A water-soluble CBCS microsphere and its preparation and application

Abstract

The invention relates to water-soluble CBCS microspheres and preparation and application thereof. Chitosan is dissolved in a solvent to dissolve and swell, and then phthalic anhydride is added for reaction to obtain a reaction solution. The reaction solution is poured into water to cool, the pH value is adjusted to alkaline, and the filtrate is filtered and dialyzed. The filtrate is freeze-dried to obtain flocculent light yellow solid CBCS: N-(2-carboxybenzoyl)-chitosan. The CBCS solution is dripped into an oil phase and an emulsifier for emulsification, and then an aldehyde cross-linking agent is added to cross-link and solidify amino groups and aldehyde groups of the CBCS to generate shiff base bonds to obtain the CBCS microspheres. The CBCS microspheres can adsorb antibiotics in water in accordance with the Langmuir model, and the adsorption process conforms to pseudo-secondary adsorption kinetics. The water solubility and yield of chitosan can be increased, the chitosan microspheres are improved in terms of reusability, and the scope of application can be further broadened. The CBCS microspheres can efficiently, economically, and environmentally friendly treat antibiotic pollution in wastewater.

Description

Technical Field

[0001] The present invention belongs to the technical field of polysaccharide functional materials, and particularly relates to water-soluble CBCS microspheres and preparation and application thereof. Background Art

[0002] Antibiotics, including tetracyclines, macrolides, β-lactams, aminoglycosides, quinolones, and others, inhibit microbial growth and promote development. They primarily treat diseases caused by various pathogenic bacteria and are widely used in disease treatment, animal husbandry, and aquaculture. However, traditional antibiotic wastewater treatment technologies are subject to secondary pollution and low reuse rates. Long-term abuse of antibiotics not only poses potential damage to the natural environment but also poses a threat to human health. Therefore, to avoid the problems associated with the widespread use of antibiotics, it is necessary to find a material that can efficiently, economically, and environmentally friendlyly treat antibiotic contamination in wastewater.

[0003] Compared with other pollutant treatment methods, adsorption methods have the advantages of simple operation, high treatment efficiency, large specific surface area, and no secondary pollution. Chitosan is a high-performance, environmentally friendly natural green polymer alkaline polysaccharide material with a large number of hydroxyl and amino groups that can remove various pollutants in wastewater, degrade, resist bacteria, and integrate heavy metal ions. However, chitosan has disadvantages such as poor mechanical properties, low density, and easy agglomeration to form gels, which limit its application in removing antibiotic pollution by adsorption. To this end, using microspheres as chitosan carriers can improve the chemical stability of chitosan and broaden its application range. Existing methods for preparing chitosan microspheres include emulsification crosslinking, ion gelation, neutralization precipitation, spray drying, and complex coacervation. Many crosslinking agents are used, such as glutaraldehyde, glyoxal, and cyclopropane. However, its main drawback is that the hydrogen bond force between chitosan molecules makes it insoluble in alkaline and neutral media and can only be completely dissolved in relatively low acidic environments, which limits the application range of chitosan microspheres and affects the extension of the main chain within the chitosan molecule, reducing its adsorption capacity.

[0004] Secondly, the poor preparation process conditions result in incomplete structure of chitosan microspheres, which are prone to agglomeration and adhesion, resulting in poor spherical regularity, surface smoothness, particle size uniformity and dispersion, making it difficult for the chemical stability and reusability of chitosan microspheres to meet application requirements. Summary of the Invention

[0005] The present invention aims to solve at least one of the above-mentioned technical problems to a certain extent. The present invention provides water-soluble CBCS microspheres and their preparation and application, which can increase the water solubility and yield of chitosan, improve the adsorption capacity, chemical stability and reusability of chitosan microspheres for antibiotics, further broaden their application range, and can efficiently, economically and environmentally friendly treat antibiotic contamination in wastewater.

[0006] The technical solution adopted by the present invention to solve its technical problem is:

[0007] A water-soluble CBCS microsphere, wherein the water-soluble CBCS microsphere is chitosan modified by phthalic anhydride to CBCS: N-(2-carboxybenzoyl)-chitosan, and has the structural formula:

[0008] Then the amino group of CBCS is cross-linked and cured with the aldehyde group to produce shiff base bonds.

[0009] Furthermore, the molecular weight of the CBCS is greater than 10KD.

[0010] Furthermore, the particle size of the water-soluble CBCS microspheres is less than 5 μm, and the particle size distribution index PDI is less than 0.3.

[0011] A method for preparing water-soluble CBCS microspheres, comprising:

[0012] S1. Preparation of CBCS: Chitosan was dissolved in a solvent to allow it to swell, and then phthalic anhydride was added to react to obtain a reaction solution, which was poured into water and cooled. The pH was adjusted to alkaline, and the filtrate was filtered and dialyzed, followed by freeze-drying to obtain a flocculent light yellow solid CBCS.

[0013] S2. Preparation of CBCS microspheres: The CBCS obtained in step S1 is dissolved in water to obtain a CBCS solution, the CBCS solution is dropped into the oil phase and emulsifier for emulsification, and an aldehyde crosslinking agent is added for cross-linking reaction to obtain a reaction product, the oil phase in the reaction product is removed, and the reaction product is dried to obtain CBCS microspheres.

[0014] Furthermore, the solvent in step S1 is dimethyl sulfoxide, the molar volume ratio of chitosan to dimethyl sulfoxide is 0.12-0.13 mmol / ml, preferably 0.124 mmol / ml, and the dissolution and swelling time is 1.5-2.5 h, preferably 2 h.

[0015] Furthermore, in step S1, the molar ratio of chitosan to phthalic anhydride is 3-3.2:4-4.2, preferably 3.1:4.1, and the reaction is carried out at 70-80°C for 24 hours, preferably at 80°C for 24 hours.

[0016] Furthermore, after the reaction in step S1, the reaction solution is added to water and placed in an ice-water bath for cooling.

[0017] Furthermore, the pH is adjusted to 9-11 with a NaOH solution, preferably to 10, and the NaOH mass volume concentration in the NaOH solution is preferably 20%.

[0018] Furthermore, the molecular weight cut-off of the semipermeable membrane used in dialysis is 10KD.

[0019] Furthermore, the mass volume concentration of CBCS in the CBCS solution in step S2 is 0.8-1.5%, preferably 0.8-1%.

[0020] Furthermore, in step S2, the oil phase is liquid paraffin, the emulsifier is Span 80, and the volume ratio of Span 80 to liquid paraffin is 1:50-150, preferably 1:75.

[0021] Furthermore, in step S2, the volume ratio of the oil phase to the CBCS solution is 13-17:1, preferably 15:1.

[0022] Furthermore, the emulsification time in step S2 is 30-60 min, preferably 30 min.

[0023] Furthermore, in step S2, the aldehyde cross-linking agent is glutaraldehyde, the mass volume ratio of CBCS to glutaraldehyde is 1.5-3.5 g / ml, and the cross-linking reaction time is >3 h, preferably 4-4.5 h.

[0024] Furthermore, the reaction product in step S2 is centrifuged at 7000-8000 rpm for 4-6 minutes, and the upper oil phase is discarded. The centrifuged product is filtered and washed with petroleum ether, isopropanol, and acetone, respectively, and dried at 40-60° C. to obtain CBCS microspheres.

[0025] The invention discloses an application of water-soluble CBCS microspheres, wherein the water-soluble CBCS microspheres are used for adsorbing antibiotics in water.

[0026] Furthermore, the smaller the particle size and PDI value of the water-soluble CBCS microspheres are, the larger the specific surface area is and the better the adsorption performance is.

[0027] Furthermore, the antibiotic includes tetracycline, and the adsorption process conforms to pseudo-secondary adsorption kinetics.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) By modifying chitosan CS, phthalic anhydride is introduced into the amino group of chitosan CBCS, so that a new functional group carboxyl is introduced into the active amino group, which weakens the hydrogen bond force between chitosan molecules and stretches the chitosan molecules. At the same time, the thermal stability and crystallinity of CBCS decrease, making the solubility of CBCS much higher than that of chitosan. At the same time, due to the introduction of oxygen-containing functional group carboxyl into CBCS, the adsorption sites for tetracycline are increased. CBCS not only electrostatically binds to tetracycline in anionic state, but also the intermolecular force of CBCS is weaker than that of chitosan after the introduction of phthalic anhydride substituent, which significantly weakens the hydrogen bond between chitosan molecules and stretches the main chain of chitosan molecules, thereby greatly enhancing the adsorption capacity and achieving a higher yield.

[0030] Tests have shown that when the initial tetracycline concentrations are 100 mg / L and 30 mg, the saturated adsorption capacity of CBCS microspheres is 117.35 mg / g. The equilibrium state is basically reached at an adsorption time of 210 minutes, which matches the Langmuir model. The adsorption process conforms to quasi-secondary adsorption kinetics. At various concentrations and temperatures, the adsorption capacity of CBCS microspheres is significantly better than that of CS microspheres, and they have better stability and repeatability. After regeneration, they can be recycled multiple times, further broadening their application range and can efficiently, economically and environmentally friendly treat antibiotic pollution in wastewater.

[0031] (2) The degree of influence of various factors in the preparation process of CBCS microspheres is: CBCS solution concentration > oil-water ratio > cross-linking time. By further optimizing the process parameters, the CBCS microspheres can have a regular structure, a smooth surface, a good spherical shape, good dispersion between microspheres, basically no agglomeration or adhesion, and a uniform particle size distribution. The particle size of water-soluble CBCS microspheres can be achieved to be less than 5 μm, and the particle size distribution index PDI is less than 0.3, further improving the adsorption performance of antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0033] Figure 1 is the reaction formula of the reaction between chitosan and phthalic anhydride of the present invention;

[0034] Figure 2 This is a photo of flocculent light yellow solid CBCS obtained in Example 1 of the present invention;

[0035] Figure 3 This is the reaction mechanism of cross-linking between the amino group and the aldehyde group of the CBCS of the present invention;

[0036] Figure 4 is a scanning electron microscope image of the CBCS microspheres prepared in Example 1 of the present invention;

[0037] Figure 5 This is the reaction formula for preparing CACS in Comparative Example 1 of the present invention;

[0038] Figure 6 1 is the infrared spectra of CS, CACS, CBCS and CBCS microspheres obtained in Example 1;

[0039] Figure 7 1H NMR spectra of (a) CACS and (b) CBCS obtained in Example 1;

[0040] Figure 8 This is the carbon NMR spectrum of CBCS obtained in Example 1 of the present invention;

[0041] Figure 9 are the XRD patterns of (a) CS and (b) CBCS obtained in Example 1;

[0042] Figure 10 DSC graphs of (a) CS and (b) CBCS obtained in Example 1;

[0043] Figure 11 It is the main effect diagram of each parameter of the orthogonal experiment of the present invention;

[0044] Figure 12 It is the interaction diagram of various parameters of the orthogonal experiment of the present invention;

[0045] Figure 13 is the effect of initial tetracycline concentration on the adsorption of microspheres;

[0046] Figure 14 The adsorption isotherms of tetracycline on (a) CS microspheres and (b) CBCS microspheres at room temperature;

[0047] Figure 15 is the adsorption kinetic curve of CS microspheres and CBCS microspheres;

[0048] Figure 16 are the (a) pseudo-first-order and (b) pseudo-second-order adsorption kinetic fitting curves of CS microspheres and CBCS microspheres;

[0049] Figure 17 is the intraparticle diffusion fitting curve of adsorbed tetracycline in CS microspheres and CBCS microspheres;

[0050] Figure 18 Graph showing the effect of temperature on the adsorption of tetracycline by (a) CS microspheres and (b) CBCS microspheres.

[0051] Figure 19 This is the cyclic performance diagram of CS microspheres and CBCS microspheres in adsorbing tetracycline. DETAILED DESCRIPTION

[0052] The embodiments of the present invention are described in detail below. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0053] Example 1:

[0054] A water-soluble CBCS microsphere, the preparation method of which comprises the following steps:

[0055] S1: Preparation of CBCS: 0.5 g chitosan (3.1 mmol chitosan) was dissolved in 25 mL dimethyl sulfoxide and allowed to dissolve and swell for 2 h. Then 0.4 g phthalic anhydride (4.1 mmol phthalic anhydride) was added to a three-necked flask and stirred to dissolve. The mixture was heated until the temperature reached 80 °C. Figure 1 The phthalic anhydride was acylated with chitosan amino groups to obtain a reaction solution after 24 hours. The reaction solution was poured into 55 mL of water and placed in an ice water bath. A 20% mass volume concentration of NaOH solution was added to adjust the pH to 10. The filtrate was filtered to obtain a filtrate. The filtrate was dialyzed through a semipermeable membrane for 5 days. The molecular weight cut-off of the semipermeable membrane was 10000D. The retentate of the semipermeable membrane was freeze-dried to obtain the following: Figure 2 The flocculent light yellow solid CBCS shown: N-(2-carboxybenzoyl)-chitosan 0.79 g, with a yield of 87.6%;

[0056] S2. Preparation of CBCS microspheres: 0.5 g of the CBCS obtained in step S1 was gradually poured into 50 mL of pure water in several portions, stirring continuously to dissolve the CBCS. When only a small amount of white transparent flocs remained in the solution, stirring was stopped and the solution was allowed to stand overnight to obtain a CBCS solution with a mass volume concentration of 1%;

[0057] 75 mL of liquid paraffin and 1 mL of Span 80 were poured into a three-necked flask, heated and stirred at a rate of 1000 rpm until the temperature reached 50 °C, 5 mL of the prepared CBCS solution was slowly added dropwise, and a mixed solution was obtained after emulsification for 30 minutes. 0.3 mL of glutaraldehyde was then added to the mixed solution. Figure 3 The amino groups of the CBCS were cross-linked with the aldehyde groups to produce shiff base bonds (C=N). The cross-linking reaction was carried out for 4 hours to obtain a reaction product. The reaction product was centrifuged at 8000 rpm for 5 minutes, and the upper oil phase was discarded. The centrifuged product was filtered and washed three times with petroleum ether, isopropanol, and acetone, and then dried in a vacuum drying oven at 50°C to obtain CBCS microspheres.

[0058] The CBCS microspheres were imaged using a S-4800I scanning electron microscope. Figure 4 The scanning electron micrograph of the CBCS microspheres shown in the figure shows that the obtained CBCS microspheres have a regular structure, a smooth surface, a good spherical shape, good dispersion between the microspheres, basically no agglomeration or adhesion, and a uniform particle size distribution.

[0059] Comparative Example 1:

[0060] A CACS preparation method comprises: dissolving 0.5 g of chitosan (i.e., 3.1 mmol of chitosan) in 25 mL of dimethyl sulfoxide, allowing the mixture to dissolve and swell for 2 h, then adding 0.4 g of maleic anhydride (i.e., 4.1 mmol of maleic anhydride) into a three-necked flask and stirring and dissolving the mixture, heating the mixture until the temperature reaches 80° C., and then Figure 5The maleic anhydride was subjected to an acylation reaction with chitosan amino groups. After 24 hours of reaction, a reaction solution was obtained. The reaction solution was poured into 55 mL of water and placed in an ice-water bath. A 20% mass volume concentration of NaOH solution was added to adjust the pH to 10. The filtrate was filtered to obtain a filtrate. The filtrate was dialyzed through a semipermeable membrane with a molecular weight cutoff of 7000 Da for 5 days. The retentate of the semipermeable membrane was freeze-dried to obtain 0.53 g of N-(3-carboxyallyl)-chitosan as a flocculent white solid CACS with a yield of 71.1%. It can be seen that the yield of CBCS is higher than that of CACS.

[0061] (1) Chitosan CS, CACS obtained in Comparative Example 1, CBCS obtained in step S1 of Example 1, and CBCS microspheres obtained in step S2 were characterized by infrared spectroscopy (FT-IR) to study the properties of functional groups. The infrared conditions were as follows: Fourier transform infrared spectrometer was used, and the KBr pellet method was used for testing, with a scanning range of 4000-400 cm -1 , the sample was dried for 2 hours before testing, and the results were as follows Figure 6 shown.

[0062] Depend on Figure 6 Visible, 3400cm -1 Near the 2900cm-1, the stretching vibration absorption peaks of -NH: and -OH overlap and become a single broad peak, which indicates that chitosan CS powder, CACS, CBCS and CBCS microspheres contain functional groups amino and hydroxyl groups; -1 Around 2800cm -1 The left and right are the stretching vibration peaks of -CH2- and -CH-, 1600cm -1 The absorption peak of amino group -NH2 is at 1600-1700cm -1 The C=O stretching vibration peak of the secondary amide and the carbonyl group of the carboxylic acid, and the -NH stretching vibration peak of the amide are at 1560 cm -1 It appears around 1650cm, indicating that the acylation reaction of chitosan occurs on the amino group. Compared with CS, CACS and CBCS, CBCS microspheres have a -1 The new characteristic peak of shiff base bond (C=N bond) appears at , which is caused by the cross-linking reaction between -CHO of the cross-linking agent and -NH on chitosan to produce shiff base bond (C=N bond).

[0063] (2) Using nuclear magnetic resonance spectroscopy 1 H-NMR characterization of the CACS obtained in Comparative Example 1 and the CBCS obtained in step S1 of Example 1, conditions: 500 MHz, deuterated water D2O, the results are as follows Figure 7 As shown by Figure 7 visible:

[0064] CACS: N-(3-carboxyallyl)-chitosan 1 The H-NMR spectrum can be assigned as follows: chemical shift 4.95 is assigned to H1, chemical shift 3.24 is assigned to H2, chemical shifts 3.6-3.88 are assigned to H3, H4, H5, and H6, chemical shift 2.14 is assigned to the structural formula -NHCOCH3, and chemical shifts 5.7-6.4 are assigned to the structural formula -CO-CH=CH-COOH; a new signal peak appears around chemical shift 6.0, indicating the introduction of a double bond in the CACS structure.

[0065] CBCS: N-(2-carboxybenzoyl)-chitosan 1 The H-NMR spectrum can be attributed as follows: the chemical shift of 3.5-3.9 is attributed to H3, H4, H5, H6, the chemical shift of 2.14 is attributed to NHCOCH3, and the signal peak of chemical shift δ7-7.9 is attributed to the hydrogen on the benzene ring. Because D2O was used as the solvent, the active hydrogen on the carboxyl group did not show a peak.

[0066] (3) Using carbon nuclear magnetic resonance spectroscopy 13 C NMR characterization of the CBCS obtained in step S1 of Example 1, condition: deuterated water D2O, the results are as follows Figure 8 As shown by Figure 8 visible:

[0067] CBCS: N-(2-carboxybenzoyl)-chitosan 13 The C NMR spectrum can be assigned as follows: chemical shift 58 is assigned to C2, chemical shift 62 is assigned to C6, chemical shift 72 is assigned to C3, chemical shift 78 is assigned to C5, chemical shift 80 is assigned to C4 and 103 (C1) is the carbon signal of chitosan, the newly appeared chemical shifts 175 and 165 are assigned to the carbonyl carbon signal, and the new peaks at chemical shifts 125, 127, 131.5, 131.3, 137.6 and 141.3 are assigned to the aromatic carbon on the benzene ring.

[0068] (4) XRD and DSC were used to characterize and analyze chitosan CS and CBCS obtained in step S1 of Example 1.

[0069] The powder X-ray diffraction patterns of CS and CBCS are as follows: Figure 9 As shown by Figure 9 It can be seen that CS has diffraction peaks belonging to crystalline bands I and II at 2θ of 11° and 20° respectively, while CBCS does not have crystalline band I. The diffraction peak at 2θ = 20° is obviously transformed into a weaker broad peak, and its crystalline band almost completely disappears. This indicates that after the introduction of phthalic anhydride substituents on chitosan, the hydrogen bonding force between chitosan molecules is reduced, and the chitosan molecular chain is stretched, resulting in the intermolecular force of CBCS being weaker than that of CS, thereby significantly improving the solubility of chitosan.

[0070] The DSC diagrams of CS and CBCS are shown in Figure 2. Figure 10 As shown by Figure 10 It can be seen that the endothermic peak of CS is at 85℃, indicating that the chitosan powder contains water, and a decomposition peak appears at 306℃. The endothermic peak appears at 82℃ in the CBCS spectrum, indicating that CBCS also contains a small amount of water, but the decomposition peak drops to 301℃. This shows that due to the introduction of phthalic anhydride on the chitosan amino group, the ability of the hydrogen bonds between chitosan molecules is broken, the chitosan molecules are stretched, and at the same time the thermal stability and crystallization force of CBCS are reduced, thereby improving the solubility of CBCS, making CBCS much more soluble than chitosan.

[0071] (5) Investigate the single factor effect of CBCS solution concentration on CBCS microsphere particle size, oil-water ratio: volume ratio of liquid paraffin to CBCS solution, cross-linking time, and emulsification time:

[0072] Comparative Example 2: The difference from Example 1 is that in step S2, a CBCS solution with a mass volume concentration of 2% is prepared.

[0073] Comparative Example 3: The difference from Example 1 is that in step S2, a CBCS solution with a mass volume concentration of 3% is prepared.

[0074] Comparative Example 4: The difference from Example 1 is that the volume ratio of liquid paraffin to CBCS solution in step S2 is 10:1.

[0075] Comparative Example 5: The difference from Example 1 is that the volume ratio of liquid paraffin to CBCS solution in step S2 is 20:1.

[0076] Comparative Example 6: The difference from Example 1 is that the cross-linking time in step S2 is 3 hours.

[0077] Example 2: The difference from Example 1 is that the cross-linking time in step S2 is 5 hours.

[0078] Example 3: The difference from Example 1 is that the emulsification time in step S2 is 40 min.

[0079] Example 4: The difference from Example 1 is that the cross-linking time in step S2 is 60 min.

[0080] The CBCS microspheres obtained in the above examples and comparative examples were characterized and analyzed by a nano-laser particle size analyzer, and the results are shown in Table 1 below:

[0081] Table 1. CBCS microsphere particle size results under different conditions

[0082] serial number CBCS solution concentration / % Oil-water ratio Cross-linking time / h Emulsification time / min Microsphere particle size / μm PDI sum Example 1 1 15:1 4 30 4.15 0.216 4.366 Comparative Example 2 2 15:1 4 30 4.76 0.520 5.280 Comparative Example 3 3 15:1 4 30 5.12 0.372 5.492 Comparative Example 4 1 10:1 4 30 3.25 0.639 3.889 Comparative Example 5 1 20:1 4 30 5.83 0.345 6.275 Comparative Example 6 1 15:1 3 30 4.12 0.339 4.459 Example 2 1 15:1 5 30 4.59 0.262 4.852 Example 3 1 15:1 4 40 4.17 0.145 4.315 Example 4 1 15:1 4 60 4.18 0.203 4.383

[0083] From the results of Example 1, Comparative Example 2, and Comparative Example 3, it can be seen that as the concentration of the CBCS solution increases, the particle size of the microspheres increases, and the PDI also tends to increase, that is, the particle size distribution range of the microspheres becomes wider; when the CBCS solution concentration is 1%, the particle size and the particle PDI display value are the lowest, which indicates that when the CBCS solution concentration is small, the prepared microspheres have a relatively small particle size and are evenly distributed; as the concentration of the CBCS solution further increases, the microsphere particle size is significantly larger and the distribution range becomes wider; the main reason is that as the concentration of the CBCS solution continues to increase, the microsphere particles will agglomerate, which makes the CBCS microspheres It is difficult to stably disperse in the solution and will quickly settle, making particle size measurement more difficult. In addition, when the concentration of CBCS solution increases, the viscosity of the solution increases, making it difficult to evenly disperse CBCS in liquid paraffin. However, in order to make the solution evenly dispersed during the microsphere formation process, it is necessary to reduce the specific surface area of ​​the droplets as much as possible, thereby increasing the particle size of the CBCS microspheres and the PDI. However, even a lower CBCS solution concentration may not necessarily have the best effect, because as the CBCS concentration decreases, the amount of CBCS used for the cross-linking reaction will decrease, causing the wall of the synthesized microspheres to become thinner and more easily deformed.

[0084] From the comparison results of Example 1, Comparative Example 4, and Comparative Example 5, it can be seen that the particle size of the microspheres increases significantly with the increase of the oil-water ratio, but the PDI shows a decreasing trend, that is, the particle size distribution is uniform; when the oil-water ratio is 10:1, the oil phase accounts for a small proportion, the dispersibility with the water phase is poor, and the probability of collision and adhesion between droplets increases, resulting in irregular spherical shape and uneven particle size distribution of the prepared CBCS microspheres; when the oil phase ratio is 15:1, the CBCS has a better dispersion effect in the oil phase, the probability of collision and adhesion between droplets formed during the emulsification process is reduced, and the particle size distribution of the microspheres is uniform; continuing to increase the oil phase ratio, the oil phase distribution per unit volume of solution increases, the probability of collision between droplets increases, the microspheres are quickly prepared, and the average particle size of the prepared CBCS microspheres increases.

[0085] From the comparison results of Example 1, Comparative Example 6, and Example 2, it can be seen that when the crosslinking time is 3 h, due to insufficient crosslinking time, the obtained CBCS microspheres have lumps or flakes, and the particle size distribution is uneven; when the crosslinking time is 4 h, the chitosan microspheres are completely crosslinked, the microspheres have good sphericity, and the particle size distribution is uniform; with the extension of the crosslinking time, the change in the particle size of the microspheres is not significant; through the particle size and PDI, it can be found that when the crosslinking time is short, the structure of the microspheres is not complete, resulting in poor sphericity; with the increase of time, the amino groups on the chitosan and the aldehyde groups of the crosslinking agent can form a strong bond, making the microsphere structure more stable, the particle size distribution more uniform, and forming a better spherical structure; with the continuous extension of the crosslinking time, there is no significant effect on the particle size of the microspheres, so the preferred crosslinking time is >3 h.

[0086] From the comparison results of Example 1, Example 3, and Example 4, it can be seen that when the emulsification time is 30 minutes, the microsphere particle size is more evenly dispersed; as the emulsification time continues to increase, the microsphere particle size and PDI do not change significantly. The particle size and PDI results show that with the increase of the emulsification time, there is no significant effect on the microsphere particle size and particle size distribution range; considering the practical value of the experiment and that long-term mechanical stirring may destroy the structure of the chitosan microspheres and make the particle structure irregular, the preferred emulsification time is 30-60 minutes.

[0087] (6) According to the results of the single factor experiment, the three main factors affecting the microsphere preparation process, CBCS solution concentration, oil-water ratio, and cross-linking time, were preliminarily determined. Three levels were selected for each factor by comparing the orthogonal design table. L9(3 4 ) Orthogonal experimental design was used to explore the optimal preparation process of CBCS microspheres. The level settings of each factor are shown in Table 2:

[0088] Table 2. Orthogonal level design table

[0089]

[0090] On this basis, the average particle size of the microspheres and the particle size distribution index PDI are used as the evaluation indicators: when the average particle size of the microspheres is used as the evaluation indicator, the average particle size of the microspheres is small, the surface structure of the microspheres is regular, the particle size distribution is uniform, and there is no adhesion between the microspheres; when the particle size dispersion PDI is used as the evaluation indicator, it shows that the more uniform the particle size distribution, the more regular the microsphere structure. The orthogonal results are shown in Table 3 below:

[0091] Table 3. Orthogonal experiment results

[0092]

[0093] Orthogonal data analysis was performed using the sum of the average particle size of the microspheres and the particle size distribution index (PDI). The mean response is shown in Table 4:

[0094] Table 4. Mean response table

[0095]

[0096]

[0097] The results showed that the influence of various factors in the preparation process of CBCS microspheres was in the order of CBCS solution concentration > oil-water ratio > cross-linking time.

[0098] The sum of the orthogonal experimental results of microsphere particle size and PDI was used for variance analysis, and the total variance is shown in Table 5:

[0099] Table 4. Total variance analysis table

[0100] Sources of variance Sum of Squares of Deviations degrees of freedom F-number P-value A 0.235232 2 50.93 0.019 B 0.149528667 2 32.37 0.03 C 0.037514667 2 8.12 0.11 D(error) 0.004618667 2

[0101] F0.05(2,2)=19F 0.01 (2,2)=99, it can be seen that the CBCS solution concentration and oil-water ratio P value are less than 0.05, indicating that the CBCS solution concentration and oil-water ratio have a significant effect on the microsphere preparation process, while the cross-linking time P value is greater than 0.1, indicating that it has no significant effect on the microsphere preparation process, and from Figure 12 The interaction of the three factors showed that there was an obvious relationship between the three factors. The concentration of CBCS solution and the oil-water ratio had a significant effect on the microsphere preparation process, while the cross-linking time had no significant effect on the microsphere preparation process. Figure 11 The optimal preparation process conditions of CBCS microspheres were determined to be A1B2C3, namely, CBCS concentration of 0.8%, oil-water ratio of 15:1, and cross-linking time of 4.5h.

[0102] (7) Investigating the adsorption effect of CS microspheres and CBCS microspheres obtained in Example on tetracycline

[0103] Comparative Example 7: A CS microsphere, a preparation method thereof comprising: weighing 0.5 g of chitosan powder and gradually pouring it into 50 mL of acetic acid solution with a volume concentration of 2% acetic acid, stirring continuously to dissolve, standing to expel bubbles, and obtaining a CS solution with a mass volume concentration of 1%; pouring 75 mL of liquid paraffin and 1 mL of surfactant Span 80 into a three-necked flask, heating and stirring at a rate of 1000 rpm until the temperature reaches 50°C, then slowly adding 5 mL of the prepared CS solution dropwise thereto, emulsifying for 30 minutes to obtain a mixed solution, and then adding 0.3 mL of glutaraldehyde to the mixed solution, cross-linking reaction for 4 hours, after the reaction, centrifuging the product at 8000 rpm for 5 minutes, and then discarding the upper oil phase to obtain a centrifuge, the centrifuge was filtered and washed three times with petroleum ether, isopropanol and acetone, and then dried in a vacuum drying oven at 40°C to obtain chitosan microspheres, i.e., CS microspheres.

[0104] ① Adsorption isotherm experiment: 50 mL of tetracycline solution with concentrations of 20 mg / L, 40 mg / L, 60 mg / L, 80 mg / L, 100 mg / L, and 120 mg / L was prepared in a conical flask; 30 mg of the CS microspheres obtained in Comparative Example 7 and the CBCS microspheres obtained in Example 1 were weighed and placed in a 50 mL glass bottle, 50 mL of tetracycline solution of different concentrations was added to each, and the solution pH was adjusted to 3; the glass bottle was placed in a constant temperature oscillator at 25 ° C., the oscillation rate was 200 rpm, and after shading for 6 hours, it was centrifuged at 4000 rpm for 5 minutes, the solution was extracted with a syringe and filtered through a 0.22 μm microporous membrane. The concentration change of the test solution before and after adsorption at 6 concentrations was detected by UV-visible wind photometer at a wavelength of 357 nm.

[0105] Equilibrium adsorption capacity (Qe, mg·g -1 ) and removal rate (η%) formula are as follows:

[0106]

[0107]

[0108] In the above formulas (1) and (2), Qe is the saturated adsorption capacity of the prepared adsorbent for tetracycline (mg·g -1 ); η is the corresponding removal rate; C0 is the initial concentration of tetracycline solution (mg / L), Ce is the concentration of tetracycline solution at the end of adsorption (mg / L); V is the volume of tetracycline solution (L); m is the mass of added microspheres (mg).

[0109] The results of the effect of initial tetracycline concentration on the adsorption of microspheres are as follows Figure 13 As shown in the figure, it can be seen that with the increase of the initial concentration of tetracycline, the adsorption capacity of both microspheres increases continuously; before the initial concentration of tetracycline solution reaches 100 mg / L, the adsorption rate continues to rise, and the adsorption amount of CS microspheres and CBCS microspheres continues to increase. After the initial concentration reaches 80 mg / L, the adsorption rate becomes slow. After the initial concentration reaches 100 mg / L, the adsorption amount no longer changes and tends to equilibrium. At this time, the adsorption amount of chitosan microspheres for tetracycline solution reaches saturation when it reaches saturation. The adsorption amount of CBCS microspheres for tetracycline solution reaches saturation when it reaches saturation. The higher the initial concentration of tetracycline, the greater the driving force in the solid-liquid phase, and the adsorption capacity also increases accordingly. When the tetracycline solution reaches a certain initial concentration, the adsorption sites on the microsphere surface have basically been combined with tetracycline molecules, and the adsorption amount of the microspheres reaches saturation. This shows that the initial concentration of tetracycline is one of the factors affecting the adsorption effect of the microspheres. After the adsorption amount reaches a stable state, the initial concentration of tetracycline will no longer affect the adsorption process.

[0110] The adsorption isotherm model Langmuir model adsorption equation is as follows:

[0111]

[0112] The adsorption isotherm model Freundlich model adsorption equation is as follows:

[0113] qe=Kf*Ce^1 / n (4)

[0114] In the above formulas (3) and (4), C e is the equilibrium concentration of adsorbed tetracycline (mg / L); q e is the adsorption capacity of microspheres (mg / g); q m is the extreme adsorption capacity (mg / g); KL is the surface adsorption rate constant (L / mg), K f is the equilibrium constant of adsorption capacity; n is the adsorption constant, usually n = 0.1 to 0.5, which is easy to adsorb; when n is higher than 2, it is not easy to adsorb.

[0115] In order to verify whether the adsorption process satisfies the adsorption condition, the dimensionless separation factor R is introduced. L , the expression is as follows:

[0116]

[0117] In the above formula (5), R L Represents the separation coefficient, which reflects the characteristics of the adsorption process. When R L When R is between 0 and 1, it indicates that the adsorption process is favorable for adsorption; when R L When R is greater than 1, the adsorption process will not be realized; when R L =1, the adsorption reaction can be reversed; when R L = 0, the adsorption process will be irreversible.

[0118] The isothermal curves of tetracycline adsorption by CS microspheres and CBCS microspheres at room temperature are as follows: Figure 14 As shown, the linear correlation coefficient of the Langmuir model CS microspheres: R 2 =0.9978, CBCS microspheres: R 2 =0.9953, Freundlich linear correlation coefficient CS microspheres: R 2 =0.9297, BCS microspheres: R 2 =0.9034, which shows that the adsorption process of tetracycline by the two microspheres is more consistent with the Langmuir model, belonging to the adsorption of a single molecular layer. The R L The R of CBCS microspheres is 0.23-0.65. L The values ​​were 0.13-0.79, all between 0 and 1, indicating that the adsorption process of tetracycline by CBCS was easy.

[0119] ② Adsorption kinetics experiment: 30 mg of adsorbent was placed in 9 light-shielding volumetric flasks, and then 50 mL of 100 mg / L tetracycline solution was added to the volumetric flasks. The microspheres and tetracycline solution were fully mixed in a constant temperature oscillator. The volumetric flasks were taken out according to the set time, and the test solution was filtered through a 0.22 μm microporous filter membrane to obtain the test solution. The concentration of the solution was measured at 357 nm using an ultraviolet photometer. Finally, the adsorption amount of tetracycline solution by the microspheres at different times was calculated. The results are shown in the table. Figure 15It can be seen that the adsorption amount of tetracycline is increasing with the change of time. After 120 minutes, the adsorption rate of CS microspheres and CBCS microspheres is decreasing, the adsorption process begins to tend to equilibrium, and the adsorption amount basically stops increasing after 210 minutes.

[0120] The pseudo-first-order kinetic equation is as follows:

[0121] ln(Q e -Qt)=lnQe-K1t (6)

[0122] The pseudo-second-order kinetic equation is as follows:

[0123]

[0124] In the above formulas (6) and (7), Q e is the equilibrium adsorption capacity of tetracycline solution (mg / g); Q t is the adsorption amount of the microspheres at time t (mg / g); K1 is the pseudo-first-order model adsorption rate constant; K2 is the pseudo-second-order model adsorption rate constant.

[0125] In order to further study the effect of the intraparticle diffusion model on the adsorption process of tetracycline by two microsphere adsorbents, the experimental data were analyzed based on the pseudo-second-order model fitting, and the intraparticle diffusion equation was expressed as:

[0126] Qt=K id ×t 1 / 2 +C (8)

[0127] In formula (8), K id represents the rate constant of intraparticle diffusion (mg·g-1·min 1 / 2 ), constant C represents a constant related to the thickness of the boundary layer. Generally speaking, the larger the C value, the more significant the boundary layer effect. id and C can be used by Qt to 1 / 2 Graph the slope and intercept to find it.

[0128] Depend on Figure 16 The fitting curve shows that the fitting pseudo-first-order kinetic equation R 2 are 0.9519 and 0.9782 respectively. The Qcal is quite different from the experimental Qexp. The R 2 The Qcal values ​​were 0.9966 and 0.9968, respectively, both above 0.99. The obtained Qcal was basically close to the experimentally obtained Qexp. It can be seen that the quasi-second-order kinetic fitting effect of the adsorption process of tetracycline by CBCS microspheres was better, which was consistent with the quasi-second-order kinetic model.

[0129] Depend on Figure 17As shown in the fitting curves, the adsorption process curves of the two microspheres can be divided into three stages: the first stage is the diffusion of tetracycline on the outer surface of the particles, mainly diffusing and adsorbing on the microsphere surface and boundary layer, and the adsorption rate in this stage is relatively fast. As the adsorption reaction proceeds, the second and third stages are characterized by diffusion within the pores of the particles. As can be seen from the figure, none of the three fitting linear segments pass through the origin coordinate, indicating that the adsorption rate of the other microsphere is not only controlled by diffusion on the particle surface and within the pores, but also by diffusion in the boundary layer. This is because as the reaction proceeds, the amount of tetracycline in the solution decreases, and the adsorption points on the microsphere surface continue to decrease, causing the tetracycline molecules already adsorbed on the microsphere surface to diffuse into the pores. At this time, the adsorption rate is relatively slow, and the adsorption reaction gradually reaches equilibrium.

[0130] The parameters of the intra-particle diffusion model of tetracycline adsorbed on microspheres are shown in Table 6 below:

[0131] Table 6. Parameters of the intraparticle diffusion model for tetracycline adsorbed on microspheres

[0132]

[0133] It can be seen that the Kid1 values ​​are higher than those of Kid2 and Kid3, indicating that as the adsorption reaction time increases, the pores on the surface of the microsphere adsorbent are occupied by a large amount of tetracycline, and the adsorption rate of the adsorbent decreases.

[0134] ③ Adsorption thermodynamics experiment: When the tetracycline solution is 100 mg / L, the relationship between the adsorption temperature and the adsorption amount is investigated at three temperatures, namely 293K, 303K, and 313K. The thermodynamic equation for the effect of temperature on the adsorption process is as follows:

[0135]

[0136] ΔG 0 =-RTlnK b (10)

[0137]

[0138] In the above formulas (9)-(11), K b is the diffusion coefficient, ΔH 0 is the enthalpy change (kJ·mol -1 ), ΔS 0 is the entropy change (J / mol -1 ·K -1 ), ΔG 0 is the Gibbs free energy (kJ·mol -1 ), T (K) is the temperature, R (8.314 J / mol -1 ·K -1 ) is the molar gas constant.

[0139] See the results Figure 18 It can be seen that the unit adsorption capacity of the two microsphere adsorbents increases with the increase of temperature. High temperature promotes the swelling of the microsphere pores and accelerates the diffusion of tetracycline on the microsphere surface and in the pores, thereby increasing the adsorption rate of tetracycline by the microspheres and the adsorption capacity. At the same time, high temperature also promotes the rapid diffusion of tetracycline molecules to the surface boundary layer and internal pores.

[0140] The thermodynamic parameters of tetracycline adsorption by microspheres are shown in Table 7:

[0141] Table 7. Thermodynamic parameters of tetracycline adsorption on microspheres

[0142]

[0143] As shown in Table 7, the Buss free energy ΔG of chitosan microspheres and CBCS microspheres 0 are all negative, indicating that the adsorption reactions of the two adsorbents are mainly spontaneous reactions, and ΔG 0 Values ​​between -20 and 0 kJ·mol -1 , their adsorption is mainly physical adsorption; at the same time, with the increase of temperature, the Gibbs free energy ΔG 0 The absolute value of also increases, indicating that the change of temperature affects the adsorption behavior of the two microspheres; 11.4 and 15.1 kJ·mol-1, and 0 and 40 kJ·mol -1 The range indicates that the adsorption mode of the two microspheres is dominated by physical adsorption, and the adsorption process is an endothermic reaction. A positive value indicates that the adsorption process is irreversible.

[0144] In summary, at different concentrations, the adsorption capacity of CBCS microspheres is significantly better than that of CS microspheres, indicating that in addition to the influence of the lone pair electron orbitals on the amino and hydroxyl groups of chitosan, the introduction of carboxyl groups in CBCS increases its oxygen-containing functional groups, thereby increasing the adsorption sites for tetracycline. At the same time, CBCS not only electrostatically bonds with tetracycline in anionic state, but also the intermolecular force of CBCS is weaker than that of chitosan after the introduction of phthalic anhydride substituents, which significantly weakens the hydrogen bonds between chitosan molecules and stretches the main chain within the chitosan molecule, thereby greatly enhancing the adsorption capacity.

[0145] At various temperatures, the adsorption capacity of CBCS microspheres for tetracycline was higher than that of CS microspheres. Compared with the adsorption capacity of CS microspheres, the better adsorption capacity of CBCS microspheres was not only due to the temperature, but also due to the carboxyl groups introduced into the structure of CBCS microspheres, which increased its oxygen-containing functional groups, thereby increasing the adsorption sites for tetracycline and the adsorption amount.

[0146] (8) Investigating the multiple uses of CS microspheres and CBCS microspheres obtained in the examples when adsorbing antibiotics:

[0147] 30 mg of the CS microspheres obtained in Comparative Example 7 and the CBCS microspheres obtained in Example 1 were weighed and placed in 50 mL glass bottles, and 50 mL of tetracycline solution of different concentrations was added to each of them to adjust the solution pH to 3; the glass bottles were placed in a constant temperature oscillator at 25 ° C. and the oscillation rate was 200 rpm. After shielding for 6 hours, they were centrifuged at 4000 rpm for 5 minutes to obtain the adsorbed CS microspheres and CBCS microspheres; the adsorbed CS microspheres and CBCS microspheres were rinsed with 0.2 mol / L NaOH solution to release the tetracycline adsorbed on the CS microspheres or CBCS microspheres, and then washed with distilled water to pH = 7 and dried in a vacuum drying oven at 50 ° C. After repeated experiments, the maximum adsorption rate of each adsorption was compared with that of the first experiment.

[0148] The microspheres were used to repeatedly adsorb antibiotics to evaluate whether they were reusable. The results are shown in Figure 19 As shown in the figure, after four consecutive regeneration cycles, the TC removal rate of CBCS microspheres (Q-CBCS) is still above 80%, while the TC removal rate of chitosan microspheres (Q-CS) is only below 60% after four consecutive regeneration cycles. This shows that CBCS microspheres (Q-CBCS) have better stability and repeatability than chitosan microspheres (Q-CS), and can be recycled many times after regeneration.

[0149] The sources of reagents used in the above examples and comparative examples include:

[0150] Chitosan: CS, purchased from Shanghai Zhanyun Chemical Co., Ltd.; maleic anhydride: MA, purchased from Shanghai Aladdin Biochemical Co., Ltd.; phthalic anhydride: HHPA, purchased from Shanghai Aladdin Biochemical Co., Ltd.; sodium hydroxide, dimethyl sulfoxide DMSO, glacial acetic acid, glutaraldehyde, tetracycline: purchased from Sinopharm Chemical Reagent Co., Ltd.

[0151] The series of detailed descriptions listed above are only specific descriptions of feasible embodiments of the present invention. They are not intended to limit the scope of protection of the present invention. Any equivalent embodiments or changes that do not deviate from the technical spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. A water-soluble CBCS microsphere for adsorbing antibiotics in water, characterized in that: The water-soluble CBCS microspheres are chitosan modified by phthalic anhydride to CBCS: N-(2-carboxybenzoyl)-chitosan, and the structural formula is: Then the amino group of CBCS is cross-linked and cured with the aldehyde group to produce shiff base bonds; The molecular weight of the CBCS is greater than 10KD, and the particle size distribution index (PDI) of the water-soluble CBCS microspheres is less than 0.3; The method comprises: S1, preparing CBCS: dissolving chitosan in a solvent to dissolve and swell chitosan, then adding phthalic anhydride to react to obtain a reaction solution, pouring the reaction solution into water to cool, adjusting the pH to alkaline, filtering and dialysis the filtrate, and freeze-drying to obtain flocculent light yellow solid CBCS; S2, preparing CBCS microspheres: dissolving the CBCS obtained in step S1 in water to obtain a CBCS solution, dripping the CBCS solution into an oil phase and an emulsifier to emulsify the solution, then adding an aldehyde cross-linking agent for cross-linking reaction to obtain a reaction product, removing the oil phase in the reaction product, and drying to obtain CBCS microspheres.

2. The water-soluble CBCS microspheres for adsorbing antibiotics in water according to claim 1, characterized in that: The particle size of the water-soluble CBCS microspheres is less than 5 μm.

3. The water-soluble CBCS microspheres for adsorbing antibiotics in water according to claim 1, characterized in that: The solvent in step S1 is dimethyl sulfoxide, the molar volume ratio of chitosan to dimethyl sulfoxide is 0.12-0.13 mmol / ml, the dissolution and swelling time is 1.5-2.5 hours, the molar ratio of chitosan to phthalic anhydride is 3-3.2:4-4.2, and the reaction is carried out at 70-80° C. for 23-25 ​​hours.

4. The water-soluble CBCS microspheres for adsorbing antibiotics in water according to claim 1, characterized in that: After the reaction in step S1, the reaction solution is added to water and placed in an ice-water bath for cooling. The pH is adjusted to 9-11 with a NaOH solution. The molecular weight cut-off of the semipermeable membrane used for dialysis is 10KD.

5. The water-soluble CBCS microspheres for adsorbing antibiotics in water according to claim 1, characterized in that: The mass volume concentration of CBCS in the CBCS solution in step S2 is 0.8-1.5%.

6. The water-soluble CBCS microspheres for adsorbing antibiotics in water according to claim 1, characterized in that: In step S2, the oil phase is liquid paraffin, the emulsifier is Span 80, the volume ratio of Span 80 to liquid paraffin is 1:50-150, the volume ratio of the oil phase to the CBCS solution is 13-17:1, and the emulsification time is 30-60 min.

7. The water-soluble CBCS microspheres for adsorbing antibiotics in water according to claim 1, characterized in that: In step S2, the aldehyde cross-linking agent is glutaraldehyde, the mass volume ratio of CBCS to glutaraldehyde is 1.5-3.5 g / ml, and the cross-linking reaction time is >3 h.

8. The water-soluble CBCS microspheres for adsorbing antibiotics in water according to claim 1, characterized in that: In step S2, the reaction product is centrifuged at 7000-8000 rpm for 4-6 minutes, and the upper oil phase is discarded. The centrifuged product is filtered and washed with petroleum ether, isopropanol, and acetone, respectively, and dried at 40-60° C. to obtain CBCS microspheres.

9. The use of a water-soluble CBCS microsphere for adsorbing antibiotics in water according to claim 1, characterized in that: The water-soluble CBCS microspheres are used to adsorb antibiotics in water.

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