Green antibiotic-free feed for quails in egg producing period and preparation method of green antibiotic-free feed

Abstract

The invention discloses green antibiotic-free feed for quails in the egg producing period and a preparation method of the green antibiotic-free feed, and belongs to the technical field of animal feed. The feed additive comprises a composite additive composed of modified quercetin and modified gallic acid. The modified quercetin is subjected to acetylation, glycosylation, phosphorylation and deacetylation, so that the water solubility and bioavailability are improved; the modified gallic acid constructs a metal chelating compound through esterification, Michael addition and zinc coordination, the modified quercetin is responsible for core antioxidation, and the phytosterol component in the modified gallic acid synergistically regulates lipid metabolism, so that a multifunctional system capable of regulating antioxidation, calcium homeostasis and lipid metabolism in the egg laying period of quails is formed.

Description

Technical Field

[0001] This invention relates to the field of animal feed technology, specifically to a green antibiotic-free feed for quail during their egg-laying period and its preparation method. Background Technology

[0002] In the research and development of feed for quail during their egg-laying period, green and antibiotic-free (i.e., free of antibiotics and hormones) feed has become the main direction of industry development. Some existing patents attempt to improve the safety and functionality of feed by adding traditional Chinese medicine, probiotics, or alternative protein sources. However, these solutions still have some limitations in practical application.

[0003] Patent CN106509458A discloses a quail feed for the laying period, whose components include corn flour, soybean meal, rice bran, leaf meal, bone meal, fish meal, silkworm pupa powder, manganese sulfate, zinc sulfate, multivitamins, vitamin C, fine sand, calcium salts, and sodium bicarbonate. This feed aims to increase egg production by improving protein and calcium content, with experiments showing an annual egg production of 305–320 eggs. However, the scheme does not explicitly state whether it completely avoids the use of antibiotics or hormone additives, and the formula relies on animal-derived proteins (such as fish meal and silkworm pupa powder), which may raise questions about the sustainability and safety of raw materials under the concept of green farming. Furthermore, this feed does not introduce plant-based active ingredients with synergistic antioxidant or immunomodulatory functions, limiting its effect on improving egg quality (such as yolk color, eggshell strength, and antioxidant capacity).

[0004] Patent CN105211658A discloses a feed for adult quails during their egg-laying period, comprising corn, barley, soybeans, eggshells, peanut shells, wheat bran, and traditional Chinese medicinal herbs such as Coptis chinensis, Houttuynia cordata, dandelion, and Astragalus membranaceus. This formula aims to prevent common diseases during the egg-laying period by adding traditional heat-clearing and detoxifying herbs, and to supplement calcium through processed eggshells to enhance eggshell thickness. However, the herbs used in this feed are mostly crude extracts or directly ground, without optimization of the stability, bioavailability, or interactions with other nutrients of the active ingredients. In actual feeding, some herbal components may degrade due to the pH environment of the digestive tract or enzymatic hydrolysis, affecting their efficacy. Furthermore, the formula does not systematically integrate microecological preparations or enzyme preparations, resulting in limited synergistic regulatory effects on feed conversion rate and intestinal health.

[0005] In summary, while existing technologies have achieved antibiotic-free or partially green practices to some extent, there is still room for improvement in terms of the stability of active ingredients, synergistic effects of nutrients, overall improvement of egg quality, and adaptability to green farming throughout the entire life cycle. Therefore, it is necessary to develop a green, antibiotic-free feed for quail during their egg-laying period. This feed should ensure nutritional balance and, through scientific formulation and optimized preparation processes, enhance the bioavailability of functional components, thereby achieving the goals of high-yield, high-quality, and safe farming. Summary of the Invention

[0006] This invention provides a green antibiotic-free feed for quail during their egg-laying period and its preparation method. By constructing a modified quercetin and modified gallic acid composite system with synergistic effects, the problem of low bioavailability of natural active ingredients in the prior art is solved.

[0007] Specifically, the technical solution of the present invention includes the following steps: A method for preparing a green, antibiotic-free feed for quail during their egg-laying period, the method comprising the following steps: Quercetin, acetic anhydride, and pyridine were mixed and stirred to produce acetylated quercetin. A glycosylated complex was obtained by mixing and stirring sucrose, acetylated quercetin, and dextran sucrase enzyme solution. Sodium tripolyphosphate, sodium trimetaphosphate, glycosylation complex and sodium sulfate are mixed and stirred to react to obtain phosphorylated complex; Modified quercetin was obtained by mixing and stirring the phosphorylated complex and potassium carbonate. Modified quercetin and modified gallic acid were mixed and stirred to obtain a compound feed additive; Corn, soybean meal, fish meal, limestone powder, compound feed additives and premix are mixed together, then rapeseed oil is sprayed on, and the mixture is stirred and mixed again to obtain green antibiotic-free feed.

[0008] Furthermore, the weight ratio of quercetin, acetic anhydride and pyridine is 1:1.2~1.5:0.03~0.05.

[0009] Furthermore, the conditions for the mixed stirring reaction of quercetin, acetic anhydride and pyridine include a reaction temperature of 60-70°C and a reaction time of 1.5-2.5 h; acetic anhydride undergoes a partial phenolic hydroxyl group acylation reaction with quercetin to generate acetylated quercetin.

[0010] Furthermore, the weight ratio of sucrose, acetylated quercetin, and dextran sucrase enzyme solution is 5~7:0.01~0.03:0.01~0.02.

[0011] Furthermore, the conditions for the mixing and stirring reaction of sucrose, acetylated quercetin, and dextran sucrase include a reaction time of 20-24 h and a reaction speed of 200 r / min; utilizing the transglycosylation activity of dextran sucrase, glucose units in sucrose are regioselectively linked to the exposed phenolic hydroxyl groups of acetylated quercetin in the form of glycosidic bonds, thereby achieving glycosylation and introducing a strong hydrophilic head.

[0012] Furthermore, the weight ratio of sodium tripolyphosphate, sodium trimetaphosphate, glycosylated complex and sodium sulfate is 0.4~0.8:0.2~0.5:1:0.1~0.2.

[0013] Furthermore, the conditions for the mixed stirring reaction of sodium tripolyphosphate, sodium trimetaphosphate, glycosylation complex and sodium sulfate include a reaction pH of 8.8-9.2, a reaction temperature of 75-85℃ and a reaction time of 5-6h; the sodium tripolyphosphate / sodium trimetaphosphate mixed reagent undergoes a phosphorylation reaction with the hydroxyl groups at the end of the glycosylation product to attach phosphate groups.

[0014] Furthermore, the weight ratio of the phosphorylated complex to potassium carbonate is 10:3~4.

[0015] Furthermore, the conditions for the mixing and stirring reaction of the phosphorylated complex and potassium carbonate include a reaction temperature of 70-80°C and a reaction time of 6-8 hours.

[0016] Furthermore, the preparation method of the modified gallic acid includes the following steps: Gallic acid, phytosterol and lipase were mixed and stirred to obtain the first complex. The first complex, glycine, horseradish peroxidase and hydrogen peroxide solution were mixed and stirred to react to obtain the second complex; The second complex and zinc gluconate were mixed and stirred to obtain a complex dispersion, and then trehalose was added and stirred to obtain modified gallic acid.

[0017] Furthermore, the lipase is the immobilized lipase Novozyme 435.

[0018] Furthermore, the weight ratio of gallic acid, phytosterol and lipase is 1.5~1.7:0.8~1.0:0.15~0.17.

[0019] Furthermore, the conditions for the mixed stirring reaction of gallic acid, phytosterol and lipase include a reaction temperature of 60-65°C and a reaction time of 48-72 h; under the catalysis of lipase, the carboxyl group of gallic acid undergoes an esterification reaction with the hydroxyl group of phytosterol.

[0020] Furthermore, the concentration of the hydrogen peroxide solution is 30 wt%.

[0021] Further, the weight ratio of the first complex, glycine, horseradish peroxidase and hydrogen peroxide solution is 1:0.3~0.4:0.01~0.02:0.08~0.12.

[0022] Furthermore, the reaction conditions for mixing and stirring the first complex, glycine, horseradish peroxidase, and hydrogen peroxide solution include a reaction temperature of 35-37°C and a reaction time of 6-8 hours. Horseradish peroxidase catalyzes hydrogen peroxide to oxidize the catechol structure in the first complex into a highly reactive catechol, which then reacts with the amino group of glycine to obtain the second complex.

[0023] Furthermore, the weight ratio of the second complex to zinc gluconate is 1:0.9~1.1.

[0024] Furthermore, the mixing and stirring conditions for the second complex and zinc gluconate include a stirring speed of 800 r / min and a stirring time of 2-4 h; the divalent zinc ions coordinate with the phenolic hydroxyl, carboxylic acid and other groups on the second complex to obtain modified gallic acid.

[0025] Furthermore, the amount of trehalose used is 5% to 10% of the weight of the composite dispersion.

[0026] The weight ratio of corn, soybean meal, fish meal, stone powder, compound feed additives, premix, and rapeseed oil is 55~58:25~28:2~4:4~6:0.1~0.3:4~5:1~2.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention improves the water solubility and cell membrane affinity of quercetin derivatives by continuously modifying quercetin through acetylation of phenolic hydroxyl groups, enzymatic glucosylation, phosphorylation of glycosyl ends, and deacetylation. This solves the technical problem of low oral bioavailability of quercetin due to its high lipid solubility and rapid metabolism. However, the introduced phosphate group is prone to chelate with divalent metal ions such as calcium and iron in the complex digestive tract environment, which will interfere with the body's absorption of essential mineral elements in feed. To solve this problem, this invention prepares modified gallic acid, which is esterified with phytosterols by lipase catalysis, and then grafted with glycine by enzymatic oxidation. Finally, it coordinates with zinc ions to self-assemble into particles. Its zinc coordination network can complex calcium, iron and other metal ions in the intestine, effectively protecting the active groups of modified quercetin from interference. Its structure can jointly promote the intestinal absorption of both and form a complementary antioxidant and metabolic regulation network in vivo, achieving a synergistic effect.

[0028] (2) In this invention, modified quercetin is responsible for core antioxidant activity, while the phytosterol component in modified gallic acid synergistically regulates lipid metabolism, forming a multifunctional system that can regulate antioxidant activity, calcium homeostasis, and lipid metabolism during the quail's egg-laying period. The green antibiotic-free feed prepared based on this compound feed additive can achieve multiple benefits during the quail's egg-laying period. The compound feed additive enhances eggshell strength and reduces egg breakage rate by optimizing the dynamic utilization of minerals such as calcium. Its systemic antioxidant function helps alleviate high-production stress, maintain stable egg production performance, and improve egg quality. Furthermore, the synergistic regulatory effect of the compound on lipid metabolism supports liver health and nutrient conversion efficiency, ultimately improving overall feed efficiency. Detailed Implementation

[0029] The technical solution of the present invention will be clearly and completely described below through embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Unless otherwise stated, all raw materials and reagents used in this invention are commercially available or can be prepared by known methods.

[0031] The phytosterols were purchased from Xi'an Lantian Bioengineering Co., Ltd., with a total mass fraction of 95%, including 77% β-sitosterol, 17% rapeseed sterol, 5% stigmasterol, and 1% campesterol.

[0032] Novozyme 435 was purchased from Novozymes Enzyme Preparations Co., Ltd., and was adsorbed and immobilized on macroporous acrylic resin with an enzyme activity of 10 U / mg.

[0033] The enzyme activity of dextran sucrase is 4 U / mL.

[0034] The enzyme activity of horseradish peroxidase is 150 U / mg.

[0035] Example 1

[0036] A method for preparing a green, antibiotic-free feed for quail during their egg-laying period includes the following steps: One part by weight of quercetin was dispersed in 20 parts by weight of ethyl acetate, and 1.2 parts by weight of acetic anhydride and 0.03 parts by weight of pyridine were added. The mixture was heated to 60°C and stirred under reflux at 300 r / min for 2.5 h in a nitrogen atmosphere. After the reaction was completed, the mixture was cooled to 0°C, washed with distilled water at 0°C, allowed to stand and separate into layers, and the ethyl acetate layer was collected. The layer was washed three times with distilled water and 5 wt% sodium carbonate aqueous solution, dried with anhydrous sodium carbonate and anhydrous calcium chloride, filtered, and then subjected to vacuum distillation and vacuum drying to obtain acetylated quercetin. Five parts by weight of sucrose were dispersed in 70 parts by weight of acetate-calcium acetate buffer at pH 5.4 and stirred at 150 rpm for 10 min to obtain a sucrose solution. 0.01 parts by weight of acetylated quercetin were dispersed in 7 parts by weight of dimethyl sulfoxide and sonicated at 200 W for 5 min. This solution was then added to the sucrose solution, followed by 0.01 parts by weight of dextran sucrase enzyme solution. The mixture was stirred at 200 rpm in the dark for 20 h. After the reaction, the temperature was raised to 95°C and held for 10 min, then cooled to 25°C. Three times the volume of anhydrous ethanol was added to the reaction solution, and the mixture was stirred at 50 rpm for 20 min and allowed to stand for 2 h. The mixture was centrifuged at 5000 rpm for 20 min, and the supernatant was collected. The supernatant was concentrated to 1 / 8 of its original volume under reduced pressure at 45°C to obtain a concentrate. The concentrate was filtered through a 0.22 μm filter membrane and then passed through an AB-8 macroporous resin column and a Sephadex column. The glycosylated complex was purified by LH-20 gel column elution with distilled water and ethanol gradient, then concentrated under reduced pressure at 40°C for 4 h and freeze-dried. 0.4 parts by weight of sodium tripolyphosphate and 0.2 parts by weight of sodium trimetaphosphate were dispersed in 50 parts by weight of distilled water and stirred at 200 r / min for 15 min. Then, 1 part by weight of glycosylated complex and 0.1 part by weight of sodium sulfate were added and stirred at 250 r / min for 30 min. The pH was adjusted to 8.8 with 0.01 mol / L NaOH aqueous solution and stirred for 10 min. The temperature was then raised to 75 °C and stirred at 250 r / min for 6 h. After the reaction was completed, the temperature was cooled to 25 °C. Three times the volume of anhydrous ethanol was added and stirred at 50 r / min for 20 min. The mixture was allowed to stand for 2 h and centrifuged at 5000 r / min for 15 min. The precipitate was collected, washed three times with distilled water, dialyzed in distilled water for 72 h, and then freeze-dried to obtain the phosphorylated complex. Ten parts by weight of the phosphorylated complex were dispersed in 100 parts by weight of methanol-water mixed solvent (volume ratio 1:1), and 3 parts by weight of potassium carbonate were added. The mixture was stirred at 200 r / min at 80 °C for 8 h. After the reaction was completed, the mixture was cooled to 25 °C, and the pH was adjusted to 7.0 with 1 mol / L hydrochloric acid solution. The mixture was concentrated under reduced pressure at 40 °C for 3 h, and then extracted three times with ethyl acetate. The organic phases were combined, washed three times with saturated sodium chloride solution, dried with anhydrous sodium carbonate and anhydrous calcium chloride, and then concentrated to obtain crude product. The crude product was then purified by Sephadex LH-20 gel column chromatography, and then concentrated and freeze-dried to obtain modified quercetin. 1.5 parts by weight of gallic acid and 0.8 parts by weight of phytosterol were dispersed in 50 parts by weight of n-hexane. After stirring at 100 rpm for 10 min, 0.15 parts by weight of immobilized lipase Novozyme were added. 435, heated to 60℃, stirred at 200 r / min for 72 h. After the reaction, the immobilized lipase was recovered by hot filtration. The filtrate was collected and distilled under reduced pressure at 40℃ for 4 h to obtain the crude product. 20 parts by weight of 2 wt% sodium bicarbonate solution at 50℃ were added and stirred at 50℃ for 20 min. The mixture was then transferred to a separatory funnel, allowed to stand for separation, and the upper organic phase was collected. 40 parts by weight of anhydrous ethanol were added to the organic phase, heated to 70℃ and stirred for 10 min. The mixture was then placed at 4℃ and allowed to stand for 12 h. The supernatant was collected by centrifugation at 5000 r / min at 4℃ for 20 min. The supernatant was concentrated under reduced pressure at 50℃ for 4 h, purified by silica gel column chromatography, and then concentrated and vacuum dried to obtain the first complex. One part by weight of the first complex was dispersed in 20 parts by weight of propylene glycol, heated to 40°C and stirred for 10 min to obtain the first solution. 0.3 parts by weight of glycine was dispersed in 100 parts by weight of pH 7.5 phosphate buffer and stirred at 200 r / min for 10 min to obtain the second solution. The first solution was added to the second solution while stirring, followed by 0.01 parts by weight of horseradish peroxidase. The temperature was raised to 35°C, and 0.08 parts by weight of 30 wt% hydrogen peroxide solution was added. The mixture was stirred at 200 r / min for 8 h. After the reaction was completed, the mixture was cooled to 4°C, and the pH was adjusted to 3.0 with 1 mol / L hydrochloric acid. The mixture was centrifuged at 8000 r / min for 15 min, and the precipitate was collected. It was washed three times alternately with pH 4.0 hydrochloric acid solution and ice-cold ethanol. The washed precipitate was redissolved in 0.1 wt% ammonia water, dialyzed in distilled water for 48 h, and then freeze-dried to obtain the second complex. One part by weight of the second complex was dispersed in 80 parts by weight of distilled water, heated to 50°C, and ultrasonically dispersed for 5 min at 200 W to obtain a dispersion. 0.9 parts by weight of zinc gluconate was dispersed in 30 parts by weight of distilled water to obtain a zinc salt solution. The zinc salt solution was added dropwise to the dispersion at a speed of 2 mL / min at 800 r / min. After the addition was completed, the reaction was continued to be stirred for 4 h. After the reaction was completed, the mixture was dialyzed in distilled water for 48 h using a 5000 Da dialysis bag to obtain a composite dispersion. 5% of the weight of trehalose was added to the composite dispersion, and the mixture was stirred at 100 r / min for 60 min at 25°C and then freeze-dried to obtain modified gallic acid. 0.3 parts by weight of the above modified quercetin and 0.5 parts by weight of the above modified gallic acid were dispersed in 100 parts by weight of pH 7.4 phosphate buffer solution and stirred at 200 r / min for 6 h at 25 °C. After stirring, the mixture was concentrated by centrifugation using an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa. After washing three times with phosphate buffer solution, the mixture was freeze-dried and passed through a 30-mesh sieve to obtain a compound feed additive. Corn and soybean meal were crushed and passed through a 10-mesh sieve, fish meal through a 20-mesh sieve, and limestone powder through a 100-mesh sieve. 55 parts by weight of corn, 25 parts by weight of soybean meal, and 2 parts by weight of fish meal were placed in a mixer and mixed at 25 r / min for 6 minutes. Then, 4 parts by weight of limestone powder, 0.1 parts by weight of compound feed additive, and 4 parts by weight of 5% quail laying period premix were added and mixed for another 6 minutes. After spraying 1 part by weight of rapeseed oil at a pressure of 0.2 MPa, the mixture was mixed at 25 r / min for 12 minutes to obtain green antibiotic-free feed.

[0037] Example 2

[0038] A method for preparing a green, antibiotic-free feed for quail during their egg-laying period includes the following steps: One part by weight of quercetin was dispersed in 20 parts by weight of ethyl acetate, 1.3 parts by weight of acetic anhydride and 0.035 parts by weight of pyridine were added, the temperature was raised to 63°C, and the mixture was stirred under reflux at 300 r / min for 1.8 h in a nitrogen atmosphere. After the reaction was completed, the mixture was cooled to 2°C, washed with distilled water at 2°C, allowed to stand for separation, the ethyl acetate layer was collected, and the mixture was distilled under reduced pressure and dried under vacuum to obtain acetylated quercetin. 5.5 parts by weight of sucrose were dispersed in 70 parts by weight of acetate-calcium acetate buffer at pH 5.4 and stirred at 150 rpm for 10 min to obtain a sucrose solution. 0.02 parts by weight of acetylated quercetin were dispersed in 7 parts by weight of dimethyl sulfoxide and sonicated at 200 W for 5 min. This solution was then added to the sucrose solution. 0.0125 parts by weight of dextran sucrase enzyme solution were added, and the mixture was stirred at 200 rpm in the dark for 21 h. After the reaction, the temperature was raised to 95°C and held for 10 min, then cooled to 25°C. Three times the volume of anhydrous ethanol was added, and the mixture was stirred at 50 rpm for 20 min and allowed to stand for 2 h. The mixture was centrifuged at 5000 rpm for 20 min, and the supernatant was collected. The supernatant was concentrated under reduced pressure at 45°C to 1 / 8 of its original volume to obtain a concentrate. The concentrate was filtered through a 0.22 μm filter membrane and then passed through an AB-8 macroporous resin column and a Sephadex column. The glycosylated complex was purified by LH-20 gel column elution with distilled water and ethanol gradient, then concentrated under reduced pressure at 40°C for 4 h and freeze-dried. 0.5 parts by weight of sodium tripolyphosphate and 0.3 parts by weight of sodium trimetaphosphate were dispersed in 50 parts by weight of distilled water and stirred at 200 r / min for 15 min. Then, 1 part by weight of the glycosylated complex and 0.12 parts by weight of sodium sulfate were added and stirred at 250 r / min for 30 min. The pH was adjusted to 8.9 with 0.01 mol / L NaOH aqueous solution and stirred for 10 min. The temperature was then raised to 78 °C and stirred at 250 r / min for 5.2 h. After the reaction was completed, the temperature was cooled to 25 °C and 3 times the volume of anhydrous ethanol was added. The mixture was stirred at 50 r / min for 20 min and allowed to stand for 2 h. The mixture was then centrifuged at 5000 r / min for 15 min, the precipitate was collected, washed 3 times with distilled water, dialyzed in distilled water for 72 h, and then freeze-dried to obtain the phosphorylated complex. Ten parts by weight of the phosphorylated complex were dispersed in 100 parts by weight of methanol-water mixed solvent (volume ratio 1:1), and 3.5 parts by weight of potassium carbonate were added. The mixture was stirred at 200 r / min at 80 °C for 8 h. After the reaction was completed, the mixture was cooled to 25 °C, and the pH was adjusted to 7.0 with 1 mol / L hydrochloric acid solution. The mixture was concentrated under reduced pressure at 40 °C for 3 h, extracted three times with ethyl acetate, and the organic phases were combined. The mixture was washed three times with saturated sodium chloride solution, dried with anhydrous sodium carbonate and anhydrous calcium chloride, and then concentrated to obtain the crude product. The crude product was then purified by Sephadex LH-20 gel column chromatography, and then concentrated and freeze-dried to obtain modified quercetin. 1.55 parts by weight of gallic acid and 0.85 parts by weight of phytosterol were dispersed in 50 parts by weight of n-hexane. After stirring at 100 rpm for 10 min, 0.155 parts by weight of immobilized lipase Novozyme were added. 435, heated to 62℃, stirred at 200 r / min for 54 h. After the reaction, the immobilized lipase was recovered by hot filtration. The filtrate was collected and distilled under reduced pressure at 40℃ for 4 h to obtain the crude product. 20 parts by weight of 2 wt% sodium bicarbonate solution at 50℃ were added and stirred at 50℃ for 20 min. The mixture was then transferred to a separatory funnel, allowed to stand for separation, and the upper organic phase was collected. 40 parts by weight of anhydrous ethanol were added to the organic phase, heated to 70℃ and stirred for 10 min. The mixture was then placed at 4℃ and allowed to stand for 12 h. The supernatant was collected by centrifugation at 5000 r / min at 4℃ for 20 min. The supernatant was concentrated under reduced pressure at 50℃ for 4 h, purified by silica gel column chromatography, and then concentrated and vacuum dried to obtain the first complex. One part by weight of the first complex was dispersed in 20 parts by weight of propylene glycol, heated to 40°C and stirred for 10 min to obtain the first solution. 0.32 parts by weight of glycine was dispersed in 100 parts by weight of pH 7.5 phosphate buffer and stirred at 200 r / min for 10 min to obtain the second solution. The first solution was added to the second solution while stirring, followed by 0.012 parts by weight of horseradish peroxidase. The temperature was raised to 36°C, and 0.09 parts by weight of 30 wt% hydrogen peroxide solution was added. The mixture was stirred at 200 r / min for 6.5 h. After the reaction was completed, the mixture was cooled to 4°C, and the pH was adjusted to 3.0 with 1 mol / L hydrochloric acid. The mixture was centrifuged at 8000 r / min for 15 min, and the precipitate was collected. It was washed three times alternately with pH 4.0 hydrochloric acid solution and ice-cold ethanol. The washed precipitate was redissolved in 0.1 wt% ammonia water, dialyzed in distilled water for 48 h, and then freeze-dried to obtain the second complex. One part by weight of the second complex was dispersed in 80 parts by weight of distilled water, heated to 50°C, and ultrasonically dispersed at 200W for 5 min to obtain a dispersion. 0.95 parts by weight of zinc gluconate was dispersed in 30 parts by weight of distilled water to obtain a zinc salt solution. The zinc salt solution was added dropwise to the dispersion at a speed of 2 mL / min at 800 r / min. After the addition was completed, the reaction was stirred for 2.5 h. After the reaction was completed, the mixture was dialyzed in distilled water for 48 h using a 5000 Da dialysis bag to obtain a composite dispersion. 6% of the weight of trehalose was added to the composite dispersion, and the mixture was stirred at 100 r / min for 45 min at 25°C and then freeze-dried to obtain modified gallic acid. 0.4 parts by weight of the above modified quercetin and 0.52 parts by weight of the above modified gallic acid were dispersed in 100 parts by weight of pH 7.4 phosphate buffer solution and stirred at 200 r / min for 5 h at 25 °C. After stirring, the mixture was concentrated by centrifugation using an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa. The mixture was washed three times with phosphate buffer solution, freeze-dried, and passed through a 30-mesh sieve to obtain a compound feed additive. Corn and soybean meal were crushed and passed through a 10-mesh sieve, fishmeal through a 20-mesh sieve, and limestone powder through a 100-mesh sieve. 56 parts by weight of corn, 26 parts by weight of soybean meal, and 2.5 parts by weight of fishmeal were placed in a mixer and mixed at 30 r / min for 5 minutes. Then, 4.5 parts by weight of limestone powder, 0.15 parts by weight of compound feed additive, and 4.2 parts by weight of 5% quail laying period premix were added and mixed for another 5 minutes. After spraying 1.2 parts by weight of rapeseed oil at a pressure of 0.25 MPa, the mixture was mixed at 30 r / min for 10 minutes to obtain green antibiotic-free feed.

[0039] Example 3

[0040] A method for preparing a green, antibiotic-free feed for quail during their egg-laying period includes the following steps: One part by weight of quercetin was dispersed in 20 parts by weight of ethyl acetate, 1.35 parts by weight of acetic anhydride and 0.04 parts by weight of pyridine were added, the temperature was raised to 65°C, and the mixture was stirred under reflux at 300 r / min for 2.0 h in a nitrogen atmosphere. After the reaction was completed, the mixture was cooled to 1°C, washed with distilled water at 1°C, allowed to stand for separation, the ethyl acetate layer was collected, and the mixture was distilled under reduced pressure and dried under vacuum to obtain acetylated quercetin. Six parts by weight of sucrose were dispersed in 70 parts by weight of acetate-calcium acetate buffer at pH 5.4 and stirred at 150 rpm for 10 min to obtain a sucrose solution. 0.025 parts by weight of acetylated quercetin were dispersed in 7 parts by weight of dimethyl sulfoxide and sonicated at 200 W for 5 min. This solution was then added to the sucrose solution, followed by 0.015 parts by weight of dextran sucrase enzyme solution. The mixture was stirred at 200 rpm in the dark for 22 h. After the reaction, the temperature was raised to 95°C and held for 10 min, then cooled to 25°C. Three times the volume of anhydrous ethanol was added, and the mixture was stirred at 50 rpm for 20 min and allowed to stand for 2 h. The mixture was centrifuged at 5000 rpm for 20 min, and the supernatant was collected. The supernatant was concentrated under reduced pressure at 45°C to 1 / 8 of its original volume to obtain a concentrate. The concentrate was filtered through a 0.22 μm filter membrane and then passed through an AB-8 macroporous resin column and a Sephadex column. The glycosylated complex was purified by LH-20 gel column elution with distilled water and ethanol gradient, then concentrated under reduced pressure at 40°C for 4 h and freeze-dried. 0.6 parts by weight of sodium tripolyphosphate and 0.35 parts by weight of sodium trimetaphosphate were dispersed in 50 parts by weight of distilled water and stirred at 200 r / min for 15 min. Then, 1 part by weight of the glycosylated complex and 0.15 parts by weight of sodium sulfate were added and stirred at 250 r / min for 30 min. The pH was adjusted to 9.0 with 0.01 mol / L NaOH aqueous solution and stirred for 10 min. The temperature was then raised to 80 °C and stirred at 250 r / min for 5.4 h. After the reaction was completed, the mixture was cooled to 25 °C and 3 times the volume of anhydrous ethanol was added. The mixture was stirred at 50 r / min for 20 min and allowed to stand for 2 h. The mixture was then centrifuged at 5000 r / min for 15 min, the precipitate was collected, washed 3 times with distilled water, dialyzed in distilled water for 72 h, and then freeze-dried to obtain the phosphorylated complex. Ten parts by weight of the phosphorylated complex were dispersed in 100 parts by weight of methanol-water mixed solvent (volume ratio 1:1), and 4 parts by weight of potassium carbonate were added. The mixture was stirred at 200 r / min at 80 °C for 8 h. After the reaction was completed, the mixture was cooled to 25 °C, and the pH was adjusted to 7.0 with 1 mol / L hydrochloric acid solution. The mixture was concentrated under reduced pressure at 40 °C for 3 h, extracted three times with ethyl acetate, and the organic phases were combined. The mixture was washed three times with saturated sodium chloride solution, dried with anhydrous sodium carbonate and anhydrous calcium chloride, and then concentrated to obtain the crude product. The crude product was then purified by Sephadex LH-20 gel column chromatography, and then concentrated and freeze-dried to obtain modified quercetin. 1.6 parts by weight of gallic acid and 0.9 parts by weight of phytosterol were dispersed in 50 parts by weight of n-hexane. After stirring at 100 rpm for 10 min, 0.16 parts by weight of immobilized lipase Novozyme were added. 435, heated to 63℃, stirred at 200 r / min for 60 h. After the reaction, the immobilized lipase was recovered by hot filtration. The filtrate was collected and distilled under reduced pressure at 40℃ for 4 h to obtain the crude product. 20 parts by weight of 2 wt% sodium bicarbonate solution at 50℃ were added and stirred at 50℃ for 20 min. The mixture was then transferred to a separatory funnel, allowed to stand for separation, and the upper organic phase was collected. 40 parts by weight of anhydrous ethanol were added to the organic phase, heated to 70℃ and stirred for 10 min. The mixture was then placed at 4℃ and allowed to stand for 12 h. The supernatant was collected by centrifugation at 5000 r / min at 4℃ for 20 min. The supernatant was concentrated under reduced pressure at 50℃ for 4 h, purified by silica gel column chromatography, and then concentrated and vacuum dried to obtain the first complex. One part by weight of the first complex was dispersed in 20 parts by weight of propylene glycol, heated to 40°C and stirred for 10 min to obtain the first solution. 0.35 parts by weight of glycine was dispersed in 100 parts by weight of pH 7.5 phosphate buffer and stirred at 200 r / min for 10 min to obtain the second solution. The first solution was added to the second solution while stirring, followed by 0.015 parts by weight of horseradish peroxidase. The temperature was raised to 36°C, and 0.1 parts by weight of 30 wt% hydrogen peroxide solution was added. The mixture was stirred at 200 r / min for 7 h. After the reaction was completed, the mixture was cooled to 4°C, and the pH was adjusted to 3.0 with 1 mol / L hydrochloric acid. The mixture was centrifuged at 8000 r / min for 15 min, and the precipitate was collected. It was washed three times alternately with pH 4.0 hydrochloric acid solution and ice-cold ethanol. The washed precipitate was redissolved in 0.1 wt% ammonia water, dialyzed in distilled water for 48 h, and then freeze-dried to obtain the second complex. One part by weight of the second complex was dispersed in 80 parts by weight of distilled water, heated to 50°C, and ultrasonically dispersed at 200W for 5 min to obtain a dispersion. One part by weight of zinc gluconate was dispersed in 30 parts by weight of distilled water to obtain a zinc salt solution. The zinc salt solution was added dropwise to the dispersion at a speed of 2 mL / min at 800 r / min. After the addition was completed, the reaction was stirred for 3 h. After the reaction was completed, the mixture was dialyzed in distilled water for 48 h using a 5000 Da dialysis bag to obtain a composite dispersion. 7.5% of the weight of trehalose was added to the composite dispersion, and the mixture was stirred at 100 r / min for 45 min at 25°C and then freeze-dried to obtain modified gallic acid. 0.45 parts by weight of the above modified quercetin and 0.55 parts by weight of the above modified gallic acid were dispersed in 100 parts by weight of pH 7.4 phosphate buffer solution and stirred at 200 r / min for 5 h at 25 °C. After stirring, the mixture was concentrated by centrifugation using an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa. The mixture was washed three times with phosphate buffer solution, freeze-dried, and passed through a 30-mesh sieve to obtain a compound feed additive. Corn and soybean meal were crushed and passed through a 10-mesh sieve, fish meal through a 20-mesh sieve, and limestone powder through a 100-mesh sieve. 57 parts by weight of corn, 27 parts by weight of soybean meal, and 3 parts by weight of fish meal were placed in a mixer and mixed at 30 r / min for 5 minutes. Then, 5 parts by weight of limestone powder, 0.2 parts by weight of compound feed additive, and 4.5 parts by weight of 5% quail laying period premix were added and mixed for another 5 minutes. After spraying 1.5 parts by weight of rapeseed oil at a pressure of 0.3 MPa, the mixture was mixed at 30 r / min for 10 minutes to obtain green antibiotic-free feed.

[0041] Example 4

[0042] A method for preparing a green, antibiotic-free feed for quail during their egg-laying period includes the following steps: One part by weight of quercetin was dispersed in 20 parts by weight of ethyl acetate, 1.4 parts by weight of acetic anhydride and 0.045 parts by weight of pyridine were added, the temperature was raised to 67°C, and the mixture was stirred under reflux at 300 r / min for 2.2 h in a nitrogen atmosphere. After the reaction was completed, the mixture was cooled to 1°C, washed with distilled water at 1°C, allowed to stand for separation, the ethyl acetate layer was collected, and the mixture was distilled under reduced pressure and dried under vacuum to obtain acetylated quercetin. 6.5 parts by weight of sucrose were dispersed in 70 parts by weight of acetate-calcium acetate buffer at pH 5.4 and stirred at 150 rpm for 10 min to obtain a sucrose solution. 0.03 parts by weight of acetylated quercetin were dispersed in 7 parts by weight of dimethyl sulfoxide and sonicated at 200 W for 5 min. This solution was then added to the sucrose solution. 0.0175 parts by weight of dextran sucrase enzyme solution were added, and the mixture was stirred at 200 rpm in the dark for 23 h. After the reaction, the temperature was raised to 95°C and held for 10 min, then cooled to 25°C. Three times the volume of anhydrous ethanol was added, and the mixture was stirred at 50 rpm for 20 min. The mixture was allowed to stand for 2 h, then centrifuged at 5000 rpm for 20 min. The supernatant was collected and concentrated under reduced pressure at 45°C to 1 / 8 of the original volume to obtain a concentrate. The concentrate was filtered through a 0.22 μm filter membrane and then passed through an AB-8 macroporous resin column and a Sephadex column. The glycosylated complex was purified by LH-20 gel column elution with distilled water and ethanol gradient, then concentrated under reduced pressure at 40°C for 4 h and freeze-dried. 0.7 parts by weight of sodium tripolyphosphate and 0.4 parts by weight of sodium trimetaphosphate were dispersed in 50 parts by weight of distilled water and stirred at 200 r / min for 15 min. Then, 1 part by weight of the glycosylated complex and 0.17 parts by weight of sodium sulfate were added and stirred at 250 r / min for 30 min. The pH was adjusted to 9.1 with 0.01 mol / L NaOH aqueous solution and stirred for 10 min. The temperature was then raised to 82 °C and stirred at 250 r / min for 5.6 h. After the reaction was completed, the mixture was cooled to 25 °C and 3 times the volume of anhydrous ethanol was added. The mixture was stirred at 50 r / min for 20 min and allowed to stand for 2 h. The mixture was then centrifuged at 5000 r / min for 15 min, the precipitate was collected, washed 3 times with distilled water, dialyzed in distilled water for 72 h, and then freeze-dried to obtain the phosphorylated complex. Ten parts by weight of the phosphorylated complex were dispersed in 100 parts by weight of methanol-water mixed solvent (volume ratio 1:1), and 4 parts by weight of potassium carbonate were added. The mixture was stirred at 200 r / min at 80 °C for 8 h. After the reaction was completed, the mixture was cooled to 25 °C, and the pH was adjusted to 7.0 with 1 mol / L hydrochloric acid solution. The mixture was concentrated under reduced pressure at 40 °C for 3 h, extracted three times with ethyl acetate, and the organic phases were combined. The mixture was washed three times with saturated sodium chloride solution, dried with anhydrous sodium carbonate and anhydrous calcium chloride, and then concentrated to obtain the crude product. The crude product was then purified by Sephadex LH-20 gel column chromatography, and then concentrated and freeze-dried to obtain modified quercetin. 1.65 parts by weight of gallic acid and 0.95 parts by weight of phytosterol were dispersed in 50 parts by weight of n-hexane. After stirring and dispersing at 100 rpm for 10 min, 0.165 parts by weight of immobilized lipase Novozyme were added. 435, heated to 64℃, stirred at 200 r / min for 66 h, after the reaction was completed, the immobilized lipase was recovered by hot filtration, the filtrate was collected, and the crude product was obtained by vacuum distillation at 40℃ for 4 h. 20 parts by weight of 2 wt% sodium bicarbonate solution at 50℃ were added, and the mixture was stirred at 50℃ for 20 min. The mixture was then transferred to a separatory funnel, allowed to stand for separation, and the upper organic phase was collected. 40 parts by weight of anhydrous ethanol were added to the organic phase, the temperature was raised to 70℃ and stirred for 10 min. The mixture was then placed at 4℃ and allowed to stand for 12 h. The supernatant was collected by centrifugation at 5000 r / min for 20 min at 4℃. The supernatant was concentrated at 50℃ under reduced pressure for 4 h, purified by silica gel column chromatography, and then concentrated and vacuum dried to obtain the first complex. One part by weight of the first complex was dispersed in 20 parts by weight of propylene glycol, heated to 40°C and stirred for 10 min to obtain the first solution. 0.38 parts by weight of glycine was dispersed in 100 parts by weight of pH 7.5 phosphate buffer and stirred at 200 r / min for 10 min to obtain the second solution. The first solution was added to the second solution while stirring, followed by 0.018 parts by weight of horseradish peroxidase. The temperature was raised to 37°C, and 0.11 parts by weight of 30 wt% hydrogen peroxide solution was added. The mixture was stirred at 200 r / min for 7.5 h. After the reaction was completed, the mixture was cooled to 4°C, and the pH was adjusted to 3.0 with 1 mol / L hydrochloric acid. The mixture was centrifuged at 8000 r / min for 15 min, and the precipitate was collected. It was washed three times alternately with pH 4.0 hydrochloric acid solution and ice-cold ethanol. The washed precipitate was redissolved in 0.1 wt% ammonia water, dialyzed in distilled water for 48 h, and then freeze-dried to obtain the second complex. One part by weight of the second complex was dispersed in 80 parts by weight of distilled water, heated to 50°C, and ultrasonically dispersed for 5 min at 200 W to obtain a dispersion. One part by weight of zinc gluconate was dispersed in 30 parts by weight of distilled water to obtain a zinc salt solution. The zinc salt solution was added dropwise to the dispersion at a rate of 2 mL / min at 800 r / min. After the addition was complete, the reaction was continued with stirring for 3.5 h. After the reaction was complete, the solution was dialyzed against distilled water for 48 h using a 5000 Da dialysis bag to obtain a composite dispersion. The composite dispersion was then added... 9% trehalose was stirred at 100 r / min for 50 min at 25°C and then freeze-dried to obtain modified gallic acid; 0.5 parts by weight of the above modified quercetin and 0.58 parts by weight of the above modified gallic acid were dispersed in 100 parts by weight of pH 7.4 phosphate buffer and stirred at 200 r / min for 5.5 h at 25°C. After stirring, the mixture was concentrated by centrifugation using an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa, washed three times with phosphate buffer, freeze-dried, and passed through a 30-mesh sieve to obtain a compound feed additive. Corn and soybean meal were crushed and passed through a 10-mesh sieve, fishmeal through a 20-mesh sieve, and limestone powder through a 100-mesh sieve. 57.5 parts by weight of corn, 27.5 parts by weight of soybean meal, and 3.5 parts by weight of fishmeal were placed in a mixer and stirred at 32 r / min for 5 minutes. Then, 5.5 parts by weight of limestone powder, 0.25 parts by weight of compound feed additive, and 4.8 parts by weight of 5% quail laying period premix were added and mixed for another 5 minutes. After spraying 1.8 parts by weight of rapeseed oil at a pressure of 0.35 MPa, the mixture was stirred at 32 r / min for 11 minutes to obtain green antibiotic-free feed.

[0043] Example 5

[0044] A method for preparing a green, antibiotic-free feed for quail during their egg-laying period includes the following steps: One part by weight of quercetin was dispersed in 20 parts by weight of ethyl acetate, 1.5 parts by weight of acetic anhydride and 0.05 parts by weight of pyridine were added, the temperature was raised to 70°C, and the mixture was stirred under reflux at 300 r / min for 2.5 h in a nitrogen atmosphere. After the reaction was completed, the mixture was cooled to 0°C, washed with distilled water at 0°C, allowed to stand for separation, the ethyl acetate layer was collected, and the mixture was distilled under reduced pressure and dried under vacuum to obtain acetylated quercetin. Seven parts by weight of sucrose were dispersed in 70 parts by weight of acetate-calcium acetate buffer at pH 5.4 and stirred at 150 rpm for 10 min to obtain a sucrose solution. 0.03 parts by weight of acetylated quercetin were dispersed in 7 parts by weight of dimethyl sulfoxide and sonicated at 200 W for 5 min. This solution was then added to the sucrose solution, followed by 0.02 parts by weight of dextran sucrase enzyme solution. The mixture was stirred at 200 rpm in the dark for 24 h. After the reaction, the temperature was raised to 95°C and held for 10 min, then cooled to 25°C. Three times the volume of anhydrous ethanol was added to the reaction solution, and the mixture was stirred at 50 rpm for 20 min and allowed to stand for 2 h. The mixture was centrifuged at 5000 rpm for 20 min, and the supernatant was collected. The supernatant was concentrated under reduced pressure at 45°C to 1 / 8 of its original volume to obtain a concentrated solution. The concentrated solution was filtered through a 0.22 μm filter membrane and then passed through an AB-8 macroporous resin column and a Sephadex column. The glycosylated complex was purified by LH-20 gel column elution with distilled water and ethanol gradient, then concentrated under reduced pressure at 40°C for 4 h and freeze-dried. 0.8 parts by weight of sodium tripolyphosphate and 0.5 parts by weight of sodium trimetaphosphate were dispersed in 50 parts by weight of distilled water. After stirring at 200 r / min for 15 min, 1 part by weight of the glycosylated complex and 0.2 parts by weight of sodium sulfate were added. The mixture was stirred at 250 r / min for 30 min. The pH was adjusted to 9.2 with 0.01 mol / L NaOH aqueous solution and stirred for 10 min. The temperature was then raised to 85 °C and stirred at 250 r / min for 6 h. After the reaction was completed, the mixture was cooled to 25 °C. Three times the volume of anhydrous ethanol was added to the reaction solution and stirred at 50 r / min for 20 min. The mixture was allowed to stand for 2 h and then centrifuged at 5000 r / min for 15 min. The precipitate was collected, washed three times with distilled water, dialyzed in distilled water for 72 h, and then freeze-dried to obtain the phosphorylated complex. Ten parts by weight of the phosphorylated complex were dispersed in 100 parts by weight of methanol-water mixed solvent (volume ratio 1:1), and 4 parts by weight of potassium carbonate were added. The mixture was stirred at 200 r / min at 80 °C for 8 h. After the reaction was completed, the mixture was cooled to 25 °C, and the pH was adjusted to 7.0 with 1 mol / L hydrochloric acid solution. The mixture was concentrated under reduced pressure at 40 °C for 3 h, extracted three times with ethyl acetate, and the organic phases were combined. The mixture was washed three times with saturated sodium chloride solution, dried with anhydrous sodium carbonate and anhydrous calcium chloride, and then concentrated to obtain the crude product. The crude product was then purified by Sephadex LH-20 gel column chromatography, and then concentrated and freeze-dried to obtain modified quercetin. 1.7 parts by weight of gallic acid and 1.0 parts by weight of phytosterol were dispersed in 50 parts by weight of n-hexane. After stirring at 100 rpm for 10 min, 0.17 parts by weight of immobilized lipase Novozyme were added. 435, heated to 65℃, stirred at 200 r / min for 72 h. After the reaction, the immobilized lipase was recovered by hot filtration. The filtrate was collected and distilled under reduced pressure at 40℃ for 4 h to obtain the crude product. 20 parts by weight of 2 wt% sodium bicarbonate solution at 50℃ were added and stirred at 50℃ for 20 min. The mixture was then transferred to a separatory funnel, allowed to stand and separate into layers, and the upper organic phase was collected. 40 parts by weight of anhydrous ethanol were added to the organic phase, heated to 70℃ and stirred to disperse for 10 min. The mixture was then placed at 4℃ and allowed to stand for 12 h. The supernatant was collected by centrifugation at 5000 r / min at 4℃ for 20 min. The supernatant was concentrated under reduced pressure at 50℃ for 4 h, purified by silica gel column chromatography, and then concentrated and vacuum dried to obtain the first complex. One part by weight of the first complex was dispersed in 20 parts by weight of propylene glycol, heated to 40°C and stirred for 10 min to obtain the first solution. 0.4 parts by weight of glycine was dispersed in 100 parts by weight of pH 7.5 phosphate buffer and stirred at 200 r / min for 10 min to obtain the second solution. The first solution was added to the second solution while stirring, followed by 0.02 parts by weight of horseradish peroxidase. The temperature was raised to 37°C, and 0.12 parts by weight of 30 wt% hydrogen peroxide solution was added. The mixture was stirred at 200 r / min for 8 h. After the reaction was completed, the mixture was cooled to 4°C, and the pH was adjusted to 3.0 with 1 mol / L hydrochloric acid. The mixture was centrifuged at 8000 r / min for 15 min, and the precipitate was collected. It was washed three times alternately with pH 4.0 hydrochloric acid solution and ice-cold ethanol. The washed precipitate was redissolved in 0.1 wt% ammonia water, dialyzed in distilled water for 48 h, and then freeze-dried to obtain the second complex. One part by weight of the second complex was dispersed in 80 parts by weight of distilled water, heated to 50°C, and ultrasonically dispersed for 5 min at 200 W to obtain a dispersion. One and a half parts by weight of zinc gluconate were dispersed in 30 parts by weight of distilled water to obtain a zinc salt solution. The zinc salt solution was added dropwise to the dispersion at a rate of 2 mL / min at 800 r / min. After the addition was complete, the reaction was continued with stirring for 4 h. After the reaction was complete, the solution was dialyzed against distilled water for 48 h using a 5000 Da dialysis bag to obtain a composite dispersion. The composite dispersion was then added... 10% of trehalose was stirred at 100 r / min for 60 min at 25 °C and then freeze-dried to obtain modified gallic acid; 0.6 parts by weight of the above modified quercetin and 0.6 parts by weight of the above modified gallic acid were dispersed in 100 parts by weight of pH 7.4 phosphate buffer and stirred at 200 r / min for 6 h at 25 °C. After stirring, the mixture was concentrated by centrifugation using an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa, washed three times with phosphate buffer, freeze-dried, and passed through a 30-mesh sieve to obtain a compound feed additive; Corn and soybean meal were crushed and passed through a 10-mesh sieve, fish meal through a 20-mesh sieve, and limestone powder through a 100-mesh sieve. 58 parts by weight of corn, 28 parts by weight of soybean meal, and 4 parts by weight of fish meal were placed in a mixer and mixed at 35 r / min for 6 minutes. Then, 6 parts by weight of limestone powder, 0.3 parts by weight of compound feed additive, and 5 parts by weight of 5% quail laying period premix were added and mixed for another 6 minutes. After spraying 2 parts by weight of rapeseed oil at a pressure of 0.4 MPa, the mixture was mixed at 35 r / min for 12 minutes to obtain green antibiotic-free feed.

[0045] Comparative Example 1 A method for preparing a green, antibiotic-free feed for quail during their egg-laying period includes the following steps: Replace 0.02 parts by weight of dextran sucrase enzyme solution in Example 5 with 0.02 parts by weight of 4 U / mL β-galactosidase enzyme solution, and keep the other operation steps the same as in Example 5.

[0046] Comparative Example 2 A method for preparing a green, antibiotic-free feed for quail during their egg-laying period includes the following steps: In Example 5, 1.0 part by weight of phytosterol was replaced with 1.0 part by weight of lanosterol, and the remaining operating steps were the same as in Example 5.

[0047] Comparative Example 3 A method for preparing a green, antibiotic-free feed for quail during their egg-laying period includes the following steps: In Example 5, 0.17 parts by weight of immobilized lipase Novozyme 435 was replaced with 0.17 parts by weight of immobilized lipase Lipozyme TL IM, and the remaining operating steps were the same as in Example 5.

[0048] Comparative Example 4 A method for preparing a green, antibiotic-free feed for quail during their egg-laying period includes the following steps: In Example 5, 0.8 parts by weight of sodium tripolyphosphate and 0.5 parts by weight of sodium trimetaphosphate were replaced with 1.3 parts by weight of sodium pyrophosphate, while the remaining operating steps remained the same as in Example 5.

[0049] Comparative Example 5 A method for preparing a green, antibiotic-free feed for quail during their egg-laying period includes the following steps: In Example 5, 0.4 parts by weight of glycine was replaced with 0.4 parts by weight of DL-alanine, and the remaining operating steps were the same as in Example 5.

[0050] Quail egg production performance test: 360 healthy, laying quails of similar weight (120±5g) were randomly divided into 12 groups (Examples 1-5, Comparative Examples 1-5, and Blank Control Group), with 30 quails in each group, housed individually. The blank control group was fed a basal diet (without compound additives), while the other groups were fed the corresponding green antibiotic-free diets of the Examples and Comparative Examples, respectively. The experiment lasted for 60 days. The number of eggs laid, egg weight, and number of broken eggs were recorded daily for each group. The egg production rate (egg production rate = total number of eggs laid / (number of quails × number of experimental days) × 100%) and average egg weight were calculated. Egg quality testing: After the test, 30 qualified eggs were randomly selected from each group. The eggshell strength was measured using an eggshell strength tester, and the yolk color was measured using a colorimeter. Feed efficiency test: During the experiment, the daily feed intake and remaining amount for each group were recorded, and the average feed intake was calculated; at the beginning and end of the experiment, the weight of each group of quails was weighed, and the average weight gain was calculated. The test results are shown in Tables 1 and 2.

[0051] Table 1. Results of Quail Egg Production Performance Test Table 2. Results of Egg Quality Testing and Feed Conversion The test results in Tables 1 and 2 show that the green antibiotic-free feeds prepared in Examples 1-5 of this invention have good performance, while the performance of the green antibiotic-free feeds prepared in Comparative Examples 1-5 is reduced to varying degrees.

[0052] The decrease in Comparative Example 1 may be due to a mismatch between the transglycosylation activity and regioselectivity of the enzyme after replacing dextran sucrase with β-galactosidase. Dextran sucrase can precisely link glucose units in sucrose to the exposed phenolic hydroxyl groups of acetylated quercetin via glycosidic bonds, while β-galactosidase has low transglycosylation efficiency and cannot achieve specific site modification. This results in a limited increase in the water solubility of modified quercetin and insufficient cell membrane affinity, which in turn affects its absorption and utilization efficiency in quail, ultimately leading to a decline in the overall performance of the feed.

[0053] The decrease in Comparative Example 2 may be due to the poor compatibility between the molecular structure of lanosterol and gallic acid after replacing phytosterol with lanosterol. Under the catalysis of immobilized lipase Novozyme 435, the esterification rate of the two is slower, resulting in a lower conversion rate and affecting the yield and purity of the first complex. Furthermore, lanosterol lacks the physiological activity of phytosterols in regulating lipid metabolism and cannot form a synergistic metabolic regulatory network with modified quercetin. This weakens the protective effect on quail liver health and reduces nutrient conversion efficiency, leading to performance indicators inferior to those of Example 5.

[0054] The decrease in Comparative Example 3 may be due to differences in the catalytic properties of the immobilized lipase Lipozyme TL IM compared to Novozyme 435. Lipozyme TL IM exhibits lower catalytic efficiency in the esterification reaction of gallic acid and phytosterols, failing to efficiently promote the binding of gallic acid carboxyl groups to phytosterol hydroxyl groups. This results in a reduced amount of the first complex and a higher impurity content in the product. Subsequent oxidative grafting and zinc ion coordination reactions are also affected by poor substrate quality. The structural integrity and functional activity of the modified gallic acid decrease, making it unable to effectively chelate metal ions in the intestine to protect the modified quercetin, ultimately leading to reduced feed performance.

[0055] The decrease in Comparative Example 4 may be due to the reduced selectivity of the phosphorylation reaction after replacing the mixed system of sodium tripolyphosphate and sodium trimetaphosphate with sodium pyrophosphate as a single reagent. The mixed phosphate system can specifically undergo phosphorylation esterification with the terminal hydroxyl groups of the glycosylated complex, while the reaction site of sodium pyrophosphate is not specific, easily leading to non-specific modification of the active sites on the modified quercetin molecule, and a decrease in the grafting rate of phosphate groups. The water solubility and activity of the modified quercetin cannot achieve the expected results, and its phosphate groups are more easily chelated and inactivated by metal ions in the intestine, ultimately leading to a decline in feed performance.

[0056] The decrease in Comparative Example 5 may be due to the lower α-amino reactivity of DL-alanine compared to glycine after replacing glycine with DL-alanine. This results in reduced binding efficiency with the o-benzoquinone intermediate in the Michael addition reaction catalyzed by horseradish peroxidase, leading to insufficient grafting rate. Furthermore, the weaker hydrophilicity and coordination ability of DL-alanine compared to glycine further decreases the hydrophilicity of the second complex and worsens its coordination with zinc ions, impairing the structure and function of the modified gallic acid. Consequently, it cannot effectively complex metal ions in the intestine and is less effective at promoting synergistic absorption with modified quercetin, resulting in lower performance indicators for the feed compared to Example 5.

[0057] The embodiments described above provide a detailed explanation of the technical solutions and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Various changes and modifications can be made to the present invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed.

Claims

1. A method for preparing a green, antibiotic-free feed for quail during their egg-laying period, characterized in that, The preparation method includes the following steps: Quercetin, acetic anhydride, and pyridine were mixed and stirred to produce acetylated quercetin. A glycosylated complex was obtained by mixing and stirring sucrose, acetylated quercetin, and dextran sucrase enzyme solution. Sodium tripolyphosphate, sodium trimetaphosphate, glycosylation complex and sodium sulfate are mixed and stirred to react to obtain phosphorylated complex; Modified quercetin was obtained by mixing and stirring the phosphorylated complex and potassium carbonate. Modified quercetin and modified gallic acid were mixed and stirred to obtain a compound feed additive; Corn, soybean meal, fish meal, limestone powder, compound feed additives and premix are mixed together, then rapeseed oil is sprayed on, and the mixture is stirred and mixed again to obtain green antibiotic-free feed.

2. The method for preparing a green antibiotic-free feed for quail during their egg-laying period as described in claim 1, characterized in that, The weight ratio of quercetin, acetic anhydride and pyridine is 1:1.2~1.5:0.03~0.05; the conditions for the mixing and stirring reaction of quercetin, acetic anhydride and pyridine include a reaction temperature of 60~70℃ and a reaction time of 1.5~2.5h.

3. The method for preparing a green antibiotic-free feed for quail during their egg-laying period as described in claim 1, characterized in that, The weight ratio of sucrose, acetylated quercetin, and dextran sucrase enzyme solution is 5~7:0.01~0.03:0.01~0.02; the conditions for mixing and stirring the sucrose, acetylated quercetin, and dextran sucrase enzyme solution include a reaction time of 20~24h and a reaction speed of 200r / min.

4. The method for preparing a green antibiotic-free feed for quail during their egg-laying period as described in claim 1, characterized in that, The weight ratio of sodium tripolyphosphate, sodium trimetaphosphate, glycosylated complex, and sodium sulfate is 0.4~0.8:0.2~0.5:1:0.1~0.2; the conditions for the mixing and stirring reaction of sodium tripolyphosphate, sodium trimetaphosphate, glycosylated complex, and sodium sulfate include a reaction pH of 8.8~9.2, a reaction temperature of 75~85℃, and a reaction time of 5~6h.

5. The method for preparing a green antibiotic-free feed for quail during their egg-laying period as described in claim 1, characterized in that, The weight ratio of the phosphorylated complex to potassium carbonate is 10:3~4; the conditions for the mixing and stirring reaction of the phosphorylated complex and potassium carbonate include a reaction temperature of 70~80℃ and a reaction time of 6~8h.

6. The method for preparing a green antibiotic-free feed for quail during their egg-laying period as described in claim 1, characterized in that, The method for preparing the modified gallic acid includes the following steps: Gallic acid, phytosterol and lipase were mixed and stirred to obtain the first complex. The first complex, glycine, horseradish peroxidase and hydrogen peroxide solution were mixed and stirred to react to obtain the second complex; The second complex and zinc gluconate were mixed and stirred to obtain a complex dispersion, and then trehalose was added and stirred to obtain modified gallic acid.

7. The method for preparing a green antibiotic-free feed for quail during their egg-laying period as described in claim 6, characterized in that, The weight ratio of gallic acid, phytosterol, and lipase is 1.5~1.7:0.8~1.0:0.15~0.17; the conditions for the mixing and stirring reaction of gallic acid, phytosterol, and lipase include a reaction temperature of 60~65℃ and a reaction time of 48~72h.

8. The method for preparing a green antibiotic-free feed for quail during their egg-laying period as described in claim 6, characterized in that, The weight ratio of the first complex, glycine, horseradish peroxidase, and hydrogen peroxide solution is 1:0.3~0.4:0.01~0.02:0.08~0.12; the reaction conditions for mixing and stirring the first complex, glycine, horseradish peroxidase, and hydrogen peroxide solution include a reaction temperature of 35~37℃ and a reaction time of 6~8h; the weight ratio of the second complex and zinc gluconate is 1:0.9~1.1; the mixing and stirring conditions for the second complex and zinc gluconate include a stirring speed of 800r / min and a stirring time of 2~4h; the amount of trehalose used is 5%~10% of the weight of the composite dispersion.

9. The method for preparing a green antibiotic-free feed for quail during their egg-laying period as described in claim 1, characterized in that, The weight ratio of corn, soybean meal, fish meal, stone powder, compound feed additives, premix, and rapeseed oil is 55~58:25~28:2~4:4~6:0.1~0.3:4~5:1~2.

10. A green, antibiotic-free feed for quail during their egg-laying period, characterized in that, It is prepared by any one of the methods described in claims 1 to 9 for the preparation of a green antibiotic-free feed for quail during the egg-laying period.

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