JP3292580B2 - New liquor yeast and its uses - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a liquor yeast which is resistant to miconazole and / or econazole and a method for producing a fragrant liquor using the yeast. More particularly, the present invention provides a liquor yeast that produces a lot of isoamyl acetate of the aromatic component, a method to easily select from among many liquor yeast cells, even with a liquor yeast selected, the aroma The present invention relates to a method for producing a rich alcoholic beverage.

[0002]

2. Description of the Related Art In the production of alcoholic beverages, especially sake, it is difficult to produce a sake having a high content of fruity ginjo aroma by using low-polished rice and fermenting at about 15 ° C. ordinary sake. Hitherto, low-grade fermentation has been carried out using highly polished rice to obtain sake rich in ginjo. However, this method is time-consuming and costly. At present, low-purified rice is steamed under pressure or lipase treated.However, it is difficult to produce refined sake rich in ginjo aroma from low-polished rice with ordinary sake, so yeast The reliance on technology development from the viewpoint of breeding has been advanced, and so far 5 ', 5', 5'-trifluoro-
Method using D, L-leucine resistance (JP-A-62-66)
No. 69), a method using 4-aza-DL-leucine resistance (JP-A-1-257423), and a method of selecting a strain showing a different color tone on a solid dye plate medium (JP-A-2-1).
No. 3368) has been published. However, there are few yeasts that produce the components that make up Ginjo Aroma in a form that is functionally balanced with other components, and breeding of such yeasts has been awaited. In addition, in the production of shochu, there was a similar problem in order to produce fragrant shochu.

[0003]

In the case of Ginjo liquor, which is rich in aroma components, it has been difficult to stably produce a large amount of Ginjo liquor because the raw material cost is high and the production days are long. In addition, it is more difficult to produce refined sake rich in ginjo aroma by using ordinary sake using low-polished rice.

[0004] An object of the present invention is to solve the above-mentioned problems of the prior art by producing liquors having a high ginjo aroma or having a new ginjo aroma by using ordinary sake, and especially isoamyl acetate for that purpose. It is an object of the present invention to provide a novel liquor yeast having a high ability to produce liquor , and to provide a method for stably and in large quantities producing liquor rich in ginjo aroma from low-milled rice using the yeast.

[0005]

SUMMARY OF THE INVENTION In summary of the present invention, a first invention of the present invention comprises miconazole and / or econazo.
Alcohol-resistant liquor yeast that produces isoamyl acetate
Noh is 1.2-1.8 times the parent strain, and isoamyl alcohol
Is the same as the parent strain, and
The content ratio of mill to isoamyl alcohol is 5% or more
A second aspect of the present invention relates to a method for producing an alcoholic beverage rich in aroma components, wherein the method uses the yeast of the first aspect of the present invention. The invention relates to a liquor yeast which is obtained by mutating a liquor yeast and growing on a selective medium containing miconazole and / or econazole.
Select, and collecting the liquor yeast mutant of the first aspect of the invention relates to obtaining a method <br/> liquor yeast mutant of the first aspect of the invention.

[0006] Miconazole and econazole are known to act specifically on phospholipids, particularly unsaturated fatty acid residues, which play an important role in the construction and function of yeast cell membranes, causing membrane damage [Susumu Nagai, Methodology in yeast research-basics and applications, page 234, January 20, 1982
Published by Kagaku Shuppan Center). The present inventors have focused on the affinity of miconazole and econazole for membrane phospholipids, and the yeast that exhibits resistance to miconazole and / or econazole has a change in structure including the degree of unsaturation of cell membrane phospholipids. As a result, it was thought that the amount of acetate ester produced and the membrane permeability of the fragrance component might change.

The present inventors have grown a bubble-free yeast for sake, a mutant strain of Japan Brewing Association No. 901 (hereinafter abbreviated as K-901), in a minimal medium containing miconazole and / or econazole. The present inventors have found that a strain capable of growing in the medium can be isolated, repeated small sake preparation tests, and a sensory test of sake and instrumental analysis can select a high-producing strain of isoamyl acetate, thereby completing the present invention.

[0008] The isoamyl acetate high-producing strain in the present invention includes a malic acid and / or citric acid high producing strain, and the amount of isoamyl alcohol produced as a substrate of isoamyl acetate is equivalent to that of the parent strain. It is characterized by.

[0009] The alcoholic beverages described in the present specification include sake and baked sake.
It is a term that means both shochu. Therefore, this matter
In the detailed text, sake yeast refers to sake yeast and shochu yeast.
Means yeast summarized.

[0010] miconazole and / or econazole resistant strains obtained here, sake yeast, either baked酎酵 mother, so creating a lot of isoamyl acetate, sake using these yeasts, by producing shochu, aroma Rich products can be manufactured. Furthermore, it is also possible to produce an aromatic fragrance liquid using these yeasts.

[0011] sake, method of manufacturing shochu is not particularly limited, can be prepared according to the general procedure.

An example of the separation method will be described below. (Method for Mutation Treatment of Yeast) Mutation treatment was performed in the following steps in order to obtain an isoamyl acetate high-producing strain of K-901 strain. The K-901 strain was transformed into a YPD liquid medium (yeast extract 1w).
/ V%, polypeptone 2w / v%, glucose 2w / v
%) And cultured with shaking at 5 ° C overnight at 30 ° C. After collecting cells by centrifugation, a 0.2 M phosphate buffer (pH
8.0). The washed cells were added to 4.6 ml of 0.2 M
Suspended in phosphate buffer (pH 8.0), 40 w / v
After adding 0.25 ml of a 30% glucose solution and stirring well, 0.15 ml of a 3 w / v% aqueous solution of ethyl methanesulfonate (EMS) was added, and the mixture was gently shaken at 30 ° C. for 1 hour. 0.2ml of the 9.8ml 6w
/ V% sodium thiosulfate solution at room temperature
Maintain for 15 minutes, add 0.2 ml of it to 19.8 ml of sterile water, add 0.1 ml of SD medium containing 5 μg / ml miconazole and / or econazole [yeast nitrogen base (no amino acid) 0.67
w / v%, glucose 2 w / v%]
The plate was spread on a plate and cultured at 30 ° C. for 3 days. A strain grown on a selective medium containing miconazole (MIC17) and a strain grown on a selective medium containing econazole (ECO1)
1, ECO22) were selected as miconazole and / or econazole resistant strains.

(Method for selecting a strain having a high production of isoamyl acetate) Isoamyl acetate and isoamyl alcohol production of liquor yeast
Ability to be easily determined by the following sake small preparation test
Miconazole and / or econazo
By subjecting the resistant mutant strain to the small preparation test,
A light mutant was identified. That is, miconazole and / or
Or econazole resistant mutants (MIC17, ECO1
1, ECO22) was subjected to a small brewing test of sake with the brewing formulations shown in Table 1.

[0014]

[Table 1]

The rice used was α rice (manufactured by Seven Rice Industry Co., Ltd.) with a polishing rate of 75% (w / w). Koji rice was produced using white rice with a polishing rate of 72% (w / w). As for yeast, a yeast containing 1 × 10 ⁹ cells in 5 ml was added. The fermentation temperature was kept at 15 ° C. As a result of quantifying isoamyl acetate by gas chromatography on the filtrate of the moromi on the 13th day after distilling, any miconazole and / or econazole-resistant mutant strain was 1.2 to 1.2% of the parent strain (K-901 strain).
It was found to produce 1.8 times isoamyl acetate. The trait of high production of isoamyl acetate described above indicates that the mutant strain is a novel yeast different from the K-901 strain.

As described above, the strain according to the present invention {mutant: MIC17, ECO11, ECO22} is K-9
The mycological properties are shown below. (Bacteriological properties) Morphological properties After culturing in YPD medium at 30 ° C. for 2 days, the cells were observed with a microscope. a) Shape: oval b) Size: 4.7-7.9 μm in length, 3.8-5.5 in width
5 μm Sporulation: Yes The cells were cultured in a medium for sporulation (potassium acetate 2 w / v%, glucose 0.05 w / v%, agar 2 w / v%) at 30 ° C. for 5 days, and observed under a microscope. 3. 3. Form of growth: budding Biochemical observation a) Sugar fermentability A medium for test of carbon compound assimilation of Wickerham (manufactured by Difco) was dispensed into a test tube containing a Durham tube, the three strains were inoculated, 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 (-) Melibiose (-) Raffinose (+) b) Utilization of sugar A medium for test of carbon compound assimilation test of Wicker Ham (manufactured by Difco) Using an oxanograph method at 30 ° C., 1
The growth after 4 days was observed. Glucose (+) galactose (+) sucrose (+) maltose (+) lactose (-) c) assimilation of nitrate: (-) nitrate was potassium nitrate, and a medium for Wickerham carbon compound assimilation test (manufactured by Difco) was used. And the growth was observed by the oxanograph method. d) TTC staining: red e) Growth in β-alanine medium at 35 ° C .: (+) Formation of high bubbles As a result of small preparation of sake, formation of high bubbles was not observed in any of the mutant strains. As described above, the morphological and biochemical results indicate that the three yeast strains of the present invention are yeast belonging to Saccharomyces cerevisiae. In addition, in the small brewing test of sake,
Since no formation of high bubbles was observed, the three strains were K-
This indicates that the strain is a 901 mutant strain.

Thus, according to the present invention, the K-901 strain is mutated and then selected on a medium containing miconazole and / or econazole, whereby isoamyl acetate is produced at a high level. Yeast was provided. Representative strains MIC17 strain and ECO
The 11 strains were each Saccharomyces cerevisiae MIC
17, Saccharomyces cerevisiae ECO11 is displayed, and FERM is registered with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology.
P-14024 and FERM P-14015. Sake of the present invention, a method of manufacturing shochu liquor is characterized by using these alcoholic beverages yeast strain, brewing method is not particularly limited.

[0018]

Next, the present invention will be described in more detail with reference to specific examples of the production of alcoholic beverages using the fungus of the present invention. However, the present invention is not limited to these examples.

Example 1 Sake was produced from the three mixed strains resistant to miconazole and / or econazole 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 75% (w / w). Koji has a rice polishing rate of 72% (w /
It was manufactured using the white rice of w). 1 x 1 yeast in 5 ml
Those containing ⁰⁹ were added. The fermentation temperature was kept at 15 ° C. The parent strain K-901 was added as a control strain. Table 2 shows the analysis results of the upper tank liquid.

[0020]

[Table 2]

The sensory test was performed using a three-point method (1: good, 2: normal,
3: bad), and was expressed as the average value of 10 panelists.

As a result, the MIC17 strain and the ECO22 strain had isoamyl acetate, which is considered to greatly contribute to Ginjo aroma among the aroma components of sake, about 1.6-1.
The citric acid or malic acid having an eight-fold or refreshing acidity was produced slightly more than the parent strain, and a banana-like fruity aroma and a refreshing taste were strongly recognized functionally.

The ECO11 strain produced isoamyl acetate 1.2 to 1.3 times that of the parent strain and produced a large amount of succinic acid, and a gorgeous aroma and refreshing acidity were also organoleptically recognized. .

Example 2 Among the miconazole and / or econazole resistant mutants, two strains, MIC17 strain and ECO22 strain, were prepared as shown in Table 3.
Shochu was manufactured with the charge formulation shown in (1). As a control strain, K-701 strain, which is often used for rice shochu, was used.

[0025]

[Table 3]

The rice used was low-grade rice with a polishing rate of 70% (w / w). Koji rice was produced using white rice with a polishing rate of 72% (w / w). The yeast contained 2 × 10 ^{8 in} 5 ml. The enzyme preparation used was Spitase M (manufactured by Nagase Seikagaku Corporation). The fermentation temperature was kept constant at 20 ° C. The mash on the 14th day after the distillation was distilled under reduced pressure at a reduced pressure of -700 mmHg, and the product obtained by splitting the vertical outlet of the distilled alcohol content to 20 v / v% with 25.0 v / v% alcohol was analyzed. Table 4 shows the results.

[0027]

[Table 4]

The sensory test was performed by a three-point method (1: good, 2: normal,
3: bad), and was expressed as the average value of 10 panelists.

As a result, rice shochu produced using the MIC17 strain and the ECO22 strain had a higher content of isoamyl acetate, which is a ginjo fragrance, than the control strain K-701, and had a sensory banana-like fruity fragrance. It became a strongly recognized new type of shochu.

The above results indicate that the MIC17 strain, ECO11
This shows that the strain and the ECO22 strain have properties not found in the K-901 strain, that is, they are novel yeasts that produce a large amount of aroma esters.

[0031]

EFFECT OF THE INVENTION By using miconazole and / or econazole resistant yeast obtained by using the yeast selection method according to the present invention, it is possible to prepare ordinary sake or low-concentration rice without performing high-milled rice and low-temperature long-term rice brewing. price rice by rich aroma esters isoamyl acetate as a main in shochu charged, sake rich in aromatic, the production of shochu, at low cost, yet it is possible to perform stably in a short period of time.

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁷ Identification code FI C12R 1: 865) C12R 1: 865) (72) Inventor Toshinori Miyabe 3-4-1 Seta, Otsu City, Shiga Prefecture Takara Shuzo Co., Ltd. (56) References JP-A-62-6669 (JP, A) JP-A-3-7579 (JP, A) JP-A-1-257423 (JP, A) JP-A-4-91782 (JP) , A) Yoshihisa Noshiro, 4 outsiders, brewing dictionary, Asakura Shoten Co., Ltd., 1st print (November 10, 1988), p. 25-27 Chemotherapy (1994), Vol. 40, no. 4, p. 245-251 (58) Field surveyed (Int. Cl. ⁷ , DB name) C12N 1/00-1/38 C12G 1/00-3/14 C12C 11/02 JICST file (JOIS) BIOSIS / MEDLINE / WPID S (STN)

Claims (3)

(57) [Claims] (1) miconazole and / or econazole
A resistant liquor yeast having an ability to produce isoamyl acetate,
1.2-1.8 times of the parent strain and isoamyl alcohol
The production ability is equivalent to that of the parent strain, and the isoamyl acetate
The content ratio to isoamyl alcohol is 5% or more
A liquor yeast mutant strain, characterized in that: 2. A method for producing liquor, comprising using the liquor yeast according to claim 1. 3. An enzyme capable of mutating liquor yeast cells and growing on a selective medium containing miconazole and / or econazole.
Mother Select, and collecting the liquor yeast mutant according to claim 1, preparative liquor yeast mutant according to claim 1, wherein
How to get.