Method for producing seasoning material by mixed fermentation of different microorganisms

Abstract

[Technical Field] The present invention relates to a method for preparing a seasoning material using a process of mixing and simultaneously fermenting two or more kinds of microorganisms having different types of production substances.SOLUTION: The present invention relates to a method for preparing a seasoning material using a process of mixing and simultaneously fermenting two or more kinds of microorganisms having different kinds of production materials, and the method for preparing a seasoning material can prepare a natural seasoning material capable of improving the taste and flavor of food and improving the overall sensory characteristics through a fermentation liquid containing amino acids, nucleic acids, and / or organic acids by mixing and fermenting heterogeneous microorganisms producing different products, that is, different kinds of amino acids, nucleic acids, and / or organic acids, and such a seasoning material can be utilized in various food fields.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing a seasoning ingredient using a process of mixing and simultaneously fermenting two or more types of microorganisms that produce different types of substances. [Background technology]

[0002] L-glutamic acid, an acidic amino acid that produces a rich flavor, is naturally present in plant-based foods such as kelp, miso, and soy sauce, as well as animal-based foods such as dairy products, meat, and fish. L-glutamic acid is the most widely used food seasoning ingredient worldwide. In particular, MSG (monosodium glutamate), produced by adding salt during the purification process of L-glutamic acid, is used as a food additive in many processed foods due to its excellent flavor-enhancing properties. Recently, the market for natural seasoning ingredients such as yeast extract and natural ingredient extracts has been growing rapidly due to increased consumer awareness of health. However, MSG remains highly competitive due to its superior rich flavor and lower price compared to natural seasoning ingredients. Therefore, significant efforts are needed to develop MSG substitutes.

[0003] Meanwhile, in order to enhance the rich flavor, seasoning materials using various amino acids such as L-lysine, L-valine, and L-arginine in addition to L-glutamic acid have been developed. Seasoning materials that further contain nucleic acids such as inosinic acid and guanylic acid, or organic acids such as succinic acid and lactic acid, have also been developed. These L-glutamic acid-based seasoning materials are generally produced by blending fermentation broths produced by microorganisms that produce each product, or by blending individually produced amino acid, nucleic acid, or organic acid powders. While conventional manufacturing methods facilitate the adjustment of the concentration or content of the final substance, such as L-glutamic acid, they are costly due to the need for two separate production processes before blending. Furthermore, the resulting seasoning materials contain a large amount of culture medium components and fermentation by-products, making it difficult to resolve off-flavor and odor issues. Furthermore, they are still unable to produce a rich, rich flavor. Therefore, extensive research and development is needed to develop seasoning materials with improved rich flavor. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Korean Patent No. 10-1758332 [Patent Document 2] Korean Patent No. 10-1328091 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a method for producing a seasoning ingredient by mixed fermentation of two or more microorganisms that produce different types of substances.

[0006] Another object of the present invention is to provide a method for producing a seasoning material containing L-glutamic acid and L-lysine by mixed fermentation using a glutamic acid-producing microorganism and a lysine-producing microorganism.

[0007] A further object of the present invention is to provide a seasoning material produced by the above method.

[0008] Another object of the present invention is to provide a food composition containing the seasoning material. [Means for solving the problem]

[0009] One aspect of the present invention provides a method for producing a seasoning ingredient, comprising the steps of inoculating a first microorganism and a second microorganism into a fermentation medium, followed by fermentation to produce a fermentation liquid containing amino acids, nucleic acids, and / or organic acids, wherein the first microorganism and the second microorganism produce different products, each of which produces one selected from the group consisting of amino acids, nucleic acids, and organic acids.

[0010] The term "seasoning ingredient" as used herein refers to an ingredient added to enhance the flavor of food, and is used in a broader sense than typical seasonings that are added in small amounts at the end of a typical food cooking process. It can be used to enhance the flavor not only in home cooking but also during the production of processed foods such as ham, sausage, ramen, etc. The seasoning ingredient in the present invention refers to a substance that contains one or more amino acids, nucleic acids, and / or organic acids as flavor components, thereby enhancing the body and providing excellent sensory properties.

[0011] According to one embodiment of the present invention, the amino acid may be one or more 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.

[0012] According to one embodiment of the present invention, the nucleic acid may be one or more selected from the group consisting of inosinic acid, guanylic acid, xanthylic acid, and salt forms thereof.

[0013] For example, the nucleic acid may be, but is not limited to, inosine monophosphate (IMP), guanosine monophosphate (GMP), xanthosine monophosphate (XMP), etc.

[0014] According to one embodiment of the present invention, the organic acid may be one or more 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 salt forms thereof.

[0015] As used herein, "fermentation" refers to a biological phenomenon in which organic matter contained in a medium is decomposed or converted into other substances by microorganisms, and refers to the process by which inoculated microorganisms decompose or convert nutrients in the fermentation medium into amino acids, nucleic acids, organic acids, etc. As used herein, "fermentation broth" refers to substances produced by microorganisms through fermentation. This fermentation broth is produced by mixing and fermenting two or more microorganisms that produce different products, and includes not only useful substances such as amino acids, nucleic acids, and organic acids produced by each microorganism, but also by-products produced during the metabolic process and medium components.

[0016] Here, mixed fermentation refers to a process in which different microorganisms that produce different products, i.e., different types of amino acids, nucleic acids, or organic acids, are fermented in a single culture vessel under the same conditions during the microbial fermentation process.

[0017] In the mixed fermentation, microorganisms producing different types of amino acids, nucleic acids, or organic acids may be used, or an amino acid-producing microorganism and a nucleic acid-producing microorganism, an amino acid-producing microorganism and an organic acid-producing microorganism, or an organic acid-producing microorganism and a nucleic acid-producing microorganism may be used, but is not limited thereto.

[0018] The fermentation liquid produced by such mixed fermentation may contain, but is not limited to, two types of amino acids, nucleic acids, or organic acids, or one type of amino acid and one type of nucleic acid, one type of amino acid and one type of organic acid, or one type of organic acid and one type of nucleic acid.

[0019] For example, when the first microorganism is a glutamic 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, a guanylic acid-producing microorganism, or an inosinic acid-producing microorganism.

[0020] Furthermore, 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.

[0021] According to one embodiment of the present invention, the step may further include inoculating a third microorganism that produces a product different from the first and second microorganisms and that produces one selected from the group consisting of amino acids, nucleic acids, and organic acids.

[0022] More specifically, the fermentation broth produced from the first, second, and third microorganisms may contain three types of amino acids, nucleic acids, or organic acids, or may contain, but is not limited to, one type of amino acid and two types of nucleic acids, two types of amino acids and one type of nucleic acid, one type of amino acid and two types of organic acids, two types of amino acids and one type of organic acid, one type of organic acid and two types of nucleic acids, two types of organic acids and one type of nucleic acid, or one type of amino acid, one type of nucleic acid, and one type of organic acid.

[0023] For example, when the first microorganism is a glutamic acid-producing microorganism and the second microorganism is a guanylic acid-producing microorganism, the third microorganism may be an inosinic acid-producing microorganism.

[0024] The microorganisms used to produce these seasoning ingredients are microorganisms that produce amino acids, nucleic acids, and / or organic acids, and may be wild-type microorganisms obtained in nature or mutant strains modified to improve the production ability of wild-type microorganisms. Microorganisms that produce amino acids, nucleic acids, or organic acids may be any microorganism known in the art, including, without limitation, the genera Corynebacterium, Brevibacterium, Lactobacillus, Bifidobacterium, and Bacillus. These microorganisms may be of the same genus or species or different genera or species, and can be selected by the user.

[0025] According to one embodiment of the present invention, the first, second and third microorganisms may be microorganisms of the genus Corynebacterium.

[0026] More specifically, the microorganism of the genus Corynebacterium is selected from the group consisting of Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium callunae, Corynebacterium suranareeae, Corynebacterium lubricantis, Corynebacterium doosanense, Corynebacterium efficiens, Corynebacterium uterequii, and the like. Corynebacterium uterequi, Corynebacterium stationis, Corynebacterium pacaense, Corynebacterium singulare, Corynebacterium humireducens, Corynebacterium marinum, Corynebacterium halotolerans, Corynebacterium spheniscorum, Corynebacterium freiburgense, Corynebacterium striatum striatum, Corynebacterium canis, Corynebacterium ammoniagenes, Corynebacterium renale, Corynebacterium portisoriiThe bacterium may be, but is not limited to, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium caspium, Corynebacterium testudinoris, Corynebacterium pseudopelargi, or Corynebacterium flavescens.

[0027] For mixed fermentation of two or more microorganisms, each microorganism may be isolated and selected, or a microbial mixture obtained by mixing these may be used. Depending on the circumstances, an appropriate mixture of isolated microorganisms and a microbial mixture may be used. Each microorganism can be used for fermentation in a state where its growth and ability to produce amino acids, nucleic acids, and / or organic acids is activated, and it is preferable to perform seed culture for microbial activation.

[0028] According to one embodiment of the present invention, the first microorganism, the second microorganism, and the third microorganism may be cultured individually or may be mixed and cultured in a seed culture solution state.

[0029] As used herein, "seed culture" refers to culturing microorganisms in a small volume of medium before mass-cultivation, and "seed culture solution" refers to the culture medium components, microorganisms grown by seed culture, and their metabolic products.

[0030] The seed culture may be carried out using a suitable medium and culture conditions known in the art depending on the characteristics of each microorganism, and a person skilled in the art can easily adjust the medium and culture conditions.

[0031] More specifically, the medium used for the seed culture contains nutrients necessary for the growth and proliferation of the microorganism, and may be a liquid medium.

[0032] The culture temperature for the seed culture may generally be 20 to 45°C, for example, 25 to 40°C, or 27 to 37°C, and the culture period may be continued until the microorganisms actively grow and proliferate, for example, 10 to 160 hours, 18 to 120 hours, or 20 to 80 hours.

[0033] During cultivation, compounds such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be added to the medium or culture solution in an appropriate manner to adjust the pH of the culture solution. Furthermore, food additive antifoaming agents can be used to suppress the formation of bubbles during cultivation. Additionally, oxygen or oxygen-containing gases (e.g., air) can be injected into the culture solution to maintain an aerobic state in the medium or culture solution.

[0034] Such seed cultures can be cultivated to a desired concentration of microorganisms by the user, and the OD (optical density) value of the seed culture liquid is measured to predict the concentration of microorganisms and determine whether to continue culturing.

[0035] According to one embodiment of the present invention, the seed culture has an OD 610 =10 to 80.

[0036] In the step of producing a fermentation broth by fermenting two or more types of microorganisms, a fermentation broth containing different types of products can be produced by mixed fermentation (main fermentation) using each microorganism or its seed culture broth.

[0037] The mixed fermentation is a concept that is contrary to the conventional individual fermentation in which each microorganism is cultured individually to produce a fermentation broth containing the products of each microorganism. In the present invention, mixed fermentation is carried out to produce, for example, L-glutamic acid by glutamic acid-producing microorganisms, L-lysine by lysine-producing microorganisms, L-arginine by arginine-producing microorganisms, and IMP by inosinic acid-producing microorganisms, and the interaction between these products and the addition of flavor components such as ions, nucleic acids, organic acids, and peptides additionally produced during the microbial fermentation process can produce a fermentation broth that has a rich body and excellent sensory characteristics.

[0038] According to one embodiment of the present invention, the step preferably involves adjusting the inoculation amount of each microorganism in order to adjust the ratio of the products of each microorganism, i.e., amino acids, nucleic acids and / or organic acids, in the fermentation broth.

[0039] More specifically, when two types of microorganisms are inoculated, the inoculation ratio of the first microorganism to the second microorganism may be a ratio of first microorganism:second microorganism=0.05-99.95:99.95-0.05 in the total inoculation amount. When three types of microorganisms are inoculated, the inoculation ratio of the first microorganism, second microorganism, and third microorganism may be a ratio of first microorganism:second microorganism:third microorganism=0.05-99.95:99.95-0.05:99.95-0.05 in the total inoculation amount.

[0040] For example, when the inoculation ratio of glutamic acid-producing microorganisms to lysine-producing microorganisms is 50-99.95:50-0.05, the mixed fermentation of the two microorganisms can produce L-glutamic acid and L-lysine in a ratio of 0.83-99:1 in the fermentation broth, which is suitable for creating a flavor suitable for use as a seasoning ingredient. When the inoculation ratio of glutamic acid-producing microorganisms to arginine-producing microorganisms is 30-99.95:70-0.05, the mixed fermentation of the two microorganisms can produce L-glutamic acid and L-arginine in a ratio of 1.04-99:1. When the inoculation ratio of glutamic acid-producing microorganisms to inosinic acid-producing microorganisms is 0.05-99.95:99.95-0.05, the mixed fermentation of the two microorganisms can produce L-glutamic acid and IMP in a ratio of 0.02-99.8:1 in the fermentation broth. When the inoculation ratio of the inosinic acid-producing microorganism to the lysine-producing microorganism is 0.05-99.95:99.95-0.05, IMP and L-lysine can be produced in a ratio of 0.01-92.3:1 in the fermentation broth by mixed fermentation of the two microorganisms. When the inoculation ratio of the inosinic acid-producing microorganism to the arginine-producing microorganism is 65-99.95:35-0.05, IMP and L-arginine can be produced in a ratio of 1.04-95.1:1 in the fermentation broth by mixed fermentation of the two microorganisms.

[0041] The mixed fermentation may be carried out using an appropriate medium and fermentation conditions known in the art, taking into account the characteristics of each microorganism, and a person skilled in the art can easily adjust and use the medium and fermentation conditions.

[0042] More specifically, the fermentation medium used in the mixed fermentation contains nutrients necessary for the growth and proliferation of microorganisms and may be a liquid medium.

[0043] The fermentation medium is a medium used during the main fermentation process for mass production of amino acids, nucleic acids, and / or organic acids, 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 material without a separate purification process after fermentation, it is preferable that the fermentation medium is composed of substances usable as food ingredients and contains the minimum amount of ingredients necessary for culturing the microorganisms.

[0044] According to one embodiment of the invention, the fermentation medium may be molasses-based and contain raw sugar and / or glucose.

[0045] More specifically, the fermentation medium contains molasses, raw sugar, and glucose as sugar and nutrient sources for the microorganisms, and the molasses content of the total equivalent is 1 to 30 wt %. The molasses used here may be derived from sugar cane or sugar beet.

[0046] The fermentation medium may further contain, in addition to a sugar source, nutrients for enriching the microorganisms.

[0047] According to one embodiment of the present invention, the fermentation medium may further contain one or more selected from the group consisting of yeast extract, phosphoric acid, and betaine.

[0048] The fermentation temperature in the mixed fermentation may usually be 20 to 45°C, for example, 25 to 40°C, or 30 to 38°C, and the fermentation period may be continued until the microorganisms actively grow and proliferate, for example, 10 to 160 hours, 18 to 120 hours, or 20 to 100 hours.

[0049] During cultivation, compounds such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be added to the medium or culture solution in an appropriate manner to adjust the pH of the culture solution. Furthermore, food additive antifoaming agents can be used to suppress foam formation during cultivation. Additionally, oxygen or oxygen-containing gases (e.g., air) can be injected into the culture solution to maintain an aerobic state in the medium or culture solution.

[0050] The fermented liquid produced by such mixed fermentation is the raw material for the seasoning material and can be used as is without mixing with additional ingredients. It contains a large amount of amino acids, nucleic acids and / or organic acids produced by the microorganisms.

[0051] According to one embodiment of the present invention, the fermentation liquid may contain 3 to 90 wt % of the total microbial products in the total solid content.

[0052] More specifically, the fermentation broth may contain 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% of amino acids, nucleic acids, and / or organic acids based on the total solid content. Such a fermentation broth may contain 5 to 150 g / L of amino acids, nucleic acids, and / or organic acids.

[0053] Another aspect of the present invention provides a method for producing an L-glutamic acid- and L-lysine-containing seasoning material, which includes the steps of inoculating a fermentation medium with a glutamic acid-producing microorganism and a lysine-producing microorganism, followed by fermentation to produce an L-glutamic acid- and L-lysine-containing fermentation liquid.

[0054] The glutamic acid-producing microorganism and the lysine-producing microorganism may be wild-type microorganisms obtained in nature, or mutant strains modified to improve the ability to produce amino acids. Microorganisms known in the art may be used without limitation, including, for example, the genera Corynebacterium, Brevibacterium, Lactobacillus, Bifidobacterium, and Bacillus. The microorganisms of the present invention may be of the same genus or species or different genera or species, and can be selected by the user.

[0055] According to one embodiment of the present invention, the glutamic acid-producing microorganism and the lysine-producing microorganism may be a Corynebacterium microorganism.

[0056] In one embodiment of the present invention, Corynebacterium glutamicum was used as a glutamic acid-producing microorganism and a lysine-producing strain.

[0057] For mixed fermentation of the glutamic acid-producing microorganism and the lysine-producing microorganism, isolated and selected microorganisms may be used, or a microbial mixture obtained by mixing these microorganisms may be used. Depending on the circumstances, the isolated microorganisms may be appropriately mixed and used. Such glutamic acid- or lysine-producing microorganisms can be used for fermentation in a state where their growth and amino acid-producing abilities are activated, and it is preferable to perform seed culture for microbial activation.

[0058] According to one embodiment of the present invention, the glutamic acid-producing microorganism and the lysine-producing microorganism may be cultured individually or in a mixed culture state as a seed culture medium.

[0059] The seed culture may be carried out using a suitable medium and culture conditions known in the art depending on the characteristics of each microorganism, and a person skilled in the art can easily adjust the medium and culture conditions.

[0060] More specifically, the medium used for the seed culture contains nutrients necessary for the growth and proliferation of the microorganism, and may be a liquid medium.

[0061] According to one embodiment of the present invention, the seed culture medium for the glutamic acid-producing microorganism may contain, based on the total weight, 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 a food-grade antifoaming agent.

[0062] According to one embodiment of the present invention, the seed culture medium for the lysine-producing microorganism may contain, based on the total weight, 1.5 to 3 wt % of molasses, 9 to 12 wt % of raw sugar, 1 wt % of yeast extract paste, 1.6 wt % of (NH)SO, 0.3 wt % of HPO, 7.3 ppm of MnSO·5H, 14 ppm of nicotinamide, 2.5 ppm of thiamine HCl, 51.5 ppm of CuSO·H, 0.056 ppm of biotin, 0.045 wt % of betaine, and 0.01 wt % of a food-grade antifoaming agent.

[0063] The culture temperature for the seed culture may generally be 20 to 45°C, for example, 25 to 40°C, or 27 to 37°C, and the culture period may be continued until the microorganisms actively grow and proliferate, for example, 10 to 160 hours, 18 to 120 hours, or 20 to 80 hours.

[0064] During cultivation, compounds such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be added to the medium or culture solution in an appropriate manner to adjust the pH of the culture solution. Furthermore, food additive antifoaming agents can be used to suppress foam formation during cultivation. Additionally, oxygen or oxygen-containing gases (e.g., air) can be injected into the culture solution to maintain an aerobic state in the medium or culture solution.

[0065] Such seed cultures can be cultivated to a desired concentration of microorganisms by the user, and the OD (optical density) value of the seed culture liquid is measured to predict the concentration of microorganisms and determine whether to continue culturing.

[0066] According to one embodiment of the present invention, the seed culture has an OD 610 =10 to 80.

[0067] In the step of producing a fermentation liquid by fermentation of such glutamic acid-producing microorganisms and lysine-producing microorganisms, a fermentation liquid containing all of L-glutamic acid and L-lysine can be produced by mixed fermentation (main fermentation) using a glutamic acid-producing microorganism or a seed culture thereof and a lysine-producing microorganism or a seed culture thereof.

[0068] The mixed fermentation is a concept that is contrary to the conventional individual fermentation in which each microorganism is cultured individually to produce a fermentation broth containing the products of each microorganism for amino acid production. In the present invention, mixed fermentation produces L-glutamic acid using glutamic acid-producing microorganisms and L-lysine using lysine-producing microorganisms, and a fermentation broth that can produce a rich body and excellent sensory characteristics can be produced by adding flavor components such as ions, nucleic acids, organic acids, and peptides that are additionally produced during the microbial fermentation process through the interaction between L-glutamic acid and L-lysine.

[0069] According to one embodiment of the present invention, the step may involve adjusting the inoculation amounts of the glutamic acid-producing microorganism and the lysine-producing microorganism to adjust the ratio of L-glutamic acid to L-lysine in the fermentation broth.

[0070] More specifically, the inoculation ratio of the glutamic acid-producing microorganisms to the lysine-producing microorganisms in the total inoculation amount may be 0.05 to 99.95:99.95 to 0.05, and mixed fermentation of the two microorganisms inoculated at such a ratio can produce L-glutamic acid and L-lysine in the fermentation broth at a ratio of 1 to 99:1, so as to produce a flavor suitable for use as a seasoning ingredient.

[0071] For example, when the inoculation ratio of the glutamic acid-producing microorganism to the lysine-producing microorganism is 50 to 99.95:50 to 0.05, L-glutamic acid and L-lysine can be produced in a ratio of 0.83 to 99:1 in the fermentation broth. When the inoculation ratio of the glutamic acid-producing microorganism to the lysine-producing microorganism is adjusted to 65:35, L-glutamic acid and L-lysine can be produced in a ratio of approximately 1:1 in the fermentation broth.

[0072] The mixed fermentation may be carried out using an appropriate medium and fermentation conditions known in the art, taking into account the characteristics of each microorganism, and a person skilled in the art can easily adjust and use the medium and fermentation conditions.

[0073] More specifically, the fermentation medium used in the mixed fermentation may be a liquid medium.

[0074] The fermentation medium is a medium used in the main fermentation for mass production of L-glutamic acid and L-lysine, 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 material without a separate purification process after fermentation is completed, the fermentation medium is preferably composed of substances usable as food ingredients and contains the minimum amount of ingredients necessary for the cultivation of the microorganisms.

[0075] According to one embodiment of the invention, the fermentation medium may be molasses-based and contain raw sugar and / or glucose.

[0076] More specifically, the fermentation medium contains molasses, raw sugar, and glucose as sugar and nutrient sources for the microorganisms, and may contain molasses in an amount of 1 to 30% by weight based on the total equivalent weight. The molasses used here may be derived from sugar cane or sugar beet.

[0077] The fermentation medium may further contain, in addition to a sugar source, nutrients for enriching the microorganisms.

[0078] According to one embodiment of the present invention, the fermentation medium may further contain one or more selected from the group consisting of yeast extract, phosphoric acid, and betaine.

[0079] According to one embodiment of the present invention, the fermentation medium may contain, based on the total weight, 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 a food-grade antifoaming agent.

[0080] The fermentation temperature in the mixed fermentation may usually be 20 to 45°C, for example, 25 to 40°C, or 30 to 38°C, and the fermentation period may be continued until the desired contents or concentrations of L-glutamic acid and L-lysine are obtained, for example, 10 to 160 hours, 18 to 120 hours, or 20 to 100 hours.

[0081] During fermentation, compounds such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be added to the medium or fermentation liquor in an appropriate manner to adjust the pH of the fermentation liquor. Furthermore, food additive antifoaming agents can be used to suppress the formation of bubbles during fermentation. Additionally, oxygen or oxygen-containing gases (e.g., air) can be injected into the fermentation liquor to maintain an aerobic state in the medium or fermentation liquor.

[0082] According to one embodiment of the present invention, the fermentation may be carried out by fed-batch culture for 28 to 40 hours, starting at a temperature of 30 to 33°C and maintaining it at 36 to 39°C, maintaining a pH of 6.5 to 7.5, and maintaining a dissolved oxygen content of 20 to 70%.

[0083] Such a fermentation liquid containing L-glutamic acid and L-lysine is a raw material for an L-glutamic acid and L-lysine-containing seasoning material, and is characterized by being usable as it is without mixing with additional ingredients, and containing large amounts of L-glutamic acid and L-lysine.

[0084] According to one embodiment of the present invention, the fermentation broth may contain 3 to 90 wt % of amino acids including L-glutamic acid and L-lysine based on the total solid content.

[0085] More specifically, the content of amino acids including L-glutamic acid and L-lysine in the fermentation broth can be 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 solid content. Such a fermentation broth can contain 5 to 150 g / L of L-glutamic acid and L-lysine.

[0086] Meanwhile, the method for producing a seasoning material according to the present invention may further include an additional step for using the fermented liquid as a seasoning material or an L-glutamic acid and L-lysine-containing seasoning material.

[0087] More specifically, the method may further include the steps of separating (removing) the cells from the fermentation liquid and decolorizing the liquid.

[0088] The bacterial cell separation can be performed without limitation using a bacterial cell separation method and separation conditions known in the art, including, but not limited to, membrane separation, ultrafiltration, centrifugal filtration, etc.

[0089] The decolorization can be performed using any decolorization method and conditions known in the art, without limitation. Examples of the decolorization method include, but are not limited to, activated carbon.

[0090] The method may further include filtering the decolorized fermentation liquor.

[0091] The filtration can be performed using any filtration method and conditions known in the art, including, but not limited to, filter paper, filter mesh, membrane filtration, and ultrafiltration.

[0092] The method may further include a step of concentrating the filtered fermentation liquid.

[0093] The concentration can be performed without limitation by using a concentration method and conditions known in the art, including, but not limited to, heat concentration, vacuum concentration, freeze concentration, evaporation concentration, and vacuum low-temperature concentration.

[0094] The method may further include a step of drying and powdering the concentrated fermentation liquid.

[0095] The drying can be carried out by any drying method and under any drying conditions known in the art, including, but not limited to, freeze drying, vacuum drying, ventilation drying, hot air drying, fluidized bed drying, spray drying, infrared drying, and microwave drying.

[0096] By this method, the fermented liquid is finally obtained in a powder form, which can be used in foods as a natural seasoning material without undergoing any additional chemical refining process.

[0097] Another aspect of the present invention provides a seasoning material produced by the method for producing a seasoning material using two or more types of microorganisms described above.

[0098] According to one embodiment of the present invention, the seasoning material may be a natural seasoning material containing two or three kinds of flavor components, namely, amino acids, nucleic acids, and organic acids.

[0099] The amino acid may be one or more 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.

[0100] The nucleic acid may be one or more selected from the group consisting of inosinic acid, guanylic acid, xanthylic acid, and salt forms thereof.

[0101] For example, the nucleic acid may be, but is not limited to, inosine monophosphate (IMP), guanosine monophosphate (GMP), xanthosine monophosphate (XMP), etc.

[0102] The organic acid may be one or more 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 salt forms thereof.

[0103] For example, when two flavoring components are included, the seasoning material may contain glutamic acid and lysine, arginine, histidine, tryptophan, glycine, alanine, succinic acid, lactic acid, guanylic acid, or inosinic acid. Alternatively, the seasoning material may contain inosinic acid and lysine, arginine, histidine, tryptophan, glycine, alanine, succinic acid, lactic acid, or guanylic acid.

[0104] As another example, when the seasoning material contains three flavor components, the seasoning material may contain glutamic acid; guanylic acid; and inosinic acid.

[0105] According to one embodiment of the present invention, the seasoning material may contain 3 to 90 wt % of flavor components including amino acids, nucleic acids and / or organic acids based on the total solid content.

[0106] More specifically, the seasoning material may contain flavor components including amino acids, nucleic acids and / or organic acids 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 solid content.

[0107] Another aspect of the present invention provides an L-glutamic acid- and L-lysine-containing seasoning material produced by the method for producing an L-glutamic acid- and L-lysine-containing natural seasoning material using the aforementioned glutamic acid-producing microorganism and lysine-producing microorganism.

[0108] According to one embodiment of the present invention, the seasoning material containing L-glutamic acid and L-lysine may be a natural seasoning material.

[0109] According to one embodiment of the present invention, the seasoning ingredient may contain 3 to 90% by weight of L-glutamic acid and L-lysine based on the solid content, and may contain L-glutamic acid and L-lysine in a ratio of 0.83 to 99:1.

[0110] This seasoning ingredient contains L-lysine as well as L-glutamic acid, which creates a rich flavor, thereby increasing the solubility of L-glutamic acid and reducing the production of ammonium glutamate during the fermentation process, thereby eliminating the problem of ammonia odor.It also contains metabolic products such as organic acids, inorganic ions, proteins, peptides, and vitamins produced during the fermentation process, giving it a rich flavor and a strong body, and exhibiting excellent sensory properties, it can be added to a variety of foods to maximize the flavor of the food.

[0111] Another aspect of the present invention provides a food composition containing the above-mentioned seasoning material or the L-glutamic acid and L-lysine-containing seasoning material.

[0112] The term "food composition" as used in the present invention refers to a natural product or processed product containing one or more nutrients, preferably a product that has undergone some processing to be ready for direct consumption, and in its usual sense includes all of functional health foods, functional foods, beverages, food additives, and beverage additives.

[0113] According to one embodiment of the present invention, the food composition contains a seasoning ingredient produced without a chemical refining process or a seasoning ingredient containing natural L-glutamic acid and L-lysine, i.e., a natural seasoning ingredient, and may contain the seasoning ingredient in an amount of 0.001 to 90 wt %, more specifically 0.01 to 50 wt %, based on the total weight.

[0114] The food composition of the present invention may be provided in any dosage form suitable for food, including, for example, a solution, emulsion, viscous mixture, powder, granules, tablets, and capsules. In this case, various bases and / or additives necessary and appropriate for formulation of the dosage form may be included within a range that does not impair the main effects of the present invention. Additional additives, such as flavorings, colorants, bactericides, antioxidants, preservatives, humectants, thickeners, inorganic salts, and emulsifiers, may also be included within a range that does not impair the effects of the present invention. The amount of such additives can be selected depending on the dosage form or intended use, within a range that does not impair the objects and effects of the present invention. For example, the additives may be present in an amount of 0.01 to 70 wt %, more specifically 0.1 to 50 wt %, based on the total weight of the food composition.

[0115] Such a food composition can be used as an additive for various foods, and the types of foods to which the food composition can be added are not limited as long as they are known in the art, and examples thereof include, but are not limited to, meat, sausage, bread, chocolate, candy, snacks, sweets, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, drinking water, tea, energy drinks, alcoholic beverages, and vitamin complexes. [Effects of the Invention]

[0116] The method for producing a seasoning material according to the present invention involves fermenting a mixture of different microorganisms that produce different products, i.e., different types of amino acids, nucleic acids, and / or organic acids, to produce a natural seasoning material that can improve the taste and aroma of food and the overall sensory characteristics of the food by providing a fermentation liquid containing amino acids, nucleic acids, and / or organic acids. Such a seasoning material can be used in various food fields. [Brief explanation of the drawings]

[0117] [Figure 1] 1 is a flowchart showing a fermentation process using glutamic acid-producing microorganisms and lysine-producing microorganisms in Production Examples 1 to 3 according to one embodiment of the present invention. [Figure 2] 1 is a flowchart showing a fermentation process using glutamic acid-producing microorganisms and arginine-producing microorganisms in Production Examples 4 to 6 according to one embodiment of the present invention. [Figure 3] 1 is a flowchart showing the fermentation process using glutamic acid-producing microorganisms and inosinic acid-producing microorganisms in Production Examples 7 to 9 according to one embodiment of the present invention. [Figure 4] 1 is a flowchart showing fermentation steps using inosinic acid-producing microorganisms and lysine-producing microorganisms in Production Examples 10 to 12 according to an embodiment of the present invention. [Figure 5] 1 is a flowchart showing a fermentation process using an inosinic acid-producing microorganism and an arginine-producing microorganism in Production Examples 13 to 15 according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0118] The present invention will be described in more detail below with reference to the accompanying drawings, but such description is merely provided as an example for understanding the present invention, and the scope of the present invention is not limited by such exemplary description. [Example]

[0119] Example 1. Mixed fermentation of glutamic acid and lysine 1-1. Seed culture The glutamic acid-producing microorganism used was Corynebacterium glutamicum NFG6 (KCCM13164P), which produces L-glutamic acid (GA), and the lysine-producing microorganism used was Corynebacterium glutamicum NFL21 (KCCM13163P), which produces L-lysine (LYS).

[0120] To seed culture the glutamic acid-producing microorganism, the glutamic acid-producing microorganism was inoculated into a 2-L flask containing 0.2 L of seed culture medium and primary cultured at 30°C and 140 rpm for 22 to 24 hours (OD 610 Then, 2-3% of the primary culture solution was inoculated into a 5 L jar fermenter, and 2-2.5 L of seed culture medium was added. Secondary culture was carried out for 22-24 hours at 32°C, pH 6.9, 600 rpm, and an aeration rate of 1.0 vvm (OD ). 610 =20-60), glutamic acid seed culture medium was prepared.

[0121] To seed culture the lysine-producing microorganism, the microorganism was inoculated into a 2-L flask containing 0.2 L of seed culture medium and subjected to primary culture at 30°C and 140 rpm for 16 to 18 hours (OD 610 A 5 L jar fermenter was inoculated with 5% of the primary culture, and 2 to 2.5 L of seed culture medium was added. Secondary culture was carried out for 21 to 24 hours at 32°C, pH 7.0, 650 rpm, and an aeration rate of 2.0 vvm (OD ). 610 =20-60), lysine seed culture medium was prepared.

[0122] The composition of the seed culture medium used here is shown in Table 1 below.

[0123] [Table 1]

[0124] 1-2. Main fermentation To confirm the ratio of glutamic acid to lysine in the fermentation broth depending on the inoculation amount of glutamic acid seed culture broth and lysine seed culture broth, glutamic acid seed culture broth and lysine seed culture broth were inoculated at various ratios and fermented.

[0125] In this fermentation, 14 to 18 L of fermentation medium was added to a 50 L fermenter, and 1.2 to 1.8 L of the total seed culture was inoculated with glutamic acid seed culture and lysine seed culture at an inoculum ratio of 50 to 99.95:50 to 0.05, and mixed fermentation was carried out by fed-batch culture for 28 to 40 hours. The composition and conditions of the fermentation medium used here are shown in Table 2 below.

[0126] [Table 2]

[0127] Such mixed fermentation was carried out three times in total, and the average values ​​were calculated. The results are shown in Table 3.

[0128] [Table 3]

[0129] Referring to Table 3, it was confirmed that when the seed culture of each amino acid microorganism was inoculated with glutamic acid-producing microorganisms:lysine-producing microorganisms at a ratio of 50 to 99.95:50 to 0.05, L-glutamic acid and L-lysine were produced in the fermentation broth at a ratio of 0.83 to 99:1.

[0130] Example 2. Comparison of glutamic acid-lysine fermented powders based on differences in fermentation process 2-1. Production of glutamic acid-lysine fermentation powder In the past, a seasoning material containing glutamic acid and lysine was produced by separately fermenting glutamic acid-producing microorganisms and lysine-producing microorganisms, followed by mixing the amino acid-containing fermentation liquid or its dried product in an appropriate ratio. To compare the difference in taste between this conventional separate fermentation method and the method of mixed fermentation of glutamic acid-producing microorganisms and lysine-producing microorganisms, the components of the glutamic acid-lysine (GA-LYS) fermentation powder obtained by each production method were compared (see Figure 1).

[0131] The glutamic acid seed culture medium and lysine seed culture medium used here were prepared in the same manner as in Example 1-1.

[0132] (1) Individual fermentation (Production Example 1) In the conventional method of mixing the individual dried products, the glutamic acid seed culture solution or the lysine seed culture solution was transferred to a 50 L fermenter, and then fermented separately. The bacteria were then separated from each fermentation solution, and the solution was decolorized and filtered. The filtrate was concentrated and dried to obtain a dried product. The resulting glutamic acid and lysine dried products were mixed at an amino acid ratio of 1:1 to produce a fermented powder containing glutamic acid and lysine.

[0133] (2) Mixing of fermented liquids after individual fermentations (Production Example 2) In the conventional method of mixing separate fermentation broths, glutamic acid seed culture broth and lysine seed culture broth were transferred to 50 L fermenters, respectively, and then main fermentation was carried out separately. The fermentation broths obtained in the main fermentation were mixed so that the amino acid ratio was 1:1, and the resulting bacterial cells were separated, followed by decolorization and filtration. The filtrate was concentrated and dried to obtain glutamic acid-lysine fermentation powder.

[0134] (3) Mixed fermentation (Production Example 3) For mixed fermentation, glutamic acid seed culture and lysine seed culture were inoculated at an inoculum ratio of 65:35 in the same manner as in Examples 1-2 so that the ratio of each amino acid in the fermentation broth was approximately 1:1, as in Production Examples 1 and 2. Mixed fermentation was then carried out. The fermentation broth was then subjected to decolorization and filtration steps after separating the bacterial cells. The filtrate was concentrated and dried to obtain a glutamic acid-lysine fermentation powder.

[0135] 2-2. Comparison of ingredients in glutamic acid-lysine fermented powder The glutamic acid-lysine fermentation powders obtained by the individual fermentations of Production Examples 1 and 2, and the glutamic acid-lysine fermentation powder obtained by the mixed fermentation of Production Example 3 were subjected to component analysis.

[0136] Amino acids (L-glutamic acid and L-lysine) were measured by HPLC (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 (HPX-87H column, 214 nm, 25 min). Ions (Na, Mg, K, PO, SO, Cl, NH, etc.) were measured using an ion analyzer (Dionex IcS-1100, Thermo Scientific). The results are shown in Table 4 below.

[0137] [Table 4]

[0138] Referring to Table 4, the mixed fermentation (Preparation Example 3) showed a higher amino acid ratio than the individual fermentation (Preparation Examples 1 and 2), and the contents of organic acids, ions, and ammonium were significantly reduced.

[0139] 2-3. Sensory comparison of individual fermentation and mixed fermentation The glutamic acid-lysine fermentation powders obtained by the individual fermentations of Production Examples 1 and 2 and the glutamic acid-lysine fermentation powder obtained by the mixed fermentation of Production Example 3 were subjected to sensory evaluation.

[0140] The glutamic acid-lysine fermented powder was subjected to a sensory evaluation by a panel of 10 to 15 experts, who evaluated the full-bodied taste, duration of full-bodied taste, saltiness, sourness, bitterness, and sweetness of the sample either as is or diluted 1 to 5% with lukewarm water. The results are shown in Table 5 below.

[0141] [Table 5]

[0142] Referring to Table 5, the taste of the sample produced by the process of mixing after individual fermentation was weaker in body and more bitter and sour than the sample produced by mixed fermentation due to the difference in the components of the glutamic acid-lysine fermented powder as shown in Table 4. Furthermore, as shown in Table 4, an increase in by-products such as organic acids and an increase in ions affect sensory perception, so a mixed fermentation process is more effective in terms of taste and process simplification than mixing after individual fermentation.

[0143] Example 3. Mixed fermentation of glutamic acid and arginine 3-1. Seed culture Corynebacterium glutamicum NFG6 (KCCM13164P) was used as a glutamic acid-producing microorganism, and Corynebacterium glutamicum NFA40 (KCCM13165P), which produces L-arginine (ARG), was used as an arginine-producing microorganism.

[0144] A glutamic acid seed culture using a glutamic acid-producing microorganism was prepared in the same manner as in Example 1-1.

[0145] To seed culture the arginine-producing microorganism, the arginine-producing microorganism was inoculated into a 2-L flask containing 0.2 L of seed culture medium and subjected to primary culture at 30°C and 140 rpm for 16 to 18 hours (OD 610 A 5 L jar fermenter was inoculated with 2-4% of the primary culture, and 2-2.5 L of seed culture medium was added. Secondary culture was carried out for 16-24 hours at 32°C, pH 6.7, 600 rpm, and an aeration rate of 1.0 vvm (OD = 12-18). 610 (=20-60) and arginine seed culture medium were prepared. The composition of the seed culture medium used is shown in Table 6 below.

[0146] [Table 6]

[0147] 3-2. Main fermentation To confirm the ratio of glutamic acid to arginine in the fermentation broth depending on the inoculation amount of glutamic acid seed culture broth and arginine seed culture broth, glutamic acid seed culture broth and arginine seed culture broth were inoculated at various ratios and fermented.

[0148] In this fermentation, 14 to 18 L of fermentation medium was added to a 50 L fermenter, and 1.2 to 1.8 L of the total seed culture was inoculated with glutamic acid seed culture and arginine seed culture at an inoculum ratio of 30 to 99.95:70 to 0.05. Mixed fermentation was carried out by fed-batch culture for 31 to 60 hours. The composition and conditions of the fermentation medium used here are shown in Table 7 below.

[0149] [Table 7]

[0150] Such mixed fermentation was carried out three times in total, and the average values ​​were calculated. The results are shown in Table 8.

[0151] [Table 8]

[0152] Referring to Table 8, it was confirmed that when the seed culture of each amino acid microorganism was inoculated at a ratio of glutamic acid-producing microorganism:arginine-producing microorganism = 30 to 99.95:70 to 0.05, L-glutamic acid and L-arginine were produced in the fermentation broth at a ratio of 1.04 to 99:1.

[0153] Example 4. Comparison of glutamic acid-arginine fermented powders based on differences in fermentation process 4-1. Production of glutamic acid-arginine fermented powder In the past, a seasoning material containing glutamic acid and arginine was produced by separately fermenting glutamic acid-producing microorganisms and arginine-producing microorganisms, followed by mixing the amino acid-containing fermentation liquid or its dried product in an appropriate ratio. To compare the taste difference between this conventional separate fermentation method and the mixed fermentation method using glutamic acid-producing microorganisms and arginine-producing microorganisms, the components of the glutamic acid-arginine (GA-ARG) fermented powder obtained by each production method were compared (see Figure 2).

[0154] The glutamic acid seed culture medium and arginine seed culture medium used here were prepared in the same manner as in Example 3-1.

[0155] (1) Individual fermentation (Production Example 4) In the conventional method of mixing individual dried products, glutamic acid seed culture solution or arginine seed culture solution was transferred to a 50 L fermenter, and then fermented separately. After that, the bacteria were separated from each fermentation solution, and then decolorized and filtered. The filtrate was concentrated and dried to obtain a dried product. The resulting glutamic acid and arginine dried products were mixed at an amino acid ratio of 1:1 to produce a fermented powder containing glutamic acid and arginine.

[0156] (2) Mixing of fermented liquids after individual fermentations (Production Example 5) In the conventional method of mixing separate fermentation broths, glutamic acid seed culture broth or arginine seed culture broth was transferred to a 50 L fermenter, and then the fermentation was carried out separately. The fermentation broths obtained in the main fermentation were mixed so that the amino acid ratio was 1:1, and the resulting fermentation broths were separated, followed by decolorization and filtration. The filtrate was concentrated and dried to obtain glutamic acid-arginine fermentation powder.

[0157] (3) Mixed fermentation (Production Example 6) In mixed fermentation, glutamic acid seed culture and arginine seed culture were inoculated at an inoculum ratio of 30:70 in the same manner as in Example 3-2, so that the ratio of each amino acid in the fermentation broth was approximately 1:1, as in Production Examples 4 and 5. The fermentation broth was then subjected to decolorization and filtration steps, after which the bacterial cells were separated. The filtrate was concentrated and dried to obtain a glutamic acid-arginine fermentation powder.

[0158] 4-2. Comparison of ingredients of glutamic acid-arginine fermented powder The glutamic acid-arginine fermented powders obtained by the individual fermentations in Production Examples 4 and 5, and the glutamic acid-arginine fermented powder obtained by the mixed fermentation in Production Example 6 were subjected to component analysis.

[0159] 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.

[0160] [Table 9]

[0161] Referring to Table 9, the mixed fermentation (Preparation Example 6) showed a higher amino acid ratio than the individual fermentation (Preparation Examples 4 and 5), and the contents of organic acids, ions, and ammonium were significantly reduced.

[0162] 4-3. Sensory comparison between individual fermentation and mixed fermentation The glutamic acid-arginine fermented powders obtained by the individual fermentations in Production Examples 4 and 5 and the glutamic acid-arginine fermented powder obtained by the mixed fermentation in Production Example 6 were subjected to sensory evaluation.

[0163] The sensory evaluation was carried out in the same manner as in Example 2-3, and the results are shown in Table 10 below.

[0164] [Table 10]

[0165] Referring to Table 10, the taste of the sample produced by the process of mixing after individual fermentation was weaker in body and more bitter than the sample produced by mixed fermentation due to the difference in the components of the glutamic acid-arginine fermented powder as shown in Table 9. Furthermore, as shown in Table 9, an increase in by-products such as organic acids and an increase in ions affect sensory perception, so a mixed fermentation process is more effective in terms of taste and process simplification than mixing after individual fermentation.

[0166] Example 5. Mixed fermentation of glutamic acid and inosinic acid 5-1. Seed culture The glutamic acid-producing microorganism Corynebacterium glutamicum NFG6 (KCCM13164P) and the inosinic acid-producing microorganism Corynebacterium ammoniagenes NFI545 (KCCM13162P), which produces IMP, were used.

[0167] A glutamic acid seed culture using a glutamic acid-producing microorganism was prepared in the same manner as in Example 1-1.

[0168] To seed culture the inosinic acid-producing microorganisms, the microorganisms were inoculated into a 2-L flask containing 0.3 L of seed culture medium and primary cultured at 31°C and 150 rpm for 20 to 24 hours (OD 610 A 5 L jar fermenter was inoculated with 1% of the primary culture, and 2 to 2.5 L of seed culture medium was added. Secondary culture was carried out for 21 to 24 hours at 31°C, pH 7.1, 600 rpm, and an aeration rate of 1.0 vvm (OD ). 610 (=20-40) and inosinic acid seed culture medium were prepared. The composition of the seed culture medium used is shown in Table 11 below.

[0169] [Table 11]

[0170] 5-2. Main fermentation To confirm the ratio of glutamic acid to inosinic acid in the fermentation broth depending on the inoculation amount of glutamic acid seed culture broth and inosinic acid seed culture broth, glutamic acid seed culture broth and inosinic acid seed culture broth were inoculated at various ratios and fermented.

[0171] In this fermentation, 14 to 18 L of fermentation medium was added to a 50 L fermenter, and 1.2 to 1.8 L of the total seed culture was inoculated with glutamic acid seed culture and inosinic acid seed culture at an inoculum ratio of 0.05 to 99.95:99.95 to 0.05, and mixed fermentation was carried out by fed-batch culture for 30 to 90 hours. The composition and conditions of the fermentation medium used here are shown in Table 12 below.

[0172] [Table 12]

[0173] Such mixed fermentation was carried out three times in total, and the average values ​​were calculated. The results are shown in Table 13.

[0174] [Table 13]

[0175] Referring to Table 13, when the seed culture of each microorganism was inoculated at a ratio of glutamic acid-producing microorganisms:inosinic acid-producing microorganisms = 0.05~99.95:99.95~0.05, it was confirmed that L-glutamic acid and IMP were produced in the fermentation broth at a ratio of 0.02~99.8:1.

[0176] Example 6. Comparison of glutamic acid-inosinic acid fermented powders based on differences in fermentation process 6-1. Production of glutamic acid-inosinic acid fermentation powder In the past, a seasoning material containing glutamic acid and inosinic acid was produced by separately fermenting glutamic acid-producing microorganisms and inosinic acid-producing microorganisms, and then mixing the fermentation liquid or its dried product in an appropriate ratio. To compare the difference in taste between this conventional separate fermentation method and the mixed fermentation method using glutamic acid-producing microorganisms and inosinic acid-producing microorganisms, the components of the glutamic acid-inosinic acid (GA-IMP) fermented powder obtained by each production method were compared (see Figure 3).

[0177] The glutamic acid seed culture medium and inosinic acid seed culture medium used here were prepared in the same manner as in Example 5-1.

[0178] (1) Individual fermentation (Production Example 7) In the conventional method of mixing the individual dried products, the glutamic acid seed culture solution or the inosinic acid seed culture solution was transferred to a 50 L fermenter, and then fermented separately. The bacteria were then separated from each fermentation solution, and the solution was decolorized and filtered. The filtrate was concentrated and dried to obtain a dried product. The resulting glutamic acid and inosinic acid dried products were mixed in a 1:1 ratio to produce a fermented powder containing glutamic acid and inosinic acid.

[0179] (2) Mixing of fermented liquids after individual fermentations (Production Example 8) In the conventional method of mixing separate fermentation broths, glutamic acid seed culture broth and inosinic acid seed culture broth were transferred to 50 L fermenters, respectively, and then main fermentation was carried out separately. The fermentation broths obtained from the main fermentation were mixed in a 1:1 ratio, and the resulting fungal cells were separated, followed by decolorization and filtration. The filtrate was concentrated and dried to obtain glutamic acid-inosinic acid fermentation powder.

[0180] (3) Mixed fermentation (Production Example 9) In mixed fermentation, the glutamic acid seed culture and the inosinic acid seed culture were inoculated at an inoculum ratio of 20:80 in the same manner as in Example 5-2 so that the ratio of each amino acid in the fermentation broth was approximately 1:1, as in Production Examples 7 and 8. The fermentation broth was then subjected to decolorization and filtration steps after bacterial cells were separated. The filtrate was concentrated and dried to obtain a glutamic acid-inosinic acid fermentation powder.

[0181] 6-2. Comparison of ingredients in glutamic acid-inosinic acid fermented powder The glutamic acid-inosinic acid fermented powders obtained by individual fermentation in Production Examples 7 and 8, and the glutamic acid-inosinic acid fermented powder obtained by mixed fermentation in Production Example 9 were subjected to component analysis.

[0182] 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.

[0183] [Table 14]

[0184] 6-3. Sensory comparison of individual fermentation and mixed fermentation The glutamic acid-inosinic acid fermented powders obtained by individual fermentation in Production Examples 7 and 8, and the glutamic acid-inosinic acid fermented powder obtained by mixed fermentation in Production Example 9 were subjected to sensory evaluation.

[0185] The sensory evaluation was carried out in the same manner as in Example 2-3, and the results are shown in Table 15 below.

[0186] [Table 15]

[0187] Referring to Table 15, the taste of the sample produced by the process of mixing after individual fermentation was weaker in body and aftertaste lingering and more bitter than the sample produced by mixed fermentation due to the difference in the components of the glutamic acid-inosinic acid fermented powder as shown in Table 14. Furthermore, as shown in Table 14, an increase in by-products such as organic acids and an increase in ions also affect sensory perception, so the mixed fermentation process is more effective in terms of taste and process simplification than mixing after individual fermentation.

[0188] Example 7. Mixed fermentation of inosinic acid and lysine 7-1. Seed culture Corynebacterium ammoniagenes NFI545 (KCCM13162P) was used as an inosinic acid-producing microorganism, and Corynebacterium glutamicum NFL21 (KCCM13163P) was used as a lysine-producing microorganism.

[0189] An inosinic acid seed culture solution using an inosinic acid-producing microorganism and a lysine seed culture solution using a lysine-producing microorganism were prepared in the same manner as in Examples 5-1 and 1-1, respectively.

[0190] 7-2. Main fermentation To confirm the ratio of inosinic acid to lysine in the fermentation broth depending on the inoculation amount 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.

[0191] In this fermentation, 14 to 18 L of fermentation medium was added to a 50 L fermenter, and 1.2 to 1.8 L of the total seed culture was inoculated with the inosinic acid seed culture and the lysine seed culture at an inoculum ratio of 0.05 to 99.95:99.95 to 0.05. Mixed fermentation was carried out by fed-batch culture for 45 to 90 hours. The composition and conditions of the fermentation medium used here are shown in Table 16 below.

[0192] [Table 16]

[0193] This mixed fermentation was carried out three times in total, and the average values ​​were calculated. The results are shown in Table 17.

[0194] [Table 17]

[0195] Referring to Table 17, when the seed culture of each microorganism was inoculated at a ratio of inosinic acid-producing microorganism:lysine-producing microorganism = 0.05 to 99.95:99.5 to 0.05, it was confirmed that IMP and L-lysine were produced in the fermentation broth at a ratio of 0.01 to 92:1.

[0196] Example 8. Comparison of inosinic acid-lysine fermented powders based on differences in fermentation process 8-1. Production of inosinic acid-lysine fermented powder In the past, a seasoning material containing inosinic acid and lysine was produced by separately fermenting inosinic acid-producing microorganisms and lysine-producing microorganisms, and then mixing the fermentation liquid or its dried product in an appropriate ratio. To compare the difference in taste between this conventional separate fermentation method and the method of mixed fermentation of inosinic acid-producing microorganisms and lysine-producing microorganisms, the components of the inosinic acid-lysine (IMP-LYS) fermented powder obtained by each production method were compared (see FIG. 4).

[0197] The inosinic acid seed culture medium and lysine seed culture medium used here were prepared in the same manner as in Example 7-1.

[0198] (1) Individual fermentation (Production Example 10) In the conventional method of mixing the individual dried products, the inosinic acid seed culture solution or the lysine seed culture solution was transferred to a 50 L fermenter, and then fermented separately. Then, the cells were separated from each fermentation solution, and the solution was decolorized and filtered. The filtrate was concentrated and dried to obtain a dried product. The resulting inosinic acid and lysine dried products were mixed in a 1:1 ratio to produce a fermented powder containing inosinic acid-lysine.

[0199] (2) Mixing of fermented liquids after individual fermentations (Production Example 11) In the conventional method of mixing separate fermentation broths, the inosinic acid seed culture broth and the lysine seed culture broth were transferred to 50 L fermenters, respectively, and then main fermentation was carried out separately. The fermentation broths obtained from the main fermentation were mixed in a 1:1 ratio, and the resulting bacteria were separated, followed by decolorization and filtration. The filtrate was concentrated and dried to obtain an inosinic acid-lysine fermentation powder.

[0200] (3) Mixed fermentation (Production Example 12) In the mixed fermentation, the inosinic acid seed culture solution and the lysine seed culture solution were inoculated at an inoculum 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 broth was approximately 1:1, as in Production Examples 10 and 11. The fermentation broth was then subjected to decolorization and filtration steps after separating the bacterial cells. The filtrate was concentrated and dried to obtain an inosinic acid-lysine fermentation powder.

[0201] 8-2.Comparison of ingredients of lysine-inosinic acid fermentation powder The inosinic acid-lysine fermentation powders obtained by the individual fermentations of Production Examples 10 and 11 and the inosinic acid-lysine fermentation powder obtained by the mixed fermentation of Production Example 12 were subjected to component analysis.

[0202] 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.

[0203] [Table 18]

[0204] Referring to Table 18, the mixed fermentation (Preparation Example 12) showed a higher LYS+IMP ratio than the individual fermentation (Preparation Examples 10 and 11), and the organic acid, ion, and ammonia contents were significantly reduced.

[0205] 8-3. Sensory comparison of individual fermentation and mixed fermentation The inosinic acid-lysine fermented powders obtained by individual fermentation in Production Examples 10 and 11 and the inosinic acid-lysine fermented powder obtained by mixed fermentation in Production Example 12 were subjected to sensory evaluation.

[0206] The sensory evaluation was carried out in the same manner as in Example 2-3, and the results are shown in Table 19 below.

[0207] [Table 19]

[0208] Referring to Table 19, the taste of the sample produced by the process of mixing after individual fermentation was weaker in lingering aftertaste and more bitter than the sample produced by mixed fermentation due to the difference in the components of the inosinic acid-lysine fermented powder as shown in Table 18. Furthermore, as shown in Table 18, an increase in by-products such as organic acids and an increase in ions affect sensory perception, so the mixed fermentation process is more effective in terms of taste and process simplification than mixing after individual fermentation.

[0209] Example 9. Mixed fermentation of inosinic acid and arginine 9-1. Seed culture Corynebacterium ammoniagenes NFI545 (KCCM13162P) was used as an inosinic acid-producing microorganism, and Corynebacterium glutamicum NFA40 (KCCM13165P) was used as an arginine-producing microorganism.

[0210] An inosinic acid seed culture solution using an inosinic acid-producing microorganism and an arginine seed culture solution using an arginine-producing microorganism were prepared in the same manner as in Examples 5-1 and 3-1, respectively.

[0211] 9-2. Main fermentation In order to confirm the ratio of inosinic acid to arginine in the fermentation broth depending on the inoculation amount 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.

[0212] In this fermentation, 14 to 18 L of fermentation medium was added to a 50 L fermenter, and 1.2 to 1.8 L of the total seed culture was inoculated with the inosinic acid seed culture and the arginine seed culture at an inoculum ratio of 65 to 99.95:35 to 0.05. Mixed fermentation was carried out by fed-batch culture for 45 to 80 hours. The composition and conditions of the fermentation medium used here are shown in Table 20 below.

[0213] [Table 20]

[0214] This mixed fermentation was carried out three times in total, and the average values ​​were calculated. The results are shown in Table 21.

[0215] [Table 21]

[0216] Referring to Table 21, when the seed culture of each microorganism was inoculated at a ratio of inosinic acid-producing microorganism:arginine-producing microorganism = 65~99.95:35~0.05, it was confirmed that IMP and L-arginine were produced in the fermentation broth at a ratio of 1.04~95.1:1.

[0217] Example 10. Comparison of inosinic acid-arginine fermented powders based on differences in fermentation process 10-1. Production of inosinic acid-arginine fermented powder In the past, a seasoning material containing inosinic acid and arginine was produced by separately fermenting inosinic acid-producing microorganisms and arginine-producing microorganisms, and then mixing the fermentation liquid or its dried product in an appropriate ratio. To compare the difference in taste between this conventional separate fermentation method and the method of mixed fermentation of inosinic acid-producing microorganisms and arginine-producing microorganisms, the components of the inosinic acid-arginine (IMP-ARG) fermented powder obtained by each production method were compared (see FIG. 5).

[0218] The inosinic acid seed culture medium and arginine seed culture medium used here were prepared in the same manner as in Example 9-1.

[0219] (1) Individual fermentation (Production Example 13) In the conventional method of mixing the individual dried products, the inosinic acid seed culture solution or the arginine seed culture solution was transferred to a 50 L fermenter, and then fermented separately. The bacterial cells were then separated from each fermentation solution, followed by decolorization and filtration. The filtrate was concentrated and dried to obtain a dried product. The resulting inosinic acid and arginine dried products were mixed in a 1:1 ratio to produce a fermented powder containing inosinic acid and arginine.

[0220] (2) Mixing of fermented liquids after individual fermentations (Production Example 14) In the conventional method of mixing separate fermentation broths, the inosinic acid seed culture broth and the lysine seed culture broth were transferred to a 50 L fermenter, respectively, and then subjected to separate main fermentation. The fermentation broths obtained from the main fermentation were mixed in a 1:1 ratio, and the resulting cells were separated, followed by decolorization and filtration. The filtrate was concentrated and dried to obtain an inosinic acid-arginine fermentation powder.

[0221] (3) Mixed fermentation (Production Example 15) In the mixed fermentation, the inosinic acid seed culture and the arginine seed culture were inoculated at an inoculum 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 broth was approximately 1:1, as in Production Examples 13 and 14. The fermentation broth was then subjected to decolorization and filtration steps after bacterial cells were separated. The filtrate was concentrated and dried to obtain an inosinic acid-arginine fermentation powder.

[0222] 10-2. Comparison of ingredients of inosinic acid-arginine fermented powder The inosinic acid-arginine fermented powders obtained by the individual fermentations in Production Examples 13 and 14, and the inosinic acid-arginine fermented powder obtained by the mixed fermentation in Production Example 15 were subjected to component analysis.

[0223] L-arginine and IMP were measured by HPLC analysis (ARG-195 nm, UV detector, flow rate 1 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 22 below.

[0224] [Table 22]

[0225] 10-3. Sensory comparison of individual fermentation and mixed fermentation Sensory evaluation was carried out on the inosinic acid-arginine fermented powders obtained by individual fermentation in Production Examples 13 and 14, and the inosinic acid-arginine fermented powder obtained by mixed fermentation in Production Example 15.

[0226] The sensory evaluation was carried out in the same manner as in Example 2-3, and the results are shown in Table 23 below.

[0227] [Table 23]

[0228] Referring to Table 23, the taste of the sample produced by the process of mixing after individual fermentation was weaker in lingering aftertaste and more bitter than the sample produced by mixed fermentation due to the difference in the components of the inosinic acid-arginine fermented powder as shown in Table 22. Furthermore, as shown in Table 22, an increase in by-products such as organic acids and an increase in ions also affect sensory perception, so a mixed fermentation process is more effective in terms of taste and process simplification than mixing after individual fermentation.

[0229] The present invention has been described above with reference to its preferred embodiments. Those skilled in the art will understand that the present invention can be embodied in various modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is defined by the claims, not the above description, and all variations within the scope of the claims should be construed as being within the scope of the present invention.

[0230] [Accession number] Depository institution: Korea Center for Microorganisms (KCCM) Accession number: KCCM13162P Date of acceptance: 20220421 Depository institution: Korea Center for Microorganisms (KCCM) Accession number: KCCM13163P Date of acceptance: 20220421 Depository institution: Korea Center for Microorganisms (KCCM) Accession number: KCCM13164P Date of acceptance: 20220421 Depository institution: Korea Center for Microorganisms (KCCM) Accession number: KCCM13165P Date of acceptance: 20220421 [Deposit receipt] TIFF2026012523000024.tif249170TIFF2026012523000025.tif249170TIFF2026012523000026.tif249170TIFF2026012523000027.tif249170 TIFF2026012523000028.tif124146TIFF2026012523000029.tif123144TIFF2026012523000030.tif126145TIFF2026012523000031.tif123145

Claims

1. a step of inoculating a fermentation medium with a first microorganism and a second microorganism, and then fermenting the medium to produce a fermentation liquid containing amino acids and nucleic acids; The first microorganism and the second microorganism are Corynebacterium glutamicum and Corynebacterium ammoniagenes, respectively, and produce different products, i.e., amino acids and nucleic acids, respectively.

2. 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 one or more selected from the group consisting of inosinic acid, guanylic acid, xanthylic acid, and salt forms thereof.

4. the first microorganism is a glutamic acid-producing microorganism; The method of claim 1 , wherein the second microorganism is a guanylate-producing microorganism or an inosinate-producing microorganism.

5. the first microorganism is an inosinic acid-producing microorganism, 2. The method of claim 1, wherein the second microorganism is a lysine-producing microorganism, an arginine-producing microorganism, a histidine-producing microorganism, a tryptophan-producing microorganism, a glycine-producing microorganism, or an alanine-producing microorganism.

6. The method according to claim 1, wherein the step further comprises inoculating a third microorganism that produces a product different from the first and second microorganisms and that produces one selected from the group consisting of amino acids, nucleic acids, and organic acids.

7. 7. The method of claim 6, wherein the first microorganism is a glutamic acid-producing microorganism, the second microorganism is a guanylic acid-producing microorganism, and the third microorganism is an inosinic acid-producing microorganism.

8. The method of claim 6, wherein the third microorganism is a microorganism of the genus Corynebacterium.

9. 7. The method of claim 6, wherein the first microorganism, the second microorganism, and the third microorganism are cultured individually or in a mixed culture seed culture state.

10. The method according to claim 1 or 6, wherein the step comprises adjusting the inoculation amount of each microorganism to adjust the ratio of the product of each microorganism in the fermentation broth.

11. The method according to claim 1 or 6, wherein the fermentation liquid contains 3 to 90 wt % of the total microbial products in the total solid content.

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