JP2021159045A - Production of fission yeast schizosaccharomyces japonicus kumadai strain suitable for shochu/sake brewing - Google Patents

Abstract

To provide a novel breeding strain of the fission yeast Schizosaccharomyces japonicus by mutagenesis methods, and to provide methods for producing foods and drinks with a good flavor using the novel breeding strain.SOLUTION: Provided is the cerulenin-resistant strain of the fission yeast Schizosaccharomyces japonicus, the fission yeast having increased productivity of ethyl caproate compared to the wild strain of the fission yeast Schizosaccharomyces japonicus. The fission yeast has been deposited under accession number NITE P-03177 (Kumadai-T11 strain) or NITE P-03178 (Kumadai-U28 strain). The shochu production method comprises fermenting using the fission yeast.SELECTED DRAWING: Figure 1

Description

The present invention relates to fission yeast having a high production of ginjo aroma component and its utilization. Specifically, the present invention relates to a fission yeast that is a cerulenin-resistant strain of the fission yeast Schizosaccharomyces japonicus and has an improved ethyl caproate production ability as compared with the wild strain thereof, and its utilization. The present invention relates to a method for producing shochu, which comprises fermenting with a drug-resistant strain of fission yeast Schizosaccharomyces japonicus.

Yeast has been used in various industries such as food, brewing, and pharmaceuticals for a long time because it has excellent metabolic capacity and division ability. Conventionally, the yeast used for brewing alcoholic beverages such as sake and shochu is all budding yeast (Saccharomyces cerevisiae, Saccharomyces cerevisiae, hereinafter sometimes referred to as S. cerevisiae) strains such as Association No. 7 and Association No. 9. Has been used.

The fission yeast (Schizosaccharomyces japonicus, Schizosaccharomyces japonicus, sometimes referred to as S. japonicus) first isolated from "strawberry" collected in the field of Kyushu University, Fukuoka Prefecture in 1928 is a nutrient-rich condition. The cell cycle underneath is as short as about 3 hours, and is true, as is the other species of the genus Schizosaccharomyces pombe (isolated from African local beer) and the budding yeast S. cerevisiae. It is being studied as a model organism for nuclear cells, and the entire genome sequence has been decoded. Schizosaccharomyces cerevisiae is thought to have evolved from Saccharomyces cerevisiae 300-400 million years ago, and both yeasts are the same unicellular eukaryote but are genetically very different. Since S. japonicus has a cell body size several times larger than that of Saccharomyces cerevisiae, it is also used for studies of chromosomal dynamics.

An example of using fission yeast S. japonicus as a brewing yeast is the report "Development of a high-quality sake production method using fission yeast" (Non-Patent Document 1) by the Kofu Technical Support Center of Yamanashi Prefecture. It has been reported that alcoholic fermentation is possible using sake mash as a culture base for most of the test strains of fission yeast containing. According to the same report, sake produced by a mixed culture of fission yeast S. japonicus strain (NBRC1609 strain) and sake yeast (S. cerevisiae FJA025 strain) may be difficult to grow in a sake brewing environment with fission yeast alone. Brewing is being attempted.

On the other hand, brewer's yeast S. cerevisiae, which has been conventionally used as a brewing yeast, is actively bred. Breeding methods for Saccharomyces cerevisiae include mating and cell fusion methods, as well as mutagenesis methods and gene recombination methods. As a method of mutagenesis, a treatment method with a mutagenesis agent such as ethylmethanesulfonate or nitrosoguanidine, a method of irradiating ultraviolet rays or radiation, or the like is performed. For example, in Non-Patent Document 2, there is a technique for breeding budding yeast S. cerevisiae by inducing mutation with ethylmethane sulfonate, separating a cellrenin-resistant strain, and separating a high-producing ethyl caproate-producing strain, which is a ginjo fragrance. It is used.

Noriaki Sato, Masakazu Komatsu, Hideo Kimura, Development of high-quality sake production method using fission yeast, 2016 report of Kofu Technical Support Center, Yamanashi Prefecture, https://www.pref.yamanashi.jp/yitc/kit/ documents / report_h28_03.pdf Eiji Ichikawa et al., Breeding of a Sake yeast with improved ethyl caproate productivity, Agric. Biol. Chem., 55 (8), 2153-2154 (1991)

Conventionally, Saccharomyces cerevisiae S. cerevisiae has been widely used as a brewing yeast, so that there is a problem that the variety of tastes and flavors of the produced liquor is low. Therefore, the search for new yeasts that can be used for brewing and food production and their breeding are issues.

It has been reported that the fission yeast Schizosaccharomyces japonicus can undergo alcoholic fermentation using sake mash as a culture base (for example, Non-Patent Document 1), but there are few examples of its use as a brewing yeast. In addition, there has been no known example of obtaining a breeding strain of fission yeast S. japonicus by a mutagenesis method. There is no report that a breeding strain having useful properties for the production of food and drink was obtained for fission yeast S. japonicus.

An object of the present invention is to provide a novel breeding strain of fission yeast S. japonicus by a mutagenesis method. Another object of the present invention is to provide a method for producing a food or drink having a good flavor using the newly bred strain.

Under the above-mentioned problems, the present inventor has found that when the fission yeast Schizosaccharomyces japonicus is statically cultured (anaerobic culture), it emits a mellow scent like Ginjo scent of Japanese sake. Attempted to produce a breeding strain of fission yeast S. japonicus. In order to isolate strains that produce even better aroma in fission yeast S. japonicus, mutagenesis screening was performed by ultraviolet irradiation or treatment with nitrosoguanidine, and 36 cerulenin-resistant strains were isolated. These strains were further analyzed, and Kumadai-T11 and U28 strains, which produce high ethyl caproate, were successfully separated. The present invention has been completed based on the above findings.

According to the present invention, the following inventions are provided.
[1] A fission yeast that is a cerulenin-resistant strain of fission yeast Sizosaccharomyces japonicas and has improved ethyl caproate production ability as compared with the wild strain of fission yeast Sizosaccharomyces japonicas.
[2] Fission yeast of accession number NITE P-03177 (Kumadai-T11 strain) or NITE P-03178 (Kumadai-U28 strain).
[3] A method for producing a food or drink using the fission yeast according to [1] or [2].
[4] The production method according to [3], wherein the food or drink is an alcoholic beverage.
[5] This includes mutagenesis treatment of fission yeast Sizosaccharomyces japonicas and selection of mutagenesis fission yeast Sizosaccharomyces japonicas in a cerulenin-containing medium [1]. Alternatively, the method for producing fission yeast according to [2].
[6] Including fermentation using a drug-resistant strain of the fission yeast Sizosaccharomyces japonicas, the drug-resistant strain is derived from cerulenin, canabanine, aureobasidin A, pregnenolone, hygromycin B and p-fluorophenylalanine. A method for producing shochu, which is resistant to a drug selected from the group.
[7] The method for producing shochu according to [6], wherein the drug-resistant strain is a cellrenin-resistant strain and is the fission yeast according to [1] or [2].

According to the present invention, it is possible to provide a highly producing strain of ginjo aroma component of fission yeast Schizosaccharomyces japonicus. By using a strain with a high production of ginjo aroma components, it is possible to produce foods and drinks having a good flavor. The high-producing ginjo fragrance component strain of the present invention has properties suitable for brewing alcoholic beverages, and can produce liquor having a taste and flavor different from those using conventional brewing yeast. As a result, the variety of tastes and flavors of the produced sake can be increased.

FIG. 1 (A) shows an example of the screening results of the fission yeast S. japonicus cellrenin-resistant strain. When 500,000 to 600,000 cells of fission yeast S. japonicus NIG2028 strain was applied to a YE plate (-cerulenin) containing no cerulenin with a spreader, yeast cells proliferated over the entire surface, but on a YE plate (+ cerulenin) containing 5 μM cerulenin, no yeast cells grew. The cells did not proliferate. On the other hand, on the plate (UV + cerulenin) irradiated with ultraviolet rays, cells that became resistant to cellrenin and proliferated due to some gene mutation were observed (arrows indicate candidate strains for cellrenin resistance). FIG. 1 (B) shows a plate of 15 cerulenin resistant candidate strains obtained by screening. Cerlenin resistance candidate strains, CR1 to CR15, were streaked again into a 5 μM Cerlenin-containing YE plate to confirm resistance. The parent strain NIG208 strain (WT) could not grow on the 5 μM cellrenin-containing YE plate, but all 15 isolated strains (CR1 to CR15) grew. FIG. 2 shows the results of aroma component analysis of the aspergillus culture solution by headspace gas chromatography. The fission yeast strains (CR1 to CR36) isolated by screening using a celllenin-containing plate were cultured in a koji juice medium, and then the aroma components were analyzed by headspace gas chromatography. As control yeasts, the parent strain NIG2028 and diploid strain NIG2021 of fission yeast S. japonicus, budding yeast KF7 for shochu brewing, and brewer's yeast association No. 7 (K7) and association No. 9 (K9) for sake brewing were used. From the results of the aroma component analysis, CR11 and CR17 were identified as high-producing ethyl caproate strains, and CR28 was identified as a candidate for high-producing β-phenethyl alcohol. FIG. 3 shows the analysis result of alcohol fermentation ability by carbon dioxide gas weight loss analysis. Carbon dioxide was reduced over time to measure the alcohol fermentation ability of the three isolated CR strains (CR11, CR17, CR28) and the controls NIG2021, NIG2028, Saccharomyces cerevisiae Nos. 7, 9, KF3 and KF7. Was measured. As a result, it was estimated that CR17 had a low carbon dioxide gas loss and a weak alcohol fermentation ability. FIG. 4 shows the results of aroma component analysis of a small preparation test (300 mL flask) sample using steamed rice. A small charge test was performed using a 300 mL volumetric flask, and the aroma components after brewing were analyzed using headspace gas chromatography. CR11, CR17 and CR28 were shown to produce high ethyl caproate. Control Association No. 7 (K7), No. 9 (K9), Shochu yeast KF3, KF7 and parent strain fission yeast S. japonicus NIG2028, diploid fission yeast S. japonicus NIG2028, the production amount of ethyl caproate is the detection limit. It was as follows. It was suggested that screening for cellrenin-resistant strains in the fission yeast S. japonicus would lead to the production of strains with high production of ginjo aroma components. It was also shown that S. japonicus produced a large amount of isoamyl acetate and ethyl acetate. FIG. 5 shows the results of aroma component analysis of a small preparation test sample having a capacity of 2 liters. A scaled-up small preparation test was performed using a 2-liter flask, and the aroma components after brewing were analyzed using headspace gas chromatography. CR11 and CR28 produced ethyl caproate higher than that of Control Yeast Association No. 9 (K9), Shochu Yeast KF7, and parent strain NIG2028 even in a scale-up small preparation test. In addition, rose-like ginjo fragrance β-phenethyl alcohol is produced in the fission yeast S. japonicus to the same extent as control budding yeast, isoamyl acetate, which is a banana-like ginjo fragrance, and ethyl acetate, which is generally regarded as an off-flavor. It was shown that the production was high.

The description of the present invention described below may be based on typical embodiments or specific examples, but the present invention is not limited to such embodiments. In this specification, the numerical range represented by using "~" means a range including the numerical values before and after "~" as the lower limit value and the upper limit value.

(Highly producing fission yeast with ethyl caproate)
The present invention relates to a highly ethyl caproate-producing strain of the fission yeast Schizosaccharomyces japonicus. The present invention specifically relates to a fission yeast that is a cellrenin-resistant strain of fission yeast S. japonicus and has improved ethyl caproate production ability as compared with a wild strain of fission yeast S. japonicus. The improved ability to produce ethyl caproate as compared with the wild strain of fission yeast S. japonicus means that the production of ethyl caproate is higher than that of the wild strain. Although not limited, the static culture sample (temperature 25 ° C., 3 days) in 10 mL of aspergillus medium produced 1.1 times or more and 1.2 times or more of ethyl caproate as compared with the wild strain. , 1.3 times or more, 1.4 times or more, or 1.5 times or more higher.

Schizosaccharomyces p. It is a strain. The mutagenesis treatment method and the selection method for cerulenin-resistant strains will be described later. The wild strain of fission yeast S. japonicus means the parent strain of S. japonicus before mutagenesis treatment. The wild strain may be isolated from naturally occurring yeast, or may be distributed from domestic and foreign yeast resource organizations. When a naturally occurring yeast is used as the parent strain, the strain identified as the fission yeast Schizosaccharomyces japonicus by analyzing the characteristics of the yeast can be used as the parent strain. Representative yeast resource institutions in Japan include NBRP (National BioResource Project) yeast, Institute for Fermentation (IFO) and Schizosaccharomyces japonicus Network (JapoNet, National Institute of Genetics Microbial Function Laboratory). can. In one embodiment, the fission yeast S. japonicus wild strain is NIG2008 strain (ATCC10660; IFO1609) strain, NIG2017 strain, NIG2021 strain, NIG2025 strain, NIG2028 strain and the like, and NIG2028 strain is particularly preferable (K. Furuya and H. Niki, Yeast 2009; 26: 221-233).

The present invention relates to fission yeast of accession number NITE P-03177 (Kumadai-T11 strain) or NITE P-03178 (Kumadai-U28 strain). The Kumadai-T11 strain and the Kumadai-U28 strain are celllenin-resistant strains selected from the parent strain Schizosaccharomyces japonicus NIG2028 strain by ultraviolet irradiation or mutagenesis treatment with nitrosoguanidine, respectively, in a cellrenin-containing medium. Both the Kumadai-T11 strain and the -U28 strain have a higher ability to produce ethyl caproate than the parent strain, and the ability to produce isoamyl acetate and β-phenethyl alcohol are widely used as brewing yeasts (for example, Saccharomyces cerevisiae). It can be said that the ginjo fragrance component is highly productive because it is higher than the association No. 7 strain, the association No. 9 strain, the KF3 strain, and the KF7 strain). Furthermore, the Kumadai-T11 and -U28 strains have the same level of alcohol-producing ability as the parent strain. Ginjo aroma component productivity and alcohol production capacity will be described later.

In addition to Schizosaccharomyces japonicus, the genus Schizosaccharomyces includes S. pombe, S. octosporus, and S. cryophilus. S. japonicus has a larger cell body than S. pombe and Saccharomyces cerevisiae. In the junction type of S. japonicus, h ⁺ and h ⁻ exist, and mycelium is formed depending on the culture conditions. For culturing S. japonicus in the laboratory, in addition to the YE medium used in the examples below, YPD medium (1% yeast extract, 2% polypeptone, 2% dextrose) and YM medium (0.3). % Yeast ectact, 0.5% polypeptone, 0.3% maltoix tract, 1% glucose) and the like can be used.

The NIG2028 strain, which is the parent strain of the Kumadai-T11 strain and the Kumadai-U28 strain, was distributed from the Microbial Function Laboratory of the National Institute of Genetics. . NIG2028 strain (mating ^type: h -, ^matsj-P2028) is, S from japonicus type strains (K. Furuya and H. Niki, Yeast 2009; 26: 221-233).

(Deposit of microorganisms)
The Schizosaccharomyces japonicus Kumadai-T11 and Kumadai-U28 strains have been deposited by the national university corporation Kumamoto University (Address: 2-39-1, Kurokami, Chuo-ku, Kumamoto City, Kumamoto Prefecture) as follows.
(i) Depositary organization: National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (NITE-NPMD)
(Address: 2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture, Japan, Room 122)
(ii) Contract date: March 18, 2020
(iii) Accession Nos. NITE P-03177 (S. japonicus Kumadai-T11 strain) and NITE-03178 (S. japonicus Kumadai-U28 strain)

(Aroma component)
Ginjo incense of sake and shochu is an aroma that gives liquor gorgeousness. Various components contribute to Ginjo incense, but the main components are isoamyl acetate and ethyl caproate. Ethyl caproate has a gorgeous apple-like scent and has become a trend of ginjo aroma, and is produced by the binding of caproic acid and ethanol biosynthesized in the fatty acid synthesis pathway of yeast by esterase (Shun Mitsui, Sake). About the analysis of Ginjo incense contained in Aichi Industrial Science and Technology Center News January 2015 issue).

Isoamyl acetate has a subdued banana-like scent, and can be said to be a traditional ginjo scent before the development of high-production yeast with ethyl caproate. Isoamyl alcohol biosynthesized by sake yeast is acetylated by alcohol acetyltransferase. Generated by being done (Shun Mitsui, above). Further, β-phenethyl alcohol is known as an aroma component contained in sake and shochu. β-Phenethyl alcohol has a rose scent. Ethyl acetate, on the other hand, is present in almost all alcoholic beverages, but may be considered off-flavour when its content is high.

(Manufacturing method of mutant strain)
The present invention comprises producing a highly ethyl caproate fission yeast, which comprises mutagenesis treatment of fission yeast Schizosaccharomyces japonicus and selection of mutagenesis-treated fission yeast S. japonicus in a celllenin-containing medium. Regarding the method. In addition to the cerulenin-resistant strain, for example, a strain having resistance to canavanine, aureobasidin A, pregnenolone, hygromycin B, p-fluorophenylalanine, etc. is selected to obtain a mutant strain having high productivity of the ginjo fragrance component. You may. As described above, the strain (parent strain) of S. japonicus before mutagenesis treatment may be isolated from naturally occurring yeast, or may be distributed from domestic and foreign yeast resource institutions. good.

As the mutagenesis treatment, for example, a physical mutagenesis treatment method such as ultraviolet irradiation, irradiation and heavy ion beam irradiation, and a chemical mutagenesis treatment method using a drug such as ethylmethanesulfonate and nitrosoguanidine can be used. However, any mutagenesis treatment method can be used. Both the physical mutation treatment method and the chemical mutation treatment method can be carried out based on any method known in the art. For mutagenesis by ultraviolet irradiation, the yeast cells on the plate surface are irradiated with 12.5 mJ of ultraviolet rays using an ultraviolet irradiation device (for example, UV crosslinker CL-1000, UVP), for example, as in the examples described later. It can be carried out by. Mutagenesis by treatment with nitrosoguanidine can be carried out by adding 400 μL of 1 mg / mL nitrosoguanidine to 4 mL of yeast suspension and incubating at 30 ° C. for 30 minutes, for example, as in the examples below.

By culturing the fission yeast S. japonicus after the mutagenesis treatment in a cellrenin-containing medium, S. japonicus resistant to cellrenin can be selected. As an example of the culture method, the mutagenesis-treated cells are applied to a YE agar plate medium containing 1 to 50 μM, preferably 1 to 10 μM of cerulenin, and the cells are applied at 20 to 30 ° C. for 2 to 10 days, preferably. After culturing for 3 to 7 days, the grown one can be selected as a strain showing resistance to cerulenin.

From the cellrenin-resistant strains grown in the cellrenin-containing medium, a mutant strain having a higher ethyl caproate-producing ability than the parent strain before the mutagenesis treatment can be selected. It is known that cerulenin-resistant strains produce a large amount of caproic acid by reducing long-chain fatty acid synthesis due to the Gly1250Ser mutation of Fas2p. The high production capacity of ethyl caproate as compared with the parent strain means that the production of ethyl caproate is high as compared with the parent strain. In a static culture sample (temperature 25 ° C., 3 days) in 10 mL of aspergillus medium, the amount of ethyl caproate produced by the mutant strain was 1.1 times or more, 1.2 times or more, 1 or more, but not limited to that of the parent strain. It is preferable to select mutant strains that are 3-fold or more, 1.4-fold or more, or 1.5-fold or more higher.

From the cellrenin-resistant strains grown in the cellrenin-containing medium, a mutant strain having a higher β-phenethyl alcohol-producing ability than the parent strain before the mutagenesis treatment can be selected. The higher production capacity of β-phenethyl alcohol as compared with the parent strain means that the production of β-phenethyl alcohol is higher than that of the parent strain. In a static culture sample (temperature 25 ° C., 3 days) in 10 mL of aspergillus medium, the amount of ethyl caproate produced by the mutant strain was 1.1 times or more and 1.2 times that of the parent strain, although not limited to the above. It is preferable to select mutant strains that are 1.3 times or more, 1.4 times or more, 1.5 times or more, 1.6 times or more, 1.7 times or more, or 1.8 times or more higher.

From the cellrenin-resistant strains grown in the cellrenin-containing medium, a mutant strain having an alcohol fermentation ability equal to or higher than that of the parent strain can be selected. Having an alcohol fermentation ability equal to or higher than that of the parent strain means that the carbon dioxide weight loss, which is an index of alcohol fermentation, is equal to or higher than that of the parent strain, and the difference in carbon dioxide weight loss between the parent strain and the mutant strain is 5 If it is within% by weight, it can be judged to be equivalent.

In a preferred embodiment of the present invention, a mutant strain having a higher ability to produce ethyl caproate as compared with the parent strain and having an ability to produce β-phenethyl alcohol and an ability to ferment alcohol equal to or higher than that of the parent strain is selected. In a more preferable embodiment of the present invention, the production capacity of ethyl caproate is higher than that of the parent strain, β-phenethyl alcohol and alcohol fermentation ability are equal to or higher than those of the parent strain, and about 50% or more of the acetic acid of the parent strain. A mutant strain capable of producing isoamyl is selected. In the present application, it was clarified that the production amount of isoamyl acetate of the parent strain NIG2028 is higher than that of the brewing yeast strain of Saccharomyces cerevisiae (for example, Association No. 7, Association No. 9, KF3 strain, KF7 strain). ing. It is considered that the mutant strain selected as the high-producing ethyl caproate-producing strain provides a sufficient aroma to alcoholic beverages if it has an isoamyl acetate-producing ability of about 50% or more of that of the parent strain before the mutagenesis treatment.

In an even more preferable embodiment of the present invention, the production capacity of ethyl caproate is higher than that of the parent strain, β-phenethyl alcohol and alcohol fermentation ability are equal to or higher than those of the parent strain, and ethyl acetate is further compared with the parent strain. Variants with low production are selected. In the present application, it was clarified that the production amount of ethyl acetate of the parent strain NIG2028 was higher than that of the brewing yeast strain of Saccharomyces cerevisiae (for example, Association No. 7, Association No. 9, KF3 strain, KF7 strain). ing. Since ethyl acetate may be regarded as off-flavour when it is present in a large amount, a mutant strain in which the production amount of ethyl acetate is lower than that of the parent strain is preferable. The shochu produced using the ginjo fragrance component high-producing strain of the present invention (for example, Kumadai-T11 strain or Kumadai-U28 strain) has improved sensory evaluation of fragrance to the same extent as Saccharomyces cerevisiae No. 9 strain. It is evaluated as being. It is considered that this is because the content of ginjo aroma components such as ethyl caproate is high, and the off-flavour ethyl acetate odor is masked.

By culturing the fission yeast S. japonicus after the mutagenesis treatment in a medium containing a drug selected from the group consisting of canabanine, aureobasidin A, pregnenolone, hygromycin B and p-fluorophenylalanine, the above-mentioned drug. S. japonicus that is resistant to S. japonicus can be selected. When selecting a canavanine-resistant strain, the mutagenesis-treated cells are applied to a YE agar plate medium containing 1 to 100 ppm, preferably 5 to 20 ppm of canavanine, and the cells are applied to a YE agar plate medium containing 20 to 30 ppm. After culturing at ° C. for 2 to 10 days, preferably 3 to 7 days, the grown one can be selected as a strain exhibiting resistance to canavanine.

Among the canavanine-resistant strains grown in the above-mentioned canavanine-containing medium, a mutation having a higher production ability of a ginjo fragrance component, for example, isoamyl acetate, and an alcohol fermentation ability equal to or higher than that of the parent strain as compared with the parent strain before mutagenesis treatment. You can get the stock. The high production capacity of isoamyl acetate as compared with the parent strain means that the production of isoamyl acetate is high as compared with the parent strain. Ginjo aroma components other than isoamyl acetate may be analyzed to obtain a mutant strain having high productivity.

When selecting an aureobasidin A-resistant strain, YE containing 0.1 to 50 ppm, preferably 0.5 to 20 ppm of aureobasidin A in the mutagenesis-treated cells, is not limited to this. It can be applied to an agar plate medium and cultured at 20 to 30 ° C. for 2 to 10 days, preferably 3 to 7 days, and the grown strain can be selected as a strain exhibiting resistance to aureobasidin A.

When selecting a pregnenolone-resistant strain, the mutagenesis-treated cells are applied to a YE agar plate medium containing 1 to 100 ppm, preferably 5 to 20 ppm of pregnenolone, and the cells are applied to a YE agar plate medium containing 20 to 30 ppm. After culturing at ° C. for 2 to 10 days, preferably 3 to 7 days, the grown one can be selected as a strain exhibiting resistance to pregnenolone.

When selecting a hygromycin B resistant strain, the mutagenesis-treated cells are applied to a YE agar plate medium containing 10 to 2000 ppm, preferably 100 to 1000 ppm of hygromycin B. After culturing at 20 to 30 ° C. for 2 to 10 days, preferably 3 to 7 days, the grown strain can be selected as a strain exhibiting resistance to hygromycin B.

From the aureobasidin A, pregnenolone or hygromycin B resistant strains grown in the above-mentioned aureobasidin A, pregnenolone or hygromycin B-containing medium, respectively, a ginjo fragrance component, for example, as compared with the parent strain before mutagenesis treatment, for example. It is possible to obtain a mutant strain having high production ability of isoamyl acetate and having alcohol fermentation ability equal to or higher than that of the parent strain. Ginjo aroma components other than isoamyl acetate may be analyzed to obtain a mutant strain having high productivity.

When selecting a p-fluorophenylalanine-resistant strain, YE containing 10 to 500 μg / mL, preferably 50 to 300 μg / mL of p-fluorophenylalanine in the mutagenesis-treated cells, without limitation. It can be applied to an agar plate medium and cultured at 20 to 30 ° C. for 2 to 10 days, preferably 3 to 7 days, and the grown one can be selected as a strain exhibiting resistance to p-fluorophenylalanine.

Among the p-fluorophenylalanine-resistant strains grown in the above p-fluorophenylalanine-containing medium, the production ability of the ginjo fragrance component, for example, β-phenethyl alcohol, is higher than that of the parent strain before the mutagenesis treatment, and is equivalent to that of the parent strain. A mutant strain having the above alcohol fermentation ability can be obtained. For example, a high production capacity of β-phenethyl alcohol as compared with the parent strain means that the production of β-phenethyl alcohol is high as compared with the parent strain. Ginjo aroma components other than β-phenethyl alcohol may be analyzed to obtain a mutant strain having high productivity.

The production ability of the aroma component for selecting the mutant strain can be evaluated using the koji soup culture solution obtained by planting and culturing the yeast strain. For example, yeast strain colonies are planted in 3 mL of Jiuqu medium (for example, 500 g of rice Jiuqu with 2 L of distilled water added, saccharified at a temperature of 55 ° C. for 16 hours, and then cultured) and cultured at 30 ° C. overnight. The cells were cultured with shaking, the turbidity of this culture solution was measured, and the cells were added to 10 mL of aspergillus medium so that the number of cells to be added between the strains to be compared was almost the same, and the cells were allowed to stand at 25 ° C. for 3 days to obtain the cells. It can be carried out by analyzing the koji juice culture medium.

A small charge test may be performed to confirm or evaluate the aroma component-producing ability and the alcohol-producing ability. As an example of the small preparation test, yeast is inoculated into a koji juice medium, cultured with shaking at 25 ° C. overnight, and 24 mL of pumped water is added to the dried koji (white koji 20 g, Tokushima koji, MKS) used for the primary preparation. To prepare the primary raw material, add 1/500 of the culture solution that has been shake-cultured overnight to the primary raw material, and incubate at 25 ° C. for 5 days (primary preparation). Next, 67 g of steamed rice and 115 mL of pumped water are added to the mash after the primary preparation, and the mash is statically cultured at 25 ° C. for about 10 days (secondary preparation). In order to estimate the amount of alcohol produced from the weight loss of carbon dioxide, the medium weight can be measured before and after the primary and secondary charges, or every day or at regular intervals during the preparation period.

In one embodiment of the present invention, when the brewed sample obtained in the small preparation test was analyzed, the high ethyl caproate-producing strain of fission yeast S. japonicus produced 2 ethyl caproate as compared with the parent strain. It may be fold or more, 5 times or more, 10 times or more, 20 times or more, 30 times or more, 40 times or more, or 50 times or more higher.

The aroma component can be performed by headspace gas chromatography, but is not limited to this. For example, 1 mL of the supernatant of the Jiuqu culture solution plus 100 μL of the N-amyl alcohol internal standard solution can be used as a sample for gas chromatograph analysis. As the headspace gas chromatograph analysis system, HS-20 and GC-2010 Plus (Shimadzu Corporation) can be used without limitation. The analysis system includes, for example, an oven temperature of 70 ° C., a heat retention time of 30 minutes, a sample line temperature of 150 ° C., a transfer line temperature of 150 ° C., a trap cooling temperature of -10 ° C., a trap heating temperature of 280 ° C., a trap standby temperature of 25 ° C., and a carrier gas He. , Carrier gas pressure 99.9 kPa, purge line flow rate 3.0 mL / min, total flow rate 16.8 mL / min can be set for measurement.

Alcohol production capacity can be estimated by calculating carbon dioxide weight loss. The weight of the medium is measured before and after culturing or every day or at regular intervals during the culturing period, and the weight difference is regarded as the amount released as carbon dioxide by alcohol fermentation, and the amount of alcohol produced is estimated. By comparing the carbon dioxide weight loss between the strains, the alcohol production ability of each strain can be evaluated. Comparing the carbon dioxide weight loss of the parent strain with the carbon dioxide gas weight loss of the cellrenin-resistant strain or other drug-resistant strain, is the alcohol-producing ability of the cellrenin-resistant strain or other drug-resistant strain comparable to that of the parent strain? , Inferior or superior can be evaluated. For example, if the difference between the carbon dioxide weight loss of the cerulenin-resistant strain or other drug-resistant strain and the carbon dioxide weight loss of the parent strain is within 5% by weight of the carbon dioxide weight loss of the parent strain, it is judged that the parent strain has the same alcohol-producing ability as the parent strain. can do.

In Example 4 (FIG. 3) described later, the weight loss of carbon dioxide on the 16th day of culture was 21.6 g for the parent strain 2028 and 19.3 g for the CR17 strain, which was about 10.6% by weight ((21). There is a difference of .6-19.3) × 100 / 21.6), and it is judged that the alcohol fermentation ability of the CR17 strain is inferior (less than the same degree) to that of the parent strain. Regarding the CR11 strain (21.5 g) and the CR28 strain (22.24 g), the difference from the parent strain is within 5% by weight, and it can be judged that they are about the same level.

(Manufacturing method of yeast-based food and drink and yeast-based food and drink)
The present invention relates to a method for producing foods and drinks using a fission yeast that is a cerulenin-resistant strain of fission yeast Schizosaccharomyces japonicus and has an improved ethyl caproate production ability as compared with a wild strain of fission yeast S. japonicus. The method for producing food and drink preferably includes fermentation using yeast. The food and drink produced by the above method can be called a fermented food and drink. The fission yeast having improved ethyl caproate production ability may be a fission yeast of Accession No. NITE P-03177 (Kumadai-T11 strain) or NITE P-03178 (Kumadai-U28 strain). Examples of the food and drink produced in the present invention include, but are not limited to, alcoholic beverages, non-alcoholic beverages, other fermented beverages, bread, confectionery, miso, and soy sauce pickles. The food and drink produced in the present invention is preferably alcoholic beverages, bread, confectionery, miso, and soy sauce, and alcoholic beverages are particularly preferable. In the present specification, the alcoholic beverage refers to a beverage containing 1% or more of alcohol, and is preferably sake (including sake, turbid liquor (doburoku), turbid liquor, and moromi liquor), beer, sparkling liquor, wine, and fruit liquor. , Other brewed sake, shochu (rice shochu, barley shochu, potato shochu, brown sugar shochu, peanut shochu, buckwheat shochu, chestnut shochu, awamori, etc.), whiskey, brandy, spirits, etc. can be mentioned. The production of yeast-based foods and drinks is known using a fission yeast of the present invention, which is a cerulenin-resistant strain of the fission yeast Schizosaccharomyces japonicus and has improved ethyl caproate production ability as compared with the fission yeast S. japonicus wild strain. It can be done according to the method of. Fermentation may be carried out using fission yeast having improved ethyl caproate production ability as compared with the fission yeast S. japonicus wild strain of the present invention alone, or in combination with budding yeast (for example, S. cerevisiae). May be fermented.

The method for producing sake that can be adopted in the method for producing food and drink of the present invention is outlined below, but the method is not limited to this, and any method for producing sake can be used. The method for producing sake can include a koji making process, a sake mother making process, a mash preparation process, a mash fermentation process, a solid-liquid separation process, and the like. First, brown rice, which is the raw material, is milled to make white rice, which is then steamed to produce steamed rice. Steamed rice is used for making koji and preparing sake mother and moromi. In the process of making Jiuqu, steamed rice is planted with Jiuqu (Yellow Jiuqu) to make Jiuqu. Jiuqu is used to convert rice starch into sugar, which can be added to sake mash and mash. In the sake mother making process, yeast is added to steamed rice, water, and koji, and a large amount of yeast necessary to promote fermentation of mash is cultivated. In the mash preparation process, mash is prepared by adding koji, steamed rice, and water to the sake mother. This preparation can be performed in a fermentation tank, and usually, "stage preparation" is performed, and the preparation is performed a plurality of times (mostly three times) in four days. By stepping, the growth of yeast is promoted while suppressing the growth of various germs, and the temperature of the mash is easily controlled to produce mash. In the mash fermentation step, the mash is fermented for 20 to 40 days after the mash preparation is completed. During the fermentation period, the fermentation tank is filled with carbon dioxide gas generated from the mash, so that the vicinity of the mash is in an oxygen-free state (or a low oxygen state), and the mash is in a state where it does not come into contact with oxygen in the air. When the fermentation process of mash is completed, the "solid-liquid separation process" is performed to separate the fermented mash into sake and sake lees. In order to separate the mash into sake and sake lees, it can be squeezed using a cloth or a squeezer, but solid-liquid separation by increasing the gas pressure inside the fermentation tank can also be used.

The cerulenin-resistant strain of the fission yeast Schizosaccharomyces japonicus of the present invention, which has improved ethyl caproate production ability as compared with the fission yeast S. japonicus wild strain, is also suitable for bread production. By producing bread by a known method of fermenting with fission yeast of the present invention, bread having a good flavor can be produced. The method for producing bread that can be adopted in the method for producing food and drink of the present invention is outlined below, but the method is not limited to this, and any bread production method can be used. Add strong flour, yeast, sugar, salt and lukewarm water to make a dough, but at this time knead the dough until the dough is tight and the texture is fine. Butter may be added to the bread dough during this process. Ferment in a warm place (for example, temperature 30-40 ° C.) until the dough swells about 1.5 to 2 times (primary fermentation). Divide the dough into the size of one bread. Wait for about 5 minutes (bench time) to soften the dough hardened by this work again. After degassing and molding, ferment until the dough swells about 1.5 to 2 times (secondary fermentation, temperature 35-40 ° C, humidity 80-85%), and in an oven at 180-200 ° C, 10 to 10 Bake for about 30 minutes.

(Manufacturing method of shochu)
The present invention comprises fermenting with a drug resistant strain of the fission yeast Schizosaccharomyces japonicus, wherein the drug resistant strain consists of the group consisting of cerulenin, canabanine, aureobasidin A, pregnenolone, hygromycin B and p-fluorophenylalanine. The present invention relates to a method for producing shochu, which is resistant to the selected drug. As described above, the drug-resistant strain is selected from the group consisting of cerulenin, canabanine, aureobasidin A, pregnenolone, hygromycin B and p-fluorophenylalanine after mutagenesis treatment against the fission yeast S. japonicus. It can be obtained by using the method for producing a drug-resistant strain of S. japonicus, which comprises a step of culturing in a medium containing a drug, but is not limited thereto. The drug-resistant strain used in the present invention is preferably a high-producing strain of ginjo fragrance component, more preferably a high-producing ethyl caproate production strain, a high-producing isoamyl acetate production strain, and / or a high-producing β-phenethyl alcohol production strain. The method for producing shochu of the present invention can be carried out according to a known method using a drug-resistant strain of fission yeast Schizosaccharomyces japonicus. The method for producing shochu of the present invention may be either continuous distillation or single distillation. The method for producing pot stills by pot still that can be adopted in the method for producing shochu of the present invention is outlined below, but the method is not limited to this, and any shochu manufacturing method can be used.

The method for producing simple distilled mash may include a primary mash manufacturing step, a secondary mash manufacturing step, a distillation step, a post-distillation filtration step, a water splitting step, a post-splitting filtration step, and the like. In the primary mash manufacturing process, rice, wheat, potatoes, etc., which are the raw materials for the primary mash, are washed, soaked, steamed, and then allowed to cool. Then, seed koji (white aspergillus, black aspergillus, etc.) is seeded in the raw material after allowing to cool, and the koji is produced. After that, shochu yeast and water are added to produce the primary aged mash (sake mother). Here, as the shochu yeast, for example, the drug-resistant strain of the fission yeast Schizosaccharomyces japonicus or the fission yeast of accession number NITE P-03177 (Kumadai-T11 strain) or NITE P-03178 (Kumadai-U28 strain) can be used. can. In the secondary mash manufacturing process, water, rice, wheat, potatoes, etc., which are the main raw materials, are added to the primary aged mash produced in the primary mash manufacturing process. Then, the secondary aging mash is produced by fermenting for several days to several tens of days while controlling the temperature and the like. In the distillation step, the secondary aged mash produced in the secondary mash manufacturing process is distilled by a pot still. Distillation by a pot still includes atmospheric distillation in which the temperature in the distillation machine is around 100 ° C. and vacuum distillation in which the temperature in the distiller is 50 to 60 ° C. or lower, but any of them may be used. In the post-distillation filtration step, filtration is performed to remove oily components in the raw liquor after the distillation step. In the water splitting step, water is added to the shochu after the distillation and filtration steps in order to reduce the alcohol content. In the post-splitting filtration step, filtration is performed in order to remove the oily component that precipitates due to the lowering of the alcohol content due to the splitting water in the water splitting step. The apparatus or the like used for the filtration process is not particularly limited. The shochu that has undergone the filtration process after water splitting is bottled and shipped.

The present invention will be described in more detail based on the following examples, but the present invention is not limited to these examples. In the present specification, unless otherwise specified, "%" and the like are based on mass, and the numerical range is described as including the end points thereof.

(Example 1) Screening fission yeast Schizosaccharomyces japonicus NIG2028 strain cerulenin-resistant strains with mutations induced by ultraviolet irradiation (mating ^type: h -, ^matsj-P2028) and 10mL of YE medium (3% glucose, 0.5% Bacto The yeast extract) was inoculated using a loop loop and cultured at 26 ° C. for 18 hours with shaking. After counting the number of cells using a hemocytometer, the cells were centrifuged (2,500 rpm, 5 minutes) using a high-speed cooling centrifuge to collect the cells. After suspending the precipitate in 1 mL of YE medium, 40 to 60 YE plates (3% glucose, 0.5% bactoeast exact, 2% agar) containing 5 μM cerulenin (Fujifilm Wako Pure Chemical Industries, Ltd.) per plate. It was applied using a spreader so that it became 10,000 cells.

After the applied yeast cell suspension was dried, the yeast cells on the plate surface were irradiated with 12.5 mJ of ultraviolet rays using an ultraviolet irradiation device (UV crosslinker CL-1000, UVP). After irradiation with ultraviolet rays, the plate was placed in a dark box and kept warm at 26 ° C. for 5 days. As a result, 15 strains (CR1 to CR15) of cellrenin-resistant strains were isolated from UV-irradiated plates (screening about 25 million cells) (FIGS. 1A and 1B).

(Example 2) Screening of a celllenin-resistant strain using the mutagen nitrosoguanidine A colony of fission yeast S. japonicus NIG2028 strain was planted in 10 mL of YE medium and cultured with shaking at 26 ° C. overnight. After culturing, the cells were centrifuged in a high-speed cooling centrifuge (2,500 rpm, 5 minutes), and the precipitate was suspended in 5 mL of Tris-maleic acid buffer (50 mM Tris, 50 mM maleic acid, pH 6.0). 400 μL of 1 mg / mL nitrosoguanidine (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to 4 mL of the suspension, and the mixture was kept warm at 30 ° C. for 30 minutes and then centrifuged (10,000 rpm, 2 minutes) in a microcentrifuge. The supernatant was removed and the precipitate was washed 3 times with 1 mL Tris-maleic acid buffer and once with 1 mL YE medium. Next, the cells were suspended in 5 mL of YE medium and kept warm at 26 ° C. overnight. 50 μL of this culture solution was applied to 50 YE plates containing 5 μM celllenin, kept warm at 26 ° C., and after 3 days, the presence or absence of colonization was observed (screening for about 25 million cells). The grown colonies were streaked again on a 5 μM cellrenin-added YE plate, and the grown colonies were obtained as mutant strains. Finally, 21 cerulenin-resistant strains were isolated (CR16-CR36).

(Example 3) Analysis of aroma components of koji juice culture medium by headspace gas chromatography In Examples 1 and 2, 36 fission yeast S. japonicus cellrenin-resistant strains (CR1 to CR36) separated by ultraviolet irradiation or nitrosoguanidine treatment. ), In order to further select high-producing strains of ethyl caproate and other ginjo aroma components, these strains were cultured in a koji juice medium according to the procedure shown below, and headspace gas chromatography was used. Aroma component analysis was performed. The aroma component analysis and small preparation test were conducted at the Kumamoto Prefectural Industrial Technology Center.
The koji juice medium was prepared by adding 2 L of distilled water to 500 g of rice koji, saccharifying at a temperature of 55 ° C. for 16 hours, and then centrifuging.

Colonies of NIG2021 strain (fission yeast S. japonicus diploid strain), NIG2028 strain, and 36 cerulenin-resistant mutant strains isolated in Examples 1 and 2 were planted in 3 mL of aspergillus medium and shaken overnight at 30 ° C. It was cultured. In addition, as control yeast, Saccharomyces cerevisiae Association No. 7 strain (K7) and Association No. 9 strain (K9) used for sake brewing, and Saccharomyces cerevisiae KF7 strain used for brewing shochu were used in a koji juice medium. Similarly, it was cultured overnight. The turbidity of these cultures was measured and added to 10 mL of aspergillus medium so that the number of cells added between the strains was approximately the same. The prepared Jiuqu juice culture solution was statically cultured at 25 ° C. for 3 days. After culturing, the cells were centrifuged (KOKUSAN H-103NR, 3,000 rpm, 10 minutes), and 100 μL of N-amyl alcohol internal standard solution was added to 1 mL of the supernatant to prepare a sample for gas chromatograph analysis. The aroma component analysis of the prepared sample was performed using a headspace gas chromatograph analysis system (Shimadzu HS-20, GC-2010 Plus). The analysis system has an oven temperature of 70 ° C, a heat retention time of 30 minutes, a sample line temperature of 150 ° C, a transfer line temperature of 150 ° C, a trap cooling temperature of -10 ° C, a trap heating temperature of 280 ° C, a trap standby temperature of 25 ° C, a carrier gas He, and a carrier. The measurement was performed by setting the gas pressure to 99.9 kPa, the purge line flow rate to 3.0 mL / min, and the total flow rate to 16.8 mL / min.

FIG. 2 shows the analysis results of the aroma components. From the analysis results of the aroma components, among the 36 cerulenin-resistant strains, CR11 and CR17 were used as apple-like ginjo-scented ethyl caproate high-producing strains, and CR28 was used as rose-like ginjo-scented β-phenethyl alcohol high-producing strains. Was selected.

(Example 4) Analysis by small preparation test (300 mL flask) Next, steamed rice was used to examine whether the three cerulenin-resistant strains CR11, CR17, and CR28 selected in Example 3 were suitable for rice shochu brewing. A small brewing test was performed in a 300 mL flask. As the control yeast, Saccharomyces cerevisiae No. 7 and No. 9 and Saccharomyces cerevisiae KF3 and KF7 were used. First, yeast was inoculated into a koji juice medium and cultured with shaking at 25 ° C. overnight. To prepare the primary raw material by adding 24 mL of pumped water to the dried koji (white koji 20 g, Tokushima koji, MKS) used for the primary preparation, add 1/500 of the culture solution cultivated by shaking overnight, and at 25 ° C. It was statically cultured for 5 days (primary preparation). During this period, the weight was measured, the amount released as carbon dioxide by alcohol fermentation was calculated, and a graph was made to estimate the amount of alcohol produced (Fig. 3). Next, 67 g of steamed rice (Hitomebore) and 115 mL of pumped water were added to the mash after the primary preparation, and the cells were statically cultured at 25 ° C. for about 10 days (secondary preparation). Carbon dioxide weight loss was measured during this period as well (Fig. 3). From the measurement results of carbon dioxide weight loss, it was inferred that CR17 produced a lower amount of alcohol than other strains. Therefore, as a result of measuring the alcohol concentration of the mash after culturing using a vibrating densitometer for alcoholic beverages (DA-155, Kyoto Denshi Kogyo), as estimated from the measurement result of carbon dioxide weight loss, the CR17 strain has an alcohol concentration. Was shown to be significantly lower than other strains (11.7%). The alcohol concentrations of CR11 and CR28 were 12.93% and 13.46%, respectively, which were lower than those of the sprouting yeast strains (alcohol concentration 17.29%) such as Association No. 7 used for sake and shochu brewing. However, it was judged that it could be used sufficiently for brewing.

Next, 1 mL of the mash after culturing was dispensed into a vial, 100 μL of an internal standard solution (N-amyl alcohol) was added, and a headspace gas chromatograph analysis system (Shimadzu HS-20, GC-2010 Plus) was used. The aroma components were analyzed. As a result, even in the small preparation test using steamed rice, the production of apple-like ginjo fragrance ethyl caproate was remarkably high for CR11, CR17, and CR28, and it was found that screening for high ethyl caproate production strains based on cerulenin resistance was successful. Shown. Although the production of ethyl caproate was not high in the culture of CR28 in the koji juice medium, the small preparation test showed that the production of ethyl caproate was higher than that of the parent strains NIG2021 and 2028. On the other hand, the production of β-phenethyl alcohol was about the same as that of other strains under small preparation conditions. It was also shown that the production of ethyl acetate was higher in fission yeast S. japonicus than in Saccharomyces cerevisiae. As a result of the small preparation test, the alcohol production of CR17 was lower than that of other strains, so it was decided not to use it for future analysis.

(Example 5) Small preparation test (2L flask) Preparation of rice shochu by vacuum distillation of sample Next, using CR11 strain and CR28 strain, 134 g of rice jiuqu as the primary raw material to be used, 160.8 mL of primary pumped water, After brewing by scaling up (2 L flask) to 448 g of steamed rice (Hitomebore) as a secondary raw material and 770 mL of secondary pumped water, it was distilled under reduced pressure to prepare rice shochu. As control samples, Saccharomyces cerevisiae No. 9 strain for sake brewing and KF7 strain used for shochu brewing were used for brewing. The charging conditions were the same as in the 300 mL small charging test. As a result of aroma component analysis of the sample after brewing, the apple-like ginjo aroma ethyl caproate was highly produced in the CR11 and CR28 strains as compared with the parent strains NIG2028, Saccharomyces cerevisiae No. 9 and KF7. (Fig. 5). The reason why the production of ethyl caproate was low in Saccharomyces cerevisiae No. 9 strain was considered to be that the rice used for brewing was used in general and the rice used was not polished at all. It was also shown that the amount of isoamyl acetate, which is a banana-like ginjo fragrance, was nearly 4 to 6 times higher in the fission yeast strain (Fig. 5). Rose-like ginjo fragrance β-phenethyl alcohol was also produced at the same level as or higher than that of Saccharomyces cerevisiae No. 9 and KF7 strain (Fig. 5). On the other hand, it was shown that ethyl acetate, which is a nail polish remover-like off-flavour, is produced in a large amount in the fission yeast S. japonicus. The CR11 and CR28 strains produced lower ethyl acetate than the parent strain NIG2028, indicating that the aroma was improved.

上記専門家パネルのほか３名が上記サンプルについて官能評価を行い、表１と同じような感想を持った。 (Example 6) Sensory evaluation test of rice shochu Next, vacuum distillation was performed using the brewed sample produced in Example 5, and about 400 mL of the distillate was recovered. The recovered distillate was sealed in a sample bottle and stored at 15 ° C. for 1 month. For the distilled sample stored for 1 month, a sensory evaluation test was conducted on a 5-point scale by one expert panel for three items of aroma, taste, and total. The results are shown in Table 1. The closer the value is to 1, the higher the evaluation.

In addition to the above expert panel, three people performed sensory evaluation on the above sample and had the same impressions as in Table 1.

As a result, the parent strain, fission yeast S. japonicus NIG2028, has a strong ethyl acetate odor, and can be brewed as it is with aroma, taste, and aroma 4, taste 4, and total 4 (4 evaluation close to 5) on a 5-point scale. It was shown to be unsuitable. On the other hand, the breeding strain CR11 had a scent of 3, taste 2, and a total of 3, which was a better score than the Saccharomyces cerevisiae No. 9 strain (scent 3, taste 3, total 3), and had a flavor that could be sufficiently enjoyed as rice shochu. The CR28 strain had a taste rating of 3, which was one step lower in terms of taste than the CR11 strain.

From the results of the above small preparation test, it was shown that the CR11 strain and the CR28 strain can be used for brewing rice shochu with a new taste and flavor, and the CR11 strain was named Kumadai-T11 and the CR28 strain was named Kumadai-U28.

The present invention is useful in the field of food and drink manufacturing.

Claims (7)

A fission yeast that is a cerulenin-resistant strain of fission yeast chizosaccharomyces japonicas and has improved ethyl caproate production ability as compared with the wild strain of fission yeast chizosaccharomyces japonicas. Fission yeast of accession number NITE P-03177 (Kumadai-T11 strain) or NITE P-03178 (Kumadai-U28 strain). A method for producing a food or drink using the fission yeast according to claim 1 or 2. The production method according to claim 3, wherein the food or drink is an alcoholic beverage. Claim 1 or 2 comprising mutagenesis treatment of fission yeast Sizosaccaromyces japonicas and selection of mutagenesis yeast chizosaccharomyces japonicas in a cerulenin-containing medium. The method for producing fission yeast according to the above. Including fermentation using a drug-resistant strain of the fission yeast Sizosaccalomyces japonicas, said drug-resistant strains consisted of the group consisting of cerulenin, canabanine, aureobasidin A, pregnenolone, hygromycin B and p-fluorophenylalanine. A method for producing yeast that is resistant to the drug of choice. The method for producing shochu according to claim 6, wherein the drug-resistant strain is a cellrenin-resistant strain and is the fission yeast according to claim 1 or 2.

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