Compositions containing L-ergothioneine and their manufacturing methods, and methods for imparting color-inhibiting effects to these compositions.

Abstract

The objective of this invention is to inhibit the staining of a composition in which L-ergothioneine (EGT) and N,N-dimethyl-L-2-thiohistidine (DTH) coexist. This invention prepares a composition containing EGT and DTH, wherein the content of DTH in the composition is reduced.

Description

Technical Field

[0001] This invention relates to a composition containing L-ergothioneine (EGT). More specifically, it relates to a composition that improves the stability of L-ergothioneine (EGT) in the presence of N,N-dimethyl-L-2-thiohistidine (DTH) and a method for manufacturing the same. Prior Technology

[0002] L-ergothioneine (sometimes referred to as "EGT" in this specification) is known to be a sulfur-containing amino acid and has a variety of physiological activities, primarily antioxidant capacity.

[0003] For example, Non-Patent Literature 1 describes that EGT can enhance the efficacy of immunotherapy derived from cancer vaccines containing tumor-associated antigens (TAAs) and adjuvants. Patent Literature 1 describes that EGT can treat bacterial infections. In Experiment 1 of Patent Literature 1, it was confirmed that 5 mM and 50 mM EGT inhibited the proliferation of Escherichia coli; therefore, it can be understood that the inhibition of bacterial infections described in Patent Literature 1 is caused by EGT directly inhibiting bacterial proliferation.

[0004] Non-Patent Literature 2 describes that the production of cytokines (IL-6, IL-12p40, IL-1β, IL-10) derived from mouse bone marrow macrophages under stimulation conditions obtained from Toll-like receptor ligands is promoted by 10 mM EGT, but not by 1 mM EGT; and that in the co-culture of F4 / 80 macrophages and OT-II CD4+ T cells, Th17 polarization of OT-II CD4+ T cells is promoted by 30 mM EGT. As described above, Non-Patent Literature 2 describes that high concentrations of EGT above 10 mM activate macrophages.

[0005] Non-Patent Literature 3 is a review related to the physiological activity of EGT. It describes that EGT is absorbed / accumulated in animals and plants. [Existing technical documents] [Patent Literature]

[0006] [Patent Document 1] WO2019 / 089878 [Non-patent literature]

[0007] [Non-patent literature 1] S. Yoshida et al., Frontiers In Immunology (Front. Immunol.) 10:671 (2019) [Non-Patent Literature 2] Public Science Library, General (PLos ONE) 12(1):e0169360 [Non-Patent Document 3] Journal of Research and Education, Faculty of Agriculture, Tamagawa University, No. 1: 17-41 (2016) Summary of the Invention

[0008] [The problem that the invention aims to solve] However, the formulation stability of EGT-containing components remains insufficiently studied. In particular, the impact of other coexisting components in EGT-containing components has not been reported in detail. [Methods for solving problems]

[0009] Efforts were made to investigate the aforementioned issues, and a novel issue, previously unreported, was discovered: EGT becomes unstable in the presence of N,N-dimethyl-L-2-thiohistidine (DTH) in EGT-containing compositions, resulting in the coloration of the EGT-containing compositions.

[0010] To address this novel issue, it was discovered that by reducing the DTH content in compositions containing EGT and DTH, the stability of EGT can be improved and the coloring of the composition can be inhibited, thus completing this invention.

[0011] That is, the present invention provides the following composition.

[0012] [1] A composition comprising L-ergothioneine and N,N-dimethyl-L-2-thiohistidine, wherein in the composition, The content of N,N-dimethyl-L-2-thiohistidine was reduced.

[0013] [2] The composition as described in [1], wherein the coloring of the composition is suppressed by reducing the content of the N,N-dimethyl-L-2-thiohistamine.

[0014] [3] The composition as described in [1] or [2], wherein the content of N,N-dimethyl-L-2-thiohistamine is less than 50 parts by weight relative to 100 parts by weight of the L-ergothioneine.

[0015] [4] The composition as described in any one of [1] to [3], wherein the content of N,N-dimethyl-L-2-thiohistamine is 0.0001 parts by mass or more relative to 100 parts by mass of the L-ergothioneine.

[0016] [5] The composition as described in any one of [1] to [4], wherein the content of N,N-dimethyl-L-2-thiohistamine is 0.05 parts by mass or more relative to 100 parts by mass of the L-ergothioneine.

[0017] [6] A composition for inhibiting the color development of a composition containing L-ergothioneine and N,N-dimethyl-L-2-thiohistamine, wherein the composition contains... The content of N,N-dimethyl-L-2-thiohistamine is less than 50 parts by weight relative to 100 parts by weight of L-ergothioneine.

[0018] Furthermore, the present invention relates to the manufacturing method described below.

[0019] [7] A method for manufacturing a composition comprising L-ergothioneine and N,N-dimethyl-L-2-thiohistidine with inhibited coloring, and the method comprising: The steps to reduce the content of N,N-dimethyl-L-2-thiohistidine.

[0020] Furthermore, this invention relates to the following methods.

[0021] [8] A method that imparts a color-inhibiting effect to a composition containing L-ergothioneine and N,N-dimethyl-L-2-thiohistidine, wherein the composition, The content of N,N-dimethyl-L-2-thiohistamine is less than 50 parts by weight relative to 100 parts by weight of L-ergothioneine. [The effects of the invention]

[0022] DTH was discovered as a component that affects the stability of EGT. Therefore, by reducing the DTH content in components in which EGT and DTH coexist, the stability of the formulation can be improved and the coloration of the composition can be inhibited. Simple Explanation of the Diagram

[0023] Figure 1 is a graph showing the stability evaluation results of the composition containing EGT and DTH in Experimental Example 1. Figure 2 is a graph showing the results of the color inhibition test of the composition containing EGT and DTH in Experimental Example 2. Figure 3 is a graph showing the results of the color inhibition test of the composition containing EGT and DTH in Experimental Example 2. Figure 4 is a graph showing the results of the DTH reduction experiment in the EGT fermentation method in Experiment Example 3. Figure 5 is a graph showing the results of the DTH reduction experiment in the EGT fermentation method in Experiment Example 3. Implementation

[0024] The composition of this invention contains L-ergothioneine (EGT) and N,N-dimethyl-L-2-thiohistidine (DTH), and the content of DTH is reduced.

[0025] [L-Ergothioneine (EGT)] EGT is a histidine derivative (N,N,N-trimethyl-L-2-thiohistidine), and its structure is represented by the following formula (1).

[0026] [Chemistry 1]

[0027] EGT can also be obtained through well-known methods such as synthesis, extraction, and fermentation, and commercially available products are also available for use.

[0028] Commercially available EGT products include L-ergothioneine (manufactured by Tetraedron), among others.

[0029] [N,N-Dimethyl-L-2-thiohistidine (DTH)] DTH is an analogue of EGT, and its structure is represented by the following formula (2).

[0030] [Chemistry 2]

[0031] DTH can also be obtained through known methods such as synthesis, extraction, and fermentation, and commercially available products can also be obtained for use.

[0032] Commercially available DTH products include N,N-dimethyl-L-2-thiohistidine (manufactured by Tetraedron), etc.

[0033] EGT and / or DTH can be in free form or in salt form. Salts of EGT and / or DTH can be formed from the carboxyl groups in these structures, or from trimethylamino or dimethylamino groups. In addition, L-ergothioneine or L-ergothioneine analogues can be hydrates or other solvents.

[0034] Salts of EGT and / or DTH are not particularly restricted if they are pharmacologically or physiologically permissible. Specifically, examples include: organic acid salts, inorganic acid salts, salts with organic bases, and salts with inorganic bases. Examples of organic acid salts include: monocarboxylic acid salts such as acetate, trifluoroacetate, butyrate, palmitate, and stearate; polycarboxylic acid salts such as fumarate, maleate, succinate, and malonate; hydroxycarboxylic acid salts such as lactate, tartrate, and citrate; and organic sulfonates such as methanesulfonate, toluenesulfonate, and p-toluenesulfonate. Examples of inorganic acid salts include: hydrochloride, sulfate, nitrate, hydrobromide, and phosphate. Examples of salts with organic bases include: salts with organic amines such as methylamine, triethylamine, triethanolamine, diethanolamine, morpholine, piperazine, pyrrolidine, and ethylenediamine. Salts of inorganic bases include, for example, salts of alkali metals such as sodium or potassium, alkaline earth metals such as calcium or magnesium, and various salts of metals such as aluminum. These EGT and / or DTH salts may be used alone, or in combination of two or more. The category of "pharmaceutically or physiologically permissible salts" may also include salt solvents or hydrates.

[0035] In the following embodiments, the inventors discovered a novel problem not previously reported: when DTH coexists in a composition containing EGT, EGT becomes unstable, resulting in discoloration of the EGT-containing composition. Furthermore, the inventors discovered that by reducing the DTH content in a composition containing both EGT and DTH, the stability of EGT can be improved and discoloration of the composition can be suppressed. The manner in which DTH coexists in a composition containing EGT is not particularly limited; for example, DTH can be added to the EGT-containing composition, or it can be inherently present through impurities in extraction processes, byproducts in synthesis processes, fermentation processes, etc.

[0036] In the composition of the present invention, "suppression of coloration" means that, relative to an equal amount of a composition containing EGT and DTH, under the same pH conditions, the absorbance at 400 nm is reduced by decreasing the DTH content. The reduction in absorbance at 400 nm can be determined using a known spectrophotometer. The reduction in absorbance at 400 nm is preferably at least 5% relative to an equal amount of a composition containing EGT and DTH, and more preferably at least 10%.

[0037] In the composition of the present invention, regarding the content ratio of EGT and DTH, from the viewpoint of inhibiting the coloring of the composition and the stability of EGT, the content of DTH is preferably 50 parts by mass or less, more preferably 40 parts by mass or less, further preferably 30 parts by mass or less, particularly preferably 20 parts by mass or less, and most preferably 10 parts by mass or less, relative to 100 parts by mass of EGT.

[0038] Furthermore, regarding the content ratio of EGT and DTH in the composition of the present invention, from the viewpoint of production efficiency of the composition, the content of DTH is preferably 0.0001 parts by mass or more, more preferably 0.001 parts by mass or more, further preferably 0.005 parts by mass or more, particularly preferably 0.01 parts by mass or more, and most preferably 0.05 parts by mass or more, relative to 100 parts by mass of EGT.

[0039] Furthermore, regarding the content ratio of EGT and DTH in the composition of the present invention, from the viewpoints of inhibiting coloring of the composition, the stability of EGT, and the production efficiency of the composition, the content of DTH is preferably 0.0001 parts by mass to 50 parts by mass relative to 100 parts by mass of EGT, more preferably 0.001 parts by mass to 40 parts by mass, even more preferably 0.005 parts by mass to 30 parts by mass, particularly preferably 0.01 parts by mass to 20 parts by mass, and most preferably 0.05 parts by mass to 10 parts by mass.

[0040] Furthermore, the content of EGT in the composition of the present invention can be appropriately adjusted according to the form or use of the formulation, the type or content of other components, etc., and is not limited. For example, relative to the total amount of the composition, it can be set to 0.000001% by mass or more, such as 0.000005% by mass or more, 0.00001% by mass or more, 0.00005% by mass or more, 0.0001% by mass or more, 0.0005% by mass or more, 0.001% by mass or more, etc. Additionally, the content of EGT relative to the total amount of the composition can be set to 99.999% by mass or less, such as 99.9% by mass or less, 99.5% by mass or less, 99% by mass or less, 98.5% by mass or less, 98% by mass or less, etc. In another embodiment, when prepared in the form of a liquid formulation, there is no limitation, and the content of EGT, for example relative to the total amount of the composition, can be set to 80% by mass or less, and examples include: 70% by mass or less, 60% by mass or less, 50% by mass or less, 40% by mass or less, 30% by mass or less, 20% by mass or less, 10% by mass or less, 5% by mass or less, 1% by mass or less, etc.

[0041] [use] By reducing the DTH content in a composition containing EGT and DTH, the stability of EGT can be improved. Therefore, the present invention can be used to suppress the coloring of the composition.

[0042] Furthermore, as mentioned above, EGT is known to possess a variety of physiological activities, primarily antioxidant capacity. By using this invention in compositions containing EGT and DTH, the stability of EGT can be improved and its decomposition inhibited, thus enabling it to function without compromising the inherent physiological activities of EGT.

[0043] By using this invention, compositions containing EGT and DTH can also be better used for purposes such as anti-oxidation, improving brain function, anti-aging, treating eye diseases, whitening, absorbing ultraviolet rays, inhibiting melanin production, eliminating reactive oxygen species, inhibiting elastase activity, inhibiting wrinkle formation, inhibiting skin laxity, inhibiting the formation of skin spots, inhibiting dark circles around the eyes, reducing skin damage caused by ultraviolet rays (inhibiting photoaging), treating dry skin, treating sensitive skin, improving hair, and promoting autophagy.

[0044] The composition of the present invention can be further adapted to be used as an effective ingredient or additive in food and beverage products, functional labeled foods, specific health foods, quasi-medicines, pharmaceuticals, cosmetics, daily necessities, feed, etc. In addition, it can be adapted to be formulated using known formulation methods used in such products.

[0045] As cosmetics or daily necessities, they can be formulated into products such as toners, lotions, gels, serums, creams, sunscreens, peel-off masks, face masks, foundation creams, face powders, bath products, lotions, shampoos, conditioners, hair gels, hair sprays, hair growth oils, toothpaste, and mouthwashes.

[0046] [preparation] The components of this invention can be administered orally or non-orally (including externally) in the form of solid dosage forms such as tablets, capsules, granules, and powders; or in the form of liquid dosage forms such as liquids, syrups, injections, creams, lotions, pastes, ointments, emulsions (oil-in-water emulsions, water-in-oil emulsions, multiple emulsions, microemulsions, polyethylene terephthalate (PET) emulsions, Pickering emulsions), gels (hydrogels, alcohol gels), and suspensions. Excipients, lubricants, binders, and disintegrants can be used in solid dosage forms; solvents, dissolving aids, emulsifiers, emulsion stabilizers, thickeners, humectants, suspending agents, isotonic agents, buffers, and analgesics can be used in liquid dosage forms. Additionally, preservatives, antioxidants, colorants, sweeteners, and flavorings can be added as needed.

[0047] Examples of excipients include: sugar alcohols such as sorbitol, mannitol, and xylitol; sugars such as glucose, white sugar, lactose, and fructose; crystalline cellulose, sodium carboxymethyl cellulose, croscarmellose sodium cellulose, dicalcium phosphate, wheat starch, rice starch, corn starch, potato starch, dextrin, β-cyclodextrin, light anhydrous silicic acid, titanium dioxide, magnesium metasilicate aluminate, talc, kaolin, and olive oil.

[0048] Examples of binders include: cellulose derivatives such as methylcellulose, ethylcellulose, hydroxypropylcellulose, and hydroxypropylmethylcellulose; polyvinylpyrrolidone, polyvinyl alcohol, acrylic polymers, gelatin, gum arabic, polytriglucose, α-starch, agar, tragacanth, sodium alginate, and propylene glycol alginate.

[0049] Examples of disintegrants include: starch, low-substituted hydroxypropyl cellulose, calcium carboxymethyl cellulose, croscarmellose sodium, hydroxypropyl starch, and partially α-substituted starch.

[0050] Examples of solvents include: water, alcohol, propylene glycol, polyethylene glycol (macrogol), sesame oil, corn oil, etc.

[0051] Examples of lubricants include: stearic acid, magnesium stearate, calcium stearate, polyoxyl stearate, cetyl alcohol, talc, hardened oil, sucrose fatty acid ester, dimethyl polysiloxane, beeswax, and white beeswax.

[0052] Examples of solvent aids include: polyethylene glycol, propylene glycol, mannitol, benzyl benzoate, ethanol, tris(hydroxymethyl)aminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, etc.

[0053] Examples of suspending / emulsifying agents include: stearylamine, triethanolamine, sodium lauryl sulfate, lauryl aminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate, and other surfactants; hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, and hydroxypropyl cellulose; and waxes such as shellac wax, beeswax, carnauba wax, cetearyl wax, lanolin, liquid lanolin, reduced lanolin, hard lanolin, cyclic lanolin, lanolin wax, candelilla wax, lacquer wax, lignite wax, and rice wax.

[0054] Examples of isotropic agents include sodium chloride, glycerol, and D-mannitol.

[0055] Examples of buffers include phosphate, acetate, carbonate, and citrate buffers.

[0056] Examples of preservatives include: parabens, chlorobutanol, benzyl alcohol, phenylethanol, dehydroacetic acid, and sorbic acid.

[0057] Examples of antioxidants include sulfites and ascorbic acid.

[0058] When formulating the components of the present invention into solid dosage forms, manufacturing methods known in the art can be used. Examples include: pulverizing and granulating an extruded granule formed by mixing the components and passing it through a sieve; or adding mixing water to the components, followed by stirring granulation using a vertical granulator, and then pulverizing / sieving using a Comil pulverizer. Alternatively, examples include: compressing the dosage form using a roller compressor, then pulverizing and sieving using a roller granulator. Another example is drying the flow layer after stirring granulation. Furthermore, for example, in the case of manufacturing by direct ingot pressing, the components can be directly fed into an ingot press for ingot pressing after mixing.

[0059] [Food and Drinks] The components of this invention can also be used as food and beverage components and can be contained in food or functional food for provision. Examples of such foods or functional foods include: rice; various types of noodles including buckwheat noodles, udon noodles, glass noodles, Chinese noodles, instant noodles, and cup noodles; beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit drinks, lactic acid drinks, and sports drinks; curry cubes, stews, and various soups; ice cream, frozen fruit syrups, shaved ice, and other cold desserts; maltose, biscuits, candies, chewing gum, chocolate, tablet-shaped snacks, small round cookies, jelly, jam, cream, and other baked goods; processed aquatic / livestock products such as fish cakes, fish soup, ham, and sausages; dairy products such as processed milk and fermented milk; oils and oil-based processed foods such as salad oil, frying oil, margarine, mayonnaise, shortening, foaming cream, and salad dressing; condiments such as sauces, salad dressings, miso, soy sauce, and gravy; soups, stews, salads, cooked foods, seasonings, and pickled foods; and other forms of health / nutritional supplements / functionally labeled foods / foods for specific health purposes.

[0060] In addition, supplements (powders, granules, soft capsules, hard capsules, tablets, chewable tablets, disintegrating tablets, syrups, liquids, etc.) containing the components of the present invention can also be prepared.

[0061] Alternatively, the components of this invention can be contained in animal feed such as pet feed.

[0062] Additives may be added to food and beverage products as needed. Examples of such additives include: glucose, fructose, sucrose, maltose, sorbitol, trehalose, stevia, stevia extract, corn syrup, lactose, mannitol, dextrin, citric acid, sodium citrate, tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbic acid, tocopherol, sodium isoascorbate, glycerol, propylene glycol, glycerol fatty acid esters, polyglycerol fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, gum arabic, carrageenan, casein, gelatin, pectin, agar, B vitamins, nicotinamide, calcium pantothenate, amino acids, calcium salts, surfactants, colorings, flavorings, preservatives, etc.

[0063] The components of this invention can be used in food and beverage products licensed to improve / prevent / enhance various symptoms or conditions. Here, in this invention, food and beverage products licensed to improve / prevent / enhance symptoms or conditions are those with efficacy permitted / designated by a national or public body, such as functional foods, foods for specific health purposes, or foods for special purposes. Furthermore, names or regulations may vary depending on circumstances, era, or national systems, but those that are essentially the same are included in this invention.

[0064] In this invention, the amount of the components is not particularly limited and can be appropriately set according to the applicable purpose (type of disease or symptom), applicable body part, gender or age of the applicant, the form of food, functional food, specific health food, quasi-medicine, medicine, cosmetic, daily necessities or feed, the method or frequency of administration or intake, preferences, etc.

[0065] When using the composition of the present invention, the effective amount that can exert the physiological activity inherent in EGT can be set as the daily intake (suitable amount). For example, when a healthy adult takes it (suitable), the daily intake (suitable amount) of EGT can be, for example, 0.005 mg to 4,000 mg, preferably 0.1 mg to 3,000 mg, more preferably 0.5 mg to 2,000 mg, even more preferably 1 mg to 1,000 mg, particularly preferably 2 mg to 500 mg, and most preferably 3 mg to 300 mg.

[0066] Furthermore, when using the components of the present invention as food or beverage, there are no limitations. From the viewpoint that the physiological activity inherent in EGT can be indicated, it is preferable to make it into a functional food. Functional foods can include functional labeled foods, nutritional functional foods, nutritional supplements, and foods for specific health purposes. From the viewpoint that the use can be clearly indicated, functional labeled foods are preferred.

[0067] [Target Audience] There are no particular limitations on the age at which the components of this invention are applicable if the physiological activity of EGT is required. It can be an age at which people are easily stressed by work or other living environments, i.e., 30 years and older; or an age at which people are easily affected by changes in hormone balance or living environment, i.e., middle-aged people (45 years and older, but under 55 years old); or elderly people (55 years and older).

[0068] [pH] The pH of the composition of this invention can be appropriately set according to the type and content of other formulation ingredients, formulation form, method of use, etc. There are no restrictions if the pH is within a pharmaceutically or physiologically permissible range; for example, it can be set to pH 2-10. From the viewpoint of stably exerting the effects of this invention, the pH of the composition of this invention can be set to, for example, pH 2-10, pH 2-9, pH 2-8, pH 2-7, pH 3-10, pH 3-9, pH 3-8, pH 3-7, pH 4-10, pH 4-9, pH 4-8, pH 4-7, pH 5-10, pH 5-9, pH 5-8, pH 5-7, pH 6-10, pH 6-9, pH 6-8, pH 6-7, etc.

[0069] [Method for manufacturing a composition containing EGT and DTH with suppressed coloring] In this invention, the method for manufacturing a composition containing EGT and DTH with suppressed coloring includes a step of reducing the DTH content.

[0070] The step of reducing the DTH content can be any known method, as long as it reduces the DTH content. For example, when preparing a composition by mixing EGT and DTH, a step of reducing the DTH content ratio relative to EGT can be used. Furthermore, as mentioned above, there is no particular limitation on the form in which DTH coexists in the composition containing EGT; for example, it can be a form in which DTH exists in impurities during EGT extraction, or a form in the form of a byproduct of synthesis or fermentation.

[0071] In cases of reducing DTH in impurities from mushrooms or plants during EGT extraction, examples include: solvent extraction, separation using differences in solubility, chromatography using adsorbents such as silica gel and alumina, cation exchange chromatography, anion exchange chromatography, hydrophobic chromatography, gel filtration, thiopropyl-agarose (Sepharose) 6B, reverse phase chromatography, crystallization, activated carbon treatment, membrane treatment, and methods combining these.

[0072] Regarding methods for producing EGT via fermentation, methods using E. coli and the like are known according to International Publication No. 2019 / 163767. When reducing DTH in impurities during EGT fermentation, methods described in the examples below can be used, for example. Specifically, in EGT fermentation, by employing a step of deleting the metJ gene of bacteria belonging to the Enterobacteriaceae family that are capable of producing EGT, DTH in the culture medium can be reduced and EGT can be produced efficiently.

[0073] The steps to delete the metJ gene can use well-known methods, such as knockout or knockdown of the metJ gene by homologous recombination, mutation treatment, genome editing, etc. Examples of bacteria belonging to the Enterobacteriaceae family that possess EGT production capabilities include, but are not limited to, bacteria of the genera *Escherichia*, *Enterobacter*, *Pantoea*, *Klebsiella*, and *Salmonella*. Especially desirable enteric bacteria include *Escherichia coli* and other *Escherichia* species, and *Pantoea ananatis* and other *Pantoea* species.

[0074] Culture of bacteria belonging to the Enterobacteriaceae family can be performed using conventional methods. Specifically, LB medium, 2×YT medium, NZY medium, M9 medium, SOC medium, or YPD medium can be used. EGT and DTH can be produced using these media, but the media used are not limited to these. Furthermore, the produced EGT and DTH can accumulate within the bacteria or be secreted / accumulated outside the bacteria (in the culture medium).

[0075] The recovery of EGT and DTH from within or free within bacteria can be performed using known methods. For example, the culture can be subjected to solid-liquid separation such as centrifugation or filtration, and the extracts of EGT and DTH can be obtained from the bacteria through solvent extraction, hot water extraction, or disruption. EGT can be obtained from the extracts or culture supernatant using known chromatography methods such as ion exchange chromatography, hydrophobic chromatography, and gel filtration chromatography.

[0076] Among various methods for reducing DTH content, the preferred content ratio of EGT and DTH is based on the above.

[0077] The manufacturing method may further include a pH adjustment step. The optimal pH range is determined according to the above.

[0078] [Methods for imparting colorimetric inhibition effects to compositions containing EGT and DTH] This invention also provides a method for imparting a color-inhibiting effect to compositions containing EGT and DTH, wherein the compositions, The content of DTH is set to be less than 50 parts by weight relative to 100 parts by weight of EGT. The preferred content ratio of EGT and DTH is based on the description. [Example]

[0079] Secondly, the present invention will be specifically described through examples or test examples, but the present invention is not limited to the following examples or test examples. Furthermore, unless otherwise specified, experiments will be conducted using methods described in standard protocols related to molecular biology and applied microbiology, or methods modified or altered thereof. Additionally, unless otherwise specified, % represents w / v%.

[0080] (Experimental Example 1. Stability evaluation of components containing EGT and DTH 1) For compositions containing L-ergothioneine (EGT) and N,N-dimethyl-L-2-thiohistidine (DTH), the storage stability under various pH conditions was investigated as follows.

[0081] To achieve the final concentrations and pH of EGT and DTH as described in Table 1, EGT (manufactured by Tetraedron) and DTH (manufactured by Tetraedron) were dissolved in Britton-Robinson Buffer (final concentration 30 mM) at pH 3, 4, 5, 6, 7, 8, 9, or 10, thereby preparing 3.0 ml of mixed solutions of DTH and EGT with different pH and concentrations.

[0082] The Britton-Robinson buffer used for pH adjustment is prepared as follows: Take 20 ml of a mixed solution of boric acid, phosphoric acid, and acetic acid (final concentration 200 mM each), and adjust the pH to 3, 4, 5, 6, 7, 8, 9, or 10 using 1 N NaOH. Then, bring the volume to 40 ml with water to prepare 300 mM (100 mM each) Britton-Robinson buffers for pH 3–10. Next, to ensure a final concentration of 30 mM Britton-Robinson buffer, dissolve EGT and DTH in water at each pH level to prepare the EGT and DTH mixed solutions shown in Table 1.

[0083] For the EGT and DTH mixed solutions listed in Table 1, 1.0 ml of each sample was transferred to two tubes and incubated at 30°C or 70°C for 21 days. The color changes of the solutions were recorded by photographing the tubes immediately after the start of the process (day 0), on day 8, day 14, and day 21. 100 μl samples were then taken from each tube and used for the quantitative analysis of EGT and DTH using a high-performance liquid chromatography (HPLC) system (Shimadzu Corporation) under the conditions described in Table 2. Figure 1 shows a photograph of the solution taken on day 14.

[0084] As shown in Figure 1, in the samples stored at 70°C for 14 days, the solutions in the test areas of EGT 100 mg / L + DTH 20 mg / L, EGT 100 mg / L + DTH 50 mg / L, and EGT 100 mg / L + DTH 100 mg / L exhibited a color change from colorless and transparent to brownish-red. Furthermore, the degree of coloration increased with increasing DTH concentration. Additionally, the degree of coloration increased with increasing DTH storage time. Based on the above, this experimental example confirms for the first time the issue of coloration in mixed solutions of EGT and DTH.

[0085] [Table 1] No. EGT (mg / L) DTH (mg / L) pH No. EGT (mg / L) DTH (mg / L) pH 1 100 0 3 17 100 50 3 2 4 18 4 3 5 19 5 4 6 20 6 5 7 twenty one 7 6 8 twenty two 8 7 9 twenty three 9 8 10 twenty four 10 9 100 20 3 25 100 100 3 10 4 26 4 11 5 27 5 12 6 28 6 13 7 29 7 14 8 30 8 15 9 31 9 16 10 32 10

[0086] [Table 2] Using machines HPLC Prominence manufactured by Shimadzu Corporation tubular YMC-Pack ODS-AQ (4.6×250 mm, particle size 5 μm) YMC Company manufactures solvent 0.1% formic acid gradient condition isometry Detection wavelength 263 nm flow 0.8 mL / min Column temperature 30℃ Injection volume 5 μl Dissolution time DTH: around 5.1 min, EGT: around 6.0 min

[0087] (Experimental Example 2. Evaluation of colorimetric inhibition in compositions containing EGT and DTH 1) The results of Experiment 1 revealed a novel issue of staining in a mixed solution of EGT and DTH. Therefore, while quantifying the degree of this color change by measuring absorbance, various studies were conducted on the concentrations of EGT and DTH to explore methods for inhibiting staining.

[0088] Similar to Example 1, 2.0 ml of a mixed solution of EGT and DTH was prepared by dissolving EGT (manufactured by Tetraedron) and DTH (manufactured by Tetraedron) in Britton-Robinson Buffer (final concentration 30 mM) to achieve the final concentrations and pH values ​​shown in Tables 3 and 4.

[0089] Transfer 1.5 ml of each of the EGT and DTH mixed solutions listed in Tables 3 and 4 into 2.0 ml tubes. To prevent evaporation, add 200 μl of heavy mineral oil to the surface of each solution. Incubate the prepared samples at 70°C for 28 days. On days 0, 4, 7, 14, 21, and 28, take 200 μl of each sample and transfer it to a 96-well microplate. Measure the absorbance from 250 nm to 800 nm using a multi-wavelength microplate reader (MULTISKAN GO) (Thermo Scientific). Figure 2 shows the absorbance data at 400 nm obtained using the samples from Table 3. Furthermore, quantify EGT and DTH using 100 μl of the absorbance-measured sample under the conditions described in Table 2 using a high-performance liquid chromatography (HPLC) system (Shimadzu Corporation).

[0090] [Table 3] No. EGT (mg / L) DTH (mg / L) pH No. EGT (mg / L) DTH (mg / L) pH 1 100 50 3 37 100 50 7 2 20 38 20 3 10 39 10 4 5 40 5 5 2.5 41 2.5 6 1 42 1 7 0.1 43 0.1 8 0.01 44 0.01 9 0.001 45 0.001 10 100 50 4 46 100 50 8 11 20 47 20 12 10 48 10 13 5 49 5 14 2.5 50 2.5 15 1 51 1 16 0.1 52 0.1 17 0.01 53 0.01 18 0.001 54 0.001 19 100 50 5 55 100 50 9 20 20 56 20 21 10 57 10 22 5 58 5 23 2.5 59 2.5 24 1 60 1 25 0.1 61 0.1 26 0.01 62 0.01 27 0.001 63 0.001 28 100 50 6 64 100 50 10 29 20 65 20 30 10 66 10 31 5 67 5 32 2.5 68 2.5 33 1 69 1 34 0.1 70 0.1 35 0.01 71 0.01 36 0.001 72 0.001

[0091] [Table 4] No. EGT (mg / L) DTH (mg / L) pH No. EGT (mg / L) DTH (mg / L) pH 1 1000 500 3 37 1000 500 7 2 200 38 200 3 100 39 100 4 50 40 50 5 25 41 25 6 10 42 10 7 1 43 1 8 0.1 44 0.1 9 0.01 45 0.01 10 1000 500 4 46 1000 500 8 11 200 47 200 12 100 48 100 13 50 49 50 14 25 50 25 15 10 51 10 16 1 52 1 17 0.1 53 0.1 18 0.01 54 0.01 19 1000 500 5 55 1000 500 9 20 200 56 200 21 100 57 100 22 50 58 50 23 25 59 25 24 10 60 10 25 1 61 1 26 0.1 62 0.1 27 0.01 63 0.01 28 1000 500 6 64 1000 500 10 29 200 65 200 30 100 66 100 31 50 67 50 32 25 68 25 33 10 69 10 34 1 70 1 35 0.1 71 0.1 36 0.01 72 0.01

[0092] The results of the color inhibition evaluation test using the samples in Table 3 are shown in Figure 2. As shown in Figure 2, in the sample containing 100 mg / L EGT and 50 mg / L DTH, the absorbance at 400 nm (A400 value), representing the degree of coloration, increased over time across the entire pH range. The absorbance also confirmed the coloration of the EGT and DTH mixed solution as confirmed in Example 1. Furthermore, studies were conducted on various DTH concentrations, and the results confirmed that the A400 value decreased by reducing the DTH concentration across the entire pH range. Although not limited, it was found that setting the DTH content to 20 parts by mass or less relative to 100 parts by mass of EGT significantly inhibited coloration over time. The quantitative results of EGT for each sample also showed that, similarly to coloration, the decomposition of EGT could be inhibited by reducing the DTH concentration.

[0093] Furthermore, in the color inhibition evaluation test using the EGT and DTH mixed solution shown in Table 4, the same trend as the samples in Table 3 was obtained. The results are shown in Figure 3. Specifically, it was confirmed that the A400 value decreased by reducing the DTH concentration across the entire pH range, and it was confirmed that, in particular, setting the DTH content to 20 parts by mass or less relative to 100 parts by mass of EGT significantly inhibited coloration over time.

[0094] (Experimental Example 3. DTH Reduction Experiment in EGT Fermentation) Microbial production of EGT using *E. coli* strains exhibiting the EGT synthesis gene was attempted (Prior Art: International Publication No. 2019 / 163767). Extensive research was conducted, and it was incidentally discovered that using a strain with the metJ gene disrupted significantly reduced the DTH content in the culture medium. Examples are described below.

[0095] A strain of *E. coli* lacking the gene encoding metJ was obtained from *E. coli* strain BW25113 (NBRC103404) prepared by Nara Institute of Science and Technology according to the method described in *Molecular Systems Biology* (Mol. Syst. Biol.), 2006;2:2006.0008 (Baba, T. et al., Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection).

[0096] Furthermore, the *E. coli* ΔmetJ strain can also be prepared using the following method. It is constructed using the following known method: *E. coli* strain BW25113 is deleting gene regions using homologous recombination with a counter-selection vector (containing the sacB gene and the drug resistance gene), the counter-selection vector containing the upstream and downstream sequences of the metJ gene. The construction of the disrupted strain can be referenced in *M. Donnenberg* and *M. Kaper*, *Infection and Immunity*, 1991, 4310-4317, or in *M. Mizoguchi* et al., *Bioscience, Biotechnology and Biochemistry*, 2007, 71(12), 2905-2911.

[0097] The EGT production vector uses pACG-EGT (Prior Art: International Publication No. 2019 / 163767). EGT production strains (BW25113 / pACG-EGT, ΔmetJ / pACG-EGT) are obtained by transformation using strain BW25113 and strain ΔmetJ with pACG-EGT.

[0098] The constructed EGT production strain ΔmetJ / pACG-EGT was used to conduct fermentation production experiments of EGT. BW25113 / pACG-EGT, obtained by introducing pACG-EGT into strain BW25113, was used as the control strain. The glycerol stock solutions of BW25113 / pACG-EGT and ΔmetJ / pACG-EGT were added to test tubes containing tetracycline (final concentration 10 μg / mL) in 3 mL LB medium (10 g polypeptone, 5 g yeast extract, 10 g NaCl / 1 L medium). The tubes were then cultured at 30°C and 200 rpm with shaking for 15–18 hours until the stationary phase was reached, yielding the pre-culture medium. Subsequently, in 5 mL of 2×YT medium (polypeptone 16 g, yeast extract 10 g, NaCl 5 g, glucose 5%, L-histidine 5 mM, L-cystine 2.5 mM, L-methionine 5 mM, tetracycline 10 μg / mL / 1 L medium) in a Φ24 mm test tube, the pre-culture medium was added to achieve an optical density (OD) of 0.5, and the tube was shaken at 30°C and 300 rpm for 96 hours to initiate formal culture.

[0099] The quantification of EGT and DTH was performed as follows. During the formal culture period, 1 mL of culture medium was collected at 24, 48, 72, and 96 hours after the start of culture to evaluate the culture samples. The cell turbidity (OD600) of the collected culture medium was measured, and then centrifuged at 14,000 rpm for 10 minutes to separate the in vitro culture medium and the precipitate (cells). The EGT content in the separated in vitro culture medium (in vitro sample) was determined by high performance liquid chromatography (HPLC). The HPLC determination conditions are as described in Table 5 below, and quantification was based on the peak value of EGT generated around 10 minutes of holding time and the peak value of DTH generated around 8 minutes of holding time.

[0100] [Table 5] tubular Chromatography column (Inertsil) HILIC (5 μM; 4.6 Φ mm × 250 mm) Manufactured by GL Sciences solvent 100 mM ammonium acetate / acetonitrile / isopropanol / water (2:82:6:10) gradient condition isometry Detection wavelength 265 nm flow 1 mL / min Column temperature 40℃

[0101] The yields of EGT and DTH were investigated by culturing WT / pACG-EGT and ΔmetJ / pACG-EGT. As shown in Figures 4 and 5, when using the ΔmetJ strain, the amount of DTH in the culture medium at 96 hours was significantly reduced to about 1 / 7 compared with WT.

[0102] none

Claims

1. A composition comprising L-ergothioneine and N,N-dimethyl-L-2-thiohistamine, wherein the content of N,N-dimethyl-L-2-thiohistamine is reduced in the composition by extraction, synthesis, or fermentation, and the content of N,N-dimethyl-L-2-thiohistamine is 20 parts by weight or less relative to 100 parts by weight of L-ergothioneine.

2. The composition as described in claim 1, wherein, The coloring of the composition is inhibited by reducing the content of the N,N-dimethyl-L-2-thiohistamine.

3. The composition as described in claim 1 or claim 2, wherein, The content of N,N-dimethyl-L-2-thiohistidine is less than 10 parts by mass relative to 100 parts by mass of L-ergothioneine.

4. The composition as described in claim 1 or claim 2, wherein, The content of N,N-dimethyl-L-2-thiohistidine is 0.0001 parts by mass or more relative to 100 parts by mass of L-ergothioneine.

5. The composition as described in claim 1 or claim 2, wherein, The content of N,N-dimethyl-L-2-thiohistidine is 0.05 parts by mass or more relative to 100 parts by mass of L-ergothioneine.

6. A method for manufacturing a composition comprising L-ergothioneine and N,N-dimethyl-L-2-thiohistidine with inhibited coloring, wherein the method comprises: In the fermentation process, the step of reducing the content of N,N-dimethyl-L-2-thiohistamine is performed such that, relative to 100 parts by weight of L-ergothioneine, the content of N,N-dimethyl-L-2-thiohistamine is 20 parts by weight or less.

7. A method for manufacturing the composition as described in claim 6, wherein, The content of N,N-dimethyl-L-2-thiohistidine is less than 10 parts by mass relative to 100 parts by mass of L-ergothioneine.

8. A method of manufacturing a composition as claimed in claim 6 or claim 7, wherein the step of reducing the content of the N,N-dimethyl-L-2-thiohistidine comprises a step of deleting the metJ gene of bacteria, said step of deleting the metJ gene comprising using a method selected from homologous recombination, mutation treatment, and genome editing.

9. A method for imparting a color-inhibiting effect to a composition, wherein the composition contains L-ergothioneine and N,N-dimethyl-L-2-thiohistidine, and wherein, in an extraction, synthesis, or fermentation process, the content of N,N-dimethyl-L-2-thiohistidine is 20 parts by mass or less relative to 100 parts by mass of L-ergothioneine.

10. The method for imparting a color-inhibiting effect to a composition as described in claim 9, wherein, The content of N,N-dimethyl-L-2-thiohistidine is less than 10 parts by weight relative to 100 parts by weight of L-ergothioneine.

11. The method for imparting a staining inhibition effect to a composition as described in claim 9 or claim 10, comprising the step of deleting the metJ gene of bacteria, said step of deleting the metJ gene comprising using a method selected from homologous recombination, mutation treatment, and genome editing.

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