Method for preparing seasoning material by mixed fermentation of heterogeneous microbes

Abstract

Abstract The present invention relates to a method for manufacturing a seasoning material using a process of mixing and simultaneously fermenting two or more kinds of microorganisms having different kinds of produced materials, wherein the method for manufacturing the seasoning material is capable of manufacturing a natural seasoning material capable of improving a taste and an aroma of food and improving overall sensory characteristics through a fermentation broth containing an amino acid, a nucleic acid, and / or an organic acid by mixing and fermenting heterologous microorganisms producing different products, that is, different kinds of the amino acid, the nucleic acid, and / or the organic acid, which is utilized in various food fields. Abstract 1 / 5 Fig. 1 1 / 5 Fig. 1 Preparation Example 1 Preparation Example 2 Preparation Example 3 GA LYS GA LYS GA LYS Seed culture Seed culture Seed culture Seed culture Seed culture Seed culture Main Main Main Main Mixed fermentation fermentation fermentation fermentation fermentation (Main fermentation) Filtration Filtration Mixing Filtration (cell removal) (cell removal) (cell removal) Desorption Desorption Filtration (decolorization) (decolorization) (cell removal) Desorption (decolorization) Filtration Filtration Desorption (decolorization) Filtration Concentration Concentration Filtration Concentration Drying Drying Concentration Mixing Drying Drying GA-LYS fermented powder GA-LYS fermented powder GA-LYS fermented powder This data, for application number 2022432263, is current as of 2026-08-13 08:41 AEST 1 / 5 GA LYS GA LYS GA LYS Seed culture Seed culture Seed culture Seed culture Seed culture Seed culture Main Main Main Main Mixed fermentation Filtration Filtration Concentration Concentration Concentration Concentration 20 26 25 39 28 15 O ct 2 02 6 1 / 5 2 0 2 6 2 5 3 9 2 8 1 5 O c t 2 0 2 6 S e e d c u l t u r e S e e d c u l t u r e S e e d c u l t u r e S e e d c u l t u r e S e e d c u l t u r e S e e d c u l t u r e M a i n M a i n M a i n M a i n M i x e d f e r m e n t a t i o n F i l t r a t i o n F i l t r a t i o n F i l t r a t i o n C o n c e n t r a t i o n C o n c e n t r a t i o n C o n c e n t r a t i o n C o n c e n t r a t i o n

Description

ppm of thiamin HCl, 1.5 ppm of CuSO4^5H2O, 0.056 ppm of biotin, 0.045 wt% of betaine, and 0.01 wt% of an 10 antifoaming agent for food.

[0068] The culture temperature in the seed culture may be usually 20 to 45°C, for example, 25 to 40°C, or 27 to 37°C, and the culture may be continued until each microorganism actively grows and proliferates. The culture time may be, 15 for example, 10 to 160 hours, 18 to 120 hours, or 20 to 80 hours.

[0069] In addition, during culture, compounds such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid may be added to the 20 medium or culture broth in an appropriate manner to adjust the pH of the culture broth. In addition, it is possible to suppress the formation of air bubbles by using an antifoaming agent for food during culture. Additionally, in order to maintain the medium or culture broth in an aerobic 25 state, oxygen or oxygen-containing gas (e.g., air) may be 2026253928   15 Oct 2026 injected into the culture broth.

[0070] This seed culture may be performed depending on a microbial concentration desired by the user, and the optical density (OD) value of the seed culture broth is 5 measured to predict the microbial concentration and determine whether to continue culture.

[0071] According to one embodiment of the present invention, the seed culture broth may have OD610 = 10 to 80.

[0072] In the step of producing a fermentation broth by 10 fermentation of the glutamic acid-producing microorganism and the lysine-producing microorganism, a fermentation broth containing both L-glutamic acid and L-lysine may be produced by mixed fermentation (main fermentation) using the glutamic acid-producing microorganism or a seed culture 15 broth thereof and the lysine-producing microorganism or a seed culture broth thereof.

[0073] The mixed fermentation is a concept opposite to a conventional individual fermentation process of producing a fermentation broth containing the product of each 20 microorganism by culturing each microorganism individually for amino acid production.

[0074] In the present invention, through mixed fermentation, it is possible to produce L-glutamic acid by the glutamic acid-producing microorganism and L-lysine by the lysine- 25 producing microorganism. In addition, it is possible to 2026253928   15 Oct 2026 produce a fermentation broth capable of exhibiting a rich umami taste and excellent sensory properties due to the interaction between L-glutamic acid and L-lysine and due to taste components such as ions, nucleic acids, organic acids, 5 and other peptides, which are additionally produced in the microbial fermentation process.

[0075] According to one embodiment of the present invention, in the step, it is preferable to adjust the inoculum of each of the glutamic acid-producing microorganism and the 10 lysine-producing microorganism in order to control the ratio between L-glutamic acid and L-lysine in the fermentation broth.

[0076] More specifically, the inoculation ratio between the glutamic acid-producing microorganism and the lysine-15 producing microorganism may be a ratio of 0.05 to 99.95 (glutamic acid-producing microorganism): 99.95 to 0.05 (lysine-producing microorganism) relative to the total inoculum.   Through mixed fermentation   of the two microorganisms inoculated at this ratio, L-glutamic acid 20 and L-lysine in the fermentation broth may be produced at a ratio of 1 to 99: 1 so that they may give an appropriate taste as a flavor.

[0077] For example, when the inoculation ratio between the glutamic acid-producing microorganism and the lysine- 25 producing microorganism is 50 to 99.95: 50 to 0.05, L- 2026253928   15 Oct 2026 glutamic acid and L-lysine in the fermentation broth may be produced at a ratio of 0.83 to 99:  1, and when the inoculation ratio between the glutamic acid-producing microorganism and the lysine-producing microorganism is 5 adjusted to 65: 35, L-glutamic acid and L-lysine in the fermentation broth may be produced at a ratio of about 1: 1.

[0078] The mixed fermentation may be performed using an appropriate medium and fermentation conditions known in the art in consideration of the characteristics of each 10 microorganism, and any person skilled in the art may easily adjust the medium and fermentation conditions for use.

[0079] More specifically, the fermentation medium used for the mixed fermentation may be a liquid medium.

[0080] The fermentation medium is a medium used during the 15 main fermentation for mass production of L-glutamic acid and L-lysine, and contains nutrients necessary for the growth of the glutamic acid-producing microorganism and the lysine-producing microorganism. In the present invention, since the fermentation broth containing the fermentation 20 medium is used as a flavor without a separate purification process after completion of the fermentation, it is preferable that the fermentation medium is composed of substances usable as food substances and contains the minimum components and amounts required for culturing the 25 microorganisms. 2026253928   15 Oct 2026

[0081] According to one embodiment of the present invention, the fermentation medium may be based on molasses and contain raw sugar and / or glucose.

[0082] More specifically, the fermentation medium may 5 contain molasses, raw sugar, and glucose as both sugar sources and nutrient sources for the microorganisms, and may contain molasses in an amount of 1 to 30 wt% based on the total sugar amount. Molasses used herein may be derived from sugar cane or sugar beets. 10

[0083] The fermentation medium may further contain nutrients for nutrient enhancement of microorganisms, in addition to the sugar sources.

[0084] According to one embodiment of the present invention, the fermentation medium may further contain at least one 15 selected from the group consisting of yeast extract, phosphoric acid, and betaine.

[0085] According to one embodiment of the present invention, the fermentation medium may contain, based on the total weight of the medium, 1.5 to 3 wt% of molasses, 2.5 to 4 wt% 20 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.

[0086] The fermentation temperature in the mixed fermentation may be usually 20 to 45°C, for example, 25 to 25  40°C, or 30 to 38°C, and the fermentation may be continued 2026253928   15 Oct 2026 until the contents or concentrations of L-glutamic acid and L-lysine obtained reach desired levels. The fermentation time may be, for example, 10 to 160 hours, 18 to 120 hours, or 20 to 100 hours. 5

[0087] In addition, during fermentation, compounds such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid may be added to the medium or fermentation broth in an appropriate manner to adjust the pH of the fermentation broth. In addition, it 10 is possible to suppress the formation of air bubbles by using an antifoaming agent for food during fermentation. Additionally, in order to maintain the medium or fermentation broth in an aerobic state, oxygen or oxygencontaining gas (e.g., air) may be injected into the 15 fermentation broth.

[0088] According to one embodiment of the present invention, the fermentation may be performed by fed-batch culture, which starts at a temperature of 30 to 33°C and is maintained at a temperature of 36 to 39°C, a pH of 6.5 to 20  7.5 and a dissolved oxygen concentration of 20 to 70%, for 28 to 40 hours.

[0089] This fermentation broth containing L-glutamic acid and L-lysine is used as the raw material of a flavor containing L-glutamic acid and L-lysine, is characterized 25 by being used intact without being mixed with additional 2026253928   15 Oct 2026 components, and contains large amounts of L-glutamic acid and L-lysine.

[0090] According to one embodiment of the present invention, the fermentation broth may contain amino acids, including 5 L-glutamic acid and L-lysine, in an amount of 3 to 90 wt% based on the total solid content.

[0091] 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 10 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. This fermentation broth may contain 5 to 150 g / L of L-glutamic acid and L-lysine.

[0092] Meanwhile, the method for producing a flavor 15 according to the present invention may further comprise an additional process for using the fermentation broth as a flavor or a flavor containing L-glutamic acid and L-lysine.

[0093] More specifically, the method may further comprise a step of separating cells from the fermentation broth and 20 decolorizing the fermentation broth.

[0094] The cell separation may be performed without limitation by applying a cell separation method and separation conditions known in the art. An example of the cell separation method may be membrane separation, 25 ultrafiltration, centrifugal filtration, or the like, but 2026253928   15 Oct 2026 is not limited thereto.

[0095] The decolorization may be performed without limitation by applying a decolorization method and decolorization conditions known in the art. For example, 5 the decolorization may be performed using activated carbon or the like, without being limited thereto.

[0096] In addition, the method may further comprise a step of filtering the decolorized fermentation broth.

[0097] The filtration may be performed without limitation by 10 applying a filtration method and filtration conditions known in the art. For example, the filtration may be performed using filter paper, filter net, membrane filtration, ultrafiltration, etc., without being limited thereto. 15

[0098] In addition, the method may further comprise a step of concentrating the filtered fermentation broth.

[0099] The concentration may be performed without limitation by applying a concentration method and concentration conditions known in the art. Examples of the concentration 20 method include, but are not limited to, heat concentration, vacuum concentration, freeze concentration, evaporation concentration, vacuum low-temperature concentration, and the like.

[00100] In addition, the method may further comprise a step 25 of drying and powdering the concentrated fermentation broth. 2026253928   15 Oct 2026

[00101] The drying may be performed without limitation by applying a drying method and drying conditions known in the art. Examples of the drying method include, but are not limited to, freeze drying, vacuum drying, air drying, blown 5 air drying, hot air drying, fluidized bed drying, spray drying, infrared drying, high frequency drying, and the like.

[00102] Through this method, the fermentation broth may be finally obtained in a powder state, and may be used as a 10 natural food flavor without undergoing an additional chemical purification process.

[00103] Still another aspect of the present invention provides a flavor produced by the above-described method for producing a flavor using two or more different 15 microorganisms.

[00104] According to one embodiment of the present invention, the flavor may be a natural flavor containing two or three taste components selected from among amino acid, nucleic acid and organic acid. 20

[00105] 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- 25 lysine, L-arginine, and L-histidine. 2026253928   15 Oct 2026

[00106] The nucleic acid may be at least one selected from the group consisting of inosinic acid, guanylic acid, xanthyl acid, and salts thereof.

[00107] For example, the nucleic acid may be inosine 5 monophosphate (IMP), guanosine monophosphate (GMP), xanthosine monophosphate (XMP), etc., but is not limited thereto.

[00108] The organic acid may be at least one selected from the group consisting of succinic acid, malic acid, citric 10 acid, acetic acid, lactic acid, fumaric acid, tartaric acid, ascorbic acid, gluconic acid, and salts thereof.

[00109] For example, when the flavor contains two taste components, it may contain glutamic acid and lysine, arginine, histidine, tryptophan, glycine, alanine, succinic 15 acid, lactic acid, guanylic acid, or inosinic acid. In addition, the flavor may contain inosinic acid and lysine, arginine, histidine, tryptophan, glycine, alanine, succinic acid, lactic acid, or guanylic acid.

[00110] In another example, when the flavor contains three 20 taste components, it may contain glutamic acid, guanylic acid, and inosinic acid.

[00111] According to one embodiment of the present invention, the flavor may contain taste components, including amino acids, nucleic acids and / or organic acids, in an amount of 25  3 to 90 wt% based on the total solid content. 2026253928   15 Oct 2026

[00112] More specifically, the flavor may contain taste 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 5 to 90 wt%, 70 to 90 wt%, or 80 to 90 wt%, based on the total solid content.

[00113] Yet another aspect of the present invention provides a flavor containing L-glutamic acid and L-lysine, produced by the above-described method for producing a natural 10 flavor containing L-glutamic acid and L-lysine using a glutamic acid-producing microorganism and a lysine-producing microorganism.

[00114] According to one embodiment of the present invention, the flavor containing L-glutamic acid and L-lysine may be a 15 natural flavor.

[00115] According to one embodiment of the present invention, the flavor may have an L-glutamic acid and L-lysine content of 3 to 90 wt% based on the solid content, and contain L-glutamic acid and L-lysine at a ratio of 0.83 to 99: 1. 20

[00116] Since this flavor contains L-lysine together with L-glutamic acid that gives an umami taste, it may increase the solubility of L-glutamic acid and solve the ammonia odor problem by reducing the production of ammonium glutamate during the fermentation process. In addition, 25 since the flavor also contains metabolites such as organic 2026253928   15 Oct 2026 acids, inorganic ion components, proteins, peptides, and vitamins produced in the fermentation process, it has a rich umami taste and a strong body feeling and exhibits excellent sensory properties. Thus, the flavor may be added 5 to various foods to maximize the taste of the foods.

[00117] Still yet another aspect of the present invention provides a food composition containing the above-described flavor or the flavor containing L-glutamic acid and L-lysine. 10

[00118] As used herein, the term "food composition" refers to a natural product or processed product containing one or more nutrients, and preferably refers to a product that has undergone certain processing and is ready to be eaten directly. In a common sense, the term "food composition" 15 includes all health functional foods, functional foods, beverages, food additives, and beverage additives.

[00119] According to one embodiment of the present invention, the food composition contains a flavor produced without a chemical purification process or a flavor containing 20 natural L-glutamic acid and L-lysine, that is a natural flavor, and it may contain the flavor in an amount of 0.001 to 90 wt%, more specifically 0.01 to 50 wt%, based on the total weight of the composition.

[00120] The food composition in the present invention may be 25 provided in any formulation suitable for food, and may be, 2026253928   15 Oct 2026 for example, in the form of a solution, emulsion, viscous mixture, powder, granule, tablet, capsule, or the like. In this case, the food composition may contain various bases and / or additives necessary and appropriate for formulation 5 within the range that does not impair the main effect of the present invention. In addition, the food composition may further contain additives such as fragrance, colorants, disinfectants, antioxidants, preservatives, humectants, thickeners, inorganic salts, and emulsifiers, within the 10 range that does not impair the effect thereof. The content of these additives in the food composition may be selected depending on the  formulation or intended  use of  the composition within  the range that does not impair  the purpose and effect of the present invention. For example, 15 the content of the additives may be 0.01 to 70 wt%, more specifically 0.1 to 50 wt%, based on the total weight of the food composition.

[00121] This food composition may be used as an additive for various foods. As foods to which this food composition may 20 be added, any foods known in the art may be used without limitation. Examples of the foods include, but are not limited to, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, 25 drinks, alcoholic beverages, and vitamin complexes. 2026253928   15 Oct 2026

[00122] The method for producing a flavor according to the present invention may produce a natural flavor capable of improving the taste and aroma of food and the overall sensory properties of food through a fermentation broth 5 containing amino acids, nucleic acids and / or organic acids, which is produced by mixed fermentation of different microorganisms producing different products, that is, different kinds of amino acids, nucleic acids and / or organic acids. This flavor may be used in various food 10 fields. BRIEF DESCRIPTION OF THE DRAWINGS

[00123] FIG. 1 is a flow chart showing a fermentation process performed using a glutamic acid-producing microorganism and 15 a lysine-producing microorganism in Preparation Examples 1 to 3 according to one embodiment of the present invention.

[00124] FIG. 2 is a flow chart showing a fermentation process performed using a glutamic acid-producing microorganism and an arginine-producing microorganism in Preparation Examples 20  4 to 6 according to one embodiment of the present invention.

[00125] FIG. 3 is a flow chart showing a fermentation process performed 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 25 invention. 2026253928   15 Oct 2026

[00126] FIG. 4 is a flow chart showing a fermentation process performed using an inosinic acid-producing microorganism and a lysine-producing microorganism in Preparation Examples 10 to 12 according to one embodiment of the 5 present invention.

[00127] FIG. 5 is a flow chart showing a fermentation process performed using an inosinic acid-producing microorganism and an arginine-producing microorganism in Preparation Examples 13 to 15 according to one embodiment of the 10 present invention. DETAILED DESCRIPTION

[00128] Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings. 15 However, these descriptions are provided for illustrative purposes only to help the understanding of the present invention, and the scope of the present invention is not limited by these illustrative descriptions.

[00129] Example 1. Mixed fermentation of glutamic acid and 20 lysine

[00130] 1-1. Seed culture

[00131] Corynebacterium glutamicum NFG6 (KCCM13164P) that produces L-glutamic acid (GA) was used as a glutamic acidproducing microorganism, and Corynebacterium glutamicum 25 NFL21 (KCCM13163P) that produces L-lysine (LYS) was used as 2026253928   15 Oct 2026 a lysine-producing microorganism.

[00132] For seed culture of the glutamic acid-producing microorganism, the glutamic acid-producing microorganism was inoculated into a 2-L flask containing 0.2 L of a seed 5 culture medium, followed by primary culture for 22 to 24 hours at 30°C and 140 rpm (OD610 = 10 to 15). Next, 2 to 3% of the primary culture was inoculated into a 5-L jar fermenter, and 2 to 2.5 L of a seed culture medium was added thereto, followed by secondary culture at 32°C, pH 10   6.9, 600 rpm and an aeration rate of 1.0 vvm for 22 to 24 hours (OD610 = 20 to 60), thereby preparing a glutamic acid seed culture broth.

[00133] For seed culture of the lysine-producing microorganism, the lysine-producing microorganism was 15 inoculated into a 2-L flask containing 0.2 L of a seed culture medium, followed by primary culture for 16 to 18 hours at 30°C and 140 rpm (OD610 = 11 to 12). Next, 5% of the primary culture was inoculated into a 5-L jar fermenter, and 2 to 2.5 L of a seed culture medium was added thereto, 20 followed by secondary culture at 32°C, pH 7.0, 650 rpm and an aeration rate of 2.0 vvm for 21 to 24 hours (OD610 = 20 to 60), thereby preparing a lysine seed culture broth.

[00134] The compositions of the seed culture media used are shown in Table 1 below. 25

[00135] [Table 1] 2026253928   15 Oct 2026 Composition Seed culture medium for glutamic acid producing microorganism 4.5 to 5.5% molasses, 3% glucose, 0.85% yeast extract paste, 100 ppm methionine, 0.6% H3PO4, 0.1% sodium succinate, 50 ppm vitamin C, 12 ppm thiamine HCl, 2 0 ppb vitamin B12, 10 ppm biotin, 0.4% MgSO4, and 0.01% antifoaming agent for food Seed culture medium for lysine-producing microorganism 1.5 to 3% molasses, 9 to 12% raw sugar, 1% yeast extract paste, 1.6% (NH4)2SO4, 0.3% H3PO4, 7.3 ppm MnSO4^5H2O, 14 ppm nicotinamide, 2.5 ppm thiamine HCl, 1.5 ppm CuSO4^5H2O, 0.056 ppm biotin, 0.045% betaine, and 0.01% antifoaming agent for food

[00136] 1-2. Main fermentation

[00137] In order to examine the ratio of glutamic acid to lysine in the fermentation broth depending on the inoculums of the glutamic acid seed culture broth and the lysine seed 5 culture broth, the glutamic acid seed culture broth and the lysine seed culture broth were inoculated at various ratios and fermented.

[00138] In main fermentation, 14 to 18 L of a fermentation medium was added to a 50 L fermenter, and inoculated with 10 1.2 to 1.8 L of the total seed culture broth at an inoculation ratio of 50 to 99.95 (glutamic acid seed culture broth): 50 to 0.05 (lysine seed culture broth), 2026253928   15 Oct 2026 followed by mixed fermentation by fed-batch culture for 28 to 40 hours. The fermentation medium composition and fermentation conditions used are shown in Table 2 below.

[00139] [Table 2] Fermentation medium composition 1.5 to 3% molasses, 2.5 to 4% glucose, 0.4 to 1% yeast extract paste, 0.1 to 0.2% H3PO4, 0.05 to 0.12% betaine, and 0.005% antifoaming agent for food Fermentation conditions Temperature 32^38°C, pH 6.5 to 7.5, aeration rate 0.8 to 1.2 vvm, internal pressure 0.6 to 1.0 kg / cm3, agitation speed 320 to 350 rpm, and dissolved oxygen (DO) concentration 20 to 70%

[00140] This mixed fermentation was performed a total of three times, the average value was calculated, and the results are shown in Table 3 below.

[00141] [Table 3] Inoculation ratio between seed culture Fermentation time GA: LYS fermentat ratio in ion broth broths (GA: LYS) 99.95 : 0.05 31 hours 99 : 1 75 : 25 29 hours 1.48 : 1 70 : 30 29 hours 1.30 : 1 67 : 33 30 hours 1.10 : 1 2026253928   15 Oct 2026 65 35 30 hours 1.01 1 60 40 29 hours 0 . 93 1 50 50 28 hours 0.83 1

[00142] Referring to Table 3 above, it was confirmed that, when the seed culture broths of the amino acid-producing microorganisms were inoculated at a ratio of 50 to 99.95 (glutamic acid-producing microorganism): 50 to 0.05 5 (lysine-producing microorganism), L-glutamic acid and L-lysine in the fermentation broth were produced at a ratio of 0.83 to 99: 1.

[00143] Example 2. Comparison of glutamic acid-lysine fermented powders between fermentation processes 10

[00144] 2-1. Production of glutamic acid-lysine fermented powder

[00145] Conventionally, a flavor containing glutamic acid and lysine was produced by individually fermenting a glutamic acid-producing microorganism and a lysine-producing 15 microorganism and then mixing the amino acid-containing fermentation broths or dried products thereof at a suitable ratio. To compare the differences in taste between this conventional individual fermentation method and the method based on mixed fermentation of the glutamic acid-producing 20 microorganism and the lysine-producing microorganism, the components of glutamic acid-lysine (GA-LYS) fermented 2026253928   15 Oct 2026 powders obtained by the production methods were compared. (See FIG. 1).

[00146] The glutamic acid seed culture broth and lysine seed culture broth used here were prepared in the same manner as 5 in Example 1-1.

[00147] @ Individual fermentation (Preparation Example 1)

[00148] In the method of mixing individual dried products among the conventional methods, each of the glutamic acid seed culture broth and the lysine seed culture broth was 10 transferred into a 50-L fermenter and then individually subjected to main fermentation. Next, cells were separated from each fermentation broth, and then decolorization and filtration processes were performed. The filtrates were concentrated and dried to obtain dried products. The 15 obtained dried glutamic acid and lysine products were mixed together so that the ratio of glutamic acid to lysine was 1:1, thereby preparing fermented powder containing glutamic acid and lysine.

[00149] @ Mixing of fermentation broths after individual 20 fermentation (Preparation Example 2)

[00150] In the method of mixing individual fermentation broths among the conventional methods, each of the glutamic acid seed culture broth and the lysine seed culture broth was transferred into a 50-L fermenter and then individually 25 subjected to main fermentation. The fermentation broths 2026253928   15 Oct 2026 obtained in the main fermentation were mixed together so that the ratio of glutamic acid to lysine was 1: 1. Next, cells were separated from the fermentation broth mixture, and then decolorization and filtration processes were 5 performed. The filtrate was concentrated and dried to obtain glutamic acid-lysine fermented powder.

[00151] @ Mixed fermentation (Preparation Example 3)

[00152] In mixed fermentation, the glutamic acid seed culture broth and the lysine seed culture broth were inoculated at 10 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 broth was about 1:1 in the same manner as in Preparation Examples   1 and 2,   followed  by mixed fermentation. Next,   cells were   separated from the 15 fermentation broth, and decolorization and filtration processes were performed. The filtrate was concentrated and dried to obtain glutamic acid-lysine fermented powder.

[00153] 2-2. Comparison of components between glutamic acid-lysine fermented powders 20

[00154] Component analysis was performed on the glutamic acid-lysine fermented powder obtained through individual fermentation in each of Preparation Examples 1 and 2 and the glutamic acid-lysine fermented powder obtained through mixed fermentation in Preparation Example 3. 25

[00155] Amino acids (L-glutamic acid and L-lysine) were 2026253928   15 Oct 2026 10 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 min). Ions (Na, Mg, K, PO4, SO4, Cl, NH4, etc.) were measured by an ion analyzer (Dionex IcS-1100, Thermo Scientific). The results are shown in Table 4 below.

[00156] [Table 4] Item Preparation Example 1 Preparation Example 2 Preparation Example 3 GA 22% 35% 43% LYS 22% 35% 43% AA / TS* 47% 72% 88% Total nitrogen 9.0% 12.2% 11.8% Organic acids 3.1% 2.6% 1.5% Ions 8.1% 10.2% 2% Ammonium 3.8% 2.5% 0.7% *AA / TS: proportion of amino acids (GA+LYS) relative to total solid content

[00157] Referring to Table 4 above, mixed fermentation (Preparation Example 3) showed a higher amino acid proportion than individual fermentation (Preparation Examples 1 and 2) and showed significant decreases in the contents of organic acids, ions and ammonium. 2026253928   15 Oct 2026

[00158] 2-3. Sensory comparison between individual fermentation and mixed fermentation

[00159] Sensory evaluation was performed on the glutamic acid-lysine fermented powders obtained through individual 5 fermentation in Preparation Examples 1 and 2 and the glutamic acid-lysine fermented powder obtained through mixed fermentation in Preparation Example 3.

[00160] For sensory evaluation of the glutamic acid-lysine fermented powders, the umami taste, umami persistence, 10 salty taste, sour taste, bitter taste and sweet taste of each powder itself or a sample obtained by diluting each powder in lukewarm water at a concentration of 1 to 5% were evaluated by a trained panel consisting of 10 to 15 persons. The results are shown in Table 5 below. 15

[00161] [Table 5] Item Preparation Example 1 (individual fermentation) Preparation Example 2 (individual fermentation) Preparation Example 3 (mixed fermentation) Initial Umami +++ ++++ ++++ taste Umami ++ +++ ++++ persistence Salty taste + ++ Sour taste ++ + 2026253928   15 Oct 2026 10 15 20 Bitter taste ++ + Sweet taste + + ++

[00162] Referring to Table 5 above, due to the difference in components of the glutamic acid-lysine fermented powder as shown in Table 4 above, the sample prepared by the mixing process after individual fermentation showed a weak umami taste and an increased bitter taste and sour taste compared to the sample prepared by mixed fermentation. In addition, since the increase in by-products such as organic acids and the increase in ions as shown in Table 4 above also affect the sensory properties of the sample, the mixed fermentation process is more effective in terms of taste or process simplification than mixing after individual fermentation.

[00163] Example 3. Mixed fermentation of glutamic acid and arginine

[00164] 3-1. Seed culture

[00165] Corynebacterium glutamicum NFG6 (KCCM13164P) was used as a glutamic acid-producing microorganism, and Corynebacterium glutamicum NFA40 (KCCM13165P) that produces L-arginine (ARG) was used as an arginine-producing microorganism.

[00166] A glutamic acid seed culture broth of the glutamic acid-producing microorganism was prepared in the same manner as in Example 1-1. 2026253928   15 Oct 2026

[00167] For seed culture of the arginine-producing microorganism, the arginine-producing microorganism was inoculated into a 2-L flask containing 0.2 L of a seed culture medium, followed by primary culture for 16 to 18 5 hours at 30°C and 140 rpm (OD610 = 12 to 18). Next, 2 to 4% of the primary culture was inoculated into a 5-L jar fermenter, and 2 to 2.5 L of a seed culture medium was added thereto, followed by secondary culture at 32°C, pH 6.7, 600 rpm and an aeration rate of 1.0 vvm for 16 to 24 10 hours (OD610 = 20 to 60), thereby preparing an arginine seed culture broth. The composition of the seed culture medium used is shown in Table 6 below.

[00168] [Table 6] Composition Seed culture medium for arginineproducing microorganism 1.5 to 4.5% glucose, 2 to 6% raw sugar, 2 to 3% yeast extract paste, 0.6% (NH4)2SO4,  0.2% KH2PO4,   0.2% K2HPO4 0.2%,   15 ppm MnSO4^5H2O, 0.2 % MgSO4^7H2O 1 ppm thiamine HCl, 10 ppm ZnSO4^7H2O, 0.3 ppm biotin, 15 ppm FeSO4^7H2O, and 0.01% antifoaming agent for food

[00169] 3-2. Main fermentation 15

[00170] In order to examine the ratio of glutamic acid to arginine in the fermentation broth depending on the inoculums of the glutamic acid seed culture broth and the arginine seed culture broth, the glutamic acid seed culture 2026253928   15 Oct 2026 10 15 broth and the arginine seed culture broth were inoculated at various ratios and fermented.

[00171] In main fermentation, 14 to 18 L of a fermentation medium was added to a 50-L fermenter and inoculated with 1.2 to 1.8 L of the total seed culture broth at an inoculation ratio of 30 to 99.95 (glutamic acid seed culture broth): 70 to 0.05 (arginine seed culture broth), followed by mixed fermentation by fed-batch culture for 31 to 60 hours. The fermentation medium composition and fermentation conditions used are shown in Table 7 below.

[00172] [Table 7] Fermentation medium composition 1.5 to 3% molasses, 2.5 to 4% glucose, 0.4 to 1% yeast extract paste, 0.05 to 0.3% (NH4)2SO4, 0.1 to 0.2% H3PO4, 0.05 to 0.12% betaine, and 0.005% antifoaming agent for food Fermentation conditions Temperature 32 to 37°C, pH 6.5 to 7.5, aeration rate 0.8 to 1.2 vvm, internal pressure 0.5 to 1.0 kg / cm3, agitation speed 320 to 350 rpm, and dissolved oxygen (DO) concentration 20 to 70%

[00173] This mixed fermentation was performed a total of three times, the average value was calculated, and the results are shown in Table 8 below.

[00174] [Table 8] Inoculation ratio between Fermentation time GA: ARG ratio in 2026253928   15 Oct 2026 seed culture broths (GA: fermentation broth ARG) 99.95 : 0.05 31 hours 99.0 : 1 90 : 10 31 hours 12.2 : 1 80 : 20 34 hours 4.36 : 1 70 : 30 36 hours 2.41 : 1 60 : 40 38 hours 1.96 : 1 50 : 50 40 hours 1.57 : 1 40 : 60 44 hours 1.21 : 1 30 : 70 45 hours 1.04 : 1

[00175] Referring to Table 8 above, it was confirmed that, when the seed culture broths of the amino acid-producing microorganisms were inoculated at a ratio of 30 to 99.95 (glutamic acid-producing microorganism): 70 to 0.05 5 (arginine-producing microorganism), L-glutamic acid and L-arginine in the fermentation broth were produced at a ratio of 1.04 to 99: 1.

[00176] Example 4. Comparison of glutamic acid-arginine fermented powders between fermentation processes 10

[00177] 4-1. Production of glutamic acid-arginine fermented powder

[00178] Conventionally, a flavor containing glutamic acid and 2026253928   15 Oct 2026 arginine was produced by individually fermenting a glutamic acid-producing microorganism and an arginine-producing microorganism and then mixing the amino acid-containing fermentation broths or dried products thereof at a suitable 5 ratio. To compare the differences in taste between this conventional individual fermentation method and the method based on mixed fermentation of the glutamic acid-producing microorganism and the arginine-producing microorganism, the components of glutamic acid-arginine (GA-ARG) fermented 10 powders obtained by the production methods were compared (see FIG. 2).

[00179] The glutamic acid seed culture broth and arginine seed culture broth used here were prepared in the same manner as in Example 3-1. 15  

[00180] @ Individual fermentation (Preparation Example 4)

[00181] In the method of mixing individual dried products among the conventional methods, each of the glutamic acid seed culture broth and the arginine seed culture broth was transferred into a 50-L fermenter and then individually 20 subjected to main fermentation. Next, cells were separated from each fermentation broth, and then decolorization and filtration processes were performed. The filtrates were concentrated and dried to obtain dried products. The obtained dried glutamic acid and arginine products were 25 mixed together so that the ratio of glutamic acid to 2026253928   15 Oct 2026 arginine was 1:1, thereby preparing fermented powder containing glutamic acid and arginine.

[00182] @ Mixing of fermentation broths after individual fermentation (Preparation Example 5) 5

[00183] In the method of mixing individual fermentation broths among the conventional methods, each of the glutamic acid seed culture broth and the arginine seed culture broth was transferred into a 50-L fermenter and then individually subjected to main fermentation. The fermentation broths 10 obtained in the main fermentation were mixed together so that the ratio of glutamic acid to arginine was 1: 1. Next, cells were separated from the fermentation broth mixture, and then decolorization and filtration processes were performed. The filtrate was concentrated and dried to 15 obtain glutamic acid-arginine fermented powder.

[00184] @ Mixed fermentation (Preparation Example 6)

[00185] In mixed fermentation, the glutamic acid seed culture broth and the arginine seed culture broth were inoculated at a ratio of 30: 70 in the same manner as in Example 3-2 20 so that the ratio of glutamic acid to arginine in the fermentation broth was about 1:1 in the same manner as in Preparation Examples   4 and 5,   followed  by mixed fermentation. Next,   cells were   separated from the fermentation broth, and decolorization and filtration 25 processes were performed. The filtrate was concentrated and 2026253928   15 Oct 2026 dried to obtain glutamic acid-arginine fermented powder.

[00186] 4-2. Comparison of components between glutamic acid-arginine fermented powders

[00187] Component analysis was performed on the glutamic 5 acid-arginine fermented powder obtained through individual fermentation in each of Preparation Examples 4 and 5 and the glutamic acid-arginine fermented powder obtained through mixed fermentation in Preparation Example 6.

[00188] Amino acids (L-glutamic acid and L-arginine) were 10 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 acid and ions were measured in the same manner as in Example 2-2. The results are shown in Table 9 below. 15

[00189] [Table 9] Item Preparation Example 4 Preparation Example 5 Preparation Example 6 GA 22% 35% 42% ARG 22% 34% 42% AA / TS* 48% 71% 86% Total nitrogen 9.4% 12.0% 11.7% Organic acids 3.6% 2.5% 1.8% Ions 8.3% 9.9% 2.1% 2026253928   15 Oct 2026 Ammonium 4.0% 2.6% 0 . 9% *AA / TS: proportion of amino acids (GA+ ARG) relative to total solid content

[00190] Referring to Table 9 above, mixed fermentation (Preparation Example 6) showed a higher amino acid proportion than individual fermentation (Preparation Examples 4 and 5) and showed significant decreases in the 5 contents of organic acids, ions and ammonium.

[00191] 4-3. Sensory comparison between individual fermentation and mixed fermentation

[00192] Sensory evaluation was performed on the glutamic acid-arginine fermented powders obtained through individual 10 fermentation in Preparation Examples 4 and 5 and the glutamic acid-arginine fermented powder obtained through mixed fermentation in Preparation Example 6.

[00193] Sensory evaluation was performed in the same manner as in Example 2-3. The results are shown in Table 10 below. 15

[00194] [Table 10] Item Preparation Example 4 (individual fermentation) Preparation Example 5 (individual fermentation) Preparation Example 6 (mixed fermentation) Initial Umami taste ++ +++ ++++ Umami + ++ ++++ 2026253928   15 Oct 2026 persistence Salty taste + ++ Sour taste + + Bitter taste ++ ++ Sweet taste

[00195] Referring to Table 10 above, due to the difference in components of the glutamic acid-arginine fermented powder as shown in Table 9 above, the sample prepared by the mixing process after individual fermentation showed a weak 5 umami taste and an increased bitter taste compared to the sample prepared by mixed fermentation. In addition, since the increase in by-products such as organic acids and the increase in ions as shown in Table 9 above also affect the sensory properties of the sample, the mixed fermentation 10 process is more effective in terms of taste or process simplification than mixing after individual fermentation.

[00196] Example 5. Mixed fermentation of glutamic acid and inosinic acid

[00197] 5-1. Seed culture 15

[00198] Corynebacterium glutamicum NFG6 (KCCM13164P) was used as a glutamic acid-producing microorganism, and Corynebacterium ammoniagenes NFI545 (KCCM13162P) that produces IMP was used as an inosinic acid-producing microorganism. 20

[00199] A glutamic acid seed culture broth of the glutamic 2026253928   15 Oct 2026 acid-producing microorganism was prepared in the same manner as in Example 1-1.

[00200] For seed culture of the inosinic acid-producing microorganism, the inosinic acid-producing microorganism 5 was inoculated into a 2-L flask containing 0.3 L of a seed culture medium, followed by primary culture for 20 to 24 hours at 31°C and 150 rpm (OD610 = 15 to 20). Next, 1% of the primary culture was inoculated into a 5-L jar fermenter, and 2 to 2.5 L of a seed culture medium was added thereto, 10 followed by secondary culture at 31°C, pH 7.1, 600 rpm and an aeration rate of 1.0 vvm for 21 to 24 hours (OD610 = 20 to 40), thereby preparing an inosinic acid seed culture broth. The composition of the seed culture medium used is shown in Table 11 below. 15

[00201] [Table 11] Composition Seed culture medium for inosinic acidproducing microorganism 4 to 6% glucose, 2 to 4% yeast extract paste, 0.3% (NHJ2SO4, 0.2% KH2PO4, 0.2% K2HPO4, 200 to 300 ppm adenine, 200 to 300 ppm guanine, 0.15% MgSO4^7H2O, 10 ppm nicotinic acid, 100 ppm Ca-pantothenate, 15 ppm cysteine, 1 ppm thiamine HCl, 5 ppm ZnSO4^7H2O, 10 ppm MnSO4^5H2O, 0.1 ppm biotin, 15 ppm FeSO4^7H2O, and 0.01% antifoaming agent for food

[00202] 5-2. Main fermentation 2026253928   15 Oct 2026

[00203] In order to examine the ratio of glutamic acid to inosinic acid in the fermentation broth depending on the inoculums of the glutamic acid seed culture broth and the inosinic acid seed culture broth, the glutamic acid seed 5 culture broth and the inosinic acid seed culture broth were inoculated at various ratios and fermented.

[00204] In main fermentation, 14 to 18 L of a fermentation medium was added to a 50-L fermenter and inoculated with 1.2 to 1.8 L of the total seed culture broth at an 10 inoculation ratio of 0.05 to 99.95 (glutamic acid seed culture broth): 99.95 to 0.05 (inosinic acid seed culture broth), followed by mixed fermentation by fed-batch culture for 30 to 90 hours. The fermentation medium composition and fermentation conditions used here are shown in Table 12 15 below.

[00205] [Table 12] Fermentation medium composition 1 to 3% molasses, 5 to 7% raw sugar, 2 to 3% yeast extract paste, 0.6 to 1.2% H3PO4, 0.05 to 0.1% betaine, 100 to 200 ppm adenine, 50 to 150 ppm guanine, 0.2 to 0.5% MgSO4^7H2O, 50 to 100 ppm Ca-pantothenate, 5 to 15 ppm vitamin B3, 5 to 20 ppm thiamine HCl, 0.4 to 0.8% NaOH, 5 to 10 ppm FeSO4, 10 to 20 ppm MnSO4, 10 to 20 ppm ZnSO4, and 0.005% antifoaming agent for food Fermentation Temperature 31 to 32°C, pH 6.5 to 7.5, aeration 2026253928   15 Oct 2026 10 conditions rate 0.8 to 1.2 vvm, internal pressure 0.6 to 1.0 kg / cm3,  agitation speed 320 to 350 rpm, and dissolved oxygen (DO) concentration 20 to 70%

[00206] This mixed fermentation was performed a total of three times, the average value was calculated, and the results are shown in Table 13 below.

[00207] [Table 13] Inoculation ratio between seed culture broths (GA: IMP) Fermentation time GA: IMP ratio in fermentation broth 99.95 : 0.05 33 hours 99.8 : 1 80 : 20 42 hours 51.3 : 1 70 : 30 48 hours 31.2 : 1 50 : 50 60 hours 16.8 : 1 20 : 80 83 hours 1.04 : 1 0.05 : 99.95 90 hours 0.02 : 1

[00208] Referring to Table 13 above, it was confirmed that, when the seed culture broths of the microorganisms were inoculated at a ratio of 0.05 to 99.95 (glutamic acidproducing microorganism): 99.95 to 0.05 (inosinic acidproducing microorganism), L-glutamic acid and IMP in the fermentation broth were produced at a ratio of 0.02 to 99.8: 2026253928   15 Oct 2026 1.

[00209] Example 6. Comparison of glutamic acid-inosinic acid fermented powders between fermentation processes

[00210] 6-1. Production of glutamic acid-inosinic acid 5 fermented powder

[00211] Conventionally, a flavor containing glutamic acid and inosinic acid was produced by individually fermenting a glutamic acid-producing microorganism and an inosinic acidproducing microorganism and then mixing the fermentation 10 broths or dried products thereof at a suitable ratio. To compare the differences in taste between this conventional individual fermentation method and the method based on mixed fermentation of the glutamic acid-producing microorganism and the inosinic acid-producing microorganism, 15 the components of glutamic acid-inosinic acid (GA-IMP) fermented powders obtained by the production methods were compared (see FIG. 3).

[00212] The glutamic acid seed culture broth and inosinic acid seed culture broth used here were prepared in the same 20 manner as in Example 5-1.

[00213] @ Individual fermentation (Preparation Example 7)

[00214] In the method of mixing individual dried products among the conventional methods, each of the glutamic acid seed culture broth and the inosinic acid seed culture broth 25 was transferred into a 50-L fermenter and then individually 2026253928   15 Oct 2026 subjected to main fermentation. Next, cells were separated from each fermentation broth, and then decolorization and filtration processes were performed. The filtrates were concentrated and dried to obtain dried products. The 5 obtained dried glutamic acid and isosinic acid products were mixed together so that the ratio of glutamic acid to inosinic acid was 1:1, thereby preparing fermented powder containing glutamic acid and inosinic acid.

[00215] @ Mixing of fermentation broths after individual 10 fermentation (Preparation Example 8)

[00216] In the method of mixing individual fermentation broths among the conventional methods, each of the glutamic acid seed culture broth and the inosinic acid seed culture broth was transferred into a 50-L fermenter and then 15 individually subjected to main fermentation. The fermentation broths obtained in the main fermentation were mixed together so that the ratio of glutamic acid to inosinic acid was 1: 1. Next, cells were separated from the fermentation broth mixture, and then decolorization and 20 filtration processes were performed. The filtrate was concentrated and dried to obtain glutamic acid-inosinic acid fermented powder.

[00217] @ Mixed fermentation (Preparation Example 9)

[00218] In mixed fermentation, the glutamic acid seed culture 25 broth and the inosinic acid seed culture broth were 2026253928   15 Oct 2026 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 broth was about 1:1 in the same manner as in Preparation Examples 7 and 8, followed by 5 mixed fermentation. Next, cells were separated from the fermentation broth, and decolorization and filtration processes were performed. The filtrate was concentrated and dried to obtain glutamic acid-inosinic acid fermented powder. 10  

[00219] 6-2. Comparison of components between glutamic acid- inosinic acid fermented powders

[00220] Component analysis was performed on the glutamic acid-inosinic acid fermented powder obtained through individual fermentation in each of Preparation Examples 7 15 and 8 and the glutamic acid-inosinic acid fermented powder obtained through mixed fermentation in Preparation Example 9.

[00221] L-glutamic acid and IMP were measured by HPLC analysis (GA - 210 nm, UV detector, flow rate 0.9 ml / min; 20 IMP - 254 nm, UV detector, flow rate 0.9 ml / min). Organic acid and ions were measured in the same manner as in Example 2-2. The results are shown in Table 14 below.

[00222] [Table 14] Item Preparation Example 7 Preparation Example 8 Preparation Example 9 2026253928   15 Oct 2026 GA 22% 31% 37% IMP 20% 31% 36% (GA+IMP) / TS* 42% 62% 73% Total nitrogen 8 . 6% 9.7% 10.5% Organic acids 4.0% 3.3% 3.1% Ions 10.4% 8.8% 4.2% Ammonium 4.0% 3.0% 1.7% *(GA+IMP) / TS: Proportion of product relative to total solid content

[00223] 6-3. Sensory comparison between individual fermentation and mixed fermentation

[00224] Sensory evaluation was performed on the glutamic acid-inosinic acid fermented powders obtained through 5 individual fermentation in Preparation Examples 7 and 8 and the glutamic acid-inosinic acid fermented powder obtained through mixed fermentation in Preparation Example 9.

[00225] Sensory evaluation was performed in the same manner as in Example 2-3. The results are shown in Table 15 below. 10

[00226] [Table 15] Item Preparation Example 7 (individual fermentation) Preparation Example 8 (individual fermentation) Preparation Example 9 (mixed fermentation) Initial Umami taste +++ ++++ ++++ Umami + +++ ++++ 2026253928   15 Oct 2026 persistence Kokumi + ++ +++ Salty taste + ++ ++ Sour taste ++ ++ + Bitter taste ++ + Sweet taste + + ++

[00227] Referring to Table 15 above, due to the difference in components of the glutamic acid-inosinic acid fermented powder as in Table 14 above, the sample prepared by the mixing process after individual fermentation showed a weak 5 umami taste and umami persistence and an increased bitter taste compared to the sample prepared by mixed fermentation. In addition, since the increase in by-products such as organic acids and the increase in ions as shown in Table 14 above also affect the sensory properties of the sample, the 10 mixed fermentation process is more effective in terms of taste or process simplification than mixing after individual fermentation.

[00228] Example 7. Mixed fermentation of inosinic acid and lysine 15

[00229] 7-1. Seed culture

[00230] Corynebacterium ammoniagenes NFI545 (KCCM13162P) was used as an inosinic acid-producing microorganism, and Corynebacterium glutamicum NFL21 (KCCM13163P) was used as a lysine-producing microorganism. 2026253928   15 Oct 2026

[00231] An inosinic acid seed culture broth of the inosinic acid-producing microorganism and a lysine seed culture broth of the lysine-producing microorganism were prepared in the same manner as in Examples 5-1 and 1-1, respectively. 5  

[00232] 7-2. Main fermentation

[00233] In order to examine the ratio of inosinic acid to lysine in the fermentation broth depending on the inoculums of the inosinic acid seed culture broth and the lysine seed culture broth, the inosinic acid seed culture broth and the 10 lysine seed culture broth were inoculated at various ratios and fermented.

[00234] In main fermentation, 14 to 18 L of a fermentation medium was added to a 50-L fermenter and inoculated with 1.2 to 1.8 L of the total seed culture broth at an 15 inoculation ratio of 0.05 to 99.95 (inosinic acid seed culture broth): 99.95 to 0.05 (lysine seed culture broth), followed by mixed fermentation by fed-batch culture for 45 to 90 hours. The fermentation medium composition and fermentation conditions used here are shown in Table 16 20 below.

[00235] [Table 16] Fermentation medium composition 1 to 3% molasses, 5 to 7% raw sugar, 2 to 3% yeast extract paste, 0.6 to 1.2% H3PO4, 0.05 to 0.1% betaine, 100 to 200 ppm adenine, 50 to 150 ppm guanine, 0.2 to 2026253928   15 Oct 2026 0.5% MgSO4^7H2O, 50 to 100 ppm Ca-pantothenate, 5 to 15 ppm vitamin B3,  5 to 20 ppm thiamine HCl, 0.4% (NH4)2SO4, 0.4 to 0.8% NaOH, 5 to 10 ppm FeSO4, 10 to 20 ppm MnSO4, 10 to 20 ppm ZnSO4, and 0.005% antifoaming agent for food Fermentation conditions Temperature 31 to 32°C, pH 6.5 to 7.5, aeration rate 0.8 to 1.2 vvm, internal pressure 0.6 to 1.0 kg / cm3, agitation speed 320 to 350 rpm, and dissolved oxygen (DO) concentration 20 to 70%

[00236] This mixed fermentation was performed a total of three times, the average value was calculated, and the results are shown in Table 17 below.

[00237] [Table 17] Inoculation ratio between seed culture Fermentation time IMP: LYS fermentat ratio in ion ratio broths (IMP: LYS) 99.95 : 0.05 90 hours 92.3 : 1 80 : 20 73 hours 1.01 : 1 50 : 50 61 hours 0.05 : 1 20 : 80 48 hours 0.02 : 1 0.05 : 99.95 45 hours 0.01 : 1 5

[00238] Referring to Table 17 above, it was confirmed that, 2026253928   15 Oct 2026 when the seed culture broths of the microorganisms were inoculated at a ratio of 0.05 to 99.95 (inosinic acidproducing microorganism): 99.95 to 0.05 (lysine-producing microorganism), IMP and lysine in the fermentation broth 5 were produced at a ratio of 0.01 to 92: 1.

[00239] Example 8. Comparison of inosinic acid-lysine fermented powders between fermentation processes

[00240] 8-1. Production of inosinic acid-lysine fermented powder 10

[00241] Conventionally, a flavor containing inosinic acid and lysine was produced by individually fermenting an inosinic acid-producing microorganism and a lysine-producing microorganism and then mixing the fermentation broths or dried products thereof at a suitable ratio. To compare the 15 differences in taste between this conventional individual fermentation method and the method based on mixed fermentation of the inosinic acid-producing microorganism and the lysine-producing microorganism, the components of inosinic acid-lysine (IMP-LYS) fermented powders obtained 20 by the production methods were compared (see FIG. 4).

[00242] The inosinic acid seed culture broth and lysine seed culture broth used here were prepared in the same manner as in Example 7-1.

[00243] @ Individual fermentation (Preparation Example 10) 25

[00244] In the method of mixing individual dried products 2026253928   15 Oct 2026 among the conventional methods, each of the inosinic acid seed culture broth and the lysine seed culture broth was transferred into a 50-L fermenter and then individually subjected to main fermentation. Next, cells were separated 5 from each fermentation broth, and then decolorization and filtration processes were performed. The filtrates were concentrated and dried to obtain dried products. The obtained dried inosinic acid and lysine products were mixed together so that the ratio of inosinic acid to lysine was 10  1:1, thereby preparing fermented powder containing inosinic acid and lysine.

[00245] @ Mixing of fermentation broths after individual fermentation (Preparation Example 11)

[00246] In the method of mixing individual fermentation 15 broths among the conventional methods, each of the inosinic acid seed culture broth and the lysine seed culture broth was transferred into a 50-L fermenter and then individually subjected to main fermentation. The fermentation broths obtained in the main fermentation were mixed together so 20 that the ratio of inosinic acid to lysine was 1: 1. Next, cells were separated from the fermentation culture broth, and then decolorization and filtration processes were performed. The filtrate was concentrated and dried to obtain inosinic acid-lysine fermented powder. 25

[00247] @ Mixed fermentation (Preparation Example 12) 2026253928   15 Oct 2026

[00248] In mixed fermentation, the inosinic acid seed culture broth and the lysine seed culture broth 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 5 fermentation broth was about 1:1 in the same manner as in Preparation Examples 10 and 11, followed by mixed fermentation. Next, cells were separated from the fermentation broth, and decolorization and filtration processes were performed. The filtrate was concentrated and 10 dried to obtain inosinic acid-lysine fermented powder.

[00249] 8-2. Comparison of components between lysine-inosinic acid fermented powders

[00250] Component analysis was performed on the inosinic acid-lysine fermented powder obtained through individual 15 fermentation in each of Preparation Examples 10 and 11 and the inosinic acid-lysine fermented powder obtained through mixed fermentation in Preparation Example 12.

[00251] IMP and L-lysine were measured by HPLC analysis (IMP - 254 nm, UV detector, flow rate 0.9 ml / min; LYS - 214 nm, 20 UV detector, flow rate 0.8 ml / min). Organic acid and ions were measured in the same manner as in Example 2-2. The results are shown in Table 18 below.

[00252] [Table 18] Item Preparation Example 10 Preparation Example 11 Preparation Example 12 2026253928   15 Oct 2026 LYS 26% 32% 38% IMP 24% 30% 33% (LYS+IMP) / TS* 45% 62% 71% Total nitrogen 9.7% 11.3% 12.5% Organic acids 5.0% 4.1% 3.7% Ions 9.2% 8.2% 5.6% Ammonium 3.9% 3.5% 1. 9% *(LYS+IMP) / TS: proportion of product relative to total solid

[00253] Referring to Table 18 above, mixed fermentation (Preparation Example 12) showed a higher LYS+IMP proportion than individual fermentation (Preparation Examples 10 and 11) and showed significant decreases in the contents of 5 organic acids, ions and ammonium.

[00254] 8-3. Sensory comparison between individual fermentation and mixed fermentation

[00255] Sensory evaluation was performed on the inosinic acid-lysine fermented powders obtained through individual 10 fermentation in Preparation Examples 10 and 11 and the inosinic acid-lysine fermented powder obtained through mixed fermentation in Preparation Example 12.

[00256] Sensory evaluation was performed in the same manner as in Example 2-3. The results are shown in Table 19 below. 15

[00257] [Table 19] Item Preparation Example 10 Preparation Example 11 Preparation Example 12 2026253928   15 Oct 2026 (individual fermentation) (individual fermentation) (mixed fermentation) Initial Umami taste + ++ ++ Umami persistence + +++ ++++ Kokumi + ++ +++ Salty taste + ++ ++ Sour taste ++ ++ + Bitter taste ++ ++ Sweet taste + + ++

[00258] Referring to Table 19 above, due to the difference in components of the inosinic acid-lysine fermented powder as shown in Table 18 above, the sample prepared by the mixing process after individual fermentation showed weak umami 5 persistence and an increased bitter taste compared to the sample prepared by mixed fermentation. In addition, since the increase in by-products such as organic acids and the increase in ions as shown in Table 18 above also affect the sensory properties of the sample, the mixed fermentation 10 process is more effective in terms of taste or process simplification than mixing after individual fermentation.

[00259] Example 9. Mixed fermentation of inosinic acid and arginine

[00260] 7-1. Seed culture 2026253928   15 Oct 2026

[00261] Corynebacterium ammoniagenes NFI545 (KCCM13162P) was used as an inosinic acid-producing microorganism, and Corynebacterium glutamicum NFA40 (KCCM13165P) was used as an arginine-producing microorganism. 5

[00262] An inosinic acid seed culture broth of the inosinic acid-producing microorganism and an arginine seed culture broth of the arginine-producing microorganism were prepared in the same manner as in Examples 5-1 and 3-1, respectively.

[00263] 9-2. Main fermentation 10

[00264] In order to examine the ratio of inosinic acid to arginine in the fermentation broth depending on the inoculums 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 15 at various ratios and fermented.

[00265] In main fermentation, 14 to 18 L of a fermentation medium was added to a 50-L fermenter and inoculated with 1.2 to 1.8 L of the total seed culture broth at an inoculation ratio of 65 to 99.95 (inosinic acid seed 20 culture broth): 35 to 0.05 (arginine seed culture broth), followed by mixed fermentation by fed-batch culture for 45 to 80 hours. The fermentation medium composition and fermentation conditions used here are shown in Table 20 below. 25

[00266] [Table 20] 2026253928   15 Oct 2026 Fermentation medium composition 1 to 3% molasses, 5 to 7% raw sugar, 2 to 3% yeast extract paste, 0.6 to 1.2% H3PO4, 0.05 to 0.1% betaine, 100 to 200 ppm adenine, 50 to 150 ppm guanine, 0.2 to 0.5% MgSO4^7H2O, 50 to 100 ppm Ca-pantothenate, 5 to 15 ppm vitamin B3, 5 to 20 ppm thiamine HCl, 0.4% (NH4)2SO4, 0.4 to 0.8% NaOH, 5 to 10 ppm FeSO4, 10 to 20 ppm MnSO4, 10 to 20 ppm ZnSO4, and 0.005% antifoaming agent for food Fermentation conditions Temperature 31 to 32°C, pH 6.5 to 7.5, aeration rate 0.8 to 1.2 vvm, internal pressure 0.6 to 1.0 kg / cm3, agitation speed 320 to 350 rpm, and dissolved oxygen (DO) concentration 20 to 70%

[00267] This mixed fermentation was performed a total of three times, the average value was calculated, and the results are shown in Table 21 below.

[00268] [Table 21] Inoculation ratio between seed culture Fermentation time IMP: ARG fermentat ratio in ion broth broths (IMP: ARG) 99.95 : 0.05 80 hours 95.1 : 1 80 : 20 46 hours 5.3 : 1 70 : 30 45 hours 2.45 : 1 65 : 35 45 hours 1.04 : 1 2026253928   15 Oct 2026

[00269] Referring to Table 21 above, it was confirmed that, when the seed culture broths of the microorganisms were inoculated at a ratio of 65 to 99.95 (inosinic acidproducing microorganism): 35 to 0.05 (arginine-producing 5 microorganism), IMP and L-arginine in the fermentation broth were produced at a ratio of 1.04 to 95.1: 1.

[00270] Example 10. Comparison of inosinic acid-arginine fermented powders between fermentation processes

[00271] 10-1. Production of inosinic acid-arginine fermented 10 powder

[00272] Conventionally, a flavor containing inosinic acid and arginine was produced by individually fermenting an inosinic acid-producing microorganism and an arginineproducing microorganism and then mixing the fermentation 15 broths or dried products thereof at a suitable ratio. To compare the differences in taste between this conventional individual fermentation method and the method based on mixed fermentation of the inosinic acid-producing microorganism and the arginine-producing microorganism, the 20 components of inosinic acid-arginine (IMP-ARG) fermented powders obtained by the production methods were compared (see FIG. 5).

[00273] The inosinic acid seed culture broth and arginine seed culture broth used here were prepared in the same 25 manner as in Example 9-1. 2026253928   15 Oct 2026

[00274] @ Individual fermentation (Preparation Example 13)

[00275] In the method of mixing individual dried products among the conventional methods, each of the inosinic acid seed culture broth and the arginine seed culture broth was 5 transferred into a 50-L fermenter and then individually subjected to main fermentation. Next, cells were separated from each fermentation broth, and then decolorization and filtration processes were performed. The filtrates were concentrated and dried to obtain dried products. The 10 obtained dried inosinic acid and arginine products were mixed together so that the ratio of inosinic acid to arginine was 1:1, thereby preparing fermented powder containing inosinic acid and arginine.

[00276] @ Mixing of fermentation broths after individual 15 fermentation (Preparation Example 14)

[00277] In the method of mixing individual fermentation broths among the conventional methods, each of the inosinic acid seed culture broth and the arginine seed culture broth was transferred into a 50-L fermenter and then individually 20 subjected to main fermentation. The fermentation broths obtained in the main fermentation were mixed together so that the ratio of inosinic acid to arginine was 1: 1. Next, cells were separated from the fermentation broth mixture, and then decolorization and filtration processes were 25 performed. The filtrate was concentrated and dried to 2026253928   15 Oct 2026 obtain inosinic acid-arginine fermented powder.

[00278] @ Mixed fermentation (Preparation Example 15)

[00279] In mixed fermentation, the inosinic acid seed culture broth and the arginine seed culture broth were inoculated 5 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 broth was about 1:1 in the same manner as in Preparation  Examples 13  and 14, followed by mixed fermentation. Next, cells were separated from the 10 fermentation broth, and decolorization and filtration processes were performed. The filtrate was concentrated and dried to obtain inosinic acid-arginine fermented powder.

[00280] 10-2. Comparison of components between inosinic acid-arginine fermented powders 15

[00281] Component analysis was performed on the inosinic acid-arginine fermented powder obtained through individual fermentation in each of Preparation Examples 13 and 14 and the inosinic acid-arginine fermented powder obtained through mixed fermentation in Preparation Example 15. 20

[00282] 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 acid and ions were measured in the same manner as in Example 2-2. The results are shown in Table 22 below. 25

[00283] [Table 22] 2026253928   15 Oct 2026 Item Preparation Example 13 Preparation Example 14 Preparation Example 15 ARG 24% 31% 34% IMP 24% 31% 33% (ARG+IMP) / TS* 48% 62% 76% Total nitrogen 9.7% 11.3% 14.1% Organic acids 4.7% 3.8% 2.5% Ions 7.5% 6.2% 3.8% Ammonium 3.1% 2.5% 0.8% *(ARG+IMP) / TS: Proportion of product relative to total solid content

[00284] 10-3. Sensory comparison between individual fermentation and mixed fermentation

[00285] Sensory evaluation was performed on the inosinic acid-arginine fermented powders obtained through individual 5 fermentation in Preparation Examples 13 and 14 and the inosinic acid-arginine fermented powder obtained through mixed fermentation in Preparation Example 15.

[00286] Sensory evaluation was performed in the same manner as in Example 2-3. The results are shown in Table 23 below. 10

[00287] [Table 23] Item Preparation Example 13 (individual fermentation) Preparation Example 14 (individual fermentation) Preparation Example 15 (mixed fermentation) Initial Umami + ++ ++ 2026253928   15 Oct 2026 taste Umami persistence + +++ ++++ Kokumi + ++ +++ Salty taste + ++ ++ Sour taste ++ ++ + Bitter taste ++ ++ Sweet taste

[00288] Referring to Table 23 above, due to the difference in components of the inosinic acid-arginine fermented powder as shown in Table 22 above, the sample prepared by the mixing process after individual fermentation showed weak 5 umami persistence and an increased bitter taste compared to the sample prepared by mixed fermentation. In addition, since the increase in by-products such as organic acids and the increase in ions as shown in Table 22 above also affect the sensory properties of the sample, the mixed 10 fermentation process is more effective in terms of taste or process simplification than mixing after individual fermentation.

[00289] So far, the present invention has been described with reference to the preferred embodiments. Those of ordinary 15 skill in the art to which the present invention pertains will appreciate that the present invention may be embodied in modified forms without departing from the essential 2026253928   15 Oct 2026 characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative point of view, not from a restrictive point of view. The scope of the present invention is defined by the 5 claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the scope of the present invention.

[00290] [Accession Numbers] 10

[00291] Depository authority: Korean Culture Center of Microorganisms (KCCM)

[00292] Accession number: KCCM13162P

[00293] Deposit date: April 21, 2022

[00294] 15

[00295] Depository authority: Korean Culture Center of Microorganisms (KCCM)

[00296] Accession number: KCCM13163P

[00297] Deposit date: April 21, 2022

[00298] 20

[00299] Depository authority: Korean Culture Center of Microorganisms (KCCM)

[00300] Accession number: KCCM13164P

[00301] Deposit date: April 21, 2022

[00302] 25

[00303] Depository authority: Korean Culture Center of 2026253928   15 Oct 2026 Microorganisms (KCCM)

[00304] Accession number: KCCM13165P

[00305] Deposit date: April 21, 2022 5

Claims

1. A method for producing a flavor, the methodcomprising a step of inoculating a fermentation medium with 5 a first microorganism and a second microorganism and then producing a fermentation broth containing amino acid, nucleic acid and / or organic acid by fermentation of the microorganisms,wherein the first microorganism and the second10 microorganism produce different products and each produces one selected from the group consisting of amino acid, nucleic acid and organic acid.

2. The method of claim 1, wherein the amino acid is15 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-20 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, xanthylic acid, and saltsThis data, for application number 2022432263, is current as of 2026-08-13 08:41 AEST2026253928   15 Oct 2026thereof.

4. The method of claim 1, wherein the organic acidis at least one selected from the group consisting of5 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 first10 microorganism is a glutamic acid-producing microorganism, and the  second microorganism is a lysine-producingmicroorganism, an arginine-producing microorganism, a histidine-producing microorganism, a tryptophan-producing microorganism, a glycine-producing microorganism, an15 alanine-producing microorganism, a succinic acid-producing microorganism, a lactic acid-producing microorganism, a guanylic acid-producing microorganism, or an inosinic acidproducing microorganism.20        6.    The method of claim 1,  wherein the firstmicroorganism is an inosinic acid-producing microorganism, and the  second microorganism is a lysine-producingmicroorganism, an arginine-producing microorganism, a histidine-producing microorganism, a tryptophan-producing2026253928   15 Oct 2026microorganism, a glycine-producing microorganism, an alanine-producing microorganism, a succinic acid-producing microorganism, a lactic acid-producing microorganism, or a guanylic acid-producing microorganism.

57. The method of claim 1, wherein the step comprises further inoculating the fermentation medium with a third microorganism, which produces a product different from products produced from the first microorganism and the10 second microorganism and produces one selected from the group consisting of amino acid, nucleic acid and organic acid.

8. The method of claim 1 or 7, wherein the first15 microorganism is a glutamic acid-producing microorganism, the second microorganism is a guanylic acid-producing microorganism, the third microorganism is an inosinic acidproducing microorganism.20         9. The method of claim 1 or 7, wherein the firstmicroorganism, the second microorganism and the third microorganism are microorganisms of the genus Corynebacterium.2026253928   15 Oct 202610. The method of claim 1 or 7, wherein the first microorganism, the second microorganism and the third microorganism are in a seed culture broth state obtained by individual culture or co-culture.

511. The method of claim 1 or 7, wherein the step comprises adjusting the inoculum of each microorganism in order to control the ratio between the products of the microorganisms in the fermentation broth.1012. The method of claim 1 or 7, wherein the fermentation broth contains the products of the microorganisms in an amount of 3 to 90 wt% relative to the total solid content of the fermentation broth.1513. A method for producing a flavor containing L-glutamic acid and L-lysine, the method comprising a step of inoculating a fermentation medium with a glutamic acidproducing microorganism and a lysine-producing 20 microorganism and then producing a fermentation broth containing L-glutamic acid and L-lysine by fermentation of the microorganisms.2026253928   15 Oct 202614. The method of claim 13, wherein the glutamic acid-producing microorganism and the lysine-producing microorganism are microorganisms of the genus Corynebacterium.

515. The method of claim 13, wherein the glutamic acid-producing microorganism and the lysine-producing microorganism are in a seed culture broth state obtained by individual culture or co-culture.1016. The method of claim 13, wherein the step comprises adjusting the inoculum of each of the glutamic acid-producing microorganism and the lysine-producing microorganism in order to control the ratio of L-glutamic 15 acid 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 to 90 wt% relative to the total 20 solid content of the fermentation broth.

18. A flavor produced by the method of claim 1 or 7.2026253928   15 Oct 202619. A flavor containing L-glutamic acid and L-lysine, produced by the method of claim 13.

20. The flavor of claim 19, containing L-glutamic5 acid and L-lysine in an amount of 3 to 90 wt% relative to the total solid content of the flavor.

21. A food composition containing the flavor of claim 18.1022. A food composition containing the flavor containing L-glutamic acid and L-lysine according to claim 19.

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