Method for purifying ergothioneine

Abstract

The invention relates to the technical field of biology, in particular to a purification method of ergothioneine. The invention provides a method for purifying high-purity ergothioneine from an enzyme catalytic reaction. According to the method, high-purity ergothioneine is purified from the enzyme catalysis reaction liquid, so that the purification steps are greatly simplified, and the total purification yield is increased; and only a single organic solvent ethanol is used for crystallization, so that the ergothioneine product with high purity and uniform and thick particles is obtained, and the method is suitable for large-scale production of ergothioneine.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a method for purifying ergothioneine. Background Art

[0002] L-Ergothioneine (EGT) is an amino acid with antioxidant properties. It was first discovered in 1909 by Charles I of the University of California, Berkeley, in ergot fungi and has since been found in mushrooms, cyanobacteria, and Coprinus comatus. EGT exists in two isomers, primarily in the thiol form (carbon-sulfur double bond) at physiological pH. This prevents autooxidation, making EGT highly stable and allowing it to accumulate in the body, resulting in a wide range of cellular protective and disease-modifying properties, playing an important physiological role in human health and development. Furthermore, EGT has a higher redox potential than other naturally occurring thiols, acting as an antioxidant by scavenging reactive and hydroxyl radicals. It is more effective than other antioxidant molecules (including glutathione) in inhibiting protein nitration. In addition to its antioxidant properties, EGT can also protect cells from various apoptotic insults induced by reactive nitrogen species and ultraviolet radiation. It also inhibits peroxynitrite-mediated amino acid oxidation, demonstrating excellent efficacy in the treatment of inflammation. This allows EGT to be applied in many fields such as medicine, food, health products, cosmetics, etc., and has a wide range of uses and market prospects.

[0003] The method for preparing thioneine at present mainly contains: biological fermentation method, chemical synthesis method and natural biological extraction method.Biological fermentation method is the mainstream direction of current large-scale production of thioneine, but generally low in output, high in cost, and containing a large amount of culture medium components and microbial metabolites in the fermentation liquid, greatly improving the difficulty of separation and purification.Chemical synthesis and natural biological extraction method are more significant relative to biological fermentation rule shortcomings, and are difficult to be widely used.

[0004] Existing purification technology discloses a method for purifying thioneine from microbial fermentation liquid, by solid-liquid separation, the first solvent extraction such as ethyl acetate, n-butanol, and then protected with the second solvent such as tetrahydrofuran, methanol, and then deprotected with the third solvent after adding Boc anhydride reaction, to obtain thioneine. The method uses a variety of organic solvents, limits its application in the fields of cosmetics, food, medicine, etc., and is not suitable for industrial production.

[0005] Separate report discloses a kind of method for preparing thioneine from mushroom fruiting body or mushroom fermentation mycelium, the method is first through hot water extraction after solid-liquid separation, microfiltration, ultrafiltration, vacuum concentration, ion exchange, vacuum concentration, ion exchange, vacuum or reverse osmosis concentration, vacuum drying, and the powder thioneine purity obtained is 80%-90%.Required steps are numerous and lengthy, and final product purity is not high.Also there is purification technology that the ultrafiltration permeate of thioneine adopts HILIC filler to carry out chromatographic purification, although obtained products purity can reach 99.0%, but this HILIC filler is expensive, has caused the raising of production cost.

[0006] Since most of the current technical means for producing ergothioneine are aimed at purification and separation from fermentation broth or natural biological pathways, with the development and improvement of synthetic biology technology, the enzyme catalysis method has the characteristics of being green and environmentally friendly, with mild reaction conditions and high catalytic efficiency. Reports on the use of this method to synthesize ergothioneine are gradually increasing. However, there are relatively few reports on the subsequent purification technology of ergothioneine synthesized using the enzyme catalysis method. Summary of the Invention

[0007] In view of this, the present invention provides a method for purifying thioneine of high purity. The present invention provides a method for purifying high-purity thioneine from enzyme catalytic reaction, including: enzyme catalytic reaction solution acid adjustment is carried out ceramic membrane ultrafiltration purification to remove macromolecular substances such as enzyme, nucleic acid, polysaccharide, the ultrafiltration permeate containing thioneine is collected and carried out ion exchange chromatography to remove divalent, trivalent ions and most of pigments, concentrated through nanofiltration desalination, then anti-solvent crystallization is carried out to obtain high-purity thioneine crystal particles. The present invention purifies high-purity thioneine from enzyme catalytic reaction solution, greatly simplifies purification step, and improves purification total yield; Only single organic solvent ethanol is used for crystallization, and high-purity and uniformly thick thioneine product of particles is obtained, which is suitable for the large-scale production of thioneine.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a method for purifying thioneine, comprising the steps of:

[0010] Step 1: taking the enzyme catalytic reaction solution containing thioneine, adjusting the pH value, ultrafiltration, and collecting the permeate;

[0011] Step 2: Take the permeate described in step 1, sequentially pass it through a cation exchange resin column and an anion exchange resin column for adsorption, elute with pure water, and collect the column liquid;

[0012] Step 3, taking the column liquid described in step 2, nanofiltration, and collecting the filtrate;

[0013] Step 4, take the filtrate described in step 3, concentrate to obtain concentrated solution, crystallize, filter, wash, and dry to obtain described thioneine.

[0014] In some specific embodiments of the present invention, the purification method of thioneine comprises the steps of:

[0015] Step 1: take the enzyme-catalyzed reaction solution containing thioneine, adjust the pH value, ultrafilter, remove macromolecular substances such as enzymes, nucleic acids, polysaccharides, and collect the permeate;

[0016] Step 2, take the permeate described in step 1, adsorb through cation exchange resin column and anion exchange resin column successively, remove divalent, trivalent anion and the cation, pure water elution stays in post but not by the thioneine of resin adsorption, collects column liquid;

[0017] The anions include SO4 2- or PO4 3- ; The cations include Mg 2+ or Fe 3+ .

[0018] Step 3, take the column liquid described in step 2, nanofiltration, remove more than 95% of the monovalent salt ions, and be concentrated to thioneine concentration of 50 ~ 100g / L, collect filtrate;

[0019] Step 4, take the filtrate described in step 3, concentrate to obtain concentrated solution, crystallize, filter, wash, and dry to obtain described thioneine.

[0020] In some specific embodiments of the present invention, the concentration of ergothioneine in the enzyme-catalyzed reaction solution in step 1 is 20-40 g / L.

[0021] In some specific embodiments of the present invention, the preparation method of the enzyme-catalyzed reaction solution containing thioneine described in step 1 comprises the steps:

[0022] Step 1, preparation of Egt-1 and Egt-2 enzyme solutions: Weigh appropriate amounts of Egt-1 and Egt-2 enzyme bacteria according to the amount of enzyme required for the reaction, mix the bacteria with purified water at a ratio of 1:4 (m:m), and stir until no lumps remain, so that the bacterial solution is in a turbid liquid state; lyse the bacterial solution twice using a high-pressure homogenizer at 5-10°C and a cell lysis pressure in the range of 800-1000 bar to obtain Egt-1 and Egt-2 enzyme solutions;

[0023] Step 2, the first step enzyme reaction: configure 0.5mol / L trimethylhistidine (TMH), mix with the Egt-1 enzyme solution under 25°C stirring, adjust pH to 6.5-7.5, then mix with 0.5mol / L cysteine ​​at a certain speed, carry out the first step reaction, generate thioneine intermediate; When the reaction conversion rate reaches 95%, the first step reaction can be terminated to obtain the first step reaction solution;

[0024] Conversion rate calculation formula:

[0025]

[0026] Step 3, second step enzyme reaction: solid sodium sulfite and Egt-2 enzyme solution are added to the first step reaction solution, the pH is adjusted to 8.0-8.5, and the reaction is started to generate thioneine; when the thioneine concentration no longer continues to increase, the second step reaction can be terminated to obtain the enzyme-catalyzed reaction solution containing thioneine;

[0027] The enzyme-catalyzed reaction liquid containing thioneine contains 20 to 40 g / L of thioneine.

[0028] In some specific embodiments of the present invention, the pH adjustment in step 1 comprises adjusting the pH using 4 mol / L hydrochloric acid, and the pH value is comprised between 2.0 and 5.0.

[0029] In some specific embodiments of the present invention, the pore size of the ultrafiltration membrane in step 1 is 3 to 10 nm; the molecular weight cutoff of the ultrafiltration is 2 KDa to 20 KDa; the pressure of the ultrafiltration is 0.2 to 0.4 MPa; and the temperature of the ultrafiltration is 10 to 30°C.

[0030] In some specific embodiments of the present invention, the ultrafiltration membrane of the ultrafiltration comprises a ceramic ultrafiltration membrane.

[0031] In some specific embodiments of the present invention, the filler of the anion exchange resin column in step 2 comprises a styrene-based weakly basic resin; and / or

[0032] The filler of the cation exchange resin column in step 2 includes a styrene-based macroporous strong acid resin.

[0033] In some specific embodiments of the present invention, the types of the styrene-based weakly basic resin include but are not limited to one or more of LX-950, LX-D301, LX-67 or LX-6703; and / or

[0034] The types of the styrene-based macroporous strong acidic resin include but are not limited to one or more of LX-001, LX-35A, LX-160 or RCS72.

[0035] In some specific embodiments of the present invention, the adsorption in step 2 comprises using an anion and cation exchange resin column in which LX-950 styrene-based weakly basic resin and LX-001 styrene-based macroporous strongly acidic resin are connected in series for adsorption.

[0036] In some specific embodiments of the present invention, the adsorption in step 2 comprises using an anion and cation exchange resin column in which LX-D301 styrene-based weakly basic resin and LX-35A styrene-based macroporous strongly acidic resin are connected in series for adsorption.

[0037] In some specific embodiments of the present invention, the adsorption in step 2 comprises using an anion and cation exchange resin column in which LX-67 styrene-based weakly basic resin and LX-160 styrene-based macroporous strongly acidic resin are connected in series for adsorption.

[0038] In some specific embodiments of the present invention, the adsorption in step 2 comprises using an anion and cation exchange resin column in which LX-6703 styrene-based weakly basic resin and RCS72 styrene-based macroporous strongly acidic resin are connected in series for adsorption.

[0039] In some specific embodiments of the present invention, the adsorption in step 2 comprises using an anion and cation exchange resin column in which LX-6703 styrene-based weakly basic resin and RCS72 styrene-based macroporous strongly acidic resin are connected in series for adsorption.

[0040] In some specific embodiments of the present invention, the pH value of the permeate in step 2 is 3.0 to 5.0; and / or

[0041] The loading flow rate of the permeate is 0.5 to 2.0 times of filler volume (BV) / h, and the loading amount is 2 to 10 times of filler volume.

[0042] In some specific embodiments of the present invention, the ergothioneine concentration when the permeate is loaded in step 2 is 10 g / L.

[0043] In some specific embodiments of the present invention, the pore size of the nanofiltration membrane in step 3 is 1-2 nm; the molecular weight cutoff of the nanofiltration is 100-300 Da; the pressure of the nanofiltration is 1.0-2.0 MPa; and the temperature of the nanofiltration is 5-30°C.

[0044] In some specific embodiments of the present invention, the nanofiltration membrane material includes one or more of spiral polyamide, sulfonated polyethersulfone, sulfonated polysulfone or polyethersulfone.

[0045] In some specific embodiments of the present invention, the thioneine concentration of the filtrate in step 3 is 50-100 g / L.

[0046] In some specific embodiments of the present invention, the concentration in step 4 comprises concentrating the filtrate to a concentration of 160 to 220 g / L of ergothioneine; and / or

[0047] The crystallization in step 4 includes adding anti-solvent crystallization; the amount of the anti-solvent added includes 2 to 6 times the volume of the concentrated solution; the addition rate of the anti-solvent includes 0.2 to 0.6 times the volume of the concentrated solution / h;

[0048] The anti-solvent included 95% ethanol.

[0049] In some specific embodiments of the present invention, the crystallization in step 4 further comprises adding 0.5 to 1.0 times the volume of the concentrated solution of the anti-solvent, adding thioneine seed crystals, and then adding the remaining amount of the anti-solvent;

[0050] The added amount of the thioneine seed crystals comprises 0.1% of the actual thioneine crystal amount.

[0051] In some specific embodiments of the present invention, the crystallization in step 4 includes adding the ergothioneine seed crystals and starting to cool for 1 hour, and the cooling rate includes 2.5°C / h, or 5°C / 2h.

[0052] In some specific embodiments of the present invention, the washing in step 4 comprises washing with a 75% to 95% ethanol aqueous solution;

[0053] The drying in step 4 includes vacuum drying; the vacuum degree of the vacuum drying includes -0.1 to -0.08 MPa; the temperature of the vacuum drying includes 45 to 60°C.

[0054] The present invention includes but is not limited to the following beneficial effects:

[0055] The present invention purifies the method for thioneine from enzyme catalytic reaction liquid, and purification step is few, and product yield is high, and cost is low, and crystallized product purity content is extremely high, simple and easy to operate, without being related to other harmful solvents and reagents, has filled the purification field of producing thioneine from enzyme catalytic approach.And product bulk density is high, is suitable for the processing of downstream enterprises, is conducive to reducing the production cost of whole production chain, and the product quality obtained is stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0057] Figure 1 Shown is the structural formula of EGT;

[0058] Figure 21 shows the HPLC spectrum of the EGT crystals obtained in Example 1;

[0059] Figure 3 14 shows the HPLC spectrum of the EGT crystals obtained in Example 2;

[0060] Figure 4 14 shows the HPLC spectrum of the EGT crystals obtained in Example 3;

[0061] Figure 5 14 shows the HPLC spectrum of the EGT crystals obtained in Example 4;

[0062] Figure 6 14 shows the HPLC spectrum of the EGT crystals obtained in Example 5;

[0063] Figure 7 14 shows the HPLC spectrum of the EGT crystals obtained in Example 6;

[0064] Figure 8 14 shows the HPLC spectrum of the EGT crystals obtained in Example 7;

[0065] Figure 9 1 shows the appearance of the EGT crystal obtained in Example 1;

[0066] Figure 10 1 shows the appearance of the EGT crystal obtained in Example 2;

[0067] Figure 11 1 shows the appearance of the EGT crystal obtained in Example 3;

[0068] Figure 12 14 shows the appearance of the EGT crystal obtained in Example 4;

[0069] Figure 13 14 shows the appearance of the EGT crystal obtained in Example 5;

[0070] Figure 14 14 shows the appearance of the EGT crystal obtained in Example 6;

[0071] Figure 15 14 shows the appearance of the EGT crystal obtained in Example 7;

[0072] Figure 16 1 shows the crystal habit of the EGT crystal obtained in Example 1;

[0073] Figure 17 2 shows the crystal habit of the EGT crystal obtained in Example 2;

[0074] Figure 18 14 shows the crystal habit of the EGT crystal obtained in Example 3;

[0075] Figure 19 4 shows the crystal habit of the EGT crystal obtained in Example 4;

[0076] Figure 20 14 shows the crystal habit of the EGT crystal obtained in Example 5;

[0077] Figure 21 14 shows the crystal habit of the EGT crystal obtained in Example 6;

[0078] Figure 22 14 shows the crystal habit of the EGT crystal obtained in Example 7;

[0079] Figure 23 Shows the XRD spectrum of ergothioneine standard;

[0080] Figure 24 1 shows the XRD spectrum of the EGT crystal of Example 1;

[0081] Figure 25 2 shows the XRD spectrum of the EGT crystal of Example 2;

[0082] Figure 26 14 shows the XRD spectrum of the EGT crystal of Example 3;

[0083] Figure 27 4 shows the XRD spectrum of the EGT crystal of Example 4;

[0084] Figure 28 14 shows the XRD spectrum of the EGT crystal of Example 5;

[0085] Figure 29 14 shows the XRD spectrum of the EGT crystal of Example 6;

[0086] Figure 30 FIG2 shows the XRD spectrum of the EGT crystal of Example 7. DETAILED DESCRIPTION

[0087] The invention discloses a method for purifying thioneine, and those skilled in the art can learn from this document content, and appropriately improve process parameter and realize.It is particularly important to point out that all similar replacements and changes are apparent to those skilled in the art, and they are all considered to be included in the present invention.The method and application of the present invention have been described by preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the method and application described herein without departing from the content of the present invention, spirit and scope, to realize and apply the technology of the present invention.

[0088] In order to develop a method for the industrial production of thioneine with low cost, environmental protection, simplicity and high efficiency, fill the purification field of producing thioneine from an enzyme catalytic approach, the invention provides a method for purifying high-purity thioneine from an enzyme catalytic reaction solution. The method has few purification steps and can remove divalent and trivalent anions (such as SO4) simultaneously by a one-step ion exchange. 2- PO4 3- ), cations (such as Mg 2+ 、Fe 3+ ) and depigmentation, simplifies purification process, can well meet the industrial-scale production of thioneine. In addition, the method can obtain thioneine crystals with a purity of up to 99.9% by specific crystallization process, and crystal size is large, particles are uniform, a green, high-purity, low-price high-quality thioneine product can be provided for the downstream industries such as medicine, food, cosmetics, meet the extensive needs of people to the good life material.

[0089] Detailed plan:

[0090] Step 1, ultrafiltration: the enzyme catalytic reaction solution (thioneine concentration 20-40g / L) containing thioneine is adjusted to pH value so that protein is substantially precipitated and then filtered using a ceramic ultrafiltration membrane, the macromolecular substances such as enzyme, nucleic acid, polysaccharide are removed, and the permeate containing thioneine is collected.

[0091] Step 2, ion exchange: The ultrafiltration liquid is passed through anion and cation exchange resins for adsorption to remove divalent and trivalent anions (such as SO4 2- PO4 3- ) and cations (such as Mg 2+ 、Fe 3+ ), then use pure water to wash out the thioneine that remains in the post but is not adsorbed by the resin, and collect the column liquid containing thioneine.

[0092] Step 3, nanofiltration: using a nanofiltration membrane to remove more than 95% of the monovalent salt ions of the thioneine solution after ion exchange, and concentrating it to an EGT concentration of 50-100 g / L.

[0093] Step 4, crystallization: First, the EGT solution after nanofiltration and desalination is further concentrated to a corresponding concentration by rotary evaporation, and then an antisolvent is added to the concentrated EGT solution while stirring in a 30-60°C water bath to dissolve and crystallize. A small amount of seed crystals is added at an appropriate time and the temperature is gradually lowered to 5°C to increase the crystallization yield. After the crystallization is completed, the crystals are filtered to obtain a filter cake, and the filter cake is washed and dried to obtain pure ergothioneine.

[0094] In the above scheme, step 4-crystallization is the key step, and the EGT concentration before crystallization, water bath temperature, timing of seed addition, cooling rate, etc. are all key parameters.

[0095] The preparation method of the enzyme-catalyzed reaction solution in step 1 is as follows:

[0096] 1. Preparation of Egt-1 and Egt-2 enzyme solutions: Weigh appropriate amounts of Egt-1 and Egt-2 enzyme bacteria according to the required enzyme volume. Mix the bacteria with purified water at a ratio of 1:4 (m:m) and stir until no lumps remain and the bacterial suspension is turbid. Disrupt the bacterial suspension twice using a high-pressure homogenizer at 5-10°C and a pressure range of 800-1000 bar.

[0097] 2. First step enzyme reaction: Prepare 0.5 mol / L trimethylhistidine (TMH), add Egt-1 enzyme solution under stirring at 25°C, adjust the pH to 6.5-7.5, then add the prepared 0.5 mol / L cysteine ​​dropwise to the reaction solution at a certain speed to carry out the first step reaction to generate thioneine intermediate. The first step reaction can be terminated when the reaction conversion rate reaches 95%.

[0098] Conversion rate calculation formula:

[0099]

[0100] 3. Second step enzyme reaction: Add solid sodium sulfite and Egt-2 enzyme solution to the first step reaction solution, adjust the pH to 8.0-8.5, and start the reaction to produce thioneine. When the thioneine concentration no longer increases, the second step reaction can be terminated.

[0101] An enzyme-catalyzed reaction solution containing 20-40 g / L thioneine can be obtained by this method.

[0102] In step 1, the pH value was adjusted to 2.0-5.0 using 4 mol / L hydrochloric acid. The ultrafiltration membrane was a spiral-wound ceramic membrane material purchased from Shandong Bona Group. It is a porous, precision ceramic filtration composite material made from Al2O3, ZrO2, and TiO2, sintered at high temperatures. Its pore size was 3-10 nm, its molecular weight cut-off was 2 kDa-20 kDa, its filtration pressure was 0.2-0.4 MPa, and its filtration temperature was 10-30°C.

[0103] In step 2, the anion exchange resin column is filled with a styrene-based weakly basic resin, including but not limited to LX-950, LX-D301, LX-67, and LX-6703; the cation exchange resin is filled with a styrene-based macroporous strongly acidic resin, including but not limited to LX-001, LX-35A, LX-160, and RCS72. Specifically, the EGT concentration before loading needs to be controlled at approximately 10 g / L, the loading pH should be 3.0-5.0, the loading flow rate should be 0.5-2.0 BV (BV is the filler volume) / h, and the loading volume should be 2-10 BV.

[0104] In step 3, the nanofiltration membrane is made of a composite membrane material such as rolled polyamide, sulfonated polyethersulfone, sulfonated polysulfone, or polyethersulfone, with a molecular weight cutoff of 100-300 Da, a nanofiltration pressure of 1.0-2.0 MPa, and a filtration temperature of 5-30°C.

[0105] In step 4, it is necessary to rotary evaporate to an EGT concentration of 160-220 g / L. The anti-solvent used for crystallization is 95% ethanol, and the amount added is 2-6 times the volume of the concentrate, and the addition rate is 0.2-0.6 times the volume of the concentrate / h. The timing of adding the seed crystals is after the anti-solvent is added to 0.5-1.0 times the volume of the concentrate, at which time the EGT concentration is in the metastable zone of its solubility curve. After adding the seed crystals, stir for 1 hour and then start cooling. The cooling rate is 2.5°C per hour or 5°C every 2 hours. In particular, the stirring speed should be slow throughout the crystallization process to keep the entire solution in a flowing state.

[0106] In step 4, the solvent used for washing the filter cake is a 75%-95% ethanol aqueous solution. The drying method is vacuum drying with a vacuum degree of -0.1 to -0.08 MPa and a drying temperature of 45-60°C.

[0107] The structural formula of EGT is as follows Figure 1 As shown, the HPLC spectrum and properties of EGT crystals obtained by this method are as follows Figure 2-22 shown.

[0108] Unless otherwise specified, the raw materials and reagents used in the purification method of thioneine provided by the present invention can be purchased from the market.

[0109] The present invention will be further described below in conjunction with the embodiments:

[0110] Example 1

[0111] Preparation of enzyme-catalyzed reaction solution:

[0112] 1. Preparation of Egt-1 and Egt-2 enzyme solutions: Weigh appropriate amounts of Egt-1 and Egt-2 enzyme bacteria according to the required enzyme volume. Mix the bacteria with purified water at a ratio of 1:4 (m:m) and stir until no lumps remain and the bacterial suspension is turbid. Disrupt the bacterial suspension twice using a high-pressure homogenizer at 5-10°C and a pressure range of 800-1000 bar.

[0113] 2. First step enzyme reaction: Prepare 0.5 mol / L trimethylhistidine (TMH), add Egt-1 enzyme solution under stirring at 25°C, adjust the pH to 6.5-7.5, then add the prepared 0.5 mol / L cysteine ​​dropwise to the reaction solution at a certain speed to carry out the first step reaction to generate thioneine intermediate. The first step reaction can be terminated when the reaction conversion rate reaches 95%.

[0114] Conversion rate calculation formula:

[0115]

[0116] 3. Second step enzyme reaction: Add solid sodium sulfite and Egt-2 enzyme solution to the first step reaction solution, adjust the pH to 8.0-8.5, and start the reaction to produce thioneine. When the thioneine concentration no longer increases, the second step reaction can be terminated.

[0117] An enzyme-catalyzed reaction solution containing 20-40 g / L thioneine can be obtained by this method.

[0118] Ultrafiltration: A 5-liter volume of the enzyme-catalyzed reaction solution (EGT concentration of 25 g / L) was adjusted to pH 2.0 with 4 mol / L hydrochloric acid. Ultrafiltration was then performed using a ceramic membrane (production number 03038176), with a pore size of 3 nm and a molecular weight cutoff of 2 kDa. The pressure was set to 0.2 MPa and the temperature was controlled at 10°C. After completion of filtration, 20 L of ultrafiltrate was obtained, with an EGT yield of 95.38%.

[0119] Ion Exchange: The ultrafiltrate was adjusted to pH 3.0 and passed through an anion and cation exchange resin column consisting of LX-950 resin and LX-001 resin in series at a flow rate of 0.5 BV / h for adsorption, impurity removal, and decolorization. A sample volume of 10 BV was loaded. After the column run, 2 BV of pure water was added to the top-up, and the effluent was collected to yield approximately 30 L of a colorless EGT solution. The calculated EGT yield was 99.88%, with both anion and cation removal efficiencies exceeding 90%.

[0120] Nanofiltration: 30 L of colorless EGT solution was desalted and concentrated using a 100D polyamide nanofiltration membrane at a pressure of 1.0 MPa. The nanofiltration temperature was maintained at 5°C. By continuously adding pure water and permeating the monovalent salt solution, 2.5 L of concentrate was obtained with an EGT concentration of 44.35 g / L and an EGT yield of 93.11%.

[0121] Crystallization: First, use a rotary evaporator to further concentrate the nanofiltration solution to 200g / L, add it to a crystallization bottle preheated at 50℃, and add 6 times the volume of the concentrate dropwise with a peristaltic pump. The addition rate is 0.6 times the volume of the concentrate / h. After 95% ethanol is added to 0.75 times the volume of the concentrate, add 0.1% EGT seed crystals, stir for 1 hour, and then start gradient cooling by lowering the temperature by 5℃ every 2 hours. After the crystallization is completed, filter with 30-50μm medium-speed qualitative filter paper, wash the filter cake with 75% ethanol, and dry at 45℃ under a vacuum of -0.1MPa to obtain EGT crystals. See the results. Figure 2 、 Figure 9 、 Figure 16 、 Figure 24, the final crystallization yield is 95%, the EGT crystal purity is 99.95%, and the content is 99.82%. According to the thioneine standard ( Figure 23 ) and the X-ray powder diffraction pattern result of the obtained crystal show that the obtained crystallization is thioneine crystal.

[0122] The total purification yield was 84.27%.

[0123] The stability of the obtained crystalline product at 40°C and 75% humidity is shown in the following table:

[0124] Table 1 Stability data of the crystalline product obtained in Example 1

[0125] Detection items Properties-Color purity content 0 months White crystalline particles 99.95％ 99.82％ 1 month White crystalline particles 99.95％ 99.82％ 3 months White crystalline particles 99.93％ 99.81％ 6 months White crystalline particles 99.93％ 99.81％ 9 months White crystalline particles 99.92％ 99.80％ 12 months White crystalline particles 99.91％ 99.78％

[0126] According to the stability data of the crystalline product, the EGT crystals can maintain their properties as white crystalline particles within 12 months, with the purity and content only decreasing by 0.04%, indicating that the product can remain stable within 12 months at 40°C and 75% humidity.

[0127] Example 2

[0128] Ultrafiltration: A 5-liter volume of the enzyme-catalyzed reaction solution (EGT concentration of 30 g / L) was adjusted to pH 2.5 with 4 mol / L hydrochloric acid. Ultrafiltration was then performed using a ceramic membrane (manufactured with the product number 30800359), with a pore size of 5 nm and a molecular weight cutoff of 10 kDa. The pressure was set to 0.3 MPa and the temperature was controlled at 15°C. After filtration, 20.38 L of ultrafiltrate was obtained, with an EGT yield of 95.15%.

[0129] Ion Exchange: The ultrafiltrate was adjusted to pH 3.5 and passed through an anion and cation exchange resin column consisting of LX-D301 resin and LX-35A resin in series at a flow rate of 1.0 BV / h for adsorption, impurity removal, and decolorization. A sample volume of 10 BV was loaded. After the column run, 2 BV of pure water was added to the top-up, and the effluent was collected to yield approximately 30 L of colorless EGT solution. The calculated EGT yield was 98.53%, with both anion and cation removal efficiencies exceeding 90%.

[0130] Nanofiltration: 30 L of colorless EGT solution was desalted and concentrated using a 200D sulfonated polyethersulfone nanofiltration membrane at a pressure of 1.5 MPa. The nanofiltration temperature was maintained at 10°C. By continuously adding pure water and permeating the monovalent salt solution, approximately 2.5 L of concentrated solution was obtained with an EGT concentration of 53.42 g / L and an EGT yield of 94.96%.

[0131] Crystallization: First, use a rotary evaporator to further concentrate the nanofiltration solution to 180g / L, add it to a crystallization bottle preheated at 45℃, and add 5 times the volume of the concentrate dropwise with a peristaltic pump. The addition rate is 0.5 times the volume of the concentrate / h. When 95% ethanol is added to 1.0 times the volume of the concentrate, add 0.1% EGT seed crystals, stir for 1 hour, and then start gradient cooling by lowering the temperature by 5℃ every 2 hours. After the crystallization is completed, filter with 30-50um medium-speed qualitative filter paper, wash the filter cake with 80% ethanol, and dry at 50℃ under a vacuum of -0.09MPa to obtain EGT crystals. See the results. Figure 3 、 Figure 10 、 Figure 17 、 Figure 25 , the final crystallization yield is 92%, the EGT crystal purity is 99.96%, and the content is 99.88%. According to the thioneine standard ( Figure 23 ) and the X-ray powder diffraction pattern result of the obtained crystal show that the obtained crystallization is thioneine crystal.

[0132] The total purification yield was 81.90%.

[0133] The stability of the obtained crystalline product at 40°C and 75% humidity is shown in the following table:

[0134] Table 2 Stability data of the crystalline product obtained in Example 2

[0135] Detection items Properties-Color purity content 0 months White crystalline particles 99.96％ 99.88％ 1 month White crystalline particles 99.96％ 99.87％ 3 months White crystalline particles 99.95％ 99.85％ 6 months White crystalline particles 99.95％ 99.83％ 9 months White crystalline particles 99.93％ 99.82％ 12 months White crystalline particles 99.93％ 99.80％

[0136] According to the stability data of the crystalline product, the EGT crystals can maintain their properties as white crystalline particles within 12 months, with the purity only decreasing by 0.03% and the content only decreasing by 0.08%, indicating that the product can remain stable within 12 months at 40°C and 75% humidity.

[0137] Example 3

[0138] Ultrafiltration: A 5-liter volume of the enzyme-catalyzed reaction solution (EGT concentration of 32 g / L) was adjusted to pH 3.0 with 4 mol / L hydrochloric acid. Ultrafiltration was then performed using a ceramic membrane (manufactured with the product number 11289902), with a pore size of 10 nm and a molecular weight cutoff of 20 kDa. The pressure was set to 0.4 MPa and the temperature was controlled at 30°C. After filtration, 19.83 L of ultrafiltrate was obtained, with an EGT yield of 96.58%.

[0139] Ion Exchange: The ultrafiltrate was adjusted to pH 4.0 and passed through an anion and cation exchange resin column consisting of LX-67 and LX-160 resins in series at a flow rate of 1.5 BV / h for adsorption, impurity removal, and decolorization. An 8 BV sample volume was loaded. After the column run, 2 BV of pure water was added to the top-up, and the effluent was collected to yield approximately 30 L of a colorless EGT solution. The calculated EGT yield was 98.21%, with both anion and cation removal efficiencies exceeding 90%.

[0140] Nanofiltration: 30 L of colorless EGT solution was desalted and concentrated using a 300D sulfonated polysulfone nanofiltration membrane at a pressure of 2.0 MPa. The nanofiltration temperature was maintained at 20°C. By continuously adding pure water and permeating the monovalent salt solution, approximately 2.5 L of concentrated solution was obtained with an EGT concentration of 57.43 g / L and an EGT yield of 94.61%.

[0141] Crystallization: First, use a rotary evaporator to further concentrate the nanofiltration solution to 190g / L, add it to a crystallization bottle preheated at 40℃, and add 4 times the volume of the concentrate dropwise with 95% ethanol through a peristaltic pump, and the addition rate is 0.4 times the volume of the concentrate / h. When 95% ethanol is added to 0.9 times the volume of the concentrate, add 0.1% EGT seed crystals, stir for 1 hour, and then start gradient cooling at a temperature of 2.5℃ every 1 hour. After the crystallization is completed, filter with 30-50um medium-speed qualitative filter paper, wash the filter cake with 85% ethanol, and dry at 55℃ under a vacuum of -0.09MPa to obtain EGT crystals. See the results. Figure 4 、 Figure 11 、 Figure 18 、 Figure 26 , the final crystallization yield is 90%, the EGT crystal purity is 99.96%, and the content is 99.90%. According to the thioneine standard ( Figure 23 ) and the X-ray powder diffraction pattern result of the obtained crystal show that the obtained crystallization is thioneine crystal.

[0142] The total purification yield was 80.76%.

[0143] The stability of the obtained crystalline product at 40°C and 75% humidity is shown in the following table:

[0144] Table 3 Stability data of the crystalline product obtained in Example 3

[0145] Detection items Properties-Color purity content 0 months White crystalline particles 99.96％ 99.90％ 1 month White crystalline particles 99.95％ 99.88％ 3 months White crystalline particles 99.93％ 99.88％ 6 months White crystalline particles 99.93％ 99.85％ 9 months White crystalline particles 99.91％ 99.84％ 12 months White crystalline particles 99.90％ 99.81％

[0146] According to the stability data of the crystalline product, the EGT crystals can maintain their properties as white crystalline particles within 12 months, with the purity only decreasing by 0.06% and the content only decreasing by 0.09%, indicating that the product can remain stable within 12 months at 40°C and 75% humidity.

[0147] Example 4

[0148] Ultrafiltration: A 5 L volume of the enzyme-catalyzed reaction solution (EGT concentration of 28 g / L) was adjusted to pH 4.0 with 4 mol / L hydrochloric acid. Ultrafiltration was then performed using a ceramic membrane (manufactured with the product number 30800359), with a pore size of 5 nm and a molecular weight cutoff of 10 kDa. The pressure was set to 0.3 MPa and the temperature was controlled at 25°C. After filtration, 20 L of ultrafiltrate was obtained, and the EGT yield was 94.78%.

[0149] Ion Exchange: The ultrafiltrate was adjusted to pH 5.0 and passed through an anion and cation exchange resin column consisting of LX-6703 resin and RCS72 resin in series at a flow rate of 2.0 BV / h for adsorption, impurity removal, and decolorization. A sample volume of 6 BV was loaded. After the column run, 2 BV of pure water was added to the top-up, and the effluent was collected to yield approximately 30 L of a colorless EGT solution. The calculated EGT yield was 96.82%, with both anion and cation removal efficiencies exceeding 90%.

[0150] Nanofiltration: 30 L of colorless EGT solution was desalted and concentrated using a 200D polyethersulfone nanofiltration membrane at a pressure of 1.5 MPa. The nanofiltration temperature was maintained at 25°C. By continuously adding pure water and permeating the monovalent salt solution, 2.5 L of concentrate was obtained with an EGT concentration of 48.63 g / L and an EGT yield of 94.63%.

[0151] Crystallization: First, use a rotary evaporator to further concentrate the nanofiltration solution to 200g / L, add it to a crystallization bottle preheated at 35℃, and add 3 times the volume of the concentrate dropwise with 95% ethanol through a peristaltic pump, and the addition rate is 0.3 times the volume of the concentrate / h. After 95% ethanol is added to 0.75 times the volume of the concentrate, add 0.1% EGT seed crystals, stir for 1 hour, and then start gradient cooling at a temperature of 2.5℃ per hour. After the crystallization is completed, filter with 30-50um medium-speed qualitative filter paper, wash the filter cake with 90% ethanol, and dry at 60℃ under a vacuum of -0.08MPa to obtain EGT crystals. See the results. Figure 5 、 Figure 12 、 Figure 19 、 Figure 27 , the final crystallization yield is 87%, the EGT crystal purity is 99.96%, and the content is 99.92%. According to the thioneine standard ( Figure 23 ) and the X-ray powder diffraction pattern result of the obtained crystal show that the obtained crystallization is thioneine crystal.

[0152] The total purification yield was 75.55%.

[0153] The stability of the obtained crystalline product at 40°C and 75% humidity is shown in the following table:

[0154] Table 4 Stability data of the crystalline product obtained in Example 4

[0155] Detection items Properties-Color purity content 0 months White crystalline particles 99.96％ 99.92％ 1 month White crystalline particles 99.94％ 99.92％ 3 months White crystalline particles 99.93％ 99.92％ 6 months White crystalline particles 99.93％ 99.90％ 9 months White crystalline particles 99.93％ 99.86％ 12 months White crystalline particles 99.91％ 99.86％

[0156] According to the stability data of the crystalline product, the EGT crystals can maintain their properties as white crystalline particles within 12 months, with the purity only decreasing by 0.05% and the content only decreasing by 0.06%, indicating that the product can remain stable within 12 months at 40°C and 75% humidity.

[0157] Example 5

[0158] Ultrafiltration: A 5-liter volume of the enzyme-catalyzed reaction solution (EGT concentration of 30 g / L) was adjusted to pH 5.0 with 4 mol / L hydrochloric acid. Ultrafiltration was then performed using a ceramic membrane (manufactured with the product number 30800359), with a pore size of 5 nm and a molecular weight cutoff of 10 kDa. The pressure was set to 0.3 MPa and the temperature was controlled at 25°C. After filtration, 20 L of ultrafiltrate was obtained, with an EGT yield of 96.98%.

[0159] Ion Exchange: The ultrafiltrate was adjusted to pH 5.0 and passed through an anion and cation exchange resin column consisting of LX-6703 resin and RCS72 resin in series at a flow rate of 1.5 BV / h for adsorption, impurity removal, and decolorization. An 8 BV sample volume was loaded. After the column run, 2 BV of pure water was added to the top-up, and the effluent was collected to yield approximately 30 L of a colorless EGT solution. The calculated EGT yield was 96.66%, with both anion and cation removal efficiencies exceeding 90%.

[0160] Nanofiltration: 30 L of colorless EGT solution was desalted and concentrated using a 250D polyamide nanofiltration membrane at a pressure of 2.0 MPa. The nanofiltration temperature was maintained at 30°C. By continuously adding pure water and permeating the monovalent salt solution, 2.5 L of concentrate was obtained with an EGT concentration of 53.62 g / L and an EGT yield of 95.34%.

[0161] Crystallization: First, use a rotary evaporator to further concentrate the nanofiltration solution to 200g / L, add it to a crystallization bottle preheated at 40℃, and add 3 times the volume of the concentrate dropwise with 95% ethanol through a peristaltic pump, and the addition rate is 0.3 times the volume of the concentrate / h. When 95% ethanol is added to 0.75 times the volume of the concentrate, add 0.1% EGT seed crystals, stir for 1 hour, and then start gradient cooling at a temperature of 2.5℃ per hour. After the crystallization is completed, filter with 30-50um medium-speed qualitative filter paper, wash the filter cake with 95% ethanol, and vacuum dry at 45℃ under a vacuum degree of -0.1MPa to obtain EGT crystals. See the results. Figure 6 、 Figure 13 、 Figure 20 、 Figure 28, the final crystallization yield is 87%, the EGT crystal purity is 99.96%, and the content is 99.95%. According to the thioneine standard ( Figure 23 ) and the X-ray powder diffraction pattern result of the obtained crystal show that the obtained crystallization is thioneine crystal.

[0162] The total purification yield was 77.75%.

[0163] The stability of the obtained crystalline product at 40°C and 75% humidity is shown in the following table:

[0164] Table 5 Stability data of the crystalline product obtained in Example 5

[0165] Detection items Properties-Color purity content 0 months White crystalline particles 99.96％ 99.95％ 1 month White crystalline particles 99.93％ 99.92％ 3 months White crystalline particles 99.93％ 99.92％ 6 months White crystalline particles 99.92％ 99.92％ 9 months White crystalline particles 99.90％ 99.86％ 12 months White crystalline particles 99.89％ 99.86％

[0166] According to the stability data of the crystalline product, the EGT crystals can maintain their properties as white crystalline particles within 12 months, with the purity only decreasing by 0.07% and the content only decreasing by 0.09%, indicating that the product can remain stable within 12 months at 40°C and 75% humidity.

[0167] Example 6

[0168] Ultrafiltration: A 5-liter volume of the enzyme-catalyzed reaction solution (EGT concentration of 30 g / L) was adjusted to pH 5.0 with 4 mol / L hydrochloric acid. Ultrafiltration was then performed using a ceramic membrane (manufactured with the product number 03038176) with a pore size of 3 nm and a molecular weight cutoff of 2 kDa. The pressure was set to 0.3 MPa and the temperature was controlled at 25°C. After filtration, 20 L of ultrafiltrate was obtained, and the EGT yield was 96.34%.

[0169] Ion Exchange: The ultrafiltrate was adjusted to pH 5.0 and passed through an anion and cation exchange resin column consisting of LX-6703 resin and RCS72 resin in series at a flow rate of 0.5 BV / h for adsorption, impurity removal, and decolorization. A sample volume of 2 BV was loaded. After the column run, 2 BV of pure water was added to the top-up, and the effluent was collected to yield approximately 30 L of a colorless EGT solution. The calculated EGT yield was 95.36%, with both anion and cation removal efficiencies exceeding 90%.

[0170] Nanofiltration: 30 L of colorless EGT solution was desalted and concentrated using a 250D polyamide nanofiltration membrane at a pressure of 2.0 MPa. The nanofiltration temperature was maintained at 30°C. By continuously adding pure water and permeating the monovalent salt solution, 2.5 L of concentrate was obtained with an EGT concentration of 53.72 g / L and an EGT yield of 97.45%.

[0171] Crystallization: First, use a rotary evaporator to further concentrate the nanofiltration solution to 220g / L, add it to a crystallization bottle preheated at 40℃, and add 2 times the volume of the concentrate dropwise with a peristaltic pump. The addition rate is 0.2 times the volume of the concentrate / h. After 95% ethanol is added to 0.5 times the volume of the concentrate, add 0.1% EGT seed crystals, stir for 1 hour, and then start gradient cooling at a temperature of 2.5℃ every hour. After the crystallization is completed, filter with 30-50um medium-speed qualitative filter paper, wash the filter cake with 95% ethanol, and dry at 45℃ under a vacuum of -0.1MPa to obtain EGT crystals. See the results. Figure 7 、 Figure 14 、 Figure 21 、 Figure 29 , the final crystallization yield is 85%, the EGT crystal purity is 99.97%, and the content is 99.92%. According to the thioneine standard ( Figure 23 ) and the X-ray powder diffraction pattern result of the obtained crystal show that the obtained crystallization is thioneine crystal.

[0172] The total purification yield was 76.10%.

[0173] The stability of the obtained crystalline product at 40°C and 75% humidity is shown in the following table:

[0174] Table 6 Stability data of the crystalline product obtained in Example 6

[0175]

[0176]

[0177] According to the stability data of the crystalline product, the EGT crystals can maintain their properties as white crystalline particles within 12 months, with the purity only decreasing by 0.08% and the content only decreasing by 0.07%, indicating that the product can remain stable within 12 months at 40°C and 75% humidity.

[0178] Example 7

[0179] Ultrafiltration: A 5-liter volume of the enzyme-catalyzed reaction solution (EGT concentration of 30 g / L) was adjusted to pH 5.0 with 4 mol / L hydrochloric acid. Ultrafiltration was then performed using a ceramic membrane (manufactured with the product number 11289902), with a pore size of 5 nm and a molecular weight cutoff of 20 kDa. The pressure was set to 0.3 MPa and the temperature was controlled at 25°C. After filtration, 20 L of ultrafiltrate was obtained, and the EGT yield was 97.71%.

[0180] Ion Exchange: The ultrafiltrate was adjusted to pH 5.0 and passed through an anion and cation exchange resin column consisting of LX-6703 resin and RCS72 resin in series at a flow rate of 1.0 BV / h for adsorption, impurity removal, and decolorization. A sample volume of 4 BV was loaded. After the column run, 2 BV of pure water was added to the top-up, and the effluent was collected to yield approximately 30 L of a colorless EGT solution. The calculated EGT yield was 96.19%, with both anion and cation removal efficiencies exceeding 90%.

[0181] Nanofiltration: 30 L of colorless EGT solution was desalted and concentrated using a 250D polyamide nanofiltration membrane at a pressure of 2.0 MPa. The nanofiltration temperature was maintained at 30°C. By continuously adding pure water and permeating the monovalent salt solution, 2.5 L of concentrate was obtained with an EGT concentration of 54.60 g / L and an EGT yield of 96.82%.

[0182] Crystallization: First, use a rotary evaporator to further concentrate the nanofiltration solution to 160g / L, add it to a crystallization bottle preheated at 35℃, and add 3.5 times the volume of the concentrated solution of 95% ethanol drop by drop through a peristaltic pump, and the addition rate is 0.35 times the volume of the concentrated solution / h. After 95% ethanol is added to the volume of 0.5 times the concentrated solution, add 0.1% EGT seed crystals, stir for 1 hour, and then start gradient cooling at a temperature of 2.5℃ every 1 hour. After the crystallization is completed, filter with 30-50um medium-speed qualitative filter paper, wash the filter cake with 95% ethanol, and dry at 45℃ under a vacuum of -0.1MPa to obtain EGT crystals. See the results. Figure 8 、 Figure 15 、 Figure 22 、 Figure 30 , the final crystallization yield is 89%, the EGT crystal purity is 99.96%, and the content is 99.93%. According to the thioneine standard ( Figure 23 ) and the X-ray powder diffraction pattern result of the obtained crystal show that the obtained crystallization is thioneine crystal.

[0183] The total purification yield was 80.99%.

[0184] The stability of the obtained crystalline product at 40°C and 75% humidity is shown in the following table:

[0185] Table 7 Stability data of the crystalline product obtained in Example 7

[0186] Detection items Properties-Color purity content 0 months White crystalline particles 99.96％ 99.93％ 1 month White crystalline particles 99.96％ 99.93％ 3 months White crystalline particles 99.94％ 99.92％ 6 months White crystalline particles 99.90％ 99.88％ 9 months White crystalline particles 99.90％ 99.87％ 12 months White crystalline particles 99.89％ 99.87％

[0187] According to the stability data of the crystalline product, the EGT crystals can maintain their properties as white crystalline particles within 12 months, with the purity only decreasing by 0.07% and the content only decreasing by 0.06%, indicating that the product can remain stable within 12 months at 40°C and 75% humidity.

[0188] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. the purification method of thioneine, is characterized in that, The steps include: Step 1: taking the enzyme catalytic reaction solution containing thioneine, adjusting the pH value, ultrafiltration, and collecting the permeate; Step 2: Take the permeate described in step 1, sequentially pass it through a cation exchange resin column and an anion exchange resin column for adsorption, elute with pure water, and collect the column liquid; Step 3, taking the column liquid described in step 2, nanofiltration, and collecting the filtrate; Step 4, take the filtrate described in step 3, concentrate to obtain concentrated solution, crystallize, filter, wash, and dry to obtain described thioneine.

2. The purification method according to claim 1, wherein The pore size of the ultrafiltration membrane in step 1 is 3 to 10 nm; the molecular weight cut-off of the ultrafiltration is 2 KDa to 20 KDa; the pressure of the ultrafiltration is 0.2 to 0.4 MPa; and the temperature of the ultrafiltration is 10 to 30°C.

3. The purification method according to claim 1 or 2, wherein The filler of the anion exchange resin column in step 2 comprises a styrene-based weakly basic resin; and / or The filler of the cation exchange resin column in step 2 includes a styrene-based macroporous strong acid resin.

4. The purification method according to claim 3, wherein The types of the styrene-based weakly basic resin include but are not limited to one or more of LX-950, LX-D301, LX-67 or LX-6703; and / or The types of the styrene-based macroporous strong acidic resin include but are not limited to one or more of LX-001, LX-35A, LX-160 or RCS72.

5. The purification method according to any one of claims 1 to 4, characterized in that The pH value of the permeate in step 2 is 3.0 to 5.0; and / or The loading flow rate of the permeate is 0.5 to 2.0 times the filler volume / h, and the loading amount is 2 to 10 times the filler volume.

6. The purification method according to any one of claims 1 to 5, characterized in that The pore size of the nanofiltration membrane in step 3 is 1 to 2 nm; the molecular weight cut-off of the nanofiltration is 100 to 300 Da; the pressure of the nanofiltration is 1.0 to 2.0 MPa; and the temperature of the nanofiltration is 5 to 30° C.

7. The purification method according to any one of claims 1 to 6, wherein Concentrating described in step 4 comprises concentrating described filtrate to 160~220g / L of thioneine concentration; And / or The crystallization in step 4 includes adding anti-solvent crystallization; the amount of the anti-solvent added includes 2 to 6 times the volume of the concentrated solution; the addition rate of the anti-solvent includes 0.2 to 0.6 times the volume of the concentrated solution / h; The anti-solvent included 95% ethanol.

8. The purification method according to any one of claims 1 to 7, wherein The crystallization described in step 4 also includes adding 0.5 to 1.0 times the volume of the concentrated solution of the anti-solvent, adding thioneine seed crystals, and then adding the anti-solvent of the remainder; The added amount of the thioneine seed crystals comprises 0.1% of the mass of the thioneine.

9. The purification method according to any one of claims 1 to 8, wherein The crystallization described in step 4 includes adding the thioneine seed crystal for 1 hour and starting to cool, and the cooling rate includes 2.5°C / h, or 5°C / 2h.

10. The purification method according to any one of claims 1 to 9, characterized in that The washing in step 4 comprises washing with a 75% to 95% ethanol aqueous solution; The drying in step 4 includes vacuum drying; the vacuum degree of the vacuum drying includes -0.1 to -0.08 MPa; the temperature of the vacuum drying includes 45 to 60°C.

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