Method for producing yeast strain with low 4-vinylguaiacol production

Abstract

To provide simple and highly efficient methods for producing yeast strains with reduced 4-VG production.SOLUTION: Disclosed is a method for a producing yeast strain having low 4-vinylguaiacol productivity, the method comprising: selecting from a yeast parent strain having a wild type gene encoding wild type ferulic acid-decarboxylating enzyme and a mutant gene that encodes a mutated enzyme having no or reduced ferulic acid decarboxylating activity heterozygously, a strain having mutated gene homozygously using a drug resistance as a marker. The gene encoding an enzyme that decarboxylates ferulic acid may be a ferulic acid decarboxylase gene and / or phenylacrylic acid decarboxylase gene.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing a yeast strain that produces a reduced amount of 4-vinylguaiacol, which causes an off-flavor in sake. [Background technology]

[0002] Rice, the raw material for sake, contains ferulic acid. Many microorganisms decarboxylate ferulic acid to produce 4-vinylguaiacol (hereafter abbreviated as "4-VG"), which is then oxidized to produce vanillin. In sake, 4-VG causes off-flavors such as smoky, chemical, or spicy odors. It is known that when awamori is aged for a long period of time, 4-VG is oxidized to produce vanillin, which reduces the off-flavor caused by 4-VG and gives the sake a sweet vanillin aroma (Non-Patent Document 1). Although it has not been reported that vanillin is produced from 4-VG in significant amounts that contribute to the aroma during long-term aging of sake, it is desirable to produce sake with reduced 4-VG without performing post-brewing processing.

[0003] [ka]

[0004] In yeast, the reaction of producing 4-VG by decarboxylation of ferulic acid involves the ferulic acid decarboxylase gene (hereinafter sometimes referred to as the "FDC1 gene") and the phenylacrylic acid decarboxylase gene (hereinafter sometimes referred to as the "PAD1 gene"), and it is known that the decarboxylation reaction is possible when both genes are functioning together (Non-Patent Document 2). The sake yeast generally used in sake brewing does not have the ability to decarboxylate ferulic acid because the FDC1 gene has been disrupted. However, some laboratory yeasts and yeast strains isolated from nature have the ability to decarboxylate ferulic acid, and sake brewed using these strains has an off-flavor due to 4-VG. Sake yeast has many genes involved in high production of ethanol and desirable flavor components, and its genetic background is more complex than that of laboratory yeast. Therefore, laboratory yeast with a simple genetic background is often used for research on improving sake brewing methods. In recent years, sake has been brewed using yeast strains isolated from nature. Therefore, there is a demand to reduce the ferulic acid decarboxylation ability, i.e., the 4-VG production ability, of yeast that has the ability to decarboxylate ferulic acid, as in laboratory yeast and yeast strains isolated from nature. On the other hand, yeast bred by gene recombination technology, including genome editing technology, is not desired. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Liquor Sales Management Training Newsletter, December 2006 issue, published by the National Research Institute of Brewing [Non-Patent Document 2] National Research Institute of Brewing (NRIB) Bulletin, March 31, 2016, No. 29 Summary of the Invention [Problem to be solved by the invention]

[0006] An objective of the present invention is to provide a simple and highly efficient method for producing a yeast strain that produces reduced amounts of 4-VG. [Means for solving the problem]

[0007] The present inventors have conducted extensive research to solve the above problems and have obtained the following findings. (1) By selecting a strain that has a mutant gene in a homozygous form from a yeast parent strain that has a wild-type gene encoding an enzyme that decarboxylates ferulic acid and a mutant gene that has a lack or reduced ability to decarboxylate ferulic acid in a heterozygous form using drug resistance as an indicator, a strain that has a reduced ability to decarboxylate ferulic acid compared to the parent strain, i.e., a strain that produces reduced amounts of 4-VG, can be obtained easily and efficiently. (2) A suitable indicator is that the strain exhibits resistance to a proline analogue at a proline analogue concentration of 0.03 to 30 μg / mL, or that the strain exhibits resistance to a cinnamic acid analogue at a cinnamic acid analogue concentration of 0.03 to 10 mM. (3) Sensitivity to cinnamic acid analogues is also a suitable indicator.

[0008] The present invention has been completed based on the above findings, and provides the following [1] to [6]. [1] A method for producing a yeast strain that produces a low level of 4-vinylguaiacol, comprising a step of selecting a strain having a mutant gene in a homozygous form from a yeast parent strain having a wild-type gene encoding an enzyme that decarboxylates ferulic acid and a mutant gene encoding an enzyme that decarboxylates ferulic acid, the mutant gene being defective or reduced in the ability to decarboxylate ferulic acid, in a heterozygous form, using drug resistance as an indicator. [2] The production method according to [1], wherein the gene encoding an enzyme that decarboxylates ferulic acid is a ferulic acid decarboxylase gene and / or a phenylacrylic acid decarboxylase gene. [3] The production method according to [1] or [2], wherein a strain having the mutant gene in a homozygous form is selected using as an indicator the resistance to a proline analogue at a concentration of 0.03 to 30 μg / mL and / or the resistance to a cinnamic acid analogue at a concentration of 0.03 to 10 mM. [4] The production method according to [1] or [2], wherein a strain having the mutant gene in a homozygous form is selected using any one of the following (1) to (3) as an index: (1) A parent strain that is resistant to cinnamic acid analogs at a concentration of 0.1 mM or more is used, and the strain grows at a concentration of 0.05 mM or less of the cinnamic acid analogs. (2) A parent strain that is resistant to cinnamic acid analogs at a concentration of 0.5 mM or more is used, and the strain grows at a concentration of 0.4 mM, 0.2 mM, or 0.05 mM or less of the cinnamic acid analogs. (3) A parent strain that is resistant to cinnamic acid analogs at a concentration of 0.6 mM or 0.8 mM or more is used, and the strain grows at a concentration of 0.5 mM, 0.4 mM, 0.2 mM, or 0.05 mM or less. [5] A method for producing sake by brewing using a yeast strain that produces a low level of 4-vinylguaiacol, which is produced by any one of the methods according to [1] to [4]. [6] Bottled sake having a 4-vinylguaiacol concentration of 500 ppb or less and a vanillin concentration of 0.7 ppm or more. Effect of the Invention

[0009] As described above, the reaction of producing 4-VG by decarboxylation of ferulic acid is possible when the FDC1 gene and the PAD1 gene function. Since the wild-type genes of the FDC1 gene and the PAD1 gene are dominant, yeast strains having the wild-type gene and the mutant gene in a heterozygous form produce 4-VG. Therefore, in order to obtain a yeast strain that produces less 4-VG, which causes off-flavor, it is necessary to select a yeast strain having the mutant PAD1 gene and / or the mutant FDC1 gene in a homozygous form. According to the method of the present invention, by using drug resistance as an indicator, yeast strains with reduced 4-VG production can be obtained simply and efficiently without time-consuming comprehensive selection, etc. This makes it possible to simply and efficiently obtain laboratory yeast or yeast strains isolated from nature with reduced 4-VG production, which can contribute to the development of new sake and other products. Furthermore, since the yeast strains obtained by the method of the present invention are not obtained by genetic recombination technology, they meet the needs of consumers who are avoiding genetically modified foods. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present invention will be described in detail below. (1) Method for producing 4-VG low-producing yeast strain The method for producing a 4-VG low-producing yeast strain of the present invention includes a step of selecting a strain having a wild-type gene encoding an enzyme that decarboxylates ferulic acid and a mutant gene encoding an enzyme that decarboxylates ferulic acid, the mutant gene being defective or reduced in the ability to decarboxylate ferulic acid, in a heterozygous form from a yeast parent strain having this mutant gene in a homozygous form, using drug resistance as an indicator.

[0011] Parent strain of yeast Examples of genes encoding an enzyme that decarboxylates ferulic acid include the ferulic acid decarboxylase gene (FDC1 gene) and the phenylacrylic acid decarboxylase gene (PAD1 gene). Since both genes must function to decarboxylate ferulic acid, the method of the present invention can select a strain in which either or both of the FDC1 gene and the PAD1 gene are homozygous mutant genes. Therefore, as a parent strain, a strain in which either or both of the FDC1 gene and the PAD1 gene are heterozygous between a wild-type gene and a mutant gene can be used. Such strains can be yeasts distributed mainly as brewing yeasts by the Brewing Society of Japan, or by testing stations in each prefecture, or those provided by each country or each operating organization, such as ATCC (American Type Culture Collection), NBRC (NITE Biological Resource. Center), and RIKEN Bioresource Center, which provide various yeasts as resources. In addition, a yeast strain known to be deficient in the FDC1 gene and / or PAD1 gene provided by these organizations may be crossed with a yeast strain in which these genes are not deficient to breed a heterozygous yeast strain of the wild-type gene and the mutant gene and used as a parent strain. For example, Kyokai yeast (preferably Kyokai yeast No. 1001) distributed by the Brewing Society of Japan may be crossed with a laboratory yeast strain such as Saccharomyces cerevisiae S288C (NBRC1136) (preferably laboratory strain D458-5A (ATCC90753)).

[0012] There are no limitations on the parent yeast strain, but it may be of the genus Saccharomyces, and yeasts mainly used in brewing, such as Saccharomyces pastorianus, Saccharomyces bayanus, and Saccharomyces cerevisiae, are preferred, with Saccharomyces cerevisiae being the most preferred. Furthermore, the method of the present invention preferably includes a step of confirming by DNA sequencing that the FDC1 gene and / or PAD1 gene of the parent yeast strain are heterozygous between the wild type and the mutant type.

[0013] An example of the FDC1 gene is a gene consisting of the nucleotide sequence shown in SEQ ID NO: 1 (Saccharomyces genome data base SGD ID: S000002947). SEQ ID NO: 1 is the nucleotide sequence of the FDC1 gene of the laboratory yeast S288C strain. Alternatively, a gene may be used that has a base sequence identity of 90% or more, preferably 95% or more, more preferably 98% or more, and particularly preferably 99% or more with SEQ ID NO: 1 and that encodes an enzyme that decarboxylates ferulic acid. A more preferred gene is one that has a base sequence that differs from SEQ ID NO: 1 by 10 or less bases, and more preferably by 5 or less bases. In the present invention, the identity of a base sequence is a value calculated using genetic information processing software GENETYX (GENETYX Corporation). Also usable is a gene that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to SEQ ID NO: 1 and encodes an enzyme that decarboxylates ferulic acid. In the present invention, "stringent conditions" refers to conditions in which hybridization is carried out in a hybridization solution having a salt concentration of 6×SSC at a temperature of 50 to 60° C. for 16 hours, and washing is carried out in a solution having a salt concentration of 0.1×SSC.

[0014] Among these homologues of the gene consisting of the nucleotide sequence shown in SEQ ID NO:1, preferred are genes consisting of the nucleotide sequence in which G at nucleotide number 9 in SEQ ID NO:1 is replaced with A, G at nucleotide number 219 is replaced with A or C, T at nucleotide number 335 is replaced with C, C at nucleotide number 746 is replaced with T, T at nucleotide number 1389 is replaced with C, and / or A at nucleotide number 1485 is replaced with T. Particularly preferred among the homologues of the gene consisting of the nucleotide sequence shown in SEQ ID NO: 1 is the gene consisting of the nucleotide sequence shown in SEQ ID NO: 2. SEQ ID NO: 2 is the nucleotide sequence of the mutant FDC1 gene of Kyokai No. 7. SEQ ID NO: 2 is a sequence in which G at nucleotide number 9 of SEQ ID NO: 1 is replaced with A, G at nucleotide number 219 is replaced with A, T at nucleotide number 1389 is replaced with C, and A at nucleotide number 1485 is replaced with T.

[0015] A mutant FDC1 gene is a gene that encodes a ferulic acid decarboxylase that lacks or has reduced ferulic acid decarboxylation ability. A mutant FDC1 gene can be obtained by causing a frameshift in a wild-type FDC1 gene by base deletion or insertion, or by introducing a stop codon by base substitution. For example, substituting A at base number 160 of SEQ ID NO: 1 with T generates a stop codon at that position. Alternatively, a mutant FDC1 gene can be obtained by deleting the promoter region of the wild-type FDC1 gene or destroying it by base deletion, insertion, or substitution.

[0016] An example of the PAD1 gene is a gene consisting of the base sequence shown in SEQ ID NO: 3 (Saccharomyces genome data base SGD ID: (S000002946)). Also usable is a gene having a base sequence with 90% or more, preferably 95% or more, more preferably 98% or more, and particularly preferably 99% or more identity with SEQ ID NO: 3 and encoding an enzyme that decarboxylates ferulic acid. More preferred genes are those having a base sequence with 10 or less bases, especially 5 or less, different from SEQ ID NO: 3. Also usable is a gene that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to SEQ ID NO: 3 and encodes an enzyme that decarboxylates ferulic acid.

[0017] Among these homologues of the gene consisting of the base sequence shown in SEQ ID NO: 3, preferred are genes consisting of the base sequence in which C at base number 112 is replaced by T, C at base number 140 is replaced by T, G at base number 172 is replaced by A, C at base number 177 is replaced by T, and / or T at base number 354 is replaced by C.

[0018] The mutant PAD1 gene is a gene encoding phenylacrylic acid decarboxylase lacking or reduced ferulic acid decarboxylation ability. The mutant PAD1 gene can be obtained by frameshifting due to base deletion or insertion, introduction of a stop codon by base substitution, or deletion or destruction of the promoter region, in the same manner as described for the mutant FDC1 gene. For example, a stop codon is introduced by substituting G at base number 305 of SEQ ID NO: 3 with A.

[0019] The parent strain may be a strain obtained by subjecting a strain having a wild type and a mutant type of a gene encoding an enzyme that decarboxylates ferulic acid in a heterozygous state to a mutation treatment, which can efficiently obtain a strain having a mutant gene encoding an enzyme that decarboxylates ferulic acid in a homozygous state. Mutation treatment methods are well known, and examples thereof include chemical treatment using ethyl methanesulfonate (EMS), N-methyl-N-nitro-nitrosoguanidine (NTG), nitrous acid, acridine dyes, etc., ultraviolet irradiation, radiation irradiation, and ion beam irradiation.

[0020] Selection of strain having mutant gene in homozygous form Strains homozygous for the mutant gene encoding the enzyme that decarboxylates ferulic acid are selected using drug resistance as an indicator. The drugs include proline analogs and cinnamic acid analogs. The proline analogue may be any analogue that is toxic to yeast and inhibits its growth, and examples thereof include azetidine-2-carboxylic acid (AZC) and 3,4-dihydro-DL-proline, with azetidine-2-carboxylic acid (AZC) being preferred. The concentration of the proline analogue is preferably 0.03 μg / mL or more, more preferably 0.1 μg / mL or more, even more preferably 0.8 μg / mL or more, and is preferably 30 μg / mL or less, more preferably 10 μg / mL or less, even more preferably 3 μg / mL or less. Under these conditions, strains can be selected based on the resistance to the proline analogue. In addition, the cinnamic acid analog may be any analog (which in this specification includes cinnamic acid) that is toxic to yeast and inhibits its growth, and examples thereof include cinnamic acid, ferulic acid, trans-cinnamic acid, and α-cyano-4-hydroxycinnamic acid. The concentration of the cinnamic acid analogue is preferably 0.03 mM or more, more preferably 0.1 mM or more, even more preferably 0.3 mM or more, and is preferably 10 mM or less, more preferably 3 mM or less, even more preferably 1 mM or less. Under these conditions, strains can be selected based on the resistance to the cinnamic acid analogue.

[0021] In addition, a strain having a mutant form of the gene encoding the enzyme that decarboxylates ferulic acid in a homozygous form can be selected by using the loss or reduction of resistance to cinnamic acid analogues as an indicator. In this case, a parent strain having a heterozygous form of the wild-type and mutant FDC1 genes can be used to select a strain having a homozygous form of the mutant FDC1 gene. Preferably, the strain that has become sensitive to cinnamic acid can be selected by using as an index (1) a strain that is resistant to cinnamic acid analogues under conditions where the concentration of the cinnamic acid analogue is 0.1 mM or more as the parent strain and grows under conditions where the concentration of the cinnamic acid analogue is 0.05 mM or less, (2) a strain that is resistant to cinnamic acid analogues under conditions where the concentration of the cinnamic acid analogue is 0.5 mM or more as the parent strain and grows under conditions where the concentration of the cinnamic acid analogue is 0.4 mM, 0.2 mM, or 0.05 mM or less, or (3) a strain that is resistant to cinnamic acid analogues under conditions where the concentration of the cinnamic acid analogue is 0.6 mM or 0.8 mM or more as the parent strain and grows under conditions where the concentration of the cinnamic acid analogue is 0.5 mM, 0.4 mM, 0.2 mM, or 0.05 mM or less. The lower limit of the cinnamic acid analogue concentration used for selecting a strain that can grow can be 0 μg / mL.

[0022] A strain showing drug resistance may be selected by inoculating a parent strain into a medium containing a drug, culturing the parent strain at 25 to 37°C, preferably 28 to 30°C, for 48 to 120 hours, preferably 72 to 96 hours, and then selecting the strain that shows growth. The medium may be either a solid medium or a liquid medium, but it is simple to inoculate the parent strain into a solid medium and visually confirm the growth of the bacterial cells. The growth of a strain that has become sensitive to cinnamic acid analogues can also be confirmed under similar conditions.

[0023] The above-described selection procedure for strains homozygous for a mutant form of the gene encoding the enzyme that decarboxylates ferulic acid can be repeated two or more times, thereby increasing the occurrence of such homozygous strains. The method of the present invention may further comprise a step of confirming by sequencing whether the selected strain has a mutant gene encoding an enzyme that decarboxylates ferulic acid in a homozygous form.

[0024] According to the method of the present invention, by using a parent strain having a heterozygous wild type and mutant type of FDC1 gene, a strain having a homozygous mutant type of FDC1 gene can be selected, and by using a parent strain having a heterozygous wild type and mutant type of PAD1 gene, a strain having a homozygous mutant type of PAD1 gene can be selected. Also, by using a parent strain having a heterozygous wild type and mutant type of FDC1 gene and PAD1 gene, a strain having a homozygous mutant type of FDC1 gene and PAD1 gene can be selected.

[0025] (2) Method for producing sake The present invention provides a method for brewing alcoholic beverages such as sake (sake, shochu, wine, whiskey, beer, etc.) using the low 4-VG-producing yeast strain obtained by the above-described method of the present invention. There are no particular limitations on the method of producing sake. It can be produced by adding rice koji, rice bran, and water to the starter yeast, saccharifying and fermenting it to obtain moromi, and then performing the step of pressing (a process in which the liquid fraction of the moromi is separated from the sake lees and the liquid fraction is collected, which is the "straining" step as defined by the Liquor Tax Law). Further steps such as heat treatment, removal of sediment, and filtration may be performed. For shochu, wine, whiskey, beer, etc., grapes, grains, etc. may be used.

[0026] The rice used in brewing may be steamed rice or liquefied melted rice. Brewing using melted rice is called liquefied brewing. Melted rice is obtained by adding brewing water and α-amylase, a heat-resistant enzyme, to rice or crushed rice and liquefying it at 60-90℃. If saccharification is also performed in addition to liquefaction, glucoamylase can be added once the liquefaction is completed and the rice is cooled to about 60℃, and saccharification is carried out at about 50-55℃.

[0027] The raw rice (kakemai, kojimai) may be either rice flour or polished rice, but polished rice is preferred. There are no particular limitations on the rice variety, and it may be rice suitable for sake brewing (e.g., Yamada Nishiki, Gohyakumangoku, etc.), or general edible rice, or it may be non-glutinous rice or glutinous rice, as long as it is rice that can be used for sake brewing. In addition, either domestically produced rice or overseas rice may be used, but domestically produced rice is preferred in terms of geographical indications (standards).

[0028] Rice koji is made by growing koji mold (Aspergillus oryzae) on steamed rice. The rice koji used in sake production is stipulated in the National Tax Agency's Notice No. 8 of November 22, 1989, "Establishing standards for sake brewing and quality labeling[1]," which states that "rice koji is made by growing koji mold on white rice, and is capable of converting the starch in the white rice into sugar. Sake with a specific name is limited to sake that contains 15% or more koji rice (the ratio of the weight of koji rice to the weight of white rice; the same applies below)." The koji mold used may be any that can be used in the production of sake, such as Bioc's varieties for daiginjo, for shubo, for moromi, for machine-made koji, for junmai ginjo, for junmai sake, for honjozo, for economical sake, Yoikaori, and for liquefied brewing; and Higuchi Matsunosuke Shoten's varieties for Hikami ginjo, Hi-G, Diamond, Mototate, for moromi, Hikami Moromi No. 20, Hikami Moromi No. 30, Hikami Special Powdered A, Ace Higuchi, Higuchi Powdered Mushroom, Shiramine, Kaori, Strong Saccharification Mushroom, and for liquefied brewing.

[0029] Shubo is made by adding steamed rice, rice koji, and water to yeast to grow it in large quantities. The yeast used is a low 4-VG producing yeast strain of the present invention.

[0030] The brewing process is carried out by putting the yeast starter, rice, rice koji, and water prepared as described above into a fermentation tank. The ingredients may all be added in one stage, or may be divided into multiple stages, or may be added in four stages before the pressurized tank. A three-stage brewing process is preferred so that the yeast concentration is not diluted. The three-stage brewing process is a method of adding rice koji and rice to the yeast starter in three stages in mash making, minimizing changes to the environment of the yeast and preventing the loss of yeast activity.

[0031] The fermentation period of the mash may be 10 to 40 days, preferably 15 to 40 days, and more preferably 20 to 30 days. In the case of a three-stage brewing method, this period may be the period from the distillation (after distillation) to the separating tank. The fermentation temperature of the mash may be 5 to 25°C, and preferably 10 to 20°C. After fermentation is complete, the sake lees are removed and the sake fraction (jozoshu) is collected. For example, the sake lees and the sake fraction can be separated by squeezing, filtration, etc. The jozoshu can be further subjected to filtration, removal of sediment, heating treatment, activated charcoal treatment, etc. as necessary. Shochu and whiskey can be further distilled.

[0032] (3) Low 4-VG-containing sake The present invention provides bottled sake having a 4-VG concentration of 500 ppb or less and a vanillin concentration of 0.7 ppm or more. As shown in the Examples section, in the present invention, it has been found that when the 4-VG concentration in sake is 500 ppb or less, the smoky, chemical or spicy odor derived from 4-VG is masked if the vanillin concentration is 0.7 ppm or more, preferably 0.8 ppm or more, 0.9 ppm or more, 1 ppm or more, 1.1 ppm or more, 1.2 ppm or more, or 2 ppm or more.

[0033] The 4-VG concentration is preferably 300 ppb or less, more preferably 200 ppb or less, and even more preferably 100 ppb or less. The lower limit of the 4-VG concentration can be 0 ppb, but may be 5 ppb or more, 20 ppb or more, or 50 ppb or more. The vanillin concentration is preferably 0.8 ppm or more, more preferably 0.9 ppm or more, more preferably 1 ppm or more, more preferably 1.5 ppm or more, particularly preferably 2 ppm or more. Within this range, the smoky odor, chemical odor, or spicy odor derived from 4-VG is masked, and the resulting sake has a sweet vanillin aroma. The vanillin concentration is preferably 300 ppm or less, more preferably 100 ppm or less, and even more preferably 30 ppm or less. Within this range, the aroma derived from vanillin is not prominent, and the resulting sake has a sake-like aroma.

[0034] The sake of the present invention may be any sake fermented by yeast using rice, rice koji, and water as the main ingredients, but is preferably sake as defined by the Liquor Tax Law (particularly the Liquor Tax Law of Japan). The Liquor Tax Law and various laws and regulations related to the Liquor Tax Law (e.g., the Liquor Tax Law Enforcement Order, etc.) and notices limit the ingredients that can be used for sake to rice, rice koji, water, sake lees, brewer's alcohol, specific organic acids, etc., and do not permit the addition of flavorings that are generally recognized as food additives. In addition, the amount of enzyme preparation used and its purpose are also limited.

[0035] Furthermore, the sake of the present invention may be sake that satisfies the "Standards for Labeling Quality of Sake Production Method" established by a notice from the National Tax Agency (standards for labeling alcoholic beverages established by the Act on the Preservation of Liquor Tax and Liquor Industry Associations, etc.), and examples of sake with specific names include ginjo sake, daiginjo sake, junmai sake, junmai ginjo sake, junmai daiginjo sake, special junmai sake, honjozo sake, and special honjozo sake. Sake that does not fall under these specific names is referred to as "ordinary sake" in this specification. Furthermore, it is preferable that the bottled sake of the present invention has a word indicating sake, such as "sake" or "Nihonshu", which is a geographical indication (standard) for alcoholic beverages designated by the Commissioner of the National Tax Agency, on its container.

[0036] Alcohol content In the sake of the present invention, the alcohol content (v / v%) is not particularly limited as long as it is within the range of 1% or more and less than 22% as stipulated by the Liquor Tax Law, but is preferably 3 v / v% or more, more preferably 5 v / v% or more, and even more preferably 7 v / v% or more. Also, it is preferably 20 v / v% or less, more preferably 18 v / v% or less, and even more preferably 15 v / v% or less. If it is within this range, the sake will have no off-flavor derived from 4-VG and a sweet aroma derived from vanillin, and will have a flavor that is generally preferred by consumers. Alcohol content (alcohol content) is the percentage of the volume concentration of alcohol (ethanol) relative to the total volume of an alcoholic beverage. Alcohol content can be measured by the National Tax Agency's designated analytical method recognized by the Liquor Tax Act, or the "Standard analytical method of the National Research Institute of Brewing" (November 4, 2010, http: / / www.nrib.go.jp / data / nribanalysis.htm) established by the National Research Institute of Brewing, but the alcohol content in this invention is a value analyzed based on the provisions of "3. Sake" in the "Standard analytical method of the National Research Institute of Brewing" established by the National Research Institute of Brewing (hereinafter referred to as the "sake analytical method").

[0037] Sake meter value In the sake of the present invention, there is no particular restriction on the sake meter value, but it is preferably -90 or higher, more preferably -60 or higher, and even more preferably -30 or higher. It is also preferably +30 or lower, more preferably +20 or lower, and even more preferably +10 or lower. If it is within this range, the sake will have no off-flavor derived from 4-VG and a sweet aroma derived from vanillin, and will have an overall flavor that is favored by consumers. The sake meter value is measured using the sake analysis method described above.

[0038] Acidity In the sake of the present invention, there is no particular restriction on the acidity, but it is preferably 0.1 or higher, more preferably 0.2 or higher, and even more preferably 0.4 or higher. It is also preferably 10 or lower, more preferably 6 or lower, and even more preferably 3 or lower. If it is within this range, the sake will have no off-flavor derived from 4-VG and a sweet aroma derived from vanillin, and will have an overall flavor that is favorable to consumers. Acidity is a value that indicates the total amount of free acids (mainly lactic acid, malic acid, succinic acid, etc.) contained in sake. Specifically, acidity refers to the mL of 0.1N sodium hydroxide solution required to neutralize 10 mL of sake. The acidity was measured using the above-mentioned sake analysis method.

[0039] Examples of materials for the container include glass, plastic, paper, ceramic, wood, and combinations thereof. Examples of types of containers include cups, paper cartons, pouches, bottles, plastic tanks, and barrels. The bottled sake of the present invention is preferably stored or filled in these containers. In the case of barrels, the barrels may or may not include barrels in which alcoholic beverages are stored.

[0040] Production method Sake having a 4-VG concentration of 500 ppb or less and a vanillin concentration of 0.7 ppm or more can be produced by brewing sake using the yeast strain of the present invention that produces low levels of 4-VG as described above, and then treating the resulting sake with 4-VG oxidase (e.g., CSO2 derived from Caulobacter segnis, etc.) to convert 4-VG to vanillin. The method for brewing sake can be a general method, except that the yeast strain of the present invention that produces low levels of 4-VG is used. The bottled sake of the present invention does not exclude those that have been stored for a long period (e.g., 6 months or more, particularly 1 year or more, particularly 3 years or more), but may not be stored for a long period (e.g., 6 months or more, particularly 1 year or more, particularly 3 years or more). EXAMPLES

[0041] The present invention will be described in more detail below by way of examples, but the present invention is not limited to these.

[0042] Example 1 <Selection of parent strain> The gene sequence of the U-01 strain, a yeast (Saccharomyces cerevisiae) preserved by Gekkeikan Co., Ltd., was sequenced, and it was revealed that the FDC1 gene was a heterozygous type having two FDC1 genes, a wild-type FDC1 gene in which the base at base number 160 remains A, and a mutant FDC1 gene in which the base at base number 160 is replaced from A to T, as compared with the FDC1 gene consisting of the base sequence of SEQ ID NO: 1 registered in the Saccharomyces genome database as SGD: S000002947. The base sequences of both the wild-type FDC1 gene and the mutant FDC1 gene were sequences in which G at base number 9 was replaced with A, G at base number 219 was replaced with A, and A at base number 1485 was replaced with T in SEQ ID NO: 1.

[0043] <Homozygosis process> The U-01 strain was used as a parent strain and cultured in YPD medium (2% glucose, 2% polypeptone, 1% yeast extract) at 30°C for 1 day. The culture solution of the U-01 strain was spread on an agar medium (2% glucose, 0.67% yeast nitrogen base (containing no amino acids), 2% agar) to which AZC was added to a final concentration of 0.1 g / L, and cultured at 30°C for 4 days. AZC-resistant strains were isolated from the agar medium, and a total of 72 mutant strains were obtained. When the 160th base of the FDC1 gene was confirmed, it was found that of the 72 strains obtained, two strains (strains No. 52 and No. 57) had a mutant FDC1 gene in homozygous form, and two strains (strains No. 44 and No. 51) had a wild-type FDC1 gene in homozygous form.

[0044] <Pilot brewing> A small-scale sake brewing test was carried out using the parent strain and the four mutant strains mentioned above. The fermentation conditions were 15°C for two weeks, the raw materials were rice koji with a polishing ratio of about 70% and steamed rice, and the equipment used was a Fermograph (manufactured by ATTO Co., Ltd.). After the straining, the sake meter value, alcohol content, acidity, amino acid content, and 4-VG were measured. The 4-VG concentration, alcohol content, and acidity of each sake are shown in Table 1. [Table 1]

[0045] Compared to the U-01 strain (parent strain), the mutant homozygous strains No. 52 and No. 57 had almost the same alcohol content, acidity, amino acid content, glucose concentration, and extract concentration, but the 4-VG concentration was significantly reduced. In contrast, the wild-type homozygous strains No. 44 and No. 51 had almost the same alcohol content, acidity, amino acid content, glucose concentration, and extract concentration as the U-01 strain (parent strain), but had increased 4-VG concentration. The method of the present invention made it possible to reduce 4-VG productivity while maintaining brewing properties.

[0046] <Homozygosis of PAD1 gene sequence> The PAD1 gene of the parent strain was homozygous for the wild type, and the No. 52 strain, which had a homozygous mutant FDC1 gene, naturally had a homozygous wild type PAD1 gene. The PAD1 gene of the parent strain was heterozygous for the wild type, with a single nucleotide polymorphism of A and G at the 172nd base. In the No. 52 strain, the 172nd base was homozygous for the wild type, with only G at the 172nd base.

[0047] Example 2 It is known that sake yeast, Kyokai yeast, has only a mutant FDC1 gene with a partial deletion (FDC1 gene is homozygous for mutant). Kyokai yeast No. 7 and U-01 strain (FDC1 gene is heterozygous for wild type and mutant) were spotted on YPD agar medium containing cinnamic acid and cultured at 30°C for 6 days. The final concentration of cinnamic acid was 0.1 mM, 0.2 mM, 0.5 mM, or 1 mM. The results of visual inspection of the extent of yeast growth are shown in Table 2. [Table 2] Very good growth was indicated as ++++, almost no growth was indicated as +, and there were two levels between +++ and ++. No growth was indicated as -.

[0048] Since there was a difference in growth between the two strains at cinnamic acid concentrations in the range of 0.1 to 0.5 mM, by selecting a strain that grows equivalently to Kyokai Yeast No. 7 in this concentration range, it may be possible to obtain a strain that has a homozygous mutant FDC1 gene, such as strain No. 52 in Example 1. In addition, in a medium containing 0.6 to 0.9 mM cinnamic acid, Kyokai Yeast No. 7 may not grow, and only the U-01 strain may be able to grow. Therefore, it was suggested that a strain having the mutant FDC1 gene in homozygous form could be obtained by selecting colonies that do not grow in a medium containing 0.6 mM or more cinnamic acid, but grow in a medium containing 0.5 mM or less cinnamic acid (so-called negative selection).

[0049] Example 3 The parent strain was strain X, which was a cross between a laboratory yeast strain D458-5A (American Type Culture Collection, ATCC90753) and a haploid strain of Kyokai Yeast No. 1001. When the FDC1 gene sequence of this strain X was sequenced, it was found to be heterozygous for both the wild-type FDC1 gene, in which the 160th base remained A, and the mutant FDC1 gene, in which the 160th base was changed to T. The X strain was cultured in YPD medium in the same manner as in Example 1, and the culture solution was spread on an AZC-containing agar medium and cultured to isolate AZC-resistant strains, obtaining a total of 15 mutant strains. The FDC1 genes of the mutant strains were sequenced to confirm the 160th base, and it was found that, of the 15 obtained strains, one strain had a mutant FDC1 gene in homozygous form (strain No. 1), and three strains had a wild-type FDC1 gene in homozygous form.

[0050] Using the parent strain and No.1 strain, test brewing of sake was carried out in the same manner as in Example 1. The fermentation period was 19 days, and the sake meter value, alcohol content, acidity, amino acid content, and 4-VG concentration after transfer were measured. Compared to the parent strain, the No.1 strain had almost the same alcohol content (parent strain 17.3v / v%, No.1 strain 16.1v / v%) and acidity (parent strain 3.0, No.1 strain 2.6), but the 4-VG concentration was greatly reduced (parent strain 6784μg / L, No.1 strain 55μg / L).

[0051] Example 4 A panel of seven experienced specialists conducted a sensory evaluation of the aroma of a control sake in which 4-VG was added to a commercial sake at 500 ppb, and a test sake in which 4-VG was added to a commercial sake at 500 ppb and vanillin was added to a commercial sake at 0.6 ppm, 1.2 ppm, or 2.0 ppm. The commercial sake was classified as a normal sake under the geographical indication of Japanese sake, with an alcohol content of 13.5%, a sake meter value of 1.0, and an acidity of 1.2. The 4-VG and vanillin used in the samples were of the equivalent of special grade reagents.

[0052] The evaluation scores were determined by assessing the degree of difference compared to the control liquor, using the following criteria: 0 points was given if there was no difference in the 4-VG aroma from the control liquor (same composition as in Test 1), 3 points was given if the 4-VG aroma was almost completely masked, and the range in between was divided into thirds, giving evaluations of 1, 2, and so on. Furthermore, the average, standard deviation, and variance of the evaluation scores of the seven panelists were calculated. 0 points: No difference in scent between 4-VG (and no vanillin added). 1 point: I feel like the scent of 4-VG is slightly masked. 2 points: I feel like the scent of 4-VG is slightly masked. 3 points: 4-VG scent is almost completely masked

[0053] The results are shown in Table 3. [Table 3] The aroma of 4-VG was masked in the sake with a vanillin concentration of 1.2 ppm in Test 3 with a significant difference (p<0.01 and p<0.05, respectively, Tukey-Kramer test) compared to the sake in Tests 1 and 2, suggesting that a significant masking effect exists at vanillin concentrations of 0.7 ppm or higher. [Industrial Applicability]

[0054] According to the method for producing a low 4-VG-producing yeast strain of the present invention, laboratory yeasts and strains isolated from nature can be made into low 4-VG-producing strains, thereby contributing to research on yeast strain breeding.

Claims

1. A method for producing a yeast strain that produces a low level of 4-vinylguaiacol, comprising a step of selecting, from a yeast parent strain having, in a heterozygous form, a wild-type gene encoding an enzyme that decarboxylates ferulic acid and a mutant gene encoding an enzyme that decarboxylates ferulic acid, the mutant gene being deficient or reduced in the ability to decarboxylate ferulic acid, a strain having this mutant gene in a homozygous form, using as an indicator the presence of resistance to a proline analogue at a proline analogue concentration of 0.03 to 30 μg / mL and / or the presence of resistance to a cinnamic acid analogue at a cinnamic acid analogue concentration of 0.03 to 10 mM.

2. A method for producing a yeast strain that produces a low level of 4-vinylguaiacol, comprising a step of selecting a strain that has a wild-type gene encoding an enzyme that decarboxylates ferulic acid and a mutant gene encoding an enzyme that decarboxylates ferulic acid, the mutant gene being defective or reduced in the ability to decarboxylate ferulic acid, in a heterozygous form from a yeast parent strain that has this mutant gene in a homozygous form, using any of the following (1) to (3) as an indicator: (1) A parent strain is used that is resistant to cinnamic acid analogs at a concentration of 0.1 mM or more, and the strain is grown at a concentration of 0.05 mM or less of the cinnamic acid analogs. (2) A strain that is resistant to a cinnamic acid analogue under conditions where the concentration of the cinnamic acid analogue is 0.5 mM or more is used as a parent strain, and the strain is grown under conditions where the concentration of the cinnamic acid analogue is 0.4 mM, 0.2 mM, or 0.05 mM or less. (3) A parent strain is used that is resistant to a cinnamic acid analog at a concentration of 0.6 mM or 0.8 mM or more, and the strain is grown at a concentration of 0.5 mM, 0.4 mM, 0.2 mM, or 0.05 mM or less of the cinnamic acid analog.

3. The method according to claim 1 or 2, wherein the gene encoding an enzyme that decarboxylates ferulic acid is a ferulic acid decarboxylase gene and / or a phenylacrylic acid decarboxylase gene.

4. The method for producing sake comprises the steps of: obtaining a yeast strain having a low 4-vinylguaiacol production rate by selecting a strain having the mutant gene in a homozygous state from a yeast parent strain having, in a heterozygous state, a wild-type gene encoding an enzyme that decarboxylates ferulic acid and a mutant gene encoding an enzyme that decarboxylates ferulic acid, the mutant gene being deficient or having a reduced ability to decarboxylate ferulic acid, using as an indicator the strain exhibiting resistance to a proline analogue at a proline analogue concentration of 0.03 to 30 μg / mL and / or the strain exhibiting resistance to a cinnamic acid analogue at a cinnamic acid analogue concentration of 0.03 to 10 mM; and producing sake by brewing using the yeast strain having a low 4-vinylguaiacol production rate.

5. A method for producing sake, comprising the steps of: obtaining a yeast strain that produces a low level of 4-vinylguaiacol by selecting a strain that produces the mutant gene in a homozygous form from a yeast parent strain that has, in a heterozygous form, a wild-type gene encoding an enzyme that decarboxylates ferulic acid and a mutant gene encoding an enzyme that decarboxylates ferulic acid, the mutant gene being deficient or having reduced ability to decarboxylate ferulic acid, using any of the following (1) to (3) as an indicator; and producing sake by brewing using the yeast strain that produces a low level of 4-vinylguaiacol. (1) A parent strain is used that is resistant to cinnamic acid analogs at a concentration of 0.1 mM or more, and the strain is grown at a concentration of 0.05 mM or less of the cinnamic acid analogs. (2) A strain that is resistant to a cinnamic acid analogue under conditions where the concentration of the cinnamic acid analogue is 0.5 mM or more is used as a parent strain, and the strain is grown under conditions where the concentration of the cinnamic acid analogue is 0.4 mM, 0.2 mM, or 0.05 mM or less. (3) A parent strain is used that is resistant to a cinnamic acid analog at a concentration of 0.6 mM or 0.8 mM or more, and the strain is grown at a concentration of 0.5 mM, 0.4 mM, 0.2 mM, or 0.05 mM or less of the cinnamic acid analog.

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