Mesoporous metal organic framework-based astaxanthin selective separation and purification method

Abstract

The invention discloses an astaxanthin selective separation and purification method based on a mesoporous metal organic framework, and belongs to the technical field of astaxanthin extraction and purification. The method comprises the following steps: by taking a mesoporous metal organic framework NU-1000 with a one-dimensional pore structure as an extracting agent, adsorbing and enriching astaxanthin, then desorbing under the action of a specific eluent (a mixture of DMSO and formic acid or a mixture of DMF and formic acid), and separating and purifying to obtain astaxanthin. The purity of the separated and purified astaxanthin is improved by about 2.81 times.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of astaxanthin extraction and purification, and particularly relates to a method for selectively separating and purifying astaxanthin based on mesoporous metal organic frameworks. BACKGROUND

[0002] Astaxanthin (AXT) is a liposoluble ketocarotenoid, which is abundant in salmonids and crustaceans aquaculture. It is composed of two terminal rings connected by a polyene chain, containing conjugated double bonds, hydroxyl groups and ketones, with lipophilic and hydrophilic properties. The conjugated double bonds provide electrons and react with free radicals to convert them into more stable products and terminate the free radical chain reaction in various organisms, thereby playing the role of a strong antioxidant. Its unique structure also endows astaxanthin with the functions of anti-lipid peroxidation, anti-inflammatory, anti-diabetic, anti-cancer, cardiovascular disease prevention, and immune regulation. Due to these superior biological functions of astaxanthin, the demand for astaxanthin is increasing. Astaxanthin can be extracted from organisms or artificially synthesized by chemical synthesis. However, artificially synthesized astaxanthin is derived from petrochemical products, and is a mixture of left-handed, right-handed and meso structures. Only astaxanthin with partial left-handed structure has high biological activity, and is not allowed to be used in human health products. Therefore, extracting natural astaxanthin from organisms is the best choice.

[0003] Currently, the main methods for extracting astaxanthin include organic solvent extraction, supercritical CO2 extraction, and emerging deep eutectic solvent (DES) extraction. Organic solvent extraction mainly uses dichloromethane to extract astaxanthin from shrimp shells, which is stable at low temperature and the solvent can be recycled, but attention should be paid to solvent residues. Supercritical CO2 extraction is safe and has high product quality, but the equipment is expensive and not easy to scale up. DES low-temperature extraction technology is suitable for Antarctic krill, and as a new technology, it is still being optimized. Regardless of which extraction technology, there are still impurities such as proteins, lipids, and especially fat-soluble coexisting substances such as lutein, fucoxanthin and beta-carotene in the extracted astaxanthin, which are similar in structure to astaxanthin, making the astaxanthin purification process challenging. Therefore, it is of great significance to establish a high-extraction-rate astaxanthin extraction and purification process for the utilization and product development of astaxanthin. SUMMARY

[0004] [TECHNICAL PROBLEM]

[0005] The existing astaxanthin extraction method has problems such as low adsorption capacity, long extraction time, or poor selective adsorption. The present application constructs MOFs materials with specific mesoporous structure characteristics to improve the selective extraction efficiency of astaxanthin.

[0006] [TECHNICAL SCHEME]

[0007] To solve the above problems, the mesoporous MOFs material with one-dimensional channel structure is constructed, and through the specific structure matching between the MOFs and astaxanthin, high selective enrichment and adsorption of astaxanthin are realized; meanwhile, a faster adsorption rate can be maintained in the subsequent desorption process, and finally the separation and purification are realized.

[0008] The application provides a method for selectively separating and purifying astaxanthin, which uses the mesoporous metal organic framework NU-1000 with one-dimensional channel structure as an extractant to adsorb and enrich astaxanthin, and then desorbs under the action of an eluent.

[0009] In an embodiment of the application, the eluent is a mixture of DMSO and formic acid or a mixture of DMF and formic acid.

[0010] In an embodiment of the application, the volume percentage of formic acid in the mixture of DMSO and formic acid or the mixture of DMF and formic acid is 0.04%-1.4%. Preferably, it is 0.8%-1.0%.

[0011] In an embodiment of the application, the time for adsorption and enrichment is 3-30h.

[0012] In an embodiment of the application, the time for desorption is 4h.

[0013] In an embodiment of the application, the preparation method of the mesoporous metal organic framework NU-1000 with one-dimensional channel structure comprises the following steps: dissolving ZrOCl2·8H2O and benzoic acid in N,N-dimethylformamide and heating, then adding a ligand 1,3,6,8-tetra(4-carboxyphenyl)pyrene, obtaining a yellow solid powder through a solvothermal method, then adding hydrochloric acid as an adjuster after washing, continuing to obtain a crude product NU-1000 through the solvothermal method, and finally obtaining the mesoporous metal organic framework material NU-1000 through washing, drying, vacuum heating activation.

[0014] In an embodiment of the application, the molar ratio of ZrOCl2·8H2O, benzoic acid and the ligand 1,3,6,8-tetra(4-carboxyphenyl)pyrene is 5:318:1.

[0015] In an embodiment of the application, the first heating condition is 80℃ for 1h, the second heating condition is 100℃ for 15.5h, and the third heating condition is 100℃ for 12h.

[0016] In an embodiment of the application, the drying and vacuum heating activation are carried out in a vacuum drying box at 120℃ for 12h, and the vacuum degree is-0.1Mpa.

[0017] The application provides a method for selectively separating and purifying astaxanthin, comprising the following steps:

[0018] (1) First, astaxanthin in the shell of Penaeus vannamei is coarsely extracted to obtain crude astaxanthin;

[0019] (2) Then, mesoporous metal-organic framework NU-1000 is used as an extractant to separate and purify astaxanthin.

[0020] In an embodiment of the application, step (1) specifically comprises the following process:

[0021] Solvent extraction is used to extract astaxanthin, the shell of Penaeus vannamei is dried and physically crushed, then stirred and mixed with CH2Cl2 in the dark, and then centrifuged to obtain supernatant; the above extraction process is repeated, and CH2Cl2 is added for extraction until the supernatant becomes light in color, the supernatant is collected, filtered and rotary evaporated to obtain crude astaxanthin with ester groups;

[0022] The crude astaxanthin with ester groups is dissolved in ethanol (EtOH), then NaOH-EtOH solution is added, and the mixed solution is placed in 0 to -4 DEG C for saponification reaction; after the reaction is completed, acid is added to adjust the pH of the solution to 7, and finally rotary evaporation is performed to obtain crude astaxanthin without ester groups.

[0023] In an embodiment of the application, the separation and purification process of step (2) comprises adsorption enrichment and elution desorption.

[0024] In an embodiment of the application, NU-1000 is used to selectively adsorb and enrich astaxanthin in the crude astaxanthin in step (2), then the precipitate is collected, and an eluent is used for elution desorption.

[0025] In an embodiment of the application, the adsorption enrichment mode comprises static adsorption and dynamic adsorption.

[0026] In an embodiment of the application, the dynamic adsorption is realized by flowing the crude astaxanthin solution through a gravity column added with NU-1000 adsorption material through a peristaltic pump, and the flow rate of the astaxanthin solution is controlled to be 0.2-1.0 mL / min, and preferably 0.6-1.0 mL / min.

[0027] In an embodiment of the application, the desorption mode comprises static desorption and dynamic desorption.

[0028] In an embodiment of the application, the dynamic desorption is realized by using a mixed solution of DMSO and formic acid to elute the gravity column after complete adsorption, and the flow rate of the mixed solution of DMSO and formic acid is controlled to be 0.2-1.0 mL / min, and preferably 0.6-1.0 mL / min.

[0029] Advantages:

[0030] The mesoporous MOFs material NU-1000 of the present application has high astaxanthin adsorption capacity, fast adsorption efficiency, stable structure and no toxicity. The NU-1000 with one-way channel structure is selected as the extraction agent in the present application, which has high adsorption capacity for astaxanthin, fast adsorption rate and about 83.2% desorption rate, excellent selectivity in the astaxanthin crude extract extracted from the shell of Penaeus vannamei, and the purity after purification by NU-1000 is increased by about 2.81 times. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 is a flow chart of selective enrichment, separation and purification of astaxanthin by four mesoporous metal organic framework materials in Comparative Examples 1-3 and Example 1;

[0032] Figure 2 is a structure diagram of four mesoporous metal organic framework materials (a) NU-1000, (b) PCN-222, (c) PCN-777, (d) MIL-101-Fe-NH2 in Comparative Examples 1-3 and Example 1;

[0033] Figure 3 is an XRD spectrum of four mesoporous metal organic framework materials in Comparative Examples 1-3 and Example 1;

[0034] Figure 4 is an HRTEM diagram of NU-1000 in Example 1 and MIL-101 in Comparative Example 3, wherein (a) is NU-1000 and (b) is MIL-101;

[0035] Figure 5 is an FT-IR spectrum of four mesoporous metal organic framework materials in Comparative Examples 1-3 and Example 1;

[0036] Figure 6 is a scanning electron microscope picture of four mesoporous metal organic framework materials in Comparative Examples 1-3 and Example 1;

[0037] Figure 7 is a N2 adsorption-desorption isotherm of four mesoporous metal organic framework materials in Comparative Examples 1-3 and Example 1;

[0038] Figure 8 is a pore size distribution of four mesoporous metal organic framework materials in Comparative Examples 1-3 and Example 1;

[0039] Figure 9 is an ultraviolet-visible absorption spectrum and a high performance liquid chromatography standard curve of astaxanthin, wherein (a) is an ultraviolet-visible absorption spectrum and (b) is a high performance liquid chromatography.

[0040] Figure 10 is the adsorption performance of astaxanthin on four mesoporous metal-organic frameworks in Comparative Examples 1-3 and Example 1, wherein (a) is PCN-222, (b) is NU-1000, (c) is PCN-777, and (d) is MIL-101-Fe-NH2;

[0041] Figure 11 is the adsorption kinetics of astaxanthin on four mesoporous metal-organic frameworks in Comparative Examples 1-3 and Example 1;

[0042] Figure 12 is the intraparticle diffusion model of astaxanthin on four mesoporous metal-organic frameworks in Comparative Examples 1-3 and Example 1;

[0043] Figure 13 is a comparison chart of astaxanthin adsorption capacity of four mesoporous metal-organic frameworks in Comparative Examples 1-3 and Example 1 and other adsorbents;

[0044] Figure 14 is the Langmuir model and the Freundlich model of astaxanthin on four mesoporous metal-organic frameworks in Comparative Examples 1-3 and Example 1;

[0045] Figure 15 is the adsorption selectivity of astaxanthin on four mesoporous metal-organic frameworks in Example 2;

[0046] Figure 16 is the electrostatic potential diagram of astaxanthin, fucoxanthin, lutein and β-carotene, wherein (a) is astaxanthin, (b) is fucoxanthin, (c) is lutein, and (d) is β-carotene;

[0047] Figure 17 is the FT-IR spectra of NU-1000 and MIL-101 before and after adsorbing astaxanthin in Example 2;

[0048] Figure 18 is the O1s orbital XPS diagram of NU-1000 and MIL-101 before and after adsorbing astaxanthin in Example 2, wherein (a) is NU-1000 and (b) is MIL-101;

[0049] Figure 19 is the high performance liquid chromatogram of astaxanthin crude extract from the shell of Penaeus vannamei in Example 3;

[0050] Figure 20 is the desorption performance of astaxanthin on four mesoporous metal-organic frameworks in Example 3;

[0051] Figure 21is a high resolution mass spectrum of the eluent of NU-1000 against astaxanthin in Example 3;

[0052] Figure 22 is a dynamic adsorption-desorption curve of NU-1000 against astaxanthin in Example 4, where (a) is a dynamic adsorption curve, and (b) is a dynamic desorption curve;

[0053] Figure 23 is an HPLC spectrum of astaxanthin before and after purification in Example 4. DETAILED DESCRIPTION

[0054] Example 1

[0055] A method for preparing mesoporous metal-organic framework NU-1000 with one-way channel structure, comprising the following steps:

[0056] Dissolve 194 mg of ZrOCl2·8H2O and 5.4 g of benzoic acid in 16 mL of N,N-dimethylformamide, and after ultrasonic dissolution, place it in an 80°C oven for 1 h. After taking it out, cool it in time, then add 80 mg of ligand 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene, ultrasonic dissolution, and then place it in a 100°C oven for 15.5 h. After the reaction is completed, centrifuge at 8340 rpm for 6 min to obtain a yellow solid, then wash it with 25 mL of DMF three times, with an interval of 2 h each time. After washing, transfer the yellow solid to 26 mL of DMF, add 1 mL of 8M hydrochloric acid as a regulator, ultrasonic mix uniformly, and then place it in a 100°C oven for reaction for 12 h. After the reaction is completed, collect the yellow solid by centrifugation (8340 rpm, 6 min), wash it with 25 mL of DMF three times, with an interval of 2 h each time, and then wash it with 25 mL of anhydrous ethanol three times, with an interval of 12 h each time. Collect the solid and activate it by vacuum heating in a vacuum drying oven for 12 h, and finally obtain NU-1000 yellow solid powder.

[0057] Comparative Example 1

[0058] A method for preparing mesoporous metal-organic framework PCN-222 with one-dimensional pore, comprising the following steps:

[0059] Dissolve 75 mg of ZrCl4and 30 mg of meso-tetrakis(4-carboxyphenyl) porphyrin in 10 mL of N,N-dimethylformamide, then add 1.75 g of benzoic acid modifier, ultrasonic dissolution, and then place in a 120°C oven for 48 h; after the reaction is completed, centrifuge at 9000 rpm for 10 min to collect the purple crystalline solid, wash with 20 mL of DMF every 8 h for a total of three times, then wash with 20 mL of anhydrous ethanol every 8 h for a total of three times; finally, the solid material is activated in a vacuum drying oven at 120°C for 12 h to obtain a mesoporous metal-organic framework material PCN-222 with a unidirectional channel.

[0060] Comparative Example 2

[0061] A method for preparing a metal-organic framework PCN-777 having a cage channel structure, comprising the following steps:

[0062] Dissolve 200 mg of ZrOCl2·8H2O and 60 mg of 2,4,6-tris(4-carboxyphenyl)-1,3,5-triazine in 12 mL of N,N-dimethylformamide and 0.6 mL of trifluoroacetic acid solution, ultrasonic dissolution, and then place in a 120°C oven for 12 h; after the reaction is completed, centrifuge at 9000 rpm for 10 min to collect the white solid, wash with 20 mL of DMF every 8 h for a total of three times, then wash with 20 mL of anhydrous ethanol every 8 h for a total of three times; finally, the solid material is activated in a vacuum drying oven at 120°C for 12 h to obtain a metal-organic framework PCN-777 having a mesoporous cage channel structure.

[0063] Comparative Example 3

[0064] A method for preparing a metal-organic framework MIL-101-Fe-NH2having a mesoporous cage channel structure, comprising the following steps:

[0065] Dissolve 275 mg of FeCl3·6H2O and 180 mg of 2-amino terephthalic acid in 14 mL of N,N-dimethylformamide and 0.45 mL of acetic acid solution, ultrasonic dissolution, and then place the reaction kettle in a 120°C oven for 24 h; after the reaction is completed, centrifuge (9000 rpm, 10 min) to obtain a solid, wash with 20 mL of DMF every 8 h for a total of three times, then wash with 20 mL of anhydrous ethanol every 8 h for a total of three times; finally, the solid material is activated in a vacuum drying oven at 120°C for 12 h to obtain a metal-organic framework material MIL-101-Fe-NH2(also referred to as MIL-101) having a mesoporous cage channel structure.

[0066] Basic characterization of the four mesoporous metal-organic framework materials in Example 1 and Comparative Examples 1-3:

[0067] like Figure 2 As shown, NU-1000 has a unidirectional pore structure, and its skeleton has hexagonal mesopores with a pore size of 3.1nm and triangular micropores with a pore size of 1.2nm; PCN-222, which also has a unidirectional pore structure, has one-dimensional pores with pore sizes of 3.7nm and 1.2nm; PCN-777 has mesoporous cage-like pores with a diameter of 3.8nm; MIL-101-NH2 has spherical cages with diameters of 3.4nm and 2.9nm, and the pore windows are 1.6nm and 1.2nm, respectively.

[0068] The X-ray diffraction patterns of the samples before and after loading with AXT were measured by powder X-ray diffractometer (PXRD) (D / Max2500VB2+ / Pc, Rigaku, Japan). The detector operating voltage was 40 kV, the current was 200 mA, and the diffraction angle range was 2-40°. Figure 3 As shown in the figure, the characteristic diffraction peaks of the four MOFs are consistent with the diffraction peaks of the standard XRD card, indicating that the four mesoporous MOFs were successfully synthesized.

[0069] like Figure 4 As shown in the HRTEM image, a highly ordered structure of NU-1000 can be observed, with alternating hexagonal and triangular channels, while the MIL-101 structure is arranged in regular spheres.

[0070] The ions were analyzed by Fourier transform infrared spectroscopy (Spectrum two, PerkinElmer Instrument Co., Ltd., Japan) at 4000–400 cm -1 The structural and chemical bond changes of the four MOFs and their corresponding ligands after synthesis were analyzed under different wavenumbers. Figure 5 As shown, compared with the organic ligands, new peaks of carboxylate and metal oxygen stretching vibration appeared in the FT-IR spectra of the four MOFs, indicating the coordination of metal ions with the carboxyl groups in the ligands.

[0071] The morphology of the samples was observed by thermal field emission scanning electron microscopy (SEM, JSM-7800F) at an accelerating voltage of 5 kV. Figure 6 As shown in the figure, PCN-222 and NU-1000 have similar rod-like crystal structures, PCN-777 and MIL-101-NH2 have octahedral and spindle-shaped morphologies, and the particle size of NU-1000 is relatively small compared with the other three mesoporous MOFs.

[0072] The specific surface areas of the four MOFs were measured by a multi-station BET analyzer (Micromeritics ASAP 2460). Figure 7The BET surface areas of NU-1000, PCN-222, PCN-777 and MIL-101-NH2 and PCN-222 were 1541, 2258, 1574 and 2393 m 2 / g.

[0073] The pore size distribution of the four MOFs was determined by a multi-station BET analyzer (Micromeritics ASAP 2460), as shown in Fig. 1. Figure 8 As shown in Fig. 1, the maximum pore sizes of NU-1000, PCN-222 and PCN-777 were about 2.6, 3.5 nm and 3.0, and the pore sizes of MIL-101-NH2 were about 1.5, 1.8 and 2.3 nm.

[0074] Example 2

[0075] Method for selectively adsorbing and enriching astaxanthin:

[0076] The standard astaxanthin (Chengdu Pusibio Biotechnology Co., Ltd.) was dissolved in DMF and diluted with methanol to a concentration of 2, 4, 6, 8, 10 and 12 mg / L. The standard curve of astaxanthin was determined by a SP-2500 ultraviolet-visible spectrophotometer at a wavelength range of 300-700 nm and an Agilent 1260 high-performance liquid ultraviolet-fluorescence detector at a wavelength of 478 nm, as shown in Fig. 2. Figure 9

[0077] The astaxanthin was dissolved in acetone to prepare an astaxanthin-acetone solution with a concentration of 4 mg / L. Ten milligrams of each of the four mesoporous MOFs were weighed and added to 5 mL of the astaxanthin-acetone solution, respectively, and left to stand for adsorption. The supernatant was taken at a certain time, with acetone as a blank. The change in absorbance value of the characteristic absorption peak was detected by an ultraviolet-visible spectrophotometer, and the solution was poured back into the adsorption container.

[0078] As shown in Fig. 3, the ultraviolet absorption peak of PCN-222 for astaxanthin adsorption was close to 0 at 29 h, and the astaxanthin solution changed from orange to colorless, indicating complete adsorption. Similarly, NU-1000 adsorbed astaxanthin completely at 3 h, PCN-777 adsorbed astaxanthin completely at 0.8 h, MIL-101-NH2 adsorbed astaxanthin completely at 24 h, and PCN-777 with mesoporous cage-like pore structure had the fastest adsorption rate. Figure 10 (a) Adsorption kinetics of the four mesoporous MOFs for astaxanthin

[0079]

[0080] ​​The data of the change of solution absorbance over time when the four MOFs adsorbed astaxanthin were further processed to obtain the curves of the change of astaxanthin adsorption amount of the four MOFs over time. The calculation formula of the adsorption amount of astaxanthin by the four mesoporous MOFs is as follows:

[0081]

[0082] Where C0 and C t and are the initial and time t astaxanthin concentrations in the solution, respectively; V(L) is the volume of the solution; and m(g) is the mass of the added adsorbent.

[0083] The adsorption amount variation data over time were fitted with pseudo-first-order kinetic and pseudo-second-order kinetic models, and the kinetic fitting formulas were as follows:

[0084]

[0085]

[0086] Where q t and q e (mg g -1 ) are the amount of astaxanthin adsorbed at a certain time t and equilibrium time, k1 (min -1 ) and k2(gmg -1 min -1 ) are the rate constants of the pseudo-first-order and pseudo-second-order kinetic models, respectively.

[0087] like Figure 11 As shown in Figure 2, PCN-777 and NU-1000 have higher adsorption rates for astaxanthin due to their larger pore sizes. The fitting data show that the pseudo-second-order kinetic model has a higher R than the pseudo-first-order kinetic model. 2 The theoretical adsorption amount is closer to the actual value, indicating that the adsorption of astaxanthin by the four mesoporous MOFs is more consistent with the quasi-second-order kinetic model.

[0088] (b) Intraparticle diffusion model of astaxanthin in four mesoporous MOFs

[0089] The internal diffusion model was fitted for the adsorption amount at different times, and the fitting formula is as follows:

[0090] q t =k i t 0.5 +C i

[0091] Where k i is the intraparticle diffusion kinetic adsorption constant (nmol g -1 min 0.5 ), C i is the intercept of the linear curve.

[0092] As Figure 12 shown, the adsorption of astaxanthin by four mesoporous MOFs is divided into two stages. In the first stage, MOFs expose more active sites. With the progress of the adsorption reaction, the channels and active sites are occupied, and the adsorption rate decreases. Among them, PCN-777 and NU-1000 have larger diffusion coefficients in the initial stage, which is consistent with the change of the adsorption amount of astaxanthin with time.

[0093] (c) Maximum saturated adsorption amount of astaxanthin by different extraction materials

[0094] Astaxanthin was weighed and added to dichloromethane to prepare astaxanthin-dichloromethane solutions with concentrations of 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 mg / L. 10 mg of extraction material (MIL-101-NH2, PCN-777, NU-1000, PCN-222, UiO-67, d-UiO-67-4, and common macroporous adsorption resins such as AB-8, XDA-8, D301, D101, LX-68G, LX-68M, and LX-69B) was weighed and added to a 5 ml solution with different concentrations. After standing for 7 days, the supernatant was aspirated, and the absorbance value of astaxanthin in the supernatant was detected by SP-2500 ultraviolet-visible spectrophotometer at a wavelength range of 300-700 nm with dichloromethane as a blank. The change in the adsorption amount of astaxanthin by the four MOFs was calculated by the astaxanthin adsorption amount calculation formula, and the curve of the adsorption amount of astaxanthin by the four MOFs with the change in astaxanthin concentration was drawn.

[0095] As Figure 13 shown, the maximum saturated adsorption amount of astaxanthin by MIL-101-NH2, PCN-777, NU-1000, and PCN-222 is 416.8, 330.9, 111.3, and 40.0 mg / g, respectively. At the same time, compared with common macroporous adsorption resins such as AB-8 (~ 2 mg / g), XDA-8 (~ 2.5 mg / g), and LX-68G (~ 2.8 mg / g), as well as UiO-67 (~ 7.22 mg / g) and d-UiO-67-4 (~ 26.21 mg / g) disclosed in CN119390993A, the four mesoporous metal organic framework materials used in the present application exhibit very excellent adsorption performance for astaxanthin.

[0096] (d) Langmuir model and Freundlich model of astaxanthin adsorption by four mesoporous MOFs

[0097] Astaxanthin was weighed and added to dichloromethane to prepare astaxanthin-dichloromethane solutions with concentrations of 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 mg / L. 10 mg of each of the four mesoporous MOFs was added to 5 ml of solutions of different concentrations. After standing for 7 days, the supernatant was aspirated and the absorbance of astaxanthin in the supernatant was detected by SP-2500 UV-visible spectrophotometer in the wavelength range of 300-700 nm with dichloromethane as a blank. The Langmuir model and Freundlich model were fitted to the astaxanthin adsorption curve with concentration, and two isothermal adsorption models were obtained. The formulas used in the two models are as follows:

[0098]

[0099] Where K L and K F represent the Langmuir constant and the Freundlich constant respectively; q e (mg g -1 ) is the equilibrium adsorption capacity; C e (mg L -1 ) represents the equilibrium concentration of astaxanthin solution; Q m (mg g -1 ) is the maximum adsorption capacity; 1 / n is a dimensionless empirical constant reflecting the intensity of the adsorption process.

[0100] like Figure 14 As shown, the Langmuir model fits the data R 2 A high coefficient indicates monolayer adsorption.

[0101] (e) Selective adsorption of astaxanthin by four mesoporous MOFs

[0102] Lutein, fucoxanthin, and β-carotene solutions were prepared at concentrations of 4, 4, and 10 mg / L, respectively. 10 mg of each of the four mesoporous MOFs was added to 5 mL of these solutions for adsorption experiments. UV absorption spectra were obtained by measuring the characteristic peaks of lutein (444 nm), fucoxanthin (448 nm), and β-carotene (460 nm) in the supernatant at specific times (0, 1, 3, 6, 12, 24, 35, and 48 h) using a UV-visible spectrophotometer.

[0103] like Figure 15 As shown in the figure, all four mesoporous MOFs can completely adsorb astaxanthin within 29 hours, but the adsorption efficiency of fucoxanthin is about 30%, the adsorption rate of lutein does not exceed 30%, and the adsorption rate of β-carotene is less than 10%, indicating the high selectivity of the four MOFs for adsorbing astaxanthin. Figure 16The electrostatic potential maps for astaxanthin, fucoxanthin, lutein, and β-carotene are used to predict the interactions between the four mesoporous MOFs and molecules. Astaxanthin, lutein, and fucoxanthin, all of which contain oxygen atoms on their terminal rings, exhibit significant negative potentials. The oxygen atoms on the carbonyl and hydroxyl groups in astaxanthin have the highest negative potential, at -57.4 kcal / mol. β-carotene, which lacks oxygen atoms in its structure, exhibits no negative potential. This indicates that the carbonyl and hydroxyl groups in astaxanthin form stronger hydrogen bonds with the MOFs, leading to MOFs' higher selectivity for astaxanthin.

[0104] (f) Adsorption mechanism of astaxanthin by NU-1000 and MIL-101-NH2

[0105] The ions were analyzed by Fourier transform infrared spectroscopy (Spectrum two, PerkinElmer Instrument Co., Ltd., Japan) at 4000–400 cm -1 The structural and chemical bond changes of NU-1000 with unidirectional channels and MIL-101-NH2 with mesoporous cage channels after adsorbing astaxanthin were analyzed at different wavenumbers.

[0106] like Figure 17 3421cm shown -1 It corresponds to the -OH vibration peak on the NU-1000Zr6 node, which red-shifts to 3416 cm after adsorption of astaxanthin. -1 The carboxylates in NU-1000 are at 1605 and 1413 cm -1 The asymmetric and symmetric stretching vibrations at 1603 and 1410 cm-1 were slightly red-shifted to 1603 and 1410 cm-1 after adsorption of astaxanthin. -1 The possible interaction between the carboxylic acid group and the astaxanthin molecule is that there is an interaction between the carboxylic acid group and the astaxanthin molecule. -1 The NH stretching vibration at 3357 cm -1 , at 1558 and 1412 cm -1 The asymmetric and symmetric stretching peaks of carboxylic acid at the position of the α-D-type ... -1 , indicating that the NH and carboxylic acid groups may have molecular interactions with AXT.

[0107] The bond energy changes of NU-1000 and MIL-101-NH2 before and after astaxanthin adsorption were analyzed by X-ray photoelectron spectroscopy (Thermo Fischer K-Alpha) at a specific energy of Al Kα = 1486.6 eV.

[0108] like Figure 18As shown in FIG. 6, the O 1s XPS spectrum of NU-1000 adsorbing astaxanthin shows that the peak of O-C=O group at 533.53 eV moves to 533.27 eV, and the peak of O-C=O group of MIL-101-NH2 at 533.20 eV moves to 532.95 eV after adsorbing astaxanthin, which is consistent with the FT-IR results.

[0109] Example 3

[0110] A method for selectively separating and purifying astaxanthin:

[0111] (1) Crude extraction of astaxanthin from the shells of Penaeus vannamei

[0112] Solvent extraction was used to extract astaxanthin. The shells of Penaeus vannamei were dried in a vacuum oven for 8 hours. After physical crushing, 40 g of the dried shells were weighed and placed in a beaker. Then, 100 mL of CH2Cl2was added, and the mixture was stirred at 200 rpm in the dark for 2 hours. After centrifugation, the supernatant was collected. The above extraction process was repeated, and another 100 mL of CH2Cl2was added for extraction until the color of the supernatant became lighter. The supernatant was filtered through a 0.22 μm organic filter membrane, and then rotary evaporation was performed to obtain the crude astaxanthin with an ester group.

[0113] The crude astaxanthin with an ester group (200 mg) was dissolved in 8 mL of ethanol (EtOH), and then 2 mL of 0.105 M NaOH-EtOH solution was added. The mixed solution was placed in a refrigerator at -4°C for 3.5 hours for saponification reaction. Acetic acid was added to adjust the pH of the solution to 7. Finally, rotary evaporation was performed to obtain the crude astaxanthin without an ester group.

[0114] The extracted crude astaxanthin was analyzed by Agilent 1260 high-performance liquid chromatography with a UV-fluorescence detector. The column type was C18 Beilite Supersil ODS2 (4.6 mm x 250 mm, 5 μm), the column temperature was 25°C, the mobile phase was acetonitrile-methanol (95:5, v:v), the flow rate was 1 mL / min, and the injection volume was 10 μL.

[0115] As shown in FIG. 7, the retention time of the extracted substance was consistent with that of the standard astaxanthin, proving that the extract was astaxanthin. Figure 19

[0116] (2) Separation and purification performance of four kinds of mesoporous MOFs for astaxanthin

[0117] 2.5 mg of crude astaxanthin was weighed and added to 5 mL of EtOH to prepare a solution with a concentration of 500 mg / L. 10 mg of mesoporous MOFs was added to the solution for adsorption experiment. The solution was placed in the dark at room temperature for 48 hours until the crude astaxanthin was completely adsorbed.

[0118] ​After complete adsorption, the supernatant was removed, and the astaxanthin-loaded MOFs were vacuum dried in a vacuum drying oven for 6 h. Then, 10 mg of astaxanthin-loaded MOFs were added to 5 mL of the eluent and allowed to stand in the dark at room temperature for 4 h for desorption.

[0119] The eluents included dichloromethane (DCM), dimethyl sulfoxide (DMSO), DMF, ethyl acetate, anhydrous ethanol (EtOH), a 75 vol% ethanol-25 vol% water mixture, a DMSO-formic acid mixture (DMSO-FA, volume ratio 4.96:0.04), a DMF-formic acid mixture (DMF-FA, volume ratio 4.95:0.05), and a 60 vol% ethyl acetate-40 vol% ethanol mixture. The volume ratio of DMSO to FA in the DMSO-FA was adjusted to (4.998:0.002, 4.99:0.01, 4.98:0.02, 4.97:0.03, 4.96:0.04, 4.95:0.05, 4.94:0.06, and 4.93:0.07) to optimize the desorption of AXT from mesoporous MOFs. The astaxanthin content desorbed from mesoporous MOFs was analyzed using an Agilent 1260 high performance liquid chromatography UV-fluorescence detector, and the desorption rate was calculated by the following formula:

[0120]

[0121] Where S0 and S t are the peak areas of the initial astaxanthin solution and the peak areas of the astaxanthin solution after desorption, respectively.

[0122] The results are as follows Figure 20 and as shown in Table 1.

[0123] Table 1

[0124] Volume ratio DMSO:FA in eluent DMSO-FA Volume percentage of FA Desorption efficiency 4.998:0.002 0.04％ 49.44％ 4.99:0.01 0.2％ 62.48％ 4.98:0.02 0.4％ 70.19％ 4.97:0.03 0.6％ 66.93％ 4.96:0.04 0.8％ 83.20％ 4.95:0.05 1.0％ 77.07％ 4.94:0.06 1.2％ 74.47％ 4.93:0.07 1.4％ 71.19％

[0125] It can be seen that when the solution is DMSO-FA (DMSO:FA = 4.96:0.04), NU-1000 has the best astaxanthin desorption effect, with a maximum desorption efficiency of 83.2%, showing excellent separation and purification effects. However, the maximum desorption rate of mesoporous cage-like channels PCN-777 is only 25.4%, the maximum desorption rate of PCN-222 is only 34%, and the maximum desorption rate of MIL-101 is only 8.9%, indicating poor astaxanthin separation effects.

[0126] The astaxanthin solution desorbed from NU-1000 was characterized by high resolution mass spectrometry (HRMS).

[0127] like Figure 21 As shown, an ion with a mass-to-charge ratio of 597.39 was detected in the solution, and the detection result was consistent with that of standard astaxanthin.

[0128] Example 4

[0129] Dynamic adsorption-desorption curve of astaxanthin by NU-1000:

[0130] 150 mg of NU-1000 was added to an EZ-type gravity column (d = 9.56 mm, l = 55 mm), and a crude astaxanthin solution with a concentration of 500 mg / L (crude astaxanthin was prepared according to the method of Example 3) was driven through the gravity column by a peristaltic pump (LHZW005XT) at flow rates of 0.2, 0.6, and 1.0 mL / min, respectively. During the adsorption process, the outflowing liquid was collected every 2 mL; after the astaxanthin was completely adsorbed, the astaxanthin remaining in the pipeline and on the surface of the MOFs was washed with 20 mL of anhydrous ethanol, and then the astaxanthin was eluted with 148.8 mL of DMSO and 1.2 mL of formic acid solution at flow rates of 0.2, 0.6, and 1.0 mL / min, respectively, and every 2 ml was collected until the absorbance of the outflowing liquid was close to that of the initial astaxanthin solution. The absorbance of the collected liquid was detected at 478 nm by an enzyme marker, and the dynamic adsorption-desorption curve was drawn with time as the horizontal axis and the ratio of the absorbance A of the collected liquid to the absorbance A0 of the initial solution as the vertical axis.

[0131] like Figure 22 As shown in the figure, the breakthrough point and saturation point are respectively when the outflow solution concentration reaches 5% of the initial astaxanthin solution concentration (A t / A0=0.05) and 95%(A t / A0=0.95). As the flow rate increases, the slope of the breakthrough curve becomes steeper, and the time to reach the breakthrough point and saturation point is shortened. When the flow rates are 0.2, 0.6 and 1.0 mL / min, the breakthrough times are 61, 44 and 19 min, respectively, and the saturation times are 388, 162 and 102 min, respectively. It can be seen that 1 mL / min is the appropriate flow rate for dynamic desorption.

[0132] NU-1000's performance in separation and purification of crude astaxanthin:

[0133] Weigh 50 mg of crude astaxanthin and dissolve it in 100 mL of EtOH to prepare a crude astaxanthin solution with a concentration of 500 mg / L. After detecting the peak area S1 corresponding to astaxanthin by Agilent 1260 high performance liquid chromatography UV-fluorescence detector, the above solution was used for dynamic adsorption-desorption experiment, and the liquid of the desorption experiment was collected for dialysis, and then freeze-dried by a freeze dryer to obtain purified astaxanthin. Weigh 10 mg of purified astaxanthin and dissolve it in 20 mL of EtOH to prepare an astaxanthin ethanol solution with a concentration of 500 mg / L. The peak area S2 of purified astaxanthin was obtained by Agilent 1260 high performance liquid chromatography UV-fluorescence detector. Substitute the obtained peak area into Figure 9(b) the corresponding theoretical concentrations C1, C2 in the calibration curve, and the same method is used to obtain the theoretical concentration C3 of the standard solution with a concentration of 500 mg / L, and the purity of astaxanthin before and after purification is obtained by the ratio of C1, C2 and C3, respectively.

[0134] As shown in Figure 23 the HPLC peak area of the crude astaxanthin extracted from the South American white shrimp shell before purification is 23.016, and the HPLC peak area of the astaxanthin solution with the same concentration after NU-1000 purification is 144.198, and the purity is improved by about 2.81 times after NU-1000 purification by the above method.

[0135] The above provided examples are not intended to limit the scope covered by the present application, and the described steps are not intended to limit the execution order. Those skilled in the art make obvious improvements to the present application in combination with existing common knowledge, which also falls within the protection scope defined by the claims of the present application.

Claims

1. A method for selectively separating and purifying astaxanthin, characterized in that: The method uses a mesoporous metal-organic framework NU-1000 with a one-dimensional pore structure as an extractant to adsorb and enrich astaxanthin, and then desorb it under the action of an eluent.

2. The method according to claim 1, characterized in that The eluent is a mixture of DMSO and formic acid, or a mixture of DMF and formic acid.

3. The method according to claim 2, characterized in that The volume percentage of formic acid in the mixture of DMSO and formic acid or the mixture of DMF and formic acid is 0.04%-1.4%.

4. The method according to claim 2, characterized in that The volume percentage of formic acid in the mixture of DMSO and formic acid or the mixture of DMF and formic acid is 0.8%-1.0%.

5. The method according to claim 1, wherein The preparation method of the mesoporous metal-organic framework NU-1000 having a one-dimensional pore structure includes the following steps: dissolving ZrOCl2·8H2O and benzoic acid in N,N-dimethylformamide and heating, then adding a ligand 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene, and performing a solvent thermal method to obtain a yellow solid powder. After washing, hydrochloric acid is added as a regulator, and the solvent thermal method is continued to obtain a crude product NU-1000. The crude product is washed, dried, and vacuum-heated for activation to finally obtain the mesoporous metal-organic framework material NU-1000.

6. The method according to claim 5, characterized in that The molar ratio of ZrOCl2·8H2O, benzoic acid and ligand 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene is 5:318:

1.

7. The method according to claim 5, characterized in that The first heating condition was heating at 80°C for 1 h, the second heating condition was heating at 100°C for 15.5 h, and the third heating condition was heating at 100°C for 12 h.

8. The method according to claim 5, characterized in that Drying and vacuum heating activation are carried out by heating at 120°C for 12 hours in a vacuum drying oven with a vacuum degree of -0.1 MPa.

9. The method according to any one of claims 1 to 8, characterized in that The steps include: (1) First, crude astaxanthin is extracted from the shells of white shrimp to obtain crude astaxanthin; (2) The mesoporous metal-organic framework NU-1000 was then used as an extractant to adsorb and enrich astaxanthin, which was then desorbed under the action of an eluent.

10. The method according to claim 9, characterized in that Step (1) specifically includes the following process: Astaxanthin was extracted using a solvent extraction method. The shells of whiteleg shrimp were dried and physically crushed, then mixed with CH2Cl2 in the dark, and centrifuged to obtain a supernatant. The above extraction process was repeated, and CH2Cl2 was added for extraction until the supernatant became lighter in color. The supernatant was collected, filtered, and rotary evaporated to obtain crude astaxanthin with an ester group. The crude astaxanthin with an ester group is dissolved in ethanol, and then a NaOH-EtOH solution is added. The mixed solution is placed at 0 to -4°C for saponification reaction. After the reaction is completed, an acid is added to adjust the pH of the solution to 7. Finally, the crude astaxanthin with the ester group removed is obtained by rotary evaporation.

Images