Pet coating enzyme-based postbiotic feed additive and preparation method thereof

Abstract

The present application relates to the technical field of pet feed additives, and discloses a pet coated postbiotic feed additive containing enzymes and a preparation method, the additive comprising core particles formed by mixing postbiotics and complex enzymes coated with a slow-release coating, and an enteric layer coated on the outside of the core particles. The preparation method comprises: preparing the postbiotics and the slow-release coated complex enzymes respectively, mixing the two to form core particles, and then enteric coating the core particles. The present application protects the activity of functional components through a double-coating structure, so that the functional components are released in the intestinal tract and synergize. The additive can effectively improve the digestion of pet protein, regulate the intestinal microenvironment, significantly proliferate beneficial bacteria such as lactobacillus and prasrmanella, and inhibit escherichia coli, thereby reducing soft stool and maintaining the intestinal health of pets.

Description

Technical Field

[0001] This invention relates to the field of pet food additive technology, and in particular to a pet food additive containing enzymes and its preparation method. Background Technology

[0002] With the rapid development of the pet economy, pet health has received increasing attention. Among these concerns, gut health, as a core factor affecting the overall health of pets, has become a key area of ​​research. A healthy gut microbiota not only promotes efficient absorption of nutrients and maintains normal physiological functions, but also serves as an important immune organ, enhancing the pet's overall immunity. As animals highly sensitive to changes in the external environment, cats are prone to intestinal problems such as indigestion, soft stools, and diarrhea due to stress responses, which can even lead to intestinal inflammation in the long run.

[0003] The application of metabiotics to improve pet gut health has attracted widespread attention. Metabiotics are bioactive metabolites or functional components produced by probiotics during their growth and metabolism. They possess various beneficial effects, such as regulating the gut microbiota balance, improving intestinal barrier function, and enhancing local and systemic immunity. However, in existing technologies, metabiotics added directly are easily destroyed by gastric acid and digestive enzymes in the early stages of the animal's digestive tract (such as the stomach), resulting in a significant reduction in their activity upon reaching the target site in the intestine, thus limiting their actual efficacy.

[0004] In addition, the application of specific enzyme preparations has also attracted attention in improving the intestinal environment and digestive function. For example, glucose oxidase can consume oxygen in the animal intestine, creating an anaerobic environment for beneficial bacteria (such as Bifidobacterium and Clostridium perfringens, obligate anaerobes), while catalase can decompose its metabolic byproduct hydrogen peroxide; enterokinase, as a key protease, can initiate the cascade activation of pancreatic digestive enzymes, significantly enhancing the ability to digest proteins, thereby reducing soft stools caused by protein malabsorption. However, these enzyme preparations also face the problems of inactivation in the acidic environment of the stomach and short duration of action in the intestine.

[0005] Therefore, how to effectively combine postbiotics with various functional enzyme preparations and use appropriate technical means to protect their activity so that they can cross the gastric environment, reach the intestines simultaneously and work synergistically, thereby maximizing the improvement of intestinal health in pets, especially sensitive felines, is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] The technical problem to be solved by the present invention is that the bioactivity of active ingredients such as postbiotics or enzymes in existing pet food additives is easily lost when stored and passing through the pet's digestive tract, especially the highly acidic environment of the stomach, which reduces their effectiveness in the intestines and makes it impossible to effectively and stably regulate the balance of the pet's intestinal flora.

[0007] To solve the above-mentioned technical problems, this application provides the following technical solution:

[0008] In a first aspect, this application provides a pet-coated enzyme-containing post-biotic feed additive, which comprises:

[0009] Core particles, said core particles being a mixture of an epigenetic and a coated complex enzyme; and

[0010] An enteric coating layer covering the surface of the core particles.

[0011] By adopting the above technical solution, this feed additive forms a double-coated structure. The outer enteric coating layer resists the acidic environment of gastric juice, protecting the internal active ingredients from degradation and ensuring they can pass through the stomach intact to the intestines. In the weakly alkaline environment of the intestines, the enteric layer dissolves, releasing the core particles. The coated complex enzymes in the core particles also have a slow-release coating, which allows the enzymes to be released continuously and slowly in the intestines, prolonging their duration of action. The metabiotic, as a fermentation product, provides direct nutrients to the native beneficial intestinal flora. This structural design ensures that the metabiotic and multiple enzymes can work synergistically in specific parts of the intestines, effectively regulating the intestinal flora environment and improving the digestibility and absorption efficiency of nutrients.

[0012] Preferably, the metabiotic is a product obtained by fermenting, drying, and pulverizing the culture medium with Bacillus coagulans; the culture medium, by mass parts, comprises: 25-35 parts chicken meal, 5-15 parts yeast powder, 10 parts starch, 5 parts sodium acetate, 2 parts diammonium citrate, 2 parts dipotassium hydrogen phosphate, 0.5 parts magnesium sulfate, 0.25 parts manganese sulfate, 0.5 parts cysteine ​​hydrochloride, and 1 part Tween-80.

[0013] By adopting the above technical solution and using animal-derived proteins such as chicken meal and yeast powder as fermentation substrates, it is more in line with the physiological characteristics of carnivorous pets (such as cats). The resulting postbiotic metabolites can more effectively promote the proliferation of beneficial bacteria (such as Lactobacillus and Clostridium perfringens) in their intestines. Bacillus coagulans, as the fermentation strain, possesses good acid production and environmental tolerance, ensuring the stability of the fermentation process and the quality of the postbiotic products.

[0014] Preferably, the coated complex enzyme comprises a complex enzyme core and a sustained-release coating layer covering its surface; wherein the complex enzyme core comprises glucose oxidase, catalase, and enterokinase. In the complex enzyme core, the mass ratio of glucose oxidase, catalase, and enterokinase is (10–18):(3–7):1.

[0015] By adopting the above technical solution, the specific ratio of the three enzymes achieves functional complementarity and synergy. Glucose oxidase creates a favorable growth environment for beneficial anaerobic bacteria such as Bifidobacteria by consuming oxygen in the intestine; catalase can promptly decompose hydrogen peroxide, a byproduct of glucose oxidase reaction, preventing it from causing oxidative damage to intestinal cells; and enterokinase can activate trypsinogen, promote protein digestion and decomposition, reduce the digestive burden on the intestine, and thus reduce problems such as soft stools caused by indigestion.

[0016] Preferably, the amount of the coated complex enzyme added is 1 to 10% w / w of the total weight of the metagenics, and a specific addition ratio is 5% w / w (i.e., the mass ratio of metagenics to coated complex enzyme is 19:1).

[0017] By adopting the above technical solution, this formulation range ensures that significant technical effects are achieved while also maintaining economical production costs. Appropriate enzyme addition effectively fulfills its physiological functions, while excessive addition does not result in a proportional increase in effectiveness and may even increase costs.

[0018] Secondly, this application provides a method for preparing a pet-coated enzyme-containing biogenic feed additive, which includes the following steps:

[0019] (a) Preparation of post-genes: After fermentation by Bacillus coagulans, the culture medium is dried and pulverized to obtain post-genes;

[0020] (b) Preparation of coated complex enzyme: Granulate the complex enzyme powder and then coat the resulting particles with a slow-release coating to obtain the coated complex enzyme;

[0021] (c) Mixing and granulation: The postgenetic agent obtained in step (a) and the coated complex enzyme obtained in step (b) are mixed in a predetermined ratio and granulated to obtain core particles;

[0022] (d) Final coating: The core particles obtained in step (c) are subjected to enteric coating to obtain the feed additive.

[0023] By employing the above technical solution, this preparation method ensures the stability of each active component through stepwise preparation and coating. First, heat-sensitive enzymes are subjected to slow-release coating to form preliminary protection. Then, they are mixed with post-biotics for final enteric coating. This process avoids activity loss in unprotected enzymes due to process conditions (such as temperature and shear force) during mixing or final coating, thus guaranteeing the bioactivity of each component in the final product.

[0024] Preferably, in step (a), the fermentation is carried out at a temperature of 35-40°C for 30-40 hours, with a specific fermentation condition of 37°C for 36 hours; the drying is carried out at a temperature of 50-60°C, with a specific drying temperature of 55°C, until the moisture content of the material is below 12%, with a specific moisture content of 10%.

[0025] By adopting the above technical solutions, mild fermentation and drying conditions help maintain the activity of various beneficial metabolites in the fermentation products and avoid damage to the products caused by high temperatures. Controlling the moisture content at a specific low level is beneficial to the storage stability of the product and subsequent processing.

[0026] Preferably, the coating in step (b) and / or step (d) is achieved by spraying a coating material solution with a concentration of 1-3% onto the particulate material at a temperature of 30-40°C; a specific spraying temperature is 35°C and the concentration of the coating material solution is 2%.

[0027] By employing the above technical solution, the low-temperature spraying process is crucial for protecting enzyme activity. The temperature of 30–40°C is far below the inactivation temperature of most enzymes, ensuring that enzyme activity is not compromised during the coating process.

[0028] Preferably, the granulation in step (b) and / or step (c) is carried out by uniformly spraying a 3-7% maltodextrin solution into the material as a binder to obtain particles with a particle size of 20-100 mesh.

[0029] By adopting the above technical solution and using maltodextrin as a binder for fluidized bed micro-pellet granulation, particles with uniform size and regular structure can be obtained. This lays the physical foundation for subsequent uniform coating and ensures the quality uniformity of each final product particle.

[0030] Preferably, in step (d), after the enteric coating is completed, the process further includes drying with hot air at a temperature of 50-60°C for 20-40 minutes.

[0031] By adopting the above technical solution, the final drying step aims to remove the solvent (moisture) introduced during the coating process, so that the coating layer can be solidified and formed into a dense and complete protective film, thereby ensuring the reliability of its enteric coating function and ensuring the low moisture content of the final product for easy storage.

[0032] In summary, the present invention has at least one of the following beneficial technical effects:

[0033] 1. This invention constructs a double-coated structure by setting an enteric coating layer on the outside of the core particles and a sustained-release coating of the complex enzyme inside the core particles. This structure enables the active ingredients, such as the metabiotic and the complex enzyme, to effectively resist degradation by gastric acid and ensure their targeted and continuous release in the intestine. Compared to uncoated or single-layer coated technologies, the structure of this invention significantly improves the bioavailability of the active ingredients in the intestine, thereby maximizing its technical effects.

[0034] 2. This invention combines a metabiotic with three complementary enzymes (glucose oxidase, catalase, and enterokinase). The metabiotic provides nutrients for beneficial bacteria, glucose oxidase and catalase synergistically regulate the intestinal redox environment to promote the growth of anaerobic bacteria, and enterokinase improves digestion. This synergistic effect can simultaneously promote the proliferation of various beneficial bacteria in the intestine, such as lactobacilli, Clostridium perfringens, and Bifidobacteria, while inhibiting the growth of potentially harmful bacteria such as Escherichia coli, achieving comprehensive and effective regulation of the intestinal microecological structure.

[0035] 3. This invention, by adding enterokinase, effectively promotes the digestion and absorption of protein in pets' feed, reducing the amount of substrate entering the lower intestine due to incomplete protein digestion, thereby lowering the risk of osmotic diarrhea and soft stools. Experimental results show that the experimental group using the additive of this invention was able to completely eliminate the occurrence of soft stools, indicating that it has a direct and significant effect on maintaining normal fecal shape and improving digestive health in pets. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below with reference to specific embodiments. However, those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to the specific embodiments described in this invention without departing from the spirit and scope of this invention.

[0037] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0038] Bacillus coagulans was purchased from the China Center for Type Culture Collection (CCTCC), strain accession number CCTCC NO: M 2022195, with a viable count of not less than 1.0 × 10⁻⁶. 10 CFU / g of bacterial powder.

[0039] All enzyme preparations used were purchased from Yuanye Biotechnology Co., Ltd., specifically including: glucose oxidase (EC 1.1.3.4, CAS No.: 9001-37-0), with an enzyme activity of not less than 5,000 U / g; catalase (EC 1.11.1.6, CAS No.: 9001-05-2), with an enzyme activity of not less than 10,000 U / g; and enterokinase (EC 3.4.21.9, CAS No.: 9014-75-9), which is a recombinant bovine enterokinase light chain, with an enzyme activity of not less than 5,000 U / g.

[0040] The coating material is Ammonio Methacrylate Copolymer (CAS No.: 33434-24-1), a commercially available product from Evonik Industries, traded under the name [Brand Name Missing]. RS PO. This polymer is a copolymer of ethyl acrylate, methyl methacrylate, and trimethylammonium chloride ethyl methacrylate, with a quaternary ammonium group content of approximately 4.5% to 6.8%. It forms a pH-independent, permeable, insoluble film in an aqueous medium.

[0041] The culture medium and excipients include: chicken meal, feed grade, with a crude protein content of not less than 65%, purchased from Cargill; yeast powder, feed grade, derived from Saccharomyces cerevisiae, with a crude protein content of not less than 45%, purchased from Angel Yeast Co., Ltd.

[0042] Preparation Examples 1-2:

[0043] Preparation Example 1:

[0044] The present invention provides a method for preparing a coated complex enzyme, comprising the following steps:

[0045] (1) Granulation of the compound enzyme: By mass fraction, 14 parts of glucose oxidase (enzyme activity 5000 U / g), 5 parts of catalase (enzyme activity 10000 U / g) and 1 part of enterokinase (enzyme activity 5000 U / g) are mixed evenly to obtain compound enzyme powder. The compound enzyme powder is placed in a fluidized bed microgranulator. After the equipment has been running for 2 minutes, a 5% maltodextrin solution is sprayed into the powder at a uniform rate. The amount of solution sprayed is 10% of the total mass of the compound enzyme powder, to obtain compound enzyme particles with a particle size of 40-80 mesh.

[0046] (2) Coating of the composite enzyme: The composite enzyme particles obtained in step (1) are added to a fluidized bed. After the equipment has been running for 2 minutes, a 2% quaternary ammonium methacrylate solution is sprayed uniformly into the particles at an inlet air temperature of 35°C. The amount of sprayed is 10% of the total mass of the composite enzyme particles. After the spraying is completed, hot air at 55°C is introduced for drying for 30 minutes. After cooling, the coated composite enzyme is obtained and ready for use.

[0047] Preparation Example 2:

[0048] The present invention provides a pretreatment method for basic cat food ingredients, comprising:

[0049] The basic cat food ingredients, by weight, include: 66 parts fresh chicken, 19 parts fresh duck, 3 parts fresh chicken offal, 7 parts pumpkin, 2.6 parts oats, 2 parts fish oil, 0.2 parts multivitamins, and 0.2 parts multiminerals.

[0050] The above raw materials were pretreated as follows: the fresh chicken, fresh duck, and fresh chicken offal were cut into pieces and minced using a meat grinder into a paste with a particle size of no more than 2 mm; the pumpkin and oats were steamed for 20 minutes, cooled, and then ground into grain powder; the compound vitamins and compound minerals were premixed with a small amount of the grain powder to obtain an additive premix. The obtained meat paste, grain powder, and additive premix were placed in separate containers for later use.

[0051] Examples 1-3:

[0052] Example 1:

[0053] This embodiment provides a pet-coated enzyme-containing biogenic feed additive, the preparation steps of which are as follows:

[0054] (1) Preparation of culture medium: By weight, 25 parts chicken powder, 15 parts yeast powder, 10 parts starch, 5 parts sodium acetate, 2 parts diammonium citrate, 2 parts dipotassium hydrogen phosphate, 0.5 parts magnesium sulfate, 0.25 parts manganese sulfate, 0.5 parts L-cysteine ​​hydrochloride, and 1 part Tween-80 were mixed evenly, and distilled water was added to adjust the total moisture content of the material to 50%. The mixture was placed in a sterilized fermenter and sterilized at 121°C for 30 minutes by introducing steam. After sterilization, cooling water was introduced into the jacket of the fermenter to lower the temperature inside the tank to 37°C, thus obtaining a sterile culture medium.

[0055] (2) Inoculation and fermentation: Inoculate the sterile culture medium obtained in step (1) with activated Bacillus coagulans strain (initial concentration 1.0 × 10⁻⁶) at an inoculation rate of 0.5% of the total mass of the culture medium. 6 (cfu / g). Static fermentation was carried out at a constant temperature of 37°C for 36 hours, with the culture medium being turned over every 6 hours during the process. The fermented material was obtained after the fermentation was completed.

[0056] (3) Drying and pulverizing: The fermentation material obtained in step (2) is placed in an oven at 55°C for drying until the moisture content of the material is reduced to below 10%. The dried material is then pulverized and sieved through a screen to collect particles with a particle size of 40-100 mesh, which are the post-fermentation agents.

[0057] (4) Mixing and granulation: The post-biotic obtained in step (3) and the coated complex enzyme obtained in Preparation Example 1 are mixed evenly at a mass ratio of 19:1 to obtain an enzyme-containing post-biotic mixture. The mixture is added to a fluidized bed microparticle granulator. After the equipment has been running for 2 minutes, a 5% maltodextrin solution is sprayed into the mixture at a uniform rate as a binder. The amount of the sprayed solution is 10% of the total mass of the mixture, and core particles with a particle size of 20-80 mesh are obtained.

[0058] (5) Enteric coating: The core particles obtained in step (4) are added to a fluidized bed. After the equipment has been running for 2 minutes, a 2% quaternary ammonium methacrylate solution is sprayed into the particles at a uniform speed under an air inlet temperature of 35°C. The amount of sprayed is 10% of the total mass of the core particles. After the coating is completed, hot air at 55°C is introduced for drying for 30 minutes. After cooling, the enzymatic post-biotic feed additive for pets in this embodiment is obtained.

[0059] Example 2:

[0060] This embodiment provides a pet-coated enzyme-containing biogenic feed additive, the preparation steps of which are basically the same as those in Example 1, the only difference being the different culture medium formula in step (1).

[0061] (1) Preparation of culture medium: By mass, 30 parts of chicken powder, 10 parts of yeast powder, 10 parts of starch, and the same amount of sodium acetate, diammonium citrate, dipotassium hydrogen phosphate, magnesium sulfate, manganese sulfate, L-cysteine ​​hydrochloride and Tween-80 as in Example 1 are mixed evenly. The subsequent operations are the same as step (1) of Example 1.

[0062] The subsequent steps (2) to (5) are exactly the same as in Example 1, and finally the enzymatic post-biotic feed additive for pets in this example is obtained.

[0063] Example 3:

[0064] This embodiment provides a pet-coated enzyme-containing biogenic feed additive, the preparation steps of which are basically the same as those in Example 1, the only difference being the different culture medium formula in step (1).

[0065] (1) Preparation of culture medium: By mass, 35 parts of chicken powder, 5 parts of yeast powder, 10 parts of starch, and the same amount of sodium acetate, diammonium citrate, dipotassium hydrogen phosphate, magnesium sulfate, manganese sulfate, L-cysteine ​​hydrochloride and Tween-80 as in Example 1 are mixed evenly. The subsequent operations are the same as step (1) of Example 1.

[0066] The subsequent steps (2) to (5) are exactly the same as in Example 1, and finally the enzymatic post-biotic feed additive for pets in this example is obtained.

[0067] Experimental Examples 1-3:

[0068] Experimental Example 1:

[0069] This experimental example provides a cat food containing the feed additive of the present invention, and its preparation steps are as follows:

[0070] (1) Mixing: Add the meat paste, grain powder and 10 parts of water obtained from the pretreatment in Preparation Example 2 to a mixer and stir at low speed for 5 minutes. Then, add 2 parts of fish oil, the additive premix from Preparation Example 2 and 2 parts of the pet-coated enzyme-containing biogenic feed additive prepared in Example 1, and stir at medium speed for 10 minutes until the materials are mixed evenly to form a homogeneous viscous paste.

[0071] (2) Molding: The material obtained in step (1) is molded by a hydraulic press. The mold diameter is 4mm, the pressure is controlled at 5-8MPa, and the temperature during the molding process does not exceed 40℃.

[0072] (3) Drying and baking: The shaped material was placed in a hot air circulating drying oven and pre-dried at 60°C for 2 hours, turning it once during the process, to reduce the moisture content of the material to 20-25%. The pre-dried material was then transferred to a hot air circulating baking oven and baked at 100°C for 1 hour, turning it every 10 minutes to ensure even heating. After baking, the temperature was lowered from 60°C for 15 minutes, and after cooling, the cat food of Experimental Example 1 was obtained.

[0073] Experimental Example 2:

[0074] The preparation method of this experiment is exactly the same as that of Experiment 1. The only difference is that in the mixing process of step (1), the added "pet coated enzyme-containing biogenic feed additive prepared in Example 1" is replaced with an equal amount (2 parts) of "pet coated enzyme-containing biogenic feed additive prepared in Example 2".

[0075] Experimental Example 3:

[0076] The preparation method of this experiment is exactly the same as that of Experiment 1. The only difference is that in the mixing process of step (1), the added "pet coated enzyme-containing biogenic feed additive prepared in Example 1" is replaced with an equal amount (2 parts) of "pet coated enzyme-containing biogenic feed additive prepared in Example 3".

[0077] Comparative Examples 1-3:

[0078] Comparative Example 1:

[0079] Compared with the preparation method of Experimental Example 1, the difference is that no feed additives described in Examples 1 to 3 are added in the mixing step of this comparative example, nor are any enzyme preparations or postbiotic components added, while the remaining steps are the same.

[0080] Comparative Example 2:

[0081] Compared with the preparation method of Experimental Example 1, the difference is that in the mixing step of this comparative example, the added coated enzyme-containing biogenic feed additive is replaced with 0.1 parts of uncoated complex enzyme particles obtained by step (1) of Preparation Example 1, and the rest of the steps are the same.

[0082] Comparative Example 3:

[0083] Compared with the preparation method of Experimental Example 1, the difference is that in the mixing step of this comparative example, the added coated enzyme-containing biogenic feed additive is replaced with 0.1 parts of the coated complex enzyme finally prepared by Preparation Example 1, which has only undergone one slow-release coating. All other steps are the same.

[0084] Test Case: Application Effect Verification

[0085] To verify the practical application effect of the enzyme-containing biogenic feed additive for pets in this invention, the following animal feeding experiments were conducted.

[0086] The experimental design is as follows:

[0087] 1. Laboratory animals:

[0088] Thirty-six healthy adult Chinese domestic cats were selected, 18 males and 18 females. All cats were 48±2 months old and weighed 4±0.5 kg. Before the experiment, all animals were acclimatized for 7 days in a standard experimental environment, during which time they were fed basic cat food without any additional additives.

[0089] 2. Experimental Groups:

[0090] Thirty-six laboratory animals were randomly divided into six groups according to their weight, with six animals in each group (three males and three females). Each group was assigned a specific diet, as detailed below:

[0091] Comparative Example 1: Cats were fed the cat food prepared in Comparative Example 1.

[0092] Comparative Example 2: Cats were fed the cat food prepared in Comparative Example 2.

[0093] Comparative Example 3: Cats were fed the cat food prepared in Comparative Example 3.

[0094] Experimental Group 1: Cats were fed the cat food prepared in Experimental Group 1.

[0095] Experimental Group 2: Cats were fed the cat food prepared in Experimental Group 2.

[0096] Experimental Group 3: Cats were fed the cat food prepared in Experimental Group 3.

[0097] 3. Feeding Management:

[0098] The experiment lasted 21 days. All experimental animals were housed individually in cages, with the ambient temperature maintained at 22±2℃ and relative humidity at 55±5%, following a 12-hour light / 12-hour dark cycle. Each animal was fed 60g of its assigned group of cat food daily at 8:00 AM, and free access to water was ensured throughout the day. Daily records were kept of each animal's food and water intake, as well as its general health status.

[0099] Detection indicators and methods:

[0100] 1. Observation of fecal morphology:

[0101] From day 7 to day 21 of the experiment, the litter box was cleaned and the cats were observed at 8:00 AM and 5:00 PM daily.

[0102] (1) Fecal count: Record the total number of fecal piles excreted by each cat in 24 hours.

[0103] (2) Soft stool identification and counting: Irregular, pasty, or stool that loses its original shape upon contact with the bottom of the litter box is identified as soft stool, and the number of such piles is recorded.

[0104] (3) Calculation of the percentage of soft stool: Percentage of soft stool (%) = (Total number of soft stool piles / Total number of fecal piles) × 100%.

[0105] 2. Fecal microbiota count detection:

[0106] Fresh fecal samples were collected from all cats in each group at 8:00 AM on days 7, 14, and 21 of the experiment. Fecal samples from the same group were thoroughly mixed to create a pooled sample for that day, and immediately stored at -80°C for analysis. Specific bacterial communities were absolutely quantified using quantitative real-time PCR (qPCR), with the following specific steps:

[0107] (1) Total fecal DNA extraction: Take 200 mg of frozen mixed fecal sample and extract total microbial genomic DNA from the feces using the QIAamp DNA Stool MiniKit (QIAGEN, Germany). The specific operation is performed according to the kit instructions. The concentration and purity of the extracted DNA are determined by NanoDrop spectrophotometer and diluted to 20 ng / μL. Store at -20℃ for later use.

[0108] (2) qPCR reaction: Amplification was performed on an ABI 7500 real-time quantitative PCR instrument using primers with known sequences specific to the 16S rRNA gene of different bacterial populations (primers were designed or obtained by those skilled in the art from publicly available databases). The reaction system (20 μL) included: 10 μL of SYBR Green Pro Taq HS Premix, 0.4 μL each of forward and reverse primers (10 μM), 1 μL (20 ng) of template DNA, and 7.2 μL of sterile deionized water.

[0109] (3) Reaction procedure: pre-denaturation at 95℃ for 30 seconds; then 40 cycles of amplification (denaturation at 95℃ for 5 seconds, annealing and extension at 60℃ for 30 seconds). Three technical replicates were set for each sample.

[0110] (4) Result Calculation: Based on the standard curve constructed using standard plasmids with known copy numbers, the 16S rRNA gene copy number of the target bacterial group in the sample was calculated. The final result was converted into the logarithmic value of the target bacterial group per gram of wet feces, expressed in log10 (copy number / g). The bacterial groups detected included: Lactobacillus, Faecalibacterium prausnitzii, Bifidobacterium, and Escherichia coli.

[0111] Experimental Results and Analysis:

[0112] 1. Effects of different diet groups on fecal shape in cats:

[0113] During the 21-day feeding experiment, the fecal morphology of each group of experimental animals was observed and counted from day 7 to day 21, and the statistical results are shown in Table 1.

[0114] Table 1. Effects of different diet groups on fecal morphology in cats:

[0115] Group Total amount of feces (in piles) Number of soft stools (pile) Percentage of soft stools (%) Comparative Example 1 135 10 7.41 Comparative Example 2 129 5 3.88 Comparative Example 3 Groups 131 1 2.30 Experimental Group 1 135 0 0 Experimental Group 2 129 0 0 Experimental Group 3 135 0 0

[0116] Table 1 shows that there was no significant difference in the total number of stools among the groups during the experiment. The proportion of soft stools in Comparative Group 1 (without any additional additives) was 7.41%. Compared to Comparative Group 1, the proportion of soft stools in Comparative Group 2 (with uncoated complex enzymes) decreased to 3.88%, indicating that components such as enterokinase in the complex enzymes help improve digestion and reduce the occurrence of soft stools. The proportion of soft stools in Comparative Group 3 (with sustained-release coated complex enzymes) further decreased to 2.30%. This result indicates that the sustained-release coating structure of the complex enzymes can protect the enzyme preparation from some of the influences of the external environment, maintaining its activity in the digestive tract and thus enhancing its regulatory effect on stool morphology.

[0117] In Experimental Groups 1, 2, and 3 (which received the enzymatic postbiotic feed additive described in this invention), the percentage of soft stools was 0.00%, meaning no soft stools occurred during the observation period. This result indicates that the enteric coating layer outside the core particles of the additive described in this invention effectively prevents gastric acid from degrading the internal active ingredients (postbiotics and the coated complex enzymes); after the active ingredients reach the intestines, the slow-release coating layer of the complex enzymes allows them to continue to exert their effects. Enterokinase, by activating trypsinogen, promotes the breakdown and absorption of proteins, fundamentally reducing the problem of soft stools caused by incomplete protein digestion. This dual-coating structure ensures the targeted release and synergistic effect of the active components in the intestines, thereby achieving complete control of soft stools.

[0118] 2. Effects of different diet groups on the number of lactobacilli in cat feces:

[0119] During the experimental period, fecal samples were collected from each group on days 7, 14 and 21, and the number of lactobacilli in them was detected by qPCR. The results are expressed as log10 (copy number / g), and the specific data are shown in Table 2.

[0120] Table 2. Effects of different feed groups on the number of lactobacilli in feces (log10 copies / g):

[0121] Group 7d 14d 21d Comparative Example 1 7.2 7.3 7 Comparative Example 2 7.5 7.4 7.6 Comparative Example 3 Groups 8.0 8.3 8.2 Experimental Group 1 8.3 8.2 8.4 Experimental Group 2 8.9 8.7 8.8 Experimental Group 3 8.5 8.7 8.6

[0122] Table 2 shows that, compared with Comparative Example 1 (the baseline control group), the number of lactobacilli in the feces of the other groups increased to varying degrees. The number of lactobacilli in Comparative Example 2 (with the addition of uncoated complex enzymes) increased slightly, while the number of lactobacilli in Comparative Example 3 (with the addition of slow-release coated complex enzymes) increased significantly. This result indicates that glucose oxidase and catalase in the complex enzymes create a more suitable living environment for lactobacilli, which are facultative or obligate anaerobes, by consuming residual oxygen in the intestines. The slow-release coating structure protects the activity of these enzymes, making their effects more significant.

[0123] The number of lactobacilli in the feces of experimental groups 1, 2, and 3 was higher than that of all comparative groups, with the highest number in experimental group 2. This result indicates that the feed additive described in this invention achieves effective proliferation of lactobacilli through its unique structure and components. Its mechanism of action is as follows: First, the metabiotic components it contains, as fermentation products of Bacillus coagulans, provide direct nutrient substrates and metabolites for the growth of lactobacilli; second, the coated complex enzyme contained in its core particles continuously regulates the redox environment in the intestine, which is conducive to lactobacillus colonization; third, the additive's dual coating structure (the enteric coating layer outside the core particles and the slow-release coating layer of the complex enzyme) ensures that both the metabiotic and complex enzyme functional components can resist gastric acid degradation and simultaneously reach the posterior segment of the intestine to exert a synergistic effect, thereby maximizing the increase in the number of lactobacilli.

[0124] 3. Effects of different diet groups on the number of Clostridium perfringens in cat feces:

[0125] During the experimental period, fecal samples were collected from each group on days 7, 14 and 21, and the number of Clostridium praosporum was detected by qPCR. The results are expressed as log10 (copy number / g), and the specific data are shown in Table 3.

[0126] Table 3. Effects of different feed groups on the number of Clostridium perfringens in feces (log10 copies / g):

[0127]

[0128]

[0129] Table 3 shows that, compared with Comparative Example 1, the number of *Clostridium pluvialis* in the feces of all other groups showed an increasing trend. The number in Comparative Example 2 showed no significant change, while the number in Comparative Example 3 (with the addition of a slow-release coated complex enzyme) showed a significant increase. This result indicates that the complex enzyme creates more favorable survival conditions for *Clostridium pluvialis*, an obligate anaerobic bacterium, by consuming residual oxygen in the intestine, and the slow-release coating structure enhances this effect.

[0130] The number of *Clostridium pluvialis* in the feces of experimental groups 1, 2, and 3 was significantly higher than that of all comparative groups, with experimental group 2 showing the highest level. The mechanism of action lies in the fact that the metabiotic produced by fermentation of *Bacillus coagulans* in the feed additive of this invention contains a variety of metabolites and nutrient substrates that can be directly utilized by *Clostridium pluvialis*, forming the basis for its proliferation. Simultaneously, the coated complex enzyme contained in its core particles continuously acts in the intestine, effectively reducing the redox potential of the local environment. The dual-coating structure of this additive ensures that both the metabiotic and the complex enzyme effectively resist gastric acid degradation and simultaneously reach the lower intestinal tract. The synergistic effect of the two components—directly providing proliferation substrates and optimizing the survival microenvironment—combined leads to a significant increase in the number of *Clostridium pluvialis*.

[0131] 4. Effects of different diet groups on the number of Bifidobacteria in cat feces:

[0132] During the experimental period, fecal samples were collected from each group on days 7, 14 and 21, and the number of Bifidobacteria was detected by qPCR. The results are expressed as log10 (copy number / g), and the specific data are shown in Table 4.

[0133] Table 4. Effects of different diet groups on the number of Bifidobacteria in feces (log10 copies / g):

[0134]

[0135]

[0136] Table 4 shows that, compared with Comparative Example 1, the number of Bifidobacteria in the feces of Comparative Example 2 and Comparative Example 3 was significantly increased. This result indicates that the complex enzyme, by consuming dissolved oxygen in the intestine, provides a more suitable low-oxygen microenvironment for Bifidobacteria, which are obligate anaerobic bacteria, thereby promoting their proliferation.

[0137] The number of Bifidobacteria in the feces of Experimental Groups 1, 2, and 3 was significantly higher than that of all comparative groups, with Experimental Group 2 showing the highest level. The mechanism of this result lies in the fact that the feed additive described in this invention achieves effective proliferation of Bifidobacteria through the synergistic effect of its structure and components. First, the metagenic component (fermentation product of Bacillus coagulans) in the additive contains specific nutrient substrates that can be utilized by Bifidobacteria. Second, the coated complex enzyme contained within the core particle continuously acts in the intestine, effectively maintaining a local anaerobic environment. The additive's dual coating structure—the enteric layer outside the core particle and the slow-release layer of the complex enzyme itself—ensures that both the metagenic and complex enzyme functional components can effectively resist the degradation by gastric acid and simultaneously reach the posterior intestinal tract. The synergistic effect of the direct supply of nutrient substrates and the construction of the anaerobic microenvironment leads to a significant increase in the number of Bifidobacteria.

[0138] 5. Effects of different diet groups on the number of Escherichia coli in cat feces:

[0139] During the experimental period, fecal samples were collected from each group on days 7, 14 and 21, and the number of Escherichia coli was detected by qPCR. The results are expressed as log10 (copy number / g), and the specific data are shown in Table 5.

[0140] Table 5. Effects of different diet groups on the number of Escherichia coli in feces (log10 copies / g):

[0141]

[0142]

[0143] Table 5 shows that, compared with Comparative Example 1, the number of *E. coli* in the feces of Comparative Example 2 and Comparative Example 3 both showed a decreasing trend, with the decrease being more pronounced in Comparative Example 3. This result indicates that the complex enzyme, by consuming oxygen in the intestinal environment, creates favorable conditions for the proliferation of obligate anaerobes (such as *Clostridium plasminogen salina* and *Bifidobacterium*). These proliferating bacteria, through competitive inhibition, limit the growth of *E. coli*. The sustained-release coating structure protects the enzyme's activity, making this effect more significant.

[0144] The number of *E. coli* in the feces of experimental groups 1, 2, and 3 was significantly lower than that of all comparative groups. The mechanism of this result lies in the fact that the feed additive described in this invention achieves effective inhibition of *E. coli* through the synergistic effect of its structure and components. Firstly, the metabiotic components in the additive and the metabolites produced by the beneficial intestinal bacteria (such as *Lactobacillus* and *Clostridium plasmidonum*) that proliferate from them inhibit the growth of *E. coli* by competing for nutrients, occupying colonization sites, and lowering intestinal pH. Secondly, the complex enzymes in the additive continuously consume intestinal oxygen, providing support for the growth of anaerobic beneficial bacteria, further intensifying the competitive exclusion of *E. coli*. The dual-coating structure of this additive ensures that both the metabiotic and complex enzyme functional components can reach the intestine simultaneously to exert their effects, thereby achieving maximum inhibition of *E. coli* numbers through the synergistic effect of the above two pathways.

[0145] 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 pet-coated enzyme-containing post-biotic feed additive, characterized in that, Include: Core particles, said core particles being a mixture of an epigenetic and a coated complex enzyme; and An enteric coating layer covering the surface of the core particles; The amount of the coated complex enzyme added is 1 to 10% w / w of the total weight of the post-genetic organism; The post-generic is a product obtained by fermenting, drying, and pulverizing a culture medium containing chicken powder, yeast powder, and starch using Bacillus coagulans. The coated complex enzyme comprises a complex enzyme core and a sustained-release coating layer covering its surface; wherein the complex enzyme core comprises glucose oxidase, catalase and enterokinase; The enteric coating layer and the sustained-release coating layer are made of quaternary ammonium methacrylate. The preparation method of the pet-coated enzyme-containing biogenic feed additive includes the following steps: (a) Preparation of post-generics: The culture medium containing chicken meal, yeast powder and starch was inoculated with Bacillus coagulans for fermentation, and then dried and pulverized to obtain post-generics; (b) Preparation of coated complex enzyme: Granulation of complex enzyme powder containing glucose oxidase, catalase and enterokinase, and slow-release coating of the resulting granules to obtain coated complex enzyme; (c) Mixing and granulation: The postgenetic agent obtained in step (a) and the coated complex enzyme obtained in step (b) are mixed in a predetermined ratio and granulated to obtain core particles; (d) Final coating: The core particles obtained in step (c) are subjected to enteric coating to obtain the feed additive.

2. The pet-coated enzyme-containing post-biotic feed additive according to claim 1, characterized in that, In the core of the complex enzyme, the mass ratio of glucose oxidase, catalase and enterokinase is (10-18):(3-7):1; the enzyme activity of glucose oxidase is 1000-5000 U / g, the enzyme activity of catalase is 1000-10000 U / g, and the enzyme activity of enterokinase is 1000-5000 U / g.

3. The pet-coated enzyme-containing post-biotic feed additive according to claim 1, characterized in that, In step (a), the fermentation is carried out at a temperature of 35-40°C for 30-40 hours; the drying is carried out at a temperature of 50-60°C until the moisture content of the material is below 12%.

4. The pet-coated enzyme-containing post-biotic feed additive according to claim 1, characterized in that, In step (b), the sustained-release coating is achieved by spraying a sustained-release coating material solution onto the composite enzyme particles at a temperature of 30–40°C.

5. The pet-coated enzyme-containing post-biotic feed additive according to claim 1, characterized in that, In step (d), the enteric coating is achieved by spraying an enteric coating material solution onto the core particles at a temperature of 30–40°C, followed by hot air drying at a temperature of 50–60°C for 20–40 minutes.

6. The pet-coated enzyme-containing post-biotic feed additive according to claim 1, characterized in that, The granulation in step (b) and / or step (c) is achieved by uniformly spraying a 3-7% maltodextrin solution into the material as a binder.

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