Enzyme-containing postbiotic coating feed additive for pets and preparation method of enzyme-containing postbiotic coating feed additive

Abstract

The invention relates to the technical field of pet feed additives, and discloses a pet coated enzyme-containing metagen feed additive and a preparation method thereof, the additive comprises a core particle formed by mixing a metagen and a slow-release coated compound enzyme, and an enteric layer coated outside the core particle. The preparation method comprises the following steps: respectively preparing the metagen and the slow-release coated compound enzyme, mixing the metagen and the slow-release coated compound enzyme to prepare core particles, and then carrying out enteric coating on the core particles. The activity of functional components is protected through a double coating structure, so that the functional components are released in an intestinal tract in a targeted manner and have a synergistic effect. The additive can effectively improve pet protein digestion, regulate intestinal microenvironment, remarkably proliferate beneficial bacteria such as lactobacillus and clostridium praeparatum and inhibit escherichia coli, so that soft stool is reduced, and intestinal health of pets is maintained.

Description

Technical Field

[0001] The present invention relates to the technical field of pet feed additives, in particular to a pet-coated enzyme-containing postbiotic feed additive and a preparation method thereof. Background Art

[0002] With the rapid development of the pet economy, pet health is receiving increasing attention. Intestinal health, as a key factor affecting a pet's overall health, has become a key research area. A healthy intestinal microbiome not only promotes efficient nutrient absorption and maintains normal physiological functions, but also serves as a vital immune organ, enhancing a pet's overall immunity. Pet cats, highly sensitive to environmental changes, are prone to intestinal issues such as indigestion, loose stools, and diarrhea due to stress reactions. Long-term intestinal inflammation can even occur.

[0003] In order to improve the intestinal health of pets, the application of postbiotics has attracted widespread attention. Postbiotics, that is, the biologically active metabolites produced by probiotics during the growth and metabolism process or the functional components after bacterial lysis, have many beneficial effects such as regulating the balance of intestinal microecology, improving intestinal barrier function, and enhancing local and systemic immunity. However, the postbiotics directly added in the prior art are easily destroyed by factors such as gastric acid and digestive enzymes when passing through the front part of the animal's digestive tract (such as the stomach), resulting in a significant reduction in their activity when they reach the target site of action in the intestine, and their actual efficacy is limited.

[0004] In addition, the application of specific enzyme preparations in improving the intestinal environment and digestive function has also attracted attention. For example, glucose oxidase can consume oxygen in the animal intestine, creating an anaerobic environment for beneficial bacteria (such as Bifidobacterium, Faecalibacterium prausnitzii and other 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 digestion capacity of protein, thereby reducing the problem of soft stools caused by protein indigestion. However, these enzyme preparations also face the problem of inactivation in the acidic environment of the stomach and a short duration of action in the intestine.

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

[0006] The technical problem to be solved by the present invention is that the active ingredients such as postbiotics or enzymes in existing pet feed additives are easily lost in biological activity during storage and when passing through the pet's digestive tract, especially the highly acidic environment of the stomach, resulting in reduced effectiveness of their effects in the intestine and an inability to effectively and stably regulate the balance of the pet's intestinal flora.

[0007] To solve the above technical problems, this application provides the following technical solutions:

[0008] In a first aspect, the present application provides a pet-enzyme-coated postbiotic feed additive comprising:

[0009] Core particles, wherein the core particles are formed by mixing postbiotics and coated complex enzymes; and

[0010] An enteric coating layer is coated on the surface of the core particles.

[0011] By adopting the above technical solution, the feed additive forms a double coating structure. The outer enteric coating layer can resist the acidic environment of gastric juice, protect the internal active ingredients from degradation, and ensure that they can pass through the stomach and reach the intestine intact. In the weakly alkaline environment of the intestine, the enteric layer dissolves, releasing the core particles. The coated complex enzyme in the core particles itself also has a layer of sustained-release coating, which allows the enzyme substances to be continuously and slowly released in the intestine, prolonging their action time. As a fermentation product, postbiotics provide direct nutrients for the native beneficial flora of the intestine. This structural design ensures that postbiotics and various enzymes can work synergistically in specific parts of the intestine, achieve effective regulation of the intestinal flora environment, and improve the digestion and absorption efficiency of nutrients.

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

[0013] By adopting this technical solution, animal-derived proteins such as chicken meal and yeast powder are used as fermentation substrates, better suiting the physiological characteristics of carnivorous pets (such as cats). The resulting postbiotic metabolites can more specifically promote the proliferation of beneficial bacteria in their intestines (such as Lactobacillus and Faecalibacterium prausnitzii). The fermentation strain, Bacillus coagulans, possesses excellent acid production and environmental tolerance, ensuring a stable fermentation process and the quality of the postbiotic product.

[0014] Preferably, the coated complex enzyme comprises a complex enzyme core and a slow-release coating layer coated on the surface thereof; 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 employing this technical solution, the specific ratio of the three enzymes achieves functional complementarity and synergy. Glucose oxidase consumes oxygen in the intestine, creating a favorable growth environment for anaerobic beneficial bacteria such as Bifidobacterium. Catalase promptly decomposes hydrogen peroxide, a byproduct of the glucose oxidase reaction, preventing oxidative damage to intestinal cells. Enterokinase activates trypsinogen, promoting protein digestion and decomposition, reducing the burden on the intestine and thus reducing problems such as loose stools caused by indigestion.

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

[0017] By adopting the above technical solution, this ratio range ensures that significant technical effects are achieved while also taking into account the economical production costs. The appropriate amount of enzyme addition can effectively exert its physiological function, while excessive addition will not bring proportional effect improvement and may even increase costs.

[0018] In a second aspect, the present application provides a method for preparing a pet-enzyme-containing postbiotic feed additive, comprising the following steps:

[0019] (a) Preparing postbiotics: inoculating a culture medium with Bacillus coagulans for fermentation, drying and pulverizing the culture medium to obtain postbiotics;

[0020] (b) preparing the coated complex enzyme: granulating the complex enzyme powder, and subjecting the obtained granules to a sustained-release coating to obtain the coated complex enzyme;

[0021] (c) mixing and granulating: mixing the postbiotics obtained in step (a) and the coated complex enzyme obtained in step (b) in a predetermined ratio, and granulating to obtain core particles;

[0022] (d) Final coating: Enteric coating is performed on the core particles obtained in step (c) to obtain the feed additive.

[0023] By adopting the above technical solution, the preparation method ensures the stability of each active component through a step-by-step preparation and coating process. Heat-sensitive enzymes are first subjected to a sustained-release coating to provide initial protection, and then mixed with postbiotics for a final enteric coating. This process avoids the loss of activity of unprotected enzymes due to process conditions (such as temperature and shear force) during mixing or final coating, ensuring the biological activity 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 being 37°C for 36 hours; the drying is carried out at a temperature of 50-60°C, with a specific drying temperature being 55°C, until the moisture content of the material is lower than 12%, with a specific moisture content being 10%.

[0025] By adopting this technical solution, mild fermentation and drying conditions help maintain the activity of various beneficial metabolites in the fermentation product, avoiding damage caused by high temperatures. Keeping the moisture content at a specific low level is beneficial for product storage stability 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% into the granular material at a temperature of 30-40°C; a specific spraying temperature is 35°C and the coating material solution concentration is 2%.

[0027] By adopting the above technical solution, the low-temperature spraying process is the key to protecting enzyme activity. The temperature of 30-40°C is far below the inactivation temperature of most enzymes, ensuring that the enzyme activity is not destroyed 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 as a binder into the material 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 particle size and regular structure can be obtained, which lays a 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 step further includes drying the product by introducing hot air at a temperature of 50 to 60° C. for 20 to 40 minutes.

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

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

[0033] 1. The present invention forms a double coating structure by providing an enteric coating layer on the outside of the core particles and performing a sustained-release coating on the complex enzyme inside the core particles. This structure enables active ingredients such as postbiotics and complex enzymes to effectively resist degradation by gastric acid and ensures their targeted and sustained release in the intestine. Compared with uncoated or single-layer coated technologies, the structure of the present invention significantly improves the bioavailability of active ingredients in the intestine, thereby fully realizing its technical effects.

[0034] 2. The present invention combines postbiotics with three functionally complementary enzymes: glucose oxidase, catalase, and enterokinase. Postbiotics provide nutrition for beneficial bacteria, while glucose oxidase and catalase synergistically regulate the intestinal redox environment to facilitate the growth of anaerobic bacteria, and enterokinase improves digestion. This synergistic effect can simultaneously promote the proliferation of multiple beneficial bacteria in the intestine, such as Lactobacillus, Faecalibacterium prausnitzii, and Bifidobacterium, while inhibiting the growth of potentially harmful bacteria such as Escherichia coli, achieving comprehensive and effective regulation of the intestinal microecological structure.

[0035] 3. By adding enterokinase, the present invention effectively promotes pets' digestion and absorption of protein in feed, reducing the amount of substrate that enters the posterior intestinal tract due to incomplete protein digestion, thereby lowering the risk of osmotic diarrhea and soft stools. Experimental results showed that the experimental group using the additive completely eliminated the occurrence of soft stools, demonstrating its direct and significant effect on maintaining normal fecal morphology and improving digestive health in pets. DETAILED DESCRIPTION

[0036] To make the objectives, technical solutions, and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below with reference to specific embodiments. However, it will be understood by those skilled in the art that various changes, modifications, substitutions, and variations may be made to the specific embodiments described herein without departing from the spirit and scope of the present invention.

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

[0038] Bacillus coagulans was purchased from China Center for Type Culture Collection (CCTCC) with the accession number of CCTCC NO: M 2022195. The viable count was not less than 1.0 × 10 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 quaternary ammonium methacrylate copolymer (Ammonio Methacrylate Copolymer, CAS No.: 33434-24-1), which is a commercial product of Evonik Industries. RS PO. This polymer is a copolymer of ethyl acrylate, methyl methacrylate, and trimethylammonium ethyl methacrylate chloride. Its quaternary ammonium group content is about 4.5-6.8%. It forms an insoluble film in aqueous media that is pH-independent and has a certain degree of permeability.

[0041] The culture medium and auxiliary materials include: chicken meal, feed grade, crude protein content not less than 65%, purchased from Cargill; yeast powder, feed grade, derived from brewer's yeast (Saccharomyces cerevisiae), crude protein content not less than 45%, purchased from Angel Yeast Co., Ltd.

[0042] Preparation Examples 1-2:

[0043] Preparation Example 1:

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

[0045] (1) Compound enzyme granulation: 14 parts by mass of glucose oxidase (enzyme activity 5000 U / g), 5 parts by mass of catalase (enzyme activity 10000 U / g), and 1 part by mass of enterokinase (enzyme activity 5000 U / g) were mixed uniformly to obtain compound enzyme powder. The compound enzyme powder was placed in a fluidized bed micro-pellet granulator. After the equipment was started and operated for 2 minutes, a 5% maltodextrin solution was uniformly sprayed into the powder in an amount of 10% of the total mass of the compound enzyme powder to obtain compound enzyme granules with a particle size of 40-80 mesh.

[0046] (2) Enzyme Coating: The enzyme particles prepared in step (1) were added to a fluidized bed. After the equipment was started and operated for 2 minutes, a 2% quaternary ammonium methacrylate solution was sprayed into the particles at a constant speed at an inlet air temperature of 35°C. The amount of solution sprayed was 10% of the total mass of the enzyme particles. After spraying, hot air at 55°C was introduced for drying for 30 minutes. After cooling, the coated enzyme particles were obtained and set aside.

[0047] Preparation Example 2:

[0048] The preparation example of the present invention provides a pretreatment method for basic cat food raw materials, comprising:

[0049] The raw material components of the basic cat food are calculated by mass and include: 66 parts of fresh chicken, 19 parts of fresh duck meat, 3 parts of fresh chicken offal, 7 parts of pumpkin, 2.6 parts of oats, 2 parts of fish oil, 0.2 parts of multivitamins, and 0.2 parts of multimineral elements.

[0050] The raw materials are pre-processed as follows: the fresh chicken, fresh duck, and fresh chicken offal are diced and minced using a meat grinder to form a meat paste with a particle size no greater than 2 mm; the pumpkin and oats are steamed for 20 minutes, cooled, and ground into grain powder; and the multivitamins and multimineral elements are pre-mixed with a small amount of the grain powder to form an additive premix. The resulting meat paste, grain powder, and additive premix are placed in separate containers for later use.

[0051] Examples 1 to 3:

[0052] Example 1:

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

[0054] (1) Preparation of culture medium: By weight, mix 25 parts chicken meal, 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. Add distilled water to adjust the total moisture content of the materials to 50%. Place the mixture in a sterilized fermentation tank and sterilize it at 121°C for 30 minutes by passing steam. After sterilization, pass cooling water into the interlayer of the fermentation tank to reduce the temperature inside the tank to 37°C to obtain a sterile culture medium.

[0055] (2) Inoculation and fermentation: The activated Bacillus coagulans strain (initial concentration 1.0×10 6 cfu / g). Static fermentation was carried out at a constant temperature of 37°C for 36 hours, during which the culture medium was stirred every 6 hours, and a fermentation material was obtained after the fermentation was completed.

[0056] (3) Drying and pulverizing: The fermented 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 pulverized and sieved through a sieve to collect particles with a particle size of 40 to 100 mesh, which are the postbiotics.

[0057] (4) Mixing and granulation: The postbiotic prepared in step (3) and the coated complex enzyme prepared in Preparation Example 1 were mixed uniformly at a mass ratio of 19:1 to obtain an enzyme-containing postbiotic mixture. The mixture was added to a fluidized bed micropellet granulator. After the equipment was started and operated for 2 minutes, a 5% maltodextrin solution was uniformly sprayed into the mixture as a binder in an amount of 10% of the total mass of the mixture to obtain core particles with a particle size of 20 to 80 mesh.

[0058] (5) Enteric Coating: The core particles prepared in step (4) were added to a fluidized bed. After the equipment was started and operated for 2 minutes, a quaternary ammonium methacrylate solution with a concentration of 2% was uniformly sprayed into the particles at an inlet air temperature of 35°C, with the amount of quaternary ammonium methacrylate being 10% of the total mass of the core particles. After spraying, hot air at 55°C was introduced for drying for 30 minutes. After cooling, the enzymatically coated pet feed additive containing postbiotics was obtained.

[0059] Example 2:

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

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

[0062] The subsequent steps (2) to (5) are exactly the same as those in Example 1, and finally the pet-encapsulated enzyme-containing postbiotic feed additive of this example is obtained.

[0063] Example 3:

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

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

[0066] The subsequent steps (2) to (5) are exactly the same as those in Example 1, and finally the pet-encapsulated enzyme-containing postbiotic feed additive of 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 the preparation steps are as follows:

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

[0071] (2) Molding: The material obtained in step (1) is molded by a hydraulic press. The mold aperture used is 4 mm, the pressure is controlled at 5-8 MPa, and the molding temperature is not higher than 40°C.

[0072] (3) Drying and baking: Place the formed material in a hot air circulation drying oven and pre-dry it at 60°C for 2 hours, stirring it once during the drying process to reduce the moisture content of the material to 20-25%. Transfer the pre-dried material to a hot air circulation baking oven and bake it at 100°C for 1 hour, turning it over every 10 minutes to ensure uniform heating. After baking, cool it down at 60°C for 15 minutes. After cooling, the cat food of Experimental Example 1 is obtained.

[0073] Experimental Example 2:

[0074] The preparation method of this experimental example is exactly the same as that of Experimental Example 1, with the only difference being that during the mixing process of step (1), the added "pet encapsulated enzyme-containing postbiotic feed additive prepared in Example 1" is replaced with an equal amount (2 parts) of the "pet encapsulated enzyme-containing postbiotic feed additive prepared in Example 2".

[0075] Experimental Example 3:

[0076] The preparation method of this experimental example is exactly the same as that of Experimental Example 1, with the only difference being that during the mixing process of step (1), the added "pet encapsulated enzyme-containing postbiotic feed additive prepared in Example 1" is replaced with an equal amount (2 parts) of the "pet encapsulated enzyme-containing postbiotic feed additive prepared in Example 3".

[0077] Comparative Examples 1 to 3:

[0078] Comparative Example 1:

[0079] Compared with the preparation method of Experimental Example 1, the difference is that in this comparative example, no feed additives described in Examples 1 to 3 are added in the mixing step, nor is any form of enzyme preparation or postbiotic component added, and 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, the added coated enzyme-containing postbiotic feed additive is replaced with 0.1 parts of uncoated complex enzyme granules prepared by step (1) of Preparation Example 1, and the remaining 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, the added coated enzyme-containing postbiotic feed additive is replaced with 0.1 parts of the coated complex enzyme finally prepared by Preparation Example 1 and only subjected to one sustained-release coating, and the remaining steps are the same.

[0084] Test case: Application effect verification

[0085] In order to verify the practical application effect of the pet-encapsulated enzyme-containing postbiotic feed additive of the present invention, the following animal feeding experiment was conducted.

[0086] The experimental design is as follows:

[0087] 1. Experimental Animals

[0088] Thirty-six healthy adult Chinese rural cats (18 males and 18 females) were selected. All cats were 48 ± 2 months old and weighed 4 ± 0.5 kg. Prior to the experiment, all animals were acclimated to the experiment under standard laboratory conditions for 7 days, fed a basic cat food diet without any additives.

[0089] 2. Experimental groups:

[0090] Thirty-six experimental animals were randomly divided into six groups according to their body weight, with six animals in each group, including three males and three females. Each group corresponded to a type of feed, and the specific groups were as follows:

[0091] Comparative Example 1 group: fed with the cat food prepared in Comparative Example 1.

[0092] Comparative Example 2 group: fed with the cat food prepared in Comparative Example 2.

[0093] Comparative Example 3 group: fed with the cat food prepared in Comparative Example 3.

[0094] Experimental Example 1 group: fed with the cat food prepared in Experimental Example 1.

[0095] Experimental Example 2 group: fed with the cat food prepared in Experimental Example 2.

[0096] Experimental Example 3 group: fed with the cat food prepared in Experimental Example 3.

[0097] 3. Feeding management:

[0098] The experimental period lasted 21 days. All animals were housed individually in cages, maintained at an ambient temperature of 22 ± 2°C, a relative humidity of 55 ± 5%, and a 12-hour light / 12-hour dark cycle. Each animal was fed 60 g of cat food per group at 8:00 AM daily and had free access to water throughout the day. Each animal's food intake, water intake, and general health were recorded daily.

[0099] Testing indicators and methods:

[0100] 1. Observation of stool morphology:

[0101] From the 7th day to the 21st day of the experiment, the cat litter boxes were cleaned and observed at 8 am and 5 pm every day.

[0102] (1) Feces count: Record the total number of feces discharged by each cat within 24 hours.

[0103] (2) Soft stool identification and counting: Feces that are shapeless, mushy, and lose their original shape when touching the bottom of the cat litter box are identified as soft stools, and the number of occurrences is recorded.

[0104] (3) Calculation of the proportion of soft stools: Soft stool proportion (%) = (total number of soft stool piles / total number of feces piles) × 100%.

[0105] 2. Fecal flora detection:

[0106] Fresh fecal samples were collected from all cats in each group at 8:00 AM on the 7th, 14th, and 21st days of the experiment. Fecal samples from the same group were mixed evenly to create a pooled sample for that day and immediately stored in a -80°C refrigerator for testing. Real-time PCR (qPCR) was used to perform absolute quantitative analysis of specific bacterial communities. The specific steps were as follows:

[0107] (1) Fecal total DNA extraction: 200 mg of frozen mixed fecal samples were used to extract fecal total microbial genomic DNA using the QIAamp DNA Stool MiniKit (QIAGEN, Germany) according to the kit instructions. The extracted DNA was measured for concentration and purity using a NanoDrop spectrophotometer and diluted to 20 ng / μL and stored at −20°C until use.

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

[0109] (3) Reaction procedure: pre-denaturation at 95°C for 30 seconds, followed by 40 cycles of amplification (denaturation at 95°C for 5 seconds, annealing and extension at 60°C for 30 seconds). Three technical replicates were performed for each sample.

[0110] (4) Calculation of results: Based on the standard curve constructed from a standard plasmid 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 tested included: Lactobacillus, Faecalibacterium prausnitzii, Bifidobacterium, and Escherichia coli.

[0111] Experimental results and analysis:

[0112] 1. Effects of each feed group on cat fecal morphology:

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

[0114] Table 1 Effects of each feed group on cat feces morphology:

[0115] Group Total number of feces (pile) Number of soft stools (piles) Soft stool percentage (%) Comparative Example 1 135 10 7.41 Comparative Example 2 129 5 3.88 Comparative Example 3 131 1 2.30 Experimental Example 1 135 0 0 Experimental Example 2 129 0 0 Experimental Example 3 135 0 0

[0116] The data in Table 1 show that during the experimental period, there was no significant difference in the total number of bowel movements in each group. The proportion of soft stools in the comparative example 1 group (without any additional additives) was 7.41%. Compared with the comparative example 1 group, the proportion of soft stools in the comparative example 2 group (with the addition of uncoated composite enzyme) was reduced to 3.88%, indicating that components such as enterokinase in the composite enzyme help to improve digestion and reduce the occurrence of soft stools. The proportion of soft stools in the comparative example 3 group (with the addition of sustained-release coated composite enzyme) was further reduced to 2.30%. This result shows that the sustained-release coating structure of the composite enzyme can protect the enzyme preparation from some influences of the external environment, maintain its activity in the digestive tract, and thus enhance its regulatory effect on stool morphology.

[0117] The soft stool percentage of Experimental Example 1, Experimental Example 2 and Experimental Example 3 (added with the coated enzyme-containing postbiotic feed additive of the present invention) was 0.00%, that is, no soft stool occurred during the observation period. This result shows that the enteric coating layer on the outside of the core particles of the additive of the present invention can effectively prevent the degradation of the internal active ingredients (postbiotics and coated complex enzymes) by gastric acid; after the active ingredients reach the intestine, the sustained-release coating layer of the complex enzyme enables it to continue to function. Enterokinase promotes the decomposition and absorption of protein by activating trypsinogen, fundamentally reducing the problem of soft stool caused by incomplete protein digestion. The double coating structure ensures the targeted release and synergistic effect of the active components in the intestine, thereby achieving complete control of soft stools.

[0118] 2. Effects of each feed group on the number of Lactobacillus in cat feces:

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

[0120] Table 2 Effects of each feed group on the number of Lactobacillus 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 8.0 8.3 8.2 Experimental Example 1 8.3 8.2 8.4 Experimental Example 2 8.9 8.7 8.8 Experimental Example 3 8.5 8.7 8.6

[0122] The data display of table 2 compares with comparative example 1 group (benchmark control group), and all the other each group of lactobacillus quantity in excrement all has increase in various degrees.The lactobacillus quantity of comparative example 2 group (adding is not coated with complex enzyme) rises slightly, and the lactobacillus quantity of comparative example 3 group (adding through the complex enzyme of slow-release bag) then appears to increase significantly.This result shows, glucose oxidase and catalase in complex enzyme are by consuming oxygen remaining in intestinal tract, for the lactobacillus as facultative anaerobe or obligate anaerobe has created more suitable living environment, and the slow-release bag structure has protected the activity of these enzymes, makes its effect more remarkable.

[0123] The number of lactobacilli in the feces of experimental example 1 group, experimental example 2 group and experimental example 3 group was higher than that of all comparative example groups, and the number of experimental example 2 group was the highest. The results show that the feed additive of the present invention achieves effective proliferation of lactobacilli through its unique structure and components. Its mechanism of action is: first, the postbiotic component it contains, as a fermentation product of bacillus coagulans, provides a direct nutritional substrate 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 the colonization of lactobacilli; third, the double coating structure of the additive (enteric coating layer outside the core particles and the sustained-release coating layer of the complex enzyme) ensures that the two functional components of postbiotics and complex enzymes can resist gastric acid degradation, and synchronously reach the posterior segment of the intestine to play a synergistic role, thereby maximizing the number of lactobacilli.

[0124] 3. Effects of each feed group on the number of Faecalibacterium prausnitzii in cat feces:

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

[0126] Table 3 Effects of each feed group on the number of Faecal Clostridium prausnitzii in feces (log10 copies / g):

[0127]

[0128]

[0129] The data in Table 3 show that compared with Comparative Example 1, the number of Faecalibacterium prausnitzii in the feces of all other groups showed an upward trend. While the number of Faecalibacterium prausnitzii in Comparative Example 2 remained unchanged, the number of Faecalibacterium prausnitzii in Comparative Example 3 (which added the slow-release coated complex enzyme) increased significantly. This result suggests that the complex enzyme creates more favorable living conditions for Faecalibacterium prausnitzii, an obligate anaerobe, by consuming residual oxygen in the intestine, and that the slow-release coating enhances this effect.

[0130] The number of Faecalibacterium prausnitzii in the feces of Experimental Example 1, Experimental Example 2 and Experimental Example 3 was significantly higher than that of all the comparative example groups, among which the number of Experimental Example 2 group reached the highest level. Its mechanism of action is that the postbiotics prepared by fermentation of Bacillus coagulans in the feed additive of the present invention contain a variety of metabolites and nutritional substrates that can be directly utilized by Faecalibacterium prausnitzii, which constitute the basis for its proliferation. At the same time, the coated complex enzyme contained in its core particles continues to act in the intestine, effectively reducing the redox potential of the local environment. The dual coating structure of the additive ensures that the two functional components, postbiotics and complex enzymes, can effectively resist gastric acid degradation and reach the posterior segment of the intestine synchronously. The synergistic effect of the two components, namely directly providing proliferation substrates and optimizing the survival microenvironment, jointly leads to a significant increase in the number of Faecalibacterium prausnitzii.

[0131] 4. Effects of each feed group on the number of bifidobacteria in cat feces:

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

[0133] Table 4 Effects of each feed group on the number of bifidobacteria in feces (log10 copies / g):

[0134]

[0135]

[0136] The data in Table 4 show that the number of bifidobacteria in the feces of Comparative Example Groups 2 and 3 increased significantly compared to Comparative Example Group 1. This result indicates that the complex enzyme, by consuming dissolved oxygen in the intestine, provides a more suitable low-oxygen microenvironment for the survival of bifidobacteria, which are obligate anaerobes, thereby promoting their proliferation.

[0137] The number of bifidobacteria in the feces of Experimental Example 1, Experimental Example 2 and Experimental Example 3 groups was significantly higher than that of all comparative groups, and the number of Experimental Example 2 group reached the highest level. The mechanism of action of this result is that the feed additive of the present invention achieves effective proliferation of bifidobacteria through the synergistic effect of its structure and components. First, the postbiotic component (fermentation product of Bacillus coagulans) in the additive contains a specific nutritional substrate that can be utilized by bifidobacteria. Secondly, the coated complex enzyme contained in the core particles continues to act in the intestine, effectively maintaining a local anaerobic environment. The dual coating structure of the additive, i.e., the enteric layer outside the core particles and the sustained-release layer of the complex enzyme itself, ensures that the two functional components of postbiotics and complex enzymes can effectively resist the degradation of gastric acid and reach the posterior segment of the intestine synchronously. The direct supply of nutrient substrates and the construction of an anaerobic microenvironment work synergistically, leading to a significant increase in the number of bifidobacteria.

[0138] 5. Effects of each feed group on the number of E. coli in cat feces:

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

[0140] Table 5 Effects of each feed group on the number of Escherichia coli in feces (log10 copies / g):

[0141]

[0142]

[0143] The data in Table 5 show that compared with Comparative Example 1, the number of E. coli in the feces of Comparative Example 2 and Comparative Example 3 groups showed a downward trend, with the decrease in Comparative Example 3 being more significant. This result suggests that the complex enzyme, by consuming oxygen in the intestinal environment, creates favorable conditions for the proliferation of obligate anaerobic bacteria (such as Faecalibacterium prausnitzii and Bifidobacterium). These proliferating bacteria limit the growth of E. coli through competitive inhibition. The slow-release coating structure protects the enzyme activity, making this effect more significant.

[0144] The number of Escherichia coli in the feces of Experimental Example 1 group, Experimental Example 2 group and Experimental Example 3 group was significantly lower than that of all comparative groups. The mechanism of action of this result is that the feed additive of the present invention achieves effective inhibition of Escherichia coli through the synergistic effect of its structure and components. First, the metabolites produced by the postbiotic components in the additive and the beneficial intestinal bacteria (such as lactobacillus and Faecalibacterium prausnitzii) proliferated by it, inhibit the growth of Escherichia coli by competing for nutrition, occupying colonization sites and reducing intestinal pH. Second, the complex enzyme in the additive continuously consumes intestinal oxygen, provides support for the growth of anaerobic beneficial bacteria, and further aggravates the competitive exclusion of Escherichia coli. The dual coating structure possessed by the additive ensures that the two functional components of postbiotics and complex enzyme can reach the intestine synchronously to play a role, thereby achieving the maximum inhibition of the number of Escherichia coli through the synergistic effect of the above two approaches.

[0145] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A pet-encapsulated enzyme-containing postbiotic feed additive, characterized in that: Include: Core particles, wherein the core particles are formed by mixing postbiotics and coated complex enzymes; and an enteric coating layer coated on the surface of the core particles; Wherein, the added amount of the coated complex enzyme is 1 to 10% w / w of the total weight of the postbiotics.

2. The pet-encapsulated enzyme-containing postbiotic feed additive according to claim 1, characterized in that: The postbiotics are products obtained by fermenting a culture medium containing chicken meal, yeast powder and starch with Bacillus coagulans, followed by drying and crushing.

3. The pet-enzyme-containing postbiotic feed additive according to claim 1, characterized in that: The coated complex enzyme comprises a complex enzyme core and a slow-release coating layer coated on the surface of the complex enzyme core; wherein the complex enzyme core comprises glucose oxidase, catalase and enterokinase.

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

5. The pet-enzyme-containing postbiotic feed additive according to claim 1, characterized in that: The material of the enteric coating layer and / or the sustained-release coating layer is selected from at least one of quaternary ammonium methacrylate, polyacrylic resin latex, hydroxypropyl cellulose phthalate, and hydroxypropylmethylcellulose acetate succinate.

6. A method for preparing the pet-enzyme-containing postbiotic feed additive according to any one of claims 1 to 5, characterized in that: The following steps are involved: (a) Preparing postbiotics: inoculating a culture medium containing chicken meal, yeast powder, and starch with Bacillus coagulans for fermentation, and then drying and pulverizing the culture medium to obtain postbiotics; (b) preparing a coated complex enzyme: granulating a complex enzyme powder comprising glucose oxidase, catalase, and enterokinase, and subjecting the resulting granules to a sustained-release coating to obtain a coated complex enzyme; (c) mixing and granulating: mixing the postbiotics obtained in step (a) and the coated complex enzyme obtained in step (b) in a predetermined ratio, and granulating to obtain core particles; (d) Final coating: Enteric coating is performed on the core particles obtained in step (c) to obtain the feed additive.

7. The preparation method according to claim 6, characterized in that In step (a), the fermentation is carried out at a temperature of 35-40° C. for 30-40 hours; and the drying is carried out at a temperature of 50-60° C. until the moisture content of the material is less than 12%.

8. The preparation method according to claim 6, characterized in that In step (b), the sustained-release coating is achieved by spraying a sustained-release coating material solution onto the complex enzyme particles at a temperature of 30-40°C.

9. The preparation method according to claim 6, characterized in that In step (d), the enteric coating is achieved by spraying the 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.

10. The preparation method according to claim 6, characterized in that The granulation in step (b) and / or step (c) is achieved by uniformly spraying a maltodextrin solution with a concentration of 3-7% as a binder into the material.

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