JP5506160B2 - A novel wine yeast obtained by crossing shochu yeast and wine yeast and its production method - Google Patents

Description

  The present invention relates to a method for producing a novel and excellent wine yeast strain having excellent alcohol-producing ability, a novel wine yeast strain obtained by the production method, and a method for producing an alcoholic beverage using the strain. Further, the present invention provides a method for efficiently producing a new and excellent predetermined yeast strain, a new and excellent predetermined yeast strain obtained by the production method, and a method for producing an alcoholic beverage using the yeast strain. It is to provide.

  In brewing alcoholic beverages, specific yeast strains suitable for the type of alcoholic beverage are used. Yeasts used as wine yeasts have been selected as strains suitable for winemaking mainly in taste characteristics. On the other hand, shochu yeast selected as a strain suitable for shochu brewing is known to produce a relatively high concentration of alcohol. If the high alcohol-producing ability of shochu yeast can be imparted to wine yeast, it will be possible to brew a higher concentration of grape juice and expand the range of wine produced.

  The high alcohol-producing ability of shochu yeast is thought to be closely related to its alcohol tolerance property. However, as shown in Non-Patent Document 1, since many genes are involved in the alcohol tolerance of yeast, by introducing a gene group related to the alcohol tolerance of shochu yeast into wine yeast by genetic engineering, the alcohol tolerance is increased. Attempts to improve the performance are expected to require a great deal of effort, and even if that effort is expended, there is little possibility of realization and it is not realistic.

  In recent years, breeding by artificial means, that is, breeding by methods such as mutation, mating hybridization, protoplast fusion, transformation, etc., has been carried out in order to create excellent wine yeasts. In general, when introducing a useful trait of another yeast into wine yeast, breeding by methods such as mating hybridization, protoplast fusion, transformation, etc. is taken, but the most reliable method is by mating Cross breeding. Hybrid breeding by conjugation carried out so far includes cross breeding of wild killer yeast and thermofermentable wine yeast (Non-patent Document 2), non-hydrogen sulfide-producing yeast and wine yeast having agglutination ability. Cross breeding (nonpatent literature 3) is mentioned. However, the mating method by conjugation has a problem that the parent strains are required to have distinctly different traits in order to determine that the cells obtained by the mating are hybrid strains. Therefore, when each parent strain does not clearly have a different character, it is difficult to discriminate or detect a hybrid strain.

  Moreover, as a method of obtaining the yeast strain | stump | stock which has conjugation ability, the method obtained through sporulation like patent document 1, for example is common. When spore-forming strains are joined together and crossed, a strain exhibiting various brewing characteristics is obtained as shown in Non-Patent Document 4, but a hybrid strain having the desired trait and not having an inferior trait Therefore, it is necessary to select a good strain from a large number of hybrid strains.

  In addition, it has been attempted to obtain a strain having mating ability without undergoing spore formation from a practical yeast and use it for breeding (Patent Document 2). Such a method itself has been known for a long time as shown in Non-Patent Document 5. However, in this method, genetic differences from the practical yeast used as the parent strain do not occur so much, so it is not possible to expect diverse brewing characteristics for the strain having mating ability.

JP, 2000-245458, A Cross breeding method of wine yeast JP, 2004-16028, A Cross breeding using the auxotrophic mutant of practical yeast X. L. Hu et. Al., “Genetic dissection of ethanol tolerance in the budding yeast Saccharomyces cerevisiae.” Genetics 175: 1479-1487 (2007) S. Hara et. Al., “The breeding of cryophilic killer wine yeasts.” Agric. Biol. Chem. 45: 1327-1334 (1981) R. Romano et. Al., “Improvement of a wine Saccharomyces cerevisiae Strain by a breeding program.” Appl. Environment. Microbiol. 50: 1064-1067 (1985) Taku Kato et al. Acquisition of haploid of sake yeast No. 7 and analysis of brewing characteristics, Abstracts of Annual Meeting 2007 Annual Meeting of Japanese Society of Agricultural Chemistry: 187 N. Nakazawa et. Al., “A method for direct selection of mating-component clones from mating-incompetent industrial strains of Saccharomyces cereviae.” J. Ferment. Bioeng., 78: 6-11 (1994)

  An object of the present invention is to efficiently produce a new and excellent wine yeast strain having an excellent alcohol-producing ability, a new and excellent wine yeast strain having an excellent alcohol-producing ability obtained by the producing method, Another object of the present invention is to provide a method for producing an alcoholic beverage using the yeast strain. The subject of the present invention is a method for efficiently producing a new and excellent predetermined yeast strain, a new and excellent predetermined yeast strain obtained by the production method, and production of an alcoholic beverage using the yeast strain It is to provide a method.

  In order to solve the above problems, the present inventors used 852 strains (9 wine yeasts, 5 sake yeasts, 2 shochu yeasts, 2 top fermenting yeasts) using various yeast strains. 79 combinations) and the alcohol-producing ability of these hybrid strains was examined. Particularly when wine yeast and shochu yeast were crossed, a new and excellent wine yeast strain having high alcohol-producing ability was efficiently obtained. Based on this finding, the present invention has been completed. In the present invention, the yeast hybrid strain having high alcohol-producing ability means that the highest alcohol production concentration in the fermentation test using grape juice described in Example 4 of the present invention described later is higher than the parent strain, and The final alcohol production concentration of the culture solution after 18 days from the start of the main culture in the fermentation test using yeast that can be 16% or more or the grape juice described in Example 4 of the present invention described later is It means yeast that can be 0.1% or more, preferably 1% or more, and more preferably 5% or more in proportion to the higher parent strain. Yeast strains having a high maximum alcohol production concentration or high final alcohol production concentration inevitably have excellent alcohol resistance. In addition, for the sake of convenience, the “yeast to which a marker is added” in the present specification includes, in addition to yeast that has been artificially added a marker to a predetermined yeast that does not have the marker, and the marker obtained by isolation from the beginning. It also includes yeast that has it. In this specification, “%” means “% by mass” unless otherwise specified.

That is, the present invention is (1) a wine yeast which crosses a parent wine yeast imparted with conjugation ability with a parent shochu yeast imparted with conjugation ability, and selects a hybrid strain having a high alcohol-producing ability from the hybrid strains. A method for producing a strain, wherein the parent wine yeast imparted with the conjugation ability is a WE452 strain imparted with conjugation ability, and the parent shochu yeast imparted with the conjugation ability is used for shochu yeast yeast shochu imparted with conjugation ability No. 3 strain (S-3 strain), wherein the combination of the parent wine yeast and the parent shochu yeast is (a) a parent wine yeast provided with an auxotrophic marker, and a parent shochu yeast provided with a respiratory defect marker A combination of (b) a parent wine yeast imparted with a respiratory defect marker and a parent shochu yeast imparted with an auxotrophic marker, or (c) a different nutritional requirement for each of the parent wine yeast and the parent shochu yeast Sex marker In the case of crossing in the combination of (a) or (b), after the crossing, a hybrid strain is isolated using the phenotype of non-auxotrophic and non-respiratory deficiency as a marker, In the case of crossing in the combination of c), after the crossing, the crossing strain is separated using a non-auxotrophic phenotype as a marker, and the crossing strain having the high alcohol-producing ability is the highest alcohol production concentration or final alcohol production. characterized in that it is a hybrid strain having alcohol production ability can achieve more than 16.2% as the concentration relates mETHODS.

The present invention relates to a wine yeast strain obtained by production method described in (2) above (1).

Furthermore, the present invention relates to ( 3 ) a method for producing an alcoholic beverage characterized by inoculating a saccharide-containing material with the wine yeast strain described in ( 2 ) above.

  According to the method for producing a wine yeast strain of the present invention, a novel and excellent wine yeast strain having a high alcohol-producing ability can be efficiently produced. Moreover, since the yeast strain obtained by this production method has higher alcohol-producing ability than ordinary wine yeast, when an alcoholic beverage (especially wine) is brewed using this strain, the alcohol component is high and it is strongly rich. Taste alcoholic beverages (especially wine) can be produced.

  Moreover, according to the production method of the yeast strain of this invention, according to the use of the yeast, a novel and excellent yeast strain can be produced efficiently. In addition, if a yeast strain having properties that are more advantageous for brewing alcoholic beverages than ordinary yeast is produced by this production method, alcoholic beverages are produced by brewing alcoholic beverages using the yeast strains. It is possible to enjoy advantageous effects for brewing alcoholic beverages, such as improving the production efficiency of the product and obtaining an alcoholic beverage that is superior in flavor.

1. Method for producing novel wine yeast strain of the present invention The present inventors crossed various yeasts and measured the fermentative ability of the hybrid strain as described in Tables 2 to 6 of Example 1 described later. As a result, it was found that a new and excellent wine yeast strain having a high alcohol-producing ability can be efficiently produced by crossing wine yeast and shochu yeast. The production method of the novel wine yeast strain of the present invention (production method of the wine yeast strain of the present invention) is based on this finding.

  As a method for producing a wine yeast strain of the present invention, a parent wine yeast imparted with conjugation ability and a parent shochu yeast imparted with conjugation ability are crossed, and a hybrid strain having a high alcohol-producing ability is selected from the hybrid strains. If it is a method, it will not restrict | limit in particular, Here, wine yeast in this invention means the yeast which can ferment grape juice, and can be made into wine, Shochu yeast is fermenting shochu mash and shochu. Means yeast that can. In addition, from the viewpoint of obtaining a hybrid strain having a higher alcohol-producing ability with higher efficiency, it is preferable to use a strain having a high alcohol-producing ability as the parent wine yeast or the parent shochu yeast.

  The method for obtaining the parent wine yeast in the present invention is not particularly limited, and can be separated from nature as an indicator as to whether it can be wine when inoculated into grape juice and fermented, For example, a strain having characteristics suitable for wine brewing in properties such as low temperature fermentability, high temperature fermentability, various flavors, hydrogen sulfide generation properties, foam formation properties, metasulfite resistance, etc. should be separated from nature using the properties as indicators. It is also possible to select from existing strains having the above characteristics. Saccharomyces cerevisiae WE452 strain (Independent Administrative Institution Liquor Research Institute) and Saccharomyces cerevisiae yeast yeast that have characteristics suitable for wine brewing in the above six properties and have high alcohol-producing ability. A third example for sake (Japanese Brewing Association) can be suitably exemplified.

  The method for obtaining the parent shochu yeast in the present invention is not particularly limited, and can be separated from nature as an indicator as to whether it can be shochu when inoculated and fermented with shochu mash, It can also be appropriately selected from existing strains having characteristics suitable for shochu brewing. As an existing shochu yeast having high alcohol-producing ability in addition to characteristics suitable for shochu brewing, Saccharomyces cerevisiae yeast yeast shochu No. 3 (Japan Brewing Association) can be preferably exemplified.

  As a method for conferring conjugation ability to parent wine yeast and parent shochu yeast, conjugation ability is imparted through spore formation induction [for example, Current Protocols in Molecular Biology, Supplement 19 (John Wiley and Sons, Inc., 1992 And the method of imparting conjugation ability by spontaneous mutation without inducing sporulation induction. As a method for conferring conjugation ability by spontaneous mutation without inducing sporulation induction, a method of culturing with a tester strain used for mating type determination and selecting a strain that crosses the tester strain [for example, Current Protocols in Molecular Biology , Supplement 19 (supra)]. In addition, from the viewpoint of obtaining a hybrid strain excellent in alcohol-producing ability with higher efficiency, as a method for imparting conjugation ability in parent yeasts to cross, a method through spore formation induction, or a method by spontaneous mutation without spore formation Can be preferably exemplified. Specific combinations of strains include strains that have been imparted with conjugation ability through spore formation induction, strains that have been imparted with conjugation ability by natural mutation without inducing sporulation induction, or through spore formation induction. Any combination of a strain imparted with a conjugation ability and a strain imparted with a conjugation ability by natural mutation without induction of sporulation may be used.

  As a method of crossing the parent wine yeast and the parent shochu yeast, a method of mixing and cultivating the parent yeast in a state where they can be joined to each other can be preferably exemplified. The state that can be joined to each other means that one parent yeast has an a-type joining type and the other parent yeast has an α-type joining type. In addition, in the case of using a spore-formed strain (spore-derived strain) for any of the parent yeasts, and if the strain has homothallic properties, strains derived from spores obtained from the strain In some cases, the spore-derived strain is preferably cultured directly with the strain of the mating partner.

  As a method for determining the mating type of the parent yeast, a method (eg, the method described in the above-mentioned Patent Document 2) that uses the presence or absence of hybridization with an a-type or α-type tester strain as an index, The presence or absence of expression of a gene controlled by the promoter to be used can be used as an indicator. Preferred examples of the promoter that specifically functions for the mating type include the STE6 promoter that functions specifically in the strain having the a-type mating ability and the MFalpha1 promoter that functions specifically in the strain having the alpha-type mating ability. Can do. Therefore, when a drug resistance plasmid is used as a marker, expression of the reporter gene (for example, PHO5, for example) can be performed by further arranging a promoter functioning specifically in the above-mentioned junction type and a reporter gene such as the PHO5 gene downstream of the plasmid. It is preferable in that the yeast mating type can be easily confirmed using the acid phosphatase activity encoded by the gene as an index. In addition, since wine yeast and shochu yeast are often copper-sensitive, the determination of the presence or absence of hybridization with a tester strain is complemented by, for example, the auxotrophy of the tester strain and the copper sensitivity of the strain to which conjugation is desired. It is also possible to make a judgment based on the presence or absence of a new stock.

  In the method for producing a wine yeast strain of the present invention, after crossing a parent wine yeast and a parent shochu yeast, a hybrid strain having a high alcohol-producing ability is selected from the hybrid strains. From the viewpoint of more easily distinguishing between the hybrid strain obtained by crossing and the parent strain, yeasts with different markers are used as parent wine yeast and parent shochu yeast, and the hybrid strain is determined using the phenotype of each marker as an index. It is preferable to separate efficiently. The types of markers include drug resistance markers for resistance to drugs such as antibiotics such as geneticin (G418), blasticidin S (BS) and aureobasidin A; nutritional requirements such as amino acids, purine bases and pyrimidine bases Examples include non-drug resistance markers related to non-drug resistance such as auxotrophic markers for sex; respiratory deficiency markers; and the like. The above markers may be used in combination for each yeast.

  Examples of a method for isolating a hybrid strain using each marker given to the parent yeast as an index include, for example, a method for isolating a strain having both or complementary phenotypes of the respective markers from a mixed culture of the parent yeast. The aforementioned separation can be performed by culturing in an appropriate medium according to the properties of each marker. For example, in the case of a drug resistance marker, a medium containing the drug can be used, and in the case of an auxotrophic marker, a medium that cannot grow if it has the auxotrophy and can grow if it does not have the auxotrophy In the case of a respiratory defect marker, a medium that cannot grow if it is a respiratory defect and can grow if it is not a respiratory defect can be used. In addition, when multiple types of markers are used in combination for each yeast, it is not necessary to use all of these markers as indicators for the isolation of the hybrid strain. You can select and use those phenotypes as indicators. In addition, whether the separated strain is actually a hybrid strain is determined by the fact that DNA markers such as SNP derived from the parent strain are mixed, or the chromosome derived from the parent strain is mixed by pulse field gel electrophoresis. It can be confirmed by investigating that the DNA content is higher than that of the parent strain.

  The marker can be appropriately used depending on the purpose. For example, the introduction of a drug resistance marker gene is preferable in terms of being relatively simple and quick, but when a genetically modified yeast strain into which the marker gene has been introduced is used for the production of an alcoholic beverage, a genetically modified food Therefore, it can be suitably used for a preliminary cross test at the preparation stage (for example, a cross test for examining a particularly preferred combination of wine yeast and shochu yeast). On the other hand, the provision of auxotrophic markers and respiratory deficiency markers by natural mutations requires labor and time compared to the introduction of marker genes, but these markers cannot be used for genetically modified foods. It can use especially suitably for the crossing test for obtaining. Therefore, a step of crossing wine yeast and shochu yeast given conjugation ability and different drug resistance markers; a step of separating a hybrid strain using each of the drug resistance markers as an index; and a high alcohol-producing ability from the separated hybrid strain All of a step of selecting a hybrid strain; and a step of using a non-drug-resistant wild-type parent yeast of the selected hybrid strain as a parent wine yeast imparted with a mating ability and a parent shochu yeast imparted with a mating ability, respectively. The method for producing a wine yeast strain characterized by containing That is, first, any two types of yeast strains each imparting different drug resistances are prepared by introducing plasmids each holding a different drug resistance marker gene, and then crossed, and the above-mentioned hybrid strains are selected from the hybrid strains. By separating the drug-resistant strains that can grow in the medium containing each of these drugs, inoculating the separated strains on the saccharide-containing material, brewing the saccharide-containing material, and measuring the alcohol concentration of the brewed product Select strains with high alcohol production ability. By performing such a series of operations, a promising combination of parent yeasts capable of producing a hybrid strain having a high alcohol-producing ability can be specifically identified. In addition to selection for high alcohol-producing ability by measuring the alcohol concentration, measurements on other indicators and evaluations, for example, ester production by measuring the amount of esters such as ethyl acetate and isoamyl acetate in brewed products Specific promising combinations of parent yeasts that can produce hybrid strains that are superior in flavor and in addition to high alcohol-producing ability by performing sensory evaluation by evaluating performance and sensory evaluation of brewed products It can also be specified. Next, for the promising specific combinations of the above-mentioned parent yeasts selected, each of the yeast strains to which an auxotrophic marker and a respiratory defect marker, or a different auxotrophic marker were added, was then crossed, From the hybrid strain, isolate the strain with both markers complemented, inoculate the separated strain into the saccharide content, brew the saccharide content, and measure the alcohol concentration of the brew. It is possible to select a yeast strain having a high alcohol-producing ability and suitable for practical use. For this selected strain, by further measuring and evaluating the other indicators as described above, selecting a yeast strain for practical use that is superior in flavor in addition to high alcohol-producing ability. You can also. By using the production method as described above, it is possible to enjoy both the advantages of drug resistance markers and the advantages of non-drug resistance markers (such as auxotrophic markers and respiratory deficiency markers). Specifically, a yeast combination that yields a strain with a high alcohol-producing ability by crossing can be efficiently examined using a drug resistance marker. Based on the yeast combination, an auxotrophic marker and a respiratory defect By using a combination of markers or a combination of different auxotrophic markers, there is no problem in terms of regulation of genetically modified foods and consumer anxiety, and a novel alcohol-producing ability A wine yeast strain can be produced efficiently. When a drug resistance marker is used, a combination of geneticin resistance marker and blasticidin S resistance marker can be preferably exemplified. In addition, the non-drug-resistant wild-type parent yeast of the selected hybrid strain gives a drug-resistance marker in both yeasts used for crossing wine yeast and shochu yeast that have been given a mating ability and different drug-resistance markers. It means the previous yeast.

  In addition, as a combination of parent strain markers when producing a wine yeast strain for practical use, for example, a combination of a parent wine yeast provided with an auxotrophic marker and a parent shochu yeast provided with a respiratory defect marker, A combination of a parent wine yeast to which a respiratory deficiency marker is given and a parent shochu yeast to which an auxotrophic marker is given can be suitably exemplified, and a parent wine yeast to which a respiratory deficiency marker is given and an auxotrophic marker are given A combination of the parent wine yeast and the parent shochu yeast provided with different auxotrophic markers (for example, lysine requirement and uracil requirement) may be used. . In this case, conjugation ability to the parent yeast is induced by inducing spore formation from yeast to which an auxotrophic marker has been added, and by inducing spore formation from yeast to which a respiratory defect marker has been added. It is preferable that conjugation ability is imparted by natural mutation.

  Examples of a method for imparting the above-described drug resistance marker include a method of inducing resistance to a drug by culturing in a medium containing the drug, and a plasmid containing a drug resistance marker gene (hereinafter referred to as “drug resistance marker plasmid”). The method of transforming yeast can be exemplified by the above), but a method of transforming yeast with a drug resistant plasmid is preferably exemplified in that it can impart drug resistance more easily and efficiently. it can. Specific examples of the drug resistance marker plasmid include pYT74, pYT75, pKN12 and pKN13 shown in FIG. These four types of drug resistance marker plasmids all have the URA3 gene that complements the uracil requirement with the sequences of ARS1 and CEN3, which are necessary for autonomous replication in yeast cells. ARS1, CEN3 and URA3 sequences can all be obtained from plasmid pYT37 described in Kobayashi et al. (Mol. Gen. Genet., 251: 707-715, 1996). Among these four types of plasmids, pYT74 and pYT75 have G418 resistance as genes that confer drug resistance to the host, and pKN12 and pKN13 have blasticidin S (BS) resistance. Yes. The G418 resistance gene expression cassette can be obtained from the plasmid pZNEO described in Yamano et al. (J. Biotechnol., 32: 173-178, 1994). This G418 resistance gene expression cassette is controlled by the promoter and terminator of the budding yeast-derived PGK1 gene. On the other hand, a BS resistant gene expression cassette was obtained by PCR using pSV2bsr (manufactured by Funakoshi) by PCR with reference to the DNA base sequence described in an academic paper by Kobayashi et al. (Agric. Biol. Chem., 55: 3155-3157, 1991). The gene can be prepared by amplifying the resistance gene BSR and linking it to a budding yeast-derived TDH3 gene promoter and a PGK1 gene terminator.

  Methods for transforming yeast with the aforementioned drug resistance marker plasmid include protoplast method (Proc. Natl. Acad. Sci. USA, 75: 1929 (1978)), metal treatment method (J. Bacteriol., 153: 163 ( 1983)), electroporation (edited by Taiji Oshima, published by the Society Publishing Center, 1996), and the like. From the viewpoint of simplicity, the electroporation can be preferably exemplified.

  Examples of the method for imparting the above-mentioned auxotrophic marker include a method of selecting a strain that has become required for various amino acids, purine bases, pyrimidine bases, etc. by mutation. Specifically, a method for imparting a lysine requirement marker to a predetermined yeast strain by culturing a predetermined yeast strain in a medium containing α-aminoadipic acid and selecting a strain resistant to α-aminoadipic acid Or culturing a predetermined yeast strain in a medium containing 5-fluoroorotic acid and selecting a strain resistant to 5-fluoroorotic acid to give a uracil-requiring marker to the predetermined yeast strain be able to. The mutation may be a mutation induced by ordinary mutation treatment, but the strain used for practical use is preferably a strain obtained by natural mutation.

  As a method for imparting the above-mentioned respiratory defect marker, a predetermined yeast strain is cultured in a medium containing glycerol as a sole carbon source, and a strain that cannot grow on the medium is selected, whereby a respiratory defect marker is added to the predetermined yeast strain. And a method of imparting a respiratory defect marker to a predetermined yeast strain by culturing a predetermined yeast in a medium containing a low concentration of ethidium bromide or spontaneously mutating it to delete mitochondria. It can be illustrated.

A method for selecting a hybrid strain having a high alcohol-producing ability from the hybrid strain is not particularly limited. For example, a method of measuring the alcohol-producing ability of a hybrid strain and isolating a strain having a high alcohol-producing ability can be exemplified. . As a method for measuring the alcohol-producing ability, for example, the amount of alcohol produced under a condition in which dissolved oxygen is limited in a medium containing an appropriate amount of glucose or fructose can be measured by capillary gas chromatography or F-kit ethanol (J K. International Co., Ltd.) or Alcomate (Riken Keiki Co., Ltd.). Also, instead of directly measuring the amount of alcohol produced, the sugar content in the medium before and after culturing of the hybrid strain is measured with a digital sugar content meter (manufactured by Inoue Seieido Co., Ltd.), etc., and the amount of sugar reduced by cultivation is calculated. The amount of alcohol produced can also be examined indirectly. As a strain having a high alcohol-producing ability as described above, in a fermentation test of a strain obtained by crossing using a non-drug resistant marker described in Example 4 described later, an alcohol-producing ability higher than any of the two types of parent strains is obtained. A strain having an alcohol-producing ability that is 10% or more higher than the parent strain having a high alcohol-producing ability among the two kinds of parent strains can be exemplified more preferably.
In addition, as other strains having high alcohol-producing ability, the highest alcohol production concentration or the final alcohol production concentration in the fermentation test of the strain obtained in the cross using the non-drug resistance marker described in Example 4 described later is preferably 16.2% or more, more preferably 16.6% or more, further preferably 17.2% or more, even more preferably 17.6% or more, particularly preferably 18.0% or more, and further preferably 18.5%. More preferably, 19.0%, even more preferably 19.5% or more, and most preferably 20% or more of the strain can be mentioned. In addition, the yeast strain obtained by selection can be cultured, subcultured and maintained in a normal YPD medium or the like. Moreover, as a strain | stump | stock with the above-mentioned high alcohol-producing ability to isolate | separate, it is preferable that it is a strain | stump | stock with a quicker alcohol production | generation rate.

  As a particularly preferable embodiment in the method for producing a wine yeast strain of the present invention, “a strain having a respiratory defect marker added to WE452 strain (wine yeast) and conjugating ability by natural mutation without inducing sporulation” ”And“ a strain that gave an auxotrophic marker to Saccharomyces cerevisiae yeast strain No. 3 for shochu yeast and induced sporulation to conjugate ” A method of isolating a hybrid strain in which two properties of respiratory deficiency are complemented and selecting a strain having a high alcohol-producing ability from the obtained hybrid strain can be exemplified. As a yeast strain obtained by this production method, having a high alcohol-producing ability and excellent as a wine yeast in characteristics other than the alcohol-producing ability, the KT378 strain (strain number FERM P-21448) is most preferably exemplified. can do.

2. Production method of the novel yeast strain of the present invention Production method of the novel yeast strain of the present invention (production method of the yeast strain of the present invention) includes adding conjugation ability and different drug resistance markers to any two types of yeast. A step of crossing both yeasts, a step of separating the hybrid strain using each of the drug resistance markers as an index, and selecting a hybrid strain having a property advantageous for brewing an alcoholic beverage from the separated hybrid strain. Steps and conjugation to each of the two non-drug-resistant wild-type yeasts used as parent strains of the selected hybrid strains, followed by hybridization, and brewing of a predetermined hybrid strain, such as an alcoholic beverage, from the hybrid strains As long as it includes all of the steps for selecting a hybrid strain having advantageous properties, the properties advantageous for brewing the alcoholic beverage include high alcohol production ability and high energy. Examples include stealth generation ability and high alcohol production rate. Among them, high alcohol production ability and high ester production ability can be preferably exemplified. In addition, two types of non-drug resistant wild-type yeasts used as parent strains of the selected hybrid strains are given drug resistance markers in both yeasts used for crossing both yeasts to which different drug resistance markers have been given. It means the previous yeast.

  Furthermore, the yeast having high ester-producing ability in the present invention is any one ester (for example, ethyl acetate or isoamyl acetate) in the following ester producing ability confirmation test of a strain obtained by crossing using a non-drug resistant marker. ) Final product concentration is 5% or more, preferably 10% or more, more preferably 40% or more, even more preferably 70% or more, and even more preferably 100% or more, as a proportion of the parent strain having a high final concentration. It means yeast that can.

(Ester production ability confirmation test)
As a pre-culture, the cells of the sample yeast strain are cultured on a YPD agar medium at 25 ° C. for 1 day. Next, 60 mL of a YPD medium with a glucose concentration of 10% (w / v) is placed in a 100 ml Erlenmeyer flask, and the cells obtained by the previous culture are inoculated into one platinum loop. Pre-culture is performed by shaking culture at 25 ° C. for 72 hours. Next, filter-sterilized grape juice (straight juice for white wine (obtained from Kasuka Co., Ltd.) and concentrated fruit juice (obtained from Toshoku Co., Ltd.) were prepared, adjusted to 40 brix, and yeast extract (MERCK) as a nitrogen source. Manufactured product) 2.3 g / L and diammonium hydrogen phosphate added to 1.3 g / L) 200 mL was placed in a 250 ml wide-mouthed medium bottle, and OD600 = 1.0 (about 1.0 × 10 ⁷ cells / mL) Inoculate the cells after pre-culture so that the lid of the wide-mouthed medium bottle is loosened, and rotate at 90 rpm with MAGNETIC STIRRER F-626N (Fine) And anaerobic stirring culture (main culture) at 20 ° C. With respect to the culture solution on the seventh day from the start of the main culture, the amount of ester such as ethyl acetate or isoamyl acetate is measured using capillary gas chromatography (manufactured by Shimadzu Corporation).

  In addition, the yeast having a high alcohol production rate in the present invention means the alcohol production concentration of the culture solution after 18 days from the start of the main culture in the fermentation test using the grape juice described in Example 4 of the present invention described later. It means a yeast that can be 5% or more, preferably 10% or more, more preferably 15% or more as a proportion of the parent strain having a higher concentration.

  The two arbitrary yeasts are not particularly limited, and are described in the classification of Table 1. Wine yeast, sake yeast, shochu yeast, top fermentation yeast, bottom fermentation yeast, whiskey yeast, and other yeasts Any two types of yeast selected from the group consisting of can be used, and these yeasts may be any of natural yeast, genetically modified yeast, wild-type yeast, and mutant yeast. The combination of hybridization may be a combination of yeasts of different classifications, for example, a combination of wine yeast and shochu yeast, or a combination of yeasts of the same classification, for example, a combination of wine yeast and wine yeast. May be. Examples of the two arbitrary yeasts described above include combinations of yeasts in Tables 2 to 6 described later. In addition, when a hybrid strain having a high alcohol-producing ability is selected as a hybrid strain having advantageous properties for brewing alcoholic beverages, the column “Number of strains” in Tables 2 to 6 described below is used as any two types of yeast. The combination of yeasts with the back side of gray is preferably exemplified, and among them, “WE452 (spore) and K11 (natural mutation)”, “WE452 (natural mutation) and S− described in FIG. 3 (spore) ”,“ WE452 (spore) and L2226 (spore) ”,“ K14 (spore) and S-3 (spore) ”,“ Geisenheim 74 (spore) and DV10 (spore) ”are particularly preferably exemplified. Moreover, the combination of wine yeast and shochu yeast, and the combination of wine yeast and sake yeast can also be illustrated preferably. Here, “(spore)” means a strain through the induction of sporulation to impart conjugation ability, and “(natural mutation)” means to confer conjugation ability. This means that the strain uses natural mutation without inducing sporulation.

  Method for conferring conjugation ability, method for imparting drug resistance marker, method for crossing both yeasts, method for isolating hybrid strains using each drug resistance marker as an indicator, method for producing non-drug resistant wild-type yeast The crossing method, the method for separating the crossing strain, and preferred embodiments thereof are the same as the method for producing the wine yeast strain of the present invention described above.

  The method for selecting a hybrid strain having a property advantageous for brewing the alcoholic beverage is not particularly limited because it depends on the property, but a method for measuring the degree of the property and selecting a strain having a high level. Can be illustrated. When the property is a high alcohol-producing ability, the measurement method is as described above, and when the property is a high ester-producing ability, as in the above-described ester producing ability confirmation test, A method for measuring the concentration of the ester component in the culture solution of a predetermined yeast strain by capillary gas chromatography or the like can be exemplified.

  As a particularly preferred embodiment in the method for producing a yeast strain of the present invention, a process of crossing both yeasts after imparting conjugation ability and different drug resistance markers to any two types of yeast, using each of the above drug resistance markers as an index A step of separating the hybrid strain, a step of selecting a hybrid strain having advantageous properties for brewing an alcoholic beverage from the separated hybrid strain, and two types of non-drug resistant wild type yeast used as a parent strain of the selected hybrid strain An auxotrophic marker is given to one of the two, a respiratory deficiency marker or another auxotrophic marker is given to the other, and both yeasts are crossed after conjugating ability to both, followed by non-nutritive and non-respiratory All of the steps of isolating a hybrid strain using the deficient phenotype as a marker, and selecting a hybrid strain having a property advantageous for brewing alcoholic beverages from the hybrid strain. As a more preferred embodiment, as a combination of the above-mentioned different drug resistance markers, an embodiment using a combination of geneticin resistance marker and blasticidin S resistance marker, or the above-mentioned non-drug resistant wild type can be used. As a method for imparting conjugation ability to type yeast, by spore formation is induced from yeast to which an auxotrophic marker is imparted, and from a yeast to which a respiratory deficiency marker is imparted, by spontaneous mutation without inducing spore formation, The aspect which each provides a joining ability can be mentioned.

3. Novel yeast strain of the present invention As the novel yeast strain of the present invention (yeast strain of the present invention), the above-mentioned method for producing the wine yeast strain of the present invention and the above-described method for producing the yeast strain of the present invention (hereinafter, both production methods). In addition, the yeast strain obtained by culturing the yeast strain and the yeast strain obtained by culturing the yeast strain is not particularly limited, but has a property advantageous for brewing alcoholic beverages (high alcohol And yeast strains obtained by culturing the yeast strain and the like, and high alcohol-producing ability, high ester-producing ability, and high alcohol. More preferable examples include yeast strains having two or more properties selected from production rates and yeast strains obtained by culturing the yeast strains.

  Moreover, as a yeast strain | stump | stock which has high alcohol production ability, the strain which has alcohol production ability higher than any of two types of parent strains can be illustrated preferably in the fermentation test as described in Example 4 mentioned later. Among these parent strains, a strain having an alcohol-producing ability that is 10% or more higher than the parent strain having a high alcohol-producing ability can be exemplified more preferably. Moreover, as other strains having high alcohol-producing ability, the highest alcohol production concentration or the final alcohol production concentration in the fermentation test described in Example 4 described later is preferably 16.2% or more, more preferably 16.6% or more. More preferably 17.2% or more, still more preferably 17.6% or more, particularly preferably 18.0% or more, still more preferably 18.5% or more, more preferably 19.0%, even more preferably Examples include 19.5% or more strains, most preferably 20% or more. As a specific example of a preferable yeast strain of the present invention, KT378 strain (strain number FERM P-21448) can be particularly preferably exemplified.

4). Production method of alcoholic beverage of the present invention The production method of alcoholic beverage of the present invention is not particularly limited as long as it is a method of inoculating a saccharide-containing material with the yeast strain of the present invention, preferably a wine yeast strain, and brewing (culturing) it. In this case, the “sugar-containing material” includes grape juice, kiwi juice, apple juice, blueberry juice, apricot juice, and other cereals such as rice, wheat, straw, and corn, and sweet potatoes such as sweet potatoes. Can be illustrated. Examples of the alcoholic beverage include, but are not limited to, fruit wine such as wine; shochu; refined sake; beer; whiskey; brandy; and the like. It can illustrate suitably.

Hereinafter, the production method will be described more specifically by taking fruit wine such as wine as an example.
The yeast strain of the present invention may be directly inoculated into fruit juice, but after culturing on a normal YPD agar medium from a slant of this yeast strain, the obtained bacterial cells are inoculated into a juice medium and cultured (preculture) Alternatively, a seed culture solution for main culture may be prepared. A preferred example of the culture condition for the pre-culture is stationary culture at 25 ° C. for 2 days. A main culture (main brewing) can be performed by inoculating the above-described seed culture liquid into a pre-prepared fruit juice for main culture and culturing. The fruit juice for main culture can be prepared by adjusting the concentration using commercially available concentrated fruit juice and straight fruit juice. As conditions for the main brewing, it can be preferably exemplified by culturing at 14 to 35 ° C., preferably 15 to 25 ° C. for 8 to 14 days. Moreover, you may perform a sugar supplement in the middle of this brewing. Glucose can be used as the supplementary sugar, and it is preferable to adjust the sugar concentration of the culture solution after supplementation to about 20%.

  When the brewed fruit liquor is wine, grape juice can be mainly used as the juice used for brewing, but other fruit juices such as kiwi, apple, blueberry, and apricot can also be used without any particular limitation. Analysis of these fruit juices (specific gravity, direct reducing sugar, total acid, phenol, sugar content, etc.) can be performed according to a conventional method. In addition, the analysis of wine obtained by brewing, for example, measuring alcohol, direct reducing sugar, total acid, coloring degree (absorbance at 420 nm), phenol, hydrogen sulfide, low boiling point aroma components, etc. contained in wine, It can be performed according to a normal method such as evaluation. Further, whether or not the wine has a good taste can be determined not only by the above analysis results but also by evaluating the taste, smell, etc. by a plurality of specialized panelists. Based on the above analysis and the evaluation of specialized panelists, a strain suitable for brewing fruit wine such as wine can be selected from the strains obtained by the production method of the present invention. In addition, in the manufacturing method of the alcoholic beverage of this invention, you may use arbitrary microorganisms together with the yeast strain of this invention.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.
(Common experimental conditions)
Unless otherwise stated, yeast was cultured in YPD medium (non-selective condition) or SD medium (selective condition) described in Current Protocols in Molecular Biology, Chapter 13, Yeast (supra). YPDG medium and YPG medium (METHODS IN YEAST GENETICS, A Cold Spring Harbor Laboratory Course Manual, 1997 Edition) were used as a medium for identifying respiratory deficient strains. As a medium for selectively cultivating the hybrid strain, an SG medium in which the sugar source of the SD medium was changed from 2% glucose to 2% glycerol was used. Yeast conjugation, spore formation induction, and random spore method were all performed by the methods described in Current Protocols in Molecular Biology, Chapter 13, Yeast (supra).

(Yeast strain used in Examples)
Information on yeast strains used in the examples is listed in Table 1.

KY1381 is a yeast strain preserved by Kirin Holdings Co., Ltd., to which a part of the inventor belongs, and Kirin Holdings Co., Ltd. Frontier Technical Research Institute (1-13 Fukuura, Kanazawa-ku, Yokohama, Kanagawa Prefecture 236-0004) Obtained in 5).

[Selection of hybrid parental combinations to produce yeast hybrids with high alcohol production ability]
(1) Measurement of alcohol production ability and fermentation ability of practical yeast strains First, a combination of parent strains for producing a yeast hybrid having high alcohol production ability was selected. Specifically, the alcohol fermentation ability in grape juice was investigated by the following method about 14 types of practical yeast strains described in FIG.
As a pre-culture, each of the aforementioned yeast cells was cultured on a YPD agar medium at 25 ° C. for 3 days. Next, 15 mL of YPD medium with a glucose concentration of 10% (w / v) was placed in a test tube having a diameter of 18 mm and a height of 165 mm, and the cells after the previous culture were inoculated into one platinum loop, and 25 Pre-culture was performed by shaking culture at 48 ° C. for 48 hours. Next, in a test tube having a diameter of 25 mm and a height of 200 mm, filter-sterilized grape juice [mixed straight juice for white wine and concentrated juice to 37 Brix, and 1.3 g / L phosphorus as a nitrogen source] 30 ml of OD600 = 1.0 (about 1.0 × 10 ⁷ cells / mL) added with diammonium oxyhydrogen and 2.6 g / L Farmtech yeast extract (Merck) Inoculate the cells after the pre-culture as above, and perform the fermentation test (main culture) at 20 ° C for 14 days with a rotator RT-550 (manufactured by Taitec Co., Ltd.) at 2.5 rpm. went. The alcohol concentration of the culture solution after the main culture was measured with Alcomate (manufactured by Riken Keiki Co., Ltd.). The result is shown in FIG. As can be seen from FIG. 1, sake yeast generally showed high alcohol-producing ability, but wine yeast varied greatly depending on the strain, and shochu yeast could not find a strain showing particularly high alcohol-producing ability. .

Alcohol-producing ability when cultivated with grape juice is considered to be largely correlated with sugar-consuming ability in a 40% glucose-concentrated medium under anaerobic conditions, so it is simpler than the above-described fermentation test with grape juice. As a method for selecting a parent strain, the fermentability of a practical strain was examined by the following method.
First, as a preculture, 8 types of practical yeast strains including the yeast strain described in FIG. 2 were each cultured with shaking in a YPD medium at 25 ° C. for 1 day. Next, 60 mL of 40% glucose-containing YPD medium (hereinafter simply referred to as “YPD40 medium”) is placed in a 100 mL Erlenmeyer flask, and the cells after preculture are planted therein so that the initial OD600 = 0.16. Bacterial and static culture was performed at 25 ° C. for 7 days. After stationary culture, the appearance fermentation extract was measured with a beer analyzer (manufactured by Kyoto Electronics Industry Co., Ltd.). A part of the result is shown in FIG. Since Geisenheim 74 showed a relatively good fermentability, a strain exhibiting a fermentability equivalent to this strain was designated as “fermentation good”, and a strain having a fermentability superior to this strain by 10% or more as “fermentation excellent”. In the results of FIG. 2, for strains recognized as “fermentation good” or “fermentation excellent”, acquisition of hybrid strains was attempted as described later.

(2) Acquisition of cross strains between practical strains Fig. 3 shows a scheme for acquiring cross strains between practical strains. As can be seen from FIG. 3, different drug resistance marker plasmids were introduced into two types of parent strains (parent strain A and parent strain B), and after conjugating ability was given to the parent strains, they were mixed and cultured. Then, a cross strain was obtained using the above-mentioned drug resistance as an index. PYT74, pYT75, pKN12, and pKN13 were used as plasmids that confer drug resistance to the parent strain. A schematic diagram of these plasmids is shown in FIG. These four plasmids all have the ARS1 and CEN3 sequences necessary for autonomous replication in yeast cells and the URA3 gene that complements the uracil requirement, all of which are from Kobayashi et al. It was obtained from the plasmid pYT37 described in the paper (Mol. Gen. Genet., 251: 707-715, 1996). Among these four types of plasmids, pYT74 and pYT75 have G418 resistance as genes that confer drug resistance to the host, and pKN12 and pKN13 have blasticidin S (BS) resistance. Yes. The G418 resistance gene expression cassette was obtained from the plasmid pZNEO described in Yamano et al. (J. Biotechnol., 32: 173-178, 1994). This expression cassette is controlled by the promoter and terminator of the budding yeast-derived PGK1 gene. On the other hand, a BS resistant gene expression cassette was obtained by PCR using pSV2bsr (manufactured by Funakoshi) by PCR with reference to the DNA base sequence described in an academic paper by Kobayashi et al. (Agric. Biol. Chem., 55: 3155-3157, 1991). The resistance gene BSR was amplified and ligated to a budding yeast-derived TDH3 gene promoter and a PGK1 gene terminator. The cassette for determining the mating type of yeast was obtained from Prof. Shun Harashima, Department of Life Advanced Engineering, Graduate School of Engineering, Osaka University. This cassette is the same as the one contained in plasmids pNN63 and pNN64 described by Nakazawa (Research on Sake Yeast / Research in the 1990s, Sake Yeast / Sake Study Group, published by Japan Brewing Association: p95-99, 2003). The PHO5 gene controlled by the STE6 promoter contained in pNN63 is expressed only in strains having a-type joining ability, whereas the PHO5 gene controlled by MFalpha1 promoter contained in pNN64 is only available in strains having α-type joining ability. Expressed in Utilizing this difference in properties, the yeast zygote was detected by detecting the acid phosphatase activity encoded by the PHO5 gene according to the method described in the Biomolecular Genetics Experimental Method (edited by Taiji Oshima, published by the Academic Publishing Center, 1996). The type was determined.

  Transformation of the parent strain with the aforementioned drug resistance marker plasmid was performed by electroporation in accordance with the method described in Biomolecular Genetics Experiment Method (edited by Taiji Oshima, published by the Academic Publishing Center, 1996). The obtained transformant is subjected to liquid shaking culture in a YPD medium until stationary, the cell is applied to an agar medium, the acid phosphatase activity is examined by the method described above, and a strain expressing the activity is obtained. By isolating, the strain that had acquired conjugation ability by natural mutation was isolated from the above-mentioned transformants. The separated cells were suspended in sterilized water to obtain a spontaneous mutant suspension.

On the other hand, acquisition of a strain imparted with conjugation ability by inducing spore was performed by the following method.
Place 10 mL of spore-forming medium (potassium acetate 1.0%, Difco Yeast Extract 0.1%, glucose 0.05%) into a 100 mL Erlenmeyer flask, inoculate the yeast cells, and continue for 25 weeks or more. After culturing with shaking at 0 ° C., the culture solution is collected by centrifugation, and the obtained cells are suspended in 5 mL of sterile water and 0.25 mL of 2 mg / mL Zymolyase 100T (manufactured by Seikagaku Corporation). ) And 0.01 mL β-mercaptoethanol were added and shaken at 30 ° C. overnight. The obtained culture broth was collected by centrifugation, and the cells were suspended in 1 mL of 1.5% Nonidet P-40 (manufactured by Nacalai Tesque), and then collected by centrifugation, and then again 1 mL of 1 Suspended in 5% Nonidet P-40 (manufactured by Nacalai Tesque), treated with a water bath ultrasonic generator for 10 minutes, and then treated at 60 ° C for 10 minutes to disrupt cells other than spores. . Bacteria were collected again from this disrupted solution, then suspended again in 1 mL of 1.5% Nonidet P-40 (manufactured by Nacalai Tesque), and subjected to ultrasonic treatment for 10 minutes with a water bath ultrasonic generator. Thereafter, the cells were collected by centrifugation, and the cells were suspended in sterilized water to obtain a spore suspension.

  A cross using a parent strain imparted with conjugation ability by natural mutation or a parent strain imparted conjugation ability by inducing spores is caused by spontaneous spore suspensions derived from two parent strains. Mutant suspensions, or a spore suspension derived from one parent strain and a spontaneous mutation suspension derived from one parent strain were mixed and allowed to stand in YPD liquid medium overnight, then 0.4 mg / ML Geneticin (Gibco BRL) and 0.2 mg / mL Blasticidin S (Funakoshi) containing YPD agar medium, and select the hybrids using the resistance to the two drugs given to the parent strain as an index did. As a result, 852 hybrids were obtained from a total of 79 combinations of parent strains.

(3) Primary evaluation of the fermentability of the cross strain The cross strain obtained in Example 1 (2) was statically cultured by the method described in Example 1 (1), and the fermentability was evaluated. The evaluation of fermentability was performed using a digital sugar content meter (manufactured by Inoue Seiyodo Co., Ltd.) with the decrease in sugar content as an index. The results are shown in Tables 2-6. In the table, N in the item of “Method for imparting conjugation ability in parent strain” represents that conjugation ability was imparted without undergoing sporulation induction, and S represents that conjugation ability was imparted via sporulation induction. , The symbol on the left side of “x” represents the method of imparting the mating ability of the parent strain 1 and the symbol on the right side of the parent strain 2. A strain having a residual sugar content lower than that of Geisenheim 74 was evaluated as “fermentation good”, and a strain having a residual sugar content lower than 0.9 times that of Geisenheim 74 was evaluated as “fermentation excellent”. In addition, the strain corresponding to “fermentation excellent” was also counted as “fermentation good”. Furthermore, the back of the “Number of strains” column is shown in gray for a hybrid combination in which one or more fermented strains have been acquired.

  From these 852 hybrids, 228 strains were selected as “fermentation good” strains and 24 strains as “fermentation excellent” strains. There were 16 combinations of parental strains from which the “Fermentation Excellent” strain was obtained.

(4) Secondary Evaluation of Fermentation Ability of Cross Strains For the cross strains that showed good fermentation ability in the primary evaluation of Example 1 (3) above, the fermentability in grape juice was examined by the method described below.
As pre-culture, the cells of the hybrid strain were cultured on a YPD agar medium at 25 ° C. for 3 days. Next, 5 mL of a YPD medium with a glucose concentration of 10% (w / v) is placed in a test tube having a diameter of 18 mm and a height of 165 mm, and the cells obtained by culturing the YPD medium in advance are then inoculated into one platinum loop. After that, preculture was performed by shaking culture at 25 ° C. for 48 hours.

Next, grape juice sterilized by filtration (mixed straight juice for white wine and concentrated juice, adjusted to 37 brix, 1.3 g / L diammonium hydrogen phosphate and 2.6 g / L Farmtech yeast as nitrogen sources) 30 mL of extract (added with Merck)) was put into a test tube having a diameter of 25 mm and a height of 200 mm, and precultured so that OD600 = 1.0 (about 1.0 × 10 ⁷ cells / mL). The subsequent cells were inoculated, and subjected to a fermentation test (main culture) by rotating at 20 ° C. for 14 days with a rotator RT-550 (manufactured by Taitec Co., Ltd.) at a rotation speed of 2.5 rpm. The alcohol concentration of the culture solution after the main culture was measured with Alcomate (manufactured by Riken Keiki Co., Ltd.). The measurement result of the alcohol concentration and the combination of the parent strain of the hybrid strain are shown in FIG. As shown in FIG. 5, it was possible to produce and select a yeast having a high alcohol production capacity that exceeded the DV10 strain showing the highest alcohol production capacity in FIG. 1 and reached a maximum alcohol production concentration of 20%. From the above results, the combination of the cross parents that produces a yeast strain having a high alcohol-producing ability, such as WE452 strain (WE452) and No. 3 strain for yeast shochu (S-3 (spore)) (cross combination shown in FIG. 5) ) Moreover, when the flavor of the wine obtained by the main culture was evaluated, it was found that the wine had a strong and rich taste and an excellent flavor.

[Creation of yeast hybrids with high alcohol production ability for practical use]
The experiment of Example 1 revealed a particularly excellent hybrid parent combination for producing a yeast hybrid having high alcohol-producing ability. However, since the hybrid strain obtained in the experimental system of Example 1 is a recombinant yeast into which a drug resistance marker plasmid has been introduced, it can be used for the production of commercially available alcoholic beverages. It cannot be said that there is no problem at all in terms of anxiety. Therefore, in order to produce a practically high-alcohol-producing yeast hybrid strain suitable for production of alcoholic beverages for commercial use, the following production method that was not genetic recombination was carried out.

(1) Acquisition of tester strain used for determination of yeast conjugation ability First, a tester strain used for determination of yeast conjugation ability was acquired. Specifically, laboratory yeast, Saccharomyces cerevisiae ATCC200875 (MATa, leu2) and ATCC200883 (MATα, trp1, lys2, ura3) were joined by a conventional method, and then sporulation was induced, and a spore-derived strain was further obtained by a random spore method. Got. By applying the obtained strain to an SD medium containing 2 mM copper sulfate and culturing, the copper sulfate resistance, a-type or α-type conjugation ability, and auxotrophy (lysine requirement) from those strains. Two strains having (lys2) or uracil requirement (ura3)) were selected. These two selected strains were named MT1 (MATα, ura3) and MT2 (MATa, lys2), respectively, and used as tester strains in the following experiments. The description in parentheses after the yeast strain name indicates the genotype.

(2) Acquisition of a parent wine yeast strain imparted with a respiratory defect marker and conjugation ability As a parent wine yeast strain for hybridization, first, a parent wine yeast strain imparted with a respiratory defect marker was obtained. Specifically, WE452 strain (Independent Administrative Institution Liquor Research Institute) known as wine yeast is cultured in YPDG medium or YPG medium using glycerol as the sole sugar source, and from these strains, A respiratory-deficient strain due to spontaneous mutation, that is, a strain that formed a small colony on YPDG medium and could not grow on YPG medium with glycerol as the sole sugar source, was named KY1793 strain.

  Subsequently, a strain having further conjugation ability was obtained from the KY1793 strain by natural mutation. Specifically, after culturing the above-mentioned KY1793 strain in a YPD medium, this culture solution is diluted with a YPD medium, and dispensed into a 96-well plate so that there are about 10 cells per well, and overnight at 30 ° C. And cultured. A part of the culture solution is stored, and the culture solution of the tester strain MT2 is added to the remaining culture solution, mixed, cultured at 30 ° C. overnight, and then SD medium containing 1.5 mM copper sulfate (including lysine). And cultured at 30 ° C. for 4 days. By using the ability to grow on this medium as an indicator, a strain capable of mating with the MT2 strain, ie, a strain having a mating type of MATα, was separated from the stored culture solution and named KY2075 strain. did.

(3) Acquisition of an auxotrophic (lysine auxotrophic) marker and a parent shochu yeast strain imparted with conjugation ability As a cross partner of the KY2075 strain, Acquired. Specifically, Saccharomyces cerevisiae yeast yeast shochu No. 3 (Japan Brewing Association) was cultured in an SD medium containing 30 mg / L lysine and 2 g / L α-aminoadipic acid, and their strains. Among them, a lysine-requiring strain by natural mutation, that is, a strain resistant to α-aminoadipic acid capable of growing on the above-mentioned SD medium was obtained and named KY2069 strain. Subsequently, sporulation was induced from the KY2069 strain according to a conventional method, and conjugation ability was imparted by obtaining spores by the random spore method.

(4) Crossing of the parent wine yeast strain and the parent shochu yeast strain The KY2075 strain (a parent wine yeast strain having respiratory deficiency mutation and conjugation ability) and the spore (lysine requirement and conjugation ability) of the aforementioned KY2069 strain Having a parent shochu yeast strain). Specifically, first, mixed culture of spores derived from KY2069 strain and KY2075 strain was performed at 25 ° C. for 24 hours in a 96-well plate into which YPD medium was dispensed. The medium after mixed culture is applied to SG medium (without lysine), and from these strains, the above-mentioned conjugated strains, ie, lysine non-required and glycerol can grow as a single sugar source. Selected hybrid strains. The selected hybrid strains were subjected to pulse field gel electrophoresis by the method described by Tamai et al. (Y. Tamai, et. Al., 1998, Yeast 14: 923-933), the karyotype was examined, and these selected strains were selected. Confirmed to have both parents' chromosomes. Thus, a large number of hybrid strains (hereinafter simply referred to as “cross strains”) of wine yeast strains (KY2075 strain) and shochu yeast strains (KY2069 strain) were obtained.

(5) Selection of high-alcohol-producing yeast strains from hybrid strains A small-scale fermentation test was performed as described later for the aforementioned hybrid strains, and strains with high alcohol-producing ability were selected from the hybrid strains. Specifically, the following method was used.

  As pre-culture, the cells of the hybrid strain were cultured on a YPD agar medium at 25 ° C. for 3 days. Next, a test tube having a diameter of 18 mm and a height of 165 mm containing 15 mL of a YPD medium (hereinafter simply referred to as “YPD10 medium”) with a glucose concentration of 10% (w / v) is prepared. After inoculating the microbial cell obtained by the above in one test tube in the aforementioned test tube, preculture was performed by shaking culture at 25 ° C. for 72 hours.

Next, 30 mL of YPD medium (hereinafter, simply referred to as “YPD40 medium”) with a glucose concentration of 40% (w / v) was placed in a test tube having a diameter of 25 mm and a height of 200 mm, and OD600 = 1.0 ( The cells after pre-culture are inoculated so as to be about 1.0 × 10 ⁷ cells / mL), and at a rotation speed of 2.5 rpm with a rotator RT-550 (manufactured by Taitec Corporation) at 20 ° C. for 16 days A fermentation test (main culture) was performed by rotating culture. The alcohol concentration of the culture solution after the main culture was measured with Alcomate (manufactured by Riken Keiki Co., Ltd.). Some of the results are shown in Table 7.

  As can be seen from the results in Table 7, by crossing the parent wine yeast WE452 strain (KY2075 strain) with the parent shochu yeast Saccharomyces cerevisiae yeast yeast shochu 3 strain (KY2069 strain), It was shown that a strain (crossbred) superior in alcohol-producing ability to the parent wine yeast strain (WE452 strain) and parent shochu yeast (Kyokai yeast shochu No. 3 strain) can be obtained. Among the obtained hybrid strains, KT378 strain (alcohol concentration 18.6%) was selected as a strain excellent in alcohol-producing ability.

[Fermentation test of a selected yeast strain (KT378 strain) with high alcohol-producing ability]
In order to examine the fermentative ability of the selected yeast strain (KT378 strain) having a particularly high alcohol-producing ability obtained in Example 2, a fermentation test was conducted. Specifically, the following method was used.

  As a pre-culture, KT378 strain cells were cultured on a YPD agar medium at 25 ° C. for 3 days. Next, 15 mL of YPD medium with a glucose concentration of 10% (w / v) is placed in a test tube having a diameter of 18 mm and a height of 165 mm, and the cells obtained by the above-mentioned pre-culture are added to one platinum loop. After inoculation, preculture was performed by shaking culture at 25 ° C. for 48 hours.

Next, grape juice sterilized by filtration (mixed straight juice for white wine and concentrated juice, adjusted to 39 brix, further 1.3 g / L diammonium hydrogen phosphate and 2.6 g / L Farmtech yeast as nitrogen sources) 30 mL of extract (added with Merck)) was put into a test tube having a diameter of 25 mm and a height of 200 mm, and precultured so that OD600 = 1.0 (about 1.0 × 10 ⁷ cells / mL). The subsequent cells were inoculated and subjected to a fermentation test (main culture) by culturing at 20 ° C. for 14 days with a rotator RT-550 (manufactured by Taitec Co., Ltd.) at a rotation speed of 2.5 rpm. The alcohol production concentration of the culture solution after the main culture was measured with Alcomate (manufactured by Riken Keiki Co., Ltd.). A part of the results is shown in Table 8.

  From the results shown in Table 8, it was found that the KT378 strain exhibited an alcohol production amount that surpassed that of the parent strain (about 1.25 times that of the WE452 strain) in fermentation using actual grape juice. This KT378 stock has been deposited domestically as the deposit number FERM P-21448 (the deposit date is November 20, 2007) at the National Institute of Advanced Industrial Science and Technology (1-3, East 1-3, Tsukuba City, Ibaraki Prefecture). . Moreover, when the flavor of the wine obtained by the main culture was evaluated, it was found that the wine had a strong and rich taste and an excellent flavor.

[Fermentation test 2 of selected yeast strains with high alcohol-producing ability]
In order to examine in more detail the fermentation ability of the selected yeast strain (KT378 strain) having a high alcohol-producing ability obtained in Example 2, the strain KT378 and its parent strain, No. 3 strain for yeast yeast shochu (S −3 strain) and WE452 strain were subjected to a fermentation test by the following method.

  As a pre-culture, the cells of any of the above yeast strains were cultured on a YPD agar medium at 25 ° C. for 1 day. Next, 15 mL of YPD medium with a glucose concentration of 10% (w / v) is placed in a test tube having a diameter of 25 mm and a height of 200 mm, and the cells obtained by the above-mentioned pre-culture are added to one platinum loop. After inoculation, preculture was performed by shaking culture at 25 ° C. for 3 days.

Next, grape juice sterilized by filtration (mixed straight juice for white wine and concentrated juice, adjusted to 40 brix, 1.3 g / L diammonium hydrogen phosphate and 2.6 g / L Farmtech yeast as nitrogen sources) 30 mL of extract (added with Merck)) was put into a test tube having a diameter of 25 mm and a height of 200 mm, and precultured so that OD600 = 1.0 (about 1.0 × 10 ⁷ cells / mL). The subsequent cells were inoculated, and subjected to a fermentation test (main culture) by rotating at 20 ° C. for 18 days with a rotator RT-550 (manufactured by Taitec Corporation) at a rotation speed of 2.5 rpm. The culture solution during the main culture was sampled on the 13th, 15th, and 18th days from the start of the culture, and the alcohol concentration thereof was measured with Alcomate (manufactured by Riken Keiki Co., Ltd.). The result is shown in FIG. As can be seen from the results in FIG. 6, 18 days after the start of the culture, the hybrid strain (KT378 strain) is 15.0% higher in alcohol concentration (17.3 vol% as an actual measurement value) than the parent strain WE452. showed that. It was also found that the hybrid strain was higher in alcohol production rate than any of the parent strains. Moreover, when the flavor of the wine obtained by the main culture was evaluated, it was found that the wine had a strong and rich taste and an excellent flavor.

It is a figure which shows the alcohol production ability of a practical yeast strain. It is a figure which shows the fermentative ability in the YPD40 culture medium of a practical yeast strain. It is a figure which shows the acquisition scheme of the cross strain between practical strains using a drug resistance marker. It is a figure which shows the schematic diagram of the plasmid which provides chemical | medical agent resistance. It is a figure which shows the alcohol production ability of the selected hybrid strain. It is a figure which shows the alcohol production ability of the selected cross-bred over time.

Claims (3)

A method for producing a wine yeast strain, wherein a parent wine yeast imparted with conjugation ability and a parent shochu yeast imparted with conjugation ability are crossed, and a hybrid strain having a high alcohol-producing ability is selected from the hybrid strains,
The parent wine yeast imparted with the conjugation ability is a WE452 strain imparted with conjugation ability, and the parent shochu yeast imparted with the conjugation ability is No. 3 strain for yeast yeast shochu imparted with conjugation ability (S-3 strain) ) And
A combination of the parent wine yeast and the parent shochu yeast is (a) a combination of a parent wine yeast imparted with an auxotrophic marker and a parent shochu yeast imparted with a respiratory defect marker, and (b) a respiratory defect marker is imparted. A combination of the parent wine yeast and the parent shochu yeast given the auxotrophic marker, or (c) a combination of the parent wine yeast and the parent shochu yeast given different auxotrophic markers,
When crossed in the combination of (a) or (b), after the crossing, isolate the hybrid strain using the phenotype of non-auxotrophic and non-respiratory deficiency as a marker,
When crossed in the combination of (c), after the cross, isolate the cross strain using a non-auxotrophic phenotype as a marker,
The hybrid strain having a high alcohol-producing ability is a hybrid strain having an alcohol-producing ability capable of achieving a maximum alcohol production concentration or a final alcohol production concentration of 16.2% or more. A wine yeast strain obtained by the production method according to claim 1 . A method for producing an alcoholic beverage comprising inoculating a saccharide-containing material with the wine yeast strain according to claim 2 and brewing it.