A method for preparing large-particle high-purity ergothioneine crystals by direct crystallization from a fermentation broth and large-particle high-purity ergothioneine crystals

Abstract

This invention belongs to the field of pharmaceutical crystallization technology and substance separation and purification. It discloses a method for directly crystallizing large-particle, high-purity ergothioneine crystals from fermentation broth, and the resulting large-particle, high-purity ergothioneine crystals. The method uses ergothioneine fermentation broth as the starting material, dissolving it in a specific crystallization solvent, and then sequentially undergoing two programmed cooling and crystal growth processes to directly crystallize and obtain the target product. This method, through the synergistic selection of the solvent system and control of crystallization kinetics, simultaneously completes the purification and crystallization of ergothioneine in a single step, eliminating the complex multi-step pretreatment and purification units found in existing technologies. The obtained product has high purity (≥99.992%), good flowability, particle size >200μm, and angle of repose <25°, possessing ultra-high purity, ideal physical morphology, and excellent flowability, and can be directly used in downstream formulation production. This invention features a very simple process flow, high production efficiency, low cost, and superior overall product performance, demonstrating significant industrial application value.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical crystallization technology and substance separation and purification, specifically relating to a method for preparing ergothioneine, and more particularly to a method and product for directly preparing high-purity, large-particle, high-flowability ergothioneine crystals using ergothioneine fermentation broth as the starting material through a one-step integrated cooling crystallization process. Background Technology

[0002] L-ergothioneine (EGT), a rare natural chiral amino acid and a potent antioxidant, is an important physiologically active substance in the body. It possesses multiple functions, including antioxidation, free radical scavenging, detoxification, maintaining DNA biosynthesis, preventing UV radiation damage, regulating intracellular redox reactions, participating in intracellular energy regulation, and acting as a cellular protective agent. Given its significant and unique biological functions, scholars from various countries have long been researching its applications. Currently, ergothioneine shows promising application and market prospects in organ transplantation, cell preservation, medicine, food and beverages, functional foods, animal feed, cosmetics, and biotechnology.

[0003] On July 13, 2017, the European Commission issued Regulation (EU) 2017 / 1281, authorizing the release of L-ergothioneine as a new food ingredient in accordance with Regulation (EC) 258 / 97 of the European Parliament. With the continuous upgrading of consumer demands, consumers are paying increasingly more attention to product details, moving from focusing solely on brand and function to considering ingredients, content, and specific efficacy. Products are constantly innovating with consumer needs at the core. Antioxidants are a popular function in functional foods, and products with high stability and outstanding efficacy are more likely to gain market favor. As an "emerging" functional ingredient, ergothioneine is poised to make a significant contribution to the field of antioxidants.

[0004] Ergothioneine preparation methods include natural biological extraction, chemical synthesis, and biosynthesis. Current research on ergothioneine mainly focuses on the screening of fermentation strains (e.g., invention patents CN109439553A and CN106661585A) and product applications (e.g., invention patents CN107209173A and CN109562042A), but most of the core technologies are held by foreign companies. Furthermore, although Chinese invention patent CN117263864A has developed a method for preparing high-purity ergothioneine, this method requires multiple membrane separation and resin decolorization processes, followed by heating, evaporation, concentration, and recrystallization to obtain a solid product. This solid product must then be pulverized to obtain ergothioneine with a purity of 99.9%. Chinese invention patent CN117924184A developed a method for separating and purifying ergothioneine from fermentation broth. However, this method requires multiple steps including pretreatment, sterilization, ultrafiltration, multiple concentrations, chromatography, decolorization, and crystallization, resulting in a solid product with a purity of only 98%, unsuitable for use in cosmetics and health products. Chinese invention patent CN116143699A also developed a method for purifying ergothioneine from fermentation broth, but this method requires pretreatment with flocculants and complexation and decomplexation with metal salt solutions. This process is not only complex but also results in the incomplete removal of added substances, leading to a product purity of only 98%, hindering wider application. This further contributes to the prevalence of problems in currently commercially available ergothioneine products, such as impure crystal form, undesirable crystal morphology, easy agglomeration, uneven particle size distribution, low purity, and low color, severely affecting product quality. Furthermore, the cumbersome preparation process limits the efficiency and economic viability of the technology. These technologies all follow the traditional approach of first deeply purifying to remove impurities, and then simply crystallizing to obtain a solid. Crystallization is only the end point of the entire purification chain, and its role is merely precipitation, not refining or shaping.

[0005] On the other hand, some patents (such as CN118908894A and CN119859123A) focus on modifying the crystal form of purified ergothioneine to improve its hygroscopicity and light stability. However, these technologies all start with high-purity ergothioneine intermediates or crude products, and their processes do not involve the challenge of starting from the most complex raw material, the original fermentation broth.

[0006] Therefore, the existing technology has the following prominent problems: (1) The process flow from fermentation liquid to product is long, the equipment investment is large, the operation is complicated and the cost is high; (2) The final product often needs to be crushed to obtain powder, resulting in large dust, poor flowability and easy agglomeration, which brings difficulties to downstream mixing, tableting, packaging and other processes; (3) It is difficult to simultaneously achieve high purity (>99.9%) and ideal crystal physical properties.

[0007] In view of this, the industry urgently needs to develop a disruptive short-process technology that can directly start from the fermentation broth and simultaneously achieve efficient purification and crystal product engineering through a one-step integrated crystallization operation, thereby obtaining high-purity, large-particle, and highly fluid ergothioneine crystals. Summary of the Invention

[0008] One of the objectives of this invention is to overcome the shortcomings of existing technologies and provide a method for directly crystallizing high-purity crystals from ergothioneine fermentation broth, which has an extremely short process flow, simple operation, low equipment requirements, and is easy to industrialize. This method breaks with the traditional paradigm of purification before crystallization.

[0009] The second objective of this invention is to provide a large-particle, high-purity ergothioneine crystal product prepared by the above method. This product has ultra-high purity, a specific large-particle block morphology, and excellent flowability, and can be directly used in the production of end products without the need for crushing.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] Firstly, this invention provides a method for preparing large-particle, high-purity ergothioneine crystals directly from ergothioneine fermentation broth. The core concept of this method is to abandon multi-step pre-purification and treat the fermentation broth as a holistic system. Through a carefully designed and matched solvent system and a two-stage programmed cooling crystallization kinetics control strategy, the selective extraction, purification, and crystal growth of ergothioneine are simultaneously completed within a single crystallizer.

[0012] The specific steps are as follows:

[0013] S1. Dissolution: The ergothioneine fermentation broth is mixed with a crystallization solvent and stirred at 40–80 °C to form a homogeneous and clear initial solution; the crystallization solvent is a mixed solvent, which is a mixed system of deionized water and dimethyl sulfoxide, deionized water and acetonitrile, methanol and acetic acid, formic acid and acetonitrile, ethylene glycol and N,N-dimethylformamide, n-propanol and acetonitrile, and acetone and deionized water;

[0014] S2. First stage of programmed cooling crystallization: The initial solution is cooled to the first crystallization temperature of 25-65 ℃ at a first cooling rate of 0.05-1.0 ℃ / min, and crystals are grown at this temperature and at a stirring rate of 100-500 r / min for 0.5-1 h to induce and stabilize crystal nuclei;

[0015] S3. Second stage of programmed cooling crystallization: The system treated in step S2 is cooled to the final crystallization temperature of 0 to 20 ℃ at a second cooling rate of 0.02 to 1.5 ℃ / min, and crystals are grown at this final temperature for 0.5 to 1.5 h.

[0016] S4. Post-processing: The solid-liquid mixture obtained in step S3 is subjected to solid-liquid separation. The resulting wet crystals are washed and dried to obtain the large-particle high-purity ergothioneine crystals.

[0017] Further, in step S1, the volume ratio of the two solvents in the mixed solvent is 1:5 to 5:1.

[0018] Furthermore, in step S1, the amount of the crystallization solvent used is 1 to 4 times the volume of the ergothioneine fermentation broth.

[0019] Further, in step S4, the washing agent used is acetone, ethanol, methanol or acetonitrile; the drying is blower drying or vacuum drying, the drying temperature is 30-60 ℃, and the drying time is 2-12 h.

[0020] Secondly, this invention provides large-particle, high-purity ergothioneine crystals, prepared by the method described above. The purity is ≥99.99%, the crystals are blocky with a particle size greater than 200 μm and an angle of repose less than 25°. After storage for 30 days at 30±5℃ and 40±5% relative humidity, the purity decreases by less than 0.01%.

[0021] Thirdly, the present invention provides the application of the large-particle high-purity ergothioneine crystals in the preparation of cosmetics, health foods or pharmaceuticals.

[0022] Advantages and beneficial effects of the present invention:

[0023] 1. This invention pioneers a new path for obtaining high-quality products through one-step direct crystallization from fermentation broth. Compared with existing technologies, it eliminates multiple complex unit operations such as flocculation, ultrafiltration, chromatography, complexation, multiple concentrations, and decolorization, greatly shortening the process flow (from as many as 8-10 steps to 4 steps), reducing equipment investment, energy consumption, and operating costs, and significantly improving production efficiency.

[0024] 2. This invention controls the nucleation and growth process of ergothioneine crystals through two cooling crystallization stages, resulting in crystal products with uniform particle size, good dispersibility, and intact block shape, and rapid solid-liquid separation.

[0025] 3. The powder prepared by this invention has excellent properties, and the crystalline product has a large particle size, good flowability, a particle size > 200 μm, and an angle of repose < 25°.

[0026] 4. The method of the present invention has good process controllability, short production cycle, small batch variation, and yield of over 90%.

[0027] 5. The crystal products prepared by this invention have high purity and content, with a purity ≥99.99% and a content >99.9%. The product quality is significantly better than commercially available products and can be directly used in the production of health products and other commodities. Attached Figure Description

[0028] Figure 1 X-ray powder diffraction pattern of ergothionein crystal in Example 1 of this invention.

[0029] Figure 2 DSC-TG analysis diagram of ergothioneine crystals in Example 1 of this invention.

[0030] Figure 3 FT-IR image of ergothionein crystal in Example 1 of this invention.

[0031] Figure 4 Raman diagram of ergothioneine crystals in Example 1 of this invention.

[0032] Figure 5 An optical microscope image of ergothioneine crystals in Example 1 of this invention. Detailed Implementation

[0033] The present invention will be described more clearly and completely through the following embodiments, but the described examples are only a part of the embodiments of the present invention, and not all of them. The embodiments are provided to help understand the present invention and should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1

[0035] At 80 ℃, with a stirring rate of 400 r / min, 50 mL of ergothioneine fermentation broth was dissolved in 50 mL of deionized water-DMSO mixed solution. V 水 : V DMSO A clear solution was formed in a mixture of 1:5 (ratio of ethyl to diethyl sulfadiazine). The temperature inside the crystallizer was lowered to 65°C at a rate of 0.05°C / min, at which point a large number of crystals appeared inside the crystallizer. The stirring rate was maintained, and the crystals were cultured for 0.5 h. After the crystals were cultured, the temperature inside the crystallizer was lowered to 5°C at a rate of 0.3°C / min, and the crystals were cultured for another 1 h. The product was subjected to solid-liquid separation to obtain a filter cake, which was then washed with 20 mL of acetone. The filter cake was placed in a vacuum drying oven and dried at a constant temperature of 50°C for 6 h. After drying, ergothioneine crystals were obtained.

[0036] The final product is a blocky white crystal with a particle size of approximately 255 μm and an angle of repose of 22.4°. The product purity is 99.996%, the content is 99.92%, and the process yield is 94%. The X-ray powder diffraction pattern of the product is shown below. Figure 1 As shown, the morphology is as follows Figure 5 As shown.

[0037] Example 2

[0038] At 60 ℃, with a stirring rate of 200 r / min, 50 mL of ergothioneine fermentation broth was dissolved in 100 mL of a methanol-acetic acid mixture. V 甲醇 : V 乙酸 A clear solution was formed in a mixture of 4:1 (ratio of acetonitrile to 4.5 ℃). The temperature inside the crystallizer was lowered to 41 ℃ at a rate of 0.6 ℃ / min, at which point a large number of crystals appeared inside the crystallizer. The stirring rate was maintained, and the crystals were grown for 1 h. After the crystals were grown, the temperature inside the crystallizer was lowered to 20 ℃ at a rate of 0.02 ℃ / min, and the crystals were grown for another 1 h. The product was subjected to solid-liquid separation to obtain a filter cake, which was then washed with 30 mL of acetonitrile. The filter cake was placed in a vacuum drying oven and dried at a constant temperature of 30 ℃ for 8 h. After drying, ergothioneine crystals were obtained.

[0039] The final product is a blocky white crystal with a particle size of approximately 264 μm and an angle of repose of 21.6°. The product purity is 99.993%, the content is 99.94%, and the process yield is 96%.

[0040] Example 3

[0041] At 40 ℃, with a stirring rate of 300 r / min, 50 mL of ergothioneine fermentation broth was dissolved in 80 mL of a formic acid-acetonitrile mixed solution. V 甲酸 : V 乙腈 A clear solution was formed in a mixture of 1:1 (ratio of ethanol to ethanol). The temperature inside the crystallizer was lowered to 25°C at a rate of 0.05°C / min, at which point a large number of crystals appeared inside the crystallizer. The stirring rate was maintained, and the crystals were grown for 1 hour. After the crystals were grown, the temperature inside the crystallizer was lowered to 10°C at a rate of 0.4°C / min, and the crystals were grown for another 0.5 hours. The product was subjected to solid-liquid separation to obtain a filter cake, which was then washed with 15 mL of ethanol. The filter cake was placed in a forced-air drying oven and dried at a constant temperature of 60°C for 10 hours. After drying, ergothioneine crystals were obtained.

[0042] The final product is a blocky white crystal with a particle size of approximately 249 μm and an angle of repose of 23.1°. The product purity is 99.997%, the content is 99.93%, and the process yield is 92%.

[0043] Example 4

[0044] At 50 ℃, with a stirring rate of 500 r / min, 50 mL of ergothioneine fermentation broth was dissolved in 150 mL of ethylene glycol-DMF mixed solution. V 乙二醇 : V DMF A clear solution was formed in a mixture of 5:1 (ratio of ethyl to diethyl sulfadiazine). The temperature inside the crystallizer was lowered to 28 °C at a rate of 1.0 °C / min, at which point a large number of crystals appeared inside the crystallizer. The stirring rate was maintained, and the crystals were cultured for 0.5 h. After the crystals were cultured, the temperature inside the crystallizer was lowered to 5 °C at a rate of 1.0 °C / min, and the crystals were cultured for another 1 h. The product was subjected to solid-liquid separation to obtain a filter cake, which was then washed with 20 mL of acetone. The filter cake was placed in a vacuum drying oven and dried at a constant temperature of 40 °C for 2 h. After drying, ergothioneine crystals were obtained.

[0045] The final product is a blocky white crystal with a particle size of approximately 241 μm and an angle of repose of 22.7°. The product purity is 99.994%, the content is 99.98%, and the process yield is 95%.

[0046] Example 5

[0047] At 70 ℃, with a stirring rate of 100 r / min, 50 mL of ergothioneine fermentation broth was dissolved in 200 mL of a n-propanol-acetonitrile mixed solution. V 正丙醇 : V 乙腈 A clear solution was formed in a mixture of 4:1 (ratio of 4:1). The temperature inside the crystallizer was lowered to 49 °C at a rate of 0.6 °C / min, at which point a large number of crystals appeared inside the crystallizer. The stirring rate was maintained, and the crystals were grown for 1 h. After the crystals were grown, the temperature inside the crystallizer was lowered to 0 °C at a rate of 0.3 °C / min, and the crystals were grown for another 1.5 h. The product was subjected to solid-liquid separation to obtain a filter cake, which was then washed with 25 mL of methanol. The filter cake was placed in a forced-air drying oven and dried at a constant temperature of 50 °C for 7 h. After drying, ergothioneine crystals were obtained.

[0048] The final product is a blocky white crystal with a particle size of approximately 262 μm and an angle of repose of 21.4°. The product purity is 99.996%, the content is 99.94%, and the process yield is 93%.

[0049] Example 6

[0050] At 80 ℃, with a stirring rate of 400 r / min, 50 mL of ergothioneine fermentation broth was dissolved in 120 mL of acetone-deionized water mixture. V 丙酮 : V 水A clear solution was formed in a mixture of 3:1 (ratio of ethanol to ethanol). The temperature inside the crystallizer was lowered to 61 °C at a rate of 0.5 °C / min, at which point a large number of crystals appeared inside the crystallizer. The stirring rate was maintained, and the crystals were grown for 1 h. After the crystals were grown, the temperature inside the crystallizer was lowered to 10 °C at a rate of 1.5 °C / min, and the crystals were grown for another 1 h. The product was subjected to solid-liquid separation to obtain a filter cake, which was then washed with 20 mL of ethanol. The filter cake was placed in a vacuum drying oven and dried at a constant temperature of 50 °C for 9 h. After drying, ergothioneine crystals were obtained.

[0051] The final product is a blocky white crystal with a particle size of approximately 254 μm and an angle of repose of 22.1°. The product purity is 99.992%, the content is 99.97%, and the process yield is 96%.

[0052] The chemical properties of the ergothioneine crystal products obtained in Examples 1-6, i.e., the ergothioneine crystals of the present invention, were tested.

[0053] 1) The chemical stability of the ergothioneine crystals prepared in this invention was investigated. During storage at 30 ℃ ± 5 ℃ and RH 40 ± 5% for 30 days, the color, purity, and morphology of the product remained unchanged, indicating that the crystals have good chemical stability. The results are shown in Table 1.

[0054] Table 1. Chemical stability study of ergothioneine crystals described in this invention.

[0055]

[0056] 2) such as Figure 1 The image shows the X-ray powder diffraction pattern of ergothioneine crystals obtained in Example 1, and the X-ray powder diffraction patterns of ergothioneine crystals obtained in Examples 1-6. Figure 1 Therefore, it will not be provided again here.

[0057] 3) DSC-TG analysis was performed on the ergothioneine crystals obtained in this invention. The specific method for DSC-TG analysis is a conventional method in the prior art. The DSC-TG analysis diagram of the ergothioneine crystals obtained in Example 1 is shown below. Figure 2 As shown, its DSC analysis chromatogram reveals a melting peak at 269 °C. The information in the figure confirms the stability of the ergothiain crystals obtained in Example 1. The results of Examples 1-6 are consistent, and images are not repeated here.

[0058] 4) Figure 3 The image shown is the FT-IR image of the ergothioneine crystals obtained in Example 1. The results of Examples 1-6 are consistent, so the images are not repeated here.

[0059] 5) Figure 4The image shown is a Raman diagram of the ergothioneine crystals obtained in Example 1. The results of Examples 1-6 are consistent, and the images are not repeated here.

[0060] 6) Figure 5 The image shown is an optical microscope image of the ergothioneine crystals obtained in Example 1. The results of Examples 1-6 are consistent, and the images are not repeated here.

[0061] Comparative Example 1

[0062] The difference from Example 1 lies in the solvent system used; a single pure ethanol solvent is used for the crystallization process. The specific steps are as follows:

[0063] At 80 °C, with a stirring rate of 400 r / min, 50 mL of ergothioneine fermentation broth was dissolved in 50 mL of ethanol. The ergothioneine in the fermentation broth could not be completely dissolved, and the solution became turbid. The temperature inside the crystallizer was lowered to 65 °C at a rate of 0.05 °C / min. At this point, a mixture of yellowish-brown impurities and fine crystals appeared inside the crystallizer. The stirring rate was maintained, and crystallization was continued for 0.5 h. After crystallization, the temperature inside the crystallizer was lowered to 5 °C at a rate of 0.3 °C / min, and crystallization was continued for 1 h. The product was subjected to solid-liquid separation to obtain a filter cake, which was washed with 20 mL of acetone. The filter cake was placed in a vacuum drying oven and dried at a constant temperature of 50 °C for 6 h. After drying, a clump of yellowish-brown viscous impurities and fine crystals was obtained.

[0064] The final product was a clump of yellowish-brown viscous precipitate and fine wheat hornthion crystals, which could not be effectively separated into solid and liquid, and the experiment failed.

[0065] Comparative Example 2

[0066] The difference from Example 1 lies in the solvent system used; an ethanol-water mixed solvent is used for the crystallization process. The specific steps are as follows:

[0067] At 80 ℃, with a stirring rate of 400 r / min, 50 mL of ergothioneine fermentation broth was dissolved in 50 mL of deionized water-ethanol mixture. V 水 : V 乙醇A clear solution was formed in a mixture of 1:1 (ratio of ergothioneine to 1 part heat). The temperature inside the crystallizer was lowered to 40 °C at a rate of 0.05 °C / min. At this point, crystallization suddenly occurred in the crystallizer, resulting in a large number of extremely fine needle-like crystals. The stirring rate was maintained for 0.5 h of crystal growth. After crystal growth, the temperature inside the crystallizer was lowered to 5 °C at a rate of 0.3 °C / min, and crystal growth continued for 1 h. The product was subjected to solid-liquid separation to obtain a filter cake, which was then washed with 20 mL of acetone. The filter cake was placed in a vacuum drying oven and dried at a constant temperature of 50 °C for 6 h. After drying, ergothioneine caking powder was obtained.

[0068] The final product is difficult to filter, exhibits severe agglomeration, and forms ergothioneine caking powder after drying. The product particle size is approximately 105 μm, with an angle of repose >40°. The product purity is only 97.5%, the content is 98.2%, and the flowability is extremely poor.

[0069] Comparative Example 3

[0070] The difference from Example 1 is that the two-stage crystal growth step is omitted. The specific steps are as follows:

[0071] At 80 ℃, with a stirring rate of 400 r / min, 50 mL of ergothioneine fermentation broth was dissolved in 50 mL of deionized water-DMSO mixed solution. V 水 : V DMSO A clear solution was formed in a mixture of 1:5 (ratio of 0.3°C to 5°C). The temperature inside the crystallizer was lowered to 5°C at a rate of 0.3°C / min, during which a large number of needle-like crystals precipitated. The product was subjected to solid-liquid separation to obtain a filter cake, which was then washed with 20 mL of acetone. The filter cake was placed in a vacuum drying oven and dried at a constant temperature of 50°C for 6 h. After drying, ergothioneine powder was obtained.

[0072] The final product is difficult to wash, exhibits severe agglomeration, and forms ergothioneine powder after drying. The product particle size is less than 50 μm, and the angle of repose is 38.2°. The product purity is 99.1%, and the content is 99.2%.

[0073] Comparative Example 4

[0074] The difference from Example 1 is that the first crystallization temperature is changed from 65 °C to 20 °C. The specific steps are as follows:

[0075] At 80 ℃, with a stirring rate of 400 r / min, 50 mL of ergothioneine fermentation broth was dissolved in 50 mL of deionized water-DMSO mixed solution. V 水 : V DMSOA clear solution was formed in a mixture of 1:5 (ratio of ergothioneine to 5 ppm). The temperature inside the crystallizer was lowered to 20 ℃ at a rate of 0.05 ℃ / min. At this point, nucleation occurred inside the crystallizer, forming a large number of ergothioneine aggregates. The stirring rate was maintained, and the crystals were cultured for 0.5 h. After the crystallization was completed, the temperature inside the crystallizer was lowered to 5 ℃ at a rate of 0.3 ℃ / min, and the crystallization was continued for 1 h. The product was subjected to solid-liquid separation to obtain a filter cake, which was washed with 20 mL of acetone. The filter cake was placed in a vacuum drying oven and dried at a constant temperature of 50 ℃ for 6 h. After drying, ergothioneine granules were obtained.

[0076] The final product exhibits severe micro-agglomeration, presenting as macroscopically granular particles and containing a large amount of impurities. The particle size is approximately 195 μm, with an angle of repose of 32.4°. The product purity is 98.2%, and the content is 98.7%.

[0077] Comparative Example 5

[0078] The difference from Example 1 lies in the use of a crystallization method disclosed in the prior art, the specific steps of which are as follows:

[0079] Take 80 mL of ergothioneine fermentation broth and perform pretreatment, ultrafiltration, concentration, column chromatography, and decolorization according to the method in Example 1 of patent CN117924184A to obtain a decolorized ergothioneine concentrate (approximately 10 mL). Following the crystallization conditions of that patent, cool this concentrate to 5°C, control the stirring rate at 150 r / min, and add 30 mL of anhydrous ethanol to the crystallizer at a rate of 800 μL / min. Maintain stirring at 5°C for 10 h, collect the crystals by centrifugation, and dry to obtain ergothioneine crystalline powder.

[0080] The final product is ergothioneine crystal powder with a particle size of 15 μm and some agglomeration, and an angle of repose as high as 42°. The product purity is 98.24%, the content is 98.6%, and the flowability is poor.

[0081] Comparative Example 6

[0082] The difference from Example 1 is that the raw material used is commercially available ergothioneine standard with a purity >99.7%, dissolved in deionized water to prepare a solution with a concentration equivalent to that of the fermentation broth treated in Example 1. The crystallization procedure and solvent system are the same as in Example 1. The specific steps are as follows:

[0083] Purchase commercially available ergothioneine standard with a purity >99.7%, dissolve it in deionized water, and prepare an ergothioneine solution with a concentration equivalent to that of the fermentation broth. At 80 °C, control the stirring rate at 400 r / min, dissolve 50 mL of the ergothioneine solution in 50 mL of a deionized water-DMSO mixture. V 水 : VDMSO A clear solution was formed in a mixture of 1:5 (ratio of ethyl to diethyl sulfadiazine). The temperature inside the crystallizer was lowered to 65°C at a rate of 0.05°C / min, at which point a large number of crystals appeared inside the crystallizer. The stirring rate was maintained, and the crystals were cultured for 0.5 h. After the crystals were cultured, the temperature inside the crystallizer was lowered to 5°C at a rate of 0.3°C / min, and the crystals were cultured for another 1 h. The product was subjected to solid-liquid separation to obtain a filter cake, which was then washed with 20 mL of acetone. The filter cake was placed in a vacuum drying oven and dried at a constant temperature of 50°C for 6 h. After drying, ergothioneine crystals were obtained.

[0084] The final product is a blocky white crystal with a particle size of approximately 275 μm and an angle of repose of 21.0°. The product purity is 99.998%, the content is 99.96%, and the process yield is 96%.

[0085] This invention discloses and proposes a method for preparing large-particle, high-purity ergothioneine crystals directly from fermentation broth, as well as the large-particle, high-purity ergothioneine crystals themselves. Those skilled in the art can implement this method by appropriately modifying the raw materials, process parameters, and other aspects, based on the content of this document. The method and product of this invention have been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the methods and products described herein without departing from the content, spirit, and scope of this invention to achieve the technical results of this invention. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included within the spirit, scope, and content of this invention.

Claims

1. A method for preparing large-particle, high-purity ergothioneine crystals directly from ergothioneine fermentation broth, characterized in that, The steps are as follows: S1. Dissolution: The ergothioneine fermentation broth is mixed with a crystallization solvent and stirred at 40–80 °C to form a homogeneous and clear initial solution; the crystallization solvent is a mixed solvent, which is a mixed system of deionized water and dimethyl sulfoxide, deionized water and acetonitrile, methanol and acetic acid, formic acid and acetonitrile, ethylene glycol and N,N-dimethylformamide, n-propanol and acetonitrile, and acetone and deionized water; S2. First stage of programmed cooling crystallization: The initial solution is cooled to the first crystallization temperature of 25-65 ℃ at a first cooling rate of 0.05-1.0 ℃ / min, and crystals are grown at this temperature and at a stirring rate of 100-500 r / min for 0.5-1 h to induce and stabilize crystal nuclei; S3. Second stage of programmed cooling crystallization: The system treated in step S2 is cooled to the final crystallization temperature of 0 to 20 ℃ at a second cooling rate of 0.02 to 1.5 ℃ / min, and crystals are grown at this final temperature for 0.5 to 1.5 h. S4. Post-processing: The solid-liquid mixture obtained in step S3 is subjected to solid-liquid separation. The resulting wet crystals are washed and dried to obtain the large-particle high-purity ergothioneine crystals.

2. The method according to claim 1, characterized in that, In step S1, the volume ratio of the two solvents in the mixed solvent is 1:5 to 5:

1.

3. The method according to claim 1 or 2, characterized in that, In step S1, the amount of the crystallization solvent used is 1 to 4 times the volume of the ergothioneine fermentation broth.

4. The method according to claim 1, characterized in that, In step S4, the washing agent used is acetone, ethanol, methanol or acetonitrile; the drying is blower drying or vacuum drying, the drying temperature is 30-60 ℃, and the drying time is 2-12 h.

5. A type of large-particle, high-purity ergothioneine crystal, characterized in that, It is prepared by the method described in any one of claims 1 to 6.

6. The large-particle high-purity ergothioneine crystals according to claim 5, characterized in that, Its purity is ≥99.99%, the crystals are in a massive form, the grain size is greater than 200 μm, and the angle of repose is less than 25°.

7. The large-particle high-purity ergothioneine crystals according to claim 5, characterized in that, When stored at 30±5 ℃ and 40±5 % relative humidity for 30 days, the purity decreases by less than 0.01%.

8. The use of the large-particle high-purity ergothioneine crystals according to any one of claims 5 to 7 in the preparation of cosmetics, health foods or pharmaceuticals.

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