An additive for enhancing immunity of livestock and poultry, a preparation method and application thereof

Abstract

The application discloses an additive for enhancing immunity of livestock and poultry, a preparation method and application thereof. The additive for enhancing immunity of livestock and poultry is combined with a polymer and an antibacterial peptide, and the stability and amphiphilicity of the antibacterial peptide are increased. In the additive for enhancing immunity of livestock and poultry, a flexible polymer chain is connected to a peptide main chain to form an amphiphilic conformation, and the additive has more excellent activity than a rigid secondary structure formed by only the peptide chain. The additive for enhancing immunity of livestock and poultry has strong bioactivity and high permeability to a bacterial cell membrane, and has excellent bactericidal effect by destroying the cell membrane of the bacteria, so that the additive can be used in livestock and poultry feed, can enhance the immunity of the livestock and poultry, can effectively prevent and treat diseases, and can promote the growth of the livestock and poultry.

Description

Technical Field

[0001] This invention relates to the field of antimicrobial peptide technology, and in particular to an additive for enhancing the immunity of livestock and poultry, its preparation method, and its application. Background Technology

[0002] Antimicrobial peptides are a novel type of antibiotic alternative. They can be extracted from natural plants and animals or synthesized artificially. Antimicrobial peptides possess long-lasting antimicrobial activity and low cytotoxicity, and can alleviate the problem of bacterial resistance by replacing antibiotics.

[0003] Antimicrobial peptides, whether naturally extracted or artificially synthesized, have been put into use in aquaculture and animal husbandry. Chinese patent CN107518189A discloses a nursery pig feed with added antimicrobial peptides and traditional Chinese medicine and its preparation method. The technical problem to be solved by this invention is to provide a nursery pig feed with added antimicrobial peptides and traditional Chinese medicine, which can enhance the immunity of weaned piglets, strengthen the spleen and stomach, and prevent infectious diseases in piglets. The technical solution adopted is as follows: the feed is formulated from the following raw materials in parts by weight: 400-500 parts corn, 100-200 parts 46% soybean meal, 50-100 parts biscuit powder, 50-150 parts extruded soybeans, 20-80 parts fermented soybean meal, 15-45 parts steamed fish meal, 15-45 parts soybean oil, 5-15 parts dicalcium phosphate, 4-12 parts limestone powder, 3-7 parts montmorillonite, 2-6 parts salt, 30-80 parts chocolate powder, 15-45 parts whey powder, 3-7 parts compound traditional Chinese medicine premix, 3-7 parts antimicrobial peptide, and 30-80 parts premix. This invention, by adding antimicrobial peptides and compound traditional Chinese medicine, promotes the development of gastrointestinal function in early-weaned piglets, making it a source of power for subsequent effects and avoiding intestinal damage caused by weaning. The goal is to enable the piglets' intestinal structure to accept more non-maternal feed, thereby improving piglet production performance, enhancing their resistance, reducing morbidity and mortality, and contributing to increased daily weight gain in nursery piglets. However, as a small molecule compound, the antimicrobial peptide is structurally unstable and easily cleaved by proteases, leading to a decline in function and affecting its practical application.

[0004] Combining antimicrobial peptides with polymers to enhance their stability is a viable research direction. Chinese patent CN111110856A discloses an antimicrobial drug and its preparation method based on antimicrobial peptides and hydrophilic polymers. This patent uses flexible hydrophilic polymer molecules to covalently modify antimicrobial peptides, constructing a series of antimicrobial complex molecules with specially designed structures. Compared to the original antimicrobial peptide, molecular modification enhances the self-assembly ability of the complex, resulting in larger aggregates. These dense aggregates exhibit lipid-specific binding behavior, enabling them to target bacteria without harming mammalian cells. During subsequent membrane interaction, the antimicrobial peptide portion of the complex aggregates forms "peptide channels" within the membrane, facilitating the smooth passage of the hydrophilic polymer portion and ultimately forming stable transmembrane pores. Compared to the original antimicrobial peptide, this demonstrates enhanced antimicrobial efficiency while reducing side effects and toxicity. Although this invention covalently modifies antimicrobial peptides using hydrophilic polymer molecules, the covalent modification requires a high reaction temperature, which may reduce the activity of the antimicrobial peptides during the reaction. In addition, although the polymer can provide a certain degree of protection, the molecular chain structure of the antimicrobial peptides does not change, and they are still affected by protease hydrolysis, resulting in a decrease in actual use effect. Summary of the Invention

[0005] In view of the above-mentioned deficiencies of the prior art, the problem to be solved by the present invention is to provide an additive with good antibacterial effect to enhance the immunity of livestock and poultry, its preparation method and its application.

[0006] To improve the stability and amphiphilicity of antimicrobial peptides and enhance their efficacy, this invention uses 6-caprolactone and heptanol via ring-opening polymerization and addition reaction to obtain a polymer containing a bromine group. Subsequently, using glutamic acid 5-benzyl ester N-carboxylic acid anhydride and (S)-4-[4-(Boc-amino)butyl]oxazolidine-2,5-dione as amino acid structural monomers, an antimicrobial peptide is obtained through polymerization, functionalization, and deprotection reaction under catalysis. The thiol functional group at the end of the antimicrobial peptide molecule can undergo a substitution reaction with the bromine group in the above polymer at a relatively low temperature, thereby combining to form an antimicrobial peptide polymer, which is the additive for enhancing the immunity of livestock and poultry of this invention.

[0007] This invention discloses a method for preparing an additive to enhance the immunity of livestock and poultry, comprising the following steps:

[0008] M1. 6-Caprolactone and heptanol were subjected to ring-opening polymerization under catalysis. After the ring-opening polymerization was completed, the product was purified to obtain the ring-opening polymer.

[0009] M2. Take the ring-opening polymerization product and triethylamine in a solvent, then add 2-bromoisobutyryl bromide and carry out an addition reaction. After the addition reaction is completed, purify to obtain the addition product.

[0010] M3. Glutamic acid 5-benzyl ester N-carboxycyclic anhydride, (S)-4-[4-(Boc-amino)butyl]oxazolidine-2,5-dione, and solvent were mixed and polymerized under catalysis. After the polymerization reaction was completed, 2-(triphenylmethylthio)ethylamine was added to the polymer product to carry out a functionalization reaction. After the functionalization reaction was completed, the product was purified to obtain the peptide polymer.

[0011] M4. Take another portion of the polypeptide polymer and add it to the deprotection treatment solution to carry out the deprotection reaction; after the deprotection reaction is completed, the product is purified to obtain the deprotected product;

[0012] M5. Take the deprotected product and the addition product and mix them with a solvent to undergo a substitution reaction; after the substitution reaction is completed, the product is purified to obtain an antimicrobial peptide polymer, which is the additive for enhancing the immunity of livestock and poultry.

[0013] Specifically, the preparation method of the additive for enhancing livestock and poultry immunity includes the following steps, in parts by weight:

[0014] M1. Mix 26.7–37.5 parts of 6-caprolactone with 0.15–0.21 parts of heptanol until homogeneous. Add 0.12–0.18 parts of stannous isooctanoate to the mixture and carry out a ring-opening polymerization reaction under the catalysis of stannous isooctanoate. After the ring-opening polymerization reaction is completed, add 25–50 parts of dichloromethane to the product and mix until homogeneous. Then add the mixture to 200–400 parts of n-hexane at 0–4°C for precipitation. Filter and collect the filter cake. Wash the filter cake with n-hexane and dry it to obtain the ring-opening polymerization product for later use.

[0015] Under anaerobic conditions (M2), 2.1–2.9 parts of the ring-opening polymerization product, 0.05–0.08 parts of triethylamine, and 150–250 parts of dichloromethane were mixed evenly. Then, 0.11–0.15 parts of 2-bromoisobutyryl bromide were added to carry out an addition reaction. After the addition reaction was completed, the product was added to 200–400 parts of n-hexane at 0–4°C for precipitation. The product was filtered and the filter cake was collected. The filter cake was washed with n-hexane and dried to obtain the addition product for later use.

[0016] M3. Mix 0.38–0.53 parts of 5-benzyl glutamate N-carboxycyclic anhydride, 3.54–4.96 parts of (S)-4-[4-(Boc-amino)butyl]oxazolidine-2,5-dione, and 75–150 parts of tetrahydrofuran to obtain a reaction mixture; add 4.4–6.2 parts of hexamethyldisilamide lithium to the reaction mixture, mix well, and carry out a polymerization reaction; after the polymerization reaction is completed, add 0.83–1.16 parts of 2-(triphenylmethylthio)ethylamine to the polymerization product, mix well, and carry out a functionalization reaction; after the functionalization reaction is completed, pour the reaction product into 200–400 parts of anhydrous ethanol at 0–4°C for precipitation, filter, collect the filter cake, wash the filter cake with anhydrous ethanol, and dry to obtain a polypeptide polymer for later use;

[0017] M4. Add the polypeptide polymer to 24-40 parts of a deprotection treatment solution prepared from trifluoroacetic acid and triethylsilane, mix well, and carry out the deprotection reaction. After the deprotection reaction is completed, remove the trifluoroacetic acid in the deprotection treatment solution by nitrogen blowing. Add 15-30 parts of methanol to the remaining product and mix well. Then pour the mixture into 150-300 parts of diethyl ether at 0-4°C for precipitation. Filter and collect the filter cake. Wash the filter cake with diethyl ether and dry it to obtain the deprotected product for later use.

[0018] M5. Take 1.2 to 1.7 parts of the deprotected product and 1.6 to 2.3 parts of the addition product and add them to 50 to 75 parts of phosphate buffer solution. Mix them evenly and carry out the substitution reaction. After the substitution reaction is completed, filter and collect the filter cake. Wash the filter cake with water and dry it to obtain the antimicrobial peptide polymer, which is the additive for enhancing the immunity of livestock and poultry.

[0019] Preferably, the ring-opening polymerization reaction in step M1 is carried out at a temperature of 125–140°C for 8–24 hours.

[0020] Preferably, the temperature of the addition reaction in step M2 is 0–4°C, and the reaction time is 18–30 h.

[0021] Preferably, the polymerization reaction in step M3 is carried out at a temperature of 20–35°C and for a reaction time of 15–60 min.

[0022] Preferably, the temperature of the functionalization reaction in step M3 is 20–35°C, and the reaction time is 6–18 h.

[0023] Preferably, the deprotection treatment solution in step M4 is a mixture of trifluoroacetic acid and triethylsilane in a mass ratio of 15 to 25:1.

[0024] Preferably, the temperature of the deprotection reaction in step M4 is 20–35°C, and the reaction time is 9–24 h.

[0025] Preferably, the pH of the phosphate buffer solution in step M5 is 7.4.

[0026] Preferably, the temperature of the substitution reaction in step M5 is 25–40°C, and the reaction time is 4–12 h.

[0027] This invention combines polymers with antimicrobial peptides, increasing the stability and amphiphilicity of the antimicrobial peptides. The additive for enhancing livestock and poultry immunity in this invention uses flexible polymer chains to connect the peptide backbone, forming an amphiphilic conformation, which exhibits superior activity compared to a rigid secondary structure formed solely by peptide chains. It kills bacteria by disrupting their cell membranes. This amphiphilic additive for enhancing livestock and poultry immunity demonstrates strong biological activity, high permeability to bacterial cell membranes, and excellent bactericidal effect.

[0028] This invention uses 5-benzyl glutamate N-carboxylated intracyclic anhydride and (S)-4-[4-(Boc-amino)butyl]oxazolidine-2,5-dione as amino acid polymerization monomers. The racemic structure of the monomers gives the polymerized peptide stronger resistance to protease hydrolysis. Through the combined effects of the monomer structure and the polymer, the antimicrobial peptide polymer's resistance to enzymatic degradation is optimized. Furthermore, the antimicrobial peptide and polymer bind via a substitution reaction between thiol and bromine groups, achieving binding at a lower temperature, overcoming the problem of decreased antimicrobial peptide activity during synthesis due to the higher temperatures required for covalent bonding.

[0029] This invention discloses the application of the additive that enhances the immunity of livestock and poultry in livestock and poultry feed compositions.

[0030] Preferably, the livestock and poultry feed composition contains 0.02 to 0.08 wt% of additives that enhance the immunity of livestock and poultry.

[0031] Specifically, the pig feed composition comprises the following raw materials in parts by weight: 615-625 parts corn, 75-80 parts corn middlings, 180-195 parts soybean meal, 80-95 parts extruded soybeans, 10-15 parts fish meal, 10-15 parts limestone powder, 6.75-8.25 parts L-lysine, 1.5-2.5 parts L-threonine, 0.45-0.60 parts compound vitamins, 3.9-4.5 parts compound trace elements, 3-5 parts palatability enhancer, 0.3-0.6 parts Saccharomyces boulardii, 0.25-0.75 parts additives to enhance livestock and poultry immunity, 0.75-1.25 parts choline chloride, 5-7.5 parts salt, and 8.65-9.25 parts dicalcium phosphate.

[0032] Preferably, the compound vitamin is any one of the following: 807 compound vitamin for pigs, 915 compound vitamin for pigs, and 985 compound vitamin for pigs.

[0033] Preferably, the composite trace element is any one of Zhuangyuan 100, Zhuangyuan 102, Zhuangyuan 103, Zhuangyuan 104, and Zhuangyuan 105.

[0034] The present invention also discloses a method for preparing the pig feed composition, comprising the following steps:

[0035] Prepare the raw materials according to the formula of the pig feed composition for enhancing immunity. First, mix corn, corn middlings, soybean meal, extruded soybeans, fish meal, and limestone powder evenly at room temperature. Then, add L-lysine, L-threonine, compound vitamins, compound trace elements, palatability enhancer, Saccharomyces boulardii, additives for enhancing livestock and poultry immunity, choline chloride, salt, and dicalcium phosphate. Continue mixing until all components are evenly dispersed to obtain the pig feed composition for enhancing immunity.

[0036] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0037] The beneficial effects of this invention are:

[0038] Compared to existing technologies, this invention uses glutamic acid 5-benzyl ester N-carboxylated intracyclic anhydride and (S)-4-[4-(Boc-amino)butyl]oxazolidine-2,5-dione as amino acid polymerization monomers. By utilizing the racemic structure of the monomers, the peptide polymer formed by polymerization has stronger resistance to protease hydrolysis. Under the dual effect of monomer structure and polymer, the antimicrobial peptide polymer's resistance to enzymatic hydrolysis is optimized.

[0039] Meanwhile, this invention enables the antimicrobial peptide to bind to the polymer through a substitution reaction between thiol and bromine groups, allowing the reaction to occur at a lower temperature. This overcomes the problem that covalent bonding requires higher temperatures, which leads to a decrease in the activity of the antimicrobial peptide during synthesis.

[0040] Compared with existing technologies, this invention increases the stability and amphiphilicity of antimicrobial peptides by combining polymers with them. The additive for enhancing livestock and poultry immunity in this invention uses flexible polymer chains to connect the peptide backbone, forming an amphiphilic conformation. This results in strong biological activity, high permeability to bacterial cell membranes, and excellent bactericidal effects. When used in livestock and poultry feed, it can enhance the immunity of livestock and poultry, effectively prevent and treat diseases, and promote livestock growth. Detailed Implementation

[0041] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0042] The following are some raw material parameters in the comparative examples and embodiments of the present invention:

[0043] Stone powder, feed grade, model: NBUT901241, provided by Chifeng Hengshuo Silica Processing Co., Ltd.; Compound vitamins, model: 807 for pigs, provided by Shaanxi Jinguan Animal Husbandry Co., Ltd.; Compound trace elements, model: Zhuangyuan 100, provided by Hunan Pulemei Feed Co., Ltd.; Appetite attractant, feed grade, model: 302 Milk Sweet Fragrance, provided by Henan Dongkang Veterinary Drug Co., Ltd.; Saccharomyces cerevisiae boulardii, strain code: CNCM I-1079, source: Pasteur Laboratory, France; Phosphate buffered solution, pH=7.4; (S)-4-[4-(Boc-amino)butyl]oxazolidine-2,5-dione, CAS No.: 33043-60-6.

[0044] Example 1

[0045] The preparation method of an additive to enhance the immunity of livestock and poultry is as follows:

[0046] M1. Mix 26.7 kg of 6-caprolactone and 0.15 kg of heptanol evenly. Add 0.12 kg of stannous isooctanoate to the mixture and carry out a ring-opening polymerization reaction under the catalysis of stannous isooctanoate. The ring-opening polymerization reaction temperature is 135℃ and the reaction time is 12 h. After the ring-opening polymerization reaction is completed, add 25 kg of dichloromethane to the product and mix evenly. Then add the mixture to 200 kg of n-hexane at 0℃ for precipitation. Filter and collect the filter cake. Wash the filter cake with n-hexane and dry it to obtain the ring-opening polymerization product for later use.

[0047] Under M2 and nitrogen protection, 2.1 kg of the ring-opening polymerization product, 0.05 kg of triethylamine, and 150 kg of dichloromethane were mixed evenly. Then, 0.11 kg of 2-bromoisobutyryl bromide was added to carry out an addition reaction at 4 °C for 24 h. After the addition reaction was completed, the product was added to 200 kg of n-hexane at 0 °C for precipitation. The product was filtered and the filter cake was collected. The filter cake was washed with n-hexane and dried to obtain the addition product for later use.

[0048] M3. Mix 0.38 kg of 5-benzyl glutamate N-carboxycyclic anhydride, 3.54 kg of (S)-4-[4-(Boc-amino)butyl]oxazolidine-2,5-dione and 75 kg of tetrahydrofuran to obtain a reaction mixture. Add 4.4 kg of hexamethyldisilamide lithium to the reaction mixture, mix well, and carry out a polymerization reaction at 25°C for 30 min. After the polymerization reaction, add 0.83 kg of 2-(triphenylmethylthio)ethylamine to the polymerization product and mix well to carry out a functionalization reaction at 30°C for 9 h. After the functionalization reaction, pour the reaction product into 200 kg of anhydrous ethanol at 0°C for precipitation, filter, collect the filter cake, wash and dry the filter cake with anhydrous ethanol to obtain the polypeptide polymer for later use.

[0049] M4. The polypeptide polymer is added to 24 kg of a deprotection treatment solution prepared from trifluoroacetic acid and triethylsilane. After mixing evenly, a deprotection reaction is carried out at a temperature of 25°C for 12 h. After the deprotection reaction is completed, the trifluoroacetic acid in the deprotection treatment solution is removed by nitrogen blowing. 15 kg of methanol is added to the remaining product and mixed evenly. The mixture is then poured into 150 kg of diethyl ether at 0°C for precipitation. The mixture is filtered and the filter cake is collected. The filter cake is washed with diethyl ether and dried to obtain the deprotected product for later use.

[0050] M5. Take 1.2 kg of the deprotected product and 1.6 kg of the addition product and add them to 50 kg of phosphate buffer solution with pH = 7.4. After mixing evenly, carry out the substitution reaction at 25°C for 6 hours. After the substitution reaction is completed, filter and collect the filter cake. Wash the filter cake with water and dry it to obtain the antimicrobial peptide polymer, which is an additive to enhance the immunity of livestock and poultry.

[0051] The deprotection treatment solution in step M4 is a mixture of trifluoroacetic acid and triethylsilane in a mass ratio of 20:1.

[0052] Example 2

[0053] A pig feed composition for enhancing immunity is prepared by the following method:

[0054] Prepare the raw materials according to the formula of the pig feed composition for enhancing immunity. First, mix 615 kg of corn, 75 kg of corn middlings, 180 kg of soybean meal, 80 kg of extruded soybeans, 10 kg of fish meal, and 10 kg of limestone powder evenly at room temperature. Then add 6.75 kg of L-lysine, 1.5 kg of L-threonine, 0.45 kg of compound vitamins, 3.9 kg of compound trace elements, 3 kg of palatability enhancer, 0.3 kg of Saccharomyces boulardii, 0.75 kg of choline chloride, 5 kg of salt, and 8.65 kg of dicalcium phosphate. Continue mixing until all components are evenly dispersed to obtain the pig feed composition for enhancing immunity.

[0055] Example 3

[0056] A pig feed composition for enhancing immunity is prepared by the following method:

[0057] Prepare the raw materials according to the formula of the pig feed composition for enhancing immunity. First, mix 615 kg of corn, 75 kg of corn middlings, 180 kg of soybean meal, 80 kg of extruded soybeans, 10 kg of fish meal, and 10 kg of limestone powder evenly at room temperature. Then add 6.75 kg of L-lysine, 1.5 kg of L-threonine, 0.45 kg of compound vitamins, 3.9 kg of compound trace elements, 3 kg of palatability enhancer, 0.3 kg of Saccharomyces boulardii, 0.25 kg of antimicrobial peptides, 0.75 kg of choline chloride, 5 kg of salt, and 8.65 kg of dicalcium phosphate. Continue mixing until all components are evenly dispersed to obtain the pig feed composition for enhancing immunity.

[0058] The method for preparing the antimicrobial peptide is as follows:

[0059] M1. Mix 0.38 kg of 5-benzyl glutamate N-carboxycyclic anhydride, 3.54 kg of (S)-4-[4-(Boc-amino)butyl]oxazolidine-2,5-dione and 75 kg of tetrahydrofuran to obtain a reaction mixture. Add 4.4 kg of hexamethyldisilamide lithium to the reaction mixture, mix well, and carry out a polymerization reaction at 25°C for 30 min. After the polymerization reaction, add 0.83 kg of 2-(triphenylmethylthio)ethylamine to the polymerization product and mix well to carry out a functionalization reaction at 30°C for 9 h. After the functionalization reaction, pour the reaction product into 200 kg of anhydrous ethanol at 0°C for precipitation, filter, collect the filter cake, wash and dry the filter cake with anhydrous ethanol to obtain the polypeptide polymer for later use.

[0060] M2. The polypeptide polymer is added to 24 kg of a deprotection treatment solution prepared from trifluoroacetic acid and triethylsilane. After mixing evenly, a deprotection reaction is carried out at a temperature of 25°C for 12 h. After the deprotection reaction is completed, the trifluoroacetic acid in the deprotection treatment solution is removed by nitrogen blowing. 15 kg of methanol is added to the remaining product and mixed evenly. The mixture is then poured into 150 kg of diethyl ether at 0°C for precipitation. The mixture is filtered and the filter cake is collected. The filter cake is washed with diethyl ether and dried to obtain the antimicrobial peptide.

[0061] The deprotection treatment solution in step M2 is a mixture of trifluoroacetic acid and triethylsilane in a mass ratio of 20:1.

[0062] Example 4

[0063] A pig feed composition for enhancing immunity is prepared by the following method:

[0064] Prepare the raw materials according to the formula of the pig feed composition for enhancing immunity. First, mix 615 kg of corn, 75 kg of corn middlings, 180 kg of soybean meal, 80 kg of extruded soybeans, 10 kg of fish meal, and 10 kg of limestone powder evenly at room temperature. Then add 6.75 kg of L-lysine, 1.5 kg of L-threonine, 0.45 kg of compound vitamins, 3.9 kg of compound trace elements, 3 kg of palatability enhancer, 0.3 kg of Saccharomyces boulardii, 0.25 kg of animal immunity enhancer, 0.75 kg of choline chloride, 5 kg of salt, and 8.65 kg of dicalcium phosphate. Continue mixing until all components are evenly dispersed to obtain the pig feed composition for enhancing immunity.

[0065] The additive that enhances the immunity of livestock and poultry is prepared by the method in Example 1.

[0066] Compare with Example 1

[0067] A pig feed composition for enhancing immunity is prepared by the following method:

[0068] Prepare the raw materials according to the formula of the pig feed composition for enhancing immunity. First, mix 615 kg of corn, 75 kg of corn middlings, 180 kg of soybean meal, 80 kg of extruded soybeans, 10 kg of fish meal, and 10 kg of limestone powder evenly at room temperature. Then add 6.75 kg of L-lysine, 1.5 kg of L-threonine, 0.45 kg of compound vitamins, 3.9 kg of compound trace elements, 3 kg of palatability enhancer, 0.75 kg of choline chloride, 5 kg of salt, and 8.65 kg of dicalcium phosphate. Continue mixing until all components are evenly dispersed to obtain the pig feed composition for enhancing immunity.

[0069] The descriptions and functions of some of the raw materials in the formula of this invention are as follows:

[0070] Limestone powder: short for limestone ore powder. In animal feed, limestone powder is used as a calcium supplement. Its main component is calcium carbonate, with a calcium content of over 38%, and the content of other heavy metals is within the range of hygiene standards. Limestone powder is white, grayish-white, or gray powder or fine particles, and is used to promote animal bone growth and maintain blood calcium levels.

[0071] L-Lysine: Lysine is an important component of protein and one of the eight essential amino acids that the human body cannot synthesize but is highly required by. It is an excellent food fortifier. Because lysine is lacking in food, it is also known as the "first essential amino acid." Adding lysine to beverages, rice, flour, canned goods, and other foods can improve protein utilization, thereby greatly enhancing the nutritional value of food. This can promote growth and development, increase appetite, reduce disease, and strengthen the body. In canned goods, it also has deodorizing and preservative properties.

[0072] L-Threonine: An essential amino acid, L-Threonine is mainly used in medicine, chemical reagents, food fortifiers, and feed additives. Its use in feed additives is growing rapidly, especially in piglets and poultry feed. It is the second limiting amino acid in pig feed and the third limiting amino acid in poultry feed. Adding L-Threonine to compound feed can adjust the amino acid balance, promote livestock growth, improve meat quality, improve the nutritional value of feeds with low amino acid digestibility, and reduce feed ingredient costs.

[0073] Choline chloride: A methyl donor that promotes the reformation of amino acids, improving their utilization in animals, especially essential amino acids such as methionine, thereby promoting animal growth and improving feed utilization. Adding choline chloride to growing pig feed can significantly reduce the feed conversion ratio and improve feed utilization.

[0074] Test Example 1

[0075] The effectiveness of the pig feed composition for enhancing immunity was characterized by cure rate and effective prevention rate. The cure rate was calculated by feeding the feed composition to pigs infected with swine respiratory syndrome during treatment; the effective prevention rate was calculated by administering the feed composition at a preventative dose to healthy pigs.

[0076] The design of the cure and prevention trials was carried out in accordance with the methods and procedures in the reference (Guo Yuankui, Sun Aili. Experiment on the prevention and treatment effect of compound mycoplasma-reducing agent on porcine respiratory syndrome [J]. Shandong Animal Husbandry and Veterinary Medicine, 2010, 31(5): 6-7. DOI: 10.3969 / j.issn.1007-1733.2010.05.004.).

[0077] In the cure trial, 75 pigs suffering from porcine respiratory syndrome were used as test animals. Based on the feed composition types of the corresponding examples and control examples, the pigs were divided into four experimental groups and one control group, with 15 pigs in each group. The experimental group pigs were fed a daily feed composition supplemented with 100 ppm of gentamicin and 60 ppm of florfenicol to enhance immunity, at a dosage of 4 wt% of their body weight. The control group pigs were fed the same feed composition as control example 1 daily, but without the addition of gentamicin and florfenicol. After 10 days of continuous feeding, the number of cured pigs in each group was counted, and the cure rate was calculated.

[0078] In the prevention trial, 75 healthy pigs were used as test animals. Based on the feed composition types of the corresponding examples and control examples, the healthy pigs were divided into four experimental groups and one control group, with 15 pigs in each group. The pigs in the experimental groups were fed a feed composition daily supplemented with 100 ppm of paclobutrazol and 60 ppm of florfenicol to enhance immunity, at a dosage of 4 wt% of their body weight. The pigs in the control group were fed the same feed composition as in Control Example 1, but without the addition of paclobutrazol and florfenicol. After 10 days of continuous feeding, the number of pigs in each group that did not develop swine respiratory syndrome was counted, and the effective prevention rate was calculated.

[0079] Note: Clinically, "cured" means that symptoms such as coughing, wheezing, abdominal breathing, ataxia, groaning, and cyanosis of mucous membranes and skin have disappeared, and the pig's body temperature, heart rate, respiratory rate, and mental state and appetite have returned to normal. "Effective" means that the disease progression has been controlled, the pig's mental state and appetite have recovered to some extent, and further comprehensive treatment is needed to improve the condition.

[0080] The experimental results for the cure rate are shown in Table 1, and the experimental results for the effective prevention rate are shown in Table 2.

[0081] Table 1:

[0082]

[0083]

[0084] Table 2:

[0085]

[0086] The nutrients and bioactive components in feed can help improve the immunity of pigs. Pigs with optimized immunity can achieve better curative effects when they are treated with medication. Similarly, during the prevention stage, pigs with strong immunity have a stronger ability to resist diseases and have a higher effective prevention rate under the same medication conditions.

[0087] The test results in Tables 1 and 2 show that, under the same administration conditions, pigs fed with different feed compositions from the examples or control examples exhibited different cure rates and effective prevention rates in the cure and prevention trials, with Example 4 showing the best effect. This may be because different feed compositions have different effects on enhancing pig immunity. Example 4 uses 5-benzyl glutamate N-carboxylated intracyclic anhydride and (S)-4-[4-(Boc-amino)butyl]oxazolidine-2,5-dione as amino acid polymer monomers. The racemic structure of the monomers gives the polymerized peptide stronger resistance to protease hydrolysis. Under the dual effect of monomer structure and polymer, the anti-enzymatic ability of the additive enhancing livestock and poultry immunity is optimized. In this example, the antimicrobial peptide and polymer undergo a substitution reaction between thiol and bromine groups, allowing them to bind at a lower temperature, overcoming the problem of decreased antimicrobial peptide activity during synthesis due to the need for higher temperatures for covalent bonding. The additive used in Example 4 exhibits the strongest activity and the best effect on improving pig immunity.

[0088] Test Example 2

[0089] The antimicrobial activity of additives or antimicrobial peptides for enhancing livestock and poultry immunity, prepared in pig feed compositions for enhancing immunity, was tested according to the specific methods and procedures in the national standard GB / T 39101-2020 "Determination of Antimicrobial Activity of Polypeptides - Inhibition Zone Method". The culture medium used was nutrient agar (NA), and the indicator strain was Staphylococcus aureus CICC 10473. Test results are expressed as the arithmetic mean of three parallel determinations, retained to two significant figures. The antimicrobial activity test results of additives or antimicrobial peptides for enhancing livestock and poultry immunity, prepared in pig feed compositions for enhancing immunity, are shown in Table 3.

[0090] Table 3:

[0091] name Antibacterial potency (AU / mg) Example 3 9100 Example 4 13000

[0092] The test results in Table 3 show that the antibacterial potency of the additive used in Example 4 to enhance livestock and poultry immunity is higher than that in Example 3. This phenomenon may be because Example 4 combines polymers with antimicrobial peptides, increasing the stability and amphiphilicity of the antimicrobial peptides; the flexible polymer chains in the additive enhancer connect the peptide backbone, forming an amphiphilic conformation, which exhibits superior activity compared to the rigid secondary structure formed solely by peptide chains. Antimicrobial peptide polymers kill bacteria by disrupting their cell membranes. Amphiphilic antimicrobial peptide polymers possess strong biological activity, high permeability to bacterial cell membranes, and excellent bactericidal effects.

Claims

1. A method of preparing an additive for enhancing immunity of livestock and poultry, characterized in that, Comprise the following steps: in parts by weight: M1, 26.7~37.5 parts of 6-caprolactone and 0.15~0.21 parts of heptanol are mixed uniformly, 0.12~0.18 parts of isooctanoic acid stannous is added to the mixture, and ring-opening polymerization is carried out under the catalysis of isooctanoic acid stannous; after the ring-opening polymerization reaction is completed, 25~50 parts of dichloromethane is added to the product, and after being mixed uniformly, the mixture is added to 200~400 parts of 0~4℃ n-hexane for precipitation, and the filter cake is collected by filtration and washing, and the filter cake is dried to obtain a ring-opening polymerization product, which is ready for use; M2, under anaerobic conditions, 2.1~2.9 parts of the ring-opening polymerization product, 0.05~0.08 parts of triethylamine and 150~250 parts of dichloromethane are mixed uniformly, then 0.11~0.15 parts of 2-bromoisobutyryl bromide is added to carry out addition reaction; after the addition reaction is completed, the product is added to 200~400 parts of 0~4℃ n-hexane for precipitation, and the filter cake is collected by filtration and washing, and the filter cake is dried to obtain an addition product, which is ready for use; M3, 0.38~0.53 parts of glutamic acid 5-benzyl ester N-carboxylic anhydride, 3.54~4.96 parts of (S)-4-[4-(Boc-amino)butyl]oxazolidine-2,5-diketone and 75~150 parts of tetrahydrofuran are mixed uniformly to obtain a reaction mixture; 4.4~6.2 parts of lithium hexamethyldisilazide is added to the reaction mixture, mixed uniformly and subjected to polymerization reaction; after the polymerization reaction is completed, 0.83~1.16 parts of 2-(tritylthio)ethylamine is continuously added to the polymerization product, mixed uniformly to carry out functionalization reaction; after the functionalization reaction is completed, the reaction product is poured into 200~400 parts of 0~4℃ anhydrous ethanol for precipitation, and the filter cake is collected by filtration and washing, and the filter cake is dried to obtain a polypeptide polymer, which is ready for use; M4, the polypeptide polymer is added to 24~40 parts of a deprotection treatment solution prepared from trifluoroacetic acid and triethylsilane, mixed uniformly and subjected to deprotection reaction; after the deprotection reaction is completed, the trifluoroacetic acid in the deprotection treatment solution is removed by nitrogen blowing, 15~30 parts of methanol is added to the remaining product and mixed uniformly, and then the mixture is poured into 150~300 parts of 0~4℃ diethyl ether for precipitation, and the filter cake is collected by filtration and washing, and the filter cake is dried to obtain a deprotection product, which is ready for use; M5, 1.2~1.7 parts of the deprotection product and 1.6~2.3 parts of the addition product are added to 50~75 parts of a phosphate buffered saline solution, mixed uniformly and subjected to substitution reaction; after the substitution reaction is completed, the filter cake is collected by filtration and washing, and the filter cake is dried to obtain an antibacterial peptide polymer, which is the additive for enhancing the immunity of livestock and poultry.

2. The method of claim 1, wherein the additive for enhancing immunity of livestock and poultry is prepared by the steps of: The temperature of the ring-opening polymerization reaction in step M1 is 125~140℃, and the reaction time is 8~24h. ​ 3. The method for preparing the additive for enhancing livestock and poultry immunity as described in claim 1, characterized in that: The temperature of the addition reaction in step M2 is 0~4℃, and the reaction time is 18~30h.

4. The method for preparing the additive for enhancing livestock and poultry immunity as described in claim 1, characterized in that: The temperature of the polymerization reaction in step M3 is 20-35℃, and the reaction time is 15-60 min; the temperature of the functionalization reaction is 20-35℃, and the reaction time is 6-18 h.

5. The method for preparing the additive for enhancing the immunity of livestock and poultry as described in claim 1, characterized in that: The deprotection treatment liquid in step M4 is a mixture of trifluoroacetic acid and triethylsilane in a mass ratio of 15-25:1; the temperature of the deprotection reaction is 20-35℃, and the reaction time is 9-24 h.

6. The method for preparing the additive for enhancing livestock and poultry immunity as described in claim 1, characterized in that: The pH of the phosphate buffered saline solution in step M5 is 7.4; the temperature of the substitution reaction is 25-40℃, and the reaction time is 4-12 h.

7. An additive for enhancing immunity of livestock and poultry, characterized in that, Prepared by the method of any one of claims 1-6.

8. Use of the additive for enhancing immunity of livestock and poultry in claim 7 in a livestock and poultry feed composition.

9. Use according to claim 8, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The livestock and poultry feed composition contains 0.02-0.08 wt% of the additive for enhancing immunity of livestock and poultry.

10. The use according to claim 8, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The livestock and poultry feed composition is prepared by the following method: Prepare raw materials according to the formula of the pig feed composition for enhancing immunity, first mix corn 615 kg, corn meal 75 kg, soybean meal 180 kg, puffed soybean 80 kg, fish meal 10 kg, stone powder 10 kg uniformly at room temperature, then add L-lysine 6.75 kg, L-threonine 1.5 kg, compound vitamin 0.45 kg, compound trace element 3.9 kg, attractant 3 kg, Blady yeast 0.3 kg, additive for enhancing immunity of livestock and poultry 0.25 kg, choline chloride 0.75 kg, salt 5 kg, calcium hydrogen phosphate 8.65 kg, continue to mix until the components are uniformly dispersed, to obtain the pig feed composition for enhancing immunity; A method for preparing an additive for enhancing immunity of livestock and poultry is as follows: M1, uniformly mix 26.7 kg of 6-hexanolactone and 0.15 kg of heptanol, add 0.12 kg of isooctanoic acid stannous to the mixture, and perform ring-opening polymerization under the catalysis of isooctanoic acid stannous, the temperature of the ring-opening polymerization is 135℃, and the reaction time is 12 h; after the ring-opening polymerization is completed, add 25 kg of dichloromethane to the product, uniformly mix, and then add the mixture to 200 kg of n-hexane at 0℃ to perform precipitation, filter and collect the filter cake, and the filter cake is washed with n-hexane and dried to obtain a ring-opening polymerization product, which is reserved for use; M2, under nitrogen protection, uniformly mix 2.1 kg of the ring-opening polymerization product, 0.05 kg of triethylamine, and 150 kg of dichloromethane, then add 0.11 kg of 2-bromoisobutyryl bromide to perform addition reaction, the temperature of the addition reaction is 4℃, and the reaction time is 24 h; after the addition reaction is completed, add the product to 200 kg of n-hexane at 0℃ to perform precipitation, filter and collect the filter cake, and the filter cake is washed with n-hexane and dried to obtain an addition product, which is reserved for use; M3, 0.38 kg of 5-benzyl glutamate N-carboxyl cyclic anhydride, 3.54 kg of (S)-4-[4-(Boc-amino)butyl]oxazolidine-2,5-dione and 75 kg of tetrahydrofuran are uniformly mixed to obtain a reaction mixture; 4.4 kg of lithium hexamethyldisilazide is added to the reaction mixture, which is uniformly mixed and subjected to a polymerization reaction, wherein the polymerization reaction is carried out at 25°C for 30 min; after the polymerization reaction, 0.83 kg of 2-(tritylthio)ethylamine is continuously added to the polymerization product, which is uniformly mixed to perform a functionalization reaction, wherein the functionalization reaction is carried out at 30°C for 9 h; after the functionalization reaction, the reaction product is poured into 200 kg of 0°C anhydrous ethanol for precipitation, and the filter cake is collected by filtration, washed with anhydrous ethanol and dried to obtain a polypeptide polymer, which is ready for use; M4, the polypeptide polymer is added to 24 kg of a deprotection treatment solution prepared from trifluoroacetic acid and triethylsilane, which is uniformly mixed and subjected to a deprotection reaction, wherein the deprotection reaction is carried out at 25°C for 12 h; after the deprotection reaction, the trifluoroacetic acid in the deprotection treatment solution is removed by nitrogen blowing, 15 kg of methanol is added to the remaining product and uniformly mixed, and then the mixture is poured into 150 kg of 0°C diethyl ether for precipitation, and the filter cake is collected by filtration, washed with diethyl ether and dried to obtain a deprotection product, which is ready for use; M5, 1.2 kg of the deprotection product and 1.6 kg of the addition product are added to 50 kg of a phosphate buffer solution with pH = 7.4, which is uniformly mixed and subjected to a substitution reaction, wherein the substitution reaction is carried out at 25°C for 6 h; after the substitution reaction, the filter cake is collected by filtration, washed with water and dried to obtain an antibacterial peptide polymer, which is an additive for enhancing the immunity of livestock and poultry; The deprotection treatment solution in step M4 is a mixture of trifluoroacetic acid and triethylsilane in a mass ratio of 20:1.

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