Slow-release antibacterial feed additive, preparation method and application thereof

Abstract

This invention relates to a slow-release antibacterial feed additive, its preparation method, and its application, belonging to the field of feed additive technology. The invention uses quercetin as a polyhydroxy core scaffold. First, a stearic acid hydrophobic chain is introduced at the 3' position of quercetin through regioselective esterification with porcine pancreatic lipase. Then, gallic acid is introduced at the 4' position through regioselective esterification with Candida antarcticus lipase B. Following citric anhydride esterification and hydrolytic ring-opening reactions, a quercetin-citric acid cross-linked ester containing a stearic acid hydrophobic chain, a gallic acid ester bond, and a citric acid cross-linking arm is obtained. This ester is then cross-linked with drug-loaded calcium alginate microspheres in the presence of a dehydrating agent and a catalyst to construct a covalently cross-linked shell on the surface of the microspheres, thus obtaining the slow-release antibacterial feed additive. This additive can reduce gastric burst release rate, achieve intestinal slow release, improve processing tolerance, and has targeted release to the colon and cecum with dual antibacterial functions.

Description

Technical Field

[0001] This invention belongs to the field of feed additive technology, specifically relating to a slow-release antibacterial feed additive, its preparation method, and its application. Background Technology

[0002] Thymol is a natural phenolic monoterpene isolated from thyme and oregano, possessing a variety of physiological functions including broad-spectrum antibacterial, antioxidant, and anti-inflammatory effects. In recent years, with the restriction of growth-promoting antibiotics in animal husbandry, thymol has attracted considerable attention as a promising alternative to feed antibiotics. However, thymol is a fat-soluble small molecule with high volatility and oxidative sensitivity. When used directly as a feed additive, it suffers from poor stability, rapid absorption or degradation in the early digestive tract, and difficulty reaching the large intestine and cecum to exert its effects.

[0003] To overcome the aforementioned shortcomings, researchers have attempted to encapsulate thymol in microspheres or microcapsules to achieve sustained-release delivery. Sodium alginate is a common natural polysaccharide carrier material; its molecular chains are rich in carboxyl groups, which can form an egg-box structured three-dimensional gel network through ionic cross-linking with calcium ions, making it a commonly used material for constructing oral sustained-release carriers.

[0004] However, existing calcium alginate drug-loaded microsphere technology still has the following shortcomings: Sodium alginate molecules lack hydrophobic sites for effective binding with fat-soluble thymol, resulting in a natural thermodynamic incompatibility between the two. This leads to a low thymol loading in the microspheres. Furthermore, the gel network relies solely on calcium ions for cross-linking, exhibiting inherent defects in mechanical strength and thermal stability, making it prone to breakage and deformation under the high temperatures and screw extrusion conditions of feed pelleting. Simultaneously, the monolayer calcium alginate gel readily swells and bursts release in the acidic environment of gastric juice, causing a large amount of thymol to concentrate in the stomach. This not only damages the gastric mucosa but also fails to effectively reach susceptible sites of harmful bacteria such as the large intestine and cecum, making it difficult to maintain antibacterial activity throughout the entire feeding interval.

[0005] Therefore, it is necessary to provide a slow-release antibacterial feed additive that can simultaneously increase load, enhance gastric acid stability, and improve the targeted release effect in the large intestine and cecum. Summary of the Invention

[0006] The purpose of this invention is to provide a slow-release antibacterial feed additive, its preparation method, and its application, in order to solve the problems mentioned in the background art.

[0007] The objective of this invention can be achieved through the following technical solutions: A method for preparing a slow-release antibacterial feed additive includes the following steps: S1: Quercetin and stearic acid are esterified under the catalysis of porcine pancreatic lipase to obtain quercetin 3-stearate. The chemical reaction equation is: S2: Quercetin 3-stearate and gallic acid were esterified under the catalysis of immobilized Candida antarcticis lipase B to obtain quercetin 3-stearate 4'-gallic acid. The chemical reaction equation is: S3: Quercetin 3-stearate and 4'-gallic acid ester are esterified with citric anhydride in the presence of a dehydrating agent and a catalyst to obtain quercetin citric anhydride ester; The chemical reaction equation is: S4: Quercetin citrate anhydride ester is subjected to a hydrolysis and ring-opening reaction under alkaline conditions to obtain quercetin citrate cross-linked ester; The chemical reaction equation is: S5: Dissolve sodium alginate in deionized water, add thymol oil solution, emulsify by high-speed shearing, and then solidify by dripping into calcium chloride aqueous solution to obtain drug-loaded calcium alginate microspheres. S6: Quercetin citrate cross-linked ester and drug-loaded calcium alginate microspheres are subjected to esterification and cross-linking reaction in the presence of dehydrating agent and catalyst to obtain a slow-release antibacterial feed additive.

[0008] Furthermore, the molar ratio of quercetin to stearic acid is 1.0:1.0-1.2, and the amount of porcine pancreatic lipase used is 2.0-2.6 times the quality of quercetin.

[0009] Furthermore, the molar ratio of quercetin 3-stearate to gallic acid is 1.0:1.0-1.2, and the amount of immobilized Candida antarcticis lipase B is 2.0-3.5 times the mass of quercetin 3-stearate.

[0010] Furthermore, the molar ratio of quercetin 3-stearate to 4'-gallate to citric anhydride is 1.0:6.0-6.6, the dehydrating agent is N,N'-dicyclohexylcarbodiimide, the catalyst is 4-dimethylaminopyridine, the molar ratio of N,N'-dicyclohexylcarbodiimide to citric anhydride is 1.0-1.5:1, and the molar ratio of 4-dimethylaminopyridine to citric anhydride is 0.05-0.2:1.

[0011] Furthermore, the hydrolysis ring-opening reaction in S4 is carried out in a mixed solvent of dioxane and water, with a volume ratio of dioxane to water of 2.5–3.5:1.

[0012] Furthermore, the mass ratio of thymol to sodium alginate is 0.1–0.2:1, the mass ratio of quercetin citrate cross-linked ester to drug-loaded calcium alginate microspheres is 0.05–0.10:1, the dehydrating agent is N,N'-dicyclohexylcarbodiimide, the catalyst is 4-dimethylaminopyridine, the molar ratio of N,N'-dicyclohexylcarbodiimide to quercetin citrate cross-linked ester is 6.0–9.0:1, and the molar ratio of 4-dimethylaminopyridine to quercetin citrate cross-linked ester is 0.3–1.2:1.

[0013] Furthermore, the esterification reaction temperature in S1 is 35–40℃ and the reaction time is 36–60 hours; the esterification reaction temperature in S2 is 55–65℃ and the reaction time is 6–10 hours; and the esterification reaction temperature in S3 is 20–30℃ and the reaction time is 18–24 hours.

[0014] Furthermore, in S4, the hydrolysis reaction has a pH of 7.8–8.2, a reaction temperature of 20–30°C, and a reaction time of 2–4 hours; in S6, the esterification crosslinking reaction has a temperature of 20–30°C and a reaction time of 45–75 minutes.

[0015] A slow-release antibacterial feed additive is prepared by any of the above preparation steps.

[0016] A slow-release antibacterial feed additive, wherein the above-mentioned slow-release antibacterial feed additive is used to prepare feed for the prevention or treatment of animal diarrhea.

[0017] The beneficial effects of this invention are: 1) This invention uses quercetin as a multi-hydroxyl core scaffold. Through stepwise regioselective catalysis by porcine pancreatic lipase and Candida antarcticis lipase B, a stearic acid hydrophobic chain is introduced at the 3' position of quercetin, and gallic acid antibacterial is introduced at the 4' position. Then, through citric anhydride esterification and hydrolytic ring-opening, a quercetin-citric acid cross-linked ester containing a stearic acid hydrophobic chain, a gallic acid ester bond, and a citric acid cross-linking arm is obtained. The free carboxyl group provided by the citric acid arm in this cross-linked ester molecule undergoes an esterification reaction with the hydroxyl groups on the surface of calcium alginate microspheres under the catalysis of the dehydrating agent N,N'-dicyclohexylcarbodiimide, forming a covalently cross-linked shell linked by ester bonds. This shell is covalently anchored to the microsphere surface by ester bonds, which helps to enhance the binding stability between the shell and the core, reducing the risk of the shell peeling off during digestive tract peristalsis and chyme flushing. The long-chain alkyl stearate in the shell increases the hydrophobicity of the shell, which can limit the permeation rate of water molecules in gastric juice and help reduce the burst release rate of thymol in the stomach.

[0018] 2) In this invention, the outer shell uses ester bonds as the cross-linking and grafting linkage. These ester bonds hydrolyze slowly in the acidic environment of gastric juice, maintaining the integrity of the shell. This helps reduce the concentrated release of thymol in the stomach, lowering the irritation of high-concentration phenolic substances to the gastric mucosa and reducing their killing effect on beneficial bacteria such as lactobacilli and bifidobacteria in the intestines. Upon entering the intestines, the ester bonds gradually hydrolyze under the weakly alkaline environment and the action of esterases. The outer shell degrades layer by layer from the outside in, releasing covalently linked gallic acid with each layer hydrolyzed, achieving programmed release of the antibacterial components and helping to extend the duration of antibacterial action throughout the entire feeding interval. As the outer shell degrades layer by layer, the thymol in the core is gradually exposed and released, forming a dual antibacterial system with gallic acid. This helps the antibacterial components reach more susceptible sites of Clostridium perfringens, such as the large intestine and cecum, to exert their effects. The synergistic mechanism of the shell barrier and gradual degradation makes the shell degradation rate controllable, which helps to improve the release defects of monolayer calcium alginate microspheres, which rely solely on passive diffusion due to concentration difference, resulting in a large release of essential oils in the early stage of feeding and a cliff drop in concentration in the later stage of feeding interval.

[0019] 3) In this invention, quercetin citrate cross-linked esters construct a shell on the surface of drug-loaded calcium alginate microspheres via covalent ester bonds. This covalently cross-linked shell forms a synergistic reinforcing structure with the calcium ion cross-linking network of the core. The covalently cross-linked shell maintains structural integrity under feed pelleting temperatures and screw extrusion conditions, helping to reduce the breakage and deformation of the microsphere skeleton during processing and lowering the risk of thymol volatilization and oxidation due to direct exposure to high-temperature hot air. The aromatic ring structure of the quercetin core in the shell provides π-π stacking forces, helping to anchor thymol inside the microspheres and reducing essential oil leakage during storage. Quercetin itself has auxiliary antibacterial and antioxidant activities after degradation and release in the intestine, forming a triple functional synergy with gallic acid and thymol. Detailed Implementation

[0020] All raw materials used in this invention are not particularly restricted in their source; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0021] Preparation Example 1 The preparation method of citric anhydride includes the following steps: Under nitrogen protection, anhydrous citric acid and acetic anhydride were added to a reactor and stirred until homogeneous. The mixture was then reacted at 36°C for 18 hours. After the reaction was completed, the reaction solution was cooled to room temperature, and acetic acid and excess acetic anhydride were removed by vacuum distillation. The residue was dissolved in chloroform, frozen overnight, filtered, and dried to obtain citric anhydride. The molar ratio of anhydrous citric acid to acetic anhydride is 1.0:1.5.

[0022] Example 1 A method for preparing a slow-release antibacterial feed additive includes the following steps: S1: Under nitrogen protection, quercetin and stearic acid were dissolved in anhydrous acetone. After stirring and dissolving, porcine pancreatic lipase was added, and the mixture was stirred at 130 rpm for 36 hours at 35°C. After the reaction was completed, the mixture was filtered, and the filtrate was washed twice with cold sodium bicarbonate aqueous solution and extracted three times with diethyl ether. The organic layers were combined, dried with anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum distillation. The residue was recrystallized with a mixed solvent of ethanol and water in a volume ratio of 3:1 to obtain quercetin stearate at the 3-position. The molar ratio of quercetin to stearic acid was 1.0:1.0, the amount of porcine pancreatic lipase was 2.0 times the mass of quercetin, and the ratio of anhydrous acetone to the total mass of quercetin and stearic acid was 25 mL:1 g.

[0023] S2: Under nitrogen protection, quercetin 3-stearate and gallic acid were dissolved in anhydrous acetonitrile. After stirring and dissolving, activated molecular sieves and immobilized Candida antarcticis lipase B were added, and the reaction was stirred at 55°C for 6 hours. After the reaction was completed, the mixture was filtered, and the solvent was removed by vacuum distillation. The residue was purified by silica gel column chromatography (eluting with a gradient of dichloromethane and methanol in a volume ratio of 15:1) to obtain quercetin 3-stearate and 4'-gallic acid ester. The molar ratio of quercetin 3-stearate to gallic acid was 1.0:1.0; the amount of immobilized Candida antarctica lipase B was 2.0 times the mass of quercetin 3-stearate; the amount of activated molecular sieve was 1.0 times the total mass of quercetin 3-stearate and gallic acid; and the ratio of anhydrous acetonitrile to the total mass of quercetin 3-stearate and gallic acid was 15 mL:1 g.

[0024] S3: Under nitrogen protection and anhydrous conditions, quercetin 3-stearate and 4'-gallate were dissolved in anhydrous N,N-dimethylformamide. After stirring and dissolving, N,N'-dicyclohexylcarbodiimide (a dehydrating agent) and 4-dimethylaminopyridine (a catalyst) were added, and the mixture was stirred at 20°C for 18 hours. After the reaction was completed, the N,N'-dicyclohexylcarbodiimide byproduct was removed by filtration, and most of the solvent was removed by vacuum distillation. The residue was extracted with ethyl acetate and water, and the organic phase was washed twice with saturated sodium bicarbonate solution and twice with saturated sodium chloride solution. After drying with anhydrous sodium sulfate, the mixture was filtered, and the solvent was removed by vacuum distillation to obtain quercetin citric anhydride ester. The molar ratio of quercetin 3-stearate 4'-gallate to citric anhydride is 1.0:6.0, the molar ratio of N,N'-dicyclohexylcarbodiimide to citric anhydride is 1.0:1, the molar ratio of 4-dimethylaminopyridine to citric anhydride is 0.05:1, and the mass ratio of anhydrous N,N-dimethylformamide to the total mass of quercetin 3-stearate 4'-gallate and citric anhydride is 8 mL:1 g.

[0025] S4: Quercetin citrate anhydride ester was dissolved in a mixed solvent of dioxane and water. A 5% (w / w) sodium hydroxide aqueous solution was added to adjust the pH to 7.8, and the mixture was stirred and hydrolyzed at 20°C for 2 hours. After hydrolysis, the pH was adjusted to 2 with dilute hydrochloric acid, and the mixture was extracted three times with ethyl acetate. The organic layers were combined, dried with anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum distillation to obtain quercetin citrate cross-linked ester. The volume ratio of dioxane to water is 2.5:1, and the volume ratio of quercetin citrate anhydride ester to the mixed solvent is 1g:10mL.

[0026] S5: Dissolve sodium alginate in deionized water and stir until completely dissolved. Then add thymol oil solution and emulsify at 10,000 rpm for 5 minutes using high-speed shearing. Drop the emulsion into a 5% (w / w) calcium chloride aqueous solution at a rate of 5 mL / min and stir and solidify at 20°C for 25 minutes. After solidification, filter and collect the microspheres, and wash them repeatedly with deionized water three times to obtain drug-loaded calcium alginate microspheres. It should be further noted that the thymol oil solution is prepared as a 20% by mass solution using medium-chain fatty acid glycerides. The ratio of sodium alginate to deionized water is 1g:40mL, the ratio of sodium alginate to calcium chloride aqueous solution is 1g:15mL, and the mass ratio of thymol to sodium alginate is 0.1:1.

[0027] S6: Quercetin citrate cross-linked ester was dissolved in phosphate buffer, and the pH was adjusted to 6.0 with dilute hydrochloric acid. Drug-loaded calcium alginate microspheres were then added and soaked at 20°C for 20 minutes. After soaking, N,N'-dicyclohexylcarbodiimide (a dehydrating agent) and 4-dimethylaminopyridine (a catalyst) were added, and the mixture was stirred at 20°C for 45 minutes. After the reaction, the microspheres were collected by filtration and washed three times sequentially with deionized water, ethanol, and then deionized water. The microspheres were then vacuum-dried at 40°C for 12 hours to obtain a slow-release antibacterial feed additive. The mass ratio of quercetin citrate cross-linked ester to drug-loaded calcium alginate microspheres was 0.05:1, the molar ratio of N,N'-dicyclohexylcarbodiimide to quercetin citrate cross-linked ester was 6.0:1, the molar ratio of 4-dimethylaminopyridine to quercetin citrate cross-linked ester was 0.3:1, the concentration of phosphate buffer was 0.1 mol / L, and the volume ratio of phosphate buffer to quercetin citrate cross-linked ester was 20 mL: 1 g.

[0028] Example 2 A method for preparing a slow-release antibacterial feed additive includes the following steps: S1: Under nitrogen protection, quercetin and stearic acid were dissolved in anhydrous acetone. After stirring and dissolving, porcine pancreatic lipase was added, and the mixture was stirred at 130 rpm for 40 hours at 36°C. After the reaction was completed, the mixture was filtered, and the filtrate was washed twice with cold sodium bicarbonate aqueous solution and extracted three times with diethyl ether. The organic layers were combined, dried with anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum distillation. The residue was recrystallized with a mixed solvent of ethanol and water in a volume ratio of 3:1 to obtain quercetin stearate at the 3-position. The molar ratio of quercetin to stearic acid was 1.0:1.05, the amount of porcine pancreatic lipase was 2.1 times the mass of quercetin, and the ratio of anhydrous acetone to the total mass of quercetin and stearic acid was 27 mL:1 g.

[0029] S2: Under nitrogen protection, quercetin 3-stearate and gallic acid were dissolved in anhydrous acetonitrile. After stirring and dissolving, activated molecular sieves and immobilized Candida antarcticis lipase B were added, and the reaction was stirred at 57°C for 7 hours. After the reaction was completed, the mixture was filtered, and the solvent was removed by vacuum distillation. The residue was purified by silica gel column chromatography (eluting with a gradient of dichloromethane and methanol in a volume ratio of 15:1) to obtain quercetin 3-stearate and 4'-gallic acid ester. The molar ratio of quercetin 3-stearate to gallic acid was 1.0:1.05; the amount of immobilized Candida antarctica lipase B was 2.4 times the mass of quercetin 3-stearate; the amount of activated molecular sieve was 1.25 times the total mass of quercetin 3-stearate and gallic acid; and the ratio of anhydrous acetonitrile to the total mass of quercetin 3-stearate and gallic acid was 17 mL:1 g.

[0030] S3: Under nitrogen protection and anhydrous conditions, quercetin 3-stearate and 4'-gallic acid anhydride were dissolved in anhydrous N,N-dimethylformamide. After stirring and dissolving, N,N'-dicyclohexylcarbodiimide (a dehydrating agent) and 4-dimethylaminopyridine (a catalyst) were added, and the mixture was stirred at 22°C for 19 hours. After the reaction was completed, the N,N'-dicyclohexylcarbodiimide byproduct was removed by filtration, and most of the solvent was removed by vacuum distillation. The residue was extracted with ethyl acetate and water, and the organic phase was washed twice with saturated sodium bicarbonate solution and twice with saturated sodium chloride solution. After drying with anhydrous sodium sulfate, the mixture was filtered, and the solvent was removed by vacuum distillation to obtain quercetin citric anhydride ester. The molar ratio of quercetin 3-stearate 4'-gallate to citric anhydride is 1.0:6.15, the molar ratio of N,N'-dicyclohexylcarbodiimide to citric anhydride is 1.1:1, the molar ratio of 4-dimethylaminopyridine to citric anhydride is 0.075:1, and the mass ratio of anhydrous N,N-dimethylformamide to the total mass of quercetin 3-stearate 4'-gallate and citric anhydride is 9 mL:1 g.

[0031] S4: Quercetin citrate anhydride ester was dissolved in a mixed solvent of dioxane and water. A 5% (w / w) sodium hydroxide aqueous solution was added to adjust the pH to 7.9. The mixture was stirred and hydrolyzed at 22°C for 2.5 hours. After hydrolysis, the pH was adjusted to 2 with dilute hydrochloric acid, and the mixture was extracted three times with ethyl acetate. The organic layers were combined, dried with anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum distillation to obtain quercetin citrate cross-linked ester. The volume ratio of dioxane to water is 2.7:1, and the volume ratio of quercetin citrate anhydride ester to the mixed solvent is 1g:10mL.

[0032] S5: Dissolve sodium alginate in deionized water and stir until completely dissolved. Then add thymol oil solution and emulsify at 10,000 rpm for 5 minutes using high-speed shearing. Drop the emulsion into a 5% (w / w) calcium chloride aqueous solution at a rate of 5 mL / min and stir to solidify at 22°C for 27 minutes. After solidification, filter and collect the microspheres, and wash them repeatedly with deionized water three times to obtain drug-loaded calcium alginate microspheres. It should be further noted that the thymol oil solution is prepared as a 20% by mass solution using medium-chain fatty acid glycerides. The ratio of sodium alginate to deionized water is 1g:45mL, the ratio of sodium alginate to calcium chloride aqueous solution is 1g:17mL, and the mass ratio of thymol to sodium alginate is 0.12:1.

[0033] S6: Quercetin citrate cross-linked ester was dissolved in phosphate buffer, and the pH was adjusted to 6.0 with dilute hydrochloric acid. Drug-loaded calcium alginate microspheres were then added and soaked at 22°C for 20 minutes. After soaking, N,N'-dicyclohexylcarbodiimide (a dehydrating agent) and 4-dimethylaminopyridine (a catalyst) were added, and the mixture was stirred at 22°C for 50 minutes. After the reaction, the microspheres were collected by filtration and washed three times sequentially with deionized water, ethanol, and then deionized water. The microspheres were then vacuum-dried at 40°C for 12 hours to obtain a slow-release antibacterial feed additive. The mass ratio of quercetin citrate cross-linked ester to drug-loaded calcium alginate microspheres was 0.06:1, the molar ratio of N,N'-dicyclohexylcarbodiimide to quercetin citrate cross-linked ester was 6.5:1, the molar ratio of 4-dimethylaminopyridine to quercetin citrate cross-linked ester was 0.5:1, the concentration of phosphate buffer was 0.1 mol / L, and the volume ratio of phosphate buffer to quercetin citrate cross-linked ester was 22 mL:1 g.

[0034] Example 3 A method for preparing a slow-release antibacterial feed additive includes the following steps: S1: Under nitrogen protection, quercetin and stearic acid were dissolved in anhydrous acetone. After stirring and dissolving, porcine pancreatic lipase was added, and the mixture was stirred at 130 rpm for 48 hours at 37.5°C. After the reaction was completed, the mixture was filtered, and the filtrate was washed twice with cold sodium bicarbonate aqueous solution and extracted three times with diethyl ether. The organic layers were combined, dried with anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum distillation. The residue was recrystallized with a mixed solvent of ethanol and water in a volume ratio of 3:1 to obtain quercetin stearate at the 3-position. The molar ratio of quercetin to stearic acid was 1.0:1.1, the amount of porcine pancreatic lipase was 2.3 times the mass of quercetin, and the ratio of anhydrous acetone to the total mass of quercetin and stearic acid was 30 mL:1 g.

[0035] S2: Under nitrogen protection, quercetin 3-stearate and gallic acid were dissolved in anhydrous acetonitrile. After stirring and dissolving, activated molecular sieves and immobilized Candida antarcticis lipase B were added, and the reaction was stirred at 60°C for 8 hours. After the reaction was completed, the mixture was filtered, and the solvent was removed by vacuum distillation. The residue was purified by silica gel column chromatography (eluting with a gradient of dichloromethane and methanol in a volume ratio of 15:1) to obtain quercetin 3-stearate and 4'-gallic acid ester. The molar ratio of quercetin 3-stearate to gallic acid was 1.0:1.1; the amount of immobilized Candida antarctica lipase B was 2.75 times the mass of quercetin 3-stearate; the amount of activated molecular sieve was 1.5 times the total mass of quercetin 3-stearate and gallic acid; and the ratio of anhydrous acetonitrile to the total mass of quercetin 3-stearate and gallic acid was 20 mL:1 g.

[0036] S3: Under nitrogen protection and anhydrous conditions, quercetin 3-stearate and 4'-gallate were dissolved in anhydrous N,N-dimethylformamide. After stirring and dissolving, N,N'-dicyclohexylcarbodiimide (a dehydrating agent) and 4-dimethylaminopyridine (a catalyst) were added, and the mixture was stirred at 25°C for 21 hours. After the reaction was completed, the N,N'-dicyclohexylcarbodiimide byproduct was removed by filtration, and most of the solvent was removed by vacuum distillation. The residue was extracted with ethyl acetate and water, and the organic phase was washed twice with saturated sodium bicarbonate solution and twice with saturated sodium chloride solution. After drying with anhydrous sodium sulfate, the mixture was filtered, and the solvent was removed by vacuum distillation to obtain quercetin citric anhydride ester. The molar ratio of quercetin 3-stearate 4'-gallate to citric anhydride is 1.0:6.3, the molar ratio of N,N'-dicyclohexylcarbodiimide to citric anhydride is 1.25:1, the molar ratio of 4-dimethylaminopyridine to citric anhydride is 0.125:1, and the mass ratio of anhydrous N,N-dimethylformamide to the total mass of quercetin 3-stearate 4'-gallate and citric anhydride is 10 mL:1 g.

[0037] S4: Quercetin citrate anhydride ester was dissolved in a mixed solvent of dioxane and water, and a 5% (w / w) sodium hydroxide aqueous solution was added to adjust the pH to 8.0. The mixture was then stirred and hydrolyzed at 25°C for 3 hours. After hydrolysis, the pH was adjusted to 2 with dilute hydrochloric acid, and the mixture was extracted three times with ethyl acetate. The organic layers were combined, dried with anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum distillation to obtain quercetin citrate cross-linked ester. The volume ratio of dioxane to water is 3.0:1, and the volume ratio of quercetin citrate anhydride ester to the mixed solvent is 1g:10mL.

[0038] S5: Dissolve sodium alginate in deionized water and stir until completely dissolved. Then add thymol oil solution and emulsify at 10,000 rpm for 5 minutes using high-speed shearing. Drop the emulsion into a 5% (w / w) calcium chloride aqueous solution at a rate of 5 mL / min and stir and solidify at 25°C for 30 minutes. After solidification, filter and collect the microspheres, and wash them repeatedly with deionized water three times to obtain drug-loaded calcium alginate microspheres. It should be further noted that the thymol oil solution is prepared as a 20% by mass solution using medium-chain fatty acid glycerides. The ratio of sodium alginate to deionized water is 1g:50mL, the ratio of sodium alginate to calcium chloride aqueous solution is 1g:20mL, and the mass ratio of thymol to sodium alginate is 0.15:1.

[0039] S6: Quercetin citrate cross-linked ester was dissolved in phosphate buffer, and the pH was adjusted to 6.0 with dilute hydrochloric acid. Drug-loaded calcium alginate microspheres were then added and soaked at 25°C for 20 minutes. After soaking, N,N'-dicyclohexylcarbodiimide (a dehydrating agent) and 4-dimethylaminopyridine (a catalyst) were added, and the mixture was stirred at 25°C for 60 minutes. After the reaction, the microspheres were collected by filtration and washed three times sequentially with deionized water, ethanol, and then deionized water. The microspheres were then vacuum-dried at 40°C for 12 hours to obtain a slow-release antibacterial feed additive. The mass ratio of quercetin citrate cross-linked ester to drug-loaded calcium alginate microspheres was 0.075:1, the molar ratio of N,N'-dicyclohexylcarbodiimide to quercetin citrate cross-linked ester was 7.5:1, the molar ratio of 4-dimethylaminopyridine to quercetin citrate cross-linked ester was 0.75:1, the concentration of phosphate buffer was 0.1 mol / L, and the volume ratio of phosphate buffer to quercetin citrate cross-linked ester was 25 mL:1 g.

[0040] Example 4 A method for preparing a slow-release antibacterial feed additive includes the following steps: S1: Under nitrogen protection, quercetin and stearic acid were dissolved in anhydrous acetone. After stirring and dissolving, porcine pancreatic lipase was added, and the mixture was stirred at 130 rpm for 56 hours at 39°C. After the reaction was completed, the mixture was filtered, and the filtrate was washed twice with cold sodium bicarbonate aqueous solution and extracted three times with diethyl ether. The organic layers were combined, dried with anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum distillation. The residue was recrystallized with a mixed solvent of ethanol and water in a volume ratio of 3:1 to obtain quercetin stearate at the 3-position. The molar ratio of quercetin to stearic acid was 1.0:1.15, the amount of porcine pancreatic lipase was 2.5 times the amount of quercetin, and the ratio of anhydrous acetone to the total mass of quercetin and stearic acid was 33 mL:1 g.

[0041] S2: Under nitrogen protection, quercetin 3-stearate and gallic acid were dissolved in anhydrous acetonitrile. After stirring and dissolving, activated molecular sieves and immobilized Candida antarcticis lipase B were added, and the reaction was stirred at 63°C for 9 hours. After the reaction was completed, the mixture was filtered, and the solvent was removed by vacuum distillation. The residue was purified by silica gel column chromatography (eluting with a gradient of dichloromethane and methanol in a volume ratio of 15:1) to obtain quercetin 3-stearate and 4'-gallic acid ester. The molar ratio of quercetin 3-stearate to gallic acid was 1.0:1.15; the amount of immobilized Candida antarctica lipase B was 3.1 times the mass of quercetin 3-stearate; the amount of activated molecular sieve was 1.75 times the total mass of quercetin 3-stearate and gallic acid; and the ratio of anhydrous acetonitrile to the total mass of quercetin 3-stearate and gallic acid was 23 mL:1 g.

[0042] S3: Under nitrogen protection and anhydrous conditions, quercetin 3-stearate and 4'-gallate were dissolved in anhydrous N,N-dimethylformamide. After stirring and dissolving, N,N'-dicyclohexylcarbodiimide (a dehydrating agent) and 4-dimethylaminopyridine (a catalyst) were added, and the mixture was stirred at 28°C for 23 hours. After the reaction was completed, the N,N'-dicyclohexylcarbodiimide byproduct was removed by filtration, and most of the solvent was removed by vacuum distillation. The residue was extracted with ethyl acetate and water, and the organic phase was washed twice with saturated sodium bicarbonate solution and twice with saturated sodium chloride solution. After drying with anhydrous sodium sulfate, the mixture was filtered, and the solvent was removed by vacuum distillation to obtain quercetin citric anhydride ester. The molar ratio of quercetin 3-stearate 4'-gallate to citric anhydride is 1.0:6.45, the molar ratio of N,N'-dicyclohexylcarbodiimide to citric anhydride is 1.4:1, the molar ratio of 4-dimethylaminopyridine to citric anhydride is 0.175:1, and the mass ratio of anhydrous N,N-dimethylformamide to the total mass of quercetin 3-stearate 4'-gallate and citric anhydride is 11 mL:1 g.

[0043] S4: Quercetin citrate anhydride ester was dissolved in a mixed solvent of dioxane and water, and a 5% (w / w) sodium hydroxide aqueous solution was added to adjust the pH to 8.1. The mixture was then stirred and hydrolyzed at 28°C for 3.5 hours. After hydrolysis, the pH was adjusted to 2 with dilute hydrochloric acid, and the mixture was extracted three times with ethyl acetate. The organic layers were combined, dried with anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum distillation to obtain quercetin citrate cross-linked ester. The volume ratio of dioxane to water is 3.3:1, and the volume ratio of quercetin citrate anhydride ester to the mixed solvent is 1g:10mL.

[0044] S5: Dissolve sodium alginate in deionized water and stir until completely dissolved. Then add thymol oil solution and emulsify at 10,000 rpm for 5 minutes using high-speed shearing. Drop the emulsion into a 5% (w / w) calcium chloride aqueous solution at a rate of 5 mL / min and stir and solidify at 28°C for 33 minutes. After solidification, filter and collect the microspheres, and wash them repeatedly with deionized water three times to obtain drug-loaded calcium alginate microspheres. It should be further noted that the thymol oil solution is prepared as a 20% by mass solution using medium-chain fatty acid glycerides. The ratio of sodium alginate to deionized water is 1g:55mL, the ratio of sodium alginate to calcium chloride aqueous solution is 1g:23mL, and the mass ratio of thymol to sodium alginate is 0.18:1.

[0045] S6: Quercetin citrate cross-linked ester was dissolved in phosphate buffer, and the pH was adjusted to 6.0 with dilute hydrochloric acid. Drug-loaded calcium alginate microspheres were then added and soaked at 28°C for 20 minutes. After soaking, N,N'-dicyclohexylcarbodiimide (a dehydrating agent) and 4-dimethylaminopyridine (a catalyst) were added, and the mixture was stirred at 28°C for 70 minutes. After the reaction, the microspheres were collected by filtration and washed three times sequentially with deionized water, ethanol, and then deionized water. The microspheres were then vacuum-dried at 40°C for 12 hours to obtain a slow-release antibacterial feed additive. The mass ratio of quercetin citrate cross-linked ester to drug-loaded calcium alginate microspheres was 0.09:1, the molar ratio of N,N'-dicyclohexylcarbodiimide to quercetin citrate cross-linked ester was 8.5:1, the molar ratio of 4-dimethylaminopyridine to quercetin citrate cross-linked ester was 1.0:1, the concentration of phosphate buffer was 0.1 mol / L, and the volume ratio of phosphate buffer to quercetin citrate cross-linked ester was 28 mL:1 g.

[0046] Example 5 A method for preparing a slow-release antibacterial feed additive includes the following steps: S1: Under nitrogen protection, quercetin and stearic acid were dissolved in anhydrous acetone. After stirring and dissolving, porcine pancreatic lipase was added, and the mixture was stirred at 130 rpm for 60 hours at 40°C. After the reaction was completed, the mixture was filtered, and the filtrate was washed twice with cold sodium bicarbonate aqueous solution and extracted three times with diethyl ether. The organic layers were combined, dried with anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum distillation. The residue was recrystallized with a mixed solvent of ethanol and water in a volume ratio of 3:1 to obtain quercetin stearate at the 3-position. The molar ratio of quercetin to stearic acid was 1.0:1.2, the amount of porcine pancreatic lipase was 2.6 times the mass of quercetin, and the ratio of anhydrous acetone to the total mass of quercetin and stearic acid was 35 mL:1 g.

[0047] S2: Under nitrogen protection, quercetin 3-stearate and gallic acid were dissolved in anhydrous acetonitrile. After stirring and dissolving, activated molecular sieves and immobilized Candida antarcticis lipase B were added, and the reaction was stirred at 65°C for 10 hours. After the reaction was completed, the mixture was filtered, and the solvent was removed by vacuum distillation. The residue was purified by silica gel column chromatography (eluting with a gradient of dichloromethane and methanol in a volume ratio of 15:1) to obtain quercetin 3-stearate and 4'-gallic acid ester. The molar ratio of quercetin 3-stearate to gallic acid was 1.0:1.2; the amount of immobilized Candida antarctica lipase B was 3.5 times the mass of quercetin 3-stearate; the amount of activated molecular sieve was 2.0 times the total mass of quercetin 3-stearate and gallic acid; and the ratio of anhydrous acetonitrile to the total mass of quercetin 3-stearate and gallic acid was 25 mL:1 g.

[0048] S3: Under nitrogen protection and anhydrous conditions, quercetin 3-stearate and 4'-gallate were dissolved in anhydrous N,N-dimethylformamide. After stirring and dissolving, N,N'-dicyclohexylcarbodiimide (a dehydrating agent) and 4-dimethylaminopyridine (a catalyst) were added, and the mixture was stirred at 30°C for 24 hours. After the reaction was completed, the N,N'-dicyclohexylcarbodiimide byproduct was removed by filtration, and most of the solvent was removed by vacuum distillation. The residue was extracted with ethyl acetate and water, and the organic phase was washed twice with saturated sodium bicarbonate solution and twice with saturated sodium chloride solution. After drying with anhydrous sodium sulfate, the mixture was filtered, and the solvent was removed by vacuum distillation to obtain quercetin citric anhydride ester. The molar ratio of quercetin 3-stearate 4'-gallate to citric anhydride is 1.0:6.6, the molar ratio of N,N'-dicyclohexylcarbodiimide to citric anhydride is 1.5:1, the molar ratio of 4-dimethylaminopyridine to citric anhydride is 0.2:1, and the mass ratio of anhydrous N,N-dimethylformamide to the total mass of quercetin 3-stearate 4'-gallate and citric anhydride is 12 mL:1 g.

[0049] S4: Quercetin citrate anhydride ester was dissolved in a mixed solvent of dioxane and water, and a 5% (w / w) sodium hydroxide aqueous solution was added to adjust the pH to 8.2. The mixture was then stirred and hydrolyzed at 30°C for 4 hours. After hydrolysis, the pH was adjusted to 2 with dilute hydrochloric acid, and the mixture was extracted three times with ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum distillation to obtain quercetin citrate cross-linked ester. The volume ratio of dioxane to water is 3.5:1, and the volume ratio of quercetin citrate anhydride ester to the mixed solvent is 1g:10mL.

[0050] S5: Dissolve sodium alginate in deionized water and stir until completely dissolved. Then add thymol oil solution and emulsify at 10,000 rpm for 5 minutes using high-speed shearing. Drop the emulsion into a 5% (w / w) calcium chloride aqueous solution at a rate of 5 mL / min and stir and solidify at 30°C for 35 minutes. After solidification, filter and collect the microspheres, and wash them repeatedly with deionized water three times to obtain drug-loaded calcium alginate microspheres. It should be further noted that the thymol oil solution is prepared as a 20% by mass solution using medium-chain fatty acid glycerides. The ratio of sodium alginate to deionized water is 1g:60mL, the ratio of sodium alginate to calcium chloride aqueous solution is 1g:25mL, and the mass ratio of thymol to sodium alginate is 0.2:1.

[0051] S6: Quercetin citrate cross-linked ester was dissolved in phosphate buffer, and the pH was adjusted to 6.0 with dilute hydrochloric acid. Drug-loaded calcium alginate microspheres were then added and soaked at 30°C for 20 minutes. After soaking, N,N'-dicyclohexylcarbodiimide (a dehydrating agent) and 4-dimethylaminopyridine (a catalyst) were added, and the mixture was stirred at 30°C for 75 minutes. After the reaction, the microspheres were collected by filtration and washed three times sequentially with deionized water, ethanol, and then deionized water. The microspheres were then vacuum-dried at 40°C for 12 hours to obtain a slow-release antibacterial feed additive. The mass ratio of quercetin citrate cross-linked ester to drug-loaded calcium alginate microspheres was 0.10:1, the molar ratio of N,N'-dicyclohexylcarbodiimide to quercetin citrate cross-linked ester was 9.0:1, the molar ratio of 4-dimethylaminopyridine to quercetin citrate cross-linked ester was 1.2:1, the concentration of phosphate buffer was 0.1 mol / L, and the volume ratio of phosphate buffer to quercetin citrate cross-linked ester was 30 mL: 1 g.

[0052] Comparative Example 1 The difference between this comparative example and Example 1 is that step S6 is omitted. That is, after S5, the drug-loaded calcium alginate microspheres are directly vacuum dried at 40°C for 12 hours to obtain the feed additive. The remaining steps are the same as in Example 1.

[0053] Comparative Example 2 The difference between this comparative example and Example 1 is that step S2 is omitted, while the remaining steps are the same as in Example 1.

[0054] Comparative Example 3 The difference between this comparative example and Example 1 is that step S1 is omitted, while the remaining steps are the same as in Example 1.

[0055] Experimental Example 1 The following performance tests were conducted on the slow-release antibacterial feed additives obtained in Examples 1-5 and Comparative Examples 1-3: (1) Evaluation of in vitro release performance: 100 mg of each sample was placed in a dialysis bag, and 30 mL of simulated gastric fluid (pH 1.2, containing pepsin) was added. The mixture was shaken at 37℃ and 100 rpm, and samples were taken periodically to determine the cumulative release rate. After 2 hours, the mixture was transferred to simulated intestinal fluid (pH 6.8, containing trypsin) and shaken for further determination. The determination was performed using the paddle method based on the principle of "Dissolution and Release Determination" in General Chapter 0931 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, combined with the dialysis bag method. The thymol content in the release medium was determined by gas chromatography, and the gallic acid content was determined by high performance liquid chromatography (C18 column, gradient elution of methanol-0.1% phosphoric acid solution, detection wavelength 273 nm).

[0056] (2) In vitro antibacterial activity determination: Following the paper disc diffusion method (Oxford cup method) in the "Inhibition Test" section of the "Disinfection Technical Specifications" (2002 edition), *Clostridium perfringens* (ATCC 13124), *Escherichia coli* (ATCC 25922), and *Staphylococcus aureus* (ATCC 25923) were used as indicator bacteria. The bacteria were anaerobically cultured at 37°C for 24 hours, and the diameter of the inhibition zone was measured. The bacterial culture conditions were performed in accordance with the "National Food Safety Standard for Microbiological Examination of Food" (GB 4789 series).

[0057] (3) Determination of thymol retention rate after high temperature granulation: Each sample was placed in an 85℃ oven for 10 minutes. The thymol content was determined before and after treatment according to the method in (1), and the retention rate was calculated.

[0058] (4) Weaned piglet feeding experiment: 120 healthy weaned piglets aged 28 days were randomly divided into 8 groups of 15 piglets each. The groups were: blank control group (basal diet), comparative example group 1 (basal diet + comparative example 1 sample), comparative example group 2 (basal diet + comparative example 2 sample), comparative example group 3 (basal diet + comparative example 3 sample), example 1 group, example 2 group, example 3 group, example 4 group, and example 5 group. The experiment lasted for 28 days, and the daily weight gain was recorded. The fecal condition was observed daily. The diarrhea scoring criteria were as follows: no diarrhea (strip or granular, moisture <70%): 0 points; mild diarrhea (soft, formed, moisture 70%–75%): 1 point; moderate diarrhea (pasty, moisture 75%–80%): 2 points; severe diarrhea (watery, moisture >80%): 3 points. The diarrhea index of each group was calculated (sum of daily scores). After the experiment, five animals from each group were randomly selected for slaughter, and the contents of the cecum were collected to determine the concentrations of thymol, gallic acid, and bacterial count. The experimental procedures were performed in accordance with the "Guidelines for Ethical Review of Laboratory Animal Welfare" (GB / T 35892-2018).

[0059] (5) Drug release kinetic model fitting: Based on the cumulative release rate data of thymol measured at each time point in the in vitro release experiments of Examples 1-5 (0.5h, 1h, 1.5h, 2h, 4h, 8h, 12h, 24h), zero-order release model, first-order release model, Higuchi model and Ritger-Peppas model were used for fitting, respectively. The correlation coefficient (R) was used as the model. 2 The model closest to 1 is taken as the optimal release model. In the Ritger-Peppas model, the release mechanism is determined based on the release index n: n≤0.43 indicates Fickian diffusion (driven by concentration difference), 0.43<n<0.85 indicates non-Fickian diffusion (synergistic diffusion and skeleton dissolution), and n≥0.85 indicates skeleton dissolution control.

[0060] The test results are shown in Tables 1 and 2.

[0061] Table 1 Table 2 As shown in Tables 1 and 2, the release rates of thymol in simulated gastric fluid in Examples 1-5 were all at a low level, significantly lower than that in Comparative Example 1, while the release rates in Comparative Examples 3 and 2 were higher than those in Examples 1-5. Examples 1-5 exhibited a stable release characteristic in simulated intestinal fluid, with release index n values ​​ranging from 0.65 to 0.72, between 0.43 and 0.85. The n value for Comparative Example 1 was 0.38. Gallic acid in Examples 1-5 was gradually released in simulated intestinal fluid, with a relatively high release rate over 24 hours. The release rate in simulated gastric fluid in Comparative Example 3 was higher than that in Examples 1-5, but the gallic acid release rate in simulated intestinal fluid was comparable to that in Examples 1-5. Examples 1-5 all showed strong antibacterial activity against the three tested bacteria, while the inhibition zone of Comparative Example 1 was [not specified]. The inhibitory zone of Comparative Example 2 was significantly smaller than that of Examples 1-5, while the inhibitory effect of Comparative Example 3 was comparable to that of Examples 1-5. After high-temperature treatment, the thymol retention rate of Examples 1-5 was at a high level, significantly higher than that of Comparative Example 1, while the retention rates of Comparative Examples 2 and 3 were slightly lower than those of Examples 1-5. In the weaned piglet feeding experiment, the daily weight gain of Examples 1-5 was higher than that of the blank control group and Comparative Examples 1-3, and the diarrhea index was lower than that of the blank control group and Comparative Examples 1-3. The concentrations of thymol and gallic acid in the cecum of Examples 1-5 were higher than those of Comparative Examples 1-3. The number of Clostridium perfringens in the cecum of Examples 1-5 was lower than that of the blank control group and Comparative Examples 1-3, while the number of Lactobacillus was higher than that of the blank control group and Comparative Examples 1-3. These results indicate that the slow-release antibacterial feed additives prepared in Examples 1-5 of this invention exhibit excellent performance in terms of gastric acid stability, slow-release properties, antibacterial activity, and processing tolerance, and all parameters within a given range can achieve good comprehensive performance.

[0062] The descriptions of the above embodiments are merely illustrative of the methods and core ideas of the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a slow-release antibacterial feed additive, characterized in that, Includes the following steps: S1: Quercetin and stearic acid are esterified under the catalysis of porcine pancreatic lipase to obtain quercetin 3-stearate. S2: Quercetin 3-stearate and gallic acid were esterified under the catalysis of immobilized Candida antarcticis lipase B to obtain quercetin 3-stearate 4'-gallic acid. S3: Quercetin 3-stearate and 4'-gallic acid ester are esterified with citric anhydride in the presence of a dehydrating agent and a catalyst to obtain quercetin citric anhydride ester; S4: Quercetin citrate anhydride ester is subjected to a hydrolysis and ring-opening reaction under alkaline conditions to obtain quercetin citrate cross-linked ester; S5: Dissolve sodium alginate in deionized water, add thymol oil solution, emulsify by high-speed shearing, and then solidify by dripping into calcium chloride aqueous solution to obtain drug-loaded calcium alginate microspheres. S6: Quercetin citrate cross-linked ester and drug-loaded calcium alginate microspheres are subjected to esterification and cross-linking reaction in the presence of dehydrating agent and catalyst to obtain a slow-release antibacterial feed additive.

2. The method for preparing a slow-release antibacterial feed additive according to claim 1, characterized in that, The molar ratio of quercetin to stearic acid is 1.0:1.0-1.2, and the amount of porcine pancreatic lipase used is 2.0-2.6 times the amount of quercetin.

3. The method for preparing a slow-release antibacterial feed additive according to claim 1, characterized in that, The molar ratio of quercetin 3-stearate to gallic acid is 1.0:1.0-1.2, and the amount of immobilized Candida antarcticis lipase B is 2.0-3.5 times the mass of quercetin 3-stearate.

4. The method for preparing a slow-release antibacterial feed additive according to claim 1, characterized in that, The molar ratio of quercetin 3-stearate to 4'-gallate to citric anhydride is 1.0:6.0–6.

6. The dehydrating agent is N,N'-dicyclohexylcarbodiimide, and the catalyst is 4-dimethylaminopyridine. The molar ratio of N,N'-dicyclohexylcarbodiimide to citric anhydride is 1.0–1.5:1, and the molar ratio of 4-dimethylaminopyridine to citric anhydride is 0.05–0.2:

1.

5. The method for preparing a slow-release antibacterial feed additive according to claim 1, characterized in that, The hydrolysis ring-opening reaction in S4 is carried out in a mixed solvent of dioxane and water, with a volume ratio of dioxane to water of 2.5 to 3.5:

1.

6. The method for preparing a slow-release antibacterial feed additive according to claim 1, characterized in that, The mass ratio of thymol to sodium alginate is 0.1–0.2:1, the mass ratio of quercetin citrate cross-linked ester to drug-loaded calcium alginate microspheres is 0.05–0.10:1, the dehydrating agent is N,N'-dicyclohexylcarbodiimide, the catalyst is 4-dimethylaminopyridine, the molar ratio of N,N'-dicyclohexylcarbodiimide to quercetin citrate cross-linked ester is 6.0–9.0:1, and the molar ratio of 4-dimethylaminopyridine to quercetin citrate cross-linked ester is 0.3–1.2:

1.

7. The method for preparing a slow-release antibacterial feed additive according to claim 1, characterized in that, The esterification reaction temperature in S1 is 35–40℃ and the reaction time is 36–60 hours; the esterification reaction temperature in S2 is 55–65℃ and the reaction time is 6–10 hours; and the esterification reaction temperature in S3 is 20–30℃ and the reaction time is 18–24 hours.

8. The method for preparing a slow-release antibacterial feed additive according to claim 1, characterized in that, In S4, the hydrolysis reaction occurs at a pH of 7.8–8.2, a reaction temperature of 20–30°C, and a reaction time of 2–4 hours. In S6, the esterification crosslinking reaction occurs at a temperature of 20–30°C and a reaction time of 45–75 minutes.

9. A slow-release antibacterial feed additive, characterized in that, The slow-release antibacterial feed additive is prepared by the preparation method described in any one of claims 1 to 8.

10. The application of a slow-release antibacterial feed additive, characterized in that, The slow-release antibacterial feed additive described in claim 9 can be used to prepare feed for the prevention or treatment of animal diarrhea.

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