Construction method of livestock and poultry indigenous probiotics and application of livestock and poultry indigenous probiotics in antibiotic-free feed

Abstract

The invention relates to a construction method of livestock and poultry indigenous probiotics and application of the livestock and poultry indigenous probiotics in antibiotic-free feed.The construction method comprises the steps that homologous initial strains are separated from corresponding digestive tract parts of healthy livestock and poultry, adaptive domestication is conducted in the simulated digestive tract environment, candidate bacteria are screened with the inhibition zone diameter larger than or equal to 11 mm as the standard, and the livestock and poultry indigenous probiotics are obtained. Culture conditions are optimized through metabolic engineering, a digestive tract targeting expression system is constructed by utilizing an environmental signal response type promoter, and livestock and poultry origin probiotics capable of specifically expressing the antibacterial peptide are obtained through genetic transformation. The livestock and poultry origin probiotics are further embedded by a sodium alginate-chitosan double-layer microcapsule to prepare a preparation of which the survival rate in gastric acid is greater than 95%. According to the present invention, the antibiotic-free feed is added according to the addition amount of 0.05-0.5%, such that the daily gain of livestock and poultry is improved, the diarrhea rate is reduced, the nitrogen emission is reduced, the long-acting digestive tract field planting is achieved, the systematic process from the strain directional breeding and the intelligent function design to the terminal product application is achieved, and the effective solution is provided for the antibiotic-free healthy breeding.

Description

Technical Field

[0001] This invention relates to the fields of agricultural biotechnology and feed processing technology, and in particular to a method for constructing probiotics native to livestock and poultry and their application in antibiotic-free feed. Background Technology

[0002] Animal husbandry is a pillar industry of my country's agricultural economy, with pig farming being of paramount importance. However, the industry currently faces two major challenges: first, disease control and antibiotic dependence. Highly lethal diseases such as African swine fever pose a significant threat, while the traditional reliance on antibiotics leads to drug resistance and residue risks; second, environmental pressure and manure treatment difficulties. Large-scale farming generates massive amounts of manure, and traditional treatment methods are inefficient and prone to secondary pollution. Probiotic technology, as a key pathway to replace antibiotics and promote ecological farming, suffers from significant shortcomings in existing products: the strains used are mostly derived from human sources or laboratories, resulting in poor colonization in the digestive tract of livestock and poultry, essentially becoming "transit bacteria"; their functional expression lacks intelligent responses to the digestive environment, and ordinary formulations cannot effectively protect the strains from the high temperatures of feed processing and the stomach acid of animals, leading to a sharp drop in viable bacteria count and efficacy after feeding. Existing technologies mostly treat animal health and manure treatment in isolation, lacking a systematic solution from the feed end to the environmental end. Summary of the Invention

[0003] The main objective of this invention is to provide a method for constructing probiotics native to livestock and poultry and their application in antibiotic-free feed. By integrating local strain screening, intelligent targeted genetic engineering modification, and double-layer microcapsule protection technology, this invention aims to construct a highly efficient, stable probiotic feed system native to livestock and poultry that can completely replace antibiotics, ultimately achieving the goals of ensuring livestock and poultry health, improving breeding efficiency, and promoting environmentally friendly antibiotic-free farming.

[0004] To achieve the above objectives, the present invention provides a method for constructing probiotics derived from livestock and poultry, comprising the following steps: Contents were collected from a specific digestive tract region of target healthy livestock and poultry, and initial microbial strains were isolated; wherein, for pigs and poultry, the specific digestive tract region is the intestine, and for ruminants, the specific digestive tract region is the rumen; According to the target healthy livestock and poultry species, prepare the corresponding simulated digestive tract environment culture medium, and place the initial microbial strain under the corresponding simulated conditions for multiple generations of adaptive subculturing. In vitro antibacterial tests were conducted on microbial strains after adaptive culture to screen strains with inhibition zones against Staphylococcus aureus and Escherichia coli as candidate probiotics. Metabolic engineering techniques were used to regulate the expression of genes related to carbon and nitrogen source utilization in the selected probiotic candidates, and the culture conditions of the selected probiotic candidates were optimized. An expression vector containing a digestive tract-specific promoter and an antimicrobial peptide encoding gene was constructed, and the expression vector was introduced into the candidate probiotics through genetic transformation to obtain a livestock-derived probiotic that can specifically express antimicrobial peptides in response to the digestive tract environment.

[0005] Furthermore, the preparation of the culture medium for the simulated digestive tract environment includes: simulating the digestive tract of livestock and poultry with a pH of 5.0 to 7.0 and a bile acid concentration of 0.05% to 0.3%; simulating the intestinal tract of poultry with a pH of 6.0 to 7.5 and a bile acid concentration of 0.05% to 0.2%; and simulating the rumen of ruminants with a pH of 5.5 to 7.0 and a total volatile fatty acid content of 60 mM to 120 mM.

[0006] Furthermore, in the in vitro antibacterial test, the diameter of the inhibition zone against Staphylococcus aureus or Escherichia coli is not less than 11 mm.

[0007] Furthermore, the metabolic engineering methods include: controlling the culture temperature between 35°C and 42°C, and optimizing the types and concentrations of carbon and nitrogen sources in the culture medium.

[0008] Furthermore, based on the target livestock and poultry species, for pigs or poultry, the livestock and poultry digestive tract-specific promoter is a promoter that is responsive to bile acids; or for ruminants, the livestock and poultry digestive tract-specific promoter is a pH-responsive promoter.

[0009] Furthermore, the genetic transformation method is electroporation or chemical transformation.

[0010] The present invention also provides an antibiotic-free feed, comprising the livestock and poultry native probiotics obtained by the screening method, wherein the livestock and poultry native probiotics are subjected to microcapsule encapsulation treatment with a sodium alginate inner layer and a chitosan outer layer.

[0011] Furthermore, the probiotics derived from livestock and poultry are isolated from the digestive tract contents of healthy animals; specifically, for pigs and poultry, they are isolated from intestinal contents; and for ruminants, they are isolated from rumen contents.

[0012] Further, by weight percentage, the antibiotic-free feed comprises: 45%–75% energy feed ingredients, 9%–30% plant protein feed ingredients, 2%–15% wheat bran or bran-like ingredients, 3%–10% premix of essential nutrients for livestock and poultry, and 0.05%–0.5% microcapsule preparation of the probiotics derived from livestock and poultry. The energy feed ingredients include one or more of corn, wheat, and barley; and / or the plant protein feed ingredients include one or more of soybean meal, rapeseed meal, cottonseed meal, and protein grass.

[0013] Furthermore, the microcapsule encapsulation is prepared by the following steps: mixing the probiotics derived from livestock and poultry with a sodium alginate solution of 1% to 3%, then dripping the mixture into a calcium chloride solution to form an inner layer of gel microspheres, and then placing the gel microspheres in a chitosan solution of pH 4.0 to 5.0 and a concentration of 0.5% to 1.5% to form an outer layer coating structure.

[0014] The method for constructing probiotics native to livestock and poultry and its application in antibiotic-free feed provided by this invention have the following beneficial effects: This invention directly isolates probiotics from the digestive tract of healthy livestock and poultry. The obtained native strains are naturally adapted to the host environment and can achieve long-term colonization for more than 28 days, fundamentally solving the problem of difficult colonization of exogenous bacteria. A specific environmental signal-responsive expression system constructed through genetic engineering enables the probiotics to efficiently secrete antibacterial substances only in a specific digestive tract environment, achieving precise targeting and intelligent control of function, thus improving the safety and effectiveness of the effect. The use of sodium alginate-chitosan double-layer microcapsule encapsulation technology provides robust protection for the probiotics, ensuring a survival rate of over 95% in feed processing and gastric acid environments, guaranteeing sufficient live bacteria to reach the specific digestive tract site and exert a sustained-release effect. Antibiotic-free feed using this probiotic preparation has been validated through feeding and shows significant improvement in livestock and poultry health and growth performance, increasing weight gain, reducing diarrhea rates, and improving fecal formation rates, thereby reducing ammonia concentration in the barn and improving the farming environment from the source. Attached Figure Description

[0015] Figure 1 This is a flowchart illustrating a method for constructing probiotics derived from livestock and poultry in one embodiment of the present invention. Figure 2 This is a schematic diagram of the process for preparing antibiotic-free feed in one embodiment of the present invention; The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0017] Reference Figure 1 This invention proposes a method for constructing probiotics derived from livestock and poultry, comprising the following steps: S1, collect contents from a specific digestive tract site of the target healthy livestock and poultry, and isolate the initial microbial strain; wherein, for pigs and poultry, the specific digestive tract site is the intestine, and for ruminants, the specific digestive tract site is the rumen; S2, according to the type of target healthy livestock and poultry, prepare the corresponding simulated digestive tract environment culture medium, and place the initial microbial strain under the corresponding simulated conditions for multi-generation adaptive subculturing; S3. In vitro antibacterial tests were conducted on microbial strains after adaptive culture to screen strains with inhibition zones against Staphylococcus aureus and Escherichia coli as candidate probiotics. S4. Using metabolic engineering techniques, the expression of genes related to carbon and nitrogen source utilization in the selected probiotics is regulated, and the culture conditions of the selected probiotics are optimized. S5. Construct an expression vector containing a livestock and poultry digestive tract-specific promoter and an antimicrobial peptide encoding gene, and introduce the expression vector into the candidate probiotics through genetic transformation to obtain livestock and poultry-derived probiotics that can specifically express antimicrobial peptides in response to the digestive tract environment.

[0018] The preparation of the culture medium for the simulated digestive tract environment includes: simulating the digestive tract of livestock and poultry with a pH of 5.0 to 7.0 and a bile acid concentration of 0.05% to 0.3%; simulating the intestinal tract of poultry with a pH of 6.0 to 7.5 and a bile acid concentration of 0.05% to 0.2%; and simulating the rumen of ruminants with a pH of 5.5 to 7.0 and a total volatile fatty acid content of 60 mM to 120 mM.

[0019] In the in vitro antibacterial test, the diameter of the inhibition zone against Staphylococcus aureus or Escherichia coli is not less than 11 mm.

[0020] The metabolic engineering methods include: controlling the culture temperature between 35°C and 42°C, and optimizing the types and concentrations of carbon and nitrogen sources in the culture medium.

[0021] Based on the target livestock and poultry species, for pigs or poultry, the livestock and poultry digestive tract specific promoter is a promoter that is responsive to bile acids; or for ruminants, the livestock and poultry digestive tract specific promoter is a pH-responsive promoter.

[0022] The genetic transformation method is either electroporation or chemical transformation.

[0023] Example 1, This embodiment provides a method for constructing probiotics specific to the intestinal (or rumen) environment of livestock and poultry such as pigs, poultry, and ruminants. The aim is to obtain probiotic strains that can efficiently colonize the digestive tract of the target animal and exert targeted antibacterial functions. This method organically combines multiple steps, including isolating strains from specific digestive tract sites of the target animal, adaptive acclimatization to the intestinal environment, screening based on clearly defined antibacterial indicators, metabolic engineering optimization, and targeted genetic engineering modification, ultimately obtaining high-performance probiotics native to livestock and poultry. The specific steps are as follows: Step S1: Aseptically collect samples from specific digestive tract sites of healthy individuals according to the target animal species: for pigs and poultry, collect intestinal contents; for ruminants such as cattle and sheep, collect rumen contents. After serially diluting the samples with sterile phosphate-buffered saline (PBS), plate them onto MRS agar plates (suitable for lactic acid bacteria screening) or brain heart extract (BHI) agar plates (suitable for broad-spectrum bacterial screening), and incubate them anaerobically at 37°C for 48 hours. Select morphologically diverse and well-grown single colonies for purification and culture to obtain the initial microbial strain library. Step S2: To improve the adaptability of the strains to the complex environment of the livestock and poultry digestive tract, perform targeted acclimatization on the isolated initial strains. Depending on the target animal species, prepare appropriate culture media simulating the digestive tract environment: For pigs and poultry, use MRS broth as a base, adjusting the initial pH of the culture medium to 5.0–7.0 (simulated pigs) or 6.0–7.5 (simulated poultry) using hydrochloric acid or sodium hydroxide solution, and adding 0.05%–0.3% (simulated pigs) or 0.05%–0.2% (simulated poultry) of a mixture of pig (or poultry) bile salts. For ruminants, prepare a culture medium simulating the rumen environment, adjusting its pH to 5.5–7.0, and adding a mixture of volatile fatty acids to achieve a total concentration of 60 mM–120 mM. Inoculate the purified initial strain into this acclimation medium and culture it in an anaerobic workstation at 37°C (or create an anaerobic environment using anaerobic culture bags). Every 24–48 hours, transfer the inoculum to fresh, identical acclimation medium at a rate of 1%–5%, and continue subculturing for at least 20 generations. This process applies stable environmental selection pressure (acidic pH, bile acids, anaerobic conditions) to enrich individuals with stronger adaptability in the bacterial community, thereby completing multiple generations of adaptive subculturing to obtain domesticated strains.

[0024] Step S3 involves preliminary screening of the antibacterial efficacy of each strain after acclimatization in Step S2 to assess their potential to inhibit common pathogens. An in vitro antibacterial test was conducted using the agar diffusion method (perforation method): The activated bacterial suspensions of indicator bacteria (Staphylococcus aureus ATCC25923 and Escherichia coli ATCC 25922) were adjusted to 0.5 McFarland turbidity and evenly spread onto MHA agar plates. Perforations were made on the plates, and 100 μL of the centrifuged, sterilized fermentation supernatant of the test strain was added to each plate. The plates were incubated at 37°C for 18-24 hours, and the diameter of the inhibition zone was measured. Strains that produced clear inhibition zones against one or both of the above indicator bacteria, with inhibition zone diameters not less than 11 mm, were selected as candidate probiotics with potential beneficial functions. For example, a strain was screened with an inhibition zone diameter of 12.50±0.10 mm against Staphylococcus aureus and 11.63±0.15 mm against Escherichia coli.

[0025] Step S4 involves optimizing the culture conditions and physiological regulation of candidate probiotics to further enhance their growth performance and antimicrobial substance synthesis capabilities. Culture conditions were optimized by culturing the selected candidate probiotics at different temperatures (35℃, 37℃, 39℃, and 42℃), measuring their growth curves and the antimicrobial activity of the fermentation supernatant, and determining the optimal temperature range (35℃ to 42℃) for growth and antimicrobial substance production. Culture medium composition was optimized by systematically optimizing the types and concentrations of carbon sources (such as glucose, lactose, and sucrose) and nitrogen sources (such as peptone, yeast extract, and soybean peptides) based on the determined optimal temperature, using methods such as single-factor and orthogonal experiments. The optimal carbon and nitrogen source combination and ratio that maximizes bacterial growth and antimicrobial peptide secretion were determined by measuring indicators such as bacterial biomass (OD600), pH changes, and inhibition zone diameter. This step aims to optimize the culture conditions and maximize functionality by regulating nutrient supply and optimizing the metabolic flux of the strains.

[0026] Step S5 involves precise genetic engineering to ensure the probiotic's antimicrobial function is specifically activated within the target digestive tract and to prevent ineffective expression in non-target sites such as the stomach. Promoter selection and vector construction: Based on the target animal species, a digestive tract-specific promoter is selected. For pigs or poultry, a bile acid-responsive promoter with high sensitivity and activity to bile acids is selected as the intestinal-specific promoter. For example, the bile acid-sensitive promoter P16090 from *Lactobacillus*. For ruminants, a pH-responsive promoter is selected as the rumen-specific promoter. This promoter is linked to one or more genes encoding broad-spectrum antimicrobial peptides (such as bacteriocins) to construct an expression cassette. This expression cassette is then cloned into an expression vector (such as a shuttle plasmid or an integrative plasmid) that can stably replicate or integrate into the target host bacteria, completing the expression vector construction. Genetic transformation and engineered strain acquisition: The constructed recombinant expression vector is introduced into the candidate probiotic cells optimized in step S4 using genetic transformation methods such as electroporation or chemical transformation. Specifically, electroporation can be used: prepared competent cells are mixed with recombinant plasmid DNA, and electroporation is performed under specific voltage and capacitance parameters to allow the DNA to enter the cells. Alternatively, a chemical transformation method suitable for this strain (such as the CaCl2 method) can be used. The transformed cells are plated on selection plates containing the corresponding antibiotics, and positive clones are picked. PCR verification and sequencing confirm the correct integration or presence of the expression cassette. Finally, a genetically engineered strain—a probiotic derived from livestock and poultry—is obtained. This strain is characterized by the activation of its specific promoter in the target digestive tract (such as the intestine containing bile acids or the rumen at a specific pH), driving the specific expression of downstream antimicrobial peptide genes, thereby efficiently secreting antimicrobial substances at the target site (intestine); while in non-target environments, this expression system remains silent, achieving targeted functional expression.

[0027] This embodiment designs differentiated strain isolation sources and simulated domestication conditions for different livestock and poultry species (pigs, poultry, and ruminants). By isolating from the source (the specific digestive tract of healthy livestock and poultry), the natural affinity of the obtained strains to the target host is ensured. Using an inhibition zone diameter of not less than 11 mm as a quantitative screening criterion ensures that the selected candidate bacteria possess clear in vitro antibacterial functions. Metabolic engineering optimization further explores the production potential of the strains. By introducing specific promoters (bile acid-responsive or pH-responsive) that match the digestive tract environment of the target animal to construct a targeted expression system, the technical problems of non-specific functional expression and easy inactivation in the stomach in traditional probiotics are overcome. This achieves "on-demand, on-site" expression of antibacterial function, improving the efficiency and safety of probiotic application.

[0028] Reference Figure 2 This is a schematic diagram of the process for preparing antibiotic-free feed in one embodiment of the present invention. It includes an antibiotic-free feed prepared using probiotics derived from livestock and poultry, wherein the probiotics are encapsulated in microcapsules having an inner layer of sodium alginate and an outer layer of chitosan.

[0029] The probiotics derived from livestock and poultry are isolated from the digestive tract contents of healthy animals; specifically, for pigs and poultry, they are isolated from intestinal contents; and for ruminants, they are isolated from rumen contents.

[0030] The antibiotic-free feed comprises: 45%–75% energy feed ingredients, 9%–30% plant protein feed ingredients, 2%–15% wheat bran or bran-like ingredients, 3%–10% premix of essential nutrients for livestock and poultry, and 0.05%–0.5% microencapsulated preparations of the probiotics native to livestock and poultry. The energy feed ingredients include one or more of corn, wheat, and barley; and / or the plant protein feed ingredients include one or more of soybean meal, rapeseed meal, cottonseed meal, and protein grass.

[0031] The microcapsule encapsulation is prepared by the following steps: the probiotics derived from livestock and poultry are mixed with a sodium alginate solution with a concentration of 1% to 3%, and then dropped into a calcium chloride solution to form an inner layer of gel microspheres. The gel microspheres are then placed in a chitosan solution with a pH of 4.0 to 5.0 and a concentration of 0.5% to 1.5% to form an outer layer coating structure.

[0032] Example 2: This embodiment provides a method for preparing highly efficient and stable antibiotic-free feed and microencapsulated probiotics native to livestock and poultry. The feed is prepared by processing the probiotics native to livestock and poultry with targeted antibacterial function obtained in Example 1 using a specific microencapsulation technique, and then compounding them with scientifically proportioned conventional feed ingredients. The aim is to achieve high survival rates and targeted release of the probiotics into the digestive tract during feed processing, storage, and animal feeding, thereby effectively replacing antibiotics and promoting healthy growth at the livestock end. The specific steps for preparing the microencapsulated probiotic formulation are as follows: The fermentation broth of the livestock and poultry-derived probiotics obtained in Example 1 (which are native strains isolated from the corresponding digestive tracts of healthy animals and genetically engineered to possess specific responsive (e.g., bile acid responsive or pH responsive) antimicrobial peptide expression characteristics) was collected by centrifugation, and the cells were resuspended in sterile physiological saline to prepare a high-concentration bacterial suspension (e.g., 10^9-10^10 CFU / mL). Inner layer sodium alginate gel microsphere formation: A sodium alginate solution with a concentration of 1% to 3% (w / v) was prepared, and 0% to 5% (w / v) of prebiotics (e.g., fructooligosaccharides) could be selectively added to provide nutrients for the subsequent growth of the strain. The bacterial suspension and sodium alginate solution were mixed evenly at a volume ratio of 1:9 to 3:7 to obtain a bacterial-sodium alginate mixture. Using a syringe or dropper, the mixture was dripped into a 1.5% (w / v) calcium chloride (CaCl2) solution at a constant rate. Sodium alginate undergoes ionic cross-linking with calcium ions, instantly forming a robust gel network on the droplet surface, encapsulating probiotics and forming uniformly sized inner gel microspheres. After standing and solidifying for 10-15 minutes, the microspheres are collected by filtration and gently rinsed with sterile deionized water to remove residual CaCl2 solution. Outer chitosan coating: Prepare a 0.5%-1.5% (w / v) chitosan solution, adjusting the pH to 4.0-5.0 using dilute acetic acid. Transfer the obtained sodium alginate gel microspheres to the chitosan solution and gently stir for 10-20 minutes. During this time, positively charged chitosan molecules tightly bind to the negatively charged sodium alginate gel microsphere surface through electrostatic adsorption, forming a dense outer coating structure. The coated microcapsules are collected by filtration and gently rinsed with sterile deionized water. Drying and Storage: The moistened microcapsules were placed in a fluidized bed dryer and dried under a low-temperature airflow at a temperature range of 40-80℃ until the moisture content was below 10%. The final product was a dried microencapsulated probiotic preparation with a sodium alginate inner layer and a chitosan outer layer. This bilayer structure effectively resists the acidic environment of the stomach. Testing showed a survival rate of over 95% in simulated gastric juice (pH 2.0, 2 hours) and slow disintegration releasing live bacteria in simulated intestinal juice, achieving targeted delivery and long-term colonization in the digestive tract (colonization time can reach over 28 days).

[0033] Formulating and processing antibiotic-free feed: The above-mentioned microencapsulated probiotic preparations are mixed with bulk feed ingredients in an optimized ratio to prepare complete antibiotic-free feed.

[0034] Formula composition (by weight): Energy feed ingredients: 60% (selected from one or more of corn, wheat, and barley, with an adjustment range of 45% to 75%), providing the main energy.

[0035] Plant protein feed ingredients: 20% (selected from one or more of soybean meal, rapeseed meal, cottonseed meal, and forage protein, with an adjustment range of 9% to 30%), providing high-quality plant protein.

[0036] Wheat bran or bran-like raw materials: 8% (adjustment range 2%~15%), providing dietary fiber and some phosphorus source.

[0037] Fish lysate protein or similar animal protein source: 2% (range 0%~5%, selected in this example) to improve palatability and amino acid balance.

[0038] Lysine (e.g.): 0.2% (range 0%~0.5%), supplementing limiting amino acids.

[0039] Essential nutrients premix for livestock and poultry (vitamins, minerals, etc.): 1.0% (adjustment range 3%~10%), which contains at least calcium, phosphorus, vitamins and trace elements to meet the basic nutritional needs of animals.

[0040] Animal and poultry native probiotic microcapsule preparation: 0.1% (range 0.05%~0.5%), is the core functional additive of this invention.

[0041] Processing technology flow: Raw material crushing: Crush raw materials such as corn and soybean meal to a suitable particle size (pass through a 40-mesh sieve).

[0042] Mixing and stirring: Put all raw materials (including microcapsule probiotic preparations) into a biaxial paddle mixer and mix thoroughly and evenly for 3-5 minutes. The coefficient of variation (CV) of the mixing uniformity should be ≤7%.

[0043] Steam conditioning: The mixed materials are fed into a conditioner and saturated steam is introduced to condition the materials at 75-80℃ (range 70-85℃) for 3-4 minutes to improve starch gelatinization and kill some pathogenic microorganisms.

[0044] Extrusion pelleting: The conditioned material is extruded through a ring die pellet mill under appropriate pressure to produce pellet feed with a diameter of 3-4 mm.

[0045] Cooling: Hot pellet feed is cooled to room temperature (within ±3℃ of ambient temperature) by a counter-current cooler to reduce moisture and prevent mold growth.

[0046] (Optional) Post-probiotic spraying: To maximize the protection of probiotic activity, the microcapsule probiotic preparation can be dispersed in a small amount of vegetable oil to form a suspension, which is then evenly sprayed onto the surface of the pellets after the pellet feed has cooled using a precision spraying device.

[0047] Low-temperature drying: Dry the feed at a low temperature of 45-80℃ to a safe moisture content (≤12.5%), and then package and store it.

[0048] This embodiment utilizes microencapsulation double-layer encapsulation technology to significantly enhance the probiotics' tolerance to the high temperatures and harsh acidic environment of feed processing (survival rate >95%), and also achieves intelligent release in the intestine. The probiotics, derived from the corresponding digestive tract contents of the target animal, naturally possess stronger intestinal mucosal adhesion and colonization capabilities (lasting for more than 28 days). The optimized feed formulation ensures basic nutrition, while the addition amount of 0.05%-0.5% controls costs while guaranteeing effectiveness. Feeding trials show that using antibiotic-free feed can significantly improve the health status of pigs, poultry, and other animals (e.g., significantly reduce diarrhea rates), enhance growth performance (e.g., increase daily weight gain), and reduce ammonia concentration in feces (<20ppm), truly achieving the goal of "using bacteria to control bacteria, healthy farming," and responding to the strategic needs of green biomanufacturing and healthy farming.

[0049] In summary, this invention relates to a method for constructing probiotics native to livestock and poultry and their application in antibiotic-free feed. The method includes isolating homologous initial strains from corresponding digestive tract sites of healthy livestock and poultry, adaptively acclimating them in a simulated digestive tract environment, screening candidate strains based on an inhibition zone diameter ≥11mm, optimizing culture conditions through metabolic engineering, constructing a digestive tract-targeted expression system using an environmental signal-responsive promoter, and obtaining livestock and poultry native probiotics that specifically express antimicrobial peptides through genetic transformation. These probiotics are further encapsulated in sodium alginate-chitosan bilayer microcapsules to prepare a formulation with a survival rate >95% in gastric acid. This formulation is added to antibiotic-free feed at a dosage of 0.05-0.5% to improve daily weight gain, reduce diarrhea rate, reduce nitrogen emissions, and achieve long-term digestive tract colonization. This method realizes a systematic process from targeted strain selection and intelligent functional design to end-product application, providing an effective solution for antibiotic-free healthy farming.

[0050] The above description is only a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for constructing probiotics derived from livestock and poultry, characterized in that, Includes the following steps: Contents were collected from a specific digestive tract region of target healthy livestock and poultry, and initial microbial strains were isolated; wherein, for pigs and poultry, the specific digestive tract region is the intestine, and for ruminants, the specific digestive tract region is the rumen; According to the target healthy livestock and poultry species, prepare the corresponding simulated digestive tract environment culture medium, and place the initial microbial strain under the corresponding simulated conditions for multiple generations of adaptive subculturing. In vitro antibacterial tests were conducted on microbial strains after adaptive culture to screen strains with inhibition zones against Staphylococcus aureus and Escherichia coli as candidate probiotics. Metabolic engineering techniques were used to regulate the expression of genes related to carbon and nitrogen source utilization in the selected probiotic candidates, and the culture conditions of the selected probiotic candidates were optimized. An expression vector containing a digestive tract-specific promoter and an antimicrobial peptide encoding gene was constructed, and the expression vector was introduced into the candidate probiotics through genetic transformation to obtain a livestock-derived probiotic that can specifically express antimicrobial peptides in response to the digestive tract environment.

2. The method for constructing probiotics of livestock and poultry origin according to claim 1, characterized in that, The preparation of the culture medium for the simulated digestive tract environment includes: simulating the digestive tract of livestock and poultry with a pH of 5.0 to 7.0 and a bile acid concentration of 0.05% to 0.3%; simulating the intestinal tract of poultry with a pH of 6.0 to 7.5 and a bile acid concentration of 0.05% to 0.2%; and simulating the rumen of ruminants with a pH of 5.5 to 7.0 and a total volatile fatty acid content of 60 mM to 120 mM.

3. The method for constructing probiotics derived from livestock and poultry according to claim 1, characterized in that, In the in vitro antibacterial test, the diameter of the inhibition zone against Staphylococcus aureus or Escherichia coli is not less than 11 mm.

4. The method for constructing probiotics derived from livestock and poultry according to claim 1, characterized in that, The metabolic engineering methods include: controlling the culture temperature between 35°C and 42°C, and optimizing the types and concentrations of carbon and nitrogen sources in the culture medium.

5. The method for constructing probiotics derived from livestock and poultry according to claim 1, characterized in that, Based on the target livestock and poultry species, for pigs or poultry, the livestock and poultry digestive tract specific promoter is a promoter that is responsive to bile acids; or for ruminants, the livestock and poultry digestive tract specific promoter is a pH-responsive promoter.

6. The method for constructing probiotics derived from livestock and poultry according to claim 1, characterized in that, The genetic transformation method is either electroporation or chemical transformation.

7. An antibiotic-free feed, characterized in that, The product comprises livestock and poultry-derived probiotics obtained by the screening method according to any one of claims 1-6, wherein the livestock and poultry-derived probiotics are subjected to microcapsule encapsulation treatment having a sodium alginate inner layer and a chitosan outer layer.

8. The antibiotic-free feed according to claim 7, characterized in that, The probiotics derived from livestock and poultry are isolated from the digestive tract contents of healthy animals; specifically, for pigs and poultry, they are isolated from intestinal contents; and for ruminants, they are isolated from rumen contents.

9. The antibiotic-free feed according to claim 7, characterized in that, By weight percentage, the antibiotic-free feed comprises: 45%–75% energy feed ingredients, 9%–30% plant protein feed ingredients, 2%–15% wheat bran or bran-like ingredients, 3%–10% premix of essential nutrients for livestock and poultry, and 0.05%–0.5% microcapsule preparation of the probiotics native to livestock and poultry. The energy feed ingredients include one or more of corn, wheat, and barley; and / or the plant protein feed ingredients include one or more of soybean meal, rapeseed meal, cottonseed meal, and protein forage.

10. The antibiotic-free feed according to claim 7, characterized in that, The microcapsule encapsulation is prepared by the following steps: the probiotics derived from livestock and poultry are mixed with a sodium alginate solution with a concentration of 1% to 3%, and then dropped into a calcium chloride solution to form an inner layer of gel microspheres. The gel microspheres are then placed in a chitosan solution with a pH of 4.0 to 5.0 and a concentration of 0.5% to 1.5% to form an outer layer coating structure.

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