Fermented flavoring for dogs and method for preparing the same

Abstract

This invention provides a canine fermented flavoring agent and its preparation method, comprising the following steps: (1) Raw material pretreatment: Animal source treatment: Take 40-50 parts of canine-preferred animal source raw materials, pound them, coat them with animal fat ointment, and then grind them; Plant source treatment: Take 15-25 parts of plant source raw materials and ultrafine grind them to 60-120 mesh; (2) Targeted enzymatic hydrolysis: Enzymatically hydrolyze the animal source raw materials with a compound enzyme at 40-55℃ and pH 5.8-6.8 for 1.5-3 hours. The compound enzyme is composed of flavor protease, papain, and neutral protease in a ratio of 1:(1.5-2.5):(0.3-0.7); (3) Three-stage fermentation. The canine fermented flavoring agent prepared by this invention has high palatability, strong digestive aid, excellent intestinal protection, and good storage stability, making it suitable for canine needs and industrial applications.

Description

Technical Field

[0001] This invention belongs to the field of animal feed technology, and in particular relates to a fermented flavoring agent for dogs and its preparation method. Background Technology

[0002] In the pet food industry, canine flavorings are key additives for improving palatability, and their performance directly affects dogs' willingness to eat and their nutrient intake efficiency. With the industrialization of pet food, the market demand for flavorings has shifted from simply improving aroma to a synergistic improvement of palatability, digestibility, and functionality. In particular, more efficient technological solutions are urgently needed to address common issues in dogs such as insufficient appetite and weak intestinal digestion.

[0003] In existing technologies, traditional canine flavorings generally suffer from the limitation of "separation of flavor and digestive function." Most products generate flavor substances through a single Maillard reaction or simple enzymatic hydrolysis. Although this can increase feed intake in the short term, the raw materials are not fully hydrolyzed (the degree of protein hydrolysis is often below 35%), and there is a lack of targeted digestive aids. This results in a high level of undigested protein residue after dogs consume the food, easily causing digestive problems such as bloating and diarrhea. At the same time, these products often rely on chemically synthesized flavorings or single animal-derived extracts, making it difficult to balance the naturalness of the flavor with the ease of nutrient absorption, which is significantly different from the "healthy" requirements of modern pet food.

[0004] Another prominent issue is the low survival rate and insufficient storage stability of probiotics. Most existing probiotic flavoring products use unencapsulated free bacteria, resulting in a survival rate of less than 20% in the acidic environment of the stomach, making it difficult to effectively colonize the intestines. Furthermore, due to the lack of optimized encapsulation and drying processes, flavor substances are prone to oxidation and deterioration. After three months of storage, flavor retention generally drops below 60%, and rancidity exceeds 1.0 mg / kg, severely impacting the product's practical application and shelf life. These technical deficiencies prevent traditional flavorings from meeting dogs' comprehensive needs for "high palatability, strong digestibility, and long-term stability," becoming a significant bottleneck restricting the upgrading of pet food quality. Summary of the Invention

[0005] To solve the above-mentioned technical problems, the present invention provides a canine fermented flavoring agent and its preparation method. The prepared canine fermented flavoring agent has high palatability, strong digestive aid, excellent intestinal protection and good storage stability, and is suitable for the needs of dogs and industrial applications.

[0006] This invention provides a method for preparing a fermented flavoring agent for dogs, comprising the following steps: (1) Raw material pretreatment: Animal-derived processing: Take 40-50 portions of dog-preferred animal-derived raw materials, pound them, coat them with animal fat ointment, and then grind them. Plant-derived processing: Take 15-25 parts of plant-derived raw materials and pulverize them into 60-120 mesh; (2) Targeted enzymatic hydrolysis: Animal-derived raw materials are enzymatically hydrolyzed with a compound enzyme at 40-55℃ and pH 5.8-6.8 for 1.5-3 hours. The compound enzyme is composed of flavor protease, papain and neutral protease in a ratio of 1:(1.5-2.5):(0.3-0.7). (3) Three-stage fermentation: First stage: Add Lactobacillus strains and ferment at 28-34℃ for 10-18 hours; Second stage: Add yeast strains and ferment at 32-38℃ for 15-24 hours; Third stage: Add Bacillus strains and ferment at 35-40℃ for 5-10 hours; (4) Maillard reaction: Add reducing sugar, creatine and sulfur-containing precursor to the fermentation mixture and react with stepwise temperature increase; (5) Post-encapsulation treatment: After adding functional excipients, spray dry and mix in canine probiotic microspheres; The functional excipients include intestinal regulators, plant extracts, and embedding wall materials.

[0007] Furthermore, the animal-derived raw materials are selected from at least one of chicken liver, spleen, and salmonid fish scraps, and the plant-derived raw materials are selected from at least two of germinated brown rice, mulberry, hawthorn, and dandelion root.

[0008] Furthermore, the amount of the compound enzyme added is 1.0-1.5% of the animal-derived raw material, and the degree of protein hydrolysis at the end of the enzymatic hydrolysis is ≥42% and the amount of free amino acids is ≥2.2mg / g.

[0009] Furthermore, the three fermentation endpoint indicators are as follows: lactic acid ≥ 0.6 mg / g, ethyl acetate ≥ 0.12 mg / g, and amylase activity ≥ 100 U / g.

[0010] Furthermore, the sulfur-containing precursor of the Maillard reaction is cysteine ​​or its hydrochloride, and the reaction product contains ≥0.3wt% 2-acetylfuran and ≥0.25wt% 4-ethylguaiacol.

[0011] Furthermore, the embedding wall material is a natural polysaccharide or alcohol-soluble protein with an encapsulation rate of ≥80% and a 2-hour survival rate of ≥80% in canine gastric fluid at pH 1.2-3.0.

[0012] Furthermore, the plant extract is a mixture of Astragalus membranaceus and Glycyrrhiza uralensis water extracts at a ratio of 1:(0.5-1), and the intestinal regulator is sodium butyrate with a coating rate of ≥85%.

[0013] Furthermore, the spray drying parameters are: inlet air temperature 200-220℃, outlet air temperature 80-90℃, finished product moisture content ≤8%, crude protein ≥15%.

[0014] This invention provides a canine fermented flavoring agent prepared by the above method, comprising: amylase activity ≥100 U / g, branched-chain amino acids ≥1.5 wt%, and canine probiotics ≥3 × 10⁻⁶. 7 cfu / g, flavor retention rate ≥80% and rancidity value ≤0.8mg / kg after 6 months of storage.

[0015] Furthermore, the amount of flavoring agent added is 1.8-3.0% of the dog food, the feed intake rate is ≥90% 2 hours after feeding, and the fecal SCFA is ≥1.5mg / g.

[0016] The advantages and positive effects of this invention are: 1. Excellent palatability and digestibility: Through targeted enzymatic hydrolysis, three-stage fermentation and Maillard reaction, meat-flavored substances such as 2-acetylfuran are generated, resulting in a feed intake rate of ≥90% within 2 hours after the finished product is added to dog food; at the same time, Bacillus fermentation produces amylase (≥100U / g), which helps dogs digest carbohydrates and reduces the digestive burden.

[0017] 2. Protects the gut and ensures stable probiotics: Contains coated sodium butyrate (coating rate ≥85%), astragalus-licorice water extract, and encapsulated canine probiotics (2-hour survival rate in gastric juice ≥80%). After feeding, the SCFA in dog feces is ≥1.5mg / g, effectively maintaining the balance of the intestinal microecology. Detailed Implementation

[0018] The present disclosure will now be described in more detail, including exemplary embodiments thereof. The technical solutions in the embodiments of the present disclosure will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of the present disclosure, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present disclosure without creative effort are within the scope of protection of the present disclosure.

[0019] The specific implementation method of this application is as follows: I. Material Preparation 1. Raw materials and reagents Animal-derived raw materials: Chicken liver: Select healthy and fresh chicken livers, freeze at -20℃, and store for no more than 3 months. When thawing, place in a 4℃ refrigerator to thaw naturally until the core temperature is 2℃. After thawing, manually remove the surface fascia and connective tissue, and use sterile degreased gauze to absorb the surface moisture. Store at 4℃ for no more than 24 hours.

[0020] Spleen: The processing method is the same as that for chicken liver. In addition, use a scalpel to scrape off the fatty tissue at the splenic hilum (the amount of residual fat is ≤5% as detected by an infrared fat analyzer) to avoid affecting the enzymatic hydrolysis efficiency.

[0021] Salmon scraps: By-products of salmon processing (including fish skin, minced meat, and fish bones with a particle size ≤5mm); first rinse 3 times with running purified water (each time the amount of water is twice the weight of the raw material) to remove blood and surface impurities; then blanch in a 60℃ constant temperature water bath for 1 minute, stirring gently with a glass rod to ensure even heating; after blanching, quickly remove and cool to 25℃ with room temperature purified water, drain and place on a stainless steel tray for later use.

[0022] Plant-derived raw materials: Germinated brown rice: Indica brown rice produced in the current year; cultured in a constant temperature and humidity incubator at 25℃ and 90% relative humidity for 48 hours, turning it once every 12 hours with a sterile spatula, the germination rate was tested by random sampling and was ≥85%; after germination, it was dried in a hot air drying oven at 60℃ until the moisture content was ≤12%, and after cooling, it was screened with a 10-mesh standard sieve to remove ungerminated particles, with a screening pass rate of ≥98%.

[0023] Mulberries: Dried product; First, manually pick out the fruit stems, moldy and insect-damaged particles, then place them in an 80℃ hot air drying oven for 4 hours, turning them over once every hour to ensure uniform moisture content; after drying, use a universal pulverizer to initially pulverize to 20 mesh, and then pulverize them again using an ultra-fine pulverizer. During the pulverization process, use an infrared thermometer to monitor the material temperature in real time to ensure it is ≤40℃.

[0024] Hawthorn: Dried product; after manual pitting, it is dried in a 50℃ drying oven for 2 hours. After drying, residual pits and impurities are removed by a 10-mesh standard sieve. Before pulverizing, it is irradiated in a 254nm ultraviolet sterilization box for 15 minutes (irradiation distance 30cm). After sterilization, the total bacterial count is ≤100cfu / g.

[0025] Dandelion root: Dried product; use a soft brush to manually remove surface dirt and sand (avoid damaging the root structure), wash and dry in a 60℃ drying oven until constant weight (moisture content ≤10%), crush and seal in an aluminum-plastic composite bag, and store in a cool, dry place (temperature 25℃±2℃, humidity ≤60%).

[0026] Enzyme preparations: Flavor protease: 500 LAPU / g, purchased from Novozymes (China) Biotechnology Co., Ltd. (Product Model: Flavorzyme500MG, Production Batch No.: FN202403); Enzyme activity detection method: using 1% casein solution as substrate, react at 40℃ and pH 7.5 for 10 min, and calculate enzyme activity by determining the amount of released amino acids using the ninhydrin colorimetric method; dilute to 100 LAPU / mL with 0.05 mol / L phosphate buffer (pH 6.5) before use, and prepare fresh before use.

[0027] Papain: Activity 800,000 U / g, purchased from Guangzhou Huamei Biotechnology Co., Ltd. (batch number: MG202404); Activity is defined as "the amount of enzyme required to break down casein to produce 1 μg of tyrosine per minute is 1 U"; Detection conditions: 2% casein solution (pH 7.0), reaction at 37℃ for 5 min, absorbance measured at 280 nm using a UV spectrophotometer.

[0028] Neutral protease: activity 100,000 U / g, purchased from Beijing Solarbio Science & Technology Co., Ltd. (batch number: ZP202403); enzyme activity detection conforms to GB / T23527-2009 standard: using casein as substrate, reacting at 40℃ and pH7.0 for 30 min, and determining the amino nitrogen content by formaldehyde titration method; filtered through a 0.22μm aqueous filter membrane for sterilization before use.

[0029] Fermentation starter: Lactobacillus species (Lactobacillus plantarum): viable count 1×10 10 The cfu / g was purchased from Beijing Ketuo Biotechnology Co., Ltd. (batch number: LB202405). Before use, the cells were activated for two generations in MRS medium (containing 1% glucose) in an anaerobic incubator at 37℃, with each generation incubated for 18h. After activation, the viable count was verified by plate counting (MRS medium anaerobic incubation at 37℃ for 48h).

[0030] Yeast species (Saccharomyces cerevisiae): viable count 1×10 9 cfu / g was purchased from Shanghai Yinqi Biotechnology Co., Ltd. (batch number: YA202405); it was activated for one generation in YPD medium (1% yeast extract, 2% peptone, 2% glucose) in a constant temperature shaker at 30℃ and cultured for 12h. After activation, the viability was confirmed by counting with a hemocytometer.

[0031] Bacillus species (Bacillus subtilis): viable count 1×10⁻⁶ 9 CFU / g was purchased from Jiangsu Suwei Microbial Research Co., Ltd. (batch number: BS202405). The bacteria were activated for two generations using LB medium at 37°C in a constant temperature shaker, with each generation incubated for 24 hours. After activation, the purity of the strain was verified by Gram staining, and the purity was ≥99%.

[0032] Maillard reaction feedstock: Reducing sugars: glucose (purity ≥99%, Sinopharm Chemical Reagent Co., Ltd., batch number: GT20240310), xylose (purity ≥98%, Sinopharm Chemical Reagent Co., Ltd., batch number: MT20240405); dried in a 105℃ forced-air drying oven for 2 hours before use to remove water of crystallization; dissolved in 50℃ purified water, stirred with a magnetic stirrer at 100r / min until completely dissolved, and cooled to room temperature before use.

[0033] Creatine: 98% purity, food grade, purchased from Jiangsu Tiancheng Biochemical Products Co., Ltd. (batch number: JS202402); before use, the purity was tested by high performance liquid chromatography (Agilent 1260 model). Chromatographic conditions: C18 column (250mm×4.6mm), mobile phase methanol-water = 10:90, detection wavelength 210nm, purity ≥98%; before addition, mix with a small amount of reducing sugar solution to avoid direct contact with high temperature and decomposition.

[0034] Cysteine ​​hydrochloride: purity ≥99%, purchased from Sinopharm Chemical Reagent Co., Ltd. (batch number: BY20240315); dissolve in 0.1mol / L hydrochloric acid solution to a concentration of 10%, prepare immediately and use within 2 hours after preparation to avoid oxidation.

[0035] Functional excipients: Intestinal regulator (coated sodium butyrate): Coating rate 88%, the coating material is hydrogenated vegetable oil, purchased from Jiangsu Zhengchang Group Co., Ltd. (batch number: DS202403); the coated sodium butyrate showed a stable coating rate of ≥88% as determined by Soxhlet extraction, suitable for the sustained-release requirements of canines in acidic gastric environments. The specific testing method was as follows: the coating material was extracted with petroleum ether (boiling range 60-90℃, Sinopharm Group), and the remaining sodium butyrate was determined by titration with 0.1 mol / L sodium hydroxide solution. This method meets the requirements of the standard "Sodium Butyrate as Feed Additive" (GB / T25883-2010).

[0036] Plant extracts: Astragalus membranaceus aqueous extract (astragaloside A content 0.28wt%) and Glycyrrhiza uralensis aqueous extract (glycyrrhizic acid content 0.32wt%) were purchased from Xi'an Lvtian Biotechnology Co., Ltd. (batch numbers HQ202404 and GC202404, respectively); Preparation process: Astragalus membranaceus slices were boiled with 10 times the amount of purified water and then refluxed for 2 hours; Glycyrrhiza uralensis slices were boiled with 8 times the amount of purified water and then refluxed for 1.5 hours. After filtration, the solid content was concentrated to 50% by rotary evaporator and then spray dried into powder.

[0037] Encapsulation wall material: Gum arabic (food grade, 25℃ 10% aqueous solution viscosity 3000 cP), purchased from Sinopharm Chemical Reagent Co., Ltd. (batch number: AL20240220); Zeatin (purity ≥90%, ethanol solubility ≥95%), purchased from Shandong Zhongchuang Biotechnology Co., Ltd. (batch number: ZP202403); The gum arabic solution was prepared using 60℃ purified water, magnetically stirred until completely dissolved, and then filtered through a 0.45μm aqueous filter membrane after cooling.

[0038] Canine probiotics: Canine Enterococcus (live count 1×10⁻⁶) 9 cfu / g), canine Bifidobacterium (live count 1×10⁻⁶) 9All CFU / g were purchased from Jiangsu Hengfengqiang Biotechnology Co., Ltd. (batch numbers CE202404 and CB202404, respectively); they were stored by freeze-drying (-80℃ ultra-low temperature freezer); before use, they were activated for one generation in canine intestinal simulated culture medium (containing 0.5% bile salts) in a 37℃ anaerobic incubator. After activation, gastric acid tolerance was tested: after incubation in pH 1.2 simulated gastric juice (containing 0.3% pepsin) for 2 hours, the survival rate was ≥80%.

[0039] Other reagents: Citric acid (analytical grade, Sinopharm Chemical Reagent Co., Ltd., batch number: NL202403), sodium hydroxide (analytical grade, Sinopharm Chemical Reagent Co., Ltd., batch number: QY202403); carbon dioxide-free purified water (boiled and cooled to room temperature) was used to prepare 0.1 mol / L solution, and the pH error was ±0.01 after pH meter calibration.

[0040] Maltodextrin (DE15): Food grade; DE value was tested using Fehling's reagent method, and the result DE15±1 met the requirements for drying agents.

[0041] 2. Instruments and Equipment Ultrafine pulverizer: rotation speed 8000r / min, water-cooled temperature control of the pulverizing chamber (chamber temperature ≤40℃); equipped with 60 mesh and 120 mesh standard sieves, and the pulverizing accuracy is verified periodically with standard glass microspheres (particle size 75-106μm), with a particle size deviation ≤5%.

[0042] Meat grinder: JR-120 model, 3mm aperture, blade assembly material 304 stainless steel; wipe and disinfect with 75% ethanol before use, disassemble the blade assembly and feed cylinder after each use, rinse with 60℃ hot water and air dry; check the aperture with a calibration block (3mm±0.02mm) every month, replace the blade assembly if the deviation exceeds the limit.

[0043] Reactor: GR-50 type, 50L volume, made of 316L stainless steel; equipped with intelligent temperature control system, online pH monitor and anchor-type stirring paddle (speed 0-100r / min); stirring speed is 75r / min during enzymatic hydrolysis to ensure that the enzymatic hydrolysis system is uniform and without stratification.

[0044] Fermenter: FB-10 type, with anaerobic / aerobic switching system; nitrogen gas (purity ≥99.99%) is introduced during anaerobic operation, and the oxygen content is monitored in real time by an oxygen content detector, with an oxygen concentration ≤0.5%; sterile air (filtered through a 0.22μm air filter membrane) is introduced during aerobic operation, and the air flow rate is adjusted by a rotor flow meter; temperature and humidity control accuracy is ±2%, and it is equipped with a sterile sampling port (with a 0.22μm filter membrane).

[0045] Spray dryer: LPG-5 type, inlet air temperature range 180-250℃, outlet air temperature range 70-90℃; atomizer is centrifugal (speed 15000r / min); before use, clean the spray chamber with purified water, and after drying, run it under no-load for 30 minutes (inlet air 210℃, outlet air 85℃) to ensure temperature stability.

[0046] High-performance liquid chromatograph (HPLC): Agilent 1260 model from Agilent Technologies, equipped with a UV detector; for lactic acid detection, an Aminex HPX-87H column (300 mm × 7.8 mm) was used, with a mobile phase of 0.05 mol / L sulfuric acid (pH 2.7), a flow rate of 0.6 mL / min, a column temperature of 60 °C, and a detection wavelength of 210 nm; for amino acid detection, a C18 column (250 mm × 4.6 mm) was used, with a mobile phase of methanol-0.1% phosphoric acid water = 5:95, a flow rate of 1 mL / min, and a detection wavelength of 220 nm.

[0047] Ultraviolet spectrophotometer: UV-2600, wavelength range 200-800nm, accuracy ±0.5nm; when used for amylase activity detection, wavelength 540nm, cuvette is 1cm quartz cuvette, blank control is a mixture of DNS reagent and 0.05mol / L phosphate buffer (pH 6.0).

[0048] Microbial incubator: MJ-150 model, which can create an anaerobic environment (anaerobic bag: oxygen concentration ≤0.1%); temperature control 37℃±1℃, humidity 90%±5%; when used for probiotic culture and counting, anaerobic culture for 48 hours.

[0049] Example 1 1. Raw material pretreatment Animal-derived processing: Weigh 20 portions of chicken liver (100g each, accurate to 0.1g) and place them in a CJ-2024 type pounding machine. Set the pounding frequency to 30 times / min and the pounding time to 5min. After pounding, randomly select 3 samples (10g each) and observe the muscle fiber breakage using an optical microscope (XSP-2CA type, 10×40x objective lens): Randomly select 10 fields of view for each sample, count 50 muscle fibers in each field of view, and the proportion of broken fibers ≥80% is considered qualified (the average breakage rate measured in this example is 86%).

[0050] Weigh out two portions of chicken fat (100g each, from the same batch of chicken) and place them in a 304 stainless steel pot. Turn on the 500W heating mantle and stir with a magnetic stirrer at 50r / min. Heat to 120℃ and maintain for 1.5h. During this period, take a sample with a glass rod every 15 minutes to observe and avoid local scorching. After cooking, filter with a 300-mesh nylon filter cloth to remove the oil residue (after drying the oil residue in a 60℃ drying oven, grind it into 80-mesh powder with a universal grinder, and seal it in a wide-mouth glass bottle for later use). Collect the oil paste and cool it to 50℃.

[0051] Apply chicken fat paste evenly to the surface of the pounded chicken liver using a soft brush, at a rate of 10g per 100g of chicken liver, ensuring that no part of the chicken liver is exposed. Then, weigh out 20 portions of salmon scraps (100g each) and put them into a meat grinder. Set the grinding speed to 10kg / h. After grinding, check the particle size using a 3mm standard sieve. The passing rate should be ≥98% (99.2% in this example). Mix the oil-coated chicken liver with the ground salmon scraps and place them in a 304 stainless steel container. Refrigerate at 4°C for 1.5 hours (not exceeding the 24-hour limit).

[0052] Plant-derived treatment: Weigh 8 parts germinated brown rice (100g each), 5 parts dried mulberry (100g each), and 4 parts dried hawthorn (100g each), mix them, and put them into an ultrafine pulverizer; set the pulverizing speed to 8000r / min and the pulverizing time to 5min, and after pulverizing, sieve the material through an 80-mesh standard sieve and collect the sieve-passing material (pass rate ≥96%, the pass rate measured in this embodiment was 97.5%); take 3 samples (5g each), and use a laser particle size analyzer to detect the particle size distribution, and control the D50 to be within 70-90μm (the D50 measured in this embodiment was 83μm); place the pulverized plant-derived powder in an aluminum-plastic composite sealed bag and store it at room temperature (25℃±1℃) for later use.

[0053] 2. Targeted enzymatic hydrolysis The animal-derived mixture (total mass 4000g) was put into a 50L reactor, and 6000mL of purified water was added (solid-liquid ratio 1:1.5). The anchor-type agitator was turned on (speed 75r / min) and stirred for 10min to make the mixture uniformly dispersed and without obvious particle agglomeration.

[0054] Weigh 1.2 portions of the compound enzyme (total mass 48g, flavor protease 16g, papain 32g, neutral protease 8g, mass ratio 1:2:0.5), dissolve in 50mL of purified water at 48℃ (temperature controlled by a constant temperature water bath), stir until no enzyme particles are visible to the naked eye, and then slowly add to the reaction vessel; keep stirring during the addition process to avoid local aggregation of enzyme solution leading to uneven enzymatic hydrolysis.

[0055] Turn on the temperature control system of the reactor and raise the temperature to 48℃ at a rate of 5℃ / min. Adjust the pH to 6.3 with 0.1mol / L citric acid solution: after each addition of 0.5mL citric acid solution, stir for 1min and check the pH with a pH meter. Repeat the adjustment until the pH is stable at 6.3±0.1. Set the stirring program: stir for 5min every 30min at a speed of 75r / min to avoid the enzyme hydrolysis system from standing for a long time and causing stratification.

[0056] After 2.5 hours of enzymatic hydrolysis, samples were taken for endpoint testing: Degree of protein hydrolysis (DH): 10 mL of enzymatic hydrolysate was placed in a 250 mL Erlenmeyer flask, 2 drops of neutral red indicator (0.1% ethanol solution) were added, and titrated with 0.1 mol / L sodium hydroxide solution until the solution turned pale red (pH 7.0). The volume consumed was recorded as V1 = 4.3 mL. 5 mL of neutral formaldehyde solution (adjusted to pH 7.0 with 0.1 mol / L sodium hydroxide beforehand) was added, and after shaking, titration was continued until the solution turned pale red. The volume consumed was recorded as V2 = 8.6 mL. The total nitrogen content of the enzymatic hydrolysate was determined using a Kjeldahl nitrogen analyzer = 2.52 g / 100 mL. The result was calculated using the formula: DH = (V2 × 0.1 × 7.5) / (total nitrogen × 100) × 100% = (8.6 × 0.1 × 7.5) / (2.52 × 100) × 100% = 45.3%, which meets the requirement of DH ≥ 42%.

[0057] Free amino acids: Take 5 mL of enzymatic hydrolysate, centrifuge at 12000 r / min for 10 min, take the supernatant and filter it through a 0.22 μm aqueous filter membrane; detect it with high performance liquid chromatography, and quantify it using the external standard method (mixture of amino acid standards). The free amino acid content was found to be 2.45 mg / g, which meets the requirement of ≥2.2 mg / g.

[0058] 3. Three-stage fermentation Stage 1 (Lactobacillus fermentation): Add the enzymatically hydrolyzed material (approximately 10,000 g total mass) and plant-derived powder (1,700 g total mass) to a 10L fermenter. Turn on the stirrer (30 rpm) and stir for 10 minutes to ensure uniform mixing. Weigh 0.6 parts of the *Lactobacillus plantarum* suspension (60 mL total mass, diluted with sterile physiological saline to 1×10⁻⁶). 9 Add the bacterial suspension (cfu / mL) slowly along the inner wall of the fermenter and continue stirring for 5 minutes to ensure that the suspension is evenly dispersed.

[0059] Close the fermenter's air inlet valve, introduce nitrogen (at a flow rate of 2 L / min, controlled by a rotor flow meter) to replace the air in the tank for 5 minutes, then close the nitrogen valve, set the fermentation temperature to 31℃ and the relative humidity to 50%, and start anaerobic fermentation; during fermentation, take a sample every 4 hours using a sterile sampling tube (10 mL each time), and use a high-performance liquid chromatograph to detect the lactic acid content. After 14 hours of fermentation, the lactic acid content was measured to be 0.73 mg / g, which meets the requirement of ≥0.6 mg / g.

[0060] Second stage (yeast fermentation): Open the air inlet valve of the fermenter and introduce sterile air (air flow rate 0.5 L / (L·min)). At the same time, weigh 0.4 parts of the brewer's yeast suspension (total mass 40 mL, diluted with sterile physiological saline to 1×10⁻⁶). 8 Add cfu / mL to the fermenter; adjust the fermentation temperature to 35℃ and relative humidity to 52%, and start aerobic fermentation; take a sample every 4 hours (10mL each time), centrifuge at 12000r / min for 10min, and take the supernatant to detect the ethyl acetate content using gas chromatography. Chromatographic conditions: DB-WAX capillary column (30m×0.25mm×0.25μm), column temperature 60℃ for 3min, then increase to 180℃ at 5℃ / min, detector FID; after 20h of fermentation, the ethyl acetate content was measured to be 0.15mg / g, which meets the requirement of ≥0.12mg / g.

[0061] Third stage (Bacillus fermentation): Adjust the aeration rate of the fermenter to 0.3 L / (L·min), weigh 0.3 parts of Bacillus subtilis suspension (total mass 30 mL, diluted with sterile physiological saline to 1×10⁻⁶). 8 Add cfu / mL to the fermenter; adjust the fermentation temperature to 38℃ and relative humidity to 55%, and continue aerobic fermentation; take a sample every 2 hours (10mL each time), immediately place it in a boiling water bath for 10min to inactivate (stop enzyme activity), and after cooling to room temperature, detect amylase activity using the DNS method: take 1mL of inactivated solution, add 1mL of 1% soluble starch solution (pH 6.0), react in a constant temperature water bath at 55℃ for 30min, add 2mL of DNS reagent, boil in a water bath for 5min, cool and make up to 10mL, measure the absorbance at 540nm with a UV spectrophotometer, and calculate the enzyme activity by referring to the glucose standard curve (0.1-1.0mg / mL); after 8h of fermentation, the amylase activity was measured to be 116U / g, which meets the requirement of ≥100U / g; stop fermentation, collect the fermentation mixture (total mass about 11200g), and transfer it to a clean stainless steel container for later use.

[0062] 4. Maillard reaction: Transfer the fermentation mixture to a 50L reactor and start stirring (50 rpm); weigh 4 parts of reducing sugar (total mass 160g, glucose 53.3g, xylose 106.7g, mass ratio 1:2), dissolve in 200mL of 50℃ purified water and add to the reactor; weigh 0.4 parts of creatine (total mass 16g), add to 10mL of reducing sugar solution, mix well and add to the reactor; weigh 0.2 parts of cysteine ​​hydrochloride (total mass 8g), dissolve in 20mL of 0.1mol / L hydrochloric acid solution and add to the reactor; stir for 10min to ensure all raw materials are completely dissolved and there is no solid precipitate.

[0063] Adjust the pH of the material in the reactor to 6.0 using 0.1 mol / L citric acid solution. Turn on the temperature control system and set a stepwise heating program: increase the temperature to 95℃ at a rate of 5℃ / min and hold for 1.5 h; then continue to increase the temperature to 115℃ at a rate of 5℃ / min and hold for 1 h. Stir continuously during the heating process (50 r / min) to avoid local overheating and scorching. After the reaction is complete, take a 10 g sample, centrifuge at 12000 r / min for 10 min, and filter the supernatant through a 0.22 μm filter membrane. Analyze the characteristic flavor compounds using gas chromatography-mass spectrometry: use an HP-5MS column (30 m × 0.25 mm × 0.25 μm), program the temperature to 50℃ and hold for 2 min, then increase the temperature to 280℃ at a rate of 10℃ / min and hold for 5 min, with an ion source temperature of 230℃. The results showed that 2-acetylfuran = 0.36 wt% and 4-ethylguaiacol = 0.29 wt%, both meeting the requirements. Close the reactor and allow it to cool naturally to 50℃ for later use.

[0064] 5. Post-embedding treatment Mixing of functional excipients: Weigh 0.4 parts of coated sodium butyrate (total mass 17.6 g), slowly add it to the reactor (avoid adding it all at once to prevent agglomeration), and stir for 5 min; weigh 0.8 parts of plant extract (total mass 35.2 g, Astragalus membranaceus water extract 19.6 g, Glycyrrhiza uralensis water extract 15.6 g, mass ratio 1:0.8), dilute with 100 mL of 50℃ purified water to 10 wt%, stir evenly, and add it to the reactor; weigh 0.3 parts of gum arabic (total mass 13.2 g), dissolve it with 120 mL of 60℃ purified water to 10 wt%, cool to 50℃, and add it to the reactor; turn on the high-speed disperser (speed 1500 r / min), stir for 20 min, and take samples every 5 min to ensure that the excipients are completely dispersed (no visible particles, uniform dispersion observed under a microscope).

[0065] Spray drying: Turn on the spray dryer, set the inlet air temperature to 210℃, the outlet air temperature to 85℃, and the feed rate to 18mL / min; transport the mixture (temperature 50℃) in the reactor to the feed port of the spray dryer through a peristaltic pump, and set the atomizer speed to 15000r / min; collect the dried powder (intermediate), pulverize it with an 80-mesh standard sieve, take 3 samples (5g each), and test the moisture content in a vacuum drying oven (105℃, 4h), which shows a moisture content of 6.1%; test the crude protein content in a Kjeldahl nitrogen analyzer, which shows a crude protein content of 16.6%, which meets the requirements of moisture ≤8% and crude protein ≥15%.

[0066] Probiotic mixture: Weigh 0.8 portions of canine Enterococcus microspheres (total mass 35.2g, viable count 1×10⁻⁶). 9The intermediate (cfu / g) was placed in a sterile 304 stainless steel container. The spray-dried intermediate (approximately 1020g total mass) was slowly added to the container, and a V-type mixer was turned on at 60 rpm for 10 minutes. During mixing, the material temperature was monitored in real-time using an infrared thermometer, with a maximum temperature of 27℃ (≤30℃ to avoid inactivation of probiotics). After mixing, three samples (10g each) were taken and anaerobically cultured on MRS medium for 48 hours (microbial incubator). The viable probiotic count was determined by plate counting to be 4.3 × 10⁻⁶. 7 cfu / g indicates the finished product of fermented flavoring agent for dogs.

[0067] 6. Performance Testing Key indicators of finished products: Amylase activity: Take 1g of the finished product, add 10mL of 0.05mol / L phosphate buffer (pH 6.0), place it on a shaker (150r / min) and shake for 30min, centrifuge at 12000r / min for 10min, take the supernatant and detect it by DNS method. The amylase activity was measured to be 113U / g, which meets the requirement of ≥100U / g.

[0068] Branched-chain amino acids (leucine + isoleucine): Take 0.5g of the finished product, dissolve it in 10mL of 6mol / L hydrochloric acid solution, place it in a sealed tube and hydrolyze it in a hydrolysis furnace at 110℃ for 24h. Neutralize the hydrolysate to pH 7.0 with 0.1mol / L sodium hydroxide solution and filter it through a 0.22μm filter membrane. The amino acid content was measured to be 1.72wt% using an automatic amino acid analyzer, which meets the requirement of ≥1.5wt%.

[0069] Storage stability: Take 100g of the finished product, place it in an aluminum-plastic composite sealed bag, heat-seal it, and store it in a constant temperature and humidity chamber (25℃, relative humidity 65%) for 6 months; take samples once a month (10g each time), and use gas chromatography to detect the content of 2-acetylfuran and 4-ethylguaiacol. Calculate the flavor retention rate = (content after storage / initial content) × 100%. After 6 months, the flavor retention rate was measured to be 83.5%. Oxidative rancidity value was detected by TBARS method: Take 5g of sample, add 20mL of 7.5% trichloroacetic acid solution, homogenize for 2min, filter, take 5mL of filtrate, add 5mL of 0.02mol / L thiobarbituric acid solution, incubate at 95℃ for 40min, cool, and measure the absorbance at 532nm. The oxidative rancidity value was measured to be 0.58mg / kg, which meets the requirements of flavor retention rate ≥80% and rancidity value ≤0.8mg / kg.

[0070] Feeding effect: Prepare basic dog food (compliant with GB / T31216-2014 standard, crude protein 25.2%, crude fat 12.1%). Add the finished product to the basic dog food at an addition rate of 2.5%, and stir with a V-type mixer at 100r / min for 5 minutes to ensure uniform mixing (sampling and testing, the deviation of the finished product content ≤2%).

[0071] Ten adult Beagles (weighing 10-12 kg, half male and half female, in good health, and acclimatized for 7 days – fed only basic dog food, with free access to water, and observed daily without abnormalities) were selected. A feeding experiment was then conducted, with feeding twice daily at 8:00 AM and 6:00 PM, providing 200g of mixed dog food each time (accurate to 0.1g). After 30 minutes, any remaining food was collected, dried in a 105℃ drying oven until constant weight, and then weighed. The feed intake rate was calculated as (provided amount - remaining amount) / provided amount × 100%, with an average feed intake rate of 92.5% over 2 hours.

[0072] The dogs were fed continuously for 30 days. Fresh feces were collected daily (10g per dog per feeding, under aseptic conditions). 10mL of pH 7.0 phosphate buffer was added, homogenized for 3min, centrifuged at 12000r / min for 10min, and the supernatant was filtered through a 0.22μm filter membrane. The short-chain fatty acids (acetic acid and propionic acid) were detected by gas chromatography. The result showed that acetic acid + propionic acid = 1.82mg / g, which meets the requirement of ≥1.5mg / g.

[0073] Example 2 1. Raw material pretreatment 1.1 Animal-derived treatment Spleen preparation: Weigh 25 healthy spleens and place them in a CJ-2024 type percussion machine. Set the percussion frequency to 30 times / min and the percussion time to 4min. Randomly select 3 10g samples and observe the muscle fiber breakage using an XSP-2CA type optical microscope (10×40x objective lens). Select 10 fields of view for each sample and count 50 muscle fibers in each field of view. The average breakage rate is 75%.

[0074] Preparation of beef fat ointment: Weigh 1.5 portions of beef fat (100g each) and place them in a 304 stainless steel pot. Heat the pot with a DK-98-II type 500W electric heating mantle and stir with a magnetic stirrer at 50r / min. Maintain the temperature at 130℃ for 1 hour. Take samples every 15 minutes during this period to avoid local scorching. After cooking, filter the ointment through a 300-mesh nylon filter cloth to remove the oil residue. Collect the ointment and let it cool naturally to 50℃. Apply the ointment evenly to the surface of the pounded spleen with a soft brush (10g / 100g spleen), ensuring that no bare tissue is exposed.

[0075] Salmon scraps processing: Weigh 15 portions of salmon scraps (100g each), put them into a meat grinder, set the grinding speed to 8kg / h, and after grinding, check the particle size with a 3mm standard sieve, with a passing rate of 95%; mix the oil-coated spleen with the ground salmon scraps, and place them in a refrigerator at 4℃ for 1 hour (the storage time is ≤24 hours, which meets the requirements for raw material freshness).

[0076] 1.2 Plant-derived treatment Weigh 10 portions of germinated brown rice (100g each, from the same source as in Example 1) and 5 portions of dandelion root (100g each), mix them, and put them into an ultrafine pulverizer. Set the pulverizing speed to 8000r / min and the pulverizing time to 4min. After pulverizing, sieve the powder through a 60-mesh standard sieve and collect the sieve material. The passing rate is 95%. Take 3 5g samples and use a laser particle size analyzer to detect the particle size distribution. The measured D50 is 110μm. Pack the pulverized plant-derived powder into an aluminum-plastic composite bag and seal it at room temperature (25℃±1℃) to avoid moisture absorption.

[0077] 2. Targeted enzymatic hydrolysis 2.1 Construction of the enzymatic hydrolysis system - Add the pretreated animal-derived mixture (total mass 4000g: 2500g spleen + 1500g salmon offal) into a GR-50 50L reactor, add 6000mL of purified water (solid-liquid ratio 1:1.5 to ensure uniform dispersion of the enzymatic hydrolysis system), turn on the anchor-type stirrer, set the speed to 65r / min, and stir for 10min until there is no obvious particle aggregation.

[0078] Weigh 1.0 part of the compound enzyme (total mass 35g, prepared in a mass ratio of 1:1.5:0.7: 10g of flavor protease, purchased from Novozymes (China) Biotechnology Co., Ltd., batch number FN202403; 15g of papain, purchased from Guangzhou Huamei Biotechnology Co., Ltd., batch number MG202404; 10g of neutral protease, purchased from Beijing Solarbio Technology Co., Ltd., batch number ZP202403), dissolve it in 50mL of 45℃ purified water (dissolved in an RO-400 water treatment equipment, stirred until no visible enzyme particles are visible, and then slowly added to the reaction vessel; keep stirring during the addition process to avoid local aggregation of enzyme solution leading to uneven enzymatic hydrolysis).

[0079] 2.2 Control of Enzymatic Hydrolysis Conditions Turn on the temperature control system of the reactor and raise the temperature to 45℃ at a rate of 5℃ / min. Adjust the pH of the system to 6.0 with 0.1mol / L citric acid solution (purchased from Sinopharm Chemical Reagent Co., Ltd., batch number NL202403). After each addition of 0.5mL of citric acid solution, stir for 1min and use a PHS-3C pH meter to detect the pH. Repeat the adjustment until the pH stabilizes at 6.0±0.1.

[0080] Set the stirring program: stir for 4 minutes every 25 minutes (65 r / min) to avoid prolonged standing and stratification of the enzymatic hydrolysis system; the total enzymatic hydrolysis time is 2 hours, and samples are taken every 30 minutes to observe the status of the system. There is no stratification or precipitation.

[0081] 2.3 Detection of the endpoint of enzymatic digestion Degree of protein hydrolysis (DH) detection: Take 10 mL of enzymatic hydrolysate into a 250 mL Erlenmeyer flask, add 2 drops of neutral red indicator (0.1% ethanol solution), and titrate with 0.1 mol / L sodium hydroxide solution (purchased from Sinopharm Chemical Reagent Co., Ltd., batch number QY202403) until the solution turns light red (pH 7.0), and record the volume consumed V1 = 3.8 mL; add 5 mL of neutral formaldehyde solution (adjusted to pH 7.0 with 0.1 mol / L sodium hydroxide beforehand), shake well, and continue titrating until light red, and record the volume consumed V2 = 7.9 mL; determine the total nitrogen content of the enzymatic hydrolysate using a Kjeldahl nitrogen analyzer = 2.48 g / 100 mL, and calculate according to the formula: DH = (7.9 × 0.1 × 7.5) / (2.48 × 100) × 100% = 42.5%.

[0082] Detection of free amino acids: Take 5 mL of enzymatic hydrolysate, centrifuge at 12000 r / min for 10 min using a TGL-16M centrifuge, and filter the supernatant through a 0.22 μm aqueous filter membrane; detect using an Agilent 1260 high performance liquid chromatograph (Agilent Technologies). Chromatographic conditions: C18 column (250 mm × 4.6 mm), mobile phase methanol-0.1% phosphoric acid water = 5:95, flow rate 1 mL / min, detection wavelength 220 nm, quantification using external standard method (amino acid standard mixture), the free amino acid content was determined to be 2.2 mg / g.

[0083] 3. Three-stage fermentation 3.1 First stage (Lactobacillus fermentation) Transfer the enzymatically hydrolyzed material (approximately 10,000 g total mass) and plant-derived powder (1,500 g total mass: 1,000 g germinated brown rice + 500 g dandelion root) into a 10L FB-10 fermentation tank. Turn on the agitator (30 rpm) and stir for 10 minutes to ensure uniform mixing. Weigh 0.5 parts of the *Lactobacillus plantarum* suspension (50 mL total mass, purchased from Wecon Probiotics (Suzhou) Co., Ltd., batch number: LB202405) and dilute with sterile physiological saline to a final concentration of 1×10⁻⁶. 9Add the cfu / mL solution slowly along the inner wall of the fermenter and continue stirring for 5 minutes to ensure the bacterial suspension is evenly dispersed. Close the fermenter's air inlet valve and purge the air inside the tank with nitrogen (99.99% purity) for 5 minutes (air flow rate 2L / min, controlled by an LZB-6 type rotor flow meter). Then close the nitrogen valve, set the fermentation temperature to 30℃ and the relative humidity to 50% (adjusted by the fermenter's built-in temperature and humidity control system), and start anaerobic fermentation. After 12 hours of fermentation, take 10mL of sample with a sterile sampling tube, centrifuge at 12000r / min for 10 minutes, filter the supernatant through a 0.22μm filter membrane, and determine the lactic acid content by HPLC: 0.65mg / g.

[0084] 3.2 Second stage (yeast fermentation) Open the air inlet valve of the fermenter and introduce sterile air filtered through a 0.22μm air filter membrane, adjusting the ventilation rate to 0.5L / (L·min); weigh 0.3 parts of the Saccharomyces cerevisiae suspension (total mass 30mL, diluted with sterile physiological saline to 1×10⁻⁶). 8 (cfu / mL) was added to the fermenter.

[0085] Adjust the fermenter temperature to 36℃ and relative humidity to 52%, and start aerobic fermentation. After 18 hours of fermentation, take a 10 mL sample, centrifuge at 12000 r / min for 10 min, and take the supernatant to detect the ethyl acetate content using a GC-2014 gas chromatograph. Chromatographic conditions: DB-WAX capillary column (30 m × 0.25 mm × 0.25 μm), column temperature 60℃ for 3 min, then increase to 180℃ at 5℃ / min, detector FID, and the ethyl acetate content was measured to be 0.13 mg / g.

[0086] 3.3 Third stage (Bacillus fermentation) Adjust the aeration rate of the fermenter to 0.3 L / (L·min), and weigh 0.2 portions of Bacillus subtilis suspension (total mass 20 mL) and dilute with sterile physiological saline to 1×10⁻⁶. 8 (cfu / mL) was added to the fermenter.

[0087] Adjust the fermenter temperature to 37℃ and relative humidity to 55%, and continue aerobic fermentation. After 6 hours of fermentation, take a 10mL sample and immediately place it in a boiling water bath for 10 minutes to inactivate the enzyme (to terminate enzyme activity). After cooling to room temperature, detect amylase activity using the DNS method: Take 1mL of the inactivated solution, add 1mL of 1% soluble starch solution (pH 6.0), react in a 55℃ constant temperature water bath for 30 minutes, add 2mL of DNS reagent, boil in a water bath for 5 minutes, cool, and bring the volume to 10mL. Measure the absorbance at 540nm using a UV-2600 ultraviolet spectrophotometer, and calculate the enzyme activity by referring to the glucose standard curve (0.1-1.0mg / mL). The measured amylase activity is 105U / g. Stop fermentation, collect the fermentation mixture (total mass approximately 11000g), and transfer it to a clean stainless steel container for later use.

[0088] 4. Maillard reaction 4.1 Reaction System Construction: The fermentation mixture was transferred to a GR-50 50L reactor, and the stirrer was turned on (50 r / min). 3.5 parts of reducing sugar (total mass 122.5 g, prepared at a mass ratio of 1:1.8: glucose 43.75 g and xylose 78.75 g, both purchased from Sinopharm Chemical Reagent Co., Ltd., glucose batch number GT20240310, xylose batch number MT20240405) were weighed, dissolved in 150 mL of 50℃ purified water, and added to the reactor. 0.3 parts of creatine (total mass 10.5 g) were weighed, added to 10 mL of the reducing sugar solution, mixed thoroughly, and added to the reactor. 0.15 parts of cysteine ​​hydrochloride (total mass 5.25 g, purchased from Sinopharm Chemical Reagent Co., Ltd., batch number BY20240315) were weighed, dissolved in 15 mL of 0.1 mol / L hydrochloric acid solution, and added to the reactor. The mixture was stirred for 10 min to ensure that all raw materials were completely dissolved and no solid precipitate remained.

[0089] 4.2 Stepwise heating reaction Adjust the pH of the reaction system to 5.8 with 0.1 mol / L citric acid solution, turn on the temperature control system of the reactor, and set a step heating program: raise the temperature to 100℃ at a rate of 5℃ / min and hold for 1 h; then continue to raise the temperature to 120℃ at a rate of 5℃ / min and hold for 0.5 h; stir continuously during the heating process (50 r / min) to avoid local overheating and scorching.

[0090] 4.3 Detection of reaction products After the reaction was completed, a 10g sample was taken, centrifuged at 12000r / min for 10min, and the supernatant was filtered through a 0.22μm filter membrane. Characteristic flavor compounds were detected using a GC-MS7890A-5975C gas chromatograph-mass spectrometer. Chromatographic conditions: HP-5MS capillary column (30m×0.25mm×0.25μm), programmed temperature rise to 50℃ and hold for 2min, then rise to 280℃ at 10℃ / min and hold for 5min, ion source temperature 230℃. The results showed that 2-acetylfuran = 0.32wt% and 4-ethylguaiacol = 0.26wt%. The reaction vessel was then closed and allowed to cool naturally to 50℃ for later use.

[0091] 5. Post-embedding treatment 5.1 Mixing of functional excipients: Weigh 0.3 parts of coated sodium butyrate (total mass 10.5 g), slowly add it to the reaction vessel, and stir for 5 min to avoid agglomeration; weigh 0.6 parts of plant extract (total mass 21 g, prepared at a mass ratio of 1:0.5: 14 g of Astragalus membranaceus aqueous extract and 7 g of Glycyrrhiza uralensis aqueous extract), dilute with 60 mL of 50℃ purified water to 10 wt%, stir evenly, and add it to the reaction vessel; weigh 0.2 parts of zein (total mass 7 g), dissolve it in 56 mL of 8 wt% ethanol solution (ethanol purchased from Sinopharm Chemical Reagent Co., Ltd., analytical grade), and add it to the reaction vessel; turn on the GFJ-0.5 high-speed disperser, set the speed to 1500 r / min, stir for 20 min, and take samples every 5 min to ensure that the excipients are completely dispersed (no visible particles under the microscope).

[0092] 5.2 Spray drying Turn on the LPG-5 spray dryer, set the inlet air temperature to 205℃, the outlet air temperature to 82℃, and the feed rate to 15mL / min (controlled by a BT100-1F peristaltic pump); transfer the mixture (temperature 50℃) in the reactor to the spray dryer inlet via the peristaltic pump, and set the atomizer speed to 15000r / min; collect the dried powder (intermediate) and pulverize it using an 80-mesh standard sieve; take three 5g samples and dry them in a DZF-6050 vacuum drying oven at 105℃ for 4 hours, and the moisture content was measured to be 6.8%; the crude protein content was determined to be 15.8% using a KDN-08A Kjeldahl nitrogen analyzer (meeting the requirements of "moisture ≤ 8%, crude protein ≥ 15%)".

[0093] 5.3 Probiotic Mix Weigh 0.6 portions of canine Bifidobacterium microspheres (total mass 21g) and place them in a sterile 304 stainless steel container. Slowly add the spray-dried intermediate (total mass approximately 980g) into the container, turn on a VH-5 V-type mixer, set the speed to 60r / min, and mix for 10min. During the mixing process, monitor the material temperature in real time using an IT-300 infrared thermometer, with a maximum temperature of 28℃ (≤30℃ to avoid inactivation of probiotics). After mixing, take three 10g samples and anaerobic culture them for 48h in an MJ-150 microbial incubator (MRS medium). The viable probiotic count was determined by plate counting to be 3.8 × 10⁻⁶. 7 cfu / g.

[0094] 6. Performance Testing 6.1 Testing of core indicators of finished products Amylase activity: Take 1g of the product, add 10mL of 0.05mol / L phosphate buffer (pH 6.0), place it on a TS-2000 shaker at 150r / min for 30min, centrifuge at 12000r / min for 10min, take the supernatant and detect it by DNS method, the amylase activity was measured to be 108U / g.

[0095] Branched-chain amino acid content: Take 0.5g of the finished product, dissolve it in 10mL of 6mol / L hydrochloric acid solution, place it in an SH220N type hydrolysis furnace at 110℃ for 24h, neutralize the hydrolysate with 0.1mol / L sodium hydroxide solution to pH 7.0, and filter it through a 0.22μm filter membrane; use an L-8900 type automatic amino acid analyzer to detect the content, and the content of leucine + isoleucine = 1.6wt%.

[0096] 6.2 Storage stability test Take 100g of the finished product, pack it into an aluminum-plastic composite sealed bag and heat-seal it. Store it in an HS-100 constant temperature and humidity chamber (25℃, 65% relative humidity) for 6 months. Take 10g of the sample every month and use GC to detect the content of 2-acetylfuran and 4-ethylguaiacol. Calculate the flavor retention rate = (content after storage / initial content) × 100%. After 6 months, the flavor retention rate is measured to be 81%. Detect the oxidative rancidity value using the TBARS method: Take 5g of sample, add 20mL of 7.5% trichloroacetic acid solution, homogenize for 2min using an FJ200-S homogenizer, filter, take 5mL of filtrate, add 5mL of 0.02mol / L thiobarbituric acid solution, incubate at 95℃ for 40min, cool, and then measure the absorbance at 532nm using a UV-2600 ultraviolet spectrophotometer. The oxidative rancidity value is measured to be 0.7mg / kg.

[0097] 6.3 Feeding effect detection Basic dog food preparation: Basic dog food conforming to GB / T31216-2014 standard (25.0% crude protein, 12.0% crude fat) was used. The finished product was added to the basic dog food at an addition rate of 2.0%, and stirred at 100r / min for 5min using a VH-5 V-type mixer. The deviation of the finished product content was ≤2%.

[0098] Feeding experiment: Eight adult Beagles (weighing 10-12kg, half male and half female, in good health) were selected and acclimatized for 7 days (feeding only basic dog food, with free access to water); then fed twice a day at 8:00 am and 6:00 pm, each time providing 200g of mixed dog food (accurate to 0.1g). After 30 minutes, the remaining dog food was collected, dried at 105℃ to constant weight, and weighed. The feed intake rate was calculated as (amount provided - amount remaining) / amount provided × 100%, and the average feed intake rate within 2 hours was 90%.

[0099] Intestinal metabolism assay: After feeding for 30 consecutive days, 10g of fresh feces were collected from each dog daily (aseptic operation), 10mL of pH 7.0 phosphate buffer was added, homogenized for 3min, centrifuged at 12000r / min for 10min, and the supernatant was filtered through a 0.22μm filter membrane; short-chain fatty acids (acetic acid and propionic acid) were detected by GC, and the result was acetic acid + propionic acid = 1.6mg / g.

[0100] Example 3 1. Raw material pretreatment 1.1 Animal-derived treatment Weigh out 15 portions of chicken liver (100g each), 10 portions of spleen (100g each), and 15 portions of salmon offal (100g each, including fish skin, minced meat, and fish bones with a particle size ≤5mm). All raw materials are temporarily stored at 4℃ for ≤12 hours to ensure freshness.

[0101] Hammering and oiling treatment: Chicken liver and spleen were mixed and placed in a CJ-2024 hammering machine. The hammering frequency was set to 35 times / min and the hammering time was 6min. Three 10g samples were randomly selected and the muscle fiber breakage was observed using an XSP-2CA optical microscope (10×40x objective lens). Ten fields of view were selected for each sample, and 50 muscle fibers were counted in each field of view. The average breakage rate was ≥90% (to ensure that the tissue is loose and conducive to subsequent enzymatic digestion).

[0102] Weigh 2.5 portions of chicken fat (100g per portion, purchased from the same slaughterhouse, batch number: ZY202405), place them in a 304 stainless steel pot, heat with a DK-98-II type 500W electric heating mantle, stir with a magnetic stirrer at 60r / min, and maintain the temperature at 125℃ for 1.8h; take samples every 20min during this period to avoid local scorching; after cooking, filter with a 300-mesh nylon filter cloth to remove oil residue (after drying the oil residue, grind it into 80-mesh powder for later use), collect the oil paste and cool it to 48℃, then use a soft brush to evenly coat the surface of the pounded chicken liver-spleen mixture, with a coating amount of 12g / 100g of raw material, ensuring that no raw material surface is exposed.

[0103] Grinding and particle size control: The oiled chicken liver-spleen mixture was mixed with salmon offal and fed into a JR-120 meat grinder. The grinding speed was set to 12 kg / h. After grinding, the particle size was checked with a 2 mm standard sieve. The passing rate was 99% (counted with an electronic particle counter, the proportion of undersize material was ≥99%). The mixed animal source material was placed in a 304 stainless steel container and refrigerated at 4℃ for 0.5 h to avoid spoilage due to prolonged storage.

[0104] 1.2 Plant-derived treatment Raw material preparation and source: Weigh out 6 parts germinated brown rice (100g per part, germination rate ≥88%), 4 parts mulberry (100g per part), 3 parts hawthorn (100g per part), and 2 parts dandelion root (100g per part). All plant raw materials are selected and impurities and moldy particles are removed.

[0105] Ultrafine grinding and particle size detection: The plant raw materials were mixed and put into an XDW-60 ultrafine grinder. The grinding speed was set to 8500 r / min and the grinding time to 6 min. After grinding, the material was sieved through a 100-mesh standard sieve. The sieve-undersized material was collected and the passing rate was tested using an electronic sieve analyzer. The passing rate was 98% (the mass percentage of the sieve-undersized material was ≥98%). Three 5g samples were taken and the particle size distribution was detected using a Mastersizer3000 laser particle size analyzer. The detection parameters were set as follows: refractive index 1.52, dispersion medium water, and the measured D50 was 70μm. The pulverized plant-derived powder was placed in an aluminum-plastic composite sealed bag and stored at room temperature (25℃±1℃) and relative humidity ≤60% to avoid moisture absorption.

[0106] 2. Targeted enzymatic hydrolysis 2.1 Construction of the enzymatic hydrolysis system Raw material input and mixing: The pretreated animal-derived mixture (total mass 4000g: 1500g chicken liver + 1000g spleen + 1500g salmon offal) was put into a GR-50 type 50L reactor, and 6000mL of purified water (prepared by RO-400 type water treatment equipment, conductivity ≤10μS / cm) was added, with a solid-liquid ratio of 1:1.5; the anchor-type agitator was turned on, the speed was set to 80r / min, and the mixture was stirred for 15min to ensure that the animal-derived raw materials were completely dispersed and there were no obvious agglomerated particles.

[0107] Preparation and addition of the compound enzyme: Weigh 1.5 portions of the compound enzyme (total mass 60g, prepared at a mass ratio of 1:2.5:0.7): 15g of flavor protease (purchased from Novozymes (China) Biotechnology Co., Ltd., model: Flavorzyme500MG, batch number: FN202403, activity 500 LAPU / g); 37.5g of papain (purchased from Guangzhou Huamei Biotechnology Co., Ltd., batch number: MG202404, activity 800,000 U / g); 7.5g of neutral protease (purchased from Beijing Solarbio Technology Co., Ltd., batch number: ZP202403, activity 100,000 U / g); Dissolve the compound enzyme in 80mL of purified water at 50℃, stir until no enzyme particles are visible to the naked eye, and slowly add it through the feed inlet at the top of the reactor. Keep stirring during the addition process to avoid local aggregation of enzyme solution, which would lead to uneven enzymatic hydrolysis.

[0108] 2.2 Control of Enzymatic Hydrolysis Conditions Temperature and pH adjustment: Turn on the temperature control system of the reactor and raise the temperature to 50℃ at a rate of 5℃ / min. Adjust the pH of the system to 6.5 with 0.1mol / L citric acid solution (purchased from Sinopharm Chemical Reagent Co., Ltd., batch number: NL202403). After each addition of 0.5mL of citric acid solution, stir for 2min and use a PHS-3C pH meter (accuracy ±0.01) to detect the pH. Repeat the adjustment until the pH is stable at 6.5±0.1 to ensure stable enzymatic hydrolysis conditions.

[0109] Stirring program settings: adopt the "intermittent stirring" mode: stir for 7 minutes every 35 minutes, stirring speed 80 r / min, to avoid the enzyme hydrolysis system from standing for a long time and causing stratification; the total enzymatic hydrolysis time is 3 hours, during which samples are taken every 1 hour to observe the status of the system and record the color change of the enzyme hydrolysate (from light pink to light brown), with no stratification and no precipitation.

[0110] 2.3 Detection of the endpoint of enzymatic digestion Degree of protein hydrolysis (DH) detection: Take 10 mL of enzymatic hydrolysate into a 250 mL Erlenmeyer flask, add 2 drops of neutral red indicator (0.1% ethanol solution), and titrate with 0.1 mol / L sodium hydroxide solution (purchased from Sinopharm Chemical Reagent Co., Ltd., batch number: QY202403) until the solution turns pale red (pH 7.0), and record the volume consumed V1 = 5.2 mL; add 5 mL of neutral formaldehyde solution (adjusted to pH 7.0 with 0.1 mol / L sodium hydroxide beforehand), shake well, and continue titrating until pale red, and record the volume consumed V2 = 10.8 mL; determine the total nitrogen content of the enzymatic hydrolysate using a KDN-08A Kjeldahl nitrogen analyzer = 2.65 g / 100 mL, and calculate according to the formula: DH = (V2 × 0.1 × 7.5) / (Total Nitrogen × 100) × 100% = (10.8 × 0.1 × 7.5) / (2.65 × 100) × 100% = 48.5%.

[0111] Detection of free amino acids: Take 5 mL of enzymatic hydrolysate, centrifuge at 12000 r / min for 10 min using a TGL-16M centrifuge, and filter the supernatant through a 0.22 μm aqueous filter membrane; detect using an Agilent 1260 high performance liquid chromatograph (Agilent Technologies). Chromatographic conditions: column: C18 column (250 mm × 4.6 mm, 5 μm); mobile phase: methanol-0.1% phosphoric acid water = 5:95 (V / V); flow rate: 1 mL / min; detection wavelength: 220 nm; column temperature: 30 ℃; quantification was performed using the external standard method (amino acid standard mixture, concentration 1 mg / mL), and the free amino acid content was determined to be 2.8 mg / g.

[0112] 3. Three-stage fermentation 3.1 First Phase Preparation and inoculation of bacterial strain: Weigh 0.7 portions of Lactobacillus plantarum suspension (total mass 70 mL): Add Lactobacillus plantarum (live count 1 × 10⁻⁶) to the inoculation solution. 10 The cfu / g concentration was diluted to 1×10⁻⁶ with sterile physiological saline (0.9% sodium chloride solution). 9 CFU / mL, the concentration was verified by counting with a hemocytometer during dilution. The enzymatic hydrolysate (total mass approximately 10000g) and plant-derived powder (total mass 1500g: 600g germinated brown rice + 400g mulberry + 300g hawthorn + 200g dandelion root) were transferred into an FB-10 type 10L fermenter. The agitator was turned on (speed 35r / min) and stirred for 10min to ensure uniformity of the materials. The Lactobacillus plantarum suspension was slowly added along the inner wall of the fermenter and stirred for another 5min to ensure uniform dispersion of the suspension.

[0113] Fermentation conditions and endpoint detection: The inlet valve of the fermenter was closed, and nitrogen (99.99% purity) was introduced to replace the air in the tank for 6 minutes (air flow rate 2.5 L / min, controlled by an LZB-6 type rotor flow meter). Then, the nitrogen valve was closed, and the fermentation temperature was set to 33℃ and the relative humidity to 55% (monitored in real-time by the fermenter's built-in temperature and humidity sensors). Anaerobic fermentation was then initiated. After 16 hours of fermentation, 10 mL of sample was taken using a sterile sampling tube, centrifuged at 12000 r / min for 10 minutes, and the supernatant was filtered through a 0.22 μm filter membrane. The lactic acid content was determined by HPLC. Chromatographic column: Aminex HPX-87H column (300mm×7.8mm); mobile phase: 0.05mol / L sulfuric acid solution; flow rate: 0.6mL / min; column temperature: 60℃; detection wavelength: 210nm; external standard method (lactic acid standard, concentration 0.1-5mg / mL) was used for quantification, and the lactic acid concentration was determined to be 0.85mg / g.

[0114] 3.2 Second Phase Strain preparation and inoculation: Weigh 0.5 part of the Saccharomyces cerevisiae suspension (total mass 50 mL): Add Saccharomyces cerevisiae (live count 1×10⁻⁶) 9 (cfu / g) was diluted to 1×10⁻⁶ with sterile saline. 8 The concentration was verified by plate counting after dilution (cfu / mL). The inlet valve of the fermenter was opened to introduce sterile air filtered through a 0.22 μm air filter membrane, and the aeration rate was adjusted to 0.6 L / (L·min). The Saccharomyces cerevisiae suspension was added to the fermenter and stirred for 5 min to disperse the inoculum.

[0115] Fermentation conditions and endpoint detection: The fermenter temperature was adjusted to 34℃ and relative humidity to 53%, and aerobic fermentation was started. After 22 hours of fermentation, 10 mL of sample was taken, centrifuged at 12000 r / min for 10 min, and the supernatant was used to detect the ethyl acetate content using a GC-2014 gas chromatograph: Column: DB-WAX capillary column (30 m × 0.25 mm × 0.25 μm); Column temperature program: 60℃ for 3 min, increased to 180℃ at 5℃ / min, and held for 5 min; Injector temperature: 250℃; Detector (FID) temperature: 280℃; Carrier gas: Nitrogen (purity 99.999%); Quantification was performed using the external standard method (ethyl acetate standard, concentration 0.01-0.2 mg / mL), and the ethyl acetate content was determined to be 0.18 mg / g.

[0116] 3.3 Third Stage Strain preparation and inoculation: Weigh 0.4 portions of Bacillus subtilis suspension (total mass 40 mL): Add Bacillus subtilis (live count 1 × 10⁻⁶) 9 (cfu / g) was diluted to 1×10⁻⁶ with sterile saline. 8CFU / mL, diluted and Gram-stained to verify purity (purity ≥99%). Adjust the aeration rate of the fermenter to 0.4 L / (L·min), add the Bacillus subtilis suspension to the fermenter, and stir for 5 min to disperse the strain.

[0117] Fermentation conditions and endpoint detection: Adjust the fermenter temperature to 39℃ and relative humidity to 58%, and continue aerobic fermentation; after 9 hours of fermentation, take a 10mL sample and immediately place it in a boiling water bath for 12 minutes to inactivate the enzyme (to terminate enzyme activity). After cooling to room temperature, detect amylase activity using the DNS method. (1) Take 1 mL of inactivation solution and add 1 mL of 1% soluble starch solution (pH 6.0, prepared with 0.05 mol / L phosphate buffer); (2) React in a constant temperature water bath at 55℃ for 30 min, and add 2 mL of DNS reagent (concentration 0.1 mol / L); (3) Boil in a water bath for 5 min, cool, and then make up to 10 mL with purified water; (4) Measure the absorbance at 540 nm using a UV-2600 ultraviolet spectrophotometer, and calculate the enzyme activity by referring to the glucose standard curve (0.1-1.0 mg / mL); the amylase activity was measured to be 135 U / g; stop fermentation, collect the fermentation mixture (total mass about 11500 g), transfer it to a clean stainless steel bucket, and let it cool naturally to 55℃ for later use.

[0118] 4. Maillard reaction 4.1 Construction of the reaction system Raw material preparation and addition: Transfer the fermentation mixture into a GR-50 50L reactor and turn on the stirrer (55 r / min); add the following raw materials in sequence: 5 parts reducing sugar (total mass 175g, prepared at a mass ratio of 1:2.2: glucose 54.7g, xylose 120.3g, both purchased from Sinopharm Chemical Reagent Co., Ltd., glucose batch number GT20240310, xylose batch number MT20240405), dissolved in 200mL of 55℃ purified water and then added. 0.5 parts creatine (total mass 25g, purity 98%, verified by HPLC) were added to 15mL of reducing sugar solution and mixed thoroughly before being added; 0.3 parts cysteine ​​hydrochloride (total mass 15g, purchased from Sinopharm Chemical Reagent Co., Ltd., batch number: BY20240315, purity 99%) were dissolved in 30mL of 0.1mol / L hydrochloric acid solution and then added; the mixture was stirred for 15min to ensure that all raw materials were completely dissolved and the reaction system was a homogeneous paste without any solid particle precipitate. pH adjustment: The pH of the reaction system was adjusted to 6.3 with 0.1mol / L citric acid solution. After stirring for 5min, the pH was retested to ensure that it was stable at 6.3±0.1, providing a suitable acid-base environment for the Maillard reaction.

[0119] 4.2 Stepwise heating reaction Temperature program settings: Turn on the reactor temperature control system and adopt the "two-stage stepped heating" mode: 1. First stage: heat up from 55℃ to 98℃ at a rate of 5℃ / min and hold for 2 hours; 2. Second stage: heat up from 98℃ to 118℃ at a rate of 5℃ / min and hold for 1.2 hours; during the heating process, stir continuously (55r / min) and use an infrared thermometer to monitor the material temperature in real time to avoid local overheating and scorching.

[0120] 4.3 Detection of reaction products Detection of characteristic flavor compounds: After the reaction, take a 15g sample, centrifuge at 12000r / min for 15min, and filter the supernatant through a 0.22μm organic phase filter membrane. Detect the compounds using a GC-MS7890A-5975C gas chromatograph-mass spectrometer: Column: HP-5MS capillary column (30m×0.25mm×0.25μm); Column temperature program: 50℃ for 2min, increase to 280℃ at 10℃ / min, hold for 5min; Ion source: EI source, electron energy 70eV; Ion source temperature: 230℃; Detector temperature: 250℃; Quantification was performed using external standard method (2-acetylfuran and 4-ethylguaiacol standards, concentration 0.1-1wt%), and the results showed 2-acetylfuran = 0.43wt% and 4-ethylguaiacol = 0.32wt%. The reaction vessel was closed, and the reaction product was cooled to 50℃ with cooling water for later use.

[0121] 5. Post-embedding treatment 5.1 Mixing of functional excipients Preparation and addition of excipients: The following functional excipients were added to the reactor in sequence, and stirred for 10 min after each addition to ensure uniformity: (1) 0.5 parts of coated sodium butyrate (total mass 25g, coating rate 89%): The coating rate was tested by Soxhlet extraction (coating material was extracted with petroleum ether, and sodium butyrate content was titrated with 0.1mol / L sodium hydroxide) to ensure coating effect; (2) 1.0 part of plant extract (total mass 50g, prepared at a mass ratio of 1:1: 25g of Astragalus membranaceus water extract and 25g of Glycyrrhiza uralensis water extract): Astragaloside A content in Astragalus membranaceus water extract was 0.28wt %, the glycyrrhizic acid content in the licorice water extract was 0.32wt%, verified by HPLC detection; (3) 0.4 parts of gum arabic (total mass 20g, purchased from Sinopharm Chemical Reagent Co., Ltd., batch number: AL20240220): dissolved in 160mL of 60℃ purified water to prepare a 12wt% aqueous solution, stirred until completely dissolved and then added; turn on the GFJ-0.5 high-speed disperser, set the speed to 1800r / min, stir for 25min, take a sample every 5min during the period, observe the dispersion of excipients with a microscope, and ensure that there are no visible agglomerated particles.

[0122] 5.2 Spray drying Equipment parameter settings: Turn on the LPG-5 spray dryer, preheat for 30 minutes, and set the following parameters: Inlet air temperature: 215℃; outlet air temperature: 88℃; atomizer speed: 16000r / min; feed rate: 20mL / min; induced draft fan frequency: 50Hz; Intermediate collection and detection: The mixture in the reactor (temperature 50℃) was pumped to the feed inlet of the spray dryer through a peristaltic pump. After atomization drying, the dried powder (intermediate) was collected and pulverized using a 100-mesh standard sieve. Three 5g samples were taken and dried in a DZF-6050 vacuum drying oven at 105℃ for 4 hours. The moisture content was measured to be 5.9%. The crude protein content was determined to be 17.3% using a KDN-08A Kjeldahl nitrogen analyzer.

[0123] 5.3 Probiotic Mix Probiotic preparation: Weigh 0.9 portions of canine probiotic mixed microspheres (total mass 45g): composed of canine enterococci (live count 1×10⁻⁶). 9 cfu / g) and canine Bifidobacterium (live count 1×10⁻⁶) 9 The mixture (cfu / g) was prepared by embedding in a 1:1 ratio. The embedding wall material was a gum arabic-zein composite wall material. The survival rate was 88% after 2 hours in simulated gastric juice at pH 1.2.

[0124] Mixing and Quality Control: Transfer the intermediate (total mass approximately 1050g) into a VH-5 type V-type mixer, add the probiotic microspheres, set the speed to 70 rpm, and mix for 12 minutes. During the mixing process, monitor the material temperature in real time with an infrared thermometer, ensuring the maximum temperature is ≤28℃ (to avoid inactivation of probiotics due to high temperature). After mixing, take three 10g samples and anaerobic culture them for 48 hours in an MJ-150 type microbial incubator (MRS medium). The viable probiotic count was determined by plate counting to be 5.2 × 10⁻⁶. 7 CFU / g indicates the finished product of fermented flavoring agent for dogs.

[0125] 6. Performance Testing 6.1 Testing of core indicators of finished products Amylase activity assay: Take 1g of the finished product, add 10mL of 0.05mol / L phosphate buffer (pH 6.0), place it on a TS-2000 shaker at 180r / min for 35min, centrifuge at 12000r / min for 10min, take the supernatant and detect it using the DNS method, the amylase activity was measured to be 132U / g.

[0126] Branched-chain amino acid (leucine + isoleucine) detection: Dissolve 0.5g of the finished product in 10mL of 6mol / L hydrochloric acid solution and hydrolyze at 110℃ for 24h in a hydrolysis furnace; neutralize the hydrolysate to pH 7.0 with 0.1mol / L sodium hydroxide solution and filter through a 0.22μm filter membrane; detect using an automated amino acid analyzer under the following chromatographic conditions: Chromatographic column: cation exchange column (4.6 mm × 250 mm); column temperature: 55 ℃; mobile phase: sodium citrate buffer (pH 3.2-4.9); detection wavelength: 570 nm; the measured leucine + isoleucine = 1.9 wt%.

[0127] 6.2 Storage stability test Sample storage conditions: Take 100g of the finished product and put it into an aluminum-plastic composite sealed bag (oxygen permeability ≤5mL / (m)). 2 After heat sealing (24h·atm), the sample was placed in an HS-100 constant temperature and humidity chamber at 25℃ and 65% relative humidity for 6 months; 10g samples were taken monthly for the following tests: Flavor retention rate test: The contents of 2-acetylfuran and 4-ethylguaiacol were detected by GC. The flavor retention rate was calculated as (content after storage / initial content) × 100%. The flavor retention rate was 86% after 6 months.

[0128] Oxidative rancidity test: The TBARS method was used: 5g of sample was taken and 20mL of 7.5% trichloroacetic acid solution was added. The sample was homogenized for 3min at 8000r / min using an FJ200-S homogenizer. After filtration, 5mL of filtrate was taken and 5mL of 0.02mol / L thiobarbituric acid solution was added. The sample was then heated in a water bath at 95℃ for 40min. After cooling, the absorbance was measured at 532nm using a UV-2600 ultraviolet spectrophotometer. The oxidative rancidity value was calculated to be 0.5mg / kg.

[0129] 6.3 Feeding effect detection Basic dog food and mixed preparation: Basic dog food conforming to GB / T31216-2014 standard (25.5% crude protein, 12.3% crude fat) is used. The finished product is mixed into the basic dog food at an addition rate of 3.0%. The mixture is stirred for 8 minutes at 120r / min using a VH-5 V-type mixer. The deviation of the finished product content is ≤1.5% to ensure uniform mixing.

[0130] Feeding experiment design: Twelve adult Beagles (weighing 11-13 kg, half male and half female, in good health) were selected and acclimatized for 7 days (fed only basic dog food, with free access to water, and no abnormalities observed daily); followed by a 30-day feeding experiment: Feeding frequency: once at 8:00 AM and once at 6:00 PM daily; Feeding amount: 220g of mixed dog food per feeding (accurate to 0.1g); Feed intake calculation: collect the remaining dog food after 30 minutes, dry it at 105℃ to constant weight, and then weigh it. Feed intake = (amount provided - amount remaining) / amount provided × 100%. The average feed intake within 2 hours is 95%.

[0131] Intestinal metabolic index detection: After 30 days of continuous feeding, 10g of fresh feces were collected from each dog daily (aseptic operation), 10mL of pH7.0 phosphate buffer was added, homogenized for 3min, centrifuged at 12000r / min for 10min, and the supernatant was filtered through a 0.22μm filter membrane; short-chain fatty acids (acetic acid and propionic acid) were detected by GC, and the result was acetic acid + propionic acid = 2.1mg / g.

[0132] Comparative Example 1 1. Different process: Steps 1-2 (enzymatic hydrolysis) and 4-5 (Maillard reaction + encapsulation post-treatment) are the same as in Example 1; only the third stage of the three-stage fermentation (Bacillus subtilis fermentation) is omitted, and the mixture after yeast fermentation is directly put into the Maillard reaction.

[0133] 2. Performance Testing Amylase activity detection: 1g of the finished product was tested using the DNS method in Example 1, and the activity was found to be 32U / g. Detection details: After centrifugation of the enzyme extract, the supernatant was taken and reacted with 1% starch solution for 30min. After DNS color development, the absorbance at 540nm was 0.12 (0.35 in Example 1). The enzyme activity was calculated by referring to the glucose standard curve, confirming that it was far below the target value. Fecal undigested protein detection: 10 Beagles (same as in Example 1) were fed for 30 days, and 5g of feces were collected. The total nitrogen was determined by the Kjeldahl method. After subtracting the crude fiber bound nitrogen, the undigested protein content was found to be 8.6% (7.1% in Example 1). Feed intake detection: With a 2.5% addition amount, the feed intake rate after 2 hours was 82%. Specifically, each dog was given 200g of mixed food each time, and an average of 36g remained. The feed intake rate was calculated as (200-36) / 200×100%=82%.

[0134] 3. Results and Analysis Bacillus subtilis is the core source of amylase. The absence of this stage will lead to a sharp drop in the digestibility of the finished product (increased undigested protein), insufficient release of flavor substances, and decreased palatability.

[0135] Comparative Example 2 1. Different process: Steps 1-3 (enzymatic hydrolysis + three-stage fermentation) and 5 (embedding post-treatment) are the same as in Example 1; only creatine is removed in the Maillard reaction, and the other raw materials (reducing sugar, cysteine ​​hydrochloride) and reaction conditions remain unchanged.

[0136] 2. Performance Testing Characteristic flavor compound detection: GC-MS parameters were the same as in Example 1, with 2-acetylfuran = 0.21 wt% and 4-ethylguaiacol = 0.18 wt%. Compared with Example 1 (0.35 wt% / 0.28 wt%), creatine deficiency led to insufficient amino donors, incomplete Maillard reaction, and reduced formation of characteristic flavor compounds. Storage stability test: After storage at 25℃ / 65% humidity for 6 months, GC-analyzed flavor retention rate was 68%. Detailed testing: Monthly sampling was conducted to measure flavor compound content. After 6 months, 2-acetylfuran residue was 0.14 wt%, and the retention rate was calculated as 0.14 / 0.21 × 100% = 68%, lower than the 83% in Example 1, proving that creatine can improve flavor stability. Feed intake test: At a 2.5% addition level, the feed intake rate after 2 hours was 80%. Due to insufficient flavor intensity, the dogs' willingness to eat decreased.

[0137] 3. Results and Analysis Creatine is a cooperating amino donor in the Maillard reaction. Its absence leads to a reduction in the formation of characteristic flavor compounds, decreased storage stability, and reduced palatability.

[0138] Comparative Example 3 1. Different process: Steps 1-4 (enzymatic hydrolysis + fermentation + Maillard reaction) are the same as in Example 1; only unencapsulated canine Enterococcus faecalis powder is used in the probiotic mixing stage, and the other excipients and mixing conditions remain unchanged.

[0139] 2. Performance Testing Gastric juice viability assay: Prepare pH 1.2 simulated gastric juice (0.3% pepsin + 0.1 mol / L hydrochloric acid), add 0.1 g sample to 9 mL of gastric juice, and shake at 37℃ for 2 h; Plate count: serially diluted to 10⁻⁶. -6 After being coated on MRS medium and anaerobic cultured for 48 hours, the survival rate was measured to be 23%. In contrast, in Example 1 (85%), the unencapsulated probiotics were directly exposed to a strong acid environment, resulting in cell wall damage and a sharp drop in the number of viable bacteria.

[0140] Probiotic count after storage: After 6 months of storage at 25℃ / 65% humidity, the viable bacteria count obtained by plate counting is 1.2 × 10⁻⁶. 6 CFU / g; Detection details: During storage, probiotics are affected by oxygen and humidity, and are easily oxidized and inactivated in the unencapsulated state, resulting in a level far lower than 4.2 × 10⁻⁶ in Example 1. 7 cfu / g.

[0141] 3. Results and Analysis: The embedding wall material (gum arabic) can form a protective film on the surface of probiotics, resisting the strong acid environment of gastric acid and oxidation during storage. Without embedding, probiotics have difficulty passing through the gastric barrier and are easily inactivated during storage, failing to reach the concentration required for intestinal colonization. This demonstrates the crucial role of embedding treatment in the survival of probiotics.

[0142] Comparative Example 4 Differences in process: Steps 1 (raw material pretreatment) and 3-5 (fermentation + Maillard reaction + encapsulation posttreatment) are the same as in Example 1; only in the directional enzymatic hydrolysis stage, 48g of papain (single enzyme) is used to replace the complex enzyme (flavor protease + papain + neutral protease), and the enzymatic hydrolysis conditions remain unchanged.

[0143] 2. Performance Testing Degree of protein hydrolysis (DH): Titration method yielded DH = 35%; Detection details: V1 = 3.1 mL, V2 = 6.3 mL, total nitrogen = 2.5 g / 100 mL, calculated DH = (6.3 × 0.1 × 7.5) / (2.5 × 100) × 100% = 35%, lower than 45.2% in Example 1, because papain only hydrolyzes specific peptide bonds, resulting in incomplete protein degradation. Free amino acid detection: HPLC measured content = 1.8 mg / g; compared to Example 1 (2.4 mg / g), single enzymatic hydrolysis cannot fully break protein chains, resulting in insufficient release of free amino acids. Amylase activity detection: DNS method yielded = 80 U / g, due to insufficient enzymatic hydrolysis, insufficient substrate for Bacillus enzyme production during fermentation, leading to reduced amylase production.

[0144] 3. Results and Analysis: The three proteases in the complex enzyme can synergistically hydrolyze proteins with different structures. Papain alone has strong hydrolysis specificity but low efficiency, which leads to insufficient protein decomposition and thus affects subsequent enzyme production and the function of the finished product.

[0145] Comparative Example 5 1. Specific operating steps Difference in process: Steps 1-4 (enzymatic hydrolysis + fermentation + Maillard reaction) are the same as in Example 1; only gum arabic is added in the post-embedding treatment stage, and the coated sodium butyrate and plant extracts (Astragalus membranaceus aqueous extract + Glycyrrhiza uralensis aqueous extract) are removed, while the other conditions remain unchanged.

[0146] 2. Performance Testing Fecal SCFA detection: After 30 days of continuous feeding, GC analysis showed that acetic acid + propionic acid = 1.2 mg / g. Detection details: After homogenization and centrifugation of feces, the supernatant was filtered through a membrane. GC parameters were the same as in Example 1: acetic acid = 0.8 mg / g, propionic acid = 0.4 mg / g, lower than the 1.8 mg / g in Example 1. This is because sodium butyrate coating promotes the proliferation of intestinal acid-producing bacteria, and its absence reduces SCFA production. Intestinal motility frequency monitoring: The number of daily defecations and fecal form of the dogs were observed. The average peristaltic frequency decreased by 15% compared to Example 1. Specific data: In Example 1, dogs defecate an average of 2.2 times per day with formed feces; in this control group, the average was 1.87 times per day, with some feces being harder, indicating decreased intestinal motility.

[0147] 3. Results and Analysis: Sodium butyrate coating can promote the proliferation of intestinal acid-producing bacteria, and plant extracts can regulate intestinal mucosal function; the absence of both leads to a decline in intestinal metabolic function, reduced production of short-chain fatty acids, and weakened peristalsis.

[0148] In summary, through gradient optimization in Examples 1-3 (adjusting the types of animal / plant sources, dosage of compound enzymes, three-stage fermentation parameters, Maillard reaction conditions, and spray drying parameters), the functionality of the finished product was gradually improved. Among them, Example 3 (chicken liver + spleen + salmon offal, 4 kinds of plant sources, 1.5% compound enzyme, optimized fermentation and Maillard process) showed the best performance, with a protein hydrolysis degree of 48.5%, amylase activity of 132 U / g, 2-acetylfuran of 0.43 wt%, flavor retention rate of 86% after 6 months, feed intake rate of 95% after 2 hours, and fecal short-chain fatty acid content of 2.1 mg / g. Comparative examples 1-5 further validated the necessity of key processes: the absence of Bacillus subtilis fermentation leads to a sharp drop in amylase and decreased digestive capacity; the removal of creatine reduces flavor substances and storage stability; the lack of encapsulation of probiotics significantly reduces their gastric juice survival rate and the number of viable bacteria in storage; the replacement of complex enzymes with a single papain reduces protein hydrolysis efficiency; and the removal of coated sodium butyrate and plant extracts weakens intestinal metabolism and peristalsis. In summary, this canine fermented flavoring agent requires the combination of multiple raw materials and the synergy of the entire process to achieve the comprehensive effects of "superior flavor, aided digestion, good gut health, and stable storage".

[0149] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. A method for preparing a fermented flavoring agent for dogs, characterized in that, Includes the following steps: (1) Raw material pretreatment: Animal-derived processing: Take 40-50 portions of dog-preferred animal-derived raw materials, pound them, coat them with animal fat ointment, and then mince them; the animal-derived raw materials are selected from at least one of chicken liver, spleen, and salmonid fish scraps; Plant-derived processing: Take 15-25 parts of plant-derived raw materials and pulverize them into 60-120 mesh to obtain plant-derived powder; the plant-derived raw materials are selected from a combination of germinated brown rice and at least one of mulberry, hawthorn and dandelion root; (2) Targeted enzymatic hydrolysis: Animal-derived raw materials are enzymatically hydrolyzed with a compound enzyme at 40-55℃ and pH 5.8-6.8 for 1.5-3 hours. The compound enzyme is composed of flavor protease, papain and neutral protease in a ratio of 1:(1.5-2.5):(0.3-0.7). (3) Three-stage fermentation: The plant-based powder obtained in step (1) is mixed with the enzymatically hydrolyzed animal-based raw material, and the following fermentation is carried out in sequence: First stage: Add Lactobacillus strains and ferment at 28-34℃ for 10-18 hours; Second stage: Add yeast strains and ferment at 32-38℃ for 15-24 hours; Third stage: Add Bacillus strains and ferment at 35-40℃ for 5-10 hours; (4) Maillard reaction: Add reducing sugar, creatine and sulfur-containing precursor to the fermentation mixture and react with stepwise temperature increase; (5) Post-encapsulation treatment: After adding functional excipients, spray dry and mix in canine probiotic microspheres; The functional excipients include intestinal regulators, plant extracts, and embedding wall materials; wherein the plant extracts are a mixture of Astragalus membranaceus and Glycyrrhiza uralensis aqueous extracts at a ratio of 1:(0.5-1), and the intestinal regulators are sodium butyrate with an encapsulation rate of ≥85%.

2. The method according to claim 1, characterized in that, The amount of the compound enzyme added is 1.0-1.5% of the animal-derived raw material, and the degree of protein hydrolysis at the end of the enzymatic hydrolysis is ≥42% and the free amino acid content is ≥2.2 mg / g.

3. The method according to claim 1, characterized in that, The three fermentation endpoint indicators are as follows: lactic acid ≥ 0.6 mg / g, ethyl acetate ≥ 0.12 mg / g, and amylase activity ≥ 100 U / g.

4. The method according to claim 1, characterized in that, The Maillard reaction uses cysteine ​​or its hydrochloride as the sulfur-containing precursor, and the reaction product contains ≥0.3wt% 2-acetylfuran and ≥0.25wt% 4-ethylguaiacol.

5. The method according to claim 1, characterized in that, The embedding wall material is a natural polysaccharide or alcohol-soluble protein with an encapsulation rate of ≥80% and a survival rate of ≥80% in canine gastric fluid at pH 1.2-3.0 for 2 hours.

6. The method according to claim 1, characterized in that, The spray drying parameters are: inlet air temperature 200-220℃, outlet air temperature 80-90℃, finished product moisture ≤8%, crude protein ≥15%.

7. The canine fermented flavoring agent prepared by the method according to any one of claims 1-6, characterized in that, Includes: amylase activity ≥100U / g, branched-chain amino acids ≥1.5wt%, canine probiotics ≥3×10 7 cfu / g, flavor retention rate ≥80% and rancidity value ≤0.8mg / kg after 6 months of storage.

8. The flavoring agent according to claim 7, characterized in that, The flavoring agent is added at a rate of 1.8-3.0% of the dog food, with an intake rate of ≥90% 2 hours after feeding and fecal SCFA ≥1.5mg / g.