A method for purifying ergothioneine from fermentation broth

Abstract

The invention discloses a method for separating and purifying thioneine from a fermentation broth, and belongs to the field of biochemical engineering. The method of the invention comprises: S1: adding a flocculant to the thioneine fermentation broth, and filtering to obtain a filtrate; S2: filtering the filtrate obtained in step S1 with an ultrafiltration membrane to obtain an ultrafiltrate; S3: applying a macroporous resin column to the ultrafiltrate obtained in step S2, and collecting the effluent; S4: concentrating the effluent obtained in step S3, adding a certain concentration of a metal salt solution to the concentrated solution, standing and centrifuging to obtain a precipitate, dripping a competitive complexing agent into the precipitate, releasing thioneine, and centrifuging to obtain a supernatant; S5: concentrating the supernatant of step S4 and placing it for crystallization, filtering, washing and drying the filter cake to obtain the pure thioneine. The method has simple process, large processing capacity, and less industrial waste production, and the obtained thioneine is pure white, purity>98% and recovery>90%.

Description

Technical Field

[0001] The invention belongs to the field of biochemical engineering, and particularly relates to a method for separating and purifying ergothioneine from a fermentation broth. Background Art

[0002] Ergothioneine (EGT) is scientifically known as 2-mercapto-histidine-trimethyl salt, with a relative molecular mass of 229.3. It is a colorless, tasteless and rare natural chiral small molecule amino acid. In 1909, Tanret C first isolated ergothioneine from ergot (a fungus parasitic on rye, a grass plant). Ergothioneine mainly exists in some bacteria and fungi and cannot be synthesized by the animal body itself (Li Yiqun et al., 2010). The human body itself cannot synthesize ergothioneine, but after eating foods containing ergothioneine, especially edible fungi and meat, the level of ergothioneine in human blood increases. It exists in the human brain, red blood cells, liver, kidneys, eye tissues, etc. (Lee et al., 2009). Among the microorganisms that can synthesize ergothioneine, edible fungi have the highest content, and many scholars have investigated its content in edible fungi.

[0003] The preparation method of ergothioneine includes: extraction method / chemical synthesis method and biological fermentation synthesis method. The extraction method is mainly extracted from edible fungi. The extraction method has low yield, is susceptible to seasonal influences, lacks raw materials, and regional restrictions, which become the main bottlenecks restricting large-scale production. In addition, the content of ergothioneine in edible fungi is not high. Therefore, the ergothioneine extracted from edible fungi has low purity, is difficult to mass produce, cannot meet market demand, has high cost, and is difficult for general consumers to accept. Compared with plant extraction, the biological fermentation synthesis method has good benefits, and compared with the chemical synthesis method, the biological fermentation synthesis method is greener and more natural. Therefore, biological fermentation technology will inevitably become the mainstream method for industrial mass production of ergothioneine.

[0004] CN110372597A discloses a method for extracting thioneine from shimeji mushroom, which sequentially performs vacuum freeze drying, crushing and sieving, microwave extraction and ultrasonic crushing on shimeji mushroom, and finally takes supernatant to obtain thioneine extract. The yield of thioneine described in the method is higher, but shimeji mushroom is directly extracted as raw material, the raw material cost is higher, and the obtained extract is not purified.

[0005] CN104774182A discloses a method for extracting and purifying thioneine from mushroom mycelium fermentation liquid, the method includes fermentation liquid centrifugation, water extraction mycelium, ultrafiltration, concentration, preparative liquid chromatography separation and freeze drying, and thioneine with a purity of more than 98% can be obtained by the method. The thioneine obtained by the method has high purity, but using mushroom as substrate fermentation, the raw material cost is relatively high, the equipment used is expensive and the preparation amount is very small, it is impossible to meet the needs of industrial production, and the thioneine processed by the method does not have a higher recovery rate.

[0006] In addition, the extraction routes of ergothioneine in fermentation broth mostly use cation exchange resin to adsorb ergothioneine, and use alkaline solution as eluent to obtain ergothioneine analytical solution. A large amount of acid and alkaline wastewater is generated in the process, causing huge pressure on environmental wastewater treatment. Summary of the invention

[0007] [Technical issues]

[0008] The technical problem to be solved by the present invention is that the existing method for separating and purifying thioneine from fermentation broth produces a large amount of acid-base wastewater, causing huge environmental wastewater treatment pressure.

[0009] [Technical solution]

[0010] The invention provides a method for purifying ergothioneine from a fermentation broth. The method has the advantages of simple process, large processing capacity and less generation of three industrial wastes. The obtained ergothioneine is pure white, has a purity greater than 98%, and a recovery rate greater than 90%.

[0011] The method comprises the following steps:

[0012] S1. Fermentation broth pretreatment. The purpose of pretreatment is to effectively change the dispersion state of colloidal particles such as cells, bacteria and proteins, aggregate them, and increase the volume for easy filtration. It is often used in the pretreatment of fermentation broth with small bacteria and high viscosity;

[0013] The pretreatment method is to add a flocculant to the ergothioneine fermentation broth, and after flocculation, filter with a plate and frame filter press to obtain filter residue and filtrate; compared with direct filtration, flocculation first and then filter pressing can remove more solid impurities and reduce the pressure of subsequent processes;

[0014] The fermentation liquid is fermented with a recombinant Escherichia coli capable of synthesizing ergothioneine as a fermentation strain, and with histidine, methionine, glycerol and trace elements as substrates, and the main components in the fermentation liquid are ergothioneine, methionine, histidine (trace), pigment, sugar, salt, protein, polypeptide and bacterial cells;

[0015] The flocculant is composed of modified activated clay and chitosan. The modified activated clay is prepared by adding aluminum sulfate to water, heating and stirring to obtain an aluminum sulfate solution, adding activated clay to the aluminum sulfate solution for impregnation, drying and activating pyrolysis to obtain the modified activated clay, and mixing the modified activated clay with chitosan to obtain the flocculant. The mass fraction of the aluminum sulfate solution is 5-15%, the activation pyrolysis temperature is 80-100° C., the time is 3-5 hours, and the modified activated clay is obtained. The mass ratio of the modified activated clay to the chitosan is (1:2)-(1:8).

[0016] S2, ultrafiltration

[0017] The filtrate obtained in step S1 is filtered through an ultrafiltration membrane to remove impurities such as macromolecular proteins, and the membrane liquid is collected to obtain an ultrafiltrate;

[0018] S3, column chromatography

[0019] The ultrafiltrate obtained in step S2 is subjected to a non-polar or weakly polar macroporous resin column, the effluent containing thioneine is collected, and the thioneine remaining in the post but not adsorbed by the resin is washed out with water after the loading is completed, and the washing liquid is collected, and the washing liquid of the effluent containing thioneine is merged; the non-polar or weakly polar macroporous resin can adsorb small and medium polar components in the fermentation broth, thereby removing some impurities;

[0020] S4. Complexation and decomplexation

[0021] The effluent obtained by step S3 is concentrated, a certain density of metal salt solution is added to the concentrated solution for complexing, fully stirred, centrifuged after standing for a period of time, and the precipitation is collected, and a competitive complexing agent (decomplexing agent) is dripped into the precipitation, thioneine is discharged, and the supernatant is collected by centrifugation;

[0022] S5, concentration, crystallization and drying

[0023] The supernatant obtained in step S4 is adjusted to a pH value that is nearly neutral, concentrated to a certain volume, and then crystallized. The crystals are filtered to obtain a filter cake, and the filter cake is washed and dried to obtain pure thioneine, wherein the drying needs to reach a moisture content of less than 4%.

[0024] In one embodiment of the present invention, the amount of flocculant added to the fermentation broth in step S1 is 8-10 g / L.

[0025] In one embodiment of the present invention, the flocculation temperature in step S1 is 30-50° C., the flocculation pH is 4-6, and the flocculation time is 2-4 hours.

[0026] In one embodiment of the present invention, the filter medium of the plate and frame filter press in step S1 is a 300-500 mesh polyester filter cloth, and the plate and frame feed pressure is 0.4-0.6 MPa.

[0027] In one embodiment of the present invention, the molecular weight cut-off of the ultrafiltration membrane in step S2 is 1000-5000 Daltons, and ultrafiltration can remove most impurities with a molecular weight greater than the above molecular weight cut-off.

[0028] In one embodiment of the present invention, the filler used in the non-polar or weakly polar macroporous resin column in step S3 is a non-polar or weakly polar macroporous adsorption resin with styrene as the skeleton, and the models include but are not limited to D101, AB 8, DM130 and HP20 macroporous resins.

[0029] In one embodiment of the present invention, the column chromatography in step S3 at least meets the following conditions: the sample loading volume is 5-50 BV, and the sample loading flow rate is 1.0-5.0 BV / h.

[0030] In one embodiment of the present invention, the metal salt used for complexation in step S4 is a copper salt or a zinc salt, the end point of adding the metal salt is when no new precipitation is generated in the system, and the complexation time is 20-40 minutes.

[0031] In one embodiment of the present invention, in step S4, the decomplexing agent is EDTA, hydrochloric acid or acetic acid, and the end point of adding the decomplexing agent is that the content of ergothioneine in the supernatant no longer changes.

[0032] In one embodiment of the present invention, the concentration in step S5 is to 1 / 10-1 / 50 of the original volume.

[0033] In one embodiment of the present invention, the crystallization temperature of step S5 is room temperature or a low temperature of 2-10° C., and the crystallization time is 12-24 hours.

[0034] In one embodiment of the present invention, the solvent used for washing the filter cake in step S5 is 70-100% ethanol aqueous solution.

[0035] In one embodiment of the present invention, the drying in step S5 is performed by vacuum drying, spray drying, hot air drying, freeze drying, or freeze drying.

[0036] [Beneficial Effects]

[0037] 1. The present invention proposes a novel composite flocculant, compared to a single flocculant, the composite flocculant has a larger flocculation mass after reaction, can clarify and preliminarily decolorize thioneine fermented liquid, remove 40-50% of protein and residual sugar in the fermented liquid without the loss of thioneine, the process reduces the processing pressure in the subsequent purification process, and the filtrate can directly pass through an ultrafiltration membrane to obtain an ultrafiltrate.

[0038] 2. The thioneine extraction and purification method of the present invention solves the problem of difficult purification of thioneine produced by microbial fermentation, and the extraction process is simple to operate, has few steps, and has high purification efficiency; the obtained thioneine is pure white, has a purity of >98%, and a recovery rate of >90%.

[0039] 3. The method of the present invention has low equipment requirements, reduces purification costs, and improves extraction efficiency; the product extraction purity is high, the recovery rate is considerable, and the product competitiveness and production profit are improved.

[0040] 4. The method of the present invention has low energy consumption, low pollution and simple process route. DETAILED DESCRIPTION

[0041] The fermented liquid used in the following examples is a fermentation strain with a recombinant Escherichia coli capable of synthesizing ergothioneine, and fermented with histidine, methionine, glycerol and trace elements as substrates, and the main components in the fermented liquid include ergothioneine, methionine, histidine (trace), pigment, sugar, salt, protein, polypeptide and bacterial cell.

[0042] Detection method of ergothioneine:

[0043] References for detection methods of ergothioneine: Wang Li, Wang Yang, Li Jianghua, et al. Construction and optimization of ergothioneine-producing Escherichia coli engineering strains[J]. Chinese Journal of Biotechnology, 2022, 38(2): 796-806.

[0044] The HPLC detection of ergothioneine uses an Agilent 1200 high performance liquid phase detection system, a UV detector, and a C18 column (Agilent ZORBAX Eclipse Plus, 250mm×4.6mm, 5μm). Mobile phase ratio: water: methanol = 99:1, flow rate: 0.7mL / min, column temperature: 30°C, UV absorption: 257nm, injection volume: 5μL, detection time: 20min. Ergothioneine standards with concentrations of 5mg / L, 25mg / L, 50mg / L, 100mg / L, 200mg / L and 500mg / L were prepared, and the standard curve was drawn using the above HPLC detection method, with the concentration of ergothioneine standard as the horizontal coordinate and the peak area as the vertical coordinate. The linear equation of the standard curve is y=35.484x–49.415, R 2 =1.0.

[0045] Example 1

[0046] The method for extracting and separating thioneine from fermented liquid of the present invention, the steps of the method are as follows:

[0047] S1. Fermentation broth pretreatment

[0048] The volume of 20L of thioneine fermentation broth was adjusted to pH=5, and a mixed flocculant (modified activated clay: chitosan=1:3) was added at a ratio of 8:1 (g / L) at 40°C, and the mixture was stirred for 2h to fully settle, and then filtered and washed with a plate and frame filter press to obtain filter residue and filtrate. The feed pressure of the plate and frame filter press was 0.4MPa, the filter cloth was a 400-mesh polyester filter cloth, and the washing volume was 3L. After treatment, the filter residue can be used as a high-quality protein raw material for feed, and the filtrate is 21.3L in total, which is used to extract and separate thioneine.

[0049] S2, ultrafiltration

[0050] The filtrate obtained in step S1 was ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 1000 Daltons to remove macromolecular impurities such as protein and residual sugar, and the permeate was collected to obtain a total of 27.5 L of ultrafiltrate.

[0051] S3, column chromatography

[0052] The ultrafiltrate obtained in step S2 is passed through the D-101 type macroporous resin column with a flow velocity of 2.0BV / h and a column volume of 1.5L. Ergothioneine has a large polarity, and will not be adsorbed when passing through a nonpolar or weakly polar macroporous resin, and will flow out under gravity. The small and medium polar components (impurities) in the ultrafiltrate can be adsorbed by resins, so that thioneine is played a role of purification. When thioneine can be detected from the effluent, the effluent is collected, after loading is completed, deionized water is used to wash with a flow velocity of 2.0BV / h, to wash out the thioneine remaining in the resin gap, and washing to water washing liquid stops washing when there is no thioneine detected, collect and merge effluent and water washing liquid 29.4L.

[0053] S4. Complexation and decomplexation

[0054] The ultrafiltrate obtained in step S3 was concentrated to 1.5 L, and ZnSO with a concentration of 0.02 mol / L was added to the concentrate. 4 The solution is stirred until no new precipitate is precipitated, the complex reaction time is 30 minutes, the complex reaction temperature is 50° C., and the precipitate generated by the complex reaction is separated by centrifugation; a competitive complexing agent EDTA solution is added dropwise to the precipitate, and the solution is stirred sufficiently to release ergothioneine, and the supernatant obtained by decomplexation is separated by centrifugation.

[0055] S5, concentration, crystallization and drying

[0056] The supernatant obtained in step S4 was adjusted to pH=7.5 and then concentrated to 200 mL, and placed in a refrigerator at 4°C for crystallization for 10 h. After crystallization was completed, suction filtration was performed to separate crystals and mother liquor, and the filter cake was washed with 80% ethanol until the filter cake was pure white. The obtained filter cake was dried in a vacuum drying oven at 50°C to a moisture content of less than 4%, to obtain pure white thioneine pure product 41.56 g, and HPLC measured its purity to be 99.14%, and the thioneine recovery rate in the fermentation broth was 92.31%.

[0057] Example 2

[0058] The method for extracting and separating thioneine from fermented liquid of the present invention, the steps of the method are as follows:

[0059] S1. Fermentation broth pretreatment

[0060] The volume of 50L of thioneine fermentation broth was adjusted to pH = 4.5, and a mixed flocculant (modified activated clay: chitosan = 1: 2.5) was added at a ratio of 10: 1 (g / L) at 40°C. After stirring, the reaction was allowed to react for 2h to fully settle. The fermentation broth was separated after filtering and washing with a plate and frame filter press to obtain filter residue and filtrate. The feed pressure of the plate and frame filter press was 0.40MPa, the filter cloth was a 400-mesh polyester filter cloth, and the washing volume was 8L. The filter residue can be used as a high-quality protein raw material for feed after treatment, and 54.9L of the filtrate is to be extracted and separated from thioneine.

[0061] S2, ultrafiltration

[0062] The plate-and-frame filtrate obtained in step S1 was passed through an ultrafiltration membrane with a molecular weight cutoff of 2000 Daltons to remove macromolecular impurities such as protein and residual sugar, and the permeate was collected to obtain 66.3 L of ultrafiltrate.

[0063] S3, column chromatography

[0064] The ultrafiltrate obtained in step S2 is passed through an AB-8 type macroporous resin column with a column volume of 3.0L at a flow velocity of 2.2BV / h, and collection is started when thioneine can be detected in the effluent, and after loading is completed, deionized water is used to wash with a flow velocity of 2.2BV / h to wash out the thioneine remaining in the resin gap, and washing is stopped when washing liquid does not detect thioneine, and effluent and washing liquid 68.7L are collected and combined.

[0065] S4. Complexation and decomplexation

[0066] The ultrafiltrate obtained in step S3 was concentrated to 1.5 L, and ZnCl with a concentration of 0.03 mol / L was added to the concentrate. 2The solution is stirred until no new precipitate is precipitated, the complex reaction time is 40 minutes, the complex reaction temperature is 50°C, and the precipitate generated by the complex reaction is separated by centrifugation; competitive complexing agent acetic acid is added dropwise to the precipitate to release ergothioneine, and the supernatant obtained by decomplexation is separated by centrifugation.

[0067] S5, concentration, crystallization and drying

[0068] The supernatant obtained in step S4 was adjusted to pH=6.5 and then concentrated to 450 mL, and placed in a refrigerator at 10°C for crystallization for 12 h. After crystallization was completed, suction filtration was performed to separate crystals and mother liquor, and the filter cake was washed with 80% ethanol until the filter cake was pure white. The obtained filter cake was dried in a vacuum drying oven at 45°C to a moisture content of less than 4%, to obtain 108.91 g of pure thioneine, and HPLC measured its purity to be 98.84%, and the recovery of thioneine in the fermentation broth was 93.67%.

[0069] Example 3

[0070] The method for extracting and separating thioneine from fermented liquid of the present invention, the steps of the method are as follows:

[0071] S1. Fermentation broth pretreatment

[0072] The volume of 200L of thioneine fermentation liquid was adjusted to pH=5.0, and a mixed flocculant (modified activated clay: chitosan=1:3) was added at a ratio of 8:1 (g / L) at 40°C, and the mixture was stirred for 2h to fully settle. The filter residue and filtrate were obtained after filtering and washing with a plate and frame filter press. The feed pressure of the plate and frame filter press was 0.45MPa, the filter cloth was a 500-mesh polyester filter cloth, and the washing volume was 35L. The filter residue can be used as a high-quality protein raw material for feed after treatment, and 228.3L of the filtrate is to be extracted and separated from thioneine;

[0073] S2, ultrafiltration

[0074] The plate-and-frame filtrate obtained in step S1 was passed through an ultrafiltration membrane with a molecular weight cutoff of 1000 Daltons to remove macromolecular impurities such as protein and residual sugar, and the permeate was collected to obtain 273.4 L of ultrafiltrate;

[0075] S3, column chromatography

[0076] The ultrafiltrate obtained in step S2 is passed through the DM130 type macroporous resin column with a flow velocity of 3BV / h and a column volume of 5L, and thioneine is detected from the effluent and collected, and washed with deionized water at a flow velocity of 3.0BV / h after loading, to wash out the thioneine remaining in the resin gap, and the washing liquid is stopped when thioneine is detected without thioneine, and the effluent and washing liquid 281.5L are collected and merged;

[0077] S4. Complexation and decomplexation

[0078] The ultrafiltrate obtained in step S3 was concentrated to 10 L, and CuCl with a concentration of 0.02 mol / L was added to the concentrate. 2 The solution is stirred until no new precipitation is precipitated, the complex reaction time is 30 minutes, the complex reaction temperature is 50° C., and the precipitate generated by the complex reaction is centrifuged; dilute hydrochloric acid as a competitive complexing agent is added dropwise to the precipitate, stirred sufficiently to release ergothioneine, and the supernatant obtained by decomplexation is centrifuged;

[0079] S5, concentration, crystallization and drying

[0080] The supernatant obtained in step S4 is adjusted to pH=7.5, and the volume is concentrated to 1600mL, and crystallization is placed in a refrigerator at 4°C for 10h. After crystallization is completed, suction filtration is performed to separate crystals and mother liquor, and the filter cake is washed with 80% ethanol until the filter cake is pure white. The obtained filter cake is dissolved in water (concentration is 15 degrees Baume) and spray-dried to obtain pure white thioneine pure product 418.96g. HPLC determines its purity to be 98.97%, and the recovery rate of thioneine in the fermentation broth is 91.95%.

[0081] Comparative Example 1

[0082] This comparative example provides a method for purifying thioneine from a fermentation broth, and the specific steps are the same as those in Example 1, except that in step S1, the flocculant is a composite flocculant: unmodified activated clay: chitosan = 1:3 mass ratio. 37.19 g of thioneine was obtained, and its purity was 93.14% by HPLC, the recovery rate of thioneine in the fermentation broth was 80.31%, and the color was light yellow.

[0083] Comparative Example 2

[0084] This comparative example provides a method for purifying thioneine from a fermentation broth, and the specific steps are the same as those in Example 1, except that in step S1, the flocculant is a single flocculant: chitosan. 36.65 g of pure thioneine was obtained, and its purity was 95.01% by HPLC, and the recovery rate of thioneine in the fermentation broth was 76.39%.

[0085] Comparative Example 3

[0086] This comparative example provides a method for purifying thioneine from a fermentation broth, and the specific steps are the same as those in Example 2, except that in step S1, the flocculant is a single flocculant: modified activated clay. The obtained thioneine pure product 93.25g, the purity of which is 88.41% by HPLC, and the recovery rate of thioneine in the fermentation broth is 75.41%.

[0087] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for purifying thioneine from a fermented liquid, characterized in that, The following steps are involved: S1. Fermentation broth pretreatment Add a flocculant to the thioneine fermented liquid, and after flocculation, filter with a plate and frame filter press to obtain a filter residue and a filtrate; The flocculant is composed of modified activated clay and chitosan, wherein the modified activated clay is obtained by modification with aluminum sulfate, and the mass ratio of the modified activated clay to the chitosan is (1:2)-(1:8); S2, ultrafiltration The filtrate obtained in step S1 is filtered through an ultrafiltration membrane, and the membrane liquid is collected to obtain an ultrafiltrate; S3, column chromatography The ultrafiltrate obtained in step S2 is passed through a non-polar or weakly polar macroporous resin column, and the effluent containing thioneine is collected. After loading, the thioneine remaining in the post but not adsorbed by the resin is washed out with water, and the washing liquid is collected, and the washing liquid of the effluent containing thioneine is merged; The non-polar or weakly polar macroporous resin can adsorb small and medium polar components in the fermentation broth, thereby removing some impurities; S4. Complexation and decomplexation The effluent and washing liquid obtained in step S3 are concentrated, a metal salt solution is added to the concentrated solution for complexation, stirring, standing, centrifuging, collecting precipitation, dripping a competitive complexing agent into the precipitation, releasing thioneine, centrifuging, and collecting the supernatant; S5, concentration, crystallization and drying The supernatant obtained in step S4 is adjusted to a neutral pH, concentrated, and crystallized, and the crystals are filtered to obtain a filter cake, and the filter cake is washed and dried to obtain a pure thioneine product.

2. The method according to claim 1, characterized in that: The modified activated clay is prepared by adding aluminum sulfate to water, heating and stirring to obtain an aluminum sulfate solution, impregnating activated clay in the aluminum sulfate solution, drying and activating pyrolysis to obtain the modified activated clay, and mixing the modified activated clay with chitosan to obtain a flocculant; wherein the mass fraction of the aluminum sulfate solution is 5-15%, the temperature of the activated pyrolysis is 80-100°C, and the time is 3-5h.

3. The method according to claim 1 or 2, characterized in that: In step S1, the amount of flocculant added to the fermentation liquid is 8-10 g / L.

4. The method according to claim 1, characterized in that In the step S1, the flocculation temperature is 30-50° C., the flocculation pH is 4-6, and the flocculation time is 2-4 hours.

5. The method according to claim 1, characterized in that In step S1, the filter medium of the plate and frame filter press is a 300-500 mesh polyester filter cloth, and the plate and frame feed pressure is 0.4-0.6 MPa.

6. The method according to claim 1, characterized in that The ultrafiltration membrane in step S2 has a molecular weight cut-off of 1000-5000 Daltons.

7. The method according to claim 1, characterized in that The filler used in the non-polar or weakly polar macroporous resin column in step S3 is a non-polar or weakly polar macroporous adsorption resin with styrene as the skeleton, and the models include but are not limited to D101, AB-8, DM130 and HP20 macroporous resins.

8. The method according to claim 1, characterized in that The column chromatography in step S3 at least meets the following conditions: the sample loading volume is 5-50 BV, and the sample loading flow rate is 1.0-5.0 BV / h.

9. The method according to claim 1, characterized in that: The metal salt used for complexation in step S4 is a copper salt or a zinc salt. The end point of adding the metal salt is when no new precipitation is generated in the system. The complexation time is 20-40 minutes. The competitive complexing agent is EDTA, hydrochloric acid or acetic acid, and the end point of adding the competitive complexing agent is that the content of ergothioneine in the supernatant no longer changes.

10. The method according to claim 1, characterized in that The concentration in step S5 is concentrated to 1 / 10-1 / 50 of the original volume, the crystallization temperature is room temperature or a low temperature of 2-10° C., and the crystallization time is 12-24 hours; the solvent used for washing the filter cake is 70-100% ethanol aqueous solution.