JP2014143964A - Method for producing fermented food of high hemf content - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new and useful method for breeding HEMF-hyper-producing microorganisms by clarifying HEMF production-relevant genes in yeast, and also provide an HEMF production method and a method for producing fermented food of higher HEMF content in a simple manner not much different from the conventional fermented food production process.SOLUTION: Provided is a HEMF production method utilizing an yeast being deficient in ADH1 gene function. Also provided is a method for producing fermented food of high HEMF content characterized in that the yeast being deficient in ADH1 function is added during the initial to middle stage of fermentation process in the manufacturing process of fermented food.

Description

  The present invention relates to a method for producing 4-hydroxy-2 (or 5) -ethyl-5 (or 2) -methyl-3 (2H) -furanone (hereinafter referred to as “HEMF”), and a HEMF-rich fermented food. It relates to the manufacturing method.

  HEMF is a kind of aroma component found in various fermented foods such as soy sauce, miso, Japanese sake, cheese, etc., and has a caramel-like sweet scent, and is known as an effective component for improving the flavor especially in miso and soy sauce. In addition to miso and soy sauce, it is also expected as a raw material for imparting excellent flavor and flavor to foods.

Conventionally, HEMF has been produced using microorganisms, and various attempts have been made to improve the flavor and flavor of fermented foods and the like by using the HEMF. For example, as a method of causing HEMF to be produced by a microorganism, a method of inoculating yeast into an enzyme degradation solution obtained by degrading a protein in the presence of 5 units or more of peptidase activity per gram of protein and culturing the yeast (Patent Document 1) And a method of inoculating yeast after heating a liquid medium containing pentose and amino acids and culturing the yeast (Patent Document 2).
In addition, as a method for improving the flavor and flavor of fermented foods, a method of adding the HEMF-rich yeast culture solution obtained by the above-mentioned conventional method as a flavor improving agent, or a yeast strain with high HEMF-producing ability obtained by selective breeding There is known a method of adding fermented miso to fermented miso and fermenting it again (Patent Document 3).

  On the other hand, regarding the biosynthetic pathway of HEMF and related compounds in yeast, several findings have been obtained in Tigosaccharomyces roxy (Non-patent Document 1).

JP-A-8-116983 JP 2001-120293 A Japanese Patent Laid-Open No. 11-18759

Sasaki et.al., J.Agric.Food.Chem., Vol.39,934-938 (1991)

  However, in the production of fermented foods and the like, sufficient knowledge has not been obtained as to what genes of yeast contribute to the production of HEMF. For biosynthesis pathways of HEMF and related compounds in yeast, The detailed control mechanism was not elucidated. In addition, the conventional method for improving the flavor and flavor of fermented foods requires a yeast culture step in addition to the original fermented food production step, which increases the number of steps compared to the conventional fermented food production method. In many cases, it was not necessarily suitable for practical use.

  Accordingly, an object of the present invention is to clarify HEMF production-related genes in microorganisms, and to include HEMF easily in a method that is not greatly different from the production process of ordinary fermented foods, as well as a new and useful method for breeding HEMF high-producing microorganisms. An object of the present invention is to provide a method for producing a HEMF-rich fermented food product that can increase the amount.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have dramatically increased HEMF production ability by using a strain deficient in the alcohol dehydrogenase 1 (ADH1) gene in yeast used in the production of fermented foods. And the present invention was completed by finding that a fermented food with a very rich aroma can be produced by using the yeast having a deficiency of ADH1 gene function.

  Specifically, in Saccharomyces cerevisiae which is a kind of yeast used for the production of fermented foods, the ability to produce HEMF is dramatically improved by using a strain lacking the alcohol dehydrogenase 1 (ADH1) gene, Furthermore, it discovered that a fermented food with a very rich fragrance could be produced by using the ADH1 gene function-deficient yeast.

  In addition, the present inventors similarly use the genome database (http://www.genolevures.org/) of Tigo Saccharomyces luxi, which is a kind of yeast used in the production of fermented foods, to use ADH1 of Saccharomyces cerevisiae. We searched for genes with high amino acid sequence homology. As a result, a gene having a high sequence homology with ADH1 of Saccharomyces cerevisiae (hereinafter referred to as “ZrADH1”) was also found in the genome database of Tigo Saccharomyces luxi.

  Therefore, the present inventors have analyzed the function of the ZrADH1 gene. As a result, even in Tigosaccharomyces luxi, the ability to produce HEMF is dramatically improved by deleting the function of ZrADH1, and further, the ZrADH1 gene is further improved. It has been found completely new that fermented foods with a high aroma can be produced by using yeast lacking function. Further, in addition to the deletion of the ZrADH1 gene, if a gene that was also found to have a high amino acid sequence homology with Saccharomyces cerevisiae ADH1 (hereinafter referred to as “ZrADH2”) is simultaneously deleted, the ability to produce HEMF I found out that it will increase further.

That is, the present invention relates to the following (1) to (9).
(1) A method for producing HEMF, wherein a yeast lacking the function of the ADH1 gene is used.
(2) The production method according to (1), wherein the yeast is Saccharomyces cerevisiae or Tigosaccharomyces luxi.
(3) The method for producing HEMF according to (1), wherein the yeast is Saccharomyces cerevisiae and the ADH1 gene is a gene comprising the base sequence represented by SEQ ID NO: 8.
(4) The method for producing HEMF according to (1), wherein the yeast is Tigosaccharomyces luxi and the ADH1 gene is a gene comprising a base sequence represented by SEQ ID NO: 21.
(5) The method for producing HEMF according to (4), wherein the yeast further lacks the function of a gene comprising the base sequence represented by SEQ ID NO: 22.
(6) A method for producing a fermented food containing a high amount of HEMF, wherein yeast lacking the function of the ADH1 gene is added from the start of fermentation to the middle of fermentation in the fermented food production process.
(7) The production method according to (6), wherein the yeast is Saccharomyces cerevisiae or Tigosaccharomyces luxi.
(8) The production method according to (6) or (7), wherein a Maillard reaction product is further added.
(9) The production method according to any one of (6) to (8), wherein the fermented food is selected from the group consisting of soy sauce, miso, brewed sake, or vinegar.

  In the present invention, a gene involved in the production of HEMF in yeast can be clarified for the first time, and a new and useful method for selecting and producing a HEMF high-producing yeast can be provided. Moreover, if the manufacturing method of this invention is used, it will become possible to manufacture the fermented food with rich flavor which highly contained HEMF in the manufacturing process of fermented food very easily.

FIG. 1 shows intracellular ADH activity in Saccharomyces cerevisiae BY4743 strain in wild strain (WT) and ADH1 gene function deficient strain (adh1Δ). FIG. 2 shows the HEMF production ability of two types of Saccharomyces cerevisiae BY4743 strain and X2180 strain in the wild strain (WT) and the ADH1 gene function-deficient strain (adh1Δ). FIG. 3 shows the HEMF content in the brewed liquor when the brewed liquor was produced using the Saccharomyces cerevisiae wild strain (WT) and the ADH1 gene function deficient strain (adh1Δ), respectively. FIG. 4 shows intracellular ADH activity in the parent strain (Zr WT), ZrADH1 gene disruption strain (Zr adh1Δ), ZrADH2 gene disruption strain (Zr adh2Δ), and ZrADH gene double disruption strain (Zr adh1Δadh2Δ). Is shown. FIG. 5 shows that when using HEMF production medium, T. saccharomyces luxy parent strain (Zr WT), ZrADH1 gene single disruption strain (Zr adh1Δ), ZrADH2 gene single disruption strain (Zr adh2Δ), ZrADH gene double disruption strain The amount of HEMF produced in Zr adh1Δadh2Δ) is shown. FIG. 6 shows the amount of HEMF produced in the T. saccharomyces luxy parent strain (Zr WT) and the ZrADH gene double disruption strain (Zr adh1Δadh2Δ) when using a high-temperature digested liquid.

  The yeast used in the present invention is not particularly limited as long as it is a yeast generally used in the production of fermented foods, and examples thereof include Saccharomyces cerevisiae and Zygosaccharomyces rouxii.

  In the present invention, HEMF productivity can be improved by deleting the function of the yeast alcohol dehydrogenase 1 (ADH1) gene.

  The ADH1 gene in the present invention can be defined by a yeast gene database or the like. For example, in Saccharomyces cerevisiae, it refers to a gene represented by “YOL086C” in Saccharomyces Genome Database (http://www.yeastgenome.org/). The base sequence of the gene is shown in SEQ ID NO: 8 in the sequence listing.

  Further, the ADH1 gene in the present invention refers to a gene represented by “ZYRO0B05940g” (hereinafter referred to as “ZrADH1”) in the genome database (http://www.genolevures.org/) of Tigosaccharomyces luxi, for example. The base sequence of the ZrADH1 gene is shown in SEQ ID NO: 21 in the sequence listing.

  In the present invention, when the ADH1 gene of Tigo Saccharomyces luxi is deleted, not only the ZrADH1 gene is deleted, but also indicated by “ZYRO0C12562g” in the genome database of Tigo Saccharomyces luxi (http://www.genolevures.org/). Gene (hereinafter referred to as “ZrADH2”) may be deleted. The base sequence of the gene is shown in SEQ ID NO: 22 in the sequence listing.

  In the present invention, as a method for deleting a gene function, a known method for causing mutation or deletion in a gene may be used, and a gene disruption method by homologous recombination, a mutation introduction method, or the like can be used.

  As a method of screening for a strain in which a mutation or deletion has occurred in a gene among strains that have been mutated by such a method, for example, in the case of gene disruption by homologous recombination, For example, a method may be used in which another gene conferring resistance to a specific antibiotic is simultaneously incorporated, and only strains that have grown normally in a medium supplemented with the antibiotic are selected. However, since it is not possible to confirm whether or not the target locus has been replaced with the introduced marker gene only by selection as described above, the target locus can be determined by using the PCR method, Southern hybridization method or the like as appropriate. It is recommended to make sure that it is replaced by Furthermore, it can also be confirmed that the function of alcohol dehydrogenase is deficient. For confirmation, the alcohol dehydrogenase activity may be measured using a known method, for example, a method of measuring the concentration of NADH produced in the reaction process due to the action of alcohol dehydrogenase.

  As a method for confirming the productivity of HEMF, a known method may be followed. For example, the method described in Patent Document 2 can be used. Specifically, a method of culturing the strain in a medium suitable for HEMF production (medium composed of pentose, amino acid, glucose, etc.) and quantifying the amount of HEMF on the medium by high performance liquid chromatography or gas chromatography Etc. can be used.

In the present invention, HEMF can be produced by culturing the ADH1 gene function-deficient yeast obtained by the method as described above. Specific culture conditions for producing HEMF include, for example, HEMF production medium (416 mM glucose, 200 mM glycine, 200 mM ribose, 7.3 mM KH ₂ PO ₄ , 20 mM MgSO ₄ .7H ₂ O, 0.5% yeast extract. , PH 6.0) at 25 to 35 ° C. for about 1 to 5 days.

  Further, HEMF obtained separately from the production of fermented food by culturing the ADH1 gene function-deficient yeast can be added to the fermented food. Various fermented foods can be produced by adding them. At this time, the ADH1 gene-deficient yeast is added at the start of the fermented food production process to the middle of the fermentation, so that a sufficient amount of HEMF can be generated even in the ADH1 gene-deficient yeast during the fermentation process of the food. It is preferable because it is possible.

  Examples of fermented foods include soy sauce, miso, brewed sake, various seasonings (such as cooking liquor) produced using brewed liquor, vinegar, etc., and fermented foods produced using yeast. If there is, it is not limited to these. When producing soy sauce, miso, etc., it is preferable to use Tigosaccharomyces ruxii as yeast, and when producing brewed sake, vinegar, etc., Saccharomyces cerevisiae as yeast.

  As a method for producing fermented food, a conventional method may be used. When the fermented food is soy sauce, inoculated and mixed koji molds into a normal koji material, for example, a mixture of soy material that has been boiled and steamed and wheat material that has been cracked and fried Then, the koji is prepared, and the koji obtained is charged into a normal charging tank with a suitable concentration of saline, and the above ADH1 gene function-deficient yeast is added as appropriate, and fermented and matured for about 3 to 6 months with stirring. The soy sauce moromi can be obtained by making it so that it can be compressed, refined, and fired as necessary to obtain a product soy sauce (raw soy sauce or fired soy sauce). In addition, although ADH1 gene function deficient yeast can be added from the start of fermentation of moromi to the middle of fermentation, it is particularly preferable to add at the start of fermentation.

  In addition, when the fermented food is a brewed liquor using rice as a raw material, for example, rice of any milling ratio, dried koji, water and 90% lactic acid are mixed to make koji (moromi), and the above ADH1 gene is used here. The target brewed liquor can be obtained by adding a function-deficient yeast and fermenting it at 10 to 15 ° C. for about 20 to 30 days, followed by fermentation. ADH1 gene function-deficient yeast can be added from the start of fermentation of the koji to the middle of the fermentation, but is particularly preferably added at the start of fermentation.

The addition amount of the ADH1 gene function-deficient yeast may be appropriately added in an amount suitable for the characteristics of each fermented food. For example, in the case of soy sauce or miso, about 10 ⁴ to 10 ⁵ (cells / mL) at the end of lactic acid fermentation. In addition, if it is brewed liquor or vinegar, it is preferable to add so that it may become about 10 < ⁶ > -10 < ⁸ > (cells / mL) per pumping water at the time of preparation.

  In addition, in the manufacturing method of fermented food which adds an ADH1 gene function defect yeast strain to the raw material of fermented food, at the start of fermentation, in addition to addition of Saccharomyces yeast lacking the function of ADH1 gene, Maillard reaction product is a precursor of HEMF. It may be added as a body. In particular, when producing a fermented food having a low content of precursors such as brewed sake and vinegar, it is preferable to add a Maillard reaction product.

  Here, the Maillard reaction product refers to a reaction product of an amino acid and a sugar. For example, an arbitrary amino acid aqueous solution and a sugar aqueous solution are mixed and heated at 80 to 140 ° C. for 10 to 20 minutes. . As the amino acid aqueous solution, any amino acid that can be used in foods may be used. For example, an aqueous solution of glycine, alanine, glutamic acid, or the like can be used. As the sugar aqueous solution, any sugar can be used as long as it can be used in foods. In particular, pentose is preferred because it is said that the Maillard reaction easily proceeds. As the pentose, for example, ribose, xylose, arabinose, ribulose and the like can be used.

  The Maillard reaction product can be added before or simultaneously with the addition of the ADH1 gene-deficient yeast. The addition amount of the Maillard reaction product can be appropriately adjusted depending on the type of fermented food and the desired HEMF concentration. For example, when the fermented food is brewed sake, 2M amino acid aqueous solution and sugar are used for 100 g of koji. It is preferable to add 0.2 to 15 mL of the Maillard reaction product produced from the aqueous solution.

  Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.

(Example 1) Production and evaluation of Saccharomyces cerevisiae ADH1 gene function deficient strain (Example 1-1) Acquisition of Saccharomyces cerevisiae ADH1 gene disruption strain Saccharomyces cerevisiae diploid yeast BY4743 and its ADH1 gene disruption strain are I got what is provided by Funakoshi Corporation. The method for producing the ADH1 gene disrupted strain of Saccharomyces cerevisiae diploid yeast BY4743 is described on the website of Saccharomyces Genome Deletion Project (http://sequence-www.stanford.edu/group/yeast_deletion_project/deletions3.html) Was used.

  Moreover, it was confirmed whether the ADH1 gene disrupted strain surely lacks the function of ADH1. The measurement is carried out according to a known method (U. Lutstorf et.al., Arch. Biochem. Biophys., 126, 933-944 (1968)), and the NADH concentration produced by the action of alcohol dehydrogenase is measured using an absorptiometer. Was done by.

1 mL of yeast cells cultured in a HEMF production medium were collected, 300 μL of 32 mM sodium pyrophosphate (pH 8.8) and an equal amount of glass beads were added, and disrupted at 4 ° C. for 15 minutes. After centrifugation at 4 ° C. and 10,000 rpm for 10 minutes, the supernatant was collected to obtain a cell disruption solution. For ADH activity, a certain amount of cell lysate was added to assay buffer (0.2 mM ethanol, 5 mM NAD, 32 mM sodium pyrophosphate (pH 8.8)), and the absorbance at a wavelength of 340 nm was measured. As a result, the protein concentration in the cell lysate was quantified and calculated as specific activity.
As a result, as shown in FIG. 1, it was confirmed that the function of the ADH1 gene was certainly lost in the ADH1-disrupted strain.

(Example 1-2) Preparation of ADH1 gene disruption strain using Saccharomyces cerevisiae X2180 strain (1) Disruption of ADH1 gene in X2180 haploid In order to clarify the contribution of ADH1 gene to HEMF production, the above-mentioned BY4743 strain Separately from the disrupted strain, the diploid yeast strain X2180 of Saccharomyces cerevisiae was used as a parent strain, and an ADH1 gene disrupted strain was obtained by the following method. The X2180-1A and X2180-1B strains, which are haploids of X2180, were obtained from the American Type Culture Collection (ATCC) as ATCC 204504 and ATCC 26787, respectively.

  The gene disruption of the X2180 strain was performed using kanMX as a marker gene. Since the X2180 strain is diploid, as described below, the disruption of the gene disrupts the haploid X2180-1A and -1B genes with kanMX, and crosses them into a diploid. Acquired by doing. In addition, the yeast introduced with the kanMX gene has resistance to the antibiotic geneticin.

  Using genomic DNA prepared from Saccharomyces cerevisiae BY4743 ADH1 gene disruption strain as a template, PCR was performed using primers ADH1 del cassette Fw (SEQ ID NO: 1) and ADH1 del cassette Rv (SEQ ID NO: 2). Thereafter, DNA fragments having DNA in the upstream and downstream portions adjacent to the ORF of ADH1 were prepared on both sides of the marker kanMX. Using this DNA fragment, the X2180-1A strain and the X2180-1B strain were transformed, and strains that grew on a geneticin-containing medium were selected. PCR was performed using the genomic DNA of each of the selected strains as a template, primers ADH1-A Fw (SEQ ID NO: 3) and KANB (SEQ ID NO: 4), and the size of the resulting product was measured. It was confirmed that the polyploid ADH1 gene was disrupted.

(2) Production of diploid A diploid of yeast can be obtained by a conventional method. The obtained haploid gene-disrupted strains were cultured overnight at 30 ° C. in a YPD medium, collected and resuspended in a new YPD medium. Thereafter, equal amounts of this cell suspension were mixed and left overnight at 30 ° C. Since the recombinant strain exhibits resistance to the antibiotic geneticin, the overnight strain was inoculated into a geneticin-containing medium, and a strain that grew on the geneticin-containing medium was selected.

As a method for confirming the diploid of yeast, a method of amplifying MAT locus responsible for sex determination by PCR and judging from the size of the PCR product can be used (T. Katou et al, Yeast, 25, 799). -807 (2008)).
Using the genomic DNA extracted from the selected diploid-disrupted strain as a template, three primers, MAT specific primer (SEQ ID NO: 5), MATa specific primer (SEQ ID NO: 6), and MATα specific primer (SEQ ID NO: 7) were used. PCR was performed, and the size of the obtained product was measured to confirm that it was a diploid X2180 ADH1 gene disruption strain.

(Example 1-3) Confirmation of HEMF productivity of ADH1 gene disruption strain (1) Generation of HEMF BY4743 ADH1 gene disruption strain obtained in Example 1-1, or X2180 ADH1 gene disruption prepared in Example 1-2 The strain and each parent strain were cultured in 5 mL of 2 × YPD medium (containing 2% by weight of yeast extract, 4% by weight of peptone and 4% by weight of glucose) at 30 ° C. for 60 hours. Then, 1 mL of HEMF production medium (416 mM glucose, 200 mM glycine, 200 mM ribose, 7.3 mM KH ₂ PO ₄ , 20 mM MgSO ₄ .7H ₂ O, 0.5% yeast extract, pH 6 so that OD600 = 2 / mL. 0), and HEMF was produced by shaking culture at 30 ° C. for 48 hours.

(2) Extraction of HEMF 600 μL of the culture supernatant was collected from the culture solution in which HEMF was produced, mixed with an equal amount of ethyl acetate, and vigorously stirred for 3 minutes. After stirring, the mixture was centrifuged at 15000 rpm for 3 minutes. After separation, 500 μL of the supernatant was recovered, and the solvent was removed by a vacuum centrifugal concentrator to dry it. This was redissolved with 500 μL of methanol, and the HEMF content was quantified by high performance liquid chromatography (HPLC) using this as a sample.

(3) Determination of HEMF Analysis of HEMF by HPLC was performed under the conditions shown in Table 1 below. The HEMF contained in the culture supernatant was calculated from a standard line created based on the peak height of a known concentration of HEMF (available for purchase from Tokyo Kasei). The result is shown in FIG.

  As shown in the results of FIG. 2, in all of the ADH1 gene disrupted strains, HEMF productivity was remarkably improved as compared with the wild type.

(Example 1-4) Production of brewed liquor to which ADH1 gene function deficient strain was added In the preparation test, 72.8 g of alpha rice with 70% milling rate as the kake rice, and dry koji with 70% milling rate as koji. 2 g, 174 g of pumped water, and 100 μL of 90% lactic acid were added. At this time, as a HEMF precursor, the Maillard reaction product was added to 1.16 mL of the condition 1 and 17.4 mL of the pumped water in the condition 2. As the Maillard reaction product, 2M glycine and 2M ribose solution heated in an autoclave at 121 ° C. for 15 minutes was used. After the BY4743 ADH1 gene disruption strain obtained in Example 1-1 and its parent strain (WT) are appropriately cultured at 30 ° C. in a YPD medium (containing 1% by mass of yeast extract, 2% by mass of peptone, and 2% by mass of glucose). The yeast number was previously added to the pumped water so that the number of yeast was 0.5 × 10 ⁸ cells / mL. The fermentation temperature was set to 15 ° C., and the tank was placed on the 20th day after the preparation. The result is shown in FIG.

  From the results shown in FIG. 3, the brewed liquor produced by adding the ADH1 gene-deficient strain in both conditions 1 and 2 has a significantly improved HEMF content compared to the brewed liquor produced by adding the wild strain. Was. Further, the degree of increase in the HEMF amount was dependent on the added amount of the Maillard reactant, which is a precursor of HEMF. Furthermore, the brewed liquor produced by adding the ADH1 gene function-deficient strain and increasing the HEMF content had a very good aroma.

  From the above results, it was clarified that, in Saccharomyces cerevisiae, by using a yeast strain deficient in the function of the ADH1 gene, a high-quality fermented food with high HEMF content and high HEMF content can be easily produced.

(Example 2) Production and evaluation of Tigo Saccharomyces luxi ADH1 gene function-deficient strain (Example 2-1) Production of Tigo Saccharomyces luxi, ADH1 gene disruption strain As a parent strain, available from the American Type Culture Collection (ATCC) Using a certain ATCC2623 strain, a single disruption strain lacking the function of either the ZrADH1 gene or the ZrADH2 gene and a double disruption strain in which both the ZrADH1 gene and the ZrADH2 gene were disrupted were obtained as follows.

  A single disruption of the ZrADH1 gene or the ZrADH2 gene of ATCC2623 was performed using loxP-kanMX-lox-P as a marker gene. Regarding ZrADH1 and ZrADH2 gene double disruption, the ZrADH1 gene was used as a marker gene using loxP-kanMX-loxP, and the ZrADH2 gene was used as a marker gene using loxP-zeoMX-loxP.

(1) Preparation of a ZrADH1 gene-only disruption strain of ATCC2623 strain Using pUG6 (U. Guldener et al, Nucleic Acids Res, 24, 2519-2524 (1996)) as a template, primers ADH1disConst-F1 (SEQ ID NO: 9) and ADH1disConst- PCR was performed using R1 (SEQ ID NO: 10). Using the obtained PCR reaction product as a template, PCR was performed using primers ADH1disConst-F2 (SEQ ID NO: 11) and ADH1disConst-R2 (SEQ ID NO: 12), so that ZrADH1 was placed on both sides of the marker loxP-kanMX-loxP. A DNA having upstream and downstream DNA adjacent to the ORF was prepared. Using this DNA, the ATCC2623 strain was transformed, and a strain that grew on a geneticin-containing medium was selected. By using the genomic DNA extracted from the selected strain as a template, PCR was performed using the primers ADH1disConf-Fw (SEQ ID NO: 13) and ADH1disConf-Rv (SEQ ID NO: 14), and the size of the resulting product was measured, whereby ZrADH1 It was confirmed that the gene was destroyed. As a result, a ZrADH1 gene-only disruption strain of ATCC2623 was obtained.

(2) Preparation of ZrADH2 gene single disruption strain of ATCC2623 strain PCR was performed using pUG6 as a template and primers ADH2disConst-F1 (SEQ ID NO: 15) and ADH2disConst-R1 (SEQ ID NO: 16). By using the obtained PCR reaction product as a template and performing PCR using primers ADH2disConst-F2 (SEQ ID NO: 17 in the sequence listing) and ADH2disConst-R2 (SEQ ID NO: 18), both sides of the marker, loxP-kanMX-loxP A DNA having upstream and downstream DNA adjacent to the ORF of ZrADH2 was prepared. Using this DNA, the ATCC2623 strain was transformed, and a strain that grew on a geneticin-containing medium was selected. Using genomic DNA extracted from the selected strain as a template, PCR was performed using primers ADH2disConf-Fw (SEQ ID NO: 19 in the sequence listing) and zeoMX-intra-Rv (SEQ ID NO: 20), and the size of the resulting product was measured. It was confirmed that the ZrADH2 gene was destroyed. As a result, a ZrADH2 gene single disruption strain of ATCC2623 was obtained.

(3) Preparation of ZrADH1 and ZrADH2 Gene Double Disrupted Strains of ATCC2623 Strain PCR was performed using pUZ6 as a template and primers ADH2disConst-F1 (SEQ ID NO: 15) and ADH2disConst-R1 (SEQ ID NO: 16). PUZ6 was prepared by amplifying a Zeocin resistance gene (zeoMX) by PCR using pCR-Blunt (Invitrogen) as a template, and then substituting the amplified fragment with kanMX of pUG6. Using the obtained PCR reaction product as a template, PCR was performed using primers ADH2disConst-F2 (SEQ ID NO: 17) and ADH2disConst-R2 (SEQ ID NO: 18), so that ADH2 was added to both sides of the marker, loxP-zeoMX-loxP. A DNA having upstream and downstream DNA adjacent to the ORF was prepared. Using this DNA, the ATCC2623 strain ZrADH1 gene disrupted strain prepared in (2) above was transformed, and a strain that grew on a geneticin and zeocin-containing medium was selected. By performing PCR using the genomic DNA extracted from the selected strain as a template and using primers ADH2disConf-Fw (SEQ ID NO: 19) and zeoMX-intra-Rv (SEQ ID NO: 20), and measuring the size of the resulting product A strain in which the ZrADH1 gene and the ZrADH2 gene are simultaneously disrupted (hereinafter referred to as a ZrADH gene double disruption strain) was obtained.

(4) Confirmation of ADH1 gene function deficiency Furthermore, it was confirmed whether the ADH1 gene disruption strain surely lacks ADH1 function. The measurement is carried out according to a known method (U. Lutstorf et.al., Arch. Biochem. Biophys., 126, 933-944 (1968)), and the NADH concentration produced by the action of alcohol dehydrogenase is measured using an absorptiometer. Was done by. The method was the same as that described in Example 1-1.
As a result, as shown in FIG. 4, it was confirmed that the function of the ADH1 gene was certainly lost in the ZrADH1-disrupted strain and the ADH gene double-disrupted strain in which both the ZrADH1 gene and the ZrADH2 gene were disrupted.

(Example 2-2) HEMF productivity of ZrADH gene-disrupted strain 5 ml each of the ATCC2623 strain ZrADH1 single-disrupted strain, ZrADH2 single-disrupted strain, and ZrADH gene double-disrupted strain prepared in Example 2-1 X YPD medium (2% yeast extract, 4% glucose, 4% peptone) was cultured at 28 ° C for about 60 hours. Then, 2 mL of HEMF production medium (416 mM glucose, 200 mM glycine, 200 mM ribose, 7.3 mM KH ₂ PO ₄ , 20 mM MgSO ₄ .7H ₂ O, 0.5% yeast extract, pH 6.0 so that OD600 = 2. ) And culturing with shaking at 28 ° C. for 48 hours to generate HEMF.

  600 μL of the culture supernatant was collected from the culture solution in which HEMF was produced, mixed with an equal amount of ethyl acetate, and vigorously stirred for 3 minutes. After stirring, the mixture was centrifuged at 15000 × rpm for 3 minutes to recover 500 μL of the supernatant, and the solvent was removed with a vacuum centrifugal concentrator to dryness. This was redissolved with 500 μL of methanol, and the HEMF content was quantified by high performance liquid chromatography (HPLC) using this as a sample. HEMF was quantified by the same method as shown in Example 1-3 (3).

  As a result, as shown in FIG. 5, it was revealed that when a single disruption strain in which the function of the ZrADH1 gene was disrupted was used, the HEMF production ability was significantly increased as compared with the wild strain. Further, it was found that when an ADH gene double disruption strain in which both the ZrADH1 gene and the ZrADH2 gene were disrupted was used, the HEMF production ability was further improved as compared with a strain in which only ZrADH1 was disrupted.

(Example 2-3) Preparation of a high-temperature digestive juice containing a ZrADH gene double-disrupted strain A mixture of soybeans that had been submerged and steamed and wheat that had been cracked with fried rice was inoculated and mixed to prepare soy sauce cake. Three times the amount of distilled water heated to 50 ° C. was added to the soy sauce cake, and left overnight in a constant temperature bath at 50 ° C., followed by filter paper filtration to recover the filtrate. Sodium chloride was added to the collected filtrate so as to have a concentration of 5%, and heat sterilized one was used as a high-temperature digestion liquid for the test.

  The ZrADH gene double disruption strain of ATCC2623 prepared in Example 2-1 was cultured in 5 mL of 2 × YPD medium (2% yeast extract, 4% glucose, 4% peptone) at 28 ° C. for about 60 hours. Thereafter, it was added to 2 mL of high-temperature digestive juice so that OD600 = 2, and HEMF was generated by shaking culture at 28 ° C. for 48 hours.

  600 μL of the culture supernatant was collected from the culture solution in which HEMF was produced, mixed with an equal amount of ethyl acetate, and vigorously stirred for 3 minutes. After stirring, the mixture was centrifuged at 15000 × rpm for 3 minutes to recover 500 μL of the supernatant, and the solvent was removed with a vacuum centrifugal concentrator to dryness. This was redissolved with 500 μL of methanol, and the HEMF content was quantified by high performance liquid chromatography (HPLC) using this as a sample. The result is shown in FIG. The HEMF analysis by HPLC was performed under the same conditions as in Example 1-3 (3). The parent strain (WT) was used as a control.

  As shown in FIG. 6, the amount of HEMF produced in the high-temperature digestive juice increased by adding the ADH gene double disruption strain in which the functions of both the ZrADH1 gene and the ZrADH2 gene were disrupted. Moreover, when the aroma of each digestion liquid was compared, the high-temperature digestion liquid added with the ADH gene double disruption strain is milder and has a favorable aroma compared with the high-temperature digestion liquid added with the wild strain. It was a thing.

(Summary)
From the above, it has been clarified that the ability to produce HEMF is significantly improved by deleting the ADH1 gene in yeasts commonly used in the production of fermented foods such as Saccharomyces cerevisiae and Tigosaccharomyces luxi. .

Claims (9)

  A method for producing HEMF, comprising using yeast lacking the function of ADH1 gene.   The production method according to claim 1, wherein the yeast is Saccharomyces cerevisiae or Tigosaccharomyces luxi.   The method for producing HEMF according to claim 1, wherein the yeast is Saccharomyces cerevisiae and the ADH1 gene is a gene comprising a base sequence represented by SEQ ID NO: 8.   The method for producing HEMF according to claim 1, wherein the yeast is Tigosaccharomyces luxi, and the ADH1 gene is a gene comprising a base sequence represented by SEQ ID NO: 21.   The method for producing HEMF according to claim 4, wherein the yeast further lacks the function of a gene comprising the base sequence represented by SEQ ID NO: 22.   A method for producing a HEMF-rich fermented food comprising adding yeast lacking the function of the ADH1 gene from the start of fermentation to the middle of fermentation in the fermented food production process.   The method according to claim 6, wherein the yeast is Saccharomyces cerevisiae or Tigosaccharomyces luxi.   Furthermore, the manufacturing method of Claim 6 or 7 which adds a Maillard reaction material.   The production method according to any one of claims 6 to 8, wherein the fermented food is selected from the group consisting of soy sauce, miso, brewed sake, or vinegar.