Plant antibacterial agent and preparation method thereof

Abstract

The invention belongs to the field of plant protection, and particularly relates to a plant antibacterial agent and a preparation method thereof.The preparation method comprises the steps that p-methoxycinnamic acid and hexamethylenediamine are grafted, and then an intermediate is obtained; amidating amino on the intermediate and gallic acid to obtain a complex ligand; complexing the complexing ligand with copper ions to obtain an antibacterial agent; the plant antibacterial agent disclosed by the invention is good in antibacterial effect, long in antibacterial time and environment-friendly.

Description

Technical Field

[0001] The invention belongs to the field of plant protection, and specifically relates to a plant antibacterial agent and a preparation method thereof. Background Art

[0002] Microbial infections such as bacteria and fungi are the main causes of disease in animals and plants. The economic losses of crops caused by plant fungal diseases amount to hundreds of billions of dollars each year. Chemical control is popular among farmers because of its quick effect and low cost. However, while pesticides increase crop yields and agricultural productivity, they also expose a series of serious problems. The large-scale use of pesticides to control plant diseases not only seriously damages the ecological balance, but also leads to the emergence of drug-resistant strains. The drug resistance of agricultural pathogens increases year by year, and crop yields decrease significantly. Therefore, there is an urgent need to develop new pesticide substitutes. Therefore, plant antimicrobial agents that are safe for humans, animals and natural enemies, environmentally friendly, and have diverse biological activities have attracted more and more attention. Summary of the invention

[0003] In order to solve the problem in the prior art that the frequent use of traditional chemical fungicides increases the resistance of plant pathogens and reduces the bactericidal effect of fungicides, the present invention mainly provides a plant antibacterial agent with high efficiency, safety and long antibacterial effect, and a preparation method of the plant antibacterial agent, and the technical scheme is as follows:

[0004] A plant antibacterial agent comprises a complex ligand formed by p-methoxycinnamic acid and gallic acid linked by hexamethylenediamine, and a copper ion complexed with the complex ligand.

[0005] A preparation method of the above-mentioned plant antibacterial agent comprises the following steps: grafting p-methoxycinnamic acid with hexamethylenediamine to obtain an intermediate; amidating the amino group on the intermediate with gallic acid to obtain a complex ligand; and complexing the complex ligand with copper ions to obtain the product.

[0006] Further, the following steps are included:

[0007] a. Disperse p-methoxycinnamic acid and potassium hydroxide in water, add hexamethylenediamine, reflux at 70-80°C for 3-5h, filter, wash and dry to obtain the intermediate;

[0008] b. placing the intermediate and gallic acid in a catalytic solvent, fully dispersing them, and adjusting the pH of the system to a weakly acidic state using hydrochloric acid to obtain a reaction solution;

[0009] c. Under a nitrogen atmosphere, the reaction solution was stirred at a low speed and then at a high speed at 50-60°C; after the reaction was completed, it was cooled to room temperature, the mixed product after the reaction was filtered, washed, and then freeze-dried to obtain a complex ligand;

[0010] d. Dissolve copper sulfate in dilute ethanol, add the complexing ligand and mix thoroughly, then stir at a low speed for 10 to 30 minutes and let stand for 0.5 to 1 hour, freeze-dry the system, and then fully disperse it in anhydrous ethanol. After removing the precipitate, freeze-dry the solution to obtain the product.

[0011] Furthermore, the molar ratio of p-methoxycinnamic acid to potassium hydroxide in step a is 1:(1.8-2.2); the molar ratio of p-methoxycinnamic acid to hexamethylenediamine in step a is 1:(0.8-1.2).

[0012] Furthermore, the molar ratio of the intermediate to gallic acid in step b is 1:(1.1-1.3); and the pH of the system is adjusted to 5.5-6.5 using hydrochloric acid in step b.

[0013] Furthermore, in step c, the first low-speed stirring reaction and then high-speed stirring reaction is to first stir the reaction at a speed of 300-500 r / min for 10-20 min, and then stir the reaction at a speed of 1200-1500 r / min for 0.5-1 h.

[0014] Furthermore, the low-speed stirring in step d is stirring at a speed of 100 to 200 r / min; and the molar ratio of the complexing ligand to copper sulfate in step d is 1:(2 to 2.5).

[0015] Furthermore, the preparation of the catalytic solvent includes the following steps: taking zinc chloride and tetrabutylammonium chloride and mixing them thoroughly at 70-80° C. until the system becomes clear, and the molar ratio of the zinc chloride to tetrabutylammonium chloride is 1:(1.1-1.2).

[0016] Furthermore, the prepared plant antibacterial agent is loaded on a carrier, specifically comprising the following steps: adding xanthan gum to 80°C water while stirring, mixing evenly to obtain a 0.5-1.5% w / v solution, heating to 90°C and keeping warm for 1-2 hours, cooling to room temperature and adding the plant antibacterial agent to a concentration of 10-100 mg / L, and obtaining the product after homogenization.

[0017] A plant antibacterial method using the plant antibacterial agent allows the plant antibacterial agent to be evenly covered on the surface of a plant that can receive light.

[0018] By adopting the above scheme, the method of the present invention has the following advantages:

[0019] 1. After irradiation with light, the present invention can lead to the generation of reactive oxygen with high antibacterial activity, which can diffuse through the cell membrane and react with many biological macromolecules in the cell, destroying the normal structure and function of the cell, thereby causing the death of pathogens. Compared with traditional bactericidal effects, the antibacterial method of the present invention has high selectivity and less damage to the plant itself.

[0020] 2. The present invention complexes copper ions with ligands, which can destroy the membrane structure and membrane lipids of plant pathogens on the surface of plants, damage the internal genetic material of the pathogens, thereby affecting the growth of the plant pathogens and hindering their reproduction.

[0021] 3. The antibacterial ligand of the present invention has multiple ortho-phenolic groups and amide groups that can complex with copper ions, and can bind more copper ions to improve the antibacterial ability of the antibacterial agent.

[0022] 4. The polyphenols of the present invention are complexed with metal ions and have high biocompatibility, and the amide groups and phenol groups on the complexing ligands can interact with the phospholipid membrane to destroy the cell membranes of surface microbial pathogens.

[0023] 5. The catalytic solvent of the present invention is a low eutectic solvent, which forms hydrogen bonds after synthesis and can more effectively attack reactants, making the reaction easier to occur.

[0024] 6. The antibacterial agent of the present invention has a long chain connection in the middle, is spindle-shaped, and has copper ions complexed at both ends, which can be easily embedded in the convex structure on the surface of microorganisms, can be tightly combined with the surface of microorganisms, is not easy to fall off, and has a long antibacterial effect and action time.

[0025] 7. The preparation method of the present invention is simple, the production cost and threshold are low, and it is driven by light as energy during application, which is environmentally friendly and healthy. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. 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.

[0027] Example 1: (1) Zinc chloride and tetrabutylammonium chloride are mixed at a molar ratio of 1:1.2 at 70-80° C. until the system becomes clear, thereby obtaining a catalytic solvent;

[0028] (2) dispersing p-methoxycinnamic acid and potassium hydroxide in water at a molar ratio of 1:1.8, adding hexamethylenediamine in an amount equal to 1 times the amount of p-methoxycinnamic acid, and reacting under reflux at 70-80° C. for 3 h, filtering, washing, and drying to obtain an intermediate;

[0029] (3) placing the intermediate and gallic acid in a catalytic solvent at a molar ratio of 1:1.1, fully dispersing them, and adjusting the pH of the system to 5.5 with hydrochloric acid to obtain a reaction solution;

[0030] (4) Under a nitrogen atmosphere, the reaction solution was stirred at 50-60° C. at a speed of 300 r / min for 15 min, and then at a speed of 1200 r / min for 0.5 h; after the reaction was completed, the mixture was cooled to room temperature, filtered, washed, and freeze-dried to obtain a complex ligand;

[0031] (5) Dissolve copper sulfate in dilute ethanol, add 0.45 times the amount of complexing ligand as copper sulfate, mix thoroughly, stir at 150 r / min for 10 min, let stand for 0.5 h, freeze-dry the system, and then fully disperse it in anhydrous ethanol. After removing the precipitate, freeze-dry the solution to obtain;

[0032] (6) Add xanthan gum to 80°C water while stirring, mix well to obtain a 0.5% w / v solution, heat to 90°C and keep warm for 1 hour, cool to room temperature and add the plant antibacterial agent obtained in step (5) to a concentration of 50 mg / L. After homogenization, the loading is completed.

[0033] Example 2: (1) Zinc chloride and tetrabutylammonium chloride are mixed at a molar ratio of 1:1.2 at 70-80° C. until the system becomes clear, thereby obtaining a catalytic solvent;

[0034] (2) dispersing p-methoxycinnamic acid and potassium hydroxide in water at a molar ratio of 1:2.2, adding hexamethylenediamine in an amount equal to 1 times the amount of p-methoxycinnamic acid, and reacting under reflux at 70-80° C. for 3 h, filtering, washing, and drying to obtain an intermediate;

[0035] (3) placing the intermediate and gallic acid in a catalytic solvent at a molar ratio of 1:1.1, fully dispersing them, and adjusting the pH of the system to 5.5 with hydrochloric acid to obtain a reaction solution;

[0036] (4) Under a nitrogen atmosphere, the reaction solution was stirred at 50-60° C. at a speed of 300 r / min for 15 min, and then at a speed of 1200 r / min for 0.5 h; after the reaction was completed, the mixture was cooled to room temperature, filtered, washed, and freeze-dried to obtain a complex ligand;

[0037] (5) Dissolve copper sulfate in dilute ethanol, add 0.45 times the amount of complexing ligand as copper sulfate, mix thoroughly, stir at 150 r / min for 10 min, let stand for 0.5 h, freeze-dry the system, and then fully disperse it in anhydrous ethanol. After removing the precipitate, freeze-dry the solution to obtain;

[0038] (6) Add xanthan gum to 80°C water while stirring, mix well to obtain a 0.5% w / v solution, heat to 90°C and keep warm for 1 hour, cool to room temperature and add the plant antibacterial agent obtained in step (5) to a concentration of 50 mg / L. After homogenization, the loading is completed.

[0039] Example 3: (1) Zinc chloride and tetrabutylammonium chloride are mixed at a molar ratio of 1:1.2 at 70-80° C. until the system becomes clear, thereby obtaining a catalytic solvent;

[0040] (2) dispersing p-methoxycinnamic acid and potassium hydroxide in water at a molar ratio of 1:1.8, adding hexamethylenediamine in an amount equal to 1 times the amount of p-methoxycinnamic acid, and reacting under reflux at 70-80° C. for 5 h, filtering, washing, and drying to obtain an intermediate;

[0041] (3) placing the intermediate and gallic acid in a catalytic solvent at a molar ratio of 1:1.1, fully dispersing them, and adjusting the pH of the system to 5.5 with hydrochloric acid to obtain a reaction solution;

[0042] (4) Under a nitrogen atmosphere, the reaction solution was stirred at 50-60° C. at a speed of 300 r / min for 15 min, and then at a speed of 1200 r / min for 0.5 h; after the reaction was completed, the mixture was cooled to room temperature, filtered, washed, and freeze-dried to obtain a complex ligand;

[0043] (5) Dissolve copper sulfate in dilute ethanol, add 0.45 times the amount of complexing ligand as copper sulfate, mix thoroughly, stir at 150 r / min for 10 min, let stand for 0.5 h, freeze-dry the system, and then fully disperse it in anhydrous ethanol. After removing the precipitate, freeze-dry the solution to obtain;

[0044] (6) Add xanthan gum to 80°C water while stirring, mix well to obtain a 0.5% w / v solution, heat to 90°C and keep warm for 1 hour, cool to room temperature and add the plant antibacterial agent obtained in step (5) to a concentration of 50 mg / L. After homogenization, the loading is completed.

[0045] Example 4: (1) Zinc chloride and tetrabutylammonium chloride are taken in a molar ratio of 1:1.2 and mixed thoroughly at 70-80° C. until the system becomes clear, thereby obtaining a catalytic solvent;

[0046] (2) dispersing p-methoxycinnamic acid and potassium hydroxide in water at a molar ratio of 1:1.8, adding hexamethylenediamine in an amount equal to 1 times the amount of p-methoxycinnamic acid, and reacting under reflux at 70-80° C. for 3 h, filtering, washing, and drying to obtain an intermediate;

[0047] (3) placing the intermediate and gallic acid in a catalytic solvent at a molar ratio of 1:1.3, fully dispersing them, and adjusting the pH of the system to 5.5 with hydrochloric acid to obtain a reaction solution;

[0048] (4) Under a nitrogen atmosphere, the reaction solution was stirred at 50-60° C. at a speed of 300 r / min for 15 min, and then at a speed of 1200 r / min for 0.5 h; after the reaction was completed, the mixture was cooled to room temperature, filtered, washed, and freeze-dried to obtain a complex ligand;

[0049] (5) Dissolve copper sulfate in dilute ethanol, add 0.45 times the amount of complexing ligand as copper sulfate, mix thoroughly, stir at 150 r / min for 10 min, let stand for 0.5 h, freeze-dry the system, and then fully disperse it in anhydrous ethanol. After removing the precipitate, freeze-dry the solution to obtain;

[0050] (6) Add xanthan gum to 80°C water while stirring, mix well to obtain a 0.5% w / v solution, heat to 90°C and keep warm for 1 hour, cool to room temperature and add the plant antibacterial agent obtained in step (5) to a concentration of 50 mg / L. After homogenization, the loading is completed.

[0051] Example 5: (1) Zinc chloride and tetrabutylammonium chloride are taken in a molar ratio of 1:1.2 and mixed thoroughly at 70-80° C. until the system becomes clear, thereby obtaining a catalytic solvent;

[0052] (2) dispersing p-methoxycinnamic acid and potassium hydroxide in water at a molar ratio of 1:1.8, adding hexamethylenediamine in an amount equal to 1 times the amount of p-methoxycinnamic acid, and reacting under reflux at 70-80° C. for 3 h, filtering, washing, and drying to obtain an intermediate;

[0053] (3) placing the intermediate and gallic acid in a catalytic solvent at a molar ratio of 1:1.1, fully dispersing them, and adjusting the pH of the system to 6.5 with hydrochloric acid to obtain a reaction solution;

[0054] (4) Under a nitrogen atmosphere, the reaction solution was stirred at 50-60° C. at a speed of 300 r / min for 15 min, and then at a speed of 1200 r / min for 0.5 h; after the reaction was completed, the mixture was cooled to room temperature, filtered, washed, and freeze-dried to obtain a complex ligand;

[0055] (5) Dissolve copper sulfate in dilute ethanol, add 0.45 times the amount of complexing ligand as copper sulfate, mix thoroughly, stir at 150 r / min for 10 min, let stand for 0.5 h, freeze-dry the system, and then fully disperse it in anhydrous ethanol. After removing the precipitate, freeze-dry the solution to obtain;

[0056] (6) Add xanthan gum to 80°C water while stirring, mix well to obtain a 0.5% w / v solution, heat to 90°C and keep warm for 1 hour, cool to room temperature and add the plant antibacterial agent obtained in step (5) to a concentration of 50 mg / L. After homogenization, the loading is completed.

[0057] Example 6: (1) Zinc chloride and tetrabutylammonium chloride are mixed at a molar ratio of 1:1.2 at 70-80° C. until the system becomes clear, thereby obtaining a catalytic solvent;

[0058] (2) dispersing p-methoxycinnamic acid and potassium hydroxide in water at a molar ratio of 1:1.8, adding hexamethylenediamine in an amount equal to 1 times the amount of p-methoxycinnamic acid, and reacting under reflux at 70-80° C. for 3 h, filtering, washing, and drying to obtain an intermediate;

[0059] (3) placing the intermediate and gallic acid in a catalytic solvent at a molar ratio of 1:1.1, fully dispersing them, and adjusting the pH of the system to 5.5 with hydrochloric acid to obtain a reaction solution;

[0060] (4) Under a nitrogen atmosphere, the reaction solution was stirred at 50-60° C. at a speed of 500 r / min for 15 min, and then at a speed of 1200 r / min for 0.5 h; after the reaction was completed, the mixture was cooled to room temperature, filtered, washed, and freeze-dried to obtain a complex ligand;

[0061] (5) Dissolve copper sulfate in dilute ethanol, add 0.45 times the amount of complexing ligand as copper sulfate, mix thoroughly, stir at 150 r / min for 10 min, let stand for 0.5 h, freeze-dry the system, and then fully disperse it in anhydrous ethanol. After removing the precipitate, freeze-dry the solution to obtain;

[0062] (6) Add xanthan gum to 80°C water while stirring, mix well to obtain a 0.5% w / v solution, heat to 90°C and keep warm for 1 hour, cool to room temperature and add the plant antibacterial agent obtained in step (5) to a concentration of 50 mg / L. After homogenization, the loading is completed.

[0063] Example 7: (1) Zinc chloride and tetrabutylammonium chloride are mixed at a molar ratio of 1:1.2 at 70-80° C. until the system becomes clear, thereby obtaining a catalytic solvent;

[0064] (2) dispersing p-methoxycinnamic acid and potassium hydroxide in water at a molar ratio of 1:1.8, adding hexamethylenediamine in an amount equal to 1 times the amount of p-methoxycinnamic acid, and reacting under reflux at 70-80° C. for 3 h, filtering, washing, and drying to obtain an intermediate;

[0065] (3) placing the intermediate and gallic acid in a catalytic solvent at a molar ratio of 1:1.1, fully dispersing them, and adjusting the pH of the system to 5.5 with hydrochloric acid to obtain a reaction solution;

[0066] (4) Under a nitrogen atmosphere, the reaction solution was stirred at 50-60° C. at a speed of 300 r / min for 15 min, and then at a speed of 1500 r / min for 0.5 h; after the reaction was completed, the mixture was cooled to room temperature, filtered, washed, and freeze-dried to obtain a complex ligand;

[0067] (5) Dissolve copper sulfate in dilute ethanol, add 0.45 times the amount of complexing ligand as copper sulfate, mix thoroughly, stir at 150 r / min for 10 min, let stand for 0.5 h, freeze-dry the system, and then fully disperse it in anhydrous ethanol. After removing the precipitate, freeze-dry the solution to obtain;

[0068] (6) Add xanthan gum to 80°C water while stirring, mix well to obtain a 0.5% w / v solution, heat to 90°C and keep warm for 1 hour, cool to room temperature and add the plant antibacterial agent obtained in step (5) to a concentration of 50 mg / L. After homogenization, the loading is completed.

[0069] Example 8: (1) Zinc chloride and tetrabutylammonium chloride are taken in a molar ratio of 1:1.2 and mixed thoroughly at 70-80° C. until the system becomes clear, thereby obtaining a catalytic solvent;

[0070] (2) dispersing p-methoxycinnamic acid and potassium hydroxide in water at a molar ratio of 1:1.8, adding hexamethylenediamine in an amount equal to 1 times the amount of p-methoxycinnamic acid, and reacting under reflux at 70-80° C. for 3 h, filtering, washing, and drying to obtain an intermediate;

[0071] (3) placing the intermediate and gallic acid in a catalytic solvent at a molar ratio of 1:1.1, fully dispersing them, and adjusting the pH of the system to 5.5 with hydrochloric acid to obtain a reaction solution;

[0072] (4) Under a nitrogen atmosphere, the reaction solution was stirred at 50-60° C. at a speed of 300 r / min for 15 min, and then at a speed of 1200 r / min for 1 h; after the reaction was completed, the mixture was cooled to room temperature, filtered, washed, and freeze-dried to obtain a complex ligand;

[0073] (5) Dissolve copper sulfate in dilute ethanol, add 0.45 times the amount of complexing ligand as copper sulfate, mix thoroughly, stir at 150 r / min for 10 min, let stand for 0.5 h, freeze-dry the system, and then fully disperse it in anhydrous ethanol. After removing the precipitate, freeze-dry the solution to obtain;

[0074] (6) Add xanthan gum to 80°C water while stirring, mix well to obtain a 0.5% w / v solution, heat to 90°C and keep warm for 1 hour, cool to room temperature and add the plant antibacterial agent obtained in step (5) to a concentration of 50 mg / L. After homogenization, the loading is completed.

[0075] Example 9: (1) Zinc chloride and tetrabutylammonium chloride are taken in a molar ratio of 1:1.2 and mixed thoroughly at 70-80° C. until the system becomes clear, thereby obtaining a catalytic solvent;

[0076] (2) dispersing p-methoxycinnamic acid and potassium hydroxide in water at a molar ratio of 1:1.8, adding hexamethylenediamine in an amount equal to 1 times the amount of p-methoxycinnamic acid, and reacting under reflux at 70-80° C. for 3 h, filtering, washing, and drying to obtain an intermediate;

[0077] (3) placing the intermediate and gallic acid in a catalytic solvent at a molar ratio of 1:1.1, fully dispersing them, and adjusting the pH of the system to 5.5 with hydrochloric acid to obtain a reaction solution;

[0078] (4) Under a nitrogen atmosphere, the reaction solution was stirred at 50-60° C. at a speed of 300 r / min for 15 min, and then at a speed of 1200 r / min for 0.5 h; after the reaction was completed, the mixture was cooled to room temperature, filtered, washed, and freeze-dried to obtain a complex ligand;

[0079] (5) Dissolve copper sulfate in dilute ethanol, add 0.45 times the amount of complexing ligand as copper sulfate, mix thoroughly, stir at 150 r / min for 30 min, let stand for 0.5 h, freeze-dry the system, and then fully disperse it in anhydrous ethanol. After removing the precipitate, freeze-dry the solution to obtain;

[0080] (6) Add xanthan gum to 80°C water while stirring, mix well to obtain a 0.5% w / v solution, heat to 90°C and keep warm for 1 hour, cool to room temperature and add the plant antibacterial agent obtained in step (5) to a concentration of 50 mg / L. After homogenization, the loading is completed.

[0081] Example 10: (1) Zinc chloride and tetrabutylammonium chloride are mixed at a molar ratio of 1:1.2 at 70-80° C. until the system becomes clear, thereby obtaining a catalytic solvent;

[0082] (2) dispersing p-methoxycinnamic acid and potassium hydroxide in water at a molar ratio of 1:1.8, adding hexamethylenediamine in an amount equal to 1 times the amount of p-methoxycinnamic acid, and reacting under reflux at 70-80° C. for 3 h, filtering, washing, and drying to obtain an intermediate;

[0083] (3) placing the intermediate and gallic acid in a catalytic solvent at a molar ratio of 1:1.1, fully dispersing them, and adjusting the pH of the system to 5.5 with hydrochloric acid to obtain a reaction solution;

[0084] (4) Under a nitrogen atmosphere, the reaction solution was stirred at 50-60° C. at a speed of 300 r / min for 15 min, and then at a speed of 1200 r / min for 0.5 h; after the reaction was completed, the mixture was cooled to room temperature, filtered, washed, and freeze-dried to obtain a complex ligand;

[0085] (5) Dissolve copper sulfate in dilute ethanol, add 0.45 times the amount of complexing ligand as copper sulfate, mix thoroughly, stir at 150 r / min for 10 min, let stand for 1 h, freeze-dry the system, and then fully disperse it in anhydrous ethanol. After removing the precipitate, freeze-dry the solution to obtain;

[0086] (6) Add xanthan gum to 80°C water while stirring, mix well to obtain a 0.5% w / v solution, heat to 90°C and keep warm for 1 hour, cool to room temperature and add the plant antibacterial agent obtained in step (5) to a concentration of 50 mg / L. After homogenization, the loading is completed.

[0087] Example 11: (1) Zinc chloride and tetrabutylammonium chloride are mixed at a molar ratio of 1:1.2 at 70-80° C. until the system becomes clear, thereby obtaining a catalytic solvent;

[0088] (2) dispersing p-methoxycinnamic acid and potassium hydroxide in water at a molar ratio of 1:1.8, adding hexamethylenediamine in an amount equal to 1 times the amount of p-methoxycinnamic acid, and reacting under reflux at 70-80° C. for 3 h, filtering, washing, and drying to obtain an intermediate;

[0089] (3) placing the intermediate and gallic acid in a catalytic solvent at a molar ratio of 1:1.1, fully dispersing them, and adjusting the pH of the system to 5.5 with hydrochloric acid to obtain a reaction solution;

[0090] (4) Under a nitrogen atmosphere, the reaction solution was stirred at 50-60° C. at a speed of 300 r / min for 15 min, and then at a speed of 1200 r / min for 0.5 h; after the reaction was completed, the mixture was cooled to room temperature, filtered, washed, and freeze-dried to obtain a complex ligand;

[0091] (5) Dissolve copper sulfate in dilute ethanol, add 0.45 times the amount of complexing ligand as copper sulfate, mix thoroughly, stir at 150 r / min for 10 min, let stand for 0.5 h, freeze-dry the system, and then fully disperse it in anhydrous ethanol. After removing the precipitate, freeze-dry the solution to obtain;

[0092] (6) Add xanthan gum to 80°C water while stirring, mix well to obtain a 1.5% w / v solution, heat to 90°C and keep warm for 1 hour, cool to room temperature and add the plant antibacterial agent obtained in step (5) to a concentration of 50 mg / L. After homogenization, the loading is completed.

[0093] Comparative Example 1: (1) Zinc chloride and tetrabutylammonium chloride in a molar ratio of 1:1.2 were mixed at 70-80° C. until the system was clear, thereby obtaining a catalytic solvent;

[0094] (2) dispersing p-methoxycinnamic acid and potassium hydroxide in water at a molar ratio of 1:1.8, adding hexamethylenediamine in an amount equal to 1 times the amount of p-methoxycinnamic acid, and reacting under reflux at 70-80° C. for 3 h, filtering, washing, and drying to obtain an intermediate;

[0095] (3) placing the intermediate and gallic acid in a catalytic solvent at a molar ratio of 1:1.1, fully dispersing them, and adjusting the pH of the system to 5.5 with hydrochloric acid to obtain a reaction solution;

[0096] (4) Under a nitrogen atmosphere, the reaction solution was stirred at 50-60° C. at a speed of 300 r / min for 15 min, and then at a speed of 1200 r / min for 0.5 h; after the reaction was completed, the mixture was cooled to room temperature, filtered, washed, and freeze-dried to obtain a complex ligand;

[0097] (5) Dissolve copper sulfate in dilute ethanol, add 0.45 times the amount of complexing ligand as copper sulfate, mix thoroughly, then stir at 150 r / min for 10 min and let stand for 0.5 to 1 h, freeze-dry the system, then fully disperse in anhydrous ethanol, remove the precipitate, and freeze-dry the solution to obtain the product.

[0098] Comparative Example 2: (1) Zinc chloride and tetrabutylammonium chloride in a molar ratio of 1:1.2 were mixed at 70-80° C. until the system was clear, thereby obtaining a catalytic solvent;

[0099] (2) dispersing p-methoxycinnamic acid and potassium hydroxide in water at a molar ratio of 1:1.8, adding hexamethylenediamine in an amount equal to 1 times the amount of p-methoxycinnamic acid, and reacting under reflux at 70-80° C. for 3 h, filtering, washing, and drying to obtain an intermediate;

[0100] (3) placing the intermediate and gallic acid in a catalytic solvent at a molar ratio of 1:1.1, fully dispersing them, and adjusting the pH of the system to 5.5 with hydrochloric acid to obtain a reaction solution;

[0101] (4) Under a nitrogen atmosphere, the reaction solution was stirred at 50-60° C. at a speed of 300 r / min for 15 min, and then at a speed of 1200 r / min for 0.5 h; after the reaction was completed, the mixture was cooled to room temperature, filtered, washed, and freeze-dried to obtain a complex ligand;

[0102] (5) Add xanthan gum to 80°C water while stirring, mix well to obtain a 0.5% w / v solution, heat to 90°C and keep warm for 1 hour, cool to room temperature and add the complexing ligand obtained in step (4) to make the concentration of the plant antibacterial agent 50 mg / L, and complete the loading after homogenization.

[0103] Example sample test:

[0104] The mycelium linear growth rate method of GB / T38480-2020 was used, and the mass concentration of the antimicrobial agent was 50 mg / L, using a xenon lamp (light intensity of 9×10 4 The samples were irradiated with 100 lux for 45 min and then placed in a constant temperature incubator for culture. The inhibition rates of each embodiment and comparative example against Botrytis cinerea, Rhizoctonia solani and Fusarium oxysporum were determined to evaluate the in vitro inhibition activity of each antibacterial agent. The results are as follows:

[0105]

[0106]

[0107] As can be seen from the above table, the inhibition rate of the three fungi in each embodiment of the present invention is maintained at more than 80%, and the inhibition rate of most embodiments is more than 90%, and the antibacterial activity is significant. Among them, compared with Example 1, in the process of grafting with hexamethylenediamine, the inhibition rate of the samples of Example 2 with more potassium hydroxide added and Example 3 with longer reaction time decreased. It may be that more potassium hydroxide or longer reaction time easily lead to the production of by-products that are difficult to separate. In the step of connecting the amino group with gallic acid, Example 4 has more gallic acid added, but its inhibition rate has not changed significantly, which may be that the reaction is almost complete; and Example 5, in which the reaction pH is raised to 6.5, has an inhibition rate of 100% for the three pathogens, indicating that the acidity of the reaction system has a significant effect on the progress of the reaction and the amount of by-products produced. In the slow stirring reaction process of Example 6, the stirring speed is faster, and the inhibition rate decreases, while in the high-speed stirring reaction process, the inhibition rates of Example 7 with a faster stirring speed and Example 8 with a longer high-speed stirring reaction time are significantly increased. It may be that in the reaction process, the early reaction requires a low-speed stirring ratio to reduce the production of by-products, while the later stage requires high-speed stirring to promote the full and uniform reaction, avoid product polymerization, and promote the production of the target product. In Example 9 and Example 10, when copper ion complexation treatment is carried out, the stirring and standing time are respectively extended. The inhibition rates of the antibacterial samples of the two embodiments on the three fungi are close to 90%, and even reach 100%, indicating that longer stirring and standing treatment time are conducive to the complexation. The concentration of the load xanthan gum in Example 11 is greater, the inhibition rate decreases, and more loads will also have a certain obstacle to the antibacterial effect of the antibacterial agent; while in the comparative example 1 that is not loaded at all, the inhibition rate is higher than that of Example 11, but its antibacterial ability is not significantly different from the other embodiments, and the concentration relationship between the load and the antibacterial agent can be balanced within a suitable range. The antibacterial rate of Comparative Example 2 without copper ion complexation decreased, but the inhibition rates against Botrytis cinerea and Rhizoctonia solani were both above 50%, indicating that the complexing ligand itself can also achieve moderate antibacterial activity.

[0108] The mycelium dry weight method was used to determine the sustained inhibitory effect of each embodiment and comparative example on Rhizoctonia solani. 0.1 ml of the mixture was placed in a concentration of 1×10 5 The spore suspension of cfu / ml was inoculated in a V8 liquid culture medium containing 50 mg / L of the antimicrobial concentration of each embodiment and comparative example. The culture was carried out at 25°C, 120 r / min speed, and light. The control group without antimicrobial agent was used, and the dry weight of mycelium was weighed once at 6h, 12h, 20h, 36h, and 60h. The difference between each weighing time and the initial dry weight was calculated, and the antibacterial rate of each embodiment and comparative example antimicrobial sample was calculated using the following formula:

[0109] Bacterial inhibition rate = (dry weight difference of control group - dry weight difference of test sample) / dry weight difference of control group. The results are as follows:

[0110]

[0111]

[0112] As can be seen from the above table, the antibacterial rate of each embodiment of the present invention can be maintained at more than 70% within 60h, with a long antibacterial time and slow decay. In the preparation process of the complex ligand, the antibacterial rate of Examples 5, 7 and 8, which react more fully, can be maintained at 100% for a long period of time, while Examples 1 to 4 and 6 begin to decay at 20h, indicating that the complex ligand of the present invention has a long-term antibacterial ability. In the process of copper ion complexation, the degree of complexation of Examples 9 and 10 is higher, and the long-term antibacterial ability of their samples is also stronger. The continuous antibacterial effect of Comparative Example 2 without copper ion complexation is significantly worse. The coating of the embodiment has a higher concentration and the best long-term antibacterial effect. Compared with Comparative Example 1, the coating with a higher concentration is conducive to the continuous action of the antibacterial agent, but it is also necessary to comprehensively consider the selection of concentration based on its overall antibacterial ability.

[0113] For those skilled in the art, various other corresponding changes and deformations can be made according to the technical solutions and concepts described above, and all of these changes and deformations should fall within the protection scope of the claims of the present invention.

Claims

1. A plant antibacterial agent, characterized in that: The invention comprises a complex ligand formed by p-methoxycinnamic acid and gallic acid connected by hexamethylenediamine, and a copper ion complexed with the complex ligand.

2. A method for preparing the plant antibacterial agent according to claim 1, characterized in that: The following steps are involved: After p-methoxycinnamic acid is grafted with hexamethylenediamine, an intermediate is obtained; the amino group on the intermediate is amidated with gallic acid to obtain a complex ligand; The complexing ligand is complexed with copper ions to obtain.

3. The method for preparing the plant antibacterial agent according to claim 1, characterized in that: The following steps are involved: a. Disperse p-methoxycinnamic acid and potassium hydroxide in water, add hexamethylenediamine, reflux at 70-80°C for 3-5h, filter, wash and dry to obtain the intermediate; b. placing the intermediate and gallic acid in a catalytic solvent, fully dispersing them, and adjusting the pH of the system to a weakly acidic state using hydrochloric acid to obtain a reaction solution; c. Under a nitrogen atmosphere, the reaction solution was stirred at a low speed and then at a high speed at 50-60°C; after the reaction was completed, it was cooled to room temperature, the mixed product after the reaction was filtered, washed, and then freeze-dried to obtain a complex ligand; d. Dissolve copper sulfate in dilute ethanol, add the complexing ligand and mix thoroughly, then stir at a low speed for 10 to 30 minutes and let stand for 0.5 to 1 hour, freeze-dry the system, and then fully disperse it in anhydrous ethanol. After removing the precipitate, freeze-dry the solution to obtain the product.

4. The method for preparing the plant antibacterial agent according to claim 3, characterized in that: The molar ratio of p-methoxycinnamic acid to potassium hydroxide in step a is 1:(1.8-2.2); the molar ratio of p-methoxycinnamic acid to hexamethylenediamine in step a is 1:(0.8-1.2).

5. The method for preparing the plant antibacterial agent according to claim 3, characterized in that: The molar ratio of the intermediate in step b to gallic acid is 1: (1.1-1.3); in step b, the pH of the system is adjusted to 5.5-6.5 using hydrochloric acid.

6. The method for preparing the plant antibacterial agent according to claim 3, characterized in that: In step c, the first low-speed stirring reaction and then high-speed stirring reaction is to first stir the reaction at a speed of 300-500 r / min for 10-20 min, and then stir the reaction at a speed of 1200-1500 r / min for 0.5-1 h.

7. The method for preparing the plant antibacterial agent according to claim 3, characterized in that: The low-speed stirring in step d is stirring at a speed of 100 to 200 r / min; the molar ratio of the complexing ligand to copper sulfate in step d is 1:(2 to 2.5).

8. The method for preparing the plant antibacterial agent according to claim 3, characterized in that: The preparation of the catalytic solvent comprises the following steps: Zinc chloride and tetrabutylammonium chloride are mixed at 70-80° C. until the system becomes clear, thereby obtaining the product; the molar ratio of the zinc chloride to the tetrabutylammonium chloride is 1:(1.1-1.2).

9. The method for preparing the plant antibacterial agent according to claim 2, characterized in that: The prepared plant antibacterial agent is loaded on a carrier, which specifically includes the following steps: adding xanthan gum into 80°C water while stirring, mixing evenly to obtain a 0.5-1.5% w / v solution, heating to 90°C and keeping warm for 1-2 hours, cooling to room temperature and then adding the plant antibacterial agent to make the concentration of the plant antibacterial agent 10-100 mg / L, and obtaining the product after homogenization.

10. A plant antibacterial method using the plant antibacterial agent according to claim 1, characterized in that: Make sure the plant antibacterial agent is evenly covered on the surface of the plant that can receive light.

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