Method for improving flavor

Abstract

Provided is a technique for improving the flavor of food. In the present invention, Bacillus bacteria are used to produce components such as 2-methylbutyric acid, diacetyl and acetoin that are effective for improving the flavor of food.

Description

How to improve flavor

[0001] The present invention relates to a technique for improving the flavor of food. Specifically, the present invention can relate to a food flavor improving agent and a method for improving the flavor of food.

[0002] Aliphatic carboxylic acids such as 2-methylbutyric acid, 3-methylbutyric acid, and isobutyric acid, as well as diacetyl and acetoin, are known to be components of cheese (Non-Patent Document 1).

[0003] A method for producing (S)-2-methylbutyric acid from L-isoleucine using Bacillus bacteria has been reported (Patent Document 1). Also, a method for producing aliphatic carboxylic acids using yeast has been reported (Patent Documents 2 to 5, Non-Patent Document 2).

[0004] A method for producing diacetyl and acetoin using bacteria of the genus Bacillus has been reported (Non-Patent Document 3).

[0005] Patent Publication No. 2009-284861, Patent Publication No. 2020-156343, Patent Publication No. 2013-223485, Patent Publication No. 06-504447, Patent Publication No. 61-092582

[0006] TK Singh et al., Flavor of Cheddar Cheese: A Chemical and Sensory Perspective, COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY - Vol. 2, 2003. Weerawat Runguphan and Jay D Keasling, Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals, Metab Eng. 2014 Jan;21:103-13.Kaloyan Petrov and Penka Petrova, Current Advances in Microbial Production of Acetoin and 2,3-Butanediol by Bacillus spp. Fermentation 2021, 7(4), 307.

[0007] An object of the present invention is to provide a technique for improving the flavor of food.

[0008] As a result of intensive research to solve the above problems, the present inventors discovered that components effective in improving the flavor of food can be produced using Bacillus bacteria, and thus completed the present invention.

[0009] That is, the present invention can be exemplified as follows: [1] A method for producing a carboxylic acid, diacetyl, and acetoin, comprising: culturing a Bacillus bacterium having carboxylic acid, diacetyl, and acetoin production abilities in a medium containing a precursor of the carboxylic acid and a sugar to obtain a culture containing the carboxylic acid, diacetyl, and acetoin, wherein the carboxylic acid is selected from the group consisting of 2-methylbutyric acid, 3-methylbutyric acid, isobutyric acid, and a combination thereof, and the precursor of 2-methylbutyric acid is isoleucine, the precursor of 3-methylbutyric acid is leucine, and the precursor of isobutyric acid is selected from the group consisting of isoleucine, leucine, valine, and a combination thereof. [2] The method (specifically, the method described in [1]), wherein the 2-methylbutyric acid is (S)-2-methylbutyric acid and / or (R)-2-methylbutyric acid, and the precursor of (S)-2-methylbutyric acid and the precursor of (R)-2-methylbutyric acid are L-isoleucine and D-isoleucine, respectively. [3] The method (specifically described in [1] or [2]) wherein the carboxylic acid, diacetyl, and acetoin are produced as a composition containing the carboxylic acid, diacetyl, and acetoin. [4] The method (specifically described in [3]) wherein the composition contains the culture or a processed product thereof. [5] The method (specifically described in [3] or [4]) wherein the composition contains a dried product of the culture or a dried product of the supernatant of the culture. [6] The method (specifically described in any of [3] to [5]) wherein the content of the carboxylic acid in the composition is 10 ppm (w / w) or more. [7] The method (specifically described in any of [3] to [6]) wherein the content of diacetyl in the composition is 0.5 ppm (w / w) or more. [8] The method (specifically described in any of [3] to [7]) wherein the content of acetoin in the composition is 10 ppm (w / w) or more.[9] The method (specifically, the method according to any one of [1] to [8]) above, wherein the bacterium is Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus polymixa, Bacillus stearothermophilus, or Bacillus velezensis.

[10] The method (specifically, according to any one of [1] to [9]) wherein the bacterium is Bacillus subtilis or Bacillus amyloliquefaciens.

[11] The method (specifically, according to any one of [1] to

[10] ) wherein the isoleucine content in the medium is 0.1 to 5% (w / w).

[12] The method (specifically, according to any one of [1] to

[11] ) wherein the leucine content in the medium is 0.1 to 5% (w / w).

[13] The method (specifically, according to any one of [1] to

[12] ) wherein the valine content in the medium is 0.1 to 5% (w / w).

[14] The method (specifically, according to any one of [1] to

[13] ) wherein the sugar content in the medium is 1 to 50% (w / w).

[15] The method (specifically, according to any one of [1] to

[14] ) wherein the sugar is glucose.

[16] The method (specifically, according to any one of [3] to

[15] ) wherein the composition is a composition for improving the flavor of food.

[17] The method (specifically, according to

[16] ) wherein the flavor improvement is the imparting of a cheese flavor.

[18] The method (specifically, according to any one of [3] to

[17] ) wherein the composition is a seasoning.

[19] The method (specifically, described in any one of [1] to

[18] ) wherein at least 2-methylbutyric acid is produced.

[20] The method (specifically, described in any one of [1] to

[19] ) wherein 3-methylbutyric acid is further produced.

[21] The method (specifically, described in

[19] or

[20] ) wherein isobutyric acid is further produced.

[22] A composition produced by the method (specifically, described in any one of [3] to

[21] ).

[23] A composition for improving the flavor of food, comprising the following components (A), (B), and (C): (A) a carboxylic acid selected from the group consisting of 2-methylbutyric acid, 3-methylbutyric acid, isobutyric acid, and combinations thereof; (B) diacetyl; (C) acetoin.

[24] The composition (specifically, described in

[23] ) wherein the components (A), (B), and (C) are produced by the method (specifically, described in any one of [1] to

[21] ).

[25] The composition (specifically, the composition described in

[23] or

[24] ) containing at least 2-methylbutyric acid.

[26] The composition (specifically, the composition described in

[25] ) further containing 3-methylbutyric acid.

[27] The composition (specifically, the composition described in

[25] or

[26] ) further containing isobutyric acid.

[28] The composition (specifically, the composition described in any of

[23] to

[27] ) wherein the flavor improvement is the imparting of a cheese flavor.

[29] A method for improving the flavor of food, comprising the step of adding the following components (A), (B), and (C) to raw materials of the food: (A) a carboxylic acid selected from the group consisting of 2-methylbutyric acid, 3-methylbutyric acid, isobutyric acid, and combinations thereof; (B) diacetyl; (C) acetoin.

[30] The method (specifically the method described in

[29] ) wherein the components (A), (B), and (C) are produced by the method (specifically, the method described in any of [1] to

[21] ).

[31] The method (specifically, the method described in

[29] or

[30] ) comprising, prior to the step, a step of producing the components (A), (B), and (C) by the method (specifically, the method described in any one of [1] to

[21] ).

[32] The method (specifically, described in any one of

[29] to

[31] ) wherein the component (A) is added to a concentration of 0.001 to 5000 ppm (w / w) for consumption.

[33] The method (specifically, described in any one of

[29] to

[32] ) wherein the component (B) is added to a concentration of 0.0001 to 100 ppm (w / w) for consumption.

[34] The method (specifically, described in any one of

[29] to

[33] ) wherein the component (C) is added to a concentration of 0.001 to 5000 ppm (w / w) for consumption.

[35] The method (specifically, described in any one of

[29] to

[34] ) wherein at least 2-methylbutyric acid is added.

[36] The method (specifically, described in

[35] ) wherein 3-methylbutyric acid is further added.

[37] The method (specifically, the method described in

[35] or

[36] ) further comprising adding isobutyric acid.

[38] The method (specifically, the method described in any of

[29] to

[37] ) further comprising adding a cheese flavor to the product.

[0010] According to the present invention, the flavor of food can be improved.

[0011] The present invention will be described in detail below.

[0012] <1> Active Ingredient <1-1> Active Ingredient In the present invention, the following components (A), (B), and (C) are used as active ingredients: (A) a carboxylic acid selected from the group consisting of 2-methylbutyric acid, 3-methylbutyric acid, isobutyric acid, and combinations thereof; (B) diacetyl; (C) acetoin.

[0013] Components (A), (B), and (C) are collectively referred to as the "active ingredients." Component (A) is also referred to as the "carboxylic acid."

[0014] The use of an active ingredient can improve the flavor of food, i.e., an effect of improving the flavor of food is obtained. This effect is also referred to as a "flavor improving effect." In other words, the active ingredient has the function of improving the flavor of food. This function is also referred to as a "flavor improving function." Improving the flavor of food is also simply referred to as "flavor improvement." An example of flavor improvement is imparting a cheese flavor to food. That is, an example of the flavor improving effect is imparting a cheese flavor to food. This effect is also referred to as a "cheese flavor imparting effect." Furthermore, an example of the flavor improving function is imparting a cheese flavor to food. This function is also referred to as a "cheese flavor imparting function." Imparting a cheese flavor to food is also simply referred to as "imparting cheese flavor." Examples of imparting cheese flavor include imparting a cheese flavor to foods that do not have a cheese flavor (e.g., foods that do not contain cheese) and enhancing the cheese flavor of foods that have a cheese flavor (e.g., foods that contain cheese). Examples of cheese flavor include a rich cheese-like flavor and a rich cheese-like taste. Specifically, the use of an active ingredient can improve the flavor of a food (e.g., impart a cheese flavor to a food) compared to when the active ingredient is not used. Therefore, the flavor-improving effect (e.g., cheese flavor-imparting effect) can be determined by measuring and comparing the flavor of a food (e.g., cheese flavor in a food) when the active ingredient is used and when the active ingredient is not used. That is, a flavor-improving effect can be determined if the flavor of a food is more preferable when the active ingredient is used compared to when the active ingredient is not used. Specifically, for example, a cheese flavor-imparting effect can be determined if the cheese flavor of a food is stronger when the active ingredient is used compared to when the active ingredient is not used. Examples of when an active ingredient is not used include when none of the components (A) to (C) are used or when some of the components (A) to (C) are not used. That is, by using at least the components (A) to (C) in combination, the flavor of a food can be improved (e.g., cheese flavor can be imparted to a food) compared to when none of the components (A) to (C) are used.In one embodiment, the combined use of components (A) to (C) may improve the flavor of a food (e.g., impart a cheese flavor to a food) compared to when at least one component selected from components (A) to (C) is not used. In one embodiment, the combined use of components (A) to (C) may improve the flavor of a food (e.g., impart a cheese flavor to a food) compared to when component (A) is used alone. Measurement and comparison of the flavor of a food (e.g., the cheese flavor of a food) can be performed, for example, by sensory evaluation by a specialist panel.

[0015] Flavors (e.g., cheese flavor) may be divided into, for example, initial taste, middle taste, and aftertaste. The terms "initial taste," "middle taste," and "aftertaste" refer to the flavors perceived, respectively, from 0 to 1 second, 1 to 3 seconds, and 3 to 5 seconds after ingestion (after placing the food in the mouth) in the case of a liquid (liquid food). Furthermore, the terms "initial taste," "middle taste," and "aftertaste" refer to the flavors perceived, respectively, from 0 to 4 seconds, 4 to 10 seconds, and 10 to 15 seconds after ingestion (after placing the food in the mouth) in the case of a solid (solid food). In the present invention, "solid" refers to forms other than liquids, including pastes and gels. For example, the initial taste, middle taste, aftertaste, or a combination thereof may be improved by using an active ingredient. That is, by utilizing the active ingredient, specifically, for example, a cheese initial flavor, a cheese middle flavor, a cheese aftertaste, or a combination thereof may be imparted.

[0016] The carboxylic acid is selected from the group consisting of 2-methylbutyric acid, 3-methylbutyric acid, isobutyric acid, and combinations thereof. The combination is not particularly limited. Examples of combinations include a combination of 2-methylbutyric acid and 3-methylbutyric acid, a combination of 3-methylbutyric acid and isobutyric acid, a combination of 2-methylbutyric acid and isobutyric acid, and a combination of 2-methylbutyric acid, 3-methylbutyric acid, and isobutyric acid. Examples of 2-methylbutyric acid include (S)-2-methylbutyric acid and (R)-2-methylbutyric acid. Examples of 2-methylbutyric acid include, in particular, (S)-2-methylbutyric acid. For example, at least 2-methylbutyric acid may be selected as the carboxylic acid. For example, at least 2-methylbutyric acid may be selected as the carboxylic acid, and further, 3-methylbutyric acid and / or isobutyric acid may be selected. That is, the carboxylic acid may be, for example, 2-methylbutyric acid or a combination containing it, in other words, it may include 2-methylbutyric acid. For example, at least (S)-2-methylbutyric acid may be selected as the carboxylic acid. That is, the carboxylic acid may be, for example, (S)-2-methylbutyric acid or a combination containing it, in other words, it may contain (S)-2-methylbutyric acid. In other words, at least (S)-2-methylbutyric acid may be selected as the 2-methylbutyric acid. For example, at least (S)-2-methylbutyric acid may be selected as the 2-methylbutyric acid, and (R)-2-methylbutyric acid may also be selected. That is, the 2-methylbutyric acid may be, for example, (S)-2-methylbutyric acid or a combination containing it, in other words, it may contain (S)-2-methylbutyric acid.

[0017] Each active ingredient may be commercially available or may be obtained by appropriate manufacturing. The manufacturing method for each active ingredient is not particularly limited. Each active ingredient can be manufactured, for example, by chemical synthesis, enzymatic reaction, fermentation, extraction, or a combination thereof. Specifically, each active ingredient can be manufactured, for example, using Bacillus bacteria, as described below. Each active ingredient may be purified to a desired degree or not. That is, each active ingredient may be a purified product, or a material containing the active ingredient may be used. Examples of materials containing each active ingredient include fermentation products such as culture broth, bacterial cells, and culture supernatant obtained by culturing a microorganism capable of producing the active ingredient, as well as processed products thereof. Examples of processed products include materials such as the fermentation products described above that have been subjected to processes such as concentration, dilution, drying, fractionation, extraction, and purification. For each active ingredient, for example, a material containing the active ingredient at a content of 1% (w / w) or more, 5% (w / w) or more, 10% (w / w) or more, 30% (w / w) or more, 50% (w / w) or more, 70% (w / w) or more, 90% (w / w) or more, or 95% (w / w) or more may be used.

[0018] The carboxylic acid may be used in a free form, may be used as a salt, or may be produced as a combination thereof. That is, unless otherwise specified, the term "carboxylic acid" may refer to a carboxylic acid in a free form, a salt thereof, or a combination thereof. With respect to the salt of a carboxylic acid, the description of the salt of an acidic group in a precursor of the carboxylic acid described below can be applied mutatis mutandis.

[0019] When a material containing the active ingredient is used, the amount of each active ingredient (e.g., content (concentration) or amount used) is calculated based on the amount of the active ingredient itself in the material. When the active ingredient forms a salt, the amount of each active ingredient (e.g., content (concentration) or amount used) is calculated based on the mass of the salt converted to the mass of an equimolar free form.

[0020] <1-2> Method for producing active ingredient Hereinafter, a method for producing an active ingredient using Bacillus bacteria will be described. That is, the active ingredient may be produced by this production method. This production method is also referred to as the "production method of the present invention."

[0021] Bacillus bacteria may have one, two, or all three of the abilities to produce carboxylic acid, diacetyl, and acetoin. Bacillus bacteria may particularly have all of the abilities to produce carboxylic acid, diacetyl, and acetoin. Bacillus bacteria having the ability to produce carboxylic acid are also called "carboxylic acid-producing bacteria." Bacillus bacteria having the ability to produce diacetyl acid are also called "diacetyl-producing bacteria." Bacillus bacteria having the ability to produce acetoin are also called "acetoin-producing bacteria." For example, Bacillus bacteria having all of the abilities to produce carboxylic acid, diacetyl, and acetoin are carboxylic acid-producing bacteria, diacetyl-producing bacteria, and acetoin-producing bacteria.

[0022] The carboxylic acid producing ability, diacetyl producing ability, and acetoin producing ability are also collectively referred to as "active ingredient producing ability." The carboxylic acid producing bacteria, diacetyl producing bacteria, and acetoin producing bacteria are also collectively referred to as "active ingredient producing bacteria." The active ingredient producing bacteria have the corresponding active ingredient producing ability (i.e., carboxylic acid producing ability for carboxylic acid producing bacteria, diacetyl producing ability for diacetyl producing bacteria, and acetoin producing ability for acetoin producing bacteria).

[0023] For example, each active ingredient may be produced individually by the production method of the present invention. Alternatively, for example, two or all three active ingredients may be produced together by the production method of the present invention. In particular, all active ingredients may be produced together by the production method of the present invention.

[0024] The active ingredient can be produced by culturing the corresponding active ingredient-producing bacteria (i.e., carboxylic acid-producing bacteria for carboxylic acids, diacetyl-producing bacteria for diacetyl, and acetoin-producing bacteria for acetoin) in a medium. However, when at least carboxylic acids are produced (i.e., when carboxylic acids are produced alone or in combination with diacetyl and / or acetoin), a medium containing a carboxylic acid precursor is used. Also, when at least diacetyl is produced (i.e., when diacetyl is produced alone or in combination with carboxylic acids and / or acetoin), a medium containing sugar is used. Also, when at least acetoin is produced (i.e., when acetoin is produced alone or in combination with carboxylic acids and / or diacetyl), a medium containing sugar is used.

[0025] That is, the production method of the present invention is a method for producing an active ingredient, which comprises the step of culturing the corresponding active ingredient-producing bacteria in a medium to obtain a culture containing the active ingredient.

[0026] For example, component (A) (carboxylic acid) can be produced by culturing a carboxylic acid-producing bacterium in a medium containing a precursor of the carboxylic acid.

[0027] That is, one embodiment of the production method of the present invention is a method for producing a carboxylic acid, comprising: a step of culturing a carboxylic acid-producing bacterium in a medium containing a precursor of the carboxylic acid to obtain a culture containing the carboxylic acid, wherein the carboxylic acid is selected from the group consisting of 2-methylbutyric acid, 3-methylbutyric acid, isobutyric acid, and combinations thereof.

[0028] Furthermore, for example, component (B) (diacetyl) can be produced by culturing a diacetyl-producing bacterium in a medium containing sugar.

[0029] That is, one embodiment of the production method of the present invention is a method for producing diacetyl, comprising the step of culturing a diacetyl-producing bacterium in a medium containing sugar to obtain a culture containing diacetyl.

[0030] Furthermore, for example, component (C) (acetoin) can be produced by culturing an acetoin-producing bacterium in a medium containing sugar.

[0031] That is, one aspect of the production method of the present invention is a method for producing acetoin, which includes a step of culturing an acetoin-producing bacterium in a medium containing sugar to obtain a culture containing acetoin.

[0032] Furthermore, for example, components (A), (B), and (C) (carboxylic acid, diacetyl, and acetoin) can be produced collectively by culturing Bacillus bacteria having the ability to produce carboxylic acid, diacetyl, and acetoin in a medium containing a precursor of the carboxylic acid and sugar.

[0033] That is, one embodiment of the production method of the present invention is a method for producing a carboxylic acid, diacetyl, and acetoin, comprising the steps of: culturing a Bacillus bacterium having an ability to produce a carboxylic acid, diacetyl, and acetoin in a medium containing a precursor of the carboxylic acid and a sugar to obtain a culture containing the carboxylic acid, diacetyl, and acetoin; and the carboxylic acid is selected from the group consisting of 2-methylbutyric acid, 3-methylbutyric acid, isobutyric acid, and combinations thereof.

[0034] "Bacteria capable of producing carboxylic acids" refers to bacteria capable of producing carboxylic acids and accumulating them in the medium and / or within the bacterial cells when cultured in a medium containing a precursor of the carboxylic acid. Bacteria capable of producing carboxylic acids may accumulate at least the carboxylic acid in the medium. Bacteria capable of producing carboxylic acids may accumulate carboxylic acids in the medium in an amount of, for example, 100 ppm (w / w) or more, 300 ppm (w / w) or more, 500 ppm (w / w) or more, 1000 ppm (w / w) or more, 2000 ppm (w / w) or more, 3000 ppm (w / w) or more, 4000 ppm (w / w) or more, 5000 ppm (w / w) or more, 6000 ppm (w / w) or more, 7000 ppm (w / w) or more, or 8000 ppm (w / w) or more. Bacteria capable of producing carboxylic acids may accumulate carboxylic acids in the medium in an amount of, for example, 50,000 ppm (w / w) or less, 20,000 ppm (w / w) or less, or 10,000 ppm (w / w) or less. When carboxylic acid-producing bacteria produce two or more carboxylic acids, the carboxylic acid-producing bacteria may accumulate these carboxylic acids in the medium in the amounts exemplified above, either independently or in total. For example, the carboxylic acid-producing bacteria may accumulate 2-methylbutyric acid in the medium in the amount exemplified above. When two or more carboxylic acids accumulate in the medium, the term "accumulated amount of carboxylic acids in the medium" refers to the total accumulated amount of these two or more carboxylic acids in the medium, unless otherwise specified.

[0035] "Diacetyl-producing bacteria" refers to bacteria that, when cultured in a sugar-containing medium, produce diacetyl and accumulate it in the medium and / or within the bacterial cells. Diacetyl-producing bacteria may accumulate at least diacetyl in the medium. Diacetyl-producing bacteria may accumulate diacetyl in an amount of, for example, 50 ppm (w / w) or more, 100 ppm (w / w) or more, 200 ppm (w / w) or more, 300 ppm (w / w) or more, 400 ppm (w / w) or more, or 500 ppm (w / w) or more in the medium. Diacetyl-producing bacteria may accumulate diacetyl in an amount of, for example, 5000 ppm (w / w) or less, 2000 ppm (w / w) or less, or 1000 ppm (w / w) or less in the medium.

[0036] "Bacteria capable of producing acetoin" refers to bacteria capable of producing acetoin and accumulating it in the medium and / or within the bacterial cells when cultured in a medium containing sugar. Bacteria capable of producing acetoin may accumulate at least acetoin in the medium. Bacteria capable of producing acetoin may accumulate acetoin in an amount of, for example, 500 ppm (w / w) or more, 1000 ppm (w / w) or more, 2000 ppm (w / w) or more, 3000 ppm (w / w) or more, 4000 ppm (w / w) or more, 5000 ppm (w / w) or more, 6000 ppm (w / w) or more, 7000 ppm (w / w) or more, 8000 ppm (w / w) or more, 9000 ppm (w / w) or more, or 10000 ppm (w / w) or more in the medium. A bacterium capable of producing acetoin may accumulate acetoin in the medium in an amount of, for example, 50,000 ppm (w / w) or less, 20,000 ppm (w / w) or less, or 10,000 ppm (w / w) or less.

[0037] The carboxylic acid is as described above. As the carboxylic acid, for example, at least 2-methylbutyric acid may be produced. As the carboxylic acid, for example, at least 2-methylbutyric acid may be produced, and further, 3-methylbutyric acid and / or isobutyric acid may be produced. As the carboxylic acid, for example, at least (S)-2-methylbutyric acid may be produced. In other words, as the 2-methylbutyric acid, for example, at least (S)-2-methylbutyric acid may be produced. As the 2-methylbutyric acid, for example, at least (S)-2-methylbutyric acid may be produced, and further, (R)-2-methylbutyric acid may be produced.

[0038] The precursor of the carboxylic acid is selected depending on the type of the carboxylic acid. The precursor of the carboxylic acid may also be simply referred to as a "precursor."

[0039] An example of a precursor for producing 2-methylbutyric acid is isoleucine (Ile). In other words, the precursor of 2-methylbutyric acid may be isoleucine. 2-Methylbutyric acid may also be a carboxylic acid corresponding to isoleucine.

[0040] An example of a precursor for producing 3-methylbutyric acid is leucine (Leu). In other words, the precursor of 3-methylbutyric acid may be leucine. 3-methylbutyric acid may also be a carboxylic acid corresponding to leucine.

[0041] Precursors for producing isobutyric acid include isoleucine (Ile), leucine (Leu), and valine (Val). In other words, the precursor of isobutyric acid may be selected from the group consisting of isoleucine, leucine, valine, and combinations thereof. Isobutyric acid may also be a carboxylic acid corresponding to isoleucine, leucine, valine, or a combination thereof. The combination is not particularly limited. Examples of combinations include a combination of isoleucine and leucine, a combination of leucine and valine, a combination of isoleucine and valine, and a combination of isoleucine, leucine, and valine. The combination may, for example, include at least valine. Precursors for producing isobutyric acid include, in particular, valine (Val). In other words, the precursor of isobutyric acid may, in particular, be valine. Isobutyric acid may also be, in particular, a carboxylic acid corresponding to valine.

[0042] Precursors for producing (S)-2-methylbutyric acid and (R)-2-methylbutyric acid include L-isoleucine and D-isoleucine, respectively. In other words, the precursors of (S)-2-methylbutyric acid and (R)-2-methylbutyric acid may be L-isoleucine and D-isoleucine, respectively. Furthermore, (S)-2-methylbutyric acid and (R)-2-methylbutyric acid may be the carboxylic acid corresponding to L-isoleucine and the carboxylic acid corresponding to D-isoleucine, respectively.

[0043] Unless otherwise specified, the precursor may be of the D-configuration, the L-configuration, or a combination thereof. There are no particular limitations on the ratio of the D-configuration to the L-configuration in the combination. The ratio of the D-configuration or the L-configuration in the combination may be, for example, 20 to 80%, 30 to 70%, 40 to 60%, or 45 to 55% in terms of molar ratio. The precursor may particularly be of the L-configuration. Note that when a D-configuration precursor is selected, it is sufficient to use the D-configuration precursor, and this does not preclude the use of an L-configuration precursor in combination. Note that when an L-configuration precursor is selected, it is sufficient to use the L-configuration precursor, and this does not preclude the use of a D-configuration precursor in combination.

[0044] The precursor may be a commercially available product or may be obtained by appropriate production. The method for producing the precursor is not particularly limited. The precursor can be produced, for example, by chemical synthesis, enzymatic reaction, fermentation, extraction, or a combination thereof. Specifically, the precursor can be produced, for example, by culturing a microorganism capable of producing the precursor and recovering the precursor from the culture medium or cells. The precursor may be purified to a desired degree or not. That is, the precursor may be a purified product, or a precursor-containing material. Examples of precursor-containing materials include fermentation products such as culture medium, cells, and culture supernatant obtained by culturing a microorganism capable of producing the precursor, as well as processed products thereof. Examples of processed products include materials such as the fermentation products described above that have been subjected to processes such as concentration, dilution, drying, fractionation, extraction, and purification. Examples of precursor-containing materials include organic nitrogen sources such as yeast extract and peptone. As the precursor, for example, a material having a precursor content of 1% (w / w) or more, 5% (w / w) or more, 10% (w / w) or more, 30% (w / w) or more, 50% (w / w) or more, 70% (w / w) or more, 90% (w / w) or more, or 95% (w / w) or more may be used.

[0045] The precursor may be used in a free form, a salt, or a combination thereof. That is, unless otherwise specified, the term "precursor" may refer to the precursor in a free form, a salt thereof, or a combination thereof. "Free form" refers to a form in which no salt is formed. The precursor may be in any form, such as an ion, when used (e.g., during the culture step).

[0046] The salt is not particularly limited as long as a carboxylic acid is produced. Orally ingestible salts may be used. Specific examples of salts of acidic groups such as carboxyl groups include ammonium salts, salts with alkali metals such as sodium and potassium, salts with alkaline earth metals such as calcium and magnesium, aluminum salts, zinc salts, salts with organic amines such as triethylamine, ethanolamine, morpholine, pyrrolidine, piperidine, piperazine, and dicyclohexylamine, and salts with basic amino acids such as arginine and lysine. Specific examples of salts with basic groups such as amino groups include salts with inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, and hydrobromic acid, salts with organic carboxylic acids such as acetic acid, citric acid, benzoic acid, maleic acid, fumaric acid, tartaric acid, succinic acid, tannic acid, butyric acid, hybenzic acid, pamoic acid, enanthic acid, decanoic acid, teoclic acid, salicylic acid, lactic acid, oxalic acid, mandelic acid, malic acid, methylmalonic acid, and adipic acid, and salts with organic sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid. One type of salt may be used, or two or more types of salts may be used in combination.

[0047] In addition, when a material containing a precursor is used, the amount of precursor (e.g., content (concentration) or amount used) is calculated based on the amount of precursor itself in the material unless otherwise specified. In addition, when the precursor forms a salt, the amount of precursor (e.g., content (concentration) or amount used) is calculated based on the value obtained by converting the mass of the salt into the mass of an equimolar free form unless otherwise specified.

[0048] The sugar is not particularly limited as long as it can be assimilated by the active ingredient-producing bacteria. Examples of sugars include monosaccharides and oligosaccharides such as glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, maltose, isomaltose, and fructooligosaccharides. Purified sugars may be used, or sugar-containing materials may be used. For example, materials with a sugar content of 1% (w / w) or more, 5% (w / w) or more, 10% (w / w) or more, 30% (w / w) or more, 50% (w / w) or more, 70% (w / w) or more, 90% (w / w) or more, or 95% (w / w) or more may be used. Examples of sugar-containing materials (e.g., monosaccharides and oligosaccharides) include invert sugar, blackstrap molasses, starch hydrolysates, and plant biomass hydrolysates. Examples of blackstrap molasses include cane molasses, beet molasses, high-test molasses, and citrus molasses. Examples of starches include corn starch, tapioca starch, cassava starch, potato starch, and various other grain starches. Examples of biomass include cellulose and hemicellulose. Plant biomass hydrolysates can be obtained by subjecting plant biomass to treatments such as steam treatment, concentrated acid hydrolysis, dilute acid hydrolysis, hydrolysis with enzymes such as cellulase, and alkali treatment. Because hemicellulose is generally more easily hydrolyzed than cellulose, hemicellulose in plant biomass may be pre-hydrolyzed to liberate pentoses, and then cellulose may be hydrolyzed to produce hexoses. Examples of sugars include glucose. Commercially available sugars may be used, or sugars obtained by appropriate manufacturing may be used. A single type of sugar may be used, or two or more types of sugars may be used in combination. For example, at least glucose may be used. That is, as the sugar, for example, glucose may be used alone, or glucose may be used in combination with one or more other sugars. Note that when a material containing sugar is used, the amount of sugar (e.g., content (concentration) or amount used) is calculated based on the amount of sugar itself in the material unless otherwise specified.

[0049] The active ingredient-producing bacteria may be those that inherently have the ability to produce the active ingredient, or may be those that have been modified to have the ability to produce the active ingredient. For example, the active ingredient-producing bacteria can be obtained by imparting the ability to produce the active ingredient to any Bacillus bacterial strain, or by enhancing the ability to produce the active ingredient of any Bacillus bacterial strain.

[0050] The method for imparting or enhancing the ability to produce an active ingredient is not particularly limited. For example, known methods can be used as the method for imparting or enhancing the ability to produce an active ingredient. The ability to produce an active ingredient can be imparted or enhanced, for example, by mutation or genetic engineering techniques.

[0051] For example, methods for imparting or enhancing carboxylic acid production ability include modifying bacteria to increase the activity of one or more enzymes selected from enzymes that catalyze the conversion of precursors to carboxylic acids. Examples of such enzymes include, but are not limited to, branched-chain amino acid transaminase (EC 2.6.1.42), leucine dehydrogenase (EC 1.4.1.9), L-amino acid oxidase (EC 1.4.3.2), phenylpyruvate decarboxylase (EC 4.1.1.43), and pyruvate decarboxylase (EC 4.1.1.1). Enzyme activity can be increased, for example, by increasing the expression of the gene encoding the enzyme. Gene expression can be increased, for example, by increasing the copy number of the gene. Gene expression can be increased, for example, by modifying the gene's expression regulatory sequence, such as a promoter (e.g., by replacing it with a stronger one). Gene expression can be increased, for example, by obtaining a strain in which expression of the target gene is increased by mutagenesis. Enzyme activity can also be increased, for example, by modifying bacteria to have a gene with a mutation that increases the specific activity of the enzyme. The mutation that increases the specific activity of the enzyme may be a mutation in a gene encoding the enzyme, or may be a mutation in a gene other than the gene encoding the enzyme. Bacteria having a gene with a mutation that increases enzyme activity can be obtained, for example, by obtaining bacteria with such a mutation by mutagenesis. Specific methods for increasing enzyme activity are disclosed, for example, in WO2015 / 060391 and WO2018 / 030507.

[0052] The active ingredient-producing bacteria may be those that inherently have the ability to produce the active ingredient, or those that have been modified to have the ability to produce the active ingredient by a method other than genetic engineering (e.g., mutation). The active ingredient-producing bacteria may more particularly be those that inherently have the ability to produce the active ingredient. The active ingredient-producing bacteria may more particularly be those that inherently have the ability to produce a carboxylic acid, diacetyl, and acetoin.

[0053] Examples of Bacillus bacteria that can be used as active ingredient-producing bacteria or parent strains for constructing such bacteria include Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus polymixa, Bacillus stearothermophilus, and Bacillus velezensis. Examples of Bacillus bacteria include Bacillus subtilis and Bacillus amyloliquefaciens. Examples of Bacillus bacteria include Bacillus subtilis. Specific examples of Bacillus subtilis include the 168 Marburg strain (ATCC 6051), the PY79 strain (Plasmid, 1984, 12, 1-9), and the IAM1114 strain (JCM20057).Specific examples of Bacillus amyloliquefaciens include the F strain (ATCC 23350), the T strain (ATCC 23842), the N strain (ATCC 23845), the AJ11708 strain (NITE BP-02609), and the FZB42 strain (DSM 23117).

[0054] Various strains of the genus Bacillus can be obtained, for example, from the American Type Culture Collection (address: 12301 Parklawn Drive, Rockville, Maryland 20852 PO Box 1549, Manassas, VA 20108, United States of America). Each strain is assigned a corresponding accession number, and can be obtained using this accession number (see http: / / www.atcc.org / ). The accession numbers corresponding to each strain are listed in the catalog of the American Type Culture Collection. Various strains of the genus Bacillus can also be obtained, for example, from the depository institution where the strain was deposited.

[0055] The step of culturing an active ingredient-producing bacterium in a medium to obtain a culture containing the active ingredient is also referred to as the "culturing step." One embodiment of the culturing step is a step of culturing a carboxylic acid-producing bacterium in a medium containing a precursor to obtain a culture containing a carboxylic acid. One embodiment of the culturing step is a step of culturing a diacetyl-producing bacterium in a medium containing a sugar to obtain a culture containing diacetyl. One embodiment of the culturing step is a step of culturing an acetoin-producing bacterium in a medium containing a sugar to obtain a culture containing acetoin. One embodiment of the culturing step is a step of culturing an active ingredient-producing bacterium having the ability to produce carboxylic acid, diacetyl, and acetoin in a medium containing a precursor and a sugar to obtain a culture containing a carboxylic acid, diacetyl, and acetoin. The culture obtained in the culturing step is also referred to as the "fermentation liquid" or "fermented product."

[0056] The following describes the production method of the present invention when all active ingredients are produced together. That is, in the following description of the production method of the present invention, "active ingredient" means all active ingredients (carboxylic acid, diacetyl, and acetoin) unless otherwise specified. Furthermore, in the following description of the production method of the present invention, "active ingredient-producing bacteria" means active ingredient-producing bacteria having carboxylic acid production ability, diacetyl production ability, and acetoin production ability unless otherwise specified.

[0057] The following description of the production method of the present invention can be applied to cases other than when all active ingredients are produced at once. When the following description of the production method of the present invention is applied to cases other than when all active ingredients are produced at once, the term "active ingredient" shall be read as the active ingredient to be produced, the term "active ingredient-producing bacteria" as the active ingredient-producing bacteria corresponding to the active ingredient to be produced, the term "culture medium containing a precursor" as "culture medium optionally containing a precursor", and the term "culture medium containing a sugar" as "culture medium optionally containing a sugar". The term "culture medium optionally containing a precursor" means that the culture medium contains a precursor when the production method of the present invention is carried out to produce at least a carboxylic acid, but in other cases the culture medium may or may not contain a precursor. The term "culture medium optionally containing a sugar" means that the culture medium contains a sugar when the production method of the present invention is carried out to produce at least diacetyl or acetoin, but in other cases the culture medium may or may not contain a sugar. If the culture medium does not contain sugar, it is preferable to use a component that can replace the carbon source, such as a carbon source other than sugar or an organic nitrogen source, as appropriate.

[0058] The medium used is not particularly limited as long as it contains a precursor and sugar, allows the active ingredient-producing bacteria to grow, and produces the active ingredient. For example, a medium prepared by adding a precursor and sugar to a conventional medium used for culturing bacteria such as Bacillus bacteria can be used. For example, a medium containing, in addition to the precursor and sugar, a nitrogen source, a phosphate source, a sulfur source, and components selected from various other organic and inorganic components as needed can be used. The types and concentrations of medium components can be appropriately determined depending on various conditions, such as the type of active ingredient-producing bacteria used.

[0059] Specific examples of nitrogen sources include ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate; organic nitrogen sources such as peptone, yeast extract, meat extract, and hydrolyzed vegetable protein (HVP; e.g., soy protein hydrolyzate, soybean soy sauce, and pea soy sauce); ammonia; and urea. Ammonia gas or aqueous ammonia, used for pH adjustment, may also be used as a nitrogen source. Furthermore, the medium may be reused, as described in "Fundamentals of Fermentation Engineering," Academic Press, 1988. A single nitrogen source may be used, or two or more nitrogen sources may be used in combination.

[0060] Specific examples of the phosphate source include phosphate salts such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphate polymers such as pyrophosphate. As the phosphate source, one type of phosphate source may be used, or two or more types of phosphate sources may be used in combination.

[0061] Specific examples of sulfur sources include inorganic sulfur compounds such as sulfates, thiosulfates, and sulfites, and sulfur-containing amino acids such as cysteine, cystine, and glutathione. As the sulfur source, one type of sulfur source may be used, or two or more types of sulfur sources may be used in combination.

[0062] Other organic and inorganic components include, for example, inorganic salts such as sodium chloride and potassium chloride; trace metals such as iron, manganese, magnesium, and calcium; vitamins such as vitamin B1, vitamin B2, vitamin B6, nicotinic acid, nicotinamide, vitamin B12, biotin, and folic acid; amino acids; nucleic acids; and organic components containing these, such as peptone, casamino acids, yeast extract, and hydrolyzed vegetable protein (HVP; e.g., soy protein hydrolyzate, soy sauce, and pea sauce). Other organic and inorganic components also include antifoaming agents, medium osmotic pressure regulators, and osmotic pressure compensation substances. Examples of antifoaming agents include silicone-based antifoaming agents (oil-type, solution-type, oil-compound-type, emulsion-type, self-emulsifying type, etc.), alcohol-based antifoaming agents, oil-based antifoaming agents, polyether-based antifoaming agents, and vegetable oils (cottonseed oil, linseed oil, soybean oil, olive oil, castor oil, coconut oil, etc.). Antifoaming agents can be used in any form, including liquid, paste, solid, powder, emulsion, and wax. Examples of osmotic pressure regulators for the medium include salts such as sodium chloride and potassium chloride, and polysaccharides (such as sorbitol and dextrin) that cannot be assimilated by microorganisms. Examples of osmotic pressure compensation substances include potassium ions, betaine (glycine betaine), blackstrap molasses (particularly sugar beet blackstrap molasses), glutamic acid, and trehalose. Other components that may be added to the medium include polymers selected from the group consisting of water-soluble cellulose derivatives, water-soluble polyvinyl compounds, polar organic solvent-soluble polyvinyl compounds, water-soluble starch derivatives, alginates, and polyacrylates. These and other various organic and inorganic components may be used alone or in combination of two or more.

[0063] When using an auxotrophic mutant strain that requires amino acids or the like for growth, it is preferable to supplement the required nutrients in the medium.

[0064] The culture conditions are not particularly limited as long as the active ingredient-producing bacteria can grow and the active ingredient can be produced, except that a medium containing a precursor and sugar is used. The culture can be carried out under normal conditions used for culturing bacteria such as Bacillus bacteria. The culture conditions may be appropriately set depending on various conditions such as the type of active ingredient-producing bacteria used.

[0065] Cultivation can be carried out using a liquid medium (i.e., by liquid culture). Examples of liquid culture methods include those described in "Biotechnology Textbook Series 13: Culture Engineering" by Toshiomi Yoshida, published by Corona Publishing in 1998. Specifically, liquid culture can be performed using, for example, surface culture, submerged culture, membrane (e.g., dialysis membrane or for-ferber) separation culture, or immobilized microbial culture. Furthermore, examples of culture devices that can be used include aeration and agitation culture devices, airlift culture devices, packed-bed culture devices, and fluidized-bed culture devices. Cultivation can be performed using the methods described in "Fermentation Engineering Fundamentals" published by the Academic Press in 1988. Cultivation can be performed in two stages: seed culture and main culture. The active ingredient may be produced in the main culture. Both the precursor and sugar may be contained in the medium in the main culture. In other words, when the culture is performed in two stages, the "culturing step" refers to the step of culturing the active ingredient-producing bacteria in the main culture in the medium, unless otherwise specified. The culture conditions for seed culture and main culture may or may not be the same. At least, the main culture may be carried out using a liquid medium. For example, the active ingredient-producing bacteria may be cultured in a solid medium such as an agar medium and then directly inoculated into the liquid medium, or the active ingredient-producing bacteria may be seed cultured in a liquid medium and then inoculated into the liquid medium for main culture. The amount of active ingredient-producing bacteria contained in the medium at the start of culture is not particularly limited. For example, the main culture may be carried out by inoculating the medium for main culture with 1 to 50% (v / v) of the seed culture solution. Furthermore, the seed culture step may include, for example, two or more seed culture steps to obtain the amount of bacteria required for the main culture step. Furthermore, the seed culture solution may be inoculated only at the start of main culture, or may be inoculated at the start of main culture and then during main culture.

[0066] Cultivation can be carried out by batch culture, fed-batch culture, continuous culture, or a combination thereof. Examples of combinations include two or more stages of connected fed-batch cultures and two or more stages of connected continuous cultures. The medium at the start of culture is also called the "initial medium." The medium supplied to a culture system (e.g., a fermenter) in fed-batch culture or continuous culture is also called the "fed-batch medium." Supplying a fed-batch medium to a culture system in fed-batch culture or continuous culture is also called "fed-batch." When culture is divided into a seed culture and a main culture, both the seed culture and the main culture may be performed by batch culture, for example. Alternatively, the seed culture may be performed by batch culture, and the main culture may be performed by fed-batch or continuous culture. Alternatively, the seed culture may be performed by fed-batch culture, and the main culture may be performed by batch culture. The feed medium may be supplied, for example, from a location in the upper part of the culture tank that is not in contact with the liquid surface of the culture medium, or from a location inside the culture tank such as the middle or lower part of the culture tank, or from both the upper and middle parts of the culture tank. An embodiment in which the feed medium is supplied from a location inside the culture tank is disclosed, for example, in Japanese Patent No. 6097869.

[0067] Each medium component may be contained in the initial medium, the feed medium, or both. The type of component contained in the initial medium may or may not be the same as the type of component contained in the feed medium. Furthermore, the concentration of each component contained in the initial medium may or may not be the same as the concentration of each component contained in the feed medium. Furthermore, two or more feed media containing different types and / or concentrations of components may be used. For example, when multiple feeds are performed intermittently, the type and / or concentration of components contained in the feed medium for each feed may or may not be the same.

[0068] The medium may or may not be sterilized. Sterilization of the medium may be carried out, for example, for the purpose of preventing contamination. Sterilization of the medium may also be referred to as "sterilization" or "removal of bacteria." Methods for sterilizing the medium include sterilization under high temperature and high pressure conditions, sterilization by UV irradiation, and sterilization using a filter or membrane. Sterilization of the medium may be carried out batchwise or continuously. For example, methods for batchwise sterilization under high temperature and high pressure conditions include autoclave sterilization and batch sterilization in a culture tank. Furthermore, for example, methods for continuous sterilization under high temperature and high pressure conditions include continuous sterilization equipped with a plate-type heat exchanger. Furthermore, sterilization of the sugar may be carried out simultaneously with other medium components, or may be carried out separately from the other components. Preferably, the sugar and the other components may be sterilized separately.

[0069] Cultivation may be carried out under aerobic conditions, for example, using a liquid medium. "Aerobic conditions" may refer to conditions in which the dissolved oxygen concentration in the medium is 0.18 ppm or higher, 0.33 ppm or higher, or 1.5 ppm or higher. The dissolved oxygen concentration can be measured using a sensor such as a PL electrode or a DO electrode. Cultivation under aerobic conditions can be carried out, for example, by aerobic culture or shaking culture. The pH of the medium may be, for example, 3 to 10, preferably 4.0 to 9.5. The pH of the medium can be adjusted as needed during cultivation. The pH of the medium can be adjusted using various alkaline or acidic substances such as ammonia gas, ammonia water, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, or magnesium hydroxide. The culture temperature may be, for example, 20 to 40°C, preferably 25 to 37°C, and more preferably 28 to 30°C. The culture period may be, for example, 10 to 120 hours. The culture may be continued, for example, until the sugar in the medium is consumed or until the activity of the active ingredient-producing bacteria is lost.

[0070] The content of medium components such as precursors and sugars in the medium is not particularly limited as long as the active ingredient-producing bacteria can grow and the active ingredient can be produced.

[0071] The content of the precursor in the medium may be, for example, 0.1% (w / w) or more, 0.2% (w / w) or more, 0.3% (w / w) or more, 0.5% (w / w) or more, 0.7% (w / w) or more, 1% (w / w) or more, 1.5% (w / w) or more, 2% (w / w) or more, 2.5% (w / w) or more, 3% (w / w) or more, 3.5% (w / w) or more, 4% (w / w) or more, or 4.5% (w / w) or more, and may be 20% (w / w) or less, 1 It may be 0% (w / w) or less, 5% (w / w) or less, 4.5% (w / w) or less, 4% (w / w) or less, 3.5% (w / w) or less, 3% (w / w) or less, 2.5% (w / w) or less, 2% (w / w) or less, 1.5% (w / w) or less, 1% (w / w) or less, 0.7% (w / w) or less, 0.5% (w / w) or less, 0.3% (w / w) or less, or 0.2% (w / w) or less, or any compatible combination thereof. The content of the precursor in the medium may be, for example, 0.1 to 0.2% (w / w), 0.2 to 0.3% (w / w), 0.3 to 0.5% (w / w), 0.5 to 0.7% (w / w), 0.7 to 1% (w / w), 1 to 1.5% (w / w), 1.5 to 2% (w / w), 2 to 2.5% (w / w), 2.5 to 3% (w / w), 3 to 3.5% (w / w), 3.5 to 4% (w / w), 4 to 4.5% (w / w), 4.5 to 5% (w / w), or 10 to 20% (w / w), or any compatible combination thereof. The content of the precursor in the medium may be, for example, 0.1 to 20% (w / w), 0.2 to 10% (w / w), 0.3 to 4% (w / w), or 0.5 to 3% (w / w). When the medium contains two or more precursors, the content of those two or more precursors in the medium may be set independently or in total within the range of the precursor content in the medium exemplified above. When the medium contains two or more precursors, the "content of precursors in the medium" refers to the total content of those two or more precursors in the medium, unless otherwise specified.

[0072] When the medium contains two or more precursors, the contents of the two or more precursors in the medium may or may not be identical to one another. When the medium contains a first and a second precursor, the content of the second precursor in the medium may be, for example, 0.1 parts by weight or more, 0.2 parts by weight or more, 0.5 parts by weight or more, 1 part by weight or more, 2 parts by weight or more, 5 parts by weight or more, 10 parts by weight or more, 20 parts by weight or more, 30 parts by weight or more, 50 parts by weight or more, or 70 parts by weight or more, or 100 parts by weight or less, 70 parts by weight or less, 50 parts by weight or less, 30 parts by weight or less, 20 parts by weight or less, 10 parts by weight or less, 5 parts by weight or less, 2 parts by weight or less, 1 part by weight or less, 0.5 parts by weight or less, or 0.2 parts by weight or less, per 100 parts by weight of the first precursor contained in the medium, or any compatible combination thereof. The content of the second precursor in the medium may be, for example, 0.1 to 0.2 parts by weight, 0.2 to 0.5 parts by weight, 0.5 to 1 part by weight, 1 to 2 parts by weight, 2 to 5 parts by weight, 5 to 10 parts by weight, 10 to 20 parts by weight, 20 to 30 parts by weight, 30 to 50 parts by weight, 50 to 70 parts by weight, or 70 to 100 parts by weight, per 100 parts by weight of the first precursor contained in the medium. The content of the second precursor in the medium may be, for example, 0.1 to 100 parts by weight, 0.5 to 50 parts by weight, or 2 to 20 parts by weight, per 100 parts by weight of the first precursor contained in the medium. The first precursor may be any precursor, for example, isoleucine. The first precursor may be, for example, L-isoleucine, in particular. The second precursor may be any precursor other than the first precursor, such as leucine or valine. For example, when the medium contains isoleucine, leucine, and valine, the leucine and valine contents in the medium may be the amounts exemplified above, independently or in total, per 100 parts by weight of isoleucine contained in the medium.

[0073] The content of the precursor in the medium may be set, for example, so as to obtain the desired production amount of the corresponding carboxylic acid. The content of the precursor in the medium may be, for example, 1-fold or more in molar ratio relative to the desired production amount of the corresponding carboxylic acid. The content of the precursor in the medium may be, for example, 1-fold or more, 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.5-fold or more, 1.7-fold or more, 2-fold or more, 2.5-fold or more, 3-fold or more, 5-fold or more, or 7-fold or more in molar ratio relative to the desired production amount of the corresponding carboxylic acid, or 10-fold or less, 7-fold or less, 5-fold or less, 3-fold or less, 2.5-fold or less, 2-fold or less, 1.7-fold or less, 1.5-fold or less, 1.3-fold or less, 1.2-fold or less, or 1.1-fold or less, or a compatible combination thereof. The content of the precursor in the medium may be, for example, 1 to 1.1 times, 1.1 to 1.2 times, 1.2 to 1.3 times, 1.3 to 1.5 times, 1.5 to 1.7 times, 1.7 to 2 times, 2 to 2.5 times, 2.5 to 3 times, 3 to 5 times, 5 to 7 times, or 7 to 10 times the molar ratio of the desired production amount of the corresponding carboxylic acid. The content of the precursor in the medium may be, for example, 1 to 10 times, 1 to 7 times, or 1 to 5 times the molar ratio of the desired production amount of the corresponding carboxylic acid.

[0074] The sugar content in the medium is, for example, 0.1% (w / w) or more, 0.7% (w / w) or more, 1% (w / w) or more, 2% (w / w) or more, 3% (w / w) or more, 5% (w / w) or more, 7% (w / w) or more, 10% (w / w) or more, 15% (w / w) or more, 20% (w / w) or more, 25% (w / w) or more, 30% (w / w) or more, or 40% (w / w) or more. and may be 50% (w / w) or less, 40% (w / w) or less, 30% (w / w) or less, 25% (w / w) or less, 20% (w / w) or less, 15% (w / w) or less, 10% (w / w) or less, 7% (w / w) or less, 5% (w / w) or less, 3% (w / w) or less, or 2% (w / w) or less, or any compatible combination thereof. The sugar content in the medium may be, for example, 0.1-0.7% (w / w), 1-2% (w / w), 2-3% (w / w), 3-5% (w / w), 5-7% (w / w), 7-10% (w / w), 10-15% (w / w), 15-20% (w / w), 20-25% (w / w), 25-30% (w / w), 30-40% (w / w), or 40-50% (w / w). The sugar content in the medium may be, for example, 1-50% (w / w), 1-30% (w / w), or 3-10% (w / w). The sugar content in the medium may be as high as possible, for example, within the range that does not inhibit the production of the active ingredient. Furthermore, additional sugar may be added to the medium as appropriate. For example, sugar may be added to the medium in accordance with the consumption of sugar as the fermentation progresses. In addition, in fed-batch culture or continuous culture, the amount of sugar supplied may be sufficient (i.e., an amount of sugar supplied that is in excess of the sugar assimilation capacity of the active ingredient-producing bacteria) or may be limiting (i.e., an amount of sugar supplied that is insufficient to the sugar assimilation capacity of the active ingredient-producing bacteria).

[0075] Any of the medium components, such as precursors and sugars, may be present in the medium throughout the entire culture period, or only during a portion of the culture period. In other words, "culture is carried out in a medium containing a certain component" or "a certain component is contained in the medium during culture" means that the component is contained in the medium during at least a portion of the culture period, and does not necessarily have to be present in the medium throughout the entire culture period. Any of the medium components, such as precursors and sugars, may be present in the medium at the start of culture, or may be supplied to the medium after the start of culture. Any of the medium components, such as precursors and sugars, may be present in the medium at the start of culture, and may be further supplied to the medium after the start of culture (e.g., after the component has been consumed).

[0076] Each of the medium components, such as precursors and sugars, may be present in the medium at the concentrations exemplified above throughout the entire culture period, or only during a portion of the culture period. That is, "culture is performed in a medium containing a certain component at a certain concentration," "a certain component is contained in the medium at a certain concentration during culture," or "the concentration of a certain component in the medium during culture is a certain concentration" means that the concentration of the component in the medium is within the range for at least a portion of the culture period, but does not necessarily need to be within the range for the entire culture period. Each of the medium components, such as precursors and sugars, may be present in the medium at the concentrations exemplified above, for example, at the start of culture, or may be supplied to the medium after the start of culture to achieve the concentrations exemplified above. Furthermore, each of the medium components, such as precursors and sugars, may be present in the medium at the concentrations exemplified above, and may be further supplied to the medium after the start of culture (e.g., after the component has been consumed) to achieve the concentrations exemplified above.

[0077] The length of the "partial culture period" is not particularly limited as long as it allows the production of an active ingredient. The length of the "partial culture period" can be appropriately set depending on various conditions, such as the type of medium components, the type of active ingredient-producing bacteria used, the length of the culture period, and the desired production amount of the active ingredient. The "partial period" may be, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more of the total culture period. Furthermore, the "partial period" may be, for example, 10 hours or more, 20 hours or more, 40 hours or more, 60 hours or more, 80 hours or more, 100 hours or more, 120 hours or more, or 150 hours or more. Note that the "total culture period" refers to the total period of the main culture when the culture is divided into a seed culture and a main culture.

[0078] The content of medium components such as precursors and sugars in the medium can be measured, for example, by known methods used for detecting or identifying compounds. Such methods include, for example, HPLC, UPLC, LC / MS, GC / MS, and NMR. These methods can also be used to confirm the production of active ingredients. These methods may be used alone or in appropriate combinations.

[0079] By culturing the active ingredient-producing bacteria in this manner, the active ingredient accumulates in the medium and / or within the bacteria, thereby obtaining a culture (i.e., fermentation broth) containing the active ingredient. In one embodiment, 2-methylbutyric acid and 3-methylbutyric acid can be detected and quantified as single components. In one embodiment, the quantitative ratio (e.g., weight ratio or molar ratio) of 2-methylbutyric acid to 3-methylbutyric acid contained in the culture may be conveniently considered to be the same as the quantitative ratio (e.g., weight ratio or molar ratio) of isoleucine to leucine consumed in the culture.

[0080] The carboxylic acid may be produced in a free form, a salt, or a combination thereof. That is, unless otherwise specified, the term "carboxylic acid" may refer to a carboxylic acid in a free form, a salt thereof, or a combination thereof. The same description of the salt of the acidic group in the precursor can be applied mutatis mutandis to the salt of the carboxylic acid.

[0081] The active ingredient may be produced (e.g., obtained and utilized) as a composition containing the active ingredient. That is, the produced active ingredient may be a composition containing the active ingredient. In one embodiment, the quantitative ratio (e.g., weight ratio or molar ratio) of 2-methylbutyric acid to 3-methylbutyric acid contained in the composition may be conveniently considered to be the same as the quantitative ratio (e.g., weight ratio or molar ratio) of isoleucine to leucine consumed in the culture. The composition containing the active ingredient may be, for example, the composition of the present invention described below.

[0082] That is, the production method of the present invention may be a production method for a composition containing an active ingredient.

[0083] Specifically, the production method of the present invention may be a method for producing a composition containing a carboxylic acid, diacetyl, and acetoin, comprising the steps of culturing a Bacillus bacterium having the ability to produce a carboxylic acid, diacetyl, and acetoin in a medium containing a precursor of the carboxylic acid and a sugar to obtain a culture containing the carboxylic acid, diacetyl, and acetoin, wherein the carboxylic acid is selected from the group consisting of 2-methylbutyric acid, 3-methylbutyric acid, isobutyric acid, and combinations thereof.

[0084] A composition containing an active ingredient may consist solely of the active ingredient, or may contain ingredients other than the active ingredient. The ingredients other than the active ingredient are not particularly limited. The ingredients other than the active ingredient can be appropriately selected depending on various conditions, such as the intended use of the active ingredient. The ingredients other than the active ingredient may be derived from a culture (i.e., a fermentation liquid) containing the active ingredient, or may be separately blended. Examples of ingredients other than the active ingredient include bacterial cells, medium components, water, and bacterial metabolic by-products. Examples of ingredients other than the active ingredient include ingredients blended in foods or pharmaceuticals. Specific examples of ingredients blended in foods or pharmaceuticals include additives such as excipients.

[0085] The active ingredient may be produced (e.g., obtained and utilized) as, for example, a culture (i.e., a fermentation broth) containing the active ingredient. That is, the produced active ingredient (e.g., a composition containing the active ingredient) may be, for example, a culture (i.e., a fermentation broth) containing the active ingredient. The active ingredient may also be recovered, for example, from the culture (i.e., the fermentation broth). That is, the production method of the present invention may further include a step of recovering the active ingredient from the culture (i.e., the fermentation broth). The active ingredient may be recovered as a suitable fraction containing the active ingredient. An example of such a fraction is a culture supernatant. The culture supernatant can be obtained, for example, by subjecting the culture to centrifugation. The active ingredient may also be further separated and purified from such a fraction. For example, the cells of the active ingredient-producing bacteria may be separated from the culture to obtain a culture supernatant, and the active ingredient may be recovered from the culture supernatant. Furthermore, when the active ingredient accumulates within the cells, for example, the cells may be disrupted by ultrasound or the like, the supernatant may be obtained by centrifugation or the like, and the active ingredient may be recovered from the supernatant. The recovery of the active ingredient can be carried out, for example, by known methods used for separating and purifying compounds. Such methods include ion exchange resin methods (Nagai, H. et al., Separation Science and Technology, 39(16), 3691-3710), precipitation methods, membrane separation methods (JP-A-9-164323, JP-A-9-173792), and crystallization methods (WO2008 / 078448, WO2008 / 078646). Furthermore, fractions containing the active ingredient (e.g., cultures or culture supernatants) may be appropriately treated before use. Examples of such treatments include concentration, drying, and heating. Concentration, drying, and heating can all be carried out by known methods, for example. Known drying methods include spray drying, freeze drying, drum drying, and vacuum drum drying. That is, examples of the active ingredient (e.g., a composition containing the active ingredient) to be produced include a culture of an active ingredient-producing bacterium, a culture supernatant recovered from the culture, a processed product thereof, and the active ingredient recovered from the processed product. Specific examples of the processed product include dried products (e.g., a dried product of the culture or a dried product of the culture supernatant).Specific examples of dried materials include dry powders (e.g., dry powders of cultures and dry powders of culture supernatants). The dried material (e.g., dry powders) may or may not have been subjected to processing other than drying. The dried material (e.g., dry powders) may or may not contain ingredients other than the active ingredient (e.g., additives such as excipients).

[0086] The recovered active ingredient may contain, in addition to the active ingredient, bacterial cells, medium components, water, and / or bacterial metabolic by-products. The recovered active ingredient may particularly contain bacterial cells of the active ingredient-producing bacteria. The recovered active ingredient may be purified to a desired degree. The purity of the recovered active ingredient may be, for example, 50% (w / w) or more, preferably 85% (w / w) or more, and particularly preferably 95% (w / w) or more (JP1214636B, USP5,431,933, USP4,956,471, USP4,777,051, USP4,946,654, USP5,840,358, USP6,238,714, US2005 / 0025878).

[0087] The produced active ingredient may be used, for example, as is or in combination with ingredients other than the active ingredient. Specifically, the produced active ingredient may be used, for example, as is or in combination with ingredients other than the active ingredient, as a composition containing the active ingredient. Furthermore, ingredients other than the active ingredient may be blended during the production of the active ingredient (for example, during the processing of a fraction containing the active ingredient). For example, additives such as excipients may be blended during processing such as drying.

[0088] The use of the produced active ingredient (e.g., a composition containing an active ingredient) is not particularly limited. The produced active ingredient (e.g., a composition containing an active ingredient) can be used, for example, in the use of the composition of the present invention described below. Furthermore, the produced active ingredient (e.g., a composition containing an active ingredient) can be used, for example, in the use of the method of the present invention described below.

[0089] <2> Composition of the Present Invention The composition of the present invention is a composition containing an active ingredient (i.e., carboxylic acid, diacetyl, and acetoin). The active ingredient may be one produced by the production method of the present invention. That is, the composition of the present invention may be a composition containing an active ingredient (i.e., carboxylic acid, diacetyl, and acetoin), where the active ingredient is one produced by the production method of the present invention. When the active ingredient is produced as a composition containing the active ingredient in the production method of the present invention, the composition is an example of the composition of the present invention. That is, the composition of the present invention may be a composition containing an active ingredient (i.e., carboxylic acid), where the composition is one produced by the production method of the present invention.

[0090] As described above, the active ingredient can be used to improve the flavor of food (e.g., to impart a cheese flavor to food). Thus, the composition of the present invention may be, for example, a composition for improving the flavor of food (e.g., a composition for imparting a cheese flavor to food).

[0091] Furthermore, by utilizing the active ingredient, it is possible to produce foods with improved flavor (e.g., foods imparted with a cheese flavor). Thus, the composition of the present invention may be, for example, a composition for use in producing foods (specifically, producing foods with improved flavor, such as foods imparted with a cheese flavor).

[0092] The composition of the present invention may be, for example, a seasoning. Specifically, the composition of the present invention may be, for example, a seasoning for improving the flavor of food (e.g., a seasoning for imparting a cheese flavor to food), or a seasoning for food production (specifically, production of a food with an improved flavor, such as a food imparted with a cheese flavor).

[0093] The composition of the present invention may be used, for example, to improve the flavor of food or to manufacture food in the manner described in the method of the present invention below.

[0094] The composition of the present invention can be produced, for example, by producing all of the active ingredients together by the production method of the present invention, or by producing the active ingredients separately by the production method of the present invention and combining them.

[0095] The composition of the present invention may contain one carboxylic acid as component (A), or may contain two or more carboxylic acids. A composition of the present invention containing two or more carboxylic acids can be produced, for example, by simultaneously producing two or more carboxylic acids using the production method of the present invention (i.e., by carrying out the production method of the present invention using a medium containing precursors of two or more carboxylic acids). Alternatively, a composition of the present invention containing two or more carboxylic acids can be produced, for example, by separately producing two or more carboxylic acids using the production method of the present invention and combining them.

[0096] The composition of the present invention may consist of an active ingredient, or may contain ingredients other than the active ingredient. The ingredients other than the active ingredient are not particularly limited. The ingredients other than the active ingredient can be appropriately selected depending on various conditions, such as the use of the active ingredient. The ingredients other than the active ingredient may be derived from a culture (i.e., a fermentation liquid) containing the active ingredient, or may be separately blended. Examples of ingredients other than the active ingredient include bacterial cells, medium components, water, and bacterial metabolic by-products. Examples of ingredients other than the active ingredient include ingredients blended in foods or pharmaceuticals. Specific examples of ingredients blended in foods or pharmaceuticals include additives such as excipients.

[0097] The composition of the present invention may be formulated as appropriate, for example. When formulating, additives may be used as appropriate. Examples of additives include excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, diluents, surfactants, and solvents. The additives can be selected as appropriate depending on various conditions, such as the shape of the composition of the present invention.

[0098] The form of the composition of the present invention is not particularly limited, and the composition of the present invention may be in any form, such as powder, flakes, tablets, paste, or liquid.

[0099] The content and content ratio of each component (i.e., the active ingredient and optionally other components) in the composition of the present invention are not particularly limited as long as the desired effect, such as a flavor improving effect, is obtained. The content and content ratio of each component in the composition of the present invention can be appropriately set depending on various conditions, such as the mode of use of the composition of the present invention.

[0100] The total content of the active ingredients in the composition of the present invention is more than 0% (w / w) and less than 100% (w / w). The content of each active ingredient in the composition of the present invention is more than 0% (w / w) and less than 100% (w / w).

[0101] The content of carboxylic acid in the composition of the present invention may be, for example, 10 ppm (w / w) or more, 50 ppm (w / w) or more, 100 ppm (w / w) or more, 500 ppm (w / w) or more, 1000 ppm (w / w) or more, 5000 ppm (w / w) or more, 10000 ppm (w / w) or more, 20000 ppm (w / w) or more, or 50000 ppm (w / w) or more, or 90000 ppm (w / w) or less, 50000 ppm (w / w) or less, 20000 ppm (w / w) or less, 10000 ppm (w / w) or less, 5000 ppm (w / w) or less, 1000 ppm (w / w) or less, 500 ppm (w / w) or less, 100 ppm (w / w) or less, or 50 ppm (w / w) or less, or a compatible combination thereof. The content of the carboxylic acid in the composition of the present invention may be, for example, 10 to 50 ppm (w / w), 50 to 100 ppm (w / w), 100 to 500 ppm (w / w), 500 to 1000 ppm (w / w), 1000 to 5000 ppm (w / w), 5000 to 10,000 ppm (w / w), 10,000 to 20,000 ppm (w / w), 20,000 to 50,000 ppm (w / w), or 50,000 to 90,000 ppm (w / w), or any combination thereof. When the composition of the present invention contains two or more carboxylic acids, the content of those two or more carboxylic acids in the composition of the present invention may be set independently or in total within the range of the carboxylic acid content in the composition of the present invention exemplified above (provided that the total content of those two or more carboxylic acids in the composition of the present invention is 100% (w / w) or less). In one embodiment, for example, the content of 2-methylbutyric acid in the composition of the present invention may be 10 to 90,000 ppm (w / w), 50 to 50,000 ppm (w / w), or 100 to 20,000 ppm (w / w).In one embodiment, for example, the content of (S)-2-methylbutyric acid in the composition of the present invention may be 10 to 90,000 ppm (w / w), 50 to 50,000 ppm (w / w), or 100 to 20,000 ppm (w / w). Furthermore, in one embodiment, for example, the total content of 2-methylbutyric acid and 3-methylbutyric acid in the composition of the present invention may be 10 to 90,000 ppm (w / w), 50 to 50,000 ppm (w / w), or 100 to 20,000 ppm (w / w). In one embodiment, for example, the contents of 3-methylbutyric acid and isobutyric acid in the composition of the present invention may each independently be 10 to 20,000 ppm (w / w), 10 to 5,000 ppm (w / w), or 10 to 1,000 ppm (w / w). In addition, when the composition of the present invention contains two or more carboxylic acids, the "content of carboxylic acids in the composition of the present invention" means the total content of those two or more carboxylic acids in the composition of the present invention, unless otherwise specified.

[0102] When the composition of the present invention contains two or more carboxylic acids, the contents of the two or more carboxylic acids in the composition may or may not be the same. When the composition of the present invention contains a first and a second carboxylic acid, the content of the second carboxylic acid in the composition may be, for example, 0.1 parts by weight or more, 0.2 parts by weight or more, 0.5 parts by weight or more, 1 part by weight or more, 2 parts by weight or more, 5 parts by weight or more, 10 parts by weight or more, 20 parts by weight or more, 30 parts by weight or more, 50 parts by weight or more, or 70 parts by weight or more, or 100 parts by weight or less, 70 parts by weight or less, 50 parts by weight or less, 30 parts by weight or less, 20 parts by weight or less, 10 parts by weight or less, 5 parts by weight or less, 2 parts by weight or less, 1 part by weight or less, 0.5 parts by weight or less, or 0.2 parts by weight or less, or any combination thereof, based on 100 parts by weight of the first carboxylic acid contained in the composition. The content of the second carboxylic acid in the composition may be, for example, 0.1 to 0.2 parts by weight, 0.2 to 0.5 parts by weight, 0.5 to 1 part by weight, 1 to 2 parts by weight, 2 to 5 parts by weight, 5 to 10 parts by weight, 10 to 20 parts by weight, 20 to 30 parts by weight, 30 to 50 parts by weight, 50 to 70 parts by weight, or 70 to 100 parts by weight, relative to 100 parts by weight of the first carboxylic acid contained in the composition. The content of the second carboxylic acid in the composition may be, for example, 0.1 to 100 parts by weight, 0.5 to 50 parts by weight, or 2 to 20 parts by weight, relative to 100 parts by weight of the first carboxylic acid contained in the composition. The first carboxylic acid may be any carboxylic acid, for example, 2-methylbutyric acid. The first carboxylic acid may be, for example, particularly (S)-2-methylbutyric acid. The second carboxylic acid may be any carboxylic acid other than the first carboxylic acid, such as 3-methylbutyric acid or isobutyric acid. For example, when the composition of the present invention contains 2-methylbutyric acid, 3-methylbutyric acid, and isobutyric acid, the contents of 3-methylbutyric acid and isobutyric acid in the composition may be, independently or in total, in the amounts exemplified above per 100 parts by weight of 2-methylbutyric acid contained in the composition.In one aspect, when the composition of the present invention or the active ingredient contained therein is produced by the production method of the present invention, the quantitative ratio (e.g., weight ratio or molar ratio) of 2-methylbutyric acid to 3-methylbutyric acid contained in the composition of the present invention may be conveniently considered to be the same as the quantitative ratio (e.g., weight ratio or molar ratio) of isoleucine to leucine consumed in the culture in the production method of the present invention.

[0103] Furthermore, the content of the carboxylic acid in the composition of the present invention may be, for example, 0.5 parts by weight or more, 1 part by weight or more, 2 parts by weight or more, 3 parts by weight or more, 4 parts by weight or more, 5 parts by weight or more, 6 parts by weight or more, 8 parts by weight or more, 10 parts by weight or more, 12 parts by weight or more, 15 parts by weight or more, or 20 parts by weight or less, or 15 parts by weight or less, 12 parts by weight or less, 10 parts by weight or less, 8 parts by weight or less, 6 parts by weight or less, 5 parts by weight or less, 4 parts by weight or less, 3 parts by weight or less, 2 parts by weight or less, or 1 part by weight or less, or any combination thereof that does not contradict. The content of the carboxylic acid in the composition of the present invention may be, for example, 0.5 to 1 part by weight, 1 to 2 parts by weight, 2 to 3 parts by weight, 3 to 4 parts by weight, 4 to 5 parts by weight, 5 to 6 parts by weight, 6 to 8 parts by weight, 8 to 10 parts by weight, 10 to 12 parts by weight, 12 to 15 parts by weight, or 15 to 20 parts by weight, per 100 parts by weight of diacetyl contained in the composition. The content of the carboxylic acid in the composition of the present invention may be, for example, 0.5 to 20 parts by weight, 1 to 15 parts by weight, or 2 to 12 parts by weight, per 100 parts by weight of diacetyl contained in the composition. The content of the carboxylic acid in the composition of the present invention may be, for example, 0.5 to 8 parts by weight, 1 to 6 parts by weight, or 2 to 4 parts by weight, per 100 parts by weight of diacetyl contained in the composition.

[0104] Furthermore, the content of carboxylic acid in the composition of the present invention may be, for example, 10 parts by weight or more, 20 parts by weight or more, 50 parts by weight or more, 70 parts by weight or more, 100 parts by weight or more, 150 parts by weight or more, 200 parts by weight or more, 300 parts by weight or more, 400 parts by weight or more, 500 parts by weight or more, or 700 parts by weight or more, relative to 100 parts by weight of acetoin contained in the composition, or 1000 parts by weight or less, 700 parts by weight or less, 500 parts by weight or less, 400 parts by weight or less, 300 parts by weight or less, 200 parts by weight or less, 150 parts by weight or less, 100 parts by weight or less, 70 parts by weight or less, 50 parts by weight or less, or 20 parts by weight or less, or a compatible combination thereof. The content of the carboxylic acid in the composition of the present invention, relative to 100 parts by weight of acetoin contained in the composition, specifically, for example, 10 parts by weight to 20 parts by weight, 20 parts by weight to 50 parts by weight, 50 parts by weight to 70 parts by weight, 70 parts by weight to 100 parts by weight, 100 parts by weight to 150 parts by weight, 150 parts by weight to 200 parts by weight, 200 parts by weight to 300 parts by weight, 300 parts by weight to 400 parts by weight, 400 parts by weight to 500 parts by weight, 500 parts by weight to 700 parts by weight, or 700 parts by weight to 1000 parts by weight. The content of the carboxylic acid in the composition of the present invention, relative to 100 parts by weight of acetoin contained in the composition, specifically, for example, 10 parts by weight to 1000 parts by weight, 20 parts by weight to 700 parts by weight, or 50 parts by weight to 500 parts by weight. The content of carboxylic acid in the composition of the present invention may be, for example, 10 to 300 parts by weight, 20 to 200 parts by weight, or 50 to 150 parts by weight per 100 parts by weight of acetoin contained in the composition.

[0105] The diacetyl content of the compositions of the present invention may be 0.5 ppm (w / w) or more, 1 ppm (w / w) or more, 5 ppm (w / w) or more, 10 ppm (w / w) or more, 50 ppm (w / w) or more, 100 ppm (w / w) or more, 500 ppm (w / w) or more, 1000 ppm (w / w) or more, or 2000 ppm (w / w) or more, or 5000 ppm (w / w) or less, 2000 ppm (w / w) or less, 1000 ppm (w / w) or less, 500 ppm (w / w) or less, 100 ppm (w / w) or less, 50 ppm (w / w) or less, 10 ppm (w / w) or less, 5 ppm (w / w) or less, or 1 ppm (w / w) or less, or any compatible combination thereof. The content of diacetyl in the composition of the present invention may be, for example, 0.5 to 1 ppm (w / w), 1 to 5 ppm (w / w), 5 to 10 ppm (w / w), 10 to 50 ppm (w / w), 50 to 100 ppm (w / w), 100 to 500 ppm (w / w), 500 to 1000 ppm (w / w), 1000 to 2000 ppm (w / w), or 2000 to 5000 ppm (w / w). The content of diacetyl in the composition of the present invention may be, for example, 0.5 to 5000 ppm (w / w), 1 to 2000 ppm (w / w), or 5 to 1000 ppm (w / w).

[0106] The content of acetoin in the composition of the present invention may be, for example, 10 ppm (w / w) or more, 50 ppm (w / w) or more, 100 ppm (w / w) or more, 500 ppm (w / w) or more, 1000 ppm (w / w) or more, 5000 ppm (w / w) or more, 10000 ppm (w / w) or more, 20000 ppm (w / w) or more, or 50000 ppm (w / w) or more, or 90000 ppm (w / w) or less, 50000 ppm (w / w) or less, 20000 ppm (w / w) or less, 10000 ppm (w / w) or less, 5000 ppm (w / w) or less, 1000 ppm (w / w) or less, 500 ppm (w / w) or less, 100 ppm (w / w) or less, or 50 ppm (w / w) or less, or a compatible combination thereof. The content of acetoin in the composition of the present invention may be, for example, 10 to 50 ppm (w / w), 50 to 100 ppm (w / w), 100 to 500 ppm (w / w), 500 to 1000 ppm (w / w), 1000 to 5000 ppm (w / w), 5000 to 10000 ppm (w / w), 10000 to 20000 ppm (w / w), 20000 to 50000 ppm (w / w), or 50000 to 90000 ppm (w / w), or a compatible combination thereof. The content of acetoin in the composition of the present invention may be, for example, 10 to 90000 ppm (w / w), 50 to 50000 ppm (w / w), or 100 to 20000 ppm (w / w).

[0107] The content of each component (i.e., the active ingredient and optionally other ingredients) in the composition of the present invention can be set, for example, so as to obtain the amount of each component added in the method of the present invention described below.

[0108] The components (i.e., the active ingredient and optional other ingredients) contained in the composition of the present invention may be mixed together and contained in the composition of the present invention, or may be contained separately or in any combination. For example, the composition of the present invention may be provided as a set of components each packaged separately. In such a case, the components contained in the set can be used together as appropriate when used.

[0109] <3> Method of the Present Invention The method of the present invention is a method comprising a step of utilizing active ingredients (i.e., carboxylic acid, diacetyl, and acetoin). The active ingredients may be those produced by the production method of the present invention. That is, the method of the present invention may be a method comprising a step of utilizing active ingredients (i.e., carboxylic acid, diacetyl, and acetoin), in which the active ingredients are those produced by the production method of the present invention.

[0110] The method of the present invention, specifically by utilizing the active ingredient, can improve the flavor of food (e.g., impart a cheese flavor to food). Thus, the method of the present invention may be carried out to improve the flavor of food (e.g., impart a cheese flavor to food). That is, the method of the present invention may be, for example, a method for improving the flavor of food (e.g., a method for imparting a cheese flavor to food). This method is also referred to as the "flavor improving method of the present invention."

[0111] Furthermore, the method of the present invention, specifically by utilizing the active ingredient, allows the production of a food product with an improved flavor (e.g., a food product imparted with a cheese flavor). Thus, the method of the present invention may be carried out for the production of a food product (specifically, the production of a food product with an improved flavor, such as a food product imparted with a cheese flavor). That is, the method of the present invention may be, for example, a method for producing a food product (specifically, the production of a food product with an improved flavor, such as a food product imparted with a cheese flavor). This method is also referred to as the "food product production method of the present invention."

[0112] The active ingredient can be added to food ingredients during food production to improve flavor or to be used in food production. That is, an example of using the active ingredient is adding the active ingredient to food ingredients. Specifically, the method of the present invention may be, for example, a method for improving food flavor (e.g., a method for imparting a cheese flavor to food) that includes adding the active ingredient to food ingredients. Furthermore, the method of the present invention may be, for example, a method for producing food (e.g., a food with an improved flavor, such as a food imparted with a cheese flavor) that includes adding the active ingredient to food ingredients. "Addition" may also be referred to as "blending." In the method of the present invention, the amount of each active ingredient added to the food ingredients may be an amount that can achieve the content of each active ingredient in the composition of the present invention.

[0113] The active ingredient may be utilized in the method of the present invention, for example, in the form of a composition of the present invention. That is, "utilization of an active ingredient" also includes utilization of a composition of the present invention. For example, "addition of an active ingredient" also includes addition of a composition of the present invention.

[0114] The food obtained by the method of the present invention is also referred to as the "food of the present invention." Specifically, the food of the present invention is a food with an improved flavor (for example, a food imparted with a cheese flavor). In other words, the food of the present invention is a food to which an active ingredient has been added. In other words, the food of the present invention is a food containing an active ingredient.

[0115] The flavor improvement or food production may be carried out in the same manner as the production of ordinary foods, except for the use of an active ingredient. That is, the flavor improvement or food production may be carried out using the same raw materials and under the same production conditions as ordinary foods, except for the use of an active ingredient. Furthermore, the raw materials and production conditions of the food may both be appropriately modified for use in the flavor improvement or food production.

[0116] The food is not particularly limited as long as it exhibits the desired effect, such as a flavor-improving effect. Examples of foods include cheese-containing foods. Examples of foods include cheese-free foods. Examples of foods include foods that typically contain cheese but have a reduced cheese content. Foods also include beverages. Foods also include seasonings. Foods may be liquid or solid, for example. Specific examples of foods include beverages such as soft drinks, alcoholic beverages, and soups; cooked rice dishes such as chicken rice, omelet rice, and paella; stews such as curry, beef stew, hayashi rice, and hash brown; roux such as curry roux; sauces such as cheese sauce and meat sauce; pizzas such as pizza Margherita; pasta such as carbonara and bolognese; and sweets such as ice cream, yogurt, mousse, cakes, and snacks. The term "soft drink" may refer to non-alcoholic beverages (drinks with an alcohol concentration of less than 1%) excluding milk and dairy products.

[0117] The manner in which the food is provided is not particularly limited. For example, the food may be provided in a form that can be consumed as is, or in a form that requires preparation before or at the time of consumption, such as a concentrated product or a dried product. The food may also be provided in any container, such as a retort pouch, a paper pack, a plastic bottle such as a PET bottle, a metal can such as a steel can or an aluminum can, or a glass bottle. Foods are not limited to general foods, but also include so-called health foods or medical foods such as nutritional supplements (supplements), nutritionally functional foods, and foods for specified health uses. That is, for example, the foods exemplified above may be provided as general foods, health foods, or medical foods.

[0118] "Food ingredients" refers to food materials used to produce food. There are no particular limitations on food ingredients, as long as they can be used to produce food. Food ingredients can be selected appropriately depending on various conditions, such as the type of food. Food ingredients include ingredients that can be commonly used in the production of foods, such as those exemplified above. Specific examples of food ingredients include grains such as rice and wheat flour; seasoning ingredients such as sugars, inorganic salts, organic acids, nucleic acids, amino acids, and protein hydrolysates; dairy products such as milk, cheese, and butter; fruits; vegetables; meat; fish; eggs; spices; flavorings; oils and fats; alcohol; dietary fiber; and pH buffers.

[0119] The active ingredient may be added to food ingredients at any stage of the food manufacturing process, as long as the desired effect, such as flavor improvement, is obtained. In other words, the "food ingredients" to which the active ingredient is added may be those at any stage of the food manufacturing process. For example, the "food ingredients" to which the active ingredient is added may include finished foods before the active ingredient is added. The active ingredient may be added to the food ingredients either as is or after being prepared into a desired form, such as a solution. "Addition of an active ingredient" may refer collectively to the process of coexisting the active ingredient with the food ingredients. Ingredients other than the active ingredient (e.g., cheese) may also be added to the food ingredients as appropriate. The description of the addition of an active ingredient also applies mutatis mutandis to the addition of ingredients other than the active ingredient. The ingredients (i.e., the active ingredient and optionally other ingredients) may be added to the food ingredients all at the same time, separately, or in any combination. The order in which the ingredients are added to the food ingredients is not particularly limited.

[0120] The amount and ratio of each component (i.e., the active ingredient and optionally other components) added in the method of the present invention are not particularly limited as long as the desired effect, such as a flavor-improving effect, is obtained. The amount and ratio of each component added in the method of the present invention can be appropriately set depending on various conditions, such as the type of food raw material and the type of food.

[0121] The active ingredient may be added to the raw materials of the food product so that the ingestible concentration of the active ingredient falls within a desired range (for example, the range of ingestible concentration of the active ingredient described below).

[0122] The ingested concentration of the carboxylic acid may be, for example, 0.001 ppm (w / w) or more, 0.01 ppm (w / w) or more, 0.1 ppm (w / w) or more, 0.2 ppm (w / w) or more, 0.5 ppm (w / w) or more, 1 ppm (w / w) or more, 2 ppm (w / w) or more, 5 ppm (w / w) or more, 10 ppm (w / w) or more, 20 ppm (w / w) or more, 50 ppm (w / w) or more, 100 ppm (w / w) or more, 200 ppm (w / w) or more, 500 ppm (w / w) or more, 1000 ppm (w / w) or more, or 2000 ppm (w / w) or more, and may be 5000 ppm (w / w) or less, 2000 ppm (w / w) or less, 1000 ppm (w / w) or less, 500 ppm (w / w) or less, 200 ppm (w / w) or less, 100 ppm (w / w) or less, 50 ppm (w / w) or less, 20 ppm (w / w) or less, 10 ppm (w / w) or less, 5 ppm (w / w) or less, 2 ppm (w / w) or less, 1 ppm (w / w) or less, 0.5 ppm (w / w) or less, or 0.2 ppm (w / w) or less, or any compatible combination thereof. The ingested concentration of the carboxylic acid may be, for example, 0.001 to 0.01 ppm (w / w), 0.1 to 0.2 ppm (w / w), 0.2 to 0.5 ppm (w / w), 0.5 to 1 ppm (w / w), 1 to 2 ppm (w / w), 2 to 5 ppm (w / w), 5 to 10 ppm (w / w), 10 to 20 ppm (w / w), 20 to 50 ppm (w / w), 50 to 100 ppm (w / w), 100 to 200 ppm (w / w), 200 to 500 ppm (w / w), 500 to 1000 ppm (w / w), 1000 to 2000 ppm (w / w), or 2000 to 5000 ppm (w / w). The ingested concentration of the carboxylic acid may be, for example, 0.001 to 5000 ppm (w / w), 0.01 to 4000 ppm (w / w), 0.1 to 3000 ppm (w / w), or 1.0 to 2000 ppm (w / w).When the food product of the present invention contains two or more carboxylic acids (e.g., when two or more carboxylic acids are added), the ingestible concentrations of the two or more carboxylic acids, either independently or in total, may be set within the range of the ingestible concentrations of the carboxylic acids listed above. In one embodiment, for example, the ingestible concentration of 2-methylbutyric acid may be 0.001 to 5000 ppm (w / w), 0.01 to 4000 ppm (w / w), 0.1 to 3000 ppm (w / w), or 1.0 to 2000 ppm (w / w). In one embodiment, for example, the ingestible concentration of (S)-2-methylbutyric acid in particular may be 0.001 to 5000 ppm (w / w), 0.01 to 4000 ppm (w / w), 0.1 to 3000 ppm (w / w), or 1.0 to 2000 ppm (w / w). In one embodiment, for example, the ingestible concentrations of 3-methylbutyric acid and isobutyric acid may each independently be 0.1 to 1000 ppm (w / w), 0.1 to 100 ppm (w / w), or 0.1 to 10 ppm (w / w). When the food product of the present invention contains two or more carboxylic acids (e.g., when two or more carboxylic acids are added), the "ingestible concentration of carboxylic acids" refers to the total ingestible concentration of those two or more carboxylic acids, unless otherwise specified.

[0123] When the food product of the present invention contains two or more carboxylic acids, the contents of the two or more carboxylic acids in the food product may or may not be the same. When the food product of the present invention contains a first and a second carboxylic acid, the content of the second carboxylic acid in the food product may be, for example, 0.1 parts by weight or more, 0.2 parts by weight or more, 0.5 parts by weight or more, 1 part by weight or more, 2 parts by weight or more, 5 parts by weight or more, 10 parts by weight or more, 20 parts by weight or more, 30 parts by weight or more, 50 parts by weight or more, or 70 parts by weight or more, or 100 parts by weight or less, 70 parts by weight or less, 50 parts by weight or less, 30 parts by weight or less, 20 parts by weight or less, 10 parts by weight or less, 5 parts by weight or less, 2 parts by weight or less, 1 part by weight or less, 0.5 parts by weight or less, or 0.2 parts by weight or less, per 100 parts by weight of the first carboxylic acid contained in the food product, or any compatible combination thereof. The content of the second carboxylic acid in the food product may be, for example, 0.1 to 0.2 parts by weight, 0.2 to 0.5 parts by weight, 0.5 to 1 part by weight, 1 to 2 parts by weight, 2 to 5 parts by weight, 5 to 10 parts by weight, 10 to 20 parts by weight, 20 to 30 parts by weight, 30 to 50 parts by weight, 50 to 70 parts by weight, or 70 to 100 parts by weight, relative to 100 parts by weight of the first carboxylic acid contained in the food product. The content of the second carboxylic acid in the food product may be, for example, 0.1 to 100 parts by weight, 0.5 to 50 parts by weight, or 2 to 20 parts by weight, relative to 100 parts by weight of the first carboxylic acid contained in the food product. The first carboxylic acid may be any carboxylic acid, for example, 2-methylbutyric acid. The first carboxylic acid may be, for example, particularly (S)-2-methylbutyric acid. The second carboxylic acid may be any carboxylic acid other than the first carboxylic acid, such as 3-methylbutyric acid or isobutyric acid. For example, when the food product of the present invention contains 2-methylbutyric acid, 3-methylbutyric acid, and isobutyric acid, the contents of 3-methylbutyric acid and isobutyric acid in the food product may be, independently or in total, in the amounts exemplified above per 100 parts by weight of 2-methylbutyric acid contained in the food product.In one aspect, when the active ingredient contained in the food of the present invention is produced by the production method of the present invention, the quantitative ratio (e.g., weight ratio or molar ratio) of 2-methylbutyric acid to 3-methylbutyric acid contained in the food of the present invention may be conveniently considered to be the same as the quantitative ratio (e.g., weight ratio or molar ratio) of isoleucine to leucine consumed in the culture by the production method of the present invention.

[0124] Furthermore, the content of carboxylic acid in the food of the present invention may be, for example, 0.5 parts by weight or more, 1 part by weight or more, 2 parts by weight or more, 3 parts by weight or more, 4 parts by weight or more, 5 parts by weight or more, 6 parts by weight or more, 8 parts by weight or more, 10 parts by weight or more, 12 parts by weight or more, 15 parts by weight or more, or 20 parts by weight or less, or 15 parts by weight or less, 12 parts by weight or less, 10 parts by weight or less, 8 parts by weight or less, 6 parts by weight or less, 5 parts by weight or less, 4 parts by weight or less, 3 parts by weight or less, 2 parts by weight or less, or 1 part by weight or less, or any compatible combination thereof, per 100 parts by weight of diacetyl contained in the food. The content of the carboxylic acid in the food product of the present invention may be, for example, 0.5 to 1 part by weight, 1 to 2 parts by weight, 2 to 3 parts by weight, 3 to 4 parts by weight, 4 to 5 parts by weight, 5 to 6 parts by weight, 6 to 8 parts by weight, 8 to 10 parts by weight, 10 to 12 parts by weight, 12 to 15 parts by weight, or 15 to 20 parts by weight, per 100 parts by weight of diacetyl contained in the food product. The content of the carboxylic acid in the food product of the present invention may be, for example, 0.5 to 20 parts by weight, 1 to 15 parts by weight, or 2 to 12 parts by weight, per 100 parts by weight of diacetyl contained in the food product. The content of the carboxylic acid in the food product of the present invention may be, for example, 0.5 to 8 parts by weight, 1 to 6 parts by weight, or 2 to 4 parts by weight, per 100 parts by weight of diacetyl contained in the food product.

[0125] Furthermore, the content of carboxylic acid in the food of the present invention may be, for example, 10 parts by weight or more, 20 parts by weight or more, 50 parts by weight or more, 70 parts by weight or more, 100 parts by weight or more, 150 parts by weight or more, 200 parts by weight or more, 300 parts by weight or more, 400 parts by weight or more, 500 parts by weight or more, or 700 parts by weight or more, relative to 100 parts by weight of acetoin contained in the food, or 1000 parts by weight or less, 700 parts by weight or less, 500 parts by weight or less, 400 parts by weight or less, 300 parts by weight or less, 200 parts by weight or less, 150 parts by weight or less, 100 parts by weight or less, 70 parts by weight or less, 50 parts by weight or less, or 20 parts by weight or less, or any compatible combination thereof. The content of the carboxylic acid in the food of the present invention, relative to 100 parts by weight of acetoin contained in the food, specifically, for example, 10 parts by weight to 20 parts by weight, 20 parts by weight to 50 parts by weight, 50 parts by weight to 70 parts by weight, 70 parts by weight to 100 parts by weight, 100 parts by weight to 150 parts by weight, 150 parts by weight to 200 parts by weight, 200 parts by weight to 300 parts by weight, 300 parts by weight to 400 parts by weight, 400 parts by weight to 500 parts by weight, 500 parts by weight to 700 parts by weight, or 700 parts by weight to 1000 parts by weight. The content of the carboxylic acid in the food of the present invention, relative to 100 parts by weight of acetoin contained in the food, specifically, for example, 10 parts by weight to 1000 parts by weight, 20 parts by weight to 700 parts by weight, or 50 parts by weight to 500 parts by weight. The content of carboxylic acid in the food of the present invention may be, for example, 10 to 300 parts by weight, 20 to 200 parts by weight, or 50 to 150 parts by weight per 100 parts by weight of acetoin contained in the food.

[0126] The ingested concentration of diacetyl may be, for example, 0.0001 ppm (w / w) or more, 0.001 ppm (w / w) or more, 0.01 ppm (w / w) or more, 0.02 ppm (w / w) or more, 0.05 ppm (w / w) or more, 0.1 ppm (w / w) or more, 0.2 ppm (w / w) or more, 0.5 ppm (w / w) or more, 1 ppm (w / w) or more, 2 ppm (w / w) or more, 5 ppm (w / w) or more, 10 ppm (w / w) or more, 20 ppm (w / w) or more, or 50 ppm (w / w) or more, and may be 100 ppm (w / w) or less, 50 ppm (w / w) or less, 20 ppm (w / w) or less, 10 ppm (w / w) or less, 5 ppm (w / w) or less, 2 ppm (w / w) or less, 1 ppm (w / w) or less, 0.5 ppm (w / w) or less, 0.2 ppm (w / w) or less, 0.1 ppm (w / w) or less, 0.05 ppm (w / w) or less, or 0.02 ppm (w / w) or less, or any consistent combination thereof. Specific examples of the diacetyl intake concentration include 0.0001 to 0.001 ppm (w / w), 0.01 to 0.02 ppm (w / w), 0.02 to 0.05 ppm (w / w), 0.05 to 0.1 ppm (w / w), 0.1 to 0.2 ppm (w / w), 0.2 to 0.5 ppm (w / w), 0.5 to 1 ppm (w / w), 1 to 2 ppm (w / w), 2 to 5 ppm (w / w), 5 to 10 ppm (w / w), 10 to 20 ppm (w / w), 20 to 50 ppm (w / w), and 50 to 100 ppm (w / w). The ingested concentration of diacetyl may be, for example, 0.0001 to 100 ppm (w / w), 0.001 to 80 ppm (w / w), 0.01 to 60 ppm (w / w), or 0.1 to 40 ppm (w / w).

[0127] The ingested concentration of acetoin may be, for example, 0.001 ppm (w / w) or more, 0.01 ppm (w / w) or more, 0.1 ppm (w / w) or more, 0.2 ppm (w / w) or more, 0.5 ppm (w / w) or more, 1 ppm (w / w) or more, 2 ppm (w / w) or more, 5 ppm (w / w) or more, 10 ppm (w / w) or more, 20 ppm (w / w) or more, 50 ppm (w / w) or more, 100 ppm (w / w) or more, 200 ppm (w / w) or more, 500 ppm (w / w) or more, 1000 ppm (w / w) or more, or 2000 ppm (w / w) or more, and may be 5000 ppm (w / w) or less, 2000 ppm (w / w) or less, 1000 ppm (w / w) or less, 500 ppm (w / w) or less, 200 ppm (w / w) or less, 100 ppm (w / w) or less, 50 ppm (w / w) or less, 20 ppm (w / w) or less, 10 ppm (w / w) or less, 5 ppm (w / w) or less, 2 ppm (w / w) or less, 1 ppm (w / w) or less, 0.5 ppm (w / w) or less, or 0.2 ppm (w / w) or less, or any compatible combination thereof. The acetoin intake concentration may be, for example, 0.001 to 0.01 ppm (w / w), 0.1 to 0.2 ppm (w / w), 0.2 to 0.5 ppm (w / w), 0.5 to 1 ppm (w / w), 1 to 2 ppm (w / w), 2 to 5 ppm (w / w), 5 to 10 ppm (w / w), 10 to 20 ppm (w / w), 20 to 50 ppm (w / w), 50 to 100 ppm (w / w), 100 to 200 ppm (w / w), 200 to 500 ppm (w / w), 500 to 1000 ppm (w / w), 1000 to 2000 ppm (w / w), or 2000 to 5000 ppm (w / w). The ingested concentration of acetoin may be, for example, 0.001 to 5000 ppm (w / w), 0.01 to 4000 ppm (w / w), 0.1 to 3000 ppm (w / w), or 1.0 to 2000 ppm (w / w).

[0128] The description of the addition of an active ingredient also applies mutatis mutandis to the addition of the composition of the present invention. For example, the composition of the present invention can be added so as to obtain the amount of the active ingredient exemplified above.

[0129] In one embodiment, the food product of the present invention may be cheese-free, i.e., the food product of the present invention may be manufactured so as to be cheese-free.

[0130] In one embodiment, the food of the present invention may contain cheese. That is, the food of the present invention may be produced so as to contain cheese. The cheese-containing food can be produced, for example, by adding cheese. That is, the method of the present invention may further comprise adding cheese to the ingredients of the food. The addition of cheese can be carried out in the same manner as the addition of the active ingredient. Furthermore, the cheese-containing food can be produced, for example, using ingredients of a cheese-containing food. That is, the ingredients of the food may contain cheese.

[0131] The food of the present invention is typically a cheese-containing food, but may have a reduced cheese content. A "reduced cheese content food" refers to a food with a lower cheese content than normal (i.e., a food with a lower cheese content than normal cheese), and also includes food that does not contain any cheese at all. A food that contains a normal amount of cheese (i.e., a food with a normal cheese content) is also referred to as a "normal cheese-containing food." Specifically, a "reduced cheese content food" may refer to a food of the same type as a normal cheese-containing food, but with a lower cheese content than normal cheese-containing foods. More specifically, a "reduced cheese content food" may refer to a food of the same type as a normal cheese-containing food, but with a cheese content that is 0.9 times or less, 0.8 times or less, 0.7 times or less, 0.6 times or less, 0.5 times or less, 0.4 times or less, 0.3 times or less, 0.2 times or less, or 0.1 times or less of the normal cheese-containing food, and may also include food that does not contain any cheese at all. Foods with reduced cheese content include foods that typically contain cheese, such as the foods exemplified above, but are manufactured to have a lower cheese content than normal or to contain no cheese at all. Normal cheese-containing foods include foods that contain cheese, such as the foods exemplified above, but are manufactured to contain a normal amount of cheese. According to the present invention, for example, a reduction in cheese flavor due to a reduction in cheese content may be compensated for. In other words, "imparting cheese flavor" may also include compensating for a reduction in cheese flavor due to a reduction in cheese content.

[0132] The method of the present invention may include a step of producing the active ingredient prior to the step of utilizing the active ingredient (e.g., a step of adding the active ingredient to a food ingredient). The active ingredient utilized in the method of the present invention is produced by the production method of the present invention. Thus, the step of producing the active ingredient in the method of the present invention may be a step of producing the active ingredient by the production method of the present invention. Specifically, the step of producing the active ingredient in the method of the present invention may be the culturing step of the production method of the present invention. The step of producing the active ingredient in the method of the present invention may be, in particular, a step of culturing an active ingredient-producing bacterium having carboxylic acid production ability, diacetyl production ability, and acetoin production ability in a medium containing precursors to obtain a culture containing carboxylic acid, diacetyl, and acetoin. The active ingredient utilized in the method of the present invention may be produced, for example, as the composition of the present invention. Thus, the step of producing the active ingredient in the method of the present invention may be, for example, a step of producing the composition of the present invention by the production method of the present invention.

[0133] <4> Use of Active Ingredient The present invention also discloses the use of the active ingredient in the applications exemplified above. That is, the present invention discloses, for example, the use of the active ingredient for improving the flavor of food or producing food, and the use of the active ingredient in producing a composition for improving the flavor of food or producing food.

[0134] The present invention also discloses active ingredients for use in the above-exemplified applications, i.e., active ingredients for use in improving the flavor of food or in producing food, and active ingredients for use in producing a composition for improving the flavor of food or producing food.

[0135] The present invention will now be described in more detail with reference to the following non-limiting examples, in which all precursors (amino acids) used are L-amino acids.

[0136] Example 1 Fermentation Test Using Bacillus Bacteria (Evaluation of Strain Differences) The two strains of Bacillus bacteria listed in Table 1 were used. A liquid medium (1% yeast extract, 1% peptone, 1% Glc) was inoculated from a glycerol stock and cultured at 30°C for 20 hours with shaking at 120 rpm to prepare a preculture solution. Next, main fermentation medium 1 was prepared with the composition shown in Table 2. A 500-mL Sakaguchi flask was charged with 1 g of calcium carbonate for pH buffering and 10 mL of main fermentation medium 1, to which 1 mL of the preculture solution was added. Main fermentation was then carried out by culturing at 30°C for 48.5 hours with shaking at 120 rpm.

[0137] The results are shown in Table 3. Both Bacillus subtilis and Bacillus amyloliquefaciens consumed sugar (glucose) and Ile and simultaneously produced 2-methylbutyric acid (2MB), acetoin, and diacetyl. The balance of the components produced differed between the two strains. Because it was difficult to separate 2MB and 3-methylbutyric acid (IVA), IVA may have been included in the quantified 2MB. However, because Ile is a precursor of 2MB, it is likely that the majority of the quantified 2MB was 2MB. These findings demonstrate that active ingredients can be produced simultaneously using Bacillus bacteria.

[0138]

[0139]

[0140]

[0141] Example 2 Fermentation Test Using Bacillus Bacteria (Evaluation of Differences in Medium Composition) (1) Bacillus subtilis (Table 1) was used as the Bacillus bacterium. A liquid medium (1% yeast extract, 1% peptone, 1% Glc) was inoculated from a glycerol stock and cultured at 30°C for 20 hours with shaking at 120 rpm to prepare a preculture solution. Main fermentation medium 1, 2, and 3 were then prepared with the compositions shown in Table 4. For medium 1 and medium 3 containing added sugar, 1 g of calcium carbonate for pH buffering and 10 mL of main fermentation medium were placed in a 500 mL Sakaguchi flask, and 1 mL of the preculture solution was added. The main fermentation was carried out by culturing at 30°C for 48.5 hours with shaking at 120 rpm. In the case of medium 2 without added sugar, 10 mL of the main fermentation medium was placed in one 500 mL Sakaguchi flask without adding calcium carbonate, and 1 mL of the preculture solution was added thereto. The main fermentation was carried out by shaking culture at 30°C and 120 rpm for 48.5 hours.

[0142] The results are shown in Table 5. Data in No. 1 in Table 5 are a reprint of data in No. 1 in Table 3. The initial concentrations of each amino acid include those from yeast extract and peptone. Acetoin and diacetyl were produced in Medium 1 and Medium 3, which contained sugar, but not in Medium 2, which contained no sugar. In Medium 3, which contained Ile, Leu, and Val, increased production of 2-methylbutyric acid (2MB) and isobutyric acid was observed compared to Medium 1 and Medium 2, which contained no Leu or Val. Because it was difficult to separate 2MB from 3-methylbutyric acid (IVA), IVA may have been included in the quantified 2MB. However, since Ile is a precursor of 2MB, it is likely that the majority of the quantified 2MB was 2MB. Furthermore, since Leu is a precursor of IVA, it is likely that the addition of Leu also increased IVA production. These results demonstrate that the addition of sugar is effective for the production of acetoin and diacetyl. It was also revealed that the addition of carboxylic acid precursors was effective for the production of carboxylic acids.

[0143]

[0144]

[0145] (2) Isobutyric acid can be produced from Val, but the consumption of Val was small in (1). Therefore, we evaluated the possibility of isobutyric acid being produced from Ile or Leu.

[0146] Cultivation was carried out in the same manner as in (1) using main fermentation medium 1 and main fermentation medium 4 shown in Table 4. The amount of each amino acid consumed after 48 hours of fermentation was quantified. The amount of isobutyric acid produced after 24 and 48 hours of fermentation was quantified as the peak area by GC / MS.

[0147] The results are shown in Table 6. In all media, amino acids (especially the added Ile or Leu) were consumed, and isobutyric acid was produced. Main Medium 1 (medium supplemented with Ile) showed a greater total consumption of amino acids and a greater production of isobutyric acid. These results suggest that isobutyric acid may be produced from Ile and / or Leu.

[0148]

[0149] Example 3: Sensory Evaluation of Fermented Broth (Evaluation of Differences in Bacterial Strains) A sensory evaluation was performed on the two types of fermented broth prepared in Example 1. The evaluation system used was a cheese-containing product, "Knorr (registered trademark) Cup Soup, Thick and Rich Potage with Four Cheeses (Ajinomoto Co., Inc.)," ​​and a cheese-flavored mock sauce simulating plant-based cheese. The mock sauce was prepared according to the composition shown in Table 7. The ingredients were mixed in a blender for approximately 1 minute and heated at 60°C for 3 minutes to prepare the mock sauce. The fermented broth was added to the potage and mock sauce at a concentration of 0.3%, and the results were evaluated by a panel of five experts. Kerry's Cheddar Cheese Powder (CR-15270) was also used as a positive control (PC).

[0150] The results are shown in Tables 8 and 9. The cheese flavor was enhanced when either the B. subtilis or B. amyloliquefaciens fermentation liquid was added. In an evaluation of a potage that already had a cheese flavor, the B. amyloliquefaciens fermentation liquid had a strong yogurt flavor and was unbalanced, while the B. subtilis fermentation liquid enhanced the cheese flavor in a well-balanced manner. In an evaluation of a simulated sauce without a cheese flavor, both fermentation liquids imparted a well-balanced cheese flavor. These results demonstrate that the fermentation liquid of Bacillus bacteria is effective in imparting a cheese flavor.

[0151]

[0152]

[0153]

[0154] Example 4: Sensory Evaluation of Fermented Broth (Evaluation of Differences in Culture Medium Composition) A sensory evaluation was performed on the three types of fermented broth prepared in Example 2. A simulated sauce without a cheese flavor, which imitated plant-based cheese, was used as the evaluation system. The simulated sauce was prepared with the composition shown in Table 7. The ingredients were mixed in a mixer for approximately 1 minute and heated at 60°C for 3 minutes to prepare the simulated sauce. The fermented broth was added to the simulated sauce to a concentration of 0.3%, and the sauce was evaluated by a panel of five experts. Kerry's Cheddar Cheese Powder (CR-15270) was also used as a positive control (PC).

[0155] The results are shown in Table 10. A cheese flavor was imparted to all fermentation broths. However, the fermentation broth obtained with medium 2 (containing neither diacetyl nor acetoin) was less effective in imparting cheese flavor than the fermentation broths obtained with medium 1 and medium 3 (containing diacetyl and acetoin). Both the fermentation broths obtained with medium 1 and medium 3 (containing diacetyl and acetoin) imparted a well-balanced cheese flavor, with the effect being particularly pronounced in the fermentation broth obtained with medium 3. These results demonstrate that the addition of a combination of carboxylic acid, diacetyl, and acetoin is more effective in imparting a cheese flavor than the addition of a carboxylic acid alone. Furthermore, it was revealed that the fermentation broth of Bacillus bacteria produced in a medium supplemented with sugar, Ile, Leu, and Val was particularly effective in imparting a cheese flavor.

[0156]

Claims

1. A method for producing a carboxylic acid, diacetyl, and acetoin, comprising: culturing a Bacillus bacterium having carboxylic acid, diacetyl, and acetoin production abilities in a medium containing a precursor of the carboxylic acid and a sugar to obtain a culture containing the carboxylic acid, diacetyl, and acetoin, wherein the carboxylic acid is selected from the group consisting of 2-methylbutyric acid, 3-methylbutyric acid, isobutyric acid, and combinations thereof, the precursor of 2-methylbutyric acid is isoleucine, the precursor of 3-methylbutyric acid is leucine, and the precursor of isobutyric acid is selected from the group consisting of isoleucine, leucine, valine, and combinations thereof.

2. The method of claim 1, wherein the 2-methylbutyric acid is (S)-2-methylbutyric acid and / or (R)-2-methylbutyric acid, and the precursors of (S)-2-methylbutyric acid and (R)-2-methylbutyric acid are L-isoleucine and D-isoleucine, respectively.

3. The method of claim 1, wherein the carboxylic acid, diacetyl, and acetoin are prepared as a composition containing the carboxylic acid, diacetyl, and acetoin.

4. The method according to claim 3, wherein the composition comprises the culture or a processed product thereof.

5. The method of claim 3, wherein the composition comprises a dried product of the culture or a dried product of the supernatant of the culture.

6. The method according to claim 3, wherein the content of said carboxylic acid in said composition is 10 ppm (w / w) or more.

7. The method according to claim 3, wherein the content of diacetyl in said composition is 0.5 ppm (w / w) or more.

8. The method according to claim 3, wherein the content of acetoin in the composition is 10 ppm (w / w) or more.

9. The method of any one of claims 1 to 8, wherein the bacterium is Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus polymixa, Bacillus stearothermophilus, or Bacillus velezensis.

10. The method according to any one of claims 1 to 8, wherein the bacterium is Bacillus subtilis or Bacillus amyloliquefaciens.

11. The method according to any one of claims 1 to 8, wherein the isoleucine content in the medium is 0.1 to 5% (w / w).

12. The method according to any one of claims 1 to 8, wherein the leucine content in the medium is 0.1 to 5% (w / w).

13. The method according to any one of claims 1 to 8, wherein the valine content in the medium is 0.1 to 5% (w / w).

14. The method according to any one of claims 1 to 8, wherein the content of the sugar in the medium is 1 to 50% (w / w).

15. The method according to any one of claims 1 to 8, wherein the sugar is glucose.

16. The method of claim 3, wherein the composition is a composition for improving the flavor of food.

17. The method of claim 16, wherein the flavor improvement is the imparting of a cheese flavor.

18. The method of claim 3, wherein the composition is a seasoning.

19. The method according to any one of claims 1 to 8, wherein at least 2-methylbutyric acid is produced.

20. The method of claim 19, further comprising producing 3-methylbutyric acid.

21. The method of claim 19, further comprising producing isobutyric acid.

22. A composition made by the method of claim 3.

23. A composition for improving the flavor of food, comprising the following components (A), (B), and (C): (A) a carboxylic acid selected from the group consisting of 2-methylbutyric acid, 3-methylbutyric acid, isobutyric acid, and combinations thereof; (B) diacetyl; and (C) acetoin.

24. The composition of claim 23, wherein components (A), (B), and (C) are produced by the method of any one of claims 1 to 8.

25. The composition of claim 23, containing at least 2-methylbutyric acid.

26. The composition of claim 25, further comprising 3-methylbutyric acid.

27. The composition of claim 25, further comprising isobutyric acid.

28. The composition of claim 23, wherein the flavor improvement is the imparting of a cheese flavor.

29. A method for improving the flavor of a food product, comprising the step of adding the following components (A), (B), and (C) to a food ingredient: (A) a carboxylic acid selected from the group consisting of 2-methylbutyric acid, 3-methylbutyric acid, isobutyric acid, and combinations thereof; (B) diacetyl; (C) acetoin.

30. The method of claim 29, wherein components (A), (B), and (C) are produced by the method of any one of claims 1 to 8.

31. The method of claim 29, further comprising, prior to said step, the step of preparing said components (A), (B), and (C) by the method of any one of claims 1 to 8.

32. The method according to claim 29, wherein the component (A) is added to give an ingestible concentration of 0.001 to 5000 ppm (w / w).

33. The method according to claim 29, wherein the component (B) is added to give an ingestible concentration of 0.0001 to 100 ppm (w / w).

34. The method according to claim 29, wherein the component (C) is added to give an ingestible concentration of 0.001 to 5000 ppm (w / w).

35. The method of claim 29, wherein at least 2-methylbutyric acid is added.

36. The method of claim 35, further comprising the addition of 3-methylbutyric acid.

37. The method of claim 35 further comprising adding isobutyric acid.

38. The method of claim 29, wherein the flavor improvement is the imparting of a cheese flavor.

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