JP2008054560A - New yeast and method for producing sake using the yeast - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To grow and prepare a new mutant yeast suppressing generation of isoamyl alcohol but highly producing other perfume components by hybridization method. <P>SOLUTION: The new mutant yeast having new sake-producing suitability with strong fermentation potency and producing little amount of acids with bringing the produced amount of isoamyl acetate which is called a precursor material of "Mureka", an undesirable, specific sweetish smell of sake, to be small is grown and prepared by treating various fermentation yeasts to mutate by hybridizing a mutant yeast obtained from strains grown on a cerulenin-containing medium and a 5'-5'-5'-trifluoro-D,L-leucine-containing medium, and producing a large amount of ethyl capronate and isoamyl acetate, e.g., Kyokai No.1701 yeast (K-1701), especially Kyokai No.1601 yeast (K-1601) producing a large amount of ethyl capronate and little acids with Kyokai No.9 yeast (K-9). <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to novel yeast and production of alcoholic beverages using the same.

  In recent years, the demand for diversification of alcoholic beverages has increased particularly in recent years. For example, consumer demand for aromatic sake rich in alcohol has increased.

  In order to respond to such needs, breeding of fragrant yeast is performed. For example, one of the methods for obtaining mutants of fragrant yeast is a cerulenin-resistant strain known as an antibiotic that inhibits fatty acid synthase. High yield of isoamyl acetate obtained from a method for obtaining a mutant yeast producing high caproate-producing mutant yeast (Patent Document 1) and a leucine analog 5'-5'-5'-trifluoro-D, L-leucine resistant strain Using two methods of obtaining mutant yeast (Patent Document 2), breeding of mutant yeast that produces isoamyl acetate and ethyl caproate with high production and low acid production has been successful. No. 1601 (K-1601: Non-Patent Documents 1 and 2) and Kyokai Sake Yeast No. 1701 (K-1701: Non-Patent Document 3).

  These mutant yeasts are excellent yeasts, but the disadvantage of low fermentability, which is a disadvantage of mutant yeasts, is unavoidable. Naturally, isoamyl acetate high-producing mutant yeasts are isoamyl acetate, a precursor of isoamyl acetate. A lot of alcohol is also produced. However, isoamyl alcohol is a precursor of isovaleraldehyde, which is said to be a raw chick odor, “Mure smell”, which is one of the causative substances of sake odor.

Therefore, when isoamyl acetate, which is an aroma component, is produced at a high rate, isoamyl alcohol is also produced at a high rate, and in sake made with such a mutant yeast, isoamyl alcohol is oxidized and is said to have a “smelling odor”. It changes into isovaleraldehyde and deterioration of quality, especially scent, is inevitable. This disadvantage is amplified with longer storage periods and higher storage temperatures.
Japanese Patent No. 2632654 Japanese Patent Publication No. 7-14335 Journal of the Japan Brewing Association, 90 (10) 751-758 (1995) Journal of Japan Brewing Association, 8 (7) 565-569 (1993) Journal of the Japan Brewing Association, 96 (10) 679-687 (2001)

  The present invention is a novel mutant yeast having a low production amount of isoamyl alcohol compared to various yeasts, a high yield of ethyl caproate and a well-balanced production of isoamyl acetate, a strong fermenting power and a low acid production amount. The purpose is to create a new. Another object of the present invention is to newly produce alcoholic beverages such as high-scented sake that suppresses the production of isovaleraldehyde, which is one of the raw scents of “green odor”.

  The present invention has been made to achieve the above object, and yeast that produces high amounts of isoamyl acetate usually produces high amounts of isoamyl alcohol, while isoamyl acetate produces high amounts while isoamyl alcohol is produced. However, the present invention has been made in order to breed new yeasts having such properties that are difficult to achieve at the same time.

  Moreover, ethyl caproate was produced in order to breed a new yeast having high properties, high fermentability, and low acid production.

  In order to achieve the above-mentioned object, an object that is difficult to achieve, the present inventors have studied artificially in view of the fact that one of the uses of the bred yeast is the brewing of alcoholic beverages, that is, the production of food and drink. The target mutant yeast was newly bred and constructed by crossing existing yeasts, not by gene manipulation.

  As a result of intensive studies, the present inventors obtained a mutation from brewing yeast and obtained from a strain grown on a cerulenin-containing medium and a 5′-5′-5′-trifluoro-D, L-leucine-containing medium. When a high ester-producing mutant yeast that produces a large amount of ethyl caproate and isoamyl acetate was crossed with sake yeast for brewing sake, the production of isoamyl alcohol was low, but isoamyl acetate was produced in a well-balanced manner, and ethyl caproate was produced. For the first time, we succeeded in obtaining a new mutant yeast that produced a large amount, produced less acid, had a strong fermenting power, and had a good flavor.

  As described above, the mutant yeast according to the present invention has been used as a high-yeast-producing yeast that has been bred in recent years and an excellent yeast that produces excellent sake. For example, yeast that has been widely used as a sake yeast for ginjo for many years. Can be obtained by crossing.

  As a highly scented high ester-producing mutant yeast, an ethyl caproate high-producing mutant yeast obtained from a cerulenin-resistant strain known as an antibiotic that inhibits fatty acid synthase, and a leucine analog 5′-5′-5 Both isoamyl acetate and ethyl caproate, which have been bred using two methods of obtaining a highly isoamyl acetate-producing mutant yeast obtained from a strain resistant to '-trifluoro-D, L-leucine, have already been put into practical use. Examples are K-1601 yeast and K-1701 yeast, which produce a high amount of ester and have low acid production. These mutant yeasts maintain isoamyl alcohol productivity.

  As the latter yeast, yeasts that have been widely used as sake yeast for ginjo are conventionally used as appropriate. Examples include yeast No. 7 yeast (K-7) and yeast No. 701 yeast (K-701). And No. 9 yeast (K-9), No. 901 yeast (K-901), No. 1001 yeast (K-1001), and the like.

  The target mutant yeast can be obtained by crossing the high ester-producing mutant yeast with a commonly used excellent yeast such as K-9 according to the mass-mating method or other conventional methods.

  In this way, the amount of isoamyl alcohol produced can be increased by crossing the K-1601 and K-1701 yeasts with high production of isoamyl acetate and ethyl caproate with the excellent regular yeasts such as K-9. It is a hybrid strain that has less flavor than yeast, etc., has high production of ethyl caproate, and also produces isoamyl acetate in a well-balanced manner, and eliminates the weak point that fermenting power is weak, which is a disadvantage of general mutant yeast. A mutant yeast with less production was constructed, and finally a hybrid strain of K-1601 yeast and K-9 yeast was selected as a particularly excellent mutant yeast. In addition, the yeasts prepared were K-1601, K-1701, Kyoto 7 yeast (K-7), Kyoto 701 yeast (K-701), K9, Kyoto 901 yeast (K- 901) and Kyoto 1001 yeast (K-1001) and the like, and can be easily distinguished from various types of sake yeast for sake. In addition to storage management of yeast, quality control such as mash mash management It is a sake yeast having excellent new brewing aptitude.

  According to the present invention, for example, K-1601 yeast was used as one parent strain (that is, mutant yeast that produced high amounts of ethyl caproate and isoamyl acetate and high amounts of isoamyl alcohol was used. Regardless, the resulting hybrid strain produces ethyl caproate higher than the parent strain, while isoamyl acetate also produces a good balance, although it is somewhat inferior to the parent strain, but the production of isoamyl alcohol is as high as K-1601. Hybrid strains can be obtained that have very characteristic properties that are much lower than esterogenic mutant yeast.

  Such a hybrid strain has not been known so far, and therefore the mutant yeast according to the present invention can be referred to as a novel mutant yeast.

  In order to carry out the present invention, a commercially available yeast such as K-1601 or K-9 may be used as a parent strain, crossed by a known hybridization method (for example, mass mating method), and the target strain may be screened. . According to the method described later, the method for confirming the hybrid strain can be carried out promptly and accurately, so that the present invention can be sufficiently implemented according to the description in the present specification.

  In addition to the above-mentioned yeast, high-ester high-producing mutant yeast can also be bred as appropriate by, for example, the following operation. Therefore, the present invention can be sufficiently implemented from this point of view. .

  That is, first, various brewing yeasts are subjected to mutation treatment, and conventional methods are used as the mutation treatment. Examples of physical methods of mutation include ultraviolet irradiation and radiation irradiation, and chemical methods include mutation agents such as ethyl methanesulfonate, N-methyl-N-nitro-N-nitrosoguanidine, nitrous acid, and acridine. There is a mutation method in which the pigment is suspended in a solution such as a dye.

  Mutant yeast is cultured in a cerulenin-containing medium. The addition of cerulenin may be either a solid medium or a liquid medium, and is used by adding about 10 to 100 μM, preferably about 25 μM. As the treated yeast, a cerulenin-resistant strain can be obtained by any yeast of sake yeast, shochu yeast, brewer's yeast, wine yeast, and baker's yeast. After mutating various yeasts, they are transferred to a cerulenin-containing medium, and a strain grown by culturing at 30 ° C. for about 1 week is isolated, and yeasts that generate caproic acid well from this can be employed.

  The mutant yeast thus obtained is then added to a medium such as a solid medium to which leucine analog (5′-5′-5′-trifluoro-D, L-leucine) is added at about 0.5 to 5 mM, for example, about 1 mM. Incubate at 30 ° C. for about 2 days, isolate the resulting colonies, and use yeast that produces well isoamyl alcohol and isoamyl acetate.

  In this way, a high ester-producing yeast that produces a high amount of ethyl caproate and isoamyl acetate can be obtained, and this yeast can be sufficiently obtained. Moreover, if desired, the order of the treatment with the cerulenin-containing medium and the treatment with the leucine analog medium may be reversed or simultaneously performed.

  A hybrid strain of a high ester-producing yeast and a commercially available yeast such as K-9 has a remarkable effect that its production is remarkably suppressed even though the parent strain produces high isoamyl alcohol. On the other hand, isoamyl acetate is also produced in a well-balanced manner compared to the parent strain, and ethyl caproate is highly produced. In addition, this hybrid strain also has the remarkable effect that one of the parent strains has a strong fermenting power inherently and a low acid production amount is maintained.

  The novel mutant yeast thus bred and constructed can be used to brew liquors with excellent flavor. The obtained liquor is said to be a precursor of isovaleraldehyde, which is said to have a high balance of ethyl caproate and isoamyl acetate in a well-balanced manner, compared to the case of using the parent strain, while it is said to have a fresh scent of cypress “Mure smell”. The amount of isoamyl alcohol produced is very low, and alcoholic beverages such as sake that exhibit such effects are not found in the past and are novel. In fact, despite the fact that it was stored at a high temperature of 20 ° C. for a long period of 3 months, it was actually confirmed that the amount of isovaleraldehyde produced was significantly lower than when the parent strain was used.

  Therefore, according to the present invention, it is possible to effectively produce alcoholic beverages such as sake and shochu, which have no flavor and have a flavor.

  The present invention is a high ester-producing mutant yeast that highly produces ethyl caproate and isoamyl acetate, and a yeast that has excellent characteristics for brewing as well as flavor and fermentability (for example, sake yeast for ginjo: One of the preferred examples is K-9 yeast), which produces little isoamyl alcohol, high ester production such as ethyl caproate, and excellent acidity and fermenting power. A new yeast is produced, and an alcoholic beverage having a low stuffy odor and excellent flavor is produced using the new yeast.

  Hereinafter, the present invention will be described more specifically by using sake yeast and sake brewing as examples, but the present invention is not limited to these examples.

(Creation of hybrid strains)
A large number of cultivated strains were randomly separated from the yeasts of K-1601 and K-1701. Among them, ethyl caproate and isoamyl acetate were produced at a high rate, and the production of isoamyl alcohol and acid was low. The 1601-Hp10 (junction type (MATa)) having a strong culture medium without bubbles was selected.

On the other hand, many haploid strains were randomly separated from the K-9 yeast and K-901 yeast in the same manner as the method for separating the haploid strains of the K-1601 and K-1701 yeasts. 9-Hp5 (joining type (MATα)) having no foam was selected as a haploid strain with less isoamyl alcohol and acid production and excellent fermentative flavor. From many of the hybrids of the selected haploid strain of K-1601 yeast and the haploid strain of K-9 yeast, the non-foaming cross of the diploid strain that was the most aimed by the small preparation test The strain (9-Hp5 × 1601-Hp10) Dp-12 was selected.

(1) Method for separating haploid strains of K-1601, K-1701, K-9 and K-901 yeast, and method for determining mating type The test yeast was treated with Bllg. After washing yeast cells cultured with shaking in 5 ° wort medium at 30 ° C. for 2 days with sterilized water, they were applied to an agar medium (spore-forming medium) containing 0.4% potassium acetate and cultured at 30 ° C. for 2 days. After washing the spore formed with sterilized water and adding 0.05% cell wall lytic enzyme (Zymolase 100T Seikagaku) to this, it was allowed to act at 30 ° C. for 1 hour to dissolve the cell wall and release the spore. And sterilizing the diploid yeast, followed by Bllg. The haploid yeast was grown by spreading on a 10 ° broth agar plate medium and culturing at 30 ° C. for 7 to 10 minutes. Saccharomyces cerevisiae standard strains Saccharomyces cerevisiae IFO 10175 (MATa) and Saccharomyces cerevisiae IFO 10176 (Saccharomyces cerevisiae) previously cultured in a wort medium at 30 ° C. for 3 days and then cultured in the same manner. MATα) yeast and wort agar plate medium, cross-streaked and cultured at 30 ° C. for 3 days. After culturing at 30 ° C. for 2 days in a 5 ° broth medium, spore formation was carried out in a spore formation medium as described above, and the haploid yeast mating type of the test bacterium was determined by the presence or absence of zygote formation after crossing. .

(2) K-1601 yeast haploid strain 1601-Hp10 (mating type (MATa)) and K-9 yeast haploid strain 9-Hp5 (mating type MATα)) cross-mass method That is, the haploid strains of K-1601 and K-9 yeast determined above were determined according to Bllg. After pre-cultured at 5 ° C. juice at 30 ° C. for 2 days, Bllg. Cross cross streaks on 10 ° wort agar plate medium, and the presence or absence of sporulation among many hybrid strains, comparison of mycological properties of each haploid strain and hybrid diploid strain, etc. The cross was confirmed by the above, and the target strain (9-Hp5 × 1601-Hp10) Dp-12 was finally selected by a small preparation test.

(3) Crossing confirmation method of breeding strain
i) Composition of hybridization confirmation medium The test yeast was inoculated into 10 ml of YM medium (yeast extract 0.3%, malt extract 0.3%, polypeptone 0.5%, glucose 2%, agar 2.5%), 30 After culturing at 2 ° C. for 2 days, galactose identification medium (yeast extract 0.15%, polypeptone 0.2%, KH ₂ PO ₄ 0.1%, MgSO ₄ .7H ₂ O 0.04%, galactose 2%, agar 25 %), Alanine identification medium (Difco. Yeast Nitrogen Base W / O Amino Acids and Ammonium Sulfate, glucose 2%, alanine 0.5%), cerulenin identification medium (Difco. Yeast Nitrogen Base, glucose 2%, agar 2.5%) %, Cerulenin 1 ppm) in each agar plate identification medium 200 to 3 per dish It is applied so that the number of cells is 0, the galactose identification medium is 30 ° C. for 3 to 4 days, the alanine identification medium is 38 ° C. for 3 to 4 days, and the cerulenin identification medium is 30 ° C. and 35 ° C. for 3 to 7 days. Each TTC upper layer medium (galactose identification medium: galactose 0.5%, 2,3,5-Triphenyltetrazolium chloride 0.05%, agar 1.5%), alanine identification medium and cerulenin In the identification medium, 0.5% glucose, 2,3,5-Triphenyltetrazole chloride 0.05%, agar 1.5%) were overlaid, and cultured at 30 ° C. for 3 to 4 hours, followed by TTC staining and identification of each. Comparing the growth in the medium, Dp-12 is 9-Hp5 ((MATα)) and 1601-Hp10 ( (MATa)).

ii) Cross confirmation method K-9 yeast haploid strain 9-Hp5 (conjugated MATα)) grows well in each of the galactose identification medium at 30 ° C. and the alanine identification medium at 38 ° C. and each TTC. While the staining properties are white and red, respectively, the haploid strain 1601-Hp10 (mating type (MATa)) of the K-1601 yeast has a characteristic that it cannot grow at all in the above-mentioned two identification media. This hybrid of haploid strains (9-Hp5 × 1601-Hp10) Dp-12 grows well at 30 ° C. in galactose discrimination medium, shows red in TTC staining, and grows well at 38 ° C. in alanine discrimination medium. And since TTC dyeing | staining property exhibits white, since it has the characteristic which both haploid strains do not have, it can confirm that it was crossed clearly. Further, 9-Hp5 cannot grow at all at 35 ° C. in cerulenin discrimination medium, whereas 1601-Hp10 grows well and TTC staining exhibits a white color, whereas a hybrid strain (9-Hp5 × 1601- Hp10) Dp-12 grows well and the TTC staining property is red. This also confirms that the hybrid strain has the characteristics of both haploid strains and is resistant to cerulenin. It was also confirmed that was inherited by the hybrid strain.

  Table 1 below shows a list of various confirmation methods for hybrid strains.

(4) Small preparation results of cultivated and hybrid strains of K-1601 yeast and K-9 yeast In the hybrid preparation results of hybrid and haploid strains, the hybrid strain is the parent strain K-9 yeast and Compared to K-1601 yeast, the amount of ethyl caproate produced is high, but isoamyl acetate is also produced in a relatively large amount. On the other hand, a diploid strain with low production of isoamyl alcohol and acid and a very strong fermentative ability was constructed. The obtained results are shown in Table 2.

The small preparation was performed as follows.
Small preparation method;
α rice (dried steamed rice) 115g, A koji (alcohol dehydrated koji) 35g, pumped water 270ml 15 ° C, constant fermentation.

(5) Pure rice ginjo total rice 8t preparation result Ginjo liquor was manufactured with the preparation composition shown in Table 3 using pure rice ginjo total rice 8 ton. Table 4 shows the analysis results of the general components of the sake.

(Table 3)
Pure rice Ginjo 8 tons of total rice blending ――――――――――――――――――――――――――――――――――――
Ingredients Sake Mother First-time Addition Nakazoe Total Addendum ――――――――――――――――――――――――――――――――――――
Total rice (kg) 560 1,110 2,220 4,110 8,000
Steamed rice (kg) 380 800 1,800 3,570 6,550
Sticky rice (kg) 180 310 420 540 1,450
Pumped water (L) 650 1,350 3,000 6,200 11,200
――――――――――――――――――――――――――――――――――――
Raw material rice: 60% polished rice

(Table 4)
General ingredients ――――――――――――――――――――――――――――――――――――
K-901 K-1701 K-1601 Hybrid strain ――――――――――――――――――――――――――――――――――――
Alcohol content (%) 18.2 18.1 17.8 17.9
Sake degree +3.0 +1.5 +1.0 +0.5
Acidity 2.00 2.15 1.55 1.45
Amino acid degree 1.25 1.35 1.4 1.20
――――――――――――――――――――――――――――――――――――

  The aromatic component of the obtained sake was analyzed by gas chromatography. The aroma component analysis conditions are shown below, and the obtained aroma component results are shown in Table 5.

(GLC analysis conditions)
Shimadzu GC-9A (FID)
Column PEG 1540 10% 60/80 Uniport R Glass 3φ × 3m
Carrier gas 0.6ml / min
H ₂ gas 0.8ml / min
Air 0.8ml / min
INJ / DET Tem 180 ° C
Column Tem 60 ° C. (10 min) → 100 ° C. (5 ° C./min)→100° C. (hold)
Range 10 ²
Atten 1
Measurement method Headspace method (52 ° C / 30min)

(Table 5)
Odor component ――――――――――――――――――――――――――――――――――――
K-901 K-1701 K-1601 Hybrid strain ――――――――――――――――――――――――――――――――――――
Acetaldehyde 28 40 21 39
n-propyl alcohol 72 59 65 55
Isobutyl alcohol 49 28 22 37
Isoamyl alcohol 133 215 184 112
Ethyl acetate 65 44 46 29
Isoamyl acetate 3.1 4.8 3.2 2.1
Ethyl caproate 2.1 4.0 5.8 9.2
――――――――――――――――――――――――――――――――――――
Unit: ppm

  Moreover, about the obtained sake, the organic acid composition was analyzed on the following organic acid composition analysis conditions, and the result of Table 6 was obtained.

(Organic acid composition analysis conditions)
Column: Shin-pack SCR-102 H (7.9 mmφ × 30 cm)
Mobile phase: 0.85% phosphoric Acid: Water (7:93, v / v)
Column temperature: 50 ° C, 65 ° C
Flow rate: 1.0 ml / min
Injection volume: 15μl
Detection: UV 210 nm
Determination: peak height method

(Table 6)
Organic acid composition ――――――――――――――――――――――――――――――――――――
K-901 K-1701 K-1601 Hybrid strain ――――――――――――――――――――――――――――――――――――
Citric acid 97 82 93 101
Pyruvate 8 99 9 9
Malic acid 334 121 197 185
Succinic acid 278 232 186 186
Lactic acid 304 342 266 232
Fumaric acid 7 4 4 8
Acetic acid trace trace trace
Pyroglutamic acid 24 19 10 29
――――――――――――――――――――――――――――――――――――
Total 1052 899 765 750
――――――――――――――――――――――――――――――――――――
Unit: ppm

Analysis of the mashing process, aroma components, and acidity of the produced sake, the hybrid strain has a strong fermenting power and can shorten the number of days of mashing, producing less isoamyl alcohol, producing a high ester such as ethyl caproate, and producing acid. The results of the sensory test showed that the scented gorgeous sake had a gorgeous and well-flavored swell, and bitterness was not particularly pointed out for astringency and miscellaneous taste, and there were no problems such as off-flavor. .

(6) Comparison of storage temperature and storage time and production amount of isovaleraldehyde (smellar odor component) Next, the obtained sake is divided into those that have been fired and those that are not to be fired, and 5 ° C and 20 ° C, respectively. Were stored for 0 to 3 months, and the amount of isovaleraldehyde produced was compared.

  The analysis of isovaleraldehyde was performed under the analysis conditions shown in Table 7 below. The obtained results are shown in Table 8 and FIG.

(Table 7)
(GLC analysis conditions)
Shimadzu GC-1700 (FID)
DB-WAX capillary column (0.25 mm × 0.25 μm × 30 m) manufactured by Column J & W
Carrier gas He, 0.9ml / min
INJ / DET Tem 250 ° C
Column Tem 50-70 ° C (1 ° C / min), 70-220 ° C (5 ° C / min)
Range 4
Atten 1
Spirit ratio 1/100
Sample 2 μl
Measuring method Ethyl acetate extraction method

(Table 8)
(result of analysis)
Storage temperature and isovaleraldehyde production
Raw 5 ℃ Raw 20 ℃
K-901 K-1601 Hybrid strain K-901 K-1601 Hybrid strain 0 months 0.002 0.004 0.004 0.002 0.004 0.004
1 month 0.02 0.08 0.02 0.2 0.5 0.2
2 months 0.11 0.16 0.09 0.8 1.1 0.6
3 months 0.15 0.25 0.13 1.2 1.9 0.9

Firing at 5 ° C Firing at 20 ° C
K-901 K-1601 Hybrid strain K-901 K-1601 Hybrid strain 0 months 0.002 0.004 0.004 0.002 0.004 0.004
1 month 0.002 0.004 0.004 0.002 0.004 0.004
2 months 0.002 0.004 0.004 0.002 0.004 0.004
3 months 0.002 0.004 0.004 0.002 0.004 0.004

  The discrimination threshold of isovaleraldehyde, which is the main ingredient of “Mureka” which is a deteriorated odor of raw sake, is said to be 1.7-1.8 ppm for Kamidate and 2.4 ppm for incense, which is common in sake. Is contained in 0.1 to 2 ppm. This component was found to be isoamyl alcohol in sake, and the enzymatic oxidation reaction derived from koji found that isovaleraldehyde was accelerated when the storage temperature of raw sake was higher and the storage time was longer ( Literature: Akira Nishimura, Brewery 88 (11) 852 (1993)), but as shown in FIG. 1, the amount of isovaleraldehyde produced in the sake storage liquor of K-1601 yeast that has not been fired At a storage temperature of 20 ° C., the greater the number of months of storage, the greater the number of months. On the other hand, the amount of K-701 yeast and the hybrid strain isovaleraldehyde is small, and the amount of the hybrid strain is particularly small.

(7) Hybrid strain identification method Hybrid strains and various yeasts were identified as follows. That is, the identification method described below was performed using an identification medium having the following composition.

1) Identification medium composition of hybrid strain Inoculate the test yeast into 10 ml of YM medium (yeast extract 0.3%, malt extract 0.3%, polypeptone 0.5%, glucose 2%, agar 2.5%). After culturing at 30 ° C. for 2 days, maltose, galactose and α-methyl-D-glucoside discrimination medium ((α-MG discrimination medium) (yeast extract 0.15%, polypeptone 0.2%, KH ₂ PO ₄ . 1%, MgSO ₄ · 7H ₂ O 0.04%, maltose, galactose, and α-MG, each of the carbon source sugars corresponding to the identification medium is 2%, agar 2.5%)), alanine identification medium (Difco Yeast Nitrogen Base W / O Amino Acids and Ammonium Sulfate, glucose 2%, alanine 0.5%), cerulenin discrimination medium (Difco. Y) (as Nitrogen Base, glucose 2%, agar 2.5%, cerulenin 1 ppm), β-alanine identification medium (manufactured by Japan Brewing Association), each agar plate identification medium (200-300 cells per petri dish) The galactose identification medium is cultured at 30 ° C. for 3-4 days, the alanine identification medium is cultured at 38 ° C., 3-4 days, the β-alanine identification medium and the cerulenin identification medium are cultured at 30 ° C. and 35 ° C. for 3-7 days, respectively. The TTC upper layer culture medium (galactose identification medium: 0.5%, 2,3,5-Triphenyltetrazole chloride 0.05%, agar 1.5% for galactose identification medium), alanine identification medium and cerulenin identification medium Contains 0.5% glucose, 2,3,5-Triphenyltetrazo Imm Chlorid 0.05%, agar 1.5%), and after culturing at 30 ° C. for 3-4 hours, the TTC stainability and the growth in each identification medium were compared. And was identified.

2) Discrimination method between various yeasts and hybrid strains The bacteriological properties of each yeast are shown in Table 9, and can be identified using this property.

  As yeasts for sake production, representative K-7, K-701, K-9, K-901, K-1001, and high ester-producing yeasts K-1601, K-1701 yeast and new breeding hybrids (9 × 1601) As a method for distinguishing from Dp-12, as shown in FIG. 2, K-1601 yeast that grows well at 30 ° C. in a cellurenin discrimination medium containing 1 ppm of cerulenin and is stained red in TTC staining, K-9701 yeast and hybrid strain (9 × 1601) Dp-12, on the other hand, it is not able to grow at 30 ° C. at all in the cellulenin identification medium at K-7, K-701, K-9, K-901 and K-1001. It is a traditional representative yeast of No. yeast.

  K-9, K-901 and K-1001 yeasts that show viability in this β-alanine identification medium at 35 ° C. and have a TTC staining color of red, and K-7 and K- It is distinguished from No. 701 yeast. High ester-producing yeasts of K-1601, K-1701 and hybrid (9 × 1601) Dp-12 yeast that grow well at 30 ° C. in cerulenin discrimination medium and show red color of TTC are maltose, galactose, leucine And K-1701 yeast and a hybrid (9 × 1601) Dp-12 that grows well on various identification media of α and MG and shows red coloration are very slight even when they do not grow or grow. The dyeing property of K-1601 yeast is white.

  In addition, K-1701 yeast and a cross strain (9 × 1601) Dp-12, which grow well at 30 ° C. in galactose identification medium and show red color of TTC, grow well in galactose identification medium at 35 ° C. and red color of TTC. However, the hybrid strain (9 × 1601) Dp-12 does not show any growth at all, and the hybrid strain can be identified.

  In addition, as shown in FIG. 3, all the test yeasts including the hybrids grow well and the TTC staining property shows red in leucine identification medium at 30 ° C., but only K-1601 yeast grows well and TTC staining. The color is white. However, under the culturing conditions on the leucine identification medium at 35 ° C., it grows well and the TTC staining is stained red in the K-9, K-901 and K-1001 yeasts including the hybrid strains. On the other hand, the K-7, K-701 and K-1701 yeasts grow well and exhibit white TTC staining, and only the K-1601 yeast cannot grow at all.

  By using the identification method described above, the hybrid strain (9 × 1601) Dp-12 is used for sake with a new brewing aptitude that is different in nature from the various yeasts that are widely used nationwide as yeast for sake brewing. It is a novel sake yeast that is a yeast and can be distinguished from other excellent sake yeasts. Since it is possible to confirm the presence or absence of natural mutation and contaminating yeast during storage, it is a novel yeast excellent in quality control such as yeast storage management, liquor and moromi management.

The relationship between storage temperature and the production amount of isovaleraldehyde is shown. The identification method is shown. The identification method is shown.

Claims (7)

  High-ester-producing mutant yeast that produces a large amount of ethyl caproate and isoamyl acetate from a strain that grows on cerrenin-containing medium and 5'-5'-5'-trifluoro-D, L-leucine-containing medium by mutating brewing yeast The high ester producing mutant yeast and the sake yeast for brewing sake are crossed, producing less isoamyl alcohol, producing a large amount of ethyl caproate, producing less acid, and having a strong fermenting power, A method for breeding mutant yeast with excellent flavor.   The method according to claim 1, wherein the high ester-producing mutant yeast is Kyokai No. 1601 yeast (K-1601), and the sake sake yeast for ginjo is Kyokai No. 9 yeast (K-9).   A mutant yeast produced by the method according to claim 1 or 2.   Use of the mutant yeast according to claim 3, characterized in that the production of isoamyl alcohol is low, the production of ethyl caproate is high, and the acid production is low, and the alcoholic beverage selected from sake, shochu, beer, wine is selected for a short time. How to manufacture with.   An alcoholic beverage with excellent flavor, which is produced by the method according to claim 4 and has a suppressed odor.   Compared to sake produced with sake yeast, the amount of isoamyl alcohol produced is low, ethyl caproate is highly produced, and isoamyl acetate is produced in a well-balanced manner, producing acid. The liquor according to claim 5, wherein the liquor has a scent with a fragrant and scented bulge.   The alcoholic beverage according to claim 6, wherein the amount of isovaleraldehyde, which is one of stuffy odors, is small even when stored at a high temperature for a long time.

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