CN-122004369-A - Preparation method and application of charcoal-based probiotic microspheres for targeted field planting of intestinal tracts of broilers

CN122004369ACN 122004369 ACN122004369 ACN 122004369ACN-122004369-A

Abstract

The invention discloses a preparation method and application of a charcoal-based probiotic microsphere for targeted planting of intestinal tracts of broilers, and relates to the technical field of feed additive preparation, wherein the charcoal is prepared by preparing iron-carbon composite catalytic biochar with a hierarchical pore structure and active catalytic sites, and the surface performance of the charcoal-based probiotic microsphere is optimized by modifying citric acid, so that the affinity of the charcoal-based probiotic microsphere to probiotics and the adsorption specificity of the charcoal toxin are enhanced; and then preparing high-concentration probiotics suspension, and constructing a core-shell structured composite microsphere by adopting a chitosan/sodium alginate layer-by-layer assembly process, so as to realize embedding protection of probiotics and targeted delivery of intestinal tracts of broilers. The preparation method has the advantages of clear process steps, controllable parameters, readily available raw materials, low cost and easy industrialized mass production, and the prepared biochar-based probiotic microsphere has stable structure and excellent performance, and can effectively solve the technical problems of gastric acid inactivation, low field planting rate and poor performance of the biochar carrier of the traditional probiotic preparation.

Inventors

ZHANG YU
YANG JIANCHENG
WANG XING
LI XIAOHONG
LIANG XIAO
WANG SHUJUN

Assignees

沈阳农业大学
辽东学院

Dates

Publication Date: 20260512
Application Date: 20260302

Claims (10)

1. The preparation method of the charcoal-based probiotic microsphere for targeted planting of the intestinal tracts of broilers is characterized by comprising the following specific steps: s1, taking agricultural wastes as biomass raw materials, carrying out pretreatment, carrying out precursor impregnation complexing with ferric salt and nitrogen source according to the mass ratio of 1:0.1-1.0:0.5-3.0, drying, carrying out sectional temperature programming pyrolysis, and carrying out acid washing, water washing and drying to obtain the Fe/N-HPC material; S2, taking the prepared Fe/N-HPC as a matrix, and carrying out dipping, dehydration condensation reaction, cleaning and drying by using a citric acid aqueous solution to obtain the CA-BC material with the carboxyl grafted on the surface; S3, selecting acid-resistant and bile-salt-resistant lactobacillus strains, performing activation culture on the lactobacillus strains by using an MRS (media dependent control) culture medium, centrifugally collecting thalli, washing and resuspension, and adjusting the concentration of a bacterial liquid to 10 9 -10 11 CFU/mL to obtain a high-concentration bacterial suspension; S4, dispersing CA-BC in a sodium alginate solution by adopting a layer-by-layer assembly process of internal phase dispersion-ionic crosslinking-external phase coating, inoculating probiotics to prepare an internal phase mixed solution, forming primary gel beads by CaCl 2 ionic crosslinking, and then carrying out electrostatic complexing coating by a chitosan solution to obtain the biochar-based probiotic microsphere.
2. The method for preparing the charcoal-based probiotic micro-spheres for targeted colonization of the intestinal tracts of broilers according to claim 1, wherein in the step S1, ferric salt is preferably selected And the nitrogen source is preferably urea, melamine, dicyandiamide or thiourea.
3. The preparation method of the biochar-based probiotic microspheres for targeted planting of intestinal tracts of broilers, which is characterized in that in the step S1, biomass powder, ferric salt and a nitrogen source are added into deionized water or ethanol aqueous solution together according to the mass ratio of 1 (0.1-1.0) (0.5-3.0), continuously stirring or ultrasonically dispersing is carried out for 4-24 hours at the room temperature to 80 ℃, and after precursor dipping and complexing are completed, the mixed slurry is placed into an oven at 80-105 ℃ for drying, so that a solid precursor is obtained.
4. The preparation method of the biochar-based probiotic microspheres for targeted planting of intestinal tracts of broilers according to claim 3, wherein in the step S1, a solid precursor is placed in a quartz boat or a corundum boat of a tube furnace, high-purity N 2 or Ar with the flow rate of 100-500mL/min is introduced as a shielding gas, pyrolysis is carried out by adopting a sectional heating strategy, the temperature is firstly increased to 400-600 ℃ at the rate of 2-10 ℃ per min, the temperature is kept for 30-60 min, the temperature is further increased to 700-1000 ℃ and kept for 1-3H, a pyrolysis product is naturally cooled to room temperature and then ground into powder, the powder is soaked in 0.1-2.0M HCl or H 2 SO 4 solution and stirred for 6-24H, after pickling is completed, the powder is repeatedly washed with deionized water until the pH value of filtrate is=7.0, and finally the powder is placed in a vacuum drying box with the temperature of 60-80 ℃ for drying, so that the Fe/N-HPC material is prepared.
5. The method for preparing the biochar-based probiotic microspheres for targeted colonization of intestinal tracts of broilers according to claim 1, wherein in the step S2, prepared Fe/N-HPC is used as a matrix, citric acid aqueous solution with the concentration of 0.5-3.0M is prepared, fe/N-HPC powder is added into the citric acid aqueous solution according to the solid-to-liquid ratio of 1 (5-20) g/mL, stirring is carried out for 12-24 hours at room temperature or 40-60 ℃, after citric acid soaking is finished, biochar is separated through a suction filtration or centrifugation mode, the separated biochar is placed into a 50-60 ℃ oven for drying until moisture volatilizes, then the separated biochar is placed into a 100-160 ℃ oven or a tubular oven for heat treatment for 60-120 minutes, dehydration condensation reaction is completed, finally the biochar is fully washed by deionized water, free citric acid which does not participate in the reaction is removed, and the CA-material is prepared after drying.
6. The method for preparing the biochar-based probiotic micro-spheres for targeted colonization of the intestinal tracts of broilers according to claim 1, wherein in the step S3, lactobacillus strain is selected from one or more of lactobacillus plantarum, lactobacillus fermentum, lactobacillus casei and lactobacillus rhamnosus.
7. The method for preparing the charcoal-based probiotic micro-spheres for targeted colonization of the intestinal tracts of broilers according to claim 1, wherein in the step S3, the activation culture is anaerobic static culture at 37 ℃ for 18-24 hours and passage is carried out for 2-3 generations to the final stage of logarithmic growth or the initial stage of stationary phase, the collection of thalli is 4 ℃, centrifugation is carried out at 3000-5000rpm for 10-15 minutes, and the washing is carried out for 2-3 times by adopting sterile normal saline or PBS buffer solution with pH of 7.2.
8. The method for preparing the biochar-based probiotic microspheres for targeted colonization of intestinal tracts of broilers according to claim 1, wherein in the step S4, sodium alginate powder is dissolved in distilled water to prepare a colloid solution with the concentration of 1.5% -3.5% (w/v), the colloid solution is cooled to room temperature after high-temperature sterilization, the prepared CA-BC powder is added into the colloid solution, the adding amount of the CA-BC powder in the sodium alginate colloid solution is 0.5% -3.0% (w/v), the CA-BC powder is uniformly dispersed through strong mechanical stirring and ultrasonic dispersion for 30-60 minutes to form SA-BC suspension, the prepared high-concentration probiotic suspension is inoculated into the SA-BC suspension according to the volume ratio of 1:4-10, the mixture is gently stirred and uniformly mixed, the viable count in the mixed solution is controlled to be 10 8 -10 9 CFU/mL, the dissolved anhydrous calcium chloride is added into distilled water to prepare a hardening solution with the concentration of 1.0% -5.0% (w/v), the pH value of the CA-BC powder is adjusted to be slightly mixed with the neutral solution, the hardening solution is subjected to a micro-injection molding device for forming an electrostatic crosslinking gel, and the micro-injection is performed for forming the primary gel after the micro-injection is gradually cooled for 45 minutes.
9. The preparation method of the biochar-based probiotic microspheres for targeted colonization of intestinal tracts of broilers, which is characterized by comprising the steps of filtering out primary gel beads in S4 by using a filter screen, rinsing 2-3 times by using sterile distilled water, selecting chitosan powder with the deacetylation degree of more than 85%, dissolving the chitosan powder in 0.5-2.0% (v/v) acetic acid solution to prepare chitosan solution with the concentration of 0.2-1.5% (w/v), regulating the pH value of the chitosan solution to 4.5-5.5 by using NaOH solution, adding a small amount of CaCl 2 , immersing rinsed primary gel beads in the chitosan solution, placing the primary gel beads on a shaking table to shake and coat at the speed of 50-100rpm for 10-60 minutes, filtering out coated composite microspheres by using the filter screen, rapidly cleaning by using sterile water to prepare wet biochar-based probiotic microspheres, and preparing the wet biochar-based probiotic microspheres by soaking the cleaned composite microspheres in cryoprotectants such as freeze-dried seaweed microspheres after vacuum drying.
10. The biochar-based probiotic microsphere prepared by the preparation method of any one of claims 1-9 is characterized in that the prepared biochar-based probiotic microsphere is used as a feed additive, is directly added into basic feed of broilers in an additive amount of 0.5% -2% of the mass of the basic feed of the broilers, is uniformly mixed and then is used for feeding broilers in different growth stages, and can be applied to links of intestinal flora regulation, mycotoxin detoxification and intestinal inflammation prevention and control in the broiler breeding process, and is suitable for broiler breeding scenes with mycotoxin pollution and intestinal flora imbalance.

Description

Preparation method and application of charcoal-based probiotic microspheres for targeted field planting of intestinal tracts of broilers Technical Field The invention relates to the technical field of feed additive preparation, in particular to a preparation method and application of charcoal-based probiotic microspheres for targeted planting of intestinal tracts of broilers. Background In the intensive broiler raising industry, along with the continuous expansion of raising scale and the improvement of raising density, the intestinal health problem of broiler chickens is increasingly prominent, and becomes a key factor for restricting the sustainable development of the industry. The mycotoxin pollution and the antibiotic abuse are two main causes of intestinal flora imbalance and chronic inflammation frequent of the broiler chickens, the mycotoxin is used as a common pollutant in the feed, can destroy intestinal mucosa barriers of the broiler chickens and cause immunosuppression and metabolic disturbance, and the long-term use of the antibiotic can lead to reduction of beneficial flora in intestinal tracts and growth of pathogenic bacteria and further aggravate intestinal health problems, so that the safe and efficient feed additive is developed to replace the antibiotic and effectively prevent and control the mycotoxin pollution, and becomes a research hot spot in the current broiler chicken cultivation field. Aiming at the problems, the traditional probiotic preparation is expected to improve the health condition of the intestinal canal by regulating the intestinal canal flora of the broiler chickens, but in practical application, the traditional probiotic preparation has a plurality of defects, on one hand, gastric acid and digestive enzymes of the broiler chickens have strong acid environment and high activity, most probiotics are easily inactivated by the gastric acid and the digestive enzymes before entering the intestinal canal, so that the number of effective viable bacteria reaching the intestinal canal is greatly reduced, the effect of regulating the intestinal canal flora cannot be fully exerted, on the other hand, the field planting rate of the traditional probiotic preparation in the intestinal canal is lower, stable flora is difficult to form in the intestinal canal, the regulation effect on the intestinal canal flora cannot be continuously exerted, and the regulation efficiency of the intestinal canal flora is low. In addition, although the common biochar has a certain adsorption performance, mycotoxin can be adsorbed to a certain extent, but the surface functional groups are single, the active sites are few, the affinity to probiotics is poor, the adsorption specificity to the mycotoxin is low, the efficient adsorption to the specific mycotoxin is difficult to realize, in addition, the complex mode of the biochar and the probiotics in the prior art is mainly simple physical adsorption, the prepared preparation has insufficient structural stability, is easy to disintegrate in the complex environment of the digestive tract of the broiler, can not realize the intestinal targeted delivery and slow-release field planting of the probiotics of the broiler, and also has difficulty in fully playing the synergistic effect of the biochar and the probiotics, and the problems of mycotoxin pollution, intestinal flora imbalance and the like in broiler cultivation are difficult to effectively solve. Disclosure of Invention The invention aims to make up the defects of the prior art, and provides a preparation method and application of a charcoal-based probiotic microsphere for targeted planting of broiler intestinal tracts, which can improve the affinity of the charcoal-based probiotic microsphere for probiotics and the adsorption specificity for mycotoxin by preparing iron-carbon composite catalytic charcoal with a hierarchical pore structure and active catalytic sites and optimizing the surface performance of the charcoal through citric acid modification; and then preparing high-concentration probiotics suspension, and constructing a core-shell structured composite microsphere by adopting a chitosan/sodium alginate layer-by-layer assembly process, so as to realize embedding protection of probiotics and targeted delivery of intestinal tracts of broilers. The preparation method has the advantages of clear process steps, controllable parameters, readily available raw materials, low cost and easy industrialized mass production, and the prepared biochar-based probiotic microsphere has stable structure and excellent performance, can effectively solve the technical problems of gastric acid inactivation, low field planting rate and poor performance of a biochar carrier of the traditional probiotic preparation, and provides a new solution for the green broiler breeding industry. The invention provides a preparation method of charcoal-based probiotic microspheres for targeted colonization of intestinal tracts of br