Compound feed for improving disease resistance of breeding ducks and preparation process of compound feed

Abstract

The invention provides a compound feed for improving disease resistance of breeding ducks and a preparation process of the compound feed, and belongs to the technical field of breeding feeds. The preparation process comprises the following steps: preparing modified resistant starch acetate; preparing a bifidobacterium complex microbial inoculant; preparing a composite nano-carrier; preparing an extract composite additive; and preparing the compound feed. The method comprises the following steps: activating resistant starch acetate by using 1, 1-carbonyl diimidazole, adding a concanavalin A solution, reacting to obtain modified resistant starch acetate, and loading bifidobacteria by using the modified resistant starch acetate, so that the bifidobacteria can be protected from being damaged by gastric acid, digestive enzyme and the like, and the number of the bifidobacteria reaching the large intestine of the breeding duck is further increased; according to the present invention, with the application of the concanavalin A, the breeding of harmful bacteria in the large intestine of the breeding duck can be well inhibited, the disease resistance of the breeding duck can be improved, and in addition, after the concanavalin A is adopted to modify the resistant starch acetate, the loading rate of the resistant starch acetate on the bifidobacterium can be easily improved.

Description

Technical Field

[0001] The present invention relates to the technical field of breeding feed, and particularly relates to a compound feed for improving the disease resistance of breeding ducks and a preparation process thereof. Background Art

[0002] In the current breeding of breeding ducks, laying breeding ducks are often in open or semi-open duck houses, and the sanitary conditions are difficult to effectively control. The humid environment in the duck house provides a breeding ground for bacteria and viruses, resulting in viral diseases such as duck plague and avian influenza and bacterial infections such as Escherichia coli as the main health threats. In the past, the method of adding antibiotics to feed to prevent and treat diseases not only makes pathogens develop drug resistance, leading to a gradual decrease in the therapeutic effect of antibiotics, but also causes drug residue problems.

[0003] As a dominant probiotic in the intestine, Bifidobacterium can reduce the risk of intestinal infection, promote the proliferation and differentiation of lymphocytes, enhance the phagocytic ability of macrophages, and strengthen the humoral and cellular immune functions. However, when directly adding Bifidobacterium to feed, the gastric acid secretion of laying breeding ducks is strong. After Bifidobacterium enters the stomach through oral administration, the strong acid environment will cause the denaturation of the bacterial protein and the rupture of the cell membrane, resulting in a significant decrease in the viable count.

[0004] Resistant starch is a type of carbohydrate that is not easily hydrolyzed by amylase in the digestive tract. Through physical embedding, structural barriers, and microenvironment regulation, etc., it can significantly improve the survival rate of Bifidobacterium in the gastric acid environment. However, its loading of Bifidobacterium usually relies on van der Waals forces, electrostatic interactions, or hydrogen bonds, with relatively weak binding forces. Moreover, natural resistant starch has a high crystallinity, a dense molecular arrangement, and an internal pore diameter smaller than the size of Bifidobacterium, so bacteria cannot enter the interior of the carrier and can only adsorb on the surface, resulting in a low loading rate of Bifidobacterium and limited effect on enhancing the immunity of breeding ducks.

[0005] Therefore, it is necessary to propose a compound feed for improving the disease resistance of breeding ducks and a preparation process thereof that can enhance immunity. Summary of the Invention

[0006] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a compound feed for improving the disease resistance of breeding ducks and a preparation process thereof.

[0007] A preparation process of a compound feed for improving the disease resistance of breeding ducks includes the following steps: S1: Prepare modified resistant starch acetate First, use resistant starch and acetic anhydride as raw materials to prepare resistant starch acetate. After activating it, react it with concanavalin A to make modified resistant starch acetate; S2: Prepare Bifidobacterium complex bacterium agent The bifidobacterium freeze-dried powder is activated to prepare a bifidobacterium suspension, which is then mixed with a modified resistant starch acetate solution and then added to a mixture of Span 80 and liquid paraffin. After homogenization and emulsification, a calcium chloride solution is added for cross-linking to prepare a bifidobacterium composite bacterial agent. S3: Preparation of composite nanocarriers After bacterial cellulose is acidified, it is dispersed in PBS buffer and then mixed with activated oleic acid to react and prepare a composite nanocarrier; S4: Preparation of extract compound additives The composite nanocarrier is dissolved, mixed with verbena extract solution and Moringa leaf extract solution, and then added into Tween 80 chloroform solution, homogenized and emulsified to prepare an extract composite additive; S5: Preparation of compound feed Corn, broken rice, soybean meal and eggshell powder are mixed and crushed, and then the extract compound additive, bifidobacterium compound bacterial agent, oligoxylose, compound microorganism and spirulina powder are added, fully mixed and granulated to obtain compound feed.

[0008] Furthermore, step S1 specifically includes the following steps: S1.1: Add resistant starch to deionized water at a ratio of 1 g to (10-20) mL, stir thoroughly to form a suspension, and cool in an ice bath to 3-5°C to obtain a resistant starch suspension. S1.2: Add 0.1 mol / L sodium hydroxide solution to the resistant starch suspension to adjust the pH to 8-10, and stir to swell for 20-30 minutes. Then, add acetic anhydride, stir and react at 20-30°C for 2-3 hours, then add 0.1 mol / L hydrochloric acid solution to adjust the pH to neutral. Then, collect the precipitate by centrifugation, wash, and vacuum dry to obtain resistant starch acetate. S1.3: Suspend the resistant starch acetate in acetone at a ratio of 1 g:(20-30) mL, add 1,1-carbonyldiimidazole, and activate with stirring at 25-35°C for 10-12 h to obtain an activated resistant starch acetate solution, wherein the mass ratio of 1,1-carbonyldiimidazole to resistant starch acetate is 1:(4-5). S1.4: Dissolve concanavalin A in PBS buffer at a ratio of 1 g: (150-200) mL, then add to the activated resistant starch acetate solution. Stir and react at 3-5°C for 12-16 hours. Purify by dialysis and freeze-dry to obtain modified resistant starch acetate.

[0009] Furthermore, S2 specifically includes the following steps: S2.1: Inoculate the freeze-dried Bifidobacterium powder into MRS medium, anaerobically culture at 37 °C for 40 - 48 h, then centrifuge at 4 °C, discard the supernatant, wash 2 - 3 times with sterile physiological saline, and then resuspend with sterile PBS buffer to obtain a Bifidobacterium suspension with a concentration of 10 10 CFU / mL; S2.2: Dissolve the modified resistant starch acetate prepared in step S1.4 in sterile PBS buffer at a ratio of 1 g : (20 - 30) mL, then add the above Bifidobacterium suspension and stir to mix evenly to obtain an aqueous phase, where the concentration of Bifidobacterium in the aqueous phase is (4 - 5)×10 9 CFU / mL; S2.3: Add Span 80 to liquid paraffin at a ratio of 1 g : (40 - 50) mL, stir to dissolve, then add the above aqueous phase, and homogenize and emulsify at 1500 - 2500 rpm for 10 - 20 min to obtain an emulsion, where the volume ratio of the aqueous phase to liquid paraffin is 1:2; S2.4: Add 0.1 mol / L calcium chloride solution to the above emulsion, heat and stir at 37 °C for crosslinking for 20 - 30 min, then centrifuge at 3 - 5 °C and discard the supernatant. After washing with sterile petroleum ether and sterile PBS buffer, freeze-dry to obtain a Bifidobacterium complex bactericide, where the volume ratio of the calcium chloride solution to the aqueous phase is 1 : (6 - 8).

[0010] Further, S3 specifically includes the following steps: S3.1: Add bacterial cellulose to a phosphate buffer with a pH of 5 - 6 at a ratio of 1 g : (20 - 30) mL, and ultrasonically treat for 30 - 40 min to obtain a bacterial cellulose suspension; S3.2: Add a sulfuric acid solution with a volume fraction of 64% to the above bacterial cellulose suspension, stir and hydrolyze at 40 - 50 °C for 14 - 16 h, then add 1 mol / L sodium hydroxide solution to adjust the pH to neutral. After centrifugal separation, washing and freeze-drying, obtain acidified bacterial cellulose, where the volume ratio of the sulfuric acid solution to the bacterial cellulose suspension is 1 : (2 - 3); S3.3: Add oleic acid to chloroform at a volume ratio of 1 : (8 - 10) mL, stir and mix well, then add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, stir and activate in the dark for 1 - 2 h to obtain an activated oleic acid solution; S3.4: Disperse the above acidified bacterial cellulose in dimethyl sulfoxide at a ratio of 1 g : (16 - 20) mL, then add the above activated oleic acid solution, and under nitrogen protection, stir and react at 60 - 70 °C for 24 - 36 h. After alcohol precipitation, centrifugal separation, washing and freeze-drying, obtain a composite nanocarrier, where the mass ratio of bacterial cellulose to oleic acid is 1 : (2 - 2.2).

[0011] Further, S4 specifically includes the following steps: S4.1: Dissolve the composite nanocarrier prepared in step S3.4 in dimethyl sulfoxide at a ratio of 1 g : (20 - 30) mL to obtain a composite nanocarrier solution, then add verbena extract solution and moringa leaf extract solution, and stir and mix evenly to obtain a mixed solution; S4.2: Dissolve Tween 80 in chloroform at a ratio of 1 g : (40 - 50) mL, then add the above mixed solution accounting for 3 / 5 of the volume of chloroform, and homogenize and emulsify at 2000 - 3000 rpm for 10 - 20 min to obtain a mixed emulsion; S4.3: Rotavaporize the above mixed emulsion to remove chloroform, then filter through a 0.45 μm membrane, centrifuge, wash, and freeze-dry to obtain an extract composite additive.

[0012] Further, the mass ratio of acetic anhydride to resistant starch is (1.6 - 1.8) : 1, and the mass ratio of concanavalin A to resistant starch acetate is 1 : (95 - 105).

[0013] Further, the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is 1:2, and the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to oleic acid is 1 : (3.6 - 4).

[0014] Further, the volume ratios of the verbena extract solution and the moringa leaf extract solution to the composite nanocarrier solution are 1 : (3 - 4) and 1 : (4 - 6) respectively, and the concentrations of both the verbena extract solution and the moringa leaf extract solution are 50 mg / mL.

[0015] Further, by mass, the raw material composition of the compound feed is: 40 - 50 parts of corn, 10 - 20 parts of broken rice, 10 - 20 parts of soybean meal, 10 - 15 parts of eggshell powder, 10 - 20 parts of spirulina powder, 8 - 14 parts of xylo-oligosaccharide, 8 - 10 parts of the extract composite additive, 3 - 5 parts of bifidobacterium complex bacteria agent, and 2 - 4 parts of compound vitamins, wherein the compound vitamins are composed of vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin C, vitamin D, and vitamin E in a mass ratio of (1 - 2) : (3 - 5) : (3 - 4) : 1 : (2 - 3) : (1 - 1.2) : (1.3 - 1.5).

[0016] Further, a compound feed for improving the disease resistance of breeding ducks is prepared by the preparation process of a compound feed for improving the disease resistance of breeding ducks described in any one of the above.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects: 1. In the present invention, after activating resistant starch acetate with 1,1-carbonyldiimidazole, concanavalin A solution is added for reaction to form amide bonds, enabling concanavalin A to graft onto the resistant starch acetate molecules, obtaining modified resistant starch acetate. After loading Bifidobacterium with it, it can protect Bifidobacterium from being damaged by gastric acid and digestive enzymes, etc., thereby increasing the number of Bifidobacterium reaching the large intestine of breeding ducks, facilitating better inhibition of the reproduction of harmful bacteria in the large intestine of breeding ducks, reducing the risk of intestinal inflammation, and further enhancing the immunity of breeding ducks and improving their disease resistance. In addition, after modifying resistant starch acetate with concanavalin A, concanavalin A can form specific binding with the surface of Bifidobacterium through sugar-protein interaction, thus reducing the shedding of Bifidobacterium during the loading process and helping to improve the loading rate of resistant starch acetate for Bifidobacterium.

[0018] 2. In the present invention, first, bacterial cellulose is hydrolyzed and acidified with sulfuric acid and oleic acid is activated, then the acidified bacterial cellulose reacts with the activated oleic acid to form covalent bonds, preparing a composite nanocarrier. After loading verbena extract and moringa leaf extract with this composite nanocarrier, due to the introduction of oleic acid molecules, the specific surface area of the nanocarrier can be increased and the pore volume can be expanded, enabling it to accommodate more extract molecules, thus effectively improving the encapsulation rate of the composite nanocarrier for verbena extract and moringa leaf extract, facilitating the improvement of the disease resistance efficacy of the extract composite additive, and further enhancing the disease resistance of breeding ducks.

[0019] 3. In the present invention, on the one hand, verbena extract can neutralize reactive oxygen species and reduce oxidative damage to immune cells, while the active antioxidant components in moringa leaf extract can further scavenge free radicals. The combination of the two can significantly enhance the antioxidant capacity of the body, reduce immune cell apoptosis, and maintain the efficient operation of the immune system. On the other hand, verbena extract can enhance the activity of immune cells by regulating cytokines, while moringa leaf extract can promote the proliferation and differentiation of lymphocytes and enhance cellular immunity. The combination of the two can significantly increase the level of immunoglobulins, enhance the synergistic effect of humoral immunity and cellular immunity, and improve the clearance efficiency of pathogenic microorganisms. Therefore, verbena extract and moringa leaf extract can synergistically enhance the immunity of breeding ducks and further improve their disease resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The drawings incorporated herein and constituting a part of the specification illustrate embodiments of the present disclosure and, together with the specification, are further used to explain the principles of the present disclosure and enable those skilled in the relevant art to implement and use the present disclosure.

[0021] Figure 1 It is a process flow chart of the preparation of the composite feed used in the embodiments of the present invention for improving the disease resistance of breeding ducks. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0022] The following is a detailed description of a compound feed for improving the disease resistance of breeding ducks and its preparation process provided by the present invention in conjunction with the accompanying drawings and specific embodiments.

[0023] Example 1 A preparation process of a compound feed for improving the disease resistance of breeding ducks, as Figure 1 shown, includes the following steps: S1: Prepare modified resistant starch acetate S1.1: Add resistant starch to deionized water at a ratio of 1 g: 10 mL, stir well to form a suspension, and cool it to 3 °C in an ice bath to obtain a resistant starch suspension; S1.2: Add 0.1 mol / L sodium hydroxide solution to the above-mentioned resistant starch suspension to adjust the pH to 8, stir and swell for 20 min, then add acetic anhydride, stir and react at 20 °C for 2 h, add 0.1 mol / L hydrochloric acid solution to adjust the pH to neutral, and then collect the precipitate by centrifugation, wash and vacuum dry to obtain resistant starch acetate, wherein the mass ratio of acetic anhydride to resistant starch is 1.6: 1; S1.3: Suspend the above-mentioned resistant starch acetate in acetone at a ratio of 1 g: 20 mL, and add 1,1-carbonyldiimidazole, stir and activate at 25 °C for 10 h to obtain an activated resistant starch acetate solution, wherein the mass ratio of 1,1-carbonyldiimidazole to resistant starch acetate is 1: 4; S1.4: Dissolve concanavalin A in PBS buffer at a ratio of 1 g: 150 mL, and then add it to the above-mentioned activated resistant starch acetate solution, stir and react at 3 °C for 12 h, and then dialyze, purify and freeze-dry to obtain modified resistant starch acetate, wherein the mass ratio of concanavalin A to resistant starch acetate is 1: 95; S2: Prepare a Bifidobacterium complex bacterium agent S2.1: Inoculate the freeze-dried powder of Bifidobacterium into MRS medium, anaerobically culture at 37 °C for 40 h, centrifuge at 4 °C, discard the supernatant, wash twice with sterile physiological saline, and then resuspend with sterile PBS buffer to obtain a Bifidobacterium suspension with a concentration of 10 10 CFU / mL; S2.2: Dissolve the modified resistant starch acetate prepared in step S1.4 in sterile PBS buffer at a ratio of 1 g: 20 mL, and then add the above-mentioned Bifidobacterium suspension, stir and mix evenly to obtain an aqueous phase, wherein the concentration of Bifidobacterium in the aqueous phase is about 4×10 9 CFU / mL; S2.3: Add Span 80 to liquid paraffin at a ratio of 1 g:40 mL. Stir to dissolve. Then add the aqueous phase and homogenize at 1500 rpm for 10 min to obtain an emulsion. The volume ratio of aqueous phase to liquid paraffin is 1:2. S2.4: Add 0.1 mol / L calcium chloride solution to the emulsion, heat and stir at 37°C for 20 minutes to crosslink, then centrifuge at 3°C, discard the supernatant, wash with sterile petroleum ether and sterile PBS buffer, and freeze-dry to obtain a bifidobacterium composite bacterial agent, wherein the volume ratio of calcium chloride solution to aqueous phase is 1:6; S3: Preparation of composite nanocarriers S3.1: Add BC to phosphate buffer (pH 5) at a ratio of 1 g to 20 mL and sonicate for 30 min to obtain a BC suspension. S3.2: Add 64% by volume sulfuric acid solution to the bacterial cellulose suspension, stir and hydrolyze at 40°C for 14 hours, then add 1 mol / L sodium hydroxide solution to adjust the pH to neutral, centrifuge, wash and freeze-dry to obtain acidified bacterial cellulose, wherein the volume ratio of sulfuric acid solution to bacterial cellulose suspension is 1:2; S3.3: Add oleic acid to chloroform in a volume ratio of 1:8 mL. After thorough stirring, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide. Stir and activate in the dark for 1 h to obtain an activated oleic acid solution, wherein the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is 1:2, and the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to oleic acid is 1:3.6. S3.4: Disperse the acidified bacterial cellulose in dimethyl sulfoxide at a ratio of 1 g:16 mL, then add the activated oleic acid solution. Under nitrogen, stir and react at 60°C for 24 h. Precipitate with alcohol, centrifuge, wash, and freeze-dry to obtain a composite nanocarrier, wherein the mass ratio of bacterial cellulose to oleic acid is 1:2. S4: Preparation of extract compound additives S4.1: Dissolve the composite nanocarrier prepared in step S3.4 in dimethyl sulfoxide at a ratio of 1 g:20 mL to obtain a composite nanocarrier solution, then add the verbena extract solution and the Moringa leaf extract solution, stirring and mixing to obtain a mixed solution, wherein the volume ratios of the verbena extract solution and the Moringa leaf extract solution to the composite nanocarrier solution are 1:3 and 1:4, respectively, and the concentrations of the verbena extract solution and the Moringa leaf extract solution are both 50 mg / mL; The preparation method of the verbena extract solution is as follows: crushing verbena leaves at a ratio of 1 g to 20 mL, adding the crushed leaves to a 70% ethanol solution, heating and refluxing at 70° C. for 2-3 hours, repeating the extraction twice, combining the extracts, concentrating under reduced pressure until there is no ethanol, extracting the extracts twice with petroleum ether, extracting the aqueous phase with ethyl acetate three times, combining the ethyl acetate phases, and evaporating under reduced pressure to dryness to obtain the verbena extract, and then dissolving the verbena extract in a 50% ethanol solution to obtain the verbena extract solution; The preparation method of the Moringa leaf extract solution is as follows: adding Moringa leaf powder to a 70% ethanol solution at a ratio of 1 g:10 mL, ultrasonically treating at 300 W and 40° C. for 30 min, centrifuging to obtain the supernatant, concentrating under reduced pressure to a paste, then adding deionized water for re-dissolution, freeze-drying to obtain the Moringa leaf extract, and finally dissolving the Moringa leaf extract in a 50% ethanol solution to obtain the Moringa leaf extract; S4.2: Dissolve Tween 80 in chloroform at a ratio of 1 g:40 mL. Add the above mixed solution in a volume ratio of 3 / 5 to chloroform. Homogenize and emulsify at 2000 rpm for 10 min to obtain a mixed emulsion. S4.3: Rotary evaporation is performed on the mixed emulsion to remove chloroform, and the mixed emulsion is then filtered through a 0.45 μm membrane, centrifuged, washed, and freeze-dried to obtain an extract composite additive; S5: Preparation of compound feed Corn, broken rice, soybean meal and eggshell powder are mixed and crushed, and the above-mentioned extract compound additive, bifidobacterium compound bacterial agent, oligoxylose, composite microorganism and spirulina powder are added, and the mixture is fully mixed and granulated to obtain a compound feed, wherein the raw materials of the compound feed are composed of 40 parts of corn, 10 parts of broken rice, 10 parts of soybean meal, 10 parts of eggshell powder, 10 parts of spirulina powder, 8 parts of oligoxylose, 8 parts of extract compound additive, 3 parts of bifidobacterium compound bacterial agent and 2 parts of compound vitamins, wherein the compound vitamins are composed of vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin C, vitamin D and vitamin E in a mass ratio of 1:3:3:1:2:1:1.3.

[0024] Example 2 A preparation process of compound feed for improving disease resistance of breeding ducks, such as Figure 1 As shown, the following steps are included: S1: Preparation of modified resistant starch acetate S1.1: Add resistant starch to deionized water at a ratio of 1 g:15 mL, stir thoroughly to form a suspension, and cool to 4°C in an ice bath to obtain a resistant starch suspension. S1.2: Add 0.1 mol / L sodium hydroxide solution to the above-mentioned resistant starch suspension to adjust the pH to 9, stir and swell for 25 min, then add acetic anhydride, stir and react at 25 °C for 2.5 h, add 0.1 mol / L hydrochloric acid solution to adjust the pH to neutral, and then collect the precipitate by centrifugation, wash and vacuum dry to obtain resistant starch acetate, where the mass ratio of acetic anhydride to resistant starch is 1.7:1; S1.3: Suspend the above-mentioned resistant starch acetate in acetone at a ratio of 1 g:25 mL, and add 1,1-carbonyldiimidazole, stir and activate at 30 °C for 11 h to obtain an activated resistant starch acetate solution, where the mass ratio of 1,1-carbonyldiimidazole to resistant starch acetate is 1:4.5; S1.4: Dissolve concanavalin A in PBS buffer at a ratio of 1 g:175 mL, and then add it to the above-mentioned activated resistant starch acetate solution, stir and react at 4 °C for 14 h, and then dialyze, purify and freeze-dry to obtain modified resistant starch acetate, where the mass ratio of concanavalin A to resistant starch acetate is 1:100; S2: Prepare the Bifidobacterium complex bacterium agent S2.1: Inoculate the freeze-dried powder of Bifidobacterium into MRS medium, anaerobically culture at 37 °C for 44 h, centrifuge at 4 °C, discard the supernatant, wash twice with sterile physiological saline, and then resuspend with sterile PBS buffer to obtain a Bifidobacterium suspension with a concentration of 10 10 CFU / mL; S2.2: Dissolve the modified resistant starch acetate prepared in step S1.4 in sterile PBS buffer at a ratio of 1 g:25 mL, and then add the above-mentioned Bifidobacterium suspension, stir and mix evenly to obtain an aqueous phase, where the concentration of Bifidobacterium in the aqueous phase is about 4.5×10 9 CFU / mL; S2.3: Add Span 80 to liquid paraffin at a ratio of 1 g:45 mL, stir and dissolve, then add the above-mentioned aqueous phase, and homogenize and emulsify at 2000 rpm for 15 min to obtain an emulsion, where the volume ratio of the aqueous phase to liquid paraffin is 1:2; S2.4: Add 0.1 mol / L calcium chloride solution to the above-mentioned emulsion, heat and stir at 37 °C for crosslinking for 25 min, then centrifuge at 4 °C, discard the supernatant, wash with sterile petroleum ether and sterile PBS buffer, and then freeze-dry to obtain the Bifidobacterium complex bacterium agent, where the volume ratio of the calcium chloride solution to the aqueous phase is 1:7; S3: Prepare the composite nanocarrier S3.1: Add bacterial cellulose to phosphate buffer with a pH of 5.5 at a ratio of 1 g:25 mL, and ultrasonically treat for 35 min to obtain a bacterial cellulose suspension; S3.2: Add 64% by volume sulfuric acid solution to the bacterial cellulose suspension, stir and hydrolyze at 45°C for 15 hours, then add 1 mol / L sodium hydroxide solution to adjust the pH to neutral. Centrifuge, wash and freeze-dry to obtain acidified bacterial cellulose, wherein the volume ratio of sulfuric acid solution to bacterial cellulose suspension is 1:2.5; S3.3: Add oleic acid to chloroform in a volume ratio of 1:9 mL. After thorough stirring, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide. Stir and activate in the dark for 1.5 h to obtain an activated oleic acid solution, wherein the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is 1:2, and the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to oleic acid is 1:3.8. S3.4: Disperse the acidified bacterial cellulose in dimethyl sulfoxide at a ratio of 1 g:18 mL, then add the activated oleic acid solution. Under nitrogen, stir and react at 65°C for 30 h. Precipitate with alcohol, centrifuge, wash, and freeze-dry to obtain a composite nanocarrier, wherein the mass ratio of bacterial cellulose to oleic acid is 1:2.1. S4: Preparation of extract compound additives S4.1: Dissolve the composite nanocarrier prepared in step S3.4 in dimethyl sulfoxide at a ratio of 1 g:25 mL to obtain a composite nanocarrier solution, then add the verbena extract solution and the Moringa leaf extract solution, stirring and mixing to obtain a mixed solution, wherein the volume ratios of the verbena extract solution and the Moringa leaf extract solution to the composite nanocarrier solution are 1:3.5 and 1:5, respectively, and the concentrations of the verbena extract solution and the Moringa leaf extract solution are both 50 mg / mL; The preparation method of the verbena extract solution is as follows: crushing verbena leaves at a ratio of 1 g to 25 mL, adding the crushed leaves to a 70% ethanol solution, heating and refluxing at 75° C. for 2.5 hours, repeating the extraction twice, combining the extracts, concentrating under reduced pressure until there is no ethanol, extracting the extracts twice with petroleum ether, extracting the aqueous phase with ethyl acetate three times, combining the ethyl acetate phases, and evaporating under reduced pressure to dryness to obtain the verbena extract, and then dissolving the verbena extract in a 50% ethanol solution to obtain the verbena extract solution; The preparation method of the Moringa leaf extract solution is as follows: adding Moringa leaf powder to 70% ethanol solution at a ratio of 1 g:15 mL, ultrasonically treating at 350 W and 45° C. for 35 min, centrifuging to obtain the supernatant, concentrating under reduced pressure to a paste, adding deionized water for re-dissolution, freeze-drying to obtain the Moringa leaf extract, and finally dissolving the Moringa leaf extract in 50% ethanol solution to obtain the Moringa leaf extract; S4.2: Dissolve Tween 80 in chloroform at a ratio of 1 g: 45 mL, then add the above-mentioned mixed solution accounting for 3 / 5 of the volume of chloroform, and homogenize and emulsify at 2500 rpm for 15 min to obtain a mixed emulsion; S4.3: Rotavaporize the above-mentioned mixed emulsion to remove chloroform, then filter through a 0.45 μm membrane, centrifuge, wash, and freeze-dry to obtain an extract composite additive; S5: Prepare compound feed After mixing and pulverizing corn, broken rice, soybean meal, and eggshell powder, add the above-mentioned extract composite additive, Bifidobacterium composite bacterium agent, xylooligosaccharide, compound microorganism, and spirulina powder, fully mix and granulate to obtain compound feed. Among them, by mass, the raw material composition of the compound feed is: 45 parts of corn, 15 parts of broken rice, 15 parts of soybean meal, 12 parts of eggshell powder, 15 parts of spirulina powder, 11 parts of xylooligosaccharide, 9 parts of extract composite additive, 4 parts of Bifidobacterium composite bacterium agent, and 3 parts of compound vitamin. Among them, the compound vitamin is composed of vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin C, vitamin D, and vitamin E in a mass ratio of 1.5: 4: 3.5: 1: 2.5: 1.1: 1.4.

[0025] Example 3 A preparation process of a compound feed for improving the disease resistance of breeding ducks, as Figure 1 shown, includes the following steps: S1: Prepare modified resistant starch acetate S1.1: Add resistant starch to deionized water at a ratio of 1 g: 20 mL, stir well to form a suspension, and cool it to 5 °C in an ice bath to obtain a resistant starch suspension; S1.2: Add 0.1 mol / L sodium hydroxide solution to the above-mentioned resistant starch suspension to adjust the pH to 10, stir and swell for 30 min, then add acetic anhydride, stir and react at 30 °C for 3 h, then add 0.1 mol / L hydrochloric acid solution to adjust the pH to neutral, and then collect the precipitate by centrifugation, wash, and vacuum-dry to obtain resistant starch acetate, where the mass ratio of acetic anhydride to resistant starch is 1.8: 1; S1.3: Suspend the above-mentioned resistant starch acetate in acetone at a ratio of 1 g: 30 mL, and add 1,1-carbonyldiimidazole, stir and activate at 35 °C for 12 h to obtain an activated resistant starch acetate solution, where the mass ratio of 1,1-carbonyldiimidazole to resistant starch acetate is 1: 5; S1.4: Dissolve concanavalin A in PBS buffer at a ratio of 1 g: 200 mL, then add it to the above-mentioned activated resistant starch acetate solution, stir and react at 5 °C for 16 h, then dialyze and purify and freeze-dry to obtain modified resistant starch acetate, where the mass ratio of concanavalin A to resistant starch acetate is 1: 105; S2: Prepare the Bifidobacterium complex bacterium agent S2.1: Inoculate the Bifidobacterium freeze-dried powder into the MRS culture medium, anaerobically culture at 37 °C for 48 h, then centrifuge at 4 °C, discard the supernatant, wash 3 times with sterile physiological saline, and then resuspend with sterile PBS buffer to obtain a Bifidobacterium suspension with a concentration of 10 10 CFU / mL; S2.2: Dissolve the modified resistant starch acetate prepared in step S1.4 in sterile PBS buffer at a ratio of 1 g: 30 mL, then add the above Bifidobacterium suspension, stir and mix evenly to obtain an aqueous phase, where the concentration of Bifidobacterium in the aqueous phase is about 5×10 9 CFU / mL; S2.3: Add Span 80 to liquid paraffin at a ratio of 1 g: 50 mL, stir and dissolve, then add the above aqueous phase, and homogenize and emulsify at 2500 rpm for 20 min to obtain an emulsion, where the volume ratio of the aqueous phase to liquid paraffin is 1:2; S2.4: Add 0.1 mol / L calcium chloride solution to the above emulsion, heat and stir at 37 °C for crosslinking for 30 min, then centrifuge at 5 °C and discard the supernatant, wash with sterile petroleum ether and sterile PBS buffer, and then freeze-dry to obtain the Bifidobacterium complex bacterium agent, where the volume ratio of the calcium chloride solution to the aqueous phase is 1:8; S3: Prepare the composite nanocarrier S3.1: Add bacterial cellulose to the phosphate buffer with a pH of 6 at a ratio of 1 g: 30 mL, and ultrasonically treat for 40 min to obtain a bacterial cellulose suspension; S3.2: Add a sulfuric acid solution with a volume fraction of 64% to the above bacterial cellulose suspension, stir and hydrolyze at 50 °C for 16 h, then add 1 mol / L sodium hydroxide solution to adjust the pH to neutral, and after centrifugal separation, washing and freeze-drying, obtain acidified bacterial cellulose, where the volume ratio of the sulfuric acid solution to the bacterial cellulose suspension is 1:3; S3.3: Add oleic acid to chloroform at a volume ratio of 1: 10 mL, stir and mix well, then add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and stir and activate in the dark for 2 h to obtain an activated oleic acid solution, where the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is 1:2, and the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to oleic acid is 1:4; S3.4: Disperse the above-mentioned acidified bacterial cellulose in dimethyl sulfoxide at a ratio of 1 g: 20 mL, then add the above-mentioned activated oleic acid solution. Under nitrogen protection, stir and react at 70 °C for 36 h, and obtain the composite nanocarrier through alcohol precipitation, centrifugal separation, washing and freeze-drying. Among them, the mass ratio of bacterial cellulose to oleic acid is 1: 2.2; S4: Prepare the extract composite additive S4.1: Dissolve the composite nanocarrier prepared in step S3.4 in dimethyl sulfoxide at a ratio of 1 g: 30 mL to obtain a composite nanocarrier solution, then add the verbena extract solution and the moringa leaf extract solution, and stir and mix evenly to obtain a mixed solution. Among them, the volume ratios of the verbena extract solution and the moringa leaf extract solution to the composite nanocarrier solution are 1: 4 and 1: 6 respectively, and the concentrations of both the verbena extract solution and the moringa leaf extract solution are 50 mg / mL; The preparation method of the verbena extract solution is as follows: Crush the verbena leaves at a ratio of 1 g: 30 mL and add them to a 70% ethanol solution, heat and reflux extract at 80 °C for 3 h, repeat 2 times, then combine the extract solutions, concentrate under reduced pressure until there is no ethanol, extract with petroleum ether 2 times, extract the aqueous phase with ethyl acetate 3 times, combine the ethyl acetate phases, evaporate to dryness under reduced pressure to obtain the verbena extract, and then dissolve the verbena extract in a 50% ethanol solution to obtain the verbena extract solution; The preparation method of the moringa leaf extract solution is as follows: Add moringa leaf powder to a 70% ethanol solution at a ratio of 1 g: 20 mL, ultrasonically treat at 400 W and 50 °C for 40 min, centrifuge to take the supernatant, concentrate under reduced pressure to a paste, then add deionized water to redissolve, freeze-dry to obtain the moringa leaf extract, and finally dissolve the moringa leaf extract in a 50% ethanol solution to obtain the moringa leaf extract; S4.2: Dissolve Tween 80 in chloroform at a ratio of 1 g: 50 mL, then add 3 / 5 of the above-mentioned mixed solution by volume of chloroform, and homogenize and emulsify at 3000 rpm for 20 min to obtain a mixed emulsion; S4.3: Rotate and evaporate the above-mentioned mixed emulsion to remove chloroform, then filter through a 0.45 μm membrane, centrifuge, wash and freeze-dry to obtain the extract composite additive; S5: Prepare the composite feed After mixing and pulverizing corn, broken rice, soybean meal, and eggshell powder, add the above extract composite additive, Bifidobacterium composite bacterium agent, xylo-oligosaccharide, composite microorganism, and spirulina powder, mix well and granulate to obtain a composite feed. Among them, by mass, the raw material composition of the composite feed is: 50 parts of corn, 20 parts of broken rice, 20 parts of soybean meal, 15 parts of eggshell powder, 20 parts of spirulina powder, 14 parts of xylo-oligosaccharide, 10 parts of extract composite additive, 5 parts of Bifidobacterium composite bacterium agent, and 4 parts of composite vitamin. Among them, the composite vitamin is composed of vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin C, vitamin D, and vitamin E in a mass ratio of 2:5:4:1:3:1.2:1.5.

[0026] Comparative Example 1 The difference between this Comparative Example 1 and Example 1 is that step S1 is removed, and the modified resistant starch acetate in step S2.2 is replaced with an equal amount of resistant starch acetate.

[0027] Comparative Example 2 The difference between this Comparative Example 2 and Example 1 is that step S1 is removed, and the modified resistant starch acetate in step S2.2 is replaced with an equal amount of resistant starch.

[0028] Comparative Example 3 The difference between this Comparative Example 3 and Example 1 is that step S3 is removed, and the composite nanocarrier in step S4 is replaced with an equal amount of acidified bacterial cellulose.

[0029] Comparative Example 4 The difference between this Comparative Example 4 and Example 1 is that the moringa leaf extract solution in step S4 is replaced with an equal amount of verbena extract solution.

[0030] Comparative Example 5 The difference between this Comparative Example 5 and Example 1 is that the verbena extract solution in step S4 is replaced with an equal amount of moringa leaf extract solution.

[0031] Test Example Test 1: Test the Bifidobacterium loading rate in the Bifidobacterium composite bacterium agent prepared in Examples 1-3 and Comparative Examples 1-2, and the results are shown in Table 1.

[0032] Table 1: Test results of Bifidobacterium loading rate

[0033] As shown in Table 1, when directly loading Bifidobacterium with resistant starch acetate or resistant starch in Comparative Example 1 and Comparative Example 2, the loading rates of Bifidobacterium were lower than those in Example 1. Thus, it can be seen that esterifying resistant starch and modifying resistant starch acetate with concanavalin A can improve the loading rate of resistant starch on Bifidobacterium, which is beneficial to better inhibiting the reproduction of harmful bacteria in the large intestine of breeding ducks, reducing the risk of intestinal inflammation, and further enhancing the immunity of breeding ducks and improving their disease resistance.

[0034] Test 2: The encapsulation rates of the total amount of verbena extract and moringa leaf extract in the extract composite additives prepared in Examples 1 - 3 and Comparative Example 3 were tested, and the results are shown in Table 2.

[0035] Table 2: Test results of the encapsulation rates of the total amount of verbena extract and moringa leaf extract

[0036] As shown in Table 2, in Comparative Example 3, without reacting with oleic acid and directly loading verbena extract and moringa leaf extract with acidified bacterial cellulose, the encapsulation rates of verbena extract and moringa leaf extract were much lower than those in Example 1. Thus, it can be seen that by first hydrolyzing and acidifying bacterial cellulose with sulfuric acid and activating oleic acid, then reacting acidified bacterial cellulose with activated oleic acid to form covalent bonds to prepare a composite nanocarrier, and then loading verbena extract and moringa leaf extract with the composite nanocarrier, the encapsulation rate of the composite nanocarrier on verbena extract and moringa leaf extract can be effectively improved, which is beneficial to improving the disease resistance efficacy of the extract composite additive and further improving the disease resistance of breeding ducks.

[0037] Test 3: Randomly select 600 white - feather parent - stock breeding ducks and divide them into 6 groups, with 100 ducks in each group. Each group was fed the composite feeds prepared in Examples 1 - 3 and Comparative Examples 4 - 5 according to the conventional breeding process of the farm. The remaining 1 group was the blank group, which was only fed with the conventional commercially available basic feed. After 4 weeks of feeding, 10 ducks were randomly selected from each group for wing - vein blood collection, with 5 mL collected from each duck. Then, it was centrifuged at 3000 r / min for 15 min, and the serum immunoglobulin (IgG, IgM, IgA) indexes were measured by the ELISA method, recorded and analyzed, and the average value was taken. The results are shown in Table 3.

[0038] Table 3: Test results of immunoglobulin indexes

[0039] As shown in Table 3, when only one of verbena extract and moringa leaf extract was used in Comparative Example 4 and Comparative Example 5, although the immune indexes of the breeding ducks fed with the prepared compound feed were higher than those of the blank group, they were lower than those of Example 1. Thus, it can be seen that verbena extract and moringa leaf extract can synergistically enhance the immunity of breeding ducks, thereby improving their disease resistance.

[0040] The above embodiments are only illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes completed by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A preparation process of a compound feed for improving the disease resistance of breeding ducks, characterized in that, It includes the following steps: S1: Prepare modified resistant starch acetate, S1.1: Add resistant starch into deionized water to form a suspension, and cool it in an ice bath to 3 - 5 °C to obtain a resistant starch suspension; S1.2: Add a sodium hydroxide solution to the above - mentioned resistant starch suspension to adjust the pH to 8 - 10, then add acetic anhydride, stir and react, and then adjust the pH to neutral to obtain resistant starch acetate; S1.3: Suspend the resistant starch acetate in acetone, and add 1,1 - carbonyldiimidazole, stir and activate at 25 - 35 °C for 10 - 12 h to obtain an activated resistant starch acetate solution; S1.4: Dissolve concanavalin A in PBS buffer solution, then add it to the activated resistant starch acetate solution, stir and react at 3 - 5 °C for 12 - 16 h, and then carry out dialysis purification and freeze - drying to obtain modified resistant starch acetate; S2: Prepare a Bifidobacterium complex agent, After activating the freeze - dried Bifidobacterium, mix it with the modified resistant starch acetate solution to make a Bifidobacterium complex agent; S3: Prepare a composite nanocarrier, After acidifying bacterial cellulose, react it with activated oleic acid to make a composite nanocarrier; S4: Prepare an extract composite additive, Load the composite nanocarrier with verbena extract and moringa leaf extract to make an extract composite additive; S5: Prepare a composite feed, Mix and crush corn, broken rice, soybean meal and eggshell powder, then add the above - mentioned extract composite additive, Bifidobacterium complex agent, xylo - oligosaccharide, composite microorganism and spirulina powder, fully mix and granulate to obtain a composite feed.

2. The preparation process of a compound feed for improving the disease resistance of breeding ducks according to claim 1, characterized in that, S2 specifically includes the following steps: S2.1: Inoculate the freeze-dried bifidobacterium powder into MRS medium, anaerobically culture it at 37 °C for 40 - 48 h, then centrifuge at 4 °C, discard the supernatant, wash and resuspend it with sterile normal saline to obtain a bifidobacterium suspension with a concentration of 10 10 CFU / mL; S2.2: After dissolving the modified resistant starch acetate prepared in step S1.4, add the above-mentioned Bifidobacterium suspension and stir to mix evenly to obtain an aqueous phase, wherein the concentration of Bifidobacterium in the aqueous phase is (4-5)×10 9 CFU / mL; S2.3: Add Span 80 to liquid paraffin at a ratio of 1 g:(40 - 50) mL, stir to dissolve and add the above - mentioned aqueous phase, homogenize and emulsify for 10 - 20 min to obtain an emulsion, where the volume ratio of the aqueous phase to liquid paraffin is 1:2; S2.4: Add 0.1 mol / L calcium chloride solution to the above - mentioned emulsion, heat and stir at 37 °C for cross - linking for 20 - 30 min, and then carry out centrifugation, discard the supernatant, wash and freeze - dry to obtain a Bifidobacterium complex agent, where the volume ratio of the calcium chloride solution to the aqueous phase is 1:(6 - 8).

3. The preparation process of a compound feed for improving the disease resistance of breeding ducks according to claim 2, characterized in that, S3 specifically includes the following steps: S3.1: Add bacterial cellulose to a phosphate buffer solution with a pH of 5 - 6 at a ratio of 1 g:(20 - 30) mL, and ultrasonically treat for 30 - 40 min to obtain a bacterial cellulose suspension; S3.2: Add a sulfuric acid solution to the above - mentioned bacterial cellulose suspension, stir and hydrolyze, and then adjust the pH to neutral to make acidified bacterial cellulose, where the volume ratio of the sulfuric acid solution to the bacterial cellulose suspension is 1:(2 - 3); S3.3: Add oleic acid to chloroform at a volume ratio of 1:(8 - 10) mL, fully stir and mix, then add 1 - ethyl - 3 - (3 - dimethylaminopropyl)carbodiimide and N - hydroxysuccinimide with a mass ratio of 1:2, stir and activate in the dark for 1 - 2 h to obtain an activated oleic acid solution; S3.4: Disperse the acidified bacterial cellulose in dimethyl sulfoxide, then add the activated oleic acid solution. Under nitrogen protection, stir and react at 60 - 70 °C for 24 - 36 h. After alcohol precipitation, centrifugal separation, washing and freeze-drying, a composite nanocarrier is obtained, wherein the mass ratio of bacterial cellulose to oleic acid is 1:(2 - 2.2).

4. The preparation process of a compound feed for improving the disease resistance of breeding ducks according to claim 3, characterized in that, S4 specifically includes the following steps: S4.1: Dissolve the composite nanocarrier prepared in step S3.4 in dimethyl sulfoxide at a ratio of 1 g:(20 - 30) mL to obtain a composite nanocarrier solution. Then add the verbena extract solution and the moringa leaf extract solution, and stir and mix evenly to obtain a mixed solution; S4.2: Dissolve Tween 80 in chloroform at a ratio of 1 g:(40 - 50) mL, and then add 3 / 5 of the above mixed solution by volume of chloroform. Homogenize and emulsify for 10 - 20 min to obtain a mixed emulsion; S4.3: Rotavaporize the mixed emulsion to remove chloroform, and then through membrane filtration, centrifugal separation, washing and freeze-drying to obtain the extract composite additive.

5. The preparation process of a compound feed for improving the disease resistance of breeding ducks according to claim 1, characterized in that, The mass ratio of 1,1-carbonyldiimidazole to resistant starch acetate is 1:(4 - 5).

6. The preparation process of a compound feed for improving the disease resistance of breeding ducks according to claim 1, characterized in that, The mass ratio of acetic anhydride to resistant starch is (1.6 - 1.8):1, and the mass ratio of concanavalin A to resistant starch acetate is 1:(95 - 105).

7. The preparation process of a compound feed for improving the disease resistance of breeding ducks according to claim 3, characterized in that, The mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to oleic acid is 1:(3.6 - 4).

8. The preparation process of a compound feed for improving the disease resistance of breeding ducks according to claim 4, characterized in that, The volume ratio of the verbena extract solution to the composite nanocarrier solution is 1:(3 - 4), and the volume ratio of the moringa leaf extract solution to the composite nanocarrier solution is 1:(4 - 6), and the concentrations of both the verbena extract solution and the moringa leaf extract solution are 50 mg / mL.

9. The preparation process of a compound feed for improving the disease resistance of breeding ducks according to claim 1, characterized in that, By mass, the raw material composition of the compound feed is: 40 - 50 parts of corn, 10 - 20 parts of broken rice, 10 - 20 parts of soybean meal, 10 - 15 parts of eggshell powder, 10 - 20 parts of spirulina powder, 8 - 14 parts of xylo-oligosaccharide, 8 - 10 parts of the extract composite additive, 3 - 5 parts of the bifidobacterium complex bacterium agent and 2 - 4 parts of the compound vitamin.

10. A compound feed for improving the disease resistance of breeding ducks, characterized in that, It is prepared by the preparation process of a compound feed for improving the disease resistance of breeding ducks described in any one of claims 1 - 9.

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