CN113666873A - Method for industrially preparing high-purity ergothioneine - Google Patents
Method for industrially preparing high-purity ergothioneine Download PDFInfo
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- ergothioneine
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- SSISHJJTAXXQAX-ZETCQYMHSA-N L-ergothioneine Chemical compound C[N+](C)(C)[C@H](C([O-])=O)CC1=CNC(=S)N1 SSISHJJTAXXQAX-ZETCQYMHSA-N 0.000 title claims abstract description 169
- 229940093497 ergothioneine Drugs 0.000 title claims abstract description 168
- 238000000034 method Methods 0.000 title claims abstract description 47
- 239000007788 liquid Substances 0.000 claims abstract description 50
- 238000002425 crystallisation Methods 0.000 claims abstract description 43
- 230000008025 crystallization Effects 0.000 claims abstract description 43
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 37
- 239000000523 sample Substances 0.000 claims abstract description 36
- 238000003795 desorption Methods 0.000 claims abstract description 35
- 238000011033 desalting Methods 0.000 claims abstract description 34
- 239000013078 crystal Substances 0.000 claims abstract description 24
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims abstract description 18
- 238000001035 drying Methods 0.000 claims abstract description 15
- 238000001953 recrystallisation Methods 0.000 claims abstract description 13
- 238000011097 chromatography purification Methods 0.000 claims abstract description 12
- 239000003456 ion exchange resin Substances 0.000 claims abstract description 12
- 229920003303 ion-exchange polymer Polymers 0.000 claims abstract description 12
- 238000000926 separation method Methods 0.000 claims abstract description 11
- 235000007685 Pleurotus columbinus Nutrition 0.000 claims abstract description 7
- 240000001462 Pleurotus ostreatus Species 0.000 claims abstract description 7
- 235000001603 Pleurotus ostreatus Nutrition 0.000 claims abstract description 7
- 238000001914 filtration Methods 0.000 claims abstract description 7
- 239000000843 powder Substances 0.000 claims abstract description 3
- 239000012488 sample solution Substances 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 98
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical group CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 81
- 235000019441 ethanol Nutrition 0.000 claims description 25
- 238000005406 washing Methods 0.000 claims description 23
- 239000012528 membrane Substances 0.000 claims description 19
- 238000011068 loading method Methods 0.000 claims description 17
- 238000002013 hydrophilic interaction chromatography Methods 0.000 claims description 15
- 238000001816 cooling Methods 0.000 claims description 14
- 238000000855 fermentation Methods 0.000 claims description 14
- 230000004151 fermentation Effects 0.000 claims description 14
- 238000012856 packing Methods 0.000 claims description 10
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 9
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 9
- 239000000919 ceramic Substances 0.000 claims description 8
- 239000012510 hollow fiber Substances 0.000 claims description 8
- 239000011347 resin Substances 0.000 claims description 8
- 229920005989 resin Polymers 0.000 claims description 8
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 6
- 239000007864 aqueous solution Substances 0.000 claims description 6
- 239000003729 cation exchange resin Substances 0.000 claims description 6
- 238000004587 chromatography analysis Methods 0.000 claims description 6
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 6
- 230000002829 reductive effect Effects 0.000 claims description 6
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 4
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 4
- 239000003957 anion exchange resin Substances 0.000 claims description 4
- 238000010828 elution Methods 0.000 claims description 4
- 239000011148 porous material Substances 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 3
- 239000003960 organic solvent Substances 0.000 claims description 3
- 235000019270 ammonium chloride Nutrition 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims description 2
- 239000012141 concentrate Substances 0.000 claims description 2
- 238000010438 heat treatment Methods 0.000 claims description 2
- 238000001471 micro-filtration Methods 0.000 claims description 2
- 239000012798 spherical particle Substances 0.000 claims description 2
- 230000002378 acidificating effect Effects 0.000 claims 1
- 238000010612 desalination reaction Methods 0.000 claims 1
- 235000013305 food Nutrition 0.000 abstract description 7
- 239000002537 cosmetic Substances 0.000 abstract description 6
- 239000003814 drug Substances 0.000 abstract description 6
- 229940079593 drug Drugs 0.000 abstract description 5
- 230000036541 health Effects 0.000 abstract description 3
- -1 health care Substances 0.000 abstract description 3
- 238000011031 large-scale manufacturing process Methods 0.000 abstract description 2
- 239000000047 product Substances 0.000 description 15
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 12
- 238000003756 stirring Methods 0.000 description 12
- 238000000967 suction filtration Methods 0.000 description 12
- 238000011084 recovery Methods 0.000 description 11
- 230000008569 process Effects 0.000 description 9
- 238000003786 synthesis reaction Methods 0.000 description 8
- 238000000605 extraction Methods 0.000 description 7
- 238000005342 ion exchange Methods 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 239000002244 precipitate Substances 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 239000000945 filler Substances 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 239000012535 impurity Substances 0.000 description 5
- 239000010413 mother solution Substances 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 239000002994 raw material Substances 0.000 description 5
- 239000012043 crude product Substances 0.000 description 4
- 238000012544 monitoring process Methods 0.000 description 4
- 230000001502 supplementing effect Effects 0.000 description 4
- 238000000108 ultra-filtration Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 241000233866 Fungi Species 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 239000007900 aqueous suspension Substances 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 3
- 239000012452 mother liquor Substances 0.000 description 3
- 238000001694 spray drying Methods 0.000 description 3
- 239000001888 Peptone Substances 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 239000005018 casein Substances 0.000 description 2
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 2
- 235000021240 caseins Nutrition 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000011049 filling Methods 0.000 description 2
- 239000000049 pigment Substances 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000004382 Amylase Substances 0.000 description 1
- 244000164418 Curcuma xanthorrhiza Species 0.000 description 1
- 235000003393 Curcuma xanthorrhiza Nutrition 0.000 description 1
- 235000019764 Soybean Meal Nutrition 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- SKOLWUPSYHWYAM-UHFFFAOYSA-N carbonodithioic O,S-acid Chemical compound SC(S)=O SKOLWUPSYHWYAM-UHFFFAOYSA-N 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 238000004042 decolorization Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000003631 expected effect Effects 0.000 description 1
- 239000012527 feed solution Substances 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- NBGMRMDAEWWFIR-UHFFFAOYSA-N imidazole-2-thione Chemical compound S=C1N=CC=N1 NBGMRMDAEWWFIR-UHFFFAOYSA-N 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000006340 racemization Effects 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000002000 scavenging effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000004455 soybean meal Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 125000002813 thiocarbonyl group Chemical group *C(*)=S 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
- 238000003809 water extraction Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
- C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
- C07D233/66—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D233/84—Sulfur atoms
Abstract
The invention provides a method for industrially preparing high-purity ergothioneine, which comprises the following steps: performing hot water treatment on the mycelia, performing solid-liquid separation, and collecting a feed liquid containing ergothioneine; vacuum concentrating the ergothioneine-containing feed liquid, filtering, ultrafiltering, decolorizing, desorbing, and concentrating; carrying out chromatographic purification on the ergothioneine desorption concentrated solution, collecting a target peak, concentrating to be dry, and dissolving in water to obtain an ergothioneine sample solution; desalting and decoloring the ergothioneine sample liquid by ion exchange resin, and collecting to obtain an ergothioneine desalting concentrated solution; and (3) carrying out coarse crystallization and recrystallization on the ergothioneine desalting concentrated solution, and drying to obtain high-purity ergothioneine crystal powder. The invention prepares high-purity ergothioneine in large quantity from mycelium of pleurotus ostreatus CGMCC No.23071, ensures the product quality of the ergothioneine, is particularly suitable for large-scale production of the ergothioneine, and meets the requirements of the product in the fields of food, cosmetics, health care, medicines and the like.
Description
Technical Field
The invention belongs to the fields related to biochemical products, foods and medicines, and relates to a method for industrially preparing high-purity ergothioneine.
Background
Ergothioneine (L-Ergothionine, EGT) is a rare natural chiral amino acid strong antioxidant, the only natural 2-thioimidazole amino acid (2-thio-imidazole) discovered so far exists mainly in the form of thioketone under physiological pH environment, and the stability of thiocarbonyl is higher than that of sulfydryl, so that EGT can not be spontaneously oxidized and has good pH stability and thermal stability. Ergothioneine has effects of scavenging free radicals, removing toxic substances, maintaining DNA growth, resisting radiation and aging, and is a unique cell physiological protectant; and secondly, the existence of the transporter ETT enables the EGT to become the only antioxidant which can be transported among cells, and the EGT can play roles in protecting the nervous system, inhibiting developmental defect, protecting the liver and the like. Due to the physiological activity of the EGT, the EGT has wide application prospect in the fields of food, cosmetics, medicines and the like.
There are three methods for the preparation of EGT: chemical synthesis, natural biological extraction and biological synthesis. EGT is chiral amino acid, and the synthesis of levorotatory EGT is difficult by chemical method, and several synthetic methods do not achieve the expected effect due to partial or complete racemization. The OXIS corporation first developed a commercial chemical synthesis method for EGT, but the raw materials for EGT synthesis are expensive and the synthesis cost is high. The extraction of EGT from natural organism has the problems of low content of EGT in raw materials, more impurities, high extraction cost, popular contraindication and the like, and the content of EGT in animal tissue pig blood is only as high as 60mg/L as known at present. The content of EGT in the fruiting body of the large fungi is high, but the EGT is limited by the cost of raw materials, and the price of the EGT obtained by extraction is high. Compared with a chemical synthesis method and an extraction method, the EGT synthesized by fermenting large edible fungi has the advantages of low production cost, wide raw material source, no custom contraindication problem, and more importantly, the safety of EGT products can be ensured, and the method is the mainstream direction of the EGT synthesis technology at present.
Most of ergothioneine synthesized by submerged fermentation of large fungi is accumulated in mycelia, a technology for separating and purifying the ergothioneine from the mycelia is required to be provided, a mycelium treatment solution has the characteristics of deep color value, complex mycelium release product and low EGT mass ratio, and the stable and sustainable process technology for industrially preparing the high-purity ergothioneine is urgent. The method for extracting ergothioneine from mycelium established by Curcuma xanthorrhiza comprises mycelium hot water extraction, leaching liquor ultrafiltration, ultrafiltrate drying or concentrating, acetonitrile solution dissolving and ultrasonic treatment, HILIC column purification, collecting component containing EGT peak, concentrating and drying to obtain ergothioneine with purity of 96-99% and recovery rate of 28-47% (CN104774182A 8). In the purification method, an acetonitrile solution is adopted as a mobile phase, the acetonitrile with toxicity is strictly limited in the fields of cosmetics, foods, medicines and the like, then the HILIC column is directly used for purifying the extraction ultrafiltration concentrated solution, a large amount of pigments and impurities can cause pollution of HILIC fillers, the service life of the HILIC column is seriously influenced, the single batch treatment amount of the HILIC column is reduced by directly carrying out HILIC column purification on the extraction ultrafiltration concentrated solution, the comprehensive treatment batch is increased, the use amount of organic solvents is increased, and the cost is increased suddenly; finally, the dried samples after HILIC purification have different colors and unstable quality; the final sample obtained by the method limits the application of the ergothioneine product in the main field.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for industrially preparing high-purity ergothioneine, which is used for preparing the high-purity ergothioneine in large quantities from mycelium, ensures the quality of the ergothioneine product, is particularly suitable for large-scale production of the ergothioneine, and meets the requirements of the product in the fields of food, cosmetics, health care, medicine and the like.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
a method for industrially preparing high-purity ergothioneine, comprising:
1) performing solid-liquid separation after mycelium hot water treatment, and collecting to obtain feed liquid containing ergothioneine;
2) vacuum concentrating the ergothioneine-containing feed liquid, adjusting pH to 8-10, and filtering to obtain ergothioneine concentrated solution;
3) respectively clarifying the ergothioneine concentrated solution by adopting ceramic membrane microfiltration and ultrafiltering by adopting a hollow fiber membrane to obtain ergothioneine ultrafiltrate;
4) decolorizing ergothioneine ultrafiltrate with anion exchange resin, washing with water, and collecting to obtain ergothioneine decolorized solution;
5) adsorbing the ergothioneine destaining solution with cation exchange resin, washing with water, desorbing, collecting the desorption solution, and concentrating to obtain an ergothioneine desorption concentrated solution;
6) carrying out chromatographic purification on the ergothioneine desorption concentrated solution, collecting a target peak, concentrating to be dry, and dissolving in water to obtain an ergothioneine chromatographic sample solution;
7) desalting and decoloring the ergothioneine chromatography sample liquid by ion exchange resin, and collecting to obtain an ergothioneine desalting concentrated solution;
8) and (3) carrying out coarse crystallization and recrystallization on the ergothioneine desalting concentrated solution, and drying to obtain high-purity ergothioneine crystal powder.
Preferably, step 1) comprises: performing solid-liquid centrifugal separation on mycelium fermentation liquor of pleurotus ostreatus CGMCC No.23071, washing the mycelium in the centrifugal separation process, centrifuging, collecting the mycelium, adding 8-10 times of water into the mycelium, heating to 70-90 ℃, treating for 20-30min, performing solid-liquid centrifugal separation, and respectively collecting the mycelium and feed liquid containing ergothioneine.
Preferably, in the step 2), vacuum concentration is carried out according to the concentration multiple of 5-10 times, the pH of the concentrated solution is adjusted to 8-10 by adopting sodium hydroxide or ammonia water, and the concentrated solution is kept stand for 10-30min after the pH is adjusted.
Preferably, in step 3), the pore size of the ceramic membrane is 0.1 μm to 0.5 μm, preferably 0.1 μm; the hollow fiber membrane has a molecular weight cut-off of 1kDa to 6kDa, preferably 3 kDa to 4 kDa.
Preferably, in step 4), the anion exchange resin is a styrene strongly basic resin, preferably HZ214 resin and D296 resin; the washing volume is 2-3 BV.
Preferably, in the step 5), the cation exchange resin is a strong acid cation exchange resin, preferably HZ001, and the washing volume is 2-3 BV; desorbing with ammonium chloride and ammonia water buffer solution at pH of 8-9, preferably pH of 8.5-8.8, and desorbing solution concentration of 0.2-1mol/L, preferably 0.5-0.8 mol/L; vacuum concentrating the collected desorption solution at 60-80 deg.C under 0.08-0.096Mpa to obtain ergothioneine desorption concentrate with ergothioneine concentration of 10-100 g/L, preferably 40-70 g/L.
Preferably, in the step 6), a HILIC column is adopted for chromatographic purification, wherein the filler of the HILIC column is spherical particles with the diameter of 20-35 μm, the pore diameter is 100 angstroms, and the height of the filler is not less than 30cm, and the chromatographic purification column comprises, but is not limited to, a chromatographic column, a manual DSC column and a dynamic axial DAC column; the mobile phase composition of chromatographic purification is ethanol-water, the proportion of the mobile phase is selected from 90:10-95:5, and the ethanol reagent is not limited to analytically pure and chromatographically pure, preferably analytically pure ethanol reagent; the sample concentration for chromatographic purification is 10g/L-90g/L, preferably 40g/L-60g/L, and the sample loading amount is 0.15-0.6g ergothioneine per 100g packing material.
Preferably, in the step 7), desalting is performed by using three desalting columns consisting of serial columns, namely weak-base macroporous ion exchange resin, weak-acid macroporous ion exchange resin and weak-base macroporous ion exchange resin, preferably weak-base macroporous ion exchange resin is D315 resin, D301 resin, preferably weak-acid macroporous ion exchange resin is D152 resin, D155 resin.
Preferably, step 7) further comprises: the collected desalinized concentrated ergothioneine solution is dried to obtain crude ergothioneine, and the drying method can be, but is not limited to, spray drying, vacuum drying or freeze drying.
Preferably, in the step 8), a cooling crystallization or a elution crystallization mode is adopted for coarse crystallization and recrystallization, wherein the cooling crystallization can be natural cooling crystallization or gradient cooling crystallization, the natural cooling crystallization is to place the ergothioneine desalting concentrated solution at 0-10 ℃ for natural cooling crystallization, stir for 3-5h once, stand for 15-48h, wash the crystals to be 90% -100% ethanol water solution, preferably, the crystallization temperature condition is 3-5 ℃, preferably, the ergothioneine concentration is 100-; the initial concentration of the ergothioneine desalting concentrated solution in the gradient cooling crystallization is 160g/L, the temperature is kept constant at 30-50 ℃ for 0.5-2h, the temperature gradient is 0.1-0.5K/min, the temperature is reduced to 1-5 ℃, the crystal growing time is 2-15h, and the crystal is washed to be 90-100% ethanol water solution; in the elution crystallization, the concentration of the ergothioneine desalting concentrated solution is 80-150g/L, the fed-in organic solvent is absolute ethyl alcohol or 95 ethyl alcohol, the fed-in amount of the ethyl alcohol is 2-6 times of the ergothioneine desalting concentrated solution in volume, the crystal growing time is 2-20h, and the crystal is washed to be 90% -100% ethyl alcohol aqueous solution.
More preferably, in the step 8), the drying temperature is 60-90 ℃ and the drying time is 4-12 h.
The invention has the beneficial effects that:
(1) the process of each step is stable to implement, easy to control, simple to operate, high in overall recovery rate and capable of being industrially prepared in large scale.
(2) After the pH value of the ergothioneine-containing concentrated feed solution is adjusted, a large amount of precipitates are separated out, the method for removing the precipitates is simple, the treatment capacity of a subsequent membrane process is increased, the use of the membrane area is reduced, and the service life of the membrane is prolonged.
(3) The decolorization process obviously reduces the color value of the ultrafiltration feed liquid at the early stage, removes a large amount of impurities, reduces dead adsorption pollution to the subsequent ion exchange resin and chromatographic packing, plays an obvious role in protecting, prolongs the service life of the two kinds of packing, and improves the stability of the whole process.
(4) The addition of the ion exchange process enables most of the sugar and impurities with larger molecular weight to be removed, amino acid micromolecular substances are reserved, the removal of the sugar and the impurities with larger molecular weight remarkably increases the ergothioneine concentration in the ergothioneine concentrated solution, the concentration of the ergothioneine in the ion exchange collected concentrated solution can reach 100g/L through experimental measurement, and the highest concentration of the ergothioneine decolored solution or ultrafiltrate ergothioneine which does not pass through the ion exchange process is only 8 g/L. Finally, the single treatment capacity of chromatographic purification is obviously increased to reach the industrial level, about 0.3-0.6g of filler can be treated per 100g of filler, and the increase of the single treatment capacity obviously reduces the chromatographic purification cost.
(5) The mobile phase in the chromatographic process adopts ethanol, the process is stable, the recovery rate is high, and compared with other solvents such as acetonitrile, the cost is low, the method has no toxicity, and the method can be widely applied to the fields of cosmetics, foods, health products, medicines and the like.
(6) The desalting process can obviously remove the salt and the pigment of the ergothioneine chromatography sample liquid, obviously improve the stability of the product of the crystallization process, ensure that the final high-purity product has high quality and purity and is stable in batches, and the finally obtained ergothioneine crystal meets the standards of cosmetic raw materials and European Union food.
Detailed Description
The following examples of the present invention are merely illustrative of specific embodiments for carrying out the present invention and are not to be construed as limiting the invention. Rather, the invention is intended to cover not only these embodiments, but also any other changes, modifications, substitutions, combinations, and simplifications which may be made without departing from the spirit and principles of the invention.
The strain used in the present application was Pleurotus ostreatus (Pleurotus ostreatus) obtained by laboratory self-screening and named FM 2021003.
The strain FM 2021003 has been preserved in China general microbiological culture Collection center (CGMCC) at 29 th.07 th.2021, with the preservation address: west road No. 1, north west of the republic of kyo, yang, institute of microbiology, academy of sciences of china, zip code: 100101. the preservation number of the strain is CGMCC No. 23071.
Slant culture medium: PDA medium (Becton, Dickinson and Company).
First-stage seeding tank liquid seed culture medium: 40g/L of corn flour, 25g/L of soybean meal and 54U/L, KH of alpha-amylase2PO45g/L、MgSO4·7H2O3.5 g/L and the balance of water, sterilizing at 121 ℃ for 20min, and filling the liquid into 50L seed tanks for 20L.
A secondary seed tank liquid fermentation culture medium: 40g/L of glycerol and 35g/L, KH of casein peptone2PO4 5g/L、MgSO4·7H2O3.5 g/L and water in balance, sterilizing at 121 ℃ for 20min, and filling 500L of secondary seed tank with liquid volume of 300L.
Fermentation tank liquid fermentation culture medium: 50g/L of glycerol and 35g/L, KH of casein peptone2PO4 5g/L、MgSO4·7H2O3.5 g/L and the balance of water, and the mixture is sterilized at 121 ℃ for 20min, and the liquid loading of the 3 ton fermentation tank is 2100L.
Pleurotus ostreatus (Pleurotus ostreatus) CGMCC No.23071 mycelium fermentation broth is prepared by the following method: selecting thallus Porphyrae of slant culture medium, inoculating into a first-stage seed tank, and culturing at 25 deg.C and 150rpm for 4 days to obtain first-stage seed solution. Transferring the primary seed solution to a secondary seed tank, culturing at 25 deg.C and 150rpm for 3 days, transferring the secondary seed solution to a 3 ton fermentation tank, and culturing at 25 deg.C and 150rpm for 12 days to obtain mycelium fermentation broth.
The ergothioneine adopts the following detection method:
high Performance Liquid Chromatography (HPLC), wherein the chromatographic column is an Agilent ZORBAX-SB-aq chromatographic column, the volume ratio of the components of the mobile phase is 1: 99 of methanol and pure water, and the pH is adjusted to 5 by using acetic acid and ammonia water; the detection wavelength is 257nm, the column temperature is 30 ℃, the sample injection volume is 5 mu L, and the flow rate is 0.7 mL/min.
Example 1
Separating mycelium fermentation liquor in a 3 ton tank by using an industrial centrifuge (separation factor is 800g), transferring a pipeline after mycelium is suspended by water into a feed liquid tank, supplementing water to 2000L, stirring, treating at 90 ℃ for 30min, and separating by using the industrial centrifuge again (separation factor is 800g) to obtain 1800L of feed liquid containing ergothioneine; double-effect concentrating the ergothioneine-containing feed liquid to 200L, adjusting the pH of the concentrated solution to 9 with ammonia water, standing for 30min, and filtering with a pipeline to remove precipitate to obtain an ergothioneine concentrated solution; the ergothioneine concentrated solution is filtered by a 0.1 mu m ceramic membrane and a 4KD hollow fiber membrane in sequence to obtain 180L of ergothioneine ultrafiltrate.
Treating the ergothioneine ultrafiltrate with HZ214 decolorizing column at flow rate of 1BV/h and water washing volume of 2BV, and collecting flow-through liquid to obtain 300L of ergothioneine decolorizing liquid; injecting the ergothioneine decolored solution into an HZ001 ion exchange chromatographic column, wherein the sample loading amount is 50%, the sample loading flow rate is 1BV/h, the water washing volume is 2BV, the concentration of desorption solution is 0.5mol/L, the pH value is 8.0, the desorption flow rate is 0.1BV/h, the monitoring wavelength is 280nm, collecting 50L desorption solution, the temperature is 75 ℃, the vacuum degree is 0.085Mpa, and the desorption solution is concentrated in vacuum to the ergothioneine concentration of 60g/L to obtain the ergothioneine desorption concentrated solution.
The height of a chromatographic column HILIC packing is 35cm, the diameter is 30cm, the single sample loading amount is 600mL ergothioneine desorption concentrated solution, and the mobile phase ethanol: the method comprises the following steps of (1) collecting target peaks, concentrating until ethanol is removed, processing the water through a desalting series column D315-D152-D315 after redissolving, obtaining a ergothioneine desalting concentrated solution by collecting flow-through liquid and concentrating, obtaining an ergothioneine crude product by spray drying the ergothioneine desalting concentrated solution, wherein the purity is detected to be 75.02%, and the recovery rate reaches 60.50%.
And (3) carrying out constant volume on the ergothioneine desalting concentrated solution to 150g/L, standing in a refrigeration house at 4 ℃ for 20h, stirring and shaking uniformly, continuing standing for 10h, carrying out suction filtration and draining, washing with 90% ethanol and absolute ethanol in sequence, carrying out suction filtration and draining, drying at 85 ℃ for 10h, treating the crude crystallization mother liquor twice by the same method, combining to obtain an ergothioneine crude crystallization sample, and detecting the purity to be 99%.
After the ergothioneine crude crystallization sample aqueous solution is redissolved, the volume is determined to 135g/L, the stirring speed of a crystallization reaction kettle is 200rpm, the temperature is kept at 25 ℃, the dropping amount of absolute ethyl alcohol is 3 times of the volume, the dropping time is 5 hours, the crystal growing time is 15 hours, the crystal is filtered and drained, 90% ethyl alcohol and absolute ethyl alcohol are sequentially used for washing, the crystal is filtered and drained, the crystal is dried at 85 ℃ for 10 hours, the recrystallization mother solution is treated once by the same method, the ergothioneine recrystallization sample is obtained by combining, the purity is detected to be 100.51%, and the overall purification recovery rate is 49.56%.
Example 2
Separating 3 tons of tank mycelium fermentation liquor by an industrial centrifuge, transferring a pipeline after mycelium water suspension to a feed liquid tank, supplementing water to 2000L, stirring, treating at 90 ℃ for 30min, and separating by the industrial centrifuge again to obtain 1800L of feed liquid containing ergothioneine; double-effect concentrating the ergothioneine-containing feed liquid to 200L, adjusting the pH of the concentrated solution to 8.5 with ammonia water, standing for 30min, and filtering with a pipeline to remove precipitate to obtain an ergothioneine concentrated solution; the ergothioneine concentrated solution is filtered by 0.1 μm ceramic membrane and 4KD hollow fiber membrane to obtain 200L ergothioneine ultrafiltrate.
Treating the ergothioneine ultrafiltrate with HZ214 decolorizing column at flow rate of 1BV/h and water washing volume of 2BV, and collecting flow-through liquid to obtain 380L ergothioneine decolorizing liquid; injecting the ergothioneine decolored solution into an HZ001 ion exchange chromatographic column, wherein the sample loading amount is 75%, the sample loading flow rate is 1BV/h, the water washing volume is 2BV, the concentration of desorption solution is 0.5mol/L, the pH value is 8.7, the desorption flow rate is 0.1BV/h, the monitoring wavelength is 280nm, collecting 58L desorption solution, the temperature is 60 ℃, the vacuum degree is 0.08Mpa, and the desorption solution is concentrated in vacuum to the ergothioneine concentration of 60g/L to obtain the ergothioneine desorption concentrated solution.
The chromatography column HILIC packing has the column height of 35cm, the diameter of 30cm, the single sample loading amount of 500ml ergothioneine desorption concentrated solution, and the mobile phase ethanol: the method comprises the following steps of (1) collecting target peaks, concentrating until ethanol is removed, processing the water by a desalting series column D315-D152-D315 after redissolving, washing for 2BV with water at a flow rate of 1BV/h, collecting flow-through liquid, concentrating to obtain a ergothioneine desalting concentrated solution, and spray-drying the ergothioneine desalting concentrated solution to obtain a crude product of the ergothioneine, wherein the water flow rate is 2000ml/min, the detection wavelength and the collection wavelength are respectively 280nm and 254nm, wherein the purity is detected to be 72.59%, and the recovery rate is up to 59.85%.
And (3) fixing the volume of the ergothioneine desalted concentrated solution to 150g/L, keeping the temperature of 30 ℃ for 1h, reducing the temperature at the rate of 0.5k/min to 4 ℃, growing the crystals for 8h, performing suction filtration and draining, washing with 90% ethanol and absolute ethanol in sequence, performing suction filtration and draining, drying at 85 ℃ for 8h, treating the crude crystal mother solution twice by the same method, combining to obtain a crude crystal sample of the ergothioneine, and detecting the purity to 98.98%.
After the ergothioneine crude crystallization sample aqueous solution is redissolved, the volume is determined to 130g/L, the stirring speed of a crystallization reaction kettle is 200rpm, the temperature is kept at 25 ℃ for 2h, the dropping amount of absolute ethyl alcohol is 4 times the volume, the dropping time is 5h, the crystal growing time is 15h, suction filtration and draining are carried out, 90% ethyl alcohol and absolute ethyl alcohol are sequentially used for washing, suction filtration and draining are carried out, drying is carried out at 85 ℃ for 10h, a recrystallization mother solution is treated by the same method once to obtain an ergothioneine recrystallization sample, the purity is detected to be 100.21%, and the overall recovery rate is 52.13%.
Example 3
Separating 3 tons of tank mycelium fermentation liquor by an industrial centrifuge, transferring a pipeline after mycelium water suspension to a feed liquid tank, supplementing water to 2100L, stirring, treating at 90 ℃ for 30min, and separating by the industrial centrifuge again to obtain 1800L of feed liquid containing ergothioneine; double-effect concentrating the ergothioneine-containing feed liquid to 250L, adjusting the pH of the concentrated solution to 9.1 with ammonia water, standing for 30min, and filtering with a pipeline to remove precipitates to obtain an ergothioneine concentrated solution; the ergothioneine concentrated solution is filtered by 0.1 μm ceramic membrane and 4KD hollow fiber membrane to obtain 230L ergothioneine ultrafiltrate.
Treating the ergothioneine ultrafiltrate with HZ214 decolorizing column at flow rate of 1BV/h and water washing volume of 2BV, and collecting flow-through liquid to obtain 380L ergothioneine decolorizing liquid; injecting the ergothioneine decolored solution into an HZ001 ion exchange chromatographic column, wherein the sample loading amount is 60%, the sample loading flow rate is 1BV/h, the sample loading flow rate is 2BV, the desorption solution concentration is 0.6mol/L, the pH value is 8.2, the desorption flow rate is 0.1BV/h, the monitoring wavelength is 280nm, collecting the desorption solution 46L, the temperature is 80 ℃, the vacuum degree is 0.096Mpa, and the desorption solution is concentrated in vacuum to the ergothioneine concentration of 80g/L to obtain the ergothioneine desorption concentrated solution.
The chromatography column HILIC packing has the column height of 35cm, the diameter of 30cm, the single sample loading amount of 500ml ergothioneine desorption concentrated solution, and the mobile phase ethanol: the flow rate of water is 95:5, the flow rate is 2000ml/min, the detection wavelength and the collection wavelength are respectively 280nm and 254nm, target peaks are collected and concentrated until ethanol is removed, the water is redissolved and then is treated by a desalting series column D315-D152-D315, the flow rate is 1BV/h, the water is washed for 2BV, flow-through liquid is collected and concentrated to obtain a ergothioneine desalting concentrated solution, the ergothioneine desalting concentrated solution is spray-dried to obtain a crude product of the ergothioneine, the purity is detected to be 70.31%, and the recovery rate reaches 65.35%.
And (3) fixing the volume of the obtained ergothioneine desalinized concentrated solution to 150g/L, keeping the stirring speed of a crystallization reaction kettle at 200rpm, keeping the temperature at 25 ℃, keeping the temperature for 2h, dripping 3 times of volume of absolute ethyl alcohol, keeping the dripping time for 5h, keeping the crystallization time for 12h, performing suction filtration and draining, washing with 90% ethanol and absolute ethyl alcohol in sequence, performing suction filtration and draining, drying at 85 ℃ for 10h, repeating the same method for twice on crude crystallization mother liquor, combining to obtain a crude ergothioneine crystallization sample, and detecting the purity of 98.66%.
After the ergothioneine crude crystallization sample aqueous solution is redissolved, the volume is determined to 120g/L, the stirring speed of a crystallization reaction kettle is 200rpm, the temperature is kept at 35 ℃ for 2h, the temperature reduction gradient is 0.1k/min, the temperature is reduced to 2 ℃, the crystal growth time is 15h, the product is filtered and drained, the product is washed by 90% ethanol and absolute ethanol in sequence, filtered and drained, dried at 85 ℃ for 8h, the recrystallization mother solution is treated by the same method once and combined to obtain the ergothioneine recrystallization sample, the purity is detected to be 100.12%, and the overall recovery rate is 55.61%.
Example 4
Separating 3 tons of tank mycelium fermentation liquor by an industrial centrifuge, transferring a pipeline after mycelium water suspension to a feed liquid tank, supplementing water to 2000L, stirring, treating at 90 ℃ for 30min, and separating by the industrial centrifuge again to obtain 1800L of feed liquid containing ergothioneine; double-effect concentrating the ergothioneine-containing feed liquid to 200L, adjusting the pH of the concentrated solution to 9.0 with ammonia water, standing for 30min, and filtering with a pipeline to remove precipitates to obtain an ergothioneine concentrated solution; the ergothioneine concentrated solution is filtered by 0.1 μm ceramic membrane and 4KD hollow fiber membrane to obtain 220L ergothioneine ultrafiltrate.
Treating the ergothioneine ultrafiltrate with HZ214 decolorizing column at flow rate of 1BV/h and water washing volume of 2BV, and collecting flow-through liquid to obtain 350L of ergothioneine decolorizing liquid; injecting the ergothioneine decolored solution into an HZ001 ion exchange chromatographic column, wherein the sample loading amount is 65%, the sample loading flow rate is 1BV/h, the sample loading flow rate is 2BV, the concentration of desorption solution is 0.5mol/L, the pH value is 8.6, the desorption flow rate is 0.1BV/h, the monitoring wavelength is 280nm, collecting 50L desorption solution, the temperature is 65 ℃, the vacuum degree is 0.09Mpa, and the desorption solution is concentrated in vacuum to the ergothioneine concentration of 80g/L to obtain the ergothioneine desorption concentrated solution.
The chromatography column HILIC packing has the column height of 35cm, the diameter of 30cm, the single sample loading amount of 500ml ergothioneine desorption concentrated solution, and the mobile phase ethanol: the flow rate of water is 95:5, the flow rate is 2000ml/min, the detection wavelength and the collection wavelength are respectively 280nm and 254nm, target peaks are collected and concentrated until ethanol is removed, the water is redissolved and then is treated by a desalting series column D315-D152-D315, the flow rate is 1BV/h, the water is washed for 2BV, flow-through liquid is collected and concentrated to obtain a ergothioneine desalting concentrated solution, the ergothioneine desalting concentrated solution is spray-dried to obtain a crude product of the ergothioneine, the purity is detected to be 73.31%, and the recovery rate reaches 66.57%.
And (3) fixing the volume of the obtained ergothioneine desalinized concentrated solution to 130g/L, keeping the stirring speed of a crystallization reaction kettle at 200rpm, keeping the temperature at 25 ℃, keeping the temperature for 2h, dripping 4 times of volume of absolute ethyl alcohol, keeping the dripping time for 5h, keeping the crystallization time for 12h, performing suction filtration and draining, washing with 90% ethanol and absolute ethyl alcohol in sequence, performing suction filtration and draining, drying at 85 ℃ for 12h, repeating the same method for twice on crude crystallization mother liquor, combining to obtain a crude ergothioneine crystallization sample, and detecting the purity to be 98.98%.
After the ergothioneine crude crystallization sample aqueous solution is redissolved, the volume is fixed to 130g/L, the stirring speed of a crystallization reaction kettle is 200rpm, the temperature is kept at 30 ℃ for 2h, the temperature reduction gradient is 0.1k/min, the temperature is reduced to 2 ℃, the crystal growth time is 15h, the product is subjected to suction filtration and draining, the product is washed by 90% ethanol and absolute ethanol in sequence, the product is subjected to suction filtration and draining, the product is dried at 85 ℃ for 8h, the recrystallization mother solution is treated by the same method for one time and is combined to obtain the ergothioneine recrystallization sample, the purity is detected to be 99.89%, and the overall recovery rate is 53.21%.
Claims (10)
1. A method for industrially preparing high-purity ergothioneine, comprising:
1) performing solid-liquid separation after mycelium hot water treatment, and collecting to obtain feed liquid containing ergothioneine;
2) vacuum concentrating the ergothioneine-containing feed liquid, adjusting pH to 8-10, and filtering to obtain ergothioneine concentrated solution;
3) respectively clarifying the ergothioneine concentrated solution by adopting ceramic membrane microfiltration and ultrafiltering by adopting a hollow fiber membrane to obtain ergothioneine ultrafiltrate;
4) decolorizing ergothioneine ultrafiltrate with anion exchange resin, washing with water, and collecting to obtain ergothioneine decolorized solution;
5) adsorbing the ergothioneine destaining solution with cation exchange resin, washing with water, desorbing, collecting the desorption solution, and concentrating to obtain an ergothioneine desorption concentrated solution;
6) carrying out chromatographic purification on the ergothioneine desorption concentrated solution, collecting a target peak, concentrating to be dry, and dissolving in water to obtain an ergothioneine chromatographic sample solution;
7) desalting and decoloring the ergothioneine chromatography sample liquid by ion exchange resin, and collecting to obtain an ergothioneine desalting concentrated solution;
8) and (3) carrying out coarse crystallization and recrystallization on the ergothioneine desalting concentrated solution, and drying to obtain high-purity ergothioneine crystal powder.
2. The method for industrially preparing high-purity ergothioneine according to claim 1, wherein the step 1) comprises: performing solid-liquid centrifugal separation on mycelium fermentation liquor of pleurotus ostreatus CGMCC No.23071, washing the mycelium in the centrifugal separation process, centrifuging, collecting the mycelium, adding 8-10 times of water into the mycelium, heating to 70-90 ℃, treating for 20-30min, performing solid-liquid centrifugal separation, and respectively collecting the mycelium and feed liquid containing ergothioneine.
3. The method for industrially preparing high-purity ergothioneine according to claim 1, wherein in the step 2), vacuum concentration is performed according to the concentration multiple of 5-10 times, the pH of the concentrated solution is adjusted to 8-10 by using sodium hydroxide or ammonia water, and the concentrated solution is kept standing for 10-30min after the pH is adjusted.
4. The method for industrially preparing high-purity ergothioneine according to claim 1, wherein, in the step 3), the pore diameter of the ceramic membrane is 0.1-0.5 μm; the molecular weight cut-off of the hollow fiber membrane is 1kDa-6 kDa.
5. The method for industrially preparing high-purity ergothioneine according to claim 1, wherein in the step 4), the anion exchange resin is a styrene type strong base resin, and the washing volume is 2-3 BV.
6. The method for industrially preparing high-purity ergothioneine according to claim 1 or 5, wherein in the step 5), the cation exchange resin is a strong-acid cation exchange resin, and the washing volume is 2-3 BV; desorbing with ammonium chloride and ammonia water buffer solution at pH of 8-9 and concentration of 0.2-1 mol/L; vacuum concentrating the collected desorption solution at 60-80 deg.C under 0.08-0.096Mpa to obtain ergothioneine desorption concentrate with ergothioneine concentration of 10-100 g/L.
7. The method for industrially preparing high-purity ergothioneine according to claim 1, wherein in the step 6), the chromatography purification is carried out by using HILIC column, the packing of the HILIC column is spherical particles with the diameter of 20-35 μm, the pore diameter is 100 angstroms, and the height of the packing is not less than 30 cm; the mobile phase composition of chromatographic purification is ethanol-water, and the proportion of the mobile phase is 90:10-95: 5; the sample concentration of chromatographic purification is 10g/L-90g/L, and the sample loading amount is 0.15-0.6g ergothioneine per 100g packing.
8. The method for industrially preparing high-purity ergothioneine according to claim 7, wherein in the step 7), desalting is performed by using desalting columns consisting of three series-connected columns, namely weakly basic macroporous ion exchange resin, weakly acidic macroporous ion exchange resin and weakly basic macroporous ion exchange resin.
9. The method for industrially preparing high-purity ergothioneine according to claim 1, wherein in the step 8), the crude crystallization and recrystallization are carried out by adopting a cooling crystallization or elution crystallization mode, the cooling crystallization comprises natural cooling crystallization or gradient cooling crystallization, the natural cooling crystallization is to place the concentrated solution of ergothioneine desalination at 0-10 ℃ for natural cooling crystallization, the solution is stirred for one time within 3-5h and is kept standing for 15-48h, and the crystals are washed into 90-100% ethanol water solution; the initial concentration of the ergothioneine desalting concentrated solution in the gradient cooling crystallization is 160g/L, the temperature is kept constant at 30-50 ℃ for 0.5-2h, the temperature gradient is 0.1-0.5K/min, the temperature is reduced to 1-5 ℃, the crystal growing time is 2-15h, and the crystal is washed to be 90-100% ethanol water solution; in the elution crystallization, the concentration of the ergothioneine desalting concentrated solution is 80-150g/L, the fed-in organic solvent is absolute ethyl alcohol or 95 ethyl alcohol, the fed-in amount of the ethyl alcohol is 2-6 times of the ergothioneine desalting concentrated solution in volume, the crystal growing time is 2-20h, and the crystal is washed to be 90% -100% ethyl alcohol aqueous solution.
10. The method for industrially preparing high-purity ergothioneine according to claim 1 or 9, wherein the drying temperature in step 8) is 60-90 ℃ and the drying time is 4-12 h.
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