Method for producing seasoning ingredients by mixed fermentation of different microorganisms

Abstract

The present invention relates to a method for producing a seasoning material by mixing and fermenting two or more kinds of microorganisms that produce different types of products, i.e., different types of amino acids, nucleic acids and / or organic acids, and producing a natural seasoning material that can improve the taste and aroma of food and improve the overall sensory characteristics by mixing and fermenting different kinds of microorganisms that produce different products, i.e., different types of amino acids, nucleic acids and / or organic acids, and such a seasoning material can be used in various food fields.

Description

[Technical field]

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

[0002] L-glutamic acid is an acidic amino acid that produces a rich flavor, and it is naturally found in plant foods such as kelp, miso, and soy sauce, as well as in animal foods such as dairy products, meat, and fish. L-glutamic acid is the most widely used food seasoning ingredient worldwide, and in particular, monosodium glutamate (MSG), which is 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 effect. Recently, as consumers' awareness of health has increased, the market for natural seasoning ingredients such as yeast extract and natural ingredient extracts has shown a rapid growth, but MSG still has a rich flavor and is highly competitive at a low price compared to natural seasoning ingredients. Therefore, much effort is required to develop a substitute for MSG.

[0003] Meanwhile, in order to enhance the full-bodied taste, seasoning materials using various amino acids such as L-lysine, L-valine, and L-arginine together with L-glutamic acid have been developed. Seasoning materials further containing nucleic acids such as inosinic acid and guanylic acid, and organic acids such as succinic acid and lactic acid have also been developed. Such seasoning materials based on L-glutamic acid are generally produced by mixing fermentation liquids produced from microorganisms that produce each product, or by mixing amino acid, nucleic acid, or organic acid powders produced individually. In the conventional production method, it is easy to adjust the concentration or content of the final substance such as L-glutamic acid, but it is expensive to produce because a double production process must be performed before mixing, and a large amount of medium components and fermentation by-products remain in the produced seasoning material, making it difficult to solve the problem of strange taste and odor, and it is still difficult to produce a rich full-bodied taste. Therefore, a lot of research and development is needed to develop seasoning materials with improved full-bodied taste. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Republic of Korea Patent No. 10-1758332 [Patent Document 2] Republic of Korea 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 kinds of microorganisms which 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 of 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 material, comprising the steps of inoculating a first microorganism and a second microorganism into a fermentation medium and then fermenting the medium 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 from each other, each of which produces one selected from the group consisting of amino acids, nucleic acids and organic acids.

[0010] The "seasoning ingredient" used in the present invention means an ingredient added to enhance the flavor of food, and is used in a broader sense than general seasonings that are added in small amounts at the end of a general food cooking process, and can be used to enhance the flavor not only when cooking at home, but also during the manufacturing process 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 full-bodied flavor and having 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), and the like.

[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] "Fermentation" as used in the present invention is a biological phenomenon in which organic matter contained in a medium is decomposed or changed into other substances by microorganisms, and means that the inoculated microorganisms decompose or convert the nutrients in the fermentation medium into amino acids, nucleic acids, organic acids, etc. "Fermentation liquid" as used in the present invention means containing substances produced by microorganisms through fermentation, and such a fermentation liquid is produced by mixing and fermenting two or more microorganisms that produce different types of products, and contains not only useful substances such as amino acids, nucleic acids, and organic acids produced by each microorganism, but also by-products produced in the metabolic process, medium components, etc.

[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 one 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] As an 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 produces one selected from the group consisting of amino acids, nucleic acids and organic acids.

[0022] More specifically, the fermentation liquid produced from the first microorganism, the second microorganism, and the third microorganism may contain, but is not limited to, three types of amino acids, nucleic acids, or organic acids, or 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] As an 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 such seasoning materials 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. As the amino acid, nucleic acid, or organic acid producing microorganisms, microorganisms known in the art may be used without limitation, and examples thereof may include the genus Corynebacterium, the genus Brevibacterium, the genus Lactobacillus, the genus Bifidobacterium, and the genus Bacillus. These microorganisms may be of the same genus or species, or different genuses or species, and may be selected by the user.

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

[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] In order to carry out mixed fermentation of two or more kinds of microorganisms, each of the microorganisms may be separated and selected, or a mixture of the microorganisms may be used, and depending on the circumstances, the separated microorganisms and the mixture of the microorganisms may be appropriately mixed and 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 are activated, and it is preferable to carry out 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 in a mixed culture seed culture state.

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

[0030] The seed culture may be performed 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 in the seed culture may usually be 20 to 45°C, for example, 25 to 40°C, or 27 to 37°C, and the culture period can be continued until the microorganisms actively grow and proliferate, and may be, for example, 10 to 160 hours, 18 to 120 hours, or 20 to 80 hours.

[0033] Also, during the 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, during the cultivation, a food additive antifoaming agent can be used to suppress the generation of bubbles. Additionally, oxygen or oxygen-containing gas (e.g., air) can be injected into the culture solution to maintain the medium or culture solution in an aerobic state.

[0034] Such seed cultures can be cultured 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 the microorganisms and determine whether or not to continue the culture.

[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 liquid by fermentation of two or more kinds of microorganisms, a fermentation liquid containing different types of products can be produced by mixed fermentation (main fermentation) using each microorganism or its seed culture liquid.

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

[0038] According to one embodiment of the present invention, the step preferably comprises 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 the first microorganism: the second microorganism in the total inoculation amount of 0.05 to 99.95: 99.95 to 0.05. When three types of microorganisms are inoculated, the inoculation ratio of the first microorganism, the second microorganism and the third microorganism may be a ratio of the first microorganism: the second microorganism: the third microorganism in the total inoculation amount of 0.05 to 99.95: 99.95 to 0.05: 99.95 to 0.05.

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

[0041] The mixed fermentation may be carried out using a suitable medium and fermentation conditions known in the art in consideration of 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 in the main fermentation 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 the fermentation is completed, it is preferable that the fermentation medium is composed of substances that can be used as food materials and contains the minimum components and amounts necessary for the cultivation of 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 may contain 1 to 30% by weight of molasses based on the total equivalent weight. The molasses used here may be derived from sugar cane or sugar beet.

[0046] In addition to a sugar source, the fermentation medium may further contain nutrient substances for nutritional enrichment of the microorganisms.

[0047] According to one embodiment of the present invention, the fermentation medium may further comprise 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] Also, during the 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, during the cultivation, a food additive antifoaming agent can be used to suppress the generation of bubbles. Additionally, oxygen or oxygen-containing gas (e.g., air) can be injected into the culture solution to maintain the medium or culture solution in an aerobic state.

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

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

[0052] More specifically, the fermentation liquid may contain 3-90 wt%, 10-90 wt%, 20-90 wt%, 30-90 wt%, 40-90 wt%, 50-90 wt%, 60-90 wt%, 70-90 wt%, or 80-90 wt% of amino acids, nucleic acids, and / or organic acids based on the total solid content. Such a fermentation liquid may contain 5-150 g / L of amino acids, nucleic acids, and / or organic acids.

[0053] Another aspect of the present invention provides a method for producing a seasoning material containing L-glutamic acid and L-lysine, comprising the steps of inoculating a fermentation medium with a glutamic acid-producing microorganism and a lysine-producing microorganism, and then fermenting the medium 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 amino acid production ability of wild-type microorganisms. As such glutamic acid- or lysine-producing microorganisms, microorganisms known in the art may be used without limitation, and examples thereof may include the genus Corynebacterium, the genus Brevibacterium, the genus Lactobacillus, the genus Bifidobacterium, and the genus Bacillus. The microorganisms in the present invention may be of the same genus or species, or may be of different genus or species, and may 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] In order to carry out mixed fermentation of the glutamic acid-producing microorganism and the lysine-producing microorganism, each of the microorganisms may be separated and selected, or a mixture of the microorganisms may be used, and depending on the situation, the separated microorganism and the mixture of the microorganisms may be appropriately mixed and used. Such glutamic acid- or lysine-producing microorganisms can be used for fermentation in a state in which the microbial growth and amino acid production abilities are activated, and it is preferable to carry out 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 in a seed culture liquid state.

[0059] The seed culture may be performed 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% molasses, 3 wt% glucose, 0.85 wt% yeast extract paste, 100 ppm methionine, 0.6 wt% H3PO4, 0.1 wt% sodium succinate, 50 ppm vitamin C, 12 ppm thiamine HCl, 20 ppb vitamin B12, 10 ppm biotin, 0.4 wt% MgSO4, and 0.01 wt% food grade antifoaming agent.

[0062] According to a specific example 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 (NH4)2SO4, 0.3 wt % of H3PO4, 7.3 ppm of MnSO4·5H2O, 14 ppm of nicotinamide, 2.5 ppm of thiamine HCl, 51.5 ppm of CuSO4·H2O, 0.056 ppm of biotin, 0.045 wt % of betaine, and 0.01 wt % of a food antifoaming agent.

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

[0064] Also, during the 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, during the cultivation, a food additive antifoaming agent can be used to suppress the generation of bubbles. Additionally, oxygen or oxygen-containing gas (e.g., air) can be injected into the culture solution to maintain the medium or culture solution in an aerobic state.

[0065] Such seed cultures can be cultured 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 the microorganisms and determine whether or not to continue the culture.

[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 liquid thereof and a lysine-producing microorganism or a seed culture liquid thereof.

[0068] The mixed fermentation is a concept that is opposite to individual fermentation, which is a conventional method for producing amino acids by culturing each microorganism individually to produce a fermentation liquid containing the products of each microorganism. In the present invention, mixed fermentation produces L-glutamic acid by glutamic acid-producing microorganisms and L-lysine by lysine-producing microorganisms, and a fermentation liquid that can produce a rich flavor 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 in order 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 microorganism and the lysine-producing microorganism may be such that the glutamic acid-producing microorganism:lysine-producing microorganism in the total inoculation amount is 0.05 to 99.95:99.95 to 0.05, and L-glutamic acid and L-lysine can be produced in a ratio of 1 to 99:1 in the fermentation liquid so as to produce an appropriate taste as a seasoning ingredient by mixed fermentation of the two microorganisms inoculated at such a ratio.

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

[0072] The mixed fermentation may be carried out using a suitable medium and fermentation conditions known in the art in consideration of 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, after the fermentation is completed, the fermentation liquid containing the fermentation medium is used as a seasoning material without a separate purification process, so that the fermentation medium is preferably composed of substances usable as food materials and contains the minimum components and amounts necessary for the culture of 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 1 to 30% by weight of molasses based on the total equivalent weight. The molasses used here may be derived from sugar cane or sugar beet.

[0077] In addition to a sugar source, the fermentation medium may further contain nutrient substances for nutritional enrichment of the microorganisms.

[0078] According to one embodiment of the present invention, the fermentation medium may further comprise 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, 1.5-3 wt % of molasses, 2.5-4 wt % of glucose, 0.4-1 wt % of yeast extract paste, 0.1-0.2 wt % of H3PO4, 0.05-0.12 wt % of betaine, and 0.001-0.01 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 can be continued until a desired content or concentration of L-glutamic acid and L-lysine is obtained, and may be, for example, 10 to 160 hours, 18 to 120 hours, or 20 to 100 hours.

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

[0082] According to one embodiment of the present invention, the fermentation may be carried out by feeding culture for 28 to 40 hours under conditions in which the temperature is started from 30 to 33°C and maintained at 36 to 39°C, the pH is maintained at 6.5 to 7.5, and the dissolved oxygen content is maintained at 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 in that it can be used as is without mixing with additional ingredients, and contains large amounts of L-glutamic acid and L-lysine.

[0084] According to one embodiment of the present invention, the fermentation liquid may contain 3 to 90% by weight 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 liquid may be 3 to 90% by weight, 10 to 90% by weight, 20 to 90% by weight, 30 to 90% by weight, 40 to 90% by weight, 50 to 90% by weight, 60 to 90% by weight, 70 to 90% by weight, or 80 to 90% by weight based on the solid content. Such a fermentation liquid may 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 fermentation 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 bacteria from the fermentation liquid and decolorizing it.

[0088] The bacterial cell separation can be performed without limitation by applying a bacterial cell separation method and separation conditions known in the art, and examples of the bacterial cell separation include, but are not limited to, membrane separation, ultrafiltration, centrifugal filtration, etc.

[0089] The decolorization can be performed without limitation by applying a decolorization method and decolorization conditions known in the art, for example, activated carbon can be used for the decolorization, but is not limited thereto.

[0090] The method may further include a step of filtering the decolorized fermentation liquid.

[0091] The filtration can be performed without limitation by applying a filtration method and filtration conditions known in the art. Examples of the filtration include, but are not limited to, filter paper, filter net, membrane filtration, ultrafiltration, etc.

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

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

[0094] The method may further include a step of drying the concentrated fermentation liquid to obtain a powder.

[0095] The drying can be performed without limitation by applying a drying method and drying conditions known in the art, including, but not limited to, freeze drying, vacuum drying, ventilation drying, air drying, hot air drying, fluidized bed drying, spray drying, infrared drying, and high frequency drying.

[0096] By using this method, the fermentation liquid is finally obtained in a powder form and can be used in foods as a natural flavoring ingredient without undergoing additional chemical purification processes.

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

[0098] According to one embodiment of the present invention, the seasoning material may be a natural seasoning material containing two or three kinds of taste 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), and the like.

[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 kinds of taste components are included, the seasoning material may include glutamic acid and lysine, arginine, histidine, tryptophan, glycine, alanine, succinic acid, lactic acid, guanylic acid, or inosinic acid. Also, the seasoning material may include 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 a flavor component including an amino acid, a nucleic acid and / or an organic acid, based on the total solid content.

[0106] More specifically, the seasoning material may contain 3 to 90% by weight, 10 to 90% by weight, 20 to 90% by weight, 30 to 90% by weight, 40 to 90% by weight, 50 to 90% by weight, 60 to 90% by weight, 70 to 90% by weight, or 80 to 90% by weight of flavor components including amino acids, nucleic acids and / or organic acids 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 above-mentioned glutamic acid- and L-lysine-producing microorganisms.

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

[0109] According to one embodiment of the present invention, the seasoning ingredient may contain L-glutamic acid and L-lysine in an amount of 3 to 90% by weight 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 solving the problem of ammonia odor. It also contains metabolic products such as organic acids, inorganic ion components, proteins, peptides, and vitamins produced during the fermentation process, so it has a rich flavor and a strong body, and exhibits excellent sensory properties, and can be added to various foods to maximize the flavor of the food.

[0111] Another aspect of the present invention provides a food composition comprising 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 means a natural product or processed product containing one or more nutrients, preferably one that has been processed to a certain extent 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, and may be, for example, a solution, emulsion, viscous mixture, powder, granule, tablet, capsule, etc. 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 effect of the present invention, and additives such as flavorings, colorants, bactericides, antioxidants, preservatives, moisturizers, thickeners, inorganic salts, emulsifiers, etc. may be further included within a range that does not impair the effect. The amount of such additives can be selected within a range that does not impair the object and effect of the present invention depending on the dosage form or purpose of use. For example, the additives may be 0.01 to 70% by weight, more specifically 0.1 to 50% by weight, based on the total weight of the food composition.

[0115] Such a food composition can be used as an additive for various foods. The food to which such a food composition can be added can be any type of food known in the art, including, but not limited to, meat, sausage, bread, chocolate, candy, snacks, sweets, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, drinking water, tea, energy drinks, alcoholic beverages, and vitamin complexes. Effect of the Invention

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

[0117] [Figure 1] 1 is a flow chart showing a fermentation process using a glutamic acid-producing microorganism and a lysine-producing microorganism in Production Examples 1 to 3 according to one embodiment of the present invention. [Diagram 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. [Diagram 3] 1 is a flow chart showing a fermentation process using a glutamic acid producing microorganism and an inosinic acid producing microorganism in Production Examples 7 to 9 according to one embodiment of the present invention. [Figure 4] 1 is a flow chart showing a fermentation process using an inosinic acid-producing microorganism and a lysine-producing microorganism in Production Examples 10 to 12 according to one embodiment of the present invention. [Diagram 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 PREFERRED EMBODIMENTS

[0118] The present invention will be described in more detail below with reference to the accompanying drawings. However, 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. EXAMPLES

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

[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, 2-2.5 L of seed culture medium was added, and secondary culture was performed for 22-24 hours at 32°C, pH 6.9, 600 rpm, and aeration 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 primary cultured 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 performed for 21 to 24 hours at 32°C, pH 7.0, 650 rpm, and 2.0 vvm aeration (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 In order 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 the main fermentation, 14-18 L of fermentation medium was added to a 50 L fermenter, and 1.2-1.8 L of total seed culture medium was inoculated with glutamic acid seed culture medium and lysine seed culture medium at an inoculation ratio of 50-99.95:50-0.05, and mixed fermentation was performed by fed-batch culture for 28-40 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 solution of each amino acid microorganism was inoculated at a ratio of glutamic acid producing microorganism:lysine producing microorganism = 50 to 99.95: 50 to 0.05, L-glutamic acid and L-lysine were produced in the fermentation solution at a ratio of 0.83 to 99: 1.

[0130] Example 2. Comparison of glutamic acid-lysine fermentation powders based on differences in fermentation process 2-1. Production of glutamic acid-lysine fermentation powder Conventionally, a seasoning material containing glutamic acid and lysine was produced by fermenting glutamic acid-producing microorganisms and lysine-producing microorganisms separately and then mixing the amino acid-containing fermentation liquid or its dried product in an appropriate ratio. In order to compare the difference in taste between such a conventional individual fermentation method and a method of co-fermenting 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 FIG. 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 50L fermenter, respectively, and then the main fermentation was carried out separately. After that, the bacteria were separated from each fermentation solution, and then the decolorization and filtration processes were carried out. The filtrate was concentrated and dried to obtain a dried product. The obtained glutamic acid and lysine dried products were mixed so that the amino acid ratio was 1:1, and a fermentation powder containing glutamic acid-lysine was produced.

[0133] (2) Mixing of fermented liquids after individual fermentations (Production Example 2) In the conventional method of mixing separate fermentation liquids, the glutamic acid seed culture liquid or the lysine seed culture liquid was transferred to a 50 L fermenter, and then the main fermentation was carried out separately. The fermentation liquids obtained in the main fermentation were mixed so that the amino acid ratio was 1:1, and then the bacteria 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) In the mixed fermentation, the glutamic acid seed culture solution and the lysine seed culture solution were inoculated at a ratio of 65:35 based on the inoculum amount in the same manner as in Example 1-2 so that the ratio of each amino acid in the fermentation liquid was about 1:1, as in Production Examples 1 and 2. Then, the fermentation liquid was subjected to a decolorization and filtration process in which the cells were separated. The filtrate was concentrated and dried to obtain a glutamic acid-lysine fermentation powder.

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

[0136] Amino acids (L-glutamic acid and L-lysine) were measured by HPLC analysis (GA-210 nm UV detector, flow rate 0.9 ml / min; LYS-214 nm, UV detector, flow rate 0.8 ml / min), and organic acids (citric acid, succinic acid, lactic acid, acetic acid, etc.) were measured by HPLC analysis (HPX-87H column, 214 nm, 25 min). Ions (Na, Mg, K, PO4, SO4, Cl, NH4, etc.) were measured with 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 A sensory evaluation was carried out on the glutamic acid-lysine fermentation powders obtained by the individual fermentation in Production Examples 1 and 2, and the glutamic acid-lysine fermentation powder obtained by the mixed fermentation in Production Example 3.

[0140] The sensory evaluation of the glutamic acid-lysine fermented powder was carried out 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 components of the glutamic acid-lysine fermented powder as shown in Table 4. Also, as shown in Table 4, the increase in by-products such as organic acids and the increase in ions also affect the sensory sense, so the 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 solution 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 primary cultured 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. The secondary culture was carried out for 16-24 hours at 32°C, pH 6.7, 600 rpm, and 1.0 vvm aeration (OD 610 = 20-60), arginine seed culture medium was prepared. The composition of the seed culture medium used is shown in Table 6 below.

[0146] [Table 6]

[0147] 3-2. Main fermentation In order 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 the main fermentation, 14 to 18 L of fermentation medium was added to a 50 L fermenter, and 1.2 to 1.8 L of total seed culture medium was inoculated with glutamic acid seed culture medium and arginine seed culture medium at an inoculation ratio of 30 to 99.95:70 to 0.05, and mixed fermentation was performed 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 solution 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 liquid 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 fermentation powder Conventionally, a seasoning material containing glutamic acid and arginine was produced by fermenting glutamic acid-producing microorganisms and arginine-producing microorganisms separately, and then mixing the amino acid-containing fermentation liquid or its dried product in an appropriate ratio. In order to compare the difference in taste between such a conventional individual fermentation method and a method of co-fermenting glutamic acid-producing microorganisms and arginine-producing microorganisms, the components of the glutamic acid-arginine (GA-ARG) fermentation powder obtained by each production method were compared (see FIG. 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 the individual dried products, the glutamic acid seed culture solution or the arginine seed culture solution was transferred to a 50L fermenter, respectively, 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 glutamic acid and arginine dried products obtained were mixed so that the amino acid ratio was 1:1, and a fermented powder containing glutamic acid and arginine was produced.

[0156] (2) Mixing of fermented liquids after individual fermentations (Production Example 5) In the conventional method of mixing individual fermentation liquids, the glutamic acid seed culture liquid or the arginine seed culture liquid was transferred to a 50L fermenter, and then the main fermentation was carried out separately. The fermentation liquids obtained in the main fermentation were mixed so that the amino acid ratio was 1:1, and then the bacteria 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 the mixed fermentation, the glutamic acid seed culture and the arginine seed culture were inoculated at a ratio of 30:70 based on the inoculum amount in the same manner as in Example 3-2 so that the ratio of each amino acid in the fermentation liquid was about 1:1, as in Production Examples 4 and 5. After that, the fermentation liquid was subjected to a decolorization and filtration process in which the 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 fermentation 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 of individual fermentation and mixed fermentation A sensory evaluation was carried out on the glutamic acid-arginine fermented powders obtained by the individual fermentation in Production Examples 4 and 5, and the glutamic acid-arginine fermented powder obtained by the mixed fermentation in Production Example 6.

[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. Also, as shown in Table 9, the increase in by-products such as organic acids and the increase in ions also affect the sensory perception, so the 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 Corynebacterium glutamicum NFG6 (KCCM13164P) was used as a glutamic acid producing microorganism, and Corynebacterium ammoniagenes NFI545 (KCCM13162P), which produces IMP, was used as an inosinic acid producing microorganism.

[0167] A glutamic acid seed culture solution 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 microorganism, the microorganism was 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. The secondary culture was carried out for 21 to 24 hours at 31°C, pH 7.1, 600 rpm, and 1.0 vvm aeration (OD 610 = 20-40), an inosinic acid seed culture medium was prepared. The composition of the seed culture medium used is shown in Table 11 below.

[0169] [Table 11]

[0170] 5-2. Main fermentation In order to confirm the ratio of glutamic acid and 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 the main fermentation, 14 to 18 L of fermentation medium was added to a 50 L fermenter, and 1.2 to 1.8 L of total seed culture medium was inoculated at an inoculation ratio of 0.05 to 99.95:99.95 to 0.05 between the glutamic acid seed culture medium and the inosinic acid seed culture medium, and mixed fermentation was performed 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 solution of each microorganism was inoculated at a ratio of glutamic acid producing microorganism:inosinic acid producing microorganism=0.05-99.95:99.95-0.05, it was confirmed that L-glutamic acid and IMP were produced in the fermentation solution 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. Manufacturing of glutamic acid-inosinic acid fermentation powder In the past, a seasoning material containing glutamic acid and inosinic acid was produced by fermenting glutamic acid-producing microorganisms and inosinic acid-producing microorganisms separately and then mixing the fermentation liquid or the dried product in an appropriate ratio. In order to compare the difference in taste between such a conventional individual fermentation method and a method of co-fermenting 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 FIG. 3).

[0177] The glutamic acid seed culture medium and the 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 liquid or the inosinic acid seed culture liquid was transferred to a 50L fermenter, respectively, and then fermented separately. After that, the bacteria were separated from each fermentation liquid, and then decolorized and filtered. The filtrate was concentrated and dried to obtain a dried product. The obtained glutamic acid and inosinic acid dried products were mixed in a ratio of 1:1 to produce a fermented powder containing glutamic acid-inosinic acid.

[0179] (2) Mixing of fermented liquids after individual fermentations (Production Example 8) In the conventional method of mixing separate fermentation liquids, the glutamic acid seed culture liquid or the inosinic acid seed culture liquid was transferred to a 50L fermenter, and then the main fermentation was carried out separately. The fermentation liquids obtained in the main fermentation were mixed in a ratio of 1:1, and the bacteria 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 the mixed fermentation, the glutamic acid seed culture solution and the inosinic acid seed culture solution were inoculated at a ratio of 20:80 based on the inoculum amount in the same manner as in Example 5-2 so that the ratio of each amino acid in the fermentation broth was about 1:1, as in Production Examples 7 and 8. Then, the fermentation broth was subjected to a decolorization and filtration process in which the cells were separated from the fermentation broth. The filtrate was concentrated and dried to obtain a glutamic acid-inosinic acid fermentation powder.

[0181] 6-2. Comparison of ingredients of glutamic acid-inosinic acid fermentation 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 A sensory evaluation was carried out on 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.

[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] As shown in 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 components of the glutamic acid-inosinic acid fermented powder as shown in Table 14. Also, as shown in Table 14, the increase in by-products such as organic acids and the increase in ions also affect the sensory sense, 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 In order 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 the main fermentation, 14 to 18 L of fermentation medium was added to a 50 L fermenter, and 1.2 to 1.8 L of total seed culture medium was inoculated with the inosinic acid seed culture medium and the lysine seed culture medium at an inoculation ratio of 0.05 to 99.95:99.95 to 0.05, and mixed fermentation was performed 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] Such 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 solution 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 liquid 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 fermentation powder In the past, a seasoning material containing inosinic acid and lysine was produced by fermenting inosinic acid-producing microorganisms and lysine-producing microorganisms separately and then mixing the fermentation liquid or the dried product in an appropriate ratio. In order to compare the difference in taste between such a conventional individual fermentation method and a method of co-fermenting 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 50L fermenter, respectively, and then fermented separately. Then, the bacteria were separated from each fermentation solution, and the solution was subjected to decolorization and filtration processes. The filtrate was concentrated and dried to obtain a dried product. The obtained inosinic acid and lysine dried products were mixed in a ratio of 1:1 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 liquids, the inosinic acid seed culture liquid or the lysine seed culture liquid was transferred to a 50L fermenter, and then the main fermentation was carried out separately. The fermentation liquids obtained in the main fermentation were mixed in a ratio of 1:1, and the 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, inosinic acid seed culture solution and lysine seed culture solution were inoculated at an inoculation ratio of 80:20 based on the inoculum amount in the same manner as in Example 7-2 so that the ratio of inosinic acid and lysine in the fermentation liquid was about 1:1, as in Production Examples 10 and 11. After that, the fermentation liquid was subjected to a decolorization and filtration process in which the cells were separated. 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 fermentation in Production Examples 10 and 11, and the inosinic acid-lysine fermentation powder obtained by the mixed fermentation in 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 A sensory evaluation was carried out on 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.

[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 aftertaste lingering 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. Also, as shown in Table 18, the increase in by-products such as organic acids and the increase in ions also affect the 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 the main fermentation, 14 to 18 L of fermentation medium was added to a 50 L fermenter, and 1.2 to 1.8 L of total seed culture medium was inoculated at an inoculation ratio of 65 to 99.95:35 to 0.05 between the inoculum seed culture medium and the arginine seed culture medium, and mixed fermentation was performed 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] Such 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 solution 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 fermenting inosinic acid-producing microorganisms and arginine-producing microorganisms separately and then mixing the fermentation liquid or the dried product in an appropriate ratio. In order to compare the difference in taste between such a conventional individual fermentation method and a method of co-fermenting 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 50L fermenter, respectively, 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 obtained inosinic acid and arginine dried products were mixed in a ratio of 1:1 to produce a fermented powder containing inosinic acid-arginine.

[0220] (2) Mixing of fermented liquids after individual fermentations (Production Example 14) In the conventional method of mixing individual fermentation liquids, the inosinic acid seed culture liquid or the lysine seed culture liquid was transferred to a 50L fermenter, and then the main fermentation was carried out separately. The fermentation liquids obtained in the main fermentation were mixed in a ratio of 1:1, and the bacteria 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 solution and the arginine seed culture solution were inoculated in a ratio of 65:35 based on the inoculum amount in the same manner as in Example 9-2 so that the ratio of inosinic acid and arginine in the fermentation liquid was about 1:1, as in Production Examples 13 and 14. After that, the fermentation liquid was subjected to a decolorization and filtration process in which the 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 individual fermentation in Production Examples 13 and 14, and the inosinic acid-arginine fermented powder obtained by 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 aftertaste lingering 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. Also, as shown in Table 22, the increase in by-products such as organic acids and the increase in ions also affect the sensory perception, so the 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 a focus on its preferred embodiments. Those skilled in the art will understand that the present invention can be realized in modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an explanatory perspective, not a restrictive one. The scope of the present invention is defined by the claims, not the above description, and all differences within the scope of the equivalents should be interpreted as being included in the present invention.

[0230] [Accession number] Depository institution: Korea Center for Culture Collection of Microorganisms (KCCM) Accession number: KCCM13162P Date of acceptance: 20220421 Depository institution: Korea Center for Culture Collection of Microorganisms (KCCM) Accession number: KCCM13163P Date of acceptance: 20220421 Depository institution: Korea Center for Culture Collection of Microorganisms (KCCM) Accession number: KCCM13164P Date of acceptance: 20220421 Depository institution: Korea Center for Culture Collection of Microorganisms (KCCM) Accession number: KCCM13165P Date of acceptance: 20220421 [Deposit receipt] JPEG2025502882000025.jpg239170JPEG2025502882000026.jpg239170JPEG2025502882000027.jpg240170JPEG2025502882000028.jpg239170 TIFF2025502882000029.tif122170TIFF2025502882000030.tif122170TIFF2025502882000031.tif122170TIFF2025502882000032.tif121170

Claims

1. The method includes the steps 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, nucleic acids, and / or organic acids; A method for producing a seasoning material, 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.

2. The method according to 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. 2. The method of claim 1, wherein the organic acid is 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.

5. The first microorganism is a glutamic acid producing microorganism, 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, 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.

6. 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, an alanine-producing microorganism, a succinic acid-producing microorganism, a lactic acid-producing microorganism, or a guanylic acid-producing microorganism.

7. 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.

8. The first microorganism is a glutamic acid producing microorganism, the second microorganism is a guanylic acid producing microorganism, The method according to claim 1 or 7, wherein the third microorganism is an inosinic acid producing microorganism.

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

10. The method according to claim 1 or 7, wherein the first microorganism, the second microorganism and the third microorganism are cultured individually or in a mixed culture seed culture state.

11. 10. The method according to claim 1 or 7, wherein the step comprises adjusting the inoculum amount of each microorganism to adjust the ratio of the products of each microorganism in the fermentation broth.

12. The method according to claim 1 or 7, wherein the fermentation liquid contains 3 to 90% by weight of total microbial products in the total solid content.

13. A method for producing a seasoning material containing L-glutamic acid and L-lysine, comprising the steps of inoculating a fermentation medium with a glutamic acid-producing microorganism and a lysine-producing microorganism, and then fermenting the medium to produce a fermentation liquid containing L-glutamic acid and L-lysine.

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

15. The method according to claim 13, wherein the glutamic acid producing microorganism and the lysine producing microorganism are cultured individually or in a mixed culture seed culture state.

16. 14. The method according to claim 13, wherein said step comprises 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.

17. The method according to claim 13, wherein the fermentation liquid contains 3 to 90% by weight of amino acids including L-glutamic acid and L-lysine in the total solid content.

18. A seasoning material produced by the method according to claim 1 or 7.

19. 14. A seasoning material containing L-glutamic acid and L-lysine, produced by the method according to claim 13.

20. 20. The L-glutamic acid and L-lysine-containing seasoning material according to claim 19, wherein the L-glutamic acid and L-lysine contents in the seasoning material are 3 to 90% by weight based on the solid content.

21. A food composition comprising the seasoning material according to claim 18.

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

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