Feed additive added with natural antibacterial components and preparation method of feed additive

Abstract

The invention relates to the technical field of feed additives, in particular to a feed additive added with natural antibacterial components and a preparation method of the feed additive. The feed additive added with the natural antibacterial components comprises the following raw materials in parts by mass: 200-400 parts of ramie, 20-90 parts of sodium chloride, 10-30 parts of chitosan quaternary ammonium salt, 10-30 parts of sodium alginate, 10-30 parts of pullulan, 10-50 parts of amino-terminated polyamidoamine, 50-100 parts of nano-hydroxyapatite, 10-30 parts of lecithin, 10-50 parts of lactic acid, 10-20 parts of glucose, 10-20 parts of a complex microbial inoculant, 5-15 parts of small molecule polypeptide, 5-10 parts of compound enzyme and 1-5 parts of a water-retaining agent. And 1-3 parts of composite trace elements. According to the invention, efficient utilization of active components of ramie is realized, release and absorption of the active components in intestinal tracts are ensured, the use of antibiotics is reduced, the growth performance and health level of animals are remarkably improved, and the preparation method is simple.

Description

Technical Field

[0001] This invention relates to the field of feed additive technology, and in particular to a feed additive containing natural antibacterial components and its preparation method. Background Technology

[0002] The overuse of antibiotics in feed has triggered multiple crises. Excessive use can induce the growth of drug-resistant bacteria in animals, and antibiotic residues can enter the human body through the food chain, weakening treatment effects, disrupting gut microbiota balance, affecting nutrient absorption and growth performance, and potentially damaging human immune function. Therefore, finding green, efficient, and safe feed additives has become an urgent need for the livestock industry.

[0003] Ramie, a traditional economic crop in my country, has a long history of cultivation and is widely distributed. It is rich in crude protein, carbohydrates, crude fiber, minerals, and various bioactive components (such as flavonoids and ramie polysaccharides). These components endow ramie with significant antibacterial, antiviral, and immunomodulatory activities. Studies have shown that ramie extract can effectively inhibit the growth of various pathogens, while enhancing animal immunity and promoting nutrient absorption, thereby improving animal growth performance and disease resistance.

[0004] However, the growth-promoting effect of ramie as a feed additive is not significant at present. Although ramie contains a variety of bioactive components, its effect on promoting animal growth is limited in practical applications.

[0005] If ramie can be successfully applied as a feed additive, it can not only reduce antibiotic use and lower the risk of drug resistance, but also improve animal gut health and increase feed conversion rate through its natural antibacterial components. At the same time, the addition of ramie can enhance animal immunity and reduce disease incidence, thereby improving the economic benefits of animal husbandry. How to improve its growth-promoting effects while ensuring its antibacterial effect, so as to fully realize the potential of ramie as a feed additive, has excellent research prospects. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by proposing a feed additive containing natural antibacterial components and its preparation method.

[0007] A feed additive containing natural antibacterial ingredients comprises the following raw materials by weight: 200-400 parts ramie, 20-90 parts sodium chloride, 10-30 parts chitosan quaternary ammonium salt, 10-30 parts sodium alginate, 10-30 parts pullulan polysaccharide, 10-50 parts amino-terminated polyamide amine, 50-100 parts nano hydroxyapatite, 10-30 parts lecithin, 10-50 parts lactic acid, 10-20 parts glucose, 10-20 parts compound microbial agent, 5-15 parts small molecule polypeptide, 5-10 parts compound enzyme, 1-5 parts water-retaining agent, and 1-3 parts compound trace elements.

[0008] Preferably, the compound microbial agent includes: Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Diplocos globosum, and Rhizopus nigricans.

[0009] More preferably, the viable count of Bacillus subtilis is 3-9 × 10⁻⁶. 9 CFU / g, viable count of Bacillus licheniformis is 2-4 × 10⁻⁴. 9 The cfu / g count of Bacillus amyloliquefaciens was 7-9 × 10⁻⁶. 8 The cfu / g count of *Geotrichum candida* was 2-6 × 10⁻⁶. 8 CFU / g, viable count of Rhizopus nigricans is 1-5 × 10⁻⁶. 8 cfu / g.

[0010] Preferably, the small molecule polypeptide is a mixture of oligopeptides with a molecular weight of <1000 Da.

[0011] Preferably, the complex enzyme includes: cellulase, xylanase, and β-glucanase.

[0012] More preferably, the cellulase activity is 6000-8000 U / g, the xylanase activity is 3000-5000 U / g, and the β-glucanase activity is 1000-4000 U / g.

[0013] Preferably, the water-retaining agent is sodium carboxymethyl cellulose.

[0014] Preferably, the complex trace elements are organic complexes containing calcium, magnesium, zinc, and selenium.

[0015] The preparation method of the above-mentioned feed additive includes the following steps: S1. Pulverize ramie, freeze at -40 to -50℃ for 1-2 hours, bring to room temperature, add sodium chloride solution and let stand for 4-10 hours, filter, wash the filter residue, dry to constant weight, and pulverize to obtain pretreated ramie; S2. Add chitosan quaternary ammonium salt to acetic acid solution, adjust the pH of the system to 5-6 and stir evenly. Add sodium alginate and pullulan polysaccharide, stir evenly at 40-50℃, add amino-terminated polyamide and nano hydroxyapatite and stir for 1-2 hours. Add lecithin and continue stirring for 10-30 minutes to obtain pretreated apatite. S3. Add water to the pretreated ramie, then add lactic acid, glucose, and compound bacterial agent. Stir at 25-28℃ for 6-12 days, inactivate at high temperature, cool to room temperature, and vacuum concentrate to 20-30% of the original volume. Add pretreated apatite, stir evenly, and sonicate for 1-2 hours. Dropwise add to sodium tripolyphosphate solution and let stand for 10-30 minutes. Collect microspheres, vacuum dry, and pulverize. Add small molecule peptides, compound enzymes, water-retaining agents, and compound trace elements and mix evenly.

[0016] Preferably, in S3, the ultrasonic frequency is 60-70kHz.

[0017] Preferably, in S1, the mass fraction of sodium chloride solution is 4-6%.

[0018] Preferably, in S2, the mass fraction of the acetic acid solution is 0.5-1%.

[0019] Preferably, in S3, the mass fraction of sodium tripolyphosphate solution is 1-2%.

[0020] Beneficial effects: This invention pretreats ramie with a freezing-sodium chloride solution, which disrupts its cell structure and reduces the crystallinity of macromolecules. This, combined with the action of a compound microbial agent, promotes the release of antibacterial active ingredients such as flavonoids and ramie polysaccharides. The combination of Geotrichum candida and Rhizopus nigricans produces secondary metabolites that can form a synergistic antibacterial effect with the active ingredients of ramie, inhibiting the growth of intestinal pathogens while maintaining the balance of beneficial intestinal flora.

[0021] This invention utilizes pretreated apatite to coat active ingredients, combined with a water-retaining agent. This not only slows down the passage of digesta and prolongs the action time of enzymatic hydrolysis products, but also allows the complex enzyme to continuously degrade non-starch polysaccharides in ramie under suitable humidity, releasing soluble sugars and providing additional energy for animals. Furthermore, the positive charge of chitosan quaternary ammonium salt interacts with the negative charge of the intestinal mucosa through electrostatic interaction, enhancing the adhesion of microspheres in the intestine and further prolonging the action time of active ingredients.

[0022] The small molecule peptides used in this invention are preferentially absorbed by intestinal epithelial cells, avoiding the loss caused by the precipitation of traditional inorganic salts by binding with phytic acid and phosphate in the intestine. Moreover, the small molecule peptides and organic trace elements synergistically enhance the animal's antioxidant capacity and immune function.

[0023] This invention achieves efficient utilization of ramie active ingredients, ensuring not only the release and absorption of active ingredients in the intestines, but also significantly improving animal growth performance and health while reducing antibiotic use. Furthermore, the preparation method is simple and suitable for large-scale promotion and utilization. Attached Figure Description

[0024] Figure 1 The feed conversion ratios of broilers in the blank group, Example 5 group, Comparative Example 1 group, Comparative Example 2 group, and Comparative Example 3 group are given.

[0025] Figure 2 The curves showing the changes in Escherichia coli in the cecal chyme of broilers are shown for the blank group, Example 5 group, Comparative Example 1 group, Comparative Example 2 group, and Comparative Example 3 group.

[0026] Figure 3 The curves showing the changes in Salmonella in the cecal digesta of broilers in the blank group, Example 5 group, Comparative Example 1 group, Comparative Example 2 group, and Comparative Example 3 group are shown.

[0027] Figure 4 The curves showing the changes in lactobacilli in the cecal digesta of broilers are shown for the blank group, Example 5 group, Comparative Example 1 group, Comparative Example 2 group, and Comparative Example 3 group.

[0028] Figure 5 The curves showing the changes in Bifidobacteria in the cecal chyme of broilers are shown for the blank group, Example 5 group, Comparative Example 1 group, Comparative Example 2 group, and Comparative Example 3 group. Detailed Implementation

[0029] The present invention will be further explained below with reference to specific embodiments.

[0030] The chitosan quaternary ammonium salt used below was purchased from Jiangsu Mouwei Biotechnology Co., Ltd. The small molecule peptides used below were purchased from Shandong Mourantang Biotechnology Co., Ltd., specifically fish collagen oligopeptides with a molecular weight <1000 Da. The complex trace elements used below were purchased from Shandong Mouju Biotechnology Co., Ltd., specifically a mixture of amino acid-chelated calcium, magnesium, zinc, and selenium. The terminal amino-terminated polyamide amine (PAMAM, G1.0) used below was purchased from Weihai Mouyuan Molecular New Materials Co., Ltd., with cystamine as the core.

[0031] The Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Diplostomum globosum, and Rhizopus nigricans used below are all from Bio-Biobio, with product numbers bio-093624, bio-53185, bio-53079, bio-11442, and bio-52308, respectively.

[0032] The compound enzymes used below were purchased from Sheng (Beijing) Biotechnology Development Co., Ltd., and consist of cellulase, xylanase, and β-glucanase, with cellulase having an activity of 7000 U / g, xylanase having an activity of 4000 U / g, and β-glucanase having an activity of 2500 U / g.

[0033] Example 1

[0034] A feed additive containing natural antibacterial ingredients comprises the following raw materials: 200 kg of ramie, 20 kg of sodium chloride, 10 kg of chitosan quaternary ammonium salt, 10 kg of sodium alginate, 10 kg of pullulan polysaccharide, 10 kg of amino-terminated polyamide amine, 50 kg of nano-hydroxyapatite, 10 kg of lecithin, 10 kg of lactic acid, 10 kg of glucose, 10 kg of compound microbial agent, 5 kg of small molecule polypeptide, 5 kg of compound enzyme, 1 kg of sodium carboxymethyl cellulose, and 1 kg of compound trace elements.

[0035] The compound microbial agent includes: 3 × 10⁶ live bacteria. 9 Bacillus subtilis CFU / g, viable count 2 × 10⁻⁶ 9 Bacillus licheniformis CFU / g, viable count 7 × 10⁻⁶ 8 Bacillus amyloliquefaciens cfu / g, viable count 2×10⁻⁶ 8 The viable count of *Geotrichum dipodioides* was 1 × 10⁻⁶ cfu / g. 8 CFU / g of Rhizopus nigricans.

[0036] The preparation method of the above-mentioned feed additive includes the following steps: S1. Crush the ramie, pass it through a 60-mesh sieve, freeze it at -40℃ for 1 hour, raise it to room temperature, add a 4% sodium chloride solution, let it stand for 4 hours, filter it, wash the filter residue once, dry it at 60℃ to constant weight, crush it and pass it through a 100-mesh sieve to obtain pretreated ramie. S2. Add chitosan quaternary ammonium salt to 200 kg of 0.5% acetic acid solution, adjust the pH of the system to 5-6 and stir evenly. Add sodium alginate and pullulan polysaccharide, stir evenly at 40℃, add amino-terminated polyamide amine and nano hydroxyapatite, stir at 100 r / min for 1 h, add lecithin and continue stirring for 10 min to obtain pretreated apatite. S3. Add twice the mass of water to the pretreated ramie, along with lactic acid, glucose, and compound bacterial agent. Stir at 50 r / min for 6 days at 25°C. Inactivate at high temperature, cool to room temperature, and vacuum concentrate to 20% of the original volume. Add pretreated apatite, stir evenly, and sonicate for 1 hour at 60 kHz. Dropwise add to a 1% sodium tripolyphosphate solution and let stand for 10 minutes. Collect the microspheres, vacuum dry, and pulverize through a 100-mesh sieve. Add small molecule peptides, compound enzymes, sodium carboxymethyl cellulose, and compound trace elements and mix evenly.

[0037] Example 2

[0038] A feed additive containing natural antibacterial ingredients comprises the following raw materials: 400 kg of ramie, 90 kg of sodium chloride, 30 kg of chitosan quaternary ammonium salt, 30 kg of sodium alginate, 30 kg of pullulan polysaccharide, 50 kg of amino-terminated polyamide amine, 100 kg of nano-hydroxyapatite, 30 kg of lecithin, 50 kg of lactic acid, 20 kg of glucose, 20 kg of compound microbial agent, 15 kg of small molecule polypeptide, 10 kg of compound enzyme, 5 kg of sodium carboxymethyl cellulose, and 3 kg of compound trace elements.

[0039] The compound microbial agent includes: 9 × 10⁶ live bacteria. 9 Bacillus subtilis CFU / g, viable count 4 × 10⁻⁶ 9 Bacillus licheniformis CFU / g, viable count 9 × 10⁻⁶ 8 Bacillus amyloliquefaciens cfu / g, viable count 6 × 10⁻⁶ 8 The viable count of *Geotrichum dipodioides* was 5 × 10⁻⁶ CFU / g. 8 CFU / g of Rhizopus nigricans.

[0040] The preparation method of the above-mentioned feed additive includes the following steps: S1. Crush the ramie, pass it through a 100-mesh sieve, freeze it at -50℃ for 2 hours, raise the temperature to room temperature, add a 6% sodium chloride solution, let it stand for 10 hours, filter it, wash the filter residue 3 times, dry it at 80℃ to constant weight, crush it and pass it through a 100-mesh sieve to obtain pretreated ramie. S2. Add chitosan quaternary ammonium salt to 400 kg of 1% acetic acid solution, adjust the pH of the system to 5-6 and stir evenly. Add sodium alginate and pullulan polysaccharide, stir evenly at 50℃, add amino-terminated polyamide amine and nano hydroxyapatite, stir at 500 r / min for 2 h, add lecithin and continue stirring for 30 min to obtain pretreated apatite. S3. Add 5 times the mass of water to the pretreated ramie, add lactic acid, glucose, and compound bacterial agent, stir at 100 r / min for 12 days at a stirring temperature of 28℃, inactivate at high temperature, cool to room temperature, and vacuum concentrate to 30% of the original volume. Add pretreated apatite, stir evenly, and sonicate for 2 hours at a sonic frequency of 70 kHz. Dropwise add to a 2% sodium tripolyphosphate solution and let stand for 30 minutes. Collect the microspheres, vacuum dry, and pulverize through a 100-mesh sieve. Add small molecule peptides, compound enzymes, sodium carboxymethyl cellulose, and compound trace elements and mix evenly.

[0041] Example 3

[0042] A feed additive containing natural antibacterial ingredients comprises the following raw materials: 240 kg of ramie, 70 kg of sodium chloride, 15 kg of chitosan quaternary ammonium salt, 25 kg of sodium alginate, 15 kg of pullulan polysaccharide, 40 kg of amino-terminated polyamide amine, 70 kg of nano-hydroxyapatite, 25 kg of lecithin, 20 kg of lactic acid, 18 kg of glucose, 12 kg of compound microbial agent, 12 kg of small molecule polypeptide, 7 kg of compound enzyme, 4 kg of sodium carboxymethyl cellulose, and 1.5 kg of compound trace elements.

[0043] The compound microbial agent includes: 8 × 10⁶ live bacteria. 9 Bacillus subtilis with a cfu / g concentration and a viable count of 2.5 × 10⁻⁶ 9 Bacillus licheniformis CFU / g, viable count 8.5 × 10⁻⁶ 8 Bacillus amyloliquefaciens cfu / g, viable count 3×10⁻⁶ 8 The viable count of *Geotrichum dipodioides* was 4 × 10⁻⁶ CFU / g. 8 CFU / g of Rhizopus nigricans.

[0044] The preparation method of the above-mentioned feed additive includes the following steps: S1. Pulverize the ramie, pass it through an 80-mesh sieve, freeze it at -48℃ for 80 minutes, raise it to room temperature, add a 5.5% sodium chloride solution, let it stand for 6 hours, filter it, wash the filter residue twice, dry it at 75℃ to constant weight, pulverize it and pass it through a 100-mesh sieve to obtain pretreated ramie. S2. Add chitosan quaternary ammonium salt to 250 kg of 0.9% acetic acid solution, adjust the pH of the system to 5-6 and stir evenly. Add sodium alginate and pullulan polysaccharide, stir evenly at 42℃, add amino-terminated polyamide amine and nano hydroxyapatite, stir at 400 r / min for 80 min, add lecithin and continue stirring for 25 min to obtain pretreated apatite. S3. Add 3 times the mass of water to the pretreated ramie, add lactic acid, glucose, and compound bacterial agent, stir at 90 r / min for 8 days at a stirring temperature of 26℃, inactivate at high temperature, cool to room temperature, and vacuum concentrate to 22% of the original volume. Add pretreated apatite, stir evenly, and sonicate for 100 min at a sonic frequency of 62 kHz. Dropwise add to a 1.5% sodium tripolyphosphate solution and let stand for 25 min. Collect the microspheres, vacuum dry, and pulverize through a 100-mesh sieve. Add small molecule peptides, compound enzymes, sodium carboxymethyl cellulose, and compound trace elements and mix evenly.

[0045] Example 4

[0046] A feed additive containing natural antibacterial ingredients comprises the following raw materials: 360 kg of ramie, 30 kg of sodium chloride, 25 kg of chitosan quaternary ammonium salt, 15 kg of sodium alginate, 25 kg of pullulan polysaccharide, 20 kg of amino-terminated polyamide amine, 90 kg of nano-hydroxyapatite, 15 kg of lecithin, 40 kg of lactic acid, 12 kg of glucose, 18 kg of compound microbial agent, 8 kg of small molecule polypeptide, 9 kg of compound enzyme, 2 kg of sodium carboxymethyl cellulose, and 2.5 kg of compound trace elements.

[0047] The compound microbial agent includes: 4 × 10⁶ live bacteria. 9 Bacillus subtilis with a cfu / g concentration and a viable count of 3.5 × 10⁻⁶ 9 Bacillus licheniformis CFU / g, viable count 7.5 × 10⁻⁶ 8 Bacillus amyloliquefaciens cfu / g, viable count 5 × 10⁻⁶ 8 The viable count of *Geotrichum dipodioides* was 2 × 10⁻⁶ cfu / g. 8 CFU / g of Rhizopus nigricans.

[0048] The preparation method of the above-mentioned feed additive includes the following steps: S1. Crush the ramie, pass it through an 80-mesh sieve, freeze it at -42℃ for 100 minutes, raise it to room temperature, add a 4.5% sodium chloride solution, let it stand for 8 hours, filter it, wash the filter residue twice, dry it at 65℃ to constant weight, crush it and pass it through a 100-mesh sieve to obtain pretreated ramie. S2. Add chitosan quaternary ammonium salt to 350 kg of 0.6% acetic acid solution, adjust the pH of the system to 5-6 and stir evenly. Add sodium alginate and pullulan polysaccharide, stir evenly at 48℃, add amino-terminated polyamide amine and nano hydroxyapatite, stir at 200 r / min for 100 min, add lecithin and continue stirring for 15 min to obtain pretreated apatite. S3. Add 4 times the mass of water to the pretreated ramie, add lactic acid, glucose, and compound bacterial agent, stir at 70 r / min for 10 days at a stirring temperature of 26℃, inactivate at high temperature, cool to room temperature, and vacuum concentrate to 28% of the original volume. Add pretreated apatite, stir evenly, and sonicate for 80 min at a sonic frequency of 68 kHz. Dropwise add to a 1.5% sodium tripolyphosphate solution and let stand for 15 min. Collect the microspheres, vacuum dry, and pulverize through a 100-mesh sieve. Add small molecule peptides, compound enzymes, sodium carboxymethyl cellulose, and compound trace elements and mix evenly.

[0049] Example 5

[0050] A feed additive containing natural antibacterial ingredients comprises the following raw materials: 300 kg of ramie, 50 kg of sodium chloride, 20 kg of chitosan quaternary ammonium salt, 20 kg of sodium alginate, 20 kg of pullulan polysaccharide, 30 kg of amino-terminated polyamide amine, 80 kg of nano-hydroxyapatite, 20 kg of lecithin, 30 kg of lactic acid, 15 kg of glucose, 15 kg of compound microbial agent, 10 kg of small molecule polypeptide, 8 kg of compound enzyme, 3 kg of sodium carboxymethyl cellulose, and 2 kg of compound trace elements.

[0051] The compound microbial agent includes: 6 × 10⁶ live bacteria. 9 Bacillus subtilis CFU / g, viable count 3 × 10⁻⁶ 9 Bacillus licheniformis CFU / g, viable count 8 × 10⁻⁶ 8 Bacillus amyloliquefaciens cfu / g, viable count 4 × 10⁻⁶ 8 The viable count of *Geotrichum dipodioides* was 3 × 10⁻⁶ cfu / g. 8 CFU / g of Rhizopus nigricans.

[0052] The preparation method of the above-mentioned feed additive includes the following steps: S1. Pulverize the ramie, pass it through an 80-mesh sieve, freeze it at -45℃ for 90 minutes, raise it to room temperature, add a 5% sodium chloride solution, let it stand for 7 hours, filter it, wash the filter residue twice, dry it at 70℃ to constant weight, pulverize it and pass it through a 100-mesh sieve to obtain pretreated ramie. S2. Add chitosan quaternary ammonium salt to 300 kg of 0.8% acetic acid solution, adjust the pH of the system to 5-6 and stir evenly. Add sodium alginate and pullulan polysaccharide, stir evenly at 45℃, add amino-terminated polyamide amine and nano hydroxyapatite, stir at 300 r / min for 90 min, add lecithin and continue stirring for 20 min to obtain pretreated apatite. S3. Add 3.5 times the mass of water to the pretreated ramie, along with lactic acid, glucose, and compound bacterial agent. Stir at 80 r / min for 9 days at 27°C. Inactivate at high temperature, cool to room temperature, and vacuum concentrate to 25% of the original volume. Add pretreated apatite, stir evenly, and sonicate for 90 min at 64 kHz. Dropwise add to a 1.5% sodium tripolyphosphate solution and let stand for 20 min. Collect the microspheres, vacuum dry, and pulverize through a 100-mesh sieve. Add small molecule peptides, compound enzymes, sodium carboxymethyl cellulose, and compound trace elements and mix evenly.

[0053] Comparative Example 1

[0054] A feed additive containing natural antibacterial ingredients comprises the following raw materials: 350 kg of ramie, 20 kg of chitosan quaternary ammonium salt, 20 kg of sodium alginate, 20 kg of pullulan polysaccharide, 30 kg of amino-terminated polyamide amine, 80 kg of nano-hydroxyapatite, 20 kg of lecithin, 30 kg of lactic acid, 15 kg of glucose, 15 kg of compound microbial agent, 10 kg of small molecule polypeptide, 8 kg of compound enzyme, 3 kg of sodium carboxymethyl cellulose, and 2 kg of compound trace elements.

[0055] The compound microbial agent includes: 6 × 10⁶ live bacteria. 9 Bacillus subtilis CFU / g, viable count 3 × 10⁻⁶ 9 Bacillus licheniformis CFU / g, viable count 8 × 10⁻⁶ 8 Bacillus amyloliquefaciens cfu / g, viable count 4 × 10⁻⁶ 8 The viable count of *Geotrichum dipodioides* was 3 × 10⁻⁶ cfu / g. 8 CFU / g of Rhizopus nigricans.

[0056] The preparation method of the above-mentioned feed additive includes the following steps: S1. Add chitosan quaternary ammonium salt to 300 kg of 0.8% acetic acid solution, adjust the pH of the system to 5-6 and stir evenly. Add sodium alginate and pullulan polysaccharide, stir evenly at 45℃, add amino-terminated polyamide amine and nano hydroxyapatite, stir at 300 r / min for 90 min, add lecithin and continue stirring for 20 min to obtain pretreated apatite. S2. Add 3.5 times the mass of water to ramie, add lactic acid, glucose, and compound bacterial agent, stir at 80 r / min for 9 days at a stirring temperature of 27℃, inactivate at high temperature, cool to room temperature, vacuum concentrate to 25% of the original volume, add pretreated apatite, stir evenly, sonicate for 90 min at a sonic frequency of 64 kHz, dropwise add to a 1.5% sodium tripolyphosphate solution, let stand for 20 min, collect microspheres, vacuum dry, pulverize and pass through a 100-mesh sieve; add small molecule peptides, compound enzymes, sodium carboxymethyl cellulose, and compound trace elements and mix evenly.

[0057] Comparative Example 2

[0058] A feed additive containing natural antibacterial ingredients comprises the following raw materials: 300 kg of ramie, 50 kg of sodium chloride, 20 kg of chitosan quaternary ammonium salt, 170 kg of nano hydroxyapatite, 30 kg of lactic acid, 15 kg of glucose, 15 kg of compound microbial agent, 10 kg of small molecule polypeptide, 8 kg of compound enzyme, 3 kg of sodium carboxymethyl cellulose, and 2 kg of compound trace elements.

[0059] The compound microbial agent includes: 6 × 10⁶ live bacteria. 9 Bacillus subtilis CFU / g, viable count 3 × 10⁻⁶ 9 Bacillus licheniformis CFU / g, viable count 8 × 10⁻⁶ 8 Bacillus amyloliquefaciens cfu / g, viable count 4 × 10⁻⁶ 8 The viable count of *Geotrichum dipodioides* was 3 × 10⁻⁶ cfu / g. 8 CFU / g of Rhizopus nigricans.

[0060] The preparation method of the above-mentioned feed additive includes the following steps: S1. Pulverize the ramie, pass it through an 80-mesh sieve, freeze it at -45℃ for 90 minutes, raise it to room temperature, add a 5% sodium chloride solution, let it stand for 7 hours, filter it, wash the filter residue twice, dry it at 70℃ to constant weight, pulverize it and pass it through a 100-mesh sieve to obtain pretreated ramie. S2. Add 3.5 times the mass of water to the pretreated ramie, along with lactic acid, glucose, and compound bacterial agent. Stir at 80 r / min for 9 days at 27℃. Inactivate at high temperature, cool to room temperature, and vacuum concentrate to 25% of the original volume. Add nano-hydroxyapatite, stir evenly, and sonicate for 90 min at 64 kHz. Dropwise add to a 1.5% sodium tripolyphosphate solution and let stand for 20 min. Collect the microspheres, vacuum dry, and pulverize through a 100-mesh sieve. Add small molecule peptides, compound enzymes, sodium carboxymethyl cellulose, and compound trace elements and mix evenly.

[0061] Comparative Example 3

[0062] A feed additive containing natural antibacterial ingredients comprises the following raw materials: 300 kg of ramie, 50 kg of sodium chloride, 20 kg of chitosan quaternary ammonium salt, 20 kg of sodium alginate, 20 kg of pullulan polysaccharide, 30 kg of amino-terminated polyamide amine, 80 kg of nano-hydroxyapatite, 20 kg of lecithin, 30 kg of lactic acid, 15 kg of glucose, 15 kg of compound microbial agent, 10 kg of small molecule polypeptide, 8 kg of compound enzyme, 3 kg of sodium carboxymethyl cellulose, and 2 kg of compound trace elements.

[0063] The compound microbial agent includes: 6 × 10⁶ live bacteria. 9 Bacillus subtilis CFU / g, viable count 3 × 10⁻⁶ 9 Bacillus licheniformis CFU / g, viable count 8 × 10⁻⁶ 8 CFU / g of Bacillus amyloliquefaciens.

[0064] The preparation method of the above-mentioned feed additive includes the following steps: S1. Pulverize the ramie, pass it through an 80-mesh sieve, freeze it at -45℃ for 90 minutes, raise it to room temperature, add a 5% sodium chloride solution, let it stand for 7 hours, filter it, wash the filter residue twice, dry it at 70℃ to constant weight, pulverize it and pass it through a 100-mesh sieve to obtain pretreated ramie. S2. Add chitosan quaternary ammonium salt to 300 kg of 0.8% acetic acid solution, adjust the pH of the system to 5-6 and stir evenly. Add sodium alginate and pullulan polysaccharide, stir evenly at 45℃, add amino-terminated polyamide amine and nano hydroxyapatite, stir at 300 r / min for 90 min, add lecithin and continue stirring for 20 min to obtain pretreated apatite. S3. Add 3.5 times the mass of water to the pretreated ramie, along with lactic acid, glucose, and compound bacterial agent. Stir at 80 r / min for 9 days at 27°C. Inactivate at high temperature, cool to room temperature, and vacuum concentrate to 25% of the original volume. Add pretreated apatite, stir evenly, and sonicate for 90 min at 64 kHz. Dropwise add to a 1.5% sodium tripolyphosphate solution and let stand for 20 min. Collect the microspheres, vacuum dry, and pulverize through a 100-mesh sieve. Add small molecule peptides, compound enzymes, sodium carboxymethyl cellulose, and compound trace elements and mix evenly.

[0065] The feed additives obtained in Example 5 and Comparative Examples 1-3 were added to chicken feed. The mass ratio of feed additives to chicken feed was 4:100. The chicken feed, by mass percentage, included: corn 58.70%, soybean meal 33.90%, corn oil 3.50%, methionine 0.10%, dicalcium phosphate 1.30%, salt 0.20%, limestone powder 1.30%, and premix 1.00%.

[0066] The chicken feed with added feed additives was used in a feeding experiment, as follows: This experiment was conducted in a large chicken farm in Wuhan. 42-day-old AA commercial broilers (half male, half female) were used. Natural ventilation and artificial lighting (16L:8D) were employed. During the experiment, the temperature in the chicken house was 28±2℃, and the relative humidity was 55-62%. The experiment was divided into 5 groups: a control group (using chicken feed without added feed additives), Example 5 group, Comparative Example 1 group, Comparative Example 2 group, and Comparative Example 3 group. Each group had 3 replicates, with 100 chickens per replicate. The pre-trial period was 7 days, and the formal trial period was 42 days.

[0067] After the trial period ended, the daily weight gain and average daily feed intake during the trial period were statistically calculated, and the feed conversion ratio was further calculated.

[0068] Feed conversion ratio = Daily feed intake ÷ Daily weight gain × 100%.

[0069] like Figure 1 As shown, the feed conversion ratio of broilers in Example 5 was the lowest, significantly better than the other groups.

[0070] During the trial period, 10 roosters and 10 hens were randomly selected from each group at different time points. After bleeding and slaughtering, and skinning, 0.2g of the left cecal chyme was weighed in a clean bench and diluted with 1mL of sterile water to create nine gradients. The chyme was then spread onto MRS agar for Lactobacillus, BS agar for Bifidobacterium, MacConkey agar for Escherichia coli, and bismuth sulfite agar for Salmonella, respectively. Each gradient was repeated in triplicate. Lactobacillus and Bifidobacterium were incubated at 37°C in an anaerobic incubator for 2 days, while Escherichia coli and Salmonella were incubated at 37°C in a CO2 incubator for 1 day. Plate counts were then performed.

[0071] like Figures 2 to 5 As shown, in Example 5, the number of Escherichia coli and Salmonella colonies in the cecal chyme of broilers was the lowest, while the number of Lactobacillus and Bifidobacterium colonies was the highest, significantly better than the comparative group and the blank group. This confirms that the feed additive obtained by the present invention can effectively inhibit the growth of intestinal pathogens and maintain the balance of intestinal beneficial bacteria.

[0072] The reasons for the above results are as follows: In this invention, the ramie cell structure is disrupted and the crystallinity of macromolecules is reduced after pretreatment with a freezing-sodium chloride solution. This, combined with the action of a compound microbial agent, promotes the release of antibacterial active ingredients such as flavonoids and ramie polysaccharides. Furthermore, the combination of *Geotrichum candida* and *Rhizopus nigricans* produces secondary metabolites that synergistically inhibit the growth of intestinal pathogens while maintaining the balance of beneficial intestinal flora. This invention utilizes pretreated apatite to encapsulate the active ingredients, combined with a water-retaining agent. This not only slows the passage of digesta and prolongs the action time of enzymatic hydrolysis products, but also, with the help of a compound enzyme, continuously degrades non-starch polysaccharides in ramie under suitable humidity, releasing soluble sugars and providing additional energy for animals. Additionally, the positive charge of chitosan quaternary ammonium salt interacts electrostatically with the negative charge of the intestinal mucosa, enhancing the adhesion of microspheres in the intestine and further prolonging the action time of the active ingredients.

[0073] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A feed additive containing natural antibacterial ingredients, characterized in that, The raw materials, by weight, include: 200-400 parts ramie, 20-90 parts sodium chloride, 10-30 parts chitosan quaternary ammonium salt, 10-30 parts sodium alginate, 10-30 parts pullulan polysaccharide, 10-50 parts amino-terminated polyamide amine, 50-100 parts nano hydroxyapatite, 10-30 parts lecithin, 10-50 parts lactic acid, 10-20 parts glucose, 10-20 parts compound bacterial agent, 5-15 parts small molecule polypeptides, 5-10 parts compound enzymes, 1-5 parts water-retaining agent, and 1-3 parts compound trace elements.

2. The feed additive according to claim 1, characterized in that, The compound microbial agent includes: Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Diplocos globosum, and Rhizopus nigricans.

3. The feed additive according to claim 2, characterized in that, The viable count of Bacillus subtilis is 3-9 × 10⁻⁹. 9 CFU / g, viable count of Bacillus licheniformis is 2-4 × 10⁻⁴. 9 The cfu / g count of Bacillus amyloliquefaciens was 7-9 × 10⁻⁶. 8 The cfu / g count of *Geotrichum candida* was 2-6 × 10⁻⁶. 8 CFU / g, viable count of Rhizopus nigricans is 1-5 × 10⁻⁶. 8 cfu / g.

4. The feed additive according to claim 1, characterized in that, Small molecule polypeptides are mixtures of oligopeptides with a molecular weight of <1000 Da.

5. The feed additive according to claim 1, characterized in that, Complex enzymes include: Cellulase, xylanase, β-glucanase.

6. The feed additive according to claim 5, characterized in that, The activity of cellulase is 6000-8000 U / g, the activity of xylanase is 3000-5000 U / g, and the activity of β-glucanase is 1000-4000 U / g.

7. The feed additive according to claim 1, characterized in that, The water-retaining agent is sodium carboxymethyl cellulose; the complex trace elements are an organic compound containing calcium, magnesium, zinc and selenium.

8. A method for preparing a feed additive as described in any one of claims 1-7, characterized in that, Includes the following steps: S1. Pulverize ramie, freeze at -40 to -50℃ for 1-2 hours, bring to room temperature, add sodium chloride solution and let stand for 4-10 hours, filter, wash the filter residue, dry to constant weight, and pulverize to obtain pretreated ramie; S2. Add chitosan quaternary ammonium salt to acetic acid solution, adjust the pH of the system to 5-6 and stir evenly. Add sodium alginate and pullulan polysaccharide, stir evenly at 40-50℃, add amino-terminated polyamide and nano hydroxyapatite and stir for 1-2 hours. Add lecithin and continue stirring for 10-30 minutes to obtain pretreated apatite. S3. Add water to the pretreated ramie, then add lactic acid, glucose, and compound bacterial agent. Stir at 25-28℃ for 6-12 days, inactivate at high temperature, cool to room temperature, and vacuum concentrate to 20-30% of the original volume. Add pretreated apatite, stir evenly, and sonicate for 1-2 hours. Dropwise add to sodium tripolyphosphate solution and let stand for 10-30 minutes. Collect microspheres, vacuum dry, and pulverize. Add small molecule peptides, compound enzymes, water-retaining agents, and compound trace elements and mix evenly.

9. The preparation method according to claim 8, characterized in that, In S3, the ultrasonic frequency is 60-70kHz.

10. The preparation method according to claim 8, characterized in that, In S1, the mass fraction of sodium chloride solution is 4-6%; in S2, the mass fraction of acetic acid solution is 0.5-1%; and in S3, the mass fraction of sodium tripolyphosphate solution is 1-2%.

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