Method for producing yeast containing kokumi-taste imparting substance and method for producing yeast extract containing kokumi-taste imparting substance

Abstract

The present application discloses a method for producing yeast containing a kokumi-taste imparting substance, the method comprising: a yeast proliferation step, in which yeast modified to reduce the activity of acetolactate synthase in cells and to require isoleucine and valine is cultured in a medium containing isoleucine and valine to proliferate the yeast; and a kokumi-taste imparting substance production step, in which when the content of isoleucine in the medium is less than 0.2% by mass, valine is added to the medium, the yeast is cultured, and the kokumi-taste imparting substance is produced; the kokumi-taste imparting substance is at least any one of γ-Glu-Abu and γ-Glu-Abu-Gly.

Description

Technical Field

[0001] The present invention relates to a method for producing a yeast containing a kokumi-taste imparting substance and containing at least one of γ-Glu-Abu and γ-Glu-Abu-Gly, and a method for producing a yeast extract containing a kokumi-taste imparting substance using the yeast. Background Art

[0002] Yeast extracts prepared from yeast cells have the function of imparting umami and rich flavors to food, and are widely used in the food field as food additives such as seasonings. Due to the trend of pursuing naturalness in recent years, the demand for yeast extracts has been increasing.

[0003] As one of the components that impart kokumi to food, glutathione (hereinafter sometimes referred to as “GSH”) which is a tripeptide composed of glutamic acid, cysteine, and glycine is known.

[0004] To date, technologies for increasing the GSH content in yeast cells include a technology for producing a yeast mutant strain in which at least a portion of the DOA1 gene and at least a portion of the MET30 gene are defective or mutated (see, for example, Patent Document 1), and a technology for obtaining a yeast mutant strain having a mutant MET30 gene by mutating two or more yeast mutant strains having high glutathione contents, and obtaining a yeast mutant strain having a higher GSH content by mating the obtained yeast mutant strains (see, for example, Patent Document 2).

[0005] In addition, γ-Glu-X and γ-Glu-X-Gly (X represents an amino acid or an amino acid derivative other than Cys and its derivatives) are also known as ingredients that impart kokumi to foods (see, for example, Patent Document 3).

[0006] Hitherto, with respect to techniques for increasing the content of the components imparting kokumi, there have been proposed techniques for culturing yeast in a medium supplemented with Abu (L-2-aminobutyric acid) and γ-Glu-Abu (L-γ-glutamyl-L-2-aminobutyric acid) to prepare a yeast extract containing γ-Glu-Abu from the obtained cells (see, for example, Patent Document 4), and techniques for improving the yeast cells by reducing the activity of acetolactate synthase in the cells and increasing the content of at least one selected from Abu, γ-Glu-Abu, and γ-Glu-Abu-Gly (see, for example, Patent Document 5).

[0007] As described above, various studies have been conducted on technologies for increasing the content of components that impart kokuminess to foods. However, considering industrial-scale production, the content of at least one of γ-Glu-Abu and γ-Glu-Abu-Gly in yeast or yeast extract is still insufficient, and there is a strong demand for rapid provision of technologies for further increasing the content.

[0008] Prior art literature

[0009] Patent Literature

[0010] Patent Document 1: Japanese Patent Application Publication No. 2010-29147

[0011] Patent Document 2: Japanese Patent Application Publication No. 2011-160739

[0012] Patent Document 3: Japanese Patent No. 5857973

[0013] Patent Document 4: Japanese Patent No. 5954178

[0014] Patent Document 5: International Publication No. 2015 / 005378 Summary of the invention

[0015] [Problems to be solved by the invention]

[0016] The present invention aims to solve the above-mentioned problems and achieve the following objectives. Specifically, the present invention aims to provide a method for producing a yeast containing a kokumi-imparting substance and containing a high level of at least one of γ-Glu-Abu and γ-Glu-Abu-Gly, and a method for producing a yeast extract containing a kokumi-imparting substance.

[0017] [Technical means to solve the problem]

[0018] The present inventors have conducted intensive studies to achieve the above-mentioned object, and as a result have reached the following finding: by growing yeast modified to reduce the activity of acetolactate synthase in cells and requiring isoleucine and valine, and when the content of isoleucine in the culture medium is less than 0.2% by mass, adding valine to the culture medium and culturing the yeast, the content of at least one of γ-Glu-Abu and γ-Glu-Abu-Gly in the yeast can be significantly increased.

[0019] The present invention is based on the above findings of the present inventors, and the technical means for solving the above problems are as follows. That is:

[0020] <1> A method for producing yeast containing a kokumi-taste imparting substance, characterized by comprising: a yeast proliferation step in which yeast modified to have reduced acetolactate synthase activity in cells and to require isoleucine and valine is cultured in a medium containing isoleucine and valine to proliferate the yeast; and

[0021] a step of producing a kokumi-taste-imparting substance, in which, when the content of isoleucine in the culture medium is less than 0.2% by mass, valine is added to the culture medium and the yeast is cultured to produce the kokumi-taste-imparting substance;

[0022] The kokumi-taste imparting substance is at least one of γ-Glu-Abu and γ-Glu-Abu-Gly.

[0023] <2> A method for producing a yeast extract containing a kokumi-taste imparting substance, characterized in that the yeast extract is prepared from yeast containing a kokumi-taste imparting substance, wherein the yeast containing a kokumi-taste imparting substance is obtained by the method for producing a yeast containing a kokumi-taste imparting substance as described in <1> to <4> above.

[0024] [Effects of the Invention]

[0025] According to the present invention, the above-mentioned various problems in the related art can be solved, the above-mentioned objects can be achieved, and a method for producing a yeast containing a kokumi-taste-imparting substance and containing at least one of γ-Glu-Abu and γ-Glu-Abu-Gly at a high level, and a method for producing a yeast extract containing a kokumi-taste-imparting substance can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1A This is a graph showing the results of measuring the isoleucine content in the culture supernatant in the main culture ("main culture" in Japanese) of Test Example 1.

[0027] Figure 1B This is a graph showing the results of measuring the valine content in the culture supernatant in the main culture of Test Example 1.

[0028] Figure 1C This is a graph showing the results of measuring the dry yeast cell weight in the main culture of Test Example 1.

[0029] Figure 1D This is a graph showing the results of measuring the amount of γ-Glu-Abu per unit weight of dried yeast cells in the main culture of Test Example 1.

[0030] Figure 1EThis is a graph showing the results of measuring the amount of γ-Glu-Abu-Gly per unit weight of the dried yeast cells in the main culture of Test Example 1.

[0031] Figure 1F This is a graph showing the results of measuring the amount of Abu per unit weight of dry yeast cells in the main culture of Test Example 1.

[0032] Figure 2A This is a graph showing the results of measuring the isoleucine content in the culture supernatant in the main culture of Test Example 2.

[0033] Figure 2B This is a graph showing the results of measuring the valine content in the culture supernatant in the main culture of Test Example 2.

[0034] Figure 2C This is a graph showing the results of measuring the dry yeast cell weight in the main culture of Test Example 2.

[0035] Figure 2D This is a graph showing the results of measuring the amount of γ-Glu-Abu per unit weight of dried yeast cells in the main culture of Test Example 2.

[0036] Figure 2E This is a graph showing the results of measuring the amount of γ-Glu-Abu-Gly per unit weight of the dried yeast cells in the main culture of Test Example 2.

[0037] Figure 2F This is a graph showing the results of measuring the amount of Abu per unit weight of dried yeast cells in the main culture of Test Example 2.

[0038] Figure 3A This is a graph showing the results of measuring the isoleucine content in the culture supernatant in the main culture of Test Example 3.

[0039] Figure 3B This is a graph showing the results of measuring the valine content in the culture supernatant in the main culture of Test Example 3.

[0040] Figure 3C This is a graph showing the results of measuring the dry yeast cell weight in the main culture of Test Example 3.

[0041] Figure 3D This is a graph showing the results of measuring the amount of γ-Glu-Abu per unit weight of dried yeast cells in the main culture of Test Example 3.

[0042] Figure 3EThis is a graph showing the results of measuring the amount of γ-Glu-Abu-Gly per unit weight of the dried yeast cells in the main culture of Test Example 3.

[0043] Figure 3F This is a graph showing the results of measuring the amount of Abu per unit weight of dried yeast cells in the main culture of Test Example 3.

[0044] Figure 4A FIG. 1 shows the results of measuring the isoleucine content in the culture supernatant in the main culture of Test Example 5. FIG.

[0045] Figure 4B FIG. 1 shows the results of measuring the valine content in the culture supernatant in the main culture of Test Example 5. FIG.

[0046] Figure 4C FIG. 1 is a diagram showing the results of measuring the dry yeast cell weight in the main culture of Test Example 5. FIG.

[0047] Figure 4D FIG. 1 shows the results of measuring the amount of γ-Glu-Abu per unit weight of dried yeast cells in the main culture of Test Example 5. FIG.

[0048] Figure 4E FIG. 1 shows the results of measuring the amount of γ-Glu-Abu-Gly per unit weight of dried yeast cells in the main culture of Test Example 5. FIG.

[0049] Figure 4F FIG. 1 shows the results of measuring the amount of Abu per unit weight of dried yeast cells in the main culture of Test Example 5. FIG.

[0050] Figure 4G FIG. 2 shows the results of measuring the isoleucine content in the culture supernatant in the main culture of Test Example 5. FIG.

[0051] Figure 4H FIG. 2 shows the results of measuring the valine content in the culture supernatant in the main culture of Test Example 5. FIG.

[0052] Fig. 4I FIG. 2 is a diagram showing the results of measuring the dry yeast cell weight in the main culture of Test Example 5. FIG.

[0053] Figure 4J FIG. 2 shows the results of measuring the amount of γ-Glu-Abu per unit weight of dried yeast cells in the main culture of Test Example 5. FIG.

[0054] Figure 4K FIG. 2 shows the results of measuring the amount of γ-Glu-Abu-Gly per unit weight of the dried yeast cells in the main culture of Test Example 5. FIG.

[0055] Figure 4L FIG. 2 shows the results of measuring the amount of Abu per unit weight of the dried yeast cells in the main culture of Test Example 5. FIG. DETAILED DESCRIPTION

[0056] (Method for producing yeast containing a substance imparting rich flavor)

[0057] The method for producing a yeast containing a kokumi-taste imparting substance of the present invention comprises at least a proliferation step and a kokumi-taste imparting substance producing step, and may further comprise other steps as necessary.

[0058] The kokumi-imparting substance in the present invention refers to at least one of γ-Glu-Abu and γ-Glu-Abu-Gly. In the present invention, Abu and Glu are L-forms.

[0059] <Proliferation Step>

[0060] The proliferation step is a step of culturing yeast modified to reduce the activity of acetolactate synthase in cells and to require isoleucine and valine in a medium containing isoleucine and valine to proliferate the yeast.

[0061] -yeast-

[0062] The yeast is modified so that the activity of acetolactate synthase in the cells is reduced and the yeast requires isoleucine and valine.

[0063] The yeast may be Saccharomyces cerevisiae or Saccharomyces pombe.

[0064] Examples of the cerevisiae yeast include yeast belonging to the genus Saccharomyces such as Saccharomyces cerevisiae, the genus Candida such as Candida utilis, the genus Pichia such as Pichia pastoris, the genus Hansenula such as Hansenula polymorpha, and the like.

[0065] Examples of the fission yeast include yeast belonging to the genus Schizosaccharomyces such as Schizosaccharomyces pombe.

[0066] Among them, Saccharomyces cerevisiae and Candida utilis, which are frequently used in the production of yeast extracts, are preferred.

[0067] The yeast may be haploid or may be a yeast having diploidy or higher polyploidy.

[0068] It is known that the yeast modified to reduce the intracellular acetolactate synthase activity and to require isoleucine and valine contains a high level of at least one selected from Abu, γ-Glu-Abu, and γ-Glu-Abu-Gly in the yeast body (see International Publication No. 2015 / 005378).

[0069] The yeast modified to reduce the intracellular acetolactate synthase activity and to require isoleucine and valine may be prepared by modification or a commercially available yeast.

[0070] The acetolactate synthase refers to a protein (EC 2.2.1.6) having the activity of catalyzing the reaction of generating α-acetylhydroxybutyrate and CO2 from pyruvate and α-ketobutyrate (α-KB). In addition, this activity is also referred to as "acetolactate synthase activity". In the present invention, acetolactate synthase may have the activity of catalyzing the reaction of generating acetolactate and CO2 from 2 molecules of pyruvate, or may not have this activity.

[0071] The method for modifying yeast so that the acetolactate synthase activity in the cell is reduced and the yeast requires isoleucine and valine is not particularly limited, and a known method can be appropriately selected, and examples thereof include the method described in International Publication No. 2015 / 005378.

[0072] Specifically, for example, by disrupting the ILV2 gene encoding the active subunit of acetolactate synthase, the activity of acetolactate synthase in the cell is reduced, and the cell becomes isoleucine- and valine-requiring.

[0073] The base sequence information of the ILV2 gene can be obtained from public databases and the like. For example, the base sequence of the ILV2 gene of Saccharomyces cerevisiae is disclosed in the Saccharomyces Genome Database (http: / / www.yeastgenome.org / ).

[0074] The method for disrupting the ILV2 gene is not particularly limited, and a known method can be appropriately selected.

[0075] The decrease in acetolactate synthase activity can be confirmed, for example, by preparing crude enzyme solutions from yeast before and after modification and comparing the acetolactate synthase activities of the crude enzyme solutions. Acetolactate synthase activity can be measured, for example, by a known method (FC Stormer and HE Umbarger, Biochem. Biophys. Res. Commun 17, 5, 587-592 (1964)).

[0076] The yeast may have other properties as needed, in addition to the properties that the yeast has been modified to reduce the activity of acetolactate synthase in the cells and to require isoleucine and valine.

[0077] The other properties are not particularly limited as long as they do not impair the effects of the present invention, and can be appropriately selected depending on the intended purpose. However, it is preferred that the ability to produce glutathione be enhanced, and it is more preferred that the threonine resistance be further enhanced.

[0078] The method for enhancing the glutathione production ability of the yeast is not particularly limited and can be appropriately selected according to the purpose. For example, by mating a yeast modified to enhance the glutathione production ability with the yeast modified to reduce the intracellular acetolactate synthase activity and require isoleucine and valine, a yeast modified to reduce the intracellular acetolactate synthase activity and require isoleucine and valine and having enhanced glutathione production ability can be selected.

[0079] The yeast modified to enhance the glutathione production ability may be one produced by modification or a commercially available one.

[0080] The method for improving yeast to enhance the ability to produce glutathione is not particularly limited, and a known method can be appropriately selected, for example, the following methods can be mentioned: a method such as described in Japanese Patent Laid-Open No. 2010-29147 (Japanese Patent No. 5496480) in which at least a portion of the DOA1 gene and at least a portion of the MET30 gene are deleted or mutated; a method such as described in Japanese Patent Laid-Open No. 2011-160739 (Japanese Patent No. 5667365) in which a yeast mutant having a mutant MET30 gene is subjected to mutation treatment to obtain two or more yeast mutants with high glutathione content, and the obtained yeast mutants are mated to obtain a yeast mutant with a higher glutathione content, etc.

[0081] The enhancement of the glutathione production ability can be confirmed by measuring the total glutathione amounts in the yeast before and after the improvement according to the method of Titze et al. (Analytical Biochemistry, Vol. 27, p502, 1969) and comparing the total glutathione amounts.

[0082] The method for obtaining yeast having the threonine resistance is not particularly limited, and a known method can be appropriately selected, and examples thereof include a method of adding threonine to a medium and selecting yeast that grows.

[0083] - Culture medium -

[0084] The medium used in the proliferation step is not particularly limited as long as it contains at least isoleucine and valine and the yeast can proliferate, and can be appropriately selected depending on the intended purpose.

[0085] The isoleucine content in the medium used in the proliferation step is not particularly limited as long as the yeast can proliferate, and can be appropriately selected depending on the amount of target yeast, etc., but is preferably 0.01% by mass to 2.0% by mass, and more preferably 0.1% by mass to 1.0% by mass.

[0086] The content of valine in the medium used in the proliferation step is not particularly limited as long as the yeast can proliferate, and can be appropriately selected depending on the amount of target yeast, etc., but is preferably 0.01% by mass to 2.0% by mass, and more preferably 0.1% by mass to 1.0% by mass.

[0087] The components other than isoleucine and valine in the medium used in the proliferation step and the amounts thereof are not particularly limited, and the components used for culturing microorganisms such as yeast can be appropriately selected.

[0088] For example, the carbon source includes glucose, sucrose, acetic acid, ethanol, molasses, sulfite pulp waste liquid, etc. These may be used alone or in combination of two or more.

[0089] Examples of the nitrogen source include inorganic salts such as ammonia, ammonium sulfate, ammonium chloride, and ammonium phosphate, and nitrogen-containing organic substances such as cornsteep liquor, casein, yeast extract, and peptone. These may be used alone or in combination of two or more.

[0090] Furthermore, phosphoric acid components such as superphosphate lime and ammonium phosphate, potassium components such as potassium chloride and potassium hydroxide, magnesium components such as magnesium sulfate and magnesium chloride, inorganic salts such as zinc, copper, manganese, and iron ions, vitamins, and the like may be added to the culture medium.

[0091] -nourish-

[0092] The culture form of the yeast in the proliferation step is not particularly limited, and a general yeast culture form can be appropriately selected, for example, batch culture, fed-batch culture, continuous culture, etc. Among them, fed-batch culture and continuous culture are preferred from the viewpoint of industrial-scale production.

[0093] The culture conditions of the yeast in the proliferation step are not particularly limited, and general culture conditions of yeast can be appropriately selected.

[0094] For example, the temperature is preferably 20°C to 40°C, more preferably 25°C to 35°C.

[0095] The pH is preferably 3.5 to 7.5, more preferably 4.0 to 6.9.

[0096] In addition, the culture is preferably carried out under aerobic conditions, and more preferably, the culture is carried out while being ventilated or stirred. The amount of ventilation and the stirring conditions are not particularly limited, and can be appropriately selected in consideration of the culture capacity, time, initial concentration of bacteria, etc. For example, ventilation can be carried out at about 0.2VVM (Volume per volume per minutes, ventilation ratio) to 2V.VM, and stirring can be carried out at about 50rpm to 900rpm.

[0097] The culture time is not particularly limited and can be appropriately selected according to the amount of the target yeast.

[0098] The amount of yeast in the culture after the proliferation step is not particularly limited and can be appropriately selected depending on the purpose. For example, it can be about 0.5% to 6% based on the dry yeast cell weight.

[0099] The method for measuring the dry yeast cell weight is not particularly limited, and a known method can be appropriately selected.

[0100] <Steps for producing rich flavor-imparting substances>

[0101] The kokumi-taste imparting substance producing step is a step of adding valine to the medium and culturing the yeast to produce the kokumi-taste imparting substance when the isoleucine content in the medium is less than 0.2 mass %.

[0102] -Amount of isoleucine in the medium when valine is added-

[0103] The isoleucine content in the culture medium when valine is added to the culture medium in the kokumi-taste imparting substance producing step is not particularly limited as long as it is less than 0.2 mass %, and can be appropriately selected depending on the purpose, but is preferably 0.05 mass % or less, more preferably 0.02 mass % or less, and particularly preferably 0.01 mass % or less.

[0104] The method for measuring the amount of isoleucine in the culture medium is not particularly limited, and a known method can be appropriately selected, and for example, the method described in the section of Examples described below can be mentioned.

[0105] -Amount of valine in the medium when valine is added-

[0106] The content of valine in the medium when valine is added to the medium in the kokumi-imparting substance producing step is not particularly limited and can be appropriately selected depending on the purpose, but is preferably 0.1 mass % or less, more preferably 0.05 mass % or less, and particularly preferably 0.01 mass % or less.

[0107] The method for measuring the amount of valine in the medium is not particularly limited, and a known method can be appropriately selected, and for example, the method described in the section of Examples described below can be mentioned.

[0108] -Amount of valine added to the culture medium-

[0109] The amount of valine added to the culture medium in the step of producing the kokumi-taste-imparting substance is not particularly limited as long as the kokumi-taste-imparting substance can be produced, and can be appropriately selected according to the purpose, but is preferably 0.5% or more, more preferably 1% or more, and particularly preferably 2.5% or more, per unit weight of the dry yeast cells, in terms of the amount (mass) added each time. When it is within the preferred range, the amount of valine produced can be increased, which is advantageous in that.

[0110] The upper limit of the amount of valine added to the medium is not particularly limited unless the effects of the present invention are impaired, and can be appropriately selected depending on the purpose. For example, the upper limit may be 5% per unit weight of dry yeast cells.

[0111] In the step of producing a kokumi-imparting substance, valine may be added to the culture medium once or twice or more.

[0112] -Amount of threonine added to the culture medium-

[0113] In the step of producing a kokumi-imparting substance, it is preferred that threonine is further added to the culture medium.

[0114] The threonine may be added to the culture medium at the same time as the valine or at different times. However, it is preferably added at the same time as the valine in order to increase the amount of the kokumi-imparting substance produced.

[0115] The amount of threonine added to the culture medium in the step of producing the kokumi-taste imparting substance is not particularly limited unless the effects of the present invention are impaired, and can be appropriately selected depending on the purpose, but is preferably 0.5% or more, more preferably 1% or more, and particularly preferably 2.5% or more, per unit weight of the dry yeast cells, in terms of amount (mass). When it is within the preferred range, the amount of kokumi-taste imparting substance produced can be increased, which is advantageous in that.

[0116] The upper limit of the amount of threonine added to the medium is not particularly limited unless the effects of the present invention are impaired, and can be appropriately selected depending on the purpose. For example, the upper limit may be 10% per unit weight of dry yeast cells.

[0117] In the step of producing a kokumi-imparting substance, threonine may be added to the culture medium once or twice or more.

[0118] - Culture medium -

[0119] The medium used in the kokumi-taste imparting substance producing step may be the same medium as that used in the proliferation step except for the amounts of isoleucine and valine and, if necessary, threonine.

[0120] -nourish-

[0121] The culture form and culture conditions of the yeast in the kokumi-taste imparting substance producing step are not particularly limited, and a general yeast culture form can be appropriately selected, and for example, the same culture form as in the proliferation step can be used.

[0122] -Rich flavor imparting substance-

[0123] The content of the kokumi-taste imparting substance in the yeast obtained in the kokumi-taste imparting substance producing step is not particularly limited and can be appropriately selected depending on the intended purpose. It is preferably 0.3% or more, more preferably 0.6% or more, further preferably 1.0% or more, and particularly preferably 1.3% or more, in terms of the total amount (mass) of γ-Glu-Abu and γ-Glu-Abu-Gly per unit weight of the dry yeast cells.

[0124] The upper limit of the content of the kokumi-taste imparting substance in the yeast obtained in the kokumi-taste imparting substance producing step is not particularly limited unless the effects of the present invention are impaired, and can be appropriately selected depending on the purpose. For example, the upper limit can be 5% based on the total amount (mass) of γ-Glu-Abu and γ-Glu-Abu-Gly per unit weight of the dry yeast cells.

[0125] The content ratio of the γ-Glu-Abu and the γ-Glu-Abu-Gly is not particularly limited and can be appropriately selected depending on the purpose.

[0126] The method for measuring the amount of the kokumi-imparting substance in the yeast is not particularly limited, and a known method can be appropriately selected, and for example, the method described in the section of Examples described below can be cited.

[0127] Furthermore, the yeast may further contain components that contribute to imparting kokumi other than γ-Glu-Abu and γ-Glu-Abu-Gly.

[0128] <Other steps>

[0129] The other steps are not particularly limited unless the effects of the present invention are impaired, and can be appropriately selected depending on the purpose. For example, a pre-culture step for preparing yeast used in the proliferation step can be mentioned.

[0130] The culture method, culture conditions, and medium of the yeast in the pre-culture step are not particularly limited, and the culture method, culture conditions, and medium generally used for culturing microorganisms such as yeast can be appropriately selected according to the purpose.

[0131] The yeast culture obtained in the kokumi-taste imparting substance producing step may be dried to obtain dried yeast cells having a high content of the kokumi-taste imparting substance.

[0132] The drying method is not particularly limited, and any method commonly used in preparing dried yeast cells can be appropriately selected, and examples thereof include freeze drying, spray drying, and drum drying.

[0133] In addition, the obtained dried yeast cells can also be processed into powder.

[0134] According to the method for producing yeast containing a kokumi-taste imparting substance of the present invention, yeast containing the kokumi-taste imparting substance at a high level can be produced, and the method can also be applied to industrial-scale production.

[0135] (Method for producing yeast extract containing rich flavor imparting substance)

[0136] The method for producing a yeast extract containing a kokumi-taste imparting substance of the present invention is to prepare a yeast extract from the yeast containing a kokumi-taste imparting substance obtained by the method for producing a yeast containing a kokumi-taste imparting substance of the present invention.

[0137] The method for preparing the yeast extract is not particularly limited, and a general method for preparing the yeast extract can be appropriately selected, for example: a self-decomposition method, i.e., using the proteolytic enzymes inherent in the yeast body to dissolve the yeast body; an enzyme decomposition method, i.e., adding an enzyme preparation derived from a microorganism or plant to dissolve the yeast; a hot water extraction method, i.e., dissolving the yeast body by immersing it in hot water for a certain period of time; an acid-base decomposition method, i.e., adding various acids or alkalis to dissolve the yeast body; a freeze-thaw method, i.e., breaking the yeast body by freezing and thawing once or more; and a physical disruption method, i.e., breaking the yeast body by physical stimulation; etc.

[0138] The physical stimulation is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include ultrasonic treatment; homogenization under high pressure; and grinding by mixing with solids such as glass beads.

[0139] -Rich flavor imparting substance-

[0140] The content of the kokumi-imparting substance in the yeast extract is not particularly limited and can be appropriately selected depending on the intended purpose. It is preferably more than 1%, more preferably 5% or more, and particularly preferably 10% or more, in terms of the total amount (mass) of γ-Glu-Abu and γ-Glu-Abu-Gly per unit dry weight of the yeast extract.

[0141] The upper limit of the content of the kokumi-imparting substance in the yeast extract is not particularly limited unless the effects of the present invention are impaired, and can be appropriately selected depending on the purpose. For example, the upper limit may be 17% based on the total amount (mass) of γ-Glu-Abu and γ-Glu-Abu-Gly per unit dry weight of the yeast extract.

[0142] The content ratio of the γ-Glu-Abu and the γ-Glu-Abu-Gly is not particularly limited and can be appropriately selected depending on the purpose.

[0143] The method for measuring the amount of the kokumi-taste imparting substance in the yeast extract is not particularly limited, and a known method can be appropriately selected. For example, the method can be a method similar to the method for measuring the amount of the kokumi-taste imparting substance in the yeast.

[0144] Furthermore, the yeast extract may contain components that contribute to imparting kokumi other than γ-Glu-Abu and γ-Glu-Abu-Gly.

[0145] According to the method for producing a yeast extract containing a kokumi-taste imparting substance of the present invention, a yeast extract containing a high level of the kokumi-taste imparting substance can be produced, and the method can also be applied to industrial-scale production.

[0146] The uses of the yeast containing a kokumi-imparting substance and the yeast extract containing a kokumi-imparting substance obtained by the production method of the present invention are not particularly limited and can be appropriately selected depending on the purpose. For example, the yeast can be used in various foods and beverages or supplements.

[0147] Example

[0148] Hereinafter, the present invention will be specifically described with reference to Preparation Examples and Test Examples, but the present invention is not limited to these Preparation Examples and Test Examples.

[0149] (Preparation Example 1: Preparation of Yeast)

[0150] A yeast strain was prepared in the following manner. The yeast strain was improved so that the activity of acetolactate synthase in the cell was reduced, the yeast strain required isoleucine and valine, the glutathione production ability was enhanced, and the yeast strain had threonine resistance.

[0151] <Mother plant (haploid: type a)>

[0152] As a strain modified to reduce the intracellular acetolactate synthase activity and to require isoleucine and valine, the NCYC868 strain (type a) (obtained from the National Collection of Yeast Cultures), which is an ILV2 mutant, was used.

[0153] <Mother plant (haploid: α type)>

[0154] As a strain improved to enhance the ability to produce glutathione, the Saccharomyces cerevisiae ABYC1588 strain described in Japanese Patent Publication No. 2011-160739 (Japanese Patent Publication No. 5667365) (deposit number FERM BP-10924, a strain deposited in the Patent Organism Depository of the National Institute of Advanced Industrial Science and Technology (Higashi 1-1-1, Tsukuba City, Ibaraki Prefecture), deposited on October 19, 2007) was used, and a haploid (α type) was obtained by a conventional method.

[0155] <Selection-1>

[0156] The haploid (a type) and the haploid (α type) are joined by a common method and cultured in the following manner.

[0157] - Pre-cultivation -

[0158] One platinum loop of the colony was inoculated in 5 mL of YPD (Yeast Extract Peptone Dextrose) medium and cultured overnight at 30°C. The culture solution was used as a pre-culture solution.

[0159] -Main training-

[0160] A molasses-urea medium having the following composition was prepared in a 200 mL Erlenmeyer flask with baffles, and the whole of the preculture solution was inoculated therein, followed by culturing at 30° C. and a stirring speed of 200 rpm for 48 hours.

[0161] --Molasses urea medium--

[0162]

[0163] (Made by Difco)

[0164]

[0165] The dry yeast cell weight (hereinafter sometimes referred to as "DCW") after the main culture was measured by a common method. In addition, the total glutathione content (hereinafter sometimes referred to as "GSH content") in the yeast after the main culture was measured according to the method of Titze et al. (Analytical Biochemistry, Vol. 27, p502, 1969).

[0166] Using the DCW and GSH contents obtained by the above measurement as indicators, a plurality of excellent diploid strains were selected.

[0167] <Selection-2>

[0168] The diploid strain selected in <Selection-1> was subjected to tetrad separation. The isolated bacteria were spread on the following culture medium to isolate the strain that only grows in (iv) SD (Synthetic Dropout, yeast selective auxotrophic type) medium containing isoleucine and valine (strains that require both isoleucine and valine).

[0169] - Culture medium -

[0170] (i) SD medium

[0171] (ii) SD medium containing 0.01% by mass isoleucine

[0172] (iii) SD medium containing 0.01% by mass valine

[0173] (iv) SD medium containing 0.01% by mass isoleucine and 0.01% by mass valine

[0174] The isolated strains were subjected to preliminary culture and main culture in the same manner as the culture described in <Selection-1>.

[0175] The dry yeast cell weight after the culture was measured by a common method. In addition, the contents of γ-Glu-Abu and γ-Glu-Abu-Gly, which are kokumi-imparting substances, and Abu, which is a precursor of kokumi-imparting substances, in the cultured yeast were measured as follows.

[0176] -Determination of rich taste imparting substances, etc.-

[0177] The peptide was fluorescently derivatized with 6-aminoquinoline-N-hydroxysuccinimide carbamate (AQC) and detected by LC-MS / MS (liquid chromatography tandem mass spectrometry) to measure kokumi-taste-imparting substances and the like.

[0178] Specifically, 2.5 μL of MillQ water, 5 μL of 5 μM internal standard substance solution (3-methyl-His-d2 (Sigma), Gly-d2 (Sigma); both stable isotope-labeled), and 30 μL of borate buffer (an accessory of the AccQ-Fluor (registered trademark) kit manufactured by Waters Corporation, Japan) were added to 2.5 μL of a sample diluted to an appropriate concentration or 2.5 μL of a standard solution containing 1 μM Abu, γ-Glu-Abu and γ-Glu-Abu-Gly.

[0179] To the mixture was added 10 μL of an AQC reagent solution (prepared by dissolving the reagent powder of the kit in 1 mL of acetonitrile), and the resulting mixture was heated at 55° C. for 10 minutes, followed by the addition of 100 μL of a 0.1% formic acid aqueous solution to prepare an analysis sample.

[0180] Next, the analysis sample prepared as described above was separated by the reverse phase liquid chromatography described below and then introduced into a mass spectrometer. The separation conditions were set as follows.

[0181] --Separation conditions--

[0182] (1) HPLC (High Performance Liquid Chromatography): Agilent 1200 series

[0183] (2) Separation column: Unison UK-Phenyl (inner diameter 2.0 mm, length 100 mm, particle size 3 μm (manufactured by Imtakt))

[0184] (3) Column temperature: 40°C

[0185] (4) Mobile phase A: 25 mM formic acid aqueous solution adjusted to pH 6.0 with ammonia

[0186] (5) Mobile phase B: methanol

[0187] (6) Flow rate: 0.25 mL / min

[0188] (7) Elution conditions: Elution was performed using a mixture of mobile phase A and mobile phase B. The ratio of mobile phase B to the mixture was as follows: 0 min (5%), 0 min to 17 min (5% to 40%), 17 min to 17.1 min (40% to 80%), 17.1 min to 19 min (80%), 19 min to 19.1 min (80% to 5%), 19.1 min to 27 min (5%).

[0189] Thereafter, the derivatives of Abu, γ-Glu-Abu and γ-Glu-Abu-Gly eluted under the separation conditions were introduced into a mass spectrometer and quantified by mass chromatogram. The analysis conditions were set as follows.

[0190] --Analysis conditions--

[0191] (1) Quality analysis equipment: AB Sciex API3200 QTRAP

[0192] (2) Detection mode: Selected Ion Monitoring (positive ion mode)

[0193] (3) Selected ions: Refer to Table 1 below

[0194] [Table 1]

[0195] Derivatives First mass analyzer Second mass analyzer Abu 274.2 171.1 γ-Glu-Abu 403.4 171.1 γ-Glu-Abu-Gly 460.4 171.1 3-methyl-His-d2 343.4 171.1 Gly-d2 248 171.1

[0196] The quantification of the derivatives of Abu, γ-Glu-Abu and γ-Glu-Abu-Gly was performed using the analysis software Analystver 1.4.2 (AB Sciex). As the internal standard substance used for quantification, in the case of the derivative of Abu, the derivative of 3-methyl-His-d2 was used, and in the case of the derivative of γ-Glu-Abu or γ-Glu-Abu-Gly, the derivative of Gly-d2 was used.

[0197] When γ-Glu-Abu is quantified, a mixed peak may appear due to the sample in very rare cases. In such a case, quantification is performed using 145.2 or 104.1 as the selected ion of the second mass analyzer.

[0198] The content of the kokumi-taste imparting substance and the dry yeast cell weight obtained by the above measurement are used as indicators to select excellent haploid strains.

[0199] <Obtaining diploid>

[0200] The haploid strains obtained in <Selection-2> were screened for a-type and α-type, and mated by a common method to obtain a plurality of diploid strains.

[0201] <Selection-3>

[0202] The strain obtained in the above <Obtaining a diploid> was cultured in a fed-batch manner as follows using a flask or a wide-mouthed thermos bottle.

[0203] - Culture (Flask) -

[0204] Pre-culture and main culture were carried out in the same manner as the culture described in <Selection-1>.

[0205] -Cultivation (Wide Mouth Thermos Bottle)-

[0206] --Pre-cultivation--

[0207] Two 3,000 mL culture media of the following composition were prepared (Marubishi Bioengineering small fermenter). The culture media were mixed and sterilized by high pressure steam sterilization at 121°C for 15 minutes.

[0208] [Culture medium]

[0209]

[0210] One platinum ring of the strain obtained in the above <Obtaining a diploid> grown on a YPD plate medium was inoculated in a pre-culture medium of the above composition and cultured under the following culture conditions.

[0211] After the culture was completed, all the cells were recovered by centrifugation (3,000 g × 5 minutes), and the cells were washed with an equal amount of sterile water. Thereafter, the cells were recovered by centrifugation again, and the cells were suspended in sterile water and adjusted to a solid content of 10% to 20% by mass. The resulting liquid was used as the pre-cultured cell liquid.

[0212] [Cultivation conditions]

[0213] ·Cultivation temperature: 30℃

[0214] Oscillation: 400rpm

[0215] Cultivation time: 24 hours

[0216] Ventilation volume: 3L / min (1V.VM)

[0217] --Main training--

[0218] The strain was cultured by fed-batch culture using a medium of the following composition and under the following culture conditions.

[0219] [Culture medium]

[0220]

[0221] [Cultivation conditions]

[0222] ·Cultivation temperature: 30℃

[0223] Culture conditions: 18 hours

[0224] pH: The pH was controlled with 25% caustic soda or 47% sulfuric acid so that the lower limit was 5.5 and the upper limit was 6.7.

[0225] Stirring: 600rpm~800rpm

[0226] Fed-batch medium: molasses (sugar content 36%) 870 mL to 1,000 mL

[0227] Ammonia water (10%) 100mL~200mL

[0228] Phosphoric acid (85%) 5g~20g

[0229] The dry yeast cell weight after the main culture was measured by a common method. In addition, the content of the kokumi-taste imparting substance in the yeast after the main culture was measured in the same manner as described in the item <Selection-2>.

[0230] Using the content of the kokumi-imparting substance and the dry yeast cell weight obtained by the above measurement as indicators, an excellent diploid strain (hereinafter sometimes referred to as "K16 strain") was selected.

[0231] The K16 strain is improved so that the activity of acetolactate synthase in the cell is reduced, isoleucine and valine are required, and the glutathione production ability is enhanced.

[0232] <Selection-4>

[0233] The K16 strain obtained in <Selection-3> was spread on a medium prepared by adding 2% by mass of threonine to a YPD medium, and cultured at 30°C to obtain two grown colonies (hereinafter sometimes referred to as "K16-1 strain" and "K16-2 strain").

[0234] For each of the above-mentioned colonies, preliminary culture and main culture were carried out in the same manner as the culture described in the above-mentioned <Selection-1>.

[0235] The dry yeast cell weight after the main culture was measured by a common method. In addition, the content of kokumi-imparting substances and the like in the yeast after the main culture was measured in the same manner as described in the item <Selection-2>.

[0236] As a result, the content of kokumi-imparting substances per unit weight of dried yeast cells was 0.43% for γ-Glu-Abu in K16-1 strain and 0.52% for K16-2 strain, 0.46% for γ-Glu-Abu-Gly in K16-1 strain and 0.70% for K16-2 strain, and the content of Abu per unit weight of dried yeast cells was 0.53% in both K16-1 strain and K16-2 strain.

[0237] The K16 strain obtained in the above-mentioned <Selection-3> was cultured in the same manner, and the weight of the dry yeast cells and the content of the kokumi-imparting substance were measured. As a result, the content of the kokumi-imparting substance per unit weight of the dry yeast cells was 0.08% for γ-Glu-Abu and 0.10% for γ-Glu-Abu-Gly. In addition, the content of Abu per unit weight of the dry yeast cells was 0.06%.

[0238] From the above results, it was found that the content of the kokumi-taste imparting substance increased and the content of the precursor of the kokumi-taste imparting substance also increased by imparting threonine resistance to yeast.

[0239] (Test Example 1)

[0240] The K16-2 strain obtained in the preparation example 1 was used and cultured under the conditions described in the following <Test example 1-1> and <Test example 1-2>. Pre-culture and main culture were performed in the same manner as in the case of using a wide-mouthed incubator in <Selection-3> of the preparation example 1, except that the culture was performed under the conditions described in the following <Test example 1-1> and <Test example 1-2>.

[0241] <Test Example 1-1>

[0242] In the main culture, 1,000 ppm of valine (2.9% per unit weight of dry yeast cells) was added to the medium 7 hours after the start of the culture, and the main culture was continued.

[0243] The isoleucine content in the culture supernatant immediately before the addition of valine was 118 ppm (0.0118 mass%), and the valine content was 363 ppm (0.0363 mass%). The amount of yeast in the culture medium immediately before the addition of valine was 3.45% of the dry yeast cell weight.

[0244] <Test Example 1-2>

[0245] In the main culture, 2,000 ppm of isoleucine was added to the medium 6 hours after the start of the culture, and 1,000 ppm of valine (2.9% per unit weight of dry yeast cells) was added to the medium 7 hours after the start of the culture, and the main culture was continued.

[0246] The isoleucine content in the culture supernatant immediately before the addition of valine was 1,973 ppm (0.1973 mass%), and the valine content was 899 ppm (0.0899 mass%). The amount of yeast in the culture medium immediately before the addition of valine was 3.36% of the dry yeast cell weight.

[0247] <Measurement>

[0248] -Determination of Isoleucine and Valine Contents in Culture Supernatant-

[0249] The contents of isoleucine and valine in the culture supernatant at the start of the main culture and 4 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, and 16 hours later were measured as follows.

[0250] The results are shown in Figure 1A (Isoleucine), Figure 1B (valine). In addition, Figure 1A-1B In the figure, “●, solid line” indicates the result of Test Example 1-1, and “□, dotted line” indicates the result of Test Example 1-2.

[0251] --Determination of Isoleucine and Valine Contents--

[0252] The contents of valine and isoleucine were measured by the AccQ-Tag Ultra labeling method using an "Acquity UPLC" analyzer manufactured by Waters Corporation (USA).

[0253] -Determination of dry yeast weight-

[0254] The dry cell weight of yeast (hereinafter sometimes referred to as "DCW") was measured by a conventional method at the start of the main culture and 2 hours, 4 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 16 hours and 18 hours later.

[0255] The results are shown in Figure 1C In addition, Figure 1C In the figure, “●, solid line” indicates the result of Test Example 1-1, and “□, dotted line” indicates the result of Test Example 1-2.

[0256] -Determination of rich taste imparting substances, etc.-

[0257] Yeast was recovered 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours and 16 hours after the start of the main culture, and the contents of γ-Glu-Abu and γ-Glu-Abu-Gly as kokumi-imparting substances and Abu as a precursor of the kokumi-imparting substance in the yeast (amount per unit weight of dry yeast cells) were measured in the same manner as the method described in the item <Selection-2> of (Preparation Example 1).

[0258] The results are shown in Figure 1D (γ-Glu-Abu), Figure 1E (γ-Glu-Abu-Gly) and Figure 1F (Abu). In addition, Figures 1D to 1F In the figure, “●, solid line” indicates the result of Test Example 1-1, and “□, dotted line” indicates the result of Test Example 1-2.

[0259] like Figures 1A to 1F As shown, in Test Example 1-1 in which valine was added to culture yeast when the isoleucine content in the culture supernatant was less than 0.2 mass % in the main culture, γ-Glu-Abu and γ-Glu-Abu-Gly, which are kokumi-taste imparting substances, were produced, and Abu, which is a precursor of the kokumi-taste imparting substance, was also produced. On the other hand, in Test Example 1-1 in which valine was added to culture yeast when the isoleucine content in the culture supernatant was 0.2 mass %, the production of the kokumi-taste imparting substance and its precursor was very small.

[0260] Therefore, it was confirmed that the production amount of kokumi-imparting substances can be significantly increased by the method of the present invention.

[0261] (Test Example 2)

[0262] The K16-2 strain obtained in the preparation example 1 was used and cultured under the conditions described in the following items <Test example 2-1> to <Test example 2-3>. Pre-culture and main culture were performed in the same manner as in the case of using a wide-mouthed thermos bottle in <Selection-3> of the preparation example 1.

[0263] <Test Example 2-1>

[0264] In the main culture, 1,000 ppm of valine (3.3% per unit weight of dry yeast cells) was added to the medium 6 hours after the start of the culture, and the main culture was continued.

[0265] The isoleucine content in the culture supernatant immediately before the addition of valine was 361 ppm (0.0361 mass%), and the valine content was 911 ppm (0.0911 mass%). The amount of yeast in the culture medium immediately before the addition of valine was 2.98% of the dry yeast cell weight.

[0266] <Test Example 2-2>

[0267] In the main culture, 1,000 ppm of valine (2.9% per unit weight of dry yeast cells) was added to the medium 7 hours after the start of the culture, and the main culture was continued.

[0268] The isoleucine content in the culture supernatant immediately before the addition of valine was 118 ppm (0.0118 mass%), and the valine content was 363 ppm (0.0363 mass%). The amount of yeast in the culture medium immediately before the addition of valine was 3.45% of the dry yeast cell weight.

[0269] <Test Example 2-3>

[0270] In the main culture, 1,000 ppm of valine (2.5% per unit weight of dry yeast cells) was added to the medium 8 hours after the start of the culture, and the main culture was continued.

[0271] The isoleucine content in the culture supernatant immediately before the addition of valine was 14.9 ppm (0.00149 mass%), and the valine content was 18.6 ppm (0.00186 mass%). The amount of yeast in the culture medium immediately before the addition of valine was 4.01% of the dry yeast cell weight.

[0272] <Measurement>

[0273] -Determination of Isoleucine and Valine Contents in Culture Supernatant-

[0274] The contents of isoleucine and valine in the culture supernatant at the start of the main culture, 4 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours and 16 hours later were measured in the same manner as in Experimental Example 1.

[0275] The results are shown in Figure 2A (Isoleucine), Figure 2B (valine). In addition, Figure 2A-2B In the figure, “△, solid line” represents the result of Test Example 2-1, “●, solid line” represents the result of Test Example 2-2, and “◇, solid line” represents the result of Test Example 2-3.

[0276] -Determination of dry yeast weight-

[0277] The dry yeast cell weight was measured at the start of the main culture, 2 hours, 4 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 16 hours, and 18 hours later in the same manner as in Test Example 1.

[0278] The results are shown in Figure 2C In addition, Figure 2C In the figure, “△, solid line” represents the result of Test Example 2-1, “●, solid line” represents the result of Test Example 2-2, and “◇, solid line” represents the result of Test Example 2-3.

[0279] -Determination of rich taste imparting substances, etc.-

[0280] Yeast was recovered 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours and 16 hours after the start of the main culture, and the contents of γ-Glu-Abu and γ-Glu-Abu-Gly as kokumi-imparting substances and Abu as a precursor of the kokumi-imparting substance in the yeast (amount per unit weight of dry yeast cells) were measured in the same manner as the method described in the item <Selection-2> of (Preparation Example 1).

[0281] The results are shown in Figure 2D (γ-Glu-Abu), Figure 2E (γ-Glu-Abu-Gly) and Figure 2F (Abu). In addition, Figures 2D to 2F In the figure, “△, solid line” represents the result of Test Example 2-1, “●, solid line” represents the result of Test Example 2-2, and “◇, solid line” represents the result of Test Example 2-3.

[0282] like Figures 2A to 2FAs shown, when valine is added to the main culture, the lower the content of isoleucine and valine in the culture supernatant, the more kokumi-taste-imparting substances and their precursors are produced.

[0283] (Test Example 3)

[0284] The K16-2 strain obtained in the preparation example 1 was used and cultured under the conditions described in the following <Test example 3-1> and <Test example 3-2>. Pre-culture and main culture were performed in the same manner as in the case of using a wide-mouthed thermos bottle in <Selection-3> of the preparation example 1, except that the culture was carried out under the conditions described in the following <Test example 3-1> and <Test example 3-2>.

[0285] <Test Example 3-1>

[0286] In the main culture, 1,000 ppm of valine (2.5% per unit weight of dry yeast cells) was added to the medium 8 hours after the start of the culture, and the main culture was continued.

[0287] The isoleucine content in the culture supernatant immediately before the addition of valine was 14.9 ppm (0.00149 mass%), and the valine content was 18.6 ppm (0.00186 mass%). The amount of yeast in the culture medium immediately before the addition of valine was 4.01% of the dry yeast cell weight.

[0288] <Test Example 3-2>

[0289] In the main culture, 1,000 ppm of valine (2.6% per unit weight of dry yeast cells) and 2,000 ppm of threonine (5.2% per unit weight of dry yeast cells) were added to the medium 8 hours after the start of the culture, and the main culture was continued.

[0290] The isoleucine content in the culture supernatant immediately before the addition of valine and threonine was 14.5 ppm (0.00145 mass%), and the valine content was 18.3 ppm (0.00183 mass%). In addition, the amount of yeast in the culture medium immediately before the addition of valine was 3.86% of the dry yeast cell weight.

[0291] <Measurement>

[0292] -Determination of Isoleucine and Valine Contents in Culture Supernatant-

[0293] The contents of isoleucine and valine in the culture supernatant at the start of the main culture, 4 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours and 16 hours later were measured in the same manner as in Experimental Example 1.

[0294] The results are shown in Figure 3A (Isoleucine), Figure 3B (valine). In addition, Figure 3A-3B In the figure, “◇, solid line” represents the result of Test Example 3-1, and “●, single-dot chain line” represents the result of Test Example 3-2.

[0295] -Determination of dry yeast weight-

[0296] The dry yeast cell weight was measured at the start of the main culture, 2 hours, 4 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 16 hours, and 18 hours later in the same manner as in Test Example 1.

[0297] The results are shown in Figure 3C In addition, Figure 3C In the figure, “◇, solid line” represents the result of Test Example 3-1, and “●, single-dot chain line” represents the result of Test Example 3-2.

[0298] -Determination of rich taste imparting substances, etc.-

[0299] Yeast was recovered 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours and 16 hours after the start of the main culture, and the contents of γ-Glu-Abu and γ-Glu-Abu-Gly as kokumi-imparting substances and Abu as a precursor of the kokumi-imparting substance in the yeast (amount per unit weight of dry yeast cells) were measured in the same manner as the method described in the item <Selection-2> of (Preparation Example 1).

[0300] The results are shown in Figure 3D (γ-Glu-Abu), Figure 3E (γ-Glu-Abu-Gly) and Figure 3F (Abu). In addition, Figure 3D~3F In the figure, “◇, solid line” represents the result of Test Example 3-1, and “●, single-dot chain line” represents the result of Test Example 3-2.

[0301] like Figures 3A to 3F As shown, when threonine is added together with valine in the main culture, more rich taste-imparting substances and their precursors are produced.

[0302] (Test Example 4)

[0303] The culture was carried out in the same manner as in Test Example 3-2 except that the culture time of the main culture was changed to culture for 12 hours after the addition of valine and threonine, and yeast extract was prepared as follows using the culture obtained thereafter (i.e., the culture obtained 20 hours after the start of the main culture).

[0304] First, the culture is centrifuged to recover yeast contained in the culture as a precipitate, and then washed with distilled water. Then, an appropriate amount of distilled water is added to make the bacterial cell concentration 10% to 15% by weight to prepare a yeast suspension. The yeast suspension is heated at 85°C for 70 seconds, then rapidly cooled and centrifuged to recover an extract. The recovered extract is dried to obtain a yeast extract.

[0305] <<Measurement>>

[0306] -Determination of rich taste imparting substances-

[0307] The contents of γ-Glu-Abu and γ-Glu-Abu-Gly as kokumi-imparting substances in the yeast extract (amount per dry weight of the yeast extract) were measured in the same manner as described in the item <Selection-2> of the above (Preparation Example 1). As a result, the content of γ-Glu-Abu per dry weight of the yeast extract was 9.40%, and the content of γ-Glu-Abu-Gly per dry weight of the yeast extract was 1.13%. Therefore, it was confirmed that the method of the present invention can produce a yeast extract having a kokumi-imparting substance content of more than 10% per dry weight of the yeast extract.

[0308] (Test Example 5)

[0309] The K16-2 strain obtained in the preparation example 1 was used and cultured under the conditions described in the following items <Test example 5-1> to <Test example 5-5>. Pre-culture and main culture were performed in the same manner as in the case of using a wide-mouthed thermos bottle in <Selection-3> of the preparation example 1.

[0310] <Test Example 5-1>

[0311] In the main culture, valine and threonine were not added and the main culture was continued (control).

[0312] <Test Example 5-2>

[0313] In the main culture, 500 ppm of valine (1.5% per unit weight of dry yeast cells) was added to the medium 7.5 hours after the start of the culture, and the main culture was continued.

[0314] In addition, the isoleucine content in the culture supernatant immediately before the addition of valine was 12.3 ppm (0.00123 mass%), and the valine content was 16.0 ppm (0.00160 mass%).

[0315] <Test Example 5-3>

[0316] In the main culture, 1,000 ppm of valine (3.0% per unit weight of dry yeast cells) was added to the medium 7.5 hours after the start of the culture, and the main culture was continued.

[0317] In addition, the isoleucine content in the culture supernatant immediately before the addition of valine was 0 ppm, and the valine content was 0 ppm.

[0318] <Test Example 5-4>

[0319] In the main culture, 1,500 ppm of valine (4.5% per unit weight of dry yeast cells) was added to the medium 7.5 hours after the start of the culture, and the main culture was continued.

[0320] In addition, the isoleucine content in the culture supernatant immediately before the addition of valine was 0 ppm, and the valine content was 0 ppm.

[0321] <Test Example 5-5>

[0322] In the main culture, 1,000 ppm of valine (3.0% per unit weight of dry yeast cells) and 1,000 ppm of threonine (3.0% per unit weight of dry yeast cells) were added to the medium 7.5 hours after the start of the culture, and the main culture was continued.

[0323] In addition, the isoleucine content in the culture supernatant immediately before the addition of valine and threonine was 0 ppm, and the valine content was 7.5 ppm (0.00075 mass %).

[0324] <Measurement>

[0325] -Determination of Isoleucine and Valine Contents in Culture Supernatant-

[0326] The contents of isoleucine and valine in the culture supernatant at the start of the main culture, 2 hours, 4 hours, 6 hours, 7.5 hours, 8 hours, 9 hours, 10 hours, 12 hours and 14 hours were measured in the same manner as in Experimental Example 1.

[0327] The results are shown in Figure 4A and Figure 4G(Isoleucine), Figure 4B and Figure 4H (valine). In addition, Figure 4A , 4B In 4G, 4H, "△, dotted line" represents the result of Test Example 5-1, "○, solid line" represents the result of Test Example 5-2, "■, single-point dash line" represents the result of Test Example 5-3, "□, solid line" represents the result of Test Example 5-4, and "◇, single-point dash line" represents the result of Test Example 5-5.

[0328] -Determination of dry yeast weight-

[0329] The dry yeast cell weight was measured at the start of the main culture and 2 hours, 4 hours, 6 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, and 14 hours later in the same manner as in Test Example 1.

[0330] The results are shown in Figure 4C and Fig. 4I In addition, Figure 4C and Fig. 4I In the figure, “△, dotted line” represents the result of Test Example 5-1, “○, solid line” represents the result of Test Example 5-2, “■, single-dot chain line” represents the result of Test Example 5-3, “□, solid line” represents the result of Test Example 5-4, and “◇, single-dot chain line” represents the result of Test Example 5-5.

[0331] -Determination of rich taste imparting substances, etc.-

[0332] Yeast was recovered 8 hours, 9 hours, 10 hours, 11 hours, 12 hours and 14 hours after the start of the main culture, and the contents of γ-Glu-Abu and γ-Glu-Abu-Gly as kokumi-imparting substances and Abu as a precursor of the kokumi-imparting substance (amount per unit weight of dry yeast cells) in the yeast were measured in the same manner as the method described in the item <Selection-2> of the (Preparation Example 1).

[0333] The results are shown in Figure 4D and Figure 4J (γ-Glu-Abu), Figure 4E and Figure 4K (γ-Glu-Abu-Gly) and Figure 4F and Figure 4L (Abu). In addition, Figure 4D , 4EIn 4F, 4J, 4K, and 4L, “△, dotted line” represents the result of Test Example 5-1, “○, solid line” represents the result of Test Example 5-2, “■, single-point dash line” represents the result of Test Example 5-3, “□, solid line” represents the result of Test Example 5-4, and “◇, single-point dash line” represents the result of Test Example 5-5.

[0334] like Figures 4A to 4L As shown, by increasing the amount of valine added to the main culture, more kokumi-taste imparting substances and their precursors were produced. In addition, in this test example, it was also confirmed that by adding threonine when adding valine, more kokumi-taste imparting substances and their precursors were produced.

[0335] As the aspects of the present invention, for example, the following aspects can be mentioned.

[0336] <1> A method for producing yeast containing a substance imparting a rich flavor, comprising:

[0337] a yeast proliferation step, in which yeast modified to reduce the activity of acetolactate synthase in cells and to require isoleucine and valine is cultured in a medium containing isoleucine and valine to proliferate the yeast; and

[0338] a step of producing a kokumi-taste-imparting substance, in which, when the content of isoleucine in the culture medium is less than 0.2% by mass, valine is added to the culture medium and the yeast is cultured to produce the kokumi-taste-imparting substance;

[0339] The kokumi-taste imparting substance is at least one of γ-Glu-Abu and γ-Glu-Abu-Gly.

[0340] <2> The method for producing a yeast containing a kokumi-imparting substance according to <1>, wherein the yeast is a yeast modified to enhance glutathione production ability.

[0341] <3> The method for producing a yeast containing a kokumi-taste imparting substance according to any one of <1> to <2>, wherein in the kokumi-taste imparting substance producing step, threonine is further added to the culture medium.

[0342] <4> The method for producing a yeast containing a kokumi-taste-imparting substance according to any one of <1> to <3>, wherein the content of the kokumi-taste-imparting substance in the yeast containing a kokumi-taste-imparting substance is 0.3% or more per unit weight of dry yeast cells.

[0343] <5> A method for producing a yeast extract containing a substance imparting a kokumi-taste, characterized in that the yeast extract is prepared from yeast containing a substance imparting a kokumi-taste, wherein the yeast containing a substance imparting a kokumi-taste is obtained by the method for producing a yeast containing a substance imparting a kokumi-taste according to any one of <1> to <4>.

[0344] <6> The method for producing a yeast extract containing a kokumi-taste imparting substance according to <5>, wherein the kokumi-taste imparting substance in the yeast extract containing a kokumi-taste imparting substance accounts for more than 1% of the dry weight of the yeast extract.

Claims

1. A method for producing yeast containing a substance imparting a rich taste, characterized in that: include a yeast proliferation step, in which yeast modified to have reduced acetolactate synthase activity in cells, to have isoleucine and valine requirements, to have enhanced glutathione production ability, and to have threonine resistance is cultured in a medium containing isoleucine and valine to proliferate the yeast; and a step of producing a kokumi-taste-imparting substance, in which, when the content of isoleucine in the culture medium is 0.0361 mass % or less, valine is added to the culture medium, and the yeast is cultured to produce the kokumi-taste-imparting substance; The kokumi-taste imparting substance is at least one of γ-Glu-Abu and γ-Glu-Abu-Gly, The yeast is Saccharomyces cerevisiae.

2. The method for producing yeast containing a kokumi-taste imparting substance according to claim 1, characterized in that: In the step of producing a kokumi-imparting substance, threonine is further added to the culture medium.

3. The method for producing yeast containing a kokumi-taste imparting substance according to claim 1 or 2, characterized in that: The kokumi-taste imparting substance content of the kokumi-taste imparting substance-containing yeast is 0.3% or more per unit weight of the dry yeast cells.

4. A method for producing a yeast extract containing a substance imparting a rich taste, characterized in that: include A yeast containing a kokumi-taste imparting substance is produced by the method for producing a yeast containing a kokumi-taste imparting substance according to any one of claims 1 to 3; and A yeast extract is prepared from the yeast containing the kokumi-taste imparting substance.

5. The method for producing a yeast extract containing a kokumi-flavor imparting substance according to claim 4, wherein: The yeast extract containing the kokumi-taste-imparting substance has a content of more than 1% per unit dry weight of the yeast extract.

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