A molecular imprinting extraction method for swallow's nest acid

Abstract

The present invention discloses a molecular imprinting extraction method for bird's nest acid, which belongs to the field of separation and purification technology. The present invention discloses a molecular imprinting extraction method for bird's nest acid, which comprises dissolving a bird's nest acid standard product and a functional monomer in a binary porogen-ionic liquid, adding a cross-linking agent and an initiator, polymerizing under ultraviolet light to form a high molecular polymer, washing away the bird's nest acid standard product, and preparing a molecular imprinting polymer; utilizing the molecular imprinting polymer to specifically adsorb bird's nest acid in the fermentation broth; subjecting bird's nest acid to graded elution using a methanol-water mixed solution and a methanol-acetic acid mixed solution; subjecting the bird's nest acid eluate to rotary evaporation concentration, anti-solvent crystallization, and separation and drying. The bird's nest acid purity is tested to be ≥99%, which can meet the requirements in the fields of food, health care, medicine, and cosmetics. The method of the present invention can efficiently separate and extract bird's nest acid from bird's nest acid fermentation broth, improve product yield, reduce extraction steps, and reduce production costs and three waste emissions.

Description

Technical Field

[0001] The present invention relates to the technical field of separation and purification, and more particularly to a method for extracting bird's nest acid by molecular imprinting. Background Art

[0002] Initially, sialic acid was extracted from whey, bird's nest, and egg yolk, which are relatively high in sialic acid. However, due to limitations in raw materials and yield, these methods struggled to meet the requirements for large-scale, efficient production. Enzymatic synthesis, another major method for producing sialic acid, is expensive due to the high cost of the substrates. Bacterial fermentation is a method suitable for large-scale production and readily scalable to industrialization. Compared to other methods, it offers advantages such as environmental friendliness, ease of operation, and low cost.

[0003] At present, there are many methods for extracting bird's nest acid from fermentation broth, but all of them have various defects. Like the traditional membrane filtration method, although it can achieve a certain degree of separation, there are problems such as easy membrane pollution, high cost, high energy consumption, and it is difficult to achieve extremely high purity, which is difficult to meet the stringent requirements of the high-end market for bird's nest acid purity. There are also some chemical precipitation methods, which, although relatively simple to operate, are prone to introducing impurities during the precipitation process, resulting in cumbersome subsequent purification steps and are not environmentally friendly. The application field of bird's nest acid is gradually expanding, and its market demand is also very large. It is particularly important to develop a method that is both efficient and economical and can achieve rapid and high-purity extraction of bird's nest acid.

[0004] Therefore, providing a molecular imprinting extraction method for swalnic acid is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a molecular imprinting extraction method for swallow's nest acid.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A molecularly imprinted extraction method for bird's nest acid comprises the following steps:

[0008] (1) Preparation of molecularly imprinted polymer: dissolve the standard of sialic acid and functional monomer in binary porogen-ionic liquid, stir in the dark and pre-assemble for 1-2 hours; add cross-linker and initiator, seal after deoxygenation under N2, and perform ultraviolet photopolymerization at 30-40℃ for 0.5-1.5 hours to obtain polymer; grind the polymer to a particle size of 100-200 μm, add 5-10 times the mass of polymer pure water and ultrasonically treat for 20-60 minutes; install the chromatography column, use 8:2, v / v methanol-acetic acid mixture, until sialic acid is no longer detected in the eluent, and after drying, obtain a molecularly imprinted polymer chromatography column that can specifically adsorb sialic acid.

[0009] The mass ratio of the swallow's nest acid standard to the functional monomer, binary porogen-ionic liquid, and cross-linking agent is 1:5-10:50-100:10-20; the added amount of the initiator is 3-5wt% of the total mass of the functional monomer and the cross-linking agent.

[0010] (2) Adsorption of sialic acid: The sialic acid fermentation broth was centrifuged to remove the bacteria to obtain a clarified sialic acid fermentation broth. The pH of the sialic acid fermentation broth was adjusted to 4-5 and pumped into the molecularly imprinted polymer chromatography column at a flow rate of 1-1.5 BV / h. The feeding was stopped when the sialic acid content in the effluent was the same as that in the feed, and the broth was washed with 4-5 times the column volume of water.

[0011] (3) Desorption of bird's nest acid: Use a 6:4, v / v methanol-water mixed solution and an 8:2, v / v methanol-acetic acid mixed solution to grade and elute the bird's nest acid adsorbed in the molecularly imprinted polymer chromatography column until there is no bird's nest acid in the chromatography column, and obtain a bird's nest acid eluate.

[0012] (4) Concentration and crystallization: The bird's nest acid eluate is concentrated by rotary evaporation to obtain a bird's nest acid concentrate; the bird's nest acid concentrate is crystallized by anti-solvent, separated and dried.

[0013] Furthermore, the functional monomer in step (1) is acrylamide, 4-vinylpyridine, 2-vinylpyridine or dimethylaminoethyl methacrylate.

[0014] The binary porogen is acetonitrile-methanol, acetonitrile-chloroform, acetonitrile-toluene, or acetonitrile-ethanol; the ionic liquid is 1-butyl-3-methylimidazolyl octyl sulfate; and the added mass ratio of the porogen to the ionic liquid is acetonitrile:methanol / chloroform / toluene / ethanol:1-butyl-3-methylimidazolyl octyl sulfate=7:2:1.

[0015] The crosslinking agent is ethylene glycol dimethacrylate.

[0016] The initiator is 2-hydroxy-2-methylpropiophenone or benzophenone.

[0017] The wavelength of the ultraviolet light is 365nm and the power is 50W.

[0018] The ultrasonic power is 120W, the ultrasonic frequency is 40KHz, and the temperature is 30°C.

[0019] Furthermore, the indicators of the bird's nest acid fermentation broth in step (2) are: bird's nest acid content of 30-50 g / L, conductivity of 20-30 ms / cm, purity of 50-60%, and pH of 6.95-7.10.

[0020] Furthermore, the centrifugal speed in step (2) is 8000-12000 r / min, and the time is 15-20 min.

[0021] Furthermore, the amount of the 6:4, v / v methanol-water mixed solution in step (3) is 0.5-1 times the mass of the polymer in the chromatography column.

[0022] Furthermore, the rotary evaporation concentration in step (4) uses a rotary evaporator and the control conditions are 75-85° C. to concentrate the bird's nest acid eluate to a bird's nest acid content of 300-500 g / L.

[0023] Furthermore, the anti-solvent in step (4) is isopropanol, ethanol or acetic acid; the amount of anti-solvent added is 3-5 times the volume of the bird's nest acid concentrate.

[0024] Furthermore, the crystallization method in step (4) is crystallization at 4-10°C for 8-12 hours.

[0025] Furthermore, the separation in step (4) is carried out by centrifugation at a speed of 5000-8000 r / min for 10-15 min; and the drying method is drying at 80-100° C. for 4-6 h.

[0026] It can be seen from the above technical solution that, compared with the prior art, the present invention discloses a method for extracting sialic acid by molecular imprinting, which has the following beneficial effects:

[0027] The present invention prepares a molecularly imprinted polymer that specifically adsorbs bird's nest acid, uses ultraviolet light to trigger molecular imprinting polymerization, and combines ultrasound-assisted-solvent synergistic removal of template molecules, which can save polymerization reaction time and template removal time and improve efficiency. An ionic liquid with amphiphilic properties is added to the porogen to improve the solubility and preassembly efficiency of the bird's nest acid template. The elution step uses methanol-water and methanol-acetic acid to grade and elute the polymer adsorbed by the bird's nest acid fermentation liquid. Methanol-water mainly elutes non-specifically adsorbed or weakly bound impurities, avoiding the co-elution phenomenon of bird's nest acid and impurities in the subsequent methanol-acetic acid elution process, effectively eliminating the interference of other structurally similar substances, and improving the purity of bird's nest acid. After testing, the purity of bird's nest acid is ≥99%.

[0028] The molecularly imprinted polymer of the invention can be reused, and acetonitrile, toluene, methanol and ethanol can be distilled and recovered, thereby reducing production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0030] Figure 1 This is a 1 μm scanning electron microscope image of the swalnic acid molecularly imprinted polymer of the present invention.

[0031] Figure 2 The figure is a 200 nm scanning electron microscope image of the swallow's nest acid molecularly imprinted polymer of the present invention.

[0032] Figure 3 The figure is a high performance liquid chromatogram of the bird's nest acid fermentation broth of the present invention.

[0033] Figure 4 The figure is a high performance liquid chromatogram of the bird's nest acid after purification by molecularly imprinted polymer. DETAILED DESCRIPTION

[0034] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0035] Preparation method of bird's nest acid fermentation broth: The activated bird's nest acid-producing recombinant Bacillus subtilis engineered bacteria BSnanT-pHT-BbL-veg (see patent CN201910358649.3) seed liquid is inoculated into the fermentation medium at a 1% inoculation rate, and the fermentation conditions are controlled to have dissolved oxygen of 10-25%, glucose of 5-8 g / L, and pH of 7.0 (pH adjusted with 15% ammonia water). After the bird's nest acid no longer increases, the fermentation broth is placed in a tank (the bird's nest acid content is between 30-50 g / L) to obtain bird's nest acid fermentation broth.

[0036] Fermentation medium: yeast powder 10 g / L, peptone 5 g / L, dipotassium hydrogen phosphate trihydrate 2.5 g / L, potassium dihydrogen phosphate 5 g / L, ferrous sulfate heptahydrate 0.05 g / L, manganese sulfate pentahydrate 0.03 g / L, zinc sulfate heptahydrate 0.03 g / L, polyether defoamer 0.1 mL / L.

[0037] The ultraviolet light wavelength is 365nm, the power is 50W; the ultrasonic power is 120W, the ultrasonic frequency is 40KHz, and the temperature is 30℃.

[0038] Example 1

[0039] A molecularly imprinted extraction method for bird's nest acid comprises the following steps:

[0040] (1) Preparation of molecular imprinting polymer: 5 g of sialic acid standard (Shanghai Aladdin Biochemical Technology Co., Ltd., purity 98%) and 50 g of acrylamide were dissolved in 250 g of acetonitrile-methanol-1-butyl-3-methylimidazolium octyl sulfate and preassembled in the dark with stirring for 2 h. 75 g of ethylene glycol dimethacrylate and 5 g of 2-hydroxy-2-methylpropiophenone were added, the mixture was sealed under N2 deoxygenation, and UV polymerization was carried out at 35 °C for 1 h. The polymer was ground to a particle size of 200 μm, and pure water 5 times the weight of the polymer was added for ultrasonic treatment for 40 min. The column was loaded and the sialic acid standard was eluted in countercurrent with a methanol-acetic acid mixture (8:2, v / v) until sialic acid was no longer detected in the eluate. After drying, a molecular imprinting polymer chromatography column capable of specifically adsorbing sialic acid was obtained.

[0041] (2) Adsorption of sialic acid: The sialic acid fermentation broth with a sialic acid content of 40 g / L, a conductivity of 25 ms / cm, a purity of 60%, and a pH of 6.95 was centrifuged at 10,000 r / min for 18 min to remove the bacteria and obtain a clarified sialic acid fermentation broth. The pH of the sialic acid fermentation broth was adjusted to 4.5 and pumped into a molecularly imprinted polymer chromatography column at a flow rate of 1.5 BV / h. The feeding was stopped when the sialic acid content in the effluent was the same as that in the feed, and the broth was washed with 4 times the column volume of water.

[0042] (3) Desorption of bird's nest acid: First, use a mixed solution of methanol and water (6:4, v / v) with a mass of 0.5 times that of the polymer to elute the bird's nest acid, and then use a mixed solution of methanol and acetic acid (8:2, v / v) to elute the bird's nest acid until there is no bird's nest acid in the chromatography column, and obtain the bird's nest acid eluate.

[0043] (4) Concentration and crystallization: Concentrate the sialic acid content to 400 g / L at 80°C, add acetic acid 4 times the volume of the sialic acid concentrate, place it at 4°C for crystallization for 12 hours, centrifuge at 5000 r / min for 15 minutes, and dry it at 90°C for 5 hours to obtain the sialic acid product.

[0044] in, Figure 1 This is a 1 μm scanning electron microscopy image of swalnic acid molecularly imprinted polymer; Figure 2 The 200nm scanning electron microscope image of swallow acid molecularly imprinted polymer. Figure 1 、 Figure 2 It can be seen that there are abundant pores on the surface of the sialic acid molecularly imprinted polymer, which are conducive to the adsorption of sialic acid by the molecularly imprinted polymer. Figure 3 is the HPLC chromatogram of bird's nest acid fermentation broth; Figure 3 It can be seen that in addition to bird's nest acid, the fermentation broth also contains other impurities such as salt, sugar, and some organic acids. Figure 4This is the HPLC chromatogram of bird's nest acid after purification by molecularly imprinted polymer. The purity of bird's nest acid is over 99%.

[0045] Example 2

[0046] A molecularly imprinted extraction method for bird's nest acid comprises the following steps:

[0047] (1) Preparation of molecularly imprinted polymer: 1 g of sialic acid standard and 7.5 g of 4-vinylpyridine were dissolved in 100 g of acetonitrile-chloroform-1-butyl-3-methylimidazolium octyl sulfate and stirred in the dark for pre-assembly for 1.5 h. 20 g of ethylene glycol dimethacrylate and 0.825 g of benzophenone were added, the mixture was sealed under N2 deoxygenation, and UV polymerization was carried out at 30 °C for 1.5 h. The polymer was ground to a particle size of 150 μm, and 7.5 times the mass of the polymer was added with pure water and ultrasonically treated for 40 min. The column was loaded and the sialic acid standard was countercurrently eluted with a methanol-acetic acid mixture (8:2, v / v) until sialic acid was no longer detected in the eluate. After drying, a molecularly imprinted polymer chromatography column capable of specifically adsorbing sialic acid was obtained.

[0048] (2) Adsorption of sialic acid: The sialic acid fermentation broth with a sialic acid content of 30 g / L, a conductivity of 20 ms / cm, a purity of 50%, and a pH of 7.10 was centrifuged at 12,000 r / min for 15 min to remove the bacteria and obtain a clarified sialic acid fermentation broth. The pH of the sialic acid fermentation broth was adjusted to 5 and pumped into a molecularly imprinted polymer chromatography column at a flow rate of 1 BV / h. The feeding was stopped when the sialic acid content in the effluent was the same as that in the feed, and the broth was washed with 4.5 times the column volume of water.

[0049] (3) Desorption of bird's nest acid: First, use a methanol-water mixed solution (6:4, v / v) with a mass that is 1 times the mass of the polymer to elute the bird's nest acid, and then use a methanol-acetic acid mixed solution (8:2, v / v) to elute the bird's nest acid until there is no bird's nest acid in the chromatography column, and obtain the bird's nest acid eluate.

[0050] (4) Concentration and crystallization: Concentrate the sialic acid content to 300 g / L at 75°C, add isopropanol 3 times the volume of the sialic acid concentrate, place it at 10°C for crystallization for 10 hours, centrifuge at 8000 r / min for 10 minutes, and dry it at 80°C for 6 hours to obtain the sialic acid product.

[0051] Example 3

[0052] A molecularly imprinted extraction method for bird's nest acid comprises the following steps:

[0053] (1) Preparation of molecularly imprinted polymer: 2 g of sialic acid standard and 10 g of 2-vinylpyridine were dissolved in 150 g of acetonitrile-toluene-1-butyl-3-methylimidazolium octyl sulfate and stirred in the dark for pre-assembly for 1 h. 20 g of ethylene glycol dimethacrylate and 1.5 g of 2-hydroxy-2-methylpropiophenone were added, the mixture was sealed under N2 deoxygenation, and UV polymerization was performed at 40 °C for 0.5 h. The polymer was ground to a particle size of 100 μm, and 10 times the mass of pure water was added for ultrasonic treatment for 20 min. The column was loaded and the sialic acid standard was eluted in countercurrent with a methanol-acetic acid mixture (8:2, v / v) until sialic acid was no longer detected in the eluate. After drying, a molecularly imprinted polymer chromatography column capable of specifically adsorbing sialic acid was obtained.

[0054] (2) Adsorption of sialic acid: The sialic acid fermentation broth with a sialic acid content of 50 g / L, a conductivity of 30 ms / cm, a purity of 55%, and a pH of 7.0 was centrifuged at 8000 r / min for 20 min to remove the bacteria and obtain a clarified sialic acid fermentation broth. The pH of the sialic acid fermentation broth was adjusted to 4 and pumped into a molecularly imprinted polymer chromatography column at a flow rate of 1.5 BV / h. The feeding was stopped when the sialic acid content in the effluent was the same as that in the feed, and the broth was washed with 5 times the column volume of water.

[0055] (3) Desorption of bird's nest acid: First, use a mixed solution of methanol and water (6:4, v / v) with a mass of 0.75 times that of the polymer to elute the bird's nest acid, and then use a mixed solution of methanol and acetic acid (8:2, v / v) to elute the bird's nest acid until there is no bird's nest acid in the chromatography column, and obtain the bird's nest acid eluate.

[0056] (4) Concentration and crystallization: Concentrate the sialic acid content to 500 g / L at 85°C, add 5 times the volume of ethanol of the sialic acid concentrate, place it at 8°C for crystallization for 8 hours, centrifuge at 6000 r / min for 18 minutes, and dry it at 100°C for 4 hours to obtain the sialic acid product.

[0057] Example 4

[0058] A molecularly imprinted extraction method for bird's nest acid comprises the following steps:

[0059] (1) Preparation of molecularly imprinted polymer: 0.5 g of sialic acid standard and 5 g of dimethylaminoethyl methacrylate were dissolved in 50 g of acetonitrile-ethanol-1-butyl-3-methylimidazolium octyl sulfate and stirred in the dark for pre-assembly for 2 h. 10 g of ethylene glycol dimethacrylate and 0.75 g of benzophenone were added, the mixture was sealed under N2 deoxygenation, and UV polymerization was performed at 35 °C for 0.5 h. The polymer was ground to a particle size of 200 μm, and pure water 5 times the weight of the polymer was added for ultrasonic treatment for 30 min. The column was loaded and the sialic acid standard was eluted in countercurrent with a methanol-acetic acid mixture (8:2, v / v) until sialic acid was no longer detected in the eluate. After drying, a molecularly imprinted polymer chromatography column capable of specifically adsorbing sialic acid was obtained.

[0060] (2) Adsorption of sialic acid: The sialic acid fermentation broth with a sialic acid content of 50 g / L, a conductivity of 25 ms / cm, a purity of 60%, and a pH of 7.0 was centrifuged at 12,000 r / min for 15 min to remove the bacteria and obtain a clarified sialic acid fermentation broth. The pH of the sialic acid fermentation broth was adjusted to 5 and pumped into a molecularly imprinted polymer chromatography column at a flow rate of 1.5 BV / h. The feeding was stopped when the sialic acid content in the effluent was the same as that in the feed, and the broth was washed with 4.5 times the column volume of water.

[0061] (3) Desorption of bird's nest acid: First, use a mixed solution of methanol and water (6:4, v / v) with a mass of 0.5 times that of the polymer to elute the bird's nest acid, and then use a mixed solution of methanol and acetic acid (8:2, v / v) to elute the bird's nest acid until there is no bird's nest acid in the chromatography column, and obtain the bird's nest acid eluate.

[0062] (4) Concentration and crystallization: Concentrate the sialic acid content to 350 g / L at 80°C, add acetic acid 4 times the volume of the sialic acid concentrate, place it at 4°C for crystallization for 10 hours, centrifuge at 8000 r / min for 10 minutes, and dry it at 90°C for 5 hours to obtain the sialic acid product.

[0063] Comparative Example 1 (no bird's nest acid standard added)

[0064] A method for extracting bird's nest acid comprises the following steps:

[0065] (1) Preparation of non-molecularly imprinted polymer: Dissolve 50 g of acrylamide in 250 g of acetonitrile-methanol-1-butyl-3-methylimidazolium octyl sulfate and stir in the dark for 2 h. Add 75 g of ethylene glycol dimethacrylate and 5 g of 2-hydroxy-2-methylpropiophenone, deoxygenate under N2, seal the mixture, and perform UV polymerization at 35°C for 1 h. Grind the polymer to a particle size of 200 μm, dry it, and load it onto a chromatography column to prepare a non-molecularly imprinted polymer chromatography column.

[0066] (2) Adsorption of sialic acid: The sialic acid fermentation broth with a sialic acid content of 40 g / L, a conductivity of 25 ms / cm, a purity of 60%, and a pH of 6.95 was centrifuged at 10,000 r / min for 18 min to remove the bacteria and obtain a clarified sialic acid fermentation broth. The pH of the sialic acid fermentation broth was adjusted to 4.5 and pumped into a non-molecularly imprinted polymer chromatography column at a flow rate of 1.5 BV / h. The feeding was stopped when the sialic acid content in the effluent was the same as that in the feed, and the broth was washed with 4 times the column volume of water.

[0067] (3) Desorption of bird's nest acid: First, use a mixed solution of methanol and water (6:4, v / v) with a mass of 0.5 times that of the polymer to elute the bird's nest acid, and then use a mixed solution of methanol and acetic acid (8:2, v / v) to elute the bird's nest acid until there is no bird's nest acid in the chromatography column, and obtain the bird's nest acid eluate.

[0068] (4) Concentration and crystallization: Concentrate the sialic acid content to 400 g / L at 80°C, add acetic acid 4 times the volume of the sialic acid concentrate, place it at 4°C for crystallization for 12 hours, centrifuge at 5000 r / min for 15 minutes, and dry it at 90°C for 5 hours to obtain the sialic acid product.

[0069] Comparative Example 2

[0070] A method for extracting bird's nest acid comprises the following steps:

[0071] (1) Separation of bacterial liquid: The sialic acid fermentation liquid with a sialic acid content of 30 g / L, a conductivity of 20 ms / cm, a purity of 50%, and a pH of 7.10 was filtered using a 50 nm ceramic membrane to remove the bacteria and obtain permeate A.

[0072] (2) Removal of macromolecular proteins: Filter the permeate A through a 5000Da organic ultrafiltration membrane to remove macromolecular proteins and obtain permeate B.

[0073] (3) Desalination: The permeate B is filtered through a 200Da organic nanofiltration membrane to remove small molecule salts and obtain the retentate C.

[0074] (4) Concentration and crystallization: The retentate C was concentrated by rotary evaporation, and the sialic acid content was concentrated to 300 g / L at 75 °C. Isopropanol 3 times the volume of the sialic acid concentrate was added, and the mixture was placed at 10 °C for crystallization for 10 h. After centrifugation at 8000 r / min for 10 min, the mixture was dried at 80 °C for 6 h to obtain the sialic acid product.

[0075] Comparative Example 3

[0076] A method for extracting bird's nest acid comprises the following steps:

[0077] (1) Adsorption of sialic acid: The sialic acid fermentation broth with a sialic acid content of 50 g / L, a conductivity of 30 ms / cm, a purity of 55%, and a pH of 7.0 was centrifuged at 8000 r / min for 20 min to remove the bacteria and obtain a clarified sialic acid fermentation broth. The pH of the sialic acid fermentation broth was adjusted to 4 and pumped into a D201 ion exchange resin chromatography column at a flow rate of 1.5 BV / h. The feeding was stopped when the sialic acid content in the effluent was the same as that in the feed, and the broth was washed with 5 times the column volume of water.

[0078] (2) Desorption of sialic acid: 0.6 mol / L formic acid solution was used to elute sialic acid.

[0079] (3) Concentration and crystallization: Concentrate the sialic acid content to 500 g / L at 85°C, add 5 times the volume of ethanol of the sialic acid concentrate, place it at 8°C for crystallization for 8 hours, centrifuge at 6000 r / min for 15 minutes, and dry it at 100°C for 4 hours to obtain the sialic acid product.

[0080] The amount of bird's nest acid adsorption, the bird's nest acid content of the finished bird's nest acid product, and the bird's nest acid purity of Examples 1-4 and Comparative Examples 1-3 were tested, and the bird's nest acid yield was calculated. The test results are shown in Table 1. The bird's nest acid content and purity were detected by high performance liquid chromatography; bird's nest acid adsorption amount (mg / g) = (total bird's nest acid mass - outflow bird's nest acid mass) / adsorption carrier (molecularly imprinted polymer / resin) mass; bird's nest acid yield (%) = (bird's nest acid finished product mass / (total bird's nest acid mass - outflow bird's nest acid mass)) × 100%.

[0081] Table 1 Index test results of bird's nest acid prepared in Examples 1-4 and Comparative Examples 1-3

[0082]

[0083] As can be seen from Table 1, by preparing the bird's nest acid molecularly imprinted polymer, the selectivity and adsorption effects on bird's nest acid are better, and the obtained bird's nest acid product has a higher purity; during the desorption process, there is almost no loss of bird's nest acid, and the yield of the bird's nest acid product is higher.

[0084] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A molecularly imprinted extraction method for swallow's nest acid, characterized in that: The following steps are involved: (1) Preparation of molecularly imprinted polymer: dissolve the sialic acid standard and functional monomer in a binary porogen-ionic liquid, stir and pre-assemble in the dark for 1-2 hours; add a crosslinker and an initiator, seal the mixture under N2 deoxygenation, and perform ultraviolet photopolymerization at 30-40°C for 0.5-1.5 hours to obtain a polymer; grind the polymer to a particle size of 100-200 μm, add 5-10 times the mass of pure water of the polymer and perform ultrasonic treatment for 20-60 minutes; install a chromatography column, and use a 8:2, v / v methanol-acetic acid mixture to countercurrently elute the sialic acid standard until sialic acid is no longer detected in the eluate, and dry it to obtain a molecularly imprinted polymer chromatography column; The mass ratio of the swallow acid standard to the functional monomer, binary porogen-ionic liquid, and cross-linking agent is 1:5-10:50-100:10-20; the amount of the initiator added is 3-5wt% of the total mass of the functional monomer and the cross-linking agent; The ionic liquid is 1-butyl-3-methylimidazolium octyl sulfate; (2) Adsorption of sialic acid: centrifuge the sialic acid fermentation broth to remove the bacteria and obtain a clarified sialic acid fermentation broth. Adjust the pH of the sialic acid fermentation broth to 4-5 and pump it into the molecularly imprinted polymer chromatography column at a flow rate of 1-1.5 BV / h. Stop feeding when the sialic acid content in the effluent is the same as that in the feed, and wash with 4-5 column volumes of water. The indicators of the bird's nest acid fermentation liquid are: bird's nest acid content of 30-50g / L, conductivity of 20-30ms / cm, purity of 50-60%, and pH of 6.95-7.10; (3) Desorption of bird's nest acid: Use a 6:4, v / v methanol-water mixed solution and an 8:2, v / v methanol-acetic acid mixed solution to grade and elute the bird's nest acid adsorbed in the molecularly imprinted polymer chromatography column until there is no bird's nest acid in the chromatography column, and obtain a bird's nest acid eluate; (4) Concentration and crystallization: The bird's nest acid eluate is concentrated by rotary evaporation to obtain a bird's nest acid concentrate; the bird's nest acid concentrate is crystallized by anti-solvent, separated and dried.

2. A molecularly imprinted extraction method for swallow's nest acid according to claim 1, characterized in that, The functional monomer in step (1) is acrylamide, 4-vinylpyridine, 2-vinylpyridine or dimethylaminoethyl methacrylate; The binary porogens are acetonitrile-methanol, acetonitrile-chloroform, acetonitrile-toluene, and acetonitrile-ethanol; the mass ratio of porogen to ionic liquid is acetonitrile:methanol / chloroform / toluene / ethanol:1-butyl-3-methylimidazolium octyl sulfate = 7:2:1; The cross-linking agent is ethylene glycol dimethacrylate; The initiator is 2-hydroxy-2-methylpropiophenone or benzophenone; The ultraviolet light has a wavelength of 365nm and a power of 50W; The ultrasonic power is 120W, the ultrasonic frequency is 40KHz, and the temperature is 30°C.

3. A molecularly imprinted extraction method for swallow's nest acid according to claim 1, characterized in that: The centrifugal speed in step (2) is 8000-12000 r / min, and the time is 15-20 min.

4. A molecularly imprinted extraction method for swallow's nest acid according to claim 1, characterized in that, The amount of the 6:4, v / v methanol-water mixed solution in step (3) is 0.5-1 times the mass of the polymer in the chromatography column.

5. A molecularly imprinted extraction method for swallow's nest acid according to claim 1, characterized in that, The rotary evaporation and concentration in step (4) is carried out using a rotary evaporator at a controlled temperature of 75-85° C. to concentrate the bird's nest acid eluate to a bird's nest acid content of 300-500 g / L.

6. A molecularly imprinted extraction method for swallow's nest acid according to claim 1, characterized in that: The anti-solvent in step (4) is isopropanol, ethanol or acetic acid; the amount of anti-solvent added is 3-5 times the volume of the bird's nest acid concentrate.

7. The molecularly imprinted extraction method for swallow's nest acid according to claim 1, wherein: The crystallization method in step (4) is crystallization at 4-10°C for 8-12 hours.

8. The molecularly imprinted extraction method for swallow's nest acid according to claim 1, wherein The separation in step (4) is carried out by centrifugation at a speed of 5000-8000 r / min for 10-15 min; the drying method is drying at 80-100° C. for 4-6 h.

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