Preparation method of yeast-based nitrification inhibitor sustained-release microcapsule

Abstract

The invention discloses a preparation method of a yeast-based nitrification inhibitor sustained-release microcapsule, which comprises the following steps: firstly, carrying out mass decomposition on yeast cells to obtain a mass-decomposed yeast cell solid, namely a yeast microcapsule; dissolving a nitrification inhibitor in an organic solvent to obtain a nitrification inhibitor solution; and then adding the yeast microcapsule into a nitrification inhibitor solution for embedding and carrying a drug, after adsorption and drug loading are completed, centrifugally collecting the drug-loaded yeast microcapsule, cleaning, and freeze-drying to obtain the yeast-based nitrification inhibitor sustained-release microcapsule. The yeast is taken as a microcapsule carrier and is combined with the nitrification inhibitor to form the yeast sustained-release microcapsule, so that the stability, the photolysis resistance and the sustained-release performance can be effectively improved, and the risk of secondary pollution is reduced; the method has the advantages that the entrapment rate of the nitrification inhibitor reaches 17.9-29.7%, the retention rate of the original medicine can reach 94.1-99.1% after the nitrification inhibitor is stored for 10-15 days, the light stability of the nitrification inhibitor is obviously improved, and the accumulative release amount of the nitrification inhibitor reaches 90% or above.

Description

Technical Field

[0001] The invention relates to a method for preparing sustained-release microcapsules, in particular to a method for preparing yeast-based nitration inhibitor sustained-release microcapsules. Background Art

[0002] The extensive use of agricultural fertilizers helps control pests, ensure food security, improve the quality of agricultural products, and maintain ecological and environmental safety. However, due to the large-scale use of nitrogen fertilizers around the world, a large amount of nitrogen is lost during use, leading to serious water pollution problems. In order to improve the utilization rate of nitrogen fertilizers, many nitrification inhibitors are added to fertilizers and applied to the soil. At present, the most researched method is biotechnology, that is, the use of biological inhibitors (urease inhibitors, nitrification inhibitors, ammonia stabilizers, etc.) to improve conventional fertilizers.

[0003] Nitrification inhibitors refer to a class of chemical substances that can inhibit the biological transformation process of ammonium nitrogen into nitrate nitrogen (NCT). The current mainstream nitrification inhibitors include: 2-chloro-6-(trichloromethyl)pyridine, dicyandiamide and 3,4-dimethylpyrazole phosphate. These nitrification inhibitors mainly inhibit the nitrification process of nitrogen atoms on the pyridine ring by competing with ammonia monooxygenase (AMO), reducing the generation and accumulation of nitrate nitrogen in the soil, thereby reducing the loss of nitrogen fertilizer in the form of nitrate nitrogen and its impact on the ecological environment. Some research results show that nitrification inhibitors are beneficial to reducing nitrogen leaching losses and greenhouse gas (nitrogen oxide) emissions, and have a positive effect on improving fertilizer efficiency under certain conditions. However, in the actual soil application process, nitrification inhibitors inevitably have many disadvantages after entering the soil: expensive price, high residual pollution, large toxic side effects, migration is greatly affected by the pH of the environmental medium, easy to lose, short half-life, and short effective inhibition time. Therefore, it is urgent to find a stable biological inhibitor.

[0004] At present, regarding the slow-release technology of nitrification inhibitors, Patent 2019101174484 discloses a stable nitrification inhibitor and its preparation method and application, and the nitrification inhibitor includes DMPP and a protective agent. This technology sprays DMPP and protective agents by spray drying, but there are problems such as uneven spraying and complex preparation. Patent 202011183617 discloses a soil nitrification inhibitor embedded in sodium carboxymethyl cellulose, which has stable performance, simple preparation process, and good denitrification effect, but the drug loading and encapsulation rate of this technology are low.

[0005] Existing studies have shown that microcapsule sustained-release technology effectively improves the stability, photolysis resistance and sustained-release performance of nitrification inhibitors by encapsulating active pesticide ingredients in a thin film made of natural, semi-natural or synthetic polymer materials through chemical, physical or physicochemical means. However, in actual application, microcapsule sustained-release technology is limited by factors such as preparation technology and wall materials, and has not been widely used in agricultural production. Therefore, how to improve the stability and sustained-release properties of microcapsules for agricultural chemicals with limited application in the environment has become a research hotspot in the field of microencapsulated pesticide preparation. Summary of the invention

[0006] Purpose of the invention: The purpose of the present invention is to provide a method for preparing yeast-based nitrification inhibitor sustained-release microcapsules that can improve stability, resistance to photolysis and sustained-release performance.

[0007] Technical solution: The method for preparing the yeast-based nitrification inhibitor sustained-release microcapsules of the present invention comprises the following steps:

[0008] (1) hydrolyzing yeast cells to obtain hydrolyzed yeast cell solids, namely yeast microcapsules;

[0009] (2) dissolving the nitrification inhibitor in an organic solvent to obtain a nitrification inhibitor solution;

[0010] (3) adding the yeast microcapsules into the nitrification inhibitor solution to embed and load the drug. After the drug is adsorbed and loaded, the drug-loaded yeast microcapsules are collected by centrifugation, washed, and freeze-dried to obtain yeast-based nitrification inhibitor sustained-release microcapsules.

[0011] Wherein, in step (1), the yeast cell is at least one of fresh yeast, active dry yeast or highly active dry yeast.

[0012] Wherein, in step (2), the nitrification inhibitor is at least one of 2-chloro-6-(trichloromethyl)pyridine, dicyandiamide or 3,4-dimethylpyrazole phosphate. When yeast cells are used as drug-carrying materials to load pesticides, the pesticides usually contain metal ions, which will react with polysaccharides or proteins in the yeast cell wall to form a complex, making it difficult to release them slowly, so it is necessary to add a nitrification inhibitor.

[0013] In step (2), the temperature for drug embedding is 30-60°C and the time is 1-2 days. Too low or too high drug loading temperature will lead to a reduction in drug loading amount, while a reduction in drug loading time will also lead to an insufficient reaction between the nitrification inhibitor and the yeast cells.

[0014] Wherein, in step (2), the organic solvent is a mixed solvent of water and at least one of ethanol, methanol or acetone.

[0015] Wherein, in step (3), the material is placed in a shaking table and shaken to embed the drug; the shaking table shakes at a rotation speed of 5000 to 8000 rpm.

[0016] Wherein, in step (3), the mass ratio of the nitrification inhibitor to the yeast microcapsule is 0.5 to 3.5. When the nitrification inhibitor is excessive, a large amount of nitrification inhibitor will be adsorbed on the outer wall of the yeast cell. In addition, when the content of the nitrification inhibitor is too low, the drug loading of the yeast cell will be reduced.

[0017] Wherein, in step (3), an organic solvent is used for cleaning; the organic solvent is at least one of ethanol, methanol, acetonitrile or acetone.

[0018] Beneficial effects: Compared with the prior art, the present invention achieves the following significant effects:

[0019] (1) The present invention uses yeast as a microcapsule carrier to combine with a nitrification inhibitor to form a yeast sustained-release microcapsule, which can effectively improve stability, resistance to photolysis and sustained-release performance, and reduce the risk of secondary pollution. (2) The mass ratio of the nitrification inhibitor to the yeast microcapsule of the present invention is 0.5-3.5, the optimal encapsulation rate is 25%-40%, and the retention rate of the nitrification inhibitor is above 95% at different storage temperatures (4°C-55°C). Under ultraviolet irradiation conditions, the retention rate of the nitrification inhibitor is still 50-60%, which is more than 15% higher than that of the nitrification inhibitor alone. Under different temperature (4°C-55°C) and pH (3-9) conditions, the cumulative release rate within 5 days reaches more than 90%, and is less affected by temperature and pH. (3) The present invention uses yeast microcapsules as slow-release carriers, the raw materials are easily available, the preparation method is simple and easy to control, and the purpose of waste resource utilization is achieved. At the same time, the slow-release nitrification inhibitor microcapsules are applied to the soil, which can significantly reduce the loss rate and photolysis rate of the nitrification inhibitor, improve the utilization rate of the nitrification inhibitor, avoid secondary pollution, and overcome the problems of soil ammonia nitrogen generation and unstable supply during the use of conventional nitrification inhibitors, and have good promotion and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a scanning electron micrograph of the nitrification inhibitor yeast sustained-release microcapsules prepared in Example 1;

[0021] Figure 2 is a graph showing the photodegradation efficiency of the yeast sustained-release microcapsules of the nitrification inhibitor prepared in Example 1;

[0022] Figure 3 It is the sustained-release graph of the nitrification inhibitor yeast sustained-release microcapsules prepared in Example 1 under different temperature and pH conditions. DETAILED DESCRIPTION

[0023] The present invention is described in further detail below.

[0024] Example 1

[0025] A method for preparing yeast-based nitrification inhibitor sustained-release microcapsules comprises the following steps:

[0026] (1) Preparation of yeast microcapsules

[0027] Commercially available high-activity dry yeast cells with a moisture content of 5-7% are suspended in a phosphate buffer solution at pH = 7 for washing, and then centrifuged and the yeast cells are lysed in a 10% w / w sodium chloride solution. After the lysis is completed, the cytoplasmic yeast cells are collected by low-temperature centrifugation at 4°C, and then washed with deionized water to remove cell residues and sodium chloride. After freeze-drying, the lysed yeast cell solid is obtained, which is the yeast microcapsule.

[0028] (2) Preparation of yeast sustained-release microcapsules of nitrification inhibitors

[0029] 1 g of 2-chloro-6-(trichloromethyl)pyridine was weighed and dissolved in ethanol, and then 0.8 g of yeast microcapsules were added for drug encapsulation and placed in a shaker, and the encapsulation temperature was 45°C and the temperature was kept constant at 6000 rpm for 36 hours. After the drug adsorption was completed, the drug-loaded yeast microcapsules were collected by centrifugation, washed three times with deionized water, freeze-dried, and ground into powder after drying, which was the nitrification inhibitor yeast sustained-release microcapsules.

[0030] Stability and sustained-release effect of yeast-based nitrification inhibitor sustained-release microcapsules:

[0031] like Figure 1 As shown in Figure 2, the prepared microcapsules are complete in morphology and are ellipsoidal in shape. Figure 2 As shown in FIG. 1 , the encapsulation efficiency of the nitrification inhibitor yeast sustained-release microcapsules for 2-chloro-6-(trichloromethyl)pyridine reaches 30%. Figure 3 As shown in the figure, the nitrification inhibitor yeast sustained-release microcapsules have a retention rate of 2-chloro-6-(trichloromethyl)pyridine of more than 95% at a storage temperature of 4 to 55°C, and the particle size does not change significantly at room temperature. After 10 hours of light exposure, the retention rate of 2-chloro-6-(trichloromethyl)pyridine in the yeast microcapsules is more than 50%, and under different temperature and pH conditions, the cumulative release rate within 5 days reaches more than 90%.

[0032] Example 2

[0033] A method for preparing yeast-based nitrification inhibitor sustained-release microcapsules comprises the following steps:

[0034] (1) Preparation of yeast microcapsules

[0035] Commercially available high-activity dry yeast cells with a moisture content of 5-7% are suspended in a phosphate buffer solution at pH=7 for washing, and then centrifuged, and the yeast cells are lysed in a 15% (w / w) sodium chloride solution. After the lysis of the cytoplasm, the cytoplasmic yeast cells are collected by low-temperature centrifugation at 7°C, and then washed with deionized water to remove cell residues and sodium chloride. After freeze-drying, the lysed yeast cell solid is obtained, which is the yeast microcapsule.

[0036] (2) Preparation of yeast-based nitrification inhibitor sustained-release microcapsules

[0037] Weigh 1g of a mixture of dicyandiamide and 2-chloro-6-(trichloromethyl)pyridine and dissolve them in methanol, then add 2g of yeast microcapsules for drug encapsulation and placement in a shaker, oscillate at 6000rpm for 24h at an encapsulation temperature of 35°C. After drug adsorption, collect the drug-loaded yeast microcapsules by centrifugation, wash them three times with a methanol aqueous solution, freeze-dry, and grind them into powder after drying to obtain nitrification inhibitor yeast sustained-release microcapsules.

[0038] Stability and sustained-release effect of yeast-based nitrification inhibitor sustained-release microcapsules:

[0039] The prepared microcapsules are complete and ellipsoidal in shape, and the encapsulation rate of the mixture of dicyandiamide and 2-chloro-6-(trichloromethyl)pyridine reaches 23.86%. At a storage temperature of 4 to 55°C, the retention rate of dicyandiamide is above 95%, and the particle size does not change significantly at room temperature. After 10 hours of light exposure, the retention rate of the mixture of dicyandiamide and 2-chloro-6-(trichloromethyl)pyridine in the yeast microcapsules is above 50%. Under different temperature and pH conditions, the cumulative release rate within 5 days reaches above 90%.

[0040] Example 3

[0041] A method for preparing yeast sustained-release microcapsules of nitrification inhibitors comprises the following steps:

[0042] (1) Preparation of yeast microcapsules

[0043] Commercially available high-activity dry yeast cells with a moisture content of 5-7% are suspended in a phosphate buffer solution at pH=8 for washing, and then centrifuged and the yeast cells are lysed in a 20% (w / w) sodium chloride solution. After the lysis is completed, the cytoplasmic yeast cells are collected by low-temperature centrifugation at 10°C, and then washed with deionized water to remove cell residues and sodium chloride. After freeze-drying, the lysed yeast cell solid is obtained, which is the yeast microcapsule.

[0044] (2) Preparation of nitrification inhibitor yeast sustained-release microcapsules

[0045] Weigh 1g of a mixture of 3,4-dimethylpyrazole phosphate and 2-chloro-6-(trichloromethyl)pyridine and dissolve them in acetonitrile, then add 0.5g of yeast microcapsules for drug encapsulation and placement in a shaker, oscillate at 6000rpm for 48h at an encapsulation temperature of 55°C. After the drug adsorption is completed, the drug-loaded yeast microcapsules are collected by centrifugation, washed three times with methanol, freeze-dried, and ground into powder after drying, which is the nitrification inhibitor yeast sustained-release microcapsules.

[0046] Stability and sustained-release effect of yeast-based nitrification inhibitor sustained-release microcapsules:

[0047] The prepared microcapsules are complete in ellipsoidal shape, and the encapsulation rate of the mixture of 3,4-dimethylpyrazole phosphate and 2-chloro-6-(trichloromethyl)pyridine reaches 24.41%. At a storage temperature of 4 to 55°C, the retention rate of the mixture of 3,4-dimethylpyrazole phosphate and 2-chloro-6-(trichloromethyl)pyridine is above 95%, and the particle size does not change significantly at room temperature. After 10 hours of light exposure, the retention rate of the mixture of 3,4-dimethylpyrazole phosphate and 2-chloro-6-(trichloromethyl)pyridine in the yeast microcapsules is above 50%. Under different temperature and pH conditions, the cumulative release rate within 5 days reaches above 90%.

[0048] Comparative Example 1

[0049] Based on Example 1, the difference from Example 1 is that in step (1), the yeast cells are not subjected to hydrolysis treatment, but the fresh yeast cells are only suspended in a phosphate buffer at pH = 7 for washing, and then washed with deionized water to remove cell residues, and then freeze-dried to obtain yeast microcapsules. The prepared microcapsules are complete in ellipsoidal shape, and the encapsulation rate of 2-chloro-6-(trichloromethyl)pyridine is 0%.

[0050] Comparative Example 2

[0051] Based on Example 1, the difference from Example 1 is that in step (2), the oscillation is performed at an embedding temperature of 4° C. The prepared microcapsules are complete in ellipsoidal shape, and the encapsulation rate of 2-chloro-6-(trichloromethyl)pyridine is 0%.

[0052] Comparative Example 3

[0053] Based on Example 1, the difference from Example 1 is that in step (2), 0.5 g of 3,2-chloro-6-(trichloromethyl)pyridine is weighed and 1 g of yeast microcapsules are added. The prepared microcapsules have a complete ellipsoidal shape and an encapsulation efficiency of 2-chloro-6-(trichloromethyl)pyridine of 8.2%.

[0054] Comparative Example 4

[0055] Based on Example 1, the difference from Example 1 is that in step (2), phoxim pesticide is added. The prepared microcapsules are complete in ellipsoidal shape, with an encapsulation efficiency of phoxim of 18.2% and a cumulative release rate of only 3.2% within 5 days.

[0056] Comparative Example 5

[0057] Based on Example 1, the difference from Example 1 is that in step (1), the yeast cells are lysed in 80% (w / w) sodium chloride solution. The prepared microcapsules are incomplete in morphology, the cell walls are damaged, and the encapsulation efficiency of 2-chloro-6-(trichloromethyl)pyridine is 0%.

[0058] The present invention uses yeast as a microcapsule carrier and combines it with a nitrification inhibitor to form a yeast slow-release microcapsule, which can effectively improve stability, photolysis resistance and slow-release performance and reduce the risk of secondary pollution.

Claims

1. A method for preparing yeast-based nitrification inhibitor sustained-release microcapsules, characterized in that: The following steps are involved: (1) hydrolyzing yeast cells to obtain hydrolyzed yeast cell solids, namely yeast microcapsules; (2) dissolving the nitrification inhibitor in an organic solvent to obtain a nitrification inhibitor solution; (3) adding the yeast microcapsules into the nitrification inhibitor solution to embed and load the drug. After the drug is adsorbed and loaded, the drug-loaded yeast microcapsules are collected by centrifugation, washed, and freeze-dried to obtain yeast-based nitrification inhibitor sustained-release microcapsules.

2. The method for preparing the yeast-based nitrification inhibitor sustained-release microcapsules according to claim 1, characterized in that: In step (3), the mass ratio of the nitrification inhibitor to the yeast microcapsule is 0.5 to 3.

5.

3. The method for preparing the yeast-based nitrification inhibitor sustained-release microcapsules according to claim 1, characterized in that: In step (1), the yeast cell is at least one of fresh yeast, active dry yeast or highly active dry yeast.

4. The method for preparing the yeast-based nitrification inhibitor sustained-release microcapsules according to claim 1, characterized in that: In step (2), the nitrification inhibitor is at least one of 2-chloro-6-(trichloromethyl)pyridine, dicyandiamide or 3,4-dimethylpyrazole phosphate.

5. The method for preparing the yeast-based nitrification inhibitor sustained-release microcapsules according to claim 1, characterized in that: In step (2), the temperature for embedding the drug is 30 to 60° C. and the time is 1 to 2 days.

6. The method for preparing the yeast-based nitrification inhibitor sustained-release microcapsules according to claim 1, characterized in that: In step (3), the mixture is placed in a shaker and shaken to carry out drug encapsulation.

7. The method for preparing the yeast-based nitration inhibitor sustained-release microcapsules according to claim 6, characterized in that: The shaking table oscillates at a rotation speed of 5000 to 8000 rpm.

8. The method for preparing yeast-based nitrification inhibitor sustained-release microcapsules according to claim 1, characterized in that: In step (2), the organic solvent is a mixed solvent of water and at least one of ethanol, methanol or acetone.

9. The method for preparing yeast-based nitrification inhibitor sustained-release microcapsules according to claim 1, characterized in that: In step (3), washing is performed using an organic solvent.

10. The method for preparing yeast-based nitrification inhibitor sustained-release microcapsules according to claim 9, characterized in that: The organic solvent is at least one of ethanol, methanol, acetonitrile or acetone.

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