JPH06169748A - Production of refined sake using yeast having low decomposition of ethyl caproate, method for breeding yeast having low decomposition of ethyl caproate and its yeast strain - Google Patents

Abstract

PURPOSE:To stably produce refined SAKE type rich in aroma. CONSTITUTION:A strain having low decomposition of ethyl caproate is further separated from 2-fluoroethyl caproate-resistant strains of refined sake (rice wine) yeast and used to stably produce refined rice wine rich in aroma, having highly formed ethyl caproate and a high commercial value.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing sake with rich aromaticity characterized by using ethyl caproate low-degrading yeast and breeding of yeast for separating ethyl caproate low-degrading strain from sake yeast. A method and its yeast strain.

[0002]

[Background] Ethyl caproate is one of the highly desirable aroma components that resembles the aroma of fruits and, along with isoamyl acetate, forms the main component of the scent of ginjo sake. Therefore, by increasing the concentration of ethyl caproate in sake, it becomes possible to produce sake with high aromaticity and high commercial value.

[0003]

2. Description of the Related Art "Ginjo-zukuri" is a method of imparting "Ginjo aroma" such as ethyl caproate and isoamyl acetate to sake by fermenting rice at a low temperature using highly polished raw material rice with a rice polishing rate of 60% or less. Has been done. However, this method has a drawback in that the production of ginjo aroma is not stable, and that 40% or more of the raw white rice is not used as bran for sake brewing, resulting in high raw material costs.

[0004] In recent years, as a method of stably and inexpensively imparting ginjo aroma to sake, many attempts have been made to separate yeast that highly produces ginjo aroma and use it for sake brewing. Especially for isolation of ethyl caproate high-producing yeast strain, cerulenin (Cerulenin) which is a fatty acid synthesis inhibitor
Has already been reported (Japanese Patent Laid-Open No. 63-309175). The mechanism of high production of ethyl caproate in this method is considered to be due to mutation of the fatty acid synthase due to yeast resistance to cerulenin, resulting in significant accumulation of caproic acid, a precursor of ethyl caproate. ing. However, a yeast strain having low degradability of ethyl caproate and a method for producing sake using the yeast strain are not known.

[0005]

[Problems to be improved by the present invention] In the present invention, a new method for separating and breeding mutant yeast which produces a large amount of ethyl caproate, which is an important aroma component of sake, has been newly developed. The objective is to stably produce sake with high commercial value.

[0006]

[Means for Solving the Problem] As a method for highly producing ethyl caproate in sake, the present inventors have newly developed a method using a low-decomposition strain of ethyl caproate. The mechanism of action of high production of ethyl caproate is considered as follows. In the sake mash, ethyl caproate was converted to caproyl coenzyme A by yeast alcohol acyltransferase.
It is also synthesized from ethanol and at the same time, it is decomposed by an esterase (hereinafter referred to as ethyl caproate degrading enzyme), which has high specificity for yeast ethyl caproate. Therefore, it can be expected that the production amount of ethyl caproate in sake will be increased by using the yeast strain having reduced ethyl caproate degrading enzyme activity. The present inventors newly synthesized 2-fluoroethyl caproate in order to isolate a strain with low degradation of ethyl caproate. This drug is an ester bond of caproic acid and 2-fluoroethanol, and has a growth inhibitory effect on yeast. The mechanism of action is that 2-fluoroethyl caproate is decomposed by the ethyl caproate degrading enzyme in yeast cells, and the resulting 2-fluoroethanol exhibits toxicity.
Therefore, among the mutant strains that can grow in the presence of a high concentration of 2-fluoroethyl caproate (in which the parent strain cannot grow) (hereinafter referred to as 2-fluoroethyl-caproate-resistant strain), ethyl caproate degrading enzyme activity is higher than that of the parent strain. It can be expected that there is a strain having a low γ value (hereinafter referred to as ethyl caproate low-degrading strain). Moreover, since this method is positive selection, the number of cells that can be tested per plate is 10 ⁶
To 10 ⁷ cells, the separation efficiency is good.

It has already been reported that the ethyl caproate degrading enzyme of yeast also shows high substrate specificity to aliphatic esters having different carbon chains such as ethyl caprylate and ethyl caprate [J. Inst. Brew. , 88 , 34 (198
2)], it is expected that 2-fluoroethyl caprylate, 2-fluoroethyl caprate, etc. will be degraded by the ethyl caproate degrading enzyme of yeast, and the same effect as 2-fluoroethyl caproate is obtained. You can expect to show.

Specific mutant yeasts include Saccharomyces cerevisiae strain HL-108. Such low-capacity ethyl caproate strains are other yeasts of the association which are suitable for sake brewing and are distributed by the Japan Brewing Society. , Sake yeasts such as so-called "house-made yeasts" isolated from sake breweries in various places can be isolated using this method even if they are parent strains. In general, diploid yeast is used for sake brewing from the viewpoint of fermentability and genetic stability of traits. However, since the trait of low degradation of ethyl caproate is considered to be a recessive mutation, it was expected that the appearance frequency of strains with low degradation of ethyl caproate would be low in diploid yeast. Therefore, in the following examples, a single-phase strain was separated from a diploid brewing yeast and used as a parent strain. However, this does not limit the scope of the claims at all, even for ethyl caproate low-degrading strains directly separated from diploids by this method, and for ethyl caproate low-degrading single phase with different conjugation types. It is expected that the diploid strain produced by joining two strains will have the same effect as the case of the single-phase strain.

The ethyl caproate low-degrading strain is contacted with a known mutagenesis method, for example, irradiation with ultraviolet rays or radiation, or a drug such as N-methyl-N'-nitro-N-nitrosoguanidine or ethyl methanesulfonate. It can also be obtained by applying the method to parent stocks as appropriate.
Caproic acid 2- at a concentration such that the parent strain cannot grow yeast that has been or has not been mutated by an appropriate method
Stain and culture on a minimal medium containing fluoroethyl,
The grown colonies were isolated as 2-fluoroethyl caproate-resistant strains, and then these resistant strains were cultured in an appropriate natural medium, and then the cells were collected to prepare a cell-free extract thereof, and the ethyl caproate-decomposing activity was determined. taking measurement. The activity of decomposing ethyl caproate is measured by gas chromatography by performing reaction with ethyl caproate as a substrate and producing caproic acid. As a result, a strain having a lower rate of caproic acid production than the parent strain can be selected as a strain having low degradation rate of ethyl caproate.

[0010] When a charging test was carried out using a low-decomposition strain of ethyl caproate separated by this method, the sake liquor always contained a larger amount of ethyl caproate than the parent strain and its aroma was functional. Was confirmed to be superior to the parent strain, and the present invention was completed.

Examples will be described below.

[0012]

Example 1 (Separation of 2-Fluoroethyl Caproate-Resistant Strain) The HL-69 strain, which is an excellent single-phase strain of the Japan Brewing Society No. 10 yeast, which was separated by the random spore separation method at our company, was used as a parent strain. HL-6 cultured overnight in 1 ml of YPD medium (1% yeast extract, 2% peptone, 2% glucose)
After the 9 strains were collected and washed, the cells were suspended in 1 ml of 0.1 M phosphate buffer (pH 8.0), 30 μl of ethyl methanesulfonate was added, and mutagenesis was performed at 30 ° C. for 60 minutes. YN containing 1000 ppm of 2-fluoroethyl caproate at a concentration at which the parent strain cannot grow these mutant-treated cells
After smearing on B plate medium (Difco's yeast nitrogen base 0.67%, ethanol 2%, agar 2%, pH 6), it was immediately sealed with parafilm and cultured at 30 ° C., and emerged colonies were separated. Was used as a caproic acid 2-fluoroethyl resistant strain.

(Isolation of ethyl caproate low-degrading strain) 2-fluoroethyl caproate-resistant strain was mixed with 2 ml of YPD medium.
After culturing at 1, the cells were collected and washed, and the cells were crushed using glass beads. 0.1M phosphate buffer (pH
7.5) After adding 2 ml to extract the crude enzyme, 3000
After centrifugation at xg for 5 minutes, the resulting supernatant was used as a cell-free extract for measuring ethyl caproate degrading activity. The ethyl caproate decomposing activity was measured by adding 0.1 ml of 1000 ppm ethyl caproate ethanol solution to 0.9 ml of the above cell-free extract and initiating the reaction at 30 ° C.
After reacting for 30 minutes at room temperature, 1.5 ml of ethyl acetate was added and immediately stirred vigorously to stop the reaction. 1M for this
The pH of the aqueous layer was adjusted to 3 by adding 110 μl of an aqueous phosphoric acid solution, and the produced caproic acid was extracted into the ethyl acetate layer. The ethyl acetate layer was separated from the aqueous layer by centrifugation at 3000 xg for 5 minutes, and caproic acid contained in this layer was quantified by gas chromatography. In addition, the protein concentration in the above cell-free extract was measured by a protein assay kit manufactured by Bio-Rad, and ethyl caproate degrading activity was shown by the amount of caproic acid (mg / h / mg) produced by 1 mg of protein per hour. It was

Using the above method, the strain HL-108 which was the least degrading ethyl caproate was selected. As shown in Table 1, the HL-108 strain is 66% of the parental HL-69 strain.
It showed only ethyl caproate decomposing activity. The HL-108 strain has been deposited as FERM P-13153 at the Institute of Microbial Technology, Institute of Industrial Science and Technology.

[0015]

[Table 1]

[0016]

Example 2 (Preparation test of ethyl caproate low-degrading strain) In order to understand the difference in brewing characteristics between ethyl caproate low-degrading HL-108 strain and parent strain HL-69 strain, the preparation shown in Table 2 A preparation test was conducted on the composition. 73% Nihonbare polished rice as koji rice,
Nihonbare 75% polished rice was used as steamed rice. In the preparation test, the mother of liquor was omitted, and 10 ⁷ cells / m per pumped water was added
A yeast charging method was used in which yeast was added so that the fermentation temperature was 15 ° C. Both strains were fermented in a head-like manner and placed in the upper tank 13 days after the distillation. The aroma components of the sake liquor were analyzed by head space gas chromatography, and the results are shown in Table 3.

[0017]

[Table 2]

[0018]

[Table 3]

As shown in Table 3, the HL-108 strain produced about 1.5 times as much ethyl caproate as the parent strain, and could highly produce ethyl caproate in sake as originally intended. In addition, despite the fact that the raw rice with a rice polishing rate of 75% was used and the fermentation temperature was 15 ° C.
Ethyl caproate above pm was produced. The average content of commercially available ginjo sake ethyl caproate is reported to be about 0.8 ppm [Journal of the Brewing Society, 86 , 17 (1991)].
It was considered that the HL-108 strain could be imparted with a ginjo aroma without ginjo making. In addition, the amount of ethyl caprylate produced also increased, which is considered to be because the ethyl caproate degrading enzyme shows high substrate specificity for ethyl caprylate.

Next, a general analysis of the sake liquor was conducted according to the analysis method prescribed by the National Tax Agency, and the results are shown in Table 4. HL-108
The general analysis values of the strains were almost the same as those of the parent strain, and it was clarified that the trait of low degradability of ethyl caproate did not significantly affect other brewing characteristics.

[0021]

[Table 4]

[0022] 5) and the results are shown in Table 5. HL-108
The strain's sake was sensory superior to the parent strain, and its quality was rich in aromaticity.

[0023]

[Table 5]

[0024]

[Effect] In the present invention, a method for isolating and breeding a strain degrading ethyl caproate low from a mutant strain of sake yeast having resistance to 2-fluoroethyl caproate is newly developed. By carrying out sake production using the ethyl caproate low-degrading strain isolated by this method, it became possible to stably produce sake with high aromaticity and high production of ethyl caproate.

Front page continuation (72) Inventor Junhiro Tanaka 4-5-5 Sumiyoshiminami-cho, Higashinada-ku, Kobe-shi Shiro Tsuru Sake Brewery Laboratory

Claims (3)

[Claims] 1. A method for producing sake, which comprises using a yeast strain of low degradability of ethyl caproate. 2. A method for breeding yeast, wherein a 2-fluoroethyl caproate-resistant strain is separated from sake yeast, and an ethyl caproate low-degrading strain is further separated therefrom. 3. A low-decomposition yeast strain of ethyl caproate isolated by the method according to claim 2.