Preparation method and application of charcoal-based probiotic microspheres for targeted colonization of intestinal tracts of broiler chickens

Abstract

The invention discloses a preparation method and application of biochar-based probiotic microspheres for targeted colonization of intestinal tracts of broiler chickens, and relates to the technical field of preparation of feed additives. Iron-carbon composite catalytic biochar with a hierarchical pore structure and active catalytic sites is prepared, and the surface performance of the biochar is modified and optimized through citric acid; the affinity to probiotics and the adsorption specificity to mycotoxin are enhanced; then, preparing a high-concentration probiotic suspension, and constructing a composite microsphere with a core-shell structure by adopting a chitosan / sodium alginate layer-by-layer assembly process, so as to realize embedding protection of the probiotics and targeted delivery of intestinal tracts of the broiler chickens. The preparation method has the advantages of clear process steps, controllable parameters, easily available raw materials, low cost and easiness in industrial large-scale production; the prepared biochar-based probiotic microspheres are stable in structure and excellent in performance, and can effectively solve the technical problems of gastric acid inactivation, low planting rate and poor biochar carrier performance of a traditional probiotic preparation.

Description

Technical Field

[0001] This invention relates to the field of feed additive preparation technology, specifically to a method for preparing and applying biochar-based probiotic microspheres that target and colonize the intestines of broilers. Background Technology

[0002] In the intensive broiler chicken farming industry, with the continuous expansion of farming scale and the increase in stocking density, intestinal health problems of broilers have become increasingly prominent, becoming a key factor restricting the sustainable development of the industry. Among them, mycotoxin contamination and antibiotic abuse are the two main causes of intestinal flora imbalance and frequent chronic inflammation in broilers. Mycotoxins, as common contaminants in feed, can damage the intestinal mucosal barrier of broilers, causing immunosuppression and metabolic disorders; while long-term use of antibiotics will lead to a reduction in beneficial bacteria in the intestine and the proliferation of pathogens, further aggravating intestinal health problems. Therefore, developing safe and efficient feed additives to replace antibiotics and effectively control mycotoxin contamination has become a research hotspot in the current broiler farming field.

[0003] To address the aforementioned issues, traditional probiotic preparations have been highly anticipated, with the expectation that they could improve gut health by regulating the intestinal flora of broilers. However, in practical applications, traditional probiotic preparations have several shortcomings. On the one hand, the stomach acid and digestive enzymes of broilers create a highly acidic environment with high activity. Before entering the intestines, most probiotics are easily inactivated by stomach acid and digestive enzymes, resulting in a significant reduction in the number of effective live bacteria reaching the intestines and failing to fully exert their role in regulating the intestinal flora. On the other hand, traditional probiotic preparations have a low colonization rate in the intestines, making it difficult to form a stable flora and sustainably exert their regulatory effect on the intestinal flora, resulting in low efficiency in intestinal flora regulation. In addition, although ordinary biochar has certain adsorption properties and can adsorb mycotoxins to a certain extent, its surface functional groups are singular and its active sites are few. It has poor affinity for probiotics and low adsorption specificity for mycotoxins, making it difficult to achieve efficient adsorption of specific mycotoxins. Moreover, the existing technologies for combining biochar and probiotics are mostly simple physical adsorption methods, resulting in formulations with insufficient structural stability. They are prone to disintegration in the complex environment of the broiler digestive tract, making it impossible to achieve targeted delivery and sustained-release colonization of probiotics in the broiler intestine. It is also difficult to fully utilize the synergistic effect of biochar and probiotics, and it is difficult to effectively solve the problems of mycotoxin contamination and intestinal flora imbalance in broiler farming. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for preparing and applying biochar-based probiotic microspheres for targeted colonization of broiler intestines. This method involves preparing iron-carbon composite catalytic biochar with a hierarchical porous structure and active catalytic sites, and optimizing its surface properties through citric acid modification to enhance its affinity for probiotics and its specificity for mycotoxin adsorption. Subsequently, a high-concentration probiotic suspension is prepared, and a core-shell structured composite microsphere is constructed using a chitosan / sodium alginate layer-by-layer assembly process, achieving encapsulation and protection of probiotics and targeted delivery to the broiler intestine. This preparation method features clear process steps, controllable parameters, readily available and inexpensive raw materials, and is easy to scale up for industrial production. The resulting biochar-based probiotic microspheres exhibit stable structure and excellent performance, effectively solving the technical problems of gastric acid inactivation, low colonization rate, and poor biochar carrier performance in traditional probiotic preparations, providing a new solution for the green broiler farming industry.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution: On one hand, a method for preparing biochar-based probiotic microspheres for targeted colonization of the broiler intestine, the method comprising the following specific steps:

[0006] S1: Using agricultural waste as biomass raw material, after pretreatment, it is impregnated and complexed with iron salt and nitrogen source at a mass ratio of 1:0.1-1.0:0.5-3.0. After drying, it is subjected to segmented temperature-increasing pyrolysis, and then acid washing, water washing and drying to obtain Fe / N-HPC material.

[0007] S2: Using the prepared Fe / N-HPC as a matrix, after impregnation with citric acid aqueous solution, dehydration condensation reaction, washing and drying, a CA-BC material with carboxyl groups grafted on the surface is obtained;

[0008] S3: Select acid- and bile-tolerant Lactobacillus strains, activate them in MRS medium, collect the cells by centrifugation, wash and resuspend them, and adjust the bacterial concentration to 10. 9 -10 11 CFU / mL was used to obtain a high-concentration bacterial suspension.

[0009] S4: Using a layer-by-layer assembly process of internal phase dispersion-ionic crosslinking-external phase coating, CA-BC is dispersed in sodium alginate solution, probiotics are inoculated to obtain an internal phase mixture, primary gel beads are formed by CaCl2 ionic crosslinking, and then coated by electrostatic complexation with chitosan solution to obtain the biochar-based probiotic microspheres.

[0010] Furthermore, in S1, the iron salt is preferably FeCl₂. 3• 6H₂O or Fe(NO₃) 3• The preferred nitrogen sources for 9H2O are urea, melamine, dicyandiamide, or thiourea.

[0011] Furthermore, the biomass powder, iron salt, and nitrogen source are added to deionized water or an ethanol aqueous solution at a mass ratio of 1:(0.1-1.0):(0.5-3.0), and continuously stirred or ultrasonically dispersed at room temperature to 80°C for 4-24 hours to allow the Fe... 3+ The ions coordinate with oxygen-containing functional groups such as hydroxyl and carboxyl groups on the surface of biomass and nitrogen source molecules to achieve uniform dispersion of metal ions. After the precursor impregnation and complexation are completed, the mixed slurry is dried in an oven at 80-105℃ to obtain a solid precursor.

[0012] Furthermore, in step S1, the solid precursor is placed in a quartz boat or corundum boat of a tube furnace, and high-purity N2 or Ar at a flow rate of 100-500 mL / min is introduced as a protective gas. A staged heating strategy is used for pyrolysis. First, the temperature is raised to 400-600℃ at a rate of 2-10℃ / min and held for 30-60 minutes to complete the initial carbonization of biomass and the condensation of the nitrogen source. Then, the temperature is further raised to 700-1000℃, preferably 800-900℃, and held for 1-3 hours to complete deep graphitization, causing the iron species to be carbothermally reduced to Fe. 0 Alternatively, Fe3C may recombine with doped nitrogen atoms to form thermodynamically stable Fe-N. x Active sites are formed, and the volatilization of salts creates a pore-forming effect, resulting in a hierarchical porous structure. Finally, the pyrolysis products are naturally cooled to room temperature and ground into powder. The powder is then soaked and stirred in 0.1-2.0M HCl or H2SO4 solution for 6-24 hours to remove unstable metal agglomerates and inorganic ash, exposing blocked pores while retaining stable Fe-N... x The active sites are then washed repeatedly with deionized water until the filtrate is neutral. The powder is then dried in a vacuum drying oven at 60-80℃ to obtain iron-carbon composite catalytic biochar (Fe / N-HPC) material.

[0013] Furthermore, in step S2, the obtained Fe / N-HPC is used as the matrix, and a citric acid aqueous solution with a concentration of 0.5-3.0M is prepared. Fe / N-HPC powder is added to the citric acid aqueous solution at a solid-liquid ratio of 1:(5-20)g / mL. The mixture is stirred for 12-24 hours at room temperature or 40-60℃ to complete the citric acid impregnation. The biochar is then separated by filtration or centrifugation. The separated biochar is placed in an oven at 50-60℃ to dry until the moisture evaporates. Then, it is placed in an oven or tube furnace at 100-160℃ for heat treatment for 60-120 minutes to complete the dehydration condensation reaction. Finally, the biochar is thoroughly washed with deionized water to remove unreacted free citric acid. After drying, the CA-BC material is obtained.

[0014] Furthermore, in S3, the lactobacillus strain is selected from one or more of Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus casei, and Lactobacillus rhamnosus.

[0015] Furthermore, in S3, the activation culture is carried out by anaerobic static culture at 37°C for 18-24 hours and passaged 2-3 times to the end of the logarithmic growth phase or the early stage of the stationary phase. The bacterial cells are collected by centrifugation at 3000-5000 rpm for 10-15 minutes at 4°C. The cells are washed 2-3 times with sterile physiological saline or PBS buffer at pH 7.2.

[0016] Furthermore, in step S4, sodium alginate powder is dissolved in distilled water to prepare a colloidal solution with a concentration of 1.5%-3.5% (w / v). After high-temperature sterilization and cooling to room temperature, the CA-BC powder prepared above is added to the solution, making the amount of CA-BC powder added to the sodium alginate colloidal solution 0.5%-3.0% (w / v). The CA-BC powder is uniformly dispersed by vigorous mechanical stirring and ultrasonic dispersion for 30-60 minutes to form an SA-BC suspension. The high-concentration probiotic suspension prepared is inoculated into the SA-BC suspension at a volume ratio of 1:4-10, and gently stirred to mix evenly, controlling the viable bacteria count in the mixture to be 10. 8 -10 9 CFU / mL was used to obtain an SA-BC-Pro internal phase mixture; anhydrous calcium chloride was dissolved in distilled water to prepare a hardening solution with a concentration of 1.0%-5.0% (w / v), and the pH was adjusted to neutral. Using a sterile syringe with an injection pump or a high-voltage electrostatic microcapsule forming device, the SA-BC-Pro internal phase mixture was dripped into the CaCl2 hardening solution with slight stirring. After cross-linking and curing for 15-45 minutes, primary gel beads were formed.

[0017] Furthermore, in step S4, the primary gel beads are filtered out using a filter screen and rinsed 2-3 times with sterile distilled water; chitosan powder with a deacetylation degree >85% is selected and dissolved in a 0.5%-2.0% (v / v) acetic acid solution to prepare a chitosan solution with a concentration of 0.2%-1.5% (w / v); the pH of the chitosan solution is adjusted to 4.5-5.5 with NaOH solution, and a small amount of CaCl2 can be added; the rinsed primary gel beads are immersed in the chitosan solution and placed on a shaker at a speed of 50-100 rpm for 10-60 minutes for coating; the coated composite microspheres are filtered out using a filter screen and quickly washed with sterile water to obtain wet biochar-based probiotic microspheres; alternatively, the washed composite microspheres can be soaked in cryoprotectants such as trehalose or skim milk and then freeze-dried under vacuum to prepare dry powder microspheres.

[0018] On the other hand, the application of biochar-based probiotic microspheres that target and colonize the broiler gut involves using the prepared biochar-based probiotic microspheres as a feed additive. At an addition rate of 0.5%-2% of the broiler basal feed mass, these microspheres are directly added to the broiler basal feed, mixed thoroughly, and then fed to broilers at different growth stages. These microspheres can be applied to intestinal flora regulation, mycotoxin detoxification, and intestinal inflammation control during broiler farming, making them suitable for broiler farming scenarios involving mycotoxin contamination and intestinal flora imbalance.

[0019] Compared with existing technologies, the preparation method and application of this biochar-based probiotic microspheres for targeted colonization of broiler intestines have the following beneficial effects:

[0020] This invention significantly improves the adsorption capacity and probiotic affinity of biochar carriers by preparing Fe / N-HPC materials with hierarchical porous structures and active catalytic sites, and optimizing their surface properties through citric acid modification. The core-shell composite microspheres constructed using a chitosan / sodium alginate layer-by-layer assembly process effectively solve the technical problem of traditional probiotic preparations being easily inactivated by gastric acid through a dual-layer protection mechanism formed by ionic cross-linking and electrostatic complexation. This achieves targeted delivery and sustained-release colonization of probiotics into the broiler intestine. The prepared microspheres can be produced in both wet and dry powder forms, allowing for flexible storage and transportation. As a green feed additive, the dosage is controllable, and it can be directly mixed with broiler basic feed, adapting to intensive farming models. It has significant effects on intestinal flora regulation, mycotoxin detoxification, and inflammation control.

[0021] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0023] Figure 1 A flowchart illustrating a method for preparing biochar-based probiotic microspheres that are targeted and colonized in the intestines of broilers;

[0024] Figure 2 The flowchart of method S4 is a method for preparing biochar-based probiotic microspheres that are targeted to colonize the intestines of broilers. Detailed Implementation

[0025] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0026] Example 1

[0027] like Figure 1 As shown, corn stalks free from mold and impurities were selected as biomass raw materials. They were repeatedly washed three times with deionized water to remove surface dirt and impurities, then dried in a 100℃ forced-air drying oven to constant weight. After being pulverized by a universal pulverizer and passed through an 80-mesh standard sieve, the uniformly sized powder was collected and placed in a desiccator for later use. The ratio of corn stalk powder to FeCl₂ was calculated. 3• Deionized water was added to a mixture of 6H₂O and urea at a mass ratio of 1:0.5:2.0. The mixture was then ultrasonically dispersed at 60°C and 150 rpm for 12 hours in a constant temperature water bath shaker. After impregnation and complexation, the mixture was transferred to a tray and dried at 100°C to obtain a solid precursor. The precursor was placed in a quartz boat in a tube furnace, and 300 mL / min of high-purity N₂ was introduced as a protective gas. The tube furnace was heated to 500°C at a rate of 5°C / min and held for 45 minutes. The temperature was then increased to 850°C and held for 2 hours. After pyrolysis, the mixture was allowed to cool naturally to room temperature and removed. The pyrolysis product was ground in an agate mortar and pestle, and then soaked and stirred in 1.0 M HCl solution for 12 hours, stirring every 2 hours. After acid washing, the product was repeatedly washed with deionized water until the pH of the filtrate was approximately 7.0. The product was then dried in a vacuum drying oven at 70°C for 12 hours to obtain Fe / N-HPC material, which was then sealed for later use.

[0028] Prepare a 1.5M citric acid aqueous solution. Immerse Fe / N-HPC in the citric acid aqueous solution at a solid-liquid ratio of 1:10 g / mL. Place the solution in a 50℃ constant temperature water bath shaker and stir at 150 rpm for 18 hours. Filter to separate the biochar. Dry the biochar at 55℃ until the water is completely evaporated. Transfer the biochar to a tube furnace and heat treat at 130℃ for 90 minutes to complete the dehydration condensation reaction. After cooling, wash repeatedly with deionized water to remove free citric acid. After drying, obtain CA-BC material and seal for later use.

[0029] A standard strain of *Lactobacillus plantarum* was selected. MRS liquid medium was autoclaved at 121°C for 20 min and then cooled to 37°C for later use. *Lactobacillus plantarum* strains frozen in glycerol were inoculated into MRS liquid medium and incubated statically in a 37°C anaerobic incubator for 20 hours. The medium was then passaged three times until the bacterial culture reached the late logarithmic growth phase. The activated bacterial culture was centrifuged at 4°C and 4000 rpm for 12 min, the supernatant was discarded, and the bacterial precipitate was washed three times with pH 7.2 PBS buffer. After each wash, the precipitate was resuspended and centrifuged again. Finally, the bacterial cells were resuspended in sterile physiological saline, and the bacterial concentration was adjusted to 5 × 10⁻⁶ using the plate count method. 9 CFU / mL, store temporarily at 4℃ for later use.

[0030] like Figure 2 As shown, a 2.5% (w / v) sodium alginate colloidal solution was prepared, autoclaved at 121℃ for 20 min, and then cooled to room temperature. CA-BC material was added at a dosage of 2.0% (w / v). The mixture was first magnetically stirred for 30 min for initial dispersion, and then ultrasonically dispersed at 300W for 45 min to form a uniform SA-BC suspension. Lactobacillus plantarum suspension was inoculated into the suspension at a volume ratio of 1:7, and gently magnetically stirred for 10 min to mix thoroughly. The viable cell count in the mixture was controlled to be 5 × 10⁻⁶ using the plate count method. 8 CFU / mL was used to obtain the SA-BC-Pro internal phase mixture; a 3.0% (w / v) CaCl2 hardening solution was prepared, the pH was adjusted to neutral and sterilized. Using a sterile syringe and a micro-injection pump, the internal phase mixture was dripped into the gently stirred CaCl2 hardening solution at a rate of 0.5 mL / min. Crosslinking and curing were carried out for 30 minutes to form primary gel beads. The beads were rinsed twice with sterile distilled water to remove residual Ca2+ from the surface. 2+ Prepare a 1.0% (w / v) chitosan-acetic acid solution (acetic acid volume fraction 1.0%), adjust the pH to 5.0 with 1 mol / L NaOH solution, immerse the primary gel beads in the solution, and place them in a shaker at 80 rpm for 30 minutes to coat. Quickly wash the surface with sterile water to remove residual chitosan solution, immerse the gel beads in 2% trehalose cryoprotectant for 30 minutes, and dry them in a vacuum freeze dryer for 24 hours to obtain dry powder biochar-based probiotic microspheres.

[0031] Example 2

[0032] Select mold-free rice husks and sawdust, mix them at a mass ratio of 1:1 as biomass raw materials, wash three times with deionized water, dry at 90℃ with forced air until constant weight, pulverize and pass through a 60-mesh standard sieve, and store in a desiccator for later use; mix the biomass powder with Fe(NO3). 3• Add 9H2O and melamine in a mass ratio of 1:0.8:1.5 to an aqueous ethanol solution (ethanol:water = 1:1), stir at 80℃ and 150 rpm for 8 hours, and dry at 90℃ to obtain a solid precursor. Place the precursor in a tubular furnace quartz boat, introduce 400 mL / min of high-purity Ar as a protective gas, heat to 450℃ at 8℃ / min and hold for 60 minutes, continue heating to 900℃ and hold for 1.5 hours, and allow to cool naturally to room temperature. Grind the pyrolysis product, soak and stir in 0.5M H2SO4 solution for 20 hours, wash with water until pH≈7.0, and vacuum dry at 60℃ for 12 hours to obtain Fe / N-HPC material, which is then sealed for later use.

[0033] Prepare a 2.0M citric acid aqueous solution, immerse it in Fe / N-HPC at a solid-liquid ratio of 1:15 g / mL, stir at 40℃ and 150 rpm for 24 hours, centrifuge to separate the biochar, dry it at 60℃ with forced air, heat treat it at 150℃ for 60 minutes to complete the dehydration condensation, wash it with water to remove free citric acid, and then dry it to obtain CA-BC material, which is then sealed for later use.

[0034] Standard strains of *Lactobacillus rhamnosus* and *Lactobacillus fermentum* were selected and mixed at a volume ratio of 1:1. The mixture was sterilized in MRS liquid medium and set aside. The two glycerol-frozen bacterial strains were separately inoculated into MRS liquid medium and anaerobically cultured at 37°C for 24 hours. After two passages to the early stationary phase, the two bacterial cultures were mixed in equal volumes. The mixed culture was centrifuged at 3000 rpm for 15 minutes at 4°C. The bacterial precipitate was washed twice with sterile physiological saline, resuspended in sterile physiological saline, and the concentration was adjusted to 1×10⁻⁶ using the plate count method. 10 CFU / mL, store temporarily at 4℃ for later use.

[0035] Prepare a 3.0% (w / v) sodium alginate colloidal solution, sterilize and cool, then add CA-BC at 1.5% (w / v), magnetically stir for 30 minutes and ultrasonically disperse at 300W for 60 minutes to form a uniform SA-BC suspension; inoculate the mixed bacterial suspension at a volume ratio of 1:5, gently stir to mix evenly, and control the viable count to 8 × 10⁶. 8 CFU / mL; using a 20G needle, drip 4.0% (w / v) neutral sterile CaCl2 hardening solution at a rate of 0.5 mL / min, cross-link and cure for 40 minutes, rinse twice with sterile distilled water; prepare 0.8% (w / v) chitosan acetic acid solution (acetic acid volume fraction 1.5%), add a small amount of CaCl2 to enhance stability, adjust pH to 4.5, immerse the primary gel beads in, shake at 100 rpm for 20 minutes for coating, wash with sterile water, and freeze-dry under vacuum for 24 hours to obtain dry powder biochar-based probiotic microspheres.

[0036] Example 3

[0037] Impurity-free cotton stalks were selected as biomass raw material, washed three times with deionized water, dried at 95℃ with forced air until constant weight, pulverized, and passed through a 100-mesh standard sieve. The powder was then stored in a desiccator for later use. The cotton stalk powder composition was: FeCl₂ 3• Deionized water was added to a mixture of 6H₂O and dicyandiamide at a mass ratio of 1:0.3:1.0. The mixture was stirred at 150 rpm for 24 hours at room temperature and dried at 95°C to obtain a solid precursor. The precursor was placed in a quartz boat in a tubular furnace, and 200 mL / min of high-purity N₂ was introduced. The temperature was increased to 400°C at 3°C / min and held for 60 minutes. The temperature was then increased to 800°C and held for 3 hours. The mixture was then allowed to cool naturally to room temperature. The pyrolysis product was ground and then soaked and stirred in 0.5 M HCl solution for 24 hours. The product was washed with water until the pH reached approximately 7.0 and then vacuum dried at 65°C for 12 hours to obtain Fe / N-HPC material, which was then sealed for later use.

[0038] Prepare a 1.0M citric acid aqueous solution, immerse it in Fe / N-HPC at a solid-liquid ratio of 1:8, stir at room temperature and 150 rpm for 24 hours, filter to separate biochar, dry in a forced-air dryer at 50℃, heat treat at 120℃ for 120 minutes to complete dehydration condensation, wash with water to remove free citric acid and dry to obtain CA-BC material, which is then sealed for later use.

[0039] Standard strains of *Lactobacillus casei* were selected and sterilized in MRS liquid medium for later use. Glycerol-frozen bacterial cultures were inoculated into MRS liquid medium and anaerobically cultured at 37°C for 18 hours, followed by three subcultures until the end of the logarithmic growth phase. The bacterial suspension was centrifuged at 5000 rpm for 10 minutes at 4°C, washed twice with PBS buffer (pH 7.2), resuspended in sterile physiological saline, and the concentration was adjusted to 8 × 10⁻⁶ using the plate count method. 9 CFU / mL, store temporarily at 4℃ for later use.

[0040] Prepare a 2.0% (w / v) sodium alginate colloidal solution, sterilize and cool, then add CA-BC at 1.0% (w / v), magnetically stir for 30 minutes and ultrasonically disperse at 300W for 30 minutes to form a uniform SA-BC suspension; inoculate the Lactobacillus casei suspension at a volume ratio of 1:8, gently stir and mix evenly, controlling the viable count to 3×10⁻⁶. 8 CFU / mL; 2.0% (w / v) neutral sterile CaCl2 hardening solution was added dropwise using a 25G needle at a rate of 0.5 mL / min, cross-linked and cured for 20 minutes, and rinsed 3 times with sterile distilled water; 0.5% (w / v) chitosan acetic acid solution (acetic acid volume fraction 0.8%) was prepared, pH adjusted to 5.5, the primary gel beads were immersed in the solution, and the mixture was shaken at 60 rpm for 40 minutes for coating. After rinsing with sterile water, the mixture was immersed in 5% skim milk cryoprotectant for 30 minutes, and then freeze-dried under vacuum for 24 hours to obtain dry powder biochar-based probiotic microspheres.

[0041] Comparative Example 1

[0042] Except for not modifying Fe / N-HPC with citric acid, the other preparation steps, process parameters, and raw material amounts were completely consistent with those in Example 1, and unmodified biochar-based probiotic microspheres were obtained.

[0043] Comparative Example 2

[0044] Except for the absence of any biochar material, the other preparation steps, process parameters, and raw material dosages were completely consistent with those in Example 1. The Lactobacillus plantarum suspension was directly mixed with a 2.5% (w / v) sodium alginate colloidal solution at a volume ratio of 1:7. The subsequent cross-linking, coating, and drying processes were synchronized with those in Example 1, and pure chitosan / sodium alginate probiotic microspheres were finally obtained.

[0045] Test methods

[0046] All tests were performed in triplicate, and the average value was taken. The test samples were all dry powder microspheres prepared above. Before the test, all samples were brought to room temperature and placed in a sterile environment.

[0047] Probiotic gastric acid survival rate: Prepare simulated gastric juice (containing 3g / L pepsin, 0.2% HCl, pH 1.2) and sterilize it; take an equal amount of microspheres and place them in simulated gastric juice, shake at 37℃ and 150rpm for 2h. After treatment, dilute them serially, spread them on MRS solid medium, and incubate anaerobically at 37℃ for 48h. Then count the plates and calculate the survival rate = (number of viable bacteria after treatment / number of viable bacteria at the beginning) × 100%.

[0048] Intestinal colonization rate: Healthy 1-day-old broilers were randomly divided into groups. Microspheres were mixed into the broiler's basal feed at a dosage of 1.0% and fed continuously for 7 days with free access to water and feed. After feeding, the broilers were sacrificed, and the cecal contents were collected under aseptic conditions. After serial dilution, the contents were spread on MRS solid medium and anaerobically incubated at 37°C for 48 hours for counting. The results were expressed as log... 10 CFU / g represents cecal contents.

[0049] Aflatoxin B1 (AFB1) degradation rate: Prepare and sterilize a simulated intestinal solution (containing 1 g / L trypsin, prepared with 0.2 mol / L phosphate buffer, pH 6.8), add AFB1 standard to make a final concentration of 5 μg / mL; add an equal amount of microspheres to the simulated intestinal solution, and incubate at 37℃ and 150 rpm for 4 h with shaking. Detect the residual amount of AFB1 in the culture medium by high performance liquid chromatography (HPLC), and calculate the degradation rate = (initial concentration - residual concentration) / initial concentration × 100%.

[0050] Shannon index of gut microbiota: Cecal contents of broilers were collected after 7 days of feeding. Total microbial DNA was extracted using a kit. The composition of gut microbiota was analyzed by high-throughput sequencing technology. The Shannon index was calculated to characterize the diversity of gut microbiota. The higher the index, the richer the microbiota diversity.

[0051] Group Probiotic survival rate in gastric acid (%) <![CDATA[Gut colonization rate (log 10 CFU / g cecal content)]]> <![CDATA[Degradation rate of aflatoxin B1 (%)]]> Shannon index of gut microbiota Example 1 92.5 8.96 76.8 3.85 Example 2 94.2 9.12 80.5 3.92 Example 3 89.7 8.75 72.3 3.78 Comparative Example 1 75.3 7.21 45.6 3.12 Comparative Example 2 62.8 6.58 18.9 2.85

[0052] The biochar-based probiotic microspheres prepared in this invention, after optimization of process details, exhibit excellent and stable performance in probiotic gastric acid protection, broiler intestinal colonization, mycotoxin degradation, and intestinal flora regulation. The gastric acid survival rate of the probiotics in the three embodiments was all above 89%, and the intestinal colonization rate was all above 8.75 log. 10 CFU / g and AFB1 degradation rate were both higher than 72%, and the Shannon index of intestinal flora was higher than 3.78. Among them, Example 2, due to the use of mixed biomass raw materials and compound probiotic strains, and the optimization of biochar addition and coating process, had the best performance in all aspects and the best overall effect.

[0053] As shown in Comparative Example 1, citric acid modification is a key process for improving the overall performance of microspheres. The Fe / N-HPC surface without citric acid modification has a single functional group, which significantly reduces its affinity for probiotics and its loading stability. At the same time, it has insufficient adsorption specificity for aflatoxin B1, which directly leads to a significant decrease in the survival rate of probiotics in gastric acid, the intestinal colonization rate, and the degradation rate of AFB1. The regulatory effect on the diversity of broiler intestinal flora is also significantly weakened. Compared with Comparative Example 2, the pure chitosan / sodium alginate microspheres without biochar can only achieve basic encapsulation of probiotics. They lack support for the colonization sites of probiotics and the ability to adsorb and degrade mycotoxins. Moreover, the encapsulation structure has limited resistance to gastric acid, resulting in extremely low survival rate of probiotics in gastric acid and intestinal colonization rate. They have almost no degradation effect on AFB1 and have a weak regulatory effect on the diversity of broiler intestinal flora.

[0054] This invention optimizes the preparation of iron-carbon composite catalytic biochar and citric acid modification through a dual process. Combined with a core-shell structure design using chitosan / sodium alginate layer-by-layer assembly, it achieves biochar's ability to protect probiotic load and support intestinal colonization sites, as well as its efficient adsorption and degradation of mycotoxins. At the same time, the core-shell structure effectively resists the inactivation of probiotics by gastric acid, thus solving the technical defects of traditional probiotic preparations such as gastric acid inactivation, low colonization rate, and single function.

[0055] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for preparing biochar-based probiotic microspheres for targeted colonization of the broiler gut, characterized in that, The method includes the following specific steps: S1: Using agricultural waste as biomass raw material, after pretreatment, it is impregnated and complexed with iron salt and nitrogen source at a mass ratio of 1:0.1-1.0:0.5-3.

0. After drying, it is subjected to segmented temperature-increasing pyrolysis, and then acid washing, water washing and drying to obtain Fe / N-HPC material. S2: Using the prepared Fe / N-HPC as a matrix, after impregnation with citric acid aqueous solution, dehydration condensation reaction, washing and drying, a CA-BC material with carboxyl groups grafted on the surface is obtained; S3: Select acid- and bile-tolerant Lactobacillus strains, activate them in MRS medium, collect the cells by centrifugation, wash and resuspend them, and adjust the bacterial concentration to 10. 9 -10 11 CFU / mL was used to obtain a high-concentration bacterial suspension. S4: Using a layer-by-layer assembly process of internal phase dispersion-ionic crosslinking-external phase coating, CA-BC is dispersed in sodium alginate solution, probiotics are inoculated to obtain an internal phase mixture, primary gel beads are formed by CaCl2 ionic crosslinking, and then coated by electrostatic complexation with chitosan solution to obtain the biochar-based probiotic microspheres.

2. The method for preparing biochar-based probiotic microspheres for targeted colonization of broiler intestines according to claim 1, characterized in that, In S1, iron salt is preferred. The preferred nitrogen source is urea, melamine, dicyandiamide, or thiourea.

3. The method for preparing biochar-based probiotic microspheres for targeted colonization of broiler intestines according to claim 1, characterized in that, In step S1, biomass powder, iron salt, and nitrogen source are added together in deionized water or ethanol aqueous solution at a mass ratio of 1:(0.1-1.0):(0.5-3.0). The mixture is continuously stirred or ultrasonically dispersed at room temperature to 80°C for 4-24 hours to complete the impregnation and complexation of the precursor. The mixed slurry is then dried in an oven at 80-105°C to obtain a solid precursor.

4. The method for preparing biochar-based probiotic microspheres for targeted colonization of broiler intestines according to claim 3, characterized in that, In step S1, the solid precursor is placed in a quartz boat or corundum boat in a tube furnace, and high-purity N2 or Ar is introduced as a protective gas at a flow rate of 100-500 mL / min. Pyrolysis is carried out using a segmented heating strategy. First, the temperature is raised to 400-600℃ at a rate of 2-10℃ / min and held for 30-60 minutes. Then, the temperature is raised to 700-1000℃ and held for 1-3 hours. After the pyrolysis product is naturally cooled to room temperature, it is ground into powder. The powder is soaked and stirred in 0.1-2.0M HCl or H2SO4 solution for 6-24 hours. After acid washing, the powder is repeatedly washed with deionized water until the pH of the filtrate is 7.

0. Finally, the powder is dried in a vacuum drying oven at 60-80℃ to obtain Fe / N-HPC material.

5. The method for preparing biochar-based probiotic microspheres for targeted colonization of broiler intestines according to claim 1, characterized in that, In step S2, Fe / N-HPC is used as the matrix. A citric acid aqueous solution with a concentration of 0.5-3.0M is prepared. Fe / N-HPC powder is added to the citric acid aqueous solution at a solid-liquid ratio of 1:(5-20)g / mL. The mixture is stirred for 12-24 hours at room temperature or 40-60℃ to complete the citric acid impregnation. The biochar is then separated by filtration or centrifugation. The separated biochar is dried in an oven at 50-60℃ until the moisture evaporates. Then, it is heat-treated in an oven or tube furnace at 100-160℃ for 60-120 minutes to complete the dehydration condensation reaction. Finally, the biochar is thoroughly washed with deionized water to remove unreacted free citric acid. After drying, CA-BC material is obtained.

6. The method for preparing biochar-based probiotic microspheres for targeted colonization of broiler intestines according to claim 1, characterized in that, In S3, the lactobacillus strain is selected from one or more of Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus casei, and Lactobacillus rhamnosus.

7. The method for preparing biochar-based probiotic microspheres for targeted colonization of broiler intestines according to claim 1, characterized in that, In S3, the activation culture is carried out by anaerobic static culture at 37°C for 18-24 hours and passaged 2-3 times to the end of the logarithmic growth phase or the early stage of the stationary phase. The bacterial cells are collected by centrifugation at 3000-5000 rpm for 10-15 minutes at 4°C. The cells are washed 2-3 times with sterile physiological saline or PBS buffer at pH 7.

2.

8. The method for preparing biochar-based probiotic microspheres for targeted colonization of broiler intestines according to claim 1, characterized in that, In step S4, sodium alginate powder is dissolved in distilled water to prepare a colloidal solution with a concentration of 1.5%-3.5% (w / v). After high-temperature sterilization and cooling to room temperature, the CA-BC powder prepared above is added to the solution, making the amount of CA-BC powder added to the sodium alginate colloidal solution 0.5%-3.0% (w / v). The CA-BC powder is uniformly dispersed by vigorous mechanical stirring and ultrasonic dispersion for 30-60 minutes to form an SA-BC suspension. The high-concentration probiotic suspension prepared is inoculated into the SA-BC suspension at a volume ratio of 1:4-10, and gently stirred to mix evenly, controlling the viable bacteria count in the mixture to be 10. 8 -10 9 CFU / mL was used to obtain an SA-BC-Pro internal phase mixture; anhydrous calcium chloride was dissolved in distilled water to prepare a hardening solution with a concentration of 1.0%-5.0% (w / v), and the pH was adjusted to neutral. Using a sterile syringe with an injection pump or a high-voltage electrostatic microcapsule forming device, the SA-BC-Pro internal phase mixture was dripped into the CaCl2 hardening solution with slight stirring. After cross-linking and curing for 15-45 minutes, primary gel beads were formed.

9. The method for preparing biochar-based probiotic microspheres for targeted colonization of broiler intestines according to claim 1, characterized in that, In step S4, primary gel beads are filtered out using a filter screen and rinsed 2-3 times with sterile distilled water. Chitosan powder with a deacetylation degree >85% is selected and dissolved in a 0.5%-2.0% (v / v) acetic acid solution to prepare a chitosan solution with a concentration of 0.2%-1.5% (w / v). The pH of the chitosan solution is adjusted to 4.5-5.5 with NaOH solution, and a small amount of CaCl2 can be added. The rinsed primary gel beads are immersed in the chitosan solution and placed on a shaker at a speed of 50-100 rpm for 10-60 minutes for coating. The coated composite microspheres are filtered out using a filter screen and quickly washed with sterile water to obtain wet biochar-based probiotic microspheres. Alternatively, the washed composite microspheres can be soaked in cryoprotectants such as trehalose or skim milk and then freeze-dried under vacuum to prepare dry powder microspheres.

10. A biochar-based probiotic microsphere prepared by the preparation method according to any one of claims 1-9, characterized in that, The prepared biochar-based probiotic microspheres are used as feed additives. They are added directly to the broiler basic feed at an addition rate of 0.5%-2% of the broiler basic feed and mixed evenly before being used to feed broilers at different growth stages. These microspheres can be applied to the regulation of intestinal flora, detoxification of mycotoxins, and prevention and control of intestinal inflammation in the broiler breeding process, and are suitable for broiler breeding scenarios with mycotoxin contamination and intestinal flora imbalance.

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