Compositions containing l-ergothioneine

Abstract

To provide a composition having enhanced stability of L-ergothioneine (EGT), and to provide a method for producing the same.SOLUTION: To provide a composition containing EGT in which the content of N,N-dimethyl-L-2-thiohistidine (DTH) is reduced because the EGT is destabilized and discoloration of the composition containing the EGT occurs when the DTH coexists in the composition containing the EGT.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a composition containing L-ergothioneine (EGT), and more particularly to a composition that enhances the stability of L-ergothioneine (EGT) in the presence of N,N-dimethyl-L-2-thiohistidine (DTH), and a method for producing the same. [Background technology]

[0002] L-ergothioneine (sometimes referred to as "EGT" in this specification) is a sulfur-containing amino acid that is known to have a variety of physiological activities, including antioxidant activity.

[0003] For example, Non-Patent Document 1 describes that EGT has the effect of improving the efficacy of immunotherapy using a cancer vaccine consisting of a tumor-associated antigen (TAA) and an adjuvant. Patent Document 1 also describes that EGT has the effect of treating bacterial infections. Experiment 1 in Patent Document 1 confirmed that 5 mM and 50 mM EGT inhibited the growth of Escherichia coli, and therefore it can be understood that the inhibition of bacterial infections described in Patent Document 1 is due to EGT directly inhibiting bacterial growth.

[0004] In Non-Patent Document 2, it was reported that the production of cytokines (IL-6, IL-12p40, IL-1β, IL-10) by mouse bone marrow-derived macrophages under stimulation conditions with toll-like receptor ligands was promoted by 10 mM EGT but not by 1 mM EGT, and that the production of cytokines (IL-6, IL-12p40, IL-1β, IL-10) by mouse bone marrow-derived macrophages under stimulation conditions with toll-like receptor ligands was promoted by 10 mM EGT but not by 1 mM EGT. + In co-culture with T cells, OT-II CD4 + It has been reported that 30 mM EGT promotes Th17 polarization of T cells. Thus, Non-Patent Document 2 describes that high concentrations of EGT of 10 mM or more activate macrophages.

[0005] Non-Patent Document 3 is a review of the physiological activity of EGT, and describes that EGT is absorbed and accumulated in the bodies of animals and plants. [Prior art documents] [Patent documents]

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

[0007] [Non-Patent Document 1] S. Yoshida et al., Front. Immunol. 10:671(2019) [Non-patent document 2] PLoS ONE 12(1):e0169360 [Non-patent document 3] Tamagawa University Faculty of Agriculture Research and Education Bulletin No. 1: 17-41 (2016) Summary of the Invention [Problem to be solved by the invention]

[0008] However, the formulation stability of EGT-containing compositions has not yet been fully investigated, and in particular, there have been no detailed reports on the effects of other coexisting ingredients in EGT-containing compositions. [Means for solving the problem]

[0009] In view of the above-mentioned problems, the present inventors have conducted extensive research and have discovered a novel problem that has not been reported before: when N,N-dimethyl-L-2-thiohistidine (DTH) coexists in a composition containing EGT, EGT becomes unstable, resulting in discoloration of the composition containing EGT.

[0010] To address this new problem, the present inventors discovered that by reducing the content of DTH in a composition containing EGT and DTH, the stability of EGT can be increased and discoloration of the composition can be suppressed, leading to the completion of the present invention.

[0011] That is, the present invention provides the compositions listed below.

[0012] [1] A composition comprising L-ergothioneine and N,N-dimethyl-L-2-thiohistidine, A composition having a reduced content of N,N-dimethyl-L-2-thiohistidine.

[0013] [2] The composition according to [1], wherein the content of N,N-dimethyl-L-2-thiohistidine is reduced, thereby suppressing coloration of the composition.

[0014] [3] The composition according to [1] or [2], wherein the content of the N,N-dimethyl-L-2-thiohistidine is 50 parts by mass or less per 100 parts by mass of the L-ergothioneine.

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

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

[0017] [6] A composition comprising L-ergothioneine and N,N-dimethyl-L-2-thiohistidine, the content of N,N-dimethyl-L-2-thiohistidine is 50 parts by mass or less per 100 parts by mass of L-ergothioneine; A composition for suppressing discoloration of the composition.

[0018] The present invention also relates to the following production methods.

[0019] [7] A method for producing a composition containing L-ergothioneine and N,N-dimethyl-L-2-thiohistidine and having reduced discoloration, comprising: A method comprising the step of reducing the content of N,N-dimethyl-L-2-thiohistidine.

[0020] The present invention also relates to the following methods.

[0021] [8] A composition comprising L-ergothioneine and N,N-dimethyl-L-2-thiohistidine, The content of N,N-dimethyl-L-2-thiohistidine is 50 parts by mass or less per 100 parts by mass of L-ergothioneine, A method for imparting a coloration suppression effect to the composition. [Effects of the Invention]

[0022] Since DTH has been found to be a component that affects the stability of EGT, it is possible to increase formulation stability and suppress discoloration of a composition in which EGT and DTH coexist by reducing the DTH content. [Brief explanation of the drawings]

[0023] [Figure 1] FIG. 1 is a graph showing the results of stability evaluation of a composition containing EGT and DTH in Test Example 1. [Figure 2] FIG. 2 is a graph showing the results of a coloration inhibition test for a composition containing EGT and DTH in Test Example 2. [Figure 3] FIG. 3 is a graph showing the results of a coloration inhibition test for a composition containing EGT and DTH in Test Example 2. [Figure 4] FIG. 4 is a graph showing the results of a test for reducing DTH in the EGT fermentation method in Test Example 3. [Figure 5] FIG. 5 is a graph showing the results of a test for reducing DTH in the EGT fermentation method in Test Example 3. DETAILED DESCRIPTION OF THE INVENTION

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

[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] [ka]

[0027] EGT can be obtained by known methods such as synthesis, extraction, and fermentation, and commercially available products can also be obtained and used.

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

[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] [ka]

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

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

[0033] EGT and / or DTH may be in the free form or in the form of a salt. The salt of EGT and / or DTH may be a salt formed with the carboxyl group in these structures, or a salt formed with the trimethylamino group or dimethylamino group. Furthermore, L-ergothioneine or an L-ergothioneine analog may be a solvate such as a hydrate.

[0034] Salts of EGT and / or DTH are not particularly limited as long as they are pharmacologically or physiologically acceptable, and specific 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; oxycarboxylic acid salts such as lactate, tartrate, and citrate; and organic sulfonates such as methanesulfonate, toluenesulfonate, and tosylate. 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. Examples of salts with inorganic bases include various salts with alkali metals such as sodium or potassium, alkaline earth metals such as calcium or magnesium, and metals such as aluminum. These salts of EGT and / or DTH may be used alone or in any combination of two or more. "Pharmaceutically or physiologically acceptable salts" may include solvates or hydrates of the salts.

[0035] In the following examples, the present inventors have discovered a novel problem not previously reported: when DTH coexists in a composition containing EGT, EGT becomes unstable, resulting in discoloration of the composition. Furthermore, the present inventors have discovered that by reducing the DTH content in a composition containing EGT and DTH, the stability of EGT can be increased 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 may be added to the composition containing EGT, or may be contained endogenously as a contaminant in an extraction method, or as a by-product in a synthesis method, fermentation method, or the like.

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

[0037] In the composition of the present invention, the content ratio of EGT and DTH is, from the viewpoint of suppressing discoloration of the composition and the viewpoint of EGT stability, preferably such that the content of DTH per 100 parts by mass of EGT is 50 parts by mass or less, more preferably 40 parts by mass or less, even more preferably 30 parts by mass or less, particularly preferably 20 parts by mass or less, and most preferably 10 parts by mass or less.

[0038] Furthermore, in the composition of the present invention, the content ratios of EGT and DTH are, from the viewpoint of production efficiency of the composition and the like, such that the content of DTH per 100 parts by mass of EGT is preferably 0.0001 parts by mass or more, more preferably 0.001 parts by mass or more, even more 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.

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

[0040] Furthermore, in the composition of the present invention, the content of EGT is appropriately adjusted depending on the formulation form, intended use, the type and content of other ingredients, etc., and is not limited thereto, and can be, for example, 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. Furthermore, the content of EGT can be, for example, 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., based on the total amount of the composition. In another embodiment, when prepared as a liquid formulation, the content of EGT can be, for example, but is not limited to, 80% by mass or less, relative to the total amount of the composition, including 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, and the like.

[0041] [Application] In a composition containing EGT and DTH, the stability of EGT can be increased by reducing the content of DTH, and therefore the present invention can be used to suppress coloration of the composition.

[0042] Furthermore, as described above, EGT is known to have various physiological activities, including antioxidant activity. By using the present invention, the stability of EGT can be increased and its degradation can be suppressed in compositions containing EGT and DTH, allowing EGT to exert its inherent physiological activities without impairing them.

[0043] By using the present invention, compositions containing EGT and DTH can also be suitably used for the physiological activities inherent to EGT, such as antioxidant, brain function improvement, anti-aging, eye disease treatment, skin whitening, UV absorption, melanin production inhibition, elimination of reactive oxygen species, elastase activity inhibition, wrinkle formation inhibition, skin sagging inhibition, skin blemish formation inhibition, dark circles around the eyes inhibition, UV-induced skin damage reduction (photoaging inhibition), dry skin, sensitive skin, hair improvement, and autophagy promotion.

[0044] The composition of the present invention can further be appropriately blended with active ingredients and additives that can be used in foods and beverages, functional foods, foods for specified health uses, quasi-drugs, pharmaceuticals, cosmetics, daily necessities, feed, etc., and can be appropriately formulated using known formulation methods used for the relevant items.

[0045] Cosmetics and daily necessities can be formulated into, for example, lotions, emulsions, gels, serums, creams, sunscreen creams, packs, masks, foundations, powders, bath additives, body lotions, shampoos, rinses, hair treatments, hair conditioners, hair styling products, hair tonics, toothpastes, mouthwashes, etc.

[0046] [formulation] The compositions of the present invention can be administered orally or parenterally (including topically) in solid formulations such as tablets, capsules, granules, and powders; or in liquid formulations such as solutions, syrups, injections, creams, lotions, pastes, ointments, emulsions (oil-in-water emulsions, water-in-oil emulsions, multiple emulsions, microemulsions, PET emulsions, and Pickering emulsions), gels (hydrogels and alcohol gels), and suspensions. Solid formulations may contain excipients, lubricants, binders, and disintegrants; liquid formulations may contain solvents, solubilizers, emulsifiers, emulsion stabilizers, thickeners, humectants, suspending agents, isotonicity agents, buffers, and soothing agents. Additives such as preservatives, antioxidants, colorants, sweeteners, and flavorings may also be used as needed.

[0047] Examples of excipients include sugar alcohols such as sorbitol, mannitol, and xylitol; sugars such as glucose, sucrose, lactose, and fructose; crystalline cellulose, carmellose sodium, croscarmellose sodium, calcium hydrogen phosphate, wheat starch, rice starch, corn starch, potato starch, dextrin, β-cyclodextrin, light anhydrous silicic acid, titanium oxide, magnesium aluminometasilicate, talc, kaolin, and olive oil.

[0048] Examples of binders include cellulose derivatives such as methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, and hydroxypropylmethyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, acrylic acid polymers, gelatin, gum arabic, pullulan, pregelatinized starch, agar, tragacanth, sodium alginate, and propylene glycol alginate.

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

[0050] Examples of the solvent include water, alcohol, propylene glycol, macrogol, sesame oil, and corn oil.

[0051] Examples of lubricants include stearic acid, magnesium stearate, calcium stearate, polyoxyl stearate, cetanol, talc, hydrogenated oil, sucrose fatty acid ester, dimethylpolysiloxane, beeswax, and white beeswax.

[0052] Examples of solubilizing agents include polyethylene glycol, propylene glycol, mannitol, benzyl benzoate, ethanol, tris(hydroxymethyl)aminomethane, cholesterol, triethanolamine, sodium carbonate, and sodium citrate.

[0053] Examples of suspending agents and emulsifying agents include surfactants such as stearylamine, triethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glycerin monostearate; hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose; and waxes such as shellac wax, beeswax, carnauba wax, spermaceti, lanolin, liquid lanolin, reduced lanolin, hard lanolin, cyclic lanolin, lanolin wax, candelilla wax, Japan wax, montan wax, and rice wax.

[0054] Examples of isotonic agents include sodium chloride, glycerin, and D-mannitol.

[0055] Examples of the buffering agent include buffer solutions such as phosphate, acetate, carbonate, and citrate.

[0056] Examples of preservatives include parahydroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, and sorbic acid.

[0057] Antioxidants include, for example, sulfites and ascorbic acid.

[0058] When the composition of the present invention is made into a solid formulation, a manufacturing method known in the art can be used. For example, there is a method in which the composition is kneaded, passed through a screen to form an extrusion granule, and then pulverized and sized; or there is a method in which kneading water is added to the composition, and the mixture is granulated using a vertical granulator, followed by pulverization and sieving using a Comil. Another example is a method in which the formulation composition is compressed using a roller compactor, pulverized using a roll granulator, and sieved. Another example is a method in which the composition is granulated using a fluidized bed after pulverization. For example, when the composition is produced by direct compression, the composition may be mixed and then directly charged into a tablet press for tableting.

[0059] [Food and beverages] The composition of the present invention can also be used as a food or drink composition, and can be provided by being contained in a food or functional food. Examples of such foods or functional foods include cooked rice; various noodles including soba, udon, vermicelli, Chinese noodles, instant noodles, and cup noodles; beverages such as soft drinks, carbonated drinks, energy drinks, fruit drinks, lactic acid drinks, and sports drinks; curry roux, stews, and various soups; frozen desserts such as ice cream, ice sherbet, and shaved ice; confectioneries such as candy, cookies, candy, gum, chocolate, candy tablets, snacks, biscuits, jelly, jam, cream, and other baked goods; processed seafood and livestock foods such as kamaboko, hanpen, ham, and sausage; dairy products such as processed milk and fermented milk; oils and fats and oil-based processed foods such as salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, and dressings; condiments such as sauces, dressings, miso, soy sauce, and tare sauces; soups, stews, salads, side dishes, furikake, and pickles; and various other forms of health and nutritional supplements, functional foods, and foods for specified health uses.

[0060] Furthermore, supplements (powders, granules, soft capsules, hard capsules, tablets, chewable tablets, rapidly disintegrating tablets, syrups, liquids, etc.) containing the compositions of the present invention may be prepared.

[0061] The composition of the present invention can also be added to food for animals such as pets.

[0062] Additives may be added to foods and beverages as needed, including, for example, glucose, fructose, sucrose, maltose, sorbitol, trehalose, stevioside, rubusoside, corn syrup, lactose, mannitol, dextrin, citric acid, sodium citrate, tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbic acid, tocopherol, sodium erythorbate, glycerin, propylene glycol, glycerin fatty acid esters, polyglycerin 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, colorants, flavorings, and preservatives.

[0063] The composition of the present invention can be used for foods and beverages that are permitted to label their products as improving, preventing, or improving various symptoms or conditions. In the present invention, foods and beverages that are permitted to label their products as improving, preventing, or improving symptoms or conditions are foods and beverages that have efficacy approved or designated by a country or public organization, such as functional foods, foods with health claims such as foods for specified health uses, and foods for special dietary uses. Although names and regulations may change depending on the situation, the times, and the systems of each country, foods that are essentially the same are included in the present invention.

[0064] In the present invention, the amount of the composition of the present invention to be incorporated is not particularly limited, and is set appropriately depending on the purpose of application (such as the type of target disease or symptom), the target area to be applied, the gender and age of the recipient, the product form such as food or drink, functional food, food for specified health uses, quasi-drug, pharmaceutical, cosmetic, daily necessities or feed, the method and frequency of administration or ingestion of these, preferences, etc.

[0065] When using the composition of the present invention, the daily intake (application amount) can be an effective amount that allows EGT to exert its inherent physiological activity. For example, when ingested (applied) by a healthy adult, the daily intake (application amount) of EGT can be, for example, 0.005 to 4,000 mg, preferably 0.1 to 3,000 mg, more preferably 0.5 to 2,000 mg, even more preferably 1 to 1,000 mg, particularly preferably 2 to 500 mg, and most preferably 3 to 300 mg.

[0066] Furthermore, when the composition of the present invention is used as a food or beverage, it is preferable to make it into a functional food, although this is not limited thereto, from the viewpoint of being able to display the physiological activity that EGT inherently possesses. Among functional foods, functional food, nutritional functional food, nutritional supplement, and specified health food are listed, but functional food is preferred because its use can be clearly displayed.

[0067] [Applicable persons] The subjects to whom the composition of the present invention is applied are not particularly limited, as long as they are of an age that requires the physiological activity inherent in EGT. They may be people aged around 30 or older, who are susceptible to stress from their living environment, such as work, middle-aged people (aged between around 45 and 55), who are susceptible to physical changes due to changes in hormone balance and living environment, or elderly people (55 or older).

[0068] [pH] The pH of the composition of the present invention is appropriately set depending on the type and content of other formulated ingredients, the formulation, the method of use, etc., and is not limited as long as it is within a pharmaceutically or physiologically acceptable range, but can be, for example, pH 2 to 10. From the viewpoint of stably exerting the effects of the present invention, the pH of the composition of the present invention can be, for example, pH 2 to 10, pH 2 to 9, pH 2 to 8, pH 2 to 7, pH 3 to 10, pH 3 to 9, pH 3 to 8, pH 3 to 7, pH 4 to 10, pH 4 to 9, pH 4 to 8, pH 4 to 7, pH 5 to 10, pH 5 to 9, pH 5 to 8, pH 5 to 7, pH 6 to 10, pH 6 to 9, pH 6 to 8, pH 6 to 7, etc.

[0069] [Method for producing a composition containing EGT and DTH and suppressing coloration] In the present invention, the method for producing a composition containing EGT and DTH and suppressed in coloration includes a step of reducing the content of DTH.

[0070] The step of reducing the DTH content can be carried out by any known means as long as the DTH content is reduced. For example, when EGT and DTH are mixed to prepare a composition, a step of reducing the DTH content ratio relative to EGT can be employed. As described above, the manner in which DTH coexists in a composition containing EGT is not particularly limited. For example, DTH may be present as an impurity in an EGT extraction method, or as a by-product in a synthetic or fermentation method.

[0071] In order to reduce DTH in contaminants in the extraction of EGT from mushrooms, plants, etc., methods such as solvent extraction, separation based on differences in solubility, chromatography using adsorbents such as silica gel and alumina, chromatography such as cation exchange chromatography, anion exchange chromatography, hydrophobic chromatography, gel filtration, thiopropyl-Sepharose 6B, and reverse phase chromatography, crystallization, activated carbon treatment, membrane treatment, and combinations of these methods can be used.

[0072] Methods for producing EGT by fermentation using Escherichia coli or the like are known, such as those disclosed in International Publication No. 2019 / 163767. When reducing DTH in contaminants in EGT fermentation methods, for example, methods described in the Examples below can be used. Specifically, in EGT fermentation methods, by employing a step of deleting the metJ gene of bacteria belonging to the Enterobacteriaceae family that are capable of producing EGT, it becomes possible to reduce DTH in the culture solution and efficiently produce EGT.

[0073] The step of deleting the metJ gene can be carried out by known methods, such as knocking out or knocking down the metJ gene by homologous recombination, mutation treatment, genome editing, or the like. Examples of bacteria belonging to the Enterobacteriaceae family that have the ability to produce EGT include, but are not limited to, bacteria of the genera Escherichia, Enterobacter, Pantoea, Klebsiella, and Salmonella. Particularly preferred enterobacteria include bacteria of the genus Escherichia, such as Escherichia coli, and bacteria of the genus Pantoea, such as Pantoea ananatis.

[0074] Bacteria belonging to the Enterobacteriaceae family can be cultured by conventional methods. Specifically, LB medium, 2xYT medium, NZY medium, M9 medium, SOC medium, YPD medium, or the like can be used. EGT and DTH can be produced using the above-mentioned media, but the media used are not limited to these. Furthermore, the produced EGT and DTH may be accumulated intracellularly or secreted and accumulated extracellularly (in the culture medium).

[0075] EGT and DTH released from or within the bacterial cells can be collected by known methods. For example, the culture can be subjected to solid-liquid separation such as centrifugation or filtration, and an extract of EGT and DTH can be obtained from the bacterial cells by solvent extraction, hot water extraction, disruption, or other methods. EGT can be obtained from the EGT and DTH extract or the culture supernatant by known chromatography such as ion exchange chromatography, hydrophobic chromatography, or gel filtration chromatography.

[0076] In the means for reducing the content of various DTHs, the preferable content ratio of EGT and DTH is as described above.

[0077] The above production method can further include a step of adjusting the pH. The optimum pH range is as described above.

[0078] [Method for imparting a discoloration suppression effect to a composition containing EGT and DTH] In the present invention, there is provided a composition containing EGT and DTH, It is also possible to provide a method for imparting a discoloration suppression effect to the composition by setting the content of the DTH to 50 parts by mass or less per 100 parts by mass of the EGT. The preferred content ratios of EGT and DTH are as described above. [Example]

[0079] The present invention will now be described in detail with reference to examples and test examples, but the present invention is not limited to these examples and test examples. Unless otherwise specified, experiments were performed using methods described in standard protocol collections related to molecular biology and applied microbiology, or modified or altered methods. Furthermore, % represents w / v % unless otherwise specified.

[0080] (Test Example 1. Evaluation of Stability of Composition Containing EGT and DTH 1) The storage stability of a composition containing L-ergothioneine (EGT) and N,N-dimethyl-L-2-thiohistidine (DTH) under various pH conditions was investigated as follows.

[0081] EGT (manufactured by Tetraedron) and DTH (manufactured by Tetraedron) were dissolved in Britton-Robinson buffer (final concentration 30 mM) of pH 3, 4, 5, 6, 7, 8, 9, or 10 to obtain the final concentrations and pH of EGT and DTH as shown in Table 1, and 3.0 ml of mixed solutions of DTH and EGT with different pH and concentrations were prepared.

[0082] The broad-range Britton-Robinson buffer used to adjust the pH was prepared as follows: 20 ml of a boric acid, phosphoric acid, and acetic acid mixed solution (final concentration: 200 mM each) was taken and adjusted to pH 3, 4, 5, 6, 7, 8, 9, or 10 with 1N NaOH, and then diluted to 40 ml with water to prepare a 300 mM (100 mM each) Britton-Robinson buffer with a pH of 3 to 10. Furthermore, EGT and DTH were dissolved in the 300 mM Britton-Robinson buffer of each pH and water to a final concentration of 30 mM, thereby preparing the EGT and DTH mixed solutions shown in Table 1.

[0083] For each sample number, 1.0 ml of the EGT and DTH mixed solution listed in Table 1 was transferred into two tubes and allowed to stand at 30°C or 70°C for 21 days. The tubes were photographed immediately after the start (day 0), and on days 8, 14, and 21 to record the color change of the solution. Further, 100 μl samples were taken and subjected to quantification of EGT and DTH using high-performance liquid chromatography (Shimadzu Corporation) under the conditions listed in Table 2. Figure 1 shows a photograph of the solution taken on day 14.

[0084] As shown in Figure 1, among the samples stored at 70°C for 14 days, the 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 test samples showed a change in color from clear to brownish-red. Furthermore, the degree of coloration increased with increasing DTH concentration. Furthermore, the degree of coloration increased with increasing DTH storage period. From the above, this test example confirmed for the first time the issue of coloration of EGT and DTH mixed solutions.

[0085] [Table 1]

[0086] [Table 2]

[0087] (Test Example 2. Evaluation 1 of Color Inhibition in Compositions Containing EGT and DTH) As a result of Test Example 1, a new problem was discovered in which the mixed solution of EGT and DTH became discolored. The degree of discoloration was quantified by absorbance measurement, and methods for suppressing discoloration were explored by examining various concentrations of EGT and DTH.

[0088] As in Test Example 1, EGT (manufactured by Tetraedron) and DTH (manufactured by Tetraedron) were dissolved in Britton-Robinson Buffer (final concentration 30 mM) so that the final concentrations and pH values ​​were as shown in Tables 3 and 4, to prepare 2.0 ml of an EGT and DTH mixed solution.

[0089] 1.5 ml of each EGT and DTH mixed solution listed in Tables 3 and 4 was transferred to a 2.0 ml tube, and 200 μl of mineral oil was layered on top of the solution to prevent evaporation. The prepared samples were incubated at 70°C for 28 days. 200 μl of each sample was transferred to a 96-well microplate immediately after initiation (day 0), and on days 4, 7, 14, 21, and 28. Absorbance measurements were performed at 250–800 nm using a MULTISKAN GO (Thermo Scientific). Figure 2 was generated using the absorbance data at 400 nm obtained from the absorbance measurements of the samples listed in Table 3. Furthermore, 100 μl of each sample after absorbance measurements was used to quantify EGT and DTH using high-performance liquid chromatography (Shimadzu Corporation) under the conditions listed in Table 2.

[0090] [Table 3]

[0091] [Table 4]

[0092] Figure 2 shows the results of a coloration suppression evaluation test using the samples listed in Table 3. As shown in Figure 2, the absorbance at 400 nm (A400 value), which indicates the degree of coloration, increased over time in the 100 mg / L EGT + 50 mg / L DTH sample at all pH levels. The coloration of the EGT and DTH mixed solution confirmed in Example 1 was also confirmed by the absorbance. Furthermore, various DTH concentrations were examined, and it was confirmed that the A400 value decreased with decreasing DTH concentration in all pH levels. It was found that, although not limited to, coloration over time was significantly suppressed by using a DTH content ratio of 20 parts by mass or less per 100 parts by mass of EGT. The quantitative analysis of EGT in each sample also demonstrated that, similar to coloration, decreasing the DTH concentration could suppress EGT decomposition.

[0093] Furthermore, in a discoloration suppression evaluation test using a mixed solution of EGT and DTH shown in Table 4, results similar to those of the samples in Table 3 were obtained. The results are shown in Figure 3. Specifically, it was confirmed that the A400 value decreased by lowering the DTH concentration in all pH groups, and in particular, it was confirmed that discoloration over time could be significantly suppressed by setting the content ratio of DTH to 100 parts by mass of EGT at 20 parts by mass or less.

[0094] (Test Example 3: DTH Reduction Test in EGT Fermentation Method) We attempted to produce EGT using Escherichia coli expressing an EGT synthetic gene (Prior Art: WO 2019 / 163767). After extensive research, we discovered that the DTH content in the culture medium was significantly reduced by using a strain in which the metJ gene had been disrupted. Examples of this are described below.

[0095] The E. coli ΔmetJ strain, which lacks the metJ gene, was obtained from the Nara Institute of Science and Technology according to the method described in 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). The strain was prepared from the E. coli BW25113 (NBRC103404) strain according to the method described in 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] The E. coli ΔmetJ strain can also be prepared, for example, by the following method. It was constructed by a conventional method in which each gene region of the E. coli BW25113 strain was deleted by homologous recombination using a counterselection vector (containing the sacB gene and a drug resistance gene) containing upstream and downstream sequences of the metJ gene. For the construction of the disrupted strain, see, for example, MS Donnenberg and JS Kaper, Infect. Immun., 1991, 4310-4317 and H. Mizoguchi et al., Biosci. Biotechnol. Biochem., 2007, 71(12), 2905-2911.

[0097] The EGT production vector used was pACG-EGT (Prior Art: WO 2019 / 163767). The BW25113 and ΔmetJ strains were transformed with pACG-EGT to obtain EGT-producing strains (BW25113 / pACG-EGT, ΔmetJ / pACG-EGT).

[0098] EGT fermentation production tests were performed using the EGT-producing strain ΔmetJ / pACG-EGT constructed above. BW25113 / pACG-EGT, a control strain prepared by introducing pACG-EGT into BW25113, was used as the control strain. Glycerol stocks of BW25113 / pACG-EGT and ΔmetJ / pACG-EGT were added to test tubes containing 3 mL of LB medium (10 g of polypeptone, 5 g of yeast extract, and 10 g of NaCl per liter of medium) and tetracycline (final concentration: 10 μg / mL). The strains were cultured at 30°C and 200 rpm for 15–18 hours with shaking until they reached stationary phase, yielding a preculture medium. Furthermore, the preculture solution was added to 5 mL of 2xYT 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 of medium) in a 24 mm diameter test tube to give an OD of 0.5, and the main culture was carried out by shaking at 300 rpm at 30°C for 96 hours.

[0099] EGT and DTH were quantified as follows. During the main culture, 1 mL of culture medium was collected at 24, 48, 72, and 96 hours after the start of culture, and the culture samples were evaluated. The collected culture medium was measured for bacterial cell turbidity (OD600), and then centrifuged at 14,000 rpm for 10 minutes to separate the extracellular culture medium and the precipitate (bacterial cells). The EGT content in the separated extracellular culture medium (extracellular sample) was measured by HPLC. The HPLC measurement conditions are shown in Table 5 below, and quantification was performed based on the EGT peak that appeared around 10 minutes retention time and the DTH peak that appeared around 8 minutes retention time.

[0100] [Table 5]

[0101] We cultured WT / pACG-EGT and ΔmetJ / pACG-EGT and examined the production of EGT and DTH. As shown in Figures 4 and 5, we found that the amount of DTH in the culture medium after 96 hours was significantly reduced to approximately 1 / 7 of that in the WT strain when the ΔmetJ strain was used.

Claims

[Claim 1] A composition comprising L-ergothioneine and N,N-dimethyl-L-2-thiohistidine, The composition has a reduced content of N,N-dimethyl-L-2-thiohistidine.

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