JP2017121251A - Yeast with high production capacity of ethyl caproate, and method of producing fermentation product using that yeast - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide yeast allowing a high yield of ethyl caproate even from ingredient rice having a low rice-polishing ratio.SOLUTION: A strain allowing a high yield of ethyl caproate even from ingredient rice having a rice-polishing ratio as low as 70% is obtained by subjecting Saccharomyces cerevisiae, which produces isoamyl acetate at a high yield, to mutation treatment and then selecting a strain exhibiting cerulenin resistance.SELECTED DRAWING: None

Description

  The present invention relates to a yeast having a high ability to produce ethyl caproate. Furthermore, this invention relates to the fermented material using the said yeast, and its manufacturing method.

  In recent years, consumer preferences have been diversified and high-class due to the rising level of dietary habits, and it is also rich in fermented foods and drinks such as sake, shochu, beer, miso, soy sauce, and bread. There is a growing demand for scented products. In order to follow such consumer demands, techniques for increasing the aroma of fermented foods and beverages have been studied.

  In sake, ethyl caproate is the main component of Ginjo incense and is known to be an important component that affects the aroma of sake. Therefore, it is important to increase the content of ethyl caproate in producing sake with rich ginjo aroma.

  Conventionally, it has been known that sake with a high content of ethyl caproate can be produced by fermenting at a low temperature using raw rice that is highly refined to a rice polishing ratio of 60% or less. However, in the case of using such highly refined raw material rice, there is a problem that costs and production time increase.

  Therefore, in recent years, breeding of yeasts that produce high amounts of ethyl caproate has been energetically studied. For example, Patent Document 1 discloses that an ethyl caproate high-producing strain can be obtained by crossing cells of haploid cells derived from the same origin of yeast belonging to the genus Saccharomyces. Patent Document 2 discloses that Saccharomyces cerevisiae 15BY16-9 strain (FERM AP-21235), which is spontaneously mutated in sake mash, produces a high amount of ethyl caproate. However, at present, no technology has been found for breeding yeasts that produce ethyl caproate at high yields using raw rice that has a low milling ratio of 70%.

International Publication No. 96/20272 JP 2008-228588 A

  An object of the present invention is to provide a yeast capable of producing ethyl caproate with high yield even when low-milled raw rice is used. Furthermore, another object of the present invention is to provide a method for producing a fermented product such as sake using the yeast.

  The inventors of the present invention have intensively studied to solve the above-mentioned problems. As a result, the Saccharomyces cerevisiae that produces isoamyl acetate at a high yield is subjected to a mutation treatment, and then a strain exhibiting cerulenin resistance is selected. It was found that even when using low-milled raw rice of%, a strain capable of producing ethyl caproate at a high yield can be obtained. The present invention has been completed by further studies based on these findings.

That is, this invention provides the invention of the aspect hung up below.
Item 1. A Saccharomyces cerevisiae capable of producing high amounts of ethyl caproate,
Saccharomyces cerevisiae in which the concentration of ethyl caproate in the sake obtained in the upper tank on the 15th day of distillation is 4 mg / L or more when subjected to a three-stage small preparation test of sake under the condition 1 shown in Table 1.
Item 2. Item 2. The Saccharomyces cerevisiae according to Item 1, wherein the caproic acid content in the bacterial cell is 0.01 mg / g-dry cells or more when subjected to a one-step small preparation test for sake shown in Condition 2 below.
Item 3. Item 3. The Saccharomyces cerevisiae according to Item 1 or 2, which exhibits cerulenin resistance.
Item 4. Item 4. The Saccharomyces cerevisiae according to any one of Items 1 to 3, which has a homozygous FAS2 mutant in which G at position 3748 of the FAS2 gene is mutated to A.
Item 5. Item 5. The Saccharomyces cerevisiae according to any one of Items 1 to 4, which is a hec2 strain (NITE P-02179).
Item 6. A method for breeding yeast with high caproate ethyl production, comprising a step of selecting a cerulenin-resistant strain after subjecting Saccharomyces cerevisiae having high isoamyl acetate productivity to a mutation treatment.
Item 7. Item 6. A method for producing a fermented product, comprising a step of producing a fermented product using the Saccharomyces cerevisiae according to any one of Items 1 to 5.
Item 8. Item 8. The method for producing a fermented product according to Item 7, wherein the fermented product is sake.
Item 9. Item 9. The method for producing a fermented product according to Item 8, wherein rice having a rice polishing ratio of 40 to 90% is used.
Item 10. Item 6. A sake obtained using the Saccharomyces cerevisiae according to any one of Items 1 to 5, wherein the ethyl caproate concentration is 6 mg / l or more.
Item 11. Item 11. The refined sake according to Item 10, wherein the ratio of polished rice as a raw material is 70 to 90%.

  According to the ethyl caproate high-producing yeast of the present invention, even if low-milled raw rice is used, ethyl caproate can be produced at a high level, so a fermented product having a high content of ethyl caproate and a rich ginjo aroma It becomes possible to manufacture efficiently.

In Example 1, an aureobasidin A resistant strain (hia1-6) was subjected to a three-stage small-scale preparation test for sake, and aroma components (isoamyl acetate, isoamyl alcohol), sake degree (Be '), total acidity, and amino acidity The result of having measured is shown. In Example 1, the cerulenin-resistant strain (hec 1 to 7) was subjected to a three-stage small preparation test for sake, and aroma components (ethyl caproate, isoamyl acetate), sake degree (Be ′), total acidity, and amino acidity were measured. The results are shown. In Example 3, a hia1 variant (FAS2 gene: homozygous mutation), a hec2 strain (FAS2 gene: homozygous mutation), a hia1 strain (FAS2 gene: wild type), and a K1801 strain (FAS2 gene: heterozygous mutation) were used. The results of measuring the aroma components (ethyl caproate, isoamyl acetate) are shown in a three-stage small preparation test for sake. In Example 3, a hia1 variant (FAS2 gene: homozygous mutation), a hec2 strain (FAS2 gene: homozygous mutation), a hia1 strain (FAS2 gene: wild type), and a K1801 strain (FAS2 gene: heterozygous mutation) were used. The result of having measured the amount of caproic acid in a microbial cell for the one-step small preparation test of sake is shown. In Example 4, the result of having measured the expression level of FAS1 gene and FAS2 gene in Km97 strain, hia1 strain, hec2 strain, and K1801 strain is shown. In Example 4, the result of having measured the expression level of EEB1 gene and EHT1 gene in Km97 strain, hia1 strain, hec2 strain, and K1801 strain is shown. In Example 4, the result of having measured the expression level of the ATF1 gene, the ATF2 gene, and the IAH1 gene in the Km97 strain, the hia1 strain, the hec2 strain, and the K1801 strain is shown. In Example 5, the result of having measured the alcohol acetyltransferase activity (AAT activity) in Km97 strain, hia1 strain, hec2 strain, and K1801 strain is shown. In Example 6, when the hec2 strain was used to conduct a pure rice sake production test using 70% polished rice white rice, aroma components (ethyl caproate, isoamyl acetate) contained in the koji supernatant Shows the results of measurement over time. In Example 7, the result of having measured the aromatic component (ethyl caproate, isoamyl acetate) in the sake obtained using hec2 stock | strain and commercial sake (product AH) is shown.

1. Ethyl caproate high-producing yeast The ethyl caproate high-producing yeast of the present invention is a Saccharomyces cerevisiae that can produce ethyl caproate at a high rate, It is a Saccharomyces cerevisiae in which the concentration of ethyl caproate in the sake obtained in the upper tank on the 15th day of distillation is 4 mg / L or more. Hereinafter, the ethyl caproate high production yeast of this invention is explained in full detail.

[Types of yeast producing high caproate]
The type of ethyl caproate high-producing yeast of the present invention is not particularly limited as long as it belongs to Saccharomyces cerevisiae. For example, it is used for producing sake, beer, shochu, wine, whiskey, soy sauce, miso, etc. Examples include brewer's yeast and baker's yeast used for bread making. Among these, brewery yeast is preferable, and sake yeast is more preferable.

[Ethyl caproate-producing ability of ethyl caproate high-producing yeast]
The ethyl caproate high-producing yeast of the present invention is characterized by high ability to produce ethyl caproate. Specifically, the ethyl caproate high-producing yeast of the present invention has a characteristic that the concentration of ethyl caproate in the sake obtained when subjected to the three-stage small preparation test of sake shown in the following condition 1 can be 4 mg / L or more. have.

  In the conventional yeast, when raw rice having a low milling rate of 70% is used, the production amount of ethyl caproate tends to be reduced. It has a notable feature that ethyl caproate can be produced at high yields using raw rice. As a suitable example of the ethyl caproate high-producing yeast of the present invention, the concentration of ethyl caproate in the sake obtained when subjected to the three-stage small preparation test of sake shown in Condition 1 is preferably 6 to 30 mg / L. More preferably, those having a characteristic of 9 to 30 mg / L can be mentioned.

[Amount of caproic acid in the cell of yeast with high production of ethyl caproate]
Moreover, as a suitable example of the ethyl caproate high-producing yeast of the present invention, the caproic acid content in the microbial cells is 0.01 mg / g-dry cells when subjected to a one-step small preparation test of sake shown in Condition 2 below. As mentioned above, what has the characteristic which becomes like this. Preferably it is 0.01-0.08 mg / g-dry cells, More preferably, it is 0.06-0.08 mg / g-dry cells is mentioned.

[Growth characteristics of ethyl caproate high-producing yeast]
Further, as a suitable characteristic of the ethyl caproate high-producing yeast of the present invention, those having cerulenin resistance, specifically those having characteristics capable of growing on an SD medium containing 5.0 μg / ml cerulenin. Can be mentioned.

[Genetic characteristics of yeast with high production of ethyl caproate]
One embodiment of the ethyl caproate high-producing yeast of the present invention includes one having a homozygous FAS2 mutant in which G at position 3748 of the FAS2 gene is mutated to A. FAS2 is one of the subunits of fatty acid synthase (FAS), and it is known that the yeast FAS2 gene consists of the base sequence shown in SEQ ID NO: 1. That is, in one embodiment of the ethyl caproate high-producing yeast of the present invention, it has a homozygous FAS2 mutant gene in which G at position 3748 is mutated to A in the base sequence shown in SEQ ID NO: 1, FAS2 in which the 1250th glycine in the amino acid sequence shown (wild type amino acid sequence of FAS2) is mutated to serine is produced. By having such a mutant of the FAS2 gene, the amount of caproic acid that is a precursor of ethyl caproate is increased, and it becomes possible to improve the ability to produce ethyl caproate more efficiently. Conceivable.

[Gene expression characteristics of ethyl caproate high-producing yeast]
As one aspect of the yeast with high production of ethyl caproate according to the present invention, the expression level of the FAS2 gene is high. Specifically, when subjected to a one-step small preparation test of sake shown in the above-mentioned condition 2, the amount of FAS2 mRNA is 1.5 times or more, preferably 1. Examples thereof include 5 to 2.0 times, more preferably 1.7 to 2.0 times.

  Furthermore, as one aspect of the ethyl caproate high-producing yeast of the present invention, the expression level of the FAS1 gene is high. FAS1 is one of the subunits of FAS. Specifically, when subjected to a one-step small preparation test of sake shown in the above condition 2, the amount of FAS1 mRNA is 1.3 times or more, preferably 1. The thing which becomes 3 to 2.0 times, More preferably, 1.5 to 2.0 times is mentioned.

  Moreover, as one aspect | mode of the ethyl caproate high production yeast of this invention, it is mentioned that the expression level of EEB1 gene is high. EEB1 is an ethanol O-acyltransferase, and is an enzyme that catalyzes a reaction for producing ethyl caproate from caproyl-CoA in a microbial cell. Specifically, as an embodiment of the ethyl caproate high-producing yeast of the present invention, when the EEB1 mRNA amount is Sake Yeast Kyokai No. 1801 when subjected to a one-step small preparation test of sake shown in Condition 2 above. In comparison, it may be 1.8 times or more, preferably 1.8 to 2.2 times, more preferably 2.0 to 2.2 times.

  Moreover, as an aspect of the ethyl caproate high-producing yeast of the present invention, the expression level of the ATF1 gene is high. ATF1 is an alcohol acetyltransferase, and is an enzyme that catalyzes a reaction for producing isoamyl acetate from acetyl-CoA and isoamyl alcohol in the microbial cells. Specifically, as an embodiment of the ethyl caproate high-producing yeast of the present invention, when subjected to a one-step small preparation test of sake shown in the above-mentioned condition 2, the amount of ATF1 mRNA is Sake yeast Kyokai No. 1801. In comparison, it may be 1.5 times or more, preferably 1.5 to 2.2 times, and more preferably 1.8 to 2.2 times.

[Breeding Method of Yeast Producing Yeast Caproate]
The method for breeding yeast capable of producing ethyl caproate according to the present invention is not particularly limited as long as the above-mentioned one having the ability to produce ethyl caproate can be obtained. As a preferred example, yeast having a high ability to produce isoamyl acetate is used. A method of performing a mutation treatment on the yeast (hereinafter sometimes referred to as isoamyl acetate high-producing yeast) and selecting a cerulenin-resistant strain is mentioned. Hereinafter, the breeding method will be described in detail.

-Isoamyl acetate high-producing yeast The isoamyl acetate high-producing yeast is a yeast having a higher ability to produce isoamyl acetate than ordinary yeast (such as sake yeast No. 1801). As isoamyl acetate high-producing yeast, specifically, the concentration of isoamyl acetate in the supernatant obtained when subjected to the three-stage small charge test of sake shown in the following condition 3 is 35 mg / L or more, preferably 35 to 45 mg / L, more preferably those having the characteristic of 40-45 mg / L.

  As a suitable aspect of the isoamyl acetate high-producing yeast, those having a high ratio (weight ratio, E / A ratio) of isoamyl acetate to isoamyl alcohol to be produced can be mentioned. Specifically, as a preferred embodiment of the isoamyl acetate high-producing yeast, the E / A ratio of the supernatant obtained when subjected to the three-stage small charge test of sake shown in Condition 3 is preferably 20 or more, preferably What has the characteristic which becomes 20-30, More preferably, 25-30 is mentioned.

  Moreover, as a suitable characteristic of isoamyl acetate high production yeast, it has the characteristic which can be grown on what has aureobasidin A tolerance, specifically, an SD medium containing 1.0 microgram / ml aureobasidin A. Things.

  Furthermore, as a suitable example of yeast with high production of isoamyl acetate, yeast having a high expression level of ATF1 gene and a high production amount of ATF1 can be mentioned. Specifically, as one aspect of isoamyl acetate high-producing yeast, the amount of ATF1 mRNA in the microbial cells when compared to the case of Sake Yeast Kyokai No. 1801 when subjected to the one-step small preparation test of sake shown in Condition 2 above. Then, those that are 1.5 times or more, preferably 1.5 to 2.2 times, and more preferably 2.0 to 2.2 times are mentioned. In addition, as another aspect of the yeast producing high isoamyl acetate, the amount of ATF1 in the bacterial cells when compared to the case of sake yeast No. 1801 when subjected to the one-step small preparation test of sake shown in the above condition 2 2.0 times or more, preferably 2.0 to 3.0 times, more preferably 2.5 to 3.0 times.

  Moreover, as a suitable example of yeast with high production of isoamyl acetate, one having a homozygous MGA2 mutant in which C at position 2117 of the MGA2 gene is mutated to A can be mentioned. MGA2 is a transcriptional activator of ATF1 and is known to consist of the base sequence shown in SEQ ID NO: 3. In the homozygous MGA2 mutant in which C at position 2117 of the MGA2 gene is mutated to A, the mutation causes a stop codon, and incomplete MGA2 in which about 1/3 of the C-terminal side is deleted is translated. In yeast having a homozygous MGA2 mutant in which C at position 2117 of the MGA2 gene is mutated to A, MGA2 is easily transferred into the nucleus, and is less susceptible to unsaturated fatty acids, thereby suppressing ATF1 expression. As a result, it is considered that high productivity of isoamyl acetate can be obtained.

  The isoamyl acetate high-producing yeast may be modified so as not to produce urea as necessary in order to improve the flavor of the fermented product to be produced. Modification to non-urea-producing strains includes chemical treatment using ethylmethanesulfonic acid, nitrous acid, N-methyl-N-nitro-N-nitrosoguanidine, acridine dye, etc .; irradiation treatment with ultraviolet rays, radiation, etc .; natural This can be done by mutation. In addition, since a urea non-producing strain usually shows arginase non-assimilability, growth in a CAO medium containing canavanine, which is an arginine analog, can be selected as an index.

  The isoamyl acetate high-producing yeast may be selected from known yeasts having the above-mentioned characteristics, or those obtained by breeding normal yeasts may be used.

-Breeding method of yeast producing high isoamyl acetate As a method of obtaining yeast producing high isoamyl acetate by breeding, a method of selecting an aureobasidin A resistant strain after performing a mutation treatment on the yeast as a mother strain is mentioned. It is done. As the mutation treatment, specifically, chemical treatment using ethylmethanesulfonic acid, nitrous acid, N-methyl-N-nitro-N-nitrosoguanidine, acridine dye, etc .; irradiation treatment with ultraviolet rays, radiation, etc. Is mentioned. Among these mutation treatments, a chemical treatment using ethyl methanesulfonic acid is preferable.

  In addition, in order to select an aureobasidin A resistant strain from the yeast that has been subjected to the mutation treatment, it is only necessary to cultivate in a medium containing aureobasidin A and isolate the grown strain.

  The concentration of aureobasidin A in the medium containing aureobasidin A may be appropriately set within a concentration range in which the aureobasidin A non-tolerant strain cannot grow. For example, 0.2 to 1.0 μg / ml, Preferably 0.3-1.0 microgram / ml, More preferably, 0.5-1.0 microgram / ml is mentioned.

  The medium containing aureobasidin A may be either an agar medium or a liquid medium, but an agar medium is preferable from the viewpoint of easy selection of the target strain.

  The conditions for culturing using the medium containing aureobasidin A may be appropriately set according to the growth characteristics of the yeast. For example, the culture temperature is 20 to 30 ° C, preferably 25 to 30 ° C. More preferably, it is 28-30 degreeC, and culture | cultivation time is 24-96 hours, Preferably it is 24-72 hours, More preferably, it is 24-48 hours.

  The thus obtained aureobasidin A-resistant strains frequently contain mutant strains with high isoamyl acetate production ability, and among the aureobasidin A-resistant strains, isoamyl acetate production ability is high. What is necessary is just to select a stock. As for the ability to produce isoamyl acetate, specifically, it may be subjected to a three-stage small charge test of sake shown in the above condition 3, and the concentration of isoamyl acetate in the supernatant may be measured.

-Mutation treatment of isoamyl acetate high-producing yeast The type of mutation treatment performed on isoamyl acetate high-producing yeast is not particularly limited, but for example, ethylmethanesulfonic acid, nitrous acid, N-methyl-N-nitro-N -Chemical treatment using nitrosoguanidine, acridine dye, etc .; light irradiation treatment such as ultraviolet rays and radiation. Among these mutation treatments, a chemical treatment using ethyl methanesulfonic acid is preferable.

・ Selection of cerulenin-resistant strains from isoamyl acetate high-producing yeast subjected to mutation treatment To select cerulenin-resistant strains from isoamyl acetate high-producing yeast subjected to mutation treatment, the cells are cultured in a medium containing cerulenin and grown. It can be isolated.

  The concentration of cerulenin in the medium containing cerulenin may be appropriately set within a concentration range in which the cerulenin-resistant strain cannot grow. For example, 1 to 20 μg / ml, preferably 2 to 10 μg / ml, more preferably 3 to 3 10 μg / ml is mentioned.

  The medium containing cerulenin may be either an agar medium or a liquid medium, but an agar medium is preferable from the viewpoint of easy selection of the target strain.

  The conditions for culturing using the medium containing cerulenin may be appropriately set according to the growth characteristics of the yeast. For example, the culture temperature is 20 to 30 ° C, preferably 25 to 30 ° C, more preferably. The culture time is 28 to 30 ° C., and the culture time is 24 to 96 hours, preferably 24 to 72 hours, and more preferably 24 to 48 hours.

  The cerulenin-resistant strains obtained in this way frequently contain mutant strains with high caproic acid production ability, and from among the cerulenin-resistant strains, select a strain with a high production capacity of ethyl caproate. That's fine. Regarding the ability to produce ethyl caproate, specifically, it may be subjected to a three-stage small-scale preparation test of sake shown in the above condition 1 to measure the ethyl caproate concentration in the supernatant. In addition, the obtained cerulenin-resistant strain may be confirmed for the presence or absence of other characteristics as described above, if necessary.

[Specific examples of yeast that produces ethyl caproate]
The ethyl caproate high-producing yeast of the present invention can be obtained by the above-mentioned breeding method. As a suitable strain, hec2 strain (NITE P-02179) is available at the National Institute of Technology and Evaluation Biotechnology Center as a suitable strain. ) And can be used.

2. Use of ethyl caproate high-producing yeast The ethyl caproate high-producing yeast of the present invention can be used for producing a fermented product having a high content of ethyl caproate and a rich scent. In the production of a fermented product using the ethyl caproate high-producing yeast of the present invention, the raw materials and production conditions may be appropriately set according to the type of the target fermented product.

  The type of fermented product produced using the ethyl caproate high-producing yeast of the present invention is not particularly limited. For example, alcoholic beverages such as sake, shochu, beer and wine; miso, soy sauce, bread, vinegar, bread And fermented foods and beverages. Among these fermented products, alcoholic beverages, particularly sake, are suitable production targets because ginjo aroma with ethyl caproate greatly contributes to the improvement of flavor.

  Moreover, when manufacturing refined sake using the ethyl caproate high production yeast of this invention, although it does not restrict | limit especially about the rice polishing ratio of the rice used as a raw material, For example, 40 to 90% is mentioned. Moreover, the ethyl caproate high-producing yeast of the present invention is a low-milling raw rice and has a unique property that it can produce ethyl caproate at a high rate. In view of the effect of the present invention, the rice milling ratio of the raw rice used when producing sake using the ethyl caproate high-producing yeast of the present invention is preferably 70 to 90%, more preferably 70 to 80%. %, Particularly preferably 70 to 73%.

  In addition, as a characteristic of sake produced using the ethyl caproate high-producing yeast of the present invention, the content of ethyl caproate is high. Specifically, the ethyl caproate concentration is preferably 6 mg / l or more, preferably Is 6 to 30 mg / l, more preferably 9 to 30 mg / l.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated concretely, this invention is limited to these Examples and is not interpreted. In addition, the raw material rice and glutinous rice varieties used for preparation in the following examples are Nipponbare.

Measurement conditions of aroma components In the following examples, the aroma components (isoamyl acetate, ethyl caproate, etc.) were measured by a headspace method using a gas chromatograph / hydrogen ionization detector (GC / FID). Specifically, a calibration curve was prepared by adding 1 ml of an internal standard solution to 5 ml of a standard solution containing each aromatic component at a specified concentration. Then, 1 ml of an internal standard solution was added to 5 ml of each sample and measured, and the amount of fragrance component in the sample was determined using the calibration curve obtained above. In addition, methyl hexanoate was used as an internal standard solution. Moreover, the analysis conditions by GC / FID are as follows.

(GC / FID analysis conditions)
GC-FID: GC2010 Plus (Shimadzu Corporation)
Column: DB-WAX (60 m × 0.32 mm, 0.5 μm, Agilent)
Temperature rise conditions: 40 ° C., 5 minutes → 5 ° C./minute→100° C. → 20 ° C./minute→230° C., 5 minutes Injection method: Headspace autosampler (Turbo Matrix HS40, Perkin Elmer)

Example 1: Acquisition of yeast producing high caproic acid ethyl
1. Acquisition of aureobasidin A resistant strain An aureobasidin A resistant strain was obtained using Saccharomyces cerevisiae Km97 strain. The Saccharomyces cerevisiae Km97 strain is a non-uric acid-producing strain obtained by natural mutation of a strain lacking arginase-utilizing ability that can grow in CAO medium without carrying out mutation treatment on yeast No. 9 without foam. is there.

  First, Km97 strain was cultured with shaking at 30 ° C. for 1 day using YPD medium, washed with 50 mM potassium phosphate buffer (pH 8.0), and suspended in the same buffer. Next, mutation was carried out by adding ethylmethanesulfonic acid to the obtained suspension of Km97 strain so as to be 4% by weight and shaking at 30 ° C. for 1 hour. Then, after neutralizing with a 5% by weight aqueous sodium thiosulfate solution, the yeast after the mutation treatment was suspended in sterilized water, and this was applied to an SD medium containing 1.0 μg / ml aureobasidin A, 30 ° C. And 475 colonies were isolated.

2. Acquisition of isoamyl acetate high-producing strain Each raw material shown in Table 6 was liquefied by stirring at 55 ° C. for 4 hours, diluted 1.5 times with water, and then lactic acid was added to an acidity of 4.0. A rice saccharified solution was prepared. 10 ml of the obtained rice saccharified solution was dispensed into a test tube, and 475 strains of the aureobasidin A-resistant strain obtained above were inoculated one by one, and cultured at 15 ° C. for 11 days. Subsequently, the supernatant was collected from the culture solution by centrifugation, and the amount of isoamyl acetate contained in the supernatant was measured by a headspace method using GC / FID. As a result, six strains (hia1-6) having high productivity of isoamyl acetate were selected.

  Next, the obtained 6 strains (hia 1 to 6) were subjected to a three-stage small preparation test of sake shown in the above condition 3, centrifuged after the upper tank to recover the supernatant, and the aroma components ( (Isoamyl acetate, isoamyl alcohol) was measured by GC / FID by the headspace method. In addition, the ethanol content, sake degree (Be ′), total acidity (TA, 0.1N NaOH ml), and amino acid degree (AA, 0.1N NaOH ml) in the supernatant were measured. The degree of sake (Be ′) was measured using a vibrating density specific gravity meter (DA-510). Further, the total acidity (TA) was determined by the amount of NaOH dropped when the BTB solution was added and the amount of the solution was changed from red to green with respect to the solution amount of 10 ml. Furthermore, after adding phenolphthalein to a liquid volume of 10 ml and adding NaOH until it becomes light red after adding 5 ml of formalin, the amino acid degree (AA) is until the liquid which became colorless becomes the light red color. It calculated | required with the amount of NaOH dripped. For comparison, the Km97 strain was also subjected to the same three-stage small-pickup test for sake shown in the above condition 3 and measured in the same manner.

  The obtained results are shown in FIG. In FIG. 1, the E / A ratio is the ratio (weight ratio) of isoamyl acetate to isoamyl alcohol. As a result, it was confirmed that the four strains of hia1, hia2, hia4 and hia6 have a high E / A ratio and a high isoamyl acetate production ability. Among these 4 strains, the hia1 strain having the highest sensory evaluation such as aroma was selected.

  Further, analysis of the base sequence of the MGA2 gene of the hia1 strain confirmed that the strain had a homozygous MGA2 mutant gene in which C at position 2117 of the MGA2 gene was mutated to A. .

3. Acquisition of cerulenin-resistant strain First, the hia1 strain was cultured with shaking at 30 ° C. for 1 day using YPD medium, washed with 50 mM potassium phosphate buffer (pH 8.0), and suspended in the same buffer. Next, the mutation treatment was performed by adding ethylmethanesulfonic acid to the obtained suspension of hia1 so as to be 4% by weight and shaking at 30 ° C. for 1 hour. Then, after neutralizing with a 5% by weight aqueous sodium thiosulfate solution, the mutated yeast is suspended in sterilized water, applied to an SD medium containing 5.0 μg / ml cerulenin, and cultured at 30 ° C. 44 colonies were isolated.

4). Acquisition of a high production strain of ethyl caproate A rice saccharified solution was prepared in the same manner as described above. 10 ml of the obtained rice saccharified solution was dispensed into a test tube, and 44 cerulenin-resistant strains obtained above were inoculated one by one and cultured at 15 ° C. for 11 days. Next, the supernatant was collected from the culture broth by centrifugation, and the aroma was evaluated by smell. As a result, seven strains (hec 1 to 7) with high fruity aroma were selected.

  Next, the obtained 7 strains (hec 1 to 7) were subjected to a three-stage small preparation test of sake shown in the above condition 1, centrifuged after the upper tank to recover the supernatant, and the aroma components ( Ethyl caproate, isoamyl acetate) were measured by GC / FID by the headspace method. In addition, the ethanol content, sake degree (Be ′), total acidity (TA, 0.1N NaOH ml), and amino acid degree (AA, 0.1N NaOH ml) in the supernatant were measured. For comparison, sake yeast Kyokai No. 1801 (hereinafter referred to as 1801 strain) was similarly subjected to the three-stage small preparation test for sake shown in the condition 1 and measured in the same manner.

  The obtained results are shown in FIG. As a result, in three strains of hec2, hec3, and hec6, high ethyl caproate productivity was observed. Among these 4 strains, the hia1 strain having the highest sensory evaluation such as aroma was selected. Among them, the hec2 strain with the highest sensory evaluation was deposited with the hec2 strain (NITE P-02179) at the Biotechnology Center Patent Microorganism Depositary Center, National Institute of Technology and Evaluation.

Example 2: Analysis of base sequence of FAS gene of ethyl caproate high-producing yeast hec2 strain Using the next-generation DNA sequencer, the base sequence of FAS gene of ethyl caproate high-producing yeast hec2 strain was analyzed.

  As a result, it was confirmed that the ethyl caproate high-producing yeast hec2 strain has a homozygous FAS2 mutant gene in which G at position 3748 of the FAS2 gene was mutated to A.

Example 3: Confirmation of phenotypic difference between hec2 and hia1 strains other than FAS2 In Example 2, the hec2 strain is a homozygous FAS2 mutant gene in which G at position 3748 of the FAS2 gene was mutated to A. It became clear to have. On the other hand, the hia1 strain has a wild-type FAS2 gene. Therefore, in order to confirm whether or not the high production ability of ethyl caproate possessed by the hec2 strain was dependent only on the FAS2 mutant gene, the following test was conducted.

  A homozygous FAS2 mutation was introduced into the hia1 strain by the following method. First, with the hia1 strain as a parent strain, a synthetic oligo DNA complementary to the synthetic DNA of the 3,733 to 3,762 bp region of the open reading frame of the FAS2 gene containing G3748A was annealed, and then transformed, and G A cerulenin-resistant strain having heterozygous FAS2 mutation mutated to A was obtained.

  Subsequently, the obtained cerulenin-resistant strain was used as a parent strain, and transformation was performed using the same oligo DNA as described above. The obtained transformant is applied to an SD medium containing 10 μg / ml cerulenin, and cultured at 30 ° C., whereby a hia1 having a homozygous FAS2 mutant gene in which G at position 3748 of the FAS2 gene is mutated to A A strain variant was obtained. In addition, it was confirmed by sequence analysis that a homozygous FAS2 mutant gene in which the GAS at position 3748 of the FAS2 gene was mutated to A in the hia1 strain variant was present.

  Subsequently, the obtained hia1 strain variant was subjected to a three-stage small preparation test of sake shown in the above condition 1, centrifuged after the upper tank, and the supernatant was collected. Aroma components (ethyl caproate) contained in the supernatant were collected. , Isoamyl acetate) was measured by the headspace method using GC / FID. For comparison, the hec2 strain, the hia1 strain (wild type), and the K1801 strain were also subjected to the same stage preparation test. The K1801 strain has a heterogeneous FAS2 mutant gene in which G at position 3748 of the FAS2 gene is mutated to A.

  The obtained hia1 strain variant was subjected to a one-step small preparation test of sake shown in the above condition 2, centrifuged after the upper tank to recover the supernatant and yeast, and the aroma component (caproic acid contained in the supernatant) Ethyl and isoamyl acetate) were measured by GC / FID by the headspace method, and caproic acid contained in the yeast cells was measured using a fatty acid methylation / purification kit (Nacalai Tesque).

  FIG. 3 shows the result of the three-stage small preparation test for sake shown in Condition 1, and FIG. 4 shows the result of the one-stage small preparation test for sake shown in Condition 2. In the variant of hia1 strain having a homozygous FAS2 mutant gene, the ability to produce ethyl caproate was higher than that of the hia1 strain (wild type), but the ability to produce ethyl caproate was higher than that of the hec2 strain. Was low. Moreover, the amount of intracellular caproic acid in the hec2 strain was significantly higher than that of the hia1 strain (wild type), the hia1 strain variant, and the K1801 strain. That is, these results revealed that the hec2 strain differs in phenotype from the hia1 strain even in points other than mutations in the FAS2 gene.

Example 4: Gene expression analysis of hec2 strain In order to analyze gene expression characteristics of the hec2 strain, the following test was performed. The hec2 strain was subjected to a one-step small preparation test of sake shown in the above condition 2, and the yeast was recovered by centrifugation after the upper tank. Total RNA in the microbial cells is extracted from the recovered yeast using RNasi Mini Kit (QIAGEN), and expression of FAS1, FAS2, EEB1, EHT1, ATF1, ATF2, and IAH1 genes by RT-PCR using TFC1 as a reference gene The amount was analyzed. For comparison, gene expression analysis was similarly performed for the hia1 strain (wild type), the Km97 strain, and the K1801 strain.

  The obtained results are shown in FIGS. From this result, it was revealed that the expression level of each gene of FAS1, FAS2, EEB1, and ATF1 was improved in the hec2 strain. In particular, the hec2 strain was confirmed to have a characteristic that the expression level of the EEB1 gene, which greatly contributes to the production of ethyl caproate, is much higher than that of the hia1 strain and is not recognized by the hia1 strain. .

Example 5: Measurement of alcohol acetyltransferase activity in hec2 strain The following test was conducted to measure alcohol acetyltransferase activity (AAT activity) in hec2 strain. Static culture was performed at 30 ° C. for 24 hours using an SD medium having a glucose concentration of 10%. The cultured yeast cells are collected, washed with Buffer A (25 mM imidazole HCl pH 7.5, 0.1 M NaCl, 20% glycerol, 1 mM DTT, 46 mM isoamyl alcohol, 0.1% Triton X-100), and then the yeast cells. Was dissolved in 3 ml of Buffer A, and 0.8 g of glass beads were added per 1 ml of the bacterial cell solution to disrupt the yeast (30 seconds ON / OFF 10 minutes). After collecting the supernatant, the glass beads were washed with 0.6 ml Buffer A, and the supernatant was collected. After centrifugation (15,000 g × 10 minutes), the supernatant was recovered to obtain a cell-free extract. The above cell-free extract was prepared at a temperature of 4 ° C.

  Next, after adjusting the cell concentration using Pierce BCA Protein Assay Kit (Thermo), 1 ml of BufferA containing 1.6 mM acetyl CoA was added to 1 m of the cell-free extract and allowed to react while slowly permeating at 25 ° C. for 60 minutes. It was. And the amount of produced | generated isoamyl acetate was measured by the head space method using GC / FID, and the alcohol acetyltransferase activity in the obtained cell-free extract was computed. For comparison, gene expression analysis was similarly performed for the hia1 strain (wild type), the Km97 strain, and the K1801 strain.

  The obtained result is shown in FIG. From this result, it was confirmed that the alcohol acetyltransferase in the hec2 strain was as high as the hia1 strain (wild type).

Example 6: Production of pure rice liquor using hec2 strain Using rice hec2 strain, pure rice liquor was produced using white rice with a 70% polished rice ratio. Specifically, it was charged under the conditions shown in Table 7, and after the upper tank, pure rice liquor was obtained by sequentially performing charcoal filtration, burning, storage (4 months), drooping / filtration, bottling / heating. . The soot was sampled over time from the 4th day to the 14th day of the retention, and the aroma components (ethyl caproate, isoamyl acetate) contained in the supernatant were measured by a headspace method using GC / FID. Further, aroma components (ethyl caproate, isoamyl acetate) in the obtained pure rice liquor were also measured by the headspace method using GC / FID. For comparison, eight kinds of sakes [commercial product A (pure rice liquor, 70% of rice polishing), commercial product B (pure rice liquor, 70% of rice polishing), commercial product C (pure rice liquor, polished rice) 70%), commercial product D (pure rice liquor, 65% of rice polishing rate), commercial product E (pure rice liquor, 65% of rice polishing rate), commercial product F (Daiginjo, 50% rice polishing rate), commercial product G (Daiginjo) , Rice milling ratio 50%) and commercial product H (pure rice Daiginjo, rice milling ratio 39%)] were also measured for aroma components (ethyl caproate, isoamyl acetate).

  The result of measuring the aroma component contained in the supernatant of koji over time is shown in FIG. 9, and the result of measuring the aroma component contained in sake is shown in FIG. From this result, it became clear that pure rice liquor with a high ethyl caproate content can be obtained in the actual production of 6 tons of total rice by using the hec2 strain. In addition, the pure rice sake obtained using the hec2 strain is more pure than the pure rice daiginjo (commercial product H) that uses a polished rice ratio of 39% despite using 70% polished rice. The ethyl caproate content showed a high value. In addition, the pure rice sake obtained using the hec2 strain perceived a rich ginjo aroma and had an excellent flavor.

NITE P-02179

Claims (7)

A Saccharomyces cerevisiae capable of producing high amounts of ethyl caproate,
When subjected to a three-stage small preparation test for sake under the condition 1 shown in Table 1, the concentration of ethyl caproate in the sake obtained by tanking on the 15th day of distillation is 4 mg / L or more. Saccharomyces cerevisiae, in which the amount of ATF1 mRNA is 1.5 times or more higher than that of the Sake Sake Kyokai No. 1801 strain when subjected to a one-step preparation test of sake under the condition 2 shown.
  The Saccharomyces cerevisiae according to claim 1, wherein the caproic acid content in the microbial cell is 0.01 mg / g-dry cells or more when subjected to a one-step small preparation test of sake shown in the condition 2.   The Saccharomyces cerevisiae according to claim 1 or 2, which exhibits cerulenin resistance.   The Saccharomyces cerevisiae according to any one of claims 1 to 3, which has a homozygous FAS2 mutant in which G at position 3748 of the FAS2 gene is mutated to A.   The manufacturing method of fermented material including the process of manufacturing fermented material using the Saccharomyces cerevisiae in any one of Claims 1-4.   The method for producing a fermented product according to claim 5, wherein the fermented product is sake.   The method for producing a fermented product according to claim 6, wherein rice having a rice polishing ratio of 40 to 90% is used.