Preparation and application of anionic resin for high selective adsorption of ergothioneine

Abstract

The present invention discloses a preparation and application of an anion resin for high selective adsorption of ergothioneine, belonging to the field of biochemical engineering. For the first time, a polyamine-type epoxy weak base anion resin is used as a carrier to convert Ni atoms into [NiCl4] 2‑ The form of ion is loaded on target resin carrier, then the polyamine type epoxy weak base anion resin of load metal Ni nanoparticle is prepared through in-situ reduction and washing regeneration, and preparation technology is simple, and stability is good. The present invention has strong selective adsorption to thioneine in fermented liquid, and elution is convenient, and can be widely used in the purification process that fermentation method and enzyme catalysis method prepare thioneine.

Description

Technical Field

[0001] The invention belongs to the field of biochemical engineering and relates to the preparation and application of an anion resin for highly selective adsorption of ergothioneine. Background Art

[0002] As a natural antioxidant, ergothioneine has multiple physiological functions such as scavenging free radicals, detoxification, maintaining DNA biosynthesis, normal cell growth and cellular immunity. It has a wide range of uses in medicine, cosmetics, food and beverages, and biotechnology.

[0003] Currently, ergothioneine is mainly obtained from enzyme catalysis and bio-fermentation methods. However, both enzyme catalysis solutions and fermentation broths contain a large amount of impurities such as salts, miscellaneous acids, and pigments, making separation and purification difficult. In addition, ergothioneine is a structurally very special amino acid with an internal salt structure. This makes traditional amino acid general-purpose resins unable to meet the adsorption and impurity removal requirements of ergothioneine. Traditional fillers either have too weak adsorption capacity and low separation, or too strong adsorption capacity and difficulty in elution, resulting in poor separation.

[0004] In 2005, Nanjing University applied for a patent for "A method for preparing a resin-based deionized iron adsorbent" (ZL200510095177.5). This patent involves immobilizing hydrated iron oxide (non-zero-valent iron) particles on the inner surface of anion exchange resin, mainly utilizing the synergistic adsorption effect of the resin's amino group and hydrated iron oxide particles on deionized iron ions to achieve deep purification of deionized iron ions in water bodies.

[0005] In 2009, Zhang Weiming et al. applied for patent CN101474560A for a nanocomposite resin loaded with zero-valent iron for catalytic degradation of pollutants and its preparation method. - Ions are introduced into the resin's inner and outer surfaces through ion exchange. The introduced Fe ions are then reduced to nano-zero-valent iron, which is then washed and dried to produce a zero-valent iron-loaded nanocomposite resin. This resin combines the Donnan pre-enrichment effect of inorganic anionic pollutants in water with the highly efficient catalytic degradation of environmental pollutants by nano-zero-valent iron. It overcomes the shortcomings of nano-zero-valent iron particles, such as their tendency to agglomerate, chemical instability, and high head loss due to their small size, resulting in rapid, efficient, and cost-effective catalytic degradation of micro-pollutants in the environment.

[0006] In 2009, Lu Lu et al. applied for patent CN101716525A for “Anion resin-based CdS composite material and its preparation method”, which 2- Ions are introduced into the inner and outer surfaces of the carrier resin through ion exchange: CdCl4 2-The ion precipitates as CdS, which is then filtered, placed in a reaction vessel for hydrothermal treatment, washed, and dried to yield an anionic resin-based CdS composite material. This invention addresses the significant loss of active ingredients during the subsequent separation and recovery of CdS powder, increasing its ability to pre-enrich anionic pollutants in water, thereby promoting photocatalytic degradation and ultimately enabling widespread application in practical water treatment.

[0007] In 2018, Zou Jianping et al. applied for patent CN10917419A for "A method for preparing integrated resin-loaded copper-iron bimetallic nanomaterials". The Fe-Cu / D407 bimetallic nanomaterials prepared by this invention method have a NO3 removal rate of over 99% and a N selectivity of up to 89.7%. It has extremely efficient nitrate reduction performance and highly selective reduction to nitrogen.

[0008] In 2022, Guo Jianqi et al. applied for patent CN115301297A, titled "A cation exchange resin loaded with nanosilver particles, its preparation method, and its use." This invention prepares a cation exchange resin loaded with nanosilver particles. This nanosilver particle-loaded cation exchange resin exhibits specific adsorption properties for biodiesel sulfide.

[0009] Therefore, lack a kind of anionic resin that highly selective adsorption is carried out for thioneine in the prior art, existing load metal particles Cationic exchange resin also does not have to carry out highly selective adsorption for thioneine, therefore, now be badly in need of a kind of anionic resin that can solve relatively poor for thioneine selective adsorption effect in fermented liquid, impurity removal is not thorough. Summary of the Invention

[0010] Technical issues

[0011] For existing resin fillers and other resin-based loaded metal nanomaterials, the selective adsorption effect of thioneine in fermentation liquid is relatively poor, the problem of impurity removal is not thorough, the present invention aims to provide a kind of anion resin-based loaded nano Ni composite material and preparation method thereof, not only can solve the problem that existing materials have poor adsorption of thioneine in fermentation liquid, but also can achieve high selective adsorption and efficient elution to thioneine, thus achieving the accurate purification of thioneine fermentation system.

[0012] Technical Solution

[0013] According to the invention, thioneine molecules contain imidazole structural features, and the structural properties of histidine are used for reference. Technical personnel believe that thioneine molecules should also have strong affinity adsorption capacity for metal atoms Ni, Cu, Zn, etc., and combined with the polyamine-type epoxy weak base anion resin material with a certain adsorption capacity for thioneine screened in the early stage, technical personnel in the field have developed a polyamine-type epoxy weak base anion resin loaded with metal Ni nanoparticles and a preparation method thereof. The method of the invention prepares the polyamine-type epoxy weak base anion resin loaded with metal Ni nanoparticles for the first time. The material has the characteristic of efficient selective adsorption of thioneine, can effectively remove miscellaneous salts, pigments, miscellaneous amines and fermentation substrates in a fermentation system, and is combined with other impurity removal means to finally prepare high-purity thioneine with a purity greater than 99.5%.

[0014] The present invention aims to prepare a polyamine-type epoxy weak base anion resin loaded with metal Ni nanoparticles that has high selective adsorption for thioneine, and use the resin to purify thioneine fermentation broth.

[0015] To achieve the above object, the present invention provides a method for preparing a polyamine-type epoxy weak base anion resin loaded with metal Ni nanoparticles, comprising the following steps:

[0016] Step (1) Resin pretreatment: The resin is loaded into a chromatography column, and an aqueous hydrochloric acid solution is added at a flow rate of 1 to 5 BV / h for acid washing, and then the residual hydrochloric acid is washed away with water until the pH of the effluent is 6 to 7. Then, an aqueous sodium hydroxide solution is added at the same flow rate, and then the residual sodium hydroxide is washed away with water again, and eluted until the pH of the effluent is 7 to 8. The resin is removed and the surface moisture is drained to obtain a regenerated resin;

[0017] Step (2) Resin adsorption of nickel ions: preparing a nickel ion aqueous solution, adding the resin regenerated in step (1) to the nickel ion aqueous solution, shaking at room temperature for 10 to 100 minutes, then filtering out the resin, and washing with water to remove residual solvent on the surface to obtain a treated resin;

[0018] Step (3) Reduction of nickel ions on the resin: prepare an ethanol aqueous solution, then add NaBH4 to the prepared ethanol aqueous solution to prepare an ethanol aqueous solution of NaBH4, and finally add the resin treated in (2) to the ethanol aqueous solution of NaBH4, stir for 0.5 to 5 hours, filter out the resin, rinse with water, and obtain a polyamine-type epoxy weak base anion resin loaded with metal Ni nanoparticles.

[0019] In one embodiment of the present invention, the resin in step (1) includes but is not limited to 330 type epoxy weak base anion resin, 331 type epoxy weak base anion resin and 335 type epoxy weak base anion resin.

[0020] In one embodiment of the present invention, the concentration of the hydrochloric acid aqueous solution in step (1) is 1% to 10%.

[0021] In one embodiment of the present invention, the volume ratio of the hydrochloric acid aqueous solution to the resin in step (1) is 2 to 10:1, preferably 1 to 3:1.

[0022] In one embodiment of the present invention, the concentration of the sodium hydroxide aqueous solution in step (1) is 1% to 10%.

[0023] In one embodiment of the present invention, the volume ratio of the sodium hydroxide aqueous solution to the resin in step (1) is 2 to 10:1, preferably 2 to 4:1.

[0024] In one embodiment of the present invention, the nickel salt in the nickel ion aqueous solution in step (2) includes but is not limited to K2NiCl4, K2Ni(CN)4, Na2NiCl4 and Na2Ni(CN)4.

[0025] In one embodiment of the present invention, the nickel ion aqueous solution in step (2) is prepared at a concentration of 0.1 to 1 mol / L, preferably 0.4 to 0.8 mol / L.

[0026] In one embodiment of the present invention, the volume ratio of the nickel ion aqueous solution to the resin in step (2) is 2 to 10:1, preferably 3 to 5:1.

[0027] In one embodiment of the present invention, the volume ratio of water to resin used to wash away the residual solvent on the surface in step (2) is 0.5 to 2:1.

[0028] In one embodiment of the present invention, the entire solvent preparation process and reaction process in step (3) are purged with N2 to remove dissolved oxygen in the solution.

[0029] In one embodiment of the present invention, the volume fraction of the ethanol aqueous solution in step (3) is 10% to 90%, preferably 60%.

[0030] In one embodiment of the present invention, the mass fraction of NaBH4 in the ethanol aqueous solution of NaBH4 in step (3) is 1% to 50%, preferably 20%.

[0031] In one embodiment of the present invention, the volume ratio of the ethanol aqueous solution of NaBH4 to the resin in step (3) is 0.5 to 3:1.

[0032] In one embodiment of the present invention, the volume ratio of water to resin used for flushing in step (3) is 0.5 to 2:1.

[0033] The present invention provides a polyamine-type epoxy weak base anion resin loaded with metal Ni nanoparticles prepared according to the method.

[0034] The present invention provides an application of the polyamine-type epoxy weak base anion resin loaded with metal Ni nanoparticles in the field of adsorption purification.

[0035] The present invention provides a method for preparing a polyamine-type epoxy weak base anion resin loaded with metal Ni nanoparticles, comprising the following steps:

[0036] Step (1) Resin pretreatment: The resin is loaded into a chromatography column, and a 1% to 10% hydrochloric acid aqueous solution is added at a flow rate of 1 to 5 BV / h, which is 2 to 10 times the volume of the resin, and then the residual hydrochloric acid is washed away with water until the pH of the effluent is 6 to 7. Then, a 1% to 10% sodium hydroxide aqueous solution is added at the same flow rate, which is 2 to 10 times the volume of the resin, and then the residual sodium hydroxide is washed away with water again, and the elution is carried out until the pH of the effluent is 7 to 8. The resin is removed and the surface moisture is drained to obtain a regenerated resin;

[0037] Step (2) Resin adsorption of nickel ions: preparing a nickel ion aqueous solution with a concentration of 0.1 to 1 mol / L, adding the resin regenerated in step (1) to the nickel ion aqueous solution, with the solution volume being 2 to 10 times the volume of the resin, shaking at room temperature for 10 to 100 minutes, then filtering out the resin, and washing with water to remove residual solvent on the surface to obtain a treated resin;

[0038] Step (3) Reduction of nickel ions on the resin: prepare an ethanol aqueous solution with a volume fraction of 10% to 90%, then add NaBH4 to the prepared ethanol aqueous solution to prepare an ethanol aqueous solution with a mass fraction of NaBH4 of 1% to 50%, and finally add the resin treated in (2) to the NaBH4 ethanol aqueous solution, stir for 0.5 to 5h, filter out the resin, rinse with water, and obtain a polyamine-type epoxy weak base anion resin loaded with metal Ni nanoparticles.

[0039] In one embodiment of the present invention, the resin in step (1) includes but is not limited to 330 type epoxy weak base anion resin, 331 type epoxy weak base anion resin and 335 type epoxy weak base anion resin.

[0040] In one embodiment of the present invention, the nickel salt in the nickel ion aqueous solution in step (2) includes but is not limited to K2NiCl4, K2Ni(CN)4, Na2NiCl4 and Na2Ni(CN)4.

[0041] In one embodiment of the present invention, the entire solvent preparation process and reaction process in step (3) are purged with N2 to remove dissolved oxygen in the solution.

[0042] In one embodiment of the present invention, the nickel ions on the resin in step (3) need to be washed with deionized water after reduction, and the amount of water used is 2 to 10 times the volume of the resin.

[0043] The present invention provides a purification method for highly selective adsorption of ergothioneine in a fermentation broth, comprising the following steps:

[0044] Step (1) Ceramic membrane sterilization: the fermentation liquid is passed through a ceramic membrane for microfiltration sterilization to obtain a clarified fermentation liquid;

[0045] Step (2) nanofiltration concentration and desalination: using a nanofiltration membrane to concentrate the clarified fermentation broth in step 1, collecting the concentrate to obtain a nanofiltered fermentation broth;

[0046] Step (3) column chromatography adsorption and impurity removal: first, the polyamine-type epoxy weak base anion resin loaded with metal Ni nanoparticles prepared above is loaded into a resin column, and then the fermentation broth concentrated by nanofiltration is passed through the resin column at a flow rate of 0.1 to 2 BV / h. After the sample is loaded, the waste liquid remaining in the column is rinsed with water until the conductivity is less than 0.1 ms / cm;

[0047] Step (4) eluting: eluting the ergothioneine adsorbed on the resin column with a flow rate of 0.1 to 2 BV / h of an imidazole aqueous solution, and then collecting the eluate;

[0048] Step (5) macroporous resin adsorption and impurity removal: using a macroporous resin to adsorb the imidazole in the eluate collected in step (4), and collecting the effluent;

[0049] Step (6) Concentration and crystallization: concentrating the liquid collected in step (5);

[0050] Step (7) centrifugal drying: the feed liquid after concentrating in step (6) is centrifuged, the solid obtained by centrifugation is cleaned with ethanol aqueous solution, and then dried to obtain pure thioneine.

[0051] In one embodiment of the present invention, the pore size of the ceramic membrane in step (1) is between 50 nm and 200 nm.

[0052] In one embodiment of the present invention, in step (1), the ceramic membrane has a transmembrane temperature of 35-45° C. and a transmembrane pressure of 0.03-0.05 MPa.

[0053] In one embodiment of the present invention, the content of thioneine in the fermentation broth in step (1) is 5 to 8 g / L.

[0054] In one embodiment of the present invention, the pore size of the nanofiltration membrane in step (2) is between 100 and 600 Daltons.

[0055] In one embodiment of the present invention, in step (2), the nanofiltration membrane has a transmembrane temperature of 30-40° C. and a transmembrane pressure of 0.1-0.15 MPa.

[0056] In one embodiment of the present invention, during the nanofiltration process in step (2), when the flux is reduced by half, water is added to control the flux and maintain it at a level of more than half of the original flux.

[0057] In one embodiment of the present invention, the conductivity of the nanofiltration membrane at the end point of the membrane in step (2) is less than 1.2 to 1.5 ms / cm.

[0058] In one embodiment of the present invention, the volume ratio of the fermentation broth after nanofiltration in step (3) to the polyamine-type epoxy weak base anion resin loaded with metal Ni nanoparticles is 20:0.5-3.

[0059] In one embodiment of the present invention, the concentration of imidazole in the eluent imidazole aqueous solution in step (4) is 1% to 40% (v / v).

[0060] In one embodiment of the present invention, the macroporous resin used in step (5) is any one of a polar macroporous resin, a medium polar macroporous resin or a weak polar macroporous resin.

[0061] In one embodiment of the present invention, the elution flow rate of the eluent in step (5) is 0.1 to 2 BV / h.

[0062] In one embodiment of the present invention, the concentration temperature in step (6) is 40° C. to 80° C., and the vacuum degree is -0.1 MPa to -0.08 MPa.

[0063] In one embodiment of the present invention, the water solubility of ethanol in step (7) is 70% to 90% (v / v).

[0064] In one embodiment of the present invention, in step (7), the drying temperature is 40° C. to 80° C., the vacuum degree is -0.1 MPa to -0.08 MPa, and the drying time is 2 to 12 hours.

[0065] The present invention provides ergothioneine obtained according to the purification method.

[0066] The invention relates to the application of the purification method provided by the present invention in the field of thioneine purification.

[0067] Beneficial effects

[0068] The present invention solves the problems that the impurity removal efficiency encountered in the process of preparing high-quality thioneine by enzyme catalysis or fermentation is low, impurity removal is not thorough, and investment cost is high. The polyamine-type epoxy weak base anion resin of the loaded metal Ni nanoparticles prepared by the present invention does not require too complicated equipment and technology, and strong selective adsorption and high selectivity elution to thioneine can be achieved, thereby achieving the refining and purification of thioneine fermentation liquor. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 The pure thioneine crystal morphology (electron microscope shooting) prepared in Example 2.

[0070] Figure 2 Be the thioneine pure product outward appearance prepared among embodiment 2.

[0071] Figure 3 It is the pure thioneine liquid phase spectrogram prepared in embodiment 2. DETAILED DESCRIPTION

[0072] Source of raw materials

[0073] The strain information and fermentation conditions involved in the present invention are all from patent CN114854659A "A production process of thioneine and its application", and the fermentation broth is prepared according to the fermentation conditions of Example 5 in patent CN114854659A; the resins used in the present invention are all from Xi'an Lanxiao Technology New Materials Co., Ltd.

[0074] Detection method

[0075] References for detection methods of ergothioneine: Wang Li, Wang Yang, Li Jianghua, et al. Construction and optimization of ergothioneine-producing Escherichia coli engineered strains[J]. Chinese Journal of Biotechnology, 2022, 38(2): 796-806.

[0076] The HPLC method for the 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). The mobile phase ratio is: water: methanol = 99:1, flow rate: 0.7mL / min, column temperature: 30°C, ultraviolet absorption: 257nm, injection volume: 5μL, and detection time: 20min. The 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 ergothioneine standard concentration 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.

[0077] Example 1

[0078] This embodiment is the preparation of a polyamine-type epoxy weak base anion resin loaded with metal Ni nanoparticles. In step (3), the solvent preparation process and the reaction process are both purged with N2 to remove dissolved oxygen in the solution.

[0079] (1) Resin regeneration: First, 1 L of 330 resin (polyamine-type epoxy weak base anion) was loaded into a chromatography column. The resin was acid-washed with 2 L of 4% hydrochloric acid aqueous solution at a flow rate of 1 BV / h. The residual hydrochloric acid in the resin column was then washed with pure water until the pH of the effluent reached 7. Then, 3 L of 4% sodium hydroxide aqueous solution was pumped into the column at the same flow rate (1 BV / h). The residual sodium hydroxide in the resin column was then washed with pure water until the pH of the effluent reached 7.5. The resin was removed and the surface moisture was drained to obtain the regenerated resin.

[0080] (2) Ni ion adsorption: First, prepare 4 L of a 0.6 mol / L K2NiCl4 aqueous solution, add 1 L of the regenerated resin to the K2NiCl4 aqueous solution, and then place the container on a shaker. After shaking at room temperature for 60 min, filter out the resin and wash with 1 L of pure water to remove the residual solvent on the surface, to obtain the treated resin.

[0081] (3) Reduction of Ni ions on the resin: First, prepare a 60% (V / V) ethanol aqueous solution, then add a certain amount of NaBH4 to the prepared ethanol aqueous solution to prepare a 20% (mass fraction) NaBH4 ethanol aqueous solution, and finally add the resin treated in step (2) to the NaBH4 ethanol aqueous solution (the volume of the reducing solution is about twice the volume of the resin), mechanically stir and react for 2 hours, filter out the resin, and rinse with 1 L of pure water to obtain a polyamine-type epoxy weak base anion resin loaded with metal Ni nanoparticles.

[0082] Example 2

[0083] This embodiment uses a polyamine-type epoxy weak base anion resin loaded with metal Ni nanoparticles to highly selectively adsorb and purify ergothioneine in a fermentation broth.

[0084] Fermentation strain information and fermentation conditions are all from patent CN114854659A "A kind of thioneine production technology and application thereof", first fermented according to the fermentation conditions of Example 5 in patent CN114854659A, fermentation obtained thioneine fermentation liquid 20L, content was 6.8g / L, then the fermentation liquid was purified according to the following steps.

[0085] (1) Ceramic membrane sterilization: The fermentation broth was microfiltered through a 50 nm ceramic membrane at 40°C and 0.05 MPa pressure to obtain a clarified fermentation broth;

[0086] (2) Nanofiltration concentration and desalination: The clarified fermentation broth obtained in step (1) is concentrated and desalted by passing it through a nanofiltration membrane with a pore size of 200 Daltons at 35°C and a pressure of 0.15 MPa. As the nanofiltration proceeds, the flux gradually decreases. When the flux is halved, pure water is added to control the flux and maintain it at a level of more than half of the original flux. When the conductivity reaches 1.2 ms / cm, nanofiltration is stopped to obtain the fermentation broth after nanofiltration.

[0087] (3) Column chromatography adsorption and impurity removal: 1 L of the polyamine-type epoxy weak base anion resin loaded with metal Ni nanoparticles prepared in Example 1 was loaded into a resin column, and the fermentation broth concentrated by nanofiltration was passed through the resin column at a flow rate of 1 BV / h. After loading, the residual waste liquid in the column was rinsed with a small amount of pure water until the conductivity was <0.1 ms / cm;

[0088] (4) elution: 10% imidazole aqueous solution was used to elute the thioneine adsorbed on the resin column at a flow rate of 0.8 BV / h, and liquid chromatography was used to detect whether the thioneine was eluted completely. When the liquid chromatography showed that the thioneine concentration was <0.1 g / L, elution was stopped and all imidazole eluates were collected.

[0089] (5) macroporous resin adsorption removal: take 2L middle polar macroporous resin and be packed in resin column, the imidazole eluent collected is flowed through resin column with the flow velocity of 0.8BV / h, and with a small amount of pure water elution post residual thioneine, collect discharging liquid;

[0090] (6) Concentrating and crystallizing: the collected discharge liquid is concentrated under reduced pressure at 60°C under vacuum-0.1Mpa conditions. When thioneine concentration in the feed liquid is > 150g / l, concentration is stopped, and cooling crystallization is started. The mixture is centrifuged after being cooled to 4°C and incubated for 1h.

[0091] (7) centrifugal drying: the crystallized thioneine was centrifuged with a centrifuge, and the filter cake was cleaned with 80% ethanol water for 30s during the centrifugation process, and then dried under reduced pressure for 10h at 60°C under vacuum degree-0.1 MPa to obtain pure thioneine 109.5g (yield 82.5%) with a purity of 99.73% (HPLC detection).

[0092] Comparative Example 1

[0093] The polyamine-type epoxy weak base anion resin of the load metal Ni nano-particle in embodiment 2 step 3 is changed into other unmodified common resins, when remaining operation and condition are all unchanged, according to the method in embodiment 2, thioneine fermentation liquid is purified, and finally the purity and yield of thioneine obtained by experiment are as follows:

[0094]

[0095] Due to the polyamine type epoxy weak base anion resin of the load metal Ni nano-particle of preparation has high selectivity adsorption to thioneine, it can be seen that the thioneine yield and the purity after the polyamine type epoxy weak base anion resin purification of load metal Ni nano-particles are adopted compared with other unmodified resins have obvious advantages. In addition, equally all in the case of without nano ion transformation, the raw material resin selected by the present invention--330 type polyamine epoxy weak base anion resins compared with other common resins has significantly higher yield, illustrates that its structure itself has stronger affinity to thioneine.

[0096] Comparative Example 2

[0097] The load metal element in embodiment 1 step 2 is replaced with other metallic elements, prepare the polyamine type epoxy weak base anion resin of load different kinds of nano metal, then replace the resin in embodiment 2 step 3 with the different kinds of nano metal resin prepared respectively, when remaining operation and condition are all constant, according to the condition of embodiment 2, thioneine fermentation liquid is purified, finally experiment obtains purity and yield of thioneine as follows:

[0098]

[0099] It can be seen that although the purification yield and purity of ergothioneine are significantly improved after the nano-modified resin, the purification yield of the 330 resin modified by nano-Ni is the highest compared with the 330 resin modified with other nano-ions or not modified, and the purity is also very good, indicating that nano-Ni has the strongest chelating effect on ergothioneine.

[0100] Comparative Example 3

[0101] The K in embodiment 1 step 2 is optimized by NiCl concentration, and the K of 0.2, 0.4, 0.6, 0.8, 1.0 mol / L is prepared respectively NiCl the aqueous solution, when the remaining conditions are all unchanged, thioneine fermentation liquid is purified according to the condition of embodiment 2, and finally the purity and yield of thioneine obtained by experiment are as follows:

[0102]

[0103] It can be seen that with the increase of K2NiCl4 concentration, the prepared resin effect becomes better and better. When its concentration increases to 0.6 mol / L, the resin performance basically tends to be stable. Therefore, considering the economic and yield factors, the optimal concentration of K2NiCl4 for preparing resin is 0.6 mol / L.

[0104] The embodiments provided above are not intended to limit the scope of the present invention, nor are the steps described to limit their execution order. Any obvious improvements to the present invention made by those skilled in the art in combination with existing common knowledge shall fall within the scope of protection defined by the claims of the present invention.

Claims

1. a preparation method for the polyamine-type epoxy weak base anion resin of the loaded metal Ni nanoparticles with high selectivity adsorption to thioneine, comprising the following steps: Step (1) Resin pretreatment: The resin is loaded into a chromatography column, and a hydrochloric acid aqueous solution is added at a flow rate of 1 to 5 BV / h for acid washing, and then the residual hydrochloric acid is washed away with water until the pH of the effluent is 6 to 7, and then a sodium hydroxide aqueous solution is added at the same flow rate, and then the residual sodium hydroxide is washed away with water again, and eluted until the pH of the effluent is 7 to 8, and the resin is taken out and the surface moisture is drained to obtain a regenerated resin; Step (2) Resin adsorption of nickel ions: Prepare a nickel ion aqueous solution, add the resin regenerated in step (1) to the nickel ion aqueous solution, shake at room temperature for 10 to 100 minutes, then filter out the resin and wash with water to remove residual solvent on the surface to obtain a treated resin; Step (3) Reduction of nickel ions on the resin: prepare an ethanol-water solution, then add NaBH4 to the prepared ethanol-water solution to prepare an ethanol-water solution of NaBH4, and finally add the resin treated in (2) to the ethanol-water solution of NaBH4, stir for 0.5-5h, filter out the resin, and rinse with water to obtain a polyamine-type epoxy weak base anion resin loaded with metal Ni nanoparticles.

2. The method according to claim 1, wherein The resins in step (1) include 330 type epoxy weak base anion resin, 331 type epoxy weak base anion resin and 335 type epoxy weak base anion resin.

3. The method according to claim 1, wherein The nickel salt in the nickel ion aqueous solution in step (2) includes K2NiCl4, K2Ni(CN)4, Na2NiCl4 and Na2Ni(CN)4; the nickel ion aqueous solution in step (2) is prepared at a concentration of 0.1~1 mol / L.

4. The method according to claim 1, wherein The mass fraction of NaBH4 in the ethanol aqueous solution of NaBH4 in step (3) is 1% to 50%; the volume ratio of the ethanol aqueous solution of NaBH4 to the resin in step (3) is 0.5 to 3:

1.

5. a polyamine-type epoxy weak base anion resin having the loaded metal Ni nanoparticle of high selectivity adsorption to thioneine prepared according to any one of claims 1 to 4 methods.

6. a purification process for the thioneine high-selectivity adsorption in the fermented liquid, comprising the following steps: Step (1) Ceramic membrane sterilization: the fermentation liquid is passed through a ceramic membrane for microfiltration sterilization to obtain a clarified fermentation liquid; Step (2) nanofiltration concentration and desalination: using a nanofiltration membrane to concentrate the clarified fermentation broth in step 1, collecting the concentrate to obtain a nanofiltered fermentation broth; Step (3) column chromatography adsorption and impurity removal: first, the polyamine-type epoxy weak base anion resin loaded with metal Ni nanoparticles as described in claim 5 is loaded into a resin column, and then the fermentation broth after nanofiltration concentration is passed through the resin column at a flow rate of 0.1-2 BV / h. After the sample is loaded, the waste liquid remaining in the column is washed with water until the conductivity is less than 0.1 ms / cm; Step (4) elution: eluting the ergothioneine adsorbed on the resin column with an imidazole aqueous solution at a flow rate of 0.1 to 2 BV / h, and then collecting the eluate; Step (5) macroporous resin adsorption and impurity removal: using a macroporous resin to adsorb the imidazole in the eluate collected in step (4), and collecting the effluent; Step (6) Concentration and crystallization: concentrating the liquid collected in step (5); Step (7) centrifugal drying: centrifuging the concentrated liquid in step (6), washing the solid obtained by centrifugation with an ethanol aqueous solution, and then drying to obtain pure ergothioneine.

7. The method according to claim 6, wherein The pore size of the ceramic membrane in step (1) is 50 nm to 200 nm; the membrane temperature of the ceramic membrane in step (1) is 35 to 45 ° C, and the membrane pressure is 0.03 to 0.05 MPa.

8. The method according to claim 6, wherein The pore size of the nanofiltration membrane in step (2) is 100-600 Daltons; the membrane temperature of the nanofiltration membrane in step (2) is 30-40°C, and the membrane pressure is 0.1-0.15 MPa; the conductivity of the nanofiltration membrane at the membrane end point in step (2) is <1.2-1.5 ms / cm.

9. The method according to claim 6, wherein In step (3), the volume ratio of the fermentation broth after nanofiltration to the polyamine-type epoxy weak base anion resin loaded with metal Ni nanoparticles is 20:0.5~3; in step (4), the concentration of imidazole in the eluent imidazole aqueous solution is 1%~40%.

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