Bone joint maintenance type pet food and preparation method thereof

Abstract

The invention relates to the technical field of pet food, in particular to bone joint maintenance type pet food and a preparation method thereof.The pet food is prepared from, by weight, 300-420 parts of protein sources, 110-320 parts of carbohydrates, 110-130 parts of fat, 75-85 parts of fibers and prebiotics and 95-105 parts of functional ingredients, hydrolyzed salmon protein, hermetia illucens powder, sweet potato powder, schizochytrium oil and the like are used as auxiliary materials, joint health compounds and vitamin minerals are used as auxiliary materials, and omega-3 fatty acid from schizochytrium is used for replacing traditional fish oil, so that the problem of sustainability is solved, the risk of heavy metal pollution is avoided, and meanwhile, the stability of the product is improved.

Description

Technical Field

[0001] This invention relates to the field of pet food technology, specifically to bone and joint health-promoting pet food and its preparation method. Background Technology

[0002] With the increasing number of pets and their longer lifespans, osteoarthritis has become a common problem affecting pets' quality of life. Statistics show that over 20% of adult dogs and 50% of senior dogs suffer from osteoarthritis to varying degrees, and this proportion is also rising annually among felines. Osteoarthritis not only leads to decreased mobility and pain in pets, but can also cause secondary health problems, seriously impacting their quality of life.

[0003] Currently, pet food products on the market that address pet bone and joint problems are mainly based on the following technical solutions: US Patent 10973244B2 (Applicant: Hill's Pet Nutrition, granted April 2021) discloses a composition containing omega-3 polyunsaturated fatty acids and medium-chain fatty acids. This formulation primarily provides EPA and DHA through fish oil, supplemented by MCT to improve absorption. However, the omega-3 fatty acids in this formulation are easily oxidized, exhibiting poor stability, and the high-temperature extrusion process results in significant loss of active ingredients, typically between 25% and 35%.

[0004] US Patent 6596303B1 (Applicant: Mars Incorporated, granted July 2003) discloses pet food for maintaining joint health and alleviating arthritis symptoms in companion animals. This method provides anti-inflammatory effects by directly adding green-lipped mussel powder; however, the content of active ingredients in green-lipped mussels fluctuates greatly, and the content of its key active ingredient, eicosatenic acid (ETA), is low, leading to unstable efficacy.

[0005] US Patent 6977084B2 (Applicant: MARS INCORPORATED, granted in December 2005) discloses MARS INCORPORATED, which primarily achieves joint maintenance by adding unfermented green-lipped mussel powder. The limitation of this method is that the bioavailability of the active ingredients in the green-lipped mussel powder is low, and the direct addition method is easily affected by temperature during processing.

[0006] In addition, there are many pet joint care foods on the market based on the combination of glucosamine and chondroitin sulfate. However, the effects of using glucosamine and chondroitin sulfate alone are limited, and high doses (usually 1500-2000 mg of glucosamine and 1200 mg of chondroitin sulfate per day) are required to produce clinical effects, which is difficult to achieve in pet food.

[0007] The aforementioned existing technical solutions share the following common problems: the source of ω-3 fatty acids mainly relies on fishery resources, which poses sustainability issues and risks of heavy metal pollution; the active ingredients are easily destroyed during pet food processing, resulting in low bioavailability; the synergistic effects between active ingredients have not been fully considered; a universal formula suitable for both dogs and cats has not been developed, leading to increased complexity in production and application; and traditional high-temperature extrusion processes result in significant nutrient loss.

[0008] Therefore, there is an urgent need to develop a bone and joint care pet food and its preparation method that can effectively protect active ingredients, improve bioavailability, have synergistic effects of components, and is suitable for dogs and cats. Summary of the Invention

[0009] With the increasing number of pets and their longer lifespans, osteoarthritis has become a common problem affecting pets' quality of life. Statistics show that over 20% of adult dogs and 50% of senior dogs suffer from osteoarthritis to varying degrees, and this proportion is also rising annually among felines. Osteoarthritis not only leads to decreased mobility and pain in pets, but can also cause secondary health problems, seriously impacting their quality of life.

[0010] Currently, pet food products on the market that address pet bone and joint problems are mainly based on the following technical solutions: US Patent 10973244B2 (Applicant: Hill's Pet Nutrition, granted April 2021) discloses a composition containing omega-3 polyunsaturated fatty acids and medium-chain fatty acids. This formulation primarily provides EPA and DHA through fish oil, supplemented by MCT to improve absorption. However, the omega-3 fatty acids in this formulation are easily oxidized, exhibiting poor stability, and the high-temperature extrusion process results in significant loss of active ingredients, typically between 25% and 35%.

[0011] US Patent 6596303B1 (Applicant: Mars Incorporated, granted July 2003) discloses pet food for maintaining joint health and alleviating arthritis symptoms in companion animals. This method provides anti-inflammatory effects by directly adding green-lipped mussel powder; however, the content of active ingredients in green-lipped mussels fluctuates greatly, and the content of its key active ingredient, eicosatenic acid (ETA), is low, leading to unstable efficacy.

[0012] US Patent 6977084B2 (Applicant: MARS INCORPORATED, granted in December 2005) discloses MARS INCORPORATED, which primarily achieves joint maintenance by adding unfermented green-lipped mussel powder. The limitation of this method is that the bioavailability of the active ingredients in the green-lipped mussel powder is low, and the direct addition method is easily affected by temperature during processing.

[0013] In addition, there are many pet joint care foods on the market based on the combination of glucosamine and chondroitin sulfate. However, the effects of using glucosamine and chondroitin sulfate alone are limited, and high doses (usually 1500-2000 mg of glucosamine and 1200 mg of chondroitin sulfate per day) are required to produce clinical effects, which is difficult to achieve in pet food.

[0014] The aforementioned existing technical solutions share the following common problems: the source of ω-3 fatty acids mainly relies on fishery resources, which poses sustainability issues and risks of heavy metal pollution; the active ingredients are easily destroyed during pet food processing, resulting in low bioavailability; the synergistic effects between active ingredients have not been fully considered; a universal formula suitable for both dogs and cats has not been developed, leading to increased complexity in production and application; and traditional high-temperature extrusion processes result in significant nutrient loss.

[0015] Therefore, there is an urgent need to develop a bone and joint care pet food and its preparation method that can effectively protect active ingredients, improve bioavailability, have synergistic effects of components, and is suitable for dogs and cats. Detailed Implementation

[0016] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0017] The raw materials used in this invention and their characteristics are as follows: 1. Hydrolyzed Salmon Protein: Selected from Diana Petfood's Prosense™ series, with a degree of hydrolysis of 15%–20%, a molecular weight of 3,000–5,000 Da, and a protein content of ≥85%. This product has high digestibility and low allergenicity, making it suitable for pets with sensitive joints.

[0018] 2. Black soldier fly larvae powder: Selected from the ProteinX™ series products of Protix BV, with a protein content of 55% to 65% and a fat content of 10% to 15%. It is rich in chitin and lauric acid and has natural anti-inflammatory properties.

[0019] 3. Pea protein isolate: Selected from Roquette's NUTRALYS® T70 series products, with a protein content ≥80%, anti-nutritional factor content <0.1%, and good amino acid balance and digestibility.

[0020] 4. Schizochytrium oil blend: Selected from DSM Nutritional Products' life'sOMEGA™ series products, with ω-3 content of 45% to 55% and peroxide value <0.5 meq / kg. Produced using sustainably cultured Schizochytrium ATCC 20888 strain, it is environmentally friendly and free of marine pollutants.

[0021] 5. UC-II® Undenatured Type II Collagen: Supplied by InterHealth / Lonza, extracted from chicken breast cartilage, retaining the natural triple helix structure, each 40mg contains 2mg of active type II collagen.

[0022] 6. Black cumin oil extract: Selected from the Nigellin™ series of products from Sabinsa Corporation, with a standardized content of 4.5% to 5.5% thymol, which has strong antioxidant and anti-inflammatory properties.

[0023] 7. Fermented green-lipped mussel extract: Selected from the BioLex® GLM series products of Aroma New Zealand Ltd., fermented by Lactobacillus plantarum ATCC 8014 strain, rich in 0.3% to 0.5% icosenoic acid (ETA), the fermentation process increases the bioavailability of active ingredients by 3-5 times.

[0024] In this invention, the (2-hydroxypropyl)-β-cyclopaste is selected from the CAVAMAX® W7HP series products of Wacker Chemie AG, which can effectively encapsulate hydrophobic ω-3 fatty acid molecules, improving their stability and bioavailability. Phosphatidylcholine is selected from non-GMO sunflower lecithin, which combines with cholesterol to form liposomes, protecting UC-II collagen from degradation by gastric acid.

[0025] Furthermore, the methanesulfonylmethane used in this invention is the OptiMSM® series product from Bergstrom Nutrition, with a purity >99.9%, and is co-crystallized with L-ascorbic acid to improve stability and bioavailability. The 25-hydroxyvitamin D3 is the Rovimix® HY-D series product from DSM, which has 2-3 times higher bioavailability than standard D3. The calcium citrate-malate complex and magnesium glycine chelate are provided by Jungbunzlauer AG and Albion Minerals, respectively, both being highly bioavailable chelated mineral forms.

[0026] Example 1: Basic Formula - Canine Bone and Joint Health-Promoting Dry Food This embodiment provides a dry food for canine bone and joint health, the components of which are formulated by weight as follows: The protein sources comprise 380 portions, including 180 portions of hydrolyzed salmon protein, 90 portions of black soldier fly larvae powder, and 110 portions of pea protein isolate; carbohydrate sources comprise 280 portions, including 140 portions of sweet potato flour, 90 portions of quinoa flour, and 50 portions of tapioca starch; fat sources comprise 110 portions, including 55 portions of Schizochytrium oil blend, 35 portions of medium-chain triglyceride oil, and 20 portions of black cumin oil; fiber and prebiotics comprise 75 portions, including 28 portions of chicory root inulin, 18 portions of psyllium husk powder, and 29 portions of apple pomace; and functional ingredients comprise 95 portions, including 75 portions of joint health complex, 9 portions of natural preservatives, and 11 portions of vitamin and mineral premix. The joint health complex includes 12 parts of microencapsulated Schizochytrium-derived ω-3 fatty acids, 0.035 parts of liposome-coated UC-II collagen, 1.8 parts of black cumin oil extract, 4.5 parts of fermented green-lipped mussel extract, 0.8 parts of methanesulfonylmethane, 0.00003 parts of 25-hydroxyvitamin D3, 7.5 parts of calcium citrate-malate complex, and 1.8 parts of glycine magnesium chelate.

[0027] The microencapsulated *Schizochytrium*-derived ω-3 fatty acids comprise 2.8 parts of eicosapentaenoic acid (EPA) and 2.0 parts of docosahexaenoic acid (DHA) coated with (2-hydroxypropyl)-β-cyclodextrin. The liposome-coated UC-II collagen is prepared from the following components: 0.035 parts of UC-II non-denatured type II collagen; 0.07 parts of phosphatidylcholine; and 0.02 parts of cholesterol; wherein the liposome particle size is 200 nm. The standardized *Nigeria nigra* oil extract contains 4.5% thymol, and the fermented *Mussel brevis* extract contains 0.3 parts of tetracoccal acid.

[0028] Preparation method: First, microencapsulated ω-3 fatty acids were prepared. Schizochytrium oil was heated to 35°C under a nitrogen atmosphere (oxygen content <0.1%), and 0.05 parts of α-tocopherol were added as an antioxidant. Subsequently, it was mixed with USP-grade ethanol at a weight-to-volume ratio of 1:4 and stirred at 400 rpm for 15 minutes using a Teflon stirrer. (2-hydroxypropyl)-β-cyclodextrin was dissolved in purified water at a weight-to-volume ratio of 1:10, heated to 40°C, and continuously stirred at 300 rpm. The pH was adjusted to 7.0 using 0.1N sodium hydroxide solution. The oil phase was added to the cyclodextrin solution at a molar ratio of 1:2, homogenized at 3000 rpm for 10 minutes using a high-shear mixer, and stirred continuously at 400 rpm for 2 hours at 35°C. Spray drying was performed under the conditions of an inlet temperature of 45°C, an outlet temperature of 40°C, an atomization pressure of 2.0 bar, and a feed rate of 15 mL / min. The powder was collected and stored in a sealed container under a nitrogen atmosphere.

[0029] Next, liposome UC-II collagen was prepared. UC-II powder was dissolved in 10mM phosphate buffer at pH 7.4 and gently stirred at 100 rpm for 60 minutes at 20°C, then filtered through a 0.45 μm membrane. Phosphatidylcholine and cholesterol were dissolved in chloroform:methanol (2:1 volume ratio) at a molar ratio of 7:3, and the solvent was evaporated under reduced pressure of 25 mbar at 35°C to form a thin lipid membrane. The lipid membrane was hydrated with UC-II solution under a nitrogen atmosphere and gently stirred at 22°C for 2 hours. A probe-type sonicator was used with a 35% amplitude pulse mode (5 seconds on, 5 seconds off) for a total sonication time of 10 minutes, while maintaining the temperature below 25°C using an ice bath. The membrane was extruded sequentially through 400 nm and 200 nm polycarbonate membranes, 15 times each. Trehalose was added as a cryoprotectant (lipid:trehalose = 1:2), and the membrane was rapidly frozen in liquid nitrogen for lyophilization.

[0030] Then, a cold extrusion process is performed. All powdered raw materials are sieved through a 70-mesh sieve. The protein source is mixed for 6 minutes at 15 rpm in a belt mixer, followed by the addition of the carbohydrate source and mixing for another 8 minutes. The functional ingredients are mixed for 6 minutes at 12 rpm in a separate mixer, and then all dry materials are mixed for 12 minutes at 18 rpm in the main mixer. The oils are heated to 48°C and combined in a mixing tank at 320 rpm. Liquid antioxidants, microencapsulated ω-3 fatty acids, and liposome UC-II collagen are added and stirred at 42°C for 25 minutes until homogeneous. The barrel temperatures of the twin-screw extruder are set sequentially to 40°C, 45°C, 50°C, 55°C, 53°C, and 50°C, with a screw speed of 320 rpm, a die plate opening of 4 mm, and a die pressure of 30 bar. The interim mixture is added to the main hopper, and the liquid phase is injected at position 2 of the barrel using a calibration pump. The product moisture content at the die is controlled at 25%, and the die temperature does not exceed 58°C. The extrudate is cut using a rotary cutter (speed 1300 rpm) and fed into a fluidized bed dryer. The inlet air temperature is 68°C, the product bed temperature is 52°C, the drying time is 30 minutes, the final moisture content is 9%, and it is cooled to 28°C.

[0031] Finally, the final coating application was performed. The coating solution was prepared by heating 92 parts of fermented green-lipped mussel extract, 5 parts of medium-chain triglyceride oil, and 1.5 parts of mixed tocopherols to 40°C and homogenizing at 2500 rpm for 4 minutes. The particles were preheated to 38°C in a vacuum coater, and a vacuum of 75 mbar was applied for 12 seconds. The coating solution (5% of the particle weight) was sprayed while the particles were tumbling, and the vacuum was maintained for 25 seconds after spraying. The vacuum was then slowly released over 8 seconds, and the particles were continued to tumble at atmospheric pressure for 2.5 minutes. The coated particles were transferred to a fluidized bed cooler and cooled to 25°C with ambient air, then equilibrated in a temperature-controlled chamber (21°C) for 18 hours.

[0032] The pet food in this embodiment is in brown granule form, with a slight fish and nutty aroma, and contains 23% crude protein, 12% crude fat, 9% moisture, 6% ash, and 3% fiber.

[0033] Example 2: Enhanced Formula - Canine Bone and Joint Health-Promoting Dry Food This embodiment provides a specially formulated dry food for dogs with enhanced bone and joint support, the components of which are formulated by weight as follows: The protein source comprises 400 portions, including 200 portions of hydrolyzed salmon protein, 100 portions of black soldier fly larvae powder, and 100 portions of pea protein isolate; the carbohydrate source comprises 300 portions, including 150 portions of sweet potato flour, 100 portions of quinoa flour, and 50 portions of tapioca starch; the fat source comprises 120 portions, including 60 portions of Schizochyticulata oil blend, 40 portions of medium-chain triglyceride oil, and 20 portions of black cumin oil; the fiber and prebiotics comprise 80 portions, including 30 portions of chicory root inulin, 20 portions of psyllium husk powder, and 30 portions of apple pomace; and the functional ingredients comprise 100 portions, including 80 portions of joint health complex, 10 portions of natural preservatives, and 10 portions of vitamin and mineral premix. The joint health complex includes 15 parts of microencapsulated Schizochytrium-derived ω-3 fatty acids, 0.040 parts of liposome-coated UC-II collagen, 2.0 parts of black cumin oil extract, 5.0 parts of fermented green-lipped mussel extract, 1.0 part of methanesulfonylmethane, 0.00004 parts of 25-hydroxyvitamin D3, 8.0 parts of calcium citrate-malate complex, and 2.0 parts of glycine magnesium chelate.

[0034] The microencapsulated *Schizochytrium*-derived ω-3 fatty acids comprise 3.5 parts of eicosapentaenoic acid (EPA) and 2.5 parts of docosahexaenoic acid (DHA) coated with (2-hydroxypropyl)-β-cyclodextrin. The liposome-coated UC-II collagen is prepared from the following components: 0.040 parts of UC-II non-denatured type II collagen; 0.08 parts of phosphatidylcholine; and 0.025 parts of cholesterol; wherein the liposome particle size is 190 nm. The standardized *Nigeria nigra* oil extract contains 5.0% thymol, and the fermented *Mussel brevis* extract contains 0.4 parts of tetracoccal acid.

[0035] The preparation method is basically the same as in Example 1, but in the preparation of microencapsulated ω-3 fatty acids, the heating temperature of Schizochytrium oil is adjusted to 36°C and the inlet temperature of spray drying is adjusted to 44°C; in the preparation of liposome UC-II collagen, the ultrasonic amplitude is adjusted to 38% and the total ultrasonic time is 11 minutes; in the cold extrusion process, the screw speed is adjusted to 340 rpm and the die plate opening is adjusted to 4.5 mm; in the final coating application process, the homogenization speed of the coating solution is adjusted to 2800 rpm and the coating ratio is adjusted to 5.5% of the particle weight.

[0036] The pet food in this embodiment is dark brown granules with a distinct fish and nut aroma. It contains 25% crude protein, 13% crude fat, 8.5% moisture, 6.5% ash, and 3.5% fiber.

[0037] Example 3: Premium Formula - Canine Bone and Joint Health-Promoting Dry Food This embodiment provides a high-end dry food for canine bone and joint health, the components of which are formulated by weight as follows: The protein sources comprise 420 portions, including 220 portions of hydrolyzed salmon protein, 100 portions of black soldier fly larvae powder, and 100 portions of pea protein isolate; the carbohydrate sources comprise 320 portions, including 160 portions of sweet potato flour, 110 portions of quinoa flour, and 50 portions of tapioca starch; the fat sources comprise 130 portions, including 65 portions of Schizochytrium oil blend, 45 portions of medium-chain triglyceride oil, and 20 portions of black cumin oil; the fiber and prebiotics comprise 85 portions, including 32 portions of chicory root inulin, 22 portions of psyllium husk powder, and 31 portions of apple pomace; and the functional ingredients comprise 105 portions, including 85 portions of joint health complex, 10 portions of natural preservatives, and 10 portions of vitamin and mineral premix. The joint health complex includes 18 parts of microencapsulated Schizochytrium-derived ω-3 fatty acids, 0.045 parts of liposome-coated UC-II collagen, 2.2 parts of black cumin oil extract, 5.5 parts of fermented green-lipped mussel extract, 1.2 parts of methanesulfonylmethane, 0.00005 parts of 25-hydroxyvitamin D3, 8.5 parts of calcium citrate-malate complex, and 2.2 parts of glycine magnesium chelate.

[0038] The microencapsulated *Schizochytrium*-derived ω-3 fatty acids comprise: 4.2 parts of eicosapentaenoic acid (EPA) and 3.0 parts of docosahexaenoic acid (DHA) coated with (2-hydroxypropyl)-β-cyclodextrin. The liposome-coated UC-II collagen is prepared from the following components: 0.045 parts of UC-II non-denatured type II collagen; 0.09 parts of phosphatidylcholine; and 0.03 parts of cholesterol; wherein the liposome particle size is 180 nm. The standardized *Nigeria nigra* oil extract contains 5.5% thymol, and the fermented *Mussels versicolor* extract contains 0.5 parts of tetraenoic acid.

[0039] The preparation method is basically the same as in Example 1, but in the preparation of microencapsulated ω-3 fatty acids, the amount of α-tocopherol is increased to 0.1 parts, and the atomization pressure of spray drying is adjusted to 2.2 bar; in the preparation of liposome UC-II collagen, the pH of phosphate buffer is adjusted to 7.2, and the number of extrusions is increased to 20; in the cold extrusion process, the pressure at the mold is adjusted to 35 bar, and the drying time is extended to 35 minutes; in the final coating application, the vacuum degree is increased to 100 mbar, and the vacuum holding time after coating is extended to 30 seconds.

[0040] The pet food in this embodiment is dark brown granules with a rich fish and nut aroma. It contains 27% crude protein, 14% crude fat, 8% moisture, 7% ash, and 4% fiber.

[0041] Example 4: Basic Formula - Cat Bone and Joint Care Dry Food This embodiment provides a dry food for feline bone and joint health, the components of which are formulated by weight as follows: The protein sources comprise 420 portions, including 220 portions of hydrolyzed salmon protein, 110 portions of black soldier fly larvae powder, and 90 portions of pea protein isolate; carbohydrate sources comprise 280 portions, including 140 portions of sweet potato flour, 90 portions of quinoa flour, and 50 portions of tapioca starch; fat sources comprise 120 portions, including 60 portions of Schizochytrium oil blend, 40 portions of medium-chain triglyceride oil, and 20 portions of black cumin oil; fiber and prebiotics comprise 75 portions, including 28 portions of chicory root inulin, 18 portions of psyllium husk powder, and 29 portions of apple pomace; and functional ingredients comprise 105 portions, including 85 portions of joint health complex, 10 portions of natural preservatives, and 10 portions of vitamin and mineral premix. The joint health complex includes 15 parts microencapsulated ω-3 fatty acids from Schizochytrium, 0.040 parts liposome-coated UC-II collagen, 2.0 parts black cumin oil extract, 5.0 parts fermented green-lipped mussel extract, 1.0 part methanesulfonylmethane, 0.00004 parts 25-hydroxyvitamin D3, 8.0 parts calcium citrate-malate complex, and 2.0 parts glycine magnesium chelate. It also contains 1.5 parts taurine and 0.9 parts arachidonic acid.

[0042] The microencapsulated *Schizochytrium*-derived ω-3 fatty acids comprise 3.5 parts of eicosapentaenoic acid (EPA) and 2.5 parts of docosahexaenoic acid (DHA) coated with (2-hydroxypropyl)-β-cyclodextrin. The liposome-coated UC-II collagen is prepared from the following components: 0.040 parts of UC-II non-denatured type II collagen; 0.08 parts of phosphatidylcholine; and 0.025 parts of cholesterol; wherein the liposome particle size is 190 nm. The standardized *Nigeria nigra* oil extract contains 5.0% thymol, and the fermented *Mussel brevis* extract contains 0.4 parts of tetracoccal acid.

[0043] The preparation method is basically the same as in Example 1, but the particle shape and size have been appropriately adjusted to make the particles smaller and more regular in shape, suitable for feline consumption. The mold plate opening was adjusted to 3 mm, and the cutting speed was increased to 1400 rpm, resulting in a final particle size of approximately 5-7 mm. In addition, specific nutrient ratios suitable for feline needs, including taurine and arachidonic acid, were added to the vitamin and mineral premix.

[0044] The pet food in this embodiment is in the form of small brown granules, with a moderate fish and nut aroma, and contains 30% crude protein, 15% crude fat, 8% moisture, 7% ash, and 3% fiber.

[0045] Example 5: Universal Wet Food for Dogs and Cats with Bone and Joint Health This embodiment provides a wet food suitable for bone and joint health in dogs and cats, the components of which are formulated by weight as follows: Protein sources: 300 parts, including 260 parts hydrolyzed salmon protein, 20 parts black soldier fly larvae powder, and 20 parts pea protein isolate; carbohydrate sources: 110 parts, including 60 parts sweet potato flour, 30 parts quinoa flour, and 20 parts tapioca starch; fat sources: 110 parts, including 55 parts Schizochytrium oil blend, 35 parts medium-chain triglyceride oil, and 20 parts black cumin oil; fiber and prebiotics: 75 parts, including 28 parts chicory root inulin, 18 parts psyllium husk powder, and 29 parts apple pomace; functional ingredients: 95 parts, including 75 parts joint health complex, 9 parts natural preservatives, and 11 parts vitamin and mineral premix; gelling agents: 10 parts, including 4 parts carrageenan and 3 parts locust bean gum; taurine: 1.5 parts; arachidonic acid: 0.9 parts; moisture: 700 parts. The joint health complex includes 15 parts of microencapsulated Schizochytrium-derived ω-3 fatty acids, 0.040 parts of liposome-coated UC-II collagen, 2.0 parts of black cumin oil extract, 5.0 parts of fermented green-lipped mussel extract, 1.0 part of methanesulfonylmethane, 0.00004 parts of 25-hydroxyvitamin D3, 8.0 parts of calcium citrate-malate complex, and 2.0 parts of glycine magnesium chelate.

[0046] The microencapsulated *Schizochytrium*-derived ω-3 fatty acids comprise 3.5 parts of eicosapentaenoic acid (EPA) and 2.5 parts of docosahexaenoic acid (DHA) coated with (2-hydroxypropyl)-β-cyclodextrin. The liposome-coated UC-II collagen is prepared from the following components: 0.040 parts of UC-II non-denatured type II collagen; 0.08 parts of phosphatidylcholine; and 0.025 parts of cholesterol; wherein the liposome particle size is 190 nm. The standardized *Nigeria nigra* oil extract contains 5.0% thymol, and the fermented *Mussel brevis* extract contains 0.4 parts of tetracoccal acid.

[0047] The preparation method employs a wet grain improvement process: First, microencapsulated ω-3 fatty acids and liposomal UC-II collagen were prepared according to the methods of Examples 1 and 3, respectively.

[0048] Next, hydrolyzed salmon protein, black soldier fly larvae powder, and pea protein isolate were mixed with 400 parts water in a mixing tank, heated to 60°C, and stirred for 15 minutes to form a protein paste. In another container, sweet potato flour, quinoa flour, and tapioca starch were mixed with 200 parts water, heated to 75°C, and stirred for 10 minutes to form a starch paste.

[0049] Next, carrageenan and locust bean gum are mixed with 100 parts water and heated to 85°C while stirring until completely dissolved to form a colloidal solution. The protein paste, starch paste, and colloidal solution are mixed together, and the Schizochytrium oil mixture, medium-chain triglyceride oil, and black cumin oil are added simultaneously. The mixture is then homogenized at 65°C for 10 minutes.

[0050] The mixture is filled into aluminum cans or flexible packaging bags, sealed, and then subjected to high-pressure sterilization: heating time 18 minutes, holding temperature 115℃, holding time 45 minutes, cooling time 22 minutes to 40℃, sterilization value (F0 value) 9 minutes.

[0051] After sterilization, the mixture is cooled to 42°C and then heat-sensitive ingredients are added through a sterile port: methanesulfonylmethane, vitamin and mineral premix, microencapsulated ω-3 fatty acids, and liposome UC-II collagen. The mixture is then mixed for 6 minutes at 18 rpm under HEPA-filtered air. After verifying homogeneity, the mixture is finally sealed.

[0052] The pet food in this embodiment is brown paste or meat chunks, with a delicious fishy aroma, 10% crude protein, 5% crude fat, 70% moisture, 2.5% ash, and 1% fiber.

[0053] Example 6: Special dry food for senior dogs to support bone and joint health This embodiment provides a bone and joint care dry food suitable for senior dogs, the components of which are formulated by weight as follows: The protein source comprises 380 portions, including 200 portions of hydrolyzed salmon protein, 90 portions of black soldier fly larvae powder, and 90 portions of pea protein isolate; the carbohydrate source comprises 300 portions, including 150 portions of sweet potato flour, 100 portions of quinoa flour, and 50 portions of tapioca starch; the fat source comprises 110 portions, including 60 portions of Schizochytrium oil blend, 30 portions of medium-chain triglyceride oil, and 20 portions of black cumin oil; the fiber and prebiotics comprise 85 portions, including 32 portions of chicory root inulin, 22 portions of psyllium husk powder, and 31 portions of apple pomace; and the functional ingredients comprise 105 portions, including 85 portions of joint health complex, 10 portions of natural preservatives, and 10 portions of vitamin and mineral premix. The joint health complex includes 18 parts microencapsulated omega-3 fatty acids from Schizochytrium, 0.045 parts liposome-coated UC-II collagen, 2.2 parts black cumin oil extract, 5.5 parts fermented green-lipped mussel extract, 1.2 parts methanesulfonylmethane, 0.00005 parts 25-hydroxyvitamin D3, 8.5 parts calcium citrate-malate complex, and 2.2 parts glycine magnesium chelate. It also contains 0.1 parts coenzyme Q10 and 0.5 parts green tea extract.

[0054] The microencapsulated *Schizochytrium*-derived ω-3 fatty acids comprise: 4.2 parts of eicosapentaenoic acid (EPA) and 3.0 parts of docosahexaenoic acid (DHA) coated with (2-hydroxypropyl)-β-cyclodextrin. The liposome-coated UC-II collagen is prepared from the following components: 0.045 parts of UC-II non-denatured type II collagen; 0.09 parts of phosphatidylcholine; and 0.03 parts of cholesterol; wherein the liposome particle size is 180 nm. The standardized *Nigeria nigra* oil extract contains 5.5% thymol, and the fermented *Mussels versicolor* extract contains 0.5 parts of tetraenoic acid.

[0055] The preparation method is basically the same as in Example 3, but coenzyme Q10 and green tea extract are added to the functional components to enhance the antioxidant effect, making it suitable for the special needs of senior dogs. In addition, the granules are made smaller and softer to facilitate chewing by senior dogs, the mold opening is adjusted to 3.5 mm, the drying time is shortened to 28 minutes, and the final moisture content is increased to 10%.

[0056] The pet food in this embodiment is in the form of small brown granules with a soft texture and a moderate fish and nut aroma. It contains 23% crude protein, 12% crude fat, 10% moisture, 6.5% ash, and 4% fiber.

[0057] Example 7: Dry food specifically formulated for the bone and joint health of sporting dogs This embodiment provides a bone and joint health-promoting dry food suitable for working dogs, the components of which are formulated by weight as follows: The protein sources comprise 420 portions, including 220 portions of hydrolyzed salmon protein, 110 portions of black soldier fly larvae powder, and 90 portions of pea protein isolate; the carbohydrate sources comprise 320 portions, including 160 portions of sweet potato flour, 110 portions of quinoa flour, and 50 portions of tapioca starch; the fat sources comprise 130 portions, including 65 portions of Schizochytrium oil blend, 45 portions of medium-chain triglyceride oil, and 20 portions of black cumin oil; the fiber and prebiotics comprise 80 portions, including 30 portions of chicory root inulin, 20 portions of psyllium husk powder, and 30 portions of apple pomace; and the functional ingredients comprise 105 portions, including 85 portions of joint health complex, 10 portions of natural preservatives, and 10 portions of vitamin and mineral premix. The joint health complex includes 18 parts microencapsulated ω-3 fatty acids from Schizochytrium, 0.045 parts liposome-coated UC-II collagen, 2.2 parts black cumin oil extract, 5.5 parts fermented green-lipped mussel extract, 1.2 parts methanesulfonylmethane, 0.00005 parts 25-hydroxyvitamin D3, 8.5 parts calcium citrate-malate complex, and 2.2 parts glycine magnesium chelate. It also contains 1.0 part taurine, 1.5 parts creatine, and 2.0 parts branched-chain amino acids.

[0058] The microencapsulated *Schizochytrium*-derived ω-3 fatty acids comprise: 4.2 parts of eicosapentaenoic acid (EPA) and 3.0 parts of docosahexaenoic acid (DHA) coated with (2-hydroxypropyl)-β-cyclodextrin. The liposome-coated UC-II collagen is prepared from the following components: 0.045 parts of UC-II non-denatured type II collagen; 0.09 parts of phosphatidylcholine; and 0.03 parts of cholesterol; wherein the liposome particle size is 180 nm. The standardized *Nigeria nigra* oil extract contains 5.5% thymol, and the fermented *Mussels versicolor* extract contains 0.5 parts of tetraenoic acid.

[0059] The preparation method is basically the same as in Example 3, but taurine, creatine, and branched-chain amino acids are added to the functional ingredients to enhance muscle support and energy supply, making it suitable for the special needs of sporting dogs. In addition, the pellets are made larger and harder to increase chewing time, the mold opening is adjusted to 5 mm, the drying time is extended to 35 minutes, and the final moisture content is reduced to 7%.

[0060] The pet food in this embodiment is dark brown, large-particle, hard in texture, and has a rich fish and nut aroma. It contains 28% crude protein, 15% crude fat, 7% moisture, 7% ash, and 3.5% fiber.

[0061] Example 8: General-purpose dry food for puppies and cats to support bone and joint health This embodiment provides a bone and joint care dry food suitable for young dogs and kittens, the components of which are formulated by weight as follows: The protein source comprises 420 portions, including 220 portions of hydrolyzed salmon protein, 100 portions of black soldier fly larvae powder, and 100 portions of pea protein isolate; the carbohydrate source comprises 320 portions, including 160 portions of sweet potato flour, 110 portions of quinoa flour, and 50 portions of tapioca starch; the fat source comprises 130 portions, including 65 portions of Schizochytrium oil blend, 45 portions of medium-chain triglyceride oil, and 20 portions of black cumin oil; the fiber and prebiotics comprise 80 portions, including 30 portions of chicory root inulin, 20 portions of psyllium husk powder, and 30 portions of apple pomace; and the functional ingredients comprise 100 portions, including 80 portions of joint health complex, 10 portions of natural preservatives, and 10 portions of vitamin and mineral premix. The joint health complex includes 15 parts microencapsulated Schizochytrium-derived ω-3 fatty acids, 0.040 parts liposome-coated UC-II collagen, 2.0 parts black cumin oil extract, 5.0 parts fermented green-lipped mussel extract, 1.0 part methanesulfonylmethane, 0.00004 parts 25-hydroxyvitamin D3, 8.0 parts calcium citrate-malate complex, and 2.0 parts glycine magnesium chelate. It also contains 3.0 parts DHA enhancer, 1.5 parts taurine, 0.9 parts arachidonic acid, and 2.0 parts choline.

[0062] The microencapsulated *Schizochytrium*-derived ω-3 fatty acids comprise 3.5 parts of eicosapentaenoic acid (EPA) and 2.5 parts of docosahexaenoic acid (DHA) coated with (2-hydroxypropyl)-β-cyclodextrin. The liposome-coated UC-II collagen is prepared from the following components: 0.040 parts of UC-II non-denatured type II collagen; 0.08 parts of phosphatidylcholine; and 0.025 parts of cholesterol; wherein the liposome particle size is 190 nm. The standardized *Nigeria nigra* oil extract contains 5.0% thymol, and the fermented *Mussel brevis* extract contains 0.4 parts of tetracoccal acid.

[0063] The preparation method is basically the same as in Example 2, but DHA enhancer, taurine, arachidonic acid, and choline are added to the functional components to support the nervous system and cognitive development of young animals. In addition, the granules are made smaller and of moderate softness to facilitate chewing by young animals, the mold plate opening is adjusted to 3 mm, the cutting speed is increased to 1400 rpm, and the final moisture content is increased to 9%.

[0064] The pet food in this embodiment is in the form of small brown granules with a moderate texture and a moderate fish and nut aroma. It contains 30% crude protein, 16% crude fat, 9% moisture, 7% ash, and 3% fiber.

[0065] Comparative Example 1: Bone and Joint Health-Promoting Pet Food Without Microencapsulated ω-3 Fatty Acids This comparative example is based on Example 1, but the microencapsulated ω-3 fatty acids from *Schizochytrium* were replaced with an equal amount of ordinary *Schizochytrium* oil, without microencapsulation. Other components and preparation methods are the same as in Example 1.

[0066] Comparative Example 2: Liposome-free UC-II collagen-free pet food for bone and joint health This comparative example is based on Example 1, but the liposome-coated UC-II collagen was replaced with an equal amount of ordinary UC-II collagen powder, without liposome coating. Other components and preparation methods are the same as in Example 1.

[0067] Comparative Example 3: Bone and Joint Health-Promoting Pet Food Without Black Seed Oil Synergistic System This comparative example is based on Example 1, but black cumin oil and black cumin oil extract were completely removed and replaced with an equal amount of medium-chain triglyceride oil. Other components and preparation methods are the same as in Example 1.

[0068] Comparative Example 4: Bone and Joint Care Pet Food Prepared by High-Temperature Extrusion Process This comparative example uses the same formulation as Example 1, but adopts a traditional high-temperature extrusion process, setting the extruder barrel temperature to 90-120°C and the die temperature to 100°C. Other preparation steps are the same as in Example 1.

[0069] Comparative Example 5: Traditional Glucosamine / Chondroitin Sulfate Formula This comparative example uses a traditional glucosamine / chondroitin sulfate formulation: 380 parts protein, 280 parts carbohydrates, 110 parts fat, 75 parts fiber and prebiotics, and 95 parts functional ingredients. The functional ingredients include 20 parts glucosamine, 15 parts chondroitin sulfate, 5 parts green-lipped mussel powder (unfermented), 15 parts fish oil (unmicroencapsulated), 10 parts calcium-phosphorus complex, 10 parts vitamin and mineral premix, and 10 parts natural preservatives. The preparation method uses a traditional high-temperature extrusion process, with the extruder barrel temperature at 100-130℃.

[0070] To comprehensively evaluate the efficacy of each embodiment of the present invention, the following series of systematic experimental tests were conducted to examine in detail the performance of the present invention in terms of ω-3 fatty acid stability, bioavailability, joint function improvement, and process characteristics.

[0071] Experiment 1: Stability Test of ω-3 Fatty Acids 1.1 Experimental Objective To evaluate the protective effect of microencapsulation technology on ω-3 fatty acids derived from Schizochytrium, the stability changes of ω-3 fatty acids in the examples and comparative examples under different storage conditions were examined.

[0072] 1.2 Experimental Materials and Methods Test samples: Finished product samples of Example 1, Comparative Example 1 and Comparative Example 5.

[0073] Storage conditions: Store at room temperature: 25±2℃, relative humidity 60±5%; Accelerated storage: 40±2℃, relative humidity 75±5%; Testing periods: 0, 1, 2, 3, 4, 5, 6 months; Detection method: ω-3 fatty acid content: Gas chromatography (GC-FID, Agilent 7890B) was used, following AOAC official method 991.39.

[0074] Peroxide value (POV): determined using the AOCS official method Cd 8b-90.

[0075] Fatty acid retention rate calculation: Current month's content / Initial content × 100% 1.3 Experimental Results Table 1: Retention rate of ω-3 fatty acids under normal temperature storage conditions (%)

[0076] Table 2: Changes in peroxide value under normal temperature storage conditions (meq / kg)

[0077] Table 3: Retention rate of ω-3 fatty acids under accelerated storage conditions (%)

[0078] 1.4 Results Analysis As shown in Tables 1 and 2, under normal temperature storage conditions, the retention rate of ω-3 fatty acids in Example 1 was significantly higher than that in Comparative Examples 1 and 5, with retention rates of 92.3%, 65.4%, and 58.7% respectively after 6 months. Meanwhile, the peroxide value of Example 1 increased slowly, reaching only 6.8 meq / kg after 6 months, far lower than the 23.6 meq / kg of Comparative Example 1 and the 31.2 meq / kg of Comparative Example 5. Under accelerated storage conditions (Table 3), the differences between the groups were even more significant. Example 1 maintained an ω-3 fatty acid content of 82.4% after 6 months, while Comparative Examples 1 and 5 decreased to 35.6% and 28.2%, respectively.

[0079] These results fully demonstrate that the microencapsulation technology employed in this invention has a significant protective effect on ω-3 fatty acids derived from Schizochytrium. The inclusion complex formed by (2-hydroxypropyl)-β-cyclodextrin effectively isolates oxygen, light, and heat, preventing the occurrence of lipid peroxidation chain reactions. Furthermore, microencapsulation reduces the contact between ω-3 fatty acids and other components in food, further improving stability.

[0080] It is worth noting that even under accelerated storage conditions, the retention rate of ω-3 fatty acids and the peroxide value of Example 1 after 6 months were still better than those of the comparative example after 1 month of storage at room temperature. This indicates that the microencapsulation technology of the present invention can significantly extend the shelf life of the product, maintain the activity of functional ingredients, and provide stable bone and joint care effects for pets.

[0081] Experiment 2: In vivo bioavailability test of active ingredients 2.1 Experimental Objective The in vivo bioavailability of microencapsulated ω-3 fatty acids and liposome-coated UC-II collagen was evaluated to verify the efficacy of the black cumin oil synergistic system.

[0082] 2.2 Experimental Materials and Methods Laboratory animals: 30 healthy adult beagles (weighing 9-12kg, aged 2-4 years, half male and half female), provided by a nationally certified laboratory animal center, and the experimental protocol was approved by the animal ethics committee.

[0083] Grouping scheme: The animals were randomly divided into 5 groups of 6 each, and fed the pet food of Example 1, Example 3, Comparative Example 1, Comparative Example 3 and Comparative Example 5 respectively.

[0084] Feeding plan: Each dog was fed 100g / kg of body weight per day, twice a day, morning and evening, with free access to water. The experiment was officially started after 7 days of acclimatization feeding, with a total feeding period of 28 days.

[0085] Sample collection: 5 mL of venous blood was collected from the forelimbs on days 0, 7, 14, and 28 of feeding. The blood was anticoagulated with EDTA, centrifuged at 3000 rpm for 10 minutes at 4°C to separate the plasma, and stored at -80°C for analysis.

[0086] Testing indicators: Plasma EPA and DHA levels: quantified using liquid chromatography-mass spectrometry (LC-MS / MS, Shimadzu LCMS-8060) with internal standard method.

[0087] Type II collagen antibody levels: Enzyme-linked immunosorbent assay (ELISA) was performed using a commercial kit (R&D Systems) and the instructions were followed.

[0088] Inflammatory markers, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), were measured using a multiplex enzyme-linked immunosorbent assay (Luminex) technique.

[0089] 2.3 Experimental Results Table 4: Changes in plasma EPA levels at different time points (μg / mL)

[0090] Table 5: Changes in plasma DHA levels at different time points (μg / mL)

[0091] Table 6: Changes in serum type II collagen antibody levels at different time points (ng / mL)

[0092] Table 7: Serum inflammatory marker levels (pg / mL) after 28 days

[0093] 2.4 Results Analysis and Mechanism of Action As shown in Tables 4 and 5, the plasma EPA and DHA levels in dogs in Examples 1 and 3 increased rapidly after feeding and reached stable levels at 28 days. Compared with Comparative Example 1, the final plasma EPA levels in Examples 1 and 3 were 61.4% and 89.2% higher, respectively, and the plasma DHA levels were 60.6% and 87.6% higher, respectively. Compared with Comparative Example 3 (the synergistic system without black cumin oil), the final plasma EPA levels in Examples 1 and 3 were 110.3% and 146.5% higher, respectively, and the plasma DHA levels were 105.1% and 139.6% higher, respectively. Compared with Comparative Example 5, the final plasma EPA levels in Examples 1 and 3 were 89.8% and 122.4% higher, respectively, and the plasma DHA levels were 72.8% and 101.8% higher, respectively.

[0094] The results in Table 6 show that the levels of type II collagen antibodies in the Example 1 and Example 3 groups decreased significantly after 28 days of feeding, dropping to 36.5% and 31.4% of baseline, respectively, while the decrease in the control group did not exceed 15%. This indicates that liposome-coated UC-II collagen successfully crossed the gastrointestinal barrier, was recognized by antigen-presenting cells in Pell's plaque in the small intestine, and induced the generation of regulatory T cells (Tregs), thereby establishing oral immune tolerance to type II collagen.

[0095] The inflammatory marker results in Table 7 further confirm the anti-inflammatory effect of the present invention. The IL-6 levels in Examples 1 and 3 were 38.7% and 48.4% lower than those in Comparative Example 5, respectively, and the TNF-α levels were 48.9% and 57.8% lower, respectively. This significant anti-inflammatory effect is the result of the synergistic effect of multiple mechanisms: 1. Mechanism of high bioavailability of microencapsulated ω-3 fatty acids: The inclusion complex formed by (2-hydroxypropyl)-β-cyclodextrin slowly dissociates in the alkaline environment of the small intestine, releasing ω-3 fatty acids and avoiding destruction and oxidation by gastric acid. At the same time, the hydrophilic outer surface of cyclodextrin enhances the water solubility of fat-soluble ω-3 fatty acids, promotes emulsification with bile acids, forms smaller micelles, and increases the absorption area of ​​intestinal epithelial cells.

[0096] 2. Synergistic Mechanism of Black Cumin Oil: Thymol in black cumin oil reduces the efflux of ω-3 fatty acids and prolongs their half-life in vivo by inhibiting the activity of the P-glycoprotein (P-gp) transporter. Simultaneously, thymol can also inhibit the activity of cytochrome P450 3A4, reducing the metabolic clearance rate of ω-3 fatty acids. Furthermore, the phospholipids and unsaturated fatty acids in black cumin oil can increase cell membrane fluidity and promote the passive absorption of ω-3 fatty acids.

[0097] 3. Immunomodulatory mechanism of the liposome UC-II delivery system: The particle size of liposomes (180-190 nm) is ideal for uptake by Pell's plaque in the small intestine via M cells. Their cell membrane-like structure allows them to fuse with the cell membrane, directly delivering UC-II into the cell. UC-II retains its original triple helix structure, enabling it to be recognized by specific dendritic cells, inducing the differentiation of regulatory T cells, and producing the inhibitory cytokines IL-10 and TGF-β, thus suppressing autoimmune attacks on articular cartilage.

[0098] 4. The supplementation mechanism of fermented green-lipped mussel extract: The fermentation process increases the bioavailability of icosenoic acid (ETA). ETA is an ω-3 fatty acid, but its ability to inhibit 5-lipoxygenase (5-LOX) is 3-5 times stronger than that of EPA. It selectively inhibits the production of leukotriene B4 and reduces joint inflammation.

[0099] Based on the above mechanisms, this invention forms a multi-target, multi-level joint care system that not only improves the bioavailability of each functional component, but also synergistically inhibits inflammatory responses and protects cartilage tissue through multiple pathways, achieving effects beyond the simple sum of individual components.

[0100] Experiment 3: Joint Function Evaluation 3.1 Experimental Objective The effects of various embodiments of the present invention on improving joint function in older dogs were evaluated, and their clinical efficacy in practical applications was verified.

[0101] 3.2 Experimental Materials and Methods Experimental animals: 60 senior dogs (age > 7 years) of breeds including Labrador Retriever, Golden Retriever, German Shepherd, and Border Collie, all diagnosed by veterinarians with mild to moderate arthritis symptoms and X-ray scores of 1-3 (Kellgren-Lawrence scale).

[0102] Grouping scheme: The animals were randomly divided into 6 groups of 10 each, and fed the pet food of Example 1, Example 2, Example 3 and Comparative Example 1, Comparative Example 4 and Comparative Example 5 respectively.

[0103] Feeding plan: Feed each dog 90g / kg of body weight per day, twice a day, morning and evening, with free access to water, for a total feeding period of 12 weeks.

[0104] Evaluation indicators: Veterinary joint scoring: The joints were assessed in a blinded manner by three experienced veterinarians at weeks 0, 4, 8, and 12, using the Canine Brief Pain Inventory (CBPI) scoring system.

[0105] Gait analysis: Using the electronic gait analysis system (GAITRite®), parameters such as stride length, gait speed, and load-bearing time are measured.

[0106] Owner questionnaire: including assessments of pet activity level, pain response, and quality of life.

[0107] Serum biomarkers: C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), matrix metalloproteinase-3 (MMP-3), and chondroitin oligomeric matrix protein (COMP).

[0108] Joint fluid analysis: With the consent of the dog owners, joint fluid samples were collected from a subset of dogs (3-4 dogs per group) at week 0 and week 12 to analyze hyaluronic acid concentration and white blood cell count.

[0109] 3.3 Experimental Results Table 8: Changes in Veterinary Joint Score (CBPI scale, 0-10 points, lower scores are better)

[0110] Table 9: Gait Analysis - Changes in Weight Distribution Index (%)

[0111] Table 10: Changes in joint biomarker levels (percentage relative to baseline)

[0112] Table 11: Results of synovial fluid analysis (Week 0 vs. Week 12)

[0113] 3.4 Results Analysis and Mechanism of Action The results in Tables 8-11 clearly demonstrate the significant effects of the various embodiments of the present invention on improving joint function in older dogs. Example 3 showed the most outstanding performance, with its CBPI score decreasing from a baseline of 6.9 to 1.9 at week 12, a reduction of 72.5%, while Comparative Example 5 only decreased to 5.2, a reduction of 23.5%. Gait analysis showed that the weight distribution index of Example 3 increased by 32.2%, while that of Comparative Example 5 increased by only 9.3%.

[0114] Changes in joint biomarkers further confirmed the mechanism of action of the present invention. In Example 3 group at week 12, the inflammatory markers CRP and TNF-α decreased by 47.5% and 51.8%, respectively, while in Comparative Example 5, they decreased by only 19.5% and 23.2%. Simultaneously, the cartilage degradation markers MMP-3 and COMP showed similar trends, decreasing by 42.7% and 31.5% in Example 3 group, respectively, significantly higher than the 16.4% and 12.8% in Comparative Example 5.

[0115] The synovial fluid analysis results provided direct biological evidence: the hyaluronic acid concentration in the Example 3 group increased by 81.3%, while that in the Comparative Example 5 group increased by only 15.2%; the synovial fluid white blood cell count in the Example 3 group decreased by 51.8%, while that in the Comparative Example 5 group decreased by only 14.5%. This indicates that the present invention not only alleviates joint inflammation but also promotes the reconstruction of cartilage matrix.

[0116] The above results can be attributed to the following mechanism of action: 1. Multi-target anti-inflammatory mechanism: ω-3 fatty acids inhibit the synthesis of prostaglandin E2 and leukotriene B4 by replacing arachidonic acid; UC-II collagen reduces T cell-mediated autoimmune attack by inducing immune tolerance; thymol in black cumin oil directly inhibits the NF-κB signaling pathway, reducing the expression of inflammatory factors. This multi-target anti-inflammatory strategy is more effective than a single approach, explaining the significant anti-inflammatory effect of Example 3.

[0117] 2. Cartilage protection and regeneration mechanism: ω-3 fatty acids and ETA in fermented green-lipped mussel extract inhibited the expression of MMP-3 and reduced the degradation of cartilage matrix; 25-hydroxyvitamin D3 promoted the proliferation and differentiation of chondrocytes and enhanced cartilage repair capacity; methanesulfonylmethane provided the sulfur element required for cartilage matrix synthesis and also had antioxidant properties, protecting chondrocytes from oxidative damage.

[0118] 3. Microcirculation improvement mechanism: ω-3 fatty acids and unsaturated fatty acids in black cumin oil improve blood rheological properties and increase blood flow to the synovial membrane of joints; glycosaminoglycans in fermented green mussel extract promote the secretion of synovial fluid, increase the concentration of hyaluronic acid in the joint cavity, and improve joint lubrication.

[0119] 4. Synergistic mechanism of nutrients: Microencapsulation and liposome technology ensure high bioavailability of functional components; the synergistic system of black cumin oil further improves the absorption of ω-3 fatty acids; the rational ratio of calcium, magnesium and vitamin D3 provides the necessary nutritional support for the synthesis of cartilage matrix.

[0120] The results of this experiment fully demonstrate that the present invention, through a multi-target, multi-level comprehensive mechanism of action, can effectively improve joint function, reduce pain, and improve quality of life in older dogs, and its effects are significantly superior to existing technical solutions.

[0121] Experiment 4: Comparison of Cold Extrusion and High-Temperature Extrusion Processes 4.1 Experimental Objective The differences in functional component retention rates between cold extrusion and traditional high-temperature extrusion processes were evaluated to verify the technical effectiveness of the process innovation of this invention.

[0122] 4.2 Experimental Materials and Methods Test sample: Sample A: The formulation of Example 1, prepared using a cold extrusion process (extrusion temperature <60℃); Sample B: The formulation of Comparative Example 4 was prepared using a high-temperature extrusion process (extrusion temperature 90-120℃); Test metrics: ω-3 fatty acid (EPA+DHA) content: Gas chromatography-FID UC-II collagen activity: Enzyme-linked immunosorbent assay (ELISA) was used to determine the content of type II collagen that maintains the triple helix structure; Thymol content: High performance liquid chromatography (HPLC-UV); Eicosatetetraenoic acid (ETA) content: liquid chromatography-mass spectrometry (LC-MS / MS); 25-hydroxyvitamin D3 content: liquid chromatography-mass spectrometry (LC-MS / MS); Total antioxidant capacity: Oxygen radical scavenging capacity (ORAC) method; Calculation method: Retention rate (%) = (Measured content in finished product / Theoretical addition content) × 100% 4.3 Experimental Results Table 12: Comparison of functional component retention rates between cold extrusion and high-temperature extrusion processes (%)

[0123] Table 13: Comparison of Sensory and Physical Properties of Finished Products

[0125] *Digestibility was determined using an in vitro digestion model to simulate the gastrointestinal digestion process.

[0126] 4.4 Results Analysis and Process Mechanism The results in Tables 12 and 13 clearly demonstrate the significant differences between cold extrusion and traditional high-temperature extrusion processes in terms of functional component retention and product quality. The total functional component retention rate of the cold extrusion process (sample A) is as high as 97.2%, while that of the high-temperature extrusion process (sample B) is only 69.5%, a difference of 27.7 percentage points.

[0127] Of particular note is the more significant difference in the retention rates of heat-sensitive components. The activity retention rate of UC-II collagen was 94.5% under cold extrusion, but only 32.7% under high-temperature extrusion, a difference of 61.8 percentage points. This is because the triple helix structure of UC-II collagen begins to denature above 70°C, losing its unique immunomodulatory activity. Similarly, highly unsaturated fatty acids such as EPA, DHA, and ETA, as well as the phenolic compound thymol, are easily oxidized at high temperatures, with retention rate differences of 29.3, 41.1, and 38.9 percentage points, respectively.

[0128] Comparison of the sensory and physical properties of the finished products further confirms the advantages of the cold extrusion process. Cold-extruded products retain their natural color and aroma, and their hardness and chewiness are more suitable for pets. They also have a lower density and higher water absorption rate, which facilitates rapid water absorption and expansion in the gastrointestinal tract, increasing satiety. Most importantly, the in vitro digestibility of cold-extruded products reaches 92.3%, which is 8.6 percentage points higher than that of high-temperature extruded products. This means that pets can obtain more nutrients from their food.

[0129] The advantages of cold extrusion can be attributed to the following technical mechanisms: 1. Temperature Control Mechanism: The temperature is controlled below 60℃ throughout the cold extrusion process, avoiding denaturation and oxidation of heat-sensitive components. Precise temperature control of the barrel sections during extrusion (40℃, 45℃, 50℃, 55℃, 53℃, 50℃) creates a gentle thermal gradient, ensuring that the material is fully mixed and plasticized at a suitable temperature, while avoiding localized overheating.

[0130] 2. Mechanism of mechanical shear energy utilization: The cold extrusion process mainly utilizes mechanical shear energy rather than thermal energy to plasticize materials. The twin-screw design (300-350 rpm) provides sufficient shear force and mixing effect, while minimizing the generation of frictional heat.

[0131] 3. Moisture activity control mechanism: In the cold extrusion process, the moisture content of the product at the die is controlled at 24% to 26%, which is higher than the 18% to 20% of the traditional process. This appropriately increased moisture content reduces frictional heat, while providing sufficient plasticity and reducing dependence on heat energy.

[0132] 4. Oxidation protection mechanism: The cold extrusion process is carried out in a relatively closed environment, which reduces air contact. Combined with the antioxidants (such as α-tocopherol) added in the liquid phase, it effectively prevents the oxidation of unsaturated fatty acids.

[0133] 5. Mechanism of moderate starch gelatinization: The cold extrusion temperature range (40-55℃) allows starch granules to expand moderately and partially gelatinize, forming a good physical structure, while avoiding excessive gelatinization and complex formation (such as starch-protein complexes) at high temperatures, thus maintaining the bioavailability of nutrients.

[0134] The results of this experiment fully demonstrate that the cold extrusion process of this invention is a key technology for achieving the retention of highly active ingredients in functional pet food, with a total retention rate of 97.2% of functional ingredients, providing pets with maximum nutritional value and health benefits.

[0135] Experiment 5: Evaluation of Synergistic Effect 5.1 Experimental Objective The synergistic effect among the key functional components in the formulation of this invention was evaluated, and the combined advantages of black cumin oil, microencapsulated ω-3 fatty acids and liposomal UC-II collagen were verified.

[0136] 5.2 Experimental Materials and Methods Experimental models: an in vitro inflammation model of canine mononuclear cells stimulated by LPS and an artificial joint fluid external friction test.

[0137] Test sample: Sample 1: Extract from the complete formulation of Example 2; Sample 2: Comparative Example 1 extract (without microencapsulated ω-3 fatty acids); Sample 3: Comparative Example 2 extract (liposome-free UC-II collagen); Sample 4: Comparative Example 3 extract (without black cumin oil synergistic system); Sample 5: Microencapsulated ω-3 fatty acid as a single component; Sample 6: Liposome UC-II collagen as a single component; Sample 7: Black cumin oil extract (alone component); Cellular experiments: Canine mononuclear cell isolation: peripheral blood from healthy adult dogs was isolated using density gradient centrifugation; Cell culture: RPMI 1640 medium, 10% FBS, 37℃, 5% CO2; Group treatment: After each group of cells was pretreated with different samples for 24 hours, it was stimulated with LPS (100 ng / mL) for 24 hours. Detection indicators: IL-1β, IL-6, TNF-α and PGE2 levels (ELISA); NF-κB activity (nuclear translocation assay); cell viability (MTT assay); Bioavailability experiment: Small intestinal vesicle absorption model: Fresh canine small intestine was prepared, and inverted intestinal vesicles were prepared by elution method; Processing conditions: Each sample was incubated with the inverted intestinal sac for 2 hours at 37°C. Detection indicators: ω-3 fatty acid transport rate (GC-MS method); UC-II collagen permeability (ELISA method); Artificial joint fluid friction test: Simulated synovial fluid: high molecular weight hyaluronic acid (3 mg / mL), phospholipids (0.15 mg / mL), and albumin (20 mg / mL); Friction test: UMT-3 friction and wear tester, load 0.5-5 N, reciprocating speed 20 mm / s; Group processing: Different samples (equivalent active ingredient concentration) were added to each group. Testing indicators: coefficient of friction, wear rate; 5.3 Experimental Results Table 14: Inhibition rate of inflammatory factors in canine monocytes stimulated by LPS (%)

[0138] Table 15: ω-3 fatty acid transport rate in the small intestinal vesicle absorption model (nmol / mg protein / 2h)

[0139] Table 16: Permeability of UC-II collagen in the small intestinal vesicle absorption model (ng / mg protein / 2h)

[0140] Table 17: Variation of the friction coefficient (μ) of artificial joint fluid

[0141] *Improvement rate = (Individual component plus μ + Example 2μ) / Individual component plus μ × 100% 5.4 Results Analysis and Synergistic Mechanism The results in Tables 14-17 fully demonstrate the significant synergistic effect among the key functional components in the formulation of this invention. In an LPS-stimulated canine mononuclear cell inflammation model, the full formulation of Example 2 (Sample 1) showed a significantly higher inhibition rate against various inflammatory factors than the simple additive effect of individual components. For example, the inhibition rate against IL-6 reached 72.5%, while the additive effect of individual components (Samples 5+6+7) was only 51.5%, resulting in a synergistic effect of approximately 41%.

[0142] The results from the small intestinal vesicle absorption model are even more striking: in Example 2, the total transport rate of ω-3 fatty acids was 195.1% higher than that of the ω-3 fatty acid sample alone, while the increase after removing black cumin oil (sample 4) was only 33.8%. This fully demonstrates the key role of black cumin oil in improving the bioavailability of ω-3 fatty acids. Similarly, the permeability of UC-II collagen in Example 2 was 176.5% higher than that used alone, while the permeability after removing the liposome coating (sample 3) was actually 53.6% lower than that used alone, demonstrating the necessity of the liposome delivery system.

[0143] Artificial joint fluid friction tests further validated the synergistic effect of the formulation. Under different load conditions, the friction coefficient of the added group in Example 2 was significantly lower than that of the individual component addition group, with an average improvement rate of 34.9%. This indicates that the synergistic effect of the functional components forms a more effective lubricating protective layer, reducing friction and wear between articular cartilage.

[0144] The synergistic mechanism of the formulation of this invention can be summarized in the following aspects: 1. Synergistic Mechanism of Enhanced Absorption: Thymol and unsaturated fatty acids in black cumin oil enhance the absorption of ω-3 fatty acids through multiple pathways. Thymol inhibits P-glycoprotein (P-gp) and cytochrome P450 3A4, reducing the efflux and metabolic clearance of ω-3 fatty acids; phospholipids and medium-chain fatty acids in black cumin oil increase cell membrane fluidity, promoting the passive absorption of ω-3 fatty acids. Experimental data show that this synergistic effect increases the bioavailability of ω-3 fatty acids by nearly 3 times.

[0145] 2. Synergistic Delivery System Mechanism: Liposome encapsulation protects UC-II collagen from degradation by gastric acid and proteases, while its nanoscale particle size (180-200 nm) is perfectly suited for uptake by Pellère's plaque in the small intestine via M cells. The timing and location of microencapsulated ω-3 fatty acids and liposomal UC-II collagen release in the intestine are synergistically matched, working together to act on the intestinal immune system and forming a spatiotemporal synergistic effect.

[0146] 3. Synergistic Mechanism of Anti-inflammatory Signaling Pathways: ω-3 fatty acids, UC-II collagen, and black cumin oil thyme ketone inhibit the inflammatory response through different but complementary signaling pathways. ω-3 fatty acids competitively inhibit arachidonic acid metabolism, reducing the production of prostaglandin E2 and leukotriene B4; black cumin oil thyme ketone directly inhibits the nuclear translocation of NF-κB, reducing the gene expression of inflammatory factors; UC-II collagen induces regulatory T cells to produce the inhibitory cytokines IL-10 and TGF-β, forming a negative feedback regulation. This multi-target anti-inflammatory strategy produces effects beyond simple addition, as shown in Table 14, with an anti-inflammatory synergistic rate of 40%–45%.

[0147] 4. Synergistic Mechanism of Joint Lubrication: ω-3 fatty acids and unsaturated fatty acids in black cumin oil form a more stable surfactant layer with phospholipids in synovial fluid; glycosaminoglycans in fermented green-lipped mussel extract increase the polymerization degree and viscoelasticity of hyaluronic acid; sulfur provided by methanesulfonylmethane promotes chondrocyte secretion of more proteoglycans, enhancing the lubricating capacity of synovial fluid. This multi-faceted synergistic lubrication effect significantly reduces the coefficient of friction of the joint surface, as shown in Table 17, with an average improvement rate of 34.9%.

[0148] The experimental results fully demonstrate that the formulation design of this invention possesses a scientifically sound synergistic mechanism. The functional components are not simply additive, but rather form a complementary and synergistic system, significantly improving the overall efficacy of the product. This synergistic effect is one of the core innovations of this invention and the key reason why it surpasses existing technologies.

[0149] Experiment 6: Evaluation of Canine and Feline Universality 6.1 Experimental Objective The suitability of the formulation of this invention for both dogs and cats was evaluated, and its effectiveness as a general-purpose pet food was verified.

[0150] 6.2 Experimental Materials and Methods Laboratory animals: 10 healthy adult dogs (5 Labradors, 5 Golden Retrievers, aged 2-5 years) 10 healthy adult cats (5 American Shorthairs and 5 British Shorthairs, aged 2-5 years) All animals came from licensed breeding facilities and underwent health checks before the experiment to ensure they were free of abnormalities.

[0151] Experimental Design: Crossover control experiment: Each animal was fed Example 5 (wet diet) and Comparative Example 5, respectively. The feeding cycle for each food was 14 days, with a 7-day rest period in between as the washout period.

[0152] Feeding plan: Feed according to the standard weight, once in the morning and once in the evening, and allow free access to water.

[0153] Evaluation indicators: 1. Palatability test: Preferred test: Provide two food options simultaneously and record the first food option chosen; Feed intake comparison: Comparison of feed intake within a specified time (30 minutes); Owner rating: The owner rates the pet's eating behavior (1-5 points). 2. Bioavailability test: Plasma sampling: Blood samples were collected on days 0, 7, and 14 of feeding; Detection indicators: EPA, DHA, and type II collagen antibody levels; Fecal collection: Collect fecal samples on day 13-14 of feeding; Apparent digestibility determination: Apparent digestibility of nutrients (protein, fat, carbohydrate); 3. Physiological adaptability test: Stool scoring: Stool quality was assessed using the Bristol Stool Scale; Gut microbiota analysis: 16S rRNA sequencing analysis of gut microbiota diversity and composition; Blood biochemical indicators: routine indicators such as liver and kidney function, electrolytes, and blood glucose; 6.3 Experimental Results Table 18: Palatability Test Results

[0154] Table 19: Changes in plasma EPA and DHA levels (% increase relative to baseline)

[0155] Table 20: Apparent digestibility (%)

[0156] Table 21: Gut microbiota diversity index (Shannon index)

[0157] After Experiment 3 was completed, 12 senior dogs that performed best in Example 3 were selected for a 4-week feeding test in Example 6 as an extension of Experiment 3. The results are shown in Table 22.

[0158] Table 22: Comparison results of Example 3 and Example 6 in older dogs (n=12)

[0159] The results showed that, building upon the significant improvements already achieved in Example 3, Example 6 further improved joint function and quality of life in older dogs. The CBPI joint score decreased by 15.8%, CRP and TNF-α levels decreased by 11.1% and 13.0%, respectively, and the mobility score increased by 9.8%. Although these improvements were relatively small, they were all statistically significant (p<0.05).

[0160] Through formulation optimization, Example 6 better meets the specific needs of senior dogs. Coenzyme Q10 enhances mitochondrial function and improves energy metabolism efficiency; catechins in green tea extract enhance the antioxidant defense system and reduce oxidative stress; and the smaller, softer particle design improves palatability and digestibility. These targeted improvements make Example 6 an optimal choice for senior dogs, especially those with osteoarthritis.

[0161] Table 23: Inhibition rate of NF-κB nuclear translocation in LPS-stimulated canine monocytes (%)

[0162] Table 24: Effects of different samples on the viability of LPS-treated canine monocytes (MTT assay, % control group)

[0163] *Cell protection rate = (LPS + Sample treatment value - LPS treatment value) / (Control value - LPS treatment value) × 100% The nuclear translocation experiment results showed that the full formulation of Example 2 (sample 1) inhibited NF-κB nuclear translocation by 72.6%, which was much higher than the 49.1% of the sum of individual components (samples 5+6+7), representing an increase of 47.8%. This result is consistent with the trend of inflammatory factor inhibition rates in Table 14, further confirming the synergistic anti-inflammatory mechanism of the formulation components.

[0164] MTT cell viability test results showed that LPS treatment reduced cell viability to approximately 61% of the control group, while the addition of the full formulation of Example 2 restored cell viability to 92.5%, with a cell protection rate of 80.6%, significantly higher than the 59.7% achieved by adding the individual components. This indicates that the formulation of the present invention not only inhibits the inflammatory response but also protects cells from inflammatory damage and promotes tissue repair.

[0165] The nuclear translocation experiment directly confirmed the molecular mechanism by which thymol from black cumin oil inhibits the NF-κB signaling pathway. Thymol inhibits IκB kinase activity, preventing IκB phosphorylation and degradation, thereby blocking the translocation of the NF-κB p65 subunit to the nucleus and ultimately inhibiting the expression of pro-inflammatory genes. This mechanism is complementary to the inhibition of the COX-2 and 5-LOX pathways by ω-3 fatty acids, producing a synergistic effect.

[0166] The results of cell viability testing indicate that the protective effect of the formulation of this invention is not limited to anti-inflammatory properties, but also includes direct cytoprotective effects. This may be related to multiple mechanisms: ω-3 fatty acids improve cell membrane fluidity and integrity; UC-II collagen promotes the synthesis of extracellular matrix; and the antioxidant components in black cumin oil scavenge free radicals and reduce oxidative stress. These multi-layered protective mechanisms work synergistically to give the formulation of this invention a comprehensive joint care effect.

[0167] 6.4 Results Analysis and Generality Mechanism The results in Tables 18-21 fully demonstrate that the formulation of this invention exhibits excellent palatability, bioavailability, and physiological adaptability in both dogs and cats, with no significant differences between the two animals, thus verifying its effectiveness as a general-purpose pet food.

[0168] Palatability tests (Table 18) showed that Example 5 had a first-choice rate of 87.5% and 85.0% in dogs and cats, respectively, with consumption rates of 73.2% and 70.5%, and owner ratings of 4.6 and 4.4, all significantly better than Comparative Example 5. There was no significant difference in results between the two animal groups (p>0.05), indicating that the formulation of this invention can simultaneously satisfy the taste preferences of dogs and cats.

[0169] Bioavailability tests (Tables 19-20) showed that Example 5 exhibited significantly higher increases in EPA and DHA levels in both dogs and cats than Comparative Example 5, reaching 212.6% and 195.8% respectively after 14 days, compared to only 96.3% and 92.5% in Comparative Example 5. The differences between the two animal groups were not statistically significant (p>0.05). Similarly, apparent digestibility assays confirmed that Example 5 demonstrated excellent and similar digestibility and absorption in both dogs and cats.

[0170] Gut microbiota analysis (Table 21) showed that after 14 days of feeding with Example 5, the gut microbiota diversity in both dogs and cats was significantly increased, with Shannon indices increasing to 4.23 and 4.18, respectively, while the control group (Example 5) showed only a slight increase. Detailed microbiota composition analysis (data not listed in the table) further confirmed that Example 5 promoted the growth of beneficial bacteria (such as Bifidobacteria and Lactobacillus) and inhibited the proliferation of potentially pathogenic bacteria (such as Clostridium perfringens), and this regulatory effect was consistent in both dogs and cats.

[0171] The canine and feline universality of the formulation of this invention can be attributed to the following mechanisms: 1. Mechanism for Meeting Common Needs: Although dogs and cats belong to different families and genera in evolution, they share common physiological needs in bone and joint maintenance. Key functional components such as ω-3 fatty acids and type II collagen have regulatory effects on joint inflammation and cartilage degeneration in both animals. This invention addresses these common needs by designing functional formulations suitable for both animals.

[0172] 2. Mechanism for Addressing Specific Needs: This invention adds taurine (1.5 parts) and arachidonic acid (0.9 parts), which are specifically needed by felines, to the basic formula, ensuring that the special nutritional needs of felines are met. This design concept, which prioritizes commonalities while also considering specific needs, ensures the universality and comprehensiveness of the formula.

[0173] 3. Palatability Balancing Mechanism: The hydrolyzed salmon protein and fermented green-lipped mussel extract used in this invention both contain free amino acids (such as glutamic acid and glycine) and nucleotides that are commonly preferred by dogs and cats, forming a balanced umami flavor. Simultaneously, the medium-chain triglyceride oil provides a moderate fatty aroma, enhancing the palatability of the food. Palatability test results confirm that this design successfully balances the taste preferences of dogs and cats.

[0174] 4. Universal Mechanism of Bioavailability: The application of microencapsulation and liposome technology solves the common challenges faced by functional ingredients in the digestive tracts of dogs and cats (such as gastric acid degradation and intestinal absorption disorders). In particular, the nanoscale particle size (180-220 nm) of (2-hydroxypropyl)-β-cyclodextrin inclusion complex and liposomes achieves optimal effects in the digestive physiology of both animals, ensuring high bioavailability of key ingredients.

[0175] 5. Mechanism of Gut Microbiota Regulation: The chicory root inulin (prebiotic) and fermented components in the formula provide selective nutrition to the beneficial bacteria common to both dogs and cats, promoting gut health. Gut microbiota analysis results show that this regulatory effect is consistent in both animal groups, confirming the universality of this invention in gut health.

[0176] The results of this experiment fully demonstrate that the present invention has successfully developed a universal pet food for bone and joint care that meets the common needs of dogs and cats while also taking into account their specific requirements. This solves the problem of developing separate products for different animals in the market and has significant practical value and market potential.

[0177] Through the above six systematic experiments, the core innovations and technical advantages of this invention have been fully verified: 1. The protective effect of microencapsulation technology on ω-3 fatty acids: It increases the retention rate from 65.4% to 92.3% after 6 months of storage, and reduces the peroxide value from 23.6 to 6.8 meq / kg, significantly extending the shelf life and expiration date of the product.

[0178] 2. Immunomodulatory effect of liposome-coated UC-II collagen: Successfully induced oral immune tolerance, reducing serum type II collagen antibody levels by nearly 70%, which is far superior to ordinary UC-II powder.

[0179] 3. Bioavailability enhancement effect of black cumin oil synergistic system: It increases the bioavailability of ω-3 fatty acids by about 200%, improves the anti-inflammatory effect by 40% to 45%, and creates a significant synergistic effect.

[0180] 4. Functional component retention effect of cold extrusion process: The total retention rate of functional components reaches 97.2%, which is 27.7 percentage points higher than that of traditional high temperature extrusion. In particular, the retention rate of heat-sensitive components such as UC-II collagen is increased by 61.8 percentage points.

[0181] 5. Actual improvement in joint function: Example 3 reduced the veterinary joint score from 6.9 to 1.9 in a 12-week clinical trial, a reduction of 72.5%, and reduced inflammatory markers by 45% to 50%, significantly improving the quality of life of pets.

[0182] 6. Applicability of the universal formula for dogs and cats: It exhibits excellent and similar palatability, bioavailability and physiological adaptability in both dogs and cats, with a first-choice rate of over 85% and an apparent digestibility of over 90%.

[0183] These results fully demonstrate that, through innovative formula design and process optimization, this invention has successfully solved key technical problems in existing pet joint care foods, such as poor stability of active ingredients, low bioavailability, and lack of synergistic effects between components, providing pets with an efficient, stable, and comprehensive bone and joint care solution, demonstrating significant technological innovation and practical value.

[0184] Based on the above experimental evaluations, the high-end formulation provided in Example 3 demonstrates the most outstanding performance in all aspects and is the optimal implementation scheme of this invention. This formulation has the following characteristics: 1. The highest dose of microencapsulated Schizochytrium-derived ω-3 fatty acids (18 parts) and liposomal UC-II collagen (0.045 parts) was used to achieve the best anti-inflammatory and immunomodulatory effects; 2. The black cumin oil extract contains 5.5% thymol, maximizing its synergistic effect; 3. Fermented green-lipped mussel extract contains 0.5 parts of tetracoccalanoic acid, which supplements its unique anti-inflammatory mechanism; 4. Optimized cold extrusion process parameters are used to ensure the highest retention rate of functional components; 5. Demonstrated the best joint function improvement and safety in clinical trials.

[0185] This best practice is suitable for adult to senior pets with higher requirements for joint health, and can also be used to prevent potential joint problems in young pets.

Claims

1. A pet food for bone and joint health, characterized in that, By weight, the pet food comprises the following components: Protein sources: 300-420 parts, including 180-260 parts hydrolyzed salmon protein, 20-110 parts black soldier fly larvae powder, and 20-110 parts pea protein isolate; Carbohydrate sources: 110-320 parts, including 60-160 parts sweet potato flour, 30-110 parts quinoa flour, and 20-50 parts tapioca starch; The fat source is 110-130 parts, including 55-65 parts of Schizochyticulata oil mixture, 30-45 parts of medium-chain triglyceride oil, and 20 parts of black cumin oil; 75-85 parts fiber and prebiotics, including 28-32 parts chicory root inulin, 18-22 parts psyllium husk powder, and 29-31 parts apple pomace; Functional ingredients: 95-105 parts, including 75-85 parts of joint health complex, 9-10 parts of natural preservatives, and 10-11 parts of vitamin and mineral premix. The joint health complex comprises 12-18 parts of microencapsulated Schizochytrium-derived ω-3 fatty acids, 0.035-0.045 parts of liposome-coated UC-II collagen, 1.8-2.2 parts of black cumin oil extract, 4.5-5.5 parts of fermented green-lipped mussel extract, 0.8-1.2 parts of methanesulfonylmethane, 0.00003-0.00005 parts of 25-hydroxyvitamin D3, 7.5-8.5 parts of calcium citrate-malate complex, and 1.8-2.2 parts of glycine magnesium chelate.

2. The bone and joint care pet food according to claim 1, characterized in that, The microencapsulated Schizochytrium-derived ω-3 fatty acids include 2.8-4.2 parts of eicosapentaenoic acid (EPA) and 2.0-3.0 parts of docosahexaenoic acid (DHA) coated with (2-hydroxypropyl)-β-cyclodextrin, wherein the EPA and DHA are derived from Schizochytrium strain ATCC 20888.

3. The bone and joint care pet food according to claim 1, characterized in that, The liposome-coated UC-II collagen is made from the following components: 0.035-0.045 parts of UC-II non-denatured type II collagen; Phosphatidylcholine 0.07-0.09 parts; Cholesterol 0.02-0.03 parts; The liposomes have a particle size of 180-220 nanometers.

4. The bone and joint care pet food according to claim 1, characterized in that, The black cumin oil extract is standardized to contain 4.5%–5.5% thymol, and the fermented green-lipped mussel extract is prepared by fermentation with Lactobacillus plantarum ATCC 8014 strain and contains 0.3–0.5 parts of tetracoccal acid.

5. A method for preparing the bone and joint care pet food according to any one of claims 1-4, characterized in that, Includes the following steps: Preparation of microencapsulated ω-3 fatty acids; Preparation of liposomal UC-II collagen; Cold extrusion process; Final coating application; The method is carried out at a temperature not exceeding 60°C throughout, in order to maintain biological activity.

6. The preparation method according to claim 5, characterized in that, The preparation of the microencapsulated ω-3 fatty acid includes the following steps: Under a nitrogen atmosphere, the oil of Schizochytrium was heated to 33-37°C, and 0.05-0.1 parts of α-tocopherol were added as an antioxidant. Mix with USP grade ethanol at a weight-to-volume ratio of 1:4, and stir with a Teflon stirrer at 400-450 rpm for 15-20 minutes. Dissolve (2-hydroxypropyl)-β-cyclodextrin in purified water at a weight-to-volume ratio of 1:10, heat to 38-42℃, stir continuously at 300-350 rpm, and adjust the pH to 6.8-7.2 using 0.1N sodium hydroxide solution; Add the oil phase to the cyclodextrin solution at a 1:2 molar ratio, homogenize using a high-shear mixer at 3000-4000 rpm for 10-15 minutes, and continue stirring at 400-450 rpm for 2-3 hours at 35±2℃. Spray drying is carried out under the conditions of inlet temperature 43-47℃, outlet temperature 38-42℃, atomization pressure 1.8-2.2 bar, and feed rate 12-15 ml / min. The powder is collected and stored in a sealed container under a nitrogen atmosphere.

7. The preparation method according to claim 5, characterized in that, The preparation of the liposome UC-II collagen includes the following steps: Dissolve UC-II powder in 10mM phosphate buffer solution at pH 7.2-7.6, stir gently at 100-150 rpm for 45-60 minutes at 18-22°C, and filter through a 0.45-micron membrane. Phosphatidylcholine and cholesterol were dissolved in chloroform:methanol (2:1 volume ratio) at a molar ratio of 7:

3. The solvent was evaporated under reduced pressure of 35±2℃ and 25-30 mbar to form a thin lipid film. The lipid membrane was hydrated with UC-II solution under a nitrogen atmosphere and gently stirred at 20-25°C for 2-3 hours. Use a probe-type ultrasound machine with an amplitude of 30% to 40%, a pulse mode of 5 seconds on and 5 seconds off, for a total ultrasound time of 8 to 12 minutes, while using an ice bath to maintain the temperature below 25°C. The material is extruded sequentially through 400 nm and 200 nm polycarbonate films, 15-20 times each time. Trehalose was added as a cryoprotectant (lipid: trehalose = 1:2), and the mixture was rapidly frozen in liquid nitrogen for freeze-drying.

8. The preparation method according to claim 5, characterized in that, The cold extrusion process includes the following steps: Sieve all powdered ingredients through a 60-80 mesh sieve. In a belt mixer, mix the protein source at 15-20 rpm for 5-7 minutes. Add the carbohydrate source and continue mixing for 7-10 minutes. In a separate mixer, mix the functional ingredients at 10-15 rpm for 5-7 minutes. Then, in the main mixer, mix all dry ingredients at 15-20 rpm for 10-15 minutes. Heat the oils separately to 45-50℃, combine them in a mixing tank at 300-350 rpm, add liquid antioxidants, microencapsulated ω-3 fatty acids and liposome UC-II collagen, and stir at 40-45℃ for 20-30 minutes until homogeneous; The barrel temperatures of the twin-screw extruder are set to 40℃, 45℃, 50℃, 55℃, 53℃, and 50℃ respectively; the screw speed is 300-350 rpm; the die plate opening is 3-5 mm; and the die pressure is 25-35 bar. Add the intervention mixture to the main hopper, and inject the liquid phase at position 2 of the cylinder through the calibration pump. The moisture content of the product at the mold is controlled at 24% to 26%, and the mold temperature does not exceed 58°C. The extrudate is cut using a rotary cutter at a speed of 1200-1500 rpm and conveyed to a fluidized bed dryer. The inlet air temperature is 65-70℃, the product bed temperature is 50-55℃, the drying time is 25-35 minutes, the final moisture content is 8%-10%, and it is cooled to 25-30℃.

9. The preparation method according to claim 5, characterized in that, The final coating application includes the following steps: Preparation of coating solution: 90-95 parts of fermented green-lipped mussel extract, 4-6 parts of medium-chain triglyceride oil, and 1-2 parts of mixed tocopherols, heated to 38-42℃, and homogenized at 2000-3000 rpm for 3-5 minutes; Preheat the particles to 35-40°C in a vacuum coating machine and apply a vacuum of 50-100 mbar for 10-15 seconds. Spray the coating solution (4% to 6% of the particle weight) while it is tumbling. After spraying, maintain the vacuum for 20 to 30 seconds, slowly release the vacuum within 5 to 10 seconds, and continue tumbling for 2 to 3 minutes under atmospheric pressure. The coated particles are transferred to a fluidized bed cooler and cooled to 25-30°C with ambient air. They are then equilibrated in a temperature-controlled chamber for 12-24 hours at a temperature of 20-22°C.

10. The preparation method according to claim 5, characterized in that, The method also includes a wet grain improvement step: Reduce the basic dry matter to 28%–32% (moisture content 68%–72%). Increase hydrolyzed salmon protein to 250-270 servings and reduce carbohydrate sources to 100-120 servings; Add gelling agents: 3-5 parts carrageenan, 2-3 parts locust bean gum; Microencapsulated ω-3 fatty acids were added to the fat phase, and liposome UC-II collagen was added to the protein phase. High-pressure sterilization parameters: heating time 15-20 minutes, holding temperature 113-117℃, holding time 40-50 minutes, cooling time 20-25 minutes to 40℃, sterilization value (F0 value) 8-10 minutes; For heat-sensitive ingredients such as methanesulfonylmethane and vitamins, cool the product to 40-45°C, add it through a sterile port under HEPA-filtered air, mix at 15-20 rpm for 5-7 minutes, and verify homogeneity before final sealing.

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