Liposome fish oil preparation for pets, preparation method and application thereof

Abstract

The application relates to a liposome fish oil preparation for pets and a preparation method and application thereof, and belongs to the technical field of pet nutrition and veterinary medicine. The liposome fish oil preparation for pets provided by the application comprises 10-50 parts of fish oil, 20-60 parts of hydrogenated lecithin, 4-8 parts of succinic acid modified hydroxypropyl methyl cellulose, 4-8 parts of pectin, 1-3 parts of poloxamer 188, 0.5-1 part of chitosan and 0.1-5 parts of an antioxidant. The EPA and DHA in the fish oil are effectively protected by a liposome encapsulation technology, the bioavailability and stability of the EPA and DHA are significantly improved, the palatability is improved, the efficient absorption and targeted delivery of the EPA and DHA can be realized, the pet physiological characteristics and digestive characteristics are improved, and the liposome fish oil preparation for pets has obvious market competitiveness compared with existing pet fish oil supplements.

Description

Technical Field

[0001] This application relates to a liposomal fish oil preparation for pets, its preparation method and application, belonging to the field of pet nutrition and veterinary medicine technology. Background Technology

[0002] Omega-3 polyunsaturated fatty acids, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), play a vital role in the health of dogs and cats, including anti-inflammatory effects, support for joint health, promotion of healthy skin and coat, support for cognitive function and cardiovascular health, etc.

[0003] Currently, most pet fish oil supplements on the market are traditional triglyceride (TG) or ethyl ester (EE) types. These traditional forms have significant drawbacks: 1) They require lipase secreted by the pet's pancreas to be broken down before absorption, which significantly reduces the absorption rate for older, younger, or pets with impaired pancreatic function or weak digestive systems; 2) Omega-3 fatty acids are chemically unstable and easily oxidized and rancid during processing, storage, and digestion, leading to reduced potency and an unpleasant fishy smell, affecting palatability; 3) Traditional fish oil has limited bioavailability, and its "digestion-decomposition-reassembly" process results in the loss of active ingredients.

[0004] Liposome technology, a delivery system that encapsulates active substances in microspheres composed of a phospholipid bilayer, has been widely used in the human pharmaceutical field. This technology can improve drug bioavailability, targeting, and stability.

[0005] While liposome technology is currently used in human health products, its direct application to pets faces significant challenges: 1) Differences in digestive environment: The pH value of fasting gastric acid in dogs and cats (1-2) is much lower than that in humans (1.5-3.5), posing a severe test to the stability of liposomes; 2) Fundamentally different palatability requirements: Humans can mask the taste with capsules, but pets cannot communicate, so the fishy smell problem must be fundamentally solved; 3) Differences in absorption and metabolism: Pets (especially cats) have low conversion efficiency for ethyl ester-type fish oil and generally face the risk of insufficient lipase secretion; 4) Feeding convenience: Human formulations are not suitable for pets' feeding habits.

[0006] There are no reports yet of applying liposome technology to the field of pet nutrition technology. Therefore, it is of great significance to develop a liposome fish oil formulation that can overcome many drawbacks of traditional fish oil, taking into account the unique physiological characteristics and digestive properties of pets. Summary of the Invention

[0007] To address the aforementioned issues, a liposomal fish oil formulation for pets, its preparation method, and its application are provided. This invention introduces liposome encapsulation technology into the field of pet nutrition. The liposome encapsulation structure formed by phospholipids, combined with succinic acid-modified hydroxypropyl methylcellulose, and in conjunction with poloxamer 188 and chitosan, effectively protects EPA and DHA in the fish oil, significantly improving their bioavailability and stability, and enhancing palatability. It enables efficient absorption and targeted delivery of EPA and DHA, and is improved to suit the physiological characteristics and digestive properties of pets. Compared to existing pet fish oil supplements, it has significant market competitiveness.

[0008] This application provides a liposomal fish oil formulation for pets, comprising, by weight: 10-50 parts fish oil, 20-60 parts hydrogenated lecithin, 4-8 parts succinic acid-modified hydroxypropyl methylcellulose, 4-8 parts pectin, 1-3 parts poloxamer 188, 0.5-1 parts chitosan, and 0.1-5 parts antioxidant.

[0009] Because liposomes have a structure highly similar to cell membranes, they can be directly absorbed by intestinal cells via endocytosis / membrane fusion, without relying on lipase breakdown. This makes them particularly suitable for pets with insufficient lipase secretion, offering advantages such as a shorter absorption pathway, higher efficiency, and significantly improved bioavailability. Furthermore, the relatively slow metabolism of liposomes in vivo helps maintain a more stable and sustained level of Omega-3 concentration in the blood, providing continuous health benefits. In addition, encapsulating fish oil completely within a phospholipid bilayer using liposomes physically blocks the release of fishy odor molecules, resulting in a mild-tasting formulation that can be directly mixed into food, effectively masking fishy odor and improving palatability. Moreover, by selecting hydrogenated phospholipids, the structural integrity of the liposomes is ensured in the highly acidic environment of a pet's stomach, preventing premature release and enhancing the formulation's ability to withstand strong stomach acid, thus improving its stability.

[0010] This application enhances the stability of liposomes in the pet's digestive tract by optimizing the phospholipid composition and preparation process. It fundamentally solves the palatability problem caused by the fishy smell of fish oil by utilizing the fully encapsulated structure of liposomes, and bypasses the lipase-dependent digestive bottleneck in pets through the endocytosis / membrane fusion absorption pathway, which significantly improves the bioavailability of Omega-3 fatty acids. It can be used to prevent or assist in the treatment of common health problems in pets such as osteoarthritis, skin diseases, cognitive impairment, and chronic kidney disease.

[0011] Optionally, the preparation method of the succinic acid-modified hydroxypropyl methylcellulose includes the following steps: S1. Add hydroxypropyl methylcellulose to acetic acid solvent and heat and stir to dissolve; S2. Esterification reaction is carried out after adding succinic anhydride and anhydrous sodium acetate catalyst; S3. Add deionized water to terminate the reaction, and obtain the succinic acid modified hydroxypropyl methylcellulose after separation and drying.

[0012] Optionally, the ratio of hydroxypropyl methylcellulose, succinic anhydride, and anhydrous sodium acetate is 1:(1.3~1.6):(0.2~0.4). Optionally, the ratio of hydroxypropyl methylcellulose, succinic anhydride and anhydrous sodium acetate is 1:1.5:0.3.

[0013] Optionally, the temperature of the esterification reaction in step S2 is 75~80℃; Optionally, the temperature of the esterification reaction in step S2 is 78°C.

[0014] Optionally, the pectin is short-chain pectin, and the molecular weight of the short-chain pectin is 3000~5000.

[0015] Optionally, the pet liposome fish oil formulation comprises liposomes formed by fish oil encapsulated in a phospholipid bilayer, wherein the average particle size of the liposomes is 50-500 nm. Optionally, the liposomes have an average particle size of 100-200 nm and a polydispersity index (PDI) < 0.3. This size range ensures that the liposomes can be efficiently absorbed by intestinal cells via endocytosis.

[0016] Optionally, the fish oil contains docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA); Optionally, the total content of docosahexaenoic acid and eicosapentaenoic acid in the fish oil is not less than 30 wt%. Optionally, the total content of docosahexaenoic acid and eicosapentaenoic acid in the fish oil is not less than 50 wt%. Optionally, the total content of docosahexaenoic acid and eicosapentaenoic acid in the fish oil is not less than 70 wt%.

[0017] Optionally, the antioxidant is selected from one or more of astaxanthin, Haematococcus pluvialis, tocopherol, ascorbyl palmitate, rosemary extract, tea polyphenols, butylated hydroxyanisole, butylated hydroxytoluene, and 2,6-di-tert-butyl-p-cresol.

[0018] This application provides a method for preparing the above-mentioned liposome fish oil formulation for pets, the method comprising the following steps: 1) Dissolve phospholipids, fish oil, and antioxidants in an organic solvent to form an oil phase, and dissolve poloxamer 188, pectin, and chitosan in water to form an aqueous phase; 2) The oil phase, aqueous phase, and succinic acid-modified hydroxypropyl methylcellulose are mixed and emulsified to form a primary emulsion; 3) The obtained colostrum is subjected to high-pressure homogenization to form a nanoliposome emulsion; 4) Remove the organic solvent from the nanoliposome emulsion to obtain the liposome fish oil formulation for pets; Optionally, the pressure of the high-pressure homogenization process in step 3) is 10,000 to 25,000 psi; Optionally, the high-pressure homogenization process in step 3) is repeated 3 to 7 times; Optionally, in step 2), while the oil phase is slowly added dropwise to the aqueous phase under high-speed shearing, succinic acid-modified hydroxypropyl methylcellulose is also slowly added to the aqueous phase. Optionally, in step 2), while the oil phase is slowly added dropwise to the aqueous phase under high-speed shearing at 10000±2000rpm, succinic acid-modified hydroxypropyl methylcellulose is also slowly added to the aqueous phase. Optionally, in step 2), while the oil phase is slowly added dropwise to the aqueous phase at a high-speed shearing speed of 10000±2000 rpm, succinic acid-modified hydroxypropyl methylcellulose is slowly added to the aqueous phase, and shearing is continued for 5~15 min. Optionally, the organic solvent is selected from one or more alcohol solvents; Optionally, the organic solvent is ethanol; Optionally, step 3) involves ultrasonic treatment following high-pressure homogenization. Optionally, the ultrasonic treatment conditions are: 200~600W for 5~15 minutes; Optionally, in step 4), the organic solvent is removed by rotary evaporation, thin-film evaporation, or freeze-drying.

[0019] This application provides the use of a pet liposomal fish oil formulation in the preparation of health products or functional foods for the prevention or adjunctive treatment of pet arthritis, skin diseases, cognitive impairment or chronic kidney disease, wherein the pet liposomal fish oil formulation is the aforementioned pet liposomal fish oil formulation. Optionally, the pet is a dog or a cat; This application provides the above-mentioned pet liposome fish oil formulation provided by the present invention for the preparation of products for the prevention or adjunctive treatment of any of the following canine and feline health problems: 1) Pain, stiffness, and inflammation caused by osteoarthritis; 2) Allergic skin conditions, dry skin, itching, dandruff, and dull hair; 3) Cognitive impairment, including cognitive impairment syndrome in older dogs and cats (CCDS); 4) Chronic kidney disease and cardiovascular health support; Optionally, the product may be a liquid oral preparation, a soft capsule, or a lyophilized powder.

[0020] Those skilled in the art can also use the prepared liposome fish oil emulsion as the contents, and encapsulate it with a capsule made of gelatin, glycerin and water using conventional soft capsule compression technology to make fish-shaped soft capsules with each contents of, for example, 0.5g, which is more convenient for quantitative feeding and storage.

[0021] A freeze-drying protectant, such as mannitol or trehalose, can be added during the aqueous phase preparation. The resulting liposome fish oil emulsion is then dispensed into freeze-drying trays, rapidly cooled to below -45°C and kept at that temperature. Under high vacuum (<10 Pa), the temperature is slowly increased from -45°C to -25°C and kept at that temperature for an extended period to remove free water. The temperature is then further slowly increased to 25~30°C and kept at that temperature for an extended period to remove bound water, with a target moisture content of ≤3.0%. After drying, high-purity nitrogen is introduced to ventilate the air, and the product is immediately sealed and packaged in a low-humidity environment.

[0022] The beneficial effects of this application include, but are not limited to: 1. The pet liposomal fish oil preparation of this application innovatively introduces liposome technology into the field of pet nutrition. The prepared pet liposomal fish oil preparation can be directly absorbed by intestinal cells through membrane fusion and endocytosis without relying on lipase decomposition, thereby avoiding the loss in the traditional digestion process and greatly improving absorption efficiency. In addition, the fish oil is encapsulated inside the liposomes, thus blocking the emission of fishy smell, making it highly palatable and easier for pets to accept.

[0023] 2. According to the pet liposome fish oil formulation of this application, since the gastric acid pH value of dogs and cats is 1-2 when they are fasting, it is highly acidic and very easy to cause fatty acid molecules to hydrolyze and their structure to be destroyed. The addition of succinic acid-modified hydroxypropyl methylcellulose, combined with poloxamer 188 and chitosan, can better strengthen the phospholipid bilayer structure, thereby more effectively isolating oxygen, free radicals and gastric acid. It plays an important role in protecting Omega-3 fatty acids from oxidation and hydrolysis during storage and digestion, and ensuring the activity of the ingredients.

[0024] 3. According to the pet liposome fish oil formulation of this application, the preparation method of succinic acid modified hydroxypropyl methylcellulose was optimized and screened. By controlling the amount of hydroxypropyl methylcellulose, succinic anhydride and anhydrous sodium acetate and the reaction temperature, the prepared succinic acid modified hydroxypropyl methylcellulose showed the best improvement when applied to the pet liposome fish oil formulation of this application.

[0025] 4. The pet liposomal fish oil formulation according to this application has the advantage that, due to the natural targeting properties of liposomes, nutrients can be delivered to the target tissues more effectively. In addition, because the structure of liposomes is metabolized more slowly in the body, it helps to maintain the concentration of Omega-3 in the blood at a more stable and sustained level. Moreover, phospholipids themselves are an important component of cell membranes, which has additional benefits for maintaining liver health and lipid metabolism. Therefore, it is particularly suitable for pets with weak digestive function (senior, young, and sick pets) and pets that refuse to eat traditional fish oil. Attached Figure Description

[0026] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings: Figure 1 This is a schematic diagram comparing the absorption pathways of the pet liposome fish oil formulation involved in this application with those of traditional fish oil. Figure 2 This is a schematic diagram illustrating the mechanism by which the liposome fish oil formulation for pets involved in this application releases its contents into cells through endocytosis and membrane fusion. Detailed Implementation

[0027] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments. Unless otherwise specified, the raw materials and reagents in the embodiments of the present application are all purchased through commercial channels, or can be prepared or prepared by existing technology. For example, short-chain pectin can be prepared in the manner disclosed in patent 202310252342.1.

[0028] like Figure 1 The image shows a comparison between the liposome fish oil formulation for pets in this application and the traditional fish oil absorption pathway. Figure 2 The liposome fish oil formulation of this application releases its contents into cells through endocytosis and membrane fusion. Compared with traditional fish oil formulations, the liposome fish oil formulation provided by this application has better bioavailability and is especially suitable for pets.

[0029] The present application solution will be described below through specific embodiments.

[0030] Example 1 A liposomal fish oil formulation for pets is provided, comprising, by weight, 30 parts of fish oil (Sinopharm Chemical Reagent Co., Ltd., catalog number u1270424), 40 parts of hydrogenated lecithin (Dalian Meilun Biotechnology Co., Ltd., catalog number MB2617-1, HSPC high-purity hydrogenated soybean lecithin), 6 parts of succinic acid-modified hydroxypropyl methylcellulose, 6 parts of short-chain pectin with a molecular weight of 3000-5000, 2 parts of poloxamer 188 (Beijing Boao Tuoda Technology Co., Ltd., catalog number Q60131G), 0.7 parts of chitosan (Tuoran Biotechnology, catalog number C2167), and 4 parts of antioxidant.

[0031] Preparation of succinic acid-modified hydroxypropyl methylcellulose: Hydroxypropyl methylcellulose (Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number H108815) was added to 100 mL of acetic acid solvent and heated to 65 °C. The mixture was stirred until completely dissolved. After heating to 78 °C, succinic anhydride and anhydrous sodium acetate catalyst were added for esterification. The reaction was carried out in a reflux apparatus. After 3 hours of reaction, 10 mL of deionized water was added to terminate the esterification reaction. The reactants were added to 1000 mL of deionized water and stirred to obtain a white precipitate. After filtration, the precipitate was washed with deionized water at least three times, dried, and pulverized to obtain succinic acid-modified hydroxypropyl methylcellulose. The ratio of hydroxypropyl methylcellulose, succinic anhydride, and anhydrous sodium acetate was 1:1.5:0.3. The preparation of liposome fish oil formulations for pets includes the following steps: 1) Prepare the oil phase: Weigh soybean lecithin, high-concentration fish oil, and tocopherol, dissolve them in 200 mL of anhydrous ethanol, and heat and stir in a 50 °C water bath until completely dissolved; 2) Prepare the aqueous phase: Weigh 300mL of purified water, heat to 50℃, add poloxamer 188, pectin and chitosan and stir until completely dissolved; 3) Emulsification and homogenization: Under high-speed shearing at 10,000 rpm, the oil phase was slowly added dropwise to the aqueous phase, while succinic acid modified hydroxypropyl methylcellulose was slowly added to the aqueous phase. The shearing was continued for 10 minutes to obtain the pre-emulsion. The pre-emulsion was transferred to a high-pressure homogenizer and homogenized 5 times under a pressure of 20,000 psi. 4) Post-processing: The homogenized emulsion is ultrasonically treated for 10 minutes (power 400W), and then the ethanol is removed by rotary evaporation at 40℃ to finally obtain liposome fish oil nanoemulsion, which is a liposome fish oil preparation for pets.

[0032] The average particle size was measured to be 158 nm by a laser particle size analyzer, and the PDI was 0.21, indicating uniform particle size distribution.

[0033] Example 2 A liposomal fish oil formulation for pets is provided, comprising, by weight, 10 parts fish oil (China National Pharmaceutical Group Chemical Reagent Co., Ltd., item number u1270424), 20 parts hydrogenated lecithin (Dalian Meilun Biotechnology Co., Ltd., item number MB2617-1, HSPC high-purity hydrogenated soybean lecithin), 4 parts succinic acid-modified hydroxypropyl methylcellulose, 4 parts short-chain pectin with a molecular weight of 3000-5000, 1 part poloxamer 188 (Beijing Boao Tuoda Technology Co., Ltd., item number Q60131G), 0.5 parts chitosan (Tuoran Biotechnology, item number C2167), and 0.1 parts antioxidant.

[0034] Preparation of succinic acid-modified hydroxypropyl methylcellulose: Hydroxypropyl methylcellulose (Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number H108815) was added to 100 mL of acetic acid solvent and heated to 65 °C. The mixture was stirred until completely dissolved. After heating to 75 °C, succinic anhydride and anhydrous sodium acetate catalyst were added for esterification. The reaction was carried out in a reflux apparatus. After 3 hours of reaction, 10 mL of deionized water was added to terminate the esterification reaction. The reactants were added to 1000 mL of deionized water and stirred to obtain a white precipitate. After filtration, the precipitate was washed with deionized water at least three times, dried, and pulverized to obtain succinic acid-modified hydroxypropyl methylcellulose. The ratio of hydroxypropyl methylcellulose, succinic anhydride, and anhydrous sodium acetate was 1:1.3:0.2. The preparation of liposome fish oil formulations for pets includes the following steps: 1) Prepare the oil phase: Weigh soybean lecithin, high-concentration fish oil, and astaxanthin, dissolve them in 200mL of anhydrous ethanol, and heat and stir in a 50℃ water bath until completely dissolved; 2) Prepare the aqueous phase: Weigh 300mL of purified water, heat to 50℃, add poloxamer 188, pectin and chitosan and stir until completely dissolved; 3) Emulsification and homogenization: Under high-speed shearing at 10,000 rpm, the oil phase was slowly added dropwise to the aqueous phase, while succinic acid modified hydroxypropyl methylcellulose was slowly added to the aqueous phase. The shearing was continued for 10 minutes to obtain the pre-emulsion. The pre-emulsion was then transferred to a high-pressure homogenizer and homogenized 5 times under a pressure of 10,000 psi. 4) Post-processing: The homogenized emulsion is ultrasonically treated for 10 minutes (power 400W), and then the ethanol is removed by rotary evaporation at 40℃ to finally obtain liposome fish oil nanoemulsion, which is a liposome fish oil preparation for pets.

[0035] The average particle size was measured to be 143 nm by a laser particle size analyzer, and the PDI was 0.23, indicating uniform particle size distribution.

[0036] Example 3 A liposomal fish oil formulation for pets is provided, comprising, by weight, 50 parts of fish oil (Sinopharm Chemical Reagent Co., Ltd., catalog number u1270424), 60 parts of hydrogenated lecithin (Dalian Meilun Biotechnology Co., Ltd., catalog number MB2617-1, HSPC high-purity hydrogenated soybean lecithin), 8 parts of succinic acid-modified hydroxypropyl methylcellulose, 8 parts of short-chain pectin with a molecular weight of 3000-5000, 3 parts of poloxamer 188 (Beijing Bio-Tech Technology Co., Ltd., catalog number Q60131G), 1 part of chitosan (Tuoran Biotechnology, catalog number C2167), and 5 parts of antioxidant.

[0037] Preparation of succinic acid-modified hydroxypropyl methylcellulose: Hydroxypropyl methylcellulose (Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number H108815) was added to 100 mL of acetic acid solvent and heated to 65 °C. The mixture was stirred until completely dissolved. After heating to 80 °C, succinic anhydride and anhydrous sodium acetate catalyst were added for esterification. The reaction was carried out in a reflux apparatus. After 3 hours of reaction, 10 mL of deionized water was added to terminate the esterification reaction. The reactants were added to 1000 mL of deionized water and stirred to obtain a white precipitate. After filtration, the precipitate was washed with deionized water at least 3 times, dried, and pulverized to obtain succinic acid-modified hydroxypropyl methylcellulose. The ratio of hydroxypropyl methylcellulose, succinic anhydride, and anhydrous sodium acetate was 1:1.6:0.4. The preparation of liposome fish oil formulations for pets includes the following steps: 1) Prepare the oil phase: Weigh soybean lecithin, high-concentration fish oil, and ascorbate palmitate, dissolve them in 200 mL of anhydrous ethanol, and heat and stir in a 50 °C water bath until completely dissolved; 2) Prepare the aqueous phase: Weigh 300mL of purified water, heat to 50℃, add poloxamer 188, pectin and chitosan and stir until completely dissolved; 3) Emulsification and homogenization: Under high-speed shearing at 10,000 rpm, the oil phase was slowly added dropwise to the aqueous phase, while succinic acid modified hydroxypropyl methylcellulose was slowly added to the aqueous phase. The shearing was continued for 10 minutes to obtain the pre-emulsion. The pre-emulsion was transferred to a high-pressure homogenizer and homogenized 5 times under a pressure of 25,000 psi. 4) Post-processing: The homogenized emulsion is ultrasonically treated for 10 minutes (power 400W), and then the ethanol is removed by rotary evaporation at 40℃ to finally obtain liposome fish oil nanoemulsion, which is a liposome fish oil preparation for pets.

[0038] The average particle size was measured to be 172 nm by a laser particle size analyzer, and the PDI was 0.25, indicating uniform particle size distribution.

[0039] Example 4 This embodiment is basically the same as Example 1, except that the ratio of hydroxypropyl methylcellulose, succinic anhydride and anhydrous sodium acetate is 1:1:0.6.

[0040] Example 5 This embodiment is basically the same as that of Example 1, except that the ratio of hydroxypropyl methylcellulose, succinic anhydride and anhydrous sodium acetate is 1:2:0.5.

[0041] Example 6 This embodiment is basically the same as Example 1, except that the esterification reaction temperature in the preparation of succinic acid modified hydroxypropyl methylcellulose is 72℃.

[0042] Example 7 This embodiment is basically the same as Example 1, except that the esterification reaction temperature in the preparation of succinic acid modified hydroxypropyl methylcellulose is 83℃.

[0043] Example 8 This embodiment is basically the same as Example 1, except that the pressure of the high-pressure homogenization process in the preparation of pet liposome fish oil formulation is 8000 psi.

[0044] Example 9 This embodiment is basically the same as Example 1, except that the pressure of the high-pressure homogenization process in the preparation of pet liposome fish oil formulation is 27000 psi.

[0045] Comparative Example 1 This comparative example is basically the same as Example 1, except that it does not contain chitosan.

[0046] Comparative Example 2 This comparative example is basically the same as Example 1, except that it does not contain poloxamer 188.

[0047] Comparative Example 3 This comparative example is basically the same as Example 1, except that it does not contain succinic acid-modified hydroxypropyl methylcellulose.

[0048] Comparative Example 4 The comparative example is basically the same as Example 1, except that it does not contain short-chain pectin.

[0049] Comparative Example 5 This comparative example is basically the same as Example 1, except that hydrogenated lecithin is replaced with ordinary soybean lecithin.

[0050] Test Example 1: Bioavailability Comparison Experiment Sixty beagle dogs were randomly divided into 15 groups. The experimental groups were orally administered the products obtained in Examples 1-9 and Comparative Examples 1-5 (oral dose of 50 mg EPA+DHA / kg body weight), while the control groups were orally administered the same dose (based on EPA+DHA content, dose of 50 mg EPA+DHA / kg body weight) of conventional EE-type fish oil. Blood samples were collected before administration and at 0, 2, 4, 6, 8, 12, and 24 hours after administration to measure the plasma EPA+DHA content (ω-3 index). Curves showing the change in blood drug concentration over time were plotted, and the time to peak concentration of EPA+DHA (T0) was calculated. max ) and peak concentration (C max ), calculate the area under the plasma concentration-time curve (AUC) of EPA+DHA from 0 to 24 hours. 0-24h The changes in the experimental group compared to the control group were calculated using the control group as the standard, and the results are shown in Table 1 below.

[0051] Table 1. Relative results (times) of the experimental group compared to the control group.

[0052] The results showed that the peak time of EPA+DHA in the experimental group (T) max The peak concentration (C) was shorter within 2-4 hours compared to the control group (around 6 hours). max The plasma concentration-time area (AUC) was higher than that of the control group, with a higher concentration over 0-24 hours. 0-24h The EPA+DHA ratio was significantly higher than that of the control group, indicating that the relative bioavailability of the liposomal fish oil formulation provided in this application is significantly better than that of traditional fish oil.

[0053] Further analysis of the results of Comparative Examples 1-3 and Example 1 in Table 1 shows that the introduction of the phospholipid bilayer structure has a certain improvement on the relative bioavailability of EPA+DHA, and the combination with succinic acid-modified hydroxypropyl methylcellulose has a significant improvement effect. In addition, the addition of poloxamer 188 and chitosan also has a certain auxiliary effect.

[0054] Furthermore, researchers found that the amounts of hydroxypropyl methylcellulose, succinic anhydride, and anhydrous sodium acetate, as well as the reaction temperature, all affect the structure of the prepared succinic acid-modified hydroxypropyl methylcellulose, thus impacting its performance in pet liposome fish oil formulations. In the preparation of pet liposome fish oil formulations, the pressure of the high-pressure homogenization process affects the size and uniformity of the liposome particles. The liposomes obtained by the preparation process in this application have a particle size range of approximately 100-200 nm, which is more conducive to improving the absorption effect of EPA+DHA.

[0055] Test Example 2: Palatability Comparison Experiment To verify the advantages of the liposomal fish oil formulation provided in this application in masking fishy odor and improving pet acceptance, 30 picky domestic cats with a history of refusing traditional fish oil were selected for a two-day dual-bowl test. Bowl A: 50g of basic wet food + liposomal fish oil from Example 1 (containing 50 mg of EPA + DHA); Bowl B: 50g of basic wet food + traditional TG fish oil (containing 50 mg of EPA + DHA). The food bowl positions were switched daily, and the amount of food consumed was recorded 30 minutes after feeding. The feed intake rate and first-choice rate were calculated and statistically analyzed. The results are shown in Table 2 below.

[0056] Table 2. Results of the double-bowl palatability test

[0057] The results in Table 2 show that the liposome fish oil formulation provided in this application can effectively improve palatability, and its palatability is significantly better than that of traditional fish oil, which can effectively solve the problem of picky pets refusing to eat.

[0058] Test Example 3: Verification Experiment of Gastric Acid Resistance Further, by simulating the strong acid environment of fasting stomachs in dogs and cats, the stability of the liposome fish oil formulation of this application and traditional liposome fish oil under gastric acid conditions was quantitatively evaluated and compared. The encapsulation retention rate was used as the core indicator to verify the product's tolerance to the strong acid environment in pets. The encapsulation retention rate (%) = (encapsulation rate after incubation / encapsulation rate before incubation) * 100%. The calculation process for the encapsulation rate (%) included: separating unencapsulated free fish oil by ultrafiltration centrifugation, and determining the EPA+DHA content separately using high-performance liquid chromatography, denoted as C. free The total EPA+DHA content in the sample is denoted as C. total The encapsulation rate is calculated using the following formula: Encapsulation rate (%) = (C total -C free ) / C total *100%. The encapsulation rate before incubation is the encapsulation rate of the sample before incubation, and the encapsulation rate after incubation is the encapsulation rate of the product after the incubation experiment.

[0059] A hydrochloric acid-pepsin solution (containing 2 mg / mL pepsin) with a pH of 1.5 was prepared to simulate the gastric fluid environment of dogs and cats in an empty stomach. Incubation conditions: 10 mL of each of the two test samples (the experimental group was the preparation from Example 1, and the control group was traditional EE-type fish oil) was mixed with 90 mL of gastric acid-simulating solution preheated to 37°C, for a total volume of 100 mL. The mixture was then placed in a 37°C water bath shaker and incubated at 100 rpm for 2 hours to simulate the residence time of food in the stomach. After incubation, the samples were immediately removed, and the pH of the mixture was quickly adjusted to neutral using alkaline buffer to terminate pepsin activity.

[0060] Both the experimental group and the control group were divided into three parallel experiments, and the results are shown in Table 3 below.

[0061] Table 3 Results of the simulated gastric acid stability experiment for the experimental and control groups

[0062] As shown in Table 3, the product of this application is structurally stable in a strong gastric acid environment, and most of the active ingredients are well protected. After incubation for 2 hours under harsh conditions of pH 1.5, the encapsulation rate is maintained at over 90%, and the particle size and PDI do not change significantly, indicating that its phospholipid bilayer structure is intact and can effectively resist gastric acid erosion. In contrast, traditional products have poor stability in strong gastric acid, with over 30% of the active ingredients leaking due to liposome destruction. The product is slightly turbid after incubation and contains a small amount of flocculent matter, indicating that its structure is easily damaged, leading to premature leakage of a large amount of fish oil.

[0063] Test Example 4: Stability Acceleration Experiment The oxidative stability of the liposome fish oil formulation of this application under harsh conditions was further evaluated, and its shelf life was predicted. Samples (experimental group: formulation of Example 1 of this application; control group: traditional fish oil emulsion without liposome encapsulation) were placed in a constant temperature and humidity chamber at 40°C and 75% relative humidity for one month (approximately equivalent to 6-12 months of storage at room temperature). Samples were taken at 0, 15, and 30 days for testing. Peroxide value (PV) was used to measure primary oxidation products, anisidine value (AV) was used to measure secondary oxidation products, and total oxidation value (TOTOX) was a comprehensive oxidation index. TOTOX = 2PV + AV. The results are shown in Table 4 below.

[0064] Table 4. Results of Accelerated Stability Tests

[0065] As shown in Table 4, the oxidation indicators of the liposome fish oil formulation of this application are significantly lower than those of traditional fish oil. This indicates that the phospholipid bilayer plays a good protective role for the fish oil, effectively delaying the oxidative rancidity process, and indicating that it has a longer shelf life and higher storage stability.

[0066] The above description is merely an embodiment of this application, and the scope of protection of this application is not limited to these specific embodiments, but is determined by the claims of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principles of this application should be included within the scope of protection of this application.

Claims

1. A method for preparing a liposome fish oil formulation for pets, characterized in that, The pet liposome fish oil formulation, by weight, comprises: 10-50 parts fish oil, 20-60 parts hydrogenated lecithin, 4-8 parts succinic acid-modified hydroxypropyl methylcellulose, 4-8 parts short-chain pectin with a molecular weight of 3000-5000, 1-3 parts poloxamer 188, 0.5-1 part chitosan, and 0.1-5 parts antioxidant; the pet liposome fish oil formulation comprises liposomes formed by fish oil encapsulated in a phospholipid bilayer. The method includes the following steps: 1) dissolving phospholipids, fish oil, and antioxidants in an organic solvent to form an oil phase, and dissolving poloxamer 188, short-chain pectin, and chitosan in water to form an aqueous phase; 2) mixing and emulsifying the oil phase, aqueous phase, and succinic acid-modified hydroxypropyl methylcellulose to form a primary emulsion; 3) subjecting the obtained primary emulsion to high-pressure homogenization at a pressure of 10,000 to 25,000 psi to form a nanoliposome emulsion; 4) removing the organic solvent from the nanoliposome emulsion to obtain the pet liposome fish oil formulation. The preparation method of the succinic acid modified hydroxypropyl methylcellulose includes the following steps: S1, adding hydroxypropyl methylcellulose to acetic acid solvent and heating and stirring to dissolve; S2, adding succinic anhydride and anhydrous sodium acetate catalyst and then carrying out an esterification reaction; S3, adding deionized water to terminate the reaction, and obtaining the succinic acid modified hydroxypropyl methylcellulose after separation and drying.

2. The method for preparing a liposome fish oil formulation for pets according to claim 1, characterized in that, The ratio of hydroxypropyl methylcellulose, succinic anhydride and anhydrous sodium acetate is 1:(1.3~1.6):(0.2~0.4).

3. The method for preparing a liposome fish oil formulation for pets according to claim 2, characterized in that, The ratio of hydroxypropyl methylcellulose, succinic anhydride and anhydrous sodium acetate is 1:1.5:0.

3.

4. The method for preparing a liposome fish oil formulation for pets according to claim 1, characterized in that, The molecular weight of the hydroxypropyl methylcellulose is 10,000 to 50,000.

5. The method for preparing a liposome fish oil formulation for pets according to claim 1, characterized in that, The esterification reaction in step S2 is carried out at a temperature of 75-80°C.

6. The method for preparing a liposome fish oil formulation for pets according to claim 5, characterized in that, The esterification reaction in step S2 is carried out at a temperature of 78°C.

7. The method for preparing a liposome fish oil formulation for pets according to claim 1, characterized in that, The fish oil contains docosahexaenoic acid and eicosapentaenoic acid.

8. The method for preparing a liposome fish oil formulation for pets according to claim 7, characterized in that, The total content of docosahexaenoic acid and eicosapentaenoic acid in the fish oil is not less than 30 wt%.

9. The method for preparing a liposome fish oil formulation for pets according to claim 1, characterized in that, The antioxidant is selected from one or more of astaxanthin, Haematococcus pluvialis, tocopherol, ascorbyl palmitate, rosemary extract, tea polyphenols, butylated hydroxyanisole, and 2,6-di-tert-butyl-p-cresol.

10. A liposome fish oil preparation for pets obtained by the preparation method according to any one of claims 1 to 9.

11. The application of a liposomal fish oil formulation for pets in the preparation of functional foods for the prevention or adjunctive treatment of arthritis, skin diseases, cognitive impairment, or chronic kidney disease in pets, characterized in that... The liposomal fish oil formulation for pets is the liposomal fish oil formulation for pets as described in claim 10.

12. The application according to claim 11, characterized in that, The pet is either a dog or a cat.

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