Termite killing microcapsule with pH and digestive enzyme dual response, preparation method and application

Abstract

The invention discloses a termite killing microcapsule with pH and digestive enzyme dual response, a preparation method and application. The microcapsule is prepared from imidacloprid powder, carboxymethyl chitosan and sodium lignin sulfonate. The preparation method comprises the following steps: firstly, dissolving carboxymethyl chitosan in acetic acid, then dissolving an imidacloprid active compound in a carboxymethyl chitosan solution, and centrifuging to prepare imidacloprid carboxymethyl chitosan particles; and alternately depositing the imidacloprid carboxymethyl chitosan particles and sodium lignin sulfonate under specific conditions to form a multi-layer composite structure. Through optimized centrifugation and drying processes, the microcapsule material with good load capacity is obtained. The sustained-release agent can be accurately released in intestinal tracts of the termites, meanwhile, the release amount in the external environment is extremely small, the sustained-release agent is suitable for being directly sprayed on the body surfaces of the termites, and the continuous virus transmission effect is ensured. Experimental results show that the slow-release agent shows an excellent effect in termite control, and a new solution is provided for eco-friendly agricultural pest management.

Description

Technical Field

[0001] The invention relates to the technical field of nano material synthesis, and in particular to a method for synthesizing a microcapsule material that responds to pH and digestive enzymes and an application of the microcapsule material in preventing and controlling termites. Background Art

[0002] Reticulitermes flaviceps is a kind of soil amphibious termite that causes various degrees of damage to hundreds of plants. These termites are recognized as one of the most destructive termites, causing great harm to agricultural and forestry production. In addition, the intestinal environment of most termites is alkaline (Brune, A. (2014). Symbiotic digestion of ligncocellulose in termite guts. Nature Reviews Microbiology, 12, 168-180.), and they produce various enzymes, including laccase, which plays a key role in the degradation of lignin.

[0003] Carboxymethyl chitosan (CMCS) is a cationic biopolymer. It contains a large number of highly hydrophilic amino groups, which give the biopolymer a net positive charge, which is conducive to interacting with many negatively charged polymers (Wang, K., Ma, H., Pu, S., Yan, C., Wang, M., Yu, J., Wang, X., Chu, W., & Zinchenko, A. (2019). Hybridporous magnetic bentonite-chitosan beads for selective removal of radioactive cesium in water. Journal of Hazardous Materials, 362, 160–169.). Sodium lignin sulfonate (SL) is a natural anionic polymer surfactant. Therefore, the amines in CMCS can interact with the sulfonic acid groups in SL through electrostatic forces to construct a pesticide carrier system with customized wall thickness.

[0004] However, active ingredients and pesticide adjuvants used in pesticide formulations often have negative effects on non-target organisms, ecosystems and human health (Van de Merwe, JP, Neale, PA, Melvin, SD, & Leusch, FD (2018). In vitro bioassays reveal that additives are significant contributors to the toxicity of commercial household pesticides. Aquatic Toxicology, 199, 263-268.). Summary of the invention

[0005] The technical problem to be solved by the present invention is how to prepare a microcapsule material that responds to digestive enzymes, in order to solve the problems of off-target loss, non-target accumulation and environmental pollution of chemical pesticides when controlling termites in the wild. Therefore, a method for synthesizing a microcapsule material that responds to both pH and digestive enzymes and its application in controlling termites are proposed, which can effectively control termites while also being very environmentally friendly.

[0006] The present invention also proposes a pH and enzyme responsive carboxymethyl chitosan / sodium lignin sulfonate microcapsule delivery system, which is designed to selectively release insecticides according to a specific pH value or laccase present in the termite gut.

[0007] The present invention solves the above technical problems through the following technical solutions:

[0008] 1. A pH and digestive enzyme dual-responsive termite-killing microcapsule:

[0009] It is mainly composed of imidacloprid powder IMI, carboxymethyl chitosan CMCS and sodium lignin sulfonate SL.

[0010] The mass ratio of the carboxymethyl chitosan CMCS to the sodium lignin sulfonate SL is 2:0.5-1.

[0011] Carboxymethyl chitosan CMCS and sodium lignin sulfonate SL are alternately deposited in a layer relationship of 1:1. As the layers of carboxymethyl chitosan CMCS and sodium lignin sulfonate SL increase, the mass content of imidacloprid IMI in the microcapsule is 92.63-91.67%, and the release rate of imidacloprid IMI is 66.35-52.19%.

[0012] 2. A method for preparing a pH and digestive enzyme dual-responsive termite-killing microcapsule, the method comprising the following steps:

[0013] S1, dissolving carboxymethyl chitosan CMCS in a first co-solvent to obtain a 1 wt% carboxymethyl chitosan solution, and adjusting the pH value;

[0014] S2, dissolving the imidacloprid powder IMI in the carboxymethyl chitosan solution obtained in step S1, ultrasonicating for 10 minutes, and stirring on a magnetic stirrer for 30 minutes to obtain a mixture;

[0015] S3, centrifuging the mixture obtained in step S2 at a speed of 3800 rpm and collecting the precipitate as precipitate 1, and washing the precipitate 1 with deionized water and drying under vacuum conditions to obtain imidacloprid@carboxymethyl chitosan IMI@CMCS;

[0016] S4, adding the imidacloprid@carboxymethyl chitosan IMI@CMCS obtained in step S3 to a 1 wt% sodium lignin sulfonate solution, stirring for 30 minutes, centrifuging and collecting the precipitate as precipitate 2, and drying the precipitate 2 under vacuum to obtain a layer of imidacloprid@carboxymethyl chitosan@sodium lignin sulfonate IMI@(CMCS+SL)1;

[0017] S5. Replace the imidacloprid powder IMI with one layer of IMI@(CMCS+SL) obtained in step S4, and repeat steps S2-S4 to obtain n layers of imidacloprid@carboxymethyl chitosan@sodium lignin sulfonate IMI@(CMCS+SL)n, where n is 1-3, to obtain the microcapsules, which are powders with a particle size of 335 μm.

[0018] The weakly alkaline environment and abundant laccase in the termite intestine promote the release of microcapsules after decomposition, thereby improving the targeting of insecticides and reducing toxic side effects.

[0019] In the step S1, the first auxiliary solvent is an acetic acid solution with a volume fraction of 2%.

[0020] In the step S1, the pH value is adjusted to 4 by adding HCl and NaOH dropwise to the solution.

[0021] In the step S2, the ratio of the mass of imidacloprid powder IMI to the volume of the carboxymethyl chitosan solution is 10-25 mg / ml;

[0022] In the step S4, the ratio of the mass of imidacloprid@carboxymethyl chitosan IMI@CMCS to the volume of the sodium lignin sulfonate solution is 10-25 mg / ml.

[0023] Generally speaking, in step S2 and step S4, the ratio of the mass of the added solid to the volume of the liquid before addition is 10-25 mg / ml.

[0024] 3. An application of the microcapsule in termite control.

[0025] 4. A method for killing termites using the microcapsules, the method comprising the following steps:

[0026] K1, adding the microcapsules to the second cosolvent, ultrasonically treating for 10 minutes and then placing at room temperature for 5 minutes to obtain a microcapsule suspension with a concentration of 0.2 to 5.0 ppm;

[0027] K2. The microcapsule suspension described in step K1 is placed in the living environment of termites or directly applied to termites, so that the termites contact the microcapsules. After the microcapsules enter the termites, they are basically not released in the ant nest and ant tunnel, but are specifically released in the termite intestine.

[0028] More specifically, the yellow-breasted Reticulitermes can be placed on dry filter paper containing the microcapsule solution at 28° C. and observed for 5 days.

[0029] The second auxiliary solvent in step K1 is distilled water.

[0030] The advantages of the present invention are:

[0031] 1. CMCS and SL are deposited in layers on the emulsion interface mainly through electrostatic interactions, thus developing a simple and efficient microencapsulation system for pesticide release. By adjusting the number of deposited layers n, the release behavior of IMI@(CMCS+SL)n layers can be precisely controlled.

[0032] 2. In the actual application of preventing and controlling termites, the pH-responsive and digestive enzyme materials of the present application are spread in areas where termites are infested, and termites will bring the materials into the caves through social activities. When the materials are brought into the digestive tract by termites, the alkaline conditions and laccase in the termites will decompose the carboxymethyl chitosan and sodium lignin sulfonate outside the materials, and the drugs loaded inside will be released to kill termites. This has a certain protective effect on other non-target insects.

[0033] The product of the present invention can be directly sprayed on the body surface of termites, will not cause rapid knockdown of termites, and can complete 1-2 times of poison transmission, with good poison transmission effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a Fourier transform infrared spectrum of the product prepared in the examples of this application.

[0035] Figure 2 It is an X-ray image of the product prepared in the examples of this application.

[0036] Figure 3 It is the particle size of the product prepared in the examples of this application.

[0037] Figure 4 This is the thermogravimetric diagram of the microcapsules prepared in Example 6 of the present application.

[0038] Figure 5 This is a graph showing the in vitro drug release rate of the microcapsules prepared in Example 6 of the present application.

[0039] Figure 6 This is a biological activity test chart of the microcapsules prepared in Example 6 of the present application. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are 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 creative work are within the scope of protection of the present invention.

[0041] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0042] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0043] Embodiments of the present invention are as follows:

[0044] Embodiment 1:

[0045] S1. Prepare a 1 wt % carboxymethyl chitosan (CMCS) aqueous solution by dissolving 10 g of carboxymethyl chitosan (CMCS) in a 2 % (v / v) acetic acid solution under stirring, and adjust the pH value to 4 with HCl and NaOH;

[0046] S2. Dissolve 5 g of sodium lignin sulfonate (SL) in 495 g of water to obtain a sodium lignin sulfonate solution with a weight percentage of 1. Then, weigh 1 g of IMI raw material and add it to a carboxymethyl chitosan (CMCS) aqueous solution to obtain a mixture.

[0047] S3, ultrasonicating the mixture for 10 minutes, and then stirring on a magnetic stirrer for 30 minutes. The resulting mixture was centrifuged at 3800 rpm, rinsed with deionized water, and dried under vacuum to obtain IMI@CMCS;

[0048] S4, adding IMI@CMCS to the sodium lignin sulfonate solution, stirring for 30 minutes, and then collecting the precipitate by centrifugation. Then vacuum drying the precipitate to obtain IMI@(CMCS+SL)1 layer;

[0049] S5. Replace the IMI raw material with IMI@(CMCS+SL)1 layer, repeat the cycle steps S2-S4, and obtain the product IMI@(CMCS+SL)n layer, where n is 1, 2 or 3.

[0050] In step S2 and step S4, the ratio of the mass of the solid added to the volume of the liquid before addition is maintained at 10-25 mg / ml.

[0051] The prepared products were analyzed by Fourier transform infrared spectroscopy. The synthesis of IMI, CMCS, SL, IMI@(CMCS+SL)1 layer, IMI@(CMCS+SL)2 layer and IMI@(CMCS+SL)3 layer was confirmed by Fourier transform infrared analysis. The results are shown in Figure 1 As shown in the figure, due to the presence of a large number of amino groups in the IMI molecule, the N=O bond stretching vibration occurs at 1434 cm -1 , while the characteristic absorption of NH bond stretching vibration and bending vibration occurs at 1120cm -1 The CC stretching vibration appears at 1590cm -1 , attributed to the carboxyl group in CMCS. For SL, 1130 cm -1 The peak at represents SO 3- The stretching vibration of 1512 and 1043 cm -1 The absorption bands at are due to the C9 aromatic skeleton vibration and S=O stretching vibration, respectively, which proves that the prepared materials are in line with expectations.

[0052] The prepared product was subjected to X-ray diffraction (XRD) analysis, and the results were as follows: Figure 2 As shown, IMI is highly crystalline with multiple sharp diffraction peaks, and the maximum diffraction peak is observed at about 2θ = 18.36°, but these diffraction peaks are observed in the IMI@(CMCS+SL)3 layer, indicating that the encapsulation of IMI is successful.

[0053] The particle size of the prepared product was measured and the results were as follows: Figure 3 As shown, according to the particle size data, the thickness of the multilayers gradually increases with the increase of deposition cycles, which makes it possible for the current strategy to prepare capsules with tunable shells to achieve precise drug release.

[0054] Example 2

[0055] This embodiment provides a synthesis method for increasing the drug loading of a material, comprising the following steps:

[0056] (1) preparing a 1 wt% carboxymethyl chitosan (CMCS) aqueous solution by dissolving 10 g of carboxymethyl chitosan (CMCS) in a 2% (v / v) acetic acid solution under stirring, and adjusting the pH to 4 with HCl and NaOH. Dissolving 5 g of SL in 495 g of water to obtain a sodium lignin sulfonate solution;

[0057] (2) Weighing an IMI raw material of a mass that satisfies the material drug loading amount, adding the IMI raw material to a CMCS solution to obtain a mixture, ultrasonicating the mixture, stirring it on a magnetic stirrer, centrifuging it, rinsing it with deionized water, and drying it under vacuum conditions to obtain IMI@CMCS;

[0058] (3) Add IMI@CMCS to the SL solution. Stir the mixture for 30 minutes, collect the precipitate by centrifugation, and then vacuum dry the precipitate to obtain IMI@(CMCS+SL)1 layer. Replace the IMI raw material with IMI@(CMCS+SL)1 layer, and repeat the steps S2-S4n-1 times to obtain the product IMI@(CMCS+SL)n layer, where n is 1, 2 or 3.

[0059] Example 3

[0060] This embodiment provides a synthesis method for increasing the drug loading of a material, which differs from Embodiment 2 in that the mass of imidacloprid accounts for 300 mg / g of the total mass of the microcapsule.

[0061] Example 4

[0062] This embodiment provides a synthesis method for increasing the drug loading of a material, which differs from Embodiment 2 in that the mass of imidacloprid accounts for 400 mg / g of the total mass of the microcapsule.

[0063] Example 5

[0064] This embodiment provides a synthesis method for increasing the drug loading of a material, which differs from Embodiment 2 in that the mass of imidacloprid accounts for 500 mg / g of the total mass of the microcapsule.

[0065] Example 6

[0066] This embodiment provides a synthesis method for increasing the drug loading of a material, which differs from Embodiment 2 in that the mass of imidacloprid accounts for 600 mg / g of the total mass of the microcapsule.

[0067] Example 7

[0068] This embodiment provides a synthesis method for increasing the drug loading of a material, which differs from Embodiment 2 in that the mass of imidacloprid accounts for 700 mg / g of the total mass of the microcapsule.

[0069] Example 8

[0070] This embodiment provides a synthesis method for increasing the drug loading of a material, which differs from Embodiment 2 in that the mass of imidacloprid accounts for 800 mg / g of the total mass of the microcapsule.

[0071] Example 9

[0072] This embodiment provides a synthesis method for increasing the drug loading of a material, which differs from Embodiment 2 in that the mass of imidacloprid accounts for 900 mg / g of the total mass of the microcapsule.

[0073] According to the method of Example 6, the thermal stability of the IMI@(CMCS+SL)n layer was studied. Figure 4 As shown in the figure, as the number of packaging layers increases, the weight loss rate of the IMI@(CMCS+SL)n layer decreases. The weight loss rate of the IMI@(CMCS+SL)1 layer reaches 77.97%, while the weight loss rates of the IMI@(CMCS+SL)2 layer and the IMI@(CMCS+SL)3 layer are only 73.44% and 72.58%, respectively. This shows that as the number of encapsulation layers n increases, the stability of the microcapsule IMI@(CMCS+SL)n layer is also increasing.

[0074] According to the method of Example 6, the prepared product was subjected to an in vitro release test, and the in vitro release rate was obtained. The results are as follows Figure 5 As shown in Figure 2, after calibration, the release behavior of IMI@(CMCS+SL)n layer in media with different pH values ​​is shown in Figure 2. Figure 5 AC. In the medium solution with a pH value of 9.0, IMI can be rapidly released from the IMI@(CMCS+SL)n layer, and the cumulative release rates after 24 hours are 80.15% (IMI@(CMCS+SL)1 layer), 74.41% (IMI@(CMCS+SL)2 layer) and 61.23% (IMI@(CMCS+SL)3 layer), respectively; in the medium solution with a pH value of 7.0, the release is slow, and the cumulative release rates are 64.32% (IMI@(CMCS+SL)1 layer), 56.83% (IMI@(CMCS+SL)2 layer) and 49.88% (IMI@(CMCS+SL)3 layer), respectively. Similarly, in the medium solution with a pH value of 5.0, the release of IMI was still slow, with release rates of 60.11% (IMI@(CMCS+SL)1 layer), 50.94% (IMI@(CMCS+SL)2 layer), and 41.91% (IMI@(CMCS+SL)3 layer), respectively.

[0075] In addition, the study also found that the release rate of IMI decreased with the increase in the number of layers, which was due to the multilayer wall structure hindering the release of the drug. The results showed that the prepared IMI@(CMCS+SL)n layer had good pH-controlled release performance, with rapid release in a weakly alkaline environment and slow release in a weakly acidic environment. This helps to improve the rapid control effect of nanopesticides on alkaline intestinal pests and improve the utilization rate by reducing the release of pesticides in acidic dew. This is attributed to the carboxyl groups on the CMCS chains of the IMI@(CMCS+SL)n layer, which are protonated to -COOH at low pH values ​​and form stable intermolecular hydrogen bonding forces (Shariatinia, 2018). As the pH value increases, it will be deprotonated to -COOH and dissolve and rupture due to electrostatic repulsion.

[0076] Laccase is widely present in the intestine of termites and plays a vital role in the biodegradation of lignin. The outer shell of the IMI@(CMCS+SL)n layer is composed of alkali lignin derivatives, which can be degraded by laccase. Therefore, we studied the release performance of the IMI@(CMCS+SL)n layer under the action of laccase. The release curve is shown in Figure 2. Figure 5 As shown in Figure DF, the release rate of IMI@(CMCS+SL)n layers was significantly accelerated under the action of laccase. After 24 h, the cumulative release rate increased by 82.35% (IMI@(CMCS+SL)1 layer), 75.56% (IMI@(CMCS+SL)2 layer), and 60.91% (IMI@(CMCS+SL)3 layer) in the laccase solution, respectively. In contrast, the cumulative release of IMI@(CMCS+SL)n layers without laccase was minimal.

[0077] According to the method of Example 6, the prepared product was used to test the biological activity of termites, and the insecticidal effect of the synthesized IMI@(CMCS+SL)n layer was evaluated and compared with pure IMI. The results are as follows: Figure 6 As shown. The effects of IMI@(CMCS+SL)n layers and IMI on termite mortality were dose-dependent. After 2 days of treatment, the mortality of termites exposed to IMI@(CMCS+SL)n layers was significantly lower than that of termites exposed to pure IMI. However, after 5 days of treatment, the mortality of termites treated with 1 layer of IMI@(CMCS+SL) was comparable to that of termites treated with pure IMI at the same concentration. The bioactivity of microcapsules with different layers on termites was tested, and the results are shown in Figure 6As shown in the figure, the insecticidal effect of IMI@(CMCS+SL)1 layer is significantly higher than that of IMI@(CMCS+SL)3 layer. The difference in insecticidal activity between IMI@(CMCS+SL)n layer and pure IMI can be attributed to the following factors: In the initial stage, the interaction between IMI@(CMCS+SL)n layer and termites is minimal, so only a small amount of insecticide can be released, resulting in reduced insecticidal effect. However, over time, the alkaline conditions in the termite gut combined with the microenvironmental effect of laccase promote the decomposition of IMI@(CMCS+SL)n layer. This process can release a large amount of encapsulated insecticide on demand, thereby significantly improving the insecticidal activity.

Claims

1. A pH and digestive enzyme dual-responsive termite-killing microcapsule, characterized in that: It is mainly composed of imidacloprid powder IMI, carboxymethyl chitosan CMCS and sodium lignin sulfonate SL.

2. The pH and digestive enzyme dual-responsive termite-killing microcapsule according to claim 1, characterized in that: The mass ratio of the carboxymethyl chitosan CMCS to the sodium lignin sulfonate SL is 2:0.5-1.

3. The pH and digestive enzyme dual-responsive termite-killing microcapsule according to claim 1, characterized in that: As the number of layers of the carboxymethyl chitosan CMCS and sodium lignin sulfonate SL increases, the mass content of imidacloprid IMI in the microcapsule is 92.63-91.67%, and the release rate of imidacloprid IMI is 66.35-52.19%.

4. The method for preparing the pH and digestive enzyme dual-responsive termite-killing microcapsules according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: S1, dissolving carboxymethyl chitosan CMCS in a first co-solvent to obtain a 1 wt% carboxymethyl chitosan solution, and adjusting the pH value; S2, dissolving the imidacloprid powder IMI in the carboxymethyl chitosan solution obtained in step S1, ultrasonicating for 10 minutes, and stirring for 30 minutes to obtain a mixture; S3, centrifuging the mixture obtained in step S2 and collecting the precipitate as precipitate 1, washing the precipitate 1 with deionized water and drying under vacuum conditions to obtain imidacloprid@carboxymethyl chitosan; S4, adding the imidacloprid@carboxymethyl chitosan obtained in step S3 to a 1wt% sodium lignin sulfonate solution, stirring for 30 minutes, centrifuging and collecting the precipitate as precipitate 2, and drying the precipitate 2 under vacuum to obtain a layer of imidacloprid@carboxymethyl chitosan@sodium lignin sulfonate; S5. Replace the imidacloprid powder IMI with one layer of IMI@(CMCS+SL) obtained in step S4, and repeat steps S2-S4 to obtain n layers of imidacloprid@carboxymethyl chitosan@sodium lignin sulfonate, where n is 1-3, to obtain the microcapsules.

5. The method for preparing the pH and digestive enzyme dual-responsive termite-killing microcapsules according to claim 4, characterized in that: In the step S1, the first auxiliary solvent is an acetic acid solution with a volume fraction of 2%.

6. The method for preparing the pH and digestive enzyme dual-responsive termite-killing microcapsules according to claim 4, characterized in that: In the step S1, the pH value is adjusted to 4 by adding HCl and NaOH dropwise to the solution.

7. The method for preparing the pH and digestive enzyme dual-responsive termite-killing microcapsules according to claim 4, characterized in that: In the step S2, the ratio of the mass of imidacloprid powder IMI to the volume of the carboxymethyl chitosan solution is 10-25 mg / ml; In the step S4, the ratio of the mass of imidacloprid@carboxymethyl chitosan to the volume of the sodium lignin sulfonate solution is 10-25 mg / ml.

8. Use of the microcapsule according to any one of claims 1 to 3 or the microcapsule prepared by the preparation method according to any one of claims 4 to 7 in termite control.

9. A method for killing termites using the microcapsules described in any one of claims 1 to 3 or the microcapsules prepared by the preparation method described in any one of claims 4 to 7, characterized in that: The method comprises the following steps: K1, adding the microcapsules to the second cosolvent, placing at room temperature after ultrasonic treatment, to obtain a microcapsule suspension with a concentration of 0.2 to 5.0 ppm; K2. Placing the microcapsule suspension described in step K1 in the living environment of termites or directly applying it to termites, so that the termites contact the microcapsules. After the microcapsules enter the termites, they are specifically released in the termite intestines.

10. The method for using the microcapsules in termite control according to claim 9, characterized in that: The second auxiliary solvent in step K1 is distilled water.

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