Sake, yeast breeding method, yeast, and method for producing sake

Abstract

To provide a yeast that enables high production of isobutyl acetate, a yeast breeding method that enables high production of isobutyl acetate, and a sake containing a high proportion of isobutyl acetate and a method for producing the same.SOLUTION: The present invention provides a sake with an isobutyl acetate level of at least 1.0 ppm, based on conversion to a 15% alcohol content.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to sake, a yeast breeding method, yeast, and a method for producing sake. [Background technology]

[0002] Seishu is a traditional Japanese alcoholic beverage made by brewing steamed rice, water, koji, and yeast-grown yeast starter for about 20 to 30 days. Compounds that affect the taste and aroma of seishu are mainly produced by the yeast responsible for alcoholic fermentation. Seishu is known to contain aroma compounds such as ethyl caproate and isoamyl acetate.

[0003] In the past, in research aimed at diversifying the quality of sake, strains with excellent isobutyl acetate production ability have been reported, and it has been reported that in sake produced using said strains, the isobutyl acetate concentration was 1.1 ppm when the alcohol content was 18.0% (see, for example, Non-Patent Document 1). The isobutyl acetate concentration in the sake is 0.92 ppm when converted to an alcohol content of 15.0%.

[0004] However, a yeast strain capable of producing isobutyl acetate at a higher level has not yet been provided. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] M. Watanabe, et al: JOURNAL OF FERMENTATION AND BIOENGINEERING, Vol. 76, No. 3, 229-231(1993) Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention aims to solve the above-mentioned problems and to achieve the following objectives: That is, the present invention aims to provide a yeast capable of high production of isobutyl acetate, a method for breeding a yeast capable of high production of isobutyl acetate, and sake with a high content of isobutyl acetate and a method for producing the same. [Means for solving the problem]

[0007] As a result of intensive research conducted by the present inventors to achieve the above-mentioned object, they obtained a novel yeast capable of high production of isobutyl acetate. They confirmed that the concentration of isobutyl acetate in sake produced using the above-mentioned yeast is significantly higher than that in conventional sake. Furthermore, they discovered that the above-mentioned sake has excellent pleasant aromas, such as a fruity aroma (melon aroma), a mash aroma, a freshly squeezed feeling, and a cool feeling, and that the solvent odor is suppressed.

[0008] The present invention is based on the findings of the present inventors, and the means for solving the above problems are as follows. <1> This sake is characterized by an isobutyl acetate concentration of 1.0 ppm or more when converted to an alcohol content of 15%. <2> A method for breeding a yeast that produces isobutyl acetate at a high level, comprising the steps of: selecting a strain having reduced isoamyl acetate decomposing esterase activity compared to a parent strain; selecting a strain having improved alcohol acetyltransferase activity compared to a parent strain; and selecting a strain that produces more isobutanol than a parent strain. <3> Among the strains resistant to halogen-containing carboxylate esters, a strain having a reduced isoamyl acetate decomposing esterase activity compared to a parent strain is selected. <2> This is a method for breeding the yeast described in the above. <4> Among the strains resistant to aryloxyphenoxypropionic acid-based herbicides, a strain having improved alcohol acetyltransferase activity compared to a parent strain is selected. <2> from <3> The present invention relates to a method for breeding a yeast according to any one of the above items. <5> Among the strains resistant to the valine analogue, a strain that produces a larger amount of isobutanol than the parent strain is selected. <2> from <4> The present invention relates to a method for breeding a yeast according to any one of the above items. <6> Saccharomyces cerevisiae (accession number NITE P-03894) Saccharomyces cerevisiae ) The yeast is characterized as being the HL430 strain. <7> The above <6> This is a method for producing sake, characterized in that the yeast described in the above is used. <8> The above-mentioned sake has an isobutyl acetate concentration of 1.0 ppm or more when converted to an alcohol content of 15%. <7> This is a method for producing sake as described above. Effect of the Invention

[0009] According to the present invention, the above-mentioned problems in the conventional art can be solved and the above-mentioned object can be achieved, and it is possible to provide a yeast capable of high production of isobutyl acetate, a method for breeding a yeast capable of high production of isobutyl acetate, and sake with a high content of isobutyl acetate and a method for producing the same. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] (Yeast breeding method) The yeast breeding method of the present embodiment is a method for breeding yeast that produces a large amount of isobutyl acetate, and includes at least a step of selecting a strain lacking isoamyl acetate decomposition esterase function, a step of selecting a strain with high alcohol acetyltransferase activity, and a step of selecting a strain with high isobutanol production, and further includes other steps as necessary.

[0011] According to the yeast breeding method of the present embodiment, a non-genetically modified strain that produces a large amount of isobutyl acetate can be bred by mutation induction. Since sake brewed with a genetically modified yeast that needs to be labeled as genetically modified can be clearly distinguished by consumers from non-genetically modified sake brewed with a non-genetically modified yeast and is difficult for consumers to accept, a non-genetically modified strain that produces a large amount of isobutyl acetate bred by the yeast breeding method of the present embodiment is very useful.

[0012] <Parent stock> The parent strain of yeast used in the above-mentioned yeast breeding method is not particularly limited, and any yeast such as sake yeast can be used. The parent strain may be one obtained by natural mutation, or one that has been subjected to a mutation treatment. The mutation treatment is not particularly limited and may be appropriately selected from known methods, including physical mutation treatments such as ultraviolet irradiation and radiation irradiation, and chemical mutation treatments such as addition of ethyl methanesulfonate and addition of N-methyl-N'-nitro-nitrosoguanidine, etc. These mutation treatments may be performed alone or in combination of two or more.

[0013] <Step of selecting strains lacking isoamyl acetate decomposition esterase function> The step of selecting a strain lacking the isoamyl acetate decomposing esterase function is a step of selecting a strain having reduced isoamyl acetate decomposing esterase activity compared to a parent strain.

[0014] The method for selecting a strain having reduced isoamyl acetate decomposing esterase activity compared to the parent strain is not particularly limited and can be appropriately selected depending on the purpose, but it is preferable to select a strain having reduced isoamyl acetate decomposing esterase activity compared to the parent strain from among strains having resistance to halogen-containing carboxylate esters.

[0015] The method for obtaining a strain having resistance to the halogen-containing carboxylate is not particularly limited and can be appropriately selected depending on the purpose. For example, a method for culturing yeast in a medium containing a halogen-containing carboxylate to obtain a resistant strain can be mentioned.

[0016] The yeast used to obtain the resistant strain is not particularly limited and can be appropriately selected depending on the purpose. For example, a yeast strain obtained by subjecting the parent strain to the mutation treatment can be used.

[0017] The medium containing a halogen-containing carboxylate contains at least a halogen-containing carboxylate, and further contains other components as necessary.

[0018] As the medium containing the halogen-containing carboxylate, a medium generally used for culturing yeast to which the halogen-containing carboxylate and, if necessary, other components have been added can be used. The medium generally used for culturing yeast is not particularly limited and can be appropriately selected depending on the purpose. Examples of the medium include SD medium and YPD medium.

[0019] The form of the medium is not particularly limited and can be appropriately selected depending on the purpose. Examples of the form include solid and liquid.

[0020] -Halogen-containing carboxylic acid ester- The halogen-containing carboxylic acid ester is not particularly limited and can be appropriately selected depending on the purpose, but a preferred example is a halogenated acetic acid. The halogen-containing carboxylate is not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include isoamyl monofluoroacetate, isoamyl monochloroacetate, isoamyl monobromoacetate, and isoamyl monoiodoacetate. The halogen-containing carboxylates may be used alone or in combination of two or more kinds. The halogen-containing carboxylic acid ester can be synthesized, for example, by the method described in Non-Patent Document 1 mentioned above.

[0021] The concentration of the halogen-containing carboxylate in the medium is not particularly limited and can be appropriately selected depending on the purpose, but is preferably 10 ppm or more, more preferably 100 ppm or more, and particularly preferably 250 ppm or more. The upper limit of the concentration of the halogen-containing carboxylate in the medium can be appropriately selected within a range in which a resistant strain can be obtained.

[0022] -Other ingredients- Other components in the medium are not particularly limited, and can be appropriately selected from components generally used for culturing yeast depending on the purpose, and examples thereof include nitrogen sources such as soybean flour, wheat germ, rolled barley, peptone, cottonseed meal, yeast extract, meat extract, corn steep liquor, ammonium sulfate, sodium nitrate, and urea; carbon sources such as carbohydrates and fats such as tomato paste, glycerin, starch, glucose, galactose, dextrin, and bactosytone; inorganic salts such as salt and calcium carbonate; metal salts, etc. These may be used alone or in combination of two or more. The contents of other components in the medium are not particularly limited and can be appropriately selected depending on the purpose.

[0023] The culture temperature is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include 10°C to 40°C. The culture period is not particularly limited and can be appropriately selected depending on the purpose, and may be, for example, 1 to 10 days. The culture method is not particularly limited and can be appropriately selected depending on the purpose. Examples of the culture method include static culture and shaking culture.

[0024] The method for selecting a strain having reduced isoamyl acetate decomposing esterase activity compared to a parent strain from among the strains resistant to the halogen-containing carboxylate ester is not particularly limited and can be appropriately selected depending on the purpose. For example, the isoamyl acetate decomposing esterase activity of each of the parent strain and the resistant strain can be measured, and a strain having reduced isoamyl acetate decomposing esterase activity compared to the parent strain can be selected.

[0025] The method for measuring the isoamyl acetate decomposing esterase activity is not particularly limited and can be appropriately selected depending on the purpose. For example, the activity can be measured by the method described in Test Example 1 below.

[0026] When selecting a strain having reduced isoamyl acetate decomposing esterase activity compared to the parent strain, the degree of reduction in isoamyl acetate decomposing esterase activity is not particularly limited and can be appropriately selected depending on the purpose. For example, a strain having reduced isoamyl acetate decomposing esterase activity of 35% or less of the parent strain can be selected.

[0027] <Selection process of strains with high alcohol acetyltransferase activity> The step of selecting a strain with high alcohol acetyltransferase activity is a step of selecting a strain having improved alcohol acetyltransferase activity compared to a parent strain.

[0028] The method for selecting a strain having improved alcohol acetyltransferase activity compared to the parent strain is not particularly limited and can be appropriately selected depending on the purpose, but it is preferable to select a strain having improved alcohol acetyltransferase activity compared to the parent strain from among strains having resistance to aryloxyphenoxypropionic acid-based herbicides.

[0029] The method for obtaining a strain resistant to an aryloxyphenoxypropionic acid-based herbicide is not particularly limited and can be appropriately selected depending on the purpose. For example, a method for culturing yeast in a medium containing an aryloxyphenoxypropionic acid-based herbicide and obtaining a resistant strain can be mentioned.

[0030] The yeast used to obtain the resistant strain is not particularly limited and can be appropriately selected depending on the purpose. For example, a yeast strain obtained by subjecting the parent strain to the mutation treatment can be used.

[0031] The medium containing an aryloxyphenoxypropionic acid-based herbicide contains at least an aryloxyphenoxypropionic acid-based herbicide, and further contains other components as necessary.

[0032] As the medium containing the aryloxyphenoxypropionic acid-based herbicide, a medium generally used for culturing yeast to which the aryloxyphenoxypropionic acid-based herbicide and, if necessary, other components have been added can be used. The medium generally used for culturing yeast is not particularly limited and can be appropriately selected depending on the purpose. Examples of the medium include SD medium and YPD medium.

[0033] The form of the medium is not particularly limited and can be appropriately selected depending on the purpose. Examples of the form include solid and liquid.

[0034] -Aryloxyphenoxypropionic acid herbicides- The aryloxyphenoxypropionic acid herbicide is not particularly limited and may be appropriately selected depending on the purpose. Examples thereof include fluazifop, haloxyfop, fenoxaprop, quizalofop-ethyl, diclofop-methyl, and the like. The aryloxyphenoxypropionic acid-based herbicides may be used alone or in combination of two or more kinds. As the aryloxyphenoxypropionic acid-based herbicide, commercially available products can be used.

[0035] The concentration of the aryloxyphenoxypropionic acid herbicide in the medium is not particularly limited and can be appropriately selected depending on the type of the aryloxyphenoxypropionic acid herbicide. For example, fluazifop is preferably 200 ppm or more, haloxyfop is preferably 75 ppm or more, and fenoxaprop is preferably 50 ppm or more. The upper limit of the concentration of the aryloxyphenoxypropionic acid herbicide in the medium can be appropriately selected within a range in which a resistant strain can be obtained.

[0036] -Other ingredients- Other components in the medium are not particularly limited, and can be appropriately selected from components generally used for culturing yeast depending on the purpose. For example, the other components can be the same as those described in the above section <Step of selecting a strain lacking isoamyl acetate-degrading esterase function>. The contents of other components in the medium are not particularly limited and can be appropriately selected depending on the purpose.

[0037] The culture temperature is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include 10°C to 40°C. The culture period is not particularly limited and can be appropriately selected depending on the purpose, and may be, for example, 1 to 10 days. The culture method is not particularly limited and can be appropriately selected depending on the purpose. Examples of the culture method include static culture and shaking culture.

[0038] The method for selecting a strain having improved alcohol acetyltransferase activity compared to the parent strain from among the strains resistant to the aryloxyphenoxypropionic acid-based herbicide is not particularly limited and can be appropriately selected depending on the purpose. For example, the alcohol acetyltransferase activity of each of the parent strain and the resistant strain can be measured, and a strain having improved alcohol acetyltransferase activity compared to the parent strain can be selected.

[0039] The method for measuring the alcohol acetyltransferase activity is not particularly limited and can be appropriately selected depending on the purpose. For example, the activity can be measured by the method of T. Fujii et al. (Appl. Environ. Microbiol., 60, 2786(1994)).

[0040] When selecting a strain having improved alcohol acetyltransferase activity compared to the parent strain, the degree of improvement in alcohol acetyltransferase activity is not particularly limited and can be selected appropriately depending on the purpose. For example, a strain having an improved alcohol acetyltransferase activity of 120% or more compared to the parent strain can be selected.

[0041] <Selection process of high isobutanol producing strains> The step of selecting a high isobutanol-producing strain is a step of selecting a strain that produces a larger amount of isobutanol than a parent strain.

[0042] The method for selecting a strain that produces more isobutanol than the parent strain is not particularly limited and can be appropriately selected depending on the purpose, but it is preferable to select a strain that produces more isobutanol than the parent strain from among strains that are resistant to a valine analogue.

[0043] The method for obtaining a strain resistant to a valine analogue is not particularly limited and can be appropriately selected depending on the purpose. For example, a method for culturing yeast in a medium containing a valine analogue and obtaining a resistant strain can be mentioned.

[0044] The yeast used to obtain the resistant strain is not particularly limited and can be appropriately selected depending on the purpose. For example, a yeast strain obtained by subjecting the parent strain to the mutation treatment can be used.

[0045] The medium containing a valine analog contains at least a valine analog, and further contains other components as necessary.

[0046] As the medium containing the valine analog, a medium generally used for culturing yeast may be used to which the valine analog and, if necessary, other components have been added. The medium generally used for culturing yeast is not particularly limited and can be appropriately selected depending on the purpose. Examples of the medium include SD medium and YPD medium.

[0047] The form of the medium is not particularly limited and can be appropriately selected depending on the purpose. Examples of the form include solid and liquid.

[0048] -Valine analogue- The valine analogue is not particularly limited and can be appropriately selected depending on the purpose. Examples of the valine analogue include norvaline, α-aminobutyric acid, 2-thiazolealanine, and azaleucine. The valine analogues may be used alone or in combination of two or more. The valine analogues may be commercially available.

[0049] The concentration of the valine analog in the medium is not particularly limited and can be appropriately selected depending on the purpose, but is preferably 1,000 ppm or more, more preferably 2,000 ppm or more. The upper limit of the concentration of the valine analog in the medium can be appropriately selected within a range in which a resistant strain can be obtained.

[0050] -Other ingredients- Other components in the medium are not particularly limited, and can be appropriately selected from components generally used for culturing yeast depending on the purpose. For example, the other components can be the same as those described in the above section <Step of selecting a strain lacking isoamyl acetate-degrading esterase function>. The contents of other components in the medium are not particularly limited and can be appropriately selected depending on the purpose.

[0051] The culture temperature is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include 10°C to 40°C. The culture period is not particularly limited and can be appropriately selected depending on the purpose, and may be, for example, 1 to 10 days. The culture method is not particularly limited and can be appropriately selected depending on the purpose. Examples of the culture method include static culture and shaking culture.

[0052] There is no particular limitation on the method for selecting a strain that produces more isobutanol than the parent strain from among the strains resistant to the valine analogue, and the method can be appropriately selected depending on the purpose. For example, a so-called small-scale sake brewing test in which sake production is scaled down can be carried out using each of the parent strain and the resistant strain, and the isobutanol concentration of the final sake that is transferred to the tank can be measured to select a strain that produces more isobutanol than the parent strain.

[0053] The scale, fermentation temperature, and period of the small-scale sake brewing test are not particularly limited and can be appropriately selected depending on the purpose. The analysis may be performed on a sake production scale.

[0054] The method for measuring the isobutanol concentration of the refined alcoholic beverage is not particularly limited and can be appropriately selected depending on the purpose. For example, the measurement can be performed in accordance with the National Tax Agency's Specified Analysis Method 3-18.

[0055] When selecting a strain that produces more isobutanol than the parent strain, the degree of increase in isobutanol production is not particularly limited and can be selected appropriately depending on the purpose. For example, a strain that produces more than three times the isobutanol production of the parent strain can be selected.

[0056] The order in which the isoamyl acetate decomposing esterase function-deficient strain selection step, the alcohol acetyltransferase high activity strain selection step, and the isobutanol high production strain selection step are performed is not particularly limited and can be appropriately selected depending on the purpose, but it is preferable to perform the isoamyl acetate decomposing esterase function-deficient strain selection step, the alcohol acetyltransferase high activity strain selection step, and the isobutanol high production strain selection step in this order. As the yeast to be selected in each of the selection steps, a yeast strain selected in a selection step carried out prior to the selection step can be used.

[0057] <Other processes> The other steps are not particularly limited as long as they do not impair the effects of this embodiment, and can be appropriately selected depending on the purpose. For example, there can be mentioned a step of confirming that the selected yeast strain is a strain that produces isobutyl acetate at a high level compared to the parent strain.

[0058] There are no particular limitations on the method for confirming that the selected yeast strain is a strain that produces a high amount of isobutyl acetate compared to the parent strain, and the method can be appropriately selected depending on the purpose. For example, a so-called small-scale sake brewing test, in which sake production is scaled down, can be carried out using each of the parent strain and the selected yeast strain, and the isobutyl acetate concentration of the final sake that has been transferred to the tank can be measured to confirm the result.

[0059] The scale, fermentation temperature, and period of the small-scale sake brewing test are not particularly limited and can be appropriately selected depending on the purpose. The analysis may be performed on a sake production scale.

[0060] The method for measuring the isobutyl acetate concentration in the refined alcoholic beverage is not particularly limited and can be appropriately selected depending on the purpose. For example, the concentration can be measured according to the National Tax Agency's Specified Analysis Method 3-18.

[0061] The degree of "high production of isobutyl acetate" compared to the parent strain should be at least higher than the isobutyl acetate concentration when produced using the parent strain, and can be selected appropriately depending on the purpose.

[0062] According to the yeast breeding method of this embodiment, by performing all of the above-mentioned selection steps, a yeast strain that highly produces isobutyl acetate can be obtained with a high probability.

[0063] (yeast) The yeast of this embodiment is Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) HL430 strain, which can be bred by the yeast breeding method of the present embodiment described above. When the HL430 strain is used to produce sake, it is possible to obtain sake that contains a high concentration of isobutyl acetate, has a pleasant aroma such as a fruity aroma (melon aroma), a mash aroma, a freshly squeezed feeling, and a cool and refreshing taste, and has a suppressed solvent odor.

[0064] The HL430 strain was applied for deposit at the National Institute of Technology and Evaluation, Patent Microorganism Depositary (NPMD) (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818) and was accepted as NITE P-03894 on May 25, 2023.

[0065] (Sake) The sake of this embodiment is sake having an isobutyl acetate concentration of 1.0 ppm or more when converted to an alcohol content of 15%. In this embodiment, sake refers to sake that is classified as brewed alcoholic beverages under the Liquor Tax Act.

[0066] The sake of this embodiment is preferably non-genetically modified sake. In other words, it is preferably not sake brewed with a genetically modified yeast. Since products using genetically modified organisms that require genetic modification labeling can be clearly distinguished by consumers and are difficult to accept by consumers, non-genetically modified sake is advantageous.

[0067] The concentration of the isobutyl acetate in sake is not particularly limited as long as it is 1.0 ppm or more when converted to an alcohol content of 15%, and can be appropriately selected depending on the purpose, but is preferably 1.5 ppm or more, more preferably 2 ppm or more, even more preferably 3 ppm or more, and particularly preferably 4 ppm or more. If it is within the above preferred range, it is advantageous in that it can produce sake with better pleasant aromas such as fruity aroma (melon aroma), mash aroma, freshly squeezed feeling, and cool feeling, and with less solvent odor. The upper limit of the concentration of the isobutyl acetate in sake can be appropriately selected within a range that does not impair the effects of this embodiment.

[0068] The sake of this embodiment is preferably not mixed with other alcoholic beverages such as shochu, etc. By producing isobutyl acetate using yeast, sake with a better flavor can be obtained.

[0069] There are no particular limitations on the sake meter value, alcohol content, total acidity, and amino acid content of the sake, and these can be appropriately selected depending on the purpose.

[0070] The types and concentrations of aroma components contained in the sake other than isobutyl acetate are not particularly limited as long as they do not impair the effects of this embodiment, and can be appropriately selected depending on the purpose.

[0071] The type of sake is not particularly limited and can be appropriately selected depending on the purpose. It may be a specially designated sake or an ordinary sake that is not classified as a specially designated sake. Examples of the specified name sake include ginjo sake, daiginjo sake, junmai sake, junmai ginjo sake, junmai daiginjo sake, special junmai sake, honjozo sake, and special honjozo sake.

[0072] The method for producing sake is not particularly limited and can be appropriately selected depending on the purpose, but sake can be preferably produced by the method for producing sake of the present embodiment described below.

[0073] (Sake production method) The method for producing sake according to this embodiment can be selected from ordinary methods for producing sake, as appropriate, depending on the purpose, except that the yeast according to this embodiment is used. For example, rice koji, rice for fermentation, and water are added to the starter yeast, which is then mashed and fermented to obtain moromi, which is then transferred to the tank for production.

[0074] The sake is not particularly limited and can be appropriately selected depending on the purpose, but a suitable example is the sake of this embodiment having an isobutyl acetate concentration of 1.0 ppm or more when converted to the above-mentioned alcohol content of 15%.

[0075] According to the method for producing sake of this embodiment, the yeast of this embodiment described above is used, so that it is possible to produce sake that is excellent in pleasant aromas such as a fruity aroma (melon aroma), a mash-like aroma, a freshly squeezed feeling, and a cool and refreshing taste, and that has a suppressed solvent odor. EXAMPLES

[0076] The present invention will be described below with reference to test examples, but the present invention is not limited to these test examples.

[0077] (Test Example 1: Yeast breeding) <Isoamyl acetate decomposition esterase function-deficient strain> The yeast strain was suspended in a 3% ethyl methanesulfonate solution, subjected to a mutation treatment at 30°C for 60 minutes, spread on an agar medium containing 250 ppm isoamyl monofluoroacetate, and left to stand at 30°C for 3 days to obtain a resistant strain. Note that isoamyl monofluoroacetate was synthesized by the method of Watanabe et al. (M. Watanabe, et al.: J. Ferment. Bioeng., 76, 229(1993)). The parent strain was sake yeast ( Saccharomyces cerevisiae ) A (owned by Hakutsuru Sake Brewery Co., Ltd.) was used.

[0078] From the obtained isoamyl monofluoroacetate-resistant strains, a strain (isoamyl acetate-degrading esterase function-deficient strain) was selected whose isoamyl acetate-degrading esterase activity was reduced to less than 35% (5.9 ppb / h / mg) of the parent strain's isoamyl acetate-degrading esterase activity (16.9 ppb / h / mg). Specifically, yeast cells were harvested after overnight culture in 1 mL of YPD liquid medium at 30°C, disrupted, and suspended in 5 mL of 0.1 M potassium phosphate buffer (pH 7.5). This was dispensed into HS / GC vials, and capped with isoamyl acetate to a concentration of 20 ppm. The reaction was then stopped by heating at 80°C for 3 minutes after reacting at 25°C for 5 hours. The amount of isoamyl acetate reduction before and after the reaction was divided by the reaction time (hours) and protein weight (mg) to calculate the isoamyl acetate decomposition esterase activity.

[0079] <Identification of strains with high alcohol acetyltransferase activity> SD agar medium was prepared by adding any of the aryloxyphenoxypropionic acid herbicides shown below in the amounts shown below to the agar medium and adjusting the pH to 3.0. Fluazifop 200 ppm Haloxyfop 75 ppm

[0080] The above-selected isoamyl acetate degrading esterase function-deficient strains, which had been suspended in 3% ethyl methanesulfonate solution and mutated at 30°C for 60 minutes, were spread on the SD agar medium and left to stand at 30°C for 5 to 7 days to obtain resistant strains.

[0081] From the obtained aryloxyphenoxypropionic acid herbicide-resistant strains, a strain (high alcohol acetyltransferase activity strain) was selected in which alcohol acetyltransferase (AATase) activity was improved to 124% (9.42 ppm / h / mg) compared to the AATase activity of the parent strain (7.61 ppm / h / mg). The AATase activity was measured by the method of Fujii et al. (T. Fujii et al.: Appl. Environ. Microbiol., 60, 2786(1994)).

[0082] <Isobutanol high-producing strain acquisition> The strains with high alcohol acetyltransferase activity selected above that had been mutated by UV irradiation for 3 minutes were spread on SD agar medium containing 1,000 ppm norvaline, and left to stand at 30°C for 2 days to obtain resistant strains.

[0083] The norvaline-resistant strains obtained were subjected to a small-scale sake brewing test, and a strain that produced more isobutanol than the parent strain (high isobutanol producer) was selected. Specifically, a small-scale sake brewing test was conducted using the brewing recipe in Table 1 below, and after distillation, the sake was fermented at a constant temperature of 15°C for 14 days, then transferred to a tank and the isobutanol concentration of the resulting sake was measured according to the National Tax Agency's prescribed analytical method 3-18. A strain was selected that had an isobutanol concentration of 334 ppm, 3.5 times higher than the parent strain, which had an isobutanol concentration of 94.9 ppm.

[0084] [Table 1]

[0085] The HL430 strain selected by the above methods was applied for deposit at the National Institute of Technology and Evaluation's Patent Microorganism Depositary (NPMD) (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818), and was accepted as NITE P-03894 on May 25, 2023.

[0086] (Test Example 2: Sake Production) A small-scale sake brewing test was carried out using the control strain, a common sake yeast, and the HL430 strain selected in Test Example 1 above, according to the brewing recipe shown in Table 1 above, and fermented at a constant temperature of 15°C for 14 days after distillation. Both the dried koji rice and gelatinized rice used had a polishing ratio of 70%. After the above fermentation, the sake was transferred to a tank and the sake meter value (NS), alcohol content (Alc.), total acidity (TA), and amino acid content (AA) of the resulting sake were measured according to the National Tax Agency's analytical methods 3-3 to 3-6 (Table 2: general analytical values), and the concentration of each aroma component (ppm) was measured according to the National Tax Agency's analytical method 3-18 (Table 3: aroma component concentration).

[0087] [Table 2]

[0088] [Table 3]

[0089] The aroma components in Table 3 are described as follows: AcH: Acetaldehyde n-PrOH Normal Propanol EtOAc Ethyl acetate i-BuOH Isobutanol i-AmOH: Isoamyl alcohol a-AmOH: Activated amyl alcohol i-BuOAc Isobutyl acetate Ethyl butyrate i-AmOAc Isoamyl acetate EtO Capro Ethyl caproate EtO Capryl Ethyl caprylate

[0090] As shown in Table 3, the concentration of isobutyl acetate in sake produced using the HL430 strain was 5.4 ppm when the alcohol content was 18.8% (equivalent to 4.3 ppm when the alcohol content was 15.0%).

[0091] (Test Example 3: Sensory Evaluation) Using the sake produced in Test Example 2, a sensory evaluation was carried out as follows.

[0092] <Sample preparation> A control and samples (1) to (5) were prepared as described below, and 30 mL of each was transferred into an amber vial with a stopper and sealed. -Control- Regular sake (manufactured by Hakutsuru Sake Brewery Co., Ltd.) was adjusted with water to an alcohol content of 15.0% and used as a control. -Samples (1) to (5)- The sake produced in Test Example 2 was adjusted to an alcohol content of 15.0% by adding water (hereinafter, this may be referred to as "Sake A"). In addition, sake produced using a yeast with low productivity of aroma components was adjusted to an alcohol content of 15.0% by adding water (hereinafter, this may be referred to as "Sake B"). Sake A and sake B were blended so as to obtain the isobutyl acetate concentrations shown in Table 4 below, to prepare samples (1) to (5).

[0093] [Table 4] In Table 4, the isobutyl acetate concentration is shown as the average value (N=3) obtained by analysis using HS / GC-FID (Headspace Gas Chromatography with a Flame-ionization Detector).

[0094] <Evaluation method> Each vial was returned to room temperature and the evaluation was carried out. The evaluator took 1 mL of the sample from the vial and dropped it into a 200 mL beaker, then smelled the aroma inside the beaker and evaluated the following items.

[0095] [Evaluation items] The strength of each of the following scents (i) to (v) was evaluated in comparison with the control, according to the following criteria. Note that (i) to (iv) are pleasant scents, and (v) is unpleasant scent. (i) Fruity aroma (melon aroma) (ii) Moromi-like aroma (iii) Freshness (iv) Cool sensation (a light, refreshing scent reminiscent of a highly volatile ingredient) (v) Solvent-like odor -Evaluation criteria- A: Compared to the control, the above scent is stronger. B: Compared to the control, the above scent is slightly stronger. C: Compared to the control, the above smell is unchanged or the above smell is not detected.

[0096] The results are shown in Tables 5 to 14 below. Table 5 (Fruit-like aroma), Table 7 (Morashi-like aroma), Table 9 (Freshly squeezed feeling), Table 11 (Cooling sensation), and Table 13 (Solvent-like odor) show the results of evaluation based on the above-mentioned evaluation criteria A to C. Table 6 (Fruit-like aroma), Table 8 (Morashi-like aroma), Table 10 (Freshly squeezed feeling), Table 12 (Cooling sensation), and Table 14 (Solvent-like odor) show the results of scoring based on the following criteria. -Scoring criteria- Rating A → 2 points Rating B → 1 point Rating C → 0 points In addition, for evaluations that fell between multiple evaluation criteria, such as "A to B," a score between each evaluation was assigned (for example, 1.5 points for an evaluation of "A to B").

[0097] [Table 5]

[0098] [Table 6]

[0099] [Table 7]

[0100] [Table 8]

[0101] [Table 9]

[0102] [Table 10]

[0103] [Table 11]

[0104] [Table 12]

[0105] [Table 13]

[0106] [Table 14]

[0107] As shown in Tables 5 to 14, the average scores for (i) to (iv), which are favorable aromas, increased with an increase in the concentration of isobutyl acetate. Therefore, it was confirmed that the sake of this embodiment has improved melon-like aroma, mash-like aroma, freshly squeezed feeling, and cool feeling, and suppressed solvent odor. [Accession number]

[0108] NITE P-03894

Claims

1. Sake having an isobutyl acetate concentration of 1.0 ppm or more when converted to an alcohol content of 15%.

2. A method for breeding a yeast that produces isobutyl acetate at a high level, comprising the steps of: selecting a strain having reduced isoamyl acetate decomposing esterase activity compared to a parent strain; selecting a strain having improved alcohol acetyltransferase activity compared to a parent strain; and selecting a strain that produces more isobutanol than a parent strain.

3. 3. The method for breeding yeast according to claim 2, further comprising the step of selecting a strain having reduced isoamyl acetate decomposing esterase activity as compared with a parent strain from among strains having resistance to halogen-containing carboxylates.

4. 3. The method for breeding a yeast according to claim 2, further comprising the step of selecting a strain having improved alcohol acetyltransferase activity compared to a parent strain from among strains having resistance to an aryloxyphenoxypropionic acid-based herbicide.

5. 3. The method for breeding yeast according to claim 2, wherein a strain capable of producing a larger amount of isobutanol than the parent strain is selected from among the strains resistant to the valine analogue.

6. A yeast characterized by being Saccharomyces cerevisiae HL430 strain having accession number NITE P-03894.

7. A method for producing sake, comprising using the yeast according to claim 6.

8. 8. The method for producing sake according to claim 7, wherein the sake has an isobutyl acetate concentration of 1.0 ppm or more when converted to an alcohol content of 15%.

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