Bird's nest acid molecular imprinting extraction method

Abstract

The invention discloses a sialic acid molecular imprinting extraction method, and belongs to the technical field of separation and purification. The invention discloses a sialic acid molecular imprinting extraction method which comprises the following steps: dissolving a sialic acid standard substance and a functional monomer in a binary pore-foaming agent-ionic liquid, adding a cross-linking agent and an initiator, polymerizing under ultraviolet light to prepare a high-molecular polymer, and washing off the sialic acid standard substance to prepare a molecular imprinting polymer; specifically adsorbing sialic acid in the fermentation liquor by using the molecularly imprinted polymer; performing graded elution on sialic acid by using a methanol-water mixed solution and a methanol-acetic acid mixed solution; and performing rotary evaporation concentration, anti-solvent crystallization, separation and drying on the bird's nest acid eluent. Tests show that the sialic acid purity is greater than or equal to 99%, and can meet the requirements of the sialic acid in the fields of food, health care, medicine, cosmetics and the like. According to the method, sialic acid can be efficiently separated and extracted from sialic acid fermentation liquor, the product yield is increased, the extraction procedures are reduced, and the production cost and the discharge amount of three wastes are reduced.

Description

Technical Field

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

[0002] The initial preparation of sialic acid was to extract it from whey, bird's nest, and egg yolk with relatively high sialic acid content. However, due to the limitations of raw materials and yield, these methods are difficult to meet the requirements of large-scale and high-efficiency preparation. Enzymatic synthesis is also one of the main methods for preparing sialic acid. Due to the high cost of substrates, the cost of synthesizing sialic acid by this method is very high. The bacterial fermentation method is a method suitable for large-scale production and easy to industrialize; it has the advantages of environmental friendliness, simple operation, and low cost compared with other methods.

[0003] At present, there are various methods for extracting sialic acid from fermentation broth, but all of them have various defects. For example, the traditional membrane filtration method can achieve a certain degree of separation, but there are problems such as easy membrane fouling, high cost, and large energy consumption, and it is difficult to achieve extremely high purity, which is difficult to meet the strict requirements of the high-end market for the purity of sialic acid. There are also some chemical precipitation methods. Although the operation is relatively simple, impurities are easily introduced during the precipitation process, resulting in cumbersome subsequent purification steps and being not very friendly to the environment. The application fields of sialic acid are 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 sialic acid.

[0004] Therefore, providing a method for extracting sialic acid by molecular imprinting 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 method for extracting sialic acid by molecular imprinting.

[0006] In order to achieve the above object, the present invention adopts the following technical scheme: A method for extracting sialic acid by molecular imprinting includes the following steps: (1) Preparation of molecularly imprinted polymer: Dissolve the sialic acid standard and the functional monomer in a binary porogen - ionic liquid, and stir in the dark for pre-assembly for 1 - 2 h; add the cross-linking agent and the initiator, deoxygenate with N2 and then seal, and carry out ultraviolet polymerization at 30 - 40 °C for 0.5 - 1.5 h to obtain the polymer; grind the polymer to a particle size of 100 - 200 μm, add 5 - 10 times the mass of the polymer of pure water and carry out ultrasonic treatment for 20 min - 60 min; load the chromatographic column, and use an 8:2, v / v methanol - acetic acid mixture until no sialic acid is detected in the eluate, and then dry to obtain a molecularly imprinted polymer chromatographic column capable of specifically adsorbing sialic acid.

[0007] The mass ratio of the sialic acid standard product, the functional monomer, the binary porogen - ionic liquid, and the crosslinking agent is 1:5 - 10:50 - 100:10 - 20; the addition amount of the initiator is 3 - 5 wt% of the total mass of the functional monomer and the crosslinking agent.

[0008] (2) Adsorption of sialic acid: Centrifuge the sialic acid fermentation broth to remove the cells to 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 until the sialic acid content in the effluent is the same as that in the feed, then stop feeding, and wash it with 4 - 5 times the column volume of water.

[0009] (3) Desorption of sialic acid: Use a methanol - water mixed solution with a volume ratio of 6:4 and a methanol - acetic acid mixed solution with a volume ratio of 8:2 to perform fractional elution on the sialic acid adsorbed in the molecularly imprinted polymer chromatography column until there is no sialic acid in the chromatography column to obtain a sialic acid eluate.

[0010] (4) Concentration and crystallization: Rotavapor - concentrate the sialic acid eluate to obtain a sialic acid concentrate; perform antisolvent crystallization on the sialic acid concentrate, and separate and dry it.

[0011] Further, the functional monomer in step (1) is acrylamide, 4 - vinylpyridine, 2 - vinylpyridine, or dimethylaminoethyl methacrylate.

[0012] The binary porogen is acetonitrile - methanol, acetonitrile - chloroform, acetonitrile - toluene, acetonitrile - ethanol; the ionic liquid is 1 - butyl - 3 - methylimidazolium octyl sulfate; the mass ratio of the porogen - ionic liquid addition is acetonitrile:methanol / chloroform / toluene / ethanol:1 - butyl - 3 - methylimidazolium octyl sulfate = 7:2:1.

[0013] The crosslinking agent is ethylene glycol dimethacrylate.

[0014] The initiator is 2 - hydroxy - 2 - methylpropiophenone, benzophenone.

[0015] The ultraviolet light wavelength is 365 nm and the power is 50 W.

[0016] The ultrasonic power is 120 W, the ultrasonic frequency is 40 KHz, and the temperature is 30 °C.

[0017] Further, the indexes of the sialic acid fermentation broth in step (2) are: the sialic acid content is 30 - 50 g / L, the conductivity is 20 - 30 ms / cm, the purity is 50 - 60%, and the pH is 6.95 - 7.10.

[0018] Further, the centrifugation speed in step (2) is 8000 - 12000 r / min and the time is 15 - 20 min.

[0019] Furthermore, the dosage of the 6:4, v / v methanol-water mixed solution described in step (3) is 0.5 - 1 times the mass of the polymer in the chromatography column.

[0020] Furthermore, the rotary evaporation and concentration in step (4) is carried out using a rotary evaporator, and the control conditions are 75 - 85°C, and the eluate of the bird's nest acid is concentrated to a bird's nest acid content of 300 - 500 g / L.

[0021] Furthermore, the anti-solvent in step (4) is isopropyl alcohol, ethanol or acetic acid; the addition amount of the anti-solvent is 3 - 5 times the volume of the bird's nest acid concentrate.

[0022] Furthermore, the crystallization method in step (4) is to crystallize at 4 - 10°C for 8 - 12 h.

[0023] Furthermore, the separation in step (4) adopts the centrifugation method, the centrifugation speed is 5000 - 8000 r / min, and the time is 10 - 15 min; the drying method is to dry at 80 - 100°C for 4 - 6 h.

[0024] It can be seen from the above technical solutions 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: The present invention prepares a molecularly imprinted polymer that specifically adsorbs sialic acid, uses ultraviolet light to initiate molecular imprinting polymerization, and combines ultrasonic-assisted - solvent synergistic methods to remove template molecules, which can save the 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 of the sialic acid template and the pre-assembly efficiency. In the elution step, methanol-water and methanol-acetic acid are used to fractionally elute the polymer that adsorbs the sialic acid fermentation broth. Methanol-water mainly elutes non-specifically adsorbed or weakly bound impurities, avoiding the co-elution of sialic acid and impurities during the subsequent methanol-acetic acid elution process, effectively excluding the interference of other structurally similar substances, and improving the purity of sialic acid. After testing, the purity of sialic acid is ≥99%.

[0025] The molecularly imprinted polymer of the present invention can be reused, and acetonitrile, toluene, methanol, and ethanol can be distilled and recovered, reducing the production cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained according to the provided drawings without creative efforts.

[0027] Figure 1This is the 1μm scanning electron microscope image of the sialic acid molecularly imprinted polymer of the present invention.

[0028] Figure 2 This is the 200nm scanning electron microscope image of the sialic acid molecularly imprinted polymer of the present invention.

[0029] Figure 3 This is the high performance liquid chromatography (HPLC) chart of the sialic acid fermentation broth of the present invention.

[0030] Figure 4 This is the high performance liquid chromatography (HPLC) chart of the sialic acid purified by the molecularly imprinted polymer of the present invention. Detailed implementation manners

[0031] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0032] Preparation method of sialic acid fermentation broth: The activated recombinant Bacillus subtilis engineering strain BSnanT-pHT-BbL-veg producing sialic acid (see patent CN201910358649.3) seed liquid was inoculated into the fermentation medium at an inoculation amount of 1%, and the fermentation conditions were controlled as follows: dissolved oxygen 10 - 25%, glucose controlled at 5 - 8 g / L, pH controlled at 7.0 (adjusted by 15% ammonia water), and the fermentation broth was discharged from the tank after the sialic acid no longer increased (the sialic acid content was between 30 - 50 g / L), obtaining the sialic acid fermentation broth.

[0033] 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.

[0034] The ultraviolet light wavelength is 365 nm, the power is 50 W; the ultrasonic power is 120 W, the ultrasonic frequency is 40 KHz, and the temperature is 30 °C.

[0035] Example 1 A sialic acid molecular imprinting extraction method includes the following steps: (1)Preparation of molecularly imprinted polymer: Dissolve 5 g of sialic acid standard (Shanghai Aladdin Biochemical Technology Co., Ltd., purity 98%) and 50 g of acrylamide in 250 g of acetonitrile - methanol - 1 - butyl - 3 - methylimidazolium octyl sulfate, and stir in the dark for pre - assembly for 2 h; add 75 g of ethylene glycol dimethacrylate and 5 g of 2 - hydroxy - 2 - methylpropiophenone, seal after deoxygenation with N2, and carry out ultraviolet polymerization at 35 °C for 1 h. Grind the polymer to a particle size of 200 μm, add 5 times the mass of the polymer of pure water and perform ultrasonic treatment for 40 min, load it into a chromatography column, and use a methanol - acetic acid mixed solution (8:2, v / v) to elute the sialic acid standard by counter - current until no sialic acid is detected in the eluate, and then obtain a molecularly imprinted polymer chromatography column that can specifically adsorb sialic acid after drying.

[0036] (2)Adsorption of sialic acid: Centrifuge the sialic acid fermentation broth with a sialic acid content of 40 g / L, conductivity of 25 ms / cm, purity of 60%, and pH of 6.95 at 10000 r / min for 18 min to remove the bacteria and obtain a clear 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 1.5 BV / h until the sialic acid content in the effluent is the same as that in the feed, and then stop feeding and wash it with 4 times the column volume of water.

[0037] (3)Desorption of sialic acid: First, elute with a mixed solution of 0.5 times the mass of the polymer of methanol - water (6:4, v / v), and then elute sialic acid with a mixed solution of methanol - acetic acid (8:2, v / v) until there is no sialic acid in the chromatography column to obtain a sialic acid eluate.

[0038] (4)Concentration and crystallization: Concentrate the sialic acid content to 400 g / L at 80 °C, add 4 times the volume of acetic acid of the sialic acid concentrate, place it at 4 °C for crystallization for 12 h, centrifuge at 5000 r / min for 15 min, and then dry at 90 °C for 5 h to obtain the sialic acid finished product.

[0039] Among them, Figure 1 is the 1 - μm scanning electron microscope image of the sialic acid molecularly imprinted polymer; Figure 2 is the 200 - nm scanning electron microscope image of the sialic acid molecularly imprinted polymer. It can be seen from Figure 1 and Figure 2 that there are abundant cavities on the surface of the sialic acid molecularly imprinted polymer, and these cavities are beneficial to the adsorption of sialic acid by the molecularly imprinted polymer. Figure 3 is the high - performance liquid chromatography diagram of the sialic acid fermentation broth; it can be seen from Figure 3 that in addition to sialic acid, the fermentation broth also includes other impurities such as salts, sugars, and some organic acids. Figure 4This is the high-performance liquid chromatography diagram of sialic acid after purification by molecularly imprinted polymer, and the purity of sialic acid reaches over 99%.

[0040] Example 2 A method for extracting sialic acid by molecular imprinting, comprising the following steps: (1) Preparation of molecularly imprinted polymer: Dissolve 1 g of sialic acid standard and 7.5 g of 4-vinylpyridine in 100 g of acetonitrile-chloroform-1-butyl-3-methylimidazolium octyl sulfate, and stir in the dark for pre-assembly for 1.5 h; add 20 g of ethylene glycol dimethacrylate and 0.825 g of benzophenone, seal after deoxygenation with N2, and carry out ultraviolet polymerization at 30 °C for 1.5 h. Grind the polymer to a particle size of 150 μm, add 7.5 times the mass of the polymer of pure water for ultrasonic treatment for 40 min, load it into a chromatography column, and use a methanol-acetic acid mixed solution (8:2, v / v) for countercurrent elution of the sialic acid standard until no sialic acid is detected in the eluate, and then dry to obtain a molecularly imprinted polymer chromatography column capable of specifically adsorbing sialic acid.

[0041] (2) Adsorption of sialic acid: Centrifuge 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 at 12000 r / min for 15 min to remove the bacteria, and obtain a clarified sialic acid fermentation broth. Adjust the pH of the sialic acid fermentation broth to 5, and pump it into the molecularly imprinted polymer chromatography column at a flow rate of 1 BV / h until the sialic acid content in the effluent is the same as that in the feed, and then stop feeding, and use 4.5 times the column volume of water for washing.

[0042] (3) Desorption of sialic acid: First, elute with a 1-fold methanol-water mixed solution (6:4, v / v) of the polymer mass, and then elute sialic acid with a methanol-acetic acid mixed solution (8:2, v / v) until there is no sialic acid in the chromatography column to obtain a sialic acid eluate.

[0043] (4) Concentration and crystallization: Concentrate the sialic acid content to 300 g / L at 75 °C, add 3 times the volume of isopropanol of the sialic acid concentrate, place it at 10 °C for crystallization for 10 h, centrifuge at 8000 r / min for 10 min, and then dry at 80 °C for 6 h to obtain the sialic acid finished product.

[0044] Example 3 A method for extracting sialic acid by molecular imprinting, comprising the following steps: (1)Preparation of molecularly imprinted polymer: Dissolve 2 g of sialic acid standard and 10 g of 2-vinylpyridine in 150 g of acetonitrile-toluene-1-butyl-3-methylimidazolium octyl sulfate, and stir in the dark for pre-assembly for 1 h; add 20 g of ethylene glycol dimethacrylate and 1.5 g of 2-hydroxy-2-methylpropiophenone, seal after deoxygenation with N2, and carry out ultraviolet polymerization at 40 °C for 0.5 h. Grind the polymer to a particle size of 100 μm, add 10 times the mass of the polymer of pure water and perform ultrasonic treatment for 20 min, load it into a chromatography column, and use a methanol-acetic acid mixed solution (8:2, v / v) to elute the sialic acid standard countercurrently until no sialic acid is detected in the eluate. After drying, a molecularly imprinted polymer chromatography column capable of specifically adsorbing sialic acid is prepared.

[0045] (2)Adsorption of sialic acid: Centrifuge 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 at 8000 r / min for 20 min to remove the bacteria and obtain a clear sialic acid fermentation broth. Adjust the pH of the sialic acid fermentation broth to 4 and pump it into the molecularly imprinted polymer chromatography column at a flow rate of 1.5 BV / h until the sialic acid content in the effluent is the same as that in the feed, and then stop feeding. Wash with 5 times the column volume of water.

[0046] (3)Desorption of sialic acid: First, elute with a mixed solution of methanol-water (6:4, v / v) that is 0.75 times the mass of the polymer, and then elute sialic acid with a mixed solution of methanol-acetic acid (8:2, v / v) until there is no sialic acid in the chromatography column to obtain a sialic acid eluate.

[0047] (4)Concentration and crystallization: Concentrate the sialic acid content to 500 g / L at 85 °C, add ethanol with a volume 5 times that of the sialic acid concentrate, place it at 8 °C for crystallization for 8 h, centrifuge at 6000 r / min for 18 min, and then dry at 100 °C for 4 h to obtain the sialic acid finished product.

[0048] Example 4 A method for extracting sialic acid by molecular imprinting, comprising the following steps: (1)Preparation of molecularly imprinted polymer: Dissolve 0.5 g of sialic acid standard and 5 g of dimethylaminoethyl methacrylate in 50 g of acetonitrile - ethanol - 1 - butyl - 3 - methylimidazolium octyl sulfate, and stir in the dark for pre - assembly for 2 h; add 10 g of ethylene glycol dimethacrylate and 0.75 g of benzophenone, seal after deoxygenation with N2, and carry out ultraviolet polymerization at 35 °C for 0.5 h. Grind the polymer to a particle size of 200 μm, add 5 times the mass of the polymer of pure water for ultrasonic treatment for 30 min, load it into a chromatography column, and use a methanol - acetic acid mixture (8:2, v / v) for counter - current elution of the sialic acid standard until no sialic acid is detected in the eluate, and then dry to obtain a molecularly imprinted polymer chromatography column that can specifically adsorb sialic acid.

[0049] (2)Adsorption of sialic acid: Centrifuge the sialic acid fermentation broth with a sialic acid content of 50 g / L, conductivity of 25 ms / cm, purity of 60%, and pH of 7.0 at 12000 r / min for 15 min to remove the bacteria, obtaining a clear sialic acid fermentation broth. Adjust the pH of the sialic acid fermentation broth to 5, and pump it into the molecularly imprinted polymer chromatography column at a flow rate of 1.5 BV / h until the sialic acid content in the effluent is the same as that in the feed, and then stop feeding, and use 4.5 times the column volume of water for washing.

[0050] (3)Desorption of sialic acid: First, elute with a mixed solution of 0.5 times the mass of the polymer of methanol - water (6:4, v / v), and then elute sialic acid with a mixed solution of methanol - acetic acid (8:2, v / v) until there is no sialic acid in the chromatography column to obtain a sialic acid eluate.

[0051] (4)Concentration and crystallization: Concentrate the sialic acid content to 350 g / L at 80 °C, add 4 times the volume of acetic acid of the sialic acid concentrate, place it at 4 °C for crystallization for 10 h, centrifuge at 8000 r / min for 10 min, and then dry at 90 °C for 5 h to obtain the finished product of sialic acid.

[0052] Comparative example 1 (without adding sialic acid standard) A method for extracting sialic acid, comprising the following steps: (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 pre - assembly for 2 h; add 75 g of ethylene glycol dimethacrylate and 5 g of 2 - hydroxy - 2 - methylpropiophenone, seal after deoxygenation with N2, and carry out ultraviolet polymerization at 35 °C for 1 h. Grind the polymer to a particle size of 200 μm, dry it, and load it into a chromatography column to obtain a non - molecularly imprinted polymer chromatography column.

[0053] (2)Adsorption of sialic acid: The sialic acid fermentation broth with a sialic acid content of 40 g / L, conductivity of 25 ms / cm, purity of 60%, and pH of 6.95 was centrifuged at 10,000 r / min for 18 min to remove the bacteria, obtaining a clear 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 then the column was washed with 4 times the column volume of water.

[0054] (3)Desorption of sialic acid: First, it was eluted with a mixed solution of methanol - water (6:4, v / v) that was 0.5 times the mass of the polymer, and then the sialic acid was eluted with a mixed solution of methanol - acetic acid (8:2, v / v) until there was no sialic acid in the chromatography column, obtaining a sialic acid eluate.

[0055] (4)Concentration and crystallization: The sialic acid content was concentrated to 400 g / L at 80 °C, acetic acid with a volume 4 times that of the sialic acid concentrate was added, and it was left to crystallize at 4 °C for 12 h. After centrifugation at 5000 r / min for 15 min, it was dried at 90 °C for 5 h to obtain the finished product of sialic acid.

[0056] Comparative Example 2 A method for extracting sialic acid, comprising the following steps: (1)Separation of the bacterial solution: The sialic acid fermentation broth with a sialic acid content of 30 g / L, conductivity of 20 ms / cm, purity of 50%, and pH of 7.10 was filtered through a 50 nm ceramic membrane to remove the bacteria, obtaining permeate A.

[0057] (2)Removal of macromolecular proteins: Permeate A was filtered through a 5000 Da organic ultrafiltration membrane to remove macromolecular proteins, obtaining permeate B.

[0058] (3)Desalting: Permeate B was passed through a 200 Da organic nanofiltration membrane to remove small molecular salts, obtaining retentate C.

[0059] (4)Concentration and crystallization: Retentate C was concentrated by a rotary evaporator. The sialic acid content was concentrated to 300 g / L at 75 °C, isopropanol with a volume 3 times that of the sialic acid concentrate was added, and it was left to crystallize at 10 °C for 10 h. After centrifugation at 8000 r / min for 10 min, it was dried at 80 °C for 6 h to obtain the finished product of sialic acid.

[0060] Comparative Example 3 A method for extracting sialic acid, comprising the following steps: (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, obtaining a clarified sialic acid fermentation broth. The pH of the sialic acid fermentation broth was adjusted to 4, and it was 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 then it was washed with 5 times the column volume of water.

[0061] (2)Desorption of sialic acid: The sialic acid was eluted with a 0.6 mol / L formic acid solution.

[0062] (3)Concentration and crystallization: The sialic acid content was concentrated to 500 g / L at 85 °C, and 5 times the volume of ethanol of the sialic acid concentrated solution was added. It was placed at 8 °C for crystallization for 8 h, centrifuged at 6000 r / min for 15 min, and then dried at 100 °C for 4 h to obtain the sialic acid finished product.

[0063] The sialic acid adsorption capacity, the sialic acid content and purity of the sialic acid finished product in Examples 1 - 4 and Comparative Examples 1 - 3 were detected, and the sialic acid recovery rate was calculated. The detection results are shown in Table 1. The sialic acid content and purity were detected by a high performance liquid chromatograph; sialic acid adsorption capacity (mg / g) = (total sialic acid mass - sialic acid mass in the effluent) / mass of the adsorption carrier (molecularly imprinted polymer / resin); sialic acid recovery rate (%) = (mass of the sialic acid finished product / (total sialic acid mass - sialic acid mass in the effluent)) × 100%.

[0064] Table 1 Detection results of the indexes for preparing sialic acid in Examples 1 - 4 and Comparative Examples 1 - 3

[0065] As can be seen from Table 1, by preparing the sialic acid molecularly imprinted polymer, its selectivity and adsorption effect for sialic acid are better, and the purity of the obtained sialic acid finished product is higher; during the desorption process, the loss of sialic acid is almost negligible, and the recovery rate of the sialic acid finished product is higher.

[0066] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather should be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for extracting sialic acid by molecular imprinting, characterized in that, It includes the following steps: (1) Preparation of molecularly imprinted polymer: Dissolve sialic acid standard and functional monomer in a binary porogen - ionic liquid, and stir in the dark for pre - assembly for 1 - 2 h; add cross - linker and initiator, deoxygenate with N2 and seal, and carry out ultraviolet polymerization at 30 - 40 °C for 0.5 - 1. / 5 h to obtain the polymer; grind the polymer to a particle size of 100 - 200 μm, add 5 - 10 times the mass of the polymer of pure water and carry out ultrasonic treatment for 20 min - 60 min; load the chromatography column, and use an 8:2, v / v methanol - acetic acid mixed solution to elute the sialic acid standard counter - currently until no sialic acid is detected in the eluate, and then dry to obtain a molecularly imprinted polymer chromatography column; The mass ratio of the sialic acid standard, functional monomer, binary porogen - ionic liquid, and cross - linker is 1:5 - 10:50 - 100:10 - 20; the addition amount of the initiator is 3 - 5 wt% of the total mass of the functional monomer and cross - linker; (2) Adsorption of sialic acid: Centrifuge the sialic acid fermentation broth to remove the bacteria to obtain a clear 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 until the sialic acid content in the effluent is the same as that in the feed, and then stop feeding, and use 4 - 5 times the column volume of water for washing; (3) Desorption of sialic acid: Use a 6:4, v / v methanol - water mixed solution and an 8:2, v / v methanol - acetic acid mixed solution to carry out fractional elution of the sialic acid adsorbed in the molecularly imprinted polymer chromatography column until no sialic acid remains in the chromatography column to obtain a sialic acid eluate; (4) Concentration and crystallization: Carry out rotary evaporation concentration on the sialic acid eluate to obtain a sialic acid concentrated solution; carry out anti - solvent crystallization on the sialic acid concentrated solution, and separate and dry.

2. The method for extracting sialic acid by molecular imprinting according to claim 1, wherein The functional monomer in step (1) is acrylamide, 4 - vinylpyridine, 2 - vinylpyridine, or dimethylaminoethyl methacrylate; The binary porogen is acetonitrile - methanol, acetonitrile - chloroform, acetonitrile - toluene, acetonitrile - ethanol; the ionic liquid is 1 - butyl - 3 - methylimidazolium octyl sulfate; the mass ratio of the porogen - ionic liquid addition is acetonitrile:methanol / chloroform / toluene / ethanol:1 - butyl - 3 - methylimidazolium octyl sulfate = 7:2:1; The cross - linker is ethylene glycol dimethacrylate; The initiator is 2 - hydroxy - 2 - methylpropiophenone, benzophenone; The ultraviolet light wavelength is 365 nm and the power is 50 W; The ultrasonic power is 120 W, the ultrasonic frequency is 40 KHz, 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 indicators of the sialic acid fermentation broth in step (2): the sialic acid content is 30 - 50 g / L, the conductivity is 20 - 30 ms / cm, the purity is 50 - 60%, and the pH is 6.95 - 7.

10.

4. The method for extracting sialic acid by molecular imprinting according to claim 1, wherein, The centrifugation speed in step (2) is 8000 - 12000 r / min and the time is 15 - 20 min.

5. The method for extracting sialic acid by molecular imprinting according to claim 1, wherein The usage 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.

6. The method for extracting sialic acid molecularly imprinted according to claim 1, wherein The rotary evaporation and concentration in step (4) is carried out using a rotary evaporator under the control conditions of 75 - 85 °C to concentrate the eluate of bird's nest acid to a content of 300 - 500 g / L of bird's nest acid.

7. The method for extracting sialic acid by molecular imprinting according to claim 1, characterized in that The anti-solvent in step (4) is isopropanol, ethanol or acetic acid; the addition amount of the anti-solvent is 3 - 5 times the volume of the bird's nest acid concentrate.

8. The method for extracting sialic acid molecularly imprinted according to claim 1, characterized in that, The crystallization method in step (4) is to crystallize at 4 - 10 °C for 8 - 12 h.

9. The method for extracting sialic acid molecularly imprinted according to claim 1, characterized in that, The separation in step (4) is carried out by centrifugation at a centrifugal speed of 5000 - 8000 r / min for 10 - 15 min; the drying method is to dry at 80 - 100 °C for 4 - 6 h.

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