Method for preparing flavorings by mixed fermentation of heterologous microorganisms

Abstract

The present invention relates to a method for preparing a seasoning using a process of mixing two or more microorganisms producing different products and simultaneously fermenting the microorganisms. In a method for preparing a seasoning, heterogeneous microorganisms that produce different products (i.e., different amino acids, nucleic acids and / or organic acids) are mixed and fermented, thereby making it possible to prepare a seasoning that improves the taste and flavor of a food product by means of a fermentation solution containing amino acids, nucleic acids and / or organic acids, and the overall sensory characteristics of the food are improved. The seasoning can be applied to various food fields.

Description

Background Art 1. Technical Field

[0001] The present invention relates to a method for producing flavoring agents by a co-fermentation process using mixed fermentation of two or more different microorganisms producing different products.

[0002] 2. Related technologies

[0003] L-Glutamic acid is an acidic amino acid providing umami (or salty) taste, and is naturally present in plant foods such as kelp, broad bean paste and soy sauce and animal foods such as dairy products, meat and fish. This L-Glutamic acid is the most commonly used food flavoring agent in the whole world, and especially, by adding sodium glutamate (monosodium glutamate, MSG) produced by salt during the purification of L-Glutamic acid, there is an excellent food flavor enhancement effect, and is therefore used as a food additive in many processed foods. Recently, along with the health consciousness of consumer, the market of natural flavoring agents such as yeast extract and natural substance extract has grown rapidly, but MSG still has excellent umami taste compared with natural flavoring agents and has high competitiveness at a low price. Therefore, a lot of efforts are needed to develop the substitute of MSG.

[0004] Meanwhile, the development of the flavoring agent using multiple amino acids (such as L-lysine, L-valine and L-arginine) together with L-glutamic acid has been carried out for the purpose of enhancing fresh taste. In addition, the flavoring agent also comprising nucleic acid (such as inosinic acid and guanylic acid) or organic acid (such as succinic acid and lactic acid) has also been developed. These flavoring agents based on L-glutamic acid are usually produced by mixing the fermented liquid produced by the microorganism producing the corresponding product, or by mixing the amino acid, nucleic acid or organic acid powder produced separately. In conventional production methods, the concentration or content of the final product such as L-glutamic acid are easy to control, but because the production process must be operated twice before mixing, high production cost is required. In addition, a large amount of culture medium components and fermentation byproducts are retained in the flavoring agent produced, making it difficult to solve the problem of peculiar smell and taste change, and there is still a restriction that is not enough to provide abundant fresh taste. Therefore, a large amount of research and development is needed to develop the flavoring agent with enhanced fresh taste.

[0005] [Prior art literature]

[0006] [Patent Document]

[0007] Korean Patent No. 10-1758332

[0008] Korean Patent No. 10-1328091 Summary of the invention

[0009] It is an object of the present invention to provide a method for producing flavoring agents by mixed fermentation of two or more different microorganisms producing different products.

[0010] Another object of the present invention is to provide a method for producing a seasoning containing L-glutamic acid and L-lysine by mixed fermentation of a glutamic acid-producing microorganism and a lysine-producing microorganism.

[0011] Another object of the present invention is to provide a flavoring agent produced by the above method.

[0012] Another object of the present invention is to provide a food composition containing the above seasoning.

[0013] One aspect of the present invention provides a method for producing a flavoring agent, the method comprising the steps of inoculating a fermentation medium with a first microorganism and a second microorganism, and then producing a fermentation broth containing amino acids, nucleic acids and / or organic acids by fermenting the microorganisms, wherein the first microorganism and the second microorganism produce different products and each produces one selected from amino acids, nucleic acids and organic acids.

[0014] The term "flavoring agent" as used herein refers to a substance added to enhance the flavor of food, and is used in a broader sense than general condiments (added in small amounts at the end of a general food cooking process). The flavoring agent can be used to enhance the taste in the process of manufacturing processed foods such as ham, sausages and ramen, and when cooking at home. The term "flavoring agent" in the present invention refers to a substance having a richer umami taste and excellent sensory properties by containing one or more amino acids, nucleic acids and / or organic acids as taste components.

[0015] According to one embodiment of the present invention, the amino acid may be at least one selected from the group consisting of L-glutamic acid, L-alanine, L-valine, L-leucine, L-isoleucine, L-proline, L-phenylalanine, L-tryptophan, L-methionine, L-glycine, L-serine, L-threonine, L-cysteine, L-asparagine, L-glutamine, L-aspartic acid, L-lysine, L-arginine and L-histidine.

[0016] According to one embodiment of the present invention, the nucleic acid may be at least one selected from the group consisting of inosinic acid, guanylic acid, xanthic acid, and salts thereof.

[0017] For example, the nucleic acid may be inosine monophosphate (IMP), guanosine monophosphate (GMP), xanthosine monophosphate (XMP), etc., but is not limited thereto.

[0018] According to one embodiment of the present invention, the organic acid may be at least one selected from the group consisting of succinic acid, malic acid, citric acid, acetic acid, lactic acid, fumaric acid, tartaric acid, ascorbic acid, gluconic acid, and salts thereof.

[0019] The term "fermentation" used herein is a biological phenomenon in which organic matter contained in a culture medium is decomposed or changed into other substances by microorganisms, and means that the inoculated microorganisms decompose or convert nutrients in the fermentation medium into amino acids, nucleic acids, organic acids, etc.

[0020] The term "fermentation broth" used herein refers to a liquid culture medium containing substances produced by microorganisms through fermentation. The fermentation broth is obtained by mixed fermentation of two or more types of microorganisms producing different products, and contains not only useful substances (e.g., amino acids, nucleic acids, and organic acids) produced by each microorganism, but also by-products produced in metabolic processes and culture medium components.

[0021] The term "mixed fermentation" as used herein refers to a process in which different (or heterologous) microorganisms are fermented in a microbial fermentation process under the same conditions in a single fermenter, and the different (or heterologous) microorganisms produce different products, i.e., different kinds of amino acids, nucleic acids or organic acids.

[0022] Mixed fermentation can be carried out using microorganisms that produce different kinds of amino acids, nucleic acids or organic acids, or using a combination of amino acid-producing microorganisms and nucleic acid-producing microorganisms, a combination of amino acid-producing microorganisms and organic acid-producing microorganisms, or a combination of organic acid-producing microorganisms and nucleic acid-producing microorganisms, but the present invention is not limited thereto.

[0023] The fermentation broth produced by the mixed fermentation may contain two amino acids, nucleic acids or organic acids, or one amino acid and one nucleic acid, one amino acid and one organic acid, or one organic acid and one type of nucleic acid, but the present invention is not limited thereto.

[0024] In one example, when the first microorganism is a glutamate-producing microorganism, the second microorganism can be a lysine-producing microorganism, an arginine-producing microorganism, a histidine-producing microorganism, a tryptophan-producing microorganism, a glycine-producing microorganism, an alanine-producing microorganism, a succinic acid-producing microorganism, a lactic acid-producing microorganism, a guanylate-producing microorganism, or an inosinic acid-producing microorganism.

[0025] In addition, when the first microorganism is an inosinic acid-producing microorganism, the second microorganism may be a lysine-producing microorganism, an arginine-producing microorganism, a histidine-producing microorganism, a tryptophan-producing microorganism, a glycine-producing microorganism, an alanine-producing microorganism, a succinic acid-producing microorganism, a lactic acid-producing microorganism, or a guanylic acid-producing microorganism.

[0026] According to one embodiment of the present invention, the step may include further inoculating the fermentation medium with a third microorganism that produces a product different from the products produced by the first microorganism and the second microorganism and produces one selected from amino acids, nucleic acids and organic acids.

[0027] More specifically, the fermentation broth produced by the first microorganism, the second microorganism and the third microorganism contains three amino acids, nucleic acids or organic acids, or may contain one amino acid and two nucleic acids, two amino acids and one nucleic acid, one amino acid and two organic acids, two amino acids and one organic acid, one organic acid and two nucleic acids, two organic acids and one nucleic acid, or one amino acid, one nucleic acid and one organic acid, but the present invention is not limited thereto.

[0028] For example, when the first microorganism is a microorganism that produces glutamic acid and the second microorganism is a microorganism that produces guanylate, the third microorganism may be a microorganism that produces inosinate.

[0029] These microorganisms for producing flavoring agents can be microorganisms that produce amino acids, nucleic acids and / or organic acids, and can be naturally occurring wild-type microorganisms, or modified mutant strains to improve the production ability of wild-type microorganisms. As the microorganisms that produce amino acids, nucleic acids or organic acids, microorganisms known in the art can be used without restriction, and can be microorganisms such as Corynebacterium, Brevibacterium, Lactobacillus, Bifidobacterium, Bacillus, etc. These microorganisms can be the same genus or species, or can be different genus or species, and can be selected by the user.

[0030] According to one embodiment of the present invention, the first microorganism, the second microorganism and the third microorganism may be microorganisms of the genus Corynebacterium.

[0031] More specifically, the microorganism of the genus Corynebacterium may be Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium callunae, Corynebacterium suranareeae, Corynebacterium lubricantis, Corynebacterium doosanense, Corynebacterium efficiens, Corynebacterium uterequi, Corynebacterium stationis, Corynebacterium pacaense, Corynebacterium singulare, Corynebacterium humireducens, Corynebacterium marinum, Corynebacterium halotolerans, Corynebacterium sphaeroides ... spheniscorum), Corynebacterium freiburgense, Corynebacterium striatum, Corynebacterium canis, Corynebacterium ammoniagenes, Corynebacterium renale, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium caspium, Corynebacterium testudinoris, Corynebacterium pseudopelargi or Corynebacterium flavescens, but are not limited thereto.

[0032] In order to carry out the mixed fermentation of these two or more microorganisms, the isolated and selected microorganisms can be used alone, or the microbial mixture obtained by mixing these microorganisms can be used, and in some cases, a suitable mixture of isolated microorganisms and microbial mixtures can be used. Each microorganism can be used for fermentation under the following conditions: wherein the microorganism has grown and its ability to produce amino acids, nucleic acids and / or organic acids has been activated. In addition, it is preferred that each microorganism is subjected to seed culture to carry out microbial activation.

[0033] According to one embodiment of the present invention, the first microorganism, the second microorganism, and the third microorganism may be in a seed culture solution state obtained by single culture or co-culture.

[0034] The term "seed culture" used herein means culturing a microorganism in a small volume of culture medium before culturing the microorganism in large quantities, and the term "seed culture solution" refers to a culture containing medium components, microorganisms proliferated by seed culture, and metabolites thereof.

[0035] Seed culture can be performed using an appropriate medium and culture conditions known in the art in consideration of the characteristics of each microorganism, and those skilled in the art can easily adjust the medium and culture conditions for use.

[0036] More specifically, the culture medium for seed culture may contain nutrients necessary for the growth and proliferation of microorganisms, and may be a liquid culture medium.

[0037] The culture temperature in the seed culture can be generally 20° C. to 45° C., for example 25° C. to 40° C., or 27° C. to 37° C., and the culture can be continued until each microorganism actively grows and proliferates. The culture time can be, for example, 10 to 160 hours, 18 to 120 hours, or 20 to 80 hours.

[0038] In addition, during the incubation period, compounds such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be added to the culture medium or culture solution to regulate the pH of the culture solution in an appropriate manner. In addition, the formation of bubbles can be suppressed by using a defoamer for food during the incubation period. In addition, in order to maintain the culture medium or culture solution in an aerobic state, oxygen or oxygen-containing gas (e.g., air) can be injected into the culture solution.

[0039] The seed culture can be performed according to the microorganism concentration expected by the user, and the optical density (OD) value of the seed culture solution is measured to predict the microorganism concentration and determine whether to continue the culture.

[0040] According to one embodiment of the present invention, the seed culture solution may have an OD 610=10 to 80.

[0041] In the step of producing a fermentation broth by fermentation of two or more different microorganisms, fermentation broths containing different products may be produced by mixed fermentation (main fermentation) using different microorganisms or their seed culture broths.

[0042] Mixed fermentation is a concept opposite to a conventional individual fermentation process in which a fermentation broth containing a product of each microorganism is produced by culturing each microorganism individually.

[0043] In the present invention, by mixed fermentation, for example, L-glutamic acid can be produced by a glutamic acid-producing microorganism, L-lysine can be produced by a lysine-producing microorganism, L-arginine can be produced by an arginine-producing microorganism, and IMP can be produced by an inosinic acid-producing microorganism. In addition, due to the interaction between these products and due to the taste components such as ions, nucleic acids, organic acids and other peptides that are additionally produced during the microbial fermentation process, a fermentation broth that can express a rich umami taste and excellent sensory properties can be produced.

[0044] According to one embodiment of the present invention, in the step, the inoculum of each microorganism is preferably adjusted to control the ratio between the products (ie, amino acids, nucleic acids and / or organic acids) of the microorganisms in the fermentation broth.

[0045] More specifically, when the fermentation medium is inoculated with two different microorganisms, the inoculation ratio between the first microorganism and the second microorganism can be a ratio of 0.05 to 99.95 (first microorganism): 99.95 to 0.05 (second microorganism) relative to the total inoculum. When the fermentation medium is inoculated with three different microorganisms, the inoculation ratio between the first microorganism, the second microorganism and the third microorganism can be 0.05 to 99.95 (first microorganism): 99.95 to 0.05 (second microorganism): 99.95 to 0.05 (third microorganism) relative to the total inoculum.

[0046] For example, when the inoculation ratio between the microorganism producing glutamic acid and the microorganism producing lysine is 50 to 99.95:50 to 0.05, L-glutamic acid and L-lysine in the fermentation broth can be produced at a ratio of 0.83:1 to 99:1 by the mixed fermentation of the two microorganisms, so that they can provide a suitable taste as a seasoning. When the inoculation ratio between the microorganism producing glutamic acid and the microorganism producing arginine is 30 to 99.95:70 to 0.05, L-glutamic acid and L-arginine in the fermentation broth can be produced at a ratio of 1.04:1 to 99:1 by the mixed fermentation of the two microorganisms. When the inoculation ratio between the microorganism producing glutamic acid and the microorganism producing inosinic acid is 0.05 to 99.95:99.95 to 0.05, L-glutamic acid and IMP in the fermentation broth can be produced at a ratio of 0.02:1 to 99.8:1 by the mixed fermentation of the two microorganisms. When the inoculum ratio between the inosinic acid-producing microorganism and the lysine-producing microorganism is 0.05 to 99.95:99.95 to 0.05, IMP and L-lysine in the fermentation broth can be produced by mixed fermentation of the two microorganisms at a ratio of 0.01: 1 to 92.3: 1. When the inoculum ratio between the inosinic acid-producing microorganism and the arginine-producing microorganism is 65 to 99.95:35 to 0.05, IMP and L-arginine in the fermentation broth can be produced by mixed fermentation of the two microorganisms at a ratio of 1.04: 1 to 95.1: 1.

[0047] Mixed fermentation can be performed using an appropriate medium and fermentation conditions known in the art in consideration of the characteristics of each microorganism, and those skilled in the art can easily adjust the medium and fermentation conditions for use.

[0048] More specifically, the fermentation medium used for mixed fermentation may contain nutrients necessary for the growth and proliferation of microorganisms, and may be a liquid medium.

[0049] The fermentation medium is a medium used during the main fermentation for producing amino acids, nucleic acids and / or organic acids in large quantities, and contains nutrients necessary for the growth of each microorganism. In the present invention, since the fermentation liquid containing the fermentation medium is used as a seasoning without a separate purification process after the fermentation is completed, it is preferred that the fermentation medium is composed of a substance that can be used as a food substance and contains the minimum components and amounts required for culturing the microorganism.

[0050] According to one embodiment of the invention, the fermentation medium may be based on molasses and contain raw sugar and / or glucose.

[0051] More specifically, the fermentation medium may contain molasses, raw sugar and glucose as both a sugar source and a nutrient source for the microorganism, and may contain 1 to 30 wt% molasses based on the total sugar amount. The molasses used herein may be derived from sugar cane or sugar beet.

[0052] The fermentation medium may also contain nutrients other than the sugar source for nutritional enhancement of the microorganisms.

[0053] According to one embodiment of the present invention, the fermentation medium may further contain at least one selected from the group consisting of yeast extract, phosphoric acid and betaine.

[0054] The fermentation temperature in mixed fermentation can generally be 20°C to 45°C, such as 25°C to 40°C, or 30°C to 38°C, and the fermentation can be continued until the microorganisms actively grow and proliferate. The fermentation time can be, for example, 10 to 160 hours, 18 to 120 hours, or 20 to 100 hours.

[0055] In addition, during the incubation period, compounds such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be added to the culture medium or culture solution to regulate the pH of the culture solution in an appropriate manner. In addition, the formation of bubbles can be suppressed by adding a defoamer for food during the incubation period. In addition, in order to maintain the culture medium or culture solution in an aerobic state, oxygen or oxygen-containing gas (e.g., air) can be injected into the culture solution.

[0056] The fermentation broth produced by the mixed fermentation is used as a raw material for a flavoring agent, is characterized in that it is used intact without being mixed with other components, and contains a large amount of amino acids, nucleic acids and / or organic acids produced by microorganisms.

[0057] According to one embodiment of the present invention, the fermentation broth may contain total microbial products in an amount of 3 wt % to 90 wt % based on the total solid content.

[0058] More specifically, the fermentation broth may contain the following amounts of amino acids, nucleic acids and / or organic acids: 3% to 90% by weight, 10% to 90% by weight, 20% to 90% by weight, 30% to 90% by weight, 40% to 90% by weight, 50% to 90% by weight, 60% to 90% by weight, 70% to 90% by weight, or 80% to 90% by weight based on the total solids content. The fermentation broth may contain 5 to 150 g / L of amino acids, nucleic acids and / or organic acids.

[0059] Another aspect of the present invention provides a method for producing a flavoring containing L-glutamic acid and L-lysine, the method comprising the steps of inoculating a fermentation medium with a glutamic acid-producing microorganism and a lysine-producing microorganism, and then producing a fermentation broth containing L-glutamic acid and L-lysine by fermentation of the microorganisms.

[0060] The microorganism that produces glutamic acid and the microorganism that produces lysine can be naturally occurring wild-type microorganisms or modified mutant strains to improve the amino acid production ability of wild-type microorganisms. As this microorganism that produces glutamic acid or produces lysine, microorganisms known in the art can be used without restriction. For example, the microorganism that produces glutamic acid or produces lysine can be a microorganism of coryneform bacteria, brevibacterium, lactobacillus, bifidobacterium, bacillus, etc. The microorganism in the present invention can be the same genus or species, or different genus or species, and can be selected by the user.

[0061] According to one embodiment of the present invention, the glutamic acid-producing microorganism and the lysine-producing microorganism may be microorganisms of the genus Corynebacterium.

[0062] In one example of the present invention, a Corynebacterium glutamicum strain is used as the glutamate-producing microorganism and the lysine-producing microorganism.

[0063] In order to produce the mixed fermentation of the microorganism of glutamic acid and the microorganism of lysine, the isolated and selected microorganism can be used alone, or the microorganism mixture obtained by mixing these microorganisms can be used, and in some cases, the appropriate mixture of isolated microorganism and microorganism mixture can be used. These microorganisms that produce glutamic acid or the microorganisms that produce lysine can be used for fermentation under the state that it has grown and its ability to produce amino acid has been activated. In addition, it is preferred that each microorganism is carried out seed culture to carry out microbial activation.

[0064] According to one embodiment of the present invention, the glutamic acid-producing microorganism and the lysine-producing microorganism may be in a seed culture solution state obtained by single cultivation or co-cultivation.

[0065] Seed culture can be performed using an appropriate medium and culture conditions known in the art in consideration of the characteristics of each microorganism, and any person skilled in the art can easily adjust the medium and culture conditions for use.

[0066] More specifically, the culture medium for seed culture may contain nutrients necessary for the growth and proliferation of microorganisms, and may be a liquid culture medium.

[0067] According to one embodiment of the present invention, a culture medium for seed culture of a glutamic acid-producing microorganism may contain 4.5 to 5.5 wt % of molasses, 3 wt % of glucose, 0.85 wt % of yeast extract paste, 100 ppm of methionine, 0.6 wt % of H3PO4, 0.1 wt % of sodium succinate, 50 ppm of vitamin C, 12 ppm of thiamine HCl, 20 ppb of vitamin B12, 10 ppm of biotin, 0.4 wt % of MgSO4, and 0.01 wt % of an antifoaming agent for food, based on the total weight of the culture medium.

[0068] According to one embodiment of the present invention, the culture medium for seed culture of a lysine-producing microorganism may contain 1.5 to 3 wt% of molasses, 9 to 12 wt% of raw sugar, 1 wt% of yeast extract paste, 1.6 wt% of (NH4)2SO4, 0.3 wt% of H3PO4, 7.3 ppm of MnSO4.5H2O, 14 ppm of nicotinamide, 2.5 ppm of thiamine HCl, 1.5 ppm of CuSO4.5H2O, 0.056 ppm of biotin, 0.045 wt% of betaine and 0.01 wt% of an antifoaming agent for food, based on the total weight of the culture medium.

[0069] The culture temperature in the seed culture can be generally 20° C. to 45° C., for example 25° C. to 40° C., or 27° C. to 37° C., and the culture can be continued until each microorganism actively grows and proliferates. The culture time can be, for example, 10 to 160 hours, 18 to 120 hours, or 20 to 80 hours.

[0070] In addition, during the incubation period, compounds such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be added to the culture medium or culture solution to regulate the pH of the culture solution in an appropriate manner. In addition, the formation of bubbles can be suppressed by using a defoamer for food during the incubation period. In addition, in order to maintain the culture medium or culture solution in an aerobic state, oxygen or oxygen-containing gas (e.g., air) can be injected into the culture solution.

[0071] The seed culture can be performed according to the microorganism concentration desired by the user, and the optical density (OD) value of the seed culture solution is measured to predict the microorganism concentration and determine whether to continue the culture.

[0072] According to one embodiment of the present invention, the seed culture solution may have an OD 610 =10 to 80.

[0073] In the step of producing a fermentation broth by fermentation of a glutamate-producing microorganism and a lysine-producing microorganism, a fermentation broth containing both L-glutamate and L-lysine can be produced by mixed fermentation (main fermentation) using a glutamate-producing microorganism or its seed culture broth and a lysine-producing microorganism or its seed culture broth.

[0074] Mixed fermentation is a concept opposite to a conventional individual fermentation process of producing a fermentation broth containing a product of each microorganism by culturing each microorganism individually for amino acid production.

[0075] In the present invention, by mixed fermentation, L-glutamic acid can be produced by a glutamic acid-producing microorganism and L-lysine can be produced by a lysine-producing microorganism. In addition, due to the interaction between L-glutamic acid and L-lysine and due to the taste components such as ions, nucleic acids, organic acids and other peptides additionally produced during the microbial fermentation process, a fermentation liquid that can exhibit a rich umami taste and excellent sensory properties can be produced.

[0076] According to one embodiment of the present invention, in the step, the inoculum of each of the glutamate-producing microorganism and the lysine-producing microorganism is preferably adjusted to control the ratio between L-glutamate and L-lysine in the fermentation broth.

[0077] More specifically, the inoculation ratio between the microorganism producing glutamic acid and the microorganism producing lysine can be 0.05 to 99.95 (microorganism producing glutamic acid): 99.95 to 0.05 (microorganism producing lysine) relative to the total inoculum. By mixed fermentation of the two microorganisms inoculated at this ratio, L-glutamic acid and L-lysine in the fermentation broth can be produced at a ratio of 1:1 to 99:1, so that it can provide a suitable taste as a seasoning.

[0078] For example, when the inoculation ratio between the glutamate-producing microorganism and the lysine-producing microorganism is 50 to 99.95:50 to 0.05, L-glutamate and L-lysine in the fermentation broth can be produced at a ratio of 0.83:1 to 99:1, and when the inoculation ratio between the glutamate-producing microorganism and the lysine-producing microorganism is adjusted to 65:35, L-glutamate and L-lysine in the fermentation broth can be produced at a ratio of 1:1.

[0079] Mixed fermentation can be performed using appropriate culture media and fermentation conditions known in the art in consideration of the characteristics of each microorganism, and any person skilled in the art can easily adjust the culture media and fermentation conditions for use.

[0080] More specifically, the fermentation medium used in the mixed fermentation may be a liquid medium.

[0081] The fermentation medium is a medium used during the main fermentation for producing L-glutamic acid and L-lysine in large quantities, and contains nutrients necessary for the growth of glutamic acid-producing microorganisms and lysine-producing microorganisms. In the present invention, since the fermentation liquid containing the fermentation medium is used as a seasoning without a separate purification process after the fermentation is completed, it is preferred that the fermentation medium is composed of a substance that can be used as a food substance and contains the minimum components and amounts required for culturing the microorganisms.

[0082] According to one embodiment of the invention, the fermentation medium may be based on molasses and contain raw sugar and / or glucose.

[0083] More specifically, the fermentation medium may contain molasses, raw sugar and glucose as both a sugar source and a nutrient source for the microorganism, and may contain molasses in an amount of 1 wt % to 30 wt % based on the total sugar amount. The molasses used herein may be derived from sugar cane or sugar beet.

[0084] The fermentation medium may also contain nutrients other than the sugar source for nutritional enhancement of the microorganisms.

[0085] According to one embodiment of the present invention, the fermentation medium may further contain at least one selected from the group consisting of yeast extract, phosphoric acid and betaine.

[0086] According to one embodiment of the present invention, the fermentation medium may contain 1.5 to 3 wt% of molasses, 2.5 to 4 wt% of glucose, 0.4 to 1 wt% of yeast extract paste, 0.1 to 0.2 wt% of H3PO4, 0.05 to 0.12 wt% of betaine, and 0.001 to 0.01 wt% of an antifoaming agent for food, based on the total weight of the medium.

[0087] The fermentation temperature in the mixed fermentation can generally be 20° C. to 45° C., such as 25° C. to 40° C. or 30° C. to 38° C., and the fermentation can be continued until the content or concentration of L-glutamic acid and L-lysine obtained reaches a desired level. The fermentation time can be, for example, 10 to 160 hours, 18 to 120 hours, or 20 to 100 hours.

[0088] In addition, during the fermentation, compounds such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be added to the culture medium or fermented liquid to regulate the pH of the fermented liquid in an appropriate manner. In addition, the formation of bubbles can be suppressed by using a defoamer for food during the fermentation. In addition, in order to maintain the culture medium or fermented liquid in an aerobic state, oxygen or oxygen-containing gas (e.g., air) can be injected into the fermented liquid.

[0089] According to one embodiment of the present invention, the fermentation can be carried out by fed-batch culture, which is started at a temperature of 30°C to 33°C and maintained at a temperature of 36°C to 39°C, a pH of 6.5 to 7.5, and a dissolved oxygen concentration of 20% to 70% for 28 to 40 hours.

[0090] The fermentation broth containing L-glutamic acid and L-lysine is used as a raw material for a seasoning containing L-glutamic acid and L-lysine, characterized in that it is used intact without being mixed with other components and contains a large amount of L-glutamic acid and L-lysine.

[0091] According to one embodiment of the present invention, the fermentation broth may contain amino acids including L-glutamic acid and L-lysine in an amount of 3 wt % to 90 wt % based on the total solid content.

[0092] More specifically, the fermentation broth may contain amino acids (including L-glutamic acid and L-lysine) in an amount of 3 to 90 wt %, 10 to 90 wt %, 20 to 90 wt %, 30 to 90 wt %, 40 to 90 wt %, 50 to 90 wt %, 60 to 90 wt %, 70 to 90 wt %, or 80 to 90 wt %, based on the total solids content. The fermentation broth may contain 5 to 150 g / L of L-glutamic acid and L-lysine.

[0093] Meanwhile, the method for producing a flavoring according to the present invention may further include an additional process of using a fermentation broth as a flavoring or a flavoring containing L-glutamic acid and L-lysine.

[0094] More specifically, the method may further include the steps of separating cells from the fermentation broth and decolorizing the fermentation broth.

[0095] Cell separation can be performed without limitation by using cell separation methods and separation conditions known in the art. Examples of cell separation methods may be membrane separation, ultrafiltration, centrifugal filtration, etc., but are not limited thereto.

[0096] Decolorization can be carried out without being limited to decolorization methods and decolorization conditions known in the art. For example, activated carbon etc. can be used for decolorization, but it is not limited thereto.

[0097] In addition, the method may further include the step of filtering the decolorized fermentation broth.

[0098] The filtration can be performed without limitation to the filtration methods and filtration conditions known in the art. For example, the filtration can be performed using filter paper, filter screen, membrane filtration, ultrafiltration, etc., but is not limited thereto.

[0099] In addition, the method may further include the step of concentrating the filtered fermentation broth.

[0100] Concentration can be performed without limitation to concentration methods and concentration conditions known in the art. Some examples of concentration methods include, but are not limited to, heat concentration, vacuum concentration, freeze concentration, evaporation concentration, vacuum low-temperature concentration, and the like.

[0101] In addition, the method may further include the steps of drying and powdering the concentrated fermentation broth.

[0102] Drying can be carried out without being limited to the use of drying methods and drying conditions known in the art. Some examples of drying methods include, but are not limited to, freeze drying, vacuum drying, air drying, air drying, hot air drying, fluidized bed drying, spray drying, infrared drying, high frequency drying, etc.

[0103] Through this method, the fermentation broth can be finally obtained in a powder state, and it can be used as a natural food flavoring agent without undergoing an additional chemical purification process.

[0104] Still another aspect of the present invention provides a flavoring produced by the above method for producing a flavoring using two or more different microorganisms.

[0105] According to one embodiment of the present invention, the flavoring agent may be a natural flavoring agent containing two or three flavor components selected from amino acids, nucleic acids and organic acids.

[0106] The amino acid may be at least one selected from the group consisting of L-glutamic acid, L-alanine, L-valine, L-leucine, L-isoleucine, L-proline, L-phenylalanine, L-tryptophan, L-methionine, L-glycine, L-serine, L-threonine, L-cysteine, L-asparagine, L-glutamine, L-aspartic acid, L-lysine, L-arginine, and L-histidine.

[0107] The nucleic acid may be at least one selected from the group consisting of inosinic acid, guanylic acid, xanthic acid, and salts thereof.

[0108] For example, the nucleic acid may be inosine monophosphate (IMP), guanosine monophosphate (GMP), xanthosine monophosphate (XMP), etc., but is not limited thereto.

[0109] The organic acid may be at least one selected from the group consisting of succinic acid, malic acid, citric acid, acetic acid, lactic acid, fumaric acid, tartaric acid, ascorbic acid, gluconic acid, and salts thereof.

[0110] For example, when seasoning contains two kinds of taste components, it can contain glutamic acid and lysine, arginine, histidine, tryptophan, glycine, alanine, succinic acid, lactic acid, guanylic acid or inosinic acid.In addition, seasoning can contain inosinic acid and lysine, arginine, histidine, tryptophan, glycine, alanine, succinic acid, lactic acid or guanylic acid.

[0111] In another example, when the flavoring contains three flavor components, it may contain glutamic acid, guanylic acid, and inosinic acid.

[0112] According to one embodiment of the present invention, the flavoring agent may contain a taste component including amino acids, nucleic acids and / or organic acids in an amount of 3 wt % to 90 wt % based on the total solid content.

[0113] More specifically, the flavoring agent may contain the following amounts of flavor components (including amino acids, nucleic acids and / or organic acids): 3 wt % to 90 wt %, 10 wt % to 90 wt %, 20 wt % to 90 wt %, 30 wt % to 90 wt %, 40 wt % to 90 wt %, 50 wt % to 90 wt %, 60 wt % to 90 wt %, 70 wt % to 90 wt %, or 80 wt % to 90 wt %, based on the total solids content.

[0114] Still another aspect of the present invention provides a flavoring containing L-glutamic acid and L-lysine produced by the above-mentioned method for producing a natural flavoring containing L-glutamic acid and L-lysine using a glutamic acid-producing microorganism and a lysine-producing microorganism.

[0115] According to one embodiment of the present invention, the flavoring agent containing L-glutamic acid and L-lysine may be a natural flavoring agent.

[0116] According to one embodiment of the present invention, the seasoning may have a content of L-glutamic acid and L-lysine of 3 to 90 wt % based on solid content, and contain L-glutamic acid and L-lysine at a ratio of 0.83:1 to 99:1.

[0117] Since the seasoning contains L-glutamic acid and L-lysine that provide umami taste, it can improve the solubility of L-glutamic acid and solve the ammonia smell problem by reducing the generation of ammonium glutamate during the fermentation process. In addition, since the seasoning also contains metabolites such as organic acids, inorganic ion components, proteins, peptides and vitamins produced during the fermentation process, it has rich umami taste and strong body feeling and shows excellent sensory properties. Therefore, the seasoning can be added to a variety of foods to maximize the taste of the food.

[0118] Another aspect of the present invention provides a food composition containing the above-mentioned seasoning or a seasoning containing L-glutamic acid and L-lysine.

[0119] The term "food composition" used herein refers to a natural product or processed product containing one or more nutrients, and preferably refers to a product that has undergone some processing and is ready for direct consumption. In a general sense, the term "food composition" includes all health functional foods, functional foods, beverages, food additives, and beverage additives.

[0120] According to one embodiment of the present invention, the food composition contains a flavoring produced without a chemical purification process or a flavoring containing natural L-glutamic acid and L-lysine (i.e., a natural flavoring), and the food composition may contain the flavoring in an amount of 0.001 wt % to 90 wt %, more specifically 0.01 wt % to 50 wt %, based on the total weight of the composition.

[0121] Food composition in the present invention can be provided with any preparation suitable for food, and can be for example in the form of solution, emulsion, viscous mixture, powder, granule, tablet, capsule etc.In this case, food composition can contain necessary and suitable multiple basic components (base) and / or additive of preparation in the scope of not damaging main effect of the present invention.In addition, food composition can also contain additive such as aromatic, coloring agent, disinfectant, antioxidant, preservative, humectant, thickening agent, inorganic salt and emulsifying agent in the scope of not damaging its effect.The content of these additives in food composition can be selected according to the preparation of composition or the intended use in the scope of not damaging purpose of the present invention and effect.For example, the content of additive can be 0.01 weight % to 70 weight %, more specifically 0.1 weight % to 50 weight % based on the gross weight of food composition.

[0122] The food composition can be used as an additive for a variety of foods. As the food to which the food composition can be added, any food known in the art can be used without restriction. Some examples of food include, but are not limited to, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products (including ice cream), a variety of soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes.

[0123] The method for producing the seasoning according to the present invention can produce a natural seasoning that can improve food taste and fragrance and the overall organoleptic properties of food by a fermentation broth containing amino acids, nucleic acids and / or organic acids, wherein the fermentation broth is produced by mixed fermentation of different microorganisms that produce different products (i.e. different types of amino acids, nucleic acids and / or organic acids). The seasoning can be used in multiple food fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0124] Figure 1: is a flowchart showing a fermentation process using a glutamic acid-producing microorganism and a lysine-producing microorganism in Preparation Examples 1 to 3 according to one embodiment of the present invention.

[0125] Figure 2 : is a flow chart showing a fermentation process using a glutamic acid-producing microorganism and an arginine-producing microorganism in Preparation Examples 4 to 6 according to one embodiment of the present invention.

[0126] Figure 3 : is a flow chart showing a fermentation process using a glutamic acid-producing microorganism and an inosinic acid-producing microorganism in Preparation Examples 7 to 9 according to one embodiment of the present invention.

[0127] Figure 4 : is a flowchart showing a fermentation process using an inosinic acid-producing microorganism and a lysine-producing microorganism in Preparation Examples 10 to 12 according to one embodiment of the present invention.

[0128] Figure 5 : is a flowchart showing a fermentation process using an inosinic acid-producing microorganism and an arginine-producing microorganism in Preparation Examples 13 to 15 according to one embodiment of the present invention. DETAILED DESCRIPTION

[0129] Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings. However, these descriptions are provided only for illustrative purposes to help understand the present invention, and the scope of the present invention is not limited by these illustrative descriptions.

[0130] Example 1. Mixed fermentation of glutamic acid and lysine

[0131] 1-1. Seed culture

[0132] Corynebacterium glutamicum NFG6 (KCCM13164P) producing L-glutamic acid (GA) was used as a glutamic acid-producing microorganism, and Corynebacterium glutamicum NFL21 (KCCM13163P) producing L-lysine (LYS) was used as a lysine-producing microorganism.

[0133] For seed culture of glutamate-producing microorganisms, the glutamate-producing microorganisms were inoculated into a 2-L flask containing 0.2 L of seed medium, followed by primary culture at 30° C. and 140 rpm for 22 to 24 hours (OD 610 =10 to 15). Next, 2% to 3% of the primary culture was inoculated into a 5 L jar fermenter, and 2 L to 2.5 L of seed medium was added thereto, followed by subculture at 32°C, pH 6.9, 600 rpm, and 1.0 vvm aeration for 22 to 24 hours (OD 610=20 to 60), thereby preparing a glutamic acid seed culture solution.

[0134] For the seed culture of lysine-producing microorganisms, the lysine-producing microorganisms were inoculated into a 2-L flask containing 0.2 L of seed medium, followed by primary culture at 30°C and 140 rpm for 16 to 18 hours (OD 610 =11 to 12). Next, 5% of the primary culture was inoculated into a 5 L fermenter, and 2 L to 2.5 L of seed medium was added thereto, followed by subculture at 32°C, pH 7.0, 650 rpm, and an aeration volume of 2.0 vvm for 21 to 24 hours (OD 610 =20 to 60), thereby preparing a lysine seed culture solution.

[0135] The composition of the seed medium used is shown in Table 1 below.

[0136] [Table 1]

[0137]

[0138] 1-2. Main fermentation

[0139] In order to examine the ratio of glutamate to lysine in the fermentation broth based on the inoculum of the glutamate seed culture broth and the lysine seed culture broth, the glutamate seed culture broth and the lysine seed culture broth were inoculated at various ratios and fermented.

[0140] In the main fermentation, 14L to 18L of fermentation medium is added to a 50L fermentor, and 1.2L to 1.8L of total seed culture solution is inoculated with an inoculation ratio of 50 to 99.95 (glutamic acid seed culture solution): 50 to 0.05 (lysine seed culture solution), followed by mixed fermentation for 28 to 40 hours by fed-batch culture. The fermentation medium composition and fermentation conditions used are shown in Table 2 below.

[0141] [Table 2]

[0142]

[0143] This mixed fermentation was performed a total of three times, the average value was calculated, and the results are shown in Table 3 below.

[0144] [Table 3]

[0145]

[0146]

[0147] Referring to Table 3 above, it was determined that when the seed culture broth of the amino acid-producing microorganism was inoculated at a ratio of 50 to 99.95 (glutamic acid-producing microorganism): 50 to 0.05 (lysine-producing microorganism), L-glutamic acid and L-lysine in the fermentation broth were produced at a ratio of 0.83:1 to 99:1.

[0148] Example 2. Comparison of glutamate-lysine baking powder between fermentation processes

[0149] 2-1. Production of glutamic acid-lysine baking powder

[0150] Conventionally, a seasoning containing glutamic acid and lysine is produced by fermenting a glutamic acid-producing microorganism and a lysine-producing microorganism separately and then mixing the fermented liquid containing the amino acid or its dried product in a suitable ratio. In order to compare the difference in taste between the conventional separate fermentation method and the method based on mixed fermentation of a glutamic acid-producing microorganism and a lysine-producing microorganism, the components of the glutamic acid-lysine (GA-LYS) baking powder obtained by the production method were compared. (See Figure 1 ).

[0151] The glutamate seed culture solution and the lysine seed culture solution used here were prepared in the same manner as in Example 1-1.

[0152] ① Single fermentation (Preparation Example 1)

[0153] In the method of mixing the separate dried products in the conventional method, the glutamic acid seed culture solution and the lysine seed culture solution are each transferred to a 50L fermentation tank and then subjected to main fermentation separately. Next, the cells are separated from each fermentation solution and then subjected to decolorization and filtering processes. The filtrate is concentrated and dried to obtain a dried product. The obtained dried glutamic acid and lysine products are mixed together so that the ratio of glutamic acid to lysine is 1:1, thereby preparing a baking powder containing glutamic acid and lysine.

[0154] ② Mixing of fermentation broth after separate fermentation (Preparation Example 2)

[0155] In the method of mixing separate fermentation broths in the conventional method, the glutamic acid seed culture broth and the lysine seed culture broth are transferred to a 50L fermentation tank and then the main fermentation is carried out separately. The fermentation broths obtained in the main fermentation are mixed together so that the ratio of glutamic acid to lysine is 1:1. Next, the cells are separated from the fermentation broth mixture and then decolorized and filtered. The filtrate is concentrated and dried to obtain the glutamic acid-lysine fermentation powder.

[0156] ③Mixed fermentation (Preparation Example 3)

[0157] In the mixed fermentation, glutamic acid seed culture liquid and lysine seed culture liquid were inoculated at a ratio of 65:35 in the same manner as in Example 1-2, so that the ratio of glutamic acid to lysine in the fermentation liquid was about 1:1 in the same manner as in Preparation Examples 1 and 2, followed by mixed fermentation. Next, the cells were separated from the fermentation liquid, and decolorization and filtration processes were performed. The filtrate was concentrated and dried to obtain glutamic acid-lysine fermentation powder.

[0158] 2-2. Comparison of components between glutamic acid-lysine baking powders

[0159] The glutamate-lysine baking powder obtained by the single fermentation in each of Preparation Examples 1 and 2 and the glutamate-lysine baking powder obtained by the mixed fermentation in Preparation Example 3 were subjected to component analysis.

[0160] Amino acids (L-glutamic acid and L-lysine) were measured by HPLC analysis (GA-210 nm, UV detector, flow rate 0.9 ml / min; LYS-214 nm, UV detector, flow rate 0.8 ml / min), and organic acids (citric acid, succinic acid, lactic acid, acetic acid, etc.) were measured by HPLC analysis (HPX-87H column, 214 nm, 25 minutes). Ions (Na, Mg, K, PO4, SO4, Cl, NH4, etc.) were measured by ion analyzer (Dionex IcS-1100, Thermo Scientific). The results are shown in Table 4 below.

[0161] [Table 4]

[0162]

[0163] Referring to Table 4 above, the mixed fermentation (Preparation Example 3) showed a higher amino acid ratio than the individual fermentations (Preparation Examples 1 and 2), and showed significant reductions in the contents of organic acids, ions, and ammonium.

[0164] 2-3. Sensory comparison between single fermentation and mixed fermentation

[0165] The glutamate-lysine baking powders obtained by individual fermentation in Preparation Examples 1 and 2 and the glutamate-lysine baking powder obtained by mixed fermentation in Preparation Example 3 were subjected to sensory evaluation.

[0166] For sensory evaluation of glutamic acid-lysine baking powder, umami taste, umami persistence, saltiness, sourness, bitterness and sweetness were evaluated by a trained panel consisting of 10 to 15 people for each powder itself or for samples obtained by diluting each powder in warm water at a concentration of 1% to 5%. The results are shown in Table 5 below.

[0167] [Table 5]

[0168]

[0169] Referring to Table 5 above, the samples prepared by the mixed process after the individual fermentation showed a weak umami taste and increased bitterness and sourness compared to the samples prepared by the mixed fermentation due to the component differences of the glutamic acid-lysine fermentation powder as shown in Table 4 above. In addition, since the increase of by-products (such as organic acids) and the increase of ions as shown in Table 4 above also affect the sensory properties of the samples, the mixed fermentation process is more effective in terms of taste or process simplification than mixing after the individual fermentation.

[0170] Example 3. Mixed fermentation of glutamic acid and arginine

[0171] 3-1. Seed culture

[0172] Corynebacterium glutamicum NFG6 (KCCM13164P) was used as a glutamic acid-producing microorganism, and Corynebacterium glutamicum NFA40 (KCCM13165P) producing L-arginine (ARG) was used as an arginine-producing microorganism.

[0173] A glutamate seed culture solution of a glutamate-producing microorganism was prepared in the same manner as in Example 1-1.

[0174] For seed culture of arginine-producing microorganisms, the arginine-producing microorganisms were inoculated into a 2-L flask containing 0.2 L of seed medium, followed by primary culture at 30° C. and 140 rpm for 16 to 18 hours (OD 610 =12 to 18). Next, 2% to 4% of the primary culture was inoculated into a 5 L fermenter, and 2 L to 2.5 L of seed medium was added thereto, followed by subculture at 32°C, pH 6.7, 600 rpm, and an aeration volume of 1.0 vvm for 16 to 24 hours (OD 610 =20 to 60), thereby preparing an arginine seed culture solution. The composition of the seed culture medium used is shown in Table 6 below.

[0175] [Table 6]

[0176]

[0177] 3-2. Main fermentation

[0178] In order to examine the ratio of glutamate to arginine in the fermentation broth based on the inoculum of the glutamate seed culture broth and the arginine seed culture broth, the glutamate seed culture broth and the arginine seed culture broth were inoculated at various ratios and fermented.

[0179] In the main fermentation, 14L to 18L of fermentation medium is added to a 50L fermentor, and inoculated with a total seed culture solution of 1.2L to 1.8L at an inoculation ratio of 30 to 99.95 (glutamic acid seed culture solution): 70 to 0.05 (arginine seed culture solution), followed by mixed fermentation for 31 to 60 hours by fed-batch culture. The fermentation medium composition and fermentation conditions used are shown in Table 7 below.

[0180] [Table 7]

[0181]

[0182] This mixed fermentation was performed a total of three times, the average value was calculated, and the results are shown in Table 8 below.

[0183] [Table 8]

[0184]

[0185]

[0186] Referring to Table 8 above, it was determined that when the seed culture broth of the amino acid-producing microorganism was inoculated at a ratio of 30 to 99.95 (glutamic acid-producing microorganism): 70 to 0.05 (arginine-producing microorganism), L-glutamic acid and L-arginine in the fermentation broth were produced at a ratio of 1.04:1 to 99:1.

[0187] Example 4. Comparison of glutamate-arginine baking powder between fermentation processes

[0188] 4-1. Production of glutamic acid-arginine fermentation powder

[0189] Conventionally, a seasoning containing glutamic acid and arginine is produced by fermenting a glutamic acid-producing microorganism and an arginine-producing microorganism separately and then mixing the fermented liquids containing amino acids or dried products thereof at an appropriate ratio. In order to compare the difference in taste between this conventional separate fermentation method and the method based on mixed fermentation of a glutamic acid-producing microorganism and an arginine-producing microorganism, the components of glutamic acid-arginine (GA-ARG) fermented powder obtained by this production method were compared (see Figure 2 ).

[0190] The glutamate seed culture solution and arginine seed culture solution used here were prepared in the same manner as in Example 3-1.

[0191] ① Single fermentation (Preparation Example 4)

[0192] In the method of mixing the separate dry products in the conventional method, the glutamic acid seed culture solution and the arginine seed culture solution are transferred to a 50L fermentation tank respectively, and then the main fermentation is carried out separately. Next, the cells are separated from each fermentation broth, and then a decolorization and filtering process is carried out. The filtrate is concentrated and dried to obtain a dry product. The dry glutamic acid and arginine products obtained are mixed together so that the ratio of glutamic acid to arginine is 1:1, thereby preparing a baking powder containing glutamic acid and arginine.

[0193] ② Mixing of fermentation broth after separate fermentation (Preparation Example 5)

[0194] In the method of mixing separate fermentation broths in the conventional method, glutamic acid seed culture broth and arginine seed culture broth are transferred to a 50L fermentation tank separately and then the main fermentation is carried out separately. The fermentation broths obtained in the main fermentation are mixed together so that the ratio of glutamic acid to arginine is 1:1. Next, cells are separated from the fermentation broth mixture and then decolorized and filtered. The filtrate is concentrated and dried to obtain glutamic acid-arginine fermentation powder.

[0195] ③ Mixed fermentation (Preparation Example 6)

[0196] In the mixed fermentation, glutamic acid seed culture liquid and arginine seed culture liquid were inoculated at a ratio of 30:70 in the same manner as in Example 3-2, so that the ratio of glutamic acid to arginine in the fermentation liquid was about 1:1 in the same manner as in Preparation Examples 4 and 5, followed by mixed fermentation. Next, the cells were separated from the fermentation liquid, and decolorization and filtration processes were performed. The filtrate was concentrated and dried to obtain glutamic acid-arginine fermentation powder.

[0197] 4-2. Comparison of components between glutamic acid-arginine baking powders

[0198] The glutamate-arginine baking powder obtained by the single fermentation in each of Preparation Examples 4 and 5 and the glutamate-arginine baking powder obtained by the mixed fermentation in Preparation Example 6 were subjected to component analysis.

[0199] Amino acids (L-glutamic acid and L-arginine) were measured by HPLC analysis (GA-210 nm, UV detector, flow rate 0.9 ml / min; ARG-195 nm, UV detector, flow rate 1 ml / min). Organic acids and ions were measured in the same manner as in Example 2-2. The results are shown in Table 9 below.

[0200] [Table 9]

[0201]

[0202] Referring to Table 9 above, the mixed fermentation (Preparation Example 6) showed a higher amino acid ratio than the individual fermentations (Preparation Examples 4 and 5), and showed significant reductions in the contents of organic acids, ions, and ammonium.

[0203] 4-3. Sensory comparison between single fermentation and mixed fermentation

[0204] The glutamic acid-arginine fermented powders obtained by single fermentation in Preparation Examples 4 and 5 and the glutamic acid-arginine fermented powder obtained by mixed fermentation in Preparation Example 6 were subjected to sensory evaluation.

[0205] Sensory evaluation was performed in the same manner as in Example 2-3. The results are shown in Table 10 below.

[0206] [Table 10]

[0207]

[0208] Referring to Table 10 above, the samples prepared by the mixed process after the individual fermentation showed a weak umami taste and an increased bitter taste compared to the samples prepared by the mixed fermentation due to the component differences of the glutamic acid-arginine fermentation powder as shown in Table 9 above. In addition, since the increase of by-products (e.g., organic acids) and the increase of ions as shown in Table 9 above also affect the sensory properties of the samples, the mixed fermentation process is more effective in terms of taste or process simplification than mixing after the individual fermentation.

[0209] Example 5. Mixed fermentation of glutamic acid and inosinic acid

[0210] 5-1. Seed culture

[0211] Corynebacterium glutamicum NFG6 (KCCM13164P) was used as a glutamic acid-producing microorganism, and Corynebacterium ammoniagenes NFI545 (KCCM13162P) producing IMP was used as an inosinic acid-producing microorganism.

[0212] A glutamate seed culture solution of a glutamate-producing microorganism was prepared in the same manner as in Example 1-1.

[0213] For seed culture of the inosinic acid-producing microorganism, the inosinic acid-producing microorganism was inoculated into a 2 L flask containing 0.3 L of seed medium, followed by primary culture at 31 °C and 150 rpm for 20 to 24 h (OD 610 =15 to 20). Next, 1% of the primary culture was inoculated into a 5 L fermenter, and 2 L to 2.5 L of seed medium was added thereto, followed by subculture at 31°C, pH 7.1, 600 rpm, and an aeration volume of 1.0 vvm for 21 to 24 hours (OD 610=20 to 40) to prepare an inosinic acid seed culture solution. The composition of the seed culture medium used is shown in Table 11 below.

[0214] [Table 11]

[0215]

[0216] 5-2. Main fermentation

[0217] In order to examine the ratio of glutamate to inosinic acid in the fermentation broth based on the inoculum of the glutamate seed culture broth and the inosinic acid seed culture broth, the glutamate seed culture broth and the inosinic acid seed culture broth were inoculated at various ratios and fermented.

[0218] In the main fermentation, 14L to 18L of fermentation medium was added to a 50L fermentor, and 1.2L to 1.8L of total seed culture solution was inoculated at an inoculation ratio of 0.05 to 99.95 (glutamic acid seed culture solution): 99.95 to 0.05 (inosinic acid seed culture solution), followed by mixed fermentation for 30 to 90 hours by fed-batch culture. The fermentation medium composition and fermentation conditions used herein are shown in Table 12 below.

[0219] [Table 12]

[0220]

[0221] This mixed fermentation was performed a total of three times, the average value was calculated, and the results are shown in Table 13 below.

[0222] [Table 13]

[0223]

[0224] Referring to Table 13 above, it was determined that when the seed culture of the microorganism was inoculated at a ratio of 0.05 to 99.95 (glutamic acid-producing microorganism): 99.95 to 0.05 (inosinic acid-producing microorganism), L-glutamic acid and IMP in the fermentation broth were produced at a ratio of 0.02:1 to 99.8:1.

[0225] Example 6. Comparison of glutamate-inosinic acid baking powder between fermentation processes

[0226] 6-1. Production of glutamic acid-inosinic acid fermentation powder

[0227] Conventionally, a seasoning containing glutamic acid and inosinic acid is produced by fermenting a glutamic acid-producing microorganism and an inosinic acid-producing microorganism separately and then mixing the fermentation broth or a dried product thereof at an appropriate ratio. In order to compare the difference in taste between this conventional separate fermentation method and the method based on mixed fermentation of a glutamic acid-producing microorganism and an inosinic acid-producing microorganism, the components of glutamic acid-inosinic acid (GA-IMP) fermented powder obtained by this production method were compared (see Figure 3 ).

[0228] The glutamic acid seed culture solution and the inosinic acid seed culture solution used here were prepared in the same manner as in Example 5-1.

[0229] ① Single fermentation (Preparation Example 7)

[0230] In the method of mixing the separate dried products in the conventional method, the glutamic acid seed culture solution and the inosinic acid seed culture solution are each transferred to a 50L fermentation tank, and then the main fermentation is performed separately. Next, the cells are separated from each fermentation solution, and then a decolorization and filtering process is performed. The filtrate is concentrated and dried to obtain a dried product. The obtained dry glutamic acid and inosinic acid products are mixed together so that the ratio of glutamic acid to inosinic acid is 1:1, thereby preparing a baking powder containing glutamic acid and inosinic acid.

[0231] ② Mixing of fermentation broth after separate fermentation (Preparation Example 8)

[0232] In the method of mixing separate fermentation broths in the conventional method, the glutamic acid seed culture broth and the inosinic acid seed culture broth are each transferred to a 50L fermentation tank and then subjected to a main fermentation separately. The fermentation broths obtained in the main fermentation are mixed together so that the ratio of glutamic acid to inosinic acid is 1:1. Next, the cells are separated from the fermentation broth mixture and then subjected to a decolorization and filtration process. The filtrate is concentrated and dried to obtain a glutamic acid-inosinic acid fermentation powder.

[0233] ③ Mixed fermentation (Preparation Example 9)

[0234] In the mixed fermentation, glutamic acid seed culture liquid and inosinic acid seed culture liquid were inoculated at a ratio of 20:80 in the same manner as in Example 5-2, so that the ratio of glutamic acid to inosinic acid in the fermentation liquid was about 1:1 in the same manner as in Preparation Examples 7 and 8, followed by mixed fermentation. Next, the cells were separated from the fermentation liquid, and decolorization and filtration processes were performed. The filtrate was concentrated and dried to obtain glutamic acid-inosinic acid fermentation powder.

[0235] 6-2. Comparison of components between glutamic acid-inosinic acid baking powders

[0236] The glutamate-inosinic acid fermented powder obtained by the single fermentation in each of Preparation Examples 7 and 8 and the glutamate-inosinic acid fermented powder obtained by the mixed fermentation in Preparation Example 9 were subjected to component analysis.

[0237] L-Glutamic acid and IMP were measured by HPLC analysis (GA-210 nm, UV detector, flow rate 0.9 ml / min; IMP-254 nm, UV detector, flow rate 0.9 ml / min). Organic acids and ions were measured in the same manner as in Example 2-2. The results are shown in Table 14 below.

[0238] [Table 14]

[0239]

[0240] 6-3. Sensory comparison between single fermentation and mixed fermentation

[0241] The glutamate-inosinic acid fermented powders obtained by single fermentation in Preparation Examples 7 and 8 and the glutamate-inosinic acid fermented powder obtained by mixed fermentation in Preparation Example 9 were subjected to sensory evaluation.

[0242] Sensory evaluation was performed in the same manner as in Example 2-3. The results are shown in Table 15 below.

[0243] [Table 15]

[0244]

[0245] Referring to Table 15 above, the samples prepared by the mixed process after the individual fermentation showed weak umami taste and umami persistence and increased bitterness compared to the samples prepared by the mixed fermentation due to the component differences of the glutamate-inosinic acid fermentation powder as shown in Table 14 above. In addition, since the increase of by-products (such as organic acids) and the increase of ions as shown in Table 14 above also affect the sensory properties of the samples, the mixed fermentation process is more effective in terms of taste or process simplification than mixing after the individual fermentation.

[0246] Example 7. Mixed fermentation of inosinic acid and lysine

[0247] 7-1. Seed culture

[0248] Corynebacterium ammoniagenes NFI545 (KCCM13162P) was used as the inosinic acid-producing microorganism, and Corynebacterium glutamicum NFL21 (KCCM13163P) was used as the lysine-producing microorganism.

[0249] In the same manner as in Examples 5-1 and 1-1, an inosinic acid seed culture solution of an inosinic acid-producing microorganism and a lysine seed culture solution of a lysine-producing microorganism were prepared, respectively.

[0250] 7-2. Main fermentation

[0251] In order to examine the ratio of inosinic acid to lysine in the fermentation broth based on the inoculum of the inosinic acid seed culture broth and the lysine seed culture broth, the inosinic acid seed culture broth and the lysine seed culture broth were inoculated at various ratios and fermented.

[0252] In the main fermentation, 14L to 18L of fermentation medium was added to a 50L fermentor and inoculated with 1.2L to 1.8L of total seed culture solution at an inoculation ratio of 0.05 to 99.95 (inosinic acid seed culture solution): 99.95 to 0.05 (lysine seed culture solution), followed by mixed fermentation by fed-batch culture for 45 to 90 hours. The fermentation medium composition and fermentation conditions used herein are shown in Table 16 below.

[0253] [Table 16]

[0254]

[0255] This mixed fermentation was performed a total of three times, the average value was calculated, and the results are shown in Table 17 below.

[0256] [Table 17]

[0257]

[0258] Referring to Table 17 above, it was determined that when the seed culture of the microorganism was inoculated at a ratio of 0.05 to 99.95 (microorganism producing inosinic acid):99.95 to 0.05 (microorganism producing lysinic acid), IMP and lysine in the fermentation broth were produced at a ratio of 0.01:1 to 92:1.

[0259] Example 8. Comparison of inosinic acid-lysine baking powder between fermentation processes

[0260] 8-1. Production of inosinic acid-lysine yeast powder

[0261] Conventionally, a seasoning containing inosinic acid and lysine is produced by fermenting an inosinic acid-producing microorganism and a lysine-producing microorganism separately and then mixing the fermentation broth or a dried product thereof at an appropriate ratio. In order to compare the difference in taste between this conventional separate fermentation method and the method based on mixed fermentation of an inosinic acid-producing microorganism and a lysine-producing microorganism, the components of the inosinic acid-lysine (IMP-LYS) baking powder obtained by the production method were compared (see Figure 4 ).

[0262] The inosinic acid seed culture solution and lysine seed culture solution used here were prepared in the same manner as in Example 7-1.

[0263] ① Single fermentation (Preparation Example 10)

[0264] In the method of mixing the separate dried products in the conventional method, the inosinic acid seed culture solution and the lysine seed culture solution are each transferred to a 50L fermentation tank, and then the main fermentation is performed separately. Next, the cells are separated from each fermentation broth, and then a decolorization and filtering process is performed. The filtrate is concentrated and dried to obtain a dried product. The obtained dry inosinic acid and lysine products are mixed together so that the ratio of inosinic acid to lysine is 1:1, thereby preparing a baking powder containing inosinic acid and lysine.

[0265] ② Mixing of fermentation broth after separate fermentation (Preparation Example 11)

[0266] In the method of mixing separate fermentation broths in the conventional method, the inosinic acid seed culture broth and the lysine seed culture broth are each transferred to a 50L fermentation tank and then subjected to a main fermentation separately. The fermentation broths obtained in the main fermentation are mixed together so that the ratio of inosinic acid to lysine is 1:1. Next, the cells are separated from the fermentation broth, and then a decolorization and filtering process is performed. The filtrate is concentrated and dried to obtain an inosinic acid-lysine fermentation powder.

[0267] ③ Mixed fermentation (Preparation Example 12)

[0268] In the mixed fermentation, inosinic acid seed culture liquid and lysine seed culture liquid were inoculated at a ratio of 80:20 in the same manner as in Example 7-2, so that the ratio of inosinic acid to lysine in the fermentation liquid was about 1:1 in the same manner as in Preparation Examples 10 and 11, followed by mixed fermentation. Next, the cells were separated from the fermentation liquid, and decolorization and filtration processes were performed. The filtrate was concentrated and dried to obtain inosinic acid-lysine fermentation powder.

[0269] 8-2. Comparison of components between lysine-inosinic acid baking powders

[0270] The inosinic acid-lysine fermented powder obtained by the single fermentation in each of Preparation Examples 10 and 11 and the inosinic acid-lysine fermented powder obtained by the mixed fermentation in Preparation Example 12 were subjected to component analysis.

[0271] IMP and L-lysine were measured by HPLC analysis (IMP-254 nm, UV detector, flow rate 0.9 ml / min; LYS-214 nm, UV detector, flow rate 0.8 ml / min). Organic acids and ions were measured in the same manner as in Example 2-2. The results are shown in Table 18 below.

[0272] [Table 18]

[0273]

[0274]

[0275] Referring to Table 18 above, the mixed fermentation (Preparation Example 12) showed a higher LYS+IMP ratio than the individual fermentations (Preparation Examples 10 and 11), and showed significant reductions in the contents of organic acids, ions, and ammonium.

[0276] 8-3. Sensory comparison between single fermentation and mixed fermentation

[0277] The inosinic acid-lysine fermented powders obtained by single fermentation in Preparation Examples 10 and 11 and the inosinic acid-lysine fermented powder obtained by mixed fermentation in Preparation Example 12 were subjected to sensory evaluation.

[0278] Sensory evaluation was performed in the same manner as in Example 2-3. The results are shown in Table 19 below.

[0279] [Table 19]

[0280]

[0281] Referring to Table 19 above, the samples prepared by the mixed process after separate fermentation showed weak umami persistence and increased bitterness compared to the samples prepared by mixed fermentation due to the component differences of the inosinic acid-lysine fermentation powder as shown in Table 18 above. In addition, since the increase of by-products (such as organic acids) and the increase of ions as shown in Table 18 above also affect the sensory properties of the samples, the mixed fermentation process is more effective in terms of taste or process simplification than mixing after separate fermentation.

[0282] Example 9. Mixed fermentation of inosinic acid and arginine

[0283] 9-1. Seed cultivation

[0284] Corynebacterium ammoniagenes NFI545 (KCCM13162P) was used as the inosinic acid-producing microorganism, and Corynebacterium glutamicum NFA40 (KCCM13165P) was used as the arginine-producing microorganism.

[0285] In the same manner as in Examples 5-1 and 3-1, an inosinic acid seed culture solution of an inosinic acid-producing microorganism and an arginine seed culture solution of an arginine-producing microorganism were prepared, respectively.

[0286] 9-2. Main fermentation

[0287] In order to examine the ratio of inosinic acid to arginine in the fermentation broth based on the inoculum of the inosinic acid seed culture broth and the arginine seed culture broth, the inosinic acid seed culture broth and the arginine seed culture broth were inoculated at various ratios and fermented.

[0288] In the main fermentation, 14L to 18L of fermentation medium was added to a 50L fermentor and inoculated with 1.2L to 1.8L of total seed culture solution at an inoculation ratio of 65 to 99.95 (inosinic acid seed culture solution): 35 to 0.05 (arginine seed culture solution), followed by mixed fermentation by fed-batch culture for 45 to 80 hours. The fermentation medium composition and fermentation conditions used herein are shown in Table 20 below.

[0289] [Table 20]

[0290]

[0291] This mixed fermentation was performed a total of three times, the average value was calculated, and the results are shown in Table 21 below.

[0292] [Table 21]

[0293]

[0294]

[0295] Referring to Table 21 above, it was determined that when the seed culture broth of the microorganism was inoculated at a ratio of 65 to 99.95 (microorganism producing inosinic acid):35 to 0.05 (microorganism producing arginine), IMP and L-arginine in the fermentation broth were produced at a ratio of 1.04:1 to 95.1:1.

[0296] Example 10. Comparison of inosinic acid-arginine baking powder between fermentation processes

[0297] 10-1. Production of inosinic acid-arginine fermentation powder

[0298] Conventionally, a seasoning containing inosinic acid and arginine is produced by fermenting an inosinic acid-producing microorganism and an arginine-producing microorganism separately and then mixing the fermentation broth or a dried product thereof at an appropriate ratio. In order to compare the difference in taste between this conventional separate fermentation method and the method based on mixed fermentation of an inosinic acid-producing microorganism and an arginine-producing microorganism, the components of inosinic acid-arginine (IMP-ARG) baking powder obtained by this production method were compared (see Figure 5 ).

[0299] The inosinic acid seed culture solution and arginine seed culture solution used here were prepared in the same manner as in Example 9-1.

[0300] ① Single fermentation (Preparation Example 13)

[0301] In the method of mixing the separate dried products in the conventional method, the inosinic acid seed culture solution and the arginine seed culture solution are each transferred to a 50L fermentation tank and then subjected to main fermentation separately. Next, the cells are separated from each fermentation solution, and then decolorization and filtering processes are performed. The filtrate is concentrated and dried to obtain a dried product. The obtained dried inosinic acid and arginine products are mixed together so that the ratio of inosinic acid to arginine is 1:1, thereby preparing a baking powder containing inosinic acid and arginine.

[0302] ② Mixing of fermentation broth after separate fermentation (Preparation Example 14)

[0303] In the method of mixing separate fermentation broths in the conventional method, the inosinic acid seed culture broth and the arginine seed culture broth are each transferred to a 50L fermentation tank and then subjected to a main fermentation separately. The fermentation broths obtained in the main fermentation are mixed together so that the ratio of inosinic acid to arginine is 1:1. Next, the cells are separated from the fermentation broth mixture and then subjected to a decolorization and filtration process. The filtrate is concentrated and dried to obtain an inosinic acid-arginine fermentation powder.

[0304] ③ Mixed fermentation (Preparation Example 15)

[0305] In the mixed fermentation, inosinic acid seed culture liquid and arginine seed culture liquid were inoculated at a ratio of 65:35 in the same manner as in Example 9-2, so that the ratio of inosinic acid to arginine in the fermentation liquid was about 1:1 in the same manner as in Preparation Examples 13 and 14, followed by mixed fermentation. Next, the cells were separated from the fermentation liquid, and decolorization and filtration processes were performed. The filtrate was concentrated and dried to obtain inosinic acid-arginine fermentation powder.

[0306] 10-2. Comparison of components between inosinic acid-arginine baking powders

[0307] The inosinic acid-arginine fermented powder obtained by the single fermentation in each of Preparation Examples 13 and 14 and the inosinic acid-arginine fermented powder obtained by the mixed fermentation in Preparation Example 15 were subjected to component analysis.

[0308] L-arginine and IMP were measured by HPLC analysis (ARG-195nm, UV detector, flow rate 1ml / min; IMP-254nm, UV detector, flow rate 0.9ml / min). Organic acids and ions were measured in the same manner as in Example 2-2. The results are shown in Table 22 below.

[0309] [Table 22]

[0310]

[0311] 10-3. Sensory comparison between single fermentation and mixed fermentation

[0312] The inosinic acid-arginine fermented powders obtained by single fermentation in Preparation Examples 13 and 14 and the inosinic acid-arginine fermented powder obtained by mixed fermentation in Preparation Example 15 were subjected to sensory evaluation.

[0313] Sensory evaluation was performed in the same manner as in Example 2-3. The results are shown in Table 23 below.

[0314] [Table 23]

[0315]

[0316]

[0317] Referring to Table 23 above, the samples prepared by the mixed process after the individual fermentation showed weak umami persistence and increased bitterness compared to the samples prepared by the mixed fermentation due to the component differences of the inosinic acid-arginine fermentation powder as shown in Table 22 above. In addition, since the increase of by-products (such as organic acids) and the increase of ions as shown in Table 22 above also affect the sensory properties of the samples, the mixed fermentation process is more effective in terms of taste or process simplification than mixing after the individual fermentation.

[0318] So far, the present invention has been described with reference to some preferred embodiments. It will be appreciated by those skilled in the art that the present invention may be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative perspective rather than a restrictive perspective. The scope of the present invention is defined by the appended claims rather than the above description, and all differences within the scope equivalent thereto should be interpreted as being included in the scope of the present invention.

[0319] [Registration Number]

[0320] Depository: Korean Culture Center of Microorganisms (KCCM)

[0321] Registration number: KCCM13162P

[0322] Date of deposit: April 21, 2022

[0323] Depository: Korea Collection of Microorganisms (KCCM)

[0324] Registration number: KCCM13163P

[0325] Date of deposit: April 21, 2022

[0326] Depository: Korea Collection of Microorganisms (KCCM)

[0327] Registration number: KCCM13164P

[0328] Date of deposit: April 21, 2022

[0329] Depository: Korea Collection of Microorganisms (KCCM)

[0330] Registration number: KCCM13165P

[0331] Date of deposit: April 21, 2022

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

Claims

1. A method for producing a flavoring agent, the method comprising the following steps: inoculating a fermentation medium with a first microorganism and a second microorganism, and then producing a fermentation broth containing amino acids, nucleic acids and / or organic acids by fermenting the microorganisms, The first microorganism and the second microorganism produce different products, and each produces one selected from amino acids, nucleic acids and organic acids.

2. The method of claim 1, wherein the amino acid is at least one selected from the group consisting of L-glutamic acid, L-alanine, L-valine, L-leucine, L-isoleucine, L-proline, L-phenylalanine, L-tryptophan, L-methionine, L-glycine, L-serine, L-threonine, L-cysteine, L-asparagine, L-glutamine, L-aspartic acid, L-lysine, L-arginine, and L-histidine.

3. The method of claim 1, wherein the nucleic acid is at least one selected from the group consisting of inosinic acid, guanylic acid, xanthic acid, and salts thereof.

4. The method of claim 1, wherein the organic acid is at least one selected from the group consisting of succinic acid, malic acid, citric acid, acetic acid, lactic acid, fumaric acid, tartaric acid, ascorbic acid, gluconic acid, and salts thereof.

5. The method of claim 1, wherein the first microorganism is a glutamate-producing microorganism, and the second microorganism is a lysine-producing microorganism, an arginine-producing microorganism, a histidine-producing microorganism, a tryptophan-producing microorganism, a glycine-producing microorganism, an alanine-producing microorganism, a succinate-producing microorganism, a lactate-producing microorganism, a guanylate-producing microorganism, or an inosinate-producing microorganism.

6. The method of claim 1, wherein the first microorganism is an inosinic acid-producing microorganism, and the second microorganism is a lysine-producing microorganism, an arginine-producing microorganism, a histidine-producing microorganism, a tryptophan-producing microorganism, a glycine-producing microorganism, an alanine-producing microorganism, a succinic acid-producing microorganism, a lactic acid-producing microorganism, or a guanylic acid-producing microorganism.

7. The method of claim 1, wherein the step comprises further inoculating the fermentation medium with a third microorganism that produces a product different from the products produced by the first microorganism and the second microorganism and produces one selected from the group consisting of amino acids, nucleic acids, and organic acids.

8. The method of claim 1 or 7, wherein the first microorganism is a microorganism that produces glutamate, the second microorganism is a microorganism that produces guanylate, and the third microorganism is a microorganism that produces inosinate.

9. The method of claim 1 or 7, wherein the first microorganism, the second microorganism, and the third microorganism are microorganisms of the genus Corynebacterium.

10. The method according to claim 1 or 7, wherein the first microorganism, the second microorganism, and the third microorganism are in a seed culture solution state obtained by single cultivation or co-cultivation.

11. The method of claim 1 or 7, wherein the step comprises adjusting the inoculum of each microorganism to control the ratio between the products of the microorganisms in the fermentation broth.

12. The method of claim 1 or 7, wherein the fermentation broth contains the microbial product in an amount of 3% to 90% by weight relative to the total solid content of the fermentation broth.

13. A method for producing a flavoring agent containing L-glutamic acid and L-lysine, the method comprising the following steps: A fermentation medium is inoculated with a glutamic acid-producing microorganism and a lysine-producing microorganism, and then a fermentation liquid containing L-glutamic acid and L-lysine is produced by fermentation of the microorganisms.

14. The method of claim 13, wherein the glutamic acid-producing microorganism and the lysine-producing microorganism are microorganisms of the genus Corynebacterium.

15. The method according to claim 13, wherein the glutamate-producing microorganism and the lysine-producing microorganism are in a seed culture solution state obtained by single cultivation or co-cultivation.

16. The method of claim 13, wherein the step comprises adjusting the inoculum of each of the glutamate-producing microorganism and the lysine-producing microorganism to control the ratio of L-glutamate to L-lysine in the fermentation broth.

17. The method of claim 13, wherein the fermentation broth contains amino acids including L-glutamic acid and L-lysine in an amount of 3 wt% to 90 wt% relative to the total solid content of the fermentation broth.

18. A flavoring produced by the method of claim 1 or 7.

19. A flavoring agent containing L-glutamic acid and L-lysine, produced by the method of claim 13.

20. The seasoning according to claim 19, which contains L-glutamic acid and L-lysine in an amount of 3 to 90 wt% relative to the total solid content of the seasoning.

21. A food composition comprising the flavoring agent according to claim 18.

22. A food composition comprising the seasoning comprising L-glutamic acid and L-lysine according to claim 19.

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