Purification method of ergothioneine

Abstract

The invention provides a purification method of ergothioneine, and belongs to the field of ergothioneine purification. The method comprises the steps of fermentation liquor pretreatment, ultrafiltration, column chromatography and concentration crystallization. The fermentation liquor pretreatment step comprises the following steps: adjusting the pH value of fermentation liquor containing ergothioneine to 5.8-6.0, and adding a pretreatment agent for treatment; a preparation method of the pretreatment agent comprises the following steps: putting activated carbon into a potassium permanganate solution, uniformly stirring, filtering, washing, drying, heating to 246-253 DEG C, carrying out heat preservation treatment for 3.3-3.7 hours, putting into an ethanol solution after heat preservation treatment, adding hexadecyl trimethyl ammonium bromide, carrying out ultrasonic treatment, adding glutamine, continuously stirring for 28-32 minutes, filtering, washing, drying, heating to 246-253 DEG C, carrying out heat preservation treatment for 3.3-3.7 hours, putting into an ethanol solution after heat preservation treatment is finished, and filtering to obtain the pretreatment agent. After stirring, filtering, washing and drying to obtain a pretreatment agent; by adopting the purification method provided by the invention, the purity and yield of ergothioneine are high, and the process is simple.

Description

Technical Field

[0001] The invention belongs to the field of ergothioneine purification, and particularly relates to a method for purifying ergothioneine. Background Art

[0002] Ergothioneine, chemically known as 2-mercaptohistidine trimethylol salt, is a rare natural small molecule chiral amino acid that was first discovered in a fungus. The pure product is white crystals, water-soluble, and will not self-oxidize at physiological pH and in strong alkaline solutions. Ergothioneine is a natural amino acid derived from a plant and accumulated in animals. It is a safe, non-toxic natural antioxidant that is widely used in the fields of medicine, food, health care products, and cosmetics. Specifically, ergothioneine, as a special antioxidant, can chelate heavy metal ions to protect red blood cells in the matrix from free radical damage. In terms of organ transplantation, it can be used as a substitute for glutathione to better protect transplanted organs. When added to cosmetics, it can minimize the generation of reactive oxygen species and protect cells from radiation damage, and can be used to develop outdoor skin care products and protective cosmetics. In ophthalmology, ergothioneine plays a key role in protecting the eyes. In the medical field, it can be used to treat inflammation, etc., and can be made into tablets, capsules, and oral preparations.

[0003] At present, the production methods of ergothioneine mainly include chemical synthesis, natural biological extraction, enzyme catalysis and microbial fermentation synthesis; Ergothioneine is a chiral amino acid. It is difficult to synthesize left-handed ergothioneine by chemical synthesis, and the synthesis steps are cumbersome, and the product safety is difficult to ensure; the natural biological extraction method is to extract from edible fungi rich in ergothioneine, which has many impurities and high drug residues, increases the difficulty of extraction and purification, and makes the extracted components higher, which cannot meet market demand; the enzyme catalysis method is to synthesize ergothioneine by enzyme catalysis, which is greatly affected by enzyme activity, has a long catalytic cycle, and has low yield; The microbial fermentation method has the advantages of high yield, stable production, and low cost. It can effectively control the yield of ergothioneine through metabolic regulation, culture medium optimization, and fermentation condition optimization, thereby improving the safety of the product and becoming the mainstream method for ergothioneine production. However, thioneine has good water solubility, and separation, extraction and purification are relatively difficult. The current purification process purification method is relatively complicated, and the leaching method usually adopts organic solvents or other chemical reagents, and it is necessary to remove additional organic solvents or other chemical reagents during purification; the thioneine purity obtained by high performance liquid chromatography is higher, and the finished product is high, and it is impossible to prepare on a large scale; the separation efficiency of ion exchange method and alumina chromatography method is low, and product purity and yield cannot be taken into account at the same time; Therefore, a method for purifying thioneine is provided, and the yield and purity are higher, the process is simple, and the cost is low, which is a technical problem to be solved urgently in the prior art. Summary of the invention

[0004] In order to solve the technical problems existing in the prior art, the invention provides a method for purifying ergothioneine, which has high product purity, high yield, simple process and low cost.

[0005] In view of the above technical problems, the present invention adopts the following technical solutions: A method for purifying thioneine comprises fermentation broth pretreatment, ultrafiltration, column chromatography and concentration and crystallization steps, which is specifically as follows: 1. Fermentation broth pretreatment The fermentation liquid containing ergothioneine is adjusted to pH 5.8-6.0, a pretreatment agent is added for stirring, the stirring time is 2.2-2.4h, the stirring temperature is 40-44°C, and after the stirring is completed, the pretreatment filtrate is obtained by filter pressing and washing with water through a plate and frame filter press; The mass ratio of the fermentation liquid to the pretreatment agent is 100:1.8-2.3; The preparation method of the pretreatment agent is as follows: placing activated carbon in a potassium permanganate solution, stirring at 50-54° C. for 1.3-1.8 hours, stirring evenly, filtering and washing, drying at 64-66° C. for 5.8-6.2 hours, then heating to 246-253° C. at a rate of 2.3-2.6° C. / min, and heat preservation for 3.3-3.7 hours. After the heat preservation is completed, the activated carbon is naturally restored to room temperature, placed in an ethanol solution, and hexadecyltrimethylammonium bromide is added for ultrasonic treatment, wherein the ultrasonic time is 13-17 minutes, the ultrasonic power is 114-125W, and the ultrasonic frequency is 28-32kHz. After the ultrasonic treatment is completed, glutamine is added and stirring is continued for 28-32 minutes. After the stirring is completed, the activated carbon is filtered, washed, and dried to obtain the pretreatment agent. The mass ratio of the activated carbon, potassium permanganate solution, ethanol solution, hexadecyltrimethylammonium bromide and glutamine is 10.3-10.7:76-80:89.5-90.6:1.0-1.4:3.4-3.8; The mass concentration of potassium permanganate is 25-28%; The mass concentration of the ethanol solution is 35-40%.

[0006] 2. Ultrafiltration The pre-treated filtrate is subjected to ultrafiltration, the pressure is controlled at 6.3-6.7 bar, the temperature is controlled at 24-26° C., the pore size of the ultrafiltration membrane is 1000 Da, and the permeate is collected to obtain the ultrafiltrate.

[0007] 3. Column chromatography Functional resin is loaded in a resin column, and then ultrafiltrate is flowed through the resin column by the column head, and the flow rate is controlled to be 1.2-1.8BV / h, until the ultrafiltrate enters the column completely, deionized water is adopted to wash, and the flow rate of the deionized water is controlled to be 1.4-1.7BV / h, and the effluent is collected when thioneine is detected from the effluent, and the collection is stopped when no thioneine flows out, and thioneine collected liquid is obtained; The preparation method of the functional resin comprises the following steps: placing silicon dioxide in a hydrogen peroxide solution, stirring at 42-46° C. for 38-42 minutes, filtering and washing after the stirring, placing the silicon dioxide in a reaction container, introducing toluene, adding N-[3-(triethoxysilyl)propyl]-4,5-dihydroimidazole, raising the temperature to 117-122° C. for reaction, and the reaction time is 3.8-4.2 hours. After the reaction is completed, lowering the temperature to room temperature, washing and drying, and obtaining modified silicon dioxide; The mass ratio of the silicon dioxide, hydrogen peroxide solution, toluene, and N-[3-(triethoxysilyl)propyl]-4,5-dihydroimidazole is 4.8-5.2:47-53:48-52:0.60-0.64; The mass concentration of the hydrogen peroxide solution is 23-27%; Styrene, divinylbenzene and polyvinyl pyrrolidone were mixed and stirred evenly, and then azobisisobutyronitrile was added, and ultrasonic treatment was performed at room temperature. The ultrasonic time was 27-32 minutes, the ultrasonic power was 146-153W, and the ultrasonic frequency was 47-52kHz. After the ultrasonic treatment, a mixed solution 1 was obtained; soybean lecithin, sodium lauryl sulfate and monoglycerol fatty acid ester were added to deionized water for ultrasonic treatment. The ultrasonic time was 28-32 minutes, the ultrasonic power was 120-128W, and the ultrasonic frequency was 42-4 7kHz, after the end of ultrasound, a mixed solution 2 is obtained; the mixed solution 1 is added to the mixed solution 2, and the adding time is controlled to be 27-32min, and then the modified silica is added for stirring, the stirring speed is 664-675rpm, and the stirring time is 18-22min. After the stirring is completed, the temperature is increased to 70-75℃ at a rate of 0.8-1.2℃ / min, and the temperature is kept for reaction for 7.8-8.2h. After the reaction is completed, it is filtered and washed, and then dried at 74-76℃ for 19-21h to obtain a functional resin; In the mixed solution 1, the mass ratio of styrene, divinylbenzene, polyvinyl pyrrolidone and azobisisobutyronitrile is 8.0-8.5:10.6-10.8:5.2-5.5:0.24-0.28; In the second mixed solution, the mass ratio of deionized water, soybean lecithin, sodium lauryl sulfate and monoglycerol fatty acid ester is 196-204:2.2-2.4:0.8-1.2:2.3-2.6; The mass ratio of the mixed solution 1, the mixed solution 2 and the modified silicon dioxide is 24.2-24.8:205.5-206.0:6.2-6.6.

[0008] 4. Concentration and crystallization The collected thioneine solution is concentrated under reduced pressure at a concentration temperature of 58-62° C. After being concentrated to 1 / 5 of the original volume, the temperature is lowered to 3.8-4.2° C. and allowed to stand for 9.8-10.2 h for crystallization. After crystallization is complete, it is filtered off with suction to obtain a filter cake, and the filter cake is washed with 78-82wt% ethanol solution to a pure white color and dried at a drying temperature of 58-62° C. for a drying time of 11.6-12.5 h to obtain thioneine.

[0009] Compared with the prior art, the present invention has achieved the following beneficial effects: 1. The present invention extracts and purifies thioneine from fermented liquid. First, a pretreatment agent is added to the fermented liquid for pretreatment to remove impurities such as protein, residual sugar, and thalline in the fermented liquid. The pretreatment agent is activated carbon as a pretreatment matrix. The pretreatment agent is first subjected to permanganate hydrolysis to improve its adsorption performance. The introduction of hexadecyl trimethyl ammonium bromide can improve the dispersibility of the pretreatment agent in the fermented liquid. Glutamine can enhance the adsorption of impurities. After the pretreatment is completed, after ultrafiltration, materials such as macromolecular proteins are removed. Then, functional resin is used for column chromatography. The modified diamine in the functional resin is used for the purification of the product. Silicon oxide is prepared by modifying an imidazole compound, has good affinity with the five-membered ring of thioneine, can interact with the aromatic ring part in the thioneine molecule through π-π stacking, thereby increasing the interaction force between the functional resin and thioneine, and the reaction system of the resin in the synthesis step is stable, the functional resin has good homogeneity, good stability, and excellent recycling and regeneration performance, thereby achieving the separation of thioneine from impurities, improving the yield and purity of the product, and can still maintain a high yield and purity after the functional resin is recycled and regenerated for multiple times, and is environmentally friendly and has a simple process; 2. thioneine is purified by the method of the present invention, and the purity of the thioneine product obtained is 99.46-99.85%, and the yield is 93.88-94.37%; The functional resin in the column chromatography step is recycled and regenerated for 40 times, and the recycling and regeneration is carried out after each column chromatography step. The recycling and regeneration method is as follows: the functional resin is washed with 3 times the mass of deionized water, and dried at 70° C. for 12.0 h to obtain a regenerated resin; at the 40th recycling and regeneration, the purity of the obtained ergothioneine product is 96.63-97.67%, and the yield is 91.40-92.54%. DETAILED DESCRIPTION

[0010] In order to more clearly understand the technical features, purposes and effects of the present invention, specific embodiments of the present invention are now described.

[0011] Example 1 1. Fermentation broth pretreatment The fermentation broth containing ergothioneine was adjusted to pH 5.9, a pretreatment agent was added and stirred, the stirring time was 2.3 h, the stirring temperature was 42 ° C, and after the stirring was completed, the pretreatment filtrate was obtained after filter pressing and washing with water through a plate and frame filter press; The mass ratio of the fermentation liquid to the pretreatment agent is 100:2.0; The preparation method of the pretreatment agent is as follows: 10.5g of activated carbon is placed in 78g of 26wt% potassium permanganate solution, stirred at 52°C for 1.5h, after being stirred evenly, filtered and washed, dried at 65°C for 6.0h, then heated to 250°C at a rate of 2.5°C / min, and heat-treated for 3.5h. After the heat-treatment is completed, the solution is naturally restored to room temperature, placed in 90.0g of 37wt% ethanol solution, and 1.2g of hexadecyltrimethylammonium bromide is added for ultrasonic treatment, wherein the ultrasonic time is 15min, the ultrasonic power is 120W, and the ultrasonic frequency is 30kHz. After the ultrasonic treatment is completed, 3.6g of glutamine is added and stirring is continued for 30min. After the stirring is completed, the solution is filtered, washed, and dried to obtain the pretreatment agent.

[0012] 2. Ultrafiltration The pre-treated filtrate was ultrafiltered, the pressure during the ultrafiltration process was controlled to be 6.5 bar, the temperature was controlled to be 25° C., the pore size of the ultrafiltration membrane was 1000 Da, and the permeate was collected to obtain the ultrafiltrate.

[0013] 3. Column chromatography Functional resin is loaded in resin column, then ultrafiltrate is flowed through resin column by column head, control flow rate is 1.5BV / h, treat that ultrafiltrate enters column completely, adopt deionized water to wash, control the flow rate of deionized water to be 1.6BV / h, collect effluent when thioneine is detected from effluent, stop collecting when no thioneine flows out, obtain thioneine collected liquid; The preparation method of the functional resin comprises the following steps: placing 5.0 g of silicon dioxide in 50 g of a 25 wt% hydrogen peroxide solution, stirring at 45° C. for 40 min, filtering and washing after the stirring, and then placing the mixture in a reaction container, introducing 50 g of toluene, adding 0.62 g of N-[3-(triethoxysilyl)propyl]-4,5-dihydroimidazole, raising the temperature to 120° C. for reaction, and the reaction time is 4.0 h. After the reaction is completed, lowering the temperature to room temperature, washing, and drying to obtain modified silicon dioxide; 8.3g styrene, 10.7g divinylbenzene and 5.4g polyvinylpyrrolidone were mixed and stirred evenly, and then 0.26g azobisisobutyronitrile was added and ultrasonic treatment was performed at room temperature. The ultrasonic time was 30min, the ultrasonic power was 150W, and the ultrasonic frequency was 50kHz. After the ultrasonic treatment, a mixed solution 1 was obtained. 2.3g soybean lecithin, 1.0g sodium lauryl sulfate and 2.5g monoglycerol fatty acid ester were added to 200g deionized water and ultrasonic treatment was performed. The ultrasonic time was 30min and the ultrasonic power was 150W. The rate is 125W, the ultrasonic frequency is 45kHz, and after the ultrasound is completed, a mixed solution 2 is obtained; 24.5g of the mixed solution 1 is added to 205.8g of the mixed solution 2, and the adding time is controlled to be 30min, and then 6.4g of modified silica is added for stirring, the stirring speed is 670rpm, and the stirring time is 20min. After the stirring is completed, the temperature is increased to 73°C at a rate of 1.0°C / min, and the reaction is kept warm for 8.0h. After the reaction is completed, it is filtered and washed, and then dried at 75°C for 20h to obtain a functional resin.

[0014] 4. Concentration and crystallization The collected thioneine solution was concentrated under reduced pressure at a concentration temperature of 60° C. After being concentrated to 1 / 5 of the original volume, the temperature was lowered to 4.0° C. and allowed to stand for 10.0 h for crystallization. After crystallization was completed, the filter cake was filtered off with suction to obtain a filter cake. The filter cake was washed with 80wt% ethanol solution until it was pure white and dried at a drying temperature of 60° C. for 12.0 h to obtain thioneine.

[0015] Adopt the method for embodiment 1, thioneine is purified, and the purity of the thioneine product obtained is 99.85%, and yield is 94.37%.

[0016] The functional resin in the column chromatography step is recycled and regenerated for 40 times, and recycling and regeneration are performed after each column chromatography step. The recycling and regeneration method is to wash the functional resin with 3 times the mass of deionized water, and dry it at 70° C. for 12.0 h to obtain a regenerated resin; at the 40th recycling and regeneration, the purity of the obtained thioneine product is 97.67%, and the yield is 92.54%.

[0017] Example 2 1. Fermentation broth pretreatment The fermentation liquid containing ergothioneine is adjusted to pH 5.8, a pretreatment agent is added and stirred, the stirring time is 2.2 hours, the stirring temperature is 40 ° C. After the stirring is completed, the pretreatment filtrate is obtained by filter pressing and washing with water through a plate and frame filter press; The mass ratio of the fermentation liquid to the pretreatment agent is 100:1.8; The preparation method of the pretreatment agent is as follows: 10.3g of activated carbon is placed in 76g of 25wt% potassium permanganate solution, stirred at 50°C for 1.3h, after being uniformly stirred, filtered and washed, dried at 64°C for 6.2h, then heated to 246°C at a rate of 2.3°C / min, and heat-treated for 3.3h. After the heat-treatment is completed, the solution is naturally restored to room temperature, placed in 89.5g of 35wt% ethanol solution, 1.0g of hexadecyltrimethylammonium bromide is added for ultrasonic treatment, the ultrasonic time is 13min, the ultrasonic power is 114W, and the ultrasonic frequency is 28kHz. After the ultrasonic treatment is completed, 3.4g of glutamine is added and stirring is continued for 28min. After the stirring is completed, the solution is filtered, washed, and dried to obtain the pretreatment agent.

[0018] 2. Ultrafiltration The pre-treated filtrate was ultrafiltered, the pressure during the ultrafiltration process was controlled to be 6.3 bar, the temperature was controlled to be 24° C., the pore size of the ultrafiltration membrane was 1000 Da, and the permeate was collected to obtain the ultrafiltrate.

[0019] 3. Column chromatography Functional resin is loaded in resin column, then ultrafiltrate is flowed through resin column by column cap, control flow rate is 1.2BV / h, treat that ultrafiltrate enters column completely, adopt deionized water to wash, control the flow rate of deionized water to be 1.4BV / h, collect effluent when thioneine is detected from effluent, stop collecting when no thioneine flows out, obtain thioneine collected liquid; The preparation method of the functional resin is as follows: 4.8g of silicon dioxide is placed in 47g of a 23wt% hydrogen peroxide solution, stirred at 42°C for 38min, after the stirring is completed, filtered and washed, and then put into a reaction container, 48g of toluene is introduced, 0.60g of N-[3-(triethoxysilyl)propyl]-4,5-dihydroimidazole is added, the temperature is increased to 117°C for reaction, the reaction time is 3.8h, and after the reaction is completed, the temperature is lowered to room temperature, washed and dried to obtain modified silicon dioxide; 8.0g styrene, 10.6g divinylbenzene and 5.2g polyvinylpyrrolidone were mixed and stirred evenly, and then 0.24g azobisisobutyronitrile was added and ultrasonic treatment was carried out at room temperature. The ultrasonic time was 27min, the ultrasonic power was 146W, and the ultrasonic frequency was 47kHz. After the ultrasonic treatment, a mixed solution 1 was obtained. 2.2g soybean lecithin, 0.8g sodium lauryl sulfate and 2.3g monoglycerol fatty acid ester were added to 196g deionized water and ultrasonic treatment was carried out. The ultrasonic time was 28min and the ultrasonic power was 146W. The rate is 120W, the ultrasonic frequency is 42kHz, and after the ultrasound is completed, a mixed solution 2 is obtained; 24.2g of the mixed solution 1 is added to 205.5g of the mixed solution 2, and the adding time is controlled to be 27min, and then 6.2g of modified silica is added for stirring, the stirring speed is 664rpm, and the stirring time is 18min. After the stirring is completed, the temperature is raised to 70℃ at a rate of 0.8℃ / min, and the reaction is kept warm for 7.8h. After the reaction is completed, it is filtered and washed, and then dried at 74℃ for 19h to obtain a functional resin.

[0020] 4. Concentration and crystallization The collected thioneine solution was concentrated under reduced pressure at a concentration temperature of 58° C. After being concentrated to 1 / 5 of the original volume, the temperature was lowered to 3.8° C. and allowed to stand for 9.8 h for crystallization. After crystallization was completed, the filter cake was filtered off with suction to obtain a filter cake. The filter cake was washed with 78wt% ethanol solution until it was pure white and dried at a drying temperature of 58° C. for 11.6 h to obtain thioneine.

[0021] Adopt the method for embodiment 2 to purify thioneine, the purity of the thioneine product obtained is 99.46%, and yield is 93.88%.

[0022] The functional resin in the column chromatography step is recycled and regenerated for 40 times, and recycling and regeneration are performed after each column chromatography step. The recycling and regeneration method is to wash the functional resin with 3 times the mass of deionized water, and dry it at 70° C. for 12.0 h to obtain a regenerated resin; at the 40th recycling and regeneration, the purity of the obtained thioneine product is 96.63%, and the yield is 91.40%.

[0023] Example 3 1. Fermentation broth pretreatment The fermentation liquid containing ergothioneine is adjusted to pH 6.0, a pretreatment agent is added and stirred, the stirring time is 2.4 hours, the stirring temperature is 44 ° C. After the stirring is completed, the pretreatment filtrate is obtained by filter pressing and washing with water through a plate and frame filter press; The mass ratio of the fermentation liquid to the pretreatment agent is 100:2.3; The preparation method of the pretreatment agent is as follows: 10.7g of activated carbon is placed in 80g of 28wt% potassium permanganate solution, stirred at 54°C for 1.8h, after being stirred evenly, filtered and washed, dried at 66°C for 5.8h, then heated to 253°C at a rate of 2.6°C / min, and heat-treated for 3.7h. After the heat-treatment is completed, the solution is naturally restored to room temperature, placed in 90.6g of 40wt% ethanol solution, and 1.4g of hexadecyltrimethylammonium bromide is added for ultrasonic treatment, wherein the ultrasonic time is 17min, the ultrasonic power is 125W, and the ultrasonic frequency is 32kHz. After the ultrasonic treatment is completed, 3.8g of glutamine is added and the stirring is continued for 32min. After the stirring is completed, the solution is filtered, washed, and dried to obtain the pretreatment agent.

[0024] 2. Ultrafiltration The pre-treated filtrate was ultrafiltered, the pressure during the ultrafiltration process was controlled to be 6.7 bar, the temperature was controlled to be 26° C., the pore size of the ultrafiltration membrane was 1000 Da, and the permeate was collected to obtain the ultrafiltrate.

[0025] 3. Column chromatography Functional resin is loaded in resin column, then ultrafiltrate is flowed through resin column by column head, control flow rate is 1.8BV / h, treat that ultrafiltrate enters column completely, adopt deionized water to wash, control the flow rate of deionized water to be 1.7BV / h, collect effluent when thioneine is detected from effluent, stop collecting when no thioneine flows out, obtain thioneine collected liquid; The preparation method of the functional resin is as follows: 5.2g of silicon dioxide is placed in 53g of a 27wt% hydrogen peroxide solution, stirred at 46°C for 42min, filtered and washed after the stirring, and then placed in a reaction container, 52g of toluene is introduced, 0.64g of N-[3-(triethoxysilyl)propyl]-4,5-dihydroimidazole is added, the temperature is raised to 122°C for reaction, the reaction time is 4.2h, and after the reaction is completed, the temperature is lowered to room temperature, washed and dried to obtain modified silicon dioxide; 8.5g of styrene, 10.8g of divinylbenzene and 5.5g of polyvinylpyrrolidone were mixed and stirred evenly, and then 0.28g of azobisisobutyronitrile was added, and ultrasonic treatment was performed at room temperature. The ultrasonic time was 32min, the ultrasonic power was 153W, and the ultrasonic frequency was 52kHz. After the ultrasonic treatment, a mixed solution 1 was obtained; 2.4g of soybean lecithin, 1.2g of sodium dodecyl sulfate and 2.6g of monoglycerol fatty acid ester were added to 204g of deionized water for ultrasonic treatment. The ultrasonic time was 32min, and the ultrasonic power was 153W. The rate is 128W, the ultrasonic frequency is 47kHz, and after the ultrasound is completed, a mixed solution 2 is obtained; 24.8g of the mixed solution 1 is added to 206.0g of the mixed solution 2, and the adding time is controlled to be 32min, and then 6.6g of modified silica is added for stirring, the stirring speed is 675rpm, and the stirring time is 22min. After the stirring is completed, the temperature is increased to 75℃ at a rate of 1.2℃ / min, and the reaction is kept warm for 8.2h. After the reaction is completed, it is filtered and washed, and then dried at 76℃ for 21h to obtain a functional resin.

[0026] 4. Concentration and crystallization The collected thioneine solution was concentrated under reduced pressure at a concentration temperature of 62° C. After being concentrated to 1 / 5 of the original volume, the temperature was lowered to 4.2° C. and allowed to stand for 10.2 h for crystallization. After crystallization was completed, the filter cake was filtered off with suction to obtain a filter cake. The filter cake was washed with 82wt% ethanol solution until it was pure white and dried at a drying temperature of 62° C. for 12.5 h to obtain thioneine.

[0027] Adopt the method for embodiment 2 to purify thioneine, the purity of the thioneine product obtained is 99.63%, and yield is 94.16%.

[0028] The functional resin in the column chromatography step is recycled and regenerated for 40 times, and recycling and regeneration are performed after each column chromatography step. The recycling and regeneration method is to wash the functional resin with 3 times the mass of deionized water, and dry it at 70° C. for 12.0 h to obtain a regenerated resin; at the 40th recycling and regeneration, the purity of the obtained ergothioneine product is 97.03%, and the yield is 92.06%.

[0029] The invention extracts and purifies ergothioneine from a fermentation broth. First, a pretreatment agent is added to the fermentation broth for pretreatment to remove impurities such as protein, residual sugar, and bacterial cells in the fermentation broth. The pretreatment agent uses activated carbon as a pretreatment matrix, and firstly performs permanganate hydrolysis treatment on the activated carbon to improve its adsorption performance. The introduction of hexadecyltrimethylammonium bromide can improve the dispersibility of the pretreatment agent in the fermentation broth, and glutamine can enhance the adsorption of impurities. After the pretreatment is completed, ultrafiltration is performed to remove substances such as macromolecular proteins, and then column chromatography is performed using a functional resin. The modified dioxygen in the functional resin The silicon is prepared by modifying an imidazole compound, has good affinity with the five-membered ring of ergothioneine, can interact with the aromatic ring part in the ergothioneine molecule in the manner of π-π stacking, thereby increasing the interaction force between the functional resin and ergothioneine, and the reaction system of the resin in the synthesis step is stable, the functional resin has good homogeneity, good stability, and excellent recycling and regeneration performance, thereby achieving the separation of ergothioneine from impurities, improving the yield and purity of the product, and can still maintain a high yield and purity after the functional resin is recycled and regenerated for multiple times, and is environmentally friendly and has a simple process.

[0030] Comparative Example 1 Based on Example 1, the changes are as follows: In the pretreatment step, the pretreatment agent was replaced in equal amounts with activated carbon without any treatment; In the method for preparing the functional resin in the column chromatography step, the modified silica component is omitted and an equal amount of the modified silica is replaced by mixed solution one; The rest of the operations are the same.

[0031] Comparative Example 1 omits the process of activated carbon, directly replaces pretreatment agent in equal amounts with activated carbon without any treatment, the dispersibility of activated carbon is poor, and its aggregation phenomenon is more serious in fermented liquid, and the adsorbability for impurity etc. is poor, and the removal of impurity is affected, thereby affecting the follow-up column chromatography for thioneine, and in the column chromatography step, modified silicon dioxide component is omitted, the adsorption of functional resin for thioneine is reduced, the force with thioneine is weakened, and the stability of functional resin is affected, and the yield and purity of product are finally reduced, and the regeneration performance of functional resin is affected.

[0032] Adopt the method for comparative example 1 to purify thioneine, the purity of the thioneine product obtained is 90.17%, and the yield is 83.22%.

[0033] The functional resin in the column chromatography step is recycled and regenerated for 40 times, and recycling and regeneration are performed after each column chromatography step. The recycling and regeneration method is to wash the functional resin with 3 times the mass of deionized water, and dry it at 70° C. for 12.0 h to obtain a regenerated resin; at the 40th recycling and regeneration, the purity of the obtained thioneine product is 81.16%, and the yield is 75.36%.

[0034] Comparative Example 2 Based on Example 1, the changes are as follows: In the preparation method of the functional resin in the column chromatography step, the mixed solution is replaced with deionized water in equal amounts, and the silica modification step is omitted, and the modified silica is replaced with untreated silica in equal amounts; The rest of the operations are the same.

[0035] Adopt the method for comparative example 2 to purify thioneine, the purity of the thioneine product obtained is 88.03%, and the yield is 81.15%.

[0036] The functional resin in the column chromatography step is recycled and regenerated for 40 times, and recycling and regeneration are performed after each column chromatography step. The recycling and regeneration method is to wash the functional resin with 3 times the mass of deionized water, and dry it at 70° C. for 12.0 h to obtain a regenerated resin; at the 40th recycling and regeneration, the purity of the obtained thioneine product is 76.81%, and the yield is 71.12%.

[0037] In the step of preparing the functional resin, comparative example 2 only uses silicon dioxide and styrene-divinylbenzene for cross-linking, which has poor compatibility and affinity with the resin matrix, affecting the system stability of the synthesis reaction. The obtained functional resin has poor homogeneity and stability, and the yield and purity of the product are low.

[0038] Unless otherwise specified, all ratios and percentages described in the present invention are by mass ratios and percentages are by mass percentages.

[0039] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for purifying ergothioneine, characterized in that, It includes fermentation broth pretreatment, ultrafiltration, column chromatography and concentration crystallization steps; The column chromatography step includes loading the functional resin into a resin column, and then passing the ultrafiltrate from the column head through the resin column for treatment; The preparation method of the functional resin is as follows: placing silicon dioxide in a hydrogen peroxide solution and stirring, filtering and washing, and then putting the silicon dioxide into a reaction container, introducing toluene, adding N-[3-(triethoxysilyl)propyl]-4,5-dihydroimidazole, and reacting at 117-122° C. for 3.8-4.2 hours to obtain modified silicon dioxide; Styrene, divinylbenzene and polyvinylpyrrolidone are mixed, azobisisobutyronitrile is added, and mixed solution 1 is obtained by ultrasonic treatment; soybean lecithin, sodium lauryl sulfate and monoglycerol fatty acid ester are added to deionized water, and mixed solution 2 is obtained by ultrasonic treatment; mixed solution 1 is added to mixed solution 2, modified silicon dioxide is added, the mixture is stirred evenly, and the mixture is kept warm at 70-75°C for 7.8-8.2 hours to obtain a functional resin.

2. a method for purifying thioneine according to claim 1, characterized in that, The step of pretreatment of the fermented liquid is as follows: adjusting the pH of the fermented liquid containing ergothioneine to 5.8-6.0, adding a pretreatment agent for stirring, the stirring time is 2.2-2.4h, the stirring temperature is 40-44°C, and after the stirring is completed, filtering and washing with water through a plate and frame filter press to obtain a pretreated filtrate; The mass ratio of the fermentation liquid to the pretreatment agent is 100:1.8-2.

3.

3. a method for purifying thioneine according to claim 2, characterized in that, The preparation method of the pretreatment agent is as follows: placing activated carbon in a potassium permanganate solution, stirring at 50-54° C. for 1.3-1.8 hours, stirring evenly, filtering and washing, drying at 64-66° C. for 5.8-6.2 hours, then heating to 246-253° C. at a rate of 2.3-2.6° C. / min, and heat preservation for 3.3-3.7 hours. After the heat preservation is completed, the activated carbon is naturally restored to room temperature, placed in an ethanol solution, and hexadecyltrimethylammonium bromide is added for ultrasonic treatment, wherein the ultrasonic time is 13-17 minutes, the ultrasonic power is 114-125W, and the ultrasonic frequency is 28-32kHz. After the ultrasonic treatment is completed, glutamine is added and stirring is continued for 28-32 minutes. After the stirring is completed, the activated carbon is filtered, washed, and dried to obtain the pretreatment agent. The mass ratio of the activated carbon, potassium permanganate solution, ethanol solution, hexadecyltrimethylammonium bromide and glutamine is 10.3-10.7:76-80:89.5-90.6:1.0-1.4:3.4-3.8; The mass concentration of potassium permanganate is 25-28%; The mass concentration of the ethanol solution is 35-40%.

4. a method for purifying thioneine according to claim 1, is characterized in that, The column chromatography step is as follows: functional resin is loaded into a resin column, and then ultrafiltrate is passed through the resin column from the column head, and the flow rate is controlled to be 1.2-1.8BV / h, and the ultrafiltrate is completely entered into the column, and deionized water is used for washing, and the flow rate of deionized water is controlled to be 1.4-1.7BV / h. When thioneine is detected in the effluent, the effluent is collected, and the collection is stopped when no thioneine flows out, to obtain thioneine collected liquid.

5. a method for purifying thioneine according to claim 1, is characterized in that, In the preparation method of the functional resin, the mass ratio of the silicon dioxide, hydrogen peroxide solution, toluene, and N-[3-(triethoxysilyl)propyl]-4,5-dihydroimidazole is 4.8-5.2:47-53:48-52:0.60-0.64; The mass concentration of the hydrogen peroxide solution is 23-27%.

6. a method for purifying thioneine according to claim 1, characterized in that, In the step of preparing the mixed solution, the ultrasonic treatment has an ultrasonic time of 27-32 min, an ultrasonic power of 146-153 W, and an ultrasonic frequency of 47-52 kHz; In the second step of preparing the mixed solution, the ultrasonic treatment has an ultrasonic time of 28-32 min, an ultrasonic power of 120-128 W, and an ultrasonic frequency of 42-47 kHz.

7. a method for purifying thioneine according to claim 1, characterized in that, In the mixed solution 1, the mass ratio of styrene, divinylbenzene, polyvinyl pyrrolidone and azobisisobutyronitrile is 8.0-8.5:10.6-10.8:5.2-5.5:0.24-0.28; In the second mixed solution, the mass ratio of deionized water, soybean lecithin, sodium lauryl sulfate and monoglycerol fatty acid ester is 196-204:2.2-2.4:0.8-1.2:2.3-2.6; The mass ratio of the mixed solution 1, the mixed solution 2 and the modified silicon dioxide is 24.2-24.8:205.5-206.0:6.2-6.

6.

8. a method for purifying thioneine according to claim 1, characterized in that, The ultrafiltration step is to ultrafilter the pretreated filtrate, control the pressure during the ultrafiltration process to be 6.3-6.7 bar, the temperature to be 24-26° C., the pore size of the ultrafiltration membrane to be 1000 Da, and collect the permeate to obtain the ultrafiltrate.

9. a method for purifying thioneine according to claim 1, characterized in that, The concentration and crystallization step comprises: concentrating the collected thioneine solution under reduced pressure at a concentration temperature of 58-62° C., concentrating to 1 / 5 of the original volume, lowering the temperature to 3.8-4.2° C. and standing for 9.8-10.2 h for crystallization, filtering by suction after crystallization to obtain a filter cake, washing the filter cake with a 78-82wt% ethanol solution until the filter cake is pure white and drying the filter cake, wherein the drying temperature is 58-62° C. and the drying time is 11.6-12.5 h to obtain thioneine.