Method for improving flavor of food

Abstract

A technique is provided for improving the flavor of food. The flavor of food is improved by admixing the following component (A). (A) the fraction containing cell walls of gram-positive bacteria.

Description

How to improve the flavor of food

[0001] The present invention relates to a technology for improving the flavor of food.

[0002] Various techniques have been reported to improve the flavor of food.

[0003] Patent Document 1 discloses a flavor base for enhancing taste obtained by fermentation of bacteria such as Corynebacterium glutamicum, Corynebacterium ammoniagenes, Corynebacterium casei, Corynebacterium efficiens, Brevibacterium lactofermentum, and Bacillus subtilis.

[0004] Patent Document 2 discloses a flavor improver containing yeast cells after removing the yeast contents, and gives examples of flavors such as spiciness.

[0005] Patent Document 3 discloses a yeast extract that contains peptide and nucleic acid-based flavor components and can impart or enhance full-bodied flavor to foods by simultaneously imparting a long-lasting umami flavor and a rich taste.

[0006] Patent Document 4 discloses a food aroma improver containing a yeast-derived substance such as yeast extract, and gives examples of aromas such as spice aromas.

[0007] Patent Document 5 discloses a spice sensation enhancer containing a product obtained by thermally reacting sugar and amino acid in the presence of alcohol.

[0008] Patent Document 6 discloses a fermented seasoning composition containing a fermented product of lactic acid bacteria and yeast, which can be used to enhance the stimulating sensation of spices.

[0009] Patent Publication No. 2012-521205, Patent Publication No. 2018-050562, Patent Publication No. 2007-049988, Patent Publication No. 2010-166886, Patent Publication No. 2016-158507, WO2017-014253

[0010] An object of the present invention is to provide a technology for improving the flavor of food.

[0011] The present inventors have found that the flavor of food can be improved by incorporating a fraction containing the cell wall of a gram-positive bacterium, and have completed the present invention.

[0012] That is, the present invention can be exemplified as follows. [1] A composition for improving the flavor of food, comprising the following component (A): (A) a fraction containing the cell wall of a Gram-positive bacterium. [2] The composition, wherein the improvement in flavor is an enhancement of the spicy sensation and / or the imparting of a rich flavor. [3] The composition, wherein the improvement in flavor is an enhancement of the pungency of the spice. [4] The composition, further comprising a spice. [5] A composition, comprising the following component (A) and a spice: (A) a fraction containing the cell wall of a Gram-positive bacterium. [6] The composition, wherein the component (A) is a bacterial body of the Gram-positive bacterium or a fragment thereof. [7] The composition, wherein the Gram-positive bacterium is a bacterium belonging to the phylum Actinobacteria or Firmicutes. [8] The composition, wherein the Gram-positive bacterium is a bacterium belonging to the phylum Actinobacteria. [9] The composition, wherein the Gram-positive bacteria are Corynebacterium, bacteria belonging to the family Bifidobacteriaceae, bacteria belonging to the family Dermabacteraceae, bacteria belonging to the family Bacillaceae, bacteria belonging to the family Enterococcus, or bacteria belonging to the family Lactobacillaceae.

[10] The composition, wherein the Gram-positive bacteria are bacteria of the genus Corynebacterium, bacteria of the genus Brevibacterium, bacteria of the genus Bifidobacterium, bacteria of the genus Brachybacterium, bacteria of the genus Bacillus, bacteria of the genus Enterococcus, or bacteria of the genus Lactobacillus.

[11] The composition as described above, wherein the Gram-positive bacterium is Corynebacterium casei, Corynebacterium flavescens, Brevibacterium casei, Bifidobacterium longum, Brachybacterium alimentarium, Bacillus subtilis, Enterococcus faecalis, Lactobacillus mali, Lactobacillus hilgardii, or Lactobacillus brevis.

[12] The composition, wherein the content of the component (A) is 0.1% (w / w) or more, calculated based on the dry weight of the gram-positive bacterial cells.

[13] The composition, further comprising the following component (B): (B) one or more components selected from the group consisting of L-amino acids, nucleic acids, and organic acids.

[14] The composition, wherein the L-amino acid is at least the L-amino acid, and the L-amino acid is L-glutamic acid.

[15] The composition, wherein the content of the L-glutamic acid is 0.1 to 20 parts by weight per part by weight of the component (A), calculated based on the dry weight of the gram-positive bacterial cells.

[16] The composition, wherein the content of the spice is 0.2 to 500 parts by weight per part by weight of the component (A), calculated based on the dry weight of the gram-positive bacterial cells.

[17] The composition, wherein the spice is one or more spices selected from the group consisting of spices from the Lauraceae family, spices from the Piperaceae family, spices from the Labiatae family, spices from the Umbelliferae family, spices from the Solanaceae family, spices from the Myrtleaceae family, spices from the Liliaceae family, spices from the Myrtaceae family, spices from the Mesaceae family, spices from the Fabaceae family, spices from the Polygonaceae family, spices from the Brassicaceae family, spices from the Zingiberaceae family, and spices from the Rutaceae family.

[18] The composition, wherein the spice is a spice having a pungency.

[19] The composition is a seasoning.

[20] The composition, wherein the component (A) is produced by culturing the Gram-positive bacterium in a medium containing a food raw material.

[21] The composition, wherein the raw material contained in the medium is tomato.

[22] The composition, wherein the component (A) has been heat-treated.

[23] The composition, wherein the Gram-positive bacterium is a bacterium capable of producing L-glutamic acid and has one or more mutations selected from the mutations shown in Table 1 below.

[24] The composition, further comprising a culture of Gram-positive bacteria.

[25] A method for improving the flavor of food, comprising the step of adding the following component (A) to a food raw material: (A) a fraction containing the cell wall of Gram-positive bacteria.

[26] A method for producing a food with improved flavor, comprising the step of adding the following component (A) to a food raw material: (A) a fraction containing the cell wall of Gram-positive bacteria.

[27] The method, wherein the improved flavor is enhanced spiciness and / or imparting fullness.

[28] The method, wherein the improved flavor is enhanced spice pungency.

[29] The method, wherein component (A) is a gram-positive bacterium or a fragment thereof.

[30] The method, wherein the gram-positive bacterium is a bacterium belonging to the phylum Actinobacteria or Firmicutes.

[31] The method, wherein the gram-positive bacterium is a bacterium belonging to the phylum Actinobacteria.

[32] The method as described above, wherein the Gram-positive bacterium is a coryneform bacterium, a bacterium belonging to the family Bifidobacteriaceae, a bacterium belonging to the family Dermabacteraceae, a bacterium belonging to the family Bacillaceae, a bacterium belonging to the family Enterococcaceae, or a bacterium belonging to the family Lactobacillaceae.

[33] The method as mentioned above, wherein the Gram-positive bacteria are bacteria of the genus Corynebacterium, Brevibacterium, Bifidobacterium, Brachybacterium, Bacillus, Enterococcus, or Lactobacillus.

[34] The method as mentioned above, wherein the Gram-positive bacterium is Corynebacterium casei, Corynebacterium flavescens, Brevibacterium casei, Bifidobacterium longum, Brachybacterium alimentarium, Bacillus subtilis, Enterococcus faecalis, Lactobacillus mali, Lactobacillus hilgardii, or Lactobacillus brevis.

[35] The method as mentioned above, wherein the component (A) is added so that its concentration for consumption is 0.005 to 2% (w / w) calculated as the dry weight of the gram-positive bacterial cells.

[36] The method as mentioned above, further comprising the step of adding the following component (B) to the raw materials of the food: (B) one or more components selected from the group consisting of L-amino acids, nucleic acids, and organic acids.

[37] The method as mentioned above, wherein at least the L-amino acid is added, and the L-amino acid is L-glutamic acid.

[38] The method as mentioned above, wherein L-glutamic acid is added so that its concentration for consumption is 0.01 to 2% (w / w).

[39] The method as mentioned above, wherein the content of L-glutamic acid in the food is 0.1 to 20 parts by weight per part by weight of the component (A), calculated as the dry weight of the gram-positive bacterial cells.

[40] The method, wherein the food is a food containing a spice.

[41] The method, wherein the content of the spice in the food is 0.01 to 2% (w / w) in terms of consumption concentration.

[42] The method, wherein the spice is added to the food so that the content of the spice in the food is 0.2 to 500 parts by weight per part by weight of the component (A), calculated as the dry weight of the gram-positive bacterium cells.

[43] The method, wherein the spice is one or more spices selected from the group consisting of spices from the Lauraceae family, spices from the Piperaceae family, spices from the Labiatae family, spices from the Umbelliferae family, spices from the Solanaceae family, spices from the Myristicae family, spices from the Liliaceae family, spices from the Myrtaceae family, spices from the Mesoglea family, spices from the Fabaceae family, spices from the Polygonaceae family, spices from the Brassicaceae family, spices from the Zingiberaceae family, and spices from the Rutaceae family.

[44] The method, wherein the spice is a spice with a pungency.

[45] The method as mentioned above, wherein the component (A) is produced by culturing the Gram-positive bacterium in a medium containing a food raw material.

[46] The method as mentioned above, wherein the raw material contained in the medium is tomato.

[47] The method as mentioned above, wherein the component (A) has been heat-treated.

[48] The method as mentioned above, wherein the Gram-positive bacterium is a bacterium capable of producing L-glutamic acid and has one or more mutations selected from the mutations shown in Table 1 below.

[49] The method as mentioned above, further comprising the step of adding a culture of the Gram-positive bacterium to a food raw material.

[50] A seasoning containing the following component (A): (A) a fraction containing the cell wall of a Gram-positive bacterium.

[51] The seasoning as mentioned above, wherein the component (A) is the cell body of the Gram-positive bacterium or a fragment thereof.

[52] The seasoning as mentioned above, further containing the following component (B): (B) one or more components selected from the group consisting of L-amino acids, nucleic acids, and organic acids.

[53] The seasoning as described above, which contains at least the L-amino acid, and the L-amino acid is L-glutamic acid.

[54] The seasoning as described above, which further contains a spice.

[55] The seasoning as described above, wherein the component (A) is produced by culturing the gram-positive bacterium in a medium containing a food raw material.

[56] The seasoning as mentioned above, wherein the raw material contained in the culture medium is tomato.

[57] The seasoning as mentioned above, wherein the component (A) has been heat-treated.

[58] The seasoning as mentioned above, wherein the Gram-positive bacterium is a bacterium capable of producing L-glutamic acid and has one or more mutations selected from the mutations shown in Table 1 below.

[59] The seasoning as mentioned above, further comprising a culture of a Gram-positive bacterium.

[60] A bacterium capable of producing L-glutamic acid, having one or more mutations selected from the mutations A-1 to A-135 shown in Table 1 below.

[61] The bacterium having 50 or more mutations selected from the mutations A-1 to A-135 shown in Table 1 below.

[62] The bacterium having 100 or more mutations selected from the mutations A-1 to A-135 shown in Table 1 below.

[63] The bacterium having the mutations A-1 to A-135 shown in Table 1 below.

[64] The bacterium further has one or more mutations selected from mutations B-1 to B-92 shown in Table 1 below.

[65] The bacterium has 30 or more mutations selected from mutations B-1 to B-92 shown in Table 1 below.

[66] The bacterium has 60 or more mutations selected from mutations B-1 to B-92 shown in Table 1 below.

[67] The bacterium is a coryneform bacterium.

[68] The bacterium is a bacterium of the genus Corynebacterium.

[69] The bacterium is Corynebacterium casei.

[70] The bacterium is a modified strain derived from Corynebacterium casei JCM 12072 strain.

[71] A method for producing a composition for improving the flavor of food, comprising: a step of culturing a gram-positive bacterium in a medium to obtain a culture, wherein the composition contains the following component (A): (A) a fraction containing the cell wall of the gram-positive bacterium.

[72] The method, wherein the improvement in flavor is an enhancement of spiciness and / or imparting richness.

[73] The method, wherein the improvement in flavor is an enhancement of the pungency of a spice.

[74] The method, wherein the composition further contains a spice.

[75] The method as mentioned above, wherein the component (A) is a bacterial cell of the Gram-positive bacterium or a fragment thereof.

[76] The method as mentioned above, wherein the composition further contains the following component (B): (B) one or more components selected from the group consisting of an L-amino acid, a nucleic acid, and an organic acid.

[77] The method as mentioned above, wherein the composition contains at least the L-amino acid, and the L-amino acid is L-glutamic acid.

[78] The method as mentioned above, further comprising a step of heat-treating the culture.

[79] The method as mentioned above, wherein the Gram-positive bacterium is a bacterium capable of producing L-glutamic acid and has one or more mutations selected from the mutations shown in Table 1 below.

[80] The method as mentioned above, wherein the composition further contains a culture of a Gram-positive bacterium.

[81] A method for producing a composition for enhancing the umami taste of food, comprising a step of culturing the bacterium in a medium to obtain a culture containing L-glutamic acid, wherein the composition contains the L-glutamic acid.

[82] The method, wherein the composition further contains the following component (A): (A) a fraction containing cell walls of Gram-positive bacteria.

[83] A method for producing L-glutamic acid, comprising the steps of culturing the bacterium in a medium to obtain a culture containing L-glutamic acid; and recovering the L-glutamic acid.

[84] A composition for enhancing the umami taste of food, containing L-glutamic acid, wherein the L-glutamic acid is produced by culturing the bacterium in a medium.

[85] The composition, further containing the following component (A): (A) a fraction containing cell walls of Gram-positive bacteria.

[86] A method for enhancing the umami taste of food, comprising the step of adding L-glutamic acid to raw materials for the food, wherein the L-glutamic acid is produced by culturing the bacterium in a medium.

[87] A method for producing a food product with enhanced umami, comprising the step of adding L-glutamic acid to a food material, wherein the L-glutamic acid is produced by culturing the bacterium in a medium.

[88] The method further comprises the step of adding the following component (A) to the food material: (A) a fraction containing the cell wall of a Gram-positive bacterium.

[0013] FIG. 1 shows the amount of L-glutamic acid produced by a mutant strain library.

[0014] <1> Active ingredient In the present invention, the following ingredient (A) is used as an active ingredient: (A) A fraction containing the cell wall of a gram-positive bacterium.

[0015] The above component (A) is also referred to as the "active ingredient" or "cell wall fraction."

[0016] By using an active ingredient, the flavor of food can be improved, that is, an effect of improving the flavor of food can be obtained. This effect is also called a "flavor improving effect." The improvement of the flavor of food can also be simply called "flavor improvement." Specifically, by using an active ingredient, the flavor of food can be improved compared to when the active ingredient is not used. Therefore, the active ingredient may be used to improve the flavor of food.

[0017] Furthermore, by utilizing the active ingredient, it is possible to produce food products with improved flavor, and therefore the active ingredient may be used in the production of food products (specifically, in the production of food products with improved flavor).

[0018] Flavors include taste and aroma. That is, the use of an active ingredient may improve the taste and / or aroma. Note that the term "aroma" as used herein may refer to the aroma felt from the throat to the nasal cavity when eating food (i.e., retronasal aroma) and / or the aroma smelled directly through the nose (i.e., orthonasal aroma). The term "aroma" as used herein may particularly refer to the aroma felt from the throat to the nasal cavity when eating food (i.e., retronasal aroma).

[0019] The improvement of flavor includes enhancing the spiciness and imparting a rich flavor. The improvement of flavor particularly includes enhancing the spiciness. By utilizing the active ingredient, one type of flavor may be improved alone, or two or more types of flavors may be improved in combination. That is, by utilizing the active ingredient, for example, an enhancement of the spiciness and / or imparting a rich flavor may be achieved.

[0020] "Spicy" may refer to the sensation felt due to the presence of spices. "Spicy" may also be referred to as spice flavor, spice aroma, pungency, hotness, or burning. "Spicy" may specifically refer to the sensation felt due to the presence of spices when eating a food containing spices. "Spicy" may be used interchangeably with "spice flavor." Examples of spice include the taste and aroma of spices. Examples of spice include the pungency of spices. "Enhancing spice" is not limited to enhancing the spice of a food that has a spice (e.g., a food that contains spices), but may also include imparting a spice to a food that does not have a spice (e.g., a food that does not contain spices). For example, by using an active ingredient in combination with a spice, it is possible to impart a spice to a food that does not have a spice (specifically, an enhanced spice compared to when the active ingredient is not used).

[0021] "Kokumi" refers to a sensation that cannot be expressed by the five basic tastes of sweet, salty, sour, bitter, and umami. Specifically, it may refer to a taste that not only enhances the basic tastes but also enhances marginal tastes and flavors surrounding the basic tastes, such as thickness, growth (mouthfulness), continuity, and harmony. "Imparting kokumi" includes enhancing the basic tastes and imparting or enhancing the accompanying flavors surrounding the basic tastes, such as thickness, growth, continuity, and harmony. "Imparting kokumi" also includes imparting or enhancing flavors such as complexity, maturity, richness, meatiness, milkiness, fruitiness, and fullness (such as the fullness of sugar or the fullness of wine).

[0022] Flavors (e.g., spicy or rich 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, the initial spiciness, the middle spiciness, the aftertaste spiciness, or a combination thereof may be enhanced. Also, by utilizing the active ingredient, specifically, for example, the initial full-bodied taste, the middle full-bodied taste, the aftertaste full-bodied taste, or a combination thereof may be imparted.

[0023] Measurement and comparison of flavor (e.g., spiciness or richness) can be performed, for example, by sensory evaluation by an expert panel.

[0024] The active ingredient may be utilized to improve flavor or prepare food products in the manner described in the methods of the present invention below.

[0025] The active ingredient is not particularly limited as long as it contains the cell wall of a gram-positive bacterium. The active ingredient may or may not consist of the cell wall of a gram-positive bacterium. The cell wall of a gram-positive bacterium may exist, for example, as the cell body of the gram-positive bacterium or as a fragment thereof. That is, specific examples of the active ingredient include the cell body of a gram-positive bacterium and fragments thereof. The active ingredient particularly includes the cell body of a gram-positive bacterium. The cell wall of a gram-positive bacterium may contain, for example, peptidoglycan and / or a lipid-soluble component. Examples of lipid-soluble components of the cell wall of a gram-positive bacterium include teichoic acid, lipoteichoic acid, mycolic acid, mycolic acid sugar ester, lipoarabinomannan, and glycolipids.

[0026] The gram-positive bacteria are not particularly limited. Examples of gram-positive bacteria include bacteria belonging to the phylum Actinobacteria and Firmicutes. Examples of gram-positive bacteria include bacteria belonging to the phylum Actinobacteria.

[0027] Examples of bacteria belonging to the Actinobacteria phylum include coryneform bacteria, bacteria belonging to the Bifidobacteriaceae family, and bacteria belonging to the Dermabacteraceae family. Examples of bacteria belonging to the Actinobacteria phylum include coryneform bacteria.

[0028] Examples of coryneform bacteria include bacteria of the genus Corynebacterium, Brevibacterium, and Microbacterium.

[0029] Specific examples of coryneform bacteria include the following species: Corynebacterium acetoacidophilum Corynebacterium acetoglutamicum Corynebacterium alkanolyticum Corynebacterium callunae Corynebacterium casei Corynebacterium crenatum Corynebacterium flavescens Corynebacterium glutamicum Corynebacterium lilium Corynebacterium melassecora melassecola) Corynebacterium thermoaminogenes (Corynebacterium efficiens) Corynebacterium herculis Brevibacterium casei Brevibacterium divaricatum (Corynebacterium glutamicum) Brevibacterium flavum (Corynebacterium glutamicum) Brevibacterium immariophilum Brevibacterium lactofermentum (Corynebacterium glutamicum)Brevibacterium roseum Brevibacterium saccharolyticum Brevibacterium thiogenitalis Corynebacterium ammoniagenes (Corynebacterium stationis) Brevibacterium album Brevibacterium cerinum Microbacterium ammoniaphilum

[0030] More specifically, examples of coryneform bacteria include the following strains: Corynebacterium acetoacidophilum ATCC 13870, Corynebacterium acetoglutamicum ATCC 15806, Corynebacterium alkanolyticum ATCC 21511, Corynebacterium callunae ATCC 15991, Corynebacterium casei JCM 12072, and Corynebacterium crenatum AS1.542 Corynebacterium flavescens ATCC 10340(NBRC 14136) Corynebacterium glutamicum ATCC 13020, ATCC 13032, ATCC 13060, ATCC 13869, FERM BP-734 Corynebacterium lilium ATCC 15990 Corynebacterium melassecola ATCC 17965 Corynebacterium efficiens (Corynebacterium thermoaminogenes) AJ12340 (FERM BP-1539) Corynebacterium herculis ATCC 13868 Brevibacterium casei ATCC 35513(DSM 20657) Brevibacterium divaricatum (Corynebacterium glutamicum) ATCC 14020 Brevibacterium flavum (Corynebacterium glutamicum) ATCC 13826, ATCC 14067, AJ12418 (FERM BP-2205) Brevibacterium immariophilum ATCC 14068 Brevibacterium lactofermentum (Corynebacterium glutamicum) ATCC 13869 Brevibacterium roseum ATCC 13825 Brevibacterium saccharolyticum ATCC 14066 Brevibacterium thiogenitalis ATCC 19240 Corynebacterium ammoniagenes (Corynebacterium stationis) ATCC 6871, ATCC 6872 Brevibacterium album ATCC 15111 Brevibacterium cerinum ATCC 15112 Microbacterium ammoniaphilum ATCC 15354.

[0031] Examples of coryneform bacteria include bacteria of the genus Corynebacterium and bacteria of the genus Brevibacterium. Examples of Corynebacterium bacteria include Corynebacterium casei such as Corynebacterium casei JCM 12072 and Corynebacterium flavescens such as Corynebacterium flavescens ATCC 10340. Examples of Brevibacterium bacteria include Brevibacterium casei such as Brevibacterium casei ATCC 35513.

[0032] The genus Corynebacterium also includes bacteria that were previously classified as Brevibacterium but have now been integrated into the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 255(1991)). Corynebacterium stationis also includes bacteria that were previously classified as Corynebacterium ammoniagenes but have been reclassified as Corynebacterium stationis based on 16S rRNA sequence analysis and other factors (Int. J. Syst. Evol. Microbiol., 60, 874-879(2010)).

[0033] Examples of bacteria belonging to the family Bifidobacteriaceae include bacteria of the genus Bifidobacterium. Bifidobacterium bacteria include Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium dentium, Bifidobacterium pseudocatenulatum, Bifidobacterium animalis, and Bifidobacterium pseudolongum. Examples of the genus Bifidobacterium include Bifidobacterium longum, Bifidobacterium pseudolongum, and Bifidobacterium thermophilum. Specific examples of Bifidobacterium longum include ATCC 15697, ATCC 15707, ATCC 25962, ATCC 15702, ATCC 27533, BG7, DSM 24736, SBT 2928, NCC 490 (CNCM I-2170), and NCC 2705 (CNCM I-2618). Specific examples of Bifidobacterium breve include ATCC 15700, B632 (DSM 24706), Bb99 (DSM 13692), ATCC 15698, DSM 24732, UCC2003, YIT4010, YIT4064, BBG-001, BR-03, C50, and R0070.Specific examples of Bifidobacterium bifidum include ATCC 29521, OLB6378, and BF-1.Specific examples of Bifidobacterium adolescentis include ATCC 15703. Specific examples of Bifidobacterium dentium include DSM 20436. Specific examples of Bifidobacterium pseudocatenulatum include ATCC 27919. Specific examples of Bifidobacterium animalis include DSM 10140, Bb-12, DN-173 010, GCL2505, and CNCM I-3446. Specific examples of Bifidobacterium pseudolongum include JCM 5820 and ATCC 25526. Specific examples of Bifidobacterium thermophilum include ATCC 25525.

[0034] Examples of bacteria belonging to the Dermabacteraceae family include bacteria of the genus Brachybacterium. Examples of bacteria of the genus Brachybacterium include Brachybacterium alimentarium and Brachybacterium tyrofermentans. Examples of bacteria of the genus Brachybacterium include Brachybacterium alimentarium. Examples of Brachybacterium alimentarium include ATCC 700067 (NBRC 16118). Examples of Brachybacterium tyrofermentans include DSM 10673.

[0035] Examples of bacteria belonging to the phylum Firmicutes include bacteria belonging to the family Bacillaceae, bacteria belonging to the family Enterococcaceae, and bacteria belonging to the family Lactobacillaceae.

[0036] Examples of bacteria belonging to the Bacillaceae family include bacteria of the genus Bacillus. Examples of bacteria of the genus Bacillus include Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus polymixa, Bacillus stearothermophilus, and Bacillus velezensis. Examples of bacteria of the genus Bacillus include Bacillus subtilis. Specific examples of Bacillus subtilis include the 168 Marburg strain (ATCC 6051, JCM 1465) and the PY79 strain (Plasmid, 1984, 12, 1-9).Specific examples of Bacillus amyloliquefaciens include the T strain (ATCC 23842), the N strain (ATCC 23845), the AJ11708 strain (NITE BP-02609), and the FZB42 strain (DSM 23117).

[0037] Examples of bacteria belonging to the Enterococcaceae family include bacteria of the genus Enterococcus. Examples of Enterococcus bacteria include Enterococcus faecalis and Enterococcus faecium. Examples of Enterococcus bacteria include Enterococcus faecalis. Specific examples of Enterococcus faecalis include ATCC 19433. Specific examples of Enterococcus faecium include ATCC 19434.

[0038] As the bacterium belonging to the Lactobacillaceae family, the bacterium of the genus Lactobacillus can be mentioned.As the bacterium of the genus Lactobacillus, the bacterium of the genus Lactobacillus can be mentioned Lactobacillus mali, Lactobacillus hilgardii, Lactobacillus brevis, Lactobacillus delbrueckii, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus gasseri, Lactobacillus acidophilus.As the bacterium of the genus Lactobacillus, in particular, the bacterium of the genus Lactobacillus can be mentioned Lactobacillus mali, Lactobacillus hilgardii, Lactobacillus brevis. Specific examples of Lactobacillus mali include NBRC 102159 (ATCC 27053), Lactobacillus hilgardii include NBRC 15886 (ATCC 8290), and Lactobacillus Brevis include JCM 1102 (ATCC 27305).

[0039] These strains 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. These strains can also be obtained, for example, from the depository institution where they were deposited.

[0040] Gram-positive bacteria may be modified as appropriate. That is, examples of Gram-positive bacteria include modified strains derived from the strains exemplified above. Specific examples of such modified strains include modified strains derived from Corynebacterium casei JCM 12072. The purpose of the modification is not particularly limited. Examples of modifications include modifications to impart or enhance the ability to produce a target substance. Modified strains may be, for example, strains bred by artificial modification. Examples of artificial modifications include modifications using genetic engineering techniques and mutation treatments. Examples of mutation treatments include X-ray irradiation, ultraviolet irradiation, and treatment with mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS). Furthermore, modified strains may be naturally occurring strains that arise during the use of Gram-positive bacteria. Examples of such modified strains include mutant strains that naturally arise during the cultivation of Gram-positive bacteria. The modified strain may be constructed by one type of modification or by a combination of two or more types of modifications.

[0041] The Gram-positive bacterium may have the ability to produce a target substance.

[0042] "Ability to produce a target substance" refers to the ability to produce a target substance. In other words, "bacteria capable of producing a target substance" refers to bacteria capable of producing a target substance. Specifically, "bacteria capable of producing a target substance" may refer to bacteria that, when cultured in a medium, produce a target substance and accumulate it in the medium and / or within the bacterial cells to an extent that it can be recovered. When the bacterium capable of producing a target substance is a modified strain, the bacterium capable of producing a target substance may be a bacterium that can accumulate a greater amount of the target substance in the medium and / or within the bacterial cells than a non-modified strain. Examples of non-modified strains include wild-type strains and parent strains. The bacterium capable of producing a target substance may particularly be a bacterium that can accumulate the target substance in a medium. The bacterium capable of producing a target substance may be a bacterium that can accumulate the target substance in a medium at a concentration of preferably 0.5 g / L or more, more preferably 1.0 g / L or more. The bacterium capable of producing a target substance may be capable of producing only one type of target substance, or may be capable of producing two or more types of target substances.

[0043] The target substance is not particularly limited as long as it can be produced by culturing a gram-positive bacterium. Examples of the target substance include ingredients that are used by being added to foods. Specific examples of the target substance include L-amino acids, nucleic acids, and organic acids.

[0044] Examples of L-amino acids include basic amino acids such as L-lysine, L-ornithine, L-arginine, L-histidine, and L-citrulline, aliphatic amino acids such as L-isoleucine, L-alanine, L-valine, L-leucine, and glycine, hydroxymonoaminocarboxylic acid amino acids such as L-threonine and L-serine, cyclic amino acids such as L-proline, aromatic amino acids such as L-phenylalanine, L-tyrosine, and L-tryptophan, sulfur-containing amino acids such as L-cysteine, L-cystine, and L-methionine, acidic amino acids such as L-glutamic acid and L-aspartic acid, and amino acids having an amide group in the side chain such as L-glutamine and L-asparagine. Examples of L-amino acids include, in particular, L-glutamic acid.

[0045] Nucleic acids include purine-based substances. Purine-based substances include purine nucleosides and purine nucleotides. Purine nucleosides include inosine, guanosine, xanthosine, and adenosine. Purine nucleotides include 5'-phosphate esters of purine nucleosides. 5'-phosphate esters of purine nucleosides include inosinic acid (inosine-5'-phosphate; IMP), guanylic acid (guanosine-5'-phosphate; GMP), xanthylic acid (xanthosine-5'-phosphate; XMP), and adenylic acid (adenosine-5'-phosphate; AMP). Purine-based substances particularly include inosine and guanosine. Purine-based substances, more particularly, include inosine.

[0046] Examples of organic acids include carboxylic acids. Examples of carboxylic acids include monocarboxylic acids and dicarboxylic acids. Examples of monocarboxylic acids include monocarboxylic acids having 3 to 8 carbon atoms (C3-C8 monocarboxylic acids). Specific examples of monocarboxylic acids include pyruvic acid. Examples of dicarboxylic acids include dicarboxylic acids having 3 to 8 carbon atoms (C3-C8 dicarboxylic acids). Specific examples of dicarboxylic acids include α-ketoglutaric acid (α-KG; also known as 2-oxoglutaric acid), malic acid, fumaric acid, succinic acid, itaconic acid, malonic acid, adipic acid, glutaric acid, pimelic acid, and suberic acid.

[0047] When a target substance can form a salt, the target substance may be produced and / or used as a free form, as a salt, or as a combination thereof. That is, unless otherwise specified, the term "target substance" may refer to the target substance in free form, its salt, or a combination thereof. Furthermore, when a target substance can form a hydrate, the target substance may be produced and / or used as a non-hydrate, as a hydrate, or as a combination thereof. That is, the term "target substance" (e.g., "target substance in free form" or "salt of target substance") may encompass both non-hydrate and hydrate forms unless otherwise specified. The target substance may be in any form, such as an ion, when used. In addition, when the target substance forms a salt or hydrate, the amount of the target substance (e.g., content (concentration) or amount used) is calculated based on the mass of the salt or hydrate converted to an equimolar mass of the free form.

[0048] The salt can be appropriately selected depending on various conditions such as the use of the target substance. For example, when the target substance is used for oral ingestion, an orally ingestible salt can be selected. 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. Salts of 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. Examples of salts of L-glutamic acid include sodium L-glutamate (e.g., monosodium L-glutamate; MSG) and ammonium L-glutamate (e.g., monoammonium L-glutamate). One type of salt may be used, or two or more types of salts may be used in combination.

[0049] The Gram-positive bacterium may be one that inherently has the ability to produce a target substance, or may be one that has been modified to have the ability to produce a target substance. Gram-positive bacteria capable of producing a target substance can be obtained, for example, by imparting the ability to produce the target substance to the Gram-positive bacterium described above, or by enhancing the ability of the Gram-positive bacterium described above to produce the target substance.

[0050] The method for imparting or enhancing the ability to produce a target substance is not particularly limited. For example, known methods can be used to impart or enhance the ability to produce a target substance. The ability to produce a target substance can be imparted or enhanced, for example, by mutation or genetic engineering. Methods for imparting or enhancing the ability to produce an L-amino acid (e.g., L-glutamic acid) are disclosed, for example, in WO2006 / 070944, WO2015 / 060391, and WO2018 / 030507. Methods for imparting or enhancing the ability to produce a nucleic acid are disclosed, for example, in WO2015 / 060391. Furthermore, for example, the ability to produce L-glutamic acid can be imparted or enhanced by the procedures described in the Examples.

[0051] Bacteria capable of producing L-glutamic acid (also referred to as "L-glutamic acid-producing bacteria") include bacteria having a "specific mutation."

[0052] "Specific mutations" include those shown in Table 1. The mutations shown in Table 1 consist of 135 mutations, A-1 to A-135, and 92 mutations, B-1 to B-92. Mutations A-1 to A-135 are also referred to as "group A mutations." Mutations B-1 to B-92 are also referred to as "group B mutations."

[0053]

[0054]

[0055]

[0056]

[0057]

[0058] The "specific mutation" may be one or more mutations selected from the mutations shown in Table 1. That is, the L-glutamic acid-producing bacterium may have one or more mutations selected from the mutations shown in Table 1.

[0059] The L-glutamic acid producing bacterium may have, for example, one or more mutations selected from the mutations of Group A. The L-glutamic acid producing bacterium may have, for example, one or more mutations selected from the mutations of Group B. The L-glutamic acid producing bacterium may have, for example, one or more mutations selected from the mutations of Group A and one or more mutations selected from the mutations of Group B. The L-glutamic acid producing bacterium may have, for example, one or more mutations selected from the mutations of Group A, and may further have one or more mutations selected from the mutations of Group B. The L-glutamic acid producing bacterium may have, for example, one or more mutations selected from the mutations of Group B, and may further have one or more mutations selected from the mutations of Group A. In other words, the "specific mutation" may be, for example, one or more mutations selected from the mutations of Group A, one or more mutations selected from the mutations of Group B, or a combination of one or more mutations selected from the mutations of Group A and one or more mutations selected from the mutations of Group B.

[0060] The number of mutations selected from Group A mutations possessed by L-glutamic acid-producing bacteria may be, for example, 1 or more, 5 or more, 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, 100 or more, 110 or more, 120 or more, or 130 or more; or 135 or less, 130 or less, 120 or less, 110 or less, 100 or less, 90 or less, 80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 30 or less, 20 or less, 10 or less, or 5 or less, or any compatible combination thereof. The number of mutations selected from the Group A mutations possessed by the L-glutamic acid producing bacterium may be, for example, 1 to 5, 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 110, 110 to 120, 120 to 130, or 130 to 135. The number of mutations selected from the Group A mutations possessed by the L-glutamic acid producing bacterium may be, in particular, 1 or more, 50 or more, 100 or more, 120 or more, 130 or more, or 135.

[0061] The number of mutations selected from Group B mutations possessed by L-glutamic acid-producing bacteria may be, for example, 1 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, or 90 or more, or 92 or less, 90 or less, 80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 30 or less, 20 or less, 10 or less, or 5 or less, or any compatible combination thereof. Specific examples of the number of mutations selected from Group B mutations possessed by L-glutamic acid-producing bacteria may be, for example, 1 to 5, 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, or 90 to 92. The number of mutations selected from group B mutations contained in the L-glutamic acid producing bacterium may be, in particular, 1 or more, 30 or more, 60 or more, 70 or more, 80 or more, or 92.

[0062] The L-glutamic acid producing bacterium may have, for example, 50 or more mutations selected from the mutations of Group A and 30 or more mutations selected from Group B. The L-glutamic acid producing bacterium may have, for example, 100 or more mutations selected from the mutations of Group A and 60 or more mutations selected from Group B. The L-glutamic acid producing bacterium may have, for example, 120 or more mutations selected from the mutations of Group A and 80 or more mutations selected from Group B. The L-glutamic acid producing bacterium may have, for example, 135 mutations of Group A and 92 mutations of Group B.

[0063] The L-glutamic acid producing bacterium having a "specific mutation" may particularly be a coryneform bacterium. The L-glutamic acid producing bacterium having a "specific mutation" may, more particularly, be a bacterium of the genus Corynebacterium. The L-glutamic acid producing bacterium having a "specific mutation" may, more particularly, be Corynebacterium casei. The L-glutamic acid producing bacterium having a "specific mutation" may, more particularly, be a modified strain derived from Corynebacterium casei JCM 12072.

[0064] A specific example of an L-glutamic acid-producing bacterium having a "specific mutation" is the Corynebacterium casei RUN5-2-96 strain (NITE ABP-03688). The RUN5-2-96 strain is also referred to as the AJ111891 strain. The RUN5-2-96 strain is a modified strain derived from Corynebacterium casei JCM 12072 and contains all 135 mutations of Group A. The RUN5-2-96 strain was originally deposited as an international deposit with the National Institute of Technology and Evaluation (NITE NPMD, Postal Code: 292-0818, Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) on July 7, 2022, and has been assigned the accession number NITE ABP-03688. The RUN5-2-96 strain can be obtained, for example, from NITE NPMD.

[0065] In Table 1, "genomic location" indicates the location of each mutation in the nucleotide sequence registered with NCBI (National Center for Biotechnology Information; https: / / www.ncbi.nlm.nih.gov / ) under the accession number NZ_CP004350.1. Hereinafter, this nucleotide sequence will also be referred to as the "nucleotide sequence of NZ_CP004350.1." The nucleotide sequence of NZ_CP004350.1 is the nucleotide sequence of the genome of Corynebacterium casei JCM 12072 (LMG S-19264) and has been made publicly available with annotations. The nucleotide sequence of NZ_CP004350.1 and annotations of each genomic location can be obtained, for example, from NCBI.

[0066] Each mutation shown in Table 1 should be interpreted as a mutation at a position in the genome of each bacterium corresponding to the position of the mutation shown in Table 1. For example, mutation A-1 should be interpreted as a mutation at a position corresponding to position 78,486 in the nucleotide sequence of NZ_CP004350.1 in the genome of each bacterium. The positions of each mutation shown in Table 1 are described for convenience in identifying each mutation, and do not necessarily indicate the absolute position in the genome of each bacterium. In other words, the positions of each mutation shown in Table 1 indicate relative positions based on the nucleotide sequence of NZ_CP004350.1, and the absolute positions may vary due to deletions or insertions of nucleic acid residues, etc. For example, if one nucleic acid residue is deleted or inserted at a position 5'-terminal of position X (X is a positive integer) in the nucleotide sequence of NZ_CP004350.1, the original position X becomes position X-1 or position X+1, respectively, and the mutation at the original position X is considered to be a "mutation at a position corresponding to position X in the nucleotide sequence of NZ_CP004350.1." Furthermore, the pre-mutation bases shown in Table 1 are described for convenience in identifying each mutation and do not necessarily need to be conserved in the bacterial genome before modification. In other words, if the bacterial genome before modification does not have the nucleotide sequence of NZ_CP004350.1, the pre-mutation bases shown in Table 1 may not be conserved. In other words, "introducing a mutation into a bacterium" for each mutation shown in Table 1 means modifying the base at the position of each mutation shown in Table 1 in the bacterial genome before modification (this is any base other than the mutated base) to the mutated base shown in Table 1. For example, "introducing mutation A-1 into a bacterium" means modifying the base at the position corresponding to position 78,486 of the base sequence of NZ_CP004350.1 in the genome of the bacterium before modification (which is C, G, or A) to T.

[0067] The positions in the genome of each bacterium that correspond to the positions of each mutation shown in Table 1 can be determined by aligning the nucleotide sequence of the genome of each bacterium with the nucleotide sequence of NZ_CP004350.1. Alignment can be performed, for example, using known genetic analysis software. Examples of genetic analysis software include DNASIS manufactured by Hitachi Solutions and GENETYX manufactured by Genetyx (Elizabeth C. Tyler et al., Computers and Biomedical Research, 24(1), 72-96, 1991; Barton GJ et al., Journal of molecular biology, 198(2), 327-37, 1987).

[0068] With respect to each mutation shown in Table 1, "the bacterium has a mutation" means that the base at the position of the mutation in the genome of the bacterium is the mutated base shown in Table 1, but does not necessarily mean that the bacterium was obtained by introducing the mutation. In other words, with respect to each mutation shown in Table 1, "the bacterium having a mutation" may be a bacterium in which the base at the position of the mutation originally is the mutated base shown in Table 1, or a bacterium obtained by modifying a bacterium in which the base at the position of the mutation originally is not the mutated base shown in Table 1. For example, "the bacterium has mutation A-1" means that the base at the position of mutation A-1 in the genome of the bacterium (the position corresponding to position 78,486 in the base sequence of NZ_CP004350.1) is T, but does not necessarily mean that the bacterium was obtained by introducing mutation A-1. In other words, for example, a "bacterium having mutation A-1" may be a bacterium that originally has a T nucleotide at the position of mutation A-1 in its genome (the position corresponding to position 78,486 in the nucleotide sequence of NZ_CP004350.1), or a bacterium obtained by modifying a bacterium that originally does not have a T nucleotide at the position of mutation A-1 in its genome. L-glutamic acid-producing bacteria having a "specific mutation" may, in particular, be one obtained by introducing part or all of the "specific mutation."

[0069] L-glutamic acid-producing bacteria having a "specific mutation" can be obtained, for example, by introducing the "specific mutation" into bacteria that do not have the "specific mutation." Furthermore, L-glutamic acid-producing bacteria having a "specific mutation" can be obtained, for example, by introducing the remainder of the "specific mutation" into bacteria that have a part of the "specific mutation."

[0070] Mutations can be introduced by known techniques, for example. For example, a desired mutation can be introduced at a desired position in the genome by site-directed mutagenesis. Examples of site-directed mutagenesis include PCR-based methods (Higuchi, R., 61, in PCR technology, Erlich, H.A. Eds., Stockton Press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)) and phage-based methods (Kramer, W. and Frits, H.J., Meth. in Enzymol., 154, 350 (1987); Kunkel, T.A. et al., Meth. in Enzymol., 154, 367 (1987)).

[0071] Each mutation shown in Table 1 may be a mutation that improves the L-glutamic acid-producing ability of a bacterium, particularly a mutation that improves the L-glutamic acid-producing ability of a bacterium compared to when the position of each mutation is the base before the mutation shown in Table 1.

[0072] Each mutation shown in Table 1 may be, for example, a mutation in a gene (here, a coding region of a gene), a mutation in an expression regulatory region of a gene such as a promoter, or a mutation in an intergenic region. The region (e.g., gene, gene expression regulatory region, or intergenic region) in which each mutation shown in Table 1 occurs can be confirmed, for example, by referring to the position of the mutation and the annotation in the nucleotide sequence of NZ_CP004350.1.

[0073] When each mutation shown in Table 1 is a mutation in a gene, the mutation may, for example, alter (e.g., increase or decrease) the expression of the gene. When each mutation shown in Table 1 is a mutation in a gene, the mutation may, for example, alter (e.g., increase or decrease) the activity of a protein encoded by the gene.

[0074] When each mutation shown in Table 1 is a mutation in an expression regulatory region of a gene, the mutation may, for example, change (e.g., increase or decrease) the expression of the gene.

[0075] Altered (eg, increased or decreased) expression of a gene may, for example, result in altered (eg, increased or decreased) activity of a protein encoded by the gene.

[0076] An L-glutamic acid producing bacterium having a "specific mutation" may or may not have modifications other than the "specific mutation" as long as it has the ability to produce L-glutamic acid. Modifications other than the "specific mutation" include known modifications that confer or enhance L-glutamic acid producing ability. Modifications other than the "specific mutation" also include mutations not selected as "specific mutations" among those shown in Table 1. An L-glutamic acid producing bacterium having a "specific mutation" may have the ability to produce L-glutamic acid depending on the "specific mutation", or may have the ability to produce L-glutamic acid depending on a combination of the "specific mutation" and other modifications, for example.

[0077] An L-glutamic acid-producing bacterium having a "specific mutation" may have an L-glutamic acid-producing ability significantly higher than that of Corynebacterium casei JCM 12072, for example. "An L-glutamic acid-producing ability significantly higher than that of Corynebacterium casei JCM 12072" may mean, for example, the ability to produce and accumulate L-glutamic acid in a medium in an amount at least two-fold, three-fold, five-fold, or seven-fold higher than that of JCM 12072 when cultured under appropriate culture conditions. Furthermore, an L-glutamic acid-producing bacterium having a "specific mutation" may have an L-glutamic acid-producing ability equivalent to or greater than that of Corynebacterium casei RUN5-2-96 strain (NITE ABP-03688), for example. The term "L-glutamic acid producing ability equivalent to or greater than that of Corynebacterium casei strain RUN5-2-96 (NITE ABP-03688)" may refer to the ability to produce and accumulate L-glutamic acid in a medium at an amount equal to or greater than 80%, 90%, 95%, or 100% of that of RUN5-2-96 when cultured under appropriate culture conditions. Suitable culture conditions include the culture conditions for measuring the amount of L-glutamic acid produced described in Example (1-2) below (i.e., shaking culture at 30°C for 48 hours in 500 μL of evaluation medium (Table 4) placed in a 96-deep-well plate).

[0078] The L-glutamic acid-producing bacterium having a "specific mutation" may have a genomic nucleotide sequence that is 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.7% or more, 99.9% or more, 99.95% or more, 99.97% or more, or 99.99% or more identical to that of Corynebacterium casei JCM 12072 or Corynebacterium casei RUN5-2-96 strain (NITE ABP-03688). "Identity" between nucleotide sequences refers to the identity between the nucleotide sequences calculated using blastn with default scoring parameters (Match / Mismatch Scores = 1, -2; Gap Costs = Linear).

[0079] The active ingredient may be a commercially available product or may be obtained by appropriate production. There is no particular limitation on the method for producing the active ingredient.

[0080] The active ingredient can be produced, for example, by culturing Gram-positive bacteria in a medium. That is, the method for producing the active ingredient may include, for example, a step of culturing Gram-positive bacteria in a medium. This step is also referred to as a "culturing step." Specifically, the culturing step may be a step of culturing Gram-positive bacteria in a medium to obtain a culture.

[0081] The medium used is not particularly limited as long as it allows the growth of Gram-positive bacteria. For example, a conventional medium used for culturing bacteria such as coryneform bacteria can be used. The medium may contain medium components such as a carbon source, a nitrogen source, a phosphate source, a sulfur source, and various other organic and inorganic components as needed. The types and concentrations of medium components may be appropriately determined depending on various conditions, such as the type of Gram-positive bacteria used. For specific medium compositions, reference can be made to the medium compositions described in previous reports on bacterial production of target substances (e.g., WO2015 / 060391, WO2018 / 030507, WO2015 / 060391, etc.).

[0082] The carbon source is not particularly limited as long as it can be assimilated by the Gram-positive bacterium. Specific examples of carbon sources include sugars such as glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, blackstrap molasses, starch hydrolysates, and biomass hydrolysates; organic acids such as acetic acid, citric acid, succinic acid, and gluconic acid; alcohols such as ethanol, glycerol, and crude glycerol; and fatty acids. One type of carbon source may be used, or two or more types of carbon sources may be used in combination.

[0083] 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, soy sauce, and pea sauce); ammonia; and urea. Ammonia gas or aqueous ammonia, which is used for pH adjustment, may also be used as a nitrogen source. A single nitrogen source may be used, or two or more nitrogen sources may be used in combination.

[0084] 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.

[0085] 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.

[0086] 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, and vitamin B12; 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, such as liquid, paste, solid, powder, emulsion, or wax. Examples of osmotic pressure regulators for the medium include salts such as sodium chloride and potassium chloride, and polysaccharides that cannot be assimilated by microorganisms (such as sorbitol and dextrin). Examples of osmotic pressure compensation substances include potassium ions, betaine (glycine betaine), glutamic acid, and trehalose. These and other various organic components may be used alone or in combination of two or more.

[0087] Examples of medium components include food raw materials. That is, the medium may contain the food raw materials. Cultivating gram-positive bacteria in a medium containing the food raw materials is also referred to as "fermenting the food raw materials with gram-positive bacteria." That is, the active ingredient may be, for example, a fermentation product of the food raw materials with gram-positive bacteria. The food raw materials may be used as medium components alone or in appropriate combination with other medium components.

[0088] Food ingredients used as medium components (i.e., food ingredients used to culture Gram-positive bacteria) include the food ingredients described below. The food ingredients used as medium components can be selected independently of the food ingredients used in the method of the present invention. The food ingredients used as medium components may or may not be the same as the food ingredients used in the method of the present invention. The food ingredients used as medium components may be one type of ingredient or a combination of two or more types of ingredients.

[0089] Specific examples of food raw materials used as medium components include Solanaceae plants. Solanaceae plants include plants of the genus Solanum (Solanum) and Capsicum (Capsicum). Solanaceae plants include tomatoes and eggplants. Capsicum plants include bell peppers, paprika, shishito peppers, and chili peppers. Bell peppers include green and red bell peppers. Solanaceae plants particularly include tomatoes. As Solanaceae plants, one type of plant may be used, or two or more types of plants may be used in combination.

[0090] For example, fresh solanaceous plants (specifically, fresh edible parts such as fruits) may be used for cultivation either directly or after appropriate processing. Examples of processing include cutting, crushing, straining, squeezing, fractionating, diluting, concentrating, drying, and heating. These processes may be performed alone or in appropriate combinations. Processing may involve, for example, removing the skin and / or seeds. Processed products of solanaceous plants include juice, puree, and paste of solanaceous plants. That is, for example, processed tomato products include tomato juice, tomato puree, and tomato paste. "Juice" of solanaceous plants, such as tomato juice, may refer to, for example, a product obtained by crushing an edible part such as a fruit and squeezing or straining the juice, and having a salt-free soluble solids content of less than 8% (w / w) (e.g., 4.5% (w / w) or more and less than 24% (w / w)). A "puree" of a solanaceous plant, such as tomato puree, may refer to, for example, a concentrate of solanaceous plant juice having a salt-free soluble solids content of 8% (w / w) or more and less than 24% (w / w). A "paste" of a solanaceous plant, such as tomato paste, may refer to, for example, a concentrate of solanaceous plant juice having a salt-free soluble solids content of 24% (w / w) or more. Processed solanaceous plant products may or may not contain additives such as sodium chloride. Processed solanaceous plant products may also be concentrated and reconstituted to have the salt-free soluble solids content exemplified above.

[0091] The culture conditions are not particularly limited as long as they allow the growth of Gram-positive bacteria. The culture can be carried out under normal conditions used for culturing bacteria such as coryneform bacteria. The culture conditions may be appropriately set depending on various conditions, such as the type of Gram-positive bacteria used. Specific culture conditions can be found in, for example, previous reports on the production of target substances by bacteria (WO2015 / 060391, WO2018 / 030507, WO2015 / 060391, etc.).

[0092] Cultivation may be carried out, for example, using a liquid medium under aerobic or microaerobic conditions. "Aerobic conditions" may refer to conditions in which the dissolved oxygen concentration in the medium is 0.33 ppm or higher, or 1.5 ppm or higher. "Microaerobic conditions" may refer to conditions in which the dissolved oxygen concentration in the medium is 0.35 ppm or lower. The dissolved oxygen concentration in the medium under microaerobic conditions may be 0.30 ppm or lower, 0.25 ppm or lower, 0.20 ppm or lower, 0.15 ppm or lower, 0.10 ppm or lower, or 0.05 ppm or lower. Cultivation may be carried out, for example, by aeration culture or shaking culture. The pH of the medium may be, for example, 3 to 10, or 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, and magnesium hydroxide. The culture temperature may be, for example, 20 to 45°C, or 25 to 37°C. The culture period may be, for example, 10 to 120 hours. The culture may be continued, for example, until the carbon source in the medium is consumed or until the activity of the Gram-positive bacteria is lost.

[0093] By culturing Gram-positive bacteria under such conditions, a culture of Gram-positive bacteria (specifically, a culture containing Gram-positive bacterial cells) can be obtained.

[0094] The bacterial cells may be used as an active ingredient while still contained in the culture (specifically, the medium), or may be recovered from the culture (specifically, the medium) and used as an active ingredient. That is, the method for producing an active ingredient may include, for example, a step of recovering the bacterial cells from the culture (specifically, the medium). The recovery of the bacterial cells can be carried out by, for example, a known method. Such methods include natural sedimentation, centrifugation, and filtration. The bacterial cells may also be subjected to an appropriate treatment before being used as an active ingredient. That is, the method for producing an active ingredient may include, for example, a step of subjecting the bacterial cells to treatment. Examples of the treatment include concentration, drying, and heating. Concentration, drying, and heating can all be carried out by, for example, a known method. Known drying methods include spray drying and freeze drying. The heating temperature may be, for example, 60°C or higher, 70°C or higher, 80°C or higher, 90°C or higher, 100°C or higher, 110°C or higher, or 120°C or higher. That is, examples of the active ingredient (specifically, gram-positive bacterial cells) include a culture of gram-positive bacteria, cells recovered from the culture, and processed products thereof. That is, the cells may be used as an active ingredient in the form of, for example, a culture of gram-positive bacteria, cells recovered from the culture, processed products thereof, or a combination thereof. In other words, examples of the active ingredient (specifically, gram-positive bacterial cells) include cells contained in a culture of gram-positive bacteria, cells recovered from the culture, and cells contained in processed products thereof. That is, the cells may be used as an active ingredient in the form of, for example, cells contained in a culture of gram-positive bacteria, cells recovered from the culture, cells contained in processed products thereof, or a combination thereof. Examples of processed products include cells (for example, cells contained in a culture or cells recovered from a culture) that have been subjected to treatment. The cells may be live cells, dead cells, or a combination thereof. The gram-positive bacterial body contains the gram-positive bacterial cell wall.

[0095] Furthermore, bacterial cell fragments can be prepared from the bacterial cells. For example, disruption of the bacterial cells can produce a disrupted bacterial cell material containing bacterial cell fragments. That is, the method for producing an active ingredient may include, for example, a step of disrupting the bacterial cells. Disruption of the bacterial cells can be carried out, for example, by known techniques. Such techniques include ultrasonic disruption. The bacterial cell fragments may be used as the active ingredient as they are contained in the disrupted bacterial cell material, or may be recovered from the disrupted bacterial cell material and used as the active ingredient. That is, the method for producing an active ingredient may include, for example, a step of recovering the bacterial cell fragments from the disrupted bacterial cell material. Recovery of the bacterial cell fragments can be carried out, for example, by known techniques. Such techniques include natural sedimentation, centrifugation, and filtration. Furthermore, the bacterial cell fragments may be subjected to appropriate treatment before use as the active ingredient. That is, the method for producing an active ingredient may include, for example, a step of subjecting the bacterial cell fragments to treatment. Examples of such treatments include concentration, drying, and heating. Concentration, drying, and heating can all be carried out, for example, by known techniques. Known drying techniques include spray drying and freeze drying. The heating temperature may be, for example, 60°C or higher, 70°C or higher, 80°C or higher, 90°C or higher, 100°C or higher, 110°C or higher, or 120°C or higher. Examples of the active ingredient (specifically, gram-positive bacterial cell fragments) include disrupted gram-positive bacterial cells, cell fragments recovered from the disrupted cells, and processed products thereof. Examples of the cell fragments may be disrupted gram-positive bacterial cells, cell fragments recovered from the disrupted cells, processed products thereof, or a combination thereof. In other words, examples of the active ingredient (specifically, gram-positive bacterial cell fragments) include cell fragments contained in disrupted gram-positive bacterial cells, cell fragments recovered from the disrupted cells, and cell fragments contained in processed products thereof. That is, the bacterial cell fragments may be used as an active ingredient in the form of, for example, bacterial cell fragments contained in a disrupted product of Gram-positive bacterial cells, bacterial cell fragments recovered from the disrupted product, bacterial cell fragments contained in a processed product thereof, or a combination thereof.Examples of treated materials include bacterial cell fragments (e.g., bacterial cell fragments contained in a disrupted material or bacterial cell fragments recovered from the disrupted material).Fragments of Gram-positive bacteria (e.g., disrupted Gram-positive bacteria, bacterial cell fragments recovered from the disrupted material, or processed products thereof) contain cell walls of Gram-positive bacteria.

[0096] As described above, the gram-positive bacterial cells or fragments thereof may be treated by heating or the like. That is, the active ingredient may be treated by heating or the like. That is, the method for producing an active ingredient may include, for example, a step of subjecting the active ingredient, such as gram-positive bacterial cells or fragments thereof, to a treatment such as heating or the like. The active ingredient may particularly be heat-treated. Unless otherwise specified, the phrase "subjecting the active ingredient to treatment" encompasses any form of active ingredient to treatment, in other words, the active ingredient itself or any fraction containing it to treatment. For example, the phrase "heat-treating gram-positive bacterial cells" encompasses, unless otherwise specified, the phrase "heat-treating gram-positive bacterial cells" encompasses any fraction containing gram-positive bacterial cells, such as a culture of gram-positive bacteria, cells recovered from the culture, or a processed product thereof, that is, heat-treating the culture of gram-positive bacteria. Furthermore, the method for producing an active ingredient may include, for example, a step of subjecting a culture of gram-positive bacteria to a treatment such as heating or the like. Unless otherwise specified, the phrase "subjecting a culture to treatment" includes any form of culture to treatment, in other words, the case where the culture itself or any fraction prepared therefrom is subjected to treatment. By subjecting the culture to treatment such as heating, an active ingredient that has been treated such as heated may be obtained.

[0097] When the Gram-positive bacterium has the ability to produce a target substance, the target substance may be produced by culturing the Gram-positive bacterium, i.e., the culture of the Gram-positive bacterium may contain the target substance.

[0098] The target substance may be appropriately recovered from the culture. The target substance may be recovered as a suitable fraction containing the target substance. Such a fraction may include a culture supernatant. Alternatively, the target substance may be further separated and purified from the fraction. For example, Gram-positive bacterial cells may be separated from the culture to obtain a culture supernatant, and the target substance may be recovered from the culture supernatant. Note that "recovery of the target substance" may also include removal of impurities from a fraction containing the target substance. The target substance can be recovered, for example, by known methods used for separating and purifying compounds. Examples of such methods include ion exchange resin methods, membrane treatment methods, precipitation methods, extraction methods, distillation methods, crystallization methods, and activated carbon treatment. The method for recovering the target substance can be appropriately selected depending on various conditions, such as the type of target substance. These methods can be used alone or in combination. The recovered target substance may contain other components in addition to the target substance, such as Gram-positive bacterial cells, medium components, water, and metabolic by-products of Gram-positive bacteria. The purity of the recovered target substance may be, for example, 30% (w / w) or more, 50% (w / w) or more, 70% (w / w) or more, 80% (w / w) or more, 90% (w / w) or more, or 95% (w / w) or more. The use of the recovered target substance is not particularly limited. The recovered target substance may be used separately from the active ingredient or may be used in combination with the active ingredient. The target substance may be prepared, for example, as a fermented seasoning containing the target substance and used in combination with the active ingredient.

[0099] Alternatively, a fraction containing both the target substance and the cell wall of the Gram-positive bacterium may be used as an active ingredient. For example, a culture containing both the target substance and the cells of the Gram-positive bacterium may be used as an active ingredient either directly or after appropriate drying.

[0100] The active ingredient may be used in combination with, for example, other ingredients (i.e., ingredients other than the active ingredient). Examples of other ingredients include the target substance and a culture of Gram-positive bacteria. By using the active ingredient in combination with other ingredients, the flavor-improving effect may be enhanced compared to when the active ingredient is used alone. Specifically, for example, by using the active ingredient in combination with L-glutamic acid and / or a culture of Gram-positive bacteria, the flavor-improving effect may be enhanced compared to when the active ingredient is used alone. Furthermore, for example, by using the active ingredient in combination with L-glutamic acid, the umami of a food may be enhanced compared to when the active ingredient is used alone. Unless otherwise specified, the phrase "using an active ingredient in combination with L-glutamic acid" includes cases where the active ingredient is used in combination with any form of L-glutamic acid, in other words, cases where the active ingredient is used in combination with L-glutamic acid itself or any fraction containing it (e.g., a culture containing L-glutamic acid).

[0101] The target substance is as described above. The target substance may be a commercially available product or may be obtained by appropriate production. The target substance can be produced, for example, by chemical synthesis, enzymatic reaction, fermentation, extraction, or a combination thereof. Specifically, the target substance can be produced, for example, by a fermentation method using a microorganism capable of producing the target substance. The microorganism capable of producing the target substance may or may not be the same as the Gram-positive bacterium from which the active ingredient is derived. For example, the target substance may be produced during the production of the active ingredient, or may be produced separately from the active ingredient. The target substance may or may not be purified to a desired degree. That is, the target substance may be a purified product, or a material containing the target substance. A "material containing the target substance" refers to a material containing the target substance at a content of 0.1% (w / w) or more. Specific examples of materials containing the target substance include fermentation products such as cultures, bacterial cells, and culture supernatants obtained by culturing microorganisms capable of producing the target substance, as well as processed products thereof. Examples of processed products include those obtained by subjecting materials such as the fermentation products described above to treatments such as heating, concentration, dilution, drying, fractionation, extraction, and purification. The content of the target substance in a material containing the target substance may be, for example, 1% (w / w) or more, 3% (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. The phrase "combined use of an active ingredient and a target substance" does not necessarily mean a combination of an active ingredient and a target substance obtained separately, but also includes a combination of an active ingredient and a target substance obtained together. Examples of a combination of an active ingredient and a target substance include a culture containing both the target substance and Gram-positive bacterial cells, used as an active ingredient, either directly or after appropriate drying.

[0102] The culture of Gram-positive bacteria as the other component (i.e., a component other than the active ingredient) can be obtained by culturing Gram-positive bacteria. The culturing of Gram-positive bacteria is as described above. The Gram-positive bacteria from which the culture as the other component is derived may or may not be the same as the Gram-positive bacteria from which the active ingredient is derived. The culture as the other component may, for example, be produced together with the production of the active ingredient, or may be produced separately from the active ingredient. The culture as the other component may be used as an additional component, for example, as is, or after fractionation (separation of bacterial cells or separation of target substances, etc.), concentration, drying, heating, etc. Fractionation may result in the separation (i.e., removal from the culture) of, for example, 90% or more, 95% or more, 97% or more, or 99% or more of the total number of bacterial cells. The culture as the other component may or may not contain Gram-positive bacterial cells. When the culture as the other component contains Gram-positive bacterial cells, the Gram-positive bacterial cells contained in the culture may be considered an active ingredient. The culture as another ingredient may or may not contain a target substance (e.g., L-glutamic acid). Note that "using an active ingredient and a culture of a Gram-positive bacterium in combination" is not limited to the case where an active ingredient and a culture obtained separately are used in combination, but also includes the case where an active ingredient and a culture obtained together are used. An example of the case where an active ingredient and a culture obtained together are used is when a culture containing Gram-positive bacterial cells is used as an active ingredient either as is or after being appropriately dried.

[0103] <2> Composition of the Present Invention The composition of the present invention is a composition containing an active ingredient.

[0104] That is, the composition of the present invention is a composition containing the following component (A): (A) a fraction containing the cell wall of a gram-positive bacterium.

[0105] By using the composition of the present invention, the flavor of food can be improved, i.e., a flavor improving effect can be obtained. Thus, the composition of the present invention may be used to improve the flavor of food. That is, the composition of the present invention may be, for example, a composition for improving the flavor of food. The improvement of flavor may be, for example, an enhancement of the spiciness and / or the impartation of body. The improvement of flavor may, in particular, be an enhancement of the pungency of spices.

[0106] Furthermore, by using the composition of the present invention, it is possible to produce a food product with improved flavor. Thus, the composition of the present invention may be used in the production of a food product (specifically, the production of a food product with improved flavor). That is, the composition of the present invention may be, for example, a composition for use in the production of a food product (specifically, the production of a food product with improved flavor).

[0107] 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, or a seasoning for use in food production (specifically, production of food with improved flavor).

[0108] The composition of the present invention may or may not have an improved flavor by itself. For example, when the composition of the present invention contains a spice, the composition of the present invention may have an enhanced spiciness (specifically, an enhanced spiciness compared to when the composition does not contain the active ingredient).

[0109] The compositions of the present invention may be used to improve flavor or prepare food products in the manner described in the methods of the present invention below.

[0110] The composition of the present invention may consist of an active ingredient, or may contain ingredients other than the active ingredient. In one embodiment, the composition of the present invention may exclude a composition consisting of an active ingredient. As the ingredients other than the active ingredient, one kind of ingredient may be used, or two or more kinds of ingredients may be used in combination.

[0111] The active ingredient contained in the composition of the present invention may be one produced by the above-mentioned method for producing an active ingredient. Therefore, the method for producing the composition of the present invention may include a step of producing the active ingredient by the above-mentioned method for producing an active ingredient. Specifically, the step of producing the active ingredient in the method for producing the composition of the present invention may be a step of culturing a gram-positive bacterium in a medium to obtain a culture.

[0112] The ingredients other than the active ingredient are not particularly limited as long as they do not lose the flavor improving effect (i.e., the flavor improving effect of the active ingredient is obtained). The ingredients other than the active ingredient can be appropriately selected depending on various conditions such as the type of food. Examples of ingredients other than the active ingredient include ingredients that are blended into foods or pharmaceuticals.

[0113] Specific examples of ingredients other than the active ingredient include ingredients effective in food production. Examples of ingredients effective in food production include food ingredients described below. Examples of ingredients other than the active ingredient include a culture of Gram-positive bacteria, a target substance, and a spice. That is, the composition of the present invention may be, for example, a composition containing an active ingredient and a culture of Gram-positive bacteria (e.g., a seasoning), a composition containing an active ingredient and a target substance (e.g., a seasoning), a composition containing an active ingredient and a spice (e.g., a seasoning), a composition containing an active ingredient, a culture of Gram-positive bacteria, and a spice (e.g., a seasoning), a composition containing an active ingredient, a target substance, and a spice (e.g., a seasoning), or a composition containing an active ingredient, a culture of Gram-positive bacteria, a target substance, and a spice (e.g., a seasoning).

[0114] The target substance is as described above. Note that "a composition containing an active ingredient and a target substance" is not limited to a case where the active ingredient and target substance obtained separately are contained in the composition, but also includes a case where the active ingredient and target substance obtained together are contained in the composition. An example of a case where the active ingredient and target substance obtained together are contained in the composition is a case where a culture containing both the target substance and Gram-positive bacterial cells is contained in the composition as is, or after being appropriately dried, etc.

[0115] The gram-positive bacterial culture as an ingredient other than the active ingredient is as described above. Note that "the composition contains an active ingredient and a gram-positive bacterial culture" does not limit to the case where the active ingredient and the culture obtained separately are contained in the composition, but also includes the case where the active ingredient and the culture obtained together are contained in the composition. An example of the case where the active ingredient and the culture obtained together are contained in the composition is when a culture containing gram-positive bacterial cells is contained in the composition as is, or after being appropriately dried, etc.

[0116] "Spices" may refer to the leaves, stems, bark, roots, flowers, buds, seeds, fruits, or peels of plants that are used to impart a special flavor to foods. "Spices" may also include so-called "herbs." Spices include spices from the Lauraceae family (laurel, cinnamon, etc.), spices from the Piperaceae family (black pepper, white pepper, etc.), spices from the Lamiaceae family (thyme, sage, basil, oregano, marjoram, rosemary, mint, etc.), spices from the Umbelliferae family (fennel, caraway, cumin, coriander, parsley, Italian parsley, celery, celery seed, dill, etc.), spices from the Solanaceae family (chili pepper, cayenne pepper, etc.), and spices from the Myristica family. Examples of spices include spices from the family Allium (nutmeg, mace, etc.), spices from the family Allium (garlic powder, onion powder, etc.), spices from the family Myrtaceae (allspice, cloves, etc.), spices from the family Melastomataceae (star anise, etc.), spices from the family Fabaceae (fenugreek, etc.), spices from the family Polygonaceae (polygonum, etc.), spices from the family Brassicaceae (garden cress, wasabi, Japanese mustard, etc.), spices from the family Zingiberaceae (cardamom, turmeric, ginger, etc.), and spices from the family Rutaceae (Japanese pepper, etc.). Spices particularly include spices with a pungent taste, such as spices from the family Piperaceae, spices from the family Solanaceae, spices from the family Cruciferae, and spices from the family Rutaceae. Note that, in accordance with common practice, garlic and onions, which are used in cooking as fresh vegetables in a raw state, may be excluded from the definition of spices in the present invention. In the present invention, one spice may be used, or two or more spices may be used in combination.

[0117] The composition of the present invention can be produced, for example, by appropriately mixing the active ingredient and, optionally, other ingredients.

[0118] 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.

[0119] 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, liquid, etc.

[0120] The content and content ratio of each component (i.e., the active ingredient and optionally other ingredients) in the composition of the present invention are not particularly limited as long 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.

[0121] The content of the active ingredient in the composition of the present invention is more than 0% (w / w) and not more than 100% (w / w). The content of the active ingredient in the composition of the present invention may be, for example, 0.1% (w / w) or more, 0.2% (w / w) or more, 0.5% (w / w) or more, 1% (w / w) or more, 2% (w / w) or more, 5% (w / w) or more, 10% (w / w) or more, or 20% (w / w) or more, or 100% (w / w) or less, less than 100% (w / w), 99.9% (w / w) or less, 90% (w / w) or less, 50% (w / w) or less, 20% (w / w) or less, 10% (w / w) or less, 5% (w / w) or less, 2% (w / w) or less, or 1% (w / w) or less, or any compatible combination thereof, calculated in terms of the dry weight of Gram-positive bacterial cells. Specifically, the content of the active ingredient in the composition of the present invention may be, for example, 0.2 to 99.9% (w / w), 0.2 to 90% (w / w), 0.5 to 50% (w / w), or 1 to 20% (w / w) in terms of the dry weight of Gram-positive bacterial cells. The dry weight of bacterial cells is also referred to as "dry cell weight (DCW)."

[0122] The amount of the active ingredient can be measured as a value converted to the dry weight of Gram-positive bacterial cells, for example, according to the gravimetric method, packed volume method, turbidity method, cell counting method, or indirect measurement method described on pages 57-60 of "Microbiology Basics 7, Microbial Culture Engineering, edited by Hisaji Taguchi and Shiro Nagai, Kyoritsu Shuppan Co., Ltd., 1985." That is, the dry cell weight of Gram-positive bacteria contained in a certain object (e.g., a composition or food) can be measured, for example, by separating and drying the Gram-positive bacterial cells from the object, and then measuring the weight of the resulting dried product. Specifically, the dry cell weight of Gram-positive bacteria contained in a certain object (e.g., a composition or food) can be measured by dispersing the object in water, separating the bacterial cells by solid-liquid separation means such as centrifugation or filtration, optionally washing with water and performing solid-liquid separation means such as centrifugation or filtration once or multiple times, drying the resulting product by drying means such as vacuum drying, and measuring the weight of the dried product. The dry cell weight of the gram-positive bacteria may also be calculated indirectly by converting it from a parameter that reflects the dry cell weight of the gram-positive bacteria. In other words, the "dry cell weight of the gram-positive bacteria" may refer to the value calculated indirectly in this manner. An example of a parameter that reflects the dry cell weight of the gram-positive bacteria is the cell count of the gram-positive bacteria. That is, for example, the correlation between the dry cell weight and the cell count for a selected gram-positive bacterium may be calculated, and the dry cell weight may be calculated from the cell count based on this correlation. Note that when the active ingredient is used in a form other than bacterial cells (for example, in the form of bacterial cell fragments), the dry cell weight of the gram-positive bacteria may be calculated from the dry weight of the cell wall of the gram-positive bacteria.

[0123] When the composition of the present invention contains a spice, the content of the spice in the composition of the present invention may be, for example, 0.1% (w / w) or more, 0.2% (w / w) or more, 0.5% (w / w) or more, 1% (w / w) or more, 2% (w / w) or more, 5% (w / w) or more, 10% (w / w) or more, or 20% (w / w) or more, or 90% (w / w) or less, 50% (w / w) or less, 20% (w / w) or less, 10% (w / w) or less, 5% (w / w) or less, 2% (w / w) or less, or 1% (w / w) or less, or any combination thereof that is consistent therewith. Specifically, the content of the spice in the composition of the present invention may be, for example, 0.2 to 90% (w / w), 0.5 to 50% (w / w), or 1 to 20% (w / w).

[0124] When the composition of the present invention contains a spice, the content of the spice in the composition of the present invention may be, for example, 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, 50 parts by weight or more, or 100 parts by weight or more, or 1,000 parts by weight or less, 500 parts by weight or less, 200 parts by weight or less, 100 parts by weight or less, 50 parts by weight or less, 20 parts by weight or less, 10 parts by weight or less, 5 parts by weight or less, or 2 parts by weight or less, or any compatible combination thereof, relative to 1 part by weight of the active ingredient contained in the composition of the present invention (converted to the dry weight of Gram-positive bacterial cells). The content of the spice in the composition of the present invention may be, for example, 0.2 to 500 parts by weight, 1 to 500 parts by weight, 5 to 500 parts by weight, 50 to 500 parts by weight, 0.2 to 100 parts by weight, 1 to 100 parts by weight, 5 to 100 parts by weight, 50 to 100 parts by weight, 0.2 to 100 parts by weight, 0.5 to 50 parts by weight, or 1 to 20 parts by weight, per part by weight of the active ingredient contained in the composition of the present invention (converted to the dry weight of Gram-positive bacterial cells). The content of the spice in the composition of the present invention may be, in particular, 0.2 to 500 parts by weight, or 50 to 500 parts by weight, per part by weight of the active ingredient contained in the composition of the present invention (converted to the dry weight of Gram-positive bacterial cells).

[0125] When the composition of the present invention contains a target substance (e.g., L-glutamic acid), the content of the target substance (e.g., L-glutamic acid) in the composition of the present invention may be, for example, 0.1% (w / w) or more, 0.2% (w / w) or more, 0.5% (w / w) or more, 1% (w / w) or more, 2% (w / w) or more, 5% (w / w) or more, 10% (w / w) or more, or 20% (w / w) or more, or 90% (w / w) or less, 50% (w / w) or less, 20% (w / w) or less, 10% (w / w) or less, 5% (w / w) or less, 2% (w / w) or less, or 1% (w / w) or less, or any compatible combination thereof. The content of the target substance (e.g., L-glutamic acid) in the composition of the present invention may be, specifically, for example, 0.2 to 90% (w / w), 0.5 to 50% (w / w), 1 to 50% (w / w), 1 to 20% (w / w), 5 to 50% (w / w), 10 to 50% (w / w), or 20 to 50% (w / w).

[0126] When the composition of the present invention contains a target substance (e.g., L-glutamic acid), the content of the target substance (e.g., L-glutamic acid) in the composition of the present invention 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, or 5 parts by weight or more, relative to 1 part by weight of the active ingredient contained in the composition of the present invention (converted to the dry weight of Gram-positive bacterial cells), or may be 50 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, or 1 part by weight or less, or any compatible combination thereof. The content of the target substance (e.g., L-glutamic acid) in the composition of the present invention may be, for example, 0.1 to 20 parts by weight relative to 1 part by weight of the active ingredient contained in the composition of the present invention (converted to the dry weight of Gram-positive bacterial cells).

[0127] When the composition of the present invention contains a culture of Gram-positive bacteria as an ingredient other than the active ingredient, the content of the culture of Gram-positive bacteria in the composition of the present invention may be, for example, 0.01% (w / w) or more, 0.1% (w / w) or more, 1% (w / w) or more, 5% (w / w) or more, or 10% (w / w) or more, converted into the amount of the original culture, and may be 10,000% (w / w) or less, 5,000% (w / w) or less, The concentration may be 1000% (w / w) or less, 500% (w / w) or less, 300% (w / w) or less, 200% (w / w) or less, 150% (w / w) or less, 100% (w / w) or less, 70% (w / w) or less, 50% (w / w) or less, 30% (w / w) or less, 10% (w / w) or less, 5% (w / w) or less, or 1% (w / w) or less, or any combination thereof that is compatible. The "original culture" refers to a culture in which no concentration change, such as concentration or dilution, has occurred after culture, and specifically may be a culture immediately after culture. Note that a content of more than 100% (w / w) means that the culture has been concentrated and contained in the target substance (here, the composition of the present invention). That is, for example, a content of 200% (w / w) means that the culture has been concentrated twice and contained in the target substance.

[0128] When the composition of the present invention contains a culture of Gram-positive bacteria as an ingredient other than the active ingredient, and the Gram-positive bacterial culture contains Gram-positive bacterial cells, the content of the Gram-positive bacterial culture in the composition of the present invention may be set, for example, so that the content of the active ingredient in the composition of the present invention falls within the range exemplified above.

[0129] When the composition of the present invention contains a culture of Gram-positive bacteria as an ingredient other than the active ingredient, and the Gram-positive bacterial culture contains a target substance (e.g., L-glutamic acid), the content of the Gram-positive bacterial culture in the composition of the present invention may be set, for example, so that the content of the target substance (e.g., L-glutamic acid) in the composition of the present invention falls within the range exemplified above.

[0130] 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.

[0131] 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.

[0132] <3> Method of the Present Invention The method of the present invention is a method including a step of utilizing an active ingredient.

[0133] That is, the method of the present invention is a method comprising a step of utilizing the following component (A): (A) a fraction containing cell walls of Gram-positive bacteria.

[0134] The method of the present invention, specifically by utilizing the active ingredient, can improve the flavor of food, i.e., a flavor improving effect can be obtained. Thus, the method of the present invention may be carried out to improve the flavor of food. That is, the method of the present invention may be, for example, a method for improving the flavor of food. This method is also referred to as the "flavor improving method of the present invention." The improvement of flavor may be, for example, an enhancement of spice sensation and / or imparting richness. The improvement of flavor may, in particular, be an enhancement of the pungency of spices.

[0135] Furthermore, the method of the present invention, specifically by utilizing the active ingredient, allows the production of foods with improved flavor. Thus, the method of the present invention may be carried out for the production of foods (specifically, the production of foods with improved flavor). That is, the method of the present invention may be, for example, a method for producing foods (specifically, the production of foods with improved flavor). This method is also referred to as the "food production method of the present invention."

[0136] The active ingredient can be added to food ingredients during food production to improve flavor or be used in food production. That is, the use of the active ingredient can be, for example, adding the active ingredient to food ingredients. That is, the method of the present invention can specifically be, for example, a method for improving food flavor, which includes adding the active ingredient to food ingredients. Furthermore, the method of the present invention can specifically be, for example, a method for producing food (specifically, producing food with improved flavor), which includes adding the active ingredient to food ingredients. "Addition" can also be referred to as "blending."

[0137] 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.

[0138] 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 improved flavor. In other words, the food of the present invention is a food to which an active ingredient has been added.

[0139] 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.

[0140] The type of food is not particularly limited as long as the flavor improvement is desired. Foods include beverages. Foods also include seasonings. Foods may be, for example, liquid or solid. Specific examples of foods include beverages such as milk, soft drinks, alcoholic beverages, and soups; processed meat foods such as ham, sausage, gyoza, shumai, hamburger steak, fried chicken, and pork cutlet; processed seafood foods such as salmon flakes, spicy cod roe, salted cod roe, grilled fish, dried fish, salted fish, fish sausage, kamaboko, boiled fish, tsukudani (simmered fish in soy sauce), and canned goods; sweets such as potato chips, potato snacks, corn snacks, wheat snacks, cinnamon cookies, rice crackers, and arare (crackers); noodle soups such as udon soup, soba soup, somen soup, ramen soup, champon soup, and pasta sauce; cooked rice dishes such as rice balls, pilaf, fried rice, mixed rice, rice porridge, and ochazuke (tea rice with green tea); stews such as curry, stew, chili con carne, feijoata, and mapo tofu. Examples of suitable condiments include dishes; rouxes such as stew roux and curry roux; processed vegetable products such as kimchi and pickles; other processed foods such as bread, noodles, gratin, croquettes, and mashed potatoes; sauces such as Chinese sauce, oyster sauce, cheese sauce, tomato sauce, white sauce, demi-glace sauce, curry sauce, Genoa sauce, chili sauce, and Tabasco sauce; seasoning oils such as chili oil; basic seasonings such as soy sauce and miso; flavor seasonings such as bonito flavor, chicken flavor, pork flavor, and beef flavor; spicy seasonings such as shichimi chili pepper, doubanjiang, and gochujang; menu-specific seasonings (special seasonings tailored to the menu being cooked); and other condiments such as dressings, miso, mayonnaise, tomato ketchup, and consommé. "Soft drinks" may refer to non-alcoholic beverages (drinks with an alcohol concentration of less than 1%), excluding milk and dairy products. Specific examples of soft drinks include water, fruit juice, vegetable juice, tea (chai, cinnamon tea, etc.), coffee drinks (coffee, milk drinks with coffee, etc.), carbonated drinks (ginger ale, lemon carbonated drinks, etc.), and sports drinks.Specific examples of soup include dal soup, tom yum goong, egg soup, seaweed soup, shark fin soup, Chinese-style soup, consommé soup, curry-flavored soup, clear soup, miso soup, and potage soup.Examples of foods include, in particular, foods containing spices (for example, the foods exemplified above that contain spices). Examples of foods also include spices themselves. Foods may be provided in a form that can be consumed as is, or may be provided in a form that requires preparation before or at the time of consumption, such as a concentrated product or a dried product. Furthermore, foods are not limited to general foods, but also include so-called health foods or medical foods, such as nutritional supplements, nutritionally functional foods, and foods for specified health uses. That is, for example, the foods exemplified above may be provided as general foods, or as health foods or medical foods.

[0141] "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 ingredients such as grains, vegetables, meat, seafood, and eggs; seasoning ingredients such as sugars, inorganic salts, organic acids, nucleic acids, amino acids, and protein hydrolysates; dairy products such as milk and cheese; spices; flavorings; oils and fats; and alcohol. Examples of organic acids, nucleic acids, and amino acids include those exemplified as target substances.

[0142] The active ingredient may be added to food ingredients at any stage of the food production process, as long as the flavor-improving effect is achieved. In other words, the "food ingredients" to which the active ingredient is added may be those at any stage of the food production 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. Other ingredients may also be added to the food ingredients as appropriate. In other words, the method of the present invention may further include adding other ingredients to the food ingredients. The other ingredients are not particularly limited, as long as they do not lose their flavor-improving effect (i.e., the flavor-improving effect of the active ingredient is achieved). The other ingredients can be selected appropriately depending on various conditions, such as the type of food. Examples of other ingredients include a culture of gram-positive bacteria, a target substance, and spices. 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, or may be added separately or in any combination. The order in which the ingredients are added to the food ingredients is not particularly limited.

[0143] 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 a flavor-improving effect is obtained. The amount and ratio of each component added in the method of the present invention can be appropriately determined depending on various conditions such as the type of food raw material and the type of food.

[0144] 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).

[0145] The ingested concentration of the active ingredient, converted to the dry weight of gram-positive bacterial cells, is, for example, 0.005% (w / w) or more, 0.007% (w / w) or more, 0.01% (w / w) or more, 0.02% (w / w) or more, 0.03% (w / w) or more, 0.04% (w / w) or more, 0.05% (w / w) or more, 0.07% (w / w) or more, 0.1% (w / w) or more, 0.2% (w / w) or more, 0.3% (w / w) or more. The ingested concentration of the active ingredient may be, for example, 0.005 to 2% (w / w), 0.005 to 1% (w / w), 0.005 to 0.5% (w / w), 0.01 to 2% (w / w), 0.02 to 1% (w / w), or 0.03 to 0.5% (w / w), calculated as the dry weight of the gram-positive bacterial cells.

[0146] 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.

[0147] The food of the present invention may contain a spice in addition to the active ingredient. That is, the food of the present invention may be produced so as to contain a spice. That is, the method of the present invention may further comprise adding a spice to the raw materials of the food. A food containing a spice may be produced, for example, by adding the spice itself, or by adding a spice-containing material, such as a seasoning containing a spice. Furthermore, when the composition of the present invention contains a spice, the spice may be added by adding the composition of the present invention. The addition of the spice can be carried out in the same manner as the addition of the active ingredient. The spice may be added to the raw materials of the food, for example, so that the content of the spice in the food of the present invention falls within a desired range (for example, the content range described below). Furthermore, the raw materials of the food may originally contain the spice.

[0148] When the food of the present invention contains a spice, the content of the spice in the food of the present invention may be, for example, 0.01% (w / w) or more, 0.02% (w / w) or more, 0.03% (w / w) or more, 0.04% (w / w) or more, 0.05% (w / w) or more, 0.07% (w / w) or more, 0.1% (w / w) or more, 0.2% (w / w) or more, 0.3% (w / w) or more, or 0.01% (w / w) or more, as an ingestible concentration. The spice content may be 4% (w / w) or more, or 0.5% (w / w) or more, or 5% (w / w) or less, 2% (w / w) or less, 1% (w / w) or less, 0.7% (w / w) or less, 0.5% (w / w) or less, 0.4% (w / w) or less, 0.3% (w / w) or less, 0.2% (w / w) or less, or 0.1% (w / w) or less, or any combination thereof that is not contradictory. Specific examples of the spice content in the food product of the present invention may be 0.01 to 2% (w / w), 0.02 to 1% (w / w), or 0.03 to 0.5% (w / w) in terms of consumption concentration.

[0149] When the food of the present invention contains a spice, the content of the spice in the food of the present invention may be, for example, 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, 50 parts by weight or more, or 100 parts by weight or more, or 1000 parts by weight or less, 500 parts by weight or less, 200 parts by weight or less, 100 parts by weight or less, 50 parts by weight or less, 20 parts by weight or less, 10 parts by weight or less, 5 parts by weight or less, or 2 parts by weight or less, or any compatible combination thereof, per 1 part by weight of the active ingredient contained in the food of the present invention (converted to the dry weight of gram-positive bacterial cells). Specifically, the content of the spice in the food of the present invention may be, for example, 0.2 to 500 parts by weight, 1 to 500 parts by weight, 5 to 500 parts by weight, 50 to 500 parts by weight, 0.2 to 100 parts by weight, 1 to 100 parts by weight, 5 to 100 parts by weight, 50 to 100 parts by weight, 0.2 to 100 parts by weight, 0.5 to 50 parts by weight, or 1 to 20 parts by weight, per part by weight of the active ingredient contained in the food of the present invention (converted to the dry weight of Gram-positive bacterial cells). The content of the spice in the food of the present invention may be, in particular, 0.2 to 500 parts by weight, or 50 to 500 parts by weight, per part by weight of the active ingredient contained in the food of the present invention (converted to the dry weight of Gram-positive bacterial cells).

[0150] The food of the present invention may contain a target substance in addition to the active ingredient. That is, the food of the present invention may be manufactured to contain the target substance. That is, the method of the present invention may further include adding the target substance to the raw material of the food. The target substance is as described above. A food containing a target substance may be manufactured, for example, by adding the target substance itself, or by adding a material containing the target substance, such as a seasoning containing the target substance. Note that "adding an active ingredient and a target substance" is not limited to the addition of an active ingredient and a target substance obtained separately, but also includes the addition of an active ingredient and a target substance obtained together. Examples of the addition of an active ingredient and a target substance obtained together include the addition of a culture containing both the target substance and Gram-positive bacterial cells, either as is or after appropriate drying. Furthermore, when the composition of the present invention contains the target substance, the target substance may be added by adding the composition of the present invention. The addition of the target substance can be carried out in the same manner as the addition of the active ingredient. The target substance may be added to the raw material of the food, for example, so that the content of the target substance in the food of the present invention falls within a desired range (e.g., the content range described below). Furthermore, the raw materials of the food may originally contain the target substance.

[0151] When the food of the present invention contains a target substance (e.g., L-glutamic acid), the content of the target substance (e.g., L-glutamic acid) in the food of the present invention may be, for example, 0.01% (w / w) or more, 0.02% (w / w) or more, 0.03% (w / w) or more, 0.04% (w / w) or more, 0.05% (w / w) or more, 0.07% (w / w) or more, 0.1% (w / w) or more, 0.2% (w / w) or more, as an ingestible concentration. The content of the target substance (e.g., L-glutamic acid) in the food product of the present invention may be, for example, 0.01 to 2% (w / w), 0.02 to 1% (w / w), or 0.03 to 0.5% (w / w) or more, or 5% (w / w) or less, 2% (w / w) or less, 1% (w / w) or less, 0.7% (w / w) or less, 0.5% (w / w) or less, 0.4% (w / w) or less, 0.3% (w / w) or less, 0.2% (w / w) or less, or 0.1% (w / w) or less, or a compatible combination thereof. Specifically, the content of the target substance (e.g., L-glutamic acid) in the food product of the present invention may be, for example, 0.01 to 2% (w / w), 0.02 to 1% (w / w), or 0.03 to 0.5% (w / w) in terms of consumption concentration.

[0152] When the food product of the present invention contains a target substance (e.g., L-glutamic acid), the content of the target substance (e.g., L-glutamic acid) in the food product of the present invention 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, or 5 parts by weight or more per 1 part by weight of the active ingredient contained in the food product of the present invention (converted to the dry weight of Gram-positive bacterial cells), or may be 50 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, or 1 part by weight or less, or any combination thereof that is compatible. The content of the target substance (e.g., L-glutamic acid) in the food product of the present invention may be, for example, 0.1 to 20 parts by weight per 1 part by weight of the active ingredient contained in the food product of the present invention (converted to the dry weight of Gram-positive bacterial cells).

[0153] The food of the present invention may contain a culture of Gram-positive bacteria in addition to the active ingredient. That is, the food of the present invention may be produced to contain a culture of Gram-positive bacteria. That is, the method of the present invention may further include adding a culture of Gram-positive bacteria to the raw materials of the food. The Gram-positive bacterial culture as an ingredient other than the active ingredient is as described above. A food containing a culture of Gram-positive bacteria may be produced, for example, by adding a culture of Gram-positive bacteria itself, or by adding a material containing a culture of Gram-positive bacteria. Note that "adding an active ingredient and a culture of Gram-positive bacteria" does not necessarily mean adding an active ingredient and a culture obtained separately, but also includes adding an active ingredient and a culture obtained together. Examples of adding an active ingredient and a culture obtained together include adding a culture containing Gram-positive bacterial cells as is, or after appropriate drying. Furthermore, when the composition of the present invention contains a culture of Gram-positive bacteria, the culture of Gram-positive bacteria may be added by adding the composition of the present invention. The addition of the culture of Gram-positive bacteria can be carried out in the same manner as the addition of the active ingredient. The Gram-positive bacterial culture may be added to a food ingredient so that the content of the Gram-positive bacterial culture in the food of the present invention falls within a desired range (for example, the content range described below). Alternatively, the food ingredient may originally contain the Gram-positive bacterial culture.

[0154] When the food of the present invention contains a culture of Gram-positive bacteria as an ingredient other than the active ingredient, and the Gram-positive bacterial culture contains Gram-positive bacterial cells, the content of the Gram-positive bacterial culture in the food of the present invention may be set, for example, so that the content of the active ingredient in the food of the present invention is within the range exemplified above.

[0155] When the food of the present invention contains a culture of Gram-positive bacteria as an ingredient other than the active ingredient, and the culture of Gram-positive bacteria contains a target substance (e.g., L-glutamic acid), the content of the culture of Gram-positive bacteria in the food of the present invention may be set, for example, so that the content of the target substance (e.g., L-glutamic acid) in the food of the present invention falls within the range exemplified above.

[0156] When the food of the present invention contains a culture of Gram-positive bacteria as an ingredient other than the active ingredient, the content of the culture of Gram-positive bacteria in the food of the present invention may be, for example, 0.01% (w / w) or more, 0.1% (w / w) or more, 1% (w / w) or more, 5% (w / w) or more, or 10% (w / w) or more, converted into the amount of the original culture, and may be 10,000% (w / w) or less, 5,000% (w / w) or less, ), 1000% (w / w) or less, 500% (w / w) or less, 300% (w / w) or less, 200% (w / w) or less, 150% (w / w) or less, 100% (w / w) or less, 70% (w / w) or less, 50% (w / w) or less, 30% (w / w) or less, 10% (w / w) or less, 5% (w / w) or less, or 1% (w / w) or less, or any compatible combination thereof.

[0157] <4> Use of Active Ingredient The present invention also discloses the use of the active ingredient in the above-exemplified applications. That is, the present invention discloses, for example, the use of the active ingredient for improving flavor or producing food, and the use of the active ingredient in producing a composition for improving flavor or producing food.

[0158] The present invention also discloses active ingredients for use in the above-exemplified applications, i.e., active ingredients for use in improving flavor or producing food products, and active ingredients for use in producing compositions for improving flavor or producing food products.

[0159] The present invention also discloses the use of the active ingredient in combination with other ingredients (e.g., spices). The active ingredient may be combined with other ingredients (e.g., spices) for the uses exemplified above.

[0160] <5> Another aspect of the present invention relates to the use of L-glutamic acid-producing bacteria having a "specific mutation." L-glutamic acid-producing bacteria having a "specific mutation" are as described above. In this aspect of the present invention, the L-glutamic acid-producing bacteria having a "specific mutation" are also simply referred to as "L-glutamic acid-producing bacteria."

[0161] L-glutamic acid-producing bacteria can be used, for example, to produce L-glutamic acid. L-glutamic acid is as described above. In another aspect of the present invention, L-glutamic acid is also referred to as the "active ingredient."

[0162] L-glutamic acid may be produced and / or used in its free form, in its salt form, or in a combination thereof. That is, unless otherwise specified, the term "L-glutamic acid" may refer to free L-glutamic acid, its salt, or a combination thereof. Furthermore, L-glutamic acid may be produced and / or used in its non-hydrate form, in its hydrate form, or in a combination thereof. That is, the term "L-glutamic acid" (e.g., "free L-glutamic acid" or "salt of L-glutamic acid") may encompass both the non-hydrate and the hydrate, unless otherwise specified. When used, L-glutamic acid may be in any form, such as an ion. In addition, when L-glutamic acid forms a salt or hydrate, the amount of L-glutamic acid (e.g., content (concentration) or amount used) is calculated based on the mass of the salt or hydrate converted to the equimolar mass of the free form. The above description of the salt of the target substance can be applied mutatis mutandis to salts of L-glutamic acid. Particularly, examples of salts of L-glutamic acid include sodium L-glutamate (e.g., monosodium L-glutamate; MSG) and ammonium L-glutamate (e.g., monoammonium L-glutamate).

[0163] By using L-glutamic acid, the umami of food can be enhanced, that is, an effect of enhancing the umami of food can be obtained. This effect is also called the "umami enhancement effect." Enhancement of the umami of food can also be simply called "enhancement of umami." Specifically, by using L-glutamic acid, the umami of food can be enhanced compared to when L-glutamic acid is not used. Therefore, L-glutamic acid may be used to enhance the umami of food.

[0164] Measurement and comparison of umami can be carried out, for example, by sensory evaluation by a specialist panel.

[0165] L-glutamic acid can be produced, for example, by culturing L-glutamic acid-producing bacteria in a medium. That is, a method for producing L-glutamic acid may include, for example, a step of culturing L-glutamic acid-producing bacteria in a medium. This step is also referred to as a "culturing step." Specifically, the culturing step may be a step of culturing L-glutamic acid-producing bacteria in a medium to obtain a culture. More specifically, the culturing step may be a step of culturing L-glutamic acid-producing bacteria in a medium to obtain a culture containing L-glutamic acid.

[0166] The medium and culture conditions for culturing L-glutamic acid-producing bacteria are not particularly limited, as long as the L-glutamic acid-producing bacteria can grow and L-glutamic acid can be produced. The medium and culture conditions for culturing L-glutamic acid-producing bacteria can be, for example, the same as those described above for culturing Gram-positive bacteria. For example, the medium may contain food ingredients (e.g., solanaceae plants such as tomatoes).

[0167] By culturing the L-glutamic acid-producing bacteria, a culture of the L-glutamic acid-producing bacteria (specifically, a culture containing L-glutamic acid-producing bacteria cells and L-glutamic acid) can be obtained.

[0168] L-glutamic acid may be used as an active ingredient, for example, as it is contained in the culture (specifically, the medium), or it may be recovered from the culture (specifically, the medium) and used as an active ingredient. That is, a method for producing L-glutamic acid may include, for example, a step of recovering L-glutamic acid from the culture (specifically, the medium). L-glutamic acid may be recovered as a suitable fraction containing L-glutamic acid. An example of such a fraction is the culture supernatant. L-glutamic acid may also be further separated and purified from the fraction. For example, the cells of the L-glutamic acid-producing bacterium may be separated from the culture to obtain a culture supernatant, and L-glutamic acid may be recovered from the culture supernatant. Note that "recovery of L-glutamic acid" may also include removal of impurities from the fraction containing L-glutamic acid. Recovery of L-glutamic acid can be carried out, for example, by known techniques. Examples of such methods include the ion exchange resin method (Nagai, H. et al., Separation Science and Technology, 39(16), 3691-3710), precipitation, membrane separation (JP 9-164323 A, JP 9-173792 A), and crystallization (WO 2008 / 078448 A, WO 2008 / 078646 A). Furthermore, fractions containing L-glutamic acid (e.g., cultures or culture supernatants) may be appropriately treated before use as an active ingredient. Examples of such treatments include concentration, drying, and heating. Concentration, drying, and heating can all be carried out by known methods. Specifically, examples of the active ingredient include cultures of L-glutamic acid-producing bacteria, culture supernatants recovered from the cultures, processed products thereof, and L-glutamic acid recovered from the cultures. That is, L-glutamic acid may be used as an active ingredient in the form of, for example, a culture of an L-glutamic acid-producing bacterium, a culture supernatant recovered from the culture, a processed product thereof, L-glutamic acid recovered from the culture, or a combination thereof. In other words, examples of the active ingredient include L-glutamic acid contained in a culture of an L-glutamic acid-producing bacterium, L-glutamic acid contained in a culture supernatant recovered from the culture, L-glutamic acid contained in a processed product thereof, and L-glutamic acid recovered from the culture.That is, L-glutamic acid may be used as an active ingredient in the form of, for example, L-glutamic acid contained in a culture of an L-glutamic acid-producing bacterium, L-glutamic acid contained in a culture supernatant recovered from the culture, L-glutamic acid contained in a processed product thereof, L-glutamic acid recovered from the processed product, or a combination thereof.

[0169] L-glutamic acid may be used in combination with other components (i.e., components other than L-glutamic acid). Examples of other components include fractions containing cell walls of Gram-positive bacteria. The fractions containing cell walls of Gram-positive bacteria are as described above. The Gram-positive bacteria may or may not be the same as the L-glutamic acid-producing bacteria. The fraction containing cell walls of Gram-positive bacteria may be, for example, one produced together with the production of L-glutamic acid, or one produced separately from L-glutamic acid. Note that the phrase "using a fraction containing cell walls of Gram-positive bacteria in combination" does not necessarily mean using a combination of fractions containing L-glutamic acid and cell walls of Gram-positive bacteria that have been obtained separately, but also includes using fractions containing L-glutamic acid and cell walls of Gram-positive bacteria that have been obtained together. In the case of utilizing the fractions containing L-glutamic acid and cell walls of Gram-positive bacteria obtained together, the culture of the L-glutamic acid-producing bacterium may be used as an active ingredient either directly or after appropriate drying. The combined use of L-glutamic acid and the fraction containing cell walls of Gram-positive bacteria may provide a flavor-improving effect. The flavor-improving effect is as described above.

[0170] A composition according to another embodiment of the present invention is a composition containing an active ingredient (here, L-glutamic acid produced by culturing an L-glutamic acid-producing bacterium in a medium).

[0171] By utilizing the composition of another embodiment of the present invention, the umami taste of food can be enhanced, i.e., an umami taste enhancement effect can be obtained. Thus, the composition of another embodiment of the present invention may be utilized to enhance the umami taste of food. That is, the composition of another embodiment of the present invention may be, for example, a composition for enhancing the umami taste of food.

[0172] Furthermore, by utilizing the composition according to another aspect of the present invention, it is possible to produce a food product with enhanced umami. Thus, the composition according to another aspect of the present invention may be utilized in the production of a food product (specifically, the production of a food product with enhanced umami). That is, the composition according to another aspect of the present invention may be, for example, a composition for use in the production of a food product (specifically, the production of a food product with enhanced umami).

[0173] The composition according to another embodiment of the present invention may be, for example, a seasoning. Specifically, the composition according to another embodiment of the present invention may be, for example, a seasoning for enhancing the umami flavor of food, or a seasoning for use in the production of food (specifically, the production of food with enhanced umami flavor).

[0174] The composition of another aspect of the present invention may be used to enhance umami taste or to produce food products in the manner described below in the method of another aspect of the present invention.

[0175] The composition of another embodiment of the present invention may consist of an active ingredient, or may contain ingredients other than the active ingredient. In one embodiment, the composition of another embodiment of the present invention may exclude a composition consisting of an active ingredient. As the ingredients other than the active ingredient, one kind of ingredient may be used, or two or more kinds of ingredients may be used in combination.

[0176] The active ingredient contained in the composition of another aspect of the present invention may be one produced by the method for producing an active ingredient described above. Thus, the method for producing a composition of another aspect of the present invention may include a step of producing the active ingredient by the method for producing an active ingredient described above. The step of producing the active ingredient in the method for producing a composition of another aspect of the present invention may specifically be a step of culturing an L-glutamic acid-producing bacterium in a medium to obtain a culture. The step of producing the active ingredient in the method for producing a composition of another aspect of the present invention may more specifically be a step of culturing an L-glutamic acid-producing bacterium in a medium to obtain a culture containing L-glutamic acid.

[0177] The ingredients other than the active ingredient are not particularly limited as long as they do not lose the umami enhancing effect (i.e., the umami enhancing effect of the active ingredient is obtained). The ingredients other than the active ingredient can be appropriately selected depending on various conditions such as the type of food. Examples of ingredients other than the active ingredient include ingredients that are blended into foods or pharmaceuticals.

[0178] Specific examples of ingredients other than the active ingredient include ingredients effective for food production. Examples of ingredients effective for food production include the above-mentioned food ingredients. Examples of ingredients other than the active ingredient include, in particular, a fraction containing the cell wall of a gram-positive bacterium and spices. That is, another embodiment of the composition of the present invention may be, for example, a composition containing an active ingredient and a fraction containing the cell wall of a gram-positive bacterium (e.g., a seasoning), a composition containing an active ingredient and a spice (e.g., a seasoning), or a composition containing an active ingredient, a fraction containing the cell wall of a gram-positive bacterium, and a spice (e.g., a seasoning). The fraction containing the cell wall of a gram-positive bacterium is as described above. Note that the phrase "a composition containing a fraction containing the cell wall of a gram-positive bacterium and an active ingredient and a fraction containing the cell wall of a gram-positive bacterium" does not limit the case where the composition contains individually obtained fractions containing the active ingredient and the cell wall of a gram-positive bacterium, but also encompasses a case where the composition contains a collectively obtained fraction containing the active ingredient and the cell wall of a gram-positive bacterium. An example of a composition containing a fraction containing the active ingredient and the cell wall of a gram-positive bacterium obtained together is a culture of an L-glutamic acid-producing bacterium, which is contained in the composition either directly or after being appropriately dried. Spices are as described above. In one embodiment, the composition of another aspect of the present invention may not contain L-glutamic acid produced by a method other than the above-described method for producing the active ingredient.

[0179] The composition according to another embodiment of the present invention can be produced, for example, by appropriately mixing the active ingredient and, optionally, other ingredients.

[0180] The composition of another embodiment 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 another embodiment of the present invention.

[0181] The shape of the composition according to another aspect of the present invention is not particularly limited, and the composition according to another aspect of the present invention may be in any shape, such as a powder, flakes, tablet, paste, or liquid.

[0182] The content and content ratio of each component (i.e., the active ingredient and optionally other components) in the composition of another aspect of the present invention are not particularly limited as long as the umami flavor enhancing effect is obtained. The content and content ratio of each component in the composition of another aspect of the present invention can be appropriately set depending on various conditions, such as the mode of use of the composition of another aspect of the present invention. The contents and content ratios of each component in the composition of another aspect of the present invention can be, for example, the same as those described above for the contents and content ratios of each component in the composition of the present invention. That is, the content of L-glutamic acid in the composition of another aspect of the present invention may be, for example, within the range of the content of L-glutamic acid in the composition of the present invention. Furthermore, when the composition of another aspect of the present invention contains a fraction containing the cell wall of a gram-positive bacterium, the content of the fraction containing the cell wall of a gram-positive bacterium in the composition of the present invention may be, for example, within the range of the content of the fraction containing the cell wall of a gram-positive bacterium in the composition of the present invention. Furthermore, when the composition of another aspect of the present invention contains a spice, the content of the spice in the composition of the present invention may be, for example, within the range of the content of the spice in the composition of the present invention. Furthermore, when the composition of another aspect of the present invention contains a fraction containing the cell walls of Gram-positive bacteria, the ratio of the content of L-glutamic acid to the content of the fraction containing the cell walls of Gram-positive bacteria in the composition of another aspect of the present invention may be, for example, within the range of the ratio of the content of L-glutamic acid to the content of the fraction containing the cell walls of Gram-positive bacteria in the composition of the present invention described above. Furthermore, when the composition of another aspect of the present invention contains a fraction containing the cell walls of Gram-positive bacteria and a spice, the ratio of the content of the fraction containing the cell walls of Gram-positive bacteria to the content of the spice in the composition of the present invention may be, for example, within the range of the ratio of the content of the fraction containing the cell walls of Gram-positive bacteria to the content of the spice in the composition of the present invention described above.

[0183] In another embodiment of the present invention, the content of L-glutamic acid in the composition is more than 0% (w / w) and 100% (w / w) or less. The content of L-glutamic acid in the composition of another embodiment of the present invention may be, for example, 0.1% (w / w) or more, 0.2% (w / w) or more, 0.5% (w / w) or more, 1% (w / w) or more, 2% (w / w) or more, 5% (w / w) or more, 10% (w / w) or more, or 20% (w / w) or more, or 100% (w / w) or less, less than 100% (w / w), 99.9% (w / w) or less, 90% (w / w) or less, 50% (w / w) or less, 40% (w / w) or less, 20% (w / w) or less, 10% (w / w) or less, 5% (w / w) or less, 2% (w / w) or less, or 1% (w / w) or less, or any compatible combination thereof. The content of L-glutamic acid in the composition according to another embodiment of the present invention may be, specifically, for example, 0.2 to 90% (w / w), 0.5 to 50% (w / w), 1 to 50% (w / w), 1 to 20% (w / w), 5 to 50% (w / w), 10 to 50% (w / w), 20 to 50% (w / w), 1 to 40% (w / w), 5 to 40% (w / w), 10 to 40% (w / w), or 20 to 40% (w / w).

[0184] When the composition of another embodiment of the present invention contains a fraction containing the cell wall of a gram-positive bacterium, the content of the fraction containing the cell wall of a gram-positive bacterium in the composition of another embodiment of the present invention may be, for example, 0.1% (w / w) or more, 0.2% (w / w) or more, 0.5% (w / w) or more, 1% (w / w) or more, 2% (w / w) or more, 5% (w / w) or more, 10% (w / w) or more, or 20% (w / w) or more, calculated in terms of the dry weight of the gram-positive bacterial cells, or 90% (w / w) or less, 50% (w / w) or less, 20% (w / w) or less, 10% (w / w) or less, 5% (w / w) or less, 2% (w / w) or less, or 1% (w / w) or less, or any combination thereof that does not contradict these. The content of the fraction containing the cell wall of Gram-positive bacteria in the composition of another embodiment of the present invention may be, specifically, for example, 0.2 to 99.9% (w / w), 0.2 to 90% (w / w), 0.5 to 50% (w / w), or 1 to 20% (w / w) in terms of the dry weight of Gram-positive bacterial cells.

[0185] The components (i.e., the active ingredient and optionally other ingredients) contained in the composition of another aspect of the present invention may be mixed together and contained in the composition of another aspect of the present invention, or may be contained separately or in any combination in the composition of another aspect of the present invention. For example, the composition of another aspect 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.

[0186] Another embodiment of the present invention is a method comprising a step of utilizing an active ingredient (here, L-glutamic acid produced by culturing an L-glutamic acid-producing bacterium in a medium).

[0187] According to the method of another aspect of the present invention, specifically by utilizing an active ingredient, the umami of a food can be enhanced, i.e., an umami enhancement effect can be obtained. Thus, the method of another aspect of the present invention may be carried out to enhance the umami of a food. That is, the method of another aspect of the present invention may be, for example, a method for enhancing the umami of a food. This method is also referred to as the "umami enhancement method of another aspect of the present invention."

[0188] Furthermore, a food product with enhanced umami can be produced by a method according to another aspect of the present invention, specifically by utilizing an active ingredient. Thus, the method according to another aspect of the present invention may be carried out for the production of a food product (specifically, the production of a food product with enhanced umami). That is, the method according to another aspect of the present invention may be, for example, a method for producing a food product (specifically, the production of a food product with enhanced umami). This method is also referred to as a "food product production method according to another aspect of the present invention."

[0189] The active ingredient can be added to food ingredients during food production to enhance umami or be used in food production. That is, an example of using the active ingredient is adding the active ingredient to food ingredients. That is, a method according to another aspect of the present invention may specifically be, for example, a method for enhancing the umami of food, which includes adding the active ingredient to food ingredients. Furthermore, a method according to another aspect of the present invention may specifically be, for example, a method for producing food (specifically, producing food with enhanced umami), which includes adding the active ingredient to food ingredients. "Addition" can also be referred to as "blending."

[0190] The active ingredient may be used in the method of another aspect of the present invention, for example, in the form of a composition of another aspect of the present invention. That is, "use of an active ingredient" also includes use of a composition of another aspect of the present invention. For example, "addition of an active ingredient" also includes addition of a composition of another aspect of the present invention.

[0191] A food product obtained by the method according to another aspect of the present invention is also referred to as a "food product according to another aspect of the present invention." Specifically, the food product according to another aspect of the present invention is a food product with enhanced umami flavor. In other words, the food product according to another aspect of the present invention is a food product to which an active ingredient has been added.

[0192] The enhancement of umami or the production of a food product may be carried out in the same manner as the production of a normal food product, except for the use of an active ingredient. That is, the enhancement of umami or the production of a food product may be carried out using the same raw materials and under the same production conditions as a normal food product, except for the use of an active ingredient. Furthermore, the raw materials and production conditions of the food product may be appropriately modified for use in the enhancement of umami or the production of a food product. The food product, the raw materials of the food product, and the production conditions of the food product are as described above.

[0193] The active ingredient may be added to food ingredients at any stage of the food production process, as long as the umami-enhancing effect is obtained. In other words, the "food ingredients" to which the active ingredient is added may be those at any stage of the food production 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 may also be added to the food ingredients as appropriate. In other words, another embodiment of the method of the present invention may further include adding ingredients other than the active ingredient to the food ingredients. The ingredients other than the active ingredient are not particularly limited, as long as they do not lose their umami-enhancing effect (i.e., the umami-enhancing effect of the active ingredient is obtained). The ingredients other than the active ingredient can be selected appropriately depending on various conditions, such as the type of food. Examples of ingredients other than the active ingredient include fractions containing the cell wall of Gram-positive bacteria and spices. In one embodiment, the method of another embodiment of the present invention does not necessarily include adding L-glutamic acid produced by a method other than the above-described method for producing the active ingredient to the food raw material. The description of the addition of the 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 raw material all at the same time, or may be added separately or in any combination. The order in which the ingredients are added to the food raw material is not particularly limited.

[0194] The amounts and ratios of the components (i.e., the active ingredient and optional other components) added in the method of another aspect of the present invention are not particularly limited as long as the umami flavor enhancing effect is obtained. The amounts and ratios of the components added in the method of another aspect of the present invention can be appropriately determined depending on various conditions, such as the type of raw material of the food and the type of food. The same descriptions of the amounts and ratios of the components added in the method of another aspect of the present invention as described above can be applied mutatis mutandis. That is, the amount of L-glutamic acid added in the method of another aspect of the present invention may be within the range of the amount of L-glutamic acid added in the method of another aspect of the present invention. Furthermore, when the method of another aspect of the present invention includes adding a fraction containing the cell wall of a gram-positive bacterium, the amount of the fraction containing the cell wall of a gram-positive bacterium added in the method of another aspect of the present invention may be within the range of the amount of the fraction containing the cell wall of a gram-positive bacterium added in the method of another aspect of the present invention (e.g., the amount added to achieve the content of the fraction containing the cell wall of a gram-positive bacterium in the food of the present invention described above). Furthermore, when the method of another aspect of the present invention includes adding a spice, the amount of the spice added in the method of another aspect of the present invention may be, for example, within the range of the amount of the spice added in the method of the present invention described above (e.g., the amount added to achieve the spice content in the food of the present invention described above). Furthermore, when the method of another aspect of the present invention includes adding a fraction containing the cell walls of Gram-positive bacteria, the ratio of the amount of L-glutamic acid added to the amount of the fraction containing the cell walls of Gram-positive bacteria added in the method of the present invention may be, for example, within the range of the ratio of the amount of L-glutamic acid added to the amount of the fraction containing the cell walls of Gram-positive bacteria added in the method of the present invention described above (e.g., the ratio of the amounts added to achieve the ratio of the content of L-glutamic acid to the content of the fraction containing the cell walls of Gram-positive bacteria in the food of the present invention described above).Furthermore, when the method of another embodiment of the present invention includes adding a fraction containing the cell walls of Gram-positive bacteria and a spice, the ratio of the amount of the fraction containing the cell walls of Gram-positive bacteria added to the amount of the spice added in the method of another embodiment of the present invention may be, for example, within the range of the ratio of the amount of the fraction containing the cell walls of Gram-positive bacteria added to the amount of the spice added in the method of the present invention described above (for example, the ratio of the amounts added that achieves the ratio of the content of the fraction containing the cell walls of Gram-positive bacteria to the content of the spice in the food of the present invention described above).

[0195] L-glutamic acid may be added to the raw materials of the food product so that the content of L-glutamic acid in the food product of another embodiment of the present invention falls within a desired range (for example, the content range described below).

[0196] The content of L-glutamic acid in the food product according to another embodiment of the present invention may be, for example, 0.01% (w / w) or more, 0.02% (w / w) or more, 0.03% (w / w) or more, 0.04% (w / w) or more, 0.05% (w / w) or more, 0.07% (w / w) or more, 0.1% (w / w) or more, 0.2% (w / w) or more, 0.3% (w / w) or more, 0.4% (w / w) or more, as an ingestible concentration. The L-glutamic acid content in the food product of another embodiment of the present invention may be, for example, 0.01 to 2% (w / w), 0.02 to 1% (w / w), or 0.03 to 0.5% (w / w) or more, or 0.5% (w / w) or more, or 5% (w / w) or less, 2% (w / w) or less, 1% (w / w) or less, 0.7% (w / w) or less, 0.5% (w / w) or less, 0.4% (w / w) or less, 0.3% (w / w) or less, 0.2% (w / w) or less, or 0.1% (w / w) or less, or any combination thereof that is not contradictory. Specifically, the L-glutamic acid content in the food product of another embodiment of the present invention may be, for example, 0.01 to 2% (w / w), 0.02 to 1% (w / w), or 0.03 to 0.5% (w / w) in terms of an intake concentration.

[0197] The present invention will now be described in more detail with reference to the following non-limiting examples.

[0198] Example 1: Isolation of an L-glutamic acid hyper-producing strain from Corynebacterium casei JCM 12072 (1-1) Construction of a mutant library of C. casei JCM 12072 strain. C. casei JCM 12072 strain was inoculated into a Sakaguchi flask containing 30 mL of the medium described in Table 2(A) and cultured at 30°C for 1 day with shaking. The cells were then harvested. The cells were suspended in a solution containing 0.1 M potassium phosphate buffer (pH 7.0), 6.0% dimethyl sulfoxide, and 0.1 mg / mL N-methyl-N-nitrosoguanidine (NTG) and allowed to stand at room temperature for 50 minutes. The cells were harvested and washed three times with 0.1 M potassium phosphate buffer (pH 7.0). The cells were cultured at 30°C for 2 hours in a Sakaguchi flask containing 30 mL of the recovery culture medium described in Table 2(B), and the cells were then harvested. The cells were suspended in 20% glycerol and stored at −80° C. This was used as a mutant strain library.

[0199]

[0200] (1-2) Screening of L-glutamic acid hyper-producing strains from the mutant library Next, the prepared mutant library was cultured with shaking at 30°C for 29 hours in Glu minimal medium (Table 3) supplemented with 100 mg / L ampicillin, and then plated on the agar medium (Table 2(A)). The grown colonies were inoculated onto the Glu minimal medium (Table 3) and the agar medium (Table 2(A)), and 26 clones with reduced L-glutamic acid (L-Glu) assimilation were selected as candidate strains.

[0201]

[0202] The 26 candidate strains were cultured in 500 μL of evaluation medium (Table 4) in a 96-deep-well plate at 30°C for 48 hours with shaking. The amount of L-Glu accumulated in the medium was measured using a Biotech Analyzer AS210 (Sakura SI Co., Ltd.). The measurement results are shown in Figure 1. Among the evaluated strains, strain RUN5-2-96 (NITE ABP-03688), which had the highest L-Glu concentration, was selected. This strain contains all 135 mutations in group A (i.e., all mutations A-1 to A-135).

[0203]

[0204] Example 2: Preparation of dried bacterial cells of C. casei JCM 12072 and RUN5-2-96 strains. C. casei JCM 12072 and RUN5-2-96 strains were cultured in jars using the media shown in Table 5. A 1-L fermenter was used for jar culture. The culture temperature was maintained at a constant 30°C, the culture pH was controlled to 6.8 using ammonia gas, and the dissolved oxygen concentration was controlled by stirring to be 23% or higher relative to the saturated dissolved oxygen concentration of 100%. The resulting culture solution was sterilized at 80°C for 20 minutes, and medium components were removed by centrifugation to obtain a bacterial cell pellet. The bacterial cells were then washed by adding an equal volume of water to the medium and then centrifuging to remove the supernatant, repeating this process twice. The washed bacterial pellet was frozen at −80°C and then the water was removed using a freeze dryer to obtain dried bacterial cells of C. casei JCM 12072 strain and RUN5-2-96 strain.

[0205]

[0206] Example 3: Preparation of fermented seasoning using C. casei RUN5-2-96 strain (3-1) L-Glu fermentation was carried out using C. casei RUN5-2-96 strain according to the following procedure.

[0207] First, seed culture of the RUN5-2-96 strain was performed in a jar using the seed medium shown in Table 6. The culture temperature was maintained at a constant 30°C, and the culture pH was controlled to 6.8 with ammonia gas. Next, main cultures of the RUN5-2-96 strain (n = 2; designated J1 and J2, respectively) were performed in a jar using the medium shown in Table 7. The culture pH was controlled at 6.8 with ammonia gas, as in the seed culture. The culture temperature was maintained at 30°C and the dissolved oxygen concentration was 23% or higher until the OD620 reached 40. Once the OD620 reached 40, the temperature was raised to 35°C, and the culture was continued until glucose was depleted. In this way, a culture solution containing L-Glu was obtained. The amount of L-Glu accumulated in the medium and the amount of glucose in the medium were measured using a Biotech Analyzer AS210 (Sakura SI Co., Ltd.). The dry cell weight (DCW) was measured by preparing dry cells from the culture medium in the same manner as in Example 2 (except that sterilization treatment was not performed). The culture results are shown in Table 8.

[0208]

[0209]

[0210]

[0211] (3-2) The culture obtained in (3-1) was transferred to a 2L Duran bottle, heated to 70 ° C in a water bath, and sterilized by stirring for 10 minutes. Next, the sterilized culture was dispensed into centrifuge tubes (Himac 500PA 330437A) and centrifuged at 7,000 rpm (9,400 × G) and 25 ° C for 10 minutes using a centrifuge (Hitachi CR20 GIII, PRP9-2 rotor). The supernatant was collected by decantation and an electric pipette. After the bacterial cell separation procedure, 1 / 2 the weight of pure water of the culture medium was added to the remaining bacterial cells in the centrifuge tube, and the mixture was suspended using a vortex mixer. Then, the mixture was centrifuged at 7,000 rpm (9,400 × G) and 25 ° C for 10 minutes using a centrifuge (Hitachi CR20 GIII, PRP9-2 rotor). After centrifugation, the supernatant was collected by decantation and using an electric pipette. By repeating this procedure twice, components other than the bacterial cells were separated as the supernatant, and washed bacterial cells were obtained.

[0212] (3-3) The supernatant collected in (3-2) was transferred to a 2 L Duran bottle and heated in a water bath. After reaching 60°C, an amount of granular activated carbon (NORIT® GAC1240, Cabot Norit) equal to the amount of L-Glu in the solution was added and stirred for 1 hour. The activated carbon was then filtered using a circular quantitative filter paper No. 5C (Advantec Toyo Co., Ltd.), a Buchner funnel, and a filter bottle to obtain the filtrate. The fine powder of activated carbon that leaked into the filtrate was removed by dispensing the filtrate into centrifuge tubes (Himac500PA 330437A) and centrifuging it in a centrifuge (Hitachi CR20 GIII, PRP9-2 rotor) at 7,000 rpm (9,400 × G) at 25°C for 10 minutes, followed by decantation to recover the supernatant.

[0213] (3-4) A 27% aqueous solution of sodium hydroxide was added to the filtrate obtained in (3-3) (sodium hydroxide was added in an amount 1.3 times the amount of L-Glu in the solution), and the mixture was concentrated under reduced pressure using a rotary evaporator at a reduced pressure of 90 hPa and a water bath temperature of 70°C. This procedure of adding pure water in an amount of 2 / 3 of the original volume each time the volume of the solution was reduced to approximately 1 / 3 was repeated twice, and then the mixture was concentrated under reduced pressure until the volume was reduced to approximately 1 / 3. Fine powder remaining in the concentrated solution was removed using circular quantitative filter paper No. 5C (Advantec Toyo Co., Ltd.), a Buchner funnel, and a filter bottle.

[0214] (3-5) The washed cells of the C. casei RUN5-2-96 strain obtained in (3-2) were added to the concentrate obtained in (3-4). The addition ratio was adjusted to the ratio of the culture solution before cell separation to the sterilized solution and the cells when the cells were separated. Maltodextrin (Ciranda Conventional Non-GMO Tapioca Maltodextrin DE10) was added to the concentrate so that the solid content of the product powder after spray drying was 97% and the L-Glu content was 35%. This was spray-dried using a spray dryer (B-290, manufactured by Nippon Buchi Co., Ltd.) with an inlet temperature of 180°C, a flow rate of 5 mL / min, a compressed gas flow rate of 473 L / h, and a hot air volume of 38 m. 3 / h to obtain a fermented seasoning containing C. casei RUN5-2-96 cells (hereinafter also referred to as "C. casei cell-containing seasoning"). The C. casei cell-containing seasoning contains 7% by weight of C. casei RUN5-2-96 cells.

[0215] (3-6) Maltodextrin (Ciranda Conventional Non-GMO Tapioca Maltodextrin DE10) was added to the concentrate obtained in (3-4) so ​​that the solid content in the product powder after spray drying was 97% and the L-Glu content was 35%. This was spray-dried using a spray dryer (B-290 manufactured by Nippon Buchi Co., Ltd.) at an inlet temperature of 180°C, a flow rate of 5 mL / min, a compressed gas flow rate of 473 L / h, and a hot air volume of 38 m. 3 / h to obtain a fermented seasoning that does not contain C. casei RUN5-2-96 strain cells (hereinafter also referred to as "C. casei broth seasoning").

[0216] Example 4: Effect of adding C. casei cells to cheese sauce The dried cells of the C. casei JCM 12072 strain prepared in Example 2 were added to a commercially available Alfred cheese sauce (BERUTOLLI Alfred cheese sauce; manufactured by Unilever Japan Co., Ltd.) at a concentration of 0.1% by weight to obtain an Alfred cheese sauce containing C. casei cells. A sensory evaluation of the C. casei cell-containing Alfred cheese sauce was performed by a panel of three experts using the free-word method, using a commercially available Alfred cheese sauce without added C. casei cells as a control.

[0217] Sensory evaluation confirmed that Alfredo cheese sauce containing C. casei cells had increased thickness and richness compared to Alfredo cheese sauce without C. casei cells. These results indicated that the addition of C. casei cells increased the thickness and richness of cheese, improving the palatability of cheese-containing foods. These results demonstrate that the addition of C. casei cells can improve the flavor of foods, such as imparting a richer taste.

[0218] Example 5: Effect of adding C. casei cells to tomato sauce The dried cells of the C. casei JCM 12072 strain prepared in Example 2 were added to a commercially available tomato sauce (manufactured by RAGU) at a concentration of 0.1% by weight to obtain a tomato sauce containing C. casei cells. A sensory evaluation of the C. casei cell-containing tomato sauce was performed by a panel of three experts using the free-word method, using a commercially available tomato sauce without added C. casei cells as a control.

[0219] The sensory evaluation confirmed that the tomato sauce containing C. casei cells had increased thickness, a sense of maturity, and richness compared to the tomato sauce without C. casei cells. These results indicate that the addition of C. casei cells enhances the thickness, sense of maturity, and richness of tomatoes, improving the palatability of foods containing tomatoes. These findings demonstrate that the addition of C. casei cells can improve the flavor of foods, such as imparting a richer taste.

[0220] Example 6: Effect of Adding C. casei Cells to Curry Sauce. A curry sauce was prepared by adding 9.4 parts by weight of boiling water to 1 part by weight of commercially available powdered curry roux (House Corporation) and stirring. The dried cells of C. casei JCM 12072 prepared in Example 2 were added to the curry sauce at concentrations of 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, or 1% by weight and stirring was performed to obtain a curry sauce containing C. casei cells. A sensory evaluation of the curry sauce containing C. casei cells was conducted by a panel of three experts, using a curry sauce without added C. casei cells as a control. The sensory evaluation was conducted on the thickness-imparting effect and pungency-enhancing effect using a scoring system ranging from 0 to 10 points (higher scores indicate a stronger effect, and a score of 1 or higher indicates an effective effect). The spiciness-enhancing effect was scored as 0 for the spiciness of the control product and 5 for the spiciness that was twice as strong as the control product. The thickness-enhancing effect was scored as 0 for the thickness of the control product and 5 for the thickness that was twice as strong as the control product.

[0221] The results are shown in Table 9. In the table, the "score" indicates the average score from three expert panelists. In the table, the "concentration (%)" indicates the concentration of dried C. casei JCM 12072 cells. The thickness-imparting and pungency-enhancing effects were very weak when C. casei cells were added at 0.005 wt.%, but these effects were confirmed when C. casei cells were added at concentrations of 0.01% or higher. Furthermore, when C. casei cells were added at a concentration of 1 wt.%, an off-flavor and bitterness appeared, tending to alter the flavor quality. These results demonstrate that adding 0.01 to 0.5 wt.% C. casei cells to curry sauce enhanced the thickness and pungency without significantly altering the flavor quality. These findings demonstrate that the addition of C. casei cells (e.g., 0.01 to 0.5%) can improve the flavor of foods (e.g., spice-containing foods), by imparting a richer flavor and enhancing the spicy sensation.

[0222]

[0223] Example 7: Effect of Adding C. casei Cells to Black Pepper-Containing Mashed Potatoes Mashed potatoes were obtained by adding 6 parts by weight of boiling water to 1 part by weight of commercially available dried mashed potatoes (manufactured by Calbee, Inc.) and stirring. Commercially available black pepper powder (manufactured by Gaban Co., Ltd.) was added to the mashed potatoes at a concentration of 0.15% by weight and stirred to obtain black pepper-containing mashed potatoes. The dried cells of C. casei JCM 12072 strain prepared in Example 2 were added to the black pepper-containing mashed potatoes at concentrations of 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, or 1% by weight and stirred to obtain black pepper-containing mashed potatoes with added C. casei cells. A sensory evaluation of the black pepper-containing mashed potatoes with added C. casei cells was conducted by a panel of three experts, using black pepper-containing mashed potatoes without added C. casei cells as a control. The sensory evaluation was carried out using a scoring system for the spiciness enhancement effect ranging from 0 to 10 points (the higher the score, the stronger the effect, and a score of 1 or above was considered effective). The spiciness of the control product was scored as 0 points, and the spiciness of the mashed potatoes containing 0.3 wt% black pepper was scored as 5 points.

[0224] The results are shown in Table 10. In the table, the "score" indicates the average score from three expert panelists. In the table, the "concentration (%)" indicates the concentration of dried C. casei JCM 12072 cells. The pungency-enhancing effect was very weak when 0.005 wt% C. casei cells were added, but the thickness-imparting and pungency-enhancing effects were confirmed when C. casei cells were added at concentrations of 0.01% or higher. Furthermore, when C. casei cells were added at a concentration of 1 wt%, an off-flavor and powdery texture were observed, tending to alter the flavor quality. These results demonstrate that the addition of 0.01 to 0.5 wt% C. casei cells to black pepper-containing mashed potatoes enhanced the thickness and pungency without imparting any off-flavor. These findings demonstrate that the addition of C. casei cells (e.g., 0.01 to 0.5%) can improve the flavor of foods (e.g., spice-containing foods), by imparting a richer flavor and enhancing the spicy sensation.

[0225]

[0226] Example 8: Effect of adding a seasoning containing C. casei cells to a cheese sauce The C. casei cell-containing seasoning prepared in Example 3 (3-5) was added to a commercially available Alfred cheese sauce (BERUTOLLI Alfred cheese sauce; manufactured by Unilever Japan Co., Ltd.) at a concentration of 0.1 to 0.3 wt % (corresponding to 0.007 to 0.021 wt % of the bacterial cell mass) to obtain an Alfred cheese sauce containing a seasoning containing C. casei cells. A sensory evaluation of the Alfred cheese sauce containing a seasoning containing C. casei cells was performed by a panel of three experts using the free-word method, using a commercially available Alfred cheese sauce without the seasoning containing C. casei cells as a control.

[0227] Sensory evaluation confirmed that Alfredo cheese sauce containing C. casei cells had enhanced thickness, umami, and richness compared to Alfredo cheese sauce without C. casei cells. These results indicate that the addition of C. casei cells to the seasoning enhances the thickness, umami, and richness of cheese, improving the palatability of cheese-containing foods. These findings demonstrate that the addition of C. casei cells (e.g., the addition of a fermented seasoning containing C. casei cells) can improve the flavor of foods, such as imparting a richer taste.

[0228] Example 9: Effect of adding a seasoning containing C. casei cells to tomato sauce The seasoning containing C. casei cells prepared in Example 3 (3-5) was added to a commercially available tomato sauce (manufactured by RAGU) at a concentration of 0.1 to 0.3 wt % (equivalent to 0.007 to 0.021 wt % of the bacterial cell mass) to obtain a tomato sauce containing a seasoning containing C. casei cells. A sensory evaluation of the tomato sauce containing a seasoning containing C. casei cells was performed by a panel of three experts using the free-word method, using a commercially available tomato sauce without the seasoning containing C. casei cells as a control.

[0229] Sensory evaluation confirmed that the tomato sauce containing the C. casei-containing seasoning had enhanced thickness, umami, complexity, and a sense of maturity compared to the tomato sauce without the C. casei-containing seasoning. These results indicate that the addition of the C. casei-containing seasoning enhances the thickness, umami, complexity, and maturity of tomatoes, improving the palatability of foods containing tomatoes. These findings demonstrate that the addition of C. casei (e.g., the addition of a fermented seasoning containing C. casei) can improve the flavor of foods, such as imparting a richer taste.

[0230] Example 10: Effect of adding a seasoning containing C. casei cells to curry sauce. 9.4 parts by weight of boiling water was added to 1 part by weight of commercially available powdered curry roux (manufactured by House Corporation) and stirred to obtain a curry sauce. The C. casei cell-containing seasoning prepared in Example 3 (3-5) was added to this curry sauce at 0.1 to 0.3 wt % (corresponding to 0.007 to 0.021 wt % of the bacterial mass), yielding a curry sauce containing the C. casei cell-containing seasoning. A sensory evaluation of the curry sauce containing the C. casei cell-containing seasoning was conducted by a panel of three experts using the free-word method, using a curry sauce without the C. casei cell-containing seasoning as a control.

[0231] Sensory evaluation confirmed that curry sauce containing C. casei had enhanced thickness, umami, and spiciness compared to curry sauce without C. casei. These results indicate that the addition of C. casei-containing seasonings enhances curry's thickness, umami, and spiciness, improving its palatability. These findings demonstrate that the addition of C. casei (e.g., the addition of fermented seasonings containing C. casei) can improve the flavor of foods (e.g., spice-containing foods), by imparting a richer flavor and enhancing the spicy sensation.

[0232] Example 11: Investigation of the optimal C. casei cell / broth ratio for curry sauce. 9.4 parts by weight of boiling water was added to 1 part by weight of commercially available powdered curry roux (House Corporation) and stirred to obtain a curry sauce. The C. casei broth seasoning prepared in Example 3 (3-6) was added to this curry sauce at a concentration of 0.2% by weight and stirred to obtain a curry sauce containing C. casei broth seasoning. The dried cells of C. casei JCM 12072 prepared in Example 2 were added to the curry sauce containing C. casei broth seasoning at concentrations of 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, or 1% by weight and stirred to obtain a curry sauce containing C. casei broth seasoning and C. casei cells. The weight ratio of the amount of C. casei cells added to the amount of C. casei broth seasoning added is also referred to as the "C. casei cells / broth ratio." A sensory evaluation of the curry sauce thus obtained was conducted by a panel of three experts, using a curry sauce without C. casei broth seasoning or C. casei cells as a control. The sensory evaluation was conducted using a scoring system ranging from 0 to 10 points (higher scores indicate stronger effects, with 1 or above being considered effective) to evaluate the thickness-imparting effect and the spiciness-enhancing effect. The spiciness of the control product was scored as 0, while a spiciness twice as strong as the control product was scored as 5.

[0233] The results are shown in Table 11. In the table, the "score" indicates the average score of three expert panelists. Addition of C. casei broth seasoning alone did not result in an enhanced spiciness, whereas addition of C. casei broth seasoning and C. casei cells did. Furthermore, compared with the addition of C. casei cells alone (Example 6; Table 9), addition of C. casei broth seasoning and C. casei cells resulted in an increased score for enhanced spiciness at each C. casei cell concentration, i.e., an improved spiciness enhancement effect. Specifically, when C. casei broth seasoning was used in combination with C. casei cells, an enhanced spiciness effect was observed at C. casei cell concentrations of 0.005 wt % or higher. Furthermore, when the C. casei cell concentration was increased to 1 wt %, an off-flavor and bitterness were observed, tending to alter the taste quality. These results indicate that when C. casei broth seasoning is used in combination with C. casei cells, adding 0.005 to 0.5 wt% of C. casei cells to curry sauce enhances the spiciness without imparting any off-flavor. Specifically, the spiciness-enhancing effect was confirmed when the C. casei cell / broth ratio was 0.025 to 2.5. Furthermore, a C. casei cell / broth ratio of 5 tended to result in an off-flavor and a change in flavor quality. These results indicate that when C. casei broth seasoning is used in combination with C. casei cells, adding C. casei broth seasoning and C. casei cells to curry sauce at a C. casei cell / broth ratio of 0.05 to 2.5 enhances the spiciness without imparting any off-flavor. From the above, it was revealed that the addition of C. casei cells can improve the flavor of foods (e.g., spice-containing foods), such as by enhancing the spicy flavor, and that the flavor-improving effect, such as by enhancing the spicy flavor, can be further enhanced by using C. casei broth seasoning in combination with C. casei cells (e.g., at a C. casei cell / broth ratio of 0.025 to 2.5).

[0234]

[0235] Example 12: Investigation of the optimal C. casei cell / broth ratio for black pepper-containing mashed potatoes Mashed potatoes were obtained by adding 6 parts by weight of boiling water to 1 part by weight of commercially available dried mashed potatoes (manufactured by Calbee, Inc.) and stirring. Commercially available black pepper powder (manufactured by Gaban Co., Ltd.) was added to the mashed potatoes at a concentration of 0.15% by weight and stirred to obtain black pepper-containing mashed potatoes. The C. casei broth seasoning prepared in Example 3 (3-6) was added to the black pepper-containing mashed potatoes at a concentration of 0.2% by weight and stirred to obtain black pepper-containing mashed potatoes with added C. casei broth seasoning. The dried cells of C. casei JCM 12072 prepared in Example 2 were added to black pepper-containing mashed potatoes containing C. casei broth seasoning at concentrations of 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, or 1% by weight and stirred to obtain black pepper-containing mashed potatoes containing C. casei broth seasoning and C. casei cells. A sensory evaluation of the resulting black pepper-containing mashed potatoes was conducted by a three-member panel of experts, using black pepper-containing mashed potatoes without C. casei broth seasoning or C. casei cells as a control. The sensory evaluation was conducted using a scoring system ranging from 0 to 10 points (higher scores indicate stronger effects, with scores of 1 or higher indicating positive effects) to evaluate the thickness-imparting and pungency-enhancing effects. The spiciness enhancing effect was evaluated on a scale of 0 points for the spiciness of the control product and 5 points for the spiciness of the mashed potatoes containing 0.3% by weight of black pepper.

[0236] The results are shown in Table 12. In the table, the "score" indicates the average score from three expert panelists. Addition of C. casei broth seasoning alone did not result in a pungency-enhancing effect, whereas addition of C. casei broth seasoning and C. casei cells did. Furthermore, compared with the addition of C. casei cells alone (Example 7; Table 10), addition of C. casei broth seasoning and C. casei cells resulted in a higher score for pungency enhancement at each C. casei cell concentration, i.e., an improved pungency-enhancing effect. Specifically, when C. casei broth seasoning was used in combination with C. casei cells, a pungency-enhancing effect was confirmed at C. casei cell concentrations of 0.01 wt % or higher. Furthermore, when the C. casei cell concentration was increased to 1 wt %, an off-flavor and bitterness were observed, tending to alter the taste quality. These results indicate that when C. casei broth seasoning is used in combination with C. casei cells, adding 0.01 to 0.5 wt% of C. casei cells to black pepper-containing mashed potatoes enhances pungency without imparting any off-flavor. Specifically, the pungency-enhancing effect was confirmed when the C. casei cell / broth ratio was 0.025 to 2.5. Furthermore, a C. casei cell / broth ratio of 5 tended to result in an off-flavor and alter the flavor quality. These results indicate that when C. casei broth seasoning is used in combination with C. casei cells, adding a C. casei cell / broth ratio of 0.05 to 2.5 to black pepper-containing mashed potatoes enhances pungency without imparting any off-flavor. From the above, it was revealed that the addition of C. casei cells can improve the flavor of foods (e.g., spice-containing foods), such as by enhancing the spicy flavor, and that the flavor-improving effect, such as by enhancing the spicy flavor, can be further enhanced by using C. casei broth seasoning in combination with C. casei cells (e.g., at a C. casei cell / broth ratio of 0.025 to 2.5).

[0237]

[0238] Example 13: Preparation of Dried Bacterial Cells of Various Bacteria (13-1) Bacillus subtilis JCM 1465 was cultured in a jar using the medium shown in Table 13. The culture temperature was maintained at a constant 30°C, the culture pH was controlled to 6.8 with ammonia gas, and the dissolved oxygen concentration was controlled by stirring to be 23% or higher relative to the saturated dissolved oxygen concentration of 100%. The resulting culture solution was sterilized in two stages: at 80°C for 60 minutes and at 121°C for 60 minutes. The medium components were removed by centrifugation to obtain a bacterial cell pellet. The bacterial cells were then washed twice by adding an equal amount of water to the medium and removing the supernatant by centrifugation. The washed bacterial cell pellet was frozen at -80°C and then the water was removed using a freeze dryer to obtain dried Bacillus subtilis JCM 1465 cells.

[0239]

[0240] (13-2) Gluconacetobacter kombuchae NBRC 14820 was cultured in a Sakaguchi flask containing 50 mL of the medium shown in Table 14, and the flask was immersed at 30°C for 24 hours. The resulting culture was sterilized at 80°C for 30 minutes, and the medium components were removed by centrifugation to obtain a bacterial cell pellet. The bacterial cells were then washed by adding an equal volume of water to the medium and centrifuging to remove the supernatant, repeating this process twice. The washed bacterial cell pellet was frozen at -80°C and then the water was removed using a freeze dryer to obtain dried Gluconacetobacter kombuchae NBRC 14820 bacterial cells.

[0241]

[0242] (13-3) Preparation of dried cells of Brevibacterium casei, Corynebacterium flavescens, and Brachybacterium alimentarium Brevibacterium casei DSM 20657, Corynebacterium flavescens NBRC 14136, Brachybacterium alimentarium NBRC 16118, Lactobacillus hilgardii NBRC 15886, and Lactobacillus Brevis JCM 1102 were cultured in a 300 mL pleated Erlenmeyer flask containing 100 mL of the medium shown in Table 15 at 30°C for 24 hours by rotation. Lactobacillus mali NBRC 102159 was cultured in a 300 mL fluted Erlenmeyer flask containing 100 mL of the medium shown in Table 15 supplemented with 20 g / L coconut oil, and rotated at 30°C for 48 hours. The resulting culture was sterilized at 120°C for 20 minutes, and the medium components were removed by centrifugation to obtain a bacterial cell pellet. The bacterial cells were then washed twice by adding an equal volume of water to the medium and removing the supernatant by centrifugation. The washed bacterial cell pellet was frozen at -80°C and then lyophilized in a freeze dryer to obtain dried bacterial cells.

[0243]

[0244] (13-4) Excipients were removed from sterilized Enterococcus faecalis cells EC-12 and sterilized Bifidobacterium longum cells BR-108 (both manufactured by Combi Corporation) to obtain dried cells of these bacteria.

[0245] Example 14: Effect of Adding Various Bacterial Cells to Curry Sauce Curry sauce was obtained by adding 9.4 parts by weight of boiling water to 1 part by weight of commercially available powdered curry roux (House Corporation) and stirring. The dried cells of each bacterial strain obtained in Example 13 were added to this curry sauce to a concentration of 0.1% and suspended to obtain a bacterial-added curry sauce. A sensory evaluation of the bacterial-added curry sauce was conducted by a four-member expert panel, using a curry sauce without bacterial cells as a control. The sensory evaluation was conducted using a scoring system ranging from 0 to 10 points (higher scores indicate a stronger effect, with 1 point or higher being considered effective) to evaluate the spiciness enhancement effect. A score of 0 was assigned to the spiciness of the control, and a score of 5 was assigned to a spiciness twice that of the control.

[0246] The results are shown in Table 16. In the table, the "score" indicates the average score given by four expert panelists. When gram-negative bacterial cells were added, almost no effect of enhancing spiciness was observed. On the other hand, when gram-positive bacterial cells were added, an effect of enhancing spiciness was observed. In particular, when Actinobacteria bacterial cells were added, a significant effect of enhancing spiciness was observed. From the above, it was revealed that the addition of gram-positive bacterial cells (particularly bacteria of the Actinobacteria phylum) can have the effect of improving the flavor, such as enhancing the spiciness, in foods (e.g., spice-containing foods).

[0247]

[0248] Experimental Example 14: Evaluation of the Effect of Enzyme Treatment of Bacterial Cells Dried bacterial cells of C. casei JCM 12072 strain (prepared as in Example 2) were treated with lysozyme or protease as follows: 50 mg of dried bacterial cells were suspended in 4.5 mL of distilled water, 0.5 mL of enzyme solution was added, and the pH was adjusted to 6.5 with 1 M hydrochloric acid or 1 M sodium hydroxide. The mixture was incubated at 45°C for 5 hours to allow the enzyme reaction. After the enzyme reaction, the mixture was centrifuged at 2500 rpm at 20°C to obtain a precipitate. 5 mL of distilled water was added to the obtained precipitate, and the mixture was centrifuged again under the same conditions as above. Washing with distilled water and centrifugation were repeated two more times to obtain enzyme-treated bacterial cells. The enzyme solutions used were a 1% aqueous lysozyme solution (Lysozyme BIO, manufactured by Japan Biocon Co., Ltd., dissolved in distilled water to a concentration of 1%) and a 0.1% aqueous papain solution (Papain W-40, manufactured by Amano Enzyme Inc., dissolved in distilled water to a concentration of 0.1%). The dried bacterial cells before enzyme treatment are also referred to as "untreated bacterial cells."

[0249] Mashed potatoes were obtained by adding 6 parts by weight of boiling water to 1 part by weight of commercially available dried mashed potatoes (manufactured by Calbee, Inc.) and stirring. Commercially available chili pepper powder was added to the mashed potatoes at a concentration of 0.2% by weight and stirred to obtain mashed potatoes containing 0.2% by weight of chili pepper. Untreated cells or enzyme-treated cells were added to the chili pepper-containing mashed potatoes at a concentration of 0.1% by weight, calculated as 0.1% by weight of the original amount of chili pepper, and the spiciness intensity was evaluated. The spiciness intensity was evaluated by a sensory evaluation conducted by two experienced sensory evaluation panels, with the spiciness intensity of the 0.2% by weight chili pepper-containing mashed potatoes being scored as 0 and the spiciness intensity of the 0.4% by weight chili pepper-containing mashed potatoes being scored as 100.

[0250] The results are shown in Table 17. The pungency intensity of the sample containing untreated cells was 100 points. The pungency intensity of the sample containing protease-treated cells was 90 points, indicating that the pungency-enhancing effect of the protease treatment was hardly attenuated. On the other hand, the pungency intensity of the sample containing lysozyme-treated cells was 20 points, indicating that the pungency-enhancing effect of the lysozyme treatment was significantly attenuated.

[0251] Curry sauce was prepared by adding 400 mL of boiling water to 28 g of commercially available curry roux ("Java Curry PRIME Calorie Off" manufactured by House Foods Industry Co., Ltd.) and stirring. To this curry sauce, 0.1 wt% of untreated cells or 0.1 wt% of enzyme-treated cells were added, calculated based on the original amount of cells, and the spiciness intensity was evaluated. The spiciness intensity of curry sauce without added cells was scored as 0, and the spiciness intensity of curry sauce with 0.1 wt% untreated cells was scored as 100, based on the sensory evaluation by three experienced sensory panelists.

[0252] The results are shown in Table 18. The pungency intensity of the sample containing protease-treated cells was 95 points, indicating that the protease treatment did not significantly reduce the pungency-enhancing effect. On the other hand, the pungency intensity of the sample containing lysozyme-treated cells was 20 points, indicating that the lysozyme treatment significantly reduced the pungency-enhancing effect.

[0253] These results suggest that peptidoglycan, one of the components of the cell wall of Gram-positive bacteria, contributes to the flavor-improving effect of Gram-positive bacterial cells, such as enhancing the spicy flavor.

[0254]

[0255]

[0256] Example 15: Preparation and sensory evaluation of fermented tomato juice using C. casei First, seed cultures of C. casei JCM 12072 strain and RUN5-2-96 strain (NITE ABP-03688) were performed by flask culture using the medium shown in Table 19. The culture temperature was maintained constant at 30°C, and the culture was performed for 30 hours under rotary agitation conditions of 160 rpm. After the culture was completed, the bacterial cells were separated from the culture medium by centrifugation. A washing procedure in which the bacterial cells were suspended in an equal volume of physiological saline to the medium and centrifuged again was repeated three times. This washing procedure removed medium components, and viable bacterial cells of JCM 12072 strain and RUN5-2-96 strain were obtained.

[0257]

[0258] Next, viable cells of strain JCM 12072 and strain RUN5-2-96 (AJ111891) were cultured in jars using commercially available salt-free tomato juice (Kagome). Viable cells of each strain were inoculated to achieve an OD620 of 4. The culture pH was controlled between 5.5 and 7.5 by intermittent addition of sodium hydroxide, and the culture temperature was controlled at 30°C for 16 hours. The culture medium was sampled at 0, 8, and 16 hours after the start of culture, and C. casei tomato juice fermentation products were obtained.

[0259] Each C. casei tomato juice fermentation product was added to commercially available tomato sauce (manufactured by RAGU) at a concentration of 0.5% to prepare a tomato sauce containing the C. casei tomato juice fermentation product. A sensory evaluation of the tomato sauce containing the C. casei tomato juice fermentation product was performed using a tomato sauce without the C. casei tomato juice fermentation product as a control.

[0260] The results are shown in Table 20. In the table, the "rating" is ranked in the order of A > B > C, with A representing the highest rating and C representing the lowest rating. It was confirmed that the tomato sauce containing C. casei fermented tomato juice had enhanced umami, tomato richness, flavor, meaty texture, and / or spicy flavor compared to the tomato sauce (control) without C. casei fermented tomato juice (Nos. 2-3 and 5-6). These results demonstrate that the addition of C. casei fermented tomato juice improves the palatability of foods containing tomatoes. These results are consistent with the results of Example 9. These findings demonstrate that the addition of tomato juice fermented with C. casei can improve the flavor of foods.

[0261]

[0262] According to the present invention, the flavor of food can be improved.

Claims

1. A composition for improving the flavor of food, containing the following component (A): (A) A fraction containing the cell wall of Gram-positive bacteria.

2. The composition according to claim 1, wherein the improvement of the flavor is enhancement of spiciness and / or imparting umami.

3. The composition according to claim 1, wherein the improvement of the flavor is enhancement of the pungency of spices.

4. The composition according to claim 1, further containing spices.

5. A composition containing the following component (A) and spices: (A) A fraction containing the cell wall of Gram-positive bacteria.

6. The composition according to claim 1, wherein the component (A) is the cells or fragments of the Gram-positive bacteria.

7. The composition according to claim 1, wherein the Gram-positive bacteria are bacteria belonging to the phylum Actinobacteria or the phylum Firmicutes.

8. The composition according to claim 1, wherein the Gram-positive bacteria are bacteria belonging to the phylum Actinobacteria.

9. The composition according to claim 1, wherein the Gram-positive bacteria are coryneform bacteria, bacteria belonging to the family Bifidobacteriaceae, bacteria belonging to the family Dermabacteraceae, bacteria belonging to the family Bacillaceae, bacteria belonging to the family Enterococcaceae, or bacteria belonging to the family Lactobacillaceae.

10. The composition according to claim 1, wherein the Gram-positive bacteria are bacteria belonging to the genus Corynebacterium, Brevibacterium bacteria belonging to the genus Brevibacterium, bacteria belonging to the genus Bifidobacterium , bacteria belonging to the genus Brachybacterium, bacteria belonging to the genus Bacillus, Enterococcus bacteria belonging to the genus Enterococcus, or bacteria belonging to the genus Lactobacillus .

11. The Gram-positive bacteria are Corynebacterium casei, The composition according to claim 1, which is Corynebacterium flavescens, Brevibacterium casei, Bifidobacterium longum, Brachybacterium alimentarium, Bacillus subtilis, Enterococcus faecalis, Lactobacillus mali, Lactobacillus hilgardii, or Lactobacillus Brevis.

12. The composition according to claim 1, wherein the content of the component (A) is 0.1% (w / w) or more in terms of the dry weight of the cells of the gram-positive bacterium.

13. Furthermore, the composition according to claim 1, which contains the following component (B): (B) One or more components selected from the group consisting of L-amino acids, nucleic acids, and organic acids.

14. The composition according to claim 13, which contains at least the L-amino acid, and the L-amino acid is L-glutamic acid.

15. The composition according to claim 14, wherein the content of L-glutamic acid is 0.1 to 20 parts by weight with respect to 1 part by weight of the component (A) in terms of the dry weight of the cells of the gram-positive bacterium.

16. The composition according to claim 4, wherein the content of the spice is 0.2 to 500 parts by weight with respect to 1 part by weight of the component (A) in terms of the dry weight of the cells of the gram-positive bacterium.

17. The composition according to claim 4, wherein the spice is one or more spices selected from the group consisting of spices of the Burseraceae family, spices of the Zingiberaceae family, spices of the Lamiaceae family, spices of the Apiaceae family, spices of the Solanaceae family, spices of the Buxaceae family, spices of the Alliaceae family, spices of the Myrtaceae family, spices of the Rosaceae family, spices of the Polygonaceae family, spices of the Brassicaceae family, spices of the Zingiberaceae family, and spices of the Rutaceae family.

18. The composition according to claim 4, wherein the spice is a spicy spice.

19. The composition according to any one of claims 1 to 18, which is a seasoning.

20. The composition according to any one of claims 1 to 18, wherein the component (A) is produced by culturing the Gram-positive bacterium in a medium containing a food raw material.

21. The composition according to claim 20, wherein the raw material contained in the medium is tomato.

22. The composition according to any one of claims 1 to 18, wherein the component (A) is heat-treated.

23. The composition according to any one of claims 1 to 18, wherein the Gram-positive bacterium is a bacterium having the ability to produce L-glutamic acid and having one or more mutations selected from the mutations shown in Table 1. described composition. 【Table 1-1】 【Table 1-2】 【Table 1-3】 【Table 1-4】 【Table 1-5】

24. The composition according to any one of claims 1 to 18, further containing a culture of a Gram-positive bacterium.

25. A method for improving the flavor of a food, comprising the step of adding the following component (A) to a food raw material: (A) A fraction containing the cell wall of a Gram-positive bacterium.

26. A method for producing a food with improved flavor, comprising the step of adding the following component (A) to a food raw material: (A) A fraction containing the cell wall of a Gram-positive bacterium.

27. The method according to claim 25 or 26, wherein the improvement of the flavor is enhancement of spiciness and / or imparting of umami.

28. The method according to claim 25 or 26, wherein the improvement of the flavor is enhancement of the spiciness of the spice.

29. The method according to claim 25 or 26, wherein the component (A) is the cells or fragments thereof of the Gram-positive bacterium.

30. The method according to claim 25 or 26, wherein the Gram-positive bacterium is a bacterium belonging to the phylum Actinobacteria or Firmicutes.

31. The method according to claim 25 or 26, wherein the Gram-positive bacterium is a bacterium belonging to the phylum Actinobacteria.

32. The method according to claim 25 or 26, wherein the Gram-positive bacterium is a bacterium belonging to the genus Corynebacterium, a bacterium belonging to the family Bifidobacteriaceae, a bacterium belonging to the family Dermabacteraceae, a bacterium belonging to the family Bacillaceae, a bacterium belonging to the family Enterococcaceae, or a bacterium belonging to the family Lactobacillaceae.

33. The Gram-positive bacterium is a bacterium belonging to the genus Corynebacterium, Brevibac terium, a bacterium belonging to the genus Bifidobacterium , a bacterium belonging to the genus Brachybacterium, a bacterium belonging to the genus Bacillus, Ent erococcus, or a bacterium belonging to the genus Lactobacillus The method according to claim 25 or 26.

34. The Gram-positive bacterium is Corynebacterium casei, Co rynebacterium flavescens, Brevibacterium casei, Bifidobacterium longum, Brachybacterium alimentarium, Bacillus subtilis, Enterococcus faecalis, Lactobacillus mali, Lactobacillus hilgardii, or Lactobacillus Brevis. The method according to claim 25 or 26.

35. The method according to claim 25 or 26, wherein the component (A) is added so that its consumption concentration is 0.005 to 2% (w / w) in terms of the dry weight of the cells of the Gram-positive bacterium.

36. The method according to claim 25 or 26, further comprising the step of adding the following component (B) to the raw material of the food: One or more components selected from the group consisting of (B) L-amino acids, nucleic acids, and organic acids of the component. **Claim 37** The method according to claim 36, wherein at least the L-amino acid is added and the L-amino acid is L-glutamic acid. **Claim 38** The method according to claim 37, wherein L-glutamic acid is added so that its ingestion concentration is 0.01 to 2% (w / w). **Claim 39** The method according to claim 37, wherein the content of L-glutamic acid in the food is 0.1 to 20 parts by weight with respect to 1 part by weight of the component (A) converted to the dry weight of the cells of the gram-positive bacterium. **Claim 40** The method according to claim 25 or 26, wherein the food contains a spice. **Claim 41** The method according to claim 40, wherein the content of the spice in the food is 0.01 to 2% (w / w) as the ingestion concentration. **Claim 42** The method according to claim 40, wherein the content of the spice in the food is 0.2 to 500 parts by weight with respect to 1 part by weight of the component (A) converted to the dry weight of the cells of the gram-positive bacterium. **Claim 43** The method according to claim 40, wherein the spice is one or more spices selected from the group consisting of spices of the Meliaceae family, Zingiberaceae family, Lamiaceae family, Apiaceae family, Solanaceae family, Bixaceae family, Alliaceae family, Moraceae family, Thymelaeaceae family, Fabaceae family, Polygonaceae family, Brassicaceae family, Zingiberaceae family, and Rutaceae family. **Claim 44** The method according to claim 40, wherein the spice is a spicy spice. **Claim 45** The method according to claim 25 or 26, wherein the component (A) is produced by culturing the gram-positive bacterium in a medium containing a food raw material. **Claim 46** The method according to claim 45, wherein the raw material contained in the medium is tomato. **Claim 47** The method according to claim 25 or 26, wherein the component (A) is heat-treated. **Claim 48** The method according to claim 25 or 26, wherein the gram-positive bacterium is a bacterium having the ability to produce L-glutamic acid and having one or more mutations selected from the mutations shown in Table 1. 【Table 2-1】 【Table 2-2】 【Table 2-3】 【Table 2-4】 【Table 2-5】 **Claim 49** The method according to claim 25 or 26, further comprising the step of adding a culture of Gram-positive bacteria to a food raw material.

50. A seasoning containing the following component (A): (A) A fraction containing the cell wall of Gram-positive bacteria.

51. The seasoning according to claim 50, wherein the component (A) is a cell body of the Gram-positive bacteria or a fragment thereof.

52. The seasoning according to claim 50 or 51, further containing the following component (B): (B) One or more components selected from the group consisting of L-amino acids, nucleic acids, and organic acids.

53. The seasoning according to claim 52, containing at least the L-amino acid, and the L-amino acid being L-glutamic acid.

54. The seasoning according to claim 50 or 51, further containing spices.

55. The seasoning according to claim 50 or 51, wherein the component (A) is produced by culturing the Gram-positive bacteria in a medium containing a food raw material.

56. The seasoning according to claim 55, wherein the raw material contained in the medium is tomato.

57. The seasoning according to claim 50 or 51, wherein the component (A) is heat-treated.

58. The seasoning according to claim 50 or 51, wherein the Gram-positive bacteria are bacteria having the ability to produce L-glutamic acid and having one or more mutations selected from the mutations shown in Table 1. 【Table 3-1】 【Table 3-2】 【Table 3-3】 【Table 3-4】 【Table 3-5】

59. The seasoning according to claim 50 or 51, further containing a culture of Gram-positive bacteria.

60. Bacteria having the ability to produce L-glutamic acid, Bacteria having one or more mutations selected from mutations A-1 to A-135 shown in Table 1. 【Table 4-1】 【Table 4-2】 【Table 4-3】 【Table 4-4】 【Table 4-5】

61. The bacteria according to claim 60, having 50 or more mutations selected from mutations A-1 to A-135 shown in Table 1.

62. The bacteria according to claim 60 or 61, having 100 or more mutations selected from mutations A-1 to A-135 shown in Table 1.

63. The bacteria according to claim 60 or 61, having mutations A-1 to A-135 shown in Table 1.

64. The bacteria according to claim 60 or 61, further having one or more mutations selected from mutations B-1 to B-92 shown in Table 1.

65. The bacteria according to claim 64, having 30 or more mutations selected from mutations B-1 to B-92 shown in Table 1.

66. A bacterium according to claim 64, having 60 or more mutations selected from mutations B-1 to B-92 shown in Table 1. The bacterium according to claim 1. **Claim 67** The bacterium according to claim 60 or 61, which is a coryneform bacterium. **Claim 68** The bacterium according to claim 60 or 61, which is a bacterium belonging to the genus Corynebacterium. The bacterium according to claim 60 or 61. **Claim 69** The bacterium according to claim 60 or 61, which is Corynebacterium casei. The bacterium according to claim 60 or 61. **Claim 70** The bacterium according to claim 60 or 61, which is a mutant strain derived from Corynebacterium casei JCM 12072 strain. **Claim 71** A method for producing a composition for improving the flavor of food, comprising: a step of culturing a gram-positive bacterium in a medium to obtain a culture; wherein the composition contains the following component (A): (A) A fraction containing the cell wall of the gram-positive bacterium. **Claim 72** The method according to claim 71, wherein the improvement of the flavor is enhancement of a spicy feeling and / or imparting of umami. **Claim 73** The method according to claim 71 or 72, wherein the improvement of the flavor is enhancement of the spiciness of the spice. **Claim 74** The method according to claim 71 or 72, wherein the composition further contains a spice. **Claim 75** The method according to claim 71 or 72, wherein the component (A) is the cell body of the gram-positive bacterium or a fragment thereof. **Claim 76** The method according to claim 71 or 72, wherein the composition further contains the following component (B): (B) One or more components selected from the group consisting of L-amino acids, nucleic acids, and organic acids. **Claim 77** The method according to claim 76, wherein the composition contains at least the L-amino acid, and the L-amino acid is L-glutamic acid. **Claim 78** The method according to claim 71 or 72, further comprising a step of heat-treating the culture. **Claim 79** The method according to claim 71 or 72, wherein the gram-positive bacterium is a bacterium having the ability to produce L-glutamic acid and having one or more mutations selected from the mutations shown in Table 1. 【Table 5-1】 【Table 5-2】 【Table 5-3】 【Table 5-4】 【Table 5-5】 **Claim 80** The method according to claim 71 or 72, wherein the composition further contains a culture of a gram-positive bacterium. **Claim 81** A method for producing a composition for enhancing the umami of food, comprising: A step of culturing the bacterium according to claim 60 or 61 in a medium to obtain a culture containing L-glutamic acid comprising a method, wherein the composition contains the L-glutamic acid

82. The method according to claim 81, wherein the composition further contains the following component (A): (A)A fraction containing the cell wall of Gram-positive bacteria

83. A method for producing L-glutamic acid, comprising a step of culturing the bacterium according to claim 60 or 61 in a medium to obtain a culture containing L-glutamic acid; and a step of recovering the L-glutamic acid The method comprising

84. A composition for enhancing the umami taste of food, containing L-glutamic acid wherein the L-glutamic acid is produced by culturing the bacterium according to claim 60 or 61 in a medium

85. The composition according to claim 84, further containing the following component (A): (A)A fraction containing the cell wall of Gram-positive bacteria

86. A method for enhancing the umami taste of food, comprising a step of adding L-glutamic acid to a food raw material wherein the L-glutamic acid is produced by culturing the bacterium according to claim 60 or 61 in a medium

87. A method for producing a food with enhanced umami taste, comprising a step of adding L-glutamic acid to a food raw material wherein the L-glutamic acid is produced by culturing the bacterium according to claim 60 or 61 in a medium

88. The method according to claim 86, further comprising a step of adding the following component (A) to a food raw material: (A)A fraction containing the cell wall of Gram-positive bacteria

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