As treated yeast, sake yeast, shochu yeast, brewer's yeast,
Leucine analog resistant strains can be obtained from any yeast such as wine yeast and baker's yeast.

The various yeasts were mutated and then added to a solid medium containing 5'-5'-5'-trifluoro-D, L-leucine.
Culturing is performed at 0 ° C. for about 2 days, the resulting colonies are separated, and yeast that frequently produces isoamyl alcohol and isoamyl acetate may be employed.

The mutant yeast obtained here is such that sake yeast, shochu yeast, brewer's yeast, wine yeast, and baker's yeast all produce isoamyl alcohol and isoamyl acetate well, so using them, sake, shochu, By producing beer, wine, bread, etc., it is possible to produce the respective aromatic products.

The production of sake, shochu, beer, wine, bread and the like can be carried out according to the manufacturing methods generally used in the past.

Next, examples and comparative examples of the present invention will be shown.

Example 1 (YM medium) Yeast extract 0.3% Malt extract 0.3% Polpeptone 0.5% Glucose 5% Japanese Brewing Society No. 7 yeast (hereinafter abbreviated as No. 7 yeast) was inoculated in 5 ml of the above YM medium and shake-cultured for one day. After that, it was centrifuged and washed with sterilized water.

4.5 ml of 0.2M phosphate buffer (pH 8.0) was added to the washed cells.
40% glucose 0.25 ml, ethyl methane sulphonate 0.25
After adding ml, the mixture was subjected to mutagenesis with gentle stirring at 30 ° C. for 1 hour, and the bacterial cells were treated with 5′-5′-5′-trifluoro-D,
YNB agar medium containing D-leucine (adjust the final concentration to 1 mM) (Difco yeast nitrogen base)
7-F-13 was obtained from a large number of leucine analog resistant strains which had been grown by smearing on 0.67%, glucose 2%, agar 2%). This strain has been deposited with MIC institute as FERM P-8236. The parent strain and its mutant yeast were inoculated into YM medium and 30 ℃
Table 1 shows the aroma components after static culture for 2 days.

As a result, it was found that the mutant yeast produced a larger amount of isoamyl alcohol and isoamyl acetate than the parent strain.

Comparative Example 1 Association No. 7 yeast, Saccharomyces carlsbergensis
Each of IFO 0565, Saccharomyces cerevisiae IAM 4274, and Society of Shochu Yeast No. 2 is an analog of leucine 4-
Aza-DL-leucine, DL-norleucine, DL-norvaline, 2-thiazolyl-DL-alanine alone at a final concentration of 10
It was confirmed that these analogs did not completely block the leucine biosynthesis pathway of the above yeasts, since these analogues grew in any medium when inoculated into YNB agar containing mM. Therefore, it was impossible to isolate mutant yeast from which feedback inhibition by leucine was released with these analogs.

Example 2 In order to compare the aroma-producing ability when sake is brewed with two kinds of yeasts of No. 7 yeast and mutant yeast 7-F-13 and FERM P-8236, the preparations shown in Table 2 below. I made one step preparation.

Yeast suspension is used for yeast (1.4 x 10
⁷ pieces), constant fermentation at 15 ℃, No. 7 yeast after 12 days, 7-F
-13 was in the upper tank after 14 days.

Table 3 below shows general components and aroma components obtained here.

As a result, the amount of isoamyl acetate, which is one of the most important aroma components of sake, increased by 6 times compared to the parent strain, and a sensory test gave a strong scent of fruits that had never been obtained before. Was given.

Further, isoamyl alcohol, which is a substrate of isoamyl acetate, was increased three times or more as compared with the parent strain, and isobutanol was reduced to 1/3 of the parent strain. In the pathway for producing leucine and isoamyl alcohol from glucose, leucine inhibits the reaction of converting α-ketoiso-valeric acid to α-isopropylmalate. However, it is shown that the inhibition was canceled by the mutation treatment in the mutant yeast.

Example 3 Saccharomyce
s carlsbergensis) IFO 0565 (brewer's yeast) YM Soujo 5m
The cells were inoculated in 1 l, shake-cultured for one day, centrifuged, and washed with sterilized water.

4.5 ml of 0.2M phosphate buffer (pH 8.0) was added to the washed cells.
40% glucose 0.25 ml, ethyl methane sulphonate 0.25
After adding ml to the mixture, the mixture was subjected to mutagenesis with gentle stirring at 30 ° C. for 1 hour, and the cells were mixed with 5′-5′-5′-trifluoro-D,
YNB agar medium containing D-leucine (adjusting the final concentration to 1 mM) (Difco yeast nitrogen base 0.
67%, glucose 2%, agar 2%), and B-F-3 was obtained from a large number of leucine analog resistant strains grown.

Table 4 shows the aroma components when the parent strain and this mutant yeast were inoculated in YM medium and statically cultured at 30 ° C. for 2 days. As a result, this mutant yeast was found to produce more isoamyl alcohol and isoamyl acetate among aroma components than the parent strain.

Example 4 Wine yeast, Saccharomyces cerevisiae
ces cerevisiae) IAM 4274 was inoculated into 5 ml of YM medium, shake-cultured for one day, then centrifuged and washed with sterile water.

4.5 ml of 0.2M phosphate buffer (pH 8.0) was added to the washed cells.
40% glucose 0.25 ml, ethyl methane sulphonate 0.25
After adding ml to the mixture, the mixture was subjected to mutagenesis with gentle stirring at 30 ° C. for 1 hour, and the cells were mixed with 5′-5′-5′-trifluoro-D,
YNB agar medium containing D-leucine (adjusting the final concentration to 1 mM) (Difco yeast nitrogen base 0.
67%, glucose 2%, agar 2%), and IAM-F-1 was obtained from a large number of leucine analog resistant strains grown.

Table 5 shows the aroma components when the parent strain and this mutant yeast were inoculated in YM medium and statically cultured at 30 ° C for 2 days. As a result, this mutant yeast was found to produce more isoamyl alcohol and isoamyl acetate among aroma components than the parent strain.

Example 5 Shochu Yeast No. 2 from the Society was inoculated into 5 ml of YM medium, shake-cultured for one day, then centrifuged and washed with sterilized water.

4.5 ml of 0.2M phosphate buffer (pH 8.0) was added to the washed cells.
40% glucose 0.25 ml, ethyl methane sulphonate 0.25
After adding ml to the mixture, the mixture was subjected to mutagenesis with gentle stirring at 30 ° C. for 1 hour, and the cells were mixed with 5′-5′-5′-trifluoro-D,
YNB agar medium containing D-leucine (adjusted to a final concentration of 1 mM) (Difco yeast nitrogen base 0.67)
%, Glucose 2%, agar 2%), and KS-2-F-1 was obtained from a large number of leucine analog resistant strains grown.

Table 6 shows the aroma components when the parent strain and this mutant yeast were inoculated in YM medium and statically cultured at 30 ° C. for 2 days. As a result, this mutant yeast was found to produce more isoamyl alcohol and isoamyl acetate among aroma components than the parent strain.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Shinzo Ashida 247 Minamihama-cho, Fushimi-ku, Kyoto City, Kyoto Prefecture Okura Sake Brewing Co., Ltd. (72) Koji Suginami 247, Minamihama-cho, Fushimi-ku, Kyoto Prefecture Kyoto Prefecture (56) References JP-A 55-23948 (JP, A) JP-A 50-140695 (JP, A) JP-B 49-27358 (JP, B1) JP-B 59-23788 (JP, B2)

Claims (2)

[Claims] 1. A mutant yeast which produces a large amount of aroma components isoamyl alcohol and isoamyl acetate by mutation treatment is produced, and then this is separated using a selection medium, and the isolated mutant yeast is used. A method for producing sake, shochu, beer, wine or bread characterized by: 2. A mutant yeast, which produces a large amount of isoamyl alcohol and isoamyl acetate among the aroma components obtained by the mutation treatment, is subjected to mutation treatment on various yeasts, and then used as a selection medium in 5′-5′-5′-tri. The method for producing sake, shochu, beer, wine or bread according to claim 1, which is obtained by separating from a strain grown in a fluoro-D, L-leucine-containing medium.