Piglet anti-diarrhea complete feed formula based on microecological preparation

Abstract

The invention discloses a piglet anti-diarrhea complete feed formula based on a microecological preparation, and relates to the technical field of animal nutrition and health. The microsphere has a unique three-layer core-shell structure, wherein a core layer is coated with butyric acid producing probiotics and precise prebiotics; the middle layer is a chitosan-gallic acid-vanillin terpolymer with dual functions of strong mucous membrane adsorbability and quorum sensing quenching; and the shell layer is a pH-sensitive slow-release coating and is loaded with an acidifying agent. The structure is constructed through a layer-by-layer coating process, and the technical problems that in the prior art, functional components cannot be delivered in a targeted mode, the synergistic effect is poor, and intestinal barriers cannot be actively repaired are solved. When the microspheres enter animal digestive tracts, the shell layer firstly releases an acidifying agent at the front end to inhibit pathogenic bacteria and clear obstacles for subsequent steps; after entering the small intestine, the middle layer polymer is released, adheres and repairs the damaged intestinal mucosa in the form of a biological bandage, and interferes pathogen communication at the same time.

Description

Technical Field

[0001] This invention relates to the field of animal nutrition and health technology, and in particular to a complete feed formulation for piglets to combat diarrhea based on microecological preparations. Background Technology

[0002] Gut health is the cornerstone of an animal's overall production performance and health. However, in intensive farming, animals, especially young animals, are highly susceptible to gut microbiota dysbiosis due to various stressors such as weaning, feed changes, and environmental shifts. This dysbiosis leads to the overgrowth of harmful bacteria, resulting in a range of gut health problems including diarrhea and growth retardation. To address this challenge, various intervention methods have been developed, such as directly supplementing with probiotics to competitively inhibit pathogens, using acidifiers to regulate intestinal pH, or adding prebiotics to promote the growth of beneficial bacteria.

[0003] While these methods have achieved some success, existing technologies generally have inherent limitations. Most products on the market today are simply physical mixtures of various functional components such as probiotics, acidifiers, and plant extracts. This crude preparation method results in all components being released simultaneously without targeting after entering the digestive tract, creating a functional "confusion." For example, an acidifier intended to inhibit pathogens in the lower intestine may be prematurely released and neutralized in the stomach or upper small intestine, failing to achieve the desired effect. Furthermore, the resulting acidic environment may directly kill the extremely fragile probiotics in the formula, causing internal consumption and waste of active ingredients.

[0004] Furthermore, most existing technologies remain at the level of passive defense or single-stage intervention. Even those technologies that use coating to protect probiotics only address the issue of "survival," failing to systematically consider how to create optimal conditions for their "colonization" and "functionality." The attachment and invasion of the intestinal mucosa by pathogens is an active process, often involving the coordinated action of biofilm formation and quorum sensing systems. However, existing technologies rarely offer strategies to actively interfere with this pathological process or efficiently repair damaged intestinal mucosa at the molecular level. Therefore, even if functional components reach the intestine, they may be unable to exert their effects due to severely compromised intestinal barriers and excessive pathogen load.

[0005] Current technologies lack a systematic solution for addressing complex gut health issues that can organically integrate multiple functions, achieve programmed sequential release, and proactively intervene in pathological processes. Therefore, developing a novel gut health formulation that can protect active ingredients, achieve targeted delivery, and synergistically exert multiple effects of "clearing, repairing, and rebuilding" has significant practical implications and application value. Summary of the Invention

[0006] The purpose of this invention is to provide a complete feed formula for piglets to combat diarrhea based on microecological preparations, which solves the technical problems of functional components not being able to be delivered in a targeted manner, poor synergistic effect, and inability to actively repair the damaged intestinal barrier in the prior art.

[0007] To address the aforementioned technical problems, the first aspect of this invention provides a complete feed formula for piglets to combat diarrhea based on microecological preparations.

[0008] The composition is a composite microsphere with a multilayer core-shell structure. Based on the total weight of the composition, it comprises the following components: 3–12% probiotic spores; 10–25% prebiotics; 8–20% bioadhesive polymer-quorum sensing quencher copolymer; 5–15% toxin adsorbent; 8–20% acidifier; and 2–8% fast-acting energy source.

[0009] The key is that, according to their functions, the above components are encapsulated in different structural levels of the composite microspheres: Preparation of chitosan-gallic acid-vanillin terpolymer nanoparticles 1. Preparation of chitosan-gallic acid copolymer: 2.0 g of chitosan (90% degree of deacetylation, viscosity-average molecular weight 200 kDa) was weighed and dissolved in 100 mL of 1% (v / v) acetic acid solution, and magnetically stirred until completely dissolved. Separately, 1.2 g of gallic acid, 2.2 g of EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) and 1.3 g of NHS (N-hydroxysuccinimide) were weighed and dissolved in 50 mL of methanol-water (1:1, v / v) mixed solvent, and activated at room temperature for 30 minutes. The activated gallic acid solution was added dropwise to the chitosan solution, and the reaction was carried out at 25 °C in the dark for 24 hours. After the reaction was completed, the pH of the mixed solution was adjusted to 9.0 with 1M sodium hydroxide solution, and a precipitate was formed. The precipitate was collected by centrifugation, washed three times alternately with pure water and ethanol, and freeze-dried to obtain chitosan-gallic acid copolymer (intermediate product).

[0010] 2. Preparation of chitosan-gallic acid-vanillin terpolymer: Weigh 1.5 g of the above dried intermediate product and disperse it in 100 mL of ethanol-water (9:1, v / v) mixed solvent. Weigh 0.8 g of vanillin, dissolve it in 10 mL of ethanol, and then slowly add it dropwise to the above dispersion. React at 50 °C for 12 hours to form a Schiff base. After the reaction solution is cooled to room temperature, slowly add 0.5 g of sodium borohydride aqueous solution in an ice bath and continue the reaction for 6 hours for reduction. After the reaction is complete, adjust the pH to neutral with dilute hydrochloric acid to produce a precipitate. Collect the precipitate by centrifugation, dialyze it thoroughly with pure water for 72 hours to remove unreacted small molecules, and finally freeze-dry it to obtain the target product: chitosan-gallic acid-vanillin terpolymer.

[0011] 3. Preparation of nanoparticles (ionic crosslinking method): Dissolve 100 mg of the above terpolymer in 50 mL of 0.5% acetic acid solution. While stirring vigorously, rapidly inject 10 mL of 0.1% (w / v) sodium tripolyphosphate (TPP) solution into the solution, and continue stirring for 30 minutes. The appearance of an opalescent sheen in the solution indicates the formation of nanoparticles. This suspension can be directly used for the next coating process.

[0012] Preparation of composite microspheres In this embodiment, a fluidized bed coating machine is used to complete the three-layer coating.

[0013] 1. Preparation of core particles: Weigh out Clostridium difficile subsp. butyricum spore powder (effective viable count ≥ 1 x 10¹). 0 600g of Bacillus licheniformis spore powder (effective viable count ≥ 2 x 10¹ CFU / g) 0 300g of CFU / g and 1600g of long-chain inulin were mixed evenly. This mixed powder was used as the starting material and placed in a fluidized bed. An 8% (w / v) aqueous coating solution was prepared by mixing resistant starch and gellan gum at a weight ratio of 3:1. The inlet air temperature was set to 70℃, the material temperature to 40-45℃, and the peristaltic pump spray rate to 30mL / min. The bed material was coated until the coating weight gain reached 25%. After drying, the core layer particles were obtained.

[0014] 2. Coating of the intermediate layer: The core layer particles described above are used as the new bed material. The copolymer nanoparticle suspension prepared in Example 1 is taken, and yeast cell wall polysaccharides are dispersed into it, resulting in a copolymer solid content of 2% and a yeast cell wall polysaccharide solid content of 1.5%. Sodium alginate and pectin are mixed at a weight ratio of 2:1 to prepare a 4% (w / v) aqueous solution as the binder / coating solution. The functional suspension and coating solution are sprayed simultaneously or alternately using a bottom spray method, with the inlet air temperature set at 60°C and the material temperature at 38–42°C. Coating is continued until a weight gain of 40% is achieved, followed by drying to obtain particles with a two-layer structure.

[0015] 3. Coating of the outer shell layer: The two-layer structured particles described above are used as the bed material. Microencapsulated calcium formate and benzoic acid (ratio 1:1) are mixed with glyceryl butyrate at a weight ratio of 3:1. An 8% ethanol solution of ethyl cellulose is used as the coating solution, and top spraying is employed for coating. The inlet air temperature is set at 50℃, and the material temperature at 35-40℃, with attention paid to explosion-proof measures. Coating is carried out until the weight gain reaches 25%. After thorough drying, cooling and sieving yields the final three-layer core-shell composite microsphere product.

[0016] Outer shell: As the outermost layer of the microspheres, it encapsulates an acidifier and a fast-acting energy source. In a preferred embodiment, the acidifier is microencapsulated particles of calcium formate or benzoic acid, and the fast-acting energy source is glyceryl butyrate. This outer shell is coated with ethyl cellulose, a structure designed to allow for slow degradation and release in the acidic environment of the stomach and upper small intestine (pH 2.5–5.5) after ingestion by piglets.

[0017] Intermediate layer: This layer surrounds the core layer and is covered by the outer shell layer. It contains a bioadhesive polymer-quorum sensing quencher copolymer and a toxin adsorbent. In a preferred embodiment, the copolymer is a chitosan-gallic acid-vanillin terpolymer in the form of nanoparticles; the toxin adsorbent is a yeast cell wall polysaccharide. This intermediate layer is coated with a sodium alginate-pectin complex, which is stable in gastric acid but swells or dissolves in the weakly acidic to neutral environment of the mid-small intestine (pH 6.0–7.0), thereby releasing its contents.

[0018] Core layer: As the innermost part of the microspheres, it is encapsulated by an intermediate layer and an outer shell, containing probiotic spores and prebiotics. In a preferred embodiment, the probiotic spores are Clostridium difficile subsp. butyricum spores or Bacillus licheniformis spores; the prebiotics are long-chain inulin. This core layer is coated with enteric-coating materials such as resistant starch or gellan gum to ensure that it can pass intact through the stomach and most of the small intestine, and is ultimately degraded and released in the microbial environment of the hindgut.

[0019] In one specific embodiment of the invention, the weight ratio of the bioadhesive polymer-quorum sensing quencher copolymer in the intermediate layer to the probiotic spores in the core layer is 1:1 to 5:1. This ratio ensures that sufficient copolymer is released before probiotic colonization to achieve adequate repair of the intestinal barrier and effective interference with pathogens, creating favorable conditions for subsequent probiotic colonization.

[0020] In summary, the present invention has at least one of the following beneficial technical effects: 1. This invention, through its unique three-layer core-shell structure and layer-by-layer coating process, achieves programmed and time-sequential intervention in different segments of the intestine. Unlike traditional products that simply mix all components, this invention enables each functional component to be released precisely and sequentially in the preset digestive tract location, avoiding mutual interference between components and premature failure in non-target areas. This greatly improves the bioavailability and efficacy of each active substance, thereby achieving targeted regulation and systematic treatment of the complex intestinal environment.

[0021] 2. This invention is the first to couple two mechanisms, physical barrier repair and pathogen signal interference, onto a single functional molecule. Through a self-designed chitosan-gallic acid-vanillin terpolymer, a breakthrough has been achieved in repairing the intestinal barrier. This copolymer, with its strong mucosal adsorption, can form an active biological bandage on the damaged intestinal surface, instantly sealing the damage. At the same time, its quorum sensing quenching groups can effectively disrupt the communication system of pathogens and inhibit their synergistic pathogenicity, achieving the dual effects of simultaneous repair and countermeasure, fundamentally changing the passive defense situation.

[0022] 3. This invention, through its ingenious multi-layered encapsulation design, provides unprecedented protection for the probiotics in the core layer, significantly improving their survival rate and ultimate colonization efficiency in the digestive tract. It effectively resists the erosion of various digestive fluids such as gastric acid and bile salts, ensuring that a high concentration of live bacteria can safely reach the optimal target site in the lower intestine. This overcomes the technical bottleneck of traditional probiotic products, which are prone to activity loss and unstable effects, guaranteeing the reliable realization of the microecological reconstruction function.

[0023] 4. This invention constructs a synergistic and synergistic systematic solution. The acidification of the outer shell layer removes obstacles for subsequent steps, the repair function of the middle layer paves the way for the colonization of probiotics, and the probiotics and their precise prebiotics in the core layer can deeply rebuild the microecological balance. The functions of each layer complement each other, and the overall effect can more comprehensively and thoroughly address intestinal health problems such as diarrhea caused by multiple factors, demonstrating a comprehensive advantage that single-function products cannot match.

[0024] 5. This invention introduces precise prebiotics into the core layer, achieving selective nourishment of target probiotics, thereby enabling the continuous and efficient production of key functional metabolites such as butyrate. This goes beyond simply supplementing exogenous probiotics; by providing a dedicated diet to support their growth into the dominant flora in the gut, it drives the repair and development of intestinal epithelial cells from within, fundamentally and persistently strengthening the gut's own barrier function and immune homeostasis, achieving long-term maintenance of gut health. Detailed Implementation

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example

[0026] This embodiment provides a complete feed formula for piglets to combat diarrhea based on microecological preparations. The specific steps are as follows: 1. Formulation composition (based on the preparation of 100kg of product), core layer contents (25kg); Bacillus licheniformis and Clostridium difficile subsp. butyricum complex spore powder: 7 kg, long-chain inulin: 18 kg, intermediate layer contents (24 kg), chitosan-gallic acid-vanillin terpolymer: 14 kg, yeast cell wall polysaccharide: 10 kg; outer shell contents (19 kg), microencapsulated calcium formate / benzoic acid (1:1): 14 kg, butyric acid glyceride: 5 kg, coating excipients (total 32 kg): used for coating and bonding of each layer.

[0027] 2. Preparation steps: (1) Preparation of core particles: 7 kg of compound spore powder and 18 kg of long-chain inulin were premixed in a V-type mixer for 15 minutes. This mixture was then fed into a fluidized bed coating machine as the bed material. An 8% (w / v) aqueous solution of resistant starch and gellan gum (3:1) was used as the coating solution, and coating was performed under the following parameters: inlet air temperature 70℃, material temperature 42℃, and spraying rate 80 mL / min. When the coating weight gain reached 40% of the total weight of the core layer contents (approximately 10 kg of auxiliary material was used), spraying was stopped and the mixture was dried to obtain the core particles.

[0028] (2) Covering of the intermediate layer: Using the aforementioned core particles as the bedding material, 14 kg of terpolymer and 10 kg of yeast cell wall polysaccharide were dispersed in water, and a 4% (w / v) aqueous solution of sodium alginate-pectin (2:1) was used as the binder / coating solution. Coating was performed using a bottom spray method with the following parameters: inlet air temperature 60℃, material temperature 40℃, and spray rate 100 mL / min. When the coating weight gain reached 50% of the total weight of the intermediate layer contents (approximately 12 kg of auxiliary material was used), spraying was stopped and the mixture was dried to obtain double-layer coated particles.

[0029] (3) The outer shell layer covering: Using the aforementioned double-layer coated granules as the bed material, 14 kg of microencapsulated acidifier and 5 kg of glyceryl butyrate were dry-mixed. An 8% (w / v) ethanol solution of ethyl cellulose was used as the coating solution. The dry powder mixture and coating solution were alternately sprayed in using a top spray method for coating. The parameters were set as follows: inlet air temperature 55℃, material temperature 38℃, and spray rate 90 mL / min. When the coating weight gain reached 52.6% of the total weight of the outer shell contents (approximately 10 kg of auxiliary material was used), spraying was stopped, and the mixture was thoroughly dried, cooled, and sieved to obtain the final product. Example

[0030] This embodiment provides a complete feed formula for piglets to combat diarrhea based on microecological preparations. The specific steps are as follows: 1. Formulation composition (based on the preparation of 100kg of product): Core layer contents (13kg), compound spore powder: 3kg, long-chain inulin: 10kg, intermediate layer contents (13kg), chitosan-gallic acid-vanillin terpolymer: 8kg, yeast cell wall polysaccharide: 5kg, outer shell contents (10kg), microencapsulated calcium formate / benzoic acid (1:1): 8kg, butyrate glyceryl ester: 2kg, coating excipients (total 64kg): due to the low content of active ingredients, the amount of excipients used is relatively high.

[0031] 2. Preparation steps: (1) Preparation of core particles: 3 kg of compound spore powder and 10 kg of long-chain inulin were mixed and fed into a fluidized bed. A 6% (w / v) aqueous solution of resistant starch was used as the coating solution, with the following parameters set: inlet air temperature 65℃, material temperature 40℃, and spray rate 60 mL / min. The coating weight gain reached 100% of the total weight of the core layer contents (using 13 kg of excipients), and the core particles were obtained after drying.

[0032] (2) Covering of the intermediate layer: Using the aforementioned core particles as the bed material, 8 kg of terpolymer and 5 kg of yeast cell wall polysaccharide were dispersed, and a 3% (w / v) aqueous solution of sodium alginate was used as the coating solution. The parameters were set as follows: inlet air temperature 58℃, material temperature 38℃, and spray rate 70 mL / min. The coating weight gain reached 161.5% of the total weight of the intermediate layer contents (using 21 kg of auxiliary materials). After drying, double-layer coated particles were obtained.

[0033] (3) The outer shell layer covering: Using the aforementioned double-layer coated granules as the bed material, 8 kg of microencapsulated acidifier and 2 kg of glyceryl butyrate were mixed. A 6% (w / v) ethanol solution of ethyl cellulose was used as the coating solution. The parameters were set as follows: inlet air temperature 50℃, material temperature 35℃, and spray rate 65 mL / min. The coating weight gain reached 300% of the total weight of the contents of the outer shell layer (using 30 kg of auxiliary materials). After thorough drying, the final product was obtained. Example

[0034] This embodiment provides a complete feed formula for piglets to combat diarrhea based on microecological preparations. The specific steps are as follows: 1. Formulation composition (based on the preparation of 100kg of product): Core layer contents (37kg), compound spore powder: 12kg, long-chain inulin: 25kg, intermediate layer contents (35kg), chitosan-gallic acid-vanillin terpolymer: 20kg, yeast cell wall polysaccharide: 15kg, outer shell contents (18kg), microencapsulated calcium formate / benzoic acid (1:1): 10kg, butyrate glyceryl ester: 8kg, coating excipients (total 10kg). Due to the high content of active ingredients, the amount of excipients used is relatively low.

[0035] 2. Preparation steps: (1) Preparation of core particles: 12 kg of compound spore powder and 25 kg of long-chain inulin were mixed and fed into a fluidized bed. A 10% (w / v) high-concentration aqueous solution of resistant starch and gellan gum (2:1) was used as the coating solution. The parameters were set as follows: inlet air temperature 75℃, material temperature 45℃, spray rate 90 mL / min, and the airflow was appropriately increased to ensure fluidization effect under high loading. The coating weight gain reached 10.8% of the total weight of the core layer contents (using 4 kg of excipients), and the core particles were obtained after drying.

[0036] (2) Covering of the intermediate layer: Using the aforementioned core particles as the bed material, a high-concentration dispersion was prepared by mixing 20 kg of terpolymer and 15 kg of yeast cell wall polysaccharide. A 5% (w / v) aqueous solution of sodium alginate and pectin (1:1) was used as the coating solution. The parameters were set as follows: inlet air temperature 65℃, material temperature 43℃, and spray rate 120 mL / min. The coating weight gain reached 8.6% of the total weight of the intermediate layer contents (using 3 kg of auxiliary materials). After drying, double-layer coated particles were obtained.

[0037] (3) The outer shell layer covering: Using the aforementioned double-layer coated granules as the bed material, 10 kg of microencapsulated acidifier and 8 kg of glyceryl butyrate were mixed. A 10% (w / v) ethanol solution of ethyl cellulose was used as the coating solution. The parameters were set as follows: inlet air temperature 60℃, material temperature 40℃, and spray rate 110 mL / min. The coating weight gain reached 16.7% of the total weight of the contents of the outer shell layer (using 3 kg of auxiliary materials). After thorough drying, the final product was obtained.

[0038] Comparative Example 1: Compared with Example 1, the difference is that its preparation method does not adopt a three-layer core-shell structure. Instead, all the active components contained in the core layer, middle layer and outer shell layer of Example 1 (i.e., compound spore powder, long-chain inulin, terpolymer, yeast cell wall polysaccharide, microencapsulated acidifier, butyrate glyceryl ester) are physically dry mixed, and then ethyl cellulose is used as a single packaging material to form ordinary coated particles through a fluidized bed granulation mechanism. All other aspects are the same.

[0039] Comparative Example 2: Compared with Example 1, the difference is that the functional polymer used in the intermediate layer is not "chitosan-gallic acid-vanillin terpolymer", but is replaced by an equal weight of ordinary commercially available chitosan, all the other aspects are the same.

[0040] Comparative Example 3: Compared with Example 1, the difference lies in that the coating step of the outer shell layer was omitted in its preparation process. The resulting composite microspheres only contain a core layer coated by the intermediate layer, that is, the product does not contain microencapsulated acidifier and glyceryl butyrate, and all other aspects are the same.

[0041] Comparative Example 4: Compared with Example 1, the difference is that the amount of "chitosan-gallic acid-vanillin terpolymer" in the intermediate layer was significantly reduced from 14 kg to 1 kg, and the 13 kg reduction was made up by the inert auxiliary material corn starch. All other aspects are the same.

[0042] Comparative Example 5: Compared with Example 1, the difference is that the intermediate layer uses a "chitosan-gallic acid binary copolymer", that is, a copolymer lacking the vanillin functional group, and its preparation omits the step of reacting with vanillin. The amount of this binary copolymer is the same as that of the ternary copolymer in Example 1, and all other aspects are the same.

[0043] Comparative Example 6: Compared with Example 1, the difference is that long-chain inulin is not added to the core layer, and its 18kg weight is replaced by an equal amount of glucose, while the rest are the same.

[0044] Comparative Example 7: Compared with Example 1, the difference is that its preparation method does not adopt a layer-by-layer coating process. Instead, the contents of the core layer, the contents of the middle layer and the contents of the outer shell are mixed in ethanol and then prepared into microcapsules by one-step spray drying. All other aspects are the same.

[0045] Test Example 1: Comparative Test of Release Characteristics in an In Vitro Simulated Digestive Tract I. Experimental Procedure 1. Preparation of simulated digestive fluids: Simulated gastric juice: Prepare a hydrochloric acid solution containing 0.32% pepsin and a pH of 2.5, following the pharmacopoeia method. Simulated small intestinal juice: Prepare a phosphate buffer solution containing 1% trypsin and a pH of 6.5, following the pharmacopoeia method. Simulated hind intestinal juice: In a sterile anaerobic operating room, mix fresh, healthy piglet feces with pre-reduced anaerobic culture medium at a ratio of 1:10 to prepare a diluted fecal solution as inoculum.

[0046] 2. First Stage: Simulated Gastric Release: Accurately weigh 10g each of the samples from Example 1, Comparative Example 1, and Comparative Example 7, and place them in conical flasks containing 200mL of SGF. Shake in a constant temperature shaker at 37℃ and 100rpm. At 0, 30, 60, and 120 minutes, respectively, 5mL of sample suspension was taken from each conical flask, filtered through a 0.22μm filter membrane, and the filtrate was collected. The concentration of benzoic acid in the filtrate was determined by high performance liquid chromatography (HPLC), and its cumulative release rate was calculated.

[0047] 3. Second Stage: Simulated Small Intestinal Release: At 120 minutes, the remaining contents of each conical flask were immediately collected by centrifugation, and the solid fraction was washed twice with phosphate buffer to remove residual SGF. The solid was then resuspended in 200 mL of SIF and shaken continuously at 37°C and 100 rpm. At 180, 240, and 300 minutes (i.e., 60, 120, and 180 minutes of reaction in SIF), 5 mL of the sample suspension was taken, filtered, and the filtrate was collected. The absorbance of the chitosan-gallic acid-vanillin terpolymer was measured at the characteristic absorption wavelength using UV-Vis spectrophotometry, and the cumulative release rate was calculated based on the standard curve.

[0048] 4. Third Stage: Simulated Hindgut Release: At 300 minutes, the solid fractions from each group were collected again by centrifugation. After washing with anaerobic buffer, the solid fractions were transferred to anaerobic bottles containing 200 ml of SCF, sealed, and placed in an anaerobic incubator at 37°C. Samples were aseptically taken from the anaerobic bottles at 5 hours (total time since the start of the experiment), 8 hours, 16 hours, and 24 hours. The samples were serially diluted, plated on selective culture media, and the viable cell count was determined by plate counting. Results are expressed as log CFU / g. II. Experimental Data Table 1: Cumulative release rate (%) and viable cell count (log CFU / g) of biomarkers at each stage of simulated digestion in Example 1 and Comparative Examples 1 and 7

[0049] III. Results Summary and Mechanism Elucidation As shown in Table 1, the compositions of the present invention exhibit significant programmed time-sequential release characteristics in an in vitro simulated digestive system. In the sample of Example 1, the outer shell marker benzoic acid was released steadily within 120 minutes of simulated gastric fluid, with a cumulative release rate of 55.3%. During this stage, the release of markers from the intermediate and core layers was extremely low, demonstrating the effective protective effect of the outer coating. When the environment changed to simulated small intestinal fluid, the intermediate layer marker copolymer began to be released in large quantities, while the probiotics in the core layer remained protected. Finally, in the simulated hindgut environment, the live bacteria in the core layer were released in large quantities, reaching a peak at 24 hours. In stark contrast, the samples of Comparative Example 1 (physical mixing) and Comparative Example 7 (one-step preparation) experienced severe burst release during the simulated gastric fluid stage, with most components being rapidly released within the first 120 minutes, completely losing their targeting and temporal characteristics.

[0050] This result strongly confirms that the three-layer core-shell structure constructed in this invention is the foundation for its technical effects. The slow-release design of the outer shell ensures that the acidifier and rapid energy substances can play a pretreatment role of "clearing obstacles and laying the foundation" in the front of the digestive tract, while effectively protecting the internal functional components from gastric acid damage. The pH-sensitive coating of the middle layer allows it to precisely "unlock" in the neutral environment of the small intestine, releasing functional copolymers to perform the key tasks of "active repair and signal interference". The enteric coating of the core layer ensures that probiotics can be escorted to the hindgut, the optimal site of action, with maximum activity for "deep microecological remodeling".

[0051] Therefore, this invention, through its ingenious multi-layered structural design and layer-by-layer coating process, successfully transforms the composition from a simple functional hybrid into an "intelligent" delivery system capable of executing preset programs and achieving functional synergy within the digestive tract. In the comparative example, premature release due to structural deficiencies leads to the inactivation of probiotics and other active substances, and the functional copolymers are unable to function effectively in the most severely damaged small intestine, thus failing to achieve the multi-stage, synergistic overall technical effect desired by this invention. The sequential release characteristic of this invention is a prerequisite and fundamental guarantee for the efficient performance of all its subsequent biological functions.

[0052] Test Example 2: Comparative Test of Quenching Function of Intermediate Layer Quenching I. Experimental Procedure 1. Preparation of test sample solutions: Accurately weigh 5g each of the samples from Example 1, Comparative Example 2, and Comparative Example 5, and place them in 50mL centrifuge tubes. Add 40mL of simulated small intestinal fluid (SIF, pH 6.5) to each tube, and shake in a shaker at 37℃ and 150 rpm for 2 hours to fully release the intermediate layer contents. Centrifuge the tubes at 8000 rpm for 10 minutes, and carefully aspirate the supernatant. Filter the supernatant through a 0.22μm sterile filter membrane to obtain clear test sample solutions for later use.

[0053] 2. Quorum Sensing Quenching Activity Assay: Strain Activation: The quorum sensing reporter strain *Vibrio violaceus* was inoculated into LB liquid medium and incubated overnight at 30°C. Group Co-culture: In sterile 96-well plates, the following groups were set up, with 3 replicate wells per group: Control group: Add 180 μL LLB medium + 20 μL sterile phosphate buffer + 10 μL activated bacterial suspension to each well.

[0054] Example 1 group: Add 180 μL LLB culture medium + 20 μL of Example 1 test solution + 10 μL of activated bacterial suspension to each well.

[0055] Comparative Example 2: Add 180 μL of LLB medium + 20 μL of Comparative Example 2 test solution + 10 μL of activated bacterial suspension to each well.

[0056] Comparative Example 5: Add 180 μL LLB medium + 20 μL Comparative Example 5 test solution + 10 μL activated bacterial suspension to each well.

[0057] Place the 96-well plate in a 30°C incubator and incubate for 24 hours.

[0058] 3. Data Collection: After culturing, the absorbance of each well was first measured at 600 nm using a microplate reader to assess the growth of the strain. Then, 100 μL of 1% SDS solution was added to each well, and the cells were lysed by shaking for 10 minutes. The absorbance of each well was then measured at 585 nm using a microplate reader to quantify the amount of violet pigment produced. II. Experimental Data Table 2: Effects of intermediate layer releases from different samples on the growth of *Violetella violaceum* and the production of violet pigment.

[0059] III. Results Summary and Mechanism Elucidation Table 2 shows that, compared with the control group, the intermediate layer release from Example 1 significantly inhibited the production of violet pigment in *Porphyromonas violaceus*, with its OD... 585 The value was only 0.23, while the control group was as high as 2.89. Meanwhile, the growth of the strains in Example 1 group was not significantly different from the control group (1.55) and other comparative groups. This result clearly demonstrates that the intermediate layer contents of Example 1 possess strong quorum sensing quenching activity, which does not kill bacteria but rather interferes with the bacterial signaling communication system through specific biological pathways.

[0060] The core of this experimental design lies in revealing the mechanism of action of the key functional molecules of this invention through ingenious comparison. Comparative Example 2 (using ordinary chitosan) and Comparative Example 5 (using a copolymer lacking vanillin groups) failed to show any inhibitory ability on the formation of violet pigment, and their results were essentially consistent with the control group. This directly proves two points: First, although the chitosan backbone alone has bioadhesive properties, it does not inherently possess the ability to interfere with bacterial communication; second, comparing the results of Example 1 and Comparative Example 5 confirms that the vanillin groups covalently grafted onto the polymer are the decisive chemical structure for achieving quorum sensing quenching function and disrupting the bacterial signaling system.

[0061] Therefore, this test strongly supports a major innovation of this invention: through molecular design, a biopolymer capable of physically repairing the intestinal barrier was successfully combined with a chemical entity capable of actively attacking the communication system of pathogens, creating an unprecedented dual-functional material. This material no longer passively provides physical isolation, but actively disarms pathogens while forming a biological bandage to protect the intestines, preventing them from colluding to cause harm. This simultaneous repair and countermeasure mechanism directly reflects the advanced nature and inventiveness of this invention, and is also the underlying mechanism behind its highly effective prevention and treatment of diarrhea.

[0062] Test Example 3: Verification Test of Synergistic Effect of Front-End Acidification I. Experimental Procedure 1. Preparation of bacterial strain and culture medium: Pathogenic Escherichia coli (E. coli K88) was inoculated into tryptone soybean broth (TSB) and cultured overnight at 37°C with shaking to prepare a bacterial suspension. Simulated gastric juice (SGF, pH 2.5) and simulated small intestinal juice (SIF, pH 6.5) were prepared.

[0063] 2. Grouping and In Vitro Antibacterial Experiment: Two main groups were set up: Example 1 group and Comparative Example 3 group. First stage (simulated stomach): In a sterile conical flask, 1 mL of the above bacterial suspension was inoculated into 100 mL of TSB medium to make the initial bacterial concentration approximately 10. 7 CFU / mL. Then add 10g of the samples from Example 1 and Comparative Example 3, respectively. Incubate the conical flasks in a shaker at 37°C and 120 rpm for 2 hours. Take samples at 0, 1, and 2 hours.

[0064] Phase 2 (simulated small intestine): After 2 hours, 50 mL of culture was taken from each conical flask, and acidification was terminated by adjusting the pH to 6.5 (simulating the small intestine environment). Incubation continued at 37°C and 120 rpm in a shaker. Samples were taken at 4 and 6 hours of total incubation.

[0065] Viable cell count: Samples collected at all time points were serially diluted with sterile buffer. The bacterial suspensions of appropriate dilutions were spread on MacConkey agar plates and incubated at 37°C for 18-24 hours. Colonies were counted, and the viable cell concentration (log CFU / mL) was calculated and recorded.

[0066] II. Experimental Data Table 3: Comparison of the growth inhibition effects of Example 1 and Comparative Example 3 on Escherichia coli K88 during simulated digestion.

[0067] III. Results Summary and Mechanism Elucidation Table 3 clearly reveals the key synergistic effect of the outer shell layer in this invention. Throughout the simulated digestion process, Example 1 exhibited a strong and sustained inhibitory effect on *E. coli*. Especially during the initial 2-hour simulated stomach phase, the viable bacterial concentration in the Example 1 group decreased by nearly three logarithmic levels, while the colony count in the Comparative Example 3 group, lacking the outer shell layer, only decreased slightly. Once the environment entered the neutral phase of the simulated small intestine, the number of pathogens in the Example 1 group was consistently suppressed to extremely low levels, while the number of pathogens in the Comparative Example 3 group rebounded rapidly, even exceeding the initial inoculation concentration, exhibiting uncontrolled proliferation.

[0068] This significant difference profoundly reveals the intrinsic mechanism of the programmed temporal intervention of this invention. The superiority of Example 1 stems from its ingeniously designed outer shell layer. This layer releases an acidifier at the front end of the digestive tract, strategically clearing acid-sensitive pathogens such as E. coli by lowering the local pH value, thus immediately and efficiently reducing the initial number of pathogens. This clearance step is crucial, creating a less disruptive and less stressful microenvironment for the subsequent action of intermediate layer functional molecules, making subsequent repair and intervention more effective.

[0069] In contrast, Comparative Example 3, lacking this crucial pre-treatment step, meant that its internal functional components had to directly confront a large, undiminished population of pathogens upon entering the small intestine. Even with the presence of functional copolymers, their repair and interference effects were severely weakened by the massive pathogen load, ultimately leading to antibacterial failure. Therefore, this test fully demonstrates that the outer shell is not a simple, dispensable protective layer, but rather an indispensable, foundational, and synergistic link in the entire three-step synergistic chain. It perfectly embodies the design philosophy of this invention: to systematically solve the problem of piglet diarrhea through a logically progressive and interconnected complete solution, rather than relying on the simple superposition of single functions.

[0070] Test Example 4: Dosage Effect Verification Test of Key Functional Components I. Experimental Procedure 1. Preparation of test sample solution: Accurately weigh 5g each of the samples from Example 1 and Comparative Example 4 and place them in 50mL centrifuge tubes. Add 40mL of simulated small intestinal fluid (SIF, pH 6.5) to each tube and shake in a shaker at 37℃ and 150rpm for 2 hours to fully release the intermediate layer contents. Centrifuge the tubes at 8000rpm for 10 minutes and carefully aspirate the supernatant. Filter the supernatant through a 0.22μm sterile filter membrane to obtain a clear test sample solution for later use.

[0071] 2. Dosage-response determination: Strain activation: The quorum sensing reporter strain Chromobacterium violaceum CV026 was inoculated into LB liquid medium and incubated overnight at 30°C.

[0072] Co-culture in groups: In sterile 96-well plates, set up the following groups, with 3 replicate wells per group: Control group: Add 180 μL of LB medium + 20 μL of sterile phosphate buffer + 10 μL of activated bacterial suspension to each well.

[0073] Example 1 group: Add 180 μL LB medium + 20 μL of Example 1 test solution + 10 μL of activated bacterial suspension to each well.

[0074] Comparative Example 4: Add 180 μL of LB medium + 20 μL of Comparative Example 4 test solution + 10 μL of activated bacterial suspension to each well.

[0075] Place the 96-well plate in a 30°C incubator and incubate for 24 hours.

[0076] 3. Data Acquisition and Quantification: After cultivation, the absorbance (OD) of each well was measured using a microplate reader at a wavelength of 600 nm. 600 ( ) to assess the growth of the strain.

[0077] Subsequently, 100 μL of 1% SDS solution was added to each well, and after cell lysis by shaking, the absorbance (OD) of each well was measured at 585 nm using a microplate reader. 585 (This is used to quantify the amount of purple pigment produced.)

[0078] II. Experimental Data Table 4: Effects of different doses of functional components on the quorum sensing system of *Porphyra yezoensis*

[0079] III. Results Summary and Mechanism Elucidation Table 4 clearly reveals the dose-dependent nature of the key functional components in this invention. Example 1 (containing sufficient terpolymer) exhibited an overwhelming inhibitory effect on the formation of violet pigment without affecting basic bacterial growth, with its OD... 585 The value decreased by approximately 90% compared to the control group. However, although Comparative Example 4 (with a significantly reduced terpolymer content) also showed extremely weak inhibition of violet pigment production, its OD value was significantly higher. 585 The value was as high as 2.58, which was not significantly different from the control group, indicating that its quorum quenching function was basically lost.

[0080] This result mechanistically confirms a core concept of the present invention: effective inhibition of the quorum sensing system of pathogens depends on the quencher reaching a critical threshold concentration. The terpolymer in this invention functions through its grafted vanillin groups. When the copolymer is released in the intestine, its local concentration must be sufficient for vanillin molecules to effectively compete for and block the signal receptors of pathogens. The formulation in Example 1 ensured this effective concentration was achieved, thus successfully silencing the bacterial communication network. In contrast, in Comparative Example 4, due to the excessively low content of the functional molecule, its post-release concentration was far below the effective threshold, failing to form effective competitive inhibition. Therefore, the quorum sensing system remained activated, and the bacteria continued their synergistic pathological process.

[0081] Therefore, this test strongly demonstrates that the content range of each component in the composition proposed in this invention is not arbitrarily set, but scientifically limited based on its functional mechanism and effective concentration. This highlights the inventiveness of this invention in its formulation design, not only in what it includes, but also in how much it includes. Simply introducing a functional component conceptually is insufficient; its dosage must be sufficient to achieve the expected technical effect in a complex biological environment. This experiment, from the opposite perspective, confirms that if the content of key functional components is insufficient, the entire technical solution of the invention will be significantly compromised, failing to achieve its core active interference function, thus highlighting the necessity and advancement of the quantitative design of the formulation in this invention.

[0082] Test Example 5: Comparative Test of Precise Prebiotic Effects in the Core Layer I. Experimental Procedure 1. Preparation and separation of core particles: Weigh 10g of samples from Example 1 and Comparative Example 6 respectively and place them in sterile centrifuge tubes. The outer shell and middle layer of the samples were peeled off and removed by sequential incubation and washing in simulated gastric fluid (SGF, pH 2.5) and simulated small intestinal fluid (SIF, pH 6.5), retaining only the core particles. The treated core particles were then resuspended in sterile anaerobic buffer.

[0083] 2. In vitro anaerobic fermentation: Inoculum preparation: Fresh, healthy piglet feces were mixed with pre-reduced anaerobic fermentation medium at a ratio of 1:10 in a sterile anaerobic operating table to prepare a fecal homogenate. Group culture: The following groups were set up in anaerobic serum bottles: Control group: 90 mL anaerobic fermentation medium + 10 mL fecal homogenate.

[0084] Example 1 group: 90mL anaerobic fermentation medium + 10mL fecal homogenate + treated core particle suspension of Example 1.

[0085] Comparative Example 6: 90 mL anaerobic fermentation medium + 10 mL fecal homogenate + treated core particle suspension of Comparative Example 6.

[0086] After sealing all serum bottles, they were placed in a 37°C constant temperature incubator for anaerobic fermentation.

[0087] 3. Sample Analysis: Aseptically sampled from each serum vial at 0, 12, 24, and 48 hours. Centrifuged the samples at 12,000 rpm for 10 minutes and collected the supernatant. The butyrate concentration in the supernatant was quantitatively analyzed using gas chromatography (GC).

[0088] II. Experimental Data Table 5: Effects of different core layer components on butyrate formation during in vitro anaerobic fermentation

[0089] III. Results Summary and Mechanism Elucidation The experimental data in Table 5 strongly demonstrate the synergistic effect of the core layer design of this invention. Example 1 (containing long-chain inulin) induced a large and continuously increasing amount of butyrate during in vitro fermentation, reaching a concentration of 35.7 mmol / L after 48 hours. In contrast, Comparative Example 6 (replacing long-chain inulin with glucose) also promoted butyrate production in the early stages, but its peak concentration was lower and decreased significantly after 24 hours. This result indicates that the formulation of Example 1 can drive the production of the target functional metabolite more efficiently and persistently.

[0090] Behind this significant difference lies the design mechanism of this invention regarding precise prebiotics. Long-chain inulin is a complex carbohydrate that cannot be utilized by most gut microbes, but it is the preferred food for certain probiotics such as Clostridium difficile subsp. butyrate. When the core layer of Example 1 is released in the hindgut, this precise nutrient supply creates a significant competitive advantage for the core probiotics, enabling them to selectively proliferate in large quantities and efficiently convert the substrate into butyrate. In contrast, glucose in Comparative Example 6, as a simple sugar, is quickly consumed by various bacteria in the gut, triggering non-selective and rapid fermentation. This not only leads to premature substrate depletion but also fails to efficiently guide metabolic flow to the butyrate synthesis pathway.

[0091] Therefore, this test fully demonstrates the ingenuity of the core layer design in this invention. It is not merely a simple mixture of probiotics and fiber, but rather achieves a synergistic effect greater than the sum of its parts through precise matching of probiotic strains with their preferred fermentation substrates. This design ensures that in the final step of the digestive process—the back-end reconstruction stage—the core probiotics not only successfully implant but also receive sufficient dedicated nutrients to perform their function, continuously producing butyrate to nourish intestinal epithelial cells, fundamentally repairing and strengthening the intestinal barrier. This reflects the profound understanding and creative application of this invention at the level of microecological regulation.

[0092] Test Example 6: Comparative Test of In Vitro Mucosal Adsorption Capacity I. Experimental Procedure 1. Preparation of fluorescently labeled polymers: The "chitosan-gallic acid-vanillin terpolymer" described in Example 1 of this invention and the ordinary chitosan described in Comparative Example 2 were weighed separately. Using fluorescein isothiocyanate (FITC) as a fluorescent probe, covalent labeling reactions were carried out with the two polymers under weakly alkaline conditions. After the reaction, the polymers were purified by dialysis and freeze-dried to obtain the fluorescently labeled terpolymer (FITC-terpolymer) and the fluorescently labeled chitosan (FITC-chitosan).

[0093] 2. Preparation of intestinal mucosa: Take a segment of jejunum from a healthy piglet and gently rinse it with pre-cooled physiological saline. Carefully scrape off the intestinal mucosa and prepare a homogeneous mucosal suspension with phosphate-buffered saline (PBS, pH 7.0).

[0094] 3. In vitro adsorption experiment: Grouping: Example 1: Add 1 mL of mucosal suspension and 1 mL of FITC-terpolymer solution (concentration 1 mg / mL) to a centrifuge tube.

[0095] Comparative Example 2: Add 1 mL of mucosal suspension and 1 mL of FITC-chitosan solution (concentration 1 mg / mL) to centrifuge tubes.

[0096] Incubation: Place each group of centrifuge tubes in a 37°C constant temperature shaker and incubate in the dark for 1 hour.

[0097] Separation: After incubation, centrifuge the tube at 4000 rpm for 10 minutes to allow the mucosa to precipitate. Carefully aspirate the supernatant.

[0098] 4. Data Acquisition: The fluorescence intensity (F0) of the original polymer solution before incubation and the fluorescence intensity (F1) of the supernatant after incubation were measured using a fluorescence spectrophotometer. The adsorption rate of the polymer was calculated using the formula: Adsorption rate (%) = [(F0-F1) / F0] × 100%.

[0099] II. Experimental Data Table 6: Comparison of the adsorption capacity of different polymers on porcine intestinal mucosa in vitro

[0100] III. Results Summary and Mechanism Elucidation Table 6 shows the significant advantages of the core functional material of this invention in terms of bioadhesion performance. The terpolymer used in Example 1 achieved an adsorption rate of 78.3% on the intestinal mucosa, while the adsorption rate of ordinary chitosan in Comparative Example 2 was only 31.6%. This data indicates that through the molecular design and modification of this invention, the mucosal adhesion ability of the polymer has been successfully increased by more than double, thus providing a solid physical basis for subsequent biological functions.

[0101] This significant improvement in adsorption performance stems from the unique molecular structure design of this invention. Ordinary chitosan's adsorption to mucosa primarily relies on the electrostatic attraction between its positive charge and the negatively charged mucin on the mucosal surface; this interaction is relatively weak and non-specific. However, in the terpolymer of this invention, the introduction of numerous gallic acid groups provides the polymer with abundant phenolic hydroxyl groups. These phenolic hydroxyl groups can form a large, dense network of multi-point hydrogen bonds with the mucin on the mucosal surface, with an interaction strength and stability far exceeding that of a single electrostatic attraction. It is this hydrogen-bonded, multi-interaction-dominated process that endows the terpolymer with a powerful ability to actively capture and anchor to the intestinal mucosal surface.

[0102] Therefore, this test mechanistically confirms the feasibility and advancement of the active biological bandage concept of this invention. This superior adhesive property allows the composition of this invention, upon reaching the damaged small intestine, to rapidly and firmly cover the eroded or inflamed intestinal mucosa like a smart plaster, instantly forming a physical protective barrier. This barrier not only isolates pathogens and toxins from further invasion, but more importantly, it fixes subsequently released active ingredients (such as quorum sensing quenchers) at the most needed sites of action, achieving highly efficient targeted therapy. This perfectly embodies the overall design concept of this invention, starting from material science innovation to ultimately achieve precise biological function. Test Example 7: In vitro intestinal barrier repair and probiotic synergistic effect test I. Experimental Procedure 1. Establishment and Damage of the Caco-2 Cell Model: Human colon cancer cell line Caco-2 was seeded in Transwell culture chambers and cultured for 21 days to allow it to differentiate into a dense monolayer intestinal barrier model. The initial transepithelial electrical resistance (TEER) value was measured. Lipopolysaccharide (LPS, 1 μg / mL) was added to the top layer of the culture chamber for 24 hours to induce an inflammatory response and disrupt tight junctions between cells, thus constructing an intestinal barrier damage model.

[0103] 2. Preparation of test sample solution and barrier repair: Following the aforementioned experimental methods, intermediate layer release solutions were prepared for Example 1 and Comparative Example 2 (with ordinary chitosan as the intermediate layer).

[0104] Remove the LPS culture medium and divide the cells into three groups: Control group: Add fresh serum-free culture medium.

[0105] Example 1 group: serum-free culture medium containing the intermediate layer release solution of Example 1 was added.

[0106] Comparative Example 2: Serum-free culture medium containing the intermediate layer release solution of Comparative Example 2 was added.

[0107] After cells were placed in a 37°C incubator for 12 hours for repair, the TEER values ​​of each group were measured, and their recovery rate relative to the initial TEER values ​​of healthy cells was calculated.

[0108] 3. Probiotic Adhesion Assay: After repair, the cell surface was gently washed with sterile buffer. The core particles from Example 1 were used to release the compound probiotics via an in vitro simulated digestion method, and a bacterial suspension was prepared. An equal volume of bacterial suspension was added to the top layer of the Transwell chambers for all groups, and incubation continued for 2 hours. After incubation, the cells were washed three times with buffer to remove unattached bacteria. Cell lysis buffer was added to lyse Caco-2 cells, releasing the attached bacteria. The lysis buffer was serially diluted and plated onto selective culture media, and the number of viable attached bacteria was counted using the plate count method.

[0109] II. Experimental Data Table 7: Effects of different samples on the repair of damaged Caco-2 cell barrier and subsequent probiotic adhesion

[0110] III. Results Summary and Mechanism Elucidation The experimental data in Table 7 powerfully reveal the key synergistic effects between different functional levels in this invention. Compared to the control group, which only achieved weak natural recovery, the Example 1 group not only restored the integrity of the damaged intestinal barrier to an extremely high level of 81.2%, but also significantly promoted the adhesion of subsequent core probiotics, with the number of adherent bacteria being thousands of times that of the control group. While Comparative Example 2 (using ordinary chitosan) also showed some barrier repair and adhesion promotion effects, its effect was far inferior to that of Example 1, demonstrating its functional limitations.

[0111] This result profoundly elucidates the programmed mechanism of action of the present invention, which involves repair followed by reconstruction. The intermediate layer of Example 1, thanks to the excellent bioadhesion and anti-inflammatory activity of its terpolymer, forms a functional bio-bandage on the damaged cell surface. This bandage not only physically seals the barrier defects caused by LPS, but its gallic acid groups also directly alleviate the inflammatory response, thereby actively promoting the repair of tight junctions between cells. A healthier and more intact cell surface provides an ideal environment for the subsequent colonization of probiotics.

[0112] Therefore, this test fully demonstrates that the inventiveness of this invention lies not only in the simple release of each component, but also in creating decisive favorable conditions for the subsequent step through temporal regulation. In Comparative Example 2, the limited electrostatic adsorption and weak repair capabilities of ordinary chitosan failed to effectively pave the way, making it difficult for probiotics to colonize effectively in the damaged and inflamed environment, even if they arrived. This invention, through the active repair function of the intermediate layer, transforms a pathological environment unfavorable to probiotic survival into a healthy environment conducive to probiotic adhesion. This functional synergy and enhancement is the core of the invention's outstanding technical effect, reflecting its advanced design concept of systematically solving problems.

[0113] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A complete feed formula for piglets to combat diarrhea based on microecological preparations, characterized in that, The composition comprises composite microspheres having a multilayered core-shell structure, and by total weight comprises: Probiotic spores: 3-12%; Prebiotics: 10-25%; Bioadhesive polymer-quorum sensing quencher copolymer: 8-20%; Toxin adsorbent: 5-15%; Acidifying agent: 8-20%; Rapid energy source: 2-8%; The components are distributed in different layers of the microspheres according to their functions: The core layer, coated with enteric material, contains the probiotic spores and prebiotics; the intermediate layer, surrounding the core layer, contains the bioadhesive polymer-quorum sensing quencher copolymer and toxin adsorbent; and the outer shell, surrounding the intermediate layer, contains the acidifier and fast-acting energy substance.

2. The complete feed formula for piglets with anti-diarrheal properties based on microecological preparations according to claim 1, characterized in that, The probiotic spores in the core layer are Clostridium difficile subsp. butyricum spores or Bacillus licheniformis spores, the prebiotic is long-chain inulin, and the enteric material is resistant starch or gellan gum.

3. The complete feed formula for piglets with anti-diarrheal properties based on microecological preparations according to claim 1, characterized in that, The bioadhesive polymer-quorum sensing quencher copolymer in the intermediate layer is a chitosan-gallic acid-vanillin terpolymer, and the toxin adsorbent is a yeast cell wall polysaccharide.

4. The complete feed formula for piglets with anti-diarrheal properties based on microecological preparations according to claim 3, characterized in that, The intermediate layer is coated with a sodium alginate-pectin complex that swells or dissolves in an environment of pH 6.0–7.

0.

5. The complete feed formula for piglets with anti-diarrheal properties based on microecological preparations according to claim 1, characterized in that, The acidifying agent in the outer shell is microencapsulated particles of calcium formate or benzoic acid; the rapid energy substance is glyceryl butyrate.

6. The complete feed formula for piglets with anti-diarrheal properties based on microecological preparations according to claim 5, characterized in that, The outer shell is coated with ethyl cellulose to achieve slow release in the stomach and upper small intestine at pH 2.5–5.

5.

7. The complete feed formula for piglets with anti-diarrheal properties based on microecological preparations according to claim 1, characterized in that, The composition is designed for programmed, timed release within the digestive tract of pigs. First, the outer shell releases components at the front end of the digestive tract to acidify and provide energy; Secondly, the intermediate layer releases components in the middle section of the small intestine to physically repair the intestinal mucosa and interfere with pathogen communication. Finally, the core layer releases components in the hindgut to reshape the microecology and repair the endogenous barrier.

8. The complete feed formula for piglets with anti-diarrheal properties based on microecological preparations according to claim 3, characterized in that, The chitosan-gallic acid-vanillin terpolymer exists in the form of nanoparticles and is designed to perform a dual function at the site of intestinal injury: utilizing the bioadhesive properties of chitosan to form a physical protective film, while simultaneously utilizing vanillin to partially interfere with the quorum sensing system of pathogens.

9. The complete feed formula for piglets with anti-diarrheal properties based on microecological preparations according to claim 2, characterized in that, The long-chain inulin is designed to selectively serve as a proliferation substrate for Clostridium difficile subsp. butyrate and Bacillus licheniformis to synergistically promote their colonization in the hindgut and the generation of butyrate.

10. The complete feed formula for piglets with anti-diarrheal properties based on microecological preparations according to claim 1, characterized in that, The weight ratio between the bioadhesive polymer-quorum sensing quencher copolymer in the intermediate layer and the probiotic spores in the core layer is 1:1 to 5:1.

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