Culture medium composition for corynebacterium sp. and use thereof

Abstract

The present application relates to: a culture medium composition for microorganisms of Corynebacterium species, in which part of ammonium sulfate is replaced with a different ammonium-containing compound; and a method for producing a basic amino acid by using the culture medium composition.

Description

Composition of a culture medium for microorganisms of the genus Corynebacterium and its use

[0001] This application claims the benefit of priority to Republic of Korea Patent Application No. 10-2024-0116276, dated August 28, 2024, and Republic of Korea Patent Application No. 10-2025-0101523, dated July 25, 2025, the entire contents of which are incorporated herein by reference.

[0002] Numerous papers and patents are referenced and cited throughout this application. The disclosures of these cited papers and patents are incorporated into this application in their entirety by reference, providing a clearer understanding of the state of the art and the scope of the present invention.

[0003] The present application provides a culture medium composition for a Corynebacterium genus microorganism characterized in that a portion of ammonium sulfate is replaced with another ammonium-containing compound, and a method for producing a basic amino acid using the same.

[0004]

[0005] Lysine (L-Lysine), a basic amino acid, is an essential amino acid and is used in various fields, including as a feed additive, food additive, and pharmaceutical raw material. As a bulk chemical, its market size is estimated at 2.94 million tons as of 2022. Currently, most lysine is mass-produced through direct fermentation using a medium containing a carbon source (raw sugar, sucrose, glucose) and a nitrogen source (yeast extract, soybean meal decomposition, corn steep liquor).

[0006] In the medium components supplied for lysine fermentation using coryneform microorganisms, the sulfate in the ammonium sulfate acts as a counter ion to maintain a neutral pH condition for lysine biosynthesized during fermentation, and ammonium acts as a nitrogen source required for lysine biosynthesis. As a result, lysine sulfate exists in the culture medium, and in order to commercialize it in the form of lysine hydrochloride (Lysine HCl), a powdered formulation, a process of adsorbing and eluting lysine by an ion exchange resin process is required. In this process, the complexity of the purification process and the input of auxiliary materials are essential, which significantly increases the manufacturing cost.

[0007] To overcome the above problems, the introduction of a granulated formulation (US 8916213 B2) for the fermentation liquid significantly reduced manufacturing costs through process simplification. Furthermore, the product's low hygroscopicity and good flowability allow for formulation diversification, overcoming process issues and significantly improving cost competitiveness. However, since the granulation process granulates the fermentation liquid obtained from the fermentation process without ion exchange resin treatment, byproducts other than lysine generated during fermentation are a major factor in reducing the purity of the product produced through the granulation process.

[0008] Therefore, it is necessary to develop a technology to reduce the amount of ammonium sulfate input, such as limiting the amount of ammonium sulfate input in the above fermentation process or applying it as a substitute for another candidate substance.

[0009]

[0010] One example of the present application provides a culture medium composition for a Corynebacterium sp. microorganism, comprising ammonium sulfate and other ammonium-containing compounds.

[0011] Another example provides a method for producing a basic amino acid comprising the step of culturing a Corynebacterium sp. microorganism in the above culture medium composition.

[0012] Another example provides a use of the above culture medium composition for producing basic amino acids.

[0013] Another example provides a basic amino acid-containing culture comprising a Corynebacterium sp. microorganism and the culture medium composition.

[0014] Another example provides a method for producing a culture medium for a Corynebacterium sp. microorganism or a method for improving the basic amino acid purity of a culture medium for a Corynebacterium sp. microorganism, characterized in that a part of ammonium sulfate in the culture medium for a Corynebacterium sp. microorganism is replaced with another ammonium-containing compound.

[0015]

[0016] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this application can also be applied to each other description and embodiment. That is, all combinations of various elements disclosed in this application fall within the scope of this application. In addition, the scope of this application should not be considered limited by the specific description described below. In addition, those skilled in the art will recognize or be able to identify many equivalents to the specific embodiments of this application described in this application using only routine experiments. In addition, such equivalents are intended to be included in this application. In addition, the numerical values ​​disclosed in this application can be interpreted to include numerical values ​​within an equivalent or similar range (e.g., ±20%, ±15%, ±10%, ±5%, ±3%, or ±1%, etc.).

[0017]

[0018] Hereinafter, the present application will be described in more detail:

[0019]

[0020] Composition of a culture medium for Corynebacterium sp. microorganisms

[0021] In one example of the present application, the Corynebacterium sp. microorganism culture medium composition of the present application is,

[0022] Ammonium sulfate; and at least one ammonium-containing compound selected from the group consisting of ammonium succinate, ammonium phosphate, and ammonium acetate;

[0023] One or more values ​​selected from [Formula 1] to [Formula 4] below may be greater than or equal to 2%, but is not limited thereto:

[0024] [Formula 1]

[0025] [(sum of moles (mol) of ammonium derived from ammonium succinate, ammonium phosphate, and ammonium acetate) / (sum of moles (mol) of ammonium derived from ammonium sulfate, ammonium succinate, ammonium phosphate, and ammonium acetate)]*100(%)

[0026] [Formula 2]

[0027] [(sum of the weight (g) of ammonium derived from ammonium succinate, ammonium phosphate, and ammonium acetate) / (sum of the weight (g) of ammonium derived from ammonium sulfate, ammonium succinate, ammonium phosphate, and ammonium acetate)] * 100 (%)

[0028] [Formula 3]

[0029] [(sum of moles of nitrogen derived from ammonium succinate, ammonium phosphate, and ammonium acetate) / (sum of moles of nitrogen derived from ammonium sulfate, ammonium succinate, ammonium phosphate, and ammonium acetate)]*100(%)

[0030] [Formula 4]

[0031] [(sum of the weight (g) of nitrogen derived from ammonium succinate, ammonium phosphate, and ammonium acetate) / (sum of the weight (g) of nitrogen derived from ammonium sulfate, ammonium succinate, ammonium phosphate, and ammonium acetate)]*100(%).

[0032] More specifically, the Corynebacterium sp. microbial culture medium composition of the present application comprises ammonium sulfate; and at least one ammonium-containing compound selected from the group consisting of ammonium succinate, ammonium phosphate, and ammonium acetate, wherein at least one value selected from the above [Formula 1] to [Formula 4] may be 2% or more and 100% or less, but is not limited thereto. More specifically, one or more values ​​selected from the above [Formula 1] to [Formula 4] may be a value within a range formed by one lower limit selected from 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, and 65% and one upper limit selected from 100%, 99%, 90%, 80%, 70%, 65%, 60%, 55%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, and 5%, wherein the upper limit may be set to be equal to or greater than the selected lower limit. For example, one or more values ​​selected from the above [Formula 1] to [Formula 4] may be, but are not limited to, 2% to 100%, 2% to 99%, 2% to 70%, 2% to 50%, 2% to 10%, 5% to 80%, 5% to 70%, 5% to 60%, 5% to 50%, 5% to 35%, 5% to 20%, 5% to 10%, 20% to 30%, 30% to 40%, 40% to 65%, 40% to 50%, 50% to 60%, 60% to 70%, or 65% to 70%.

[0033] In this specification, the range described as 'x to y' means x or more and y or less unless otherwise specified.

[0034] As such, the Corynebacterium microbial culture medium composition of the present application is characterized by replacing a portion of ammonium sulfate with another ammonium-containing compound. In the present application, by replacing ammonium sulfate with any one ammonium-containing compound selected from among ammonium acetate, ammonium succinate, and ammonium phosphate, which are ammonia-containing counter ion substances, or a combination of two or more ammonium-containing compounds, the amount of anionic counter ions remaining in the fermentation broth, specifically, sulfate ions, can be reduced, thereby improving the purity of the final fermentation broth. Ammonium ions act as a nitrogen source required for lysine biosynthesis, and in the case of acetic acid and succinic acid, they are completely consumed as carbon sources by microbial metabolic pathways. In the case of phosphate, by replacing phosphoric acid introduced into the existing medium with ammonium phosphate, it is consumed by the biosynthesis of nucleic acid and phospholipid in cells. Therefore, in this technology, compared to the conventional technology in which ammonium sulfate is introduced into the medium entirely, the stability of the fermentation process is significantly increased and the purity of the fermentation broth is improved, thereby improving product quality by introducing an ammonium substitute containing an anionic counterion that can be consumed by microorganisms as a carbon source or phosphate source.

[0035] The above ammonium sulfate may function as a nitrogen source and / or pH regulator in the culture medium composition, but is not limited thereto.

[0036] In the present specification, the other ammonium-containing compound refers to a substance containing an anion other than sulfate ion as an anionic counter ion of ammonium, and may be used interchangeably with “ammonium sulfate substitute” in the present specification. In one example, in order to reduce the residual amount of anionic substances in the medium, the other ammonium-containing compound may contain a substance that can be consumed as a carbon source, phosphorus, etc. in a microbial metabolic pathway as an anionic counter ion of ammonium, for example, the anionic counter ion of ammonium may be one or more anions selected from the group consisting of succinate, phosphate, acetate, etc. In one specific example, the other ammonium-containing compound may include one or more selected from the group consisting of ammonium succinate, ammonium phosphate, ammonium acetate, etc. More specifically, the other ammonium compound may include ammonium succinate, ammonium phosphate, or a mixture of ammonium succinate and ammonium phosphate, or may further include ammonium acetate in addition to ammonium succinate, ammonium phosphate, or a mixture of ammonium succinate and ammonium phosphate. In another example, the other ammonium-containing compound may not include one or more selected from the group consisting of urea, ammonium carbonate, ammonium chloride, and the like, but is not limited thereto.

[0037] The Corynebacterium microbial culture medium composition provided in the present application can improve the productivity (e.g., purity, production yield, etc.) of a target substance (e.g., basic amino acid such as L-lysine, L-arginine, and / or L-histidine) by replacing a portion of ammonium sulfate with another ammonium-containing compound, or by including ammonium sulfate together with another ammonium-containing compound, while maintaining the level of AN (ammonium nitrogen) and / or ammonium in the medium at an equivalent level compared to when no replacement was done, while reducing the amount of sulfate ions remaining in the medium after microbial culture and / or fermentation.

[0038] In the present application, basic amino acids have, in addition to the basic amino group in the molecule, one or more basic functional groups (e.g., amine group, guanidinium group, imidazole group) in the side chain, which result in a positive charge at neutral pH and the formation of sulfate ions (SO4 2- ) may refer to an amino acid that can easily form a counter ion with an anion such as L-lysine, L-arginine, and L-histidine. The basic amino acid may be at least one amino acid selected from the group consisting of L-lysine, L-arginine, and L-histidine, and more specifically, may be L-lysine, but is not limited thereto.

[0039] In the culture medium composition of the present application, the ammonium sulfate; and at least one ammonium-containing compound selected from the group consisting of ammonium succinate, ammonium phosphate, and ammonium acetate may be included as an ammonia counterion substance and / or an ammonium source and / or a pH regulator, but is not limited thereto.

[0040] In the present application, ammonium sulfate may mean a compound having the chemical formula (NH4)2SO4 and a molecular weight of 132.14. The ammonium sulfate contains two ammonium ions per molecule, and the counter ion is a sulfate ion (SO4 2- )am.

[0041] In the present application, ammonium acetate may mean a compound having a chemical formula of NH4CH3CO2 and a molecular weight of 77.0825. The ammonium acetate contains one ammonium ion per molecule, and the counter ion is acetate ion (CH3COO - )am.

[0042] In the present application, ammonium succinate may mean a compound having the chemical formula (NH4)2C4H4O4 and a molecular weight of 152.15. The ammonium succinate contains two ammonium ions per molecule, and the counter ion is the succinate ion (C4H4O4 2- )am.

[0043] In the present application, ammonium phosphate may mean a compound having the chemical formula (NH4)2HPO4 and a molecular weight of 132.06. The ammonium phosphate contains two ammonium ions per molecule, and the counter ion is a phosphate ion (HPO4 2- )am.

[0044] In one example, the above-described Corynebacterium sp. microorganism culture medium composition of the present application comprises ammonium sulfate; and at least one ammonium-containing compound selected from the group consisting of ammonium succinate, ammonium phosphate, and ammonium acetate, and at least one value selected from the following [Formula 5] to [Formula 8] may be 90% or less, but is not limited thereto:

[0045] [Formula 5]

[0046] [(Moles of ammonium derived from ammonium acetate) / (sum of moles of ammonium succinate, ammonium phosphate, and ammonium acetate derived from ammonium acetate)]*100(%)

[0047] [Formula 6]

[0048] [(Weight (g) of ammonium derived from ammonium acetate) / (Sum of weight (g) of ammonium succinate, ammonium phosphate, and ammonium derived from ammonium acetate)]*100(%)

[0049] [Formula 7]

[0050] [(mol of nitrogen derived from ammonium acetate) / (sum of mol of ammonium derived from ammonium succinate, ammonium phosphate, and ammonium acetate)]*100(%)

[0051] [Formula 8]

[0052] [(Weight (g) of nitrogen derived from ammonium acetate) / (sum of weight (g) of nitrogen derived from ammonium succinate, ammonium phosphate, and ammonium acetate)]*100(%).

[0053] More specifically, the above-described Corynebacterium sp. microorganism culture medium composition of the present application comprises ammonium sulfate; and at least one ammonium-containing compound selected from the group consisting of ammonium succinate, ammonium phosphate, and ammonium acetate, and at least one value selected from the above [Formula 5] to [Formula 8] may be 0% or more and 90% or less, but is not limited thereto. More specifically, one or more values ​​selected from the above [Formula 5] to [Formula 8] may be a value within a range formed by one lower limit selected from 0%, 2%, 5%, 10%, 20%, 30%, 34%, 40%, 50%, and 60% and one upper limit selected from 90%, 75%, 70%, 67%, 60%, 50%, 45%, 40%, 34%, 30%, 20%, 10%, 5%, 2%, and 1% or less, and the upper limit may be set to be equal to or greater than the selected lower limit. For example, one or more values ​​selected from [Formula 5] to [Formula 8] above may be 0% or more and 75% or less, 0% or more and 70% or less, 0% or more and 67% or less, 0% or more and 45% or less, 0% or more and 34% or less, 0% or more and 10% or less, or 0% or more and 1% or less, but is not limited thereto.

[0054] In this case, 0% means no ammonium acetate (absence of ammonium acetate, 0 moles or 0 g of ammonium acetate).

[0055] In one specific example, the above-described Corynebacterium sp. microbial culture medium composition of the present application may include ammonium succinate and / or ammonium phosphate as the 'other ammonium-containing compound' and may not include ammonium acetate (including 0 mol or 0 g of ammonium acetate, at least one value selected from the above [Formula 5] to [Formula 8] being 0%). In another specific example, the above-described Corynebacterium sp. microbial culture medium composition of the present application may further include ammonium acetate in addition to ammonium succinate and / or ammonium phosphate. When the above-described Corynebacterium microbial culture medium composition of the present application further comprises ammonium acetate in addition to ammonium succinate and / or ammonium phosphate, the ammonium acetate may be included such that one or more values ​​selected from the above [Formula 5] to [Formula 8] are greater than 0% and less than or equal to 90%, greater than 0% and less than or equal to 75%, greater than 0% and less than or equal to 70%, greater than 0% and less than or equal to 67%, greater than 0% and less than or equal to 60%, greater than 0% and less than or equal to 50%, greater than 0% and less than or equal to 45%, greater than 0% and less than or equal to 40%, greater than 0% and less than or equal to 34%, greater than 0% and less than or equal to 30%, greater than 0% and less than or equal to 20%, greater than 0% and less than or equal to 10%, greater than 0% and less than or equal to 5%, greater than 0% and less than or equal to 2%, or greater than 0% and less than or equal to 1%, but is not limited thereto.

[0056] In one example, the sum of the moles (mol) of ammonium derived from ammonium sulfate, ammonium succinate, ammonium phosphate, and ammonium acetate described above may be, but is not limited to, 0.1 to 0.5 moles per 1 L of the culture medium composition. More specifically, the sum of the moles (mol) of the ammonium derived from the ammonium sulfate, ammonium succinate, ammonium phosphate, and ammonium acetate is 0.1 mol to 0.5 mol, 0.1 mol to 0.47 mol, 0.1 mol to 0.465 mol, 0.1 mol to 0.46 mol, 0.1 mol to 0.4 mol, 0.1 mol to 0.3 mol, 0.1 mol to 0.275 mol, 0.1 mol to 0.265 mol, 0.1 mol to 0.26 mol, 0.1 mol to 0.225 mol, 0.1 mol to 0.2 mol, 0.15 mol to 0.5 mol, 0.15 mol to 0.47 mol, 0.15 mol to 0.465 mol, 0.15 mol to 0.46 mol, 0.15 mol to 0.4 mol, 0.15 mol to 0.3 mol, 0.15 mol to 0.275 mol, 0.15 mol to 0.265 mol, 0.15 mol to 0.26 mol, 0.15 mol to 0.225 mol, 0.15 mol to 0.2 mol, 0.18 mol to 0.5 mol, 0.18 mol to 0.47 mol, 0.18 mol to 0.465 mol, 0.18 mol to 0.46 mol, 0.18 mol to 0.4 mol, 0.18 mol to 0.3 mol, 0.18 mol to 0.275 mol, 0.18 mol to 0.265 mol, 0.18 mol to 0.26 mol, 0.18 mol to 0.225 mol, or may be 0.18 mol to 0.2 mol, but is not limited thereto.

[0057] In one example, the culture medium composition of the present application described above comprises, based on 1 mole of ammonium derived from ammonium sulfate,

[0058] 0.05 mol to 1.2 mol of ammonium derived from ammonium succinate,

[0059] 0.01 to 0.5 mol of ammonium derived from ammonium phosphate, and

[0060] It may include, but is not limited to, one or more selected from the group consisting of 0.05 mol to 1 mol of ammonium derived from ammonium acetate.

[0061] In one specific example, the culture medium composition of the present application described above, based on 1 mole of ammonium derived from ammonium sulfate,

[0062] A value within a range formed by one lower limit selected from 0.05 mol to 1.2 mol of ammonium succinate derived ammonium, more specifically 0.05 mol, 0.1 mol, 0.115 mol, 0.17 mol, 0.275 mol, 0.35 mol, 0.4 mol and 0.8 mol and one upper limit selected from 1.2 mol, 1.175 mol, 1 mol, 0.8 mol, 0.4 mol, 0.35 mol, 0.275 mol, 0.17 mol and 0.0115 mol, wherein the upper limit may be set to be equal to or greater than the selected lower limit;

[0063] A value within a range formed by one lower limit selected from 0.01 mol to 0.5 mol of ammonium phosphate derived ammonium, more specifically 0.01 mol, 0.05 mol, 0.07 mol, 0.09 mol, 0.15 mol, 0.2 mol and 0.3 mol and one upper limit selected from 0.5 mol, 0.4 mol, 0.3 mol, 0.2 mol, 0.15 mol and 0.09 mol, wherein the upper limit may be set to be equal to or greater than the selected lower limit, and

[0064] Ammonium acetate derived ammonium 0.05 mol to 1 mol, more specifically, a lower limit selected from 0.05 mol, 0.09 mol, 0.165 mol, 0.275 mol, 0.4 mol and 0.5 mol and an upper limit selected from 1 mol, 0.9 mol, 0.5 mol, 0.4 mol, 0.275 mol and 0.165 mol, wherein the upper limit may be set equal to or greater than the selected lower limit, but may include at least one selected from the group consisting of, but not limited to.

[0065] In this application, the term "medium" refers to a material containing nutrients necessary for culturing microorganisms as a main component, and supplies nutrients and growth factors, including water essential for survival and growth. Any medium that satisfies the typical culture conditions of a selected microorganism for the production of a target substance (e.g., a basic amino acid such as L-lysine, L-arginine, and / or L-histidine) may be used without particular limitation, and may contain an appropriate carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid, and / or vitamin, etc., depending on the selected microorganism, and may be one in which temperature, pH, etc. are controlled under aerobic conditions.

[0066] The carbon source may include at least one selected from the group consisting of carbohydrates (sugars) such as glucose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc. In addition, at least one natural organic nutrient source selected from the group consisting of starch hydrolysate, molasses, blackstrap molasses, rice winter, cassava, sugarcane bagasse, corn steep liquor, etc. may be used. For example, carbohydrates such as corn steep liquor, glucose, sterilized pretreated molasses (i.e., molasses converted into reducing sugar), etc. may be used as the carbon source, and in addition, various carbon sources of appropriate types and amounts may be used without limitation. These carbon sources may be used alone or in combination of two or more.

[0067] In one embodiment, the culture medium composition of the present application described above may include the carbon source (e.g., sugar) in an amount of 10 g to 100 g per 1 L of the culture medium composition, and more specifically, may include 10 g to 100 g, 10 g to 70 g, 10 g to 60 g, 10 g to 50 g, 10 g to 40 g, 20 g to 100 g, 20 g to 70 g, 20 g to 60 g, 20 g to 50 g, or 20 g to 40 g per 1 L of the culture medium composition, but is not limited thereto.

[0068] The nitrogen source may be at least one selected from the group consisting of inorganic nitrogen sources such as ammonia, ammonium chloride, ammonium carbonate, ammonium nitrate, etc.; amino acids such as glutamic acid, methionine, glutamine, etc.; organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition product, defatted soybean cake or its decomposition product, etc., but is not limited thereto. These nitrogen sources may be used alone or in combination of two or more.

[0069] The phosphorus may include at least one selected from the group consisting of phosphoric acid, monopotassium phosphate, dipotassium phosphate, or a sodium-containing salt corresponding thereto. When the culture medium composition of the present application includes ammonium phosphate as a substitute for ammonium sulfate, the content of phosphorus may be adjusted depending on the content of the ammonium phosphate. For example, the culture medium composition of the present application may include phosphorus in an amount equal to the amount obtained by deducting the amount of phosphorus contained in the ammonium phosphate from the total amount of phosphorus required for culturing a microorganism producing a target substance and / or producing a target substance, or may not include a separate phosphorus substance when the content of the ammonium phosphate is sufficient.

[0070] The above inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc., and may also include amino acids, vitamins, and / or appropriate precursors.

[0071] In the present application, the culture medium composition of the present application described above may have a pH of 6 to 9. More specifically, the culture medium composition of the application may have a pH of 6 to 9, 6 to 8.75, 6 to 8.5, 6.25 to 9, 6.25 to 8.75, 6.25 to 8.5, 6.5 to 9, 6.5 to 8.75, or 6.5 to 8.5, but is not limited thereto. In one example, ammonia gas and / or an ammonia solution may be added to the culture medium composition to maintain the pH range of the culture medium composition during the culturing step. By maintaining the pH range as described above, a metabolic environment favorable for the growth of a Corynebacterium spp. microorganism and production of a target product (e.g., a basic amino acid such as L-lysine, L-arginine, and / or L-histidine) can be provided.

[0072] The above components or precursors may be added to the medium in batch or continuous manner, but are not limited thereto.

[0073] In the present application, the medium may be a liquid medium, a solid medium, or a semi-solid medium, and in one example, may be a liquid medium, but is not limited thereto.

[0074] In one example, the Corynebacterium microbial culture medium composition provided in the present application can be used as a seed culture medium, a main culture medium, or both. Accordingly, the Corynebacterium microbial culture medium composition of the present application can be a seed culture medium composition or a main culture medium composition. In one specific example, the Corynebacterium microbial culture medium composition of the present application can be a seed culture medium composition. In another specific example, the Corynebacterium microbial culture medium composition of the present application can be a main culture medium composition. In another specific example, the Corynebacterium microbial culture medium composition of the present application can be used in both the seed culture step and the main culture step, in which case, the compositions applied to each step can be independently configured differently from each other while satisfying the conditions (or characteristics) described herein.

[0075] In one specific example, the seed culture medium composition of the present application may not contain ammonium acetate (including 0 mol or 0 g), or may additionally contain ammonium acetate in addition to ammonium succinate and / or ammonium phosphate.

[0076] More specifically, the seed culture medium composition of the present application may correspond to one or more of the following:

[0077] Contains ammonium sulfate and ammonium succinate,

[0078] Based on 1 mole of ammonium derived from the above ammonium sulfate,

[0079] The ammonium derived from the above ammonium succinate is 0.05 mol to 1.2 mol;

[0080] Contains ammonium sulfate and ammonium phosphate,

[0081] Based on 1 mole of ammonium derived from the above ammonium sulfate,

[0082] The ammonium derived from the above ammonium phosphate is 0.01 mol to 0.5 mol;

[0083] Contains ammonium sulfate, ammonium succinate and ammonium phosphate,

[0084] Based on 1 mole of ammonium derived from the above ammonium sulfate,

[0085] The ammonium derived from the above ammonium succinate is 0.05 mol to 1.2 mol,

[0086] The ammonium derived from the above ammonium phosphate is 0.01 mol to 0.5 mol;

[0087] Contains ammonium sulfate, ammonium succinate, and ammonium acetate,

[0088] Based on 1 mole of ammonium derived from the above ammonium sulfate,

[0089] The ammonium derived from the above ammonium succinate is 0.05 mol to 1.2 mol,

[0090] The ammonium derived from the above ammonium acetate is 0.05 mol to 1 mol;

[0091] Contains ammonium sulfate, ammonium phosphate, and ammonium acetate,

[0092] Based on 1 mole of ammonium derived from the above ammonium sulfate,

[0093] The ammonium derived from the above ammonium phosphate is 0.01 mol to 0.5 mol,

[0094] The ammonium derived from the ammonium acetate is 0.05 mol to 1 mol; and

[0095] Contains ammonium sulfate, ammonium succinate, ammonium phosphate and ammonium acetate,

[0096] Based on 1 mole of ammonium derived from the above ammonium sulfate,

[0097] The ammonium derived from the above ammonium succinate is 0.05 mol to 1.2 mol,

[0098] The ammonium derived from the above ammonium phosphate is 0.01 mol to 0.5 mol,

[0099] The ammonium derived from the above ammonium acetate is 0.05 mol to 1 mol.

[0100] At this time, the content (mol) of ammonium derived from ammonium succinate, ammonium derived from ammonium phosphate, and ammonium derived from ammonium acetate based on 1 mol of ammonium derived from ammonium sulfate is as described above.

[0101] In the present application, the seed culture medium may refer to a medium for growing microorganisms to secure a sufficient number of microorganisms for producing a target substance (e.g., a basic amino acid such as L-lysine, L-arginine, and / or L-histidine). The main culture medium may refer to a medium for producing a target substance (e.g., a basic amino acid such as L-lysine, L-arginine, and / or L-histidine). The culture medium composition of the present application may be used as a seed culture medium, a main culture medium, or both. In addition, the 'other ammonium-containing compound' described above may be included in the seed culture medium, the main culture medium, or both. The components of the seed culture medium and the main culture medium may be the same or different, and the specific components are as described above.

[0102] In one example, the culture medium composition of the present application may have a reduced sulfate content at the end of culture compared to a culture medium that does not include one or more ammonium-containing compounds selected from the group consisting of ammonium succinate, ammonium phosphate, and ammonium acetate, more specifically, a culture medium that includes less than 0.05 mol of ammonium derived from the 'other ammonium-containing compound' based on 1 mol of ammonium derived from ammonium sulfate, or a culture medium that includes less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 6.5%, or less than 7% of one or more of the aforementioned [Formula 1] to [Formula 4] (including a case where the 'other ammonium-containing compound' is not included).

[0103] In one example, the culture medium composition of the present application may be a seed culture medium composition, and the seed culture medium composition may include the 'other ammonium-containing compound' described above, so that compared to a culture medium containing less than 0.05 mole of ammonium derived from the 'other ammonium-containing compound' based on 1 mole of ammonium derived from ammonium sulfate or a culture medium containing less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 6.5%, or less than 7% of at least one of the above-described [Formula 1] to [Formula 4] (including a case where the 'other ammonium-containing compound' is not included), the cell number and specific growth rate at the end of seed culture (e.g., at the time of depletion of residual sugar in the medium) are maintained at the same level, while the sulfate concentration (g / L) in the medium is reduced by 10% or more, for example, 10 to 50%, 10 to 40%, 10 to 35%, 10 It can be reduced by 30%, or 10 to 20% (see Example 1 and Table 1).

[0104] In addition, the present application may be a seed culture medium composition and / or a main culture medium composition, and the seed culture medium composition and / or the main culture medium composition includes the 'other ammonium-containing compound' described above, so that compared to a culture medium that does not include one or more ammonium-containing compounds selected from the group consisting of ammonium succinate, ammonium phosphate, and ammonium acetate, more specifically, a culture medium that includes less than 0.05 mol of ammonium derived from the 'other ammonium-containing compound' based on 1 mol of ammonium sulfate derived, or a culture medium in which at least one of the above-described [Formula 1] to [Formula 4] is less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 6.5%, or less than 7% (including a case where the 'other ammonium-containing compound' is not included), the sulfate concentration (g / L) in the medium at the end of the main culture is reduced by 5% or more, for example, by 5 to The purity of basic amino acids (e.g., L-lysine, L-arginine, and / or L-histidine) in the medium may be reduced by 60%, 5 to 55%, 5 to 50%, 5 to 55%, or 5 to 40%, and / or increased by about 1% or more, about 1.5% or more, about 2% or more, about 3% or more, or about 5% or more (the upper limit is not particularly limited and may be, for example, about 30% or less, about 20% or less, about 10% or less, about 8% or less, or about 7% or less) (see Example 2 and Table 2).

[0105] In the present application, the microorganism of the genus Corynebacterium (Corynebacterium sp. or the genus Corynebacterium) may be a microorganism that produces or is capable of producing a target substance (e.g., a basic amino acid such as L-lysine, L-arginine, and / or L-histidine) by fermenting a substrate in a medium, and may include not only a microorganism (wild type) that originally has the ability to produce the target substance, but also a microorganism that has been modified to have the ability to produce the target substance, and / or a microorganism that has been modified to have the ability to produce the target substance enhanced.

[0106] In one example, the Corynebacterium genus microorganism is Corynebacterium glutamicum, Corynebacterium stationis, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium It may be Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens, and more specifically, it may be Corynebacterium glutamicum or Corynebacterium stationis, but is not limited thereto.

[0107] In a specific example, the microorganism of the genus Corynebacterium may be, but is not limited to, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14067, Corynebacterium glutamicum ATCC13869, or a strain mutated to produce the desired product using these as a parent strain (e.g., Corynebacterium glutamicum CJ3P (US registered patent US9556463 ​​B2), Corynebacterium glutamicum KCCM11016P).

[0108] In one example, the above Corynebacterium sp. microorganism culture medium composition may be a culture medium composition that can be used for culturing a Corynebacterium sp. microorganism and / or for producing a basic amino acid by the Corynebacterium sp. microorganism. The culture medium composition may be used as a main culture medium composition for the main culture that produces the basic amino acid as a target substance, a seed culture medium composition for seed culture (securing sufficient microbial proliferation or sufficient microbial cell number for producing the basic amino acid), or both. Accordingly, the culture medium composition of the present application may be a seed culture medium composition for producing a basic amino acid or a main culture medium composition for producing a basic amino acid.

[0109]

[0110] Method for producing basic amino acids

[0111] Another example of the present application provides a method for producing a basic amino acid, comprising the step of culturing a Corynebacterium sp. microorganism in the above-described Corynebacterium sp. microorganism culture medium composition.

[0112] More specifically, the above basic amino acid production method is,

[0113] A step of preparing the culture medium composition of the present application described above by adding ammonium sulfate and other ammonium-containing compounds (e.g., at least one selected from the group consisting of ammonium succinate, ammonium phosphate, and ammonium acetate); and

[0114] A step of culturing microorganisms of the genus Corynebacterium in the above medium

[0115] may include.

[0116] The above Corynebacterium sp. microbial culture medium composition (seed culture medium composition, main culture medium composition), the basic amino acid, the other ammonium-containing compound, etc. are as described above.

[0117] In the method for producing basic amino acids of the present application, the “cultivating step” may be performed by performing the proliferation of microorganisms of the genus Corynebacterium and the production of basic amino acids in a single culturing step, or may be performed by dividing into seed culture for the proliferation of microorganisms of the genus Corynebacterium and main culture for the production of basic amino acids.

[0118] In one example, when the culturing step is performed as one culturing step, the method for producing a basic amino acid may include a step of culturing a microorganism of the genus Corynebacterium in the seed culture medium composition or main culture medium composition described above.

[0119] In another example, the culturing step may include a seed culture step for proliferating a Corynebacterium genus microorganism and a main culture step for producing a basic amino acid. In this case, the method for producing a basic amino acid may include a seed culture step for culturing a Corynebacterium genus microorganism in a seed culture medium, and a main culture step for culturing the microorganism cultured in the seed culture step in a main culture medium.

[0120] In the method for producing a basic amino acid, the culturing step (seed culture or main culture) can be interpreted to mean a step of inoculating the microorganism into a culture medium composition (seed culture medium composition or main culture medium composition) and a step of culturing the inoculated microorganism under conditions suitable for the proliferation (seed culture) or production (main culture) of the microorganism.

[0121] The above method for producing a basic amino acid may further include, after the culturing step (seed culture and / or main culture), a step of recovering, separating, or purifying a basic amino acid from a culture obtained in the culturing step.

[0122] The specific composition of the above-described Corynebacterium microbial culture medium composition (seed culture medium composition, main culture medium composition) is as described above.

[0123] In one example, the method for producing a basic amino acid may further include a step of adding the 'other ammonium-containing compound' described above to the seed culture medium composition and / or the main culture medium composition. In this case, the type and amount of the 'other ammonium-containing compound' added are as described above.

[0124] In this application, the term "cultivation" refers to the growth of microorganisms under appropriately controlled environmental conditions. The culturing process of this application can be performed using appropriate media and culture conditions known in the art. These culture conditions can be appropriately adjusted by those skilled in the art depending on the microorganism being cultured. Specifically, the culturing may be batch, continuous, and / or fed-batch, but is not limited thereto.

[0125] The above culture has no particular limitation on the method, and may be performed by, for example, batch culture, fed-batch culture, continuous culture, or a combination of two or more thereof, but is not limited thereto.

[0126] When the above-mentioned culture is divided into seed culture and main culture, the seed culture and main culture may be performed in the same or different manners, and for example, each may be performed in a manner independently selected from the culture methods described above. In one specific example, the seed culture may be performed in batch culture, and the main culture may be performed in fed-batch culture or continuous culture, but is not limited thereto.

[0127] In addition, when the above-mentioned culture is divided into seed culture and main culture, the seed culture can be performed using the seed culture medium composition of the present application described above, and the main culture can be performed using the main culture medium composition of the present application described above. The ammonium sulfate, ammonium succinate, ammonium phosphate, and ammonium acetate, their contents, components, basic amino acid production, increase in basic amino acid production (production amount), increase in basic amino acid purity, etc. of the seed culture medium composition and main culture medium composition of the present application are as described in other examples above.

[0128] The above seed culture may refer to a culture whose main purpose is to proliferate microorganisms until the initial microbial concentration for the main culture is sufficiently reached. In one specific example, the seed culture may be, for example, batch culture, and may be performed under the conditions of pH 6 to 9 or pH 6.5 to 8.5, agitation speed of 350 to 550 rpm, 400 to 500 rpm, or about 450 rpm, and aeration amount of 0.1 to 2 vvm, 0.5 to 1.5 vvm, or about 1 vvm until the initial carbon source of the medium is exhausted, but is not limited thereto. The pH may be adjusted to the above range by adding ammonia gas, but is not limited thereto. The above seed culture may refer to inoculating microorganisms into the seed culture medium composition described above and culturing them under the above conditions.

[0129] The above main culture may refer to a process of culturing microorganisms grown in the above seed culture under conditions suitable for producing a target substance (e.g., a basic amino acid such as L-lysine, L-arginine, and / or L-histidine) so that the target substance is mainly produced from the microorganism (can be used interchangeably with “fermentation”), and during this process, microbial growth may not occur or may occur to a small extent. In one specific example, the main culture may be, for example, a fed-batch culture, and is performed under conditions of pH 6 to 9 or pH 6.5 to 8.5, a stirring speed of 350 to 550 rpm, 400 to 500 rpm, or about 450 rpm, and an aeration amount of 0.1 to 2 vvm, 0.5 to 1.5 vvm, or about 1 vvm, and the pH may be adjusted to the above range by introducing ammonia gas, but is not limited thereto. At this time, as the culture progresses, a feeding medium may be additionally added to the main culture medium. More specifically, when the initial carbon source in the medium is depleted, a carbon source (e.g., glucose, etc.) is additionally added to maintain the residual sugar level in the medium at about 0.1 to 2 wt%, 0.1 to 1 wt%, 0.5 to 2 wt%, or 0.5 to 1 wt%, and when the nitrogen (AN: ammonium nitrogen) content in the medium is less than about 2 g / kg, less than about 1.5 g / kg, less than about 1 g / kg, or less than about 0.5 g / kg, ammonia gas may be added to supply a nitrogen source while performing the culture, but is not limited thereto.

[0130] In the present application, the pH of the culture medium composition can be maintained at 6 to 9 in the culturing step. More specifically, the pH of the culture medium composition can be maintained at 6 to 9, 6 to 8.75, 6 to 8.5, 6.25 to 9, 6.25 to 8.75, 6.25 to 8.5, 6.5 to 9, 6.5 to 8.75, or 6.5 to 8.5, but is not limited thereto. In one example, ammonia gas and / or an ammonia solution can be added to the culture medium composition to maintain the pH range of the culture medium composition in the culturing step. By maintaining the pH range as described above, a metabolic environment favorable for the growth of Corynebacterium microorganisms and the production of target products (e.g., basic amino acids such as L-lysine, L-arginine, and / or L-histidine) can be provided.

[0131] In one example, in a method for producing a basic amino acid comprising a step of culturing a microorganism of the genus Corynebacterium in the culture medium composition of the present application described above, the culture medium composition may be a seed culture medium composition.

[0132] In one specific example, the method for producing a basic amino acid may additionally include a step of culturing the seed culture medium composition containing the Corynebacterium genus microorganism by adding the seed culture medium composition to the main culture medium.

[0133] In one specific example, the seed culture medium composition including the Corynebacterium genus microorganism may be added at 0.1 L to 0.4 L per 1 L of the main culture medium, and more specifically, at 0.1 L to 0.4 L, 0.1 L to 0.3 L, 0.1 L to 0.25 L, 0.1 L to 0.225 L, 0.1 L to 0.2 L, 0.15 L to 0.4 L, 0.15 L to 0.3 L, 0.15 L to 0.25 L, 0.15 L to 0.225 L, 0.15 L to 0.2 L, 0.175 L to 0.4 L, 0.175 L to 0.3 L, 0.175 L to 0.25 L, 0.175 L to The volume may be 0.225 L, 0.175 L to 0.2 L, 0.2 L to 0.4 L, 0.2 L to 0.3 L, 0.2 L to 0.25 L, or 0.2 L to 0.225 L, but is not limited thereto.

[0134] In one specific example, during the step of culturing by adding to the main culture medium, a feeding medium may be additionally added, but is not limited thereto. More specifically, as the main culture progresses and the initial carbon source in the medium is depleted, a carbon source (e.g., glucose, etc.) may be additionally added to maintain the residual sugar level in the medium at about 0.1 to 2 wt%, 0.1 to 1 wt%, 0.5 to 2 wt%, or 0.5 to 1 wt%, but is not limited thereto.

[0135] The production of basic amino acids by the above production method can be confirmed by known techniques commonly used for the detection and / or identification of compounds (e.g., L-lysine, L-arginine, and / or L-histidine). Such techniques include, but are not limited to, one or more selected from the group consisting of HPLC, LC / MS, GC / MS, NMR, etc.

[0136] The recovery of basic amino acids from the above culture (fermentation; which may include cells or cell lysates and / or cultured media) can be performed by known methods commonly used for the separation and / or purification of compounds (e.g., L-lysine, L-arginine, and / or L-histidine). Examples of such methods include, but are not limited to, one or more selected from the group consisting of various chromatographies such as precipitation, membrane separation, filtration (e.g., ultrafiltration), centrifugation, dialysis, salting out, gel filtration chromatography, adsorption chromatography, ion exchange chromatography, and affinity chromatography, etc. When basic amino acids accumulate in microbial cells, for example, the cells are disrupted by ultrasonication or the like, and the cells are removed by centrifugation, and the basic amino acids can be recovered from the supernatant obtained by, but are not limited to, the ion exchange resin method.

[0137] The basic amino acid to be recovered may be a free amino acid and / or a salt thereof. Examples of the salt include, but are not limited to, sulfate, hydrochloride, carbonate, ammonium salt, sodium salt, and potassium salt.

[0138] The basic amino acid production method of the present application may additionally include a purification step. The purification may be performed using any suitable method known in the art. In one example, if the basic amino acid production method of the present application includes both a recovery step and a purification step, the recovery step and the purification step may be performed sequentially or discontinuously, regardless of order, or may be performed simultaneously or integrated into a single step, but is not limited thereto.

[0139] In one example, the method for producing a basic amino acid of the present application can reduce the sulfate content at the end of the culture by using the culture medium composition of the present application described above, that is, by using the culture medium composition including the 'other ammonium-containing compound' described above, compared to a case where a culture medium does not include one or more ammonium-containing compounds selected from the group consisting of ammonium succinate, ammonium phosphate, and ammonium acetate, more specifically, a culture medium including less than 0.05 mol of ammonium derived from the 'other ammonium-containing compound' based on 1 mol of ammonium sulfate derived, or a culture medium including less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 6.5%, or less than 7% of one or more of [Formula 1] to [Formula 4] described above (including a case where the 'other ammonium-containing compound' is not included) is used.

[0140] More specifically, the method for producing a basic amino acid of the present application uses the seed culture medium composition of the present application described above in the seed culture step, that is, uses the seed culture medium composition containing the 'other ammonium-containing compound' described above, so that, compared to the case of using a culture medium that does not contain one or more ammonium-containing compounds selected from the group consisting of ammonium succinate, ammonium phosphate, and ammonium acetate, more specifically, a culture medium containing less than 0.05 mol of the 'other ammonium-containing compound' derived ammonium based on 1 mol of ammonium sulfate derived ammonium, or a culture medium containing less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 6.5%, or less than 7% of one or more of the above-described [Formula 1] to [Formula 4] (including a case of not containing the 'other ammonium-containing compound'), at the end of the seed culture (e.g., at the time of depletion of residual sugar in the medium or when the residual sugar concentration in the medium is about The cell number and specific growth rate at the point of 1% (w / v) or less can be maintained at the same level, while the sulfate concentration (g / L) in the medium can be reduced by 10% or more, for example, 10 to 50%, 10 to 40%, 10 to 35%, 10 to 30%, or 10 to 20% (see Example 1 and Table 1).

[0141] In addition, the method for producing a basic amino acid of the present application uses the seed culture medium composition of the present application described above and / or the main culture medium composition of the present application described above in the seed culture step and / or the main culture step, that is, by using the seed culture medium composition and / or the main culture medium composition containing the 'other ammonium-containing compound' described above, a culture medium that does not contain at least one ammonium-containing compound selected from the group consisting of ammonium succinate, ammonium phosphate, and ammonium acetate, more specifically, a culture medium containing less than 0.05 mol of ammonium derived from the 'other ammonium-containing compound' based on 1 mol of ammonium derived from ammonium sulfate, or a culture medium containing less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 6.5%, or less than 7% of one or more of the [Formula 1] to [Formula 4] described above (including a case where the 'other ammonium-containing compound' is not included) Compared to the case, the sulfate concentration (g / L) in the medium at the end of the main culture may be reduced by 5% or more, for example, 5 to 60%, 5 to 55%, 5 to 50%, 5 to 55%, or 5 to 40% (see Example 2 and Table 2).

[0142] In one example, the method for producing a basic amino acid of the present application uses the culture medium composition of the present application described above, that is, uses a culture medium composition containing the 'other ammonium-containing compound' described above, and, compared to a case where a culture medium does not contain one or more ammonium-containing compounds selected from the group consisting of ammonium succinate, ammonium phosphate, and ammonium acetate, a culture medium containing less than 0.05 mol of ammonium derived from the 'other ammonium-containing compound' based on 1 mol of ammonium sulfate derived ammonium, or a culture medium containing less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 6.5%, or less than 7% of one or more of the above-described [Formula 1] to [Formula 4] (including a case where the 'other ammonium-containing compound' is not included) is used, the production of a basic amino acid (e.g., L-lysine, L-arginine, and / or L-histidine) at the end of the culture is Purity can be increased.

[0143] More specifically, the basic amino acid production method of the present application uses the seed culture medium composition of the present application described above and / or the main culture medium composition of the present application described above in the seed culture step and / or the main culture step, that is, by using the seed culture medium composition and / or the main culture medium containing the 'other ammonium-containing compound' described above, a culture medium that does not contain at least one ammonium-containing compound selected from the group consisting of ammonium succinate, ammonium phosphate, and ammonium acetate, more specifically, a culture medium containing less than 0.05 mol of the ammonium derived from the 'other ammonium-containing compound' based on 1 mol of ammonium derived from ammonium sulfate, or a culture medium containing less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 6.5%, or less than 7% of one or more of the [Formula 1] to [Formula 4] described above (including a case where the 'other ammonium-containing compound' is not included) Compared to the case, the purity of basic amino acids (e.g., L-lysine, L-arginine, and / or L-histidine) in the medium at the end of the main culture may increase by about 1% or more, about 1.5% or more, about 2% or more, about 3% or more, or about 5% or more (the upper limit is not particularly limited and may be, for example, about 30% or less, about 20% or less, about 10% or less, about 8% or less, or about 7% or less) (see Example 2 and Table 2).

[0144]

[0145] Culture containing basic amino acids

[0146] Another example is,

[0147] A basic amino acid-containing culture is provided, comprising a Corynebacterium sp. microorganism and a Corynebacterium sp. microorganism culture medium composition of the present application described above.

[0148] The above-mentioned Corynebacterium genus microorganism and the above-mentioned basic amino acid are as described above.

[0149] The above Corynebacterium genus microbial culture medium composition can be used as a seed culture medium, a main culture medium, or both, and the above Corynebacterium genus microbial culture medium composition, seed culture medium, and main culture medium are as described above.

[0150] In the above basic amino acid-containing culture, the seed culture medium and / or main culture medium used in the seed culture step and / or main culture step includes the 'other ammonium-containing compound', so that the sulfate concentration (g / L) in the culture (medium) in which the main culture has been completed is reduced by 5% or more, compared to a case where a culture medium that does not include one or more ammonium-containing compounds selected from the group consisting of ammonium succinate, ammonium phosphate, and ammonium acetate, more specifically, a culture medium that includes less than 0.05 mol of ammonium derived from the 'other ammonium-containing compound' based on 1 mol of ammonium derived from ammonium sulfate, or a culture medium in which at least one of the above-described [Formula 1] to [Formula 4] is less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 6.5%, or less than 7% is used, for example, The purity of basic amino acids (e.g., L-lysine, L-arginine, and / or L-histidine) in the medium may be reduced by 5 to 60%, 5 to 55%, 5 to 50%, 5 to 55%, or 5 to 40%, and / or increased by about 1% or more, about 1.5% or more, about 2% or more, about 3% or more, or about 5% or more (the upper limit is not particularly limited and may be, for example, about 30% or less, about 20% or less, about 10% or less, about 8% or less, or about 7% or less) (see Example 2 and Table 2).

[0151] The above basic amino acid-containing culture may have one or more of the following characteristics:

[0152] (a) Compared to a case where the seed culture medium contains less than 0.05 of ammonium succinate, ammonium phosphate, and ammonium acetate derived ammonium based on 1 mol of ammonium derived from ammonium sulfate, the sulfate concentration (g / L) in the medium after the end of the seed culture is reduced by 10% or more;

[0153] (b) When the main culture medium contains less than 0.05 mol of ammonium succinate, ammonium phosphate, and ammonium acetate derived ammonium based on 1 mol of ammonium derived from ammonium sulfate, the sulfate concentration (g / L) in the medium decreases by 5% or more after the end of the main culture; and

[0154] (c) Compared to the case where the main culture medium contains less than 0.05 mol of ammonium succinate, ammonium phosphate, and ammonium acetate derived ammonium based on 1 mol of ammonium derived from ammonium sulfate, the basic amino acid purity in the medium increases by 1% or more after the end of the main culture.

[0155]

[0156] Method for preparing a composition of a culture medium for microorganisms of the genus Corynebacterium

[0157] Another example is, in a composition of a culture medium for a microorganism of the genus Corynebacterium,

[0158] A step of adjusting the content of ammonium sulfate and other ammonium-containing compounds (e.g., at least one selected from the group consisting of ammonium succinate, ammonium phosphate, and ammonium acetate) so that the value of the following formula 1 is 2% or more,

[0159] Provided is a method for preparing a culture medium composition for a microorganism of the genus Corynebacterium:

[0160] [Formula 1]

[0161] [(sum of moles (mol) of ammonium derived from ammonium succinate, ammonium phosphate, and ammonium acetate) / (sum of moles (mol) of ammonium derived from ammonium sulfate, ammonium succinate, ammonium phosphate, and ammonium acetate)]*100(%).

[0162] The medium manufactured by the above method is characterized in that the purity of basic amino acids in the medium increases by 1% or more after the completion of cultivation of a microorganism having basic amino acid production ability, compared to a medium having a value of less than 2% in the above formula 1, or a medium exceeding 2% to 70%.

[0163] In this regard, another example is a composition of a culture medium for a microorganism of the genus Corynebacterium,

[0164] A method for improving the basic amino acid purity of a microbial culture medium is provided, characterized in that a part of the ammonium sulfate is replaced with another ammonium-containing compound in a Corynebacterium sp. microbial culture medium, the method comprising the step of adjusting the content of ammonium sulfate and another ammonium-containing compound (e.g., at least one selected from the group consisting of ammonium succinate, ammonium phosphate, and ammonium acetate) so that the value of the above formula 1 is 2% or more.

[0165] The above-mentioned Corynebacterium genus microorganism and the above-mentioned basic amino acid are as described above.

[0166] The above Corynebacterium genus microbial culture medium composition can be used as a seed culture medium, a main culture medium, or both, and the above Corynebacterium genus microbial culture medium composition, seed culture medium, and main culture medium are as described above.

[0167]

[0168] For the production of basic amino acids

[0169] Other examples provide uses for producing at least one basic amino acid selected from the group consisting of ammonium sulfate, and ammonium succinate, ammonium phosphate, and ammonium acetate, increasing the production (amount) of a basic amino acid, increasing the purity of a basic amino acid, preparing a medium composition for culturing a microorganism of the genus Corynebacterium, and / or preparing a medium composition for producing a basic amino acid.

[0170] At least one selected from the group consisting of ammonium sulfate, ammonium succinate, ammonium phosphate, and ammonium acetate may have a value of 2% or more in the following [Formula 1]:

[0171] [Formula 1]

[0172] [(sum of moles (mol) of ammonium derived from ammonium succinate, ammonium phosphate, and ammonium acetate) / (sum of moles (mol) of ammonium derived from ammonium sulfate, ammonium succinate, ammonium phosphate, and ammonium acetate)]*100(%).

[0173] Other examples provide uses for producing basic amino acids, increasing basic amino acid production (production amount), increasing basic amino acid purity, producing a medium composition for culturing a Corynebacterium spp. microorganism, and / or producing a medium composition for producing basic amino acids, comprising a composition comprising at least one selected from the group consisting of ammonium sulfate, ammonium succinate, ammonium phosphate, and ammonium acetate.

[0174] The above composition may have a value of the following [Formula 1] of 2% or more:

[0175] [Formula 1]

[0176] [(sum of moles (mol) of ammonium derived from ammonium succinate, ammonium phosphate, and ammonium acetate) / (sum of moles (mol) of ammonium derived from ammonium sulfate, ammonium succinate, ammonium phosphate, and ammonium acetate)]*100(%).

[0177] In the purpose of the present application, the ammonium sulfate, ammonium succinate, ammonium phosphate, and ammonium acetate, their contents, the components of the seed culture medium and the main culture medium, production of basic amino acids, increase in production (production amount) of basic amino acids, increase in purity of basic amino acids, etc. are as described in other examples above.

[0178]

[0179] Another example provides a composition, method, product, process, or use featuring one or more elements disclosed in the present application.

[0180]

[0181] The purpose of this application is to significantly increase the stability of the fermentation process and improve the purity of the fermentation solution by introducing an ammonium substitute (an ammonium-containing compound containing an anion other than sulfate ion as an anionic counter ion) into the medium, compared to the conventional technology in which ammonium sulfate is introduced entirely into the medium, thereby improving product quality.

[0182]

[0183] The present application will be described in more detail below through examples. However, the following examples are merely preferred embodiments intended to illustrate the present application and are therefore not intended to limit the scope of the present application. Furthermore, technical details not described herein can be readily understood and implemented by those skilled in the technical field of the present application or similar fields.

[0184]

[0185] Reference example: Earning of seedlings

[0186] The culture of coryneform microbial strains with lysine production ability was carried out through solid plate medium and flask culture, and the seed culture stage was carried out in a 5 L fermenter, and the main culture stage was carried out in a 30 L fermenter.

[0187] Corynebacterium glutamicumCJ3P (US patent US9556463 ​​B2), stored in a -80°C deep-freezer in the form of glycerol stock (GS), was taken in an amount of 50 mL and inoculated onto a solid plate medium prepared based on the following medium composition by streaking, and cultured in a 30°C incubator for approximately 24 hours.

[0188] After that, to perform flask shaking culture, 100 mL of liquid medium prepared based on the flask shaking culture medium composition below was prepared, placed in a 500 mL shake flask containing a baffle, and sterilized using a small autoclave at 121°C for 30 minutes. To perform shake flask inoculation, work was performed on a clean bench. Colonies were appropriately collected from the cultured solid plate using a disposable platinum loop (10 mL), and then aseptically inoculated into the prepared shake culture medium. The inoculated 500 mL shake flask was cultured at 200 rpm and 30°C for approximately 10 hours.

[0189] For the seed culture step, the culture solution in the shake flask cultured at 30°C as described above was inoculated at a ratio of 3.0% (v / v) into a 5 L fermenter containing a medium prepared based on the following seed culture medium composition. The culture was performed for approximately 20 hours under the conditions of pH 6.5-8.5, stirring speed 800 rpm, and aeration volume 1 vvm in batch fermentation mode.

[0190]

[0191] 1) Composition of solid plate medium

[0192] Glucose 10.0 g / L, bacto tryptone 5.0 g / L, bacto yeast extract 5.0 g / L, NaCl 2.5 g / L, urea 2.0 g / L

[0193]

[0194] 2) Composition of flask shaking culture medium

[0195] Glucose 10.0 g / L, bacto tryptone 5.0 g / L, bacto yeast extract 5.0 g / L, ammonium sulfate 10.0 g / L, urea 2.0 g / L, KH2PO4 5.0 g / L, K2HPO4 10.0 g / L, MgSO4 7H2O 0.5 g / L

[0196]

[0197] 3) Fermentation tank (5 L) seed culture medium composition

[0198] Glucose 50.00 g / L, antifoaming agent (fatty acid ester series) 1 mL / L, corn steep liquor 10.0 g / L, biotin 1.0 mg / L, thiamine 10.0 mg / L, pantothenic acid 10.0 mg / L, niacinamide 10.0 mg / L

[0199]

[0200] In the examples below, the term “ammonium sulfate substitute” means an ammonium-containing compound that includes an anion other than sulfate ion as an anionic counterion.

[0201]

[0202] Example 1: Addition of ammonium sulfate substitute to fermentation medium in seed culture stage and subsequent fermentation performance

[0203] Example 1-1: Application of ammonium acetate at the seed culture stage

[0204] In the “Fermenter (5 L) seed culture medium composition” described in the above reference example, a 5 L fermenter containing a medium to which 12.75 g / L of ammonium sulfate, 4.96 g / L of ammonium acetate, and 0.86 g / L of phosphate were added was inoculated with the seed culture obtained from the flask shaking culture of the above reference example at a ratio of 3.0% (v / v). In this example, a part of the ammonium sulfate added to the control medium of Comparative Example 1 was replaced with ammonium acetate, and the ammonium sulfate replacement rate was 25.0%, and its calculation formula is as follows (weight ratio of nitrogen per 1 g of ammonium sulfate: 0.212 g; weight of nitrogen per 1 g of ammonium acetate (ammonium substitute): 0.182 g).

[0205] [Formula 1]

[0206] Ammonium sulfate replacement rate (%) = [(sum of moles (mol) of ammonium derived from ammonium succinate, ammonium phosphate, and ammonium acetate) / (sum of moles (mol) of ammonium derived from ammonium sulfate, ammonium succinate, ammonium phosphate, and ammonium acetate)]*100(%).

[0207] In batch fermentation mode, the culture was incubated for 17.1 hours under conditions of pH 6.5–8.5, agitation speed 450 rpm, and aeration volume 1 vvm. When necessary, the pH was maintained within an appropriate range by adding ammonia gas. Samples were obtained at approximately 2–3 hour intervals, and the culture was terminated when the residual sugar was depleted.

[0208] At the end of culture, the cell number (OD) was 26.1, the specific growth rate ( / h) was 0.150, and the residual sulfate concentration was 78.0% of the results of the control medium (Comparative Example 1). However, the cell number was somewhat low at 58.4%, confirming that cell activity was reduced. (See Table 1).

[0209]

[0210] Example 1-2: Application of ammonium acetate and ammonium succinate at the seed culture stage - 1

[0211] A 5 L fermenter containing a medium containing 12.75 g / L of ammonium sulfate, 3.31 g / L of ammonium acetate, 1.63 g / L of ammonium succinate, and 0.86 g / L of phosphate added to the “Fermenter (5 L) seed culture medium composition” described in the above reference example was inoculated with the seed culture solution obtained from the flask shaking culture of the above reference example at a ratio of 3.0% (v / v). In this example, part of the ammonium sulfate added to the control medium of Comparative Example 1 was replaced with ammonium acetate and ammonium succinate, and the ammonium sulfate replacement rate was 25.0% (for the calculation formula for the ammonium sulfate replacement rate, see Example 1-1; weight ratio of nitrogen per 1 g of ammonium sulfate: 0.212 g; weight of nitrogen per 1 g of ammonium acetate (ammonium substitute): 0.182 g; weight of nitrogen per 1 g of ammonium succinate (ammonium substitute): 0.184 g).

[0212] In batch fermentation mode, the culture was performed for approximately 16.0 hours under conditions of pH 6.5–8.5, agitation speed 450 rpm, and aeration volume 1 vvm. If necessary, the pH was maintained within an appropriate range by adding ammonia gas. Samples were obtained at approximately 2–3 hour intervals, and the culture was terminated when the residual sugar was depleted.

[0213] At the end of culture, the cell number (OD) was 40.8, the specific growth rate ( / h) was 0.170, and the residual sulfate concentration was confirmed to have decreased to 77.6% compared to the control medium (Comparative Example 1). The cell number of Example 1-1 was 58.4% of that of Comparative Example 1, which showed a significant decrease in activity, whereas the cell number of Example 1-2 was 91.3% of that of Comparative Example 1, showing a 32.9%p improvement compared to Example 1-1. In addition, the specific growth rate was found to have improved by 12.9% compared to Example 1-1. This suggests the possibility of improving the cell number and specific growth rate when the amount of ammonium acetate input is reduced compared to that input in Example 1-1, and shows that even if ammonium sulfate is partially replaced with ammonium acetate and ammonium succinate, culture indices equivalent to or higher can be confirmed. (See Table 1).

[0214]

[0215] Example 1-3: Application of ammonium acetate and ammonium succinate at the seed culture stage - 2

[0216] A 5 L fermenter containing a medium containing 12.75 g / L of ammonium sulfate, 1.65 g / L of ammonium acetate, 3.26 g / L of ammonium succinate, and 0.86 g / L of phosphate added to the “Fermenter (5 L) seed culture medium composition” described in the above reference example was inoculated with the seed culture solution obtained from the flask shaking culture of the above reference example at a ratio of 3.0% (v / v). In this example, part of the ammonium sulfate added to the control medium of Comparative Example 1 was replaced with ammonium acetate and ammonium succinate, and the ammonium sulfate replacement rate was 25.0% (for the calculation formula for the ammonium sulfate replacement rate, see Example 1-1; weight ratio of nitrogen per 1 g of ammonium sulfate: 0.212 g; weight of nitrogen per 1 g of ammonium acetate (ammonium substitute): 0.182 g; weight of nitrogen per 1 g of ammonium succinate (ammonium substitute): 0.184 g).

[0217] In batch fermentation mode, the culture was incubated for 16.3 hours under conditions of pH 6.5–8.5, agitation speed 450 rpm, and aeration volume 1 vvm. When necessary, the pH was maintained within an appropriate range by adding ammonia gas. Samples were obtained at approximately 2–3 hour intervals, and the culture was terminated when the residual sugar was depleted.

[0218] At the end of culture, the cell number (OD) was 41.3, the specific growth rate ( / h) was 0.179, and the residual sulfate concentration was confirmed to have decreased to 78.1% compared to the control medium (Comparative Example 1). The cell number of Example 1-1 was 58.4% of that of Comparative Example 1, which showed a significant decrease in activity, whereas the cell number of Example 1-3 was 92.4% of that of Comparative Example 1, showing an improvement of 34.0%p compared to Example 1-1 and 1.1%p compared to Example 1-2. In addition, the specific growth rate was found to have improved by 19.2% compared to Example 1-1 and by 5.6% compared to Example 1-2. This suggests that there is a gradual improvement effect in cell number and specific growth rate when the amount of ammonium acetate gradually decreases compared to that introduced in Example 1-1, and it shows that even when some of the ammonium sulfate is replaced with ammonium acetate and ammonium succinate, culture indices equivalent to or higher can be confirmed. (See Table 1).

[0219]

[0220] Example 1-4: Application of ammonium succinate at the seed culture stage

[0221] In the “Fermenter (5 L) Seed Culture Medium Composition” described in the above Reference Example, 12.80 g / L of ammonium sulfate, 4.89 g / L of ammonium succinate, and 0.86 g / L of phosphate were added to a 5 L fermenter containing the medium, and the seed culture obtained from the flask shaking culture of the above Reference Example was inoculated at a ratio of 3.0% (v / v). In this example, a portion of the ammonium sulfate added to the control medium of Comparative Example 1 was replaced with ammonium succinate, and the ammonium sulfate replacement rate was 24.9% (see Example 1-1 for the calculation formula for the ammonium sulfate replacement rate; nitrogen weight ratio per 1 g of ammonium sulfate: 0.212 g; nitrogen weight per 1 g of ammonium succinate (ammonium substitute): 0.184 g).

[0222] In batch fermentation mode, the culture was performed for 17.0 h under conditions of pH 6.5–8.5, agitation speed 450 rpm, and aeration volume 1 vvm. When necessary, the pH was maintained within an appropriate range by adding ammonia gas. Samples were obtained at approximately 2–3 h intervals, and the culture was terminated when the residual sugar was depleted.

[0223] At the end of culture, the cell number (OD) was 44.8, the specific growth rate ( / h) was 0.183, and the residual sulfate concentration was confirmed to have decreased to 76.7% compared to the results of the control medium (Comparative Example 1). The cell number and specific growth rate were confirmed to be equivalent or higher, and even when some of the ammonium sulfate was replaced with ammonium succinate, the culture index was confirmed to be equivalent or higher (see Table 1).

[0224]

[0225] Example 1-5: Application of ammonium phosphate at the seed culture stage

[0226] In the “5 L fermentation tank seed culture medium composition” described in the above reference example, 15.70 g / L of ammonium sulfate and 1.31 g / L of ammonium phosphate were added to a 5 L fermentation tank, and the seed culture obtained from the flask shaking culture of the above reference example was inoculated at a ratio of 3.0% (v / v). In this example, part of the ammonium sulfate added to the control medium of Comparative Example 1 was replaced with ammonium phosphate, and the ammonium sulfate replacement rate was 7.7% (see Example 1-1 for the calculation formula for the ammonium sulfate replacement rate; nitrogen weight ratio per 1 g of ammonium sulfate: 0.212 g; nitrogen weight per 1 g of ammonium phosphate (ammonium substitute): 0.212 g).

[0227] In batch fermentation mode, the culture was incubated for 16.6 hours under conditions of pH 6.5–8.5, agitation speed 450 rpm, and aeration volume 1 vvm. When necessary, the pH was maintained within an appropriate range by adding ammonia gas. Samples were obtained at approximately 2–3 hour intervals, and the culture was terminated when the residual sugar was depleted.

[0228] At the end of culture, the cell number (OD) was 46.2, the specific growth rate ( / h) was 0.186, and the residual sulfate concentration was confirmed to have decreased by 86.0% compared to the results of Comparative Example 1. The cell number and specific growth rate were confirmed to be equivalent or higher, and even when some of the ammonium sulfate was replaced with ammonium phosphate, the culture index was confirmed to be equivalent or higher (see Table 1).

[0229]

[0230] Comparative Example 1: Fermentation with ammonium sulfate added to the seed culture stage medium (ammonium sulfate replacement rate 0%)

[0231] A 5 L fermenter containing a medium containing 17.0 g / L ammonium sulfate and 0.86 g / L phosphate added to the “Fermenter (5 L) Seed Culture Medium Composition” described in the above Reference Example was inoculated with the seed culture obtained from the flask shaking culture of the above Reference Example at a ratio of 3.0% (v / v). The culture was performed for 17.1 hours under the conditions of pH 6.5-8.5, stirring speed 450 rpm, and aeration amount 1 vvm in batch fermentation mode. If necessary, the pH was maintained within an appropriate range by adding ammonia gas. Samples were obtained at approximately 2-3 hour intervals, and the culture was terminated when the residual sugar was depleted.

[0232] The cell count at the end of culture (OD) was 44.7, and the specific growth rate ( / h) was 0.179 (see Table 1).

[0233]

[0234] result

[0235] The results obtained in Examples 1-1 to 1-5 and Comparative Example 1 are shown in Table 1 below:

[0236] Item Unit Example 1-1 Example 1-2 Example 1-3 Example 1-4 Example 1-5 Comparative Example 1 Fermentation time hr 17.1 16.0 16.3 17.0 16.6 17.1 Number of cells OD 26.1 40.8 41.3 44.8 46.2 44.7 Specific growth rate / h 0.15 00.17 00.17 9 0.18 3 0.18 6 0.17 9 Residual sugar g / L 22.8 0.00.00.00.00.0 Sulfate*% 78.0 77.6 78.17 6.7 8 6.0 100.0

[0237] (* Normalization result value based on 100% of the result of Comparative Example 1;

[0238] Cell count: Measured by absorbance (OD) at 562 nm using a spectrophotometer;

[0239] Specific growth rate = (ln(dry weight at time tf)-ln(dry weight at time t0)) / (tf-t0) (tf: final time; t0: start time);

[0240] Dry weight: Calculated as cell count (OD*0.25=g / L)

[0241]

[0242] Summarizing the experimental results in Table 1 above,

[0243] In order to improve the purity of the final product, it is important to reduce the residual by-products in the fermentation liquid obtained in the main culture stage, but it is also essential to reduce the residual by-products in the fermentation liquid in the seed culture stage required for main culture inoculation.

[0244] In the results using ammonium acetate in Example 1-1, a 16.0% decrease in the specific growth rate and a 41.6% decrease in the cell number were confirmed compared to Comparative Example 1. Since the seed culture stage is ultimately aimed at activating seedlings and increasing the cell number within a short period of time, it was confirmed that application in the seed culture stage was not appropriate even though the residual sulfate was reduced by 22.0% in the seed culture process with a 25.0% replacement rate of ammonium sulfate using high-concentration ammonium acetate.

[0245] In the results of utilizing ammonium acetate and ammonium succinate in Example 1-2, the amount of ammonium acetate added in Example 1-1 was reduced and the amount of ammonium succinate added was increased, resulting in the same ammonium sulfate replacement rate of 25.0%, but compared to Comparative Example 1, a 5.2% decrease in specific growth rate and an 8.7% decrease in cell number were confirmed, suggesting the possibility of improving cell number and specific growth rate. Therefore, in the culture process utilizing ammonium acetate (66.6% input of Example 1-1) and ammonium succinate, a 22.4% decrease in residual sulfate and an equivalent level of culture index were confirmed, suggesting the possibility of application in the seed culture stage.

[0246] In the results of utilizing ammonium acetate and ammonium succinate in Example 1-3, the amount of ammonium acetate added in Example 1-2 was reduced and the amount of ammonium succinate added was increased, resulting in the same ammonium sulfate replacement rate of 25.0%, but a specific growth rate and a 7.6% decrease in cell number were confirmed at the same level as in Comparative Example 1, suggesting the possibility of improving cell number and specific growth rate. Therefore, in the culture process utilizing ammonium acetate (33.3% input in Example 1-1) and ammonium succinate, a 21.9% decrease in residual sulfate and a similar level of culture index were confirmed, suggesting the possibility of application in the seed culture stage.

[0247] The results of using ammonium succinate in Example 1-4 showed results equal to or better than those of Comparative Example 1 in terms of fermentation time, cell number, and specific growth rate, and the residual sulfate concentration was also reduced by 23.3%, confirming that ammonium succinate can be applied at the level of 24.9% of the ammonium sulfate replacement rate in the seed culture stage.

[0248] In the results using ammonium phosphate in Example 1-5, the fermentation time, cell number, and specific growth rate were comparable to or better than those in Comparative Example 1, and the residual sulfate concentration was also reduced by 14.0%, confirming that ammonium phosphate can be applied at a level of 7.7% of the ammonium sulfate replacement rate in the seed culture stage.

[0249]

[0250] Example 2: Combination of ammonium sulfate substitutes added to the fermentation medium in the seed culture or main culture stage and the resulting fermentation performance

[0251] Example 2-1: Application of a combination of ammonium sulfate substitutes to the fermentation medium in the seed culture stage - 1

[0252] The seed culture obtained from the flask shaking culture of the above reference example was inoculated at 3.0% (v / v) into a 5 L fermenter containing a medium in which 11.50 g / L of ammonium sulfate, 4.89 g / L of ammonium succinate, and 1.31 g / L of ammonium phosphate were added to the fermenter (5 L) seed culture medium of the above reference example (ammonium sulfate replacement rate 32.6%; see Example 1-1 for the calculation formula for the ammonium sulfate replacement rate; nitrogen weight ratio per 1 g of ammonium sulfate: 0.212 g, nitrogen weight per 1 g of ammonium succinate (ammonium substitute): 0.184 g, nitrogen weight per 1 g of ammonium phosphate (ammonium substitute): 0.212 g).

[0253] In batch fermentation mode, the culture was incubated for approximately 16 hours under conditions of pH 6.5–8.5, agitation speed 450 rpm, and aeration volume 1 vvm. If necessary, the pH was maintained within an appropriate range by adding ammonia gas. Samples were obtained approximately every 2–3 hours, and the culture was terminated when the residual sugar was depleted.

[0254] A 30 L fermenter containing a medium containing 13.00 g / L of ammonium sulfate and 0.86 g / L of phosphate added to the main culture medium composition below was inoculated with the seed culture solution obtained in the seed culture step at a ratio of 20% (v / v).

[0255]

[0256] Fermentation tank (30 L) main culture medium composition

[0257] Glucose 25.00 g / L, antifoam 1 mL / L, corn steep liquor 10.0 g / L, biotin 1.0 mg / L, thiamine 10.0 mg / L, pantothenic acid 10.0 mg / L, niacinamide 10.0 mg / L

[0258]

[0259] In this example, a portion of the ammonium sulfate added to the seed culture medium of Comparative Example 2 below was replaced with ammonium succinate and ammonium phosphate, and the ammonium sulfate replacement rate of the main culture medium was 0.0%. The total ammonium sulfate replacement rate of the seed culture medium and the main culture medium was 6.8%.

[0260] In the main cultivation stage of this example, culture was performed under the conditions of pH 6.5-8.5, stirring speed 450 rpm, and aeration volume 1 vvm in fed-batch fermentation mode. If necessary, the pH was maintained within an appropriate range by adding ammonia gas.

[0261] When the initial carbon source of the medium was exhausted, feeding medium containing 40.0% wt glucose was added to maintain the residual sugar at 0.5–1.0 wt%. Samples were obtained at intervals of approximately 2–3 hours, and the nitrogen content (AN: ammonium nitrogen, g / kg) was measured using a Kjeldahl analyzer (Foss Kjeltec 8400). If the value was less than 1.0 g / kg, the nitrogen source was additionally supplied by injecting ammonia gas.

[0262] At the end of the culture (at the point of sugar depletion), there was no residual sugar and the fermentation time was approximately 29.5 hours. The standardized residual sulfate concentration (standardized to the result of Comparative Example 2 (100%)) in the medium at the end of the culture and the standardized L-lysine purity obtained in the main culture stage were approximately 92.0% and 102.5%, respectively, indicating an 8.0% decrease in residual sulfate and a 2.5% improvement in purity compared to the result of Comparative Example 2 (see Table 2).

[0263]

[0264] Example 2-2: Application of a combination of ammonium sulfate substitutes to the fermentation medium in the seed culture stage - 2

[0265] The seed culture solution obtained from the flask shaking culture of the above reference example was inoculated at 3.0% (v / v) into a 5 L fermenter containing a medium (ammonium sulfate replacement rate 19.1%) in which 14.17 g / L of ammonium sulfate, 2.0 g / L of ammonium succinate, and 1.6 g / L of ammonium phosphate were added to the fermenter (5 L) seed culture medium of the above reference example.

[0266] In batch fermentation mode, the culture was incubated for approximately 17 hours under conditions of pH 6.5–8.5, agitation speed 450 rpm, and aeration volume 1 vvm. If necessary, the pH was maintained within an appropriate range by adding ammonia gas. Samples were obtained approximately every 2–3 hours, and the culture was terminated when the residual sugar was depleted.

[0267] A 30 L fermenter containing a medium containing 13.0 g / L of ammonium sulfate and 0.86 g / L of phosphate added to the main culture medium composition of Example 2-1 above was inoculated with the seed culture solution obtained in the seed culture step at a ratio of 20.0% (v / v). The ammonium sulfate replacement rate of the main culture medium was 0.0%, and the total ammonium sulfate replacement rate of the seed culture medium and the main culture medium was 4.0%.

[0268] In the main cultivation stage of this example, culture was performed under the conditions of pH 6.5-8.5, stirring speed 450 rpm, and aeration volume 1 vvm in fed-batch fermentation mode. If necessary, the pH was maintained within an appropriate range by adding ammonia gas.

[0269] When the initial carbon source of the medium was exhausted, feeding medium containing 40.0% wt glucose was added to maintain the residual sugar at 0.5–1.0 wt%. Samples were obtained at intervals of approximately 2–3 hours, and the nitrogen content (AN: ammonium nitrogen, g / kg) was measured using a Kjeldahl analyzer (Foss Kjeltec 8400). If the value was less than 1.0 g / kg, the nitrogen source was additionally supplied by injecting ammonia gas.

[0270] At the end of the cultivation, no residual sugar was present and the fermentation time was 29.0 hours. The standardized residual sulfate concentration derived from ammonium sulfate and the standardized purity obtained in the main cultivation stage were 94.0% and 102.1%, respectively, indicating a 6.0% decrease in residual sulfate and a 2.1% improvement in purity compared to the results of Comparative Example 2 (see Table 2).

[0271]

[0272] Example 2-3: Application of a combination of ammonium sulfate substitutes to the fermentation medium in the main culture stage.

[0273] The seed culture solution obtained from the flask shaking culture of the above reference example was inoculated at 3.0% (v / v) into a 5 L fermenter containing a medium (ammonium sulfate replacement rate 0%) in which 17 g / L of ammonium sulfate and 0.86 g / L of phosphate were added to the fermenter (5 L) seed culture medium of the above reference example.

[0274] In batch fermentation mode, the culture was incubated for approximately 17 hours under conditions of pH 6.5–8.5, agitation speed 450 rpm, and aeration volume 1 vvm. If necessary, the pH was maintained within an appropriate range by adding ammonia gas. Samples were obtained approximately every 2–3 hours, and the culture was terminated when the residual sugar was depleted.

[0275] A 30 L fermenter containing a medium containing 6.10 g / L of ammonium sulfate, 3.28 g / L of ammonium acetate, 3.24 g / L of ammonium succinate, and 1.31 g / L of ammonium phosphate added to the main culture medium composition of Example 2-1 above was inoculated with the seed culture solution obtained in the seed culture step at a ratio of 20.0% (v / v). In this example, a portion of the ammonium sulfate added to the main culture medium of Comparative Example 2 was replaced with ammonium acetate, ammonium succinate, and ammonium phosphate, and the ammonium sulfate replacement rate of the main culture medium was 53.2%, and the total ammonium sulfate replacement rate of the seed culture medium and the main culture medium was 42.2%.

[0276] In the main cultivation stage of this example, culture was performed under the conditions of pH 6.5-8.5, stirring speed 450 rpm, and aeration volume 1 vvm in fed-batch fermentation mode. If necessary, the pH was maintained within an appropriate range by adding ammonia gas.

[0277] When the initial carbon source of the medium was exhausted, feeding medium containing 40.0% wt glucose was added to maintain the residual sugar at 0.5–1.0 wt%. Samples were obtained at intervals of approximately 2–3 hours, and the nitrogen content (AN: ammonium nitrogen, g / kg) was measured using a Kjeldahl analyzer (Foss Kjeltec 8400). If the value was less than 1.0 g / kg, the nitrogen source was additionally supplied by injecting ammonia gas.

[0278] At the end of the cultivation, no residual sugar was present and the fermentation time was 30.1 hours. The standardized residual sulfate concentration derived from ammonium sulfate and the standardized purity obtained in the main cultivation stage were 72.0% and 102.9%, respectively, indicating a 28.0% decrease in residual sulfate and a 2.9% improvement in purity compared to the results of Comparative Example 2 (see Table 2).

[0279]

[0280] Example 2-4: Application of a combination of ammonium sulfate substitutes to the fermentation medium in the seed culture stage and main culture stage - 1

[0281] The seed culture solution obtained from the flask shaking culture of the above reference example was inoculated at a ratio of 3.0% (v / v) into a 5 L fermenter containing a medium (ammonium sulfate replacement rate: 32.6%) in which 11.50 g / L of ammonium sulfate, 4.89 g / L of ammonium succinate, and 1.31 g / L of ammonium phosphate were added to the fermenter (5 L) seed culture medium of the above reference example.

[0282] In batch fermentation mode, the culture was incubated for approximately 17 hours under conditions of pH 6.5–8.5, agitation speed 450 rpm, and aeration volume 1 vvm. If necessary, the pH was maintained within an appropriate range by adding ammonia gas. Samples were obtained approximately every 2–3 hours, and the culture was terminated when the residual sugar was depleted.

[0283] A 30 L fermenter containing a medium (ammonium sulfate replacement rate: 53.2%) in which 6.10 g / L of ammonium sulfate, 3.28 g / L of ammonium acetate, 3.24 g / L of ammonium succinate, and 1.31 g / L of ammonium phosphate were added to the main culture medium composition of Example 2-1 above was inoculated with the seed culture solution obtained in the seed culture step at a ratio of 20.0% (v / v).

[0284] In this example, part of the ammonium sulfate added to the seed culture medium of Comparative Example 2 was replaced with ammonium succinate and ammonium phosphate, and part of the ammonium sulfate added to the main culture medium of Comparative Example 2 was replaced with ammonium acetate, ammonium succinate, and ammonium phosphate, and the total ammonium sulfate replacement rate of the seed culture medium and the main culture medium was 48.9%.

[0285] In the main cultivation stage of this example, the culture was performed for approximately 30 hours under the conditions of pH 6.5-8.5, stirring speed 450 rpm, and aeration volume 1 vvm in fed-batch fermentation mode. If necessary, the pH was maintained within an appropriate range by adding ammonia gas.

[0286] When the initial carbon source of the medium was exhausted, feeding medium containing 40.0% wt glucose was added to maintain the residual sugar at 0.5–1.0 wt%. Samples were obtained at intervals of approximately 2–3 hours, and the nitrogen content (AN: ammonium nitrogen, g / kg) was measured using a Kjeldahl analyzer (Foss Kjeltec 8400). If the value was less than 1.0 g / kg, the nitrogen source was additionally supplied by adding ammonia gas.

[0287] At the end of the cultivation, there was no residual sugar and the fermentation time was 29.9 hours. The standardized residual sulfate concentration derived from ammonium sulfate and the standardized purity obtained in the main cultivation stage were 60.0% and 102.9%, respectively, confirming a 40.0% decrease in residual sulfate and a 2.9% improvement in purity compared to the results of Comparative Example 2 (see Table 2).

[0288]

[0289] Example 2-5: Application of a combination of ammonium sulfate substitutes to the fermentation medium in the seed culture stage and main culture stage - 2

[0290] The seed culture solution obtained from the flask shaking culture of the above reference example was inoculated at a ratio of 3.0% (v / v) into a 5 L fermenter containing a medium (ammonium sulfate replacement rate: 32.6%) in which 11.50 g / L of ammonium sulfate, 4.89 g / L of ammonium succinate, and 1.31 g / L of ammonium phosphate were added to the fermenter (5 L) seed culture medium of the above reference example.

[0291] In batch fermentation mode, the culture was incubated for approximately 17 hours under conditions of pH 6.5–8.5, agitation speed 450 rpm, and aeration volume 1 vvm. If necessary, the pH was maintained within an appropriate range by adding ammonia gas. Samples were obtained approximately every 2–3 hours, and the culture was terminated when the residual sugar was depleted.

[0292] A 30 L fermenter containing a medium (ammonium sulfate replacement rate: 69.3%) in which 3.80 g / L of ammonium sulfate, 3.28 g / L of ammonium acetate, 5.15 g / L of ammonium succinate, and 1.31 g / L of ammonium phosphate were added to the main culture medium composition of Example 2-1 above was inoculated with the seed culture solution obtained in the seed culture step at a ratio of 20.0% (v / v).

[0293] In this example, part of the ammonium sulfate added to the seed culture medium of Comparative Example 2 was replaced with ammonium succinate and ammonium phosphate, and part of the ammonium sulfate added to the main culture medium of Comparative Example 2 was replaced with ammonium acetate, ammonium succinate, and ammonium phosphate, and the total ammonium sulfate replacement rate of the seed culture medium and the main culture medium was 61.4%.

[0294] In the main cultivation stage of this example, the culture was performed for approximately 30 hours under the conditions of pH 6.5-8.5, stirring speed 450 rpm, and aeration volume 1 vvm in fed-batch fermentation mode. If necessary, the pH was maintained within an appropriate range by adding ammonia gas.

[0295] When the initial carbon source of the medium was exhausted, feeding medium containing 40.0% wt glucose was added to maintain the residual sugar at 0.5–1.0 wt%. Samples were obtained at intervals of approximately 2–3 hours, and the nitrogen content (AN: ammonium nitrogen, g / kg) was measured using a Kjeldahl analyzer (Foss Kjeltec 8400). If the value was less than 1.0 g / kg, the nitrogen source was additionally supplied by adding ammonia gas.

[0296] At the end of the cultivation, there was no residual sugar and the fermentation time was 29.1 hours. The standardized residual sulfate concentration derived from ammonium sulfate and the standardized purity obtained in the main cultivation stage were 48.0% and 105.4%, respectively, confirming a 52.0% decrease in residual sulfate and a 5.4% improvement in purity compared to the results of Comparative Example 2 (see Table 2).

[0297]

[0298] Example 2-6: Application of a combination of ammonium sulfate substitutes to the fermentation medium in the seed culture stage and main culture stage - 3

[0299] In the fermentation tank (5 L) seed culture medium composition of the above reference example, 11.50 g / L of ammonium sulfate, 4.89 g / L of ammonium succinate, and 1.31 g / L of ammonium phosphate were added to the medium (ammonium sulfate replacement rate: 32.6%). In this Example 2-6, the flask shaking culture medium used in the above reference example was used, but shaking culture was performed using a strain having the accession number KCCM11016P (Korean Patent Nos. 10-0159812 and 10-0397322), which is another coryneform microorganism with lysine production ability. The seed culture solution obtained at this time was inoculated at a ratio of 3.0% (v / v). The strain with the above-mentioned accession number KCCM11016P was disclosed as KFCC10881 and then redeposited with an international depository under the Budapest Treaty, and was assigned the accession number KCCM11016P.

[0300] In batch fermentation mode, the culture was incubated for approximately 17 hours under conditions of pH 6.5–8.5, agitation speed 450 rpm, and aeration volume 1 vvm. If necessary, the pH was maintained within an appropriate range by adding ammonia gas. Samples were obtained approximately every 2–3 hours, and the culture was terminated when the residual sugar was depleted.

[0301] A 30 L fermenter containing a medium (ammonium sulfate replacement rate: 53.2%) in which 6.10 g / L of ammonium sulfate, 3.28 g / L of ammonium acetate, 3.24 g / L of ammonium succinate, and 1.31 g / L of ammonium phosphate were added to the main culture medium composition of Example 2-1 above was inoculated with the seed culture solution obtained in the seed culture step at a ratio of 20.0% (v / v).

[0302] In this example, part of the ammonium sulfate added to the seed culture medium of Comparative Example 2 was replaced with ammonium succinate and ammonium phosphate, and part of the ammonium sulfate added to the main culture medium of Comparative Example 2 was replaced with ammonium acetate, ammonium succinate, and ammonium phosphate, and the total ammonium sulfate replacement rate of the seed culture medium and the main culture medium was 48.9%.

[0303] In the main cultivation stage of this example, the culture was performed for approximately 30 hours under the conditions of pH 6.5-8.5, stirring speed 450 rpm, and aeration volume 1 vvm in fed-batch fermentation mode. If necessary, the pH was maintained within an appropriate range by adding ammonia gas.

[0304] When the initial carbon source of the medium was exhausted, feeding medium containing 40.0% wt glucose was added to maintain the residual sugar at 0.5–1.0 wt%. Samples were obtained at intervals of approximately 2–3 hours, and the nitrogen content (AN: ammonium nitrogen, g / kg) was measured using a Kjeldahl analyzer (Foss Kjeltec 8400). If the value was less than 1.0 g / kg, the nitrogen source was additionally supplied by adding ammonia gas.

[0305] At the end of the cultivation, there was no residual sugar and the fermentation time was 28.6 hours. The standardized residual sulfate concentration derived from ammonium sulfate and the standardized purity obtained in the main cultivation stage were 59.4% and 103.2%, respectively, confirming a 40.6% decrease in residual sulfate and a 3.2% improvement in purity compared to the results of Comparative Example 2 (see Table 2).

[0306]

[0307] Comparative Example 2: Addition of ammonium sulfate to the seed culture and main culture stages.

[0308] A 5 L fermenter containing a medium containing 17.00 g / L of ammonium sulfate and 0.86 g / L of phosphate added to the seed culture medium composition of the above reference example was inoculated with the seed culture solution obtained from the flask shaking culture of the above reference example at a ratio of 3.0% (v / v).

[0309] In batch fermentation mode, the culture was incubated for approximately 17.0 hours under conditions of pH 6.5–8.5, agitation speed 450 rpm, and aeration volume 1 vvm. If necessary, the pH was maintained within an appropriate range by adding ammonia gas. Samples were obtained at approximately 2–3 hour intervals, and the culture was terminated when the residual sugar was depleted.

[0310] In a 30 L fermentation tank containing a medium containing 13.00 g / L of ammonium sulfate and 0.86 g / L of ammonium phosphate in the main culture medium composition of Example 2-1 above, the seed culture solution obtained in the seed culture step was inoculated at a ratio of 20.0% (v / v).

[0311] In the main cultivation stage of this comparative example 2, the culture was performed for 30.6 hours under the conditions of pH 6.5-8.5, stirring speed 450 rpm, and aeration volume 1 vvm in fed-batch fermentation mode. If necessary, the pH was maintained within an appropriate range by adding ammonia gas.

[0312] When the initial carbon source of the medium was exhausted, feeding medium containing 40.0% wt glucose was added to maintain the residual sugar at 0.5–1.0 wt%. Samples were obtained at intervals of approximately 2–3 hours, and the nitrogen content (AN: ammonium nitrogen, g / kg) was measured using a Kjeldahl analyzer (Foss Kjeltec 8400). If the value was less than 1.0 g / kg, the nitrogen source was additionally supplied by adding ammonia gas.

[0313]

[0314] result

[0315] The results obtained in Examples 2-1 to 2-6 and Comparative Example 2 are shown in Table 2 below:

[0316] Item Unit Example 2-1 Example 2-2 Example 2-3 Example 2-4 Example 2-5 Example 2-6 Comparative Example 2 Fermentation time hr 29.5 29.0 30.1 29.9 29.1 28.6 30.6 Residual sugar g / L 0.00.00.00.00.00.00.0 Sulfate *%92.094.072.060.048.059.4100.0Lysine (Lys)g / L218.9216.9214.2217.0221.5217.3219.4Purity * %102.5102.1102.9102.9105.4103.2100.0

[0317] ( * Normalization result value based on 100% of the results of Comparative Example 2;

[0318] Purity (%) = [Lys content (g / kg) / solid content (g / kg)] * 100)

[0319]

[0320] The experimental results in Table 2 above are summarized as follows:

[0321] In Example 2-1, ammonium sulfate added in the seed culture stage was replaced with ammonium succinate and ammonium phosphate, and culture was performed under the condition of an ammonium sulfate replacement rate of 6.8%. The fermentation time and lysine concentration were at the same level, but the standardized residual sulfate in the fermentation solution was confirmed to have decreased by 8.0%, confirming that the standardized purity was improved by 2.5%.

[0322] In Example 2-2, ammonium sulfate added in the seed culture stage was replaced with ammonium succinate and ammonium phosphate, and culture was performed under the condition of 4.0% ammonium sulfate replacement rate. The fermentation time and lysine concentration were at the same level, but a 6.0% decrease in the standardized residual sulfate in the fermentation liquid was confirmed, confirming that the standardized purity was improved by 2.1%.

[0323] In Example 2-3, ammonium sulfate added in the main cultivation stage was replaced with ammonium acetate, ammonium succinate, and ammonium phosphate, and cultured under the condition of an ammonium sulfate replacement rate of 42.2%. The fermentation time and lysine concentration were at the same level, but a 28.0% decrease in residual sulfate in the fermentation solution was confirmed, confirming that the standardized purity was improved by 2.9%.

[0324] Example 2-4 replaced ammonium sulfate added to the seed coat with ammonium succinate and ammonium phosphate, and replaced ammonium sulfate added to the main culture stage with ammonium acetate, ammonium succinate, and ammonium phosphate, and cultured under conditions of 48.9% ammonium sulfate replacement rate. The fermentation time and lysine concentration were at the same level, but a 40.0% decrease in residual sulfate in the fermentation solution was confirmed, confirming that the standardized purity was improved by 2.9%.

[0325] In Example 2-5, ammonium sulfate added to the seed coat was replaced with ammonium succinate and ammonium phosphate, and ammonium sulfate added to the main culture stage was replaced with ammonium acetate, ammonium succinate, and ammonium phosphate, and culture was conducted under the condition of an ammonium sulfate replacement rate of 61.4%. The fermentation time and lysine concentration were at the same level, but a 52.0% decrease in residual sulfate in the fermentation solution was confirmed, confirming that the standardized purity was improved by 5.4%.

[0326] Example 2-6 used KCCM11016P, a coryneform microorganism with the same lysine production ability as in the above-mentioned reference example and Example 2-4 conditions. Ammonium sulfate added to the seed coat was replaced with ammonium succinate and ammonium phosphate, and ammonium sulfate added to the main culture stage was replaced with ammonium acetate, ammonium succinate, and ammonium phosphate, and cultured under the condition of 48.9% ammonium sulfate replacement rate. As a result, the fermentation time and lysine concentration were at the same level, but the residual sulfate in the fermentation liquid was confirmed to be reduced by 40.6%, confirming that the standardized purity was improved by 3.2%. In addition, when compared to the results of Example 2-4, the residual sulfate and the standardized purity showed similar values, confirming that even if the strain was changed, similar fermentation indices and quality results were shown at the same ammonium sulfate replacement rate.

[0327] In order to improve the purity of the final product, it is essential to improve the purity of the fermentation broth by minimizing the residual sulfate derived from the ammonium sulfate added to the medium. In the lysine granule process without a separate ion exchange resin process, the problem of a decrease in product purity due to fermentation broth impurities occurs. However, when this technology was introduced and some or all of the ammonium sulfate in the seed culture medium and main culture stage medium were replaced with ammonium acetate, ammonium succinate, or ammonium phosphate, the anionic counter ions were consumed during the culture, and the sulfate concentration was reduced, confirming the effect of improving the quality of the granule product.

[0328]

[0329] From the above description, those skilled in the art will understand that the present application can be implemented in other specific forms without altering its technical concept or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of this application should be interpreted to include all changes or modifications derived from the meaning and scope of the following claims and their equivalents, rather than the detailed description above.

Claims

1. Ammonium sulfate; and at least one ammonium-containing compound selected from the group consisting of ammonium succinate, ammonium phosphate, and ammonium acetate, A composition of a culture medium for a Corynebacterium genus (Corynebacterium sp.) microorganism, wherein the value of the following [Formula 1] is 2% or more: [Formula 1] [(sum of moles (mol) of ammonium derived from ammonium succinate, ammonium phosphate, and ammonium acetate) / (sum of moles (mol) of ammonium derived from ammonium sulfate, ammonium succinate, ammonium phosphate, and ammonium acetate)]*100(%).

2. In paragraph 1, A culture medium composition in which the value of the following [Formula 5] is 90% or less: [Formula 5] [(Moles of ammonium derived from ammonium acetate) / (sum of moles of ammonium derived from ammonium succinate, ammonium phosphate, and ammonium acetate)]*100(%).

3. In paragraph 1, A culture medium composition, wherein the sum of the moles (mol) of ammonium derived from ammonium sulfate, ammonium succinate, ammonium phosphate, and ammonium acetate is 0.1 to 0.5 moles per 1 L of the culture medium composition.

4. In paragraph 1, The above culture medium composition is, based on 1 mole of ammonium derived from ammonium sulfate, 0.05 mol to 1.2 mol of ammonium derived from ammonium succinate, 0.01 to 0.5 mol of ammonium derived from ammonium phosphate, and 0.05 mol to 1 mol of ammonium derived from ammonium acetate A culture medium composition comprising at least one selected from the group consisting of:

5. In paragraph 1, The above culture medium composition is a culture medium composition having a pH of 6 to 9.

6. In paragraph 1, A culture medium composition, wherein the above Corynebacterium genus microorganism is Corynebacterium glutamicum or Corynebacterium stationis.

7. In paragraph 1, The above culture medium composition is a culture medium composition that is a seed culture medium composition or a main culture medium composition.

8. A method for producing a basic amino acid, comprising a step of culturing a microorganism of the genus Corynebacterium sp. in a culture medium composition of any one of claims 1 to 7.

9. In paragraph 8, A method for producing a basic amino acid, wherein the above culture medium composition is a seed culture medium composition.

10. In paragraph 9, A method for producing a basic amino acid, further comprising a step of adding the above-described seed culture medium composition containing a microorganism of the genus Corynebacterium to a main culture medium and culturing it.

11. In paragraph 10, A method for producing a basic amino acid, wherein the seed culture medium composition containing the above-mentioned Corynebacterium microorganism is added at 0.1 L to 0.4 L per 1 L of the main culture medium.

12. In paragraph 10, A method for producing basic amino acids, wherein a feeding medium is additionally added in the step of culturing by adding to the above main culture medium.

13. In paragraph 8, A method for producing a basic amino acid, wherein the pH of the culture medium is maintained at 6 to 9 during the above culturing step.

14. In paragraph 8, A method for producing a basic amino acid, wherein the sulfate content is reduced at the end of culture compared to when a culture medium is used that does not contain at least one ammonium-containing compound selected from the group consisting of ammonium succinate, ammonium phosphate, and ammonium acetate.

15. In paragraph 8, A method for producing a basic amino acid, wherein the purity of the basic amino acid is increased at the end of culture compared to when a culture medium is used that does not contain at least one ammonium-containing compound selected from the group consisting of ammonium succinate, ammonium phosphate, and ammonium acetate.