Composite plant extract feed additive and method for preparing the same

Abstract

The application discloses a kind of composite plant extract feed additive and preparation method thereof, it is related to animal feed additive technical field, composite plant extract feed additive has core-shell multilayer structure, from inside to outside successively includes: inner layer active carrier core, it is the porous starch of succinic anhydride modification octenyl loaded with eucommia leaf extract;Intermediate layer, it is coated in the outer surface of inner layer active carrier core;Outer membrane, it is coated in the outer surface of intermediate layer.The application solves the problems such as poor stability of traditional eucommia leaf extract feed additive, serious degradation in stomach, uncontrollable release, low bioavailability by multilayer core-shell structure design, composite plant extract feed additive in gastric juice 2h release rate <5%, intestinal fluid 4h release >95%.

Description

Technical Field

[0001] This invention belongs to the field of animal feed technology, and more specifically, this invention relates to a compound plant extract feed additive and its preparation method. Background Technology

[0002] Eucommia ulmoides ( Eucommia ulmoides Oliver Eucommia ulmoides is a monotypic plant of the Eucommia family, unique to China, and has been used as a traditional Chinese medicine for nearly two thousand years. Eucommia ulmoides leaves are rich in chlorogenic acid, flavonoids, lignans, iridoids, and polysaccharides, among other active ingredients, and possess multiple pharmacological effects, including lowering blood pressure, regulating blood lipids, lowering blood sugar, anti-inflammation, anti-oxidation, anti-tumor activity, and liver protection.

[0003] In recent years, the application of Eucommia ulmoides leaf extract as a natural plant-based feed additive in livestock production has been increasingly studied, especially showing great potential in improving the growth performance, meat quality, and antioxidant capacity of pigs. The main active components of Eucommia ulmoides leaf extract include: chlorogenic acid (CGA): typically 3%–8%, one of the most important bioactive components of Eucommia ulmoides, exhibiting significant antioxidant and anti-inflammatory activities; flavonoids: approximately 10%–30%, including quercetin and kaempferol, which have free radical scavenging and immune-regulating functions; polysaccharides: approximately 30%–45%, which can promote the proliferation of beneficial intestinal bacteria and improve intestinal microecological balance; and lignans and iridoids: which have the effects of regulating lipid metabolism and protecting the liver and kidneys.

[0004] Numerous studies have shown that adding appropriate amounts of Eucommia ulmoides leaf extract to the diet can significantly improve the growth performance of pigs. Liang Xiaoling et al. (2024) divided 160 Duroc-Landrace-Large White crossbred fattening pigs into a control group and groups supplemented with 100, 200, and 300 mg / kg Eucommia ulmoides leaf extract for 50 days. The results showed that the final weight, average daily weight gain, average daily feed intake, and feed conversion ratio of each experimental group were significantly different from those of the control group (P < 0.05), with the 200 mg / kg and 300 mg / kg supplementation levels showing the best effects. In addition, adding 0.3 g / kg Eucommia ulmoides leaf extract to the diet of weaned piglets can significantly reduce the diarrhea rate, increase the average daily weight gain, and improve the intestinal villus height, with effects comparable to antibiotics, showing potential as an antibiotic alternative. Its growth-promoting mechanisms mainly include: stimulating bile and pancreatic juice secretion, increasing digestive enzyme activity, and promoting the digestion and absorption of nutrients; regulating the intestinal flora structure and promoting the proliferation of beneficial bacteria such as Bifidobacteria and Lactobacillus; inhibiting the activation of the NF-κB signaling pathway, reducing the release of pro-inflammatory factors such as IL-6, IL-8, and TNF-α, and reducing intestinal inflammation; and the high arginine content in Eucommia ulmoides leaf extract can improve the health of the body's immune system and enhance disease defense capabilities.

[0005] The effects of Eucommia ulmoides leaf extract on pork quality improvement are multidimensional. Adding 0.050% Eucommia ulmoides leaf extract to the diet significantly reduced malondialdehyde (MDA) levels in serum and the longissimus dorsi muscle, increased glutathione (GSH) levels and superoxide dismutase (SOD) activity, reduced drip loss, and improved muscle pH, redness (a*) value, and oleic acid content, while also significantly increasing muscle fiber density. In terms of meat quality improvement, Eucommia ulmoides leaf extract's effects include: reducing drip and pressure losses, improving muscle water retention; optimizing meat color: increasing a* value (redness) and decreasing L* value (brightness), resulting in a more vibrant and appealing red color; increasing intramuscular fat: improving marbling score, enhancing muscle tenderness and flavor; regulating fatty acid composition: increasing monounsaturated fatty acid (MUFA) content, decreasing polyunsaturated fatty acid (PUFA) ratio, and optimizing the n-6 / n-3 ratio; and enhancing umami substances: increasing inosinic acid (IMP) and sweet amino acid content, improving pork flavor.

[0006] Despite the promising prospects of Eucommia ulmoides leaf extract in pig farming, several key challenges remain in its practical application: poor stability of active ingredients, susceptibility to degradation and inactivation, and sensitivity of active components such as chlorogenic acid and flavonoids to light, heat, oxygen, and the gastrointestinal environment. During the high-temperature pelleting process (typically 70-90℃) in feed processing and in the highly acidic environment of the pig's gastrointestinal tract (pH 1.5-3.5), active ingredients are prone to oxidation, hydrolysis, or degradation, leading to a significant reduction in bioavailability. Studies have shown that unprotected chlorogenic acid can be released at rates exceeding 70% within 3 hours in simulated gastric juice, but is largely inactivated before reaching the intestines. Eucommia ulmoides leaf extract has a distinctive bitter taste and pungent odor; direct addition to feed reduces palatability, resulting in decreased feed intake in pigs. While some studies have not found significant effects, palatability issues are significant at high addition levels (>0.3%), limiting the effective dosage of Eucommia ulmoides leaf extract. Currently, the effective addition levels of Eucommia ulmoides leaf extract in research are typically between 0.1% and 0.3% (i.e., 1000-3000 mg / kg), which is a relatively large dosage. Due to the complex extraction process and high raw material costs, adding large doses significantly increases feed costs, hindering its widespread application in large-scale farming. Many bioactivities of Eucommia ulmoides leaf extract (such as regulating gut microbiota and promoting nutrient absorption) require specific locations in the intestine (such as the ileum and cecum) to be effective. However, uncoated Eucommia ulmoides leaf extract is released in large quantities in the stomach, leaving very little by the time it reaches the intestine, failing to achieve targeted intestinal delivery and resulting in low actual bioavailability. Furthermore, the polyphenols in Eucommia ulmoides leaf extract easily form complexes or precipitation reactions with metal ions and proteins in feed, affecting not only its own activity but also potentially reducing the bioavailability of other nutrients, posing challenges to feed formulation design. Summary of the Invention

[0007] One object of the present invention is to solve at least the above-mentioned problems and / or defects, and to provide at least the advantages described below.

[0008] To achieve these and other advantages according to the present invention, a compound plant extract feed additive is provided, the compound plant extract feed additive having a core-shell multilayer structure, comprising, from the inside out: The inner active carrier core is a chitosan-modified octenyl succinic anhydride-modified porous starch loaded with Eucommia ulmoides leaf extract. The intermediate layer covers the outer surface of the inner active carrier core; The outer membrane covers the outer surface of the intermediate layer.

[0009] Preferably, the Eucommia ulmoides leaf extract contains chlorogenic acid, flavonoids, and Eucommia ulmoides polysaccharides, with the chlorogenic acid content ≥8%, the flavonoid content ≥10%, and the Eucommia ulmoides polysaccharide content ≥40% by dry weight.

[0010] Preferably, the intermediate layer is an ethyl cellulose film layer, and the coating weight gain of the intermediate layer is 10% to 16%.

[0011] Preferably, the outer membrane is obtained by cross-linking silica nanoparticles and chitosan with sodium tripolyphosphate.

[0012] A method for preparing a compound plant extract feed additive includes the following steps: Step 1: Add starch to deionized water to prepare a 20%~30% (w / v) starch milk, adjust the pH to 8.0~8.5, slowly add 1%~3% of octenyl succinic anhydride by dry weight of starch, maintain pH 8.0~8.5 and react at 30~40℃ for 2~4 hours; after the reaction is completed, adjust the pH to 6.5, wash and dry to obtain octenyl succinic anhydride modified starch; Octenyl succinic anhydride modified starch was re-formulated into a 25% (w / v) starch milk, the pH was adjusted to 4.5-6.0, a compound enzyme was added and enzymatically hydrolyzed at 35-45℃ for 12-18 hours, and after inactivation, it was washed, dried and sieved to obtain octenyl succinic anhydride modified porous starch. Octenyl succinic anhydride-modified porous starch was dispersed in anhydrous ethanol, and 2% to 5% (by dry weight) of chitosan and deionized water were added. The mixture was refluxed at 60 to 80°C for 4 to 6 hours to obtain chitosan-modified octenyl succinic anhydride-modified porous starch. Step 2: Dissolve Eucommia ulmoides leaf extract powder in a mixed solvent of ethanol and water at a volume ratio of 7:3 to prepare a loading solution of 250-350 mg / mL. Add the chitosan-modified octenyl succinic anhydride-modified porous starch obtained in Step 1 to the loading solution. After ultrasonic dispersion, heat to 35-45℃ and keep warm for 2-4 hours. Then, remove the solvent by rotary evaporation at 40℃ and -0.08 MPa, vacuum dry at 40℃, and sieve to obtain octenyl succinic anhydride-modified porous starch loaded with Eucommia ulmoides leaf extract. Step 3: The octenyl succinic anhydride modified porous starch loaded with Eucommia ulmoides leaf extract obtained in Step 2 is fed into a fluidized bed coating machine; a 6%~10% (w / w) anhydrous ethanol solution of ethyl cellulose is prepared, and a 5%~15% (w / w) pore-forming agent is added to it, and the mixture is stirred evenly to obtain a clear coating solution; during the coating process, the inlet air temperature is controlled at 50~60℃, the material temperature at 40~48℃, the atomization pressure at 0.15~0.25MPa, the coating solution flow rate at 2~5 mL / min, and the coating weight gain is controlled at 10%~16%; after coating, the mixture is further fluidized and dried for 20~30 min to obtain drug-loaded particles with an intermediate layer; Step 4: Dissolve chitosan with a degree of deacetylation ≥90% in a 1.5% (v / v) glacial acetic acid solution, adjust the pH to 5.0~5.5, and obtain a chitosan solution with a concentration of 1.5%~2.5% (w / v); Vaporized silica nanoparticles were ultrasonically dispersed in a chitosan solution to obtain a chitosan-silica composite suspension. Dissolve sodium tripolyphosphate in deionized water, adjust the pH to 5.0-5.5, and prepare a sodium tripolyphosphate crosslinking solution with a concentration of 1.0%-2.0% (w / v); The drug-loaded particles with an intermediate layer obtained in step three are dispersed in a chitosan-silica composite suspension and stirred at room temperature for 15-30 minutes to allow the chitosan-silica composite suspension to be uniformly adsorbed on the surface of the drug-loaded particles in the intermediate layer, thus obtaining a dispersion. Then, sodium tripolyphosphate crosslinking solution is added dropwise to the dispersion while stirring the dispersion at a speed of 80-120 rpm during the dropwise addition. After the dropwise addition is completed, the crosslinking reaction is continued for 30-60 minutes to form an outer film on the surface of the drug-loaded particles. After the reaction was completed, the product was collected by filtration, washed with deionized water to remove residual sodium tripolyphosphate and chitosan, washed with anhydrous ethanol, vacuum dried at 40-60℃ for 6-12 hours, passed through 40-mesh and 60-mesh standard sieves, and the 250-425μm particle size range was obtained to obtain the compound plant extract feed additive.

[0013] Preferably, in step one, the complex enzyme is a complex enzyme composed of α-amylase and glucoamylase in an enzyme activity ratio of 1:2 to 1:4, and the total enzyme dosage is 1500 to 2500 U / g starch.

[0014] Preferably, the pore-forming agent in step three is polyvinylpyrrolidone K30, and its addition amount is 8% to 12% of the mass of ethyl cellulose; the mass fraction of the anhydrous ethanol solution of ethyl cellulose is 6% to 8%.

[0015] Preferably, in step four, the mass ratio of the silica nanoparticles, chitosan, and sodium tripolyphosphate is 0.3~0.6:1:0.15~0.25.

[0016] Preferably, the compound plant extract feed additive is used to promote the growth and development of pigs and improve the quality and flavor of pork. The amount of compound plant extract feed additive added to weaned piglet feed is 1800-2500g per ton of complete feed, the amount added to growing pig feed is 1500-2000g per ton of complete feed, and the amount added to fattening pig feed is 1200-1800g per ton of complete feed.

[0017] This invention offers at least the following beneficial effects: Through a multi-layered core-shell structure design, it solves the problems of poor stability, severe degradation in the stomach, uncontrollable release, and low bioavailability of traditional Eucommia ulmoides leaf extract feed additives. The compound plant extract feed additive prepared by this invention exhibits a gastric juice release rate of <5% within 2 hours and an intestinal juice release rate of >95% within 4 hours. Furthermore, experimental results show that feeding piglets with feed containing this compound plant extract feed additive can significantly promote their growth and development and improve the nutritional utilization rate of the feed.

[0018] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Detailed Implementation

[0019] The present invention will now be described in further detail so that those skilled in the art can implement it based on the description.

[0020] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0021] The Eucommia ulmoides leaf extracts used in all examples and comparative examples were commercially available products purchased from Lanzhou Waterles Biotechnology Co., Ltd.

[0022] Example 1

[0023] A method for preparing a compound plant extract feed additive includes the following steps: Step 1: Preparation of octenyl succinic anhydride modified porous starch Take 500g of corn starch and add deionized water to prepare a 25% (w / v) starch slurry, stirring until evenly dispersed. Adjust the pH to 8.2 with 0.5mol / L NaOH solution, and slowly add 10.0g of octenyl succinic anhydride (2% of the dry weight of the starch), maintaining the pH at 8.0-8.5 and reacting at 35℃ for 3h. During the reaction, continuously add 0.5mol / L NaOH solution to maintain a constant pH. After the reaction is complete, adjust the pH to 6.5 with 0.5mol / L HCl to terminate the reaction. Wash the product three times with deionized water (2000mL each time) and dry under vacuum at 40℃ for 12h to obtain octenyl succinic anhydride modified starch.

[0024] Octenyl succinic anhydride modified starch was reconstituted into a 25% (w / v) starch slurry, and the pH was adjusted to 5.5. A complex enzyme (α-amylase and glucoamylase in an enzyme activity ratio of 1:3) was added, with a total enzyme dosage of 2000 U / g starch (i.e., 1.0 × 10⁻⁶). 6 The product was enzymatically hydrolyzed at 40℃ for 15 h. After enzymatic hydrolysis, it was inactivated in a water bath at 85℃ for 15 min. The product was washed three times with deionized water (2000 mL each time), vacuum dried at 40℃ for 12 h, and passed through a 100-mesh sieve to obtain octenyl succinic anhydride modified porous starch.

[0025] 200 g of octenyl succinic anhydride-modified porous starch was dispersed in 2000 mL of anhydrous ethanol. Chitosan (7.0 g), accounting for 3.5% of the dry weight of the octenyl succinic anhydride-modified porous starch, and 20 mL of deionized water were added, and the mixture was refluxed at 70 °C for 5 h. After the reaction was completed, the mixture was centrifuged (4000 rpm, 10 min), and the precipitate was washed twice with anhydrous ethanol (500 mL each time). The precipitate was then vacuum dried at 40 °C for 12 h to obtain chitosan-modified octenyl succinic anhydride-modified porous starch.

[0026] Step 2: Loading of Eucommia ulmoides leaf extract 100g of Eucommia ulmoides leaf extract powder (chlorogenic acid content ≥8%, total flavonoids ≥10%, Eucommia ulmoides polysaccharide ≥40%) was dissolved in a mixed solvent of ethanol and water at a volume ratio of 7:3 to prepare a loading solution of 300mg / mL (total volume approximately 333mL). 150g of chitosan-modified octenyl succinic anhydride-modified porous starch obtained in step one was added to the loading solution and ultrasonically dispersed (40kHz, 300W) for 30min, then heated to 40℃ and held at that temperature for 3h. Subsequently, the solvent was removed by rotary evaporation at 40℃ and -0.08MPa, and the product was vacuum dried at 40℃ for 12h. The starch was then passed through an 80-mesh sieve to obtain octenyl succinic anhydride-modified porous starch loaded with Eucommia ulmoides leaf extract.

[0027] Step 3: Encapsulation of the intermediate layer 200g of the octenyl succinic anhydride-modified porous starch loaded with Eucommia ulmoides leaf extract obtained in step two was added to a fluidized bed coating machine. An 8% (w / w) ethyl cellulose anhydrous ethanol solution was prepared (i.e., 80g of ethyl cellulose dissolved in 920g of anhydrous ethanol), and 8.0g (w / w) of polyvinylpyrrolidone K30 (a pore-forming agent) was added to the solution, along with stirring to obtain a clear coating solution. During the coating process, the inlet air temperature was controlled at 55℃, the material temperature at 44℃, the atomization pressure at 0.20MPa, and the coating solution flow rate at 3.5mL / min, controlling the coating weight gain to 13% (i.e., the mass of the coated particles is approximately 226g). After coating, fluidized bed drying was continued for 25min to obtain drug-loaded particles with an intermediate layer.

[0028] Step 4: Preparation of the outer membrane 20g of chitosan with a degree of deacetylation of 92% was dissolved in 1.5% (v / v) glacial acetic acid solution, the pH was adjusted to 5.3, and the volume was brought to 1000mL to obtain a chitosan solution with a concentration of 2.0% (w / v).

[0029] 10g of fumed silica nanoparticles (specific surface area 200m²) were added. 2 / g, native particle size 20nm) was ultrasonically dispersed (ultrasonic frequency 40kHz, ultrasonic time 20min) in chitosan solution and stirred continuously for 1.5h to obtain chitosan-silica composite suspension.

[0030] Dissolve 4g of sodium tripolyphosphate in deionized water, adjust the pH to 5.3, and bring the volume to a final concentration to obtain a 1.5% (w / v) sodium tripolyphosphate crosslinking solution.

[0031] 200g of the drug-loaded particles with an intermediate layer obtained in step three were dispersed in 1000mL of a chitosan-silica composite suspension and stirred at room temperature for 20min to allow the composite suspension to be uniformly adsorbed onto the particle surface, resulting in a dispersion. Then, a sodium tripolyphosphate crosslinking solution was added dropwise while stirring the dispersion at 100rpm. After the addition was complete, the crosslinking reaction was continued for 45min to form an outer film on the surface of the drug-loaded particles.

[0032] After the reaction was completed, the product was collected by filtration, washed three times with deionized water (2000 mL each time), and then washed twice with anhydrous ethanol (1000 mL each time). The product was then dried under vacuum at 50°C for 9 hours, passed through 40-mesh and 60-mesh standard sieves, and the 250-425 μm particle size range was selected to obtain the compound plant extract feed additive.

[0033] Example 2

[0034] A method for preparing a compound plant extract feed additive includes the following steps: Step 1: Preparation of octenyl succinic anhydride modified porous starch Take 500g of corn starch and add deionized water to prepare a 30% (w / v) starch slurry, stirring to disperse it evenly. Adjust the pH to 8.5 with 0.5mol / L NaOH solution, and slowly add 15.0g of octenyl succinic anhydride (3% of the dry weight of the starch), maintaining the pH at 8.0-8.5 and reacting at 40℃ for 4h. During the reaction, continuously add 0.5mol / L NaOH solution to maintain a constant pH. After the reaction is complete, adjust the pH to 6.5 with 0.5mol / L HCl to terminate the reaction. Wash the product three times with deionized water (1667mL each time) and dry it under vacuum at 40℃ for 12h to obtain octenyl succinic anhydride modified starch.

[0035] Octenyl succinic anhydride modified starch was reconstituted into a 25% (w / v) starch slurry (total volume approximately 2000 mL), and the pH was adjusted to 6.0. A complex enzyme (α-amylase and glucoamylase at an enzyme activity ratio of 1:4) was added, with a total enzyme dosage of 2500 U / g starch (i.e., 1.25 × 10⁻⁶). 6 The product was enzymatically hydrolyzed at 45℃ for 18 h. After enzymatic hydrolysis, it was inactivated in a water bath at 85℃ for 15 min. The product was washed three times with deionized water (2000 mL each time), vacuum dried at 40℃ for 12 h, and passed through a 100-mesh sieve to obtain octenyl succinic anhydride modified porous starch.

[0036] 200g of octenyl succinic anhydride-modified porous starch was dispersed in 2000mL of anhydrous ethanol. Chitosan (10.0g), accounting for 5% of the dry weight of the octenyl succinic anhydride-modified porous starch, and 20mL of deionized water were added, and the mixture was refluxed at 80℃ for 6h. After the reaction was completed, the mixture was centrifuged (4000rpm, 10min), and the precipitate was washed twice with anhydrous ethanol (500mL each time). The precipitate was then vacuum dried at 40℃ for 12h to obtain chitosan-modified octenyl succinic anhydride-modified porous starch.

[0037] Step 2: Loading of Eucommia ulmoides leaf extract 100g of Eucommia ulmoides leaf extract powder (chlorogenic acid content ≥8%, total flavonoids ≥10%, Eucommia ulmoides polysaccharide ≥40%) was dissolved in a mixed solvent of ethanol and water at a volume ratio of 7:3 to prepare a loading solution of 350mg / mL. 150g of chitosan-modified octenyl succinic anhydride-modified porous starch obtained in step one was added to the loading solution and ultrasonically dispersed (40kHz, 300W) for 40min, then heated to 45℃ and held at that temperature for 4h. Subsequently, the solvent was removed by rotary evaporation at 40℃ and -0.08MPa, and the product was vacuum dried at 40℃ for 12h. The starch was then passed through an 80-mesh sieve to obtain octenyl succinic anhydride-modified porous starch loaded with Eucommia ulmoides leaf extract.

[0038] Step 3: Encapsulation of the intermediate layer 200g of the octenyl succinic anhydride-modified porous starch loaded with Eucommia ulmoides leaf extract obtained in step two was added to a fluidized bed coating machine. An 8% (w / w) ethyl cellulose anhydrous ethanol solution was prepared (i.e., 80g of ethyl cellulose dissolved in 920g of anhydrous ethanol), and 9.6g (w / w) of polyvinylpyrrolidone K30 (12% of the ethyl cellulose mass) was added to the solution. The mixture was stirred until a clear coating solution was obtained. During the coating process, the inlet air temperature was controlled at 60℃, the material temperature at 48℃, the atomization pressure at 0.25MPa, and the coating solution flow rate at 5mL / min, controlling the coating weight gain to 16% (i.e., the mass of the coated particles was approximately 232g). After coating, fluidized bed drying was continued for 30min to obtain drug-loaded particles with an intermediate layer.

[0039] Step 4: Preparation of the outer membrane 25g of chitosan with a degree of deacetylation of 95% was dissolved in a 1.5% (v / v) glacial acetic acid solution, the pH was adjusted to 5.5, and the volume was brought to 1000mL to obtain a chitosan solution with a concentration of 2.5% (w / v).

[0040] 15g of fumed silica nanoparticles (specific surface area 225m²) were added. 2 / g, native particle size 7nm) was ultrasonically dispersed (ultrasonic frequency 40kHz, ultrasonic time 20min) in chitosan solution and stirred continuously for 2h to obtain chitosan-silica composite suspension.

[0041] Dissolve 5g of sodium tripolyphosphate in deionized water, adjust the pH to 5.5, and bring the volume to a final concentration to obtain a 2.0% (w / v) sodium tripolyphosphate crosslinking solution.

[0042] 200g of the drug-loaded particles with an intermediate layer obtained in step three were dispersed in 1000mL of a chitosan-silica composite suspension and stirred at room temperature for 30min to allow the composite suspension to be uniformly adsorbed onto the particle surface, thus obtaining a dispersion. Then, sodium tripolyphosphate crosslinking solution was added dropwise while continuously stirring the dispersion at 80rpm. After the addition was complete, the crosslinking reaction was continued for 60min to form an outer film on the surface of the drug-loaded particles.

[0043] After the reaction was completed, the product was collected by filtration, washed three times with deionized water (2000 mL each time), then washed twice with anhydrous ethanol (1000 mL each time), dried under vacuum at 60℃ for 6 h, and passed through 40 mesh and 60 mesh standard sieves to obtain the 250~425 μm particle size range to obtain the compound plant extract feed additive.

[0044] Example 3

[0045] A method for preparing a compound plant extract feed additive includes the following steps: Step 1: Preparation of octenyl succinic anhydride modified porous starch Take 500g of corn starch and add deionized water to prepare a 20% (w / v) starch slurry (total volume approximately 2500mL), stirring until evenly dispersed. Adjust the pH to 8.0 with 0.5mol / L NaOH solution, and slowly add 1% (i.e., 5.0g) of octenyl succinic anhydride (based on the dry weight of the starch), maintaining the pH at 8.0-8.5 and reacting at 30℃ for 2h. During the reaction, continuously add 0.5mol / L NaOH solution to maintain a constant pH. After the reaction is complete, adjust the pH to 6.5 with 0.5mol / L HCl to terminate the reaction. Wash the product three times with deionized water (2500mL each time) and dry under vacuum at 40℃ for 12h to obtain octenyl succinic anhydride modified starch.

[0046] Octenyl succinic anhydride modified starch was reconstituted into a 25% (w / v) starch slurry (total volume approximately 2000 mL), and the pH was adjusted to 4.5. A complex enzyme (α-amylase and glucoamylase at an enzyme activity ratio of 1:2) was added, with a total enzyme dosage of 1500 U / g starch (i.e., 7.5 × 10⁻⁶). 5 The product was enzymatically hydrolyzed at 35℃ for 12 h. After enzymatic hydrolysis, it was inactivated in a water bath at 85℃ for 15 min. The product was washed three times with deionized water (2000 mL each time), vacuum dried at 40℃ for 12 h, and passed through a 100-mesh sieve to obtain octenyl succinic anhydride modified porous starch.

[0047] 200g of octenyl succinic anhydride-modified porous starch was dispersed in 2000mL of anhydrous ethanol. Chitosan (4.0g), accounting for 2% of the dry weight of the octenyl succinic anhydride-modified porous starch, and 20mL of deionized water were added, and the mixture was refluxed at 60℃ for 4h. After the reaction was completed, the mixture was centrifuged at 4000rpm for 10min, and the precipitate was washed twice with anhydrous ethanol (500mL each time). The precipitate was then vacuum dried at 40℃ for 12h to obtain chitosan-modified octenyl succinic anhydride-modified porous starch.

[0048] Step 2: Loading of Eucommia ulmoides leaf extract 100g of Eucommia ulmoides leaf extract powder (chlorogenic acid content ≥8%, total flavonoids ≥10%, Eucommia ulmoides polysaccharide ≥40%) was dissolved in a mixed solvent of ethanol and water at a volume ratio of 7:3 to prepare a loading solution of 250mg / mL. 150g of chitosan-modified octenyl succinic anhydride-modified porous starch obtained in step one was added to the loading solution. The mixture was ultrasonically dispersed at 40kHz for 20min, then heated to 35℃ and held at that temperature for 2h. Subsequently, the solvent was removed by rotary evaporation at 40℃ and -0.08MPa, followed by vacuum drying at 40℃ for 12h. The starch was then passed through an 80-mesh sieve to obtain octenyl succinic anhydride-modified porous starch loaded with Eucommia ulmoides leaf extract.

[0049] Step 3: Encapsulation of the intermediate layer 200g of the octenyl succinic anhydride-modified porous starch loaded with Eucommia ulmoides leaf extract obtained in step two was added to a fluidized bed coating machine. A 6% (w / w) ethyl cellulose anhydrous ethanol solution was prepared (i.e., 60g of ethyl cellulose dissolved in 940g of anhydrous ethanol), and 4.8g (w / w) of polyvinylpyrrolidone K30 (8% of the ethyl cellulose mass) was added to the solution. The mixture was stirred until a clear coating solution was obtained. During the coating process, the inlet air temperature was controlled at 50℃, the material temperature at 40℃, the atomization pressure at 0.15MPa, the coating solution flow rate at 2mL / min, and the coating weight gain was controlled at 10% (i.e., the mass of the coated particles was approximately 220g). After coating, fluidized bed drying was continued for 20min to obtain drug-loaded particles with an intermediate layer.

[0050] Step 4: Preparation of the outer membrane Dissolve 15g of chitosan with a degree of deacetylation of 90% in a 1.5% (v / v) glacial acetic acid solution, adjust the pH to 5.0, and bring the volume to 1000mL to obtain a chitosan solution with a concentration of 1.5% (w / v).

[0051] 4.5g of fumed silica nanoparticles (specific surface area 175m²) were added. 2 / g, with a native particle size of 40nm, was ultrasonically dispersed (40kHz, 20min) in a chitosan solution and stirred continuously for 1h to obtain a chitosan-silica composite suspension.

[0052] Dissolve 3g of sodium tripolyphosphate in deionized water, adjust the pH to 5.0, and bring the volume to a final concentration to obtain a 1.0% (w / v) sodium tripolyphosphate crosslinking solution.

[0053] 200g of the drug-loaded particles with an intermediate layer obtained in step three were dispersed in 1000mL of a chitosan-silica composite suspension and stirred at room temperature for 15min to allow the composite suspension to be uniformly adsorbed onto the particle surface, resulting in a dispersion. Then, a sodium tripolyphosphate crosslinking solution was added dropwise while stirring the dispersion at 80rpm. After the addition was complete, the crosslinking reaction was continued for 30min to form an outer film on the surface of the drug-loaded particles.

[0054] After the reaction was completed, the product was collected by filtration, washed three times with deionized water (2000 mL each time), then washed twice with anhydrous ethanol (1000 mL each time), dried under vacuum at 40℃ for 12 h, and passed through 40 mesh and 60 mesh standard sieves to obtain the 250~425 μm particle size range to obtain the compound plant extract feed additive.

[0055] Comparative Example 1 A method for preparing a compound plant extract feed additive includes the following steps: Step 1: Preparation of octenyl succinic anhydride modified porous starch Take 500g of corn starch and add deionized water to prepare a 25% (w / v) starch slurry, stirring until evenly dispersed. Adjust the pH to 8.2 with 0.5mol / L NaOH solution, and slowly add 10.0g of octenyl succinic anhydride (2% of the dry weight of the starch), maintaining the pH at 8.0-8.5 and reacting at 35℃ for 3h. During the reaction, continuously add 0.5mol / L NaOH solution to maintain a constant pH. After the reaction is complete, adjust the pH to 6.5 with 0.5mol / L HCl to terminate the reaction. Wash the product three times with deionized water (2000mL each time) and dry under vacuum at 40℃ for 12h to obtain octenyl succinic anhydride modified starch.

[0056] Octenyl succinic anhydride modified starch was reconstituted into a 25% (w / v) starch slurry, and the pH was adjusted to 5.5. A complex enzyme (α-amylase and glucoamylase in an enzyme activity ratio of 1:3) was added, with a total enzyme dosage of 2000 U / g starch (i.e., 1.0 × 10⁻⁶). 6 The product was enzymatically hydrolyzed at 40℃ for 15 h. After enzymatic hydrolysis, it was inactivated in a water bath at 85℃ for 15 min. The product was washed three times with deionized water (2000 mL each time), vacuum dried at 40℃ for 12 h, and passed through a 100-mesh sieve to obtain octenyl succinic anhydride modified porous starch.

[0057] Step 2: Loading of Eucommia ulmoides leaf extract 100g of Eucommia ulmoides leaf extract powder (chlorogenic acid content ≥8%, total flavonoids ≥10%, Eucommia ulmoides polysaccharide ≥40%) was dissolved in a mixed solvent of ethanol and water at a volume ratio of 7:3 to prepare a loading solution of 300mg / mL (total volume approximately 333mL). 150g of the octenyl succinic anhydride modified porous starch obtained in step one was added to the loading solution and ultrasonically dispersed (40kHz, 300W) for 30min, then heated to 40℃ and held at that temperature for 3h. Subsequently, the solvent was removed by rotary evaporation at 40℃ and -0.08MPa, and the mixture was vacuum dried at 40℃ for 12h. The starch was then passed through an 80-mesh sieve to obtain octenyl succinic anhydride modified porous starch loaded with Eucommia ulmoides leaf extract.

[0058] Step 3: Encapsulation of the intermediate layer 200g of the octenyl succinic anhydride-modified porous starch loaded with Eucommia ulmoides leaf extract obtained in step two was added to a fluidized bed coating machine. An 8% (w / w) ethyl cellulose anhydrous ethanol solution was prepared (i.e., 80g of ethyl cellulose dissolved in 920g of anhydrous ethanol), and 8.0g (w / w) of polyvinylpyrrolidone K30 (a pore-forming agent) was added to the solution, along with stirring to obtain a clear coating solution. During the coating process, the inlet air temperature was controlled at 55℃, the material temperature at 44℃, the atomization pressure at 0.20MPa, and the coating solution flow rate at 3.5mL / min, controlling the coating weight gain to 13% (i.e., the mass of the coated particles is approximately 226g). After coating, fluidized bed drying was continued for 25min to obtain drug-loaded particles with an intermediate layer.

[0059] Step 4: Preparation of the outer membrane 20g of chitosan with a degree of deacetylation of 92% was dissolved in 1.5% (v / v) glacial acetic acid solution, the pH was adjusted to 5.3, and the volume was brought to 1000mL to obtain a chitosan solution with a concentration of 2.0% (w / v).

[0060] 10g of fumed silica nanoparticles (specific surface area 200m²) were added. 2 / g, native particle size 20nm) was ultrasonically dispersed (ultrasonic frequency 40kHz, ultrasonic time 20min) in chitosan solution and stirred continuously for 1.5h to obtain chitosan-silica composite suspension.

[0061] Dissolve 4g of sodium tripolyphosphate in deionized water, adjust the pH to 5.3, and bring the volume to a final concentration to obtain a 1.5% (w / v) sodium tripolyphosphate crosslinking solution.

[0062] 200g of the drug-loaded particles with an intermediate layer obtained in step three were dispersed in 1000mL of a chitosan-silica composite suspension and stirred at room temperature for 20min to allow the composite suspension to be uniformly adsorbed onto the particle surface, thus obtaining a dispersion. Then, sodium tripolyphosphate crosslinking solution was added dropwise to the dispersion while stirring at 100rpm during the addition. After the addition was complete, the crosslinking reaction was continued for 45min to form an outer film on the surface of the drug-loaded particles.

[0063] After the reaction was completed, the product was collected by filtration, washed three times with deionized water (2000 mL each time), and then washed twice with anhydrous ethanol (1000 mL each time). The product was then dried under vacuum at 50°C for 9 hours, passed through 40-mesh and 60-mesh standard sieves, and the 250-425 μm particle size range was selected to obtain the compound plant extract feed additive.

[0064] Comparative Example 2 A method for preparing a compound plant extract feed additive includes the following steps: Step 1: Preparation of octenyl succinic anhydride modified porous starch Take 500g of corn starch and add deionized water to prepare a 25% (w / v) starch slurry, stirring until evenly dispersed. Adjust the pH to 8.2 with 0.5mol / L NaOH solution, and slowly add 10.0g of octenyl succinic anhydride (2% of the dry weight of the starch), maintaining the pH at 8.0-8.5 and reacting at 35℃ for 3h. During the reaction, continuously add 0.5mol / L NaOH solution to maintain a constant pH. After the reaction is complete, adjust the pH to 6.5 with 0.5mol / L HCl to terminate the reaction. Wash the product three times with deionized water (2000mL each time) and dry under vacuum at 40℃ for 12h to obtain octenyl succinic anhydride modified starch.

[0065] Octenyl succinic anhydride modified starch was reconstituted into a 25% (w / v) starch slurry, and the pH was adjusted to 5.5. A complex enzyme (α-amylase and glucoamylase in an enzyme activity ratio of 1:3) was added, with a total enzyme dosage of 2000 U / g starch (i.e., 1.0 × 10⁻⁶). 6 The product was enzymatically hydrolyzed at 40℃ for 15 h. After enzymatic hydrolysis, it was inactivated in a water bath at 85℃ for 15 min. The product was washed three times with deionized water (2000 mL each time), vacuum dried at 40℃ for 12 h, and passed through a 100-mesh sieve to obtain octenyl succinic anhydride modified porous starch.

[0066] 200 g of octenyl succinic anhydride-modified porous starch was dispersed in 2000 mL of anhydrous ethanol. Chitosan (7.0 g), accounting for 3.5% of the dry weight of the octenyl succinic anhydride-modified porous starch, and 20 mL of deionized water were added, and the mixture was refluxed at 70 °C for 5 h. After the reaction was completed, the mixture was centrifuged (4000 rpm, 10 min), and the precipitate was washed twice with anhydrous ethanol (500 mL each time). The precipitate was then vacuum dried at 40 °C for 12 h to obtain chitosan-modified octenyl succinic anhydride-modified porous starch.

[0067] Step 2: Loading of Eucommia ulmoides leaf extract 100g of Eucommia ulmoides leaf extract powder (chlorogenic acid content ≥8%, total flavonoids ≥10%, Eucommia ulmoides polysaccharide ≥40%) was dissolved in a mixed solvent of ethanol and water at a volume ratio of 7:3 to prepare a loading solution of 300mg / mL (total volume approximately 333mL). 150g of chitosan-modified octenyl succinic anhydride-modified porous starch obtained in step one was added to the loading solution and ultrasonically dispersed (40kHz, 300W) for 30min, then heated to 40℃ and held at that temperature for 3h. Subsequently, the solvent was removed by rotary evaporation at 40℃ and -0.08MPa, and the product was vacuum dried at 40℃ for 12h. The starch was then passed through an 80-mesh sieve to obtain octenyl succinic anhydride-modified porous starch loaded with Eucommia ulmoides leaf extract.

[0068] Step 3: Preparation of the outer membrane 20g of chitosan with a degree of deacetylation of 92% was dissolved in 1.5% (v / v) glacial acetic acid solution, the pH was adjusted to 5.3, and the volume was brought to 1000mL to obtain a chitosan solution with a concentration of 2.0% (w / v).

[0069] 10g of fumed silica nanoparticles (specific surface area 200m²) were added. 2 / g, native particle size 20nm) was ultrasonically dispersed (ultrasonic frequency 40kHz, ultrasonic time 20min) in chitosan solution and stirred continuously for 1.5h to obtain chitosan-silica composite suspension.

[0070] Dissolve 4g of sodium tripolyphosphate in deionized water, adjust the pH to 5.3, and bring the volume to a final concentration to obtain a 1.5% (w / v) sodium tripolyphosphate crosslinking solution.

[0071] 200g of octenyl succinic anhydride-modified porous starch loaded with Eucommia ulmoides leaf extract was dispersed in 1000mL of chitosan-silica composite suspension and stirred at room temperature for 20min to obtain a dispersion. Then, sodium tripolyphosphate crosslinking solution was added dropwise to the dispersion while stirring at 100rpm. After the addition was complete, the crosslinking reaction was continued for 45min to form an outer film on the surface of the drug-loaded particles.

[0072] After the reaction was completed, the product was collected by filtration, washed three times with deionized water (2000 mL each time), and then washed twice with anhydrous ethanol (1000 mL each time). The product was then dried under vacuum at 50°C for 9 hours, passed through 40-mesh and 60-mesh standard sieves, and the 250-425 μm particle size range was selected to obtain the compound plant extract feed additive.

[0073] Comparative Example 3 A method for preparing a compound plant extract feed additive includes the following steps: Step 1: Preparation of octenyl succinic anhydride modified porous starch Take 500g of corn starch and add deionized water to prepare a 25% (w / v) starch slurry, stirring until evenly dispersed. Adjust the pH to 8.2 with 0.5mol / L NaOH solution, and slowly add 10.0g of octenyl succinic anhydride (2% of the dry weight of the starch), maintaining the pH at 8.0-8.5 and reacting at 35℃ for 3h. During the reaction, continuously add 0.5mol / L NaOH solution to maintain a constant pH. After the reaction is complete, adjust the pH to 6.5 with 0.5mol / L HCl to terminate the reaction. Wash the product three times with deionized water (2000mL each time) and dry under vacuum at 40℃ for 12h to obtain octenyl succinic anhydride modified starch.

[0074] Octenyl succinic anhydride modified starch was reconstituted into a 25% (w / v) starch slurry, and the pH was adjusted to 5.5. A complex enzyme (α-amylase and glucoamylase in an enzyme activity ratio of 1:3) was added, with a total enzyme dosage of 2000 U / g starch (i.e., 1.0 × 10⁻⁶). 6 The product was enzymatically hydrolyzed at 40℃ for 15 h. After enzymatic hydrolysis, it was inactivated in a water bath at 85℃ for 15 min. The product was washed three times with deionized water (2000 mL each time), vacuum dried at 40℃ for 12 h, and passed through a 100-mesh sieve to obtain octenyl succinic anhydride modified porous starch.

[0075] 200 g of octenyl succinic anhydride-modified porous starch was dispersed in 2000 mL of anhydrous ethanol. Chitosan (7.0 g), accounting for 3.5% of the dry weight of the octenyl succinic anhydride-modified porous starch, and 20 mL of deionized water were added, and the mixture was refluxed at 70 °C for 5 h. After the reaction was completed, the mixture was centrifuged (4000 rpm, 10 min), and the precipitate was washed twice with anhydrous ethanol (500 mL each time). The precipitate was then vacuum dried at 40 °C for 12 h to obtain chitosan-modified octenyl succinic anhydride-modified porous starch.

[0076] Step 2: Loading of Eucommia ulmoides leaf extract 100g of Eucommia ulmoides leaf extract powder (chlorogenic acid content ≥8%, total flavonoids ≥10%, Eucommia ulmoides polysaccharide ≥40%) was dissolved in a mixed solvent of ethanol and water at a volume ratio of 7:3 to prepare a loading solution of 300mg / mL (total volume approximately 333mL). 150g of chitosan-modified octenyl succinic anhydride-modified porous starch obtained in step one was added to the loading solution and ultrasonically dispersed (40kHz, 300W) for 30min, then heated to 40℃ and held at that temperature for 3h. Subsequently, the solvent was removed by rotary evaporation at 40℃ and -0.08MPa, and the product was vacuum dried at 40℃ for 12h. The starch was then passed through an 80-mesh sieve to obtain octenyl succinic anhydride-modified porous starch loaded with Eucommia ulmoides leaf extract.

[0077] Step 3: Encapsulation of the intermediate layer 200g of the octenyl succinic anhydride-modified porous starch loaded with Eucommia ulmoides leaf extract obtained in step two was added to a fluidized bed coating machine. An 8% (w / w) ethyl cellulose anhydrous ethanol solution was prepared (i.e., 80g of ethyl cellulose dissolved in 920g of anhydrous ethanol), and 8.0g (w / w) of polyvinylpyrrolidone K30 (a pore-forming agent) was added to the solution, along with stirring to obtain a clear coating solution. During the coating process, the inlet air temperature was controlled at 55℃, the material temperature at 44℃, the atomization pressure at 0.20MPa, and the coating solution flow rate at 3.5mL / min, controlling the coating weight gain to 13% (i.e., the mass of the coated particles is approximately 226g). After coating, fluidized bed drying was continued for 25min to obtain drug-loaded particles with an intermediate layer.

[0078] Step 4: Preparation of the outer cross-linked gel membrane 20g of chitosan with a degree of deacetylation of 92% was dissolved in 1.5% (v / v) glacial acetic acid solution, the pH was adjusted to 5.3, and the volume was brought to 1000mL to obtain a chitosan solution with a concentration of 2.0% (w / v).

[0079] 10g of fumed silica nanoparticles (specific surface area 200m²) were added. 2 / g, native particle size 20nm) was ultrasonically dispersed (ultrasonic frequency 40kHz, ultrasonic time 20min) in chitosan solution and stirred continuously for 1.5h to obtain chitosan-silica composite suspension.

[0080] 200g of the drug-loaded particles with an intermediate layer obtained in step three were dispersed in 1000mL of a chitosan-silica composite suspension and stirred at room temperature for 20min to allow the composite suspension to be uniformly adsorbed onto the particle surface, resulting in a dispersion. Then, 200mL of a 0.8wt% tea polyphenol solution was added dropwise to the dispersion while stirring at 100rpm. After the addition was complete, stirring was continued for 45min to allow the cross-linking reaction to proceed, forming an outer cross-linked gel film on the surface of the drug-loaded particles.

[0081] After the reaction was completed, the product was collected by filtration, washed three times with deionized water (2000 mL each time), and then washed twice with anhydrous ethanol (1000 mL each time). The product was then dried under vacuum at 50°C for 9 hours, passed through 40-mesh and 60-mesh standard sieves, and the 250-425 μm particle size range was selected to obtain the compound plant extract feed additive.

[0082] Comparative Example 4 A method for preparing a compound plant extract feed additive includes the following steps: Step 1: Preparation of octenyl succinic anhydride modified porous starch Take 500g of corn starch and add deionized water to prepare a 25% (w / v) starch slurry, stirring until evenly dispersed. Adjust the pH to 8.2 with 0.5mol / L NaOH solution, and slowly add 10.0g of octenyl succinic anhydride (2% of the dry weight of the starch), maintaining the pH at 8.0-8.5 and reacting at 35℃ for 3h. During the reaction, continuously add 0.5mol / L NaOH solution to maintain a constant pH. After the reaction is complete, adjust the pH to 6.5 with 0.5mol / L HCl to terminate the reaction. Wash the product three times with deionized water (2000mL each time) and dry under vacuum at 40℃ for 12h to obtain octenyl succinic anhydride modified starch.

[0083] Octenyl succinic anhydride modified starch was reconstituted into a 25% (w / v) starch slurry, and the pH was adjusted to 5.5. A complex enzyme (α-amylase and glucoamylase in an enzyme activity ratio of 1:3) was added, with a total enzyme dosage of 2000 U / g starch (i.e., 1.0 × 10⁻⁶).6 The product was enzymatically hydrolyzed at 40℃ for 15 h. After enzymatic hydrolysis, it was inactivated in a water bath at 85℃ for 15 min. The product was washed three times with deionized water (2000 mL each time), vacuum dried at 40℃ for 12 h, and passed through a 100-mesh sieve to obtain octenyl succinic anhydride modified porous starch.

[0084] 200 g of octenyl succinic anhydride-modified porous starch was dispersed in 2000 mL of anhydrous ethanol. Chitosan (7.0 g), accounting for 3.5% of the dry weight of the octenyl succinic anhydride-modified porous starch, and 20 mL of deionized water were added, and the mixture was refluxed at 70 °C for 5 h. After the reaction was completed, the mixture was centrifuged (4000 rpm, 10 min), and the precipitate was washed twice with anhydrous ethanol (500 mL each time). The precipitate was then vacuum dried at 40 °C for 12 h to obtain chitosan-modified octenyl succinic anhydride-modified porous starch.

[0085] Step 2: Loading of Eucommia ulmoides leaf extract 100g of Eucommia ulmoides leaf extract powder (chlorogenic acid content ≥8%, total flavonoids ≥10%, Eucommia ulmoides polysaccharide ≥40%) was dissolved in a mixed solvent of ethanol and water at a volume ratio of 7:3 to prepare a loading solution of 300mg / mL (total volume approximately 333mL). 150g of chitosan-modified octenyl succinic anhydride-modified porous starch obtained in step one was added to the loading solution and ultrasonically dispersed (40kHz, 300W) for 30min, then heated to 40℃ and held at that temperature for 3h. Subsequently, the solvent was removed by rotary evaporation at 40℃ and -0.08MPa, and the product was vacuum dried at 40℃ for 12h. The starch was then passed through an 80-mesh sieve to obtain octenyl succinic anhydride-modified porous starch loaded with Eucommia ulmoides leaf extract.

[0086] Step 3: Encapsulation of the intermediate layer 200g of the octenyl succinic anhydride-modified porous starch loaded with Eucommia ulmoides leaf extract obtained in step two was added to a fluidized bed coating machine. An 8% (w / w) ethyl cellulose anhydrous ethanol solution was prepared (i.e., 80g of ethyl cellulose dissolved in 920g of anhydrous ethanol), and 8.0g (w / w) of polyvinylpyrrolidone K30 (a pore-forming agent) was added to the solution, along with stirring to obtain a clear coating solution. During the coating process, the inlet air temperature was controlled at 55℃, the material temperature at 44℃, the atomization pressure at 0.20MPa, and the coating solution flow rate at 3.5mL / min, controlling the coating weight gain to 13% (i.e., the mass of the coated particles is approximately 226g). After coating, fluidized bed drying was continued for 25min to obtain drug-loaded particles with an intermediate layer.

[0087] Step 4: Preparation of the outer cross-linked gel membrane 20g of chitosan with a degree of deacetylation of 92% was dissolved in 1.5% (v / v) glacial acetic acid solution, the pH was adjusted to 5.3, and the volume was brought to 1000mL to obtain a chitosan solution with a concentration of 2.0% (w / v).

[0088] Dissolve 4g of sodium tripolyphosphate in deionized water, adjust the pH to 5.3, and bring the volume to a final concentration to obtain a 1.5% (w / v) sodium tripolyphosphate crosslinking solution.

[0089] 200g of the drug-loaded particles with an intermediate layer obtained in step three were dispersed in a chitosan solution (1000mL) and stirred at room temperature for 20min to obtain a dispersion. Then, sodium tripolyphosphate crosslinking solution was added dropwise while stirring the dispersion at 100rpm during the addition. After the addition was complete, the crosslinking reaction was continued for 45min to form an outer crosslinked gel film on the surface of the drug-loaded particles.

[0090] After the reaction was completed, the product was collected by filtration, washed three times with deionized water (2000 mL each time), and then washed twice with anhydrous ethanol (1000 mL each time). The product was then dried under vacuum at 50°C for 9 hours, passed through 40-mesh and 60-mesh standard sieves, and the 250-425 μm particle size range was selected to obtain the compound plant extract feed additive.

[0091] Comparative Example 5 A method for preparing a compound plant extract feed additive includes the following steps: Step 1: Preparation of octenyl succinic anhydride modified porous starch Take 500g of corn starch and add deionized water to prepare a 25% (w / v) starch slurry, stirring until evenly dispersed. Adjust the pH to 8.2 with 0.5mol / L NaOH solution, and slowly add 10.0g of octenyl succinic anhydride (2% of the dry weight of the starch), maintaining the pH at 8.0-8.5 and reacting at 35℃ for 3h. During the reaction, continuously add 0.5mol / L NaOH solution to maintain a constant pH. After the reaction is complete, adjust the pH to 6.5 with 0.5mol / L HCl to terminate the reaction. Wash the product three times with deionized water (2000mL each time) and dry under vacuum at 40℃ for 12h to obtain octenyl succinic anhydride modified starch.

[0092] Octenyl succinic anhydride modified starch was reconstituted into a 25% (w / v) starch slurry, and the pH was adjusted to 5.5. A complex enzyme (α-amylase and glucoamylase in an enzyme activity ratio of 1:3) was added, with a total enzyme dosage of 2000 U / g starch (i.e., 1.0 × 10⁻⁶). 6 The product was enzymatically hydrolyzed at 40℃ for 15 h. After enzymatic hydrolysis, it was inactivated in a water bath at 85℃ for 15 min. The product was washed three times with deionized water (2000 mL each time), vacuum dried at 40℃ for 12 h, and passed through a 100-mesh sieve to obtain octenyl succinic anhydride modified porous starch.

[0093] 200 g of octenyl succinic anhydride-modified porous starch was dispersed in 2000 mL of anhydrous ethanol. Chitosan (7.0 g), accounting for 3.5% of the dry weight of the octenyl succinic anhydride-modified porous starch, and 20 mL of deionized water were added, and the mixture was refluxed at 70 °C for 5 h. After the reaction was completed, the mixture was centrifuged (4000 rpm, 10 min), and the precipitate was washed twice with anhydrous ethanol (500 mL each time). The precipitate was then vacuum dried at 40 °C for 12 h to obtain chitosan-modified octenyl succinic anhydride-modified porous starch.

[0094] Step 2: Loading of Eucommia ulmoides leaf extract 100g of Eucommia ulmoides leaf extract powder (chlorogenic acid content ≥8%, total flavonoids ≥10%, Eucommia ulmoides polysaccharide ≥40%) was dissolved in a mixed solvent of ethanol and water at a volume ratio of 7:3 to prepare a loading solution of 300mg / mL (total volume approximately 333mL). 150g of chitosan-modified octenyl succinic anhydride-modified porous starch obtained in step one was added to the loading solution and ultrasonically dispersed (40kHz, 300W) for 30min, then heated to 40℃ and held at that temperature for 3h. Subsequently, the solvent was removed by rotary evaporation at 40℃ and -0.08MPa, and the product was vacuum dried at 40℃ for 12h. The starch was then passed through an 80-mesh sieve to obtain octenyl succinic anhydride-modified porous starch loaded with Eucommia ulmoides leaf extract.

[0095] Step 3: Encapsulation of the intermediate layer 200g of the octenyl succinic anhydride-modified porous starch loaded with Eucommia ulmoides leaf extract obtained in step two was added to a fluidized bed coating machine. An 8% (w / w) ethyl cellulose anhydrous ethanol solution was prepared (i.e., 80g of ethyl cellulose dissolved in 920g of anhydrous ethanol), and 8.0g (w / w) of polyvinylpyrrolidone K30 (a pore-forming agent) was added to the solution, along with stirring to obtain a clear coating solution. During the coating process, the inlet air temperature was controlled at 55℃, the material temperature at 44℃, the atomization pressure at 0.20MPa, and the coating solution flow rate at 3.5mL / min, controlling the coating weight gain to 13% (i.e., the weight of the coated particles is approximately 226g). After coating, fluidized bed drying was continued for 25min to obtain a composite plant extract feed additive with an intermediate layer.

[0096] The relevant properties of the compound plant extract feed additives prepared in Example 1 and Comparative Examples 1-4 were measured respectively, and the results are shown in the table below: Table 1. Comparison of relevant properties of the compound plant extract feed additives prepared in Example 1 and Comparative Examples 1-4

[0097] In all the embodiments and comparative examples, the chlorogenic acid content is calculated as 20 wt%.

[0098] The method for preparing simulated gastric juice is as follows: Take 16.4 mL of dilute hydrochloric acid, add 800 mL of water and 10 g of pepsin, shake well, dilute with water to 1000 mL, and adjust the pH to 1.2.

[0099] Three portions of the compound plant extract feed additive samples from Example 1 and each comparative example, approximately 1.0 g each, were weighed and placed in dissolution vessels. The samples were placed in 900 mL of simulated gastric fluid (pH 1.2) and stirred at 37°C and 100 rpm for 2 hours. An equal amount of unencapsulated Eucommia ulmoides leaf extract was weighed as a control group. The temperature was set to 37°C, and 5 mL samples were taken at 0.5 h, 1 h, and 2 h. The samples were filtered through a 0.45 μm microporous membrane, and 1 mL of the initial filtrate was discarded. The subsequent filtrate was collected for analysis. Weigh out chlorogenic acid standard and prepare a series of standard solutions with concentrations of 2 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, and 100 μg / mL using ethanol. Inject these solutions onto a C18 column for analysis. Use an aqueous solution of ethanol and phosphoric acid as the mobile phase, with a volume ratio of 2:8, a flow rate of 1 mL / min, and a detection wavelength of 327 nm. Perform linear regression of peak area (y) against concentration (x, μg / mL) to obtain the standard curve equation. Calculate the chlorogenic acid release rate using the following formula:

[0100] in, C t For the first t Chlorogenic acid concentration (μg / mL) in samples at each time point. V This refers to the total volume of the leaching medium. C i For the first i Chlorogenic acid concentration at each time point in the sample V s For each sampling volume, W For sample quality, f This represents the initial chlorogenic acid content in the sample.

[0101] The simulated intestinal fluid was prepared as follows: 6.8 g of potassium dihydrogen phosphate was dissolved in 500 mL of water, and the pH was adjusted to 6.8 with 0.1 mol / L sodium hydroxide solution; 10 g of pancreatic enzyme was dissolved in water, and the two solutions were mixed and diluted with water to 1000 mL. Based on the simulated gastric fluid test, after 2 hours, the medium in the dissolution vessel was completely replaced with 900 mL of simulated intestinal fluid, and stirring was continued at 37℃ and 100 rpm. 5 mL samples were taken at 0.5 h, 1 h, 2 h, 3 h, and 4 h. The calculation method for chlorogenic acid release was the same as that for simulated gastric fluid.

[0102] DPPH (1,1-diphenyl-2-picrylhydrazyl radical) was prepared into a 0.2 mmol / L DPPH solution using anhydrous ethanol. The release solutions (including the compound plant extract feed additive and dissolution solution) after 4 hours of testing with simulated gastric and intestinal fluids were used as test subjects. 2 mL of the release solutions from Example 1 and each comparative example, after testing with simulated gastric and intestinal fluids, were mixed with 2 mL of DPPH solution to form the sample group. 2 mL of the release solutions from Example 1 and each comparative example, after testing with simulated gastric and intestinal fluids, and 2 mL of anhydrous ethanol were mixed with 2 mL of anhydrous ethanol to form the blank group. 2 mL of DPPH solution and 2 mL of anhydrous ethanol were mixed to form the control group. After mixing all tubes, the tubes were allowed to stand at room temperature in the dark for 30 minutes, and the absorbance of each tube was measured at a wavelength of 517 nm. The formula for calculating the DPPH scavenging rate is:

[0103] in, A sample The absorbance of the sample group. A blank The absorbance of the blank group is... A control The absorbance is for the control group.

[0104] Each sample was placed in a constant temperature and humidity chamber (40°C, RH 75%), and samples were taken at 1, 10, 20, 30, 40, 50 and 60 days. 1.0 g of each sample was weighed, 50 mL of ethanol was added, and the samples were extracted by ultrasonication for 30 min. Filter, collect the filtrate, and determine the chlorogenic acid content using HPLC in simulated gastric and intestinal fluid tests. Calculate the chlorogenic acid retention rate: Chlorogenic acid retention rate (%) = C 0 / C t ×100%, of which C 0 This represents the initial chlorogenic acid content. C t For the first t Content at specific time points.

[0105] Weigh 1.0 g of sample into a centrifuge tube, add 10 mL of anhydrous ethanol (to dissolve only the free chlorogenic acid on the particle surface without damaging the coating layer structure), vortex for 2 min, then centrifuge at 4000 rpm for 5 min, and transfer all supernatant; wash the precipitate twice with anhydrous ethanol, combine the three supernatants, and make up to 50 mL. Filter through a 0.45 μm filter membrane and inject for analysis. Calculate the mass W of free chlorogenic acid on the particle surface. 游离 (Unit: mg)

[0106] The chlorogenic acid encapsulation efficiency is calculated using the following formula: Encapsulation efficiency (%) = (W 总 - W 游离 ) / W 理论 ×100% Among them, W 总 The total mass of chlorogenic acid in the sample, in mg and W. 总 The determination method refers to the release rate determination method in simulated gastric juice, reflecting the actual chlorogenic acid content coated in the sample; W 游离 The mass of free chlorogenic acid on the particle surface, in mg; W 理论 The theoretical total mass of chlorogenic acid in the sample, calculated based on the feeding ratio, is in mg (obtained by converting the amount of Eucommia ulmoides leaf extract added to the chlorogenic acid content).

[0107] Example 1 showed a release rate of only 4.2% in simulated gastric fluid (pH=1.2), while the release rate was as high as 95.6% in simulated intestinal fluid (pH 6.8).

[0108] The Eucommia ulmoides leaf extract in the compound plant extract feed additive prepared in Comparative Example 1 was released in large quantities (38.6%) in the gastric acid environment. The Eucommia ulmoides leaf extract is easily destroyed by gastric acid and loses its protective function as a feed additive.

[0109] Comparative Example 2: The gastric juice release rate of Eucommia ulmoides leaf extract in the compound plant extract feed additive (12.8%) was higher than that in Example 1, indicating that the intermediate layer played a key buffering and barrier role, preventing premature leakage of the drug in an acidic environment.

[0110] The antioxidant activity retention rate (91.2%) and accelerated test retention rate (94.5%) of Example 1 were significantly higher than those of Comparative Examples 1-4.

[0111] Comparative Example 4 (without silica): Its DPPH scavenging rate (76.7%) and 30-day accelerated test retention rate (78.2%) decreased significantly. This proves that the addition of silica not only enhances the mechanical strength of the gel membrane, but also forms a dense barrier network that effectively isolates oxygen and moisture, preventing the oxidative degradation of Eucommia ulmoides leaf extract.

[0112] Comparative Example 1: The worst stability (60.2%) indicates that a complete encapsulation structure is crucial for the protection of active ingredients.

[0113] Example 1 showed the highest encapsulation efficiency (93.5%), thanks to the high hydrophobicity and large specific surface area of ​​the octenyl succinic anhydride (OSA) modified porous starch, which can effectively adsorb plant extracts. Combined with subsequent multi-layer coating, it achieved efficient drug loading.

[0114] This invention, through a triple design of a modified porous starch core, a semi-permeable intermediate layer, and a chitosan-silica-pH-responsive outer layer, successfully solves the problems of easy inactivation and oxidation of plant extracts in feed processing and the acidic environment of animal stomachs, achieving precise release into the intestines, which is significantly superior to the comparative method lacking any of the structures.

[0115] Weaned piglets were used as experimental subjects to determine the growth-promoting performance of the compound plant extract feed additives prepared in Example 1 and Comparative Examples 1-4 on weaned piglets.

[0116] Seven hundred healthy, 21-day-old Duroc-Landrace-Large White crossbred weaned piglets with similar initial weights (6.50 ± 0.25 kg) were selected, with an equal number of males and females. They were randomly divided into seven treatment groups of 100 piglets each, based on weight and sex balance. The control group was fed a corn-soybean meal basal diet (by weight: corn 65%, soybean meal 22%, wheat bran 11.2%, dicalcium phosphate 1.5%, salt 0.3%) without any Eucommia ulmoides leaf extract additives. The basal diet of Example 1 group was supplemented with 2000 g / t of the compound plant extract feed additive prepared in Example 1 (the median of the recommended addition range for weaned piglets). Comparative Examples 1 to 5: The basal diets of these groups were supplemented with 2000 g / t of the corresponding samples prepared in the comparative examples.

[0117] The pre-trial period lasted 3 days, during which piglets were fed a basal diet and underwent routine procedures such as deworming and immunization. The formal trial period lasted 28 days. During the trial, piglets had free access to feed and water. The pens were kept clean, dry, and well-ventilated, and disinfection and disease prevention were carried out according to the pig farm's standard procedures. The amount of feed given and the amount of feed left over were recorded daily, and the health status of the piglets was observed.

[0118] Initial weight / final weight: Weigh yourself on an empty stomach on the morning of the start and end of the trial period, count the total weight for each repeat and calculate the average weight per head.

[0119] Average daily weight gain (ADG) = (average weight at the end of the period - average weight at the beginning) / number of days in the trial; Average daily feed intake (ADFI): The total feed intake during the experimental period, converted into daily feed intake per head; Feed conversion ratio (F / G): F / G = Average daily feed intake / Average daily weight gain Weight gain rate: Using the blank control group as a baseline, the daily weight gain rate of each experimental group was calculated. Table 2 shows the results. Table 2 Comparison of Feed Additives Containing Compound Plant Extracts for Piglets

[0120] Example 1: Best results: The complete three-layer core-shell structure achieves extremely low gastric juice leakage and efficient intestinal release, with the highest retention rate of active ingredients. Therefore, it has the most significant growth-promoting effect, the best feed conversion ratio, and a weight gain improvement rate of 37.6%.

[0121] Comparative Example 1 showed the worst results: the kernel lacked chitosan modification, resulting in low encapsulation rate, massive release and inactivation by gastric juice, and a significant decrease in the bioavailability of active ingredients, with only a 9.3% increase in weight gain compared to the blank control group.

[0122] Comparative examples 2, 4, and 5 show a mediocre result: In Comparative Example 2, which lacked an intermediate isolation layer, gastric juice leakage was significantly increased, and some active ingredients were degraded in the stomach. In Comparative Example 4, where the outer layer lacks silica, the film barrier properties decrease and the oxidation loss of active ingredients increases. Comparative Example 5, lacking an outer membrane, relies solely on a single ethyl cellulose barrier, resulting in weaker acid resistance and release controllability compared to the complete structure.

[0123] Comparative Example 3 showed the second best results: the outer membrane of the tea polyphenol crosslinking had better acid resistance, but the release rate and amount of intestinal fluid were lower than those of the silica nanoparticle, chitosan, and sodium tripolyphosphate crosslinking system in Example 1. Therefore, the overall growth-promoting effect was weaker than that of Example 1, but better than other delaminated / modified comparative examples.

[0124] The number of devices and processing scale described herein are for the purpose of simplifying the description of the invention. Applications, modifications, and variations of the invention will be readily apparent to those skilled in the art.

[0125] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and examples shown and described herein.

Claims

1. A compound plant extract feed additive, characterized in that, The compound plant extract feed additive has a core-shell multilayer structure, comprising, from the inside out: The inner active carrier core is a chitosan-modified octenyl succinic anhydride-modified porous starch loaded with Eucommia ulmoides leaf extract. The intermediate layer covers the outer surface of the inner active carrier core; The outer membrane covers the outer surface of the intermediate layer.

2. The compound plant extract feed additive according to claim 1, characterized in that, The Eucommia ulmoides leaf extract contains chlorogenic acid, flavonoids, and Eucommia ulmoides polysaccharides. On a dry weight basis, the chlorogenic acid content is ≥8%, the flavonoid content is ≥10%, and the Eucommia ulmoides polysaccharide content is ≥40%.

3. The compound plant extract feed additive according to claim 1, characterized in that, The intermediate layer is an ethyl cellulose membrane layer, and the coating weight gain of the intermediate layer is 10%~16%.

4. The compound plant extract feed additive according to claim 1, characterized in that, The outer membrane is obtained by cross-linking silica nanoparticles and chitosan with sodium tripolyphosphate.

5. A method for preparing a compound plant extract feed additive according to any one of claims 1 to 4, characterized in that, Includes the following steps: Step 1: Add starch to deionized water to prepare a 20%~30% (w / v) starch milk, adjust the pH to 8.0~8.5, slowly add 1%~3% of octenyl succinic anhydride by dry weight of starch, maintain pH 8.0~8.5 and react at 30~40℃ for 2~4 hours; after the reaction is completed, adjust the pH to 6.5, wash and dry to obtain octenyl succinic anhydride modified starch; Octenyl succinic anhydride modified starch was re-formulated into a 25% (w / v) starch milk, the pH was adjusted to 4.5-6.0, a compound enzyme was added and enzymatically hydrolyzed at 35-45℃ for 12-18 hours, and after inactivation, it was washed, dried and sieved to obtain octenyl succinic anhydride modified porous starch. Octenyl succinic anhydride-modified porous starch was dispersed in anhydrous ethanol, and chitosan and deionized water were added, accounting for 2% to 5% of the dry weight of the octenyl succinic anhydride-modified porous starch. The mixture was refluxed at 60 to 80°C for 4 to 6 hours to obtain chitosan-modified octenyl succinic anhydride-modified porous starch. Step 2: Dissolve Eucommia ulmoides leaf extract powder in a mixed solvent of ethanol and water at a volume ratio of 7:3 to prepare a loading solution of 250-350 mg / mL. Add the chitosan-modified octenyl succinic anhydride-modified porous starch obtained in Step 1 to the loading solution. After ultrasonic dispersion, heat to 35-45℃ and keep warm for 2-4 hours. Then, remove the solvent by rotary evaporation at 40℃ and -0.08 MPa, vacuum dry at 40℃, and sieve to obtain octenyl succinic anhydride-modified porous starch loaded with Eucommia ulmoides leaf extract. Step 3: The octenyl succinic anhydride modified porous starch loaded with Eucommia ulmoides leaf extract obtained in Step 2 is fed into a fluidized bed coating machine; a 6%~10% (w / w) anhydrous ethanol solution of ethyl cellulose is prepared, and a pore-forming agent accounting for 5%~15% (w / w) of the ethyl cellulose mass is added to it, and the mixture is stirred evenly to obtain a clear coating solution; during the coating process, the inlet air temperature is controlled at 50~60℃, the material temperature at 40~48℃, the atomization pressure at 0.15~0.25MPa, and the coating solution flow rate at 2~5 mL / min; after coating is completed, fluidized bed drying is continued for 20~30 min to obtain drug-loaded particles with an intermediate layer; Step 4: Dissolve chitosan with a degree of deacetylation ≥90% in a 1.5% (v / v) glacial acetic acid solution, adjust the pH to 5.0~5.5, and obtain a chitosan solution with a concentration of 1.5%~2.5% (w / v); Vaporized silica nanoparticles were ultrasonically dispersed in a chitosan solution to obtain a chitosan-silica composite suspension. Dissolve sodium tripolyphosphate in deionized water, adjust the pH to 5.0-5.5, and prepare a sodium tripolyphosphate crosslinking solution with a concentration of 1.0%-2.0% (w / v); The drug-loaded particles with an intermediate layer obtained in step three are dispersed in a chitosan-silica composite suspension and stirred at room temperature for 15-30 minutes to allow the chitosan-silica composite suspension to be uniformly adsorbed on the surface of the drug-loaded particles in the intermediate layer, thus obtaining a dispersion. Then, sodium tripolyphosphate crosslinking solution is added dropwise to the dispersion while stirring the dispersion at a speed of 80-120 rpm during the dropwise addition. After the dropwise addition is completed, the crosslinking reaction is continued for 30-60 minutes to form an outer film on the surface of the drug-loaded particles. After the reaction was completed, the product was collected by filtration, washed with deionized water, then washed with anhydrous ethanol, dried under vacuum at 40-60℃ for 6-12 hours, passed through 40-mesh and 60-mesh standard sieves, and the 250-425μm particle size range was obtained to obtain the compound plant extract feed additive.

6. The method for preparing the compound plant extract feed additive according to claim 5, characterized in that, In step one, the complex enzyme is a complex enzyme composed of α-amylase and glucoamylase in an enzyme activity ratio of 1:2 to 1:4, and the total enzyme dosage is 1500 to 2500 U / g starch.

7. The method for preparing the compound plant extract feed additive according to claim 5, characterized in that, The pore-forming agent mentioned in step three is polyvinylpyrrolidone K30, and its addition amount is 8%~12% of the mass of ethyl cellulose; the mass fraction of the anhydrous ethanol solution of ethyl cellulose is 6%~8%.

8. The method for preparing the compound plant extract feed additive according to claim 5, characterized in that, In step four, the mass ratio of silica nanoparticles, chitosan, and sodium tripolyphosphate is 0.3~0.6:1:0.15~0.

25.

9. The compound plant extract feed additive according to any one of claims 1 to 4, characterized in that, The compound plant extract feed additive is used to promote the growth and development of pigs and improve the quality and flavor of pork. The compound plant extract feed additive is added at a rate of 1800-2500g per ton of complete feed in weaned piglet feed, 1500-2000g per ton of complete feed in growing pig feed, and 1200-1800g per ton of complete feed in fattening pig feed.

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