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CN-122012557-A - Construction method of livestock and poultry indigenous probiotics and application of livestock and poultry indigenous probiotics in antibiotic-free feed

CN122012557ACN 122012557 ACN122012557 ACN 122012557ACN-122012557-A

Abstract

The invention relates to a construction method of livestock and poultry indigenous probiotics and application thereof in nonreactive feed, wherein the construction method comprises the steps of separating homologous initial strains from corresponding digestive tract parts of healthy livestock and poultry, performing adaptive domestication under simulated digestive tract environments, screening candidate bacteria by taking a bacteriostasis circle diameter of not less than 11mm as a standard, optimizing culture conditions through metabolic engineering, constructing a digestive tract targeting expression system by utilizing an environment signal responsive promoter, and obtaining the livestock and poultry indigenous probiotics capable of specifically expressing antibacterial peptides through genetic transformation. The livestock and poultry original probiotics are further embedded by sodium alginate-chitosan double-layer microcapsules, and the preparation with the survival rate of more than 95% in gastric acid is prepared. The antibiotic-free feed is added in an amount of 0.05-0.5%, so that daily gain of livestock and poultry is improved, diarrhea rate is reduced, nitrogen emission is reduced, long-acting digestive tract field planting is realized, a system flow from directional strain breeding and intelligent function design to application of terminal products is realized, and an effective solution is provided for antibiotic-free healthy cultivation.

Inventors

  • WU JIAYIN
  • ZHANG LIPING

Assignees

  • 深圳市瑞岷生物科技有限公司

Dates

Publication Date
20260512
Application Date
20260212

Claims (10)

  1. 1. The construction method of the domestic probiotics for livestock and poultry is characterized by comprising the following steps of: collecting the content from specific digestive tract parts of target healthy livestock and poultry, and separating to obtain initial microorganism strains, wherein the specific digestive tract parts are intestinal tracts for pigs and poultry, and rumen for ruminants; Preparing corresponding simulated digestive tract environment culture solution according to the types of target healthy livestock and poultry, and placing the initial microbial strain under corresponding simulated conditions for multi-generation adaptive subculture; Performing an in-vitro bacteriostasis test on the microbial strain subjected to the adaptive culture, and screening out strains with bacteriostasis areas on staphylococcus aureus and escherichia coli as candidate probiotics; Regulating and controlling the expression of the genes related to the carbon source and the nitrogen source utilization of the candidate probiotics by adopting a metabolic engineering means, and optimizing the culture conditions of the candidate probiotics; constructing an expression vector containing a specific promoter of the digestive tract of livestock and poultry and an antibacterial peptide coding gene, and introducing the expression vector into the candidate probiotics by a genetic transformation method to obtain the livestock and poultry original probiotics capable of specifically expressing the antibacterial peptide in response to the digestive tract environment.
  2. 2. The method for constructing the livestock and poultry original probiotic according to claim 1, wherein the preparation of the simulated digestive tract environment culture solution comprises the conditions of simulating the digestive tract of the livestock and poultry, wherein the pH value is 5.0-7.0, the concentration of bile acid is 0.05-0.3%, the conditions of simulating the intestinal tract of the livestock and poultry are the conditions of culturing the intestinal tract of the livestock and poultry, the pH value is 6.0-7.5, the concentration of bile acid is 0.05-0.2%, the conditions of culturing the rumen of the ruminant are the conditions of culturing the rumen of the ruminant, the pH value is 5.5-7.0, and the total amount of volatile fatty acid is 60 mM-120 mM.
  3. 3. The method for constructing the livestock and poultry original probiotics according to claim 1, wherein in the in-vitro bacteriostasis test, the diameter of a bacteriostasis ring for staphylococcus aureus or escherichia coli is not smaller than 11mm.
  4. 4. The method for constructing the natural probiotics for livestock and poultry according to claim 1, wherein the metabolic engineering means comprises the steps of controlling the culture temperature to be 35-42 ℃ and optimizing the types and the concentrations of carbon sources and nitrogen sources in a culture medium.
  5. 5. The method for constructing a native probiotic of livestock and poultry according to claim 1, wherein the livestock and poultry digestive tract specific promoter is a promoter having a response activity to bile acid for pigs or poultry or a pH-responsive promoter for ruminants based on the species of livestock and poultry of interest.
  6. 6. The method for constructing a natural probiotic of livestock and poultry according to claim 1, wherein the genetic transformation method is an electroporation method or a chemical transformation method.
  7. 7. An antibiotic-free feed, comprising the livestock and poultry original probiotics obtained by the screening method according to any one of claims 1-6, wherein the livestock and poultry original probiotics are subjected to microcapsule embedding treatment with an inner sodium alginate layer and an outer chitosan layer.
  8. 8. The antibiotic-free feed according to claim 7, wherein the probiotic of livestock origin is isolated from the digestive tract contents of healthy animals, wherein it is isolated from the intestinal contents for pigs and birds, and from the rumen contents for ruminants.
  9. 9. The antibiotic-free feed of claim 7, wherein the feed comprises, by weight, 45% -75% of an energy feed raw material, 9% -30% of a vegetable protein feed raw material, 2% -15% of a wheat bran or bran raw material, 3% -10% of a livestock and poultry essential nutrient premix, and 0.05% -0.5% of a microcapsule preparation of the livestock and poultry original probiotic. Wherein the energy feed raw material comprises one or more of corn, wheat and barley, and/or the plant protein feed raw material comprises one or more of soybean meal, rapeseed meal, cottonseed meal and protein grass.
  10. 10. The antibiotic-free feed of claim 7, wherein the microcapsule embedding is prepared by mixing the livestock and poultry original probiotics with a sodium alginate solution with the concentration of 1-3%, dripping the mixture into a calcium chloride solution to form inner gel microspheres, and then placing the gel microspheres into a chitosan solution with the pH of 4.0-5.0 and the concentration of 0.5-1.5% to form an outer coating structure.

Description

Construction method of livestock and poultry indigenous probiotics and application of livestock and poultry indigenous probiotics in antibiotic-free feed Technical Field The invention relates to the technical field of agricultural biotechnology and feed processing, in particular to a construction method of a natural probiotics for livestock and poultry and application of the natural probiotics in nonresistant feed. Background Animal husbandry is a prop industry of agricultural economy in China, and pig industry is more important. However, the current industry faces two core challenges, namely, serious threat of epidemic disease prevention and control and antibiotic dependence, serious threat of high-mortality epidemic disease such as African swine fever and the like, drug resistance and drug residue risk brought by the traditional mode depending on antibiotics, and difficult problem of environmental protection pressure and fecal sewage treatment, mass fecal sewage generated by large-scale cultivation, and low efficiency and easy secondary pollution of the traditional treatment mode. The probiotics technology is used as a key path for replacing antibiotics and promoting ecological breeding, the existing product has the remarkable defects that the used strain is sourced from human sources or laboratories, the colonization capability in the digestive tract of livestock and poultry is poor, the strain becomes 'passing bacteria', the functional expression of the strain lacks intelligent response to the digestive tract environment, and the common preparation cannot effectively protect the strain from high temperature of feed processing and animal gastric acid, so that the number of viable bacteria and the effect after feeding are suddenly reduced. The prior art is mainly used for treating animal health and fecal sewage in isolation, and lacks a systematic solution from a feed end to an environment-friendly end. Disclosure of Invention The invention mainly aims to provide a method for constructing livestock and poultry indigenous probiotics and application thereof in non-resistant feed, and aims to construct an efficient and stable livestock and poultry indigenous probiotics feed system capable of replacing antibiotics in the whole course by integrating indigenous strain screening, intelligent targeted genetic engineering improvement and double-layer microcapsule protection technology, so as to finally achieve the aims of guaranteeing livestock and poultry health, improving breeding benefit and promoting environment-friendly non-resistant breeding. In order to achieve the aim, the invention provides a construction method of a domestic probiotic for livestock and poultry, which comprises the following steps: collecting the content from specific digestive tract parts of target healthy livestock and poultry, and separating to obtain initial microorganism strains, wherein the specific digestive tract parts are intestinal tracts for pigs and poultry, and rumen for ruminants; Preparing corresponding simulated digestive tract environment culture solution according to the types of target healthy livestock and poultry, and placing the initial microbial strain under corresponding simulated conditions for multi-generation adaptive subculture; Performing an in-vitro bacteriostasis test on the microbial strain subjected to the adaptive culture, and screening out strains with bacteriostasis areas on staphylococcus aureus and escherichia coli as candidate probiotics; Regulating and controlling the expression of the genes related to the carbon source and the nitrogen source utilization of the candidate probiotics by adopting a metabolic engineering means, and optimizing the culture conditions of the candidate probiotics; constructing an expression vector containing a specific promoter of the digestive tract of livestock and poultry and an antibacterial peptide coding gene, and introducing the expression vector into the candidate probiotics by a genetic transformation method to obtain the livestock and poultry original probiotics capable of specifically expressing the antibacterial peptide in response to the digestive tract environment. Further, the preparation of the simulated digestive tract environment culture solution comprises the steps of simulating the digestive tract conditions of livestock and poultry, wherein the pH value is 5.0-7.0, the concentration of bile acid is 0.05-0.3%, the culture conditions of the intestinal tracts of the simulated livestock and poultry are 6.0-7.5, the concentration of bile acid is 0.05-0.2%, the culture conditions of the rumen of the simulated ruminant are 5.5-7.0, and the total amount of volatile fatty acid is 60-120 mM. Further, in the in vitro bacteriostasis test, the diameter of a bacteriostasis ring for staphylococcus aureus or escherichia coli is not less than 11mm. Further, the metabolic engineering means comprises controlling the culture temperature between 35 ℃ and 42 ℃ and optimizin