An antibiotic-substituted antibacterial peptide compound feed additive and a preparation method thereof

Abstract

The present application relates to the technical field of feed, and discloses an antibiotic-substituted antibacterial peptide compound feed additive and a preparation method thereof.The method separates Bacillus coagulans fermentation liquor into an antibacterial peptide component and a bacterial body component, concentrates the antibacterial peptide through membrane separation, and then performs lipidization embedding, the bacterial body is dried to form a live bacteria powder through protection, and then is compounded with carriers such as silicon dioxide and medical stone powder.The obtained product has low antibacterial peptide release in simulated gastric juice, sufficient antibacterial peptide release in simulated intestinal juice containing bile salts and pancreatin, good live bacteria stability after granulation, and sustained antibacterial effect in vitro, and can be used for reducing the use of antibiotics in pig and poultry feed.

Description

Technical Field

[0001] This invention relates to the field of feed technology, and in particular to an antimicrobial peptide compound feed additive that can replace antibiotics and its preparation method. Background Technology

[0002] With the increasing demands for antibiotic reduction and restriction in livestock and poultry farming, developing feed additives that can replace or reduce antibiotic use has become an important direction for the feed industry. Antimicrobial peptides have the characteristics of broad antibacterial spectrum, low resistance to induction, biodegradability, and low residues. Probiotics such as Bacillus coagulans have advantages such as good tolerance and the ability to regulate intestinal microecology. Therefore, preparing compound feed additives by combining antimicrobial peptides and probiotics is a promising technical approach for antibiotic-free farming.

[0003] CN121405530A discloses a method for preparing an integrated fertilizer-pesticide rich in antimicrobial peptides. The method employs a sodium alginate-chitosan biphase encapsulation core and a low-temperature spray coating onto a fertilizer substrate to form a shell, thus obtaining an integrated fertilizer-pesticide rich in antimicrobial peptides. This solution primarily targets agricultural fertilizer and plant disease control scenarios. Its core lies in the core-shell structure of the fertilizer-pesticide and the coating onto the fertilizer substrate. However, it does not address considerations for the gastrointestinal environment of livestock and poultry, the high temperatures of feed pelleting, intestinal release in pigs and poultry, or the stability of live bacteria in compound feed additives.

[0004] CN122012557A discloses a method for constructing native probiotics for livestock and poultry and their application in antibiotic-free feed. This method involves isolating native strains from the digestive tract of livestock and poultry, adaptively acclimating them, constructing a digestive tract environment-responsive antimicrobial peptide expression system, and protecting them using sodium alginate-chitosan bilayer microcapsules. This approach focuses on strain screening, genetic modification, and in vivo expression of antimicrobial peptides in probiotics. However, its process relies on genetic engineering, and the primary object of protection is the live bacteria themselves. It does not separate the antimicrobial peptide components from the bacterial components in the fermentation broth for utilization, nor does it address the targeted protection and release of exogenous antimicrobial peptides in feed pelleting, gastric acid environments, and intestinal bile salt environments.

[0005] CN122096303A discloses a method for preparing and applying a piglet anti-stress and immunity-enhancing agent. The agent is a multi-component compound containing sodium iron chlorophyll, sodium selenate, polysaccharides, functional oligosaccharides, antimicrobial peptides, and probiotics. It is prepared using processes such as gradient sugar inclusion, low-temperature microencapsulation, and adsorption-coupling of microbial peptides. The main purpose of this method is to replenish blood, resist stress, and enhance immunity. The composition is complex, with antimicrobial peptides and probiotics being only functional modules within the compound.

[0006] Therefore, although existing technologies have involved the application of antimicrobial peptides, probiotics, microencapsulation, or antibiotic-free feed, problems still exist, such as premature release or loss of activity of antimicrobial peptides in the acidic environment of the stomach, insufficient release after entering the intestines, and insufficient stability of live bacteria in compound products after feed pelleting. There is an urgent need to develop a method for preparing antimicrobial peptide compound feed additives suitable for pig and poultry feeds, which can combine gastric juice protection, intestinal juice release, high-temperature resistance to pelleting, sustained antibacterial activity, and probiotic intestinal regulation. Summary of the Invention

[0007] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is that the antimicrobial peptides in the existing antibiotic alternative feed additives are prone to premature release or loss of activity in the gastric acid environment, insufficient release in the intestinal fluid environment, insufficient synergistic utilization of antimicrobial peptides and probiotics, and insufficient stability of live bacteria after feed pelleting, resulting in insufficient synergistic utilization of antimicrobial peptides and probiotics, leading to insufficient sustained antibacterial ability and application stability of the product.

[0008] To achieve the above objectives, the present invention provides a method for preparing an antimicrobial peptide compound feed additive that can replace antibiotics, comprising the following steps: 1) Inoculate Bacillus coagulans into seed culture medium and culture at 35-39℃ and 150-220 r / min for 10-16 h to obtain seed liquid; inoculate the seed liquid into fermentation medium at 3-8% of the fermentation medium volume, and ferment at 35-39℃, aeration rate of 0.5-1.2 vvm, and stirring speed of 150-300 r / min for 20-30 h, then raise the temperature to 40-45℃ and continue to culture for 4-10 h to obtain fermentation broth containing antimicrobial peptides and Bacillus coagulans spores; 2) The fermentation broth obtained in step 1) is subjected to solid-liquid separation, and the supernatant and cell precipitate are collected separately; the supernatant is then filtered sequentially through a microporous membrane, an ultrafiltration membrane, and a nanofiltration membrane to concentrate the supernatant to obtain an antimicrobial peptide concentrate. 3) Take the antimicrobial peptide concentrate obtained in step 2), add maltodextrin and trehalose, stir evenly to obtain an antimicrobial peptide core material solution; separately take hydrogenated vegetable oil, glyceryl monostearate and soybean lecithin, melt mix them at 65-75℃ to obtain a lipid wall material; add the antimicrobial peptide core material solution to the lipid wall material, and granulate by shear emulsification and fluidized bed spray condensation to obtain lipid-encapsulated antimicrobial peptide powder; wherein, the material temperature is maintained at 60-75℃ during shear emulsification, the inlet air temperature of fluidized bed spray condensation granulation is 10-25℃, and the outlet air temperature is 25-35℃; Alternatively, take the antimicrobial peptide concentrate obtained in step 2), add maltodextrin, trehalose, sodium alginate, and low-ester pectin, and adjust the pH to 5.5-6.2 to obtain an antimicrobial peptide polysaccharide core material solution; add a calcium chloride aqueous solution with a mass fraction of 5.0-10.0% under shear conditions to allow sodium alginate and low-ester pectin to undergo calcium ion cross-linking to obtain an antimicrobial peptide polysaccharide gel core material solution; separately take hydrogenated vegetable oil, glyceryl monostearate, soybean lecithin, and glyceryl tribanilate, and melt-mix them at 65-75℃ to obtain a lipid wall material; add the antimicrobial peptide polysaccharide gel core material solution to the lipid wall material, and perform shear emulsification and fluidized bed spray condensation granulation to obtain polysaccharide gel-lipidized encapsulated antimicrobial peptide powder; wherein, the material temperature is maintained at 60-75℃ during shear emulsification, the inlet air temperature of fluidized bed spray condensation granulation is 10-25℃, and the outlet air temperature is 25-35℃; 4) Resuspend the bacterial precipitate collected in step 2) in sterile water, add trehalose and maltodextrin as protective agents, mix well to obtain a bacterial cell protective suspension; subject the bacterial cell protective suspension to low-temperature spray drying to obtain Bacillus coagulans live bacterial powder; 5) Take maifan stone powder, silicon dioxide, yeast cell wall powder and calcium stearate, grind and sieve them separately, dry them at 70-90℃ for 1-3 hours, and cool them to room temperature for later use. 6) Add the lipid-encapsulated antimicrobial peptide powder or polysaccharide gel-lipo-encapsulated antimicrobial peptide powder obtained in step 3) and the dried silica obtained in step 5) into a mixer and mix for 5-15 minutes to allow the lipid-encapsulated antimicrobial peptide powder to be adsorbed and dispersed by silica; then add maifanite powder, yeast cell wall powder and calcium stearate, and continue mixing for 10-20 minutes; after the temperature of the mixture is below 35℃, add the Bacillus coagulans live bacteria powder obtained in step 4), mix at low speed for 8-15 minutes, and sieve to obtain the antibiotic-replacing antimicrobial peptide compound feed additive.

[0009] Preferably, in step 1), the Bacillus coagulans is Bacillus coagulans BC66.

[0010] Preferably, in step 2), the pore size of the microporous filter membrane is 0.22-0.45 μm, the molecular weight cutoff of the ultrafiltration membrane is 5-15 kDa, the molecular weight cutoff of the nanofiltration membrane is 0.5-2 kDa, and the solids mass fraction of the antimicrobial peptide concentrate is 20.0-30.0%.

[0011] Preferably, in step 3) the preparation of lipo-encapsulated antimicrobial peptide powder, based on 30 parts by weight of dry matter of the antimicrobial peptide concentrate, the maltodextrin is 5-15 parts by weight, the trehalose is 1-5 parts by weight, the hydrogenated vegetable oil is 20-35 parts by weight, the glyceryl monostearate is 3-10 parts by weight, and the soybean lecithin is 2-8 parts by weight.

[0012] Preferably, in step 3), the polysaccharide gel-lipidized encapsulated antimicrobial peptide powder comprises, based on 30 parts by weight of the dry matter of the antimicrobial peptide concentrate, 5-15 parts by weight of maltodextrin, 1-5 parts by weight of trehalose, 1-5 parts by weight of sodium alginate, 0.5-3 parts by weight of low-ester pectin, 5-20 parts by weight of a 5.0-10.0% calcium chloride aqueous solution, 20-30 parts by weight of hydrogenated vegetable oil, 3-10 parts by weight of glyceryl monostearate, 3-8 parts by weight of soybean lecithin, and 2-8 parts by weight of glyceryl tartrate.

[0013] Preferably, in step 4), based on the total mass of the bacterial cell protective suspension, the trehalose mass fraction is 2.0-5.0%, and the maltodextrin mass fraction is 5.0-12.0%; the viable count of the Bacillus coagulans live bacteria powder is 1.0 × 10⁻⁶. 11 -2.0×10 11 CFU / g.

[0014] Preferably, in step 6), the lipopolymerized antimicrobial peptide powder or polysaccharide gel-lipopolymerized antimicrobial peptide powder comprises 40-100 parts by weight, silica comprises 50-120 parts by weight, maifanite powder comprises 750-850 parts by weight, yeast cell wall powder comprises 5-20 parts by weight, calcium stearate comprises 2-10 parts by weight, and Bacillus coagulans live bacteria powder comprises 10-40 parts by weight.

[0015] When the antimicrobial peptide compound feed additive of this invention is used in compound feed as an alternative to antibiotics, the addition amount in pig feed is 500-1000g / T, and the addition amount in poultry feed is 300-500g / T. During the weaning, regrouping, feed change, transportation or high temperature stress stages of animals, the staged addition amount is 1000-1500g / T. Before use, it should be premixed with a small amount of carrier or premix before being gradually added to the compound feed.

[0016] The beneficial effects of this invention are: 1) This invention separates the Bacillus coagulans fermentation broth into antimicrobial peptide components and bacterial cell components. The supernatant is concentrated through microfiltration, ultrafiltration, and nanofiltration to obtain an antimicrobial peptide concentrate. The bacterial cell precipitate is then protected and dried to produce Bacillus coagulans live bacterial powder, achieving simultaneous utilization of the antimicrobial active components and probiotic components in the fermentation product. The resulting compound feed additive combines the rapid antimicrobial effect of antimicrobial peptides with the intestinal regulatory effect of Bacillus coagulans, which is beneficial for reducing the use of antibiotic additives in pig and poultry feed.

[0017] 2) This invention protects antimicrobial peptides through lipid encapsulation, reducing premature release and activity loss in the acidic gastric environment. In particular, the use of sodium alginate, low-ester pectin, and calcium ions to construct the polysaccharide gel core, combined with a lipid wall material containing tributyrate, helps reduce premature release of antimicrobial peptides in the simulated gastric juice stage and increases their release in the simulated intestinal juice stage containing bile salts and pancreatic enzymes, thereby improving the utilization efficiency of antimicrobial peptides in the gastrointestinal environment.

[0018] 3) This invention first uses silica to pre-adsorb and disperse the lipid-encapsulated antimicrobial peptide powder, then performs low-temperature compounding with maifanite powder, yeast cell wall powder, calcium stearate, and live Bacillus coagulans powder. This improves the dispersion uniformity of the antimicrobial peptide powder in the mineral carrier, reduces the risk of material hygroscopic agglomeration, and avoids damage to the live bacteria during high-temperature mixing. The resulting product is a powdered compound feed additive, which is convenient for step-by-step premixing with compound feed and large-scale application. Detailed Implementation

[0019] The parameters and sources of some raw materials in the examples are as follows: Bacillus coagulans BC66, 3×10 11 CFU / g is sourced from Weikang Agriculture.

[0020] The seed culture medium is prepared as follows: Add approximately 800L of production water to the seed tank, start stirring, and then add 20.00kg of glucose, 10.00kg of peptone, 8.00kg of yeast extract, and 5.00kg of sodium chloride in sequence, and stir evenly; adjust the pH to 7.0±0.1 using 1mol / L sodium hydroxide aqueous solution or 1mol / L hydrochloric acid, then add production water to make up the volume to 1000L, sterilize at 121℃ for 20min, and cool to 37℃ to obtain the seed culture medium.

[0021] The fermentation medium is prepared as follows: Add approximately 800L of production water to the fermenter, start stirring, and then add 25.00kg of glucose, 30.00kg of soybean meal powder, 10.00kg of yeast extract, 1.50kg of potassium dihydrogen phosphate, 0.50kg of magnesium sulfate, and 2.00kg of calcium carbonate in sequence, stirring to disperse evenly; adjust the pH to 6.8±0.1 using 1mol / L sodium hydroxide aqueous solution or 1mol / L hydrochloric acid, then add production water to bring the volume to 1000L, sterilize at 121℃ for 20min, and cool to 37℃ to obtain the fermentation medium.

[0022] Yeast extract powder, total nitrogen ≥8.0%; amino nitrogen ≥3.0%.

[0023] Soybean meal powder, 60 mesh, crude protein ≥43.0%.

[0024] Maltodextrin, DE value 10-15.

[0025] Sodium alginate, 1% aqueous solution viscosity 100-300 mPa·s (25℃); G / M ratio 0.8-1.5.

[0026] Low-ester pectin, degree of esterification 20-40%; galacturonic acid content ≥65.0%.

[0027] Hydrogenated vegetable oil, melting point 55-65℃; acid value ≤1.0mgKOH / g; peroxide value ≤0.10g / 100g.

[0028] Glyceryl monostearate, with a monoglyceride content ≥90.0%; acid value ≤3.0mgKOH / g; melting point 55-65℃.

[0029] Soybean lecithin: acetone-insoluble matter ≥60.0%; acid value ≤30mgKOH / g.

[0030] Tributyric acid glyceride, acid value ≤1.0mgKOH / g.

[0031] Silica, specific surface area 150-250m² 2 / g; oil absorption value ≥200g / 100g.

[0032] Maifan stone powder, pulverized and passed through an 80-mesh sieve.

[0033] Yeast cell wall powder, β-glucan ≥20.0%; mannan oligosaccharide ≥18.0%.

[0034] Example 1

[0035] A method for preparing an antimicrobial peptide compound feed additive as an alternative to antibiotics includes the following steps: 1) Take Bacillus coagulans BC66, inoculate it into seed culture medium, and culture it at 37℃ and 180 r / min for 12 h to obtain seed liquid; inoculate the seed liquid into fermentation medium at a 5% inoculation rate, that is, inoculate 5 L of seed liquid into every 100 L of fermentation medium, and ferment it at 37℃, aeration rate of 0.8 vvm and stirring speed of 200 r / min for 24 h, and then raise the temperature to 42℃ and continue to culture for 6 h to obtain fermentation broth containing antimicrobial peptides and Bacillus coagulans spores; 2) Centrifuge the fermentation broth obtained in step 1) at 6000 r / min for 15 min, and collect the supernatant and cell precipitate respectively; filter the supernatant sequentially through a 0.45 μm microporous membrane and a 10 kDa ultrafiltration membrane, and collect the permeate below 10 kDa; then concentrate using a 1 kDa nanofiltration membrane, and collect the retentate above 1 kDa to obtain the antimicrobial peptide concentrate; control the temperature during the concentration process to not exceed 45℃, and the solids content of the obtained antimicrobial peptide concentrate is 25.0%; 3) Take 120.00 kg of the antimicrobial peptide concentrate obtained in step 2), the solid content of the antimicrobial peptide concentrate is 25.0%, which is equivalent to 30.00 kg of dry matter. Add 8.00 kg of maltodextrin and 2.00 kg of trehalose, and stir evenly to obtain the core material liquid. Separately take 30.00 kg of hydrogenated vegetable oil, 6.00 kg of glyceryl monostearate and 4.00 kg of soybean lecithin, and melt mix them at 70°C to obtain the lipid wall material. Slowly add the core material liquid to the lipid wall material, control the material temperature at 70°C, and emulsify it at high speed at 6000 r / min for 8 min. Then, use fluidized bed spray condensation granulation, control the inlet air temperature at 20°C and the outlet air temperature at 30°C to obtain the lipid-encapsulated antimicrobial peptide powder for later use. 4) Resuspend the bacterial precipitate collected in step 2) in sterile water, add trehalose and maltodextrin as preservatives, and mix thoroughly to obtain a bacterial cell preservative suspension; based on the total mass of the bacterial cell preservative suspension, the mass fraction of trehalose is 3.0%, and the mass fraction of maltodextrin is 8.0%; subject the bacterial cell preservative suspension to low-temperature spray drying, controlling the inlet air temperature to 90℃ and the outlet air temperature to 45℃, to obtain Bacillus coagulans live bacterial powder, the viable count of which is 1.2 × 10⁻⁶. 11 CFU / g; 5) Take 800.00 kg of maifan stone powder, 80.00 kg of silicon dioxide, 10.00 kg of yeast cell wall powder and 5.00 kg of calcium stearate, pulverize them separately and pass them through an 80-mesh sieve, dry them at 80℃ for 2 hours, and cool them to room temperature for later use. 6) Take 80.00 kg of the lipid-encapsulated antimicrobial peptide powder obtained in step 3) and 80.00 kg of the dried silica obtained in step 5) and add them to a mixer. Mix for 10 min to allow the lipid-encapsulated antimicrobial peptide powder to be uniformly adsorbed and dispersed by the silica. Then add 800.00 kg of maifanite powder, 10.00 kg of yeast cell wall powder and 5.00 kg of calcium stearate, and continue mixing for 15 min. After the temperature of the mixture is lower than 35℃, add 25.00 kg of Bacillus coagulans live bacteria powder obtained in step 4), mix at low speed for 12 min, and pass through a 40-mesh sieve to obtain an antibiotic-replacing antimicrobial peptide compound feed additive.

[0036] Example 2

[0037] A method for preparing an antimicrobial peptide compound feed additive as an alternative to antibiotics includes the following steps: 1) Take Bacillus coagulans BC66, inoculate it into seed culture medium, and culture it at 37℃ and 180 r / min for 12 h to obtain seed liquid; inoculate the seed liquid into fermentation medium at a 5% inoculation rate, that is, inoculate 5 L of seed liquid into every 100 L of fermentation medium, and ferment it at 37℃, aeration rate of 0.8 vvm and stirring speed of 200 r / min for 24 h, and then raise the temperature to 42℃ and continue to culture for 6 h to obtain fermentation broth containing antimicrobial peptides and Bacillus coagulans spores; 2) Centrifuge the fermentation broth obtained in step 1) at 6000 r / min for 15 min, and collect the supernatant and cell precipitate respectively; filter the supernatant sequentially through a 0.45 μm microporous membrane and a 10 kDa ultrafiltration membrane, and collect the permeate below 10 kDa; then concentrate using a 1 kDa nanofiltration membrane, and collect the retentate above 1 kDa to obtain the antimicrobial peptide concentrate; control the temperature during the concentration process to not exceed 45℃, and the solids content of the obtained antimicrobial peptide concentrate is 25.0%; 3) Take 120.00 kg of the antimicrobial peptide concentrate obtained in step 2), the solid content of the antimicrobial peptide concentrate is 25.0%, which is equivalent to 30.00 kg of dry matter. Add 8.00 kg of maltodextrin, 2.00 kg of trehalose, 2.50 kg of sodium alginate and 1.50 kg of low-ester pectin, stir until evenly dispersed, and adjust the pH to 5.8 ± 0.1 with 1 mol / L citric acid aqueous solution to obtain the antimicrobial peptide polysaccharide core material solution. Under the shearing condition of 1500 r / min, slowly add 10.00 kg of calcium chloride aqueous solution with a mass fraction of 8.0% to the antimicrobial peptide polysaccharide core material solution, and continue shearing and dispersing for 20 min to allow the sodium alginate and low-ester pectin to undergo calcium ion cross-linking to obtain the antimicrobial peptide polysaccharide gel core material solution. Separately, 24.00 kg of hydrogenated vegetable oil, 6.00 kg of glyceryl monostearate, 5.00 kg of soybean lecithin, and 5.00 kg of glyceryl tribaniate were melt-mixed at 70°C to obtain a lipid wall material. The antimicrobial peptide polysaccharide gel core material liquid was slowly added to the lipid wall material, and the material temperature was controlled at 70°C. High-speed shear emulsification was carried out at 6000 r / min for 8 min. Subsequently, fluidized bed spray condensation granulation was performed, and the inlet air temperature was controlled at 20°C and the outlet air temperature at 30°C to obtain polysaccharide gel-lipidized embedded antimicrobial peptide powder for later use. 4) Resuspend the bacterial precipitate collected in step 2) in sterile water, add trehalose and maltodextrin as preservatives, and mix thoroughly to obtain a bacterial cell preservative suspension; based on the total mass of the bacterial cell preservative suspension, the mass fraction of trehalose is 3.0%, and the mass fraction of maltodextrin is 8.0%; subject the bacterial cell preservative suspension to low-temperature spray drying, controlling the inlet air temperature to 90℃ and the outlet air temperature to 45℃, to obtain Bacillus coagulans live bacterial powder, the viable count of which is 1.2 × 10⁻⁶. 11 CFU / g; 5) Take 800.00 kg of maifan stone powder, 80.00 kg of silicon dioxide, 10.00 kg of yeast cell wall powder and 5.00 kg of calcium stearate, pulverize them separately and pass them through an 80-mesh sieve, dry them at 80℃ for 2 hours, and cool them to room temperature for later use. 6) Take 80.00 kg of the polysaccharide gel-lipoylation encapsulated antimicrobial peptide powder obtained in step 3) and 80.00 kg of the dried silica obtained in step 5) and add them to a mixer. Mix for 10 min to allow the polysaccharide gel-lipoylation encapsulated antimicrobial peptide powder to be uniformly adsorbed and dispersed by the silica. Then add 800.00 kg of maifanite powder, 10.00 kg of yeast cell wall powder and 5.00 kg of calcium stearate, and continue mixing for 15 min. After the temperature of the mixture is lower than 35℃, add 25.00 kg of Bacillus coagulans live bacteria powder obtained in step 4), mix at low speed for 12 min, and pass through a 40-mesh sieve to obtain an antibiotic-replacing antimicrobial peptide compound feed additive.

[0038] Comparative Example 1 A method for preparing an antimicrobial peptide compound feed additive as an alternative to antibiotics includes the following steps: 1) Take Bacillus coagulans BC66, inoculate it into seed culture medium, and culture it at 37℃ and 180 r / min for 12 h to obtain seed liquid; inoculate the seed liquid into fermentation medium at a 5% inoculation rate, that is, inoculate 5 L of seed liquid into every 100 L of fermentation medium, and ferment it at 37℃, aeration rate of 0.8 vvm and stirring speed of 200 r / min for 24 h, and then raise the temperature to 42℃ and continue to culture for 6 h to obtain fermentation broth containing antimicrobial peptides and Bacillus coagulans spores; 2) Centrifuge the fermentation broth obtained in step 1) at 6000 r / min for 15 min, and collect the supernatant and cell precipitate respectively; filter the supernatant sequentially through a 0.45 μm microporous membrane and a 10 kDa ultrafiltration membrane, and collect the permeate below 10 kDa; then concentrate using a 1 kDa nanofiltration membrane, and collect the retentate above 1 kDa to obtain the antimicrobial peptide concentrate; control the temperature during the concentration process to not exceed 45℃, and the solids content of the obtained antimicrobial peptide concentrate is 25.0%; 3) Take 120.00 kg of the antimicrobial peptide concentrate obtained in step 2), the solid content of the antimicrobial peptide concentrate is 25.0%, which is equivalent to 30.00 kg of dry matter. Add 8.00 kg of maltodextrin and 2.00 kg of trehalose, and stir evenly to obtain an antimicrobial peptide protective solution. Spray dry the antimicrobial peptide protective solution, controlling the inlet air temperature to 90℃ and the outlet air temperature to 45℃, to obtain 42.46 kg of ordinary dried antimicrobial peptide powder for later use. 4) Resuspend the bacterial precipitate collected in step 2) in sterile water, add trehalose and maltodextrin as preservatives, and mix thoroughly to obtain a bacterial cell preservative suspension; based on the total mass of the bacterial cell preservative suspension, the mass fraction of trehalose is 3.0%, and the mass fraction of maltodextrin is 8.0%; subject the bacterial cell preservative suspension to low-temperature spray drying, controlling the inlet air temperature to 90℃ and the outlet air temperature to 45℃, to obtain Bacillus coagulans live bacterial powder, the viable count of which is 1.2 × 10⁻⁶. 11 CFU / g; 5) Take 837.54 kg of maifan stone powder, 80.00 kg of silicon dioxide, 10.00 kg of yeast cell wall powder and 5.00 kg of calcium stearate, pulverize them separately and pass them through an 80-mesh sieve, dry them at 80℃ for 2 hours, and cool them to room temperature for later use. 6) Take 42.46 kg of the ordinary dried antimicrobial peptide powder obtained in step 3) and 80.00 kg of the dried silica obtained in step 5) and add them to the mixer. Mix for 10 min to allow the ordinary dried antimicrobial peptide powder to be uniformly adsorbed and dispersed by the silica. Then add 837.54 kg of maifanite powder, 10.00 kg of yeast cell wall powder and 5.00 kg of calcium stearate, and continue mixing for 15 min. After the temperature of the mixture is lower than 35℃, add 25.00 kg of Bacillus coagulans live bacteria powder obtained in step 4), mix at low speed for 12 min, and pass through a 40-mesh sieve to obtain the compound feed additive of Comparative Example 1.

[0039] The main difference between Comparative Example 1 and Example 1 is that the antimicrobial peptides in Comparative Example 1 were not encapsulated with lipid wall material, but were simply spray-dried using maltodextrin and trehalose.

[0040] Comparative Example 2 A method for preparing an antimicrobial peptide compound feed additive as an alternative to antibiotics includes the following steps: 1) Take Bacillus coagulans BC66, inoculate it into seed culture medium, and culture it at 37℃ and 180 r / min for 12 h to obtain seed liquid; inoculate the seed liquid into fermentation medium at a 5% inoculation rate, that is, inoculate 5 L of seed liquid into every 100 L of fermentation medium, and ferment it at 37℃, aeration rate of 0.8 vvm and stirring speed of 200 r / min for 24 h, and then raise the temperature to 42℃ and continue to culture for 6 h to obtain fermentation broth containing antimicrobial peptides and Bacillus coagulans spores; 2) Centrifuge the fermentation broth obtained in step 1) at 6000 r / min for 15 min, and collect the supernatant and cell precipitate respectively; filter the supernatant sequentially through a 0.45 μm microporous membrane and a 10 kDa ultrafiltration membrane, and collect the permeate below 10 kDa; then concentrate using a 1 kDa nanofiltration membrane, and collect the retentate above 1 kDa to obtain the antimicrobial peptide concentrate; control the temperature during the concentration process to not exceed 45℃, and the solids content of the obtained antimicrobial peptide concentrate is 25.0%; 3) Take 120.00 kg of the antimicrobial peptide concentrate obtained in step 2), the solids content of the antimicrobial peptide concentrate is 25.0%, equivalent to 30.00 kg of dry matter, add 12.80 kg of maltodextrin, 2.00 kg of trehalose and 9.20 kg of purified water, stir until evenly dispersed, and adjust the pH to 5.8 ± 0.1 with 1 mol / L citric acid aqueous solution to obtain a non-gel-type antimicrobial peptide core material solution; separately take 24.00 kg of hydrogenated vegetable oil and glyceryl monostearate... 6.00 kg of oleic acid ester, 5.00 kg of soybean lecithin, and 5.00 kg of tributyric acid ester were melt-mixed at 70°C to obtain a lipid wall material. The non-gel-type antimicrobial peptide core material liquid was slowly added to the lipid wall material, and the material temperature was controlled at 70°C. The mixture was then emulsified by high-speed shearing at 6000 r / min for 8 min. Subsequently, fluidized bed spray condensation granulation was performed, and the inlet air temperature was controlled at 20°C and the outlet air temperature at 30°C to obtain non-gel-type lipid-encapsulated antimicrobial peptide powder for later use. 4) Resuspend the bacterial precipitate collected in step 2) in sterile water, add trehalose and maltodextrin as preservatives, and mix thoroughly to obtain a bacterial cell preservative suspension; based on the total mass of the bacterial cell preservative suspension, the mass fraction of trehalose is 3.0%, and the mass fraction of maltodextrin is 8.0%; subject the bacterial cell preservative suspension to low-temperature spray drying, controlling the inlet air temperature to 90℃ and the outlet air temperature to 45℃, to obtain Bacillus coagulans live bacterial powder, the viable count of which is 1.2 × 10⁻⁶. 11 CFU / g; 5) Take 800.00 kg of maifan stone powder, 80.00 kg of silicon dioxide, 10.00 kg of yeast cell wall powder and 5.00 kg of calcium stearate, pulverize them separately and pass them through an 80-mesh sieve, dry them at 80℃ for 2 hours, and cool them to room temperature for later use. 6) Take 80.00 kg of the non-gel-type lipid-encapsulated antimicrobial peptide powder obtained in step 3) and 80.00 kg of the dried silica obtained in step 5) and add them to a mixer. Mix for 10 min to allow the non-gel-type lipid-encapsulated antimicrobial peptide powder to be uniformly adsorbed and dispersed by the silica. Then add 800.00 kg of maifanite powder, 10.00 kg of yeast cell wall powder and 5.00 kg of calcium stearate, and continue mixing for 15 min. After the temperature of the mixture is lower than 35℃, add 25.00 kg of Bacillus coagulans live bacteria powder obtained in step 4), mix at low speed for 12 min, and pass through a 40-mesh sieve to obtain the compound feed additive of Comparative Example 2.

[0041] Compared with Example 2, the main difference of Comparative Example 2 is that sodium alginate, low-ester pectin and calcium chloride aqueous solution are not added in step 3) of Comparative Example 2, and antimicrobial peptide polysaccharide gel core liquid is not formed. Only lipid wall material is used for embedding.

[0042] Comparative Example 3 A method for preparing an antimicrobial peptide compound feed additive as an alternative to antibiotics includes the following steps: 1) Take Bacillus coagulans BC66, inoculate it into seed culture medium, and culture it at 37℃ and 180 r / min for 12 h to obtain seed liquid; inoculate the seed liquid into fermentation medium at a 5% inoculation rate, that is, inoculate 5 L of seed liquid into every 100 L of fermentation medium, and ferment it at 37℃, aeration rate of 0.8 vvm and stirring speed of 200 r / min for 24 h, and then raise the temperature to 42℃ and continue to culture for 6 h to obtain fermentation broth containing antimicrobial peptides and Bacillus coagulans spores; 2) Centrifuge the fermentation broth obtained in step 1) at 6000 r / min for 15 min, and collect the supernatant and cell precipitate respectively; filter the supernatant sequentially through a 0.45 μm microporous membrane and a 10 kDa ultrafiltration membrane, and collect the permeate below 10 kDa; then concentrate using a 1 kDa nanofiltration membrane, and collect the retentate above 1 kDa to obtain the antimicrobial peptide concentrate; control the temperature during the concentration process to not exceed 45℃, and the solids content of the obtained antimicrobial peptide concentrate is 25.0%; 3) Take 120.00 kg of the antimicrobial peptide concentrate obtained in step 2), the solid content of the antimicrobial peptide concentrate is 25.0%, which is equivalent to 30.00 kg of dry matter. Add 8.00 kg of maltodextrin, 2.00 kg of trehalose, 2.50 kg of sodium alginate and 1.50 kg of low-ester pectin, stir until evenly dispersed, and adjust the pH to 5.8 ± 0.1 with 1 mol / L citric acid aqueous solution to obtain the antimicrobial peptide polysaccharide core material solution. Under the shearing condition of 1500 r / min, slowly add 10.00 kg of calcium chloride aqueous solution with a mass fraction of 8.0% to the antimicrobial peptide polysaccharide core material solution, and continue shearing and dispersing for 20 min to allow the sodium alginate and low-ester pectin to undergo calcium ion cross-linking to obtain the antimicrobial peptide polysaccharide gel core material solution. Separately, 29.00 kg of hydrogenated vegetable oil, 6.00 kg of glyceryl monostearate, and 5.00 kg of soybean lecithin were melt-mixed at 70°C to obtain a lipid wall material without glyceryl tartrate. The antimicrobial peptide polysaccharide gel core liquid was slowly added to the lipid wall material without glyceryl tartrate, and the material temperature was controlled at 70°C. High-speed shear emulsification was carried out at 6000 r / min for 8 min. Subsequently, fluidized bed spray condensation granulation was performed, and the inlet air temperature was controlled at 20°C and the outlet air temperature at 30°C to obtain a polysaccharide gel-lipidized embedded antimicrobial peptide powder without glyceryl tartrate, for later use. 4) Resuspend the bacterial precipitate collected in step 2) in sterile water, add trehalose and maltodextrin as preservatives, and mix thoroughly to obtain a bacterial cell preservative suspension; based on the total mass of the bacterial cell preservative suspension, the mass fraction of trehalose is 3.0%, and the mass fraction of maltodextrin is 8.0%; subject the bacterial cell preservative suspension to low-temperature spray drying, controlling the inlet air temperature to 90℃ and the outlet air temperature to 45℃, to obtain Bacillus coagulans live bacterial powder, the viable count of which is 1.2 × 10⁻⁶. 11 CFU / g; 5) Take 800.00 kg of maifan stone powder, 80.00 kg of silicon dioxide, 10.00 kg of yeast cell wall powder and 5.00 kg of calcium stearate, pulverize them separately and pass them through an 80-mesh sieve, dry them at 80℃ for 2 hours, and cool them to room temperature for later use. 6) Take 80.00 kg of the glyceryl-free polysaccharide gel-lipo-encapsulated antimicrobial peptide powder obtained in step 3) and 80.00 kg of the dried silica obtained in step 5) and add them to a mixer. Mix for 10 min to allow the glyceryl-free polysaccharide gel-lipo-encapsulated antimicrobial peptide powder to be uniformly adsorbed and dispersed by the silica. Then add 800.00 kg of maifanite powder, 10.00 kg of yeast cell wall powder and 5.00 kg of calcium stearate, and continue mixing for 15 min. After the temperature of the mixture is lower than 35℃, add 25.00 kg of Bacillus coagulans live bacteria powder obtained in step 4), mix at low speed for 12 min, and pass through a 40-mesh sieve to obtain the compound feed additive of Comparative Example 3.

[0043] Compared with Example 2, the main difference of Comparative Example 3 is that in step 3), glyceryl tartrate is not added, but 5.00 kg of glyceryl tartrate is replaced with 5.00 kg of hydrogenated vegetable oil. That is, the lipid wall material is composed of 29.00 kg of hydrogenated vegetable oil, 6.00 kg of glyceryl monostearate and 5.00 kg of soybean lecithin.

[0044] Test Example 1 Simulated gastrointestinal fluid release performance test Specific testing methods: Simulated gastric fluid and simulated intestinal fluid for pigs were prepared. The simulated gastric fluid was prepared as follows: Approximately 800 mL of purified water was added to a beaker, along with 2.00 g of sodium chloride and 3.00 g of pepsin. After stirring and dissolving, the pH was adjusted to 2.5 ± 0.1 using 1 mol / L hydrochloric acid solution. Then, purified water was added to bring the volume to 1000 mL. The solution was prepared fresh and used immediately, preheated to 37°C before use. The simulated intestinal fluid was prepared as follows: Approximately 800 mL of purified water was added to a beaker, along with 6.80 g of potassium dihydrogen phosphate, 5.00 g of sodium chloride, 1.00 g of trypsin, and 3.00 g of bile salts. After stirring and dissolving or dispersing evenly, the pH was adjusted to 6.8 ± 0.1 using sodium hydroxide solution. Then, purified water was added to bring the volume to 1000 mL. The solution was prepared fresh and used immediately, preheated to 37°C before use.

[0045] 10.00 g each of the antibiotic alternative compound feed additives obtained in the examples and comparative examples were added to 100 mL of simulated gastric fluid for pigs and treated with shaking at 37°C and 100 r / min for 2 h. After treatment, the mixture was centrifuged, the supernatant was collected, the amount of antimicrobial peptides released in the supernatant was measured, and the 2-h antimicrobial peptide release rate of the simulated gastric fluid was calculated. Subsequently, the precipitate after gastric fluid treatment was transferred to 100 mL of simulated intestinal fluid for pigs and treated with shaking at 37°C and 100 r / min for another 4 h. After treatment, the mixture was centrifuged, the released liquid of the simulated intestinal fluid was collected, the amount of antimicrobial peptides released was measured, and the 4-h cumulative release rate of the simulated intestinal fluid was calculated. The release liquid of the simulated intestinal fluid was then used to determine the inhibition rate of Escherichia coli 8099 against Escherichia coli 8099 using the plate count method. The antimicrobial peptide release rate was calculated with the total amount of antimicrobial peptides in each sample as 100%.

[0046] The method for determining the antibacterial rate of the release solution is as follows: Simulated intestinal fluid release solution is filtered through a sterile filter membrane and used as the test solution; using *Escherichia coli* 8099 as the indicator bacterium, a suspension of *E. coli* 8099 is inoculated into the culture system containing the test solution, making the initial bacterial concentration 1.0 × 10⁻⁶. 5 -5.0×10 5CFU / mL, cultured at 37℃ for 24 h, and viable bacterial count was determined using the plate count method. A blank release solution obtained without sample addition and processed using the same simulated gastric and intestinal fluid procedures served as a blank control. The inhibition rate was calculated using the following formula: Inhibition rate / % = (Viable bacterial count in blank control group - Viable bacterial count in sample group) / Viable bacterial count in blank control group × 100% Table 1. Simulated gastrointestinal fluid release performance of different samples Comparative Example 1 72.8 82.5 86.3 Comparative Example 2 34.7 66.4 94.8 Comparative Example 3 20.8 64.5 96.6 Example 1 31.5 62.7 95.6 Example 2 18.6 76.8 99.2 Comparative Example 1, without liposome encapsulation, exhibited a high antimicrobial peptide release rate of 72.8% in simulated gastric fluid, indicating premature release under acidic conditions. In Example 1, liposome encapsulation reduced the release rate in simulated gastric fluid to 31.5%, demonstrating that liposome encapsulation helps reduce premature release of antimicrobial peptides in simulated gastric fluid. Comparative Example 2 did not form sodium alginate / low-ester pectin-Ca... 2+ The polysaccharide gel core material exhibited a 34.7% antimicrobial peptide release rate in simulated gastric juice over 2 hours, higher than the 18.6% in Example 2, indicating that the polysaccharide gel core material is beneficial in reducing premature release during the gastric juice stage. Comparative Example 3, although forming a polysaccharide gel core material, did not contain tributyrate in its lipid wall material. Its 2-hour antimicrobial peptide release rate in simulated gastric juice was 20.8%, close to that of Example 2; however, its cumulative release rate in simulated intestinal juice over 4 hours was only 64.5%, significantly lower than the 76.8% in Example 2. This indicates that tributyrate mainly benefits the release of antimicrobial peptides during the intestinal juice stage containing bile salts and pancreatic enzymes, while sodium alginate / low-ester pectin-Ca... 2+ The polysaccharide gel core material is primarily beneficial for protection during the gastric juice stage. Example 2, through the combination of the polysaccharide gel core material and a lipid wall material containing tributyrate, resulted in lower release of antimicrobial peptides during the simulated gastric juice stage and more sufficient release during the simulated intestinal juice stage containing bile salts and pancreatic enzymes, demonstrating good gastric juice protection and intestinal juice release effects.

[0047] Test Example 2 Survival rate test of Bacillus coagulans after granulation The compound feed additives obtained in the examples and comparative examples were added to the same batch of basic pig compound feed at a dosage of 1000 g / T. Before addition, the compound feed additives and basic feed were premixed at a mass ratio of 1:10 for 5 minutes, and then the premix was added to the remaining basic feed and mixed for another 10 minutes to obtain a feed sample before pelleting. The basic pig compound feed was a corn-soybean meal type powdered compound feed, and the moisture content before pelleting was controlled at 11.0%-13.0%. The feed sample before pelleting was subjected to simulated pelleting treatment with a conditioning temperature of 85℃, a conditioning time of 90s, and a ring die aperture of 3.0 mm. After pelleting, the feed sample was naturally cooled to room temperature to obtain a feed sample after pelleting.

[0048] Take 10.00g of feed sample before pelleting and 10.00g of feed sample after pelleting, add them to 90mL of sterile physiological saline, shake and mix for 10min to obtain 10 -1 Homogenize the sample; prepare a series of dilutions using a 10-fold serial dilution method. Spread the appropriate dilution onto agar plates suitable for the growth of Bacillus coagulans and incubate at 37°C for 24-48 hours. Select plates with colony counts of 30-300 CFU for counting. Calculate the viable Bacillus coagulans count in the sample based on the dilution factor. Perform three parallel determinations for each sample and take the average value. The viability of Bacillus coagulans is calculated using the following formula: Bacillus coagulans survival rate / % = number of viable Bacillus coagulans in the feed sample after pelleting / number of viable Bacillus coagulans in the feed sample before pelleting × 100%.

[0049] Table 2 Survival rate of Bacillus coagulans after granulation of different samples Comparative Example 1 <![CDATA[2.7×10 6 ]]> <![CDATA[2.12×10 6 ]]> 78.5 Comparative Example 2 <![CDATA[2.8×10 6 ]]> <![CDATA[2.31×10 6 ]]> 82.5 Comparative Example 3 <![CDATA[2.9×10 6 ]]> <![CDATA[2.47×10 6 ]]> 85.2 Example 1 <![CDATA[2.8×10 6 ]]> <![CDATA[2.36×10 6 ]]> 84.3 Example 2 <![CDATA[2.9×10 6 ]]> <![CDATA[2.55×10 6 ]]> 87.9 Table 2 shows that the viable count of Bacillus coagulans decreased after simulated granulation at 85℃ for 90s in all samples. The survival rate of Bacillus coagulans was 78.5% in Comparative Example 1, 84.3% in Example 1, and 87.9% in Example 2. Example 2 maintained a high viable count of Bacillus coagulans after granulation, indicating that the low-temperature compounding method of first mixing the antimicrobial peptide encapsulated powder with silica and mineral carrier, and then adding the viable Bacillus coagulans powder after the material temperature is below 35℃, helps to reduce the adverse effects of the preparation process on the viable bacteria and ensures that the resulting compound feed additive maintains good viable bacteria stability after compound feed granulation.

[0050] Test Example 3 Sustained antibacterial performance test Antimicrobial peptide compound feed additives that replace antibiotics, obtained from the examples and comparative examples, were used to prepare sample suspensions at a ratio of 1.0 g sample to 100 mL sterile physiological saline. The suspensions were then shaken and mixed thoroughly before use. Using *Escherichia coli* 8099 (commercially available) and *Salmonella typhimurium* ATCC 13311 (commercially available) as indicator bacteria, the bacterial suspensions were inoculated into the culture system containing the sample suspensions, with an initial bacterial concentration of 1.0 × 10⁻⁶. 5 -5.0×10 5 CFU / mL, incubated at 37℃. Samples were taken at 24h and 7d, and the viable count was determined by plate counting. The inhibition rate was calculated using the following formula: Antibacterial rate / % = (Number of viable bacteria in blank control group - Number of viable bacteria in sample group) / Number of viable bacteria in blank control group × 100%.

[0051] Table 3. Sustained antibacterial properties of different samples Comparative Example 1 87.6 64.8 Comparative Example 2 96.4 89.7 Comparative Example 3 99.3 95.7 Example 1 98.6 94.1 Example 2 >99.9 99.6 Salmonella Typhimurium ATCC 1331 124h inhibition rate / % Inhibition rate of Salmonella Typhimurium ATCC 133117d / % Comparative Example 1 84.2 61.5 Comparative Example 2 95.2 87.9 Comparative Example 3 99.0 95.1 Example 1 98.2 93.5 Example 2 >99.9 99.3 Table 3 shows that, due to the lack of effective encapsulation protection for the antimicrobial peptide, the inhibition rates of Comparative Example 1 against *Escherichia coli* 8099 and *Salmonella Typhimurium* ATCC 13311 decreased to 64.8% and 61.5% respectively after 7 days, indicating insufficient sustained antimicrobial activity. In Example 1, the inhibition rate was significantly improved after 7 days by using liposome encapsulation. Comparative Example 3 formed a sodium alginate / low-ester pectin-Ca... 2+ The polysaccharide gel core material exhibited better antibacterial rates at 24h and 7d than Comparative Example 2 and Example 1, but still lower than Example 2. This indicates that the sustained in vitro antibacterial effect of the polysaccharide gel core material alone is lower than that of Example 2, which combines the polysaccharide gel core material with a tributyrate-containing lipid wall material. Example 2 used a combination of a polysaccharide gel core material and a tributyrate-containing lipid wall material to form a polysaccharide gel-lipidized embedding structure, maintaining a high antibacterial rate at both 24h and 7d. Specifically, the 24h antibacterial rate against *Escherichia coli* 8099 and *Salmonella Typhimurium* ATCC 13311 was greater than 99.9%, and the 7d antibacterial rates were 99.6% and 99.3%, respectively. This demonstrates that Example 2 exhibited better antibacterial retention in the sustained in vitro antibacterial test.

[0052] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A method for preparing an antimicrobial peptide compound feed additive as an alternative to antibiotics, characterized in that, Includes the following steps: 1) Inoculate Bacillus coagulans into seed culture medium and culture at 35-39℃ and 150-220 r / min for 10-16 h to obtain seed liquid; inoculate the seed liquid into fermentation medium at 3-8% of the fermentation medium volume, and ferment at 35-39℃, aeration rate of 0.5-1.2 vvm, and stirring speed of 150-300 r / min for 20-30 h, then raise the temperature to 40-45℃ and continue to culture for 4-10 h to obtain fermentation broth containing antimicrobial peptides and Bacillus coagulans spores; 2) The fermentation broth obtained in step 1) is subjected to solid-liquid separation, and the supernatant and cell precipitate are collected separately; the supernatant is then filtered sequentially through a microporous membrane, an ultrafiltration membrane, and a nanofiltration membrane to concentrate the supernatant to obtain an antimicrobial peptide concentrate. 3) Take the antimicrobial peptide concentrate obtained in step 2), add maltodextrin and trehalose, stir evenly to obtain an antimicrobial peptide core material solution; separately take hydrogenated vegetable oil, glyceryl monostearate and soybean lecithin, melt mix them at 65-75℃ to obtain a lipid wall material; add the antimicrobial peptide core material solution to the lipid wall material, and granulate by shear emulsification and fluidized bed spray condensation to obtain lipid-encapsulated antimicrobial peptide powder; wherein, the material temperature is maintained at 60-75℃ during shear emulsification, the inlet air temperature of fluidized bed spray condensation granulation is 10-25℃, and the outlet air temperature is 25-35℃; Alternatively, take the antimicrobial peptide concentrate obtained in step 2), add maltodextrin, trehalose, sodium alginate, and low-ester pectin, and adjust the pH to 5.5-6.2 to obtain an antimicrobial peptide polysaccharide core material solution; add a calcium chloride aqueous solution with a mass fraction of 5.0-10.0% under shear conditions to allow sodium alginate and low-ester pectin to undergo calcium ion cross-linking to obtain an antimicrobial peptide polysaccharide gel core material solution; separately take hydrogenated vegetable oil, glyceryl monostearate, soybean lecithin, and glyceryl tribanilate, and melt-mix them at 65-75℃ to obtain a lipid wall material; add the antimicrobial peptide polysaccharide gel core material solution to the lipid wall material, and perform shear emulsification and fluidized bed spray condensation granulation to obtain polysaccharide gel-lipidized encapsulated antimicrobial peptide powder; wherein, the material temperature is maintained at 60-75℃ during shear emulsification, the inlet air temperature of fluidized bed spray condensation granulation is 10-25℃, and the outlet air temperature is 25-35℃; 4) Resuspend the bacterial precipitate collected in step 2) in sterile water, add trehalose and maltodextrin as protective agents, mix well to obtain a bacterial cell protective suspension; subject the bacterial cell protective suspension to low-temperature spray drying to obtain Bacillus coagulans live bacterial powder; 5) Take maifan stone powder, silicon dioxide, yeast cell wall powder and calcium stearate, grind and sieve them separately, dry them at 70-90℃ for 1-3 hours, and cool them to room temperature for later use. 6) Add the lipid-encapsulated antimicrobial peptide powder or polysaccharide gel-lipo-encapsulated antimicrobial peptide powder obtained in step 3) and the dried silica obtained in step 5) into a mixer and mix for 5-15 minutes to allow the lipid-encapsulated antimicrobial peptide powder to be adsorbed and dispersed by silica; then add maifanite powder, yeast cell wall powder and calcium stearate, and continue mixing for 10-20 minutes; after the temperature of the mixture is below 35℃, add the Bacillus coagulans live bacteria powder obtained in step 4), mix at low speed for 8-15 minutes, and sieve to obtain the antibiotic-replacing antimicrobial peptide compound feed additive.

2. The preparation method of the antibiotic alternative antimicrobial peptide compound feed additive as described in claim 1, characterized in that: In step 2), the pore size of the microporous filter membrane is 0.22-0.45 μm, the molecular weight cutoff of the ultrafiltration membrane is 5-15 kDa, the molecular weight cutoff of the nanofiltration membrane is 0.5-2 kDa, and the solids mass fraction of the antimicrobial peptide concentrate is 20.0-30.0%.

3. The method for preparing the antibiotic alternative antimicrobial peptide compound feed additive as described in claim 1, characterized in that: In step 3) the preparation of lipo-encapsulated antimicrobial peptide powder, based on 30 parts by weight of dry matter of the antimicrobial peptide concentrate, the maltodextrin is 5-15 parts by weight, trehalose is 1-5 parts by weight, hydrogenated vegetable oil is 20-35 parts by weight, glyceryl monostearate is 3-10 parts by weight, and soybean lecithin is 2-8 parts by weight.

4. The method for preparing the antibiotic alternative antimicrobial peptide compound feed additive as described in claim 1, characterized in that: Step 3) In the polysaccharide gel-lipoylation encapsulated antimicrobial peptide powder, based on 30 parts by weight of the dry matter of the antimicrobial peptide concentrate, the following components are present: 5-15 parts by weight of maltodextrin, 1-5 parts by weight of trehalose, 1-5 parts by weight of sodium alginate, 0.5-3 parts by weight of low-ester pectin, 5-20 parts by weight of calcium chloride aqueous solution with a mass fraction of 5.0-10.0%, 20-30 parts by weight of hydrogenated vegetable oil, 3-10 parts by weight of glyceryl monostearate, 3-8 parts by weight of soybean lecithin, and 2-8 parts by weight of glyceryl tartrate.

5. The method for preparing the antibiotic alternative antimicrobial peptide compound feed additive as described in claim 1, characterized in that: In step 4), based on the total mass of the bacterial cell protective suspension, the mass fraction of trehalose is 2.0-5.0%, and the mass fraction of maltodextrin is 5.0-12.0%; the viable count of the Bacillus coagulans live bacteria powder is 1.0 × 10⁻⁶. 11 -2.0×10 11 CFU / g.

6. The method for preparing the antibiotic alternative antimicrobial peptide compound feed additive as described in claim 1, characterized in that: In step 6), the amount of lipid-encapsulated antimicrobial peptide powder or polysaccharide gel-lipid-encapsulated antimicrobial peptide powder is 40-100 parts by weight, the amount of silica is 50-120 parts by weight, the amount of maifanite powder is 750-850 parts by weight, the amount of yeast cell wall powder is 5-20 parts by weight, the amount of calcium stearate is 2-10 parts by weight, and the amount of Bacillus coagulans live bacteria powder is 10-40 parts by weight.

7. A compound feed additive containing antimicrobial peptides as an alternative to antibiotics, characterized in that: It is prepared by the method described in any one of claims 1-6.

8. The application of the antibiotic alternative antimicrobial peptide compound feed additive as described in claim 7 in feed, characterized in that: When using antibiotic-replacing antimicrobial peptide compound feed additives in compound feed, the addition amount for pig feed is 500-1000g / T, and the addition amount for poultry feed is 300-500g / T. During the weaning, regrouping, feed change, transportation, or high temperature stress stages of animals, the staged addition amount is 1000-1500g / T. Before use, it should be premixed with a small amount of carrier or premix before being gradually added to the compound feed.