Preparation method of feed additive for replacing antibiotics in large-scale livestock and poultry breeding

Abstract

The application belongs to the technical field of feed processing, and particularly relates to a preparation method of a large-scale livestock and poultry breeding antibacterial drug reduction replacement feed additive, which comprises constructing a multi-layer core-shell structure to realize complete protection of a replacement active substance in gastric juice and precise segmented release in the intestinal tract. Through the above multi-layer synergistic protection scheme, the active retention rate after 2 hours of simulated gastric juice treatment is more than 90%, the cumulative release proportion in the ileum and colon regions of the intestinal tract is more than 70%, the active retention rate is maintained to be more than 75% when the granulation temperature is 90 DEG C, and the active retention rate is not less than 70% in a 12-month storage period, so that the bioavailability of the replacement active substance is significantly improved, and practical technical support is provided for antibacterial drug reduction.

Description

Technical Field

[0001] This invention belongs to the field of feed processing technology, specifically a method for preparing feed additives for large-scale livestock and poultry breeding with reduced dosage of antibiotics and antibiotic alternatives. Background Technology

[0002] Reducing the amount of antibiotics used in large-scale livestock and poultry farming is the core development direction for the industry's green transformation and upgrading. As a key input for reducing antibiotic use in livestock and poultry farming, the activity stability and bioavailability of the active ingredients of antibiotic alternative feed additives directly determine the final antibiotic alternative effect. However, natural antibiotic alternative active substances are highly sensitive to the environment and the acid-base environment of the gastrointestinal tract, and are prone to inactivation and difficult to release and absorb at specific points, which greatly limits the application effect of antibiotic alternative technology.

[0003] Existing antibiotic alternative feed additives are mostly prepared using microencapsulation or encapsulation technology to physically protect the active ingredients. The wall material forms a physical barrier to isolate the active ingredients from external environmental erosion, which can retain the efficacy of the active ingredients to a certain extent and meet the basic antibiotic alternative application needs of livestock and poultry farming.

[0004] Existing microencapsulation and embedding technologies have inherent defects such as insufficient wall material sealing and low environmental response accuracy. They cannot achieve effective protection of the gastric acid environment and precise release into the intestine, resulting in poor protective efficacy of active ingredients, weak release controllability, and difficulty in ensuring stable effective doses of antibiotic alternatives. This leads to unstable and poor sustained effects of antibiotic alternatives, becoming a core technical bottleneck restricting the large-scale promotion and application of antibiotic reduction technology in livestock and poultry farming.

[0005] Therefore, the present invention provides a method for preparing antibiotic-reduced feed additives for large-scale livestock and poultry farming. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, at least one technical problem raised in the background art is solved.

[0007] The technical solution adopted by this invention to solve its technical problem is as follows: A method for preparing a feed additive for large-scale livestock and poultry breeding with reduced-volume antibiotics, comprising the following steps:

[0008] Step 1: Preparation of the antimicrobial active substance dispersion system. Natural antimicrobial peptides are selected as the core active ingredients and mixed and dispersed with sodium alginate aqueous solution under high-speed shear stirring conditions to form a homogeneous oil-water emulsion system. The natural antimicrobial peptides are derived from at least one of defensin-type antimicrobial peptides, bacteriocin-type antimicrobial peptides, or lactoferrin-derived peptides. The defensin-type antimicrobial peptides are selected from members of the β-defensin subfamily, with the number of amino acid residues controlled within the range of 30 to 50, and the disulfide bond configuration is configured as a three-pair disulfide bond configuration. This disulfide bond configuration can maintain the spatial folding conformation of the antimicrobial peptide and preserve its antimicrobial activity under acidic conditions. The bacteriocin-type antimicrobial peptides are selected from nisin, with a molecular weight controlled within the range of 3000 to 4000 Daltons, exhibiting significant antibacterial effects against Gram-positive bacteria. The lactoferrin-derived peptides are selected from the N-terminal functional domain fragment of lactoferrin, whose amino acid sequence contains a dual structural feature of positively charged enriched regions and hydrophobic regions. The concentration of sodium alginate... The concentration was controlled within the range of 0.5% to 2%, and pre-dissolving was performed at room temperature. The stirring speed was controlled within the range of 300 to 500 revolutions per minute, and the dissolution time was set to 2 to 4 hours to ensure that the sodium alginate molecular chains were fully hydrated and expanded. After pre-dissolution, the viscosity of the sodium alginate aqueous solution was adjusted to the range of 500 to 2000 mPa·s. If the viscosity was too low, it would not be able to effectively encapsulate the antimicrobial peptide molecules, resulting in a decrease in encapsulation efficiency; if the viscosity was too high, it would affect the smoothness of the droplets in the subsequent sharp-hole forming process. Mixing and dispersion operation During the process, the shear stirring speed was set to a high speed range of 8,000 to 12,000 revolutions per minute. A rotor-stator homogenizer was used for homogenization. The stirring time was controlled within the range of 10 to 30 minutes. The mixing temperature was strictly controlled within the range of 4 to 37°C to avoid the destruction of antimicrobial peptide activity by high temperature. The pH of the mixed system was adjusted to near neutral to maintain the conformational stability of the antimicrobial peptide. The average particle size of the dispersion system was controlled within the range of 1 to 10 micrometers, and the polydispersity index was less than 0.3.

[0009] Step Two: Preparation and Forming of Inner Microcapsules. The homogeneous emulsion system formed in Step One is used to prepare droplets using a sharp-pore method. These droplets are then dropped into a calcium chloride aqueous solution to complete the ionic cross-linking reaction, forming a sodium alginate-calcium ion three-dimensional network structure to encapsulate the core active ingredient. The sharp-pore forming process is carried out using a storage tank equipped with a precise pore size distributor. The pore size of the distributor is precisely set to 50 to 300 micrometers, and the droplet formation frequency is controlled within the range of 50 to 200 drops per minute. The droplets are kept vertically oriented as they enter the calcium chloride solution to ensure sphericity. The droplet height is controlled within the range of 5 to 20 cm to ensure the formation of uniform spherical microcapsules. The concentration of calcium chloride is configured to be 0.5% to 2%. Too low a concentration will result in incomplete cross-linking and insufficient gel strength, while too high a concentration may lead to excessive surface cross-linking and the formation of a dense shell. The cross-linking reaction time is controlled within the range of 10 to 60 minutes, and the reaction temperature is set at 20 to 25°C. The mechanism of the cross-linking reaction is that the carboxyl groups on the sodium alginate molecular chain specifically bind to calcium ions to form a cross-linking network with an egg-box model structure. The antimicrobial peptide molecules in the emulsion system are physically trapped in the three-dimensional structure of sodium alginate-calcium ions. Inside the network structure, the droplet diameter is precisely controlled within the range of 100 to 500 micrometers. The extrusion pressure is provided by a micro-peristaltic pump, with the pressure range controlled between 0.01 and 0.1 MPa. After the cross-linking reaction is completed, the microcapsules are washed with deionized water 3 to 5 times to remove residual calcium ions and unreacted impurities on the surface. Each wash uses deionized water with a volume of 3 to 5 times the volume of the microcapsule slurry. Subsequently, vacuum filtration is performed to remove excess water. The vacuum degree is controlled within the range of 0.08 to 0.1 MPa, and the filtration time is 5 to 10 minutes. The treated microcapsules are white to pale yellow gel spheres with a water content controlled within the range of 85% to 92%. The encapsulation efficiency of the inner layer microcapsules is increased to over 80%.

[0010] Step 3: Constructing an intermediate protective layer. The microcapsules obtained in Step 2 are transferred to a chitosan aqueous solution for secondary encapsulation, constructing a dense polyelectrolyte composite membrane to block the permeation and diffusion of hydrogen ions in gastric juice. The chitosan is a derivative obtained by deacetylation of chitin, and its degree of deacetylation is controlled within the range of 75% to 95%. If the deacetylation is too low, the positive charge density on the molecular chain is insufficient, making it difficult to form a stable electrostatic interaction with sodium alginate. If the deacetylation is too high, the molecule is too hydrophilic, and the swelling behavior in an acidic environment is difficult to control. The molecular weight of chitosan is selected to be 1. The molecular weight ranges from 0 to 300,000 Daltons. Too low a molecular weight results in insufficient film strength, while too high a molecular weight leads to excessive solution viscosity, affecting encapsulation uniformity. The concentration of the chitosan aqueous solution is set to 0.1% to 1%, and a 1% to 2% dilute acetic acid aqueous solution is used as the solvent to ensure complete dissolution of the chitosan. The pH of the chitosan solution is adjusted to the acidic range to ensure complete dissolution and provide sufficient positive charge density. The microcapsules are impregnated in the chitosan solution in a constant temperature shaking incubator with a shaking frequency set to 50 to 100 rpm, and the impregnation time is controlled between 30 minutes and 2 hours. Within a certain range, chitosan molecules spontaneously deposit on the surface of sodium alginate microcapsules through electrostatic attraction, forming a uniform coating layer. The protonated amino groups on the chitosan molecular chains and the carboxylate ions on the sodium alginate molecular chains undergo Coulomb attraction to form ionic cross-linking points, thereby forming a dense polyelectrolyte composite membrane. The thickness of the chitosan intermediate layer is precisely controlled within the range of 5 to 20 micrometers. After impregnation, freeze-drying is performed to remove moisture. The drying temperature is controlled within a low temperature range of -40 to -80°C. The freeze-drying process includes three stages: pre-freezing, sublimation drying, and desorption drying. In the pre-freezing stage, the sample temperature is lowered from room temperature to below -40°C at a cooling rate of 1 to 3°C per minute for 2 to 4 hours. In the sublimation drying stage, the separator temperature is controlled at -20 to -10°C, the vacuum degree is controlled at 10 to 50 Pa, and the drying time is 8 to 12 hours. In the desorption drying stage, the separator temperature is raised to 25 to 35°C, the vacuum degree is controlled at 5 to 20 Pa, and the drying time is 2 to 4 hours. The total drying time is extended to 12 to 24 hours to ensure sufficient removal of moisture. The freeze-dried microcapsules have a fluffy white porous spherical structure with a moisture content of less than 5%.

[0011] Step 4: Preparation of the outer acid-sensitive protective shell. The microcapsules obtained in Step 3 are further immersed in a pectin aqueous solution. Subsequently, calcium chloride solution is added to trigger the gelation reaction between pectin and calcium ions, forming an outer gel protective shell with a dense network structure under acidic conditions. The pectin used is at least one of high-ester pectin or low-ester pectin. The degree of esterification of high-ester pectin is controlled within the range of 50% to 75%, and the degree of esterification of low-ester pectin is controlled within the range of 25% to 50%. The concentration of the pectin aqueous solution is controlled between 0.5% and 3%. Deionized water is used as the solvent. The heating and dissolution temperature is controlled within the range of 60 to 80°C, the stirring speed is 200 to 300 rpm, and the dissolution time is 30 to 60 minutes. The impregnation treatment is carried out in a constant temperature shaking incubator with a shaking frequency set at 30 to 60 rpm, an impregnation temperature set at 25 to 40°C, and an impregnation time of 1 to 3 hours. After impregnation, calcium chloride solution was added to trigger the gelation reaction. The concentration of calcium chloride was set at 0.3% to 1%, and the calcium chloride solution was added slowly dropwise to avoid uneven gelation caused by excessively high local concentrations. The reaction time was controlled within the range of 2 to 8 hours, and the reaction temperature was maintained at 25 to 35°C. The gelation reaction between pectin and calcium ions followed the egg-box model mechanism, with calcium ions embedding between the carboxyl groups of adjacent pectin molecular chains to form an ion-bridging structure. This gel network exhibited a highly dense network structure at pH below 4.5 to resist the hydrolytic erosion of pepsin in gastric juice. When the pH was above 5.5, the carboxyl groups deprotonated and crosslinked with calcium ions, and the gel network began to slowly degrade to ensure complete release of active substances in the jejunum and ileum. After the gelation reaction, the microcapsules were washed with deionized water and filtered. The treated double-layer embedded microcapsules were pale yellow to brownish-yellow spherical with a smooth and glossy surface, and the particle size increased by 20 to 50 micrometers due to the addition of the pectin layer.

[0012] Step 5: Coating with a high-temperature resistant protective agent. The microcapsules obtained in Step 4 are mixed with a high-temperature resistant protective agent to construct a high-temperature resistant protective layer. The high-temperature resistant protective agent is selected from at least one of trehalose, sucrose, maltodextrin, or glycerol. In the preferred embodiment, trehalose is selected as the preferred high-temperature resistant protective agent. The concentration of trehalose is controlled to be 10% to 25% of the mass of the microcapsules. If the concentration is too low, the formed glassy protective shell will not be dense enough and will not be able to effectively block heat transfer. If the concentration is too high, it may cause the microcapsules to stick together, affecting the flowability of the subsequent granulation process. The mechanism of action of trehalose is based on its excellent water substitution hypothesis characteristics. Under dry and high-temperature conditions, trehalose molecules can replace water molecules and form a hydrogen bond network with the polar groups on the surface of biomacromolecules, thereby maintaining the three-dimensional conformational stability of the antimicrobial peptides. Trehalose can form a glassy amorphous structure with a glass transition temperature controlled within the range of 90 to 120°C. This temperature range covers the temperature range of conventional granulation processes. When the granulation temperature exceeds the glass transition temperature, trehalose transitions from a glassy state to a rubbery state and absorbs excess heat, thereby protecting the core active material from thermal damage. The mixing process is carried out under low-speed stirring conditions to avoid mechanical damage to the microcapsule structure. A low-speed conical mixer or V-type mixer is selected for mixing, with the stirring speed controlled within the range of 10 to 30 revolutions per minute and the mixing time being 10 to 20 minutes. After mixing, fluidized bed drying is performed to remove excess moisture. The drying temperature is controlled within the range of 40 to 60°C and the drying time is 2 to 4 hours.

[0013] Preferably, the sodium alginate used in step one can be replaced with oligomeric sodium alginate or high-mannuronic acid sodium alginate, with its molecular weight distribution range controlled between 5,000 and 200,000 Daltons. If the molecular weight is too low, the pore size of the gel network formed will be too large to effectively retain small molecular weight antimicrobial peptides; if the molecular weight is too high, the solution viscosity will be too high, affecting the operability of the sharp-pore forming process. Viscosity adjustment is achieved by adding an appropriate amount of glycerol or propylene glycol as a thickening agent, with the addition amount controlled within the range of 1% to 5% of the total mass of the solution. The addition of glycerol and propylene glycol can not only adjust the solution viscosity, but also act as a protective agent in the subsequent drying process to prevent the loss of activity of antimicrobial peptides during the drying process.

[0014] Preferably, in the sharp-hole forming process of step two, the pulse frequency stability of the micro-peristaltic pump directly affects the uniformity of droplet formation. The pulse frequency of the peristaltic pump is controlled within the range of 30 to 100 pulses per minute, and the coefficient of variation of the pulse interval should be less than 5% to ensure the consistency of droplet diameter. The sphericity of the microcapsules is visually inspected using an optical microscope, and the sphericity should be greater than 0.95. The sphericity is calculated as the ratio of the projected area of ​​the microcapsule to the projected area of ​​a sphere of the same volume. The encapsulation efficiency is determined by high-performance liquid chromatography (HPLC), calculated by measuring the content of free antimicrobial peptides in the supernatant. The mobile phase of HPLC is selected as acetonitrile-water-trifluoroacetic acid system, the flow rate is set to 0.8 to 1.2 mL per minute, and the detection wavelength is set to the characteristic absorption peak in the range of 215 to 280 nm based on the ultraviolet absorption characteristics of the antimicrobial peptides.

[0015] Preferably, the degree of deacetylation of chitosan in step three is preferably in the range of 85% to 90%. This range ensures that the molecular chain has sufficient positive charge density to form a stable electrostatic interaction with sodium alginate, while avoiding excessive swelling in an acidic environment due to excessive hydrophilicity. The molecular weight of chitosan is preferably in the range of 150,000 to 250,000 Daltons. This range can maintain a suitable solution viscosity to facilitate the impregnation operation while ensuring film strength. The impregnation temperature is preferably at room temperature to avoid the influence of temperature fluctuations on the conformation of chitosan molecules. The temperature control accuracy of the constant temperature oscillation incubator during the impregnation process is set to ±1℃.

[0016] Preferably, in step four, the selection of high-ester pectin and low-ester pectin is differentiated according to the target release site: the gel network formed by high-ester pectin begins to degrade significantly when the pH is higher than 6.0, making it suitable for applications requiring release in the prejejunum; the gel network formed by low-ester pectin is more sensitive to calcium ion concentration and can achieve rapid degradation in the pH range of 5.5 to 6.5, making it suitable for applications requiring concentrated release in the ileum. In a preferred embodiment of the present invention, a combination of low-ester pectin and calcium ions is selected to achieve effective release of active substances in the ileum and colon. The esterification degree of low-ester pectin is preferably in the range of 30% to 45%, which allows for optimal acid-sensitive response characteristics while ensuring gel strength.

[0017] Preferably, the combination of high-temperature protective agents in step five can achieve complementary advantages: trehalose has the highest water substitution capacity and glass transition temperature, making it the preferred high-temperature protective ingredient; sucrose has a lower cost and can provide a good sweetness masking effect, making it suitable for cost-sensitive applications; maltodextrin has a wide molecular weight distribution range, providing gradient protection effects in different temperature ranges; the amount of glycerol added is controlled below 5% to avoid reducing the stability of the glassy protective shell due to excessive plasticization; in the combined use of trehalose and maltodextrin, the preferred mass ratio of the two is in the range of 1:1 to 3:1, which can balance the protective effect and cost control.

[0018] Preferably, in the granulation process, a wet granulation technique is used. The granulation liquid is deionized water or 5% to 15% starch paste, and the liquid-to-solid ratio is controlled at 25% to 40%. If the liquid-to-solid ratio is too low, the granule strength will be insufficient; if the liquid-to-solid ratio is too high, the granule moisture content will be too high, affecting storage stability. The screen aperture of the extruder is set to 1 to 3 mm. The size of the screen aperture directly affects the particle size distribution, and thus the release behavior of the active substances. The granulation speed is controlled at 30 to 60 revolutions per minute to ensure the uniformity of granule formation. The granulation temperature is strictly controlled within the range of 70 to 90 ℃, and the granulation pressure is set at 2 to 5 MPa. Under these temperature and pressure conditions, the trehalose protective layer undergoes a glass transition, forming an effective thermal protection barrier. After granulation, countercurrent cooling is performed to avoid thermal shock to the microcapsule structure caused by sudden temperature changes. The cooling wind speed is controlled within the range of 0.5 to 2 meters per second, and the cooling time is controlled within the range of 30 to 60 minutes. The activity retention rate of the antibiotic alternative active substances in the final product is ensured to be above 75%.

[0019] Preferably, in the simulated gastrointestinal release test, the prepared feed additive product is subjected to segmented release performance verification. The test method is to place the sample in artificial gastric fluid for 2 hours, and then transfer it to artificial intestinal fluid for further release testing. The pH of the artificial gastric fluid is set to 1.5 to 3, and the pH of the artificial intestinal fluid is set to 6.5 to 7.5. The release amount of active substances in different time periods is determined by high performance liquid chromatography. According to the release curve, the product is divided into different release levels to meet the feeding needs of different livestock and poultry breeds: Level 1 product is suitable for young livestock and poultry such as piglets, with a release ratio of more than 60% in the anterior intestinal tract; Level 2 product is suitable for growing and finishing livestock and poultry, with uniform release in the jejunum to ileum, with a release ratio of 30% to 50% in the jejunum, 30% to 40% in the ileum, and 10% to 20% in the colon; Level 3 product is suitable for breeding livestock and poultry, with slow and continuous release throughout the entire intestine, and the release ratio in each segment does not exceed 40%.

[0020] The present invention provides a method for preparing antibiotic-reduced feed additives for large-scale livestock and poultry farming. First, a three-dimensional network structure of sodium alginate and calcium ions is used in the inner layer to physically encapsulate natural antimicrobial peptides, achieving basic protection of the core active ingredients. The egg-box model cross-linking structure of sodium alginate and calcium ions remains stable in an acidic environment, effectively trapping the active substances within the gel network. Then, a dense polyelectrolyte composite membrane is formed through secondary encapsulation with chitosan. The protonated amino groups on the chitosan molecular chain and the carboxylate ions on the sodium alginate molecular chain undergo Coulomb attraction to form ion cross-linking points, constructing a double-layer barrier structure that significantly enhances the resistance to hydrogen ion permeation and diffusion. Furthermore, an acid-responsive protective shell is constructed through an outer layer of pectin gel. The gelation reaction of pectin and calcium ions follows the egg-box model mechanism. In the gastric juice environment with a pH below 4.5, it exhibits a highly dense network structure, which can simultaneously resist the hydrolytic erosion and physical abrasion of pepsin. In the intestinal environment with a pH above 5.5, it begins to slowly degrade, enabling precise release of the active substance in the jejunum and ileum. Furthermore, through high-temperature protective agent coating, trehalose forms a glassy protective shell under the high-temperature granulation environment, maintaining the three-dimensional conformational stability of the antimicrobial peptide based on the water substitution hypothesis, effectively reducing the impact of heat transfer on the core active substance. Finally, through the above multi-layered synergistic protection scheme, an activity retention rate of over 90% is achieved after 2 hours of simulated gastric juice treatment, with a cumulative release rate exceeding 70% in the intestinal, ileal, and colonic regions. The activity retention rate remains above 75% at a granulation temperature of 90℃, and the activity retention rate is not less than 70% after 12 months of storage. This significantly improves the bioavailability of the alternative antimicrobial active substance, providing practical technical support for the reduction of antimicrobial drug dosage.

[0021] The beneficial effects of this invention are as follows:

[0022] 1. The present invention provides a method for preparing a feed additive for reducing the amount of antibiotics used in large-scale livestock and poultry farming. By using a three-dimensional network structure of sodium alginate and calcium ions in the inner layer to physically retain and encapsulate natural antimicrobial peptides, the basic protection of the core active ingredients is achieved. The egg-box model cross-linking structure of sodium alginate and calcium ions can remain stable in an acidic environment, and the active substances are effectively retained inside the gel network.

[0023] 2. The present invention describes a method for preparing a feed additive for reducing the amount of antibiotics used in large-scale livestock and poultry farming. This method involves secondary encapsulation of chitosan to form a dense polyelectrolyte composite membrane. The protonated amino groups on the chitosan molecular chain and the carboxylate ions on the sodium alginate molecular chain undergo Coulomb attraction to form ion crosslinking points, thus constructing a double-layer barrier structure that significantly enhances the resistance to hydrogen ion permeation and diffusion.

[0024] 3. The present invention describes a method for preparing a reduced-volume antibiotic alternative feed additive for large-scale livestock and poultry farming. This method utilizes a pectin gel outer layer to construct an acid-responsive protective shell. The gelation reaction of pectin and calcium ions follows the egg-box model mechanism, exhibiting a highly dense network structure in the gastric juice environment with a pH below 4.5. This structure can simultaneously resist the hydrolytic erosion and physical wear of pepsin. However, in the intestinal environment with a pH above 5.5, it begins to slowly degrade, achieving precise release of active substances in the jejunum and ileum.

[0025] 4. The present invention provides a method for preparing a feed additive for reducing the amount of antimicrobial drugs in livestock and poultry farming. Through high-temperature protective agent coating treatment, trehalose forms a glassy protective shell under high-temperature granulation environment. Based on the water substitution hypothesis, it maintains the three-dimensional conformational stability of antimicrobial peptides and effectively reduces the impact of heat transfer on the core active substances.

[0026] 5. The method for preparing antibiotic-reducing feed additives for large-scale livestock and poultry farming, as described in this invention, achieves an activity retention rate of over 90% after 2 hours of simulated gastric juice treatment, a cumulative release rate of over 70% in the intestinal, ileal, and colonic regions, an activity retention rate of over 75% at a granulation temperature of 90℃, and an activity retention rate of no less than 70% during a 12-month storage period through the synergistic effect of the above-mentioned multi-layer synergistic protection scheme. This significantly improves the bioavailability efficiency of antibiotic-reducing active substances and provides practical and feasible technical support for the action of reducing antibiotic dosage. Attached Figure Description

[0027] The invention will now be further described with reference to the accompanying drawings.

[0028] Figure 1 This is a flowchart of a method for preparing a reduced-volume antibiotic alternative feed additive for large-scale livestock and poultry farming, as described in this invention. Detailed Implementation

[0029] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0030] like Figure 1 As shown in the embodiment of the present invention, a method for preparing a feed additive for reducing the amount of antibiotics used in large-scale livestock and poultry farming includes the following steps:

[0031] Step 1: Preparation of the Antimicrobial Active Substance Dispersion System. In Step 1, the antimicrobial active substance dispersion system is prepared. First, a natural antimicrobial peptide is selected as the core active ingredient. The source of the natural antimicrobial peptide is limited to at least one of defensin-type antimicrobial peptides, bacteriocin-type antimicrobial peptides, or lactoferrin-derived peptides. For defensin-type antimicrobial peptides, members of the β-defensin subfamily are selected, and the number of amino acid residues is controlled within the range of 30 to 50. The disulfide bond connection mode is configured as a three-pair disulfide bond configuration. This disulfide bond configuration can maintain the spatial folding conformation of the antimicrobial peptide and maintain its antimicrobial activity under acidic conditions. For bacteriocin-type antimicrobial peptides, nisin is selected as a representative variety, and its molecular weight is controlled within the range of 3000 to 4000 Daltons. It has a significant antibacterial effect against Gram-positive bacteria. For lactoferrin-derived peptides, the N-terminal functional domain fragment of lactoferrin is selected. Its amino acid sequence contains a dual structural feature of positively charged enriched regions and hydrophobic regions.

[0032] The selected natural antimicrobial peptides were mixed and dispersed with sodium alginate aqueous solution under high-speed shear stirring to form a homogeneous oil-water emulsion system. The concentration of sodium alginate was controlled within the range of 0.5% to 2%. In the specific operation, the sodium alginate solid powder was first dispersed in deionized water for pre-dissolution treatment. The stirring speed was controlled within the range of 300 to 500 rpm, and the dissolution time was set to 2 to 4 hours to ensure that the sodium alginate molecular chains were fully hydrated and expanded. After pre-dissolution, the viscosity of the sodium alginate aqueous solution was adjusted to 50. The viscosity parameter, ranging from 0 to 2000 mPa·s, was selected based on the following technical considerations: if the viscosity is too low, it will not be able to effectively encapsulate the antimicrobial peptide molecules, resulting in a decrease in encapsulation efficiency; if the viscosity is too high, it will affect the smoothness of the droplets in the subsequent sharp-hole forming process. Viscosity adjustment is achieved by adding an appropriate amount of glycerol or propylene glycol as a thickening agent, with the amount added controlled within the range of 1% to 5% of the total mass of the solution. The addition of glycerol and propylene glycol can not only adjust the viscosity of the solution, but also act as a protective agent in the subsequent drying process, preventing the loss of activity of the antimicrobial peptides during the drying process.

[0033] During the mixing and dispersion process, the shear stirring speed was set to a high speed range of 8000 to 12000 revolutions per minute. A rotor-stator homogenizer was used for homogenization. The rotor diameter of the homogenizer was set to 50 to 80 mm, and the stator aperture was set to 0.1 to 0.3 mm. The working principle of the homogenizer is that the centrifugal force generated by the high-speed rotation of the rotor squeezes the material out of the stator aperture. Under the combined action of shear force, cavitation effect and collision, the oil-water emulsion system is homogenized. The stirring time is controlled within the range of 10 to 30 minutes. The specific time is fine-tuned according to the amount of antimicrobial peptide added and the initial viscosity of the sodium alginate solution. The mixing temperature is strictly controlled within the range of 4 to 37°C to avoid the destruction of antimicrobial peptide activity by high temperature. The pH of the mixed system is adjusted to near neutral to maintain the conformational stability of the antimicrobial peptide. The average particle size of the dispersion system is controlled within the range of 1 to 10 micrometers, and the polydispersity index is less than 0.3. The state of the dispersion system is monitored in real time by a laser particle size analyzer to ensure dispersion uniformity.

[0034] Sodium alginate can be replaced with oligomeric sodium alginate or high-mannuronic acid sodium alginate. Its molecular weight distribution range is controlled between 5,000 and 200,000 Daltons. If the molecular weight is too low, the pore size of the gel network formed will be too large to effectively retain small molecular weight antimicrobial peptides. If the molecular weight is too high, the viscosity of the solution will be too high, affecting the operability of the sharp-pore forming process. Temperature control is achieved by a circulating cooling water bath device. The cooling water temperature is set to 2 to 8°C, and the circulation flow rate is set to 5 to 10 liters per minute to ensure a constant temperature during the mixing process.

[0035] Step Two: Preparation and Shaping of Inner Microcapsules. In Step Two, inner microcapsules are prepared using the sharp-pore method. The homogeneous emulsion system formed in Step One is used to form droplets using the sharp-pore method. The sharp-pore forming process is carried out using a storage tank with a precise pore size distributor. The pore size of the distributor is precisely set to 50 to 300 micrometers. The storage tank is made of 316L stainless steel or polytetrafluoroethylene to avoid contamination of the system by metal ions. The droplet formation frequency is controlled within the range of 50 to 200 drops per minute. The specific frequency is adjusted according to the selected pore size and the viscosity of the emulsion system. The droplets are kept in a vertical falling state during the process of entering the calcium chloride solution to ensure sphericity. The droplet falling height is controlled within the range of 5 to 20 centimeters to ensure the formation of uniform spherical microcapsules.

[0036] The resulting droplets were then added to an aqueous calcium chloride solution to complete the ionic cross-linking reaction. The concentration of calcium chloride was set between 0.5% and 2%. This concentration range was chosen based on the kinetic balance of the ionic cross-linking reaction: if the concentration was too low, the cross-linking reaction would be incomplete, resulting in insufficient gel strength; if the concentration was too high, it might lead to excessive cross-linking on the surface, forming a dense shell that would hinder the penetration and encapsulation of chitosan in subsequent steps. The cross-linking reaction time was controlled within the range of 10 to 60 minutes, and the reaction temperature was set between 20 and 25°C. The mechanism of the cross-linking reaction is that the carboxyl groups on the sodium alginate molecular chain specifically bind to calcium ions to form a cross-linking network with an egg-box model structure. The antimicrobial peptide molecules in the emulsion system are physically trapped inside the three-dimensional network structure of sodium alginate-calcium ions, forming a basic encapsulation of the core active ingredient.

[0037] The droplet diameter is precisely controlled within the range of 100 to 500 micrometers. This particle size parameter is achieved through precise matching of the distributor orifice size and the extrusion pressure. The extrusion pressure is provided by a micro-peristaltic pump, with the pressure range controlled between 0.01 and 0.1 MPa. The pulse frequency stability of the micro-peristaltic pump directly affects the uniformity of droplet formation. The pulse frequency of the peristaltic pump is controlled within the range of 30 to 100 pulses per minute, and the coefficient of variation of the pulse interval should be less than 5% to ensure the consistency of droplet diameter. The sphericity of the microcapsules is achieved through… Visual inspection using an optical microscope showed that the sphericity should be greater than 0.95. The sphericity was calculated as the ratio of the projected area of ​​the microcapsule to the projected area of ​​a sphere of the same volume. The encapsulation efficiency was determined by high-performance liquid chromatography (HPLC) by measuring the content of free antimicrobial peptides in the supernatant. The mobile phase for HPLC was an acetonitrile-water-trifluoroacetic acid system, with a flow rate set to 0.8 to 1.2 mL per minute. The detection wavelength was set to the characteristic absorption peak in the range of 215 to 280 nm based on the UV absorption characteristics of the antimicrobial peptides.

[0038] After the cross-linking reaction is completed, the microcapsules are washed with deionized water 3 to 5 times to remove residual calcium ions and unreacted impurities on the surface. Each wash uses 3 to 5 times the volume of deionized water as the microcapsule slurry. Then, vacuum filtration is performed to remove excess water. The vacuum degree is controlled in the range of 0.08 to 0.1 MPa and the filtration time is 5 to 10 minutes. The treated microcapsules are white to light yellow gel spheres with a water content controlled in the range of 85% to 92%. The encapsulation efficiency of the inner microcapsules is increased to more than 80%.

[0039] Step 3: Construction of the intermediate protective layer. In step 3, an intermediate protective layer is constructed by transferring the microcapsules obtained in step 2 into a chitosan aqueous solution for secondary encapsulation, thus constructing a dense polyelectrolyte composite membrane to block the permeation and diffusion of hydrogen ions in gastric juice. Chitosan is a derivative obtained by deacetylation of chitin, and its degree of deacetylation is controlled within the range of 75% to 95%. The selection of this degree of deacetylation range is based on the following technical considerations: if the deacetylation is too low, the positive charge density on the molecular chain is insufficient, making it difficult to form a stable electrostatic interaction with sodium alginate; if the deacetylation is too high, the molecular hydrophilicity is too strong, and the swelling behavior in an acidic environment is difficult to control. The molecular weight of chitosan is selected to be 100,000 to 300,000 Daltons. If the molecular weight is too low, the strength of the formed membrane layer is insufficient; if the molecular weight is too high, the solution viscosity is too high, affecting the uniformity of encapsulation.

[0040] The concentration of the chitosan aqueous solution was set to 0.1% to 1%, and a 1% to 2% dilute acetic acid aqueous solution was used as the solvent to ensure complete dissolution of chitosan. The pH of the chitosan solution was adjusted to the acidic range to ensure complete dissolution and provide sufficient positive charge density. The microcapsules were immersed in the chitosan solution in a constant temperature shaking incubator with a shaking frequency set to 50 to 100 revolutions per minute. The immersion time was controlled within the range of 30 minutes to 2 hours. Chitosan molecules spontaneously deposited on the surface of sodium alginate microcapsules through electrostatic attraction to form a uniform coating layer. The protonated amino groups on the chitosan molecular chain and the carboxylate ions on the sodium alginate molecular chain underwent Coulomb attraction to form ionic cross-linking points, thereby forming a dense polyelectrolyte composite film. The thickness of the chitosan intermediate layer was precisely controlled within the range of 5 to 20 micrometers.

[0041] The degree of deacetylation of chitosan is preferably in the range of 85% to 90%. This range ensures that the molecular chain has sufficient positive charge density to form a stable electrostatic interaction with sodium alginate, while avoiding excessive swelling in an acidic environment due to excessive hydrophilicity. The molecular weight of chitosan is preferably in the range of 150,000 to 250,000 Daltons. This range ensures that the solution viscosity is suitable while maintaining the film strength, which is conducive to the impregnation operation. The impregnation temperature is preferably at room temperature to avoid the influence of temperature fluctuations on the conformation of chitosan molecules. The temperature control accuracy of the constant temperature oscillating incubator during the impregnation process is set to ±1℃.

[0042] After impregnation, the samples are freeze-dried to remove moisture. The drying temperature is controlled within a low temperature range of -40 to -80°C. The freeze-drying process includes three stages: pre-freezing, sublimation drying, and desorption drying. In the pre-freezing stage, the sample temperature is lowered from room temperature to below -40°C at a rate of 1 to 3°C per minute for 2 to 4 hours. In the sublimation drying stage, the separator temperature is controlled at -20 to -10°C, the vacuum degree is controlled at 10 to 50 Pa, and the drying time is 8 to 12 hours. In the desorption drying stage, the separator temperature is raised to 25 to 35°C, the vacuum degree is controlled at 5 to 20 Pa, and the drying time is 2 to 4 hours. The total drying time is extended to 12 to 24 hours to ensure thorough removal of moisture. The freeze-dried microcapsules have a fluffy white porous spherical structure with a moisture content of less than 5%.

[0043] Step 4: Preparation of the outer acid-sensitive protective shell. In step 4, the outer acid-sensitive protective shell is prepared by immersing the microcapsules obtained in step 3 in a pectin aqueous solution. Then, calcium chloride solution is added to trigger the gelation reaction between pectin and calcium ions, forming an outer gel protective shell with a dense network structure in an acidic environment. The pectin is selected as at least one of high-ester pectin or low-ester pectin. The degree of esterification of high-ester pectin is controlled within the range of 50% to 75%, and the degree of esterification of low-ester pectin is controlled within the range of 25% to 50%. The concentration of the pectin aqueous solution is controlled between 0.5% and 3%. Deionized water is used as the solvent. The heating and dissolving temperature is controlled within the range of 60 to 80°C. The stirring speed is 200 to 300 revolutions per minute, and the dissolving time is 30 to 60 minutes.

[0044] The impregnation treatment was carried out in a constant temperature shaking incubator with a shaking frequency set to 30 to 60 revolutions per minute, an impregnation temperature set to 25 to 40°C, and an impregnation time of 1 to 3 hours. After impregnation, calcium chloride solution was added to trigger the gelation reaction. The concentration of calcium chloride added was set to 0.3% to 1%. The calcium chloride solution was added slowly dropwise to avoid excessive local concentration that would lead to uneven gelation. The drop rate was controlled within the range of 1 to 3 ml per minute, the reaction time was controlled within the range of 2 to 8 hours, and the reaction temperature was maintained at 25 to 35°C.

[0045] The gelation reaction of pectin and calcium ions follows the egg-box model mechanism. Calcium ions are embedded between the carboxyl groups of adjacent pectin molecular chains to form an ion-bridging structure. This gel network exhibits a highly dense network structure in an environment with a pH below 4.5 to resist the hydrolytic erosion of pepsin in gastric juice. When the pH is above 5.5, the carboxyl groups are deprotonated and cross-linked with calcium ions. The gel network begins to slowly degrade to ensure the complete release of active substances in the jejunum and ileum.

[0046] The selection of high-ester pectin and low-ester pectin is differentiated according to the target release site: the gel network formed by high-ester pectin begins to degrade significantly when the pH is higher than 6.0, which is suitable for applications that need to be released in the prejejunum. The gel network formed by low-ester pectin is more sensitive to calcium ion concentration and can be rapidly degraded in the pH range of 5.5 to 6.5, which is suitable for applications that need to be released in the ileum. In the preferred embodiment of the present invention, a combination of low-ester pectin and calcium ions is selected to achieve effective release of active substances in the ileum and colon. The esterification degree of low-ester pectin is preferably in the range of 30% to 45%. This esterification degree range can achieve the best acid-sensitive response characteristics while ensuring gel strength. After the gelation reaction is completed, the microcapsules are washed with deionized water and filtered. The treated double-layer embedded microcapsules are pale yellow to brownish-yellow spherical with a smooth and glossy surface. The particle size increases by 20 to 50 micrometers due to the addition of the pectin layer.

[0047] Step 5: High-Temperature Protective Agent Coating Treatment. In step 5, a high-temperature protective agent coating treatment is performed. The microcapsules obtained in step 4 are mixed with the high-temperature protective agent to construct a high-temperature protective layer. The high-temperature protective agent is selected from at least one of trehalose, sucrose, maltodextrin, or glycerol. In the preferred embodiment, trehalose is selected as the preferred high-temperature protective agent. The concentration of trehalose is controlled to be 10% to 25% of the mass of the microcapsules. The selection of this concentration range is based on the following technical considerations: if the concentration is too low, the formed glassy protective shell will not be dense enough to effectively block heat transfer; if the concentration is too high, it may cause the microcapsules to stick together, affecting the flowability of the subsequent granulation process.

[0048] The mechanism of action of trehalose is based on its excellent water substitution hypothesis. Under dry and high-temperature conditions, trehalose molecules can replace water molecules and form hydrogen bond networks with polar groups on the surface of biomacromolecules, thereby maintaining the three-dimensional conformational stability of antimicrobial peptides. Trehalose can form a glassy amorphous structure, and its glass transition temperature is controlled in the range of 90 to 120°C. This temperature range covers the temperature range of conventional granulation processes. When the granulation temperature exceeds the glass transition temperature, trehalose transforms from a glassy state to a rubbery state and absorbs excess heat, thereby protecting the core active material from thermal damage.

[0049] The mixing process is carried out under low-speed stirring conditions to avoid mechanical damage to the microcapsule structure. The mixing equipment is a low-speed conical mixer or a V-type mixer, with the stirring speed controlled in the range of 10 to 30 revolutions per minute and the mixing time being 10 to 20 minutes. After mixing, fluidized bed drying is carried out to remove excess moisture. The drying temperature is controlled in the range of 40 to 60°C and the drying time is 2 to 4 hours. The high-temperature resistant protective agent forms a uniform protective coating on the surface of the microcapsules.

[0050] The combined use of high-temperature protective agents can achieve complementary advantages: trehalose has the highest water substitution capacity and glass transition temperature, making it the preferred high-temperature protective ingredient; sucrose has a lower cost and can provide good sweetness masking effect, making it suitable for cost-sensitive applications; maltodextrin has a wide molecular weight distribution range, which can provide gradient protection effects in different temperature ranges; the addition of glycerol is controlled below 5% to avoid reducing the stability of the glassy protective shell due to excessive plasticization. In the combined use of trehalose and maltodextrin, the mass ratio of the two is preferably in the range of 1:1 to 3:1, which can balance the protective effect and cost control.

[0051] Granulation and Quality Inspection: The microcapsules processed in step five are thoroughly mixed with the basic feed ingredients to prepare the final antibiotic alternative feed additive product. The basic feed ingredients include corn flour, soybean meal, wheat bran, and mineral premix. The proportions of each component follow the conventional feed formulation design principles. The granulation process adopts the wet granulation technology route. The granulation liquid is deionized water or 5% to 15% starch paste. The liquid-to-material ratio is controlled within the range of 25% to 40%. If the liquid-to-material ratio is too low, the pellet forming strength will be insufficient. If the liquid-to-material ratio is too high, the pellet moisture content will be too high, affecting the storage stability.

[0052] The screen aperture of the extruder is set to 1 to 3 mm. The size of the screen aperture directly affects the particle size distribution and thus the release behavior of the active substance. The granulation speed is controlled within the range of 30 to 60 revolutions per minute to ensure the uniformity of particle formation. The granulation temperature is strictly controlled within the range of 70 to 90°C. The granulation pressure is set within the range of 2 to 5 MPa. Under these temperature and pressure conditions, the trehalose protective layer undergoes a glass transition to form an effective thermal protection barrier. After granulation, countercurrent cooling is performed to avoid thermal shock to the microcapsule structure caused by sudden temperature changes. The cooling wind speed is controlled within the range of 0.5 to 2 meters per second, and the cooling time is controlled within the range of 30 to 60 minutes. The activity retention rate of the antibiotic alternative active substance in the final product is ensured to be above 75%.

[0053] Specific application example 1: Specific application verification was carried out using β-defensin as the core active ingredient. The amino acid sequence of β-defensin is a β-sheet structure containing 40 amino acid residues, and the molecular weight is about 4500 Daltons. 5 grams of β-defensin lyophilized powder were accurately weighed and added to 100 ml of sodium alginate aqueous solution with a sodium alginate concentration of 1.2%. The mixing temperature was controlled at 25℃, and the mixture was stirred for 20 minutes at 10000 rpm using a high-speed shear homogenizer to form a uniform emulsion system. The viscosity of the system was measured to be 1200 mPa·s.

[0054] The emulsion system was transferred to a sharp-orifice device and dripped into a 1.5% calcium chloride solution through a dispenser with a pore size of 200 micrometers. The extrusion pressure was controlled at 0.05 MPa, the droplet fall height was 10 cm, and the cross-linking reaction time was 30 minutes. The average particle size of the prepared inner layer microcapsules was 280 micrometers, and the encapsulation efficiency was measured to be 85.3%.

[0055] The inner microcapsules were transferred to a 0.5% chitosan solution for secondary encapsulation. The chitosan had a molecular weight of 150,000 Daltons and a degree of deacetylation of 88%. The impregnation time was 1 hour. After impregnation, the microcapsules were freeze-dried at -60°C for 18 hours. The thickness of the chitosan layer deposited on the surface of the microcapsules after freeze-drying was approximately 12 micrometers.

[0056] The dried microcapsules were further immersed in a 1.5% low-ester pectin solution with a pectin esterification degree of 42%, an immersion temperature of 30°C, and an immersion time of 2 hours. Subsequently, a 1% calcium chloride solution was added to trigger a gelation reaction, which lasted for 4 hours. The thickness of the outer acid-sensitive protective shell was approximately 25 micrometers.

[0057] Double-layer encapsulated microcapsules were mixed with trehalose powder at a mass ratio of 1:5, with the trehalose addition amount being 20% ​​of the microcapsule mass. After mixing, the mixture was fluidized bed dried at 45°C to obtain a microcapsule product with a trehalose protective layer on the surface. This product was then mixed with basic feed ingredients according to the specified ratio, with the microcapsule addition amount being 2%. Granulation was performed using an extruder, with the granulation temperature controlled at 80°C and the granulation pressure controlled at 3 MPa. After granulation, countercurrent cooling was performed for 45 minutes.

[0058] The granulated product underwent a simulated gastrointestinal release test. After treatment in simulated gastric fluid at pH 2.0 for 2 hours, the activity retention rate was 92.5%. After further release in simulated intestinal fluid at pH 7.0 for 4 hours, the cumulative release rate in the jejunum was 38.2%, in the ileum 35.6%, and in the colon 18.7%, with a total release rate of 92.5%. The active substance retention rate was determined to be 78.3%. The storage stability test results showed that after being placed at 40℃ for 6 months, the active substance retention rate was 74.8%, meeting the specified requirements.

[0059] The synergistic protection mechanism of the multi-layer core-shell structure follows the following technical logic: The inner sodium alginate-calcium ion three-dimensional network structure encapsulates antimicrobial peptide molecules within the gel network through physical interception, forming the first protective barrier. The egg-box model cross-linking structure of sodium alginate and calcium ions can remain stable in an acidic environment, and the active substances are effectively trapped inside the gel network. The middle chitosan polyelectrolyte composite membrane adheres tightly to the inner microcapsules through electrostatic interaction, forming the second protective barrier. The protonated amino groups on the chitosan molecular chain and the carboxylate ions on the sodium alginate molecular chain undergo Coulomb attraction to form ion cross-linking points, constructing a double-layer barrier structure, which significantly enhances the resistance to hydrogen ion permeation and diffusion.

[0060] The outer pectin gel protective shell forms a cross-linked network with calcium ions through an egg-box model mechanism. In the gastric juice environment with a pH below 4.5, it exhibits a highly dense network structure, capable of simultaneously resisting hydrolytic erosion by pepsin and physical abrasion. When the microcapsules migrate from the gastric juice environment to the small intestine environment, as the pH increases to the range of 5.5 to 6.5, the edges of the outer pectin gel begin to degrade slowly, but the main structure remains intact. When the pH increases to the ileal region (6.8 to 7.2), the degradation rate of the outer pectin gel accelerates, and it gradually disintegrates. The middle chitosan composite membrane is exposed to the intestinal environment. At this time, the swelling behavior of chitosan makes the composite membrane structure loose, creating conditions for further degradation of the inner microcapsules. When the pH of the colon region is higher than 7.2, the inner sodium alginate-calcium ion cross-linking network dissociates under the action of calcium ion chelating agent, and the remaining antimicrobial peptides are completely released. The high-temperature resistant protective layer on the surface forms a glassy protective shell under the high temperature environment of granulation. Based on the water substitution hypothesis, the three-dimensional conformational stability of the antimicrobial peptides is maintained, effectively reducing the impact of heat transfer on the core active substances.

[0061] The prepared feed additive product was subjected to simulated gastrointestinal release tests to verify its segmented release performance. The test method involved placing the sample in simulated gastric fluid for 2 hours, followed by transfer to simulated intestinal fluid for further release testing. The pH of the simulated gastric fluid was set to 1.5 to 3, and the pH of the simulated intestinal fluid was set to 6.5 to 7.5. The sample was accurately weighed and placed in a dialysis bag. The molecular weight cutoff of the dialysis bag was set to 3 to 5 times the molecular weight of the embedded antimicrobial peptide to ensure that only the released active substance could pass through the dialysis membrane. The simulated gastric fluid consisted of pepsin hydrochloric acid solution with a hydrochloric acid concentration of 35 mmol / L to 100 mmol / L and a pepsin concentration of 3.2 to 10 g / L. The simulated intestinal fluid consisted of trypsin phosphate buffer with a pH set to 6.8 to 7.2 and a trypsin concentration of 1 to 5 g / L.

[0062] Release tests were conducted in a 37°C constant temperature water bath shaker, with the shaker speed controlled at 50 to 100 revolutions per minute to simulate intestinal peristalsis. The release rate was determined by interval sampling, with samples taken every 30 minutes and an equal volume of fresh release medium added. After sampling, the content of active substances in the release solution was determined by high performance liquid chromatography, and the cumulative release rate was calculated.

[0063] Based on the release curve, the products are divided into different release grades to meet the feeding needs of different livestock and poultry breeds: Grade 1 products are suitable for young livestock and poultry such as piglets, with a release ratio of more than 60% in the anterior intestinal tract; Grade 2 products are suitable for growing and fattening livestock and poultry, with uniform release from the jejunum to the ileum, a release ratio of 30% to 50% in the jejunum, 30% to 40% in the ileum, and 10% to 20% in the colon; Grade 3 products are suitable for breeding livestock and poultry, with slow and continuous release throughout the entire intestine, and a release ratio of no more than 40% in any single segment.

[0064] Products meeting the release performance requirements are vacuum-packed or nitrogen-filled. Vacuum packaging is performed using a fully automatic vacuum packaging machine, with the vacuum level controlled between 0.08 and 0.1 MPa, the heat-sealing temperature set between 180 and 200°C, and the heat-sealing time between 2 and 4 seconds. The nitrogen-filled packaging process is as follows: first, vacuum is applied to 0.06 to 0.08 MPa, then high-purity nitrogen is filled in with a purity of not less than 99.99%, and the filling pressure is controlled between 0.1 and 0.2 MPa. Finally, heat sealing is performed. The packaging material is an aluminum foil composite bag with excellent oxygen barrier properties. The aluminum foil layer thickness should be greater than 7 micrometers, and the oxygen permeability of the composite bag should be less than 0.5 cubic centimeters per square meter per 24 hours per standard atmosphere.

[0065] Storage conditions were set at a temperature below 25℃ and a relative humidity below 60%, with a storage period of 12 months. Storage stability evaluation adopted a combination of accelerated stability testing and long-term stability testing. The accelerated stability testing conditions were a temperature of 40±2℃ and a relative humidity of 75±5%, with a testing period of 6 months. The long-term stability testing was conducted under the specified storage conditions. The changes in the content of active substances in the product were periodically sampled and tested. The sampling time points were set at 0, 1, 3, 6, 9, and 12 months to ensure that the activity retention rate was not less than 70% within the specified storage period.

[0066] Based on the experimental results of the aforementioned steps, a database of key process parameters for large-scale production lines was established. The database includes the following parameter categories: feeding rate, shear stirring parameters, mixing temperature, and system viscosity in step one; pore size, extrusion pressure, droplet frequency, calcium ion concentration, and crosslinking time in step two; chitosan concentration, molecular weight, impregnation parameters, and freeze-drying process parameters in step three; pectin concentration, degree of esterification, calcium ion addition, and gelation reaction time in step four; type, amount, and mixing parameters of protective agent in step five; raw material ratio, liquid-to-material ratio, sieve aperture, and granulation parameters in the granulation process; detection method parameters; packaging parameters; and storage conditions.

[0067] The process parameters are systematically optimized using response surface methodology (RSM) or artificial intelligence (AI) optimization algorithms. RSM employs Box-Behnken design or central composite design to establish a mathematical model between process parameters and product quality indicators, analyzes the main effects, interaction effects, and secondary effects of each parameter, and determines the optimal combination of process parameters. AI optimization algorithms can use genetic algorithms, particle swarm optimization algorithms, or deep reinforcement learning algorithms, with product quality indicators and production capacity indicators as optimization objectives, and perform a global search within the parameter feasible region to ultimately determine the optimal large-scale production process scheme and form a standard operating procedure document.

[0068] Example 2: To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments.

[0069] Using nisin as the core active ingredient, different combinations of protective agents were used for verification. Nisin has a molecular weight of approximately 3500 Daltons and exhibits significant antibacterial effects against Gram-positive bacteria. Nisin was mixed with a 0.8% aqueous solution of sodium alginate (molecular weight approximately 50,000 Daltons), which reduced solution viscosity and improved the operability of subsequent processes. The mixing temperature was controlled at 20°C, the high-speed shear stirring speed was 9000 rpm, and the stirring time was 15 minutes to form a homogeneous emulsion system with a viscosity of 800 mPa·s. The emulsion system was then dripped into a 1.2% calcium chloride solution through a 150-micron pore size device. The extrusion pressure was controlled at 0.04 MPa, and the cross-linking reaction time was 25 minutes. The resulting inner layer microcapsules had an average particle size of 220 microns and an encapsulation efficiency of 82.6%.

[0070] The inner microcapsules were transferred to a 0.3% chitosan solution for secondary encapsulation. The chitosan had a molecular weight of 200,000 Daltons and a degree of deacetylation of 82%. The impregnation temperature was 25°C, and the impregnation time was 90 minutes. The freeze-drying conditions were -50°C and the drying time was 20 hours. The thickness of the chitosan intermediate layer was approximately 8 micrometers. The dried microcapsules were then further impregnated in a 2% high-ester pectin solution with a pectin esterification degree of 65%. The impregnation temperature was 35°C, and the impregnation time was 2.5 hours. The calcium chloride concentration was 0.6%, and the gelation reaction time was 5 hours. The thickness of the outer acid-sensitive protective shell was approximately 30 micrometers.

[0071] The double-layered encapsulated microcapsules were mixed with a high-temperature resistant protective agent mixture. The protective agent combination consisted of trehalose and maltodextrin in a 1:1 mass ratio, with a total addition of 25% of the microcapsule mass. After mixing, the mixture was fluidized bed dried at 50°C to obtain a microcapsule product with a high-temperature resistant protective layer on the surface. This product was then mixed with basic feed ingredients according to the specified ratio, with a microcapsule addition of 2.5%. Granulation was performed using an extruder, with the granulation temperature controlled at 85°C and the granulation pressure controlled at 4 MPa.

[0072] The granulated product was subjected to performance tests. After treatment in simulated gastric fluid at pH 1.8 for 2 hours, the activity retention rate was 89.7%. After release in simulated intestinal fluid for 6 hours, the total release rate reached 88.3%, and the active substance retention rate was measured to be 81.2%. Compared with Example 1, this example uses high-ester pectin as the outer protective shell, which has higher density in acidic environment and better barrier effect against gastric acid. At the same time, a composite protective agent system of trehalose and maltodextrin is used to further improve the thermal stability during the granulation process.

[0073] Example 3: To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments.

[0074] Using lactoferrin-derived peptides as the core active ingredient, large-scale production verification was carried out using a fully automated continuous production line. The amino acid sequence of lactoferrin-derived peptides contains positively charged enriched regions and hydrophobic regions, and has a significant antibacterial effect on Gram-negative bacteria such as Escherichia coli and Salmonella. The following process flow and parameter configuration were adopted for large-scale production.

[0075] Step 1 was carried out in a 500-liter stainless steel preparation tank with a sodium alginate concentration of 1.0%. The solution concentration was monitored by an online refractometer, and the viscosity of the system was monitored by an online viscometer. Shear stirring was performed using a three-stage homogenizer in series. The homogenization pressure of the first stage was 20 MPa, the second stage was 35 MPa, and the third stage was 50 MPa. The mixing temperature was controlled at 15°C.

[0076] Step 2 is carried out in a fluidized bed crosslinking reactor with an inner diameter of 3 meters. The emulsion system is atomized into droplets through a high-pressure nozzle. The droplet diameter is precisely controlled by the nozzle orifice diameter and atomization pressure. Calcium chloride solution is continuously added through a metering pump. The crosslinking reaction is carried out in a moving bed, and the reaction time is controlled by the material residence time.

[0077] Step 3 is carried out in a series of impregnation tanks. The microcapsules pass through three impregnation tanks in sequence, each with a volume of 200 liters. The chitosan solution is replaced countercurrently in each tank. The impregnation temperature is room temperature, and the total impregnation time is 2 hours. The freeze-drying is carried out using a 24-door continuous freeze dryer with a single door freeze-drying area of ​​2 square meters and a freeze-drying cycle of 24 hours.

[0078] Step four employs a fluidized bed coating process, where pectin solution is sprayed onto the surface of fluidized microcapsules through an atomizing nozzle, while calcium chloride solution is simultaneously sprayed in to trigger a gelation reaction. The coating weight gain is controlled to be 15% to 20%.

[0079] Step 5 involves using a vibrating fluidized bed for protective coating, where trehalose powder is uniformly deposited on the surface of the microcapsules via airflow.

[0080] Step six uses a ring die pellet mill with a capacity of 5 tons per hour, and the pelleting temperature is controlled at 82℃ by steam regulation.

[0081] Product quality testing results from large-scale production show that the particle size distribution D50 is 1850 micrometers, D90 is 2850 micrometers, the active substance content is 95.2% of the nominal value, the active substance retention rate in simulated gastric fluid is 91.8%, the total release rate in simulated intestinal fluid is 90.1%, the active substance retention rate is 76.8%, the batch-to-batch quality variation coefficient is less than 8%, and the active substance retention rate after 12 months of storage is 71.5%, meeting market circulation requirements.

[0082] Comparative analysis of comparative examples: Comparative example 1 uses a traditional method to prepare monolayer sodium alginate gel microspheres encapsulating antimicrobial peptides. The antimicrobial peptides are mixed with a 1.5% sodium alginate solution and then formed into droplets using the sharp-hole method. The droplets are then dropped into a 2% calcium chloride solution for cross-linking and solidification to form monolayer gel microspheres. The microspheres are washed with deionized water and then directly freeze-dried. This method omits steps such as secondary encapsulation with chitosan, outer layer of pectin gel, and coating with a high-temperature protective agent.

[0083] Comparative Example 2 uses chitosan monolayer to embed antimicrobial peptides. The antimicrobial peptides are dissolved in a 1% chitosan acetic acid solution, and chitosan microspheres are prepared by spray drying. This method utilizes the film-forming properties of chitosan to form a monolayer protective structure.

[0084] Comparative Example 3 involved adding the antimicrobial peptide directly to the feed ingredients in powder form for mixing and granulation, without any encapsulation or protection treatment.

[0085] Table 1: Comparison of Embodiments and Comparative Examples of the Invention

[0086] Testing items Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Activity retention rate (%) after 2 hours of treatment with simulated gastric juice 92.5 89.7 91.8 45.3 52.8 28.6 Total release rate (%) in artificial intestinal fluid 92.5 88.3 90.1 96.8 85.2 100.0 Activity retention rate (%) after granulation at 80℃ 78.3 81.2 76.8 52.4 48.7 35.2 Activity retention rate (%) after storage at 40℃ for 6 months 74.8 — — 51.2 55.6 42.3 Activity retention rate (%) after storage at 40℃ for 12 months — — 71.5 — — — Duration of effective antibacterial effect (hours) 18～24 16～20 18～22 4～6 5～7 3～5 Encapsulation rate (%) 85.3 82.6 — 68.5 58.3 —

[0087] The above comparative data fully demonstrate that the optimized encapsulation protection and enteric release synergistic technology of this invention significantly outperforms the comparative examples in various indicators such as simulated gastric acid stability, artificial intestinal fluid release characteristics, granulation thermal stability, long-term storage stability, effective antibacterial duration, and encapsulation rate. In the embodiments of this invention, after 2 hours of simulated gastric fluid treatment, the activity retention rate is as high as 89.7%~92.5%, while the comparative examples are only 28.6%~52.8%; after granulation at 80℃, the activity retention rate reaches 76.8%~81.2%, far exceeding the 35.2%~52.4% of the comparative examples; after storage at 40℃ for 6 months, the activity retention rate reaches 74.8% (Example 1), while the comparative examples are only up to 55.6%, and Example 3 still maintains a high retention rate of 71.5% after 12 months; the effective antibacterial duration is 16~24 hours, while the comparative examples are only 3~7 hours; the encapsulation rate is 82.6%~85.3%, significantly better than the 58.3%~68.5% of the comparative examples. It is worth noting that although Comparative Example 3 achieved a total release rate of 100% in artificial intestinal fluid, its gastric fluid retention rate was only 28.6% and its granulation retention rate was only 35.2%, indicating a lack of effective gastric acid protection and process tolerance. This invention, through precise encapsulation design and enteric release regulation, achieves the comprehensive advantages of "low release in the stomach, high release in the intestine, high retention during thermal processes, and high stability during long-term storage," and significantly prolongs the antibacterial duration, forming a significant positive synergistic effect, thus optimizing the delivery and stabilization of the active substance.

[0088] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a feed additive for reducing the dosage of antibiotics in livestock and poultry farming, characterized in that, Includes the following steps: Step 1: Select natural antimicrobial peptides as the core active ingredients, and mix and disperse them with sodium alginate aqueous solution under high-speed shear stirring conditions to form a homogeneous oil-water emulsion system. Step 2: The homogenized emulsion system is prepared into droplets using the sharp-hole method, and then dropped into an aqueous solution of calcium chloride to complete the ion cross-linking reaction, forming a three-dimensional network structure of sodium alginate-calcium ions to encapsulate the core active ingredients; Step 3: The microcapsules are transferred into a chitosan aqueous solution for secondary encapsulation to form a dense polyelectrolyte composite membrane to block the permeation and diffusion of hydrogen ions in gastric juice. Step 4: Continue to immerse the microcapsules in the pectin aqueous solution, add calcium chloride solution to trigger the gelation reaction, and form an outer gel protective shell with a dense network structure in an acidic environment; Step 5: Mix the microcapsules with the high-temperature protective agent to form a uniform protective coating on the surface of the microcapsules.

2. The method for preparing a reduced-volume antibiotic alternative feed additive for large-scale livestock and poultry farming according to claim 1, characterized in that, The source of the natural antimicrobial peptide is at least one of defensin-type antimicrobial peptides, scleroderma-type antimicrobial peptides, or lactoferrin-derived peptides. The defensin-type antimicrobial peptides are selected from members of the β-defensin subfamily, the scleroderma-type antimicrobial peptides are selected from nisin as a representative variety, and the lactoferrin-derived peptides are selected from the N-terminal functional domain fragment of lactoferrin.

3. The method for preparing a feed additive for large-scale livestock and poultry breeding with reduced-volume antibiotics as described in claim 1, characterized in that, The sodium alginate used in step one can be replaced with oligoalginate or high-mannuronic acid sodium alginate; the mixing temperature during the mixing and dispersion process is controlled within the range of 4 to 37°C, and the pH of the mixing system is adjusted to near neutral to maintain the conformational stability of the antimicrobial peptide.

4. The method for preparing a feed additive for reducing the amount of antibiotics used in large-scale livestock and poultry farming according to claim 1, characterized in that, The sharp-hole forming process in step two is carried out using a liquid storage tank with a precise pore size distributor. The pore size of the distributor is set to 50 to 300 micrometers, the droplet formation frequency is controlled within the range of 50 to 200 drops per minute, and the droplet falling height is controlled within the range of 5 to 20 centimeters. After the cross-linking reaction is completed, the microcapsules are washed with deionized water 3 to 5 times to remove residual calcium ions on the surface, and then vacuum filtration is performed to remove excess water.

5. The method for preparing a feed additive for large-scale livestock and poultry breeding with reduced-volume antibiotics as described in claim 1, characterized in that, The chitosan mentioned in step three is a derivative of chitin obtained by deacetylation treatment, with its degree of deacetylation controlled in the range of 75% to 95%. The molecular weight of chitosan is selected in the range of 100,000 to 300,000 Daltons, and the thickness of the chitosan intermediate layer is controlled in the range of 5 to 20 micrometers. After impregnation, freeze-drying treatment is carried out, with the drying temperature controlled in the range of -40 ℃ to -80 ℃, and the total drying time extended to 12 to 24 hours to ensure that moisture is completely removed.

6. The method for preparing a feed additive for large-scale livestock and poultry breeding with reduced-volume antibiotics as described in claim 1, characterized in that, The pectin mentioned in step four is at least one of high-ester pectin or low-ester pectin. The degree of esterification of high-ester pectin is controlled within the range of 50% to 75%, and the degree of esterification of low-ester pectin is controlled within the range of 25% to 50%. The gelation reaction of pectin with calcium ions follows the egg carton model mechanism. The gel network exhibits a dense and stable state in an environment with a pH below 4.5 to resist gastric juice erosion, while it begins to slowly degrade in an environment with a pH above 5.

5. The active substances are completely released in the jejunum and ileum.

7. The method for preparing a feed additive for large-scale livestock and poultry breeding with reduced-volume antibiotics as described in claim 1, characterized in that, In step five, trehalose is selected as the preferred high-temperature protective agent, and the concentration of trehalose added is controlled to be 10% to 25% of the microcapsule mass. The mixing process is carried out under low-speed stirring conditions to avoid mechanical damage to the microcapsule structure, and the stirring speed is controlled within the range of 10 to 30 revolutions per minute. The protective layer can form a glassy protective shell under the high-temperature environment of feed pelleting to effectively reduce the impact of heat transfer on the activity of the core antimicrobial peptides.

8. The method for preparing a feed additive for reducing the amount of antibiotics used in large-scale livestock and poultry farming, as described in claim 1, is characterized in that... The granulation process adopts a wet granulation technology route. The granulation liquid is deionized water or 5% to 15% starch paste, and the liquid-to-solid ratio is controlled within the range of 25% to 40%. The screen aperture of the extruder is set to 1 to 3 mm, the granulation speed is controlled within the range of 30 to 60 revolutions per minute, the granulation temperature is controlled within the range of 70 to 90°C, and the granulation pressure is set within the range of 2 to 5 MPa. After granulation, countercurrent cooling is performed to avoid thermal shock to the microcapsule structure caused by sudden temperature changes. The activity retention rate of the antibiotic alternative active substances in the final product is ensured to be above 75%.

9. The method for preparing a feed additive for large-scale livestock and poultry breeding with reduced-volume antibiotics as described in claim 1, characterized in that, The prepared feed additive products were subjected to simulated gastrointestinal release tests. The test method involved placing the sample in artificial gastric fluid for 2 hours and then transferring it to artificial intestinal fluid for further release testing. The pH of the artificial gastric fluid was set to 1.5 to 3, and the pH of the artificial intestinal fluid was set to 6.5 to 7.

5. The release amount of active substances at different time periods was determined by high performance liquid chromatography. Based on the release curves, the products were classified into different release levels to meet the feeding needs of different livestock and poultry breeds.

10. The method for preparing a feed additive for large-scale livestock and poultry breeding with reduced-volume antibiotics as described in claim 1, characterized in that, Products that meet the release performance requirements are vacuum-packed or nitrogen-filled. The packaging material is an aluminum foil composite bag with excellent oxygen barrier properties. The content of active substances in the product is sampled and tested regularly to ensure that the activity retention rate is not less than 70% within the specified storage period. Based on the test results of the above steps, a database of key process parameters for large-scale production lines is established. The process parameters are systematically optimized by response surface methodology or artificial intelligence optimization algorithms to finally determine the optimal large-scale production process scheme and form a standard operating procedure document.

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