Extraction method of high-concentration ergothioneine

Abstract

The invention discloses an extraction method of high-concentration ergothioneine. The extraction method comprises the following extraction steps: S1, obtaining mushroom sporocarp containing ergothioneine; s2, dissolving out the ergothioneine in the mushroom sporocarp by using a solvent, and obtaining an extracting solution containing the ergothioneine; s3, carrying out centrifugal treatment on the extracting solution, and separating to obtain a supernatant and a precipitate; s4, the supernate is a high-concentration ergothioneine product; and freeze-drying the precipitate to prepare dry powder, and repeatedly carrying out dissolution and centrifugal treatment on the ergothioneine until the precipitate is not separated out. According to the invention, the ergothioneine can be efficiently produced at low cost. By means of the present invention, it is possible to provide practical, safe and low-cost ergothioneine that is suitable for use in food, medicine, dental medicine, cosmetics, feed, and the like, without relying on a chemical synthesis method, a microbial culture method, or a fermentation method.

Description

Technical Field

[0001] The present invention relates to an extraction method, in particular to a method for extracting high-concentration ergothioneine. Background Art

[0002] Ergothioneine is a rare amino acid derivative and a natural substance with strong antioxidant properties. It was first isolated and discovered by M.C. Tarnet in 1909 from the ergot fungus (Claviceps purpurea) found in rye grain. Ergothioneine is a water-soluble substance with excellent heat resistance and pH stability. It is virtually unaffected by heating at 120°C for 60 minutes and remains virtually unaffected even in acidic (pH 2) to alkaline (pH 12) conditions. Humans cannot synthesize ergothioneine naturally; it must be ingested through food and stored in the brain, liver, kidneys, red blood cells, and skin. Generally, only certain basidiomycetes, ascomycetes, and some bacteria possess the ability to synthesize ergothioneine. Edible fungi, especially the elm yellow mushroom, are rich in ergothioneine. Shiitake and oyster mushrooms also contain ergothioneine.

[0003] With the discovery of the ergothioneine transporter (OCTN1), its functions in vivo have garnered widespread attention. Ergothioneine possesses a potent ability to scavenge reactive oxygen species (ROS), with an antioxidant capacity 3 to 30 times greater than that of glutathione. It rapidly scavenges ROS, including hydroxyl radicals and singlet oxygen, and its activity is stronger than that of other antioxidants, such as vitamin C and L-cysteine. It has also been studied for its potential applications in preventing the darkening of fresh shrimp and crab, preventing oxidative spoilage of fish and meat, and preventing browning of the hematochezia of farmed fish. Furthermore, although ergothioneine is water-soluble, research reports indicate that it can cross the blood-brain barrier and be stored in the central nervous system. This suggests its potential therapeutic potential for improving memory, cognitive impairment, Alzheimer's disease, depression, and other neurological and central nervous system disorders. Ergothioneine is also present in significant quantities in the lens of the eye and skin, accumulating particularly in metabolically active cells. It is therefore believed to inhibit UV-induced oxidative reactions and genetic damage. Its transporter, OCTN1, has been implicated in a variety of diseases, including rheumatoid arthritis, ulcerative colitis, Crohn's disease, and autoimmune thyroid disease. This further suggests that ergothioneine may play a crucial role in maintaining physiological homeostasis in the human body. Consequently, the efficacy and application of ergothioneine in functional health foods, pharmaceuticals, and cosmetics has garnered widespread attention in recent years.

[0004] The traditional method of making ergothioneine is: Chemical synthesis: Ergothioneine is synthesized using expensive and toxic chemical reagents.

[0005] Fermentation method: Cultivate ascomycetes or basidiomycetes that can produce ergothioneine, and extract and purify ergothioneine from the culture medium.

[0006] Wherein, chemical synthesis uses the chemical drugs with toxicity, is therefore not suitable for food purposes.And, the method also needs to use expensive synthetic reagent, is therefore difficult to low-cost synthesis thioneine.In the production process using the mold microorganisms such as yeast, monascus to carry out fermentation extraction, microbial culture and fermentation time are longer, and culture cost is high simultaneously, is difficult to realize low-price production.

[0007] At present, confirmed, including citrinum citrinum, moon night mushroom, Agaricus bisporus, maitake mushroom, shiitake mushroom, king oyster mushroom and flat mushroom etc., all can generate thioneine.Therefore the common method of directly extracting thioneine is: the fruiting body of mushroom or its dry thing are added in water isosolv, adopt hot water etc. mode to make thioneine be dissolved in solvent, obtain the extract containing thioneine, then utilize the chromatography of adsorption chromatography, cation exchange resin or anion exchange resin, and the method such as high speed liquid chromatography to purify thioneine.But said method not only uses expensive equipment and resin, and refining step is various, and it takes time, and extraction efficiency is also low, is therefore difficult to low-cost manufacturing high-concentration thioneine solution. Summary of the Invention

[0008] In order to overcome the shortcomings of the above-mentioned technology, the present invention provides a method for extracting high-concentration ergothioneine.

[0009] In order to solve the above technical problems, the technical solution adopted in the present invention is: a kind of extraction method of high-concentration ergothioneine, including following extraction step: S1. Obtaining mushroom fruiting bodies containing ergothioneine; S2, using a solvent to dissolve thioneine in the mushroom fruiting body, and obtaining an extract containing thioneine; S3, centrifuging the extract to separate the supernatant and the precipitate; S4, described supernatant are high concentration thioneine products; Described precipitate is made into dry powder through lyophilization, to repeat the dissolving and centrifugation of thioneine, until can not separate precipitate.

[0010] Preferably, in step S1, the mushroom fruiting body is an ascomycete or basidiomycete mushroom fruiting body.

[0011] Preferably, in step S1, the mushroom fruiting body is a Pleurotus citrinopileatus fruiting body.

[0012] Preferably, in step S1, the mushroom fruiting bodies are in the form of dry powder, the moisture content of the dry powder is below 1%, and the particle size ranges from 50 μm to 200 μm.

[0013] Preferably, in step S1, the mushroom fruiting bodies are in a fresh state.

[0014] Preferably, in step S2, the solvent for thioneine extraction is: an aqueous solution containing low concentration methanol, ethanol, isopropanol, acetone or a pure aqueous solution.

[0015] Preferably, the water in the solvent is deionized water.

[0016] Preferably, in step S2, the dissolution temperature of thioneine extraction is controlled between room temperature and 121°C.

[0017] Preferably, in step S2, the dissolution temperature of thioneine extraction is 100°C.

[0018] Preferably, in step S3, the centrifugation condition is: centrifugation at a speed of 5000 rpm for 20 minutes.

[0019] The invention discloses a method for extracting high-concentration ergothioneine, which can produce ergothioneine efficiently and at low cost. By means of the present invention, ergothioneine suitable for use in food, medicine, dental medicine, cosmetics, feed, etc., can be provided, which is practical, safe, and low-cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the appearance of the extracts of the present invention with different dry powder particle sizes. DETAILED DESCRIPTION

[0021] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0022] The invention discloses a method for extracting high-concentration ergothioneine, comprising the following extraction steps: S1. Obtaining mushroom fruiting bodies containing ergothioneine; S2, using a solvent to dissolve thioneine in the mushroom fruiting body, and obtaining an extract containing thioneine; S3, centrifuging the extract to separate the supernatant and the precipitate; S4, described supernatant are high concentration thioneine products; Described precipitate is made into dry powder through lyophilization, to repeat the dissolving and centrifugation of thioneine, until can not separate precipitate.

[0023] In step S1, the mushroom fruiting bodies used for extraction are ascomycete or basidiomycete mushroom fruiting bodies, such as Pleurotus ostreatus fruiting bodies, Coprinus comatus fruiting bodies, or Pleurotus cornucopiae fruiting bodies. Among them, Pleurotus cornucopiae is preferably used. Pleurotus cornucopiae is rich in ergothioneine and is an edible and safe mushroom.

[0024] The mushroom fruiting bodies can be fresh or dried, with dried, powdered fruiting bodies being preferred as the extraction raw material. Fresh mushrooms retain their intact tissue structure, resulting in lower extraction efficiency. However, after drying and pulverizing, the tissue is broken down, resulting in higher extraction efficiency.

[0025] The moisture content of the dry powder should be controlled below 1%, and the particle size range should be 50μm to 200μm.

[0026] The range of dry particle size is obtained through a lot of experiments. Figure 1 The present invention shows some comparative results of extracting ergothioneine from powder using dry powder with a particle size of 500 μm and 50-200 μm.

[0027] The experimental method in Table 1 was to add various powders to water and boil them for 5 minutes, 10 minutes, 20 minutes, and 30 minutes, respectively. The content of ergothioneine was determined by reverse-phase HPLC. As shown in Table 1, the amount of ergothioneine extracted from powders with a particle size of 50-200 μm was more than 10 times that of powders with a particle size of 500 μm, and the extraction time of powders with a particle size of 50-200 μm was shorter, indicating that the degree of pulverization has a significant effect on the extraction efficiency.

[0028] Table 1. Effect of the particle size of dried powder of Pleurotus citrinopileatus on the ergothioneine content

[0029]

[0030] Furthermore, Figure 1 The effect of dry powder particle size differences on the extract is shown. In the figure, ① is fresh erinaceus, ② is dry powdered erinaceus with a particle size of 50 to 200 μm, and ③ is dry powdered erinaceus with a particle size of more than 500 μm. Figure 1 It can be seen that the color of the extract of dry powdered Pleurotus citrinopileatus with a particle size of 50 to 200 μm is the best.

[0031] In step S2, the solvent that thioneine leaches can adopt the aqueous solution or the pure water solution containing low concentration methanol, ethanol, isopropanol, acetone;Pure water solution can be warm water or hot water, and the water used is deionized water.Because thioneine is soluble in water, it is preferred to use pure water solution.

[0032] Furthermore, the extraction temperature must be controlled below 200°C to avoid thermal decomposition of ergothioneine; typically, the dissolution temperature is controlled between room temperature and 121°C, preferably 100°C.

[0033] For the optimization of extraction temperature, the thioneine extraction amount of fresh Pleurotus citriodora under different temperature treatments is used as an example to illustrate that the thioneine can be quantitatively determined by reversed-phase HPLC. As shown in Table 2, under boiling temperature conditions, the thioneine extraction amount can reach more than 1 mg / g in only 15 minutes.

[0034] Table 2. Ergothioneine extraction yields from fresh Pleurotus citrinopileatus treated at different temperatures

[0035] 60℃ 70℃ Boiling (100°C) 1.133 mg / g per hour 0.619 mg / g per hour 1.431 mg / g in 15 minutes 1.423 mg / g per hour 0.500 mg / g per hour 1.370 mg / g in 15 minutes

[0036] Furthermore, after adding the mushroom fruiting body obtained in S1 to a pure aqueous solution (warm water or hot water) or an aqueous solution containing low concentrations of methanol, ethanol, isopropanol, or acetone, the mixture is kept for at least 1 hour to obtain an extract, and the extract is centrifuged at 5000 rpm for 20 minutes to separate into a supernatant and a precipitate.

[0037] In step S4, gained supernatant is high concentration thioneine product, can be directly used or rotary evaporator is concentrated and used according to purposes.

[0038] In another aspect, the precipitate is freeze-dried to a dry powder, which is then placed back into a pure aqueous solution (warm or hot water) or an aqueous solution containing low concentrations of methanol, ethanol, isopropanol, or acetone for at least 39 minutes. The powder is then centrifuged again at 5,000 rpm for 20 minutes to separate the supernatant and precipitate. This process is repeated until no precipitate is separated, thereby maximizing the extraction of ergothioneine from the mushroom fruiting bodies.

[0039] It should be noted that the quantification of ergothioneine can be carried out by colorimetric quantitative method (reference Biochem. Journ. 139, 221-235 (1974)) or reversed-phase HPLC method (reference Int. Journ. Med. Mushrooms, 8, 215-222 (2006)). The separation conditions of HPLC can be appropriately selected by professionals.

[0040] The colorimetric method was performed as follows: 100 μL of 1 M glycine-sodium hydroxide buffer (pH 9.5) and 100 μL of 1 mM copper sulfate were added to 900 μL of sample, and the mixture was shaken at room temperature for 1 hour to oxidize the SH compounds. Then, 240 μL of 1 M hydrochloric acid and 1.5 mM 2,2'-dithiopyridine were added to 200 μL of oxidized sample, and the absorbance at 343 nm was measured after mixing.

[0041] The quantitative conditions of the reverse phase HPLC method are as follows: - Chromatographic column: Econosphere C18 (46 x 250 mm), two columns connected in series; - Mobile phase: 50 mM sodium phosphate buffer (pH 7.3), 3% acetonitrile, 0.1% triethylamine; - Flow rate: 1.0 ml / min;

[0042] - Detection wavelength: 254nm UV absorption.

[0043] In summary, the extraction method of high-concentration thioneine disclosed in the present invention, with the elm yellow mushroom fruiting body (or yellow flat mushroom fruiting body or coprinus comatus fruiting body) with thioneine production capacity as raw material, adopts a method completely different from the traditional method to extract thioneine, high-concentration thioneine product can be obtained only by dissolving and centrifuging, without the need for adsorption chromatography, chromatography of cation exchange resin or anion exchange resin, and methods such as high-speed liquid chromatography, with the advantages of high efficiency and low cost, is a novel method that can extract high-concentration thioneine extract.

[0044] [Example]

[0045] The extraction method of the present invention is further described below through experimental determination.

[0046] 40 grams of dried powder of Pleurotus citrinopileatus (particle size 50-200 μm) was added to a hot water solution at 100°C for at least 60 minutes. The solution was then centrifuged at 5000 rpm for 20 minutes. The ergothioneine content of the supernatant was determined using the reverse-phase HPLC method described above, revealing approximately 0.03% ergothioneine.

[0047] This method was used to determine the ergothioneine content of other commercially available mushrooms, including Pleurotus ostreatus, Lentinus edodes, Shiitake mushroom, Chestnut mushroom, King oyster mushroom, Flammulina velutipes, Pleurotus ostreatus, Hydrangea serrata, Agrocybe serrata, Coprinus comatus, and Coprinus cinereus. The measurement data were provided by Misatomushfarm. The specific measurement results are shown in Table 3 below: Table 3. Ergothioneine content in different mushrooms

[0048] According to the method of Example 1, after a treatment, the precipitate obtained by Pleurotus citriodora is freeze-dried, and then 4g of its freeze-dried powder is taken, and it is put into a pure aqueous solution such as warm water or hot water at 60°C for 30 minutes, and then centrifuged at 5,000 rpm for 20 minutes to obtain a supernatant, in which the content of thioneine is about 0.14%. That is, by the inventive method, a thioneine extract having a concentration of about 5 times that of the initial can be obtained.

[0049] The above embodiments are not limitations of the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by technicians in this technical field within the scope of the technical solution of the present invention also fall within the scope of protection of the present invention.

Claims

1. an extracting method for high-concentration thioneine, characterized in that: The extraction steps include: S1. Obtaining mushroom fruiting bodies containing ergothioneine; S2, using a solvent to dissolve thioneine in the mushroom fruiting body, and obtaining an extract containing thioneine; S3, centrifuging the extract to separate the supernatant and the precipitate; S4, described supernatant are high concentration thioneine product; The precipitate is freeze-dried to prepare dry powder, and the dissolution and centrifugation of thioneine are repeated until the precipitate cannot be separated.

2. the extracting method of high concentration thioneine according to claim 1, is characterized in that: In step S1, the mushroom fruiting body is an ascomycete or basidiomycete mushroom fruiting body.

3. the extracting method of high concentration thioneine according to claim 2, is characterized in that: In step S1, the mushroom fruiting body is a Pleurotus citrinopileatus fruiting body.

4. the extracting method of high concentration thioneine according to claim 3, is characterized in that: In step S1, the mushroom fruiting body is in the form of dry powder, the moisture content of the dry powder is below 1%, and the particle size range is between 50 μm and 200 μm.

5. the extracting method of high concentration thioneine according to claim 3, is characterized in that: In step S1, the mushroom fruiting bodies are in a fresh state.

6. according to the extracting method of high concentration thioneine according to claim 4 or 5, it is characterized in that: In step S2, the solvent extracted by described thioneine is: an aqueous solution or a pure water solution containing low concentration methanol, ethanol, isopropanol, or acetone.

7. the extracting method of high concentration thioneine according to claim 6, is characterized in that: The water in the solvent is deionized water.

8. the extracting method of high concentration thioneine according to claim 6, is characterized in that: In step S2, the dissolution temperature of the thioneine extraction is controlled between room temperature and 121°C.

9. the extracting method of high concentration thioneine according to claim 8, is characterized in that: In step S2, the dissolution temperature of the thioneine extraction is 100°C.

10. the extracting method of high concentration thioneine according to claim 9, is characterized in that: In step S3, the centrifugation condition is: centrifugation at a speed of 5000 rpm for 20 minutes.

Images