JPH0695934B2 - Yeast fusion strain - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a Saccharomyces cerevisiae yeast fusion product having acid resistance, high temperature fermentability and excellent aroma, and a method for producing shochu using the yeast fusion product.

[0002]

2. Description of the Related Art Shochu has been widely consumed in the southern Kyushu region since ancient times, and is closely related to the culture of the region. However, due to recent improvements in the manufacturing method, a soft type shochu has been developed in place of the old hard type. The image is changing from local liquor to widespread popular liquor.

Sake, which is widely consumed in Japan, is produced in cold regions and in winter. This is due to contamination of various bacteria unless careful attention is paid to the control of microorganisms in the production of sake.
This is because there is concern about rotting. However, shochu is produced in hot areas such as Okinawa in southern Kyushu, and various measures have been taken to prevent rotting. For example, using Asp. Awamori, Asp. Kawachii, etc. that produces citric acid in koji, it has acid resistance that can ferment both yeast and acid-rich moromi, and also has high-temperature fermentability that allows fermentation in hot regions. There is. It uses yeast that has a faster fermentation rate than sake yeast.

On the other hand, sake yeast has a strong acetyltransferase activity and has a property of producing a lot of fruit-like scent components (isoamyl acetate / ethyl caproate) like Ginjo sake. However, yeast for shochu has a weak enzyme activity, and so far, sake with less aroma has been produced. So far, the hard type of shochu has a lot of taste (higher fatty acid ester), so the scent was not emphasized. However, the soft type of shochu that is now popular is being drunk, and the scent has come to be emphasized.

[0005]

Under such circumstances, regarding the conventional method for producing shochu, the development of a new type of shochu by improving yeast, which is the origin of the method and has a great influence on the quality of sake, Is required. The present invention has been made in view of the above circumstances, and has excellent properties of both shochu yeast and sake yeast (acid resistance, high temperature fermentation is possible, fermentation speed is fast, and aroma is excellent. ) Is produced, and a method for producing shochu using the yeast is developed.

[0006]

That is, the present invention provides a sucker.
Sake yeast strains belonging to Romyces cerevisiae
The present invention provides a Saccharomyces cerevisiae yeast fusion product having acid resistance, high-temperature fermentability and excellent aroma, which is produced by protoplast fusion with a strain of yeast for shochu belonging to Calomyces cerevisiae. Further, the present invention provides
Upon shochu production, the production of shochu, wherein Rukoto using the Saccharomyces cerevisiae yeast fusions as yeast
It provides a method .

The present invention will be described in detail below. The present invention enhances the aromaticity of shochu, and for the purpose of obtaining a yeast suitable for the conditions of the shochu manufacturing process, yeast for ginjo sake Saccharomyces cerevisiae (Saccharomyces cerevisiae)
Kumamoto yeast for sake (Kumamoto Sake Brewing Co., Ltd.) (hereinafter referred to as Kumamoto yeast) and yeast for shochu were tried to be fused with protoplasts. Kumamoto yeast is a yeast for sake, but it is weak against acid and high temperature and is not suitable for producing shochu, but Kumamoto yeast is not suitable for producing shochu, but by combining with yeast for shochu, acid resistance and high temperature fermentability are imparted.

The yeast for shochu to be used for protoplast fusion is Saccharomyces cerevisiae S, which is an excellent yeast isolated from a shochu brewer in the Hitoyoshi region.
-4 (hereinafter referred to as S-4) was selected. In protoplast fusion, it is desirable to use complementary auxotrophic or respiratory deficient mutants for the detection of yeast fusion strains. In order to obtain such an artificial mutant strain, a conventional artificial mutagenesis treatment, for example, a physical method of irradiating with ultraviolet rays, X-rays or γ rays, ethyl methanesulfonate, N-methyl-N′-nitro-N-nitro, It is carried out by a chemical method using a mutagen such as guanidine or ethidium bromide.

The lysine-requiring mutant strain, which is one of the auxotrophic mutant strains, can grow only by culturing Kumamoto yeast and S-4 and chemically mutating with ethyl methanesulfonate. Obtained by detecting and isolating the mutant strain using the medium. For various auxotrophic strains, Kumamoto yeast and S-4 were subjected to sporulation treatment, the obtained spores were mutated with ethyl methanesulfonate to cultivate the mutated spores, and the mutated strains were cloned from the obtained colonies by the replica method. It is obtained by detecting and separating. The sporulation treatment is performed by a conventional method. Usually, yeast is cultured on YPD agar medium (Table 3) and then applied on sporulation agar medium (Table 7). Further, in order to obtain cells derived from a single spore, a method of lysing the ascites with a lytic enzyme for lysing the yeast cell wall, dispersing the spores by ultrasonication, and culturing the spores on a nutrient agar medium is used. . The respiratory deficient strain can be obtained by culturing Kumamoto yeast and S-4, chemically mutating with ethidium bromide, and separating the resulting mutant strain using a dye medium for strain identification.

Protoplast fusion is performed by standard methods. Usually, a yeast cell suspension having each marker at a concentration of 10 ⁷ to 10 ⁸ cells / ml is prepared, treated with a protoplast preparation containing yeast cell wall lysing enzyme, and then both protoplasts are treated. Mix in equal amounts and add the fusion agent
Cell fusion is performed by adding 30% PEG6000. The protoplast mixture is cultured on a hypertonic minimal agar medium, and the resulting colony is isolated as a fusion strain. Since both yeasts are mutant strains having different markers, the isolated colonies are fusion strains complementing each other.

The yeast fusion strain established by the present invention includes Saccharomyces cerevisiae.
cerevisiae) KS2, Saccharomyces cerevisiae K
S4, Saccharomyces cerevisiae KS4, and Saccharomyces cerevisiae KS5 can be mentioned. And these yeast fusion strains have been deposited at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology with the following deposit numbers. Saccharomyces cerevisiae KS2 Microindustrial Research Institute No. 12233 Saccharomyces cerevisiae KS3 Microindustrial Research Institute No. 12234 Saccharomyces cerevisiae KS4 Microindustrial Research Institute No. 12235 Saccharomyces cerevisiae KS5 Microindustrial Research Institute No. 12236 Saccharomyces ce
The mycological properties of revisiae) KS2 are shown below. Cream-colored colonies are formed on 1 YM agar, and vegetative cells germinate and grow in spherical or oval shapes. 2 Well forms ascospores. 3 No assimilation of nitrate. 4 No vitamin requirement. 5 No arbutin resolution. 6 Fermentability and assimilation of carbon sources

[0012]

[Table 1]

Saccharomyces cerevisiae (Saccharo
The two strains of myces cerevisiae) KS3 and KS5 also have the same properties as described above. Saccharomyces cerevisiae KS4 has the same properties as above, except that it has a low sporulation rate. Next, using the fusion strain obtained by the present invention, rice shochu is produced with a small preparation of 60 L scale. The manufacturing method is performed by a conventional method. That is, the fused yeast solution cultivated in a koji juice medium and water are added to koji made by mixing koji mold with steamed rice to make a primary charge, and steamed rice and water are added to make a secondary charge on the 6th day. . Two
After a week, the mash is distilled under reduced pressure to obtain the product. Analytical and sensory tests are performed to evaluate the moromi and the product.

Next, the sensory evaluation of shochu produced using the fused yeast will be shown. The score is a total score (score (1: good, 2: normal, 3: poor) x 6 nieces) evaluated by 6 shochu judges.

[0015]

[Table 2]

[0016]

EFFECTS OF THE INVENTION The fused yeast obtained by the present invention is a yeast having both the characteristics of a yeast for shochu showing a high alcohol yield in the process of producing shochu and a yeast for sake with excellent aromaticity. By using these fused yeasts, it is possible to produce scented soft type shochu.

[0017]

EXAMPLES The present invention will be specifically described below with reference to examples. However, the present invention is not limited to these examples. Example 1 (a) Preparation of lysine-requiring mutant strain Yeast Saccharomyces cerevisiae
revisiae) Kumamoto yeast and S-4 in YPD medium
After culturing at 30 ° C. for 16 hours in (Table 3), washing and harvesting of the cells, the cells were suspended in 10 ml of 0.1M phosphate buffer (pH 7.0). Then, 0.3 ml of ethyl methane sulfonate (EMS) was added to the suspension and shaken at 30 ° C. for 45 minutes. After collecting the cells, the cells were washed with 10 ml of a 5% sodium thiosulfate solution to neutralize the mutagen, and then washed twice with 10 ml of sterilized water.

[Table 3] YPD medium Yeast extract 10 g / L Polypeptone 20 g / L Glucose 20 g / L This treated cell was suspended in 10 ml of sterilized water, and 400 μl was applied to AA plate medium (Table 4) and cultured at 30 ° C for 7 days. did. Large colonies formed on the plate were isolated and inoculated on SD plate medium (Table 5) and SD + Lys plate medium (Table 6), and both plates were cultured at 30 ° C. for about 3 days. Does not grow in SD medium,
The strain grown in SD + Lys medium was used as a target lysine-requiring strain.

As a result, 6 strains of Kumamoto yeast, S-
For 4, 4 lysine-requiring strains were acquired.

[Table 4]

[Table 5]

[Table 6] (b) Preparation of auxotrophic mutant strain Yeast Saccharomyces cerevisiae (Saccharomyces ce
revisiae) Kumamoto yeast and S-4 in YPD medium
Incubate at 30 ℃ for 24 hours, then sporulate agar (Table 7)
And was cultured at 30 ° C. for 3 to 5 days to form spores.

[Table 7] Spore-forming agar medium Sodium acetate 10g / L Agar 20g / L Ascospores in sterile water 1ml so that the number of filaments is 10 ⁷ / ml.
The cells were suspended in, and the cells were collected and washed with 0.1 M phosphate buffer (pH 7.5). Then, put the ascidian 3 in 2 ml of lytic enzyme solution (Table 8).
After shaking for 1 hour at 0 ° C. to dissolve the ascites, the released spores were washed with 1 ml of sterile water and suspended in 3 ml of 0.1M phosphate buffer. To this suspension was added ethyl methanesulfonate.
After adding 1 ml and shaking at 30 ° C. for 1 hour, the cells were collected. Further 0.
Suspend in 0.2 ml of 1M phosphate buffer, add 3 ml of 5% sodium thiosulfate solution, shake at 10 ° C for 10 minutes, and EMS
Neutralized. After collecting the bacteria, the mutant spores were added to 2 ml with 1 ml of phosphate buffer.
It was washed twice and suspended in 5 ml of the same buffer, and the suspension was sonicated for 3 minutes under ice cooling to disperse the spores in the suspension. Then, the cells were collected, diluted appropriately with sterile water, and 0.1 ml of each diluted solution was applied to YPD agar medium and cultured at 30 ° C. for 48 hours.

[Table 8] Lysis enzyme solution 5 mg / ml Zymolyase 20T solution 0.2 ml 2-mercaptoethanol 0.4 μl 0.1M phosphate buffer 0.8 ml Replicate colonies to minimum medium using this YPD agar medium as a master plate, and then at 4 ° C at 30 ° C. Cultured for a day. Strains that could not grow in the minimum medium (Table 4) were picked from the master plate as auxotrophic mutants.

As a result, the desired mutant strain could not be obtained for Kumamoto yeast, but 10 tryptophan-requiring strains and 1 arginine-requiring strain were obtained for S-4. (c) Preparation of respiratory deficient mutant Saccharomyces cere
revisiae) Kumamoto yeast and S-4 were respectively inoculated into YPD medium, ethidium bromide (EB) was added so that the final concentration was 20 μg / ml, and the mixture was shaken at 30 ° C. for 24 hours. After collecting the bacteria,
After appropriately diluting with sterile water, 0.1 ml of the diluted solution was applied to PDA medium (Table 9) and cultured at 30 ° C for 2 days to prepare a master plate. Then, it was replicated in PDA medium and incubated at 30 ° C. for 1 day, while the master plate was overlaid with an overlay medium (Table 10) and incubated at 30 ° C. for 2 days. Colonies that did not turn red were plated from replica plates in minimal medium containing Glucose (Table 11).
The cells were inoculated into a minimal medium containing Glycelin (Table 12) and cultured.
A strain capable of growing in a minimal medium containing Glucose but not in a minimal medium containing Glycelin was isolated as a target strain.

As a result, two strains of Kumamoto yeast, S-
As for 4, 4 respiratory deficient strains were obtained.

[Table 9] PDA medium Poteto dextrose agar 39 g / L

[Table 10] Multilayer medium TTC (triphenyltetrazolium chloride) 500 mg / L glucose 5 g / L Bact Agar 10 g / L

[Table 11]

[Table 12] (d) Protoplast fusion of mutant strains Kumamoto yeast mutant strains and S-4 mutant strains were cultured in 10 ml of YPD medium at 30 ° C for 5 hours with shaking, and after harvesting, suspended in 2 ml of protoplast adjustment solution (Table 13). Turbid and suspend the suspension at 30 ° C for 30
Protoplast was formed by shaking for a minute. After collection, isotonic solution 0.6M
Wash twice with 1 ml of potassium chloride solution, mix both protoplasts in approximately equal amounts, and add 30% P
2 ml of EG6000 (containing 50 mM CaCl ₂ ) was added. At 30 ° C
Fusion was performed by leaving still for 15 minutes. Then, after collecting the cells, the cells were suspended in 1 ml of an equivalent isotonic solution and allowed to stand at 20 ° C. for 15 minutes. The suspension was appropriately diluted with an isotonic solution, applied to a selective medium (Table 14) and overlaid with an overlay medium (Table 15), and then cultured at 30 ° C for about 4 days. The resulting colonies were fusion strains that complemented each other for auxotrophy or respiratory deficiency. Specifically, Kumamoto yeast mutants (lysine-requiring strains / respiratory deficient strains) and S-4 mutants
About 180 fused strains were obtained by fusing various combinations of (lysine-requiring strain, tryptophan-requiring strain, arginine-requiring strain, and respiratory-deficient strain). Selection was repeated by a fermentation test using the amount of aroma component and fermentation ability as an index, and finally 4 strains (KS2, KS3, KS4) with reference to the sensory evaluation.
・ KS5) was selected as an excellent yeast. Table 16 shows the markers assigned to the parent strains of each fusion strain.

[Table 13] Protoplast adjustment solution 1.5M potassium chloride 0.8ml 0.1M phosphate buffer (pH7.5) 1.0ml 2-mercaptoethanol 1.4μl 0.25mg / ml Zymolyase 20T solution 0.2ml

[Table 14]

[Table 15]

[0021]

[Table 16]

Example 2 (a) Preparation Test Using Fusion Yeast Shochu was brewed by ordinary two-stage preparation using 4 fusion yeast strains and 2 parent strains, and the products were analyzed. The preparation test was conducted under the following conditions in a scale of 60 L for one test section. Mixing ratio: Koji ratio 40%, Pumping water ratio 150% Raw rice; Versatile rice (crushed rice) Preparation days: Secondary mash on the 6th day of the primary mash and distillation on the 14th day of the secondary mash. Room temperature was a constant temperature of 20 ° C, and product temperature was not controlled.

Distillation conditions; reduced pressure distillation Decompression degree 700 to 720 m
mHg mash temperature 35-40 ℃ The mash and product alcohol concentration were determined by gas chromatography. Two components, isoamyl acetate and ethyl caproate, which are indicators of aroma, were quantified by headspace gas chromatography. The results are in the table below
There are 17 streets.

When sake yeast was used in the production of shochu, the fermentation process was likely to be delayed due to the conditions of acid and high temperature, the alcohol yield was low, and no good aroma was produced. On the other hand, the fused yeast generally has an alcohol yield equal to or higher than that of the parent strain S-4, but also has a high scent component. The sensory evaluation of these shochu is as described above.

[0025]

[Table 17]

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Reference number within the agency FI technical display location (C12N 1/19 C12R 1: 865) (72) Inventor Yu Nakagawa 3-11 Higashimachi, Kumamoto City, Kumamoto Prefecture Kumamoto Prefectural Industrial Technology Center (72) Inventor Kimi Yawata 3-11 Higashimachi, Kumamoto City, Kumamoto Prefecture Kumamoto Prefectural Industrial Technology Center Examiner Hiroshi Kato

Claims (2)

[Claims] 1. Kiyoshi belonging to Saccharomyces cerevisiae
Sake yeast strain and belongs to Saccharomyces cerevisiae
A Saccharomyces cerevisiae yeast fusion product having acid resistance, high-temperature fermentability, and excellent aroma, which is produced by fusing a yeast strain for shochu with a protoplast. 2. A method for producing shochu, which comprises using the Saccharomyces cerevisiae yeast fusion product according to claim 1 as yeast in producing shochu.