Compound pet food antioxidant containing plant polyphenol compounds and preparation method of compound pet food antioxidant

Abstract

The invention relates to the technical field of pet food antioxidants, in particular to a compound pet food antioxidant containing plant polyphenol compounds and a preparation method of the compound pet food antioxidant. 10 to 20 parts of a grape seed extract; 8 to 15 parts of a rosemary extract; 5 to 10 parts of L-ascorbyl palmitate; 5 to 10 parts of alpha-tocopheryl acetate; 3-8 parts of citric acid; 8 to 15 parts of soybean lecithin; 20 to 30 parts of medium chain triglyceride; eGCG (epigallocatechin gallate) in the green tea contains a plurality of o-dihydroxyl structures, the grape seed procyanidine has a unique C6-C3-C6 skeleton, and carnosic acid in the rosemary belongs to a diterpenoid compound. Due to the diversity of the structure, the compound can play a role in different types of free radicals and oxidation reactions, and a broad-spectrum anti-oxidation network is formed.

Description

Technical Field

[0001] The invention relates to the technical field of pet food antioxidants, and in particular to a composite pet food antioxidant containing plant polyphenol compounds and a preparation method thereof. Background Art

[0002] In recent years, as people pay more and more attention to the health of pets, the quality and safety of pet food have become the focus of the industry. Among them, antioxidants, as important additives in pet food, play a key role in extending the shelf life, maintaining nutritional value and improving sensory quality. However, existing pet food antioxidants still have many problems that need to be solved urgently.

[0003] Although traditional synthetic antioxidants such as BHA and BHT are effective, they may pose safety risks when used for a long time and have been gradually eliminated from the market. Natural antioxidants such as vitamin E and rosemary extract are highly safe, but they often perform poorly in complex food systems and are difficult to meet the needs of long-term storage of pet food. In addition, plant extracts from a single source can usually only target specific types of oxidation reactions and are difficult to provide comprehensive protection.

[0004] At present, the technical solution commonly used in the industry is to simply mix a variety of natural antioxidants. However, this method ignores the possible interactions between different antioxidant ingredients and makes it difficult to fully utilize the potential of each component. At the same time, due to the lack of effective protection and delivery systems, these antioxidants are easily inactivated or degraded during the processing, storage and digestion of pet food, resulting in poor practical application results. Summary of the invention

[0005] In view of the above problems, the present invention proposes an innovative composite pet food antioxidant and a preparation method thereof. The core of the solution is to achieve a qualitative leap in antioxidant effect through a carefully designed multiple synergistic mechanism.

[0006] The object of the present invention is to provide a composite pet food antioxidant containing plant polyphenol compounds, which comprises the following components by weight:

[0007] Green tea extract 15-25 parts;

[0008] Grape seed extract 10-20 parts;

[0009] 8-15 parts of rosemary extract;

[0010] 5-10 parts of L-ascorbyl palmitate;

[0011] 5-10 parts of alpha-tocopherol acetate;

[0012] 3-8 parts of citric acid;

[0013] 8-15 parts of soy lecithin;

[0014] Medium chain triglycerides 20-30 parts;

[0015] 5-10 parts of β-cyclodextrin.

[0016] Preferably, the content of (-)-epigallocatechin gallate (EGCG) in the green tea extract is ≥45%.

[0017] Preferably, the content of proanthocyanidins in the grape seed extract is ≥95%.

[0018] Preferably, the content of carnosic acid in the rosemary extract is ≥30%.

[0019] The preparation method of the composite pet food antioxidant comprises the following steps:

[0020] (1) preparing β-cyclodextrin inclusion complex;

[0021] (2) preparing liposomes;

[0022] (3) Mixing the β-cyclodextrin inclusion complex obtained in step (1) with the liposome obtained in step (2) to obtain a final preparation.

[0023] Preferably, step (1) comprises:

[0024] (1.1) Dissolve β-cyclodextrin in deionized water at 50-60°C;

[0025] (1.2) dissolving green tea extract and grape seed extract in a 50% ethanol aqueous solution at 40-50° C.;

[0026] (1.3) Add the polyphenol solution dropwise into the β-cyclodextrin solution and stir for 2-3 hours;

[0027] (1.4) Let the mixture stand at 4°C for 12-24 hours;

[0028] (1.5) The obtained suspension is freeze-dried to obtain β-cyclodextrin inclusion compound powder.

[0029] Preferably, step (2) comprises:

[0030] (2.1) heating and melting medium chain triglycerides at 50-60°C;

[0031] (2.2) adding α-tocopheryl acetate and rosemary extract to the melted medium chain triglycerides;

[0032] (2.3) Add soybean lecithin and stir at 60-70°C for 30-40 minutes;

[0033] (2.4) Dissolve L-ascorbyl palmitate and citric acid in deionized water at 40-50°C;

[0034] (2.5) Slowly add the water phase into the oil phase to form a preliminary emulsified system;

[0035] (2.6) The mixture is homogenized using a high-pressure homogenizer for 3 to 5 cycles to obtain a liposome dispersion.

[0036] Preferably, step (3) comprises:

[0037] (3.1) Add β-cyclodextrin inclusion compound powder to the liposome dispersion and stir at room temperature for 1-2 hours;

[0038] (3.2) subjecting the mixture to ultrasonic treatment;

[0039] (3.3) Filter through a 60-100 mesh sieve;

[0040] (3.4) Fill under nitrogen protection and store at 4℃.

[0041] Preferably, the pressure of the high-pressure homogenization treatment is 500-800 bar.

[0042] Preferably, the ultrasonic treatment is performed at a power of 300-500 W and for a time of 10-15 minutes.

[0043] Specifically, the present invention achieves a technical breakthrough in the following ways:

[0044] First, the present invention selects green tea extract, grape seed extract and rosemary extract as the main sources of polyphenols. The active ingredients in these three extracts have significant differences in molecular structure: EGCG in green tea contains multiple ortho-dihydroxy structures, grape seed proanthocyanidins have a unique C6-C3-C6 skeleton, and carnosic acid in rosemary belongs to diterpenoid compounds. This structural diversity enables them to play a role in different types of free radicals and oxidation reactions, forming a broad-spectrum antioxidant network.

[0045] Secondly, the present invention cleverly introduces the β-cyclodextrin inclusion technology. As a cyclic oligosaccharide, β-cyclodextrin has the characteristics of internal hydrophobicity and external hydrophilicity, which enables it to form a stable inclusion complex with polyphenol compounds. This process not only improves the solubility and stability of polyphenols, but also prolongs their action time by controlling release. From the perspective of molecular orbital theory, the host-guest complex formed between β-cyclodextrin and polyphenols may involve multiple non-covalent bond interactions such as hydrogen bonds and hydrophobic interactions. This multiple interaction further enhances the stability of the complex.

[0046] Again, the present invention uses liposome technology to encapsulate fat-soluble antioxidants such as α-tocopheryl acetate and L-ascorbyl palmitate. Liposomes are composed of phospholipid bilayers, and their structure is similar to that of biological membranes. They can not only improve the dispersibility of these fat-soluble components in the aqueous phase, but also promote their absorption at the cellular level. From the perspective of chemical kinetics, the presence of liposomes may change the release kinetics of these antioxidants and achieve a sustained-release effect.

[0047] In addition, the present invention also introduces citric acid as a metal chelating agent and a pH regulator. The three carboxyl groups and one hydroxyl group in the citric acid molecule enable it to form a stable chelate with a polyvalent metal ion, thereby inhibiting the oxidation reaction catalyzed by the metal ion. At the same time, the presence of citric acid can also maintain the weakly acidic environment of the system, which is crucial to the stability of the polyphenol compound.

[0048] Finally, the present invention adopts a unique preparation process, including the steps of β-cyclodextrin inclusion, liposome preparation and ultrasonic homogenization. This multi-step preparation method not only ensures the full combination of each component, but also may further improve the dispersibility and bioavailability of the antioxidant through physical effects (such as ultrasonic cavitation effect).

[0049] Through the above-mentioned multiple synergistic mechanisms, the present invention successfully solves many problems existing in traditional pet food antioxidants. Experimental results show that the composite antioxidant exhibits excellent antioxidant properties in various types of pet foods, not only effectively inhibiting lipid oxidation, but also reducing protein degradation and microbial growth, achieving all-round quality protection. In particular, in long-term storage experiments under high temperature and high humidity conditions, the antioxidant of the present invention exhibits stability and durability far beyond expectations.

[0050] What is even more surprising is that the present invention also shows some unexpected technical effects. For example, in the storage experiment of pet snacks, the samples added with the antioxidant of the present invention not only maintained good physical and chemical indicators, but also performed well in terms of sensory quality. This discovery provides a new idea for improving the palatability of pet food. In addition, by analyzing the changing trends of oxidation indicators under different conditions, it is found that it is possible to establish an accurate shelf life prediction model, which will provide a powerful tool for quality control of pet food manufacturers.

[0051] In summary, the present invention has successfully developed a highly efficient, safe and multifunctional composite pet food antioxidant through multiple synergistic mechanisms and innovative preparation processes. This technology not only solves many problems in the prior art, but also provides a new direction for quality improvement and product innovation in the pet food industry. It can be foreseen that with the application and promotion of this technology, it will bring revolutionary changes to the pet food industry and make important contributions to improving the quality of life and health level of pets. DETAILED DESCRIPTION

[0052] The composite pet food antioxidant containing plant polyphenol compounds of the present invention comprises the following components by weight: 15-25 parts of green tea extract, main active ingredient: (-)-epigallocatechin gallate (EGCG), content: ≥45% (calculated as EGCG), trade name: 90DCF Manufacturer: Taiyo International, Inc.; Grape seed extract 10-20 parts, main active ingredient: proanthocyanidins, content: ≥95% (calculated as proanthocyanidins), trade name: Manufacturer: Polyphenolics, Inc.; Rosemary extract 8-15 parts Main active ingredient: carnosic acid, content: ≥30% (calculated as carnosic acid) Trade name: Manufacturer: Vitivad.d.; 5-10 parts of L-ascorbyl palmitate; Trade name: Manufacturer: DSM Nutritional Products, α-tocopheryl acetate 5-10 parts, content: ≥98%, trade name: F-1000, manufacturer: Cognis (now part of BASF); 3-8 parts of citric acid; 8-15 parts of soy lecithin; trade name: Lipoid S100 manufacturer: Lipoid GmbH; 20-30 parts of medium chain triglycerides, trade name: Manufacturer: Abitec Corporation; β-cyclodextrin 5-10 parts, content: ≥98%, trade name: W7PHARMA, manufacturer: Wacker Chemie AG.

[0053] This compound pet food antioxidant achieves efficient, stable and safe antioxidant performance through the synergistic effect of multiple innovative points:

[0054] 1. Multi-source plant extracts (green tea, grape seed, rosemary) provide a variety of polyphenolic compounds, forming a synergistic antioxidant effect. Green tea EGCG mainly works by scavenging free radicals and chelating metal ions, grape seed proanthocyanidins can effectively inhibit lipid peroxidation, and carnosic acid in rosemary extract has good thermal stability.

[0055] 2. β-cyclodextrin inclusion technology improves the stability and bioavailability of water-soluble polyphenol compounds and can control the release rate.

[0056] 3. Liposome technology not only improves the dispersibility of fat-soluble ingredients (such as α-tocopherol acetate and rosemary extract), but also increases their cellular uptake rate.

[0057] 4. L-ascorbyl palmitate, as a fat-soluble vitamin C, can play an antioxidant role in the oil phase, and at the same time has the ability to regenerate α-tocopherol and prolong its action time.

[0058] 5. α-Tocopherol acetate, as a fat-soluble antioxidant, forms complementary protection with water-soluble polyphenols, especially playing an important role in the chain termination stage of lipid oxidation.

[0059] 6. Citric acid, as a metal chelator and pH adjuster, can further enhance the antioxidant effect and stabilize the preparation.

[0060] 7. Soy lecithin not only acts as a natural emulsifier to improve the dispersibility and stability of each component, but the phospholipids in it also have antioxidant effects.

[0061] 8. Medium chain triglycerides as carrier oils can not only dissolve fat-soluble components but also improve the bioavailability of polyphenol compounds.

[0062] This composite antioxidant solves the stability problem of polyphenol compounds in application through multiple protection mechanisms and sustained-release systems, and achieves significant improvement in antioxidant performance through multiple synergistic effects. All raw materials meet the safety requirements of pet food additives in China and Europe and can be safely used in various pet foods.

[0063] Example 1

[0064] A composite pet food antioxidant containing plant polyphenol compounds, comprising the following components (by weight): 15 parts of green tea extract, 10 parts of grape seed extract, 8 parts of rosemary extract, 5 parts of L-ascorbyl palmitate, 5 parts of α-tocopheryl acetate, 3 parts of citric acid, 8 parts of soybean lecithin, 20 parts of medium-chain triglycerides, and 5 parts of β-cyclodextrin.

[0065] The preparation method of the composite pet food antioxidant comprises the following steps:

[0066] First, prepare the β-cyclodextrin inclusion complex. Dissolve 5 parts of β-cyclodextrin in deionized water at 50°C with a stirring speed of 300 rpm. At the same time, dissolve 15 parts of green tea extract and 10 parts of grape seed extract in a 50% ethanol aqueous solution at 40°C. Then, under continuous stirring, add the polyphenol solution dropwise to the β-cyclodextrin solution and continue stirring for 2 hours. Next, let the mixture stand at 4°C for 12 hours to allow the inclusion complex to fully form. Finally, freeze-dry the formed inclusion complex suspension at -50°C and 0.1 mbar for 48 hours to obtain a β-cyclodextrin inclusion complex powder.

[0067] Secondly, prepare liposomes. Heat 20 parts of medium-chain triglycerides at 50°C to melt. Subsequently, add 5 parts of α-tocopherol acetate and 8 parts of rosemary extract to the molten medium-chain triglycerides and stir evenly. Add 8 parts of soy lecithin and stir at 60°C for 30 minutes. At the same time, in another container, dissolve 5 parts of L-ascorbyl palmitate and 3 parts of citric acid in deionized water at 40°C. Under continuous stirring (500rpm), the aqueous phase is slowly added to the oil phase to form a preliminary emulsified system. Finally, the mixture is homogenized for 3 cycles using a high-pressure homogenizer (500bar) to obtain a nano-liposome dispersion.

[0068] Finally, prepare the final preparation. Slowly add the above-mentioned β-cyclodextrin inclusion powder to the liposome dispersion and stir at room temperature for 1 hour. Then, use ultrasonic treatment (power 300W, time 10 minutes) to further disperse and homogenize the mixture. Then, filter through a 60-mesh sieve to remove any large particles that may be present. Finally, under nitrogen protection, fill the preparation into a brown glass bottle and store it at 4°C.

[0069] Preferably, in an embodiment of the present invention, the content of EGCG in green tea extract is 45%, the content of proanthocyanidins in grape seed extract is 95%, and the content of carnosic acid in rosemary extract is 30%. This combination can provide a broad spectrum of antioxidant protection, wherein green tea EGCG mainly plays a role by scavenging free radicals and chelating metal ions, grape seed proanthocyanidins can effectively inhibit lipid peroxidation, and carnosic acid in rosemary extract has good thermal stability.

[0070] Example 2

[0071] A composite pet food antioxidant containing plant polyphenol compounds, which is composed of the following (by weight): 25 parts of green tea extract, 20 parts of grape seed extract, 15 parts of rosemary extract, 10 parts of L-ascorbyl palmitate, 10 parts of α-tocopheryl acetate, 8 parts of citric acid, 15 parts of soybean lecithin, 30 parts of medium-chain triglycerides, and 10 parts of β-cyclodextrin.

[0072] The preparation method of the composite pet food antioxidant comprises the following steps:

[0073] First, prepare the β-cyclodextrin inclusion complex. Dissolve 10 parts of β-cyclodextrin in deionized water at 60°C with a stirring speed of 400rpm. At the same time, dissolve 25 parts of green tea extract and 20 parts of grape seed extract in a 50% ethanol aqueous solution at 50°C. Then, under continuous stirring, add the polyphenol solution dropwise to the β-cyclodextrin solution and continue stirring for 3 hours. Next, let the mixture stand at 4°C for 24 hours to allow the inclusion complex to fully form. Finally, freeze-dry the formed inclusion complex suspension at -50°C and 0.1mbar for 72 hours to obtain a β-cyclodextrin inclusion complex powder.

[0074] Secondly, prepare liposomes. Heat 30 parts of medium-chain triglycerides at 60°C to melt. Subsequently, add 10 parts of α-tocopherol acetate and 15 parts of rosemary extract to the molten medium-chain triglycerides and stir evenly. Add 15 parts of soy lecithin and stir at 70°C for 40 minutes. At the same time, in another container, dissolve 10 parts of L-ascorbyl palmitate and 8 parts of citric acid in deionized water at 50°C. Under continuous stirring (600rpm), the aqueous phase is slowly added to the oil phase to form a preliminary emulsified system. Finally, the mixture is homogenized for 5 cycles using a high-pressure homogenizer (800bar) to obtain a nano-liposome dispersion.

[0075] Finally, prepare the final preparation. The above-mentioned β-cyclodextrin inclusion powder is slowly added to the liposome dispersion and stirred at room temperature for 2 hours. Then, ultrasonic treatment (power 500W, time 15 minutes) is used to further disperse and homogenize the mixture. Next, filter through a 100 mesh sieve to remove any large particles that may be present. Finally, under nitrogen protection, the preparation is filled into a brown glass bottle and stored at 4°C.

[0076] In this embodiment, L-ascorbyl palmitate, as a fat-soluble vitamin C, can play an antioxidant role in the oil phase, and at the same time has the ability to regenerate α-tocopherol and prolong its action time. α-tocopherol acetate, as a fat-soluble antioxidant, forms a complementary protection with water-soluble polyphenols, especially playing an important role in the chain termination stage of lipid oxidation.

[0077] Example 3

[0078] A composite pet food antioxidant containing plant polyphenol compounds, comprising the following components (by weight): 20 parts of green tea extract, 15 parts of grape seed extract, 12 parts of rosemary extract, 8 parts of L-ascorbyl palmitate, 8 parts of α-tocopheryl acetate, 6 parts of citric acid, 12 parts of soybean lecithin, 25 parts of medium-chain triglycerides, and 8 parts of β-cyclodextrin.

[0079] The preparation method of the composite pet food antioxidant comprises the following steps:

[0080] First, prepare the β-cyclodextrin inclusion complex. Dissolve 8 parts of β-cyclodextrin in deionized water at 55°C with a stirring speed of 350rpm. At the same time, dissolve 20 parts of green tea extract and 15 parts of grape seed extract in a 50% ethanol aqueous solution at 45°C. Then, under continuous stirring, add the polyphenol solution dropwise to the β-cyclodextrin solution and continue stirring for 2.5 hours. Next, let the mixture stand at 4°C for 18 hours to allow the inclusion complex to fully form. Finally, freeze-dry the formed inclusion complex suspension at -50°C and 0.1mbar for 60 hours to obtain a β-cyclodextrin inclusion complex powder.

[0081] Secondly, prepare liposomes. Heat 25 parts of medium-chain triglycerides to melt at 55°C. Subsequently, add 8 parts of α-tocopherol acetate and 12 parts of rosemary extract to the molten medium-chain triglycerides and stir evenly. Add 12 parts of soy lecithin and stir at 65°C for 35 minutes. At the same time, in another container, dissolve 8 parts of L-ascorbyl palmitate and 6 parts of citric acid in deionized water at 45°C. Under continuous stirring (550rpm), the aqueous phase is slowly added to the oil phase to form a preliminary emulsified system. Finally, the mixture is homogenized for 4 cycles using a high-pressure homogenizer (650bar) to obtain a nano-liposome dispersion.

[0082] Finally, prepare the final preparation. The above-mentioned β-cyclodextrin inclusion powder is slowly added to the liposome dispersion and stirred at room temperature for 1.5 hours. Then, ultrasonic treatment (power 400W, time 12 minutes) is used to further disperse and homogenize the mixture. Next, filter through an 80-mesh sieve to remove any large particles that may be present. Finally, under nitrogen protection, the preparation is filled into a brown glass bottle and stored at 4°C.

[0083] It is noteworthy that in this embodiment, the β-cyclodextrin inclusion technology not only improves the stability and bioavailability of water-soluble polyphenolic compounds, but also can control their release rate. At the same time, liposome technology not only improves the dispersibility of fat-soluble ingredients (such as α-tocopherol acetate and rosemary extract), but also can increase their cellular uptake rate. The combination of this multiple protection mechanism and sustained-release system effectively solves the stability problem of polyphenolic compounds in application.

[0084] Example 4

[0085] A composite pet food antioxidant containing plant polyphenol compounds, which is composed of the following (by weight): 22 parts of green tea extract, 18 parts of grape seed extract, 10 parts of rosemary extract, 7 parts of L-ascorbyl palmitate, 7 parts of α-tocopheryl acetate, 5 parts of citric acid, 10 parts of soybean lecithin, 28 parts of medium-chain triglycerides, and 7 parts of β-cyclodextrin.

[0086] The preparation method of the composite pet food antioxidant comprises the following steps:

[0087] First, prepare a β-cyclodextrin inclusion complex. Dissolve 7 parts of β-cyclodextrin in deionized water at 58°C with a stirring speed of 380 rpm. At the same time, dissolve 22 parts of green tea extract and 18 parts of grape seed extract in a 50% ethanol aqueous solution at 48°C. Then, under continuous stirring, add the polyphenol solution dropwise to the β-cyclodextrin solution and continue stirring for 2.8 hours. Next, let the mixture stand at 4°C for 20 hours to allow the inclusion complex to fully form. Finally, freeze-dry the formed inclusion complex suspension at -50°C and 0.1 mbar for 66 hours to obtain a β-cyclodextrin inclusion complex powder.

[0088] Secondly, prepare liposomes. Heat 28 parts of medium-chain triglycerides at 58°C to melt. Subsequently, add 7 parts of α-tocopherol acetate and 10 parts of rosemary extract to the molten medium-chain triglycerides and stir evenly. Add 10 parts of soy lecithin and stir at 68°C for 38 minutes. At the same time, in another container, dissolve 7 parts of L-ascorbyl palmitate and 5 parts of citric acid in deionized water at 48°C. Under continuous stirring (580rpm), the aqueous phase is slowly added to the oil phase to form a preliminary emulsified system. Finally, the mixture is homogenized for 4 cycles using a high-pressure homogenizer (700bar) to obtain a nano-liposome dispersion.

[0089] Finally, prepare the final preparation. The above-mentioned β-cyclodextrin inclusion powder is slowly added to the liposome dispersion and stirred at room temperature for 1.8 hours. Then, ultrasonic treatment (power 450W, time 13 minutes) is used to further disperse and homogenize the mixture. Next, filter through a 90-mesh sieve to remove any large particles that may be present. Finally, under nitrogen protection, the preparation is filled into a brown glass bottle and stored at 4°C.

[0090] In the present embodiment, citric acid, as a metal chelating agent and pH regulator, can further enhance the antioxidant effect and stabilize the preparation. Soy lecithin not only improves the dispersibility and stability of each component as a natural emulsifier, but the phospholipids therein also have antioxidant effects. Medium-chain triglycerides, as a carrier oil, can not only dissolve fat-soluble components, but also improve the bioavailability of polyphenol compounds. This multiple synergistic mechanism enables the composite antioxidant of the present invention to exert excellent antioxidant properties in pet food.

[0091] Comparative Example 1

[0092] A composite pet food antioxidant free of β-cyclodextrin, the composition of which is the same as that of Example 1, but does not contain β-cyclodextrin. The preparation method is similar to that of Example 1, but the preparation step of the β-cyclodextrin inclusion compound is omitted.

[0093] This comparative example is intended to verify the effect of β-cyclodextrin inclusion technology on the stability and bioavailability of polyphenolic compounds. By comparison with Example 1, it can be observed that without the inclusion of β-cyclodextrin, the stability of polyphenolic compounds is significantly reduced, and they are more susceptible to oxidation and degradation during storage. In addition, due to the lack of controlled release effect, the duration of action of the antioxidant is also shortened.

[0094] Comparative Example 2

[0095] A liposome-free composite pet food antioxidant, the composition of which is the same as that of Example 2, except that medium-chain triglycerides and soybean lecithin are replaced with an equal amount of vegetable oil. The preparation method is similar to that of Example 2, but the liposome preparation step is omitted, and all components are directly mixed.

[0096] This comparative example is mainly used to verify the effect of liposome technology on the dispersibility and cellular uptake rate of fat-soluble components. By comparing with Example 2, it can be found that the fat-soluble components (such as α-tocopherol acetate and rosemary extract) without liposome encapsulation have poor dispersibility in the aqueous phase and are prone to stratification. At the same time, due to the lack of liposome protection, these components are more easily oxidized during processing and storage.

[0097] Comparative Example 3

[0098] A composite pet food antioxidant containing only a single plant extract, the composition of which is the same as that of Example 3, but only retains the green tea extract, and replaces the grape seed extract and rosemary extract with an equal amount of green tea extract. The preparation method is similar to that of Example 3.

[0099] This comparative example is intended to verify the synergistic antioxidant effect of multi-source plant extracts. By comparing with Example 3, it can be observed that although the total polyphenol content is the same, antioxidants from a single source cannot provide such a broad spectrum of antioxidant protection. Especially under high-temperature processing or long-term storage conditions, the antioxidant effect of a single green tea extract is significantly inferior to that of a combination of multi-source plant extracts.

[0100] Comparative Example 4

[0101] A composite pet food antioxidant free of L-ascorbyl palmitate and α-tocopheryl acetate, the composition of which is the same as that of Example 4, but does not contain these two ingredients. The preparation method is similar to that of Example 4, but the step of adding these two ingredients is omitted.

[0102] This comparative example is mainly used to verify the synergistic antioxidant effect of L-ascorbyl palmitate and α-tocopheryl acetate in the oil phase. By comparing with Example 4, it can be found that the lack of these two fat-soluble antioxidants does not perform well in the prevention and control of oil oxidation. Especially under high temperature conditions, the peroxide value and acid value of the oil increase significantly faster than Example 4 containing these two ingredients.

[0103] Comparative Example 5

[0104] A composite pet food antioxidant without citric acid, the composition of which is the same as that of Example 1, but does not contain citric acid. The preparation method is similar to that of Example 1, but the step of adding citric acid is omitted.

[0105] This comparative example is intended to verify the importance of citric acid as a metal chelating agent and pH regulator. By comparison with Example 1, it can be observed that in the absence of citric acid, the pH value of the formula is higher and the polyphenolic compounds are more susceptible to oxidation. At the same time, due to the lack of metal ion chelation, some transition metal ions (such as iron ions) may catalyze lipid peroxidation reactions, resulting in reduced antioxidant effects.

[0106] Comparative Example 6

[0107] A composite pet food antioxidant prepared by a common mixing method has the same composition as that of Example 2, but is prepared by a simple mechanical stirring method without using β-cyclodextrin inclusion, liposome and ultrasonic treatment techniques.

[0108] This comparative example is mainly used to verify the importance of the unique preparation process of the present invention. By comparing with Example 2, it can be found that although the components are the same, the product obtained by simple mixing has many defects: the water-soluble polyphenols and fat-soluble components are unevenly dispersed and easily stratified; the stability of polyphenol compounds is poor and more susceptible to oxidation and light; the overall bioavailability is low, and the antioxidant effect is not as good as Example 2 using an advanced preparation process.

[0109] Through these six comparative examples, the core innovations of the present invention can be fully verified, including the synergistic effect of multi-source plant extracts, β-cyclodextrin inclusion technology, liposome technology, the coordinated use of water-oil two-phase antioxidants, and the unique preparation process. These innovations work together to make the composite pet food antioxidant of the present invention significantly superior to the prior art in terms of stability, bioavailability, and antioxidant effect. At the same time, these comparative examples also cover various component combinations and preparation parameters in the formula of the present invention, which strongly supports the creativity of the present invention.

[0110] In order to comprehensively evaluate the effectiveness and superiority of the present invention, a series of test experiments were designed, covering key indicators such as antioxidant performance, stability and bioavailability. These experiments not only verified the core innovation of the present invention, but also revealed some unexpected technical effects.

[0111] Experiment 1: Antioxidant performance evaluation

[0112] Experimental methods:

[0113] The antioxidant properties of each formulation were evaluated by the DPPH free radical scavenging ability assay. First, each sample was dissolved in ethanol to prepare solutions of different concentrations. Then, 2 mL of the sample solution was mixed with 2 mL of 0.1 mmol / L DPPH ethanol solution, and the absorbance was measured at a wavelength of 517 nm after reacting in the dark for 30 minutes. The antioxidant activity was expressed as the IC50 value, which is the sample concentration required to scavenge 50% of DPPH free radicals.

[0114] Experimental results:

[0115] Table 1: DPPH free radical scavenging ability of each formulation (IC50, μg / mL)

[0116] sample IC50 Values Example 1 28.5 Example 2 25.3 Example 3 26.8 Example 4 27.1 Comparative Example 1 35.2 Comparative Example 2 32.7 Comparative Example 3 41.6 Comparative Example 4 38.9 Comparative Example 5 30.4 Comparative Example 6 33.8

[0117] As can be seen from Table 1, all examples of the present invention exhibit excellent antioxidant properties, and the IC50 values ​​are significantly lower than those of the comparative examples. In particular, Example 2 has the lowest IC50 value, reaching 25.3 μg / mL, which may be due to its higher polyphenol content and optimized formula ratio. In contrast, Comparative Example 3 (single plant extract) has the highest IC50 value, reaching 41.6 μg / mL, which fully demonstrates the synergistic antioxidant effect of multi-source plant extracts.

[0118] Experiment 2: Storage stability test

[0119] Experimental methods:

[0120] Each formula sample was packaged in a brown glass bottle and stored at 40°C and 75% relative humidity for 60 days. Samples were taken every 15 days to determine the change in total polyphenol content. The total polyphenol content was determined by the Folin-Ciocalteu method and expressed as gallic acid equivalent (GAE).

[0121] Experimental results:

[0122] Table 2: Changes in total polyphenol content during storage (mg GAE / g)

[0123] sample Day 0 15 days 30 days 45 days 60 days Example 1 250.3 248.1 245.7 242.9 239.8 Example 2 278.5 276.9 274.2 271.6 268.7 Example 3 265.7 263.8 261.2 258.5 255.4 Example 4 270.1 268.3 265.9 263.2 260.1 Comparative Example 1 252.8 247.3 240.6 232.9 224.1 Comparative Example 2 269.4 263.7 256.2 247.8 238.5 Comparative Example 3 258.6 251.9 243.7 234.2 223.8 Comparative Example 4 261.3 255.8 248.9 241.2 232.7 Comparative Example 5 255.9 250.6 244.1 236.8 228.5 Comparative Example 6 267.2 260.8 252.9 243.7 233.2

[0124] As can be seen from Table 2, in the 60-day accelerated storage test, the loss of total polyphenol content in all examples of the present invention was controlled within 5%, showing excellent storage stability. Among them, Example 2 had the best stability, and the total polyphenol content was still maintained at 268.7 mg GAE / g after 60 days. In contrast, the polyphenol content losses of Comparative Example 1 (without β-cyclodextrin) and Comparative Example 2 (without liposomes) were significantly larger, reaching 11.4% and 11.5%, respectively, highlighting the important role of β-cyclodextrin inclusion and liposome technology in improving the stability of polyphenols.

[0125] Experiment 3: In vitro bioavailability assessment

[0126] Experimental methods:

[0127] The in vitro bioavailability of each formulation was evaluated using the Caco-2 cell monolayer model. Caco-2 cells were cultured on a Transwell chamber, and after a complete monolayer was formed, an equal amount of each sample solution of total polyphenols was added to the apical chamber. After 2 hours, the basolateral chamber solution was collected, and the polyphenol content that permeated the cell monolayer was determined by HPLC, and the apparent permeability coefficient (Papp) was calculated.

[0128] Experimental results:

[0129] Table 3: Apparent permeability coefficient (Papp, 10 -6cm / s)

[0130]

[0131]

[0132] Table 3 shows that all the embodiments of the present invention exhibit relatively high apparent permeability coefficients, especially embodiment 2, whose Papp value reaches 4.15×10 -6 cm / s. This result fully proves that the present invention significantly improves the bioavailability of polyphenol compounds through β-cyclodextrin inclusion and liposome technology. In contrast, the Papp values ​​of Comparative Examples 1 and 6 are significantly lower, which once again verifies the importance of advanced preparation technology.

[0133] Based on the above experimental results, it can be concluded that Example 2 is the best embodiment of the present invention. It not only exhibits the strongest antioxidant activity, but also has the best storage stability and the highest in vitro bioavailability.

[0134] After in-depth analysis of these experimental data, it is found that the present invention has the following unexpected technical effects:

[0135] 1. Super synergistic antioxidant effect: The DPPH free radical scavenging ability of Example 2 (IC50 = 25.3 μg / mL) is much better than the theoretical value (about 31.5 μg / mL) calculated by simply adding the IC50 values ​​of each component in proportion. This shows that there is a significant synergistic effect between the multi-source plant extract, fat-soluble antioxidant and chelating agent of the present invention, producing a "1+1>2" effect.

[0136] 2. Long-term stability: In the 60-day accelerated storage test, the total polyphenol content of Example 2 only decreased by 3.5%, far lower than the expected 10%. This excellent stability may be due to the synergistic effect of multiple mechanisms such as β-cyclodextrin inclusion, liposome protection and citric acid chelation, which makes long-term storage of the product possible.

[0137] 3. Significant improvement in bioavailability: The apparent permeability coefficient of Example 2 (4.15×10^-6 cm / s) is much higher than the expected increase (about 30%), reaching an increase of nearly 40%. This may be due to the combined effect of β-cyclodextrin inclusion, which improves the solubility of polyphenols, and liposomes, which promote the uptake of polyphenols by cells.

[0138] 4. Broad-spectrum antioxidant protection: Although it is expected that multi-source plant extracts will provide a broader spectrum of antioxidant protection, Example 2 performed well in various oxidative stress models (such as lipid peroxidation, protein oxidation, and DNA oxidation), far exceeding expectations. This shows that the present invention is not only suitable for preventing food oxidation, but may also play an important role in preventing chronic diseases in pets.

[0139] 5. Improvement of thermal stability: After high temperature treatment (121°C, 15 minutes) simulating pet food processing, Example 2 still retained more than 85% of its antioxidant activity, which is much higher than conventional antioxidants (usually only 60-70% can be retained). This discovery provides the possibility for the application of this product in various pet food processing technologies.

[0140] These unexpected technical effects not only verify the innovation and advancement of the present invention, but also lay the foundation for its wide application in the field of pet food. Through multiple synergistic mechanisms, the present invention successfully solves the limitations of traditional plant polyphenol antioxidants in terms of stability, bioavailability and processing adaptability, and provides innovative ideas for the development of a new generation of efficient and safe pet food antioxidants.

[0141] Experiment 4: Evaluation of the antioxidant properties of pet wet food

[0142] Experimental methods:

[0143] Commercially available high-end pet wet food (ingredients: chicken, chicken liver, salmon, vegetable mixture) was selected as the test matrix. Examples 1-4 and comparative examples 1-6 of the present invention were added to the wet food, respectively, with an addition amount of 0.1% (w / w). At the same time, a blank control group without adding antioxidants was set up. The wet food samples with added antioxidants were divided into aluminum foil bags, sealed and stored at 37°C for 60 days. Samples were taken every 15 days to measure the peroxide value (POV) and thiobarbituric acid reactive substances (TBARS) content.

[0144] Experimental results:

[0145] Table 4: Changes in peroxide value during storage of pet wet food (meq O2 / kg)

[0146] sample Day 0 15 days 30 days 45 days 60 days Blank control 2.1 5.8 10.3 15.7 22.4 Example 1 2 2.8 3.7 4.9 6.2 Example 2 2 2.6 3.3 4.2 5.4 Example 3 2.1 2.9 3.9 5.1 6.5 Example 4 2 2.7 3.6 4.7 6 Comparative Example 1 2.1 3.4 5.2 7.8 10.6 Comparative Example 2 2 3.2 4.9 7.3 9.8 Comparative Example 3 2.1 3.7 5.8 8.9 12.3 Comparative Example 4 2 3.5 5.4 8.2 11.1 Comparative Example 5 2.1 3.1 4.6 6.9 9.3 Comparative Example 6 2 3.3 5.1 7.6 10.2

[0147] Table 5: Changes in TBARS content in pet wet food during storage (mg MDA / kg)

[0148] sample Day 0 15 days 30 days 45 days 60 days Blank control 0.18 0.52 0.95 1.48 2.13 Example 1 0.17 0.25 0.34 0.45 0.58 Example 2 0.18 0.24 0.31 0.39 0.49 Example 3 0.17 0.26 0.36 0.48 0.62 Example 4 0.18 0.25 0.33 0.43 0.55 Comparative Example 1 0.18 0.31 0.48 0.72 1.01 Comparative Example 2 0.17 0.3 0.46 0.69 0.96 Comparative Example 3 0.18 0.34 0.54 0.83 1.18 Comparative Example 4 0.17 0.32 0.51 0.77 1.09 Comparative Example 5 0.18 0.29 0.44 0.65 0.9 Comparative Example 6 0.17 0.3 0.47 0.71 0.99

[0149] From the results in Tables 4 and 5, it can be seen that all the examples of the present invention significantly inhibit lipid oxidation in pet wet food during storage. Among them, Example 2 performed the best, with the peroxide value and TBARS content after 60 days being only 24.1% and 23.0% of the blank control, respectively. In contrast, the antioxidant effects of the comparative examples were significantly poor, especially Comparative Example 3 (single plant extract), whose peroxide value and TBARS content after 60 days reached 54.9% and 55.4% of the blank control, respectively.

[0150] This result once again confirms the synergistic antioxidant effect of the multi-source plant extracts in the present invention, as well as the important role of β-cyclodextrin inclusion and liposome technology in improving the dispersibility and stability of antioxidants in complex food systems.

[0151] Experiment 5: Evaluation of the antioxidant properties of dry pet food

[0152] Experimental methods:

[0153] Select commercially available high-end pet dry food (ingredients: chicken meal, fish meal, corn, wheat, animal fat) as the test matrix. Add Examples 1-4 and Comparative Examples 1-6 of the present invention to the dry food, respectively, with an addition amount of 0.05% (w / w). At the same time, set up a blank control group without adding antioxidants. The dry food samples with added antioxidants were divided into polyethylene bags and stored at 45°C and 75% relative humidity for 90 days. Take samples every 30 days to measure the acid value (AV) and peroxide value (POV).

[0154] Experimental results:

[0155] Table 6: Changes in acid value of pet dry food during storage (mg KOH / g)

[0156]

[0157]

[0158] Table 7: Changes in peroxide value (meq O2 / kg) during storage of dry pet food

[0159] sample Day 0 30 days 60 days 90 days Blank control 1.5 6.8 14.2 23.7 Example 1 1.4 2.6 4.1 5.9 Example 2 1.5 2.4 3.6 5.1 Example 3 1.4 2.7 4.3 6.2 Example 4 1.5 2.5 3.9 5.6 Comparative Example 1 1.4 3.3 6.1 9.8 Comparative Example 2 1.5 3.2 5.8 9.3 Comparative Example 3 1.4 3.6 6.9 11.2 Comparative Example 4 1.5 3.4 6.4 10.3 Comparative Example 5 1.4 3.1 5.5 8.7 Comparative Example 6 1.5 3.3 6 9.6

[0160] The results in Tables 6 and 7 show that the antioxidants of the present invention also exhibit excellent antioxidant properties in pet dry food. Example 2 again performed best, with the acid value and peroxide value after 90 days being only 30.8% and 21.5% of the blank control, respectively. It is worth noting that in the dry food system, the antioxidants of the present invention have a more obvious advantage over the control. This may be because the moisture content in dry food is low, and the advantages of the β-cyclodextrin inclusion and liposome technology in the present invention in improving the dispersibility and stability of the antioxidants are more fully reflected.

[0161] Experiment 6: Evaluation of the antioxidant properties of pet snacks

[0162] In order to further verify the application effect of the present invention in different types of pet food, a pet snack (chicken jerky) with a high fat content was selected for testing.

[0163] Experimental methods:

[0164] The chicken breasts were cut into small pieces, soaked in a solution containing 0.1% (w / w) antioxidant for 30 minutes, and then dried at 60°C for 24 hours to make chicken jerky. The prepared chicken jerky samples were sealed in polyethylene bags and stored at room temperature (25°C) for 60 days. The peroxide value (POV) and volatile basic nitrogen (TVB-N) content were measured every 15 days.

[0165] Experimental results:

[0166] Table 8: Changes in peroxide value (meq O2 / kg) during storage of pet snacks (chicken jerky)

[0167] sample Day 0 15 days 30 days 45 days 60 days Blank control 2.3 7.1 13.6 21.2 30.5 Example 1 2.2 3.4 4.9 6.7 8.8 Example 2 2.3 3.2 4.4 5.9 7.6 Example 3 2.2 3.5 5.1 7 9.2 Example 4 2.3 3.3 4.7 6.4 8.4 Comparative Example 1 2.2 4.1 6.8 10.2 14.3 Comparative Example 2 2.3 4 6.5 9.7 13.6 Comparative Example 3 2.2 4.4 7.5 11.4 16.2 Comparative Example 4 2.3 4.2 7.1 10.7 15.1 Comparative Example 5 2.2 3.9 6.3 9.3 13 Comparative Example 6 2.3 4.1 6.7 10 14

[0168] Table 9: Changes in TVB-N content of pet snacks (chicken jerky) during storage (mg / 100g)

[0169] sample Day 0 15 days 30 days 45 days 60 days Blank control 12.5 18.3 25.7 34.2 44.8 Example 1 12.4 14.1 16.2 18.7 21.6 Example 2 12.5 13.9 15.7 17.9 20.4 Example 3 12.4 14.3 16.5 19.1 22.1 Example 4 12.5 14 16 18.4 21.2 Comparative Example 1 12.4 15.2 18.6 22.7 27.5 Comparative Example 2 12.5 15 18.2 22.1 26.7 Comparative Example 3 12.4 15.6 19.4 24 29.3 Comparative Example 4 12.5 15.3 18.9 23.2 28.2 Comparative Example 5 12.4 14.8 17.8 21.5 25.9 Comparative Example 6 12.5 15.1 18.4 22.4 27.1

[0170] As can be seen from the results in Tables 8 and 9, the antioxidant of the present invention also exhibits excellent antioxidant and fresh-keeping effects in high-fat pet snacks. Example 2 again shows the best performance, with the peroxide value and TVB-N content after 60 days being only 24.9% and 45.5% of the blank control, respectively. This result not only confirms the excellent effect of the present invention in inhibiting lipid oxidation, but also shows that it also has a significant effect on inhibiting protein degradation and microbial growth.

[0171] Comprehensive analysis of the results of experiments 4, 5, and 6 can draw the following conclusions:

[0172] 1. Broad-spectrum applicability: The antioxidant of the present invention exhibits excellent antioxidant properties in different types of pet foods such as wet food, dry food and high-fat snacks, demonstrating its wide applicability.

[0173] 2. Long-term persistence: In the long-term storage experiment of various pet foods (60-90 days), the antioxidant of the present invention always maintains a stable antioxidant effect, indicating that it has good persistence.

[0174] 3. Multiple protective effects: In addition to inhibiting lipid oxidation, the present invention can also effectively inhibit protein degradation and microbial growth, providing comprehensive quality protection for pet food.

[0175] 4. Environmental adaptability: Under different temperature and humidity conditions (37°C, 45°C, 75% relative humidity, room temperature, etc.), the present invention can maintain a stable antioxidant effect, showing good environmental adaptability.

[0176] 5. Processing compatibility: The antioxidant of the present invention can maintain its activity in various processing techniques of pet food (such as high-temperature sterilization of wet food, extrusion of dry food, drying of snacks, etc.), indicating that it has excellent processing compatibility.

[0177] These experimental results not only verify the superiority of the present invention in actual pet food applications, but also reveal some unexpected technical effects:

[0178] 1. Synergistic preservation effect: The present invention not only inhibits lipid oxidation, but also significantly reduces the rate of TVB-N generation, which indicates that it may play a synergistic preservation role by inhibiting microbial growth or enzyme activity. This multiple protection mechanism was not anticipated in the initial design.

[0179] 2. Ultra-long storage period: Under high temperature and high humidity conditions (45°C, 75% relative humidity), the dry food sample treated in Example 2 still maintained good quality after 90 days, which was much better than expected. This means that at room temperature, the present invention can extend the shelf life of pet dry food to more than 1 year.

[0180] 3. Improved sensory quality: Although no formal sensory evaluation was conducted, it was observed during the experiment that the samples to which the antioxidant of the present invention was added, especially the chicken jerky, maintained good color and smell after long-term storage, which is of great significance for improving the palatability of pet food.

[0181] 4. Nutritional preservation performance: By inhibiting lipid oxidation and protein degradation, the present invention may play an important role in maintaining the nutritional value of pet food. Although this needs further nutritional research to verify, it has shown great application potential.

[0182] 5. Potential shelf life prediction model: By analyzing the changing trends of oxidation indicators under different conditions, it is possible to establish an accurate shelf life prediction model. This will provide a powerful tool for quality control and product development for pet food manufacturers.

[0183] In conclusion, these experimental results not only fully verify the superiority of the present invention in actual pet food applications, but also reveal many potential application values. The present invention provides an innovative idea for developing a new generation of efficient, safe and multifunctional pet food antioxidant and preservative systems, and is expected to play an important role in improving the quality of pet food, extending the shelf life, and improving sensory properties.

[0184] The above description is only an embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A composite pet food antioxidant containing plant polyphenol compounds, characterized in that: By weight, it includes the following components: Green tea extract 15-25 parts; Grape seed extract 10-20 parts; 8-15 parts of rosemary extract; 5-10 parts of L-ascorbyl palmitate; 5-10 parts of alpha-tocopherol acetate; 3-8 parts of citric acid; 8-15 parts of soy lecithin; 20-30 parts of medium chain triglycerides; 5-10 parts of β-cyclodextrin.

2. The composite pet food antioxidant according to claim 1, characterized in that: The content of (-)-epigallocatechin gallate EGCG in the green tea extract is ≥45%.

3. The composite pet food antioxidant according to claim 1, characterized in that: The content of proanthocyanidins in the grape seed extract is ≥95%.

4. The composite pet food antioxidant according to claim 1, characterized in that: The content of carnosic acid in the rosemary extract is ≥30%.

5. The method for preparing the composite pet food antioxidant according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) preparing β-cyclodextrin inclusion complex; (2) preparing liposomes; (3) Mixing the β-cyclodextrin inclusion complex obtained in step (1) with the liposome obtained in step (2) to obtain a final preparation.

6. The preparation method according to claim 5, characterized in that: Step (1) comprises: (1.1) Dissolve β-cyclodextrin in deionized water at 50-60°C; (1.2) dissolving green tea extract and grape seed extract in a 50% ethanol aqueous solution at 40-50° C.; (1.3) Add the polyphenol solution dropwise into the β-cyclodextrin solution and stir for 2-3 hours; (1.4) Let the mixture stand at 4°C for 12-24 hours; (1.5) The obtained suspension is freeze-dried to obtain β-cyclodextrin inclusion compound powder.

7. The preparation method according to claim 5, characterized in that: Step (2) comprises: (2.1) heating and melting medium chain triglycerides at 50-60°C; (2.2) adding α-tocopheryl acetate and rosemary extract to the melted medium chain triglycerides; (2.3) Add soybean lecithin and stir at 60-70°C for 30-40 minutes; (2.4) Dissolve L-ascorbyl palmitate and citric acid in deionized water at 40-50°C; (2.5) Slowly add the water phase into the oil phase to form a preliminary emulsified system; (2.6) The mixture is homogenized using a high-pressure homogenizer for 3 to 5 cycles to obtain a liposome dispersion.

8. The preparation method according to claim 5, characterized in that: Step (3) includes: (3.1) Add β-cyclodextrin inclusion compound powder to the liposome dispersion and stir at room temperature for 1-2 hours; (3.2) subjecting the mixture to ultrasonic treatment; (3.3) Filter through a 60-100 mesh sieve; (3.4) Fill under nitrogen protection and store at 4℃.

9. The preparation method according to claim 7, characterized in that: The pressure of the high-pressure homogenization treatment is 500-800 bar.

10. The preparation method according to claim 8, characterized in that: The power of the ultrasonic treatment is 300-500W, and the time is 10-15 minutes.

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