A pet immunization and hair beautifying composition with skin mucosal immunity enhancement function and its preparation method and application

Abstract

The application discloses a pet immune and beautiful hair composition with skin mucosa immune enhancement function and a preparation method and application thereof, and belongs to the technical field of pet food. The composition is a "core-shell-net" microcapsule structure: the lipid phase core contains functional oil and DL-methionine; a polysaccharide grid composed of sodium hyaluronate and beta-1, 3-D-glucan provides physical confinement and mechanical support; a shell layer formed by complex coagulation of lactoferrin and sodium hyaluronate realizes low release in the stomach and intestinal targeted release, and chenopodium quinoa powder is embedded in the shell layer to construct a pre-antioxidant barrier, and lactobacillus plantarum and lactobacillus acidophilus are embedded in the shell layer to realize the protection of probiotics. The application realizes high retention rate of active ingredients under baking processing, significantly prolongs the oxidation induction period and realizes targeted delivery, and ensures high survival rate of probiotics, significantly improves pet hair luster and reduces hair loss by enhancing skin mucosa immunity, and realizes the synergistic delivery of multiple components.

Description

Technical Field

[0001] This invention belongs to the field of pet food technology, specifically, it relates to a pet immune-enhancing and coat-beautifying composition with skin and mucous membrane immune enhancement function, its preparation method and application. Background Technology

[0002] With the improvement of pet ownership standards, pet owners are increasingly demanding better skin health and shinier coats for their pets. Functional oils, such as fish oil rich in Omega-3 fatty acids, evening primrose seed oil rich in Omega-6 fatty acids, and sulfur-containing amino acids like DL-methionine, are recognized as core nutrients for improving coat quality. However, these active ingredients face significant stability challenges in practical applications. Functional oils contain a large number of unsaturated bonds, making them highly susceptible to oxidative rancidity during storage and use. This not only produces unpleasant odors but also generates peroxides harmful to pets' health. Traditional antioxidant strategies typically involve directly mixing antioxidants into the oil phase. However, this internal mixing method lacks an effective external barrier before oxygen penetrates to the core of the oil phase, limiting antioxidant efficiency and making it difficult to maintain the oil's activity in the long term.

[0003] In the industrial production of pet food, dry food typically requires baking at 60-150℃. Traditional physical mixing methods or simple microencapsulation techniques, lacking internal structural support, are prone to capsule wall rupture under thermal stress, leading to leakage of internal oils and inactivation of heat-sensitive ingredients. Furthermore, water-soluble components such as DL-methionine are easily lost in the complex processing and digestive environment, and their premature release and dilution in the stomach limit their effective absorption and utilization in the intestines.

[0004] On the other hand, most existing pet coat-enhancing products focus on single nutritional supplements, neglecting the importance of the skin as the body's first line of immune defense. Modern veterinary research shows that the health of a pet's skin and the balance of its gut microbiota are closely linked through the "gut-skin axis." Lactobacillus plantarum and Lactobacillus acidophilus, as probiotics, have the function of regulating intestinal immunity and enhancing the mucosal barrier. However, probiotics are highly sensitive to the environment and are easily inactivated when baked at temperatures of 120-150°C during the pet dry food baking process. Traditional direct addition or simple encapsulation methods struggle to balance the processing survival rate of probiotics with stomach acid tolerance. Currently, there is a lack of a technological solution on the market that can deeply integrate probiotics, mucosal immune-enhancing components (such as lactoferrin and plasma proteins), and coat-enhancing nutrients, while simultaneously solving the challenges of processing stability, antioxidant interception, and targeted survival of probiotics.

[0005] In existing technologies, there are reports on the preparation of pH-responsive microcapsules using a composite coagulation method (e.g., CN111011594A). However, the wall material system (e.g., gelatin-gum arabic) suffers from insufficient thermal stress at high temperatures, and the issues of optimizing the spatial distribution of antioxidants and protecting probiotics are not resolved. Other technologies (e.g., CN120753351A) have proposed multilayer microcapsule structures, but their structures are drastically different from this invention. They do not employ a "physical grid + chemical shell" system co-constructed by neutral and charged polysaccharides, nor do they achieve an integrated design where immune components serve as both wall materials and functional ingredients. Similarly, they cannot solve the problem of probiotic survival under extreme processing conditions.

[0006] Therefore, developing an integrated pet immune and coat-enhancing composition with a stable physical structure, a highly efficient antioxidant barrier, pH-targeted release, probiotic protection, and immune-nutritional synergy has become an urgent technical problem to be solved in this field. Summary of the Invention

[0007] This invention aims to overcome the shortcomings of existing pet coat-enhancing products, such as significant loss of active ingredients during staple food processing, easy oxidation and rancidity of oils, low bioavailability, low survival rate of probiotics, and lack of synergistic immune-enhancing effects. It provides a pet immune-enhancing coat-enhancing composition with skin and mucous membrane immune-enhancing functions, its preparation method, and its applications. By constructing a polysaccharide mesh with mechanical support and a composite coagulation shell with pre-antioxidant interception function, and embedding probiotics within the shell, the stability of the composition under extreme environments, the survival rate of probiotics, and its clinical coat-enhancing effects are significantly improved.

[0008] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: A method for preparing a pet immune-enhancing and coat-beautifying composition with skin and mucous membrane immune-enhancing function includes the following steps: (1) Preparation of lipid core dispersion: The functional oil is heated to 35-45°C, DL-methionine is added, and the mixture is stirred at 2000-4000 rpm for 15-25 min to obtain a lipid core dispersion in which DL-methionine is uniformly suspended. (2) Preparation of polysaccharide mesh matrix: Sodium hyaluronate with a weight-average molecular weight of 500,000 to 1,500,000 Daltons and -1,3-D-glucan was added to water and stirred at 500-800 rpm for 30-40 minutes at 50-65°C to obtain a polysaccharide mesh matrix; (3) Core-network structure emulsification: Under high shear conditions of 8000-12000 rpm, the lipid core dispersion is added dropwise to the polysaccharide network matrix. After the addition is complete, shearing is continued for 15-30 min to obtain the primary emulsion; (4) Immunoassay complex gel shell coating and probiotic integration: The colostrum is cooled to 30-40°C, and then immunoassay protein components, wolfberry powder, Lactobacillus plantarum and Lactobacillus acidophilus are added. Citric acid solution is then added to adjust the pH of the system to 4.2-4.8, so that the positively charged lactoferrin and the negatively charged sodium hyaluronate in the system form an insoluble complex through electrostatic interaction, and co-deposit with chicken plasma protein powder, coating the surface of colostrum microdroplets to form a complex aggregate shell, and embedding wolfberry powder and probiotics in the membrane structure.

[0009] (5) Curing and drying: The system temperature is reduced to 4-10°C within 15 minutes, the structure is cured for 1-2 hours, and then spray-dried to obtain the pet immune coat beautifying composition.

[0010] Furthermore, the functional oil is a mixture of fish oil and evening primrose seed oil.

[0011] Furthermore, the immune protein component is a mixture of spray-dried chicken plasma protein powder and lactoferrin.

[0012] Further, by weight, the raw materials of the pet immune coat-enhancing composition include: 15.0-25.0 parts fish oil, 3.0-5.0 parts evening primrose seed oil, 3.0-5.0 parts DL-methionine, and 0.6-1.0 parts sodium hyaluronate. -1,3-D-glucan 3.0-5.0 parts, spray-dried chicken plasma protein powder 30.0-50.0 parts, lactoferrin 0.3-0.5 parts, wolfberry powder 3.0-5.0 parts, Lactobacillus plantarum 1.2-2.0 parts, Lactobacillus acidophilus 0.3-0.5 parts; wherein the viable count of Lactobacillus plantarum is ≥5×10 9 CFU / kg, the viable count of the Lactobacillus acidophilus is ≥1×10⁻⁶. 9 CFU / kg.

[0013] Furthermore, in step (3), the dropping rate of the lipid core dispersion is 50-100 mL / min, and the system temperature is controlled at 50-55°C during the emulsification process.

[0014] Further, in step (4), the citric acid solution has a mass percentage concentration of 0.1-0.5% and a dropping rate of 5 mL / min.

[0015] Further, in step (5), the inlet air temperature of the spray dryer is 60-70°C, the outlet air temperature is 45-55°C, and the atomizer speed is 15000-25000rpm.

[0016] The present invention also provides a pet immune-enhancing and coat-beautifying composition with skin and mucous membrane immune-enhancing function prepared by the aforementioned preparation method.

[0017] Furthermore, the pet immune coat-enhancing composition is a microencapsulated particle comprising a lipid core, a polysaccharide mesh matrix, and an immune complex coagulation shell; wherein, beneficial bacteria are embedded in the shell.

[0018] Furthermore, the lipid core comprises functional oils and DL-methionine dispersed therein; the lipid core is dispersed in a mixture of sodium hyaluronate and... The polysaccharide grid matrix is ​​composed of -1,3-D-glucan; the immune complex coagulation shell covers the surface of the polysaccharide grid matrix, the shell is mainly formed by lactoferrin in the immune protein component and sodium hyaluronate in the polysaccharide component through a complex coagulation reaction, and the shell contains wolfberry powder, Lactobacillus plantarum and Lactobacillus acidophilus.

[0019] The present invention also provides the application of the pet immune-enhancing and coat-enhancing composition with skin and mucous membrane immune enhancement function in the preparation of pet coat-enhancing food.

[0020] The core of this invention lies in constructing a synergistic system of "physical mesh support + chemical shell response + functional component space optimization", specifically embodied in: (1) Mechanical support and confinement effect of polysaccharide network: Sodium hyaluronate and β-1,3-D-glucan formed a three-dimensional interpenetrating network through hydrogen bonds and physical entanglement. This neutral polysaccharide network (mainly contributed by β-1,3-D-glucan for physical entanglement and sodium hyaluronate for viscoelasticity) serves as a physical framework, providing physical confinement and mechanical buffering for oil droplets, effectively resisting thermal stress during high-temperature processing, maintaining structural integrity, and reducing leakage. At the same time, this network increases the water molecule permeation resistance, delaying the release of water-soluble components.

[0021] (2) pH response and carrier function of the composite coagulation shell: Under pH conditions of 4.2-4.8, lactoferrin (pI≈8.5, positively charged) in the immunoprotein component interacts electrostatically with sodium hyaluronate (negatively charged) in the polysaccharide component to form an insoluble complex. Chicken plasma protein powder (pI≈4.5-5.5, with a net charge close to zero at this pH) participates in film formation as a filler component, enhancing the membrane's density. This shell is stable (low-release) in the acidic environment of the stomach and dissolves and releases in the neutral environment of the intestine, achieving targeted delivery. At the same time, this shell acts as a carrier, precisely embedding wolfberry powder and probiotics into its structure.

[0022] (3) “Pre-interception” spatial distribution of wolfberry powder: Unlike the traditional method of uniformly dispersing antioxidants in the oil phase, this invention positions wolfberry powder on the outer and shallow layers of the composite coagulation shell. This spatial distribution allows the antioxidant components in wolfberry powder to form the first line of defense on the oxygen permeation path, and are consumed in large quantities before oxygen comes into contact with the internal oil core, thus achieving efficient “pre-interception of antioxidants”.

[0023] (4) Protection of probiotics and synergy of the gut-skin axis: This invention integrates Lactobacillus plantarum and Lactobacillus acidophilus into a composite coagulation shell. By utilizing the mechanical buffering of the polysaccharide grid and the pH shielding of the shell, the high survival rate of probiotics in high-temperature processing and gastric acid environment is ensured. By improving the level of intestinal mucosal immunity (indicated by IgG), skin health and hair quality are improved from the perspective of systemic immunity.

[0024] (5) Synergy and functional integration of various structural elements: The polysaccharide grid (mainly resisting thermal stress), the composite coagulated shell (mainly controlling pH response release and carrying wolfberry powder and probiotics), and wolfberry powder (antioxidant space optimization) have clear functions, well-defined division of labor, and synergistic effects. In addition, chicken plasma protein powder and lactoferrin serve not only as film-forming materials but also as functional components for enhancing skin and mucous membrane immunity. The introduction of probiotics further strengthens the mucous membrane immunity dimension, realizing the integrated design of materials and functions. This "divide and conquer, synergistic integration" design concept solves the problem that a single structure or simple combination cannot meet multiple technical requirements.

[0025] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention uses sodium hyaluronate and The "polysaccharide grid" constructed from 1,3-D-glucan provides physical confinement and mechanical buffering for the internal lipid core. Experimental data show that, under simulated baking conditions at 130°C, the total retention rate of Omega-3 and Omega-6 fatty acids in the composition of this invention can still be maintained at over 90%.

[0026] (2) This invention breaks with the traditional practice of mixing antioxidants into the oil phase. By using a composite coagulation process, wolfberry powder is oriented to the outermost shell layer to construct a "pre-interception" barrier. Experimental data show that the oxidation induction period (OIT) is increased by more than 18 times compared with the traditional mixing method.

[0027] (3) By embedding probiotics in a composite coagulation shell, the present invention utilizes the physical shielding of the shell and the thermal buffering effect of the polysaccharide grid to achieve efficient protection of probiotics under high temperature processing (live count decrease ≤1.5 Log CFU / g) and simulated gastric acid environment (survival rate >85%), thus solving the problem of probiotic inactivation during pet dry food processing and feeding.

[0028] (4) This invention utilizes the pH response characteristics of the lactoferrin-sodium hyaluronate composite coagulation layer to achieve low-release (<10%) in the stomach and high-efficiency release (>88%) in the intestine, with high bioavailability.

[0029] (5) The chicken plasma protein powder, lactoferrin and highly active probiotics integrated in the composition prepared by the present invention not only serve as endothelial materials, but also directly participate in the enhancement of skin and mucous membrane immunity. They regulate immunity through the "gut-skin axis" mechanism. Combined with methionine and functional oils, they significantly improve the quality of pets' hair from multiple dimensions such as "immune defense", "microecological balance" and "nutritional repair". Clinical trials show that the composition of the present invention can increase the hair luster score of pets by 187.1%, reduce hair loss by 71.2%, and increase IgG level to about 3.45 mg / mL. Detailed Implementation

[0030] To enable those skilled in the art to better understand the technical solutions of this invention, the following will provide a more detailed description of this application in conjunction with embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the raw materials used in this invention are all commercially available conventional products; the technical means used, unless otherwise specified, are all conventional means well known to those skilled in the art.

[0031] Example 1 A pet immune-enhancing and coat-beautifying composition with skin and mucous membrane immune enhancement function and its preparation method: 1. Formula Composition Fish oil 15.0 kg, evening primrose seed oil 3.0 kg, DL-methionine 3.0 kg, sodium hyaluronate with a weight average molecular weight of 500,000 Daltons 0.6 kg, β-1,3-D-glucan 3.0 kg, spray-dried chicken plasma protein powder 30.0 kg, lactoferrin 0.3 kg, wolfberry powder 3.0 kg, Lactobacillus plantarum (live count ≥1.0×10⁻⁶) 12 1.2 kg of Lactobacillus acidophilus (CFU / g) and ≥1.0 × 10⁻⁶ live bacteria. 12 CFU / g) 0.3kg.

[0032] 2. Operating Procedures (1) Preparation of lipid-phase core dispersion: 15.0 kg of fish oil and 3.0 kg of evening primrose seed oil were put into a stirred tank with a constant temperature jacket, and the heating was turned on to raise the oil phase temperature to 35°C. 3.0 kg of DL-methionine was slowly added while stirring at 2000 rpm, and stirring was continued for 25 min to form a uniform suspension dispersion system of methionine microcrystals in the oil, which was then set aside for later use.

[0033] (2) Preparation of polysaccharide network matrix: 0.6 kg of sodium hyaluronate and 3.0 kg of β-1,3-D-glucan were added to 5 times their total weight (i.e., 18 kg) of deionized water. Stirring was started and the temperature was raised to 50°C, and stirred at 500 rpm for 40 min. At this temperature, the polysaccharide molecular chains fully unfolded and intertwined with each other, forming a three-dimensional interpenetrating network colloid with high viscoelasticity.

[0034] (3) Core-network structure emulsification: The polysaccharide network matrix obtained in step (2) was transferred to a high-shear emulsifier, and the rotation speed was set to 8000 rpm. Under shear conditions, the lipid core dispersion prepared in step (1) was slowly added dropwise to the polysaccharide network matrix at a rate of 50 mL / min using a peristaltic pump. After the addition was completed, shearing continued for 30 min. During this period, cooling water was introduced through the jacket to control the system temperature to be maintained at 50°C. At this time, the oil was sheared into microdroplets of 10-20 μm and initially wrapped and encapsulated by polysaccharide molecular chains, forming a proemulsion in which the oil microdroplets were initially locked by the polysaccharide network.

[0035] (4) Immunoassay Coating with a Composite Gel Shell: The colostrum was transferred to a reactor equipped with a low-speed anchor stirrer and cooled to 30°C. Then, 30.0 kg of chicken plasma protein powder, 0.3 kg of lactoferrin, 3.0 kg of wolfberry powder (80 mesh), 1.2 kg of Lactobacillus plantarum, and 0.3 kg of Lactobacillus acidophilus were added. After stirring (100 rpm) until homogeneous, a 0.1% citric acid solution was slowly added dropwise at a rate of 5 mL / min to adjust the pH of the system. The addition was stopped when the pH meter showed 4.8. At this time, the positively charged lactoferrin and the negatively charged sodium hyaluronate in the system underwent electrostatic attraction, resulting in a composite aggregation phenomenon and precipitation. A dense protein-polysaccharide composite membrane was deposited on the surface of the colostrum droplets, while the wolfberry powder particles and probiotics (Lactobacillus plantarum and Lactobacillus acidophilus) were physically embedded in the membrane structure.

[0036] (5) Cold curing and drying: Cold brine is introduced into the jacket of the reactor to rapidly reduce the system temperature to 10°C within 15 minutes. The system is then maintained at 10°C and stirred continuously for 2 hours to solidify the structure. The material obtained in step (4) is then fed into a spray dryer with the inlet air temperature set to 60°C, the outlet air temperature set to 45°C, and the atomizer speed set to 15000 rpm. The dry powder is collected to obtain microcapsule particles with a core-shell-network structure.

[0037] Example 2 A pet immune-enhancing and coat-beautifying composition with skin and mucous membrane immune enhancement function and its preparation method: 1. Formula Composition Fish oil 25.0 kg, evening primrose seed oil 5.0 kg, DL-methionine 5.0 kg, sodium hyaluronate with a weight average molecular weight of 1.5 million Daltons 1.0 kg, β-1,3-D-glucan 5.0 kg, spray-dried chicken plasma protein powder 50.0 kg, lactoferrin 0.5 kg, wolfberry powder 5.0 kg, Lactobacillus plantarum (live count ≥1.0×10⁻⁶) 12 2.0 kg of Lactobacillus acidophilus (CFU / g) and ≥1.0 × 10⁻⁶ live bacteria. 12 CFU / g) 0.5kg.

[0038] 2. Operating Procedures (1) Preparation of lipid phase core dispersion: The operation is the same as in Example 1, except that the oil phase temperature is raised to 45°C, the stirring speed is 4000 rpm, and the stirring time is 15 min.

[0039] (2) Preparation of polysaccharide mesh matrix: The operation is the same as in Example 1, except that the temperature is raised to 65°C, the stirring speed is 800 rpm, and the stirring time is 30 min.

[0040] (3) Core-network structure emulsification: The operation is the same as in Example 1, except that the high shear speed is 12000 rpm, the lipid core dispersion droplet acceleration is 100 mL / min, the shearing time is 15 min, and the system temperature is controlled at 55°C.

[0041] (4) Immunocomplex gel shell coating and probiotic integration: The operation is the same as in Example 1, except that the temperature is lowered to 40°C and the pH value is adjusted to 4.2 using 0.5% citric acid solution.

[0042] (5) Cold curing and drying: The operation is the same as in Example 1, except that the cold curing temperature is 4°C and the stirring time is 1 hour. The spray drying inlet air temperature is 70°C, the outlet air temperature is 55°C, and the atomizer speed is 25000 rpm.

[0043] Example 3 A pet immune-enhancing and coat-beautifying composition with skin and mucous membrane immune enhancement function and its preparation method: 1. Formula Composition Fish oil 20.0 kg, evening primrose seed oil 4.0 kg, DL-methionine 4.0 kg, sodium hyaluronate with a weight average molecular weight of 1 million Daltons 0.8 kg, β-1,3-D-glucan 4.0 kg, spray-dried chicken plasma protein powder 40.0 kg, lactoferrin 0.4 kg, wolfberry powder 4.0 kg, Lactobacillus plantarum (live count ≥1.0×10⁻⁶) 12 1.6 kg of Lactobacillus acidophilus (CFU / g) and ≥1.0 × 10⁻⁶ live bacteria. 12 CFU / g) 0.4kg.

[0044] 2. Operating Procedures (1) Preparation of lipid phase core dispersion: The operation is the same as in Example 1, except that the oil phase temperature is raised to 40°C, the stirring speed is 3000 rpm, and the stirring time is 20 min.

[0045] (2) Preparation of polysaccharide mesh matrix: The operation is the same as in Example 1, except that the temperature is raised to 58°C, the stirring speed is 600 rpm, and the stirring time is 35 min.

[0046] (3) Core-network structure emulsification: The operation is the same as in Example 1, except that the high shear speed is 10,000 rpm, the lipid core dispersion droplet acceleration is 80 mL / min, the shearing time is 20 min, and the system temperature is controlled at 52°C.

[0047] (4) Immunocomplex gel shell coating and probiotic integration: The operation is the same as in Example 1, except that the temperature is lowered to 35°C and the pH value is adjusted to 4.5 using 0.2% citric acid solution.

[0048] (5) Cold curing and drying: The operation is the same as in Example 1, except that the cold curing temperature is 8°C and the stirring time is 1.5h. The spray drying inlet air temperature is 65°C, the outlet air temperature is 50°C, and the atomizer speed is 20000rpm.

[0049] Comparative Example 1: Physical Mixture Ten raw materials with the same components and weights as in Example 3 were directly added into a V-type mixer and mechanically stirred at room temperature for 30 minutes. The pH of the mixture was then adjusted to 4.5 using a 0.2% citric acid solution, but no microencapsulation was performed.

[0050] Comparative Example 2: Polysaccharide-free mesh support structure Except for step (2), where sodium hyaluronate and β-1,3-D-glucan are not added (only an equal amount of deionized water is used instead), the remaining preparation steps, parameters, and formulations are the same as in Example 3 (pH adjusted to 4.5). However, due to the lack of sodium hyaluronate in the system, the lactoferrin-sodium hyaluronate composite aggregate layer cannot be formed in the subsequent step (4). Under acidic pH conditions, the added lactoferrin and chicken plasma protein powder mainly undergo self-aggregation, which may form an incomplete and non-dense protein deposition layer on the droplet surface.

[0051] Comparative Example 3: No composite condensed shell Except for step (4), in which the pH value is not adjusted (i.e., no citric acid solution is added, and the system maintains its natural pH value after mixing, which was measured to be pH 6.2), the other preparation steps, parameters and formulas are the same as in Example 3.

[0052] Comparative Example 4: Changes in the distribution location of antioxidant components Except for adding 4.0 kg of wolfberry powder directly to the oil phase in step (1) and not adding wolfberry powder in step (4), the other preparation steps, parameters and formulas are the same as in Example 3 (pH adjusted to 4.5).

[0053] Comparative Example 5: Gelatin-Carrageenan Composite Agglomerated Microcapsules 1. Formula Composition The total amount of active ingredients remained the same as in Example 3 (20.0 kg fish oil, 4.0 kg evening primrose seed oil, 4.0 kg DL-methionine, 0.8 kg spray-dried chicken plasma protein powder, 0.4 kg lactoferrin, 4.0 kg wolfberry powder, 1.6 kg Lactobacillus plantarum, and 0.4 kg Lactobacillus acidophilus). Sodium hyaluronate and β-1,3-D-glucan were replaced with an equal mass of a mixture of gelatin and carrageenan (gelatin:carrageenan = 2:1, w / w).

[0054] 2. Operating Procedures (1) Preparation of lipid-phase core dispersion: Same as in Example 3.

[0055] (2) Aqueous phase preparation: Dissolve gelatin and carrageenan in water, heat to 60°C, and stir until completely dissolved.

[0056] (3) Emulsification: The lipid dispersion was added to the aqueous phase and emulsified by high-speed shearing, as in Example 3.

[0057] (4) Composite coagulation: Cool the emulsion to 40°C, add Lactobacillus plantarum and Lactobacillus acidophilus, and then add citric acid to adjust the pH to 4.0 (near the isoelectric point of gelatin) to induce composite coagulation of gelatin (positively charged) and carrageenan (negatively charged).

[0058] (5) Curing and drying: After cold curing, spray drying is performed with the same parameters as in Example 3.

[0059] Comparative Example 6: Probiotics added later (dry mixture control group) The formulation components are the same as in Example 3. The difference is that Lactobacillus plantarum and Lactobacillus acidophilus are not added in step (4). After spray drying in step (5), the same amount of probiotic powder (1.2 kg Lactobacillus plantarum + 1.2 kg Lactobacillus acidophilus) is added to the surface of the finished granules by dry mixing.

[0060] Data Analysis and Comparison To verify the technical contribution of the "core-shell-network" structure and its specific preparation process described in this invention to improving the stability, probiotic activity and bioavailability of the pet immune coat-enhancing composition, the samples of Examples 1-3 and Comparative Examples 1-6 were subjected to comprehensive physicochemical characterization and biological effect testing.

[0061] Experimental Example 1: Evaluation of Microencapsulation Effect The free oil content on the surface of the microcapsules was determined to evaluate their integration. 2.00 g of each sample was accurately weighed and placed in a 50 mL stoppered conical flask. 20 mL of petroleum ether was added, and the mixture was shaken at 150 rpm for 60 s at room temperature, followed by rapid filtration. The filtrates were combined and placed in a constant-weight evaporating dish. The solvent was evaporated to dryness at 40°C, and the mass of the residual oil was measured as the surface oil. An equal mass of sample was taken and the total oil content was determined using the acid hydrolysis-ether extraction method. The encapsulation efficiency (%) was calculated as (total oil content - surface oil content) / total oil content × 100%. The test results are shown in Table 1.

[0062] Table 1 Encapsulation efficiency and surface oil test results for each group of samples

[0063] Table 1 shows the differences in microencapsulation effects caused by different preparation processes. The encapsulation rates of Examples 1-3 all remained between 91.9% and 94.6%, with surface oil content ranging from 1.54% to 2.31%, indicating that the "core-shell-network" three-layer structure used in this invention effectively encapsulates and isolates the oil. Among these, Example 3 had the lowest surface oil content (1.54%) and the highest encapsulation rate (94.6%), which may be related to the fact that the proportions of the raw materials used in this group (especially the ratio of polysaccharide to protein components) were conducive to forming a relatively compact composite aggregate structure.

[0064] Comparative Example 1, as the physical mixing group, had an encapsulation rate of 0%, with the oil completely exposed on the particle surface. This result was as expected, as the sample without any microencapsulation treatment lacked an encapsulation structure for the oil. Comparative Example 2 had an encapsulation rate of 63.2%, significantly lower than the example group. This difference reflects the irreplaceable role of the polysaccharide network constructed from sodium hyaluronate and β-1,3-D-glucan in the physical locking of the lipid core—without the support of the network structure and an effective composite cohesive shell, oil droplets easily migrate to the particle surface during emulsification, solidification, and drying. Comparative Example 3 failed to form a protein-polysaccharide composite cohesive layer at pH 6.2, with an encapsulation rate of only 21.8% and a surface oil content as high as 22.36%. This indicates that the lack of a composite cohesive shell results in a lack of continuous barrier on the lipid phase surface, allowing water molecules to easily penetrate into the lipid droplets and undergo interfacial rearrangement during drying.

[0065] The encapsulation efficiency of Comparative Example 4 (94.2%) was similar to that of Example 3, indicating that changes in the spatial distribution of wolfberry powder had little impact on the basic encapsulation effect of the microcapsules. This is because wolfberry powder mainly plays an antioxidant role rather than a structural support role. The encapsulation efficiency of Comparative Example 5 was 88.1%, higher than Comparative Examples 2-3 but lower than Examples 1-3, indicating that its composite cohesive shell has a certain encapsulation capacity, but due to the lack of internal mesh support, the encapsulation efficiency is still lower than that of the present invention. Comparative Example 6 was similar to Example 3 in terms of encapsulation efficiency and surface oil index, indicating that the post-addition (dry mixing) of probiotics had no significant effect on the oil encapsulation effect of the microcapsules.

[0066] The three-tiered "core-shell-network" structure constructed in this invention is key to achieving effective encapsulation of the lipid core. The polysaccharide network and the composite condensed shell respectively play the roles of physical confinement and final dense encapsulation in this process.

[0067] Experimental Example 2: Characterization of Complex Cohesion State The physical characteristics of the system were monitored using dynamic light scattering and electrophoretic light scattering techniques. Freshly prepared solutions (i.e., suspensions before spray drying) were diluted 100-fold with pre-adjusted deionized water at the same pH value as the final preparation stage of each sample (i.e., Example 1 was diluted with deionized water at pH 4.8, Example 2 with deionized water at pH 4.2, Example 3 and Comparative Examples 1, 2, and 4 with deionized water at pH 4.5, Comparative Example 3 with deionized water at pH 6.2, and Comparative Example 5 with deionized water at pH 4.0). Particle size distribution, polydispersity index (PDI), span (calculated as (D90-D10) / D50), and zeta potential were measured using a Malvern particle size analyzer at 25°C. Simultaneously, the absorbance (OD600) of the system was measured at 600 nm using a spectrophotometer to characterize the formation of the condensed phase. The test results are shown in Table 2.

[0068] Table 2 Quantitative test results of composite coagulation state of each group of samples

[0069] Table 2 shows the particle size distribution characteristics of each group of samples and their correlation with the composite aggregation state. The PDI values ​​of Examples 1-3 were all between 0.12 and 0.22, and the Span values ​​were between 0.95 and 1.35, significantly lower than the comparative group. This phenomenon indicates that under pH conditions of 4.2-4.8, sufficient charge neutralization and molecular association occurred between the protein and polysaccharide components, forming a relatively uniform integrated particle system. Example 3 exhibited the highest turbidity value (2.42), while its Zeta potential (-1.5 mV) was close to the electroneutrality point. This combination of characteristics reflects that the electrostatic interaction between the protein and polysaccharide near the isoelectric point reached a relatively balanced state.

[0070] In contrast, Comparative Examples 1 and 3 exhibited extremely low turbidity (0.12 and 0.08, respectively), PDI values ​​greater than 0.7, and strong negative Zeta potentials (-32.4 mV and -36.2 mV), indicating that in these two groups of samples, protein and polysaccharide components were mainly dispersed independently in the aqueous phase, lacking phase separation and aggregation phenomena caused by charge interactions. The Zeta potential and turbidity data of Comparative Example 3 (pH 6.2) further confirmed that at this pH, the electrostatic interaction between lactoferrin and sodium hyaluronate was weak, and they could not effectively recombine and aggregate.

[0071] The PDI (0.56) and Span (2.84) of Comparative Example 2 were at intermediate levels, a result consistent with the structural characteristics of this group lacking the polysaccharide network—due to the absence of molecular entanglement between sodium hyaluronate and β-1,3-D-glucan, lipid droplets could not be effectively locked after high shear, resulting in significant heterogeneity in particle size distribution. Furthermore, the Zeta potential of Comparative Example 2 was -18.6 mV, further confirming that the absence of sodium hyaluronate prevented the formation of electrostatically interact-based complex aggregates; although the aggregation and interfacial adsorption of proteins increased the particle size and broadened the distribution, the particle uniformity was still significantly lower than that of the Example group.

[0072] The physical parameters of the composite gel in Comparative Example 4 were basically the same as those in Example 3: D50 was 87.9 μm, PDI was 0.15, Span was 1.02, Zeta potential was -1.4 mV, and turbidity was 2.38. These data indicate that the change in the spatial distribution of wolfberry powder did not have a substantial impact on the initial physical formation of the microcapsules, which is consistent with the structural characteristics of wolfberry powder being mainly distributed on the outer shell rather than participating in the protein-polysaccharide aggregation process.

[0073] The PDI of Comparative Example 5 was 0.35, the Span was 1.98, and the turbidity was 1.52. The particle size uniformity was better than that of Comparative Examples 2-3 but worse than that of Examples 1-3, indicating that although the gelatin-carrageenan system can undergo composite aggregation, the uniformity of its particle distribution is not as good as that of the sodium hyaluronate / β-1,3-D-glucan network system of the present invention.

[0074] The physical parameters (D50, PDI, Span, Zeta potential, turbidity) of the composite gel in Comparative Example 6 were basically the same as those in Example 3, indicating that the subsequent addition of probiotics did not have a significant impact on the physicochemical state of the composite coagulation process.

[0075] The particle size distribution and Zeta potential data confirm the occurrence of the protein-polysaccharide complex aggregation reaction and its contribution to particle homogeneity from a physicochemical perspective. The pH-induced electrostatic interaction is the core mechanism for the formation of the complex aggregate shell of this invention.

[0076] Experimental Example 3: Rheological Characterization of Polysaccharide Network The products of steps (2) of Examples 1 - 3 and Comparative Examples 2 and 5 of each group of samples (i.e., polysaccharide network matrix or corresponding aqueous phase) were subjected to dynamic oscillation tests using a rotational rheometer. An appropriate amount of the sample was placed between the parallel plates of the rheometer (gap 1 mm), and within the linear strain region (1% strain), the angular frequency was scanned at 25°C (0.1 - 100 rad / s), and the storage modulus (G’, characterizing elasticity) and loss modulus (G’’, characterizing viscosity) at an angular frequency ω = 10 rad / s were recorded.

[0077] The values of G’ and G’’ at a frequency of 10 rad / s were compared. Generally, G’ > G’’ indicates that the material exhibits a solid - like gel behavior; G’ < G’’ indicates a fluid - like sol behavior.

[0078] Table 3 Comparison of Rheological Parameters of Each Group of Samples (ω = 10 rad / s)

[0079] As can be seen from Table 3, the G’ values of Examples 1 - 3 were significantly higher than the G’’ values (ratio > 6), clearly confirming that the “polysaccharide network” formed by sodium hyaluronate and β - 1,3 - D - glucan has a significant three - dimensional gel network structure and elastic solid behavior, and can provide physical support for the lipid core.

[0080] Both the G’ and G’’ values of Comparative Example 2 were extremely low and close, and the G’ / G’’ value was only 0.33, showing typical viscous fluid behavior, strongly proving from the opposite side that the lack of the two polysaccharides, sodium hyaluronate and β - 1,3 - D - glucan, cannot form an effective support network.

[0081] Although Comparative Example 5 showed certain viscoelasticity (G’ was slightly greater than G’’), its G’ value (45 Pa) was two orders of magnitude lower than that of the example group (>1200 Pa). The G’ / G’’ value was only 1.18, indicating that its gel network strength was very weak. This proves that the aqueous phase of the traditional complex coacervation system itself does not have strong gel properties, and its mechanical support mainly depends on the brittle wall material formed after drying, which is essentially different from the design of the present invention with an independent and tough “polysaccharide network”.

[0082] The network constructed by sodium hyaluronate and β - 1,3 - D - glucan in the present invention provides significant physical support and buffering ability for the lipid core, which is a structural feature not possessed by the aqueous phase of the traditional complex coacervation system.

[0083] Experimental Example 4: Analysis of Release Behavior of Water - Soluble Components The dissolution kinetics of DL-methionine were investigated to evaluate the binding effect of the network structure on water-soluble components. Each group of samples was placed in 500 mL of deionized water at 37°C and a paddle jet speed of 100 rpm. Samples were taken at 1, 5, 15, and 30 min, filtered through a 0.22 μm filter, and the concentration of DL-methionine in the filtrate was determined by HPLC, and the cumulative dissolution rate was calculated. The test results are shown in Table 4.

[0084] Table 4. Cumulative solubility (%) of DL-methionine over time

[0085] Table 4 shows that different samples exhibited distinct stratification characteristics in terms of the release behavior of water-soluble components. The cumulative dissolution rate of DL-methionine in Examples 1-3 remained between 38.6% and 45.2% within 30 minutes, with the dissolution rate below 30% at 15 minutes. Example 2, using the highest proportion of polysaccharide components, showed the most significant binding effect (dissolution rate of only 38.6% at 30 minutes). This result is consistent with the physical barrier effect of the three-dimensional network structure formed by sodium hyaluronate and β-1,3-D-glucan on the permeation pathway of water molecules and the diffusion channels of DL-methionine molecules—the dense polysaccharide network increases the resistance to water molecule penetration into the particle interior, while also prolonging the diffusion path of dissolved DL-methionine molecules into the external aqueous phase.

[0086] Comparative Example 1, as a physically mixed sample, showed that DL-methionine nearly completely dissolved (98.5%) within 1 minute and reached 100% within 5 minutes. This was due to the rapid dissolution of DL-methionine upon contact with the aqueous phase after direct exposure to the particle surface. Comparative Example 2 showed a dissolution rate of 82.1% at 15 minutes, significantly higher than the Example group. This difference confirms the crucial role of the polysaccharide mesh structure in delaying the release of water-soluble components—the lack of a physical barrier and effective composite condensation shell allows water molecules to directly contact and penetrate the lipid core region, leading to the rapid release of the internally embedded DL-methionine. The dissolution curve of Comparative Example 3 also showed a rapid upward trend, reaching 94.2% at 15 minutes. This reflects the synergistic effect of the barrier system formed by the composite condensation shell and the polysaccharide mesh in controlling the release of water-soluble components—due to unsuitable pH preventing effective shell formation, the polysaccharide mesh was directly exposed to the aqueous phase, thus weakening its blocking effect on water molecules.

[0087] The dissolution curves of Comparative Example 4 were basically consistent with those of Example 3, with dissolution rates of 6.2%, 13.2%, 21.8%, and 39.8% at 1 min, 5 min, 15 min, and 30 min, respectively. These rates were very similar to those of Example 3 (6.4%, 13.5%, 22.1%, and 40.3%). This result is in line with expectations because the wolfberry powder is mainly distributed on the outer surface of the shell, and changes in its spatial position have little impact on the grid binding effect of the small molecule DL-methionine. This further confirms that changes in the spatial distribution of wolfberry powder do not affect the release kinetics of water-soluble components.

[0088] The solubility of Comparative Example 5 at 30 min was 78.3%, which was significantly higher than that of Examples 1-3 (38.6%-45.2%). This indicates that although the gelatin-carrageenan system can form a composite aggregate shell, it lacks the physical barrier of the internal polysaccharide network, and the controlled release ability of the water-soluble components is significantly weaker than that of the present invention.

[0089] The dissolution curve of Comparative Example 6 is basically the same as that of Example 3, indicating that the subsequent addition of probiotics has no effect on the controlled release ability of water-soluble components of the polysaccharide grid.

[0090] This invention utilizes a polysaccharide network to effectively delay the release of water-soluble active ingredients by extending the pathways for water molecule penetration and component diffusion. The composite condensed shell further enhances this blocking effect. The synergy between the two is key to achieving the sustained release of water-soluble components.

[0091] Experimental Example 5: Oxidative Stability Analysis The effect of the spatial distribution of antioxidant components on the protective effect of oils was verified using a Rancimat oxidative stability analyzer. 3.0 g of each sample was weighed, the test temperature was set to 100°C, and dry air was introduced into the reaction tube at a constant flow rate (20 L / h). The time of the conductivity abrupt change was recorded as the oxidation induction period (OIT). The test results are shown in Table 5.

[0092] Table 5. Oxidation Induction Period (OIT) Test Results for Each Group of Samples

[0093] The data in Table 5 can be used to analyze the influence mechanism of different structural features on the oxidative stability of oils. The oxidation induction periods of Examples 1-3 were 45.6 h, 50.8 h, and 53.2 h, respectively, all significantly longer than those of the comparative groups. Comparative Example 1, lacking any protective structure, had an OIT of only 2.5 h. This baseline value reflects the inherent oxidative stability characteristics of the untreated oil.

[0094] Comparative Example 2 showed an OIT of 18.4 h, which was 6.36 times higher than that of Comparative Example 1. This indicates that even without the support of the polysaccharide mesh and an effective composite condensation shell, the incomplete coating of proteins on the droplet surface can still slow down the oxygen permeation rate to some extent. Comparative Example 3 showed an OIT of 8.6 h, which was 2.44 times higher than that of Comparative Example 1. This result reflects that although the polysaccharide mesh can physically encapsulate lipid droplets, its network structure itself has limited ability to block oxygen molecules in the absence of a dense composite condensation shell for synergistic protection.

[0095] Notably, the OIT (Oxidation Induction Period) of Comparative Example 4 (goji berry powder mixed into the oil phase) was 15.1 h, only 5.04 times higher than that of Comparative Example 1. Compared to Example 3 (OIT = 53.2 h, an improvement of 20.28 times) with identical components, the oxidation induction period of Comparative Example 4 was significantly shortened. This difference can be explained from the perspective of the spatial distribution of antioxidant components: In Example 3, goji berry powder was embedded in the surface and shallow layer of the aggregated shell. When external oxygen attempted to penetrate into the internal oil core, it first came into contact with the outer structure rich in goji berry powder. The antioxidant active ingredients such as polyphenols and flavonoids in goji berry powder could play a role in scavenging free radicals at this location, thus forming a "pre-interception barrier" before oxygen reached the oil core. In contrast, in Comparative Example 4, goji berry powder was uniformly dispersed inside the oil, and the antioxidant components were in direct contact with the oil molecules. Although it could also play an antioxidant role, due to the lack of spatial gradient distribution, oxygen could directly attack the oil molecules from any weak point on the particle surface, resulting in relatively low protective efficiency of the antioxidant components.

[0096] The OIT of Comparative Example 5 was 10.2h, which was only 3.08 times higher than that of Comparative Example 1. Its antioxidant capacity mainly came from the physical barrier of the composite condensed shell. However, due to the lack of directional arrangement of wolfberry powder and internal grid support, its oxidation stability was far lower than that of the embodiments of the present invention.

[0097] The OIT (52.8h) of Comparative Example 6 was comparable to that of Example 3 (53.2h), further demonstrating that the method of adding probiotics (encapsulation or post-addition) has no effect on the antioxidant properties of the oils in this system. This is because the antioxidant function is mainly determined by the spatial arrangement of the goji berry powder, and is unrelated to the location of the probiotics. At the same time, this data also rules out the possibility that probiotic encapsulation might interfere with the "pre-interception" effect of the goji berry powder.

[0098] Oxidation induction period data indicate that the spatial distribution of goji berry powder is a key factor affecting the antioxidant effect of oils. Positioning goji berry powder on the outer shell can form a pre-intercepting antioxidant barrier, which is significantly better than the traditional internal mixing method.

[0099] Experimental Example 6: Test of Total Retention Rate of Omega-3 and Omega-6 Fatty Acids and Simulated Gastric Juice Release Rate 1. Total retention rate of Omega-3 and Omega-6 after simulated processing: Each group of samples was treated at 130°C for 30 seconds to simulate the baking process of pet dry food. The total content of Omega-3 and Omega-6 fatty acids in the samples before and after treatment was detected by gas chromatography, and the total retention rate was calculated.

[0100] 2. Simulated gastric juice (SGF) release rate: Each group of samples was first immersed in simulated gastric juice (SGF, pH 2.0) and reacted for 2 hours. The lactoferrin release rate during this period was measured. Then, the pH was adjusted to 6.8, and simulated intestinal juice (SIF) was used to continue the reaction for 4 hours. The cumulative release rate of lactoferrin was measured. The test results are shown in Table 6.

[0101] Table 6. Results of Processing Stability and Targeted Release Rate Tests

[0102] Based on processing stability data, under simulated high-temperature baking conditions, the total retention rates of Omega-3 and Omega-6 fatty acids in Examples 1-3 were 90.2%, 94.5%, and 93.1%, respectively, all maintaining a high level (>90%). The total retention rate of Comparative Example 1 was only 12.4%, reflecting that the unprotected oils under extreme processing conditions underwent almost complete oxidative degradation. The total retention rates of Comparative Example 2 (41.5%) and Comparative Example 3 (35.8%) were at a moderate level, significantly lower than the Example groups. This indicates that both the lack of mechanical support from the polysaccharide network and the absence of a complex coagulated shell due to unsuitable pH (pH 6.2) lead to a decrease in the composition's ability to protect lipid-active components during processing.

[0103] Compared to Comparative Example 5 (gelatin-carrageenan, total retention of Omega-3 and Omega-6 58.2%), the total retention of Omega-3 and Omega-6 in Example 3 of this invention (93.1%) is approximately 35 percentage points higher, demonstrating the core advantages of the polysaccharide mesh of this invention in providing mechanical support and resisting processing thermal stress. The release behavior of Comparative Example 5 at the SGF and SIF stages (8.5% and 90.1%) is similar to that of the examples of this invention, indicating that both possess pH-responsive release characteristics, but this invention is superior in terms of processing stability.

[0104] Based on the gradient release data, the release rates of Examples 1-3 in simulated gastric juice (pH 2.0) were all below 10% (9.5%, 6.8%, and 7.6%, respectively), indicating that the composite coagulation shell formed by lactoferrin and sodium hyaluronate exhibits an insoluble or low-swelling state under acidic conditions, demonstrating tolerance to the gastric juice environment. Correspondingly, Comparative Examples 1, 2, and 3 achieved release rates of 98.5%, 42.6%, and 65.2% in the SGF stage, respectively. This is directly related to whether each group of samples possessed a complete pH-sensitive coagulation shell structure—Comparative Example 1 lacked an encapsulation structure, allowing the lipid-soluble components to be released freely under acidic conditions; Comparative Example 2, lacking the support of a mesh structure and unable to form an effective composite coagulation shell, had limited ability to block gastric acid due to its incomplete protein coating; Comparative Example 3, with a pH of 6.2, could not form a composite coagulation shell, only having the protection of a polysaccharide mesh, resulting in a higher release rate under acidic conditions.

[0105] When the system pH was adjusted to 6.8 (simulating intestinal fluid environment), the cumulative release rates of Examples 1-3 increased to 88.6%, 95.2%, and 92.8%, respectively. This reflects that under near-neutral pH conditions, the solubility of the protein-polysaccharide complex aggregate layer increases, the shell structure dissociates, and the active ingredients are released. The release behavior of Comparative Example 5 in the SGF and SIF stages (8.5% and 90.1%) is similar to that of the embodiments of the present invention, confirming its pH-responsive characteristics. However, due to its lack of internal mesh support, its processing stability is much lower than that of the present invention.

[0106] Comparative Examples 4 and 6 were essentially consistent with Example 3 in terms of processing stability and release behavior. For Comparative Example 3, the wolfberry powder, regardless of its distribution in the shell or oil phase, maintained its antioxidant protective function during high-temperature processing. Although Comparative Example 3 altered the position of the wolfberry powder, its pH-sensitive shell structure remained intact, enabling controlled release under acid-base gradients. For Comparative Example 6, since the probiotics were added post-processed onto the surface of the finished particles and did not participate in the construction of the "core-shell-network" microcapsule structure, it had no effect on the processing protection performance and pH-responsive release performance of the microcapsules. These results indicate that the spatial distribution of wolfberry powder and the method of probiotic addition have no impact on the processing protection and pH-responsive release functions of this system.

[0107] Environmental adaptability evaluation data show that the three-dimensional network framework provided by the polysaccharide grid is a key structural element for maintaining the retention rate of lipid components under high temperature conditions; the pH sensitivity of the composite condensation shell endows the entire system with the functional characteristics of low release in the stomach and release in the intestine.

[0108] Experiment 7: Probiotic Processing Stability and Acid Resistance Test 1. Processing Stability: Take 1.0 g of the initial sample and the sample after baking at 130°C (simulating 30 s), respectively. Under aseptic conditions, add 9.0 mL of sterile physiological saline (containing 0.1% peptone), vortex for 2 minutes to prepare a 1:10 initial homogenate. After serial 10-fold dilutions, take 100 μL of each and spread it onto MRS agar plates specifically for counting *Lactobacillus plantarum* and *Lactobacillus acidophilus*. Incubate the plates in a 37°C anaerobic incubator for 48-72 hours. Select plates with colony counts between 30-300 CFU for counting, and calculate the viable count per gram of sample (Log CFU / g). Processing survival rate is calculated by comparing the viable count after baking with the initial viable count.

[0109] 2. Acid Resistance: Take 1.0 g of the initial sample and place it in a sterile test tube. Add 9.0 mL of simulated gastric juice (containing 0.2% NaCl, 0.32% pepsin, and pH adjusted to 2.0 with hydrochloric acid) preheated to 37°C. Vortex immediately to mix well and incubate at 100 rpm for 2 hours in a 37°C constant temperature water bath. After treatment, immediately take 1.0 mL of the treatment solution and add 9.0 mL of pre-cooled sterile neutralization buffer (0.1M PBS, pH 7.2) to terminate the reaction. Subsequent dilution, plating, culturing, and counting methods are the same as above. Calculate the survival rate of probiotics after 2 hours of simulated gastric juice treatment, using the viable bacterial count of the initial sample without simulated gastric juice treatment as a baseline.

[0110] The total viable count of probiotics was the sum of the viable counts of Lactobacillus plantarum and Lactobacillus acidophilus (CFU / g), which was then converted to the common logarithm (Log CFU / g) for comparative analysis. The test results are shown in Table 7.

[0111] Table 7 Results of Probiotic Activity Test

[0112] After baking, the viable cell count in Examples 1-3 decreased to within the range of 1.2 to 1.3 logarithmic levels. Example 2 showed the highest viable cell count after processing (9.5 Log CFU / g), which was related to its higher initial viable cell count (10.7 Log CFU / g) and optimized formulation ratio.

[0113] Before baking, the probiotics in Comparative Example 6 were not mixed with the sample. Therefore, the viable count "after baking" (2.2 Log CFU / g) is actually the residual viable count of the probiotic powder after undergoing the same high-temperature conditions. Compared with Example 3 (embedded group, 9.2 Log CFU / g after processing), the viable count of Comparative Example 6 after processing was 7.0 log units lower. This significant difference quantitatively demonstrates that naked probiotics without the protection of a "core-shell-network" structure are almost completely inactivated (mortality rate >99.999%) under baking conditions at 130°C, while embedding probiotics within the shell layer achieves highly efficient thermal protection.

[0114] The viable cell counts after baking for Comparative Example 2 (without mesh support, embedded) and Comparative Example 3 (without shell, embedded) were 4.2 and 3.8 Log CFU / g, respectively, which were 5.0 and 5.4 log orders lower than those for Example 3 (9.2 Log CFU / g). This indicates that even with the embedded process, the survival rate of probiotics during high-temperature processing will still decrease significantly in the absence of a complete polysaccharide mesh support (Comparative Example 2) or a pH-responsive composite aggregated shell (Comparative Example 3). There is a synergistic effect between the mesh and the shell in providing thermal protection for probiotics.

[0115] The viable count of Comparative Example 5 (gelatin-carrageenan encapsulation) after baking was 5.6 Log CFU / g, which was 3.6 log orders lower than that of Example 3 (9.2 Log CFU / g). The data indicate that the core-shell-network structure of this invention provides significantly better heat protection for probiotics than the traditional gelatin-carrageenan composite coagulation system.

[0116] After 2 hours of treatment with simulated gastric juice (pH 2.0), the viable bacterial count in Comparative Example 6 decreased from an initial 10.5 Log CFU / g to 1.2 Log CFU / g, a total decrease of 9.3 log units, with a mortality rate approaching 100%. In contrast, the viable bacterial count in Example 3 decreased from 10.5 Log CFU / g to 8.9 Log CFU / g, a decrease of only 1.6 log units. The difference between the two is 7.7 log units. This difference quantitatively demonstrates that the composite aggregated shell with a "core-shell-network" structure forms an effective physical shield for probiotics in the acidic gastric environment, and the subsequently added naked probiotics are almost completely inactivated in the gastric acid.

[0117] The viable bacterial counts of Comparative Example 2 (without mesh) and Comparative Example 3 (without shell) after gastric juice treatment were 3.1 and 2.5 LogCFU / g, respectively, which were 5.8 and 6.4 log orders lower than those of Example 3 (8.9 Log CFU / g). This indicates that the physical confinement of the mesh and the acid shielding effect of the shell are both indispensable in protecting probiotics from gastric acid damage, and together they constitute a highly efficient protective system for probiotics.

[0118] The viable bacterial count of Comparative Example 5 (gelatin-carrageenan) after gastric juice treatment was 6.2 Log CFU / g, which was 2.7 log orders lower than that of Example 3 (8.9 Log CFU / g). The data indicate that the shell layer of this invention has a better gastric acid protection effect on probiotics than the gelatin-carrageenan system.

[0119] The viable bacterial counts (9.1 Log CFU / g) after processing and after gastric juice treatment (8.7 Log CFU / g) in Comparative Example 4 (mixed with wolfberry powder) were essentially the same as those in Example 3 (9.2 and 8.9 Log CFU / g). This indicates that changes in the spatial distribution of wolfberry powder (shell vs. oil phase) do not affect the encapsulation effect of probiotics in the "core-shell-network" structure or the protective efficacy obtained.

[0120] This experiment reveals that probiotics must be embedded within a "core-shell-network" structure to achieve effective heat protection during processing and gastric acid tolerance; simple post-addition (dry mixing) cannot achieve this goal (Comparative Example 6); the polysaccharide network and the complex aggregated shell have a synergistic effect on the protection of probiotics, and the absence of either structure will lead to a significant decrease in protective efficacy (Comparative Examples 2 and 3); the protection system of this invention is significantly superior to the traditional gelatin-carrageenan system in terms of probiotic survival rate (Comparative Example 5).

[0121] Experimental Case 8: Evaluation of the Synergistic Effect of Clinical Treatment on Hair Growth 180 adult cats with similar physical characteristics were randomly divided into 9 groups of 20 each. 5g of the sample was added to each cat's meal daily for 28 consecutive days.

[0122] Evaluation indicators included: (1) hair luster score, which was independently scored by three professional veterinarians under double-blind conditions according to a 10-point scale at the beginning and end of the experiment; (2) percentage reduction in hair loss per unit area, which was determined by standard counting method at the beginning and end of the experiment to measure the change in the number of hair roots per unit skin area (5cm×5cm) before and after treatment and to calculate the reduction rate; Immunoglobulin G assay: On the last day of the experiment, the content of immunoglobulin G (IgG) in serum was detected using an enzyme-linked immunosorbent assay (ELISA) kit, and all testing operations were strictly performed in accordance with the kit instructions. The test results are shown in Table 8.

[0123] Table 8 Evaluation results of clinical feeding trials

[0124] Clinical evaluation results showed a clear stepwise distribution in the improvement of coat luster after 28 days of feeding across the groups. The improvement rates in luster scores for Examples 1-3 were 143.8%, 180.0%, and 187.1%, respectively, corresponding to an increase in scores from the initial 3.0-3.2 to 7.8-8.9 after 28 days. In contrast, the improvement rate in Comparative Example 1 was only 35.5%, a baseline level reflecting the actual bioavailability of unencapsulated nutrients in the pet's body.

[0125] Comparative Example 4 showed a score improvement rate of 103.1% and a hair loss reduction rate of 45.6%, which was better than Comparative Examples 1-3, but still lagged behind Examples 1-3. In terms of composition, Comparative Example 4 and Example 3 were identical, differing only in the spatial distribution of the goji berry powder. This result indicates that positioning the goji berry powder on the outer shell to construct a pre-antioxidant barrier (Example 3), compared to mixing it internally into the oil phase (Comparative Example 4), demonstrates a superior synergistic effect in the final hair-beautifying effect. This may be related to the fact that goji berry powder distributed in the shell can more effectively intercept oxidative stress in the early stages of digestion, thereby maintaining the stability and bioavailability of functional oils and core active components such as DL-methionine in the digestive tract.

[0126] Comparative Example 5 showed a score improvement rate of 77.4% and a hair loss reduction rate of 30.2%, which were similar to Comparative Examples 2-3. Although Comparative Example 5 exhibited pH-responsive release characteristics (Experiment 6 confirmed that the SGF / SIF release behavior was similar to that of the present invention), its clinical hair-beautifying effect was significantly lower than that of Examples 1-3 of the present invention due to the lack of internal mesh support and pre-antioxidant protection from wolfberry powder.

[0127] Comparative Example 6 showed a 115.4% improvement in hair shine score and an IgG level of 2.40 mg / mL. In comparison, Example 3 showed a 71.7 percentage point increase in score (187.1%) and a 1.02 mg / mL increase in IgG level (3.42 mg / mL). Combined with Experiment 7—where the probiotics in Comparative Example 6 were almost completely inactivated during processing and in the gastric acid environment (2.2 Log CFU / g after processing, 1.2 Log CFU / g after gastric juice treatment), while the probiotics in Example 3 maintained high activity (9.2 Log CFU / g after processing, 8.9 Log CFU / g after gastric juice treatment)—it can be concluded that highly active probiotics can significantly increase IgG levels (from 2.40 mg / mL to 3.42 mg / mL), thereby enhancing the hair-beautifying effect by 71.7 percentage points through the gut-skin axis mechanism. This fully demonstrates that embedding probiotics in the shell to achieve high survival rates is a key technology for achieving a synergistic effect of immunity and hair beautification.

[0128] The score improvement rates of Comparative Examples 2 and 3 were 81.3% and 67.7%, respectively, which were at a moderate level, lower than the Example Groups but higher than Comparative Example 1. This result can be attributed to two factors: First, although both groups underwent partial microencapsulation, due to incomplete structures (Comparative Example 2 lacked a polysaccharide mesh and an effective complex aggregated shell; Comparative Example 3 could not form a complex aggregated shell due to pH 6.2), the retention rate of their active ingredients during processing and digestion was relatively low (Example 6), resulting in a reduction in the actual usable nutrient dose; Second, due to the lack of complete "core-shell-mesh" synergistic protection, some active components may have been released prematurely in the stomach and destroyed by gastric acid, failing to reach the intestines to perform their due physiological functions.

[0129] In terms of hair loss reduction rate, the data from Examples 1-3 (62.5%-71.2%) also showed a trend of being superior to the comparative group. This improvement may be related to the multiple mechanisms of action of the composition of the present invention: the Omega-3 and Omega-6 fatty acids in the functional oils provide essential nutrient substrates for hair follicles; DL-methionine, as a sulfur-containing amino acid, participates in the synthesis of hair keratin; lactoferrin and chicken plasma protein powder have immunomodulatory functions, which help maintain the health of the skin and mucous membranes; and the polysaccharide components in wolfberry powder may indirectly promote hair health by enhancing the body's antioxidant capacity. These nutrient components are synergistically delivered in the "core-shell-reticulate" structure, gradually released and absorbed in the intestine, thus showing a good comprehensive effect in improving hair luster and reducing hair loss.

[0130] Clinical evaluation of hair-enhancing effects indicates that the design of the "core-shell-network" integrated structure is of great significance for achieving synergistic effects of nutritional components. The complete structural hierarchy and the spatially optimized distribution of each component are key to maximizing the efficacy of the composition.

[0131] In summary, the "core-shell-network" integrated structure described in this invention exhibits significant technical advantages in pet immune and coat-enhancing compositions: the three-dimensional network constructed from sodium hyaluronate and dextran provides physical confinement and mechanical support for the lipid core, enabling the composition to maintain a high retention rate of active ingredients (>90%) even under high-temperature processing conditions and delaying the loss of water-soluble components; the pH-induced protein-polysaccharide complex coagulation shell not only achieves tolerance to the gastric acid environment but also endows the product with intestinal-targeted release characteristics; by distributing wolfberry powder on the outer side of the shell to construct a pre-intercepting antioxidant barrier, the oxidation induction period of oils is significantly prolonged compared to traditional internal mixing methods when the components are identical, while Lactobacillus plantarum and Lactobacillus acidophilus are embedded in the shell to achieve probiotic protection. This structured integration method solves the problems of physical stability and antioxidant synergy among multiple components, achieving synchronous delivery and efficient absorption of nutrients, and showing a more significant advantage over physical mixing groups in terms of clinical coat and skin-enhancing effects.

Claims

1. A method for preparing a pet immune-enhancing and coat-beautifying composition with skin and mucous membrane immune-enhancing function, characterized in that, Includes the following steps: (1) Preparation of lipid core dispersion: The functional oil is heated to 35-45°C, DL-methionine is added, and the mixture is stirred at 2000-4000 rpm for 15-25 min to obtain a lipid core dispersion in which DL-methionine is uniformly suspended. (2) Preparation of polysaccharide mesh matrix: Sodium hyaluronate with a weight-average molecular weight of 500,000 to 1,500,000 Daltons and -1,3-D-glucan was added to water and stirred at 500-800 rpm for 30-40 minutes at 50-65°C to obtain a polysaccharide mesh matrix; (3) Core-network structure emulsification: Under high shear conditions of 8000-12000 rpm, the lipid core dispersion is added dropwise to the polysaccharide network matrix. After the addition is complete, shearing is continued for 15-30 min to obtain the primary emulsion; (4) Immunoassay gel shell coating and probiotic integration: The colostrum is cooled to 30-40°C, and then immunoassay protein components, wolfberry powder, Lactobacillus plantarum and Lactobacillus acidophilus are added. Citric acid solution is then added to adjust the pH of the system to 4.2-4.8 to induce a complex coagulation reaction, so that the protein-polysaccharide composite membrane is coated on the surface of the colostrum droplets and the wolfberry powder and probiotics are integrated into the membrane structure. (5) Curing and drying: The system temperature is reduced to 4-10°C within 15 minutes, the structure is cured for 1-2 hours, and then spray-dried to obtain the pet immune coat beautifying composition.

2. The preparation method according to claim 1, characterized in that, The functional oil is a mixture of fish oil and evening primrose seed oil, and the immune protein component is a mixture of spray-dried chicken plasma protein powder and lactoferrin.

3. The preparation method according to claim 1, characterized in that, By weight, the raw materials of the pet immune coat-enhancing composition include: 15.0-25.0 parts fish oil, 3.0-5.0 parts evening primrose seed oil, 3.0-5.0 parts DL-methionine, and 0.6-1.0 parts sodium hyaluronate. -1,3-D-glucan 3.0-5.0 parts, spray-dried chicken plasma protein powder 30.0-50.0 parts, lactoferrin 0.3-0.5 parts, wolfberry powder 3.0-5.0 parts, Lactobacillus plantarum 1.2-2.0 parts, Lactobacillus acidophilus 0.3-0.5 parts; wherein the viable count of Lactobacillus plantarum is ≥5×10 9 CFU / kg, the viable count of the Lactobacillus acidophilus is ≥1×10⁻⁶. 9 CFU / kg.

4. The preparation method according to claim 1, characterized in that, In step (3), the dropping rate of the lipid core dispersion is 50-100 mL / min, and the system temperature is controlled at 50-55°C during the emulsification process.

5. The preparation method according to claim 1, characterized in that, In step (4), the citric acid solution has a mass percentage concentration of 0.1-0.5% and a dropping rate of 5 mL / min.

6. The preparation method according to claim 1, characterized in that, In step (5), the inlet air temperature of the spray dryer is 60-70°C, the outlet air temperature is 45-55°C, and the atomizer speed is 15000-25000rpm.

7. A pet immune-enhancing and coat-beautifying composition with skin and mucous membrane immune-enhancing function, characterized in that, It is prepared by the preparation method described in any one of claims 1-6.

8. The pet immune-boosting and coat-enhancing composition according to claim 7, characterized in that, The pet immune coat enhancement composition is a microencapsulated particle comprising a lipid core, a polysaccharide mesh matrix, and an immune complex coagulation shell; wherein, beneficial bacteria are embedded in the shell.

9. The pet immune-boosting and coat-enhancing composition according to claim 8, characterized in that, The lipid core comprises functional oils and DL-methionine dispersed therein; the lipid core is dispersed in a mixture of sodium hyaluronate and... In a polysaccharide network matrix composed of -1,3-D-glucan; The immune complex coagulation shell coats the surface of the polysaccharide mesh matrix. The shell is formed by the complex coagulation reaction of the immune protein component and the sodium hyaluronate, and the shell contains wolfberry powder, Lactobacillus plantarum and Lactobacillus acidophilus.

10. The use of a pet immune-enhancing and coat-enhancing composition with skin and mucous membrane immune-enhancing function as described in any one of claims 7-9 in the preparation of pet coat-enhancing foods.

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