CN118164916A - Purification method of ergothioneine - Google Patents
Purification method of ergothioneine Download PDFInfo
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- CN118164916A CN118164916A CN202410230392.4A CN202410230392A CN118164916A CN 118164916 A CN118164916 A CN 118164916A CN 202410230392 A CN202410230392 A CN 202410230392A CN 118164916 A CN118164916 A CN 118164916A
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- egt
- ergothioneine
- extract
- cation exchange
- exchange resin
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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 135
- 229940093497 ergothioneine Drugs 0.000 title claims abstract description 135
- 238000000034 method Methods 0.000 title claims abstract description 41
- 238000000746 purification Methods 0.000 title claims abstract description 34
- 239000000243 solution Substances 0.000 claims abstract description 50
- 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 28
- 239000003729 cation exchange resin Substances 0.000 claims abstract description 28
- 239000002253 acid Substances 0.000 claims abstract description 20
- 238000011068 loading method Methods 0.000 claims abstract description 20
- 239000012045 crude solution Substances 0.000 claims abstract description 12
- 239000003960 organic solvent Substances 0.000 claims abstract description 10
- 239000007788 liquid Substances 0.000 claims abstract description 9
- 239000006228 supernatant Substances 0.000 claims abstract description 9
- 238000005119 centrifugation Methods 0.000 claims abstract description 6
- 238000011049 filling Methods 0.000 claims abstract description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 32
- 239000011347 resin Substances 0.000 claims description 24
- 229920005989 resin Polymers 0.000 claims description 24
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical group CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 16
- 239000011780 sodium chloride Substances 0.000 claims description 16
- 239000008213 purified water Substances 0.000 claims description 15
- 238000010828 elution Methods 0.000 claims description 13
- 230000002378 acidificating effect Effects 0.000 claims description 11
- 230000008929 regeneration Effects 0.000 claims description 8
- 238000011069 regeneration method Methods 0.000 claims description 8
- 230000009466 transformation Effects 0.000 claims description 8
- 239000003208 petroleum Substances 0.000 claims description 7
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 6
- 230000007935 neutral effect Effects 0.000 claims description 6
- 230000008569 process Effects 0.000 claims description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 4
- 239000003480 eluent Substances 0.000 abstract description 12
- 239000004480 active ingredient Substances 0.000 abstract description 2
- 238000007670 refining Methods 0.000 abstract description 2
- 230000000052 comparative effect Effects 0.000 description 20
- 238000004128 high performance liquid chromatography Methods 0.000 description 12
- 238000001179 sorption measurement Methods 0.000 description 12
- 108090000790 Enzymes Proteins 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- GPPYTCRVKHULJH-QMMMGPOBSA-N N(alpha),N(alpha),N(alpha)-trimethyl-L-histidine Chemical compound C[N+](C)(C)[C@H](C([O-])=O)CC1=CNC=N1 GPPYTCRVKHULJH-QMMMGPOBSA-N 0.000 description 6
- 239000012535 impurity Substances 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000000605 extraction Methods 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 102100027324 2-hydroxyacyl-CoA lyase 1 Human genes 0.000 description 4
- 101001009252 Homo sapiens 2-hydroxyacyl-CoA lyase 1 Proteins 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 239000003963 antioxidant agent Substances 0.000 description 3
- 230000003078 antioxidant effect Effects 0.000 description 3
- 238000006911 enzymatic reaction Methods 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 229920001021 polysulfide Polymers 0.000 description 3
- 239000005077 polysulfide Substances 0.000 description 3
- 150000008117 polysulfides Polymers 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- HYHCSLBZRBJJCH-UHFFFAOYSA-N sodium polysulfide Chemical compound [Na+].S HYHCSLBZRBJJCH-UHFFFAOYSA-N 0.000 description 3
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- KWIUHFFTVRNATP-UHFFFAOYSA-N glycine betaine Chemical compound C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 description 2
- 229910017053 inorganic salt Inorganic materials 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- FVNKWWBXNSNIAR-BYPYZUCNSA-N (2s)-2-amino-3-(2-sulfanylidene-1,3-dihydroimidazol-4-yl)propanoic acid Chemical compound OC(=O)[C@@H](N)CC1=CNC(=S)N1 FVNKWWBXNSNIAR-BYPYZUCNSA-N 0.000 description 1
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 240000000588 Hericium erinaceus Species 0.000 description 1
- 235000007328 Hericium erinaceus Nutrition 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241001532621 Methanosalsum zhilinae Species 0.000 description 1
- 241000392443 Pleurotus citrinopileatus Species 0.000 description 1
- 240000001462 Pleurotus ostreatus Species 0.000 description 1
- 235000001603 Pleurotus ostreatus Nutrition 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 102000004385 Sulfurtransferases Human genes 0.000 description 1
- 108090000984 Sulfurtransferases Proteins 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229960003237 betaine Drugs 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 125000001453 quaternary ammonium group Chemical group 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000002000 scavenging effect Effects 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Landscapes
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention belongs to the technical field of active ingredient refining, and discloses a purification method of ergothioneine. The purification method comprises the following steps: extracting the ergothioneine crude solution by adopting an organic solvent, layering, taking a lower layer liquid, and then obtaining a supernatant by centrifugation to obtain an EGT extract; filling the chromatographic column with strong acid cation exchange resin, adjusting the pH value of the EGT extract, loading the EGT extract into the chromatographic column, and eluting with KCl solution to obtain purified ergothioneine. According to the purification method provided by the invention, through optimizing the purification step, KCl solution is selected as the eluent, and the concentration of the eluent is controlled, so that the yield and purity of ergothioneine can be greatly improved; and greatly simplifies the purification steps and improves the purification efficiency.
Description
Technical Field
The invention belongs to the technical field of active ingredient refining, and particularly relates to a purification method of ergothioneine.
Background
Ergothioneine (Ergothioneine, EGT for short) is an important active substance in the body and is a special amino acid of thiohistidine betaine. However, ergothioneine cannot be synthesized by the animal body itself and can only be taken from food. Foreign researches show that the natural antioxidant can play a role in an antioxidant in organisms, has various physiological functions of scavenging free radicals, maintaining DNA biosynthesis, normal growth of cells, cellular immunity and the like, is a natural antioxidant and a potential nutritional food, and has great application prospect in industries such as foods, cosmetics, medicines and the like.
The traditional biosynthesis of ergothioneine mainly comprises fermenting natural mushrooms such as oyster mushroom, hericium erinaceus and Pleurotus citrinopileatus. More enzymatic methods and chemical-enzymatic coupling methods have been developed for the preparation of ergothioneine, such as the enzymatic formation of ergothioneine from histidine betaine and cysteine, and the enzymatic formation of ergothioneine from histidine betaine and polysulfide. Regardless of the method used for preparing the ergothioneine, the ergothioneine is finally purified by adopting a proper method, and the purification process not only affects the yield of the product, but also affects the activity of the product. The purification method disclosed at present still has the problems of complicated process, low efficiency and low yield.
Therefore, it is highly desirable to provide a purification method of Ergothioneine (EGT) that can simplify the operation and increase the yield of ergothioneine.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the invention provides a purification method of ergothioneine, which can simplify the operation and improve the yield of ergothioneine.
The invention provides a purification method of ergothioneine.
Specifically, the purification method of ergothioneine comprises the following steps:
Extracting the ergothioneine crude solution by adopting an organic solvent, layering, taking a lower layer liquid, and then obtaining a supernatant by centrifugation to obtain an EGT extract;
Filling a chromatographic column with strong acid cation exchange resin, adjusting the pH value of the EGT extract, loading the EGT extract into the chromatographic column, and eluting with KCl solution to obtain purified ergothioneine;
the mass fraction of the KCl solution is 3.5% -8%.
It is understood that the crude ergothioneine solution is a reaction solution obtained by adopting an enzymatic method or a chemical-enzymatic coupling method for preparing the ergothioneine. Including but not limited to, the formation of ergothioneine (crude ergothioneine solution) from histidine betaine and cysteine under the action of enzymes, and the formation of ergothioneine (crude ergothioneine solution) from histidine betaine and polysulfide under the action of specific enzymes. If polysulfide is added into histidine betaine, under the catalysis of St enzyme (sulfur transfer enzyme), ergothioneine (EGT) is generated by reaction, and the obtained reaction liquid is collected after the reaction is finished; wherein the amino acid sequence of St enzyme (sulfur transferase) is shown in SEQ ID NO: 1.
SEQ ID NO：1：MKSISTDELLENLHRYKVIDIRSVDAYNGWKENGENR GGHIRSAKSLPYKWVNYIDWIEIVRSKDILPQDKLVIYGYDSEKAEEVARMFEKAGYTDLNIYPSFFEWVERNLPMDRLERYRHLVSPDWLNQLITTDNAPEYDNDKYVICHCHYRNPVDYEKGHIPGSIPLDTNSLESEDTWNRRSPEELKDALENAGISSETTVIVYGRFSYPKNDDPFPGSSAGHLGAMRCAFIMLYAGVKDVRILNGGLQSWLDAGYNVTTEPAKISKVSFGANIPLNPKIAVDLEEAKEILSDPGKKLVSVRSWREYIGEVSGYNYIEKKGRIPGSVFGDCGTDAYHMENYRNLDHTMREYHEIEDKWKELGITPEKRNAFYCGTGWRGSEAFLNAWLMGWDNAAVFDGGWFEWSNNDLPFETGVPEK( Source Methanosalsum zhilinae).
Preferably, the organic solvent is at least one selected from petroleum ether, diethyl ether, ethyl acetate and chloroform; further preferably, the organic solvent is petroleum ether.
Preferably, the centrifugation is performed by: centrifuging at 0-5deg.C at 5000-9000rpm for 10-30min; further preferably, the centrifugation is performed by: centrifuging at 7000-9000rpm and 0-5deg.C for 15-30min. According to the invention, proteins in the ergothioneine crude solution are destroyed through an extraction process, so that the ergothioneine crude solution is denatured and emulsified, and then the proteins in the crude solution can be effectively removed through low-temperature high-speed centrifugation, and the loss of EGT can be avoided.
Preferably, the EGT extract is pH-adjusted to 0.2-4 with acid liquor before loading. The acid solution may be hydrochloric acid, such as concentrated hydrochloric acid. The pH value of the EGT extract is regulated to 0.2-4 before loading, so that the binding capacity of EGT and cation exchange resin can be enhanced, and the problem that the EGT and impurities are eluted and taken away by an eluent together so as to not achieve the aim of purification is avoided.
Preferably, the strong acid cation exchange resin is one of LX-160W strong acid cation exchange resin, D001 strong acid cation exchange resin or 732 strong acid cation exchange resin.
Preferably, the strongly acidic cation exchange resin is subjected to a resin transformation treatment prior to loading. Specifically, the resin transformation process comprises the following steps: the column was loaded with a strongly acidic cation exchange resin, eluting sequentially with NaCl solution and purified water until the effluent conductivity was <5 uS/cm. The strongly acidic cation exchange resin is selective for ion adsorption in the order K +>Na+>H+. The resin is firstly transformed by NaCl solution and purified water, and the resin is transformed from H + type to Na + type. At this time, EGT can be well adsorbed, and impurities are not adsorbed; and during elution, K + (i.e. KCl) which is easier to adsorb is selected, and adsorbed EGT is replaced from the resin, so that the adsorbed EGT can be effectively separated from other impurities, and the aim of purification is fulfilled.
Preferably, the mass fraction of the NaCl solution is 10% -30%; further preferably, the mass fraction of the NaCl solution is 15% -25%.
Preferably, the KCl solution has a mass fraction of 4% -7%; further preferably, the KCl solution has a mass fraction of 4.5% -5.5%.
Preferably, after the elution is completed, the strongly acidic cation exchange resin may be subjected to a resin regeneration treatment. The resin regeneration process comprises the following steps: elution was performed sequentially with hydrochloric acid and purified water until the effluent was neutral. The regenerated resin after treatment can be reused without affecting the eluting effect.
Preferably, the mass fraction of the hydrochloric acid is 1% -10%; further preferably, the mass fraction of the hydrochloric acid is 2% -6%.
In order to further remove the inorganic salt impurities in ergothioneine, a desalting treatment may be performed later, which is a conventional treatment as long as the inorganic salt impurities can be further removed.
More specifically, a method for purifying ergothioneine comprises the following steps:
(1) Extracting the ergothioneine crude solution by adopting an organic solvent, layering, taking a lower layer liquid, centrifuging the lower layer liquid at the rotating speed of 5000-9000 and the temperature of 0-5 ℃ for 10-30min, and collecting a supernatant, namely the EGT extract;
(2) Loading the strong acid cation exchange resin into a chromatographic column, eluting by using NaCl solution and purified water in sequence until the conductivity of effluent liquid is less than 5 uS/cm; adjusting the pH value of the EGT extract to 0.2-4 by adopting acid liquor, loading the EGT extract into the chromatographic column, and eluting by adopting KCl solution with the mass fraction of 3.5-8% to obtain the purified ergothioneine.
According to the method, the ergothioneine crude solution containing the ergothioneine is extracted by adopting an organic solvent, proteins in the ergothioneine crude solution are destroyed, denatured and emulsified, and then the proteins in the ergothioneine crude solution are effectively removed by centrifugation, and the loss of EGT is reduced. Purifying EGT with strong acid cation exchange resin, utilizing amphoteric group carboxyl and quaternary ammonium group contained in EGT molecular structure, under pH condition close to its pKa 2.576, EGT shows positive electricity, can be adsorbed by cation exchange resin, eluting with KCl solution, and can effectively separate it from other impurities so as to attain the goal of purification.
Compared with the prior art, the invention has the beneficial effects that:
According to the purification method provided by the invention, the ergothioneine crude solution is extracted by an organic solvent, then a chromatographic column is filled with a strong acid cation exchange resin, and the KCl solution is used for eluting. Through optimizing the purification step, KCl solution is selected as eluent, and the concentration of the eluent is controlled, so that the yield and purity of ergothioneine are greatly improved, the purification step is greatly simplified, and the purification efficiency is improved.
Drawings
FIG. 1 is a graph showing the effect of KCl solutions of different concentrations on EGT yield.
Detailed Description
In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples will be presented. It should be noted that the following examples do not limit the scope of the invention.
The starting materials, reagents or apparatus used in the following examples and comparative examples were obtained from conventional commercial sources or by known methods unless otherwise specified.
In the following examples and comparative examples, the specific preparation method of crude ergothioneine prepared by an enzymatic method is as follows:
(1) Preparing 200g/L sodium polysulfide, regulating the pH to 9.0 by using HCl, and filtering by using a 0.22 mu m filter head to obtain a clear solution for later use; taking 2mL of sodium polysulfide solution with the concentration of 200g/L and 84mL of TMH (histidine betaine) solution (with the concentration of 51 mM), regulating the pH value to 9 by using 5M HCl, and preheating at 30 ℃ to obtain a mixed solution;
(2) 13.15g St enzyme (the concentration is 4g/L, the amino acid sequence is shown as SEQ ID NO:1, and the amino acid sequence is recorded as BZ006-DRN-1, and the enzyme load is 30.42 mg/g) is added into the mixed solution, and the pH is adjusted every 0.5h to maintain the pH at 9.0; 2mL of 200g/L sodium polysulfide solution was added every 3 hours of reaction, 7 times in total. And after the reaction is finished, obtaining the crude ergothioneine product.
Example 1
A method for purifying ergothioneine, comprising the steps of:
(1) Extraction: extracting crude ergothioneine product with petroleum ether of equal volume, standing for layering, collecting lower layer, centrifuging at 8000rpm and 4deg.C for 20min; centrifuging to obtain supernatant, which is EGT extract;
(2) Resin transformation: LX-160W strong acid cation exchange resin was packed into a chromatographic column with a column volume of 60mL. 200mL (3 BV, three column volumes) was eluted at 10mL/min using 20% NaCl. After the elution is finished, continuing to use purified water to elute until the electric conductivity of the effluent is less than 5 uS/cm;
(3) Loading: adjusting the pH of the EGT extract to be=0.3 by using concentrated hydrochloric acid, and loading 6mL (1/10 BV) of the extract into a chromatographic column;
(4) Eluting: eluting with 5% KCl solution at a flow rate of 10mL/min, eluting 300mL (5 BV), taking 10mL (1/6 BV) as a receiving unit, and performing HPCL analysis to detect EGT content to obtain purified ergothioneine. And combining a plurality of groups of single-peak receiving units with larger EGT peak areas according to the HPLC result, performing HPLC analysis, detecting to determine that the EGT is single-peak, and calculating the yield and purity. The column was eluted with 10% kcl solution under the conditions as above while detecting the EGT content in the eluate by HPLC until no EGT signal peak was detected in the effluent and the EGT dead adsorption rate was calculated.
(5) Resin regeneration: 200mL (3 BV) of the eluate was eluted at a rate of 10mL/min using 4% HCL, and the elution was continued with purified water until the effluent was neutral. The regenerated resin can be reused.
Example 2
A method for purifying ergothioneine, comprising the steps of:
(1) Extraction: extracting crude ergothioneine product with petroleum ether of equal volume, standing for layering, collecting lower layer, centrifuging at 8000rpm and 4deg.C for 20min; centrifuging to obtain supernatant, which is EGT extract;
(2) Resin transformation: LX-160W strong acid cation exchange resin was packed into a chromatographic column with a column volume of 60mL. 200mL (3 BV) was eluted at 10mL/min using 20% NaCl. After the elution is finished, continuing to use purified water to elute until the electric conductivity of the effluent is less than 5 uS/cm;
(3) Loading: adjusting the pH of the EGT extract to be=0.3 by using concentrated hydrochloric acid, and loading 6mL (1/10 BV) of the extract into a chromatographic column;
(4) Eluting: eluting with 4% KCl solution at a flow rate of 10mL/min, eluting 300mL (5 BV), taking 10mL (1/6 BV) as a receiving unit, and performing HPCL analysis to detect EGT content to obtain purified ergothioneine. And combining a plurality of groups of single-peak receiving units with larger EGT peak areas according to the HPLC result, performing HPLC analysis, detecting to determine that the EGT is single-peak, and calculating the yield and purity. The column was eluted with 10% kcl solution under the conditions as above while detecting the EGT content in the eluate by HPLC until no EGT signal peak was detected in the effluent and the EGT dead adsorption rate was calculated.
(5) Resin regeneration: 200mL (3 BV) of the eluate was eluted at a rate of 10mL/min using 4% HCL, and the elution was continued with purified water until the effluent was neutral. The regenerated resin can be reused.
Example 3
A method for purifying ergothioneine, comprising the steps of:
(1) Extraction: extracting crude ergothioneine product with petroleum ether of equal volume, standing for layering, collecting lower layer, centrifuging at 8000rpm and 4deg.C for 20min; centrifuging to obtain supernatant, which is EGT extract;
(2) Resin transformation: LX-160W strong acid cation exchange resin was packed into a chromatographic column with a column volume of 60mL. 200mL (3 BV) was eluted at 10mL/min using 20% NaCl. After the elution is finished, continuing to use purified water to elute until the electric conductivity of the effluent is less than 5 uS/cm;
(3) Loading: adjusting the pH of the EGT extract to be=0.3 by using concentrated hydrochloric acid, and loading 6mL (1/10 BV) of the extract into a chromatographic column;
(4) Eluting: eluting with 6% KCl solution at a flow rate of 10mL/min, eluting 300mL (5 BV), taking 10mL (1/6 BV) as a receiving unit, and performing HPCL analysis to detect EGT content to obtain purified ergothioneine. And combining a plurality of groups of single-peak receiving units with larger EGT peak areas according to the HPLC result, performing HPLC analysis, detecting to determine that the EGT is single-peak, and calculating the yield and purity. The column was eluted with 10% kcl solution under the conditions as above while detecting the EGT content in the eluate by HPLC until no EGT signal peak was detected in the effluent and the EGT dead adsorption rate was calculated.
(5) Resin regeneration: 200mL (3 BV) of the eluate was eluted at a rate of 10mL/min using 4% HCL, and the elution was continued with purified water until the effluent was neutral. The regenerated resin can be reused.
Example 4
A method for purifying ergothioneine, comprising the steps of:
(1) Extraction: extracting crude ergothioneine product with petroleum ether of equal volume, standing for layering, collecting lower layer, centrifuging at 8000rpm and 4deg.C for 20min; centrifuging to obtain supernatant, which is EGT extract;
(2) Resin transformation: LX-160W strong acid cation exchange resin was packed into a chromatographic column with a column volume of 60mL. 200mL (3 BV) was eluted at 10mL/min using 20% NaCl. After the elution is finished, continuing to use purified water to elute until the electric conductivity of the effluent is less than 5 uS/cm;
(3) Loading: adjusting the pH of the EGT extract to be=0.3 by using concentrated hydrochloric acid, and loading 6mL (1/10 BV) of the extract into a chromatographic column;
(4) Eluting: eluting with 7% KCl solution at a flow rate of 10mL/min, eluting 300mL (5 BV), taking 10mL (1/6 BV) as a receiving unit, and performing HPCL analysis to detect EGT content to obtain purified ergothioneine. And combining a plurality of groups of single-peak receiving units with larger EGT peak areas according to the HPLC result, performing HPLC analysis, detecting to determine that the EGT is single-peak, and calculating the yield and purity. The column was eluted with 10% kcl solution under the conditions as above while detecting the EGT content in the eluate by HPLC until no EGT signal peak was detected in the effluent and the EGT dead adsorption rate was calculated.
(5) Resin regeneration: 200mL (3 BV) was eluted using 4% HCl at a rate of 10mL/min and the elution was continued with purified water until the effluent was neutral. The regenerated resin can be reused.
Comparative examples 1 to 8
The mass fractions of the KCl solution of the eluent were changed to 0.1%, 0.5%, 0.9%, 1.2%, 1.5%, 2%, 2.5% and 3% based on example 1, respectively, and the other steps were the same as in example 1. And testing the purity after purification respectively, and calculating the EGT yield and the EGT dead adsorption rate.
Comparative example 9
On the basis of example 1, the eluent 5% KCl solution was replaced with pure water, and the rest of the procedure was the same as in example 1. And testing the purity after purification respectively, and calculating the EGT yield and the EGT dead adsorption rate.
Comparative example 10
On the basis of example 1, the eluent 5% KCl solution was replaced with 1.2% NaCl, and the rest of the procedure was the same as in example 1. And testing the purity after purification respectively, and calculating the EGT yield and the EGT dead adsorption rate.
Comparative example 11
The remainder of the procedure was as in example 1, except that the eluent 5% KCl solution was replaced with 1.5% NaCl on the basis of example 1. And testing the purity after purification respectively, and calculating the EGT yield and the EGT dead adsorption rate.
Comparative example 12
On the basis of example 1, the eluent 5% KCl solution was replaced with 5% NaCl, and the rest of the procedure was the same as in example 1. And testing the purity after purification respectively, and calculating the EGT yield and the EGT dead adsorption rate.
The results of the calculation of the purity, yield and dead adsorption rate of EGT in examples 1 to 4 and comparative examples 1 to 12 are shown in Table 1. FIG. 1 is a graph showing the effect of KCl solutions of different concentrations on EGT yield.
TABLE 1
| Group of | EGT yield/% | EGT dead adsorption rate/% | EGT purity/% |
| Example 1 | 91.96 | 6.16 | 98.95 |
| Example 2 | 75.55 | 22.29 | 98.90 |
| Example 3 | 88.89 | 5.01 | 93.43 |
| Example 4 | 88.13 | 5.08 | 94.48 |
| Comparative example 1 | 27.58 | 31.81 | 98.54 |
| Comparative example 2 | 43.21 | 21.94 | 98.46 |
| Comparative example 3 | 47.91 | 22.41 | 96.15 |
| Comparative example 4 | 66.56 | 13.13 | 98.67 |
| Comparative example 5 | 63.63 | 11.74 | 98.03 |
| Comparative example 6 | 66.44 | 11.27 | 97.66 |
| Comparative example 7 | 52.25 | 26.33 | 94.99 |
| Comparative example 8 | 58.42 | 24.18 | 98.41 |
| Comparative example 9 | 21.42 | 60.60 | 96.80 |
| Comparative example 10 | 31.87 | 30.12 | 94.43 |
| Comparative example 11 | 37.83 | 26.31 | 96.48 |
| Comparative example 12 | 51.40 | 17.69 | 95.25 |
As shown in Table 1, the purification method provided by the invention can effectively improve the yield of ergothioneine, and the yield is more than 75%, up to 91.96%, and the purity is high and is more than 93.43%. When the eluent is replaced by NaCl solution or water, the yield is obviously reduced, and the loss of ergothioneine is larger.
As can be seen from FIG. 1, the EGT yield shows a trend of increasing and decreasing with increasing KCl concentration in the eluent, the KCl can lead the yield to be more than 75% at the concentration of 4% -7%, and the KCl with the concentration of 5% is the highest after the EGT is eluted, and the yield is up to 91.96%.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. A method for purifying ergothioneine, comprising the steps of:
Extracting the ergothioneine crude solution by adopting an organic solvent, layering, taking a lower layer liquid, and then obtaining a supernatant by centrifugation to obtain an EGT extract;
Filling a chromatographic column with strong acid cation exchange resin, adjusting the pH value of the EGT extract, loading the EGT extract into the chromatographic column, and eluting with KCl solution to obtain purified ergothioneine;
the mass fraction of the KCl solution is 3.5% -8%.
2. The method according to claim 1, wherein the organic solvent is at least one selected from petroleum ether, diethyl ether, ethyl acetate, and chloroform.
3. The purification method according to claim 1 or 2, wherein the EGT extract is pH-adjusted to 0.2-4 with an acid solution before loading.
4. The purification method according to claim 1 or 2, wherein the strongly acidic cation exchange resin is one of LX-160W strongly acidic cation exchange resin, D001 strongly acidic cation exchange resin or 732 strongly acidic cation exchange resin.
5. The method according to claim 4, wherein the strongly acidic cation exchange resin is subjected to a resin transformation treatment before loading; the resin transformation process comprises the following steps: the column was loaded with a strongly acidic cation exchange resin, eluting sequentially with NaCl solution and purified water until the effluent conductivity was <5 uS/cm.
6. The method according to claim 5, wherein the mass fraction of the NaCl solution is 10% -30%.
7. The purification method according to claim 1 or 2, wherein the KCl solution has a mass fraction of 4% -7%.
8. The purification method according to claim 7, wherein the KCl solution has a mass fraction of 4.5% -5.5%.
9. The purification method according to claim 1 or 2, wherein the strongly acidic cation exchange resin is subjected to a resin regeneration treatment after the elution is completed; the resin regeneration process comprises the following steps: elution was performed sequentially with hydrochloric acid and purified water until the effluent was neutral.
10. The purification method according to any one of claims 1, 2,5, 6, 8, comprising the steps of:
(1) Extracting the ergothioneine crude solution by adopting an organic solvent, layering, taking a lower layer liquid, centrifuging the lower layer liquid at the rotating speed of 5000-9000 and the temperature of 0-5 ℃ for 10-30min, and collecting a supernatant, namely the EGT extract;
(2) Loading the strong acid cation exchange resin into a chromatographic column, eluting by using NaCl solution and purified water in sequence until the conductivity of effluent liquid is less than 5 uS/cm; adjusting the pH value of the EGT extract to 0.2-4 by adopting acid liquor, loading the EGT extract into the chromatographic column, and eluting by adopting KCl solution with the mass fraction of 3.5-8% to obtain the purified ergothioneine.
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