Functional feed additive containing tea pigment photoresponse cationic nano-liposome

Abstract

The invention relates to a functional feed additive containing tea pigment photoresponse cationic nano-liposomes, and belongs to the technical field of feed additives. The functional feed additive comprises, by mass, 25-35% of auxiliary materials and 65-75% of tea pigment photoresponse cationic nano-liposome, the tea pigment photoresponse cationic nano-liposome is prepared by taking chitosan as a shell layer and taking liposome as a core layer, the liposome is prepared by taking soybean lecithin and cholesterol as capsule wall materials, and the capsule wall materials are prepared by taking chitosan as a shell layer and taking liposome as a core layer. A photosensitizer and a tea pigment are used as core materials. According to the functional additive disclosed by the invention, the auxiliary materials and the tea pigment photoresponse cationic nano-liposome are added, and the functional additive can effectively replace antibiotics in cooperation with optimized process parameters. The functional feed additive is particularly suitable for breeding of livestock such as chickens and ducks, meanwhile, blue light irradiation is assisted, accurate release of tea pigment can be better achieved, and the multiple effects of resisting bacteria, adjusting immunity and improving meat quality are achieved.

Description

Technical Field

[0001] This invention belongs to the technical field of feed additives, and more specifically, relates to a functional feed additive containing photoresponsive cationic nanoliposomes containing tea pigments. Background Technology

[0002] In modern intensive animal husbandry, antibiotics are widely used as feed additives in poultry farming, especially in the raising of traditional livestock such as chickens and ducks. The use of antibiotics has become an important means of improving breeding success rates and economic benefits. These antibiotics mainly include tetracyclines, sulfonamides, quinolones, and β-lactams, whose main functions are disease prevention, growth promotion, and improved feed conversion ratio.

[0003] However, the negative effects of long-term, excessive use of antibiotics are becoming increasingly apparent. First, antibiotics are difficult to completely metabolize in animals, often remaining in their original form or as metabolites in meat, eggs, and other livestock products. These residues can then enter the human body through the food chain, potentially triggering allergic reactions and, more seriously, disrupting the body's normal flora and lowering immunity. Second, the overuse of antibiotics accelerates the emergence and spread of drug-resistant strains. Furthermore, antibiotic-containing wastewater and waste discharged from the livestock industry enter the ecological environment, causing soil and water pollution, disrupting the ecological balance, and creating a vicious cycle.

[0004] In recent years, plant-derived additives have received widespread attention due to their natural, safe, and residue-free characteristics. Tea pigments are a class of natural polyphenol oxidized polymers extracted from tea leaves, possessing multiple biological activities. They mainly include theaflavins, thearubigins, and theabrownins, exhibiting excellent antioxidant, antibacterial, and anti-inflammatory effects, and showing great potential in the food, pharmaceutical, and agricultural fields.

[0005] However, tea pigments face many challenges in practical applications: First, tea pigments have poor water solubility and low stability, and are easily degraded and inactivated in feed processing and animal digestive tract environments; second, conventional addition methods are difficult to achieve targeted delivery, resulting in low bioavailability; third, it is impossible to achieve precise drug administration according to different growth stages and pathological states, making it difficult to meet the needs of dynamic breeding.

[0006] Currently, there are no reports on the preparation of tea pigments into liposomes; furthermore, there are no studies combining tea pigments with visible light-responsive release technology and cationic active targeting function. Therefore, developing cationic nanoliposomes capable of releasing tea pigments in response to light stimulation and applying them to functional feed additives has significant scientific value and application prospects for solving the problem of antibiotic abuse, ensuring food safety, and promoting the green and sustainable development of the livestock industry. Summary of the Invention

[0007] The purpose of this invention is to provide a functional feed additive containing photoresponsive cationic nanoliposomes containing tea pigments, which solves the problems of poor stability and inaccurate release of tea pigments in the prior art, and has the characteristics of reducing antibiotic use and improving survival rate.

[0008] The objective of this invention can be achieved through the following technical solutions: A functional feed additive containing tea pigment photoresponsive cationic nanoliposomes comprises 25-35 wt% excipients and 65-75 wt% tea pigment photoresponsive cationic nanoliposomes. The tea pigment photoresponsive cationic nanoliposomes are prepared by using chitosan as the shell and liposomes as the core layer. The liposomes are prepared by using soybean lecithin and cholesterol as encapsulation materials and photosensitizers and tea pigments as core materials.

[0009] Among them, the excipients mainly serve as physical carriers to protect the structural stability and fluidity of the tea pigment photoresponsive cationic nanoliposomes, and to ensure that the additives have more comprehensive functions and enhance the overall efficacy.

[0010] Furthermore, the preparation method of the tea pigment photoresponsive cationic nanoliposomes includes the following steps: A1. Using black tea tree branches and leaves as raw materials, tea pigment extracts are prepared by enzymatic oxidation and macroporous resin purification technology; wherein the enzymatic oxidation reaction is carried out under the conditions of pH 4.5-5.5 and temperature 40-50℃. A2. The soybean lecithin, cholesterol, photosensitizer, and tea pigment extract prepared in step A1 are dissolved together in an organic solvent. The organic solvent is removed under reduced pressure to form a lipid film. The lipid film is hydrated with a buffer solution of pH 6.5-7.5 to obtain a primary liposome suspension loaded with tea pigment and photosensitizer. The mass ratio of soybean lecithin to cholesterol is (5:1)-(8:1). A3. Prepare a cationic chitosan acetate solution with a mass-volume concentration of 0.05-0.15%, and add it to the primary liposome suspension obtained in step A2 under stirring conditions. Utilize electrostatic interaction to coat the liposome shell with chitosan to form cationic nanoliposomes. A4. The product obtained in step A3 is homogenized and purified to obtain tea pigment photoresponsive cationic nanoliposomes.

[0011] Preferably, the photosensitizer is pheophytic acid.

[0012] In this technical solution, pheophytic chlorophyll, when excited under visible light, generates singlet oxygen, which breaks phospholipid bonds and releases the nanocapsules. Because the body absorbs little visible light, it has strong penetrating power and is safe and harmless. This technical solution encapsulates tea pigments in photoresponsive cationic nanoliposomes, enabling active targeting of bacteria. Under blue light irradiation, the tea pigments are released at the target site, enhancing drug concentration and efficacy at the infection site, thereby achieving dual-targeted infection control.

[0013] Preferably, in step A1, the raw material is pruning waste from black tea tree branches and leaves, which is a reuse of agricultural waste, reducing environmental pollution and effectively saving costs. The enzymatic oxidation method uses polyphenols in the raw material as substrates, and under the specific catalysis of enzymes, directional oxidation, coupling, and polymerization reactions occur, efficiently converting them into tea pigments. The macroporous resin purification utilizes the adsorption properties of the resin to selectively enrich the tea pigments; by using gradient elution with ethanol-water solution, impurities such as proteins and polysaccharides can be effectively removed, thereby increasing the content of the obtained tea pigment extract.

[0014] In step A1 of this technical solution, the content of tea pigments accounts for ≥80% of the total content of the tea pigment extract. The tea pigments mainly include theaflavins, thearubigins, and theabrownins. Among them, theaflavins have the most significant biological activity and are the key component that endows tea pigments with antioxidant, anti-inflammatory, and antibacterial effects; thearubigins are next, while theabrownins have relatively weaker biological activity. Based on this, the combination of enzymatic oxidation and macroporous resin purification process aims to regulate the reaction pathway to promote the formation of theaflavins and thearubigins and inhibit their excessive conversion to theabrownins, thereby obtaining an extract with a higher proportion of highly active components.

[0015] Preferably, in step A1, the pH is adjusted using a citrate-disodium hydrogen phosphate buffer solution.

[0016] Preferably, in step A1, the enzyme used in the enzymatic oxidation reaction is polyphenol oxidase and / or peroxidase, and the amount of enzyme added is 0.1%-0.5% of the raw material mass.

[0017] The determination of this range is mainly based on the balance between reaction efficiency and product composition: when the amount of enzyme added is less than 0.1%, the reaction kinetics are insufficient, resulting in incomplete substrate conversion and a significant reduction in the yield of tea pigments; when the amount added is greater than 0.5%, although it can accelerate the reaction, it will promote the excessive polymerization of intermediate quinones, leading to an increase in the proportion of high molecular weight theabrownins, which in turn reduces the relative content of highly active theaflavins and thearubigins.

[0018] Preferably, in step A1, the macroporous resin purification uses HPD-100 or AB-8 macroporous resin, and gradient elution is performed using an ethanol aqueous solution with a concentration of 30%-60%.

[0019] The selection of the above resins is based on the fact that both HPD-100 and AB-8 resins are moderately polar adsorption resins, and their surface pore size and specific surface area are suitable for adsorbing natural products such as tea pigments, which have polyphenol structures and medium molecular weights. Compared with strongly polar or non-polar resins, the above two resins have higher adsorption capacity and more suitable selectivity for tea pigments, effectively adsorbing the target analyte while allowing strongly polar impurities such as inorganic salts and some sugars to penetrate. The principle of setting the gradient elution concentration is to utilize the polarity difference between tea pigments and impurities to achieve selective separation and enrichment. First, elution is performed using approximately 30% ethanol to remove strongly polar impurities that are not firmly adsorbed by the resin; theaflavins and thearubigins in tea pigments have the best desorption efficiency at a concentration of approximately 60% ethanol. This concentration of ethanol is sufficient to disrupt the hydrophobic interactions and hydrogen bonds between tea pigment molecules and the resin skeleton, allowing them to be concentrated and eluted efficiently, thereby achieving high-purity enrichment.

[0020] Preferably, the concentrations of the ethanol-water solution used for gradient elution are 30%, 40%, 50%, and 60% respectively.

[0021] Preferably, in step A2, the total lipid mass is calculated based on the total mass of soybean lecithin and cholesterol, and the mass of the tea pigment extract accounts for 10%-20% of the total lipid mass; the mass ratio of the photosensitizer to the tea pigment extract is 1:(8-12).

[0022] The above ratio settings are based on the following considerations: Setting the mass of tea pigment extract at 10%-20% of the total lipid mass aims to balance drug loading and liposome structural stability. Too low a ratio results in insufficient drug loading, while too high a ratio will damage the lipid membrane structure, leading to a decrease in encapsulation efficiency. This range is crucial for achieving a tea pigment encapsulation efficiency of no less than 80%. Controlling the photosensitizer ratio at 1:(8-12) aims to synergistically balance photoresponse efficiency and carrier stability. Too low a ratio results in insufficient phototriggered release, while too high a ratio affects liposome stability and increases potential adverse effects. This range ensures that the nanoliposomes maintain high stability while possessing highly efficient blue light-responsive release.

[0023] Preferably, in step A2, the organic solvent is at least one of chloroform, ethanol, methanol, and dichloromethane; and the buffer solution is a phosphate buffer solution.

[0024] Preferably, in step A3, the solvent for the cationic chitosan acetic acid solution is an aqueous acetic acid solution with a concentration of 0.5%-1.5%. This concentration range is set based on the following principle: chitosan requires protonation under acidic conditions to dissolve and exhibit its cationic properties. When the acetic acid concentration is below 0.5%, the acidity is insufficient to completely dissolve it, easily producing insoluble particles and resulting in uneven coating. When the concentration is above 1.5%, excessive acidity will exacerbate the hydrolysis of chitosan molecular chains, leading to a decrease in its molecular weight and viscosity, thereby weakening the stability and integrity of the formed coating layer. Furthermore, excessively high acetic acid concentrations increase the burden and potential impact of subsequent purification. Therefore, controlling the acetic acid concentration at 0.5%-1.5% ensures that chitosan is fully dissolved and maintains its high molecular structure stability while providing the optimal cationization environment, thereby forming a stable and complete positively charged coating layer.

[0025] Preferably, in step A4, the homogenization process is performed by probe ultrasound with an ultrasound power of 200-400W and a processing time of 3-10 minutes; the purification process is performed by dialysis or ultracentrifugation.

[0026] Dialysis is based on the principle of molecular diffusion through a semipermeable membrane in solution. A cationized nanoliposome suspension is placed in a dialysis bag with a molecular weight cutoff of 10 kDa and immersed in a large amount of deionized water or buffer solution for dialysis. Free small molecules diffuse through the membrane pores to the external aqueous phase, while nanoliposomes encapsulated inside the liposomes and bound to the surface are retained inside the bag. This process is gentle, requires no high-speed shear forces, and preserves the integrity of the nanoliposome structure to the greatest extent.

[0027] Ultracentrifugation is based on the difference in sedimentation rates of different particles under a strong centrifugal force field. Centrifugation at a relative centrifugal force of at least 100,000 × g causes nanoliposomes to precipitate due to their larger particle size and density, while free drug molecules and small molecule impurities remain in the supernatant. Separation is achieved by removing the supernatant. This method is efficient and rapid, suitable for the purification and concentration of batches of samples.

[0028] The encapsulation efficiency of the tea pigment photoresponsive cationic nanoliposomes was ≥80%, as determined by high performance liquid chromatography.

[0029] Preferably, the average particle size of the tea pigment photoresponsive cationic nanoliposomes is 80-200 nm, and the Zeta potential is ≥ +30 mV.

[0030] The positive charge on the surface of nanoliposomes can specifically recognize the negatively charged bacterial cell membranes, improving the targeting of common avian pathogens such as Escherichia coli and Salmonella, and increasing the antibacterial efficiency by 3-5 times.

[0031] Further, the excipients include the following components by weight: 30-50 parts rice bran, 20-30 parts montmorillonite, 12-20 parts trehalose, 8-16 parts fructooligosaccharides, 3-5 parts choline, 0.2-0.5 parts salt, and 0.8-1.5 parts vitamin-trace element premix.

[0032] Rice bran, as the main carrier, has the functions of hygroscopic protection of nanoliposomes and slowing down the degradation by gastric acid; defatted rice bran is preferred because it has higher stability. Montmorillonite has a layered structure, which can slowly release active ingredients and protect the intestinal mucosa; trehalose, as a freeze-drying protectant and stabilizer, helps maintain the structural integrity of nanoliposomes; fructooligosaccharides, as a prebiotic, can promote the growth of lactic acid bacteria and synergistically inhibit pathogens with tea pigments; choline can promote the fusion of liposomes with cell membranes and improve the absorption rate of tea pigments.

[0033] Further, by weight, the excipients also include at least one of the following components: 0.2-0.4 parts riboflavin, 0.6-1 parts plant essential oil, 5-10 parts plant oil, 2-4 parts β-glucan, 0.1-0.2 parts organic selenium, 0.1-0.3 parts lysine, 0.05-0.2 parts methionine, and 3-5 parts nano-silica.

[0034] Among them, riboflavin, as a photosensitizing enhancer, can improve the blue light response efficiency; the preferred plant essential oils are thymol and cinnamaldehyde, which can synergistically fight bacteria; the preferred plant oils are flaxseed oil and rapeseed oil, which can increase the content of unsaturated fatty acids; β-glucan is beneficial for enhancing immune function; organic selenium and tea pigments synergistically enhance antioxidant capacity; and silicon dioxide can enhance light scattering and improve the uniformity of blue light distribution.

[0035] This invention also provides a method for preparing a functional feed additive containing tea pigment photoresponsive nanoliposomes, comprising the following steps: S1. After mixing the components of the excipients, granulate them at 35-45℃ and cool them to room temperature; S2. Mix tea pigment photoresponsive cationic nanoliposomes with microcrystalline cellulose at a weight of 10-15% to prepare a functional premix. S3. Add the functional premix to the excipient granules in step S1 and mix at 35-45℃ for 5-15 minutes. S4. Add a surfactant aqueous solution at a ratio of 0.8-1.2% of the weight of the excipients, and continue mixing for 5-10 minutes; then dry at 40-50℃ until the moisture content is 10-12%, cool in the dark, and seal in packaging.

[0036] Further, in step S4, the surfactant is a food-grade sucrose fatty acid ester or Tween-80, and its aqueous solution concentration is 5-10%.

[0037] The above-mentioned functional feed additive containing tea pigment photoresponsive nanoliposomes is used in chicken farming. The functional feed additive is added to the chicken's staple food, and the chicken is irradiated with blue light with a wavelength of 400-480nm for 5-30 minutes every day.

[0038] Preferably, during the 0-3 week stage of chickens, the proportion of the functional feed additive in the total feed mass is 4-5%; during the 4-8 week stage of chickens, the proportion of the functional feed additive in the total feed mass is 3-4%; and during the 9-14 week stage of chickens, the proportion of the functional feed additive in the total feed mass is 2-3%.

[0039] Furthermore, the blue light irradiation is performed within 20-40 minutes after the chickens are fed.

[0040] Blue light can achieve a combination of photo-targeted and active targeting by controlling the release of liposomes. Furthermore, blue light can be used to synergistically fight bacteria and promote animal growth and improve production performance.

[0041] The beneficial effects of this invention are: (1) In the prior art, the phenolic hydroxyl active groups of tea pigments are easily oxidized or hydrolyzed and destroyed in vivo and in vitro, resulting in a significant reduction in activity. In particular, the acid-base environment of the gastrointestinal tract and the action of various enzymes in vivo lead to a small amount of effective drug entering the body or a short existence time in the blood and tissues, thus failing to achieve the medicinal effect. This invention encapsulates tea pigments with liposomes. Liposomes are composed of a phospholipid bilayer, which has good biocompatibility and degradability, and can effectively encapsulate hydrophobic active ingredients, improving their stability and bioavailability. To avoid the acid-base environment of the gastrointestinal tract and the action of hydrolytic enzymes, chitosan is used to further modify the liposomes. Utilizing the characteristic that chitosan is not degraded in the gastrointestinal tract, the electrostatic interaction between the liposomes and bacterial cell membranes can be enhanced, protecting the tea pigments from destruction. This realizes a delivery system that combines photo-controlled targeting and active targeting, solving the problems of poor stability and weak targeting of tea pigments, and improving antibacterial efficiency. Furthermore, the introduction of photoresponsive materials to construct a photo-controlled release system can achieve precise control of the time and space of active ingredient release, thereby exerting a better effect.

[0042] (2) The functional additive of this invention contains excipients and tea pigment photoresponsive cationic nanoliposomes. With optimized process parameters, it can effectively replace antibiotics. This functional feed additive is particularly suitable for raising livestock such as chickens and ducks. When supplemented with blue light irradiation, it can better achieve precise release of tea pigments, exerting multiple effects such as antibacterial, immune regulation, and meat quality improvement. This technical solution has a simple process, controllable cost, and significant effects, making it easier to promote industrialization and providing a practical solution for green and healthy farming. Attached Figure Description

[0043] Figure 1 This is a schematic diagram of the structure of the tea pigment photoresponsive cationic nanoliposome in Example 1.

[0044] Figure 2 This is a process flow diagram for preparing tea pigment photoresponsive cationic nanoliposomes in Example 1.

[0045] Figure 3 This is a comparison of the in vitro cumulative release curves of the nanoliposomes prepared in Example 1 under blue light irradiation and without. Detailed Implementation

[0046] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below with reference to specific embodiments, but the scope of protection of this invention is not limited thereto. Experimental methods not specifically described in the embodiments are generally performed under conventional conditions or according to the manufacturer's recommendations. Unless otherwise specified, all reagents and materials used are commercially available.

[0047] Example 1 A functional feed additive containing tea pigment photoresponsive cationic nanoliposomes, comprising 30 wt% excipients and 70 wt% tea pigment photoresponsive cationic nanoliposomes.

[0048] The excipients include the following components by weight: 40 parts defatted rice bran, 28 parts montmorillonite, 16 parts trehalose, 12 parts fructooligosaccharides, 4 parts choline, 0.3 parts riboflavin (vitamin B2), 3 parts β-glucan, 4 parts nano silica, 0.3 parts salt, and 1 part vitamin-trace element premix.

[0049] The preparation method of tea pigment photoresponsive cationic nanoliposomes is as follows: A1. Take 1 kg of black tea tree branches and leaves, and place them in a citrate-disodium hydrogen phosphate buffer solution at pH 5.0, with a liquid-to-solid ratio of 10:1 (L / kg). Add 0.3% polyphenol oxidase (PPO) by weight of the raw materials for enzymatic oxidation. The reaction solution is purified by passing it through an HPD-100 macroporous resin column (2 L column bed volume), eluting sequentially with 5 column volumes (BV) of water, 3 BV of 30% ethanol, and 5 BV of 60% ethanol. Collect the 60% ethanol eluent, concentrate it under reduced pressure, and freeze-dry it to obtain 76 g of tea pigment extract powder. The total tea pigment content was determined by HPLC to be 83.5%.

[0050] A2. Weigh 200 mg of soybean lecithin, 40 mg of cholesterol (mass ratio 5:1), 30 mg of tea pigment extract obtained in step A1 (accounting for 12.5% ​​of the total lipid mass), and 3.0 mg of pheophoric acid (mass ratio of 1:10 to tea pigment), and dissolve them together in 20 mL of a mixed solvent of chloroform and ethanol (volume ratio 1:1). Form a lipid film using a reverse evaporation method, and hydrate it with 15 mL of pH 7.4 PBS at 45 °C to obtain a primary liposome suspension.

[0051] A3. Add 10 mL of 0.10% cationic chitosan acetate (solvent: 1% acetic acid) to the above primary liposome suspension and stir for 30 minutes for coating. Then, sonicate the mixture in an ice-water bath for 6 minutes, and transfer the solution to a dialysis bag with a molecular weight cutoff of 10 kDa. Purify by dialyzing with deionized water at 4°C for 12 hours to obtain tea pigment photoresponsive cationic nanoliposomes.

[0052] Among them, such as Figure 1 This is a schematic diagram of the structure of tea pigment photoresponsive cationic nanoliposomes; Figure 2 This is a flow chart of the preparation process of tea pigment photoresponsive cationic nanoliposomes. The encapsulation efficiency of tea pigment was 88.7%, and the encapsulation efficiency of pheophytic acid was 91.2%, determined by ultracentrifugation combined with HPLC. The average hydrodynamic particle size was 162 nm, the polydispersity index (PDI) was 0.16, and the zeta potential was +37.8 mV. Figure 3 As shown in the in vitro release experiment, the tea pigment photoresponsive cationic nanoliposomes of this embodiment achieved a cumulative release rate of 89.3% of tea pigment within 48 hours under periodic irradiation with 450nm blue light at an intensity of 50mW / cm²; while the release rate of the control group that was protected from light throughout the process was only 61.8%.

[0053] The preparation method of the above-mentioned functional feed additive containing tea pigment photoresponsive cationic nanoliposomes is as follows: S1. After mixing the components of the auxiliary materials, granulate them at 40°C with a ring die compression ratio of 1:8, and cool to room temperature. S2. Mix tea pigment photoresponsive cationic nanoliposomes with microcrystalline cellulose at 12% of their weight to prepare a functional premix. S3. Add the functional premix to the auxiliary material granules in step S1, and mix for 10 minutes in a twin-shaft paddle mixer at 30 rpm and 35-40℃. S4. Spray evenly with an 8% sucrose fatty acid ester aqueous solution, the amount of which is 1% of the weight of the excipients, and continue mixing for 5 minutes; then dry at 45℃ until the moisture content is 10-12%, cool in the dark, and seal in packaging.

[0054] Example 2 Compared with Example 1, this example differs in that the excipient mass percentage is 25wt%, and the mass percentage of tea pigment photoresponsive cationic nanoliposomes is 75wt%; in step S4, the 8% sucrose fatty acid ester aqueous solution is replaced with 8% Tween-80. All other components, preparation steps, and parameters remain the same.

[0055] Example 3 Compared with Example 1, this example differs in that the excipient mass percentage is 35wt%, and the mass percentage of tea pigment photoresponsive cationic nanoliposomes is 65wt%; in step S4, the 8% sucrose fatty acid ester aqueous solution is replaced with a 5% sucrose fatty acid ester aqueous solution. All other components, preparation steps, and parameters remain the same.

[0056] Example 4 Compared with Example 1, this example differs in that the excipients in this example include the following components by weight: 35 parts defatted rice bran, 25 parts montmorillonite, 14 parts trehalose, 10 parts fructooligosaccharides, 4 parts choline, 0.2 parts riboflavin (vitamin B2), 8 parts flaxseed oil, 4 parts nano-silica, 0.3 parts salt, and 1.2 parts vitamin-trace element premix; in step S4, the 8% sucrose fatty acid ester aqueous solution is replaced with a 10% sucrose fatty acid ester aqueous solution. All other components, preparation steps, and parameters remain the same.

[0057] Comparative Example 1 Compared with Example 1, this comparative example differs in that the functional feed additive in this comparative example does not contain tea pigment photoresponsive cationic nanoliposomes. Correspondingly, steps S2 and S3 are omitted, while the remaining components, preparation steps, and parameters are the same.

[0058] Comparative Example 2 Compared with Example 1, the difference in this comparative example is that the tea pigment photoresponsive cationic nanoliposomes in the functional feed additive of this comparative example are replaced with the tea pigment extract prepared by the method in step A1, while the other components, preparation steps and parameters are the same.

[0059] Comparative Example 3 Compared with Example 1, the difference in this comparative example is that the tea pigment photoresponsive cationic nanoliposomes in the functional feed additive of this comparative example are replaced with the primary liposome suspension prepared by the method in step A2, while the other components, preparation steps and parameters are the same.

[0060] Aquaculture Experiment Test Two hundred and ten 1-day-old Qingyuan Ma chickens were randomly divided into seven groups of 30 each, with three replicates of 10 each. The seven groups of chicks were fed the same feed as those in Examples 1-4 (groups 1-4) and Comparative Examples 1-3 (groups 5-7) for a period of 14 weeks, with identical basic feeding conditions. Half an hour after feeding, groups 1-4 and 7 were exposed to blue light for 15 minutes at a wavelength of 460±10 nm and an intensity of 4 mW / cm². Groups 5-6 received only ordinary lighting.

[0061] Weight record: Weigh yourself at a fixed time each week and calculate the average daily weight gain (ADG).

[0062] Feed conversion ratio (FCR): Record feed consumption weekly and calculate total feed consumption / total weight gain.

[0063] Survival rate: Record the number of deaths in each group and calculate the survival rate.

[0064] Disease incidence: The frequency of respiratory diseases, intestinal diseases, etc., was recorded. The results of the above tests are shown in Table 1.

[0065] Gut microbiota analysis: Cecal contents were collected at 14 weeks of age, and the number of Escherichia coli, Salmonella, Lactobacillus, and Bifidobacterium (unit: logCFU / g) was determined by plate count method. The results are shown in Table 2.

[0066] Table 1 Table 2 As shown in Table 1, compared with Comparative Examples 1-3, the chickens raised in Examples 1-4 showed better overall results in terms of average weight, feed conversion ratio, and survival rate. Table 2 shows that the example groups effectively controlled the number of pathogenic bacteria such as *E. coli* and *Salmonella*, and increased the number of *Lactobacillus* and *Bifidobacterium*. Compared with ordinary tea pigment extract and non-cationic nanoliposomes, the effect of resisting harmful bacteria was superior, demonstrating the synergistic effect of photoresponsive properties and cationic modification in targeted antibacterial activity. Therefore, the tea pigment-containing photoresponsive cationic nanoliposome feed of the present invention can significantly inhibit pathogenic bacteria, promote the growth of beneficial bacteria, and reshape a healthy intestinal microecology, showing significant advantages in promoting chicken growth and development and enhancing chicken immunity.

[0067] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A functional feed additive containing photoresponsive cationic nanoliposomes with tea pigments, characterized in that, It includes 25-35 wt% excipients and 65-75 wt% tea pigment photoresponsive cationic nanoliposomes. The tea pigment photoresponsive cationic nanoliposomes are prepared by using chitosan as the shell and liposomes as the core layer. The liposomes are prepared by using soybean lecithin and cholesterol as encapsulation materials and photosensitizers and tea pigments as core materials.

2. The functional feed additive containing tea pigment photoresponsive cationic nanoliposomes according to claim 1, characterized in that, The preparation method of the tea pigment photoresponsive cationic nanoliposomes includes the following steps: A1. Using black tea tree branches and leaves as raw materials, tea pigment extracts are prepared by enzymatic oxidation and macroporous resin purification technology; wherein the enzymatic oxidation reaction is carried out under the conditions of pH 4.5-5.5 and temperature 40-50℃. A2. The soybean lecithin, cholesterol, photosensitizer, and tea pigment extract prepared in step A1 are dissolved together in an organic solvent. The organic solvent is removed under reduced pressure to form a lipid film. The lipid film is hydrated with a buffer solution of pH 6.5-7.5 to obtain a primary liposome suspension loaded with tea pigment and photosensitizer. The mass ratio of soybean lecithin to cholesterol is (5:1)-(8:1). A3. Prepare a cationic chitosan acetate solution with a mass-volume concentration of 0.05-0.15%, and add it to the primary liposome suspension obtained in step A2 under stirring conditions. Utilize electrostatic interaction to coat the liposome shell with chitosan to form cationic nanoliposomes. A4. The product obtained in step A3 is homogenized and purified to obtain tea pigment photoresponsive cationic nanoliposomes.

3. A functional feed additive containing tea pigment photoresponsive cationic nanoliposomes according to claim 1 or 2, characterized in that, The photosensitizer is pheophytic acid.

4. The functional feed additive containing tea pigment photoresponsive cationic nanoliposomes according to claim 2, characterized in that, In step A1, the enzymes used in the enzymatic oxidation reaction are polyphenol oxidase and / or peroxidase, and the amount of enzyme added is 0.1%-0.5% of the raw material mass; The macroporous resin purification uses HPD-100 or AB-8 macroporous resin, and gradient elution is performed using an ethanol aqueous solution with a concentration of 30%-60%. In step A2, the total lipid mass is calculated based on the total mass of soybean lecithin and cholesterol, and the mass of the tea pigment extract accounts for 10%-20% of the total lipid mass; the mass ratio of the photosensitizer to the tea pigment extract is 1:(8-12). The organic solvent is at least one of chloroform, ethanol, methanol, and dichloromethane; the buffer solution is a phosphate buffer solution.

5. The functional feed additive containing tea pigment photoresponsive cationic nanoliposomes according to claim 2, characterized in that, In step A3, the solvent for the cationic chitosan acetate solution is an aqueous acetic acid solution with a concentration of 0.5%-1.5%; In step A4, the homogenization process is performed by probe ultrasound with an ultrasound power of 200-400W and a processing time of 3-10 minutes; the purification process is performed by dialysis or ultracentrifugation.

6. The functional feed additive containing tea pigment photoresponsive cationic nanoliposomes according to claim 1, characterized in that, The excipients, by weight, include the following components: 30-50 parts rice bran, 20-30 parts montmorillonite, 12-20 parts trehalose, 8-16 parts fructooligosaccharides, 3-5 parts choline, 0.2-0.5 parts salt, and 0.8-1.5 parts vitamin-trace element premix.

7. A functional feed additive containing tea pigment photoresponsive cationic nanoliposomes according to claim 6, characterized in that, By weight, the excipients also include at least one of the following components: 0.2-0.4 parts riboflavin, 0.6-1 parts plant essential oil, 5-10 parts plant oil, 2-4 parts β-glucan, 0.1-0.2 parts organic selenium, 0.1-0.3 parts lysine, 0.05-0.2 parts methionine, and 3-5 parts nano-silica.

8. A method for preparing a functional feed additive containing tea pigment photoresponsive cationic nanoliposomes as described in any one of claims 1-7, characterized in that, The preparation method includes the following steps: S1. After mixing the components of the excipients, granulate them at 35-45℃ and cool them to room temperature; S2. Mix tea pigment photoresponsive cationic nanoliposomes with microcrystalline cellulose at a weight of 10-15% to prepare a functional premix. S3. Add the functional premix to the excipient granules in step S1 and mix at 35-45℃ for 5-15 minutes. S4. Add a surfactant aqueous solution at a ratio of 0.8-1.2% of the weight of the excipients, and continue mixing for 5-10 minutes; then dry at 40-50℃ until the moisture content is 10-12%, cool in the dark, and seal in packaging.

9. The preparation method according to claim 8, characterized in that, In step S4, the surfactant is a food-grade sucrose fatty acid ester or Tween-80, and its aqueous solution concentration is 5-10%.

10. The application of the functional feed additive containing tea pigment photoresponsive nanoliposomes as described in any one of claims 1-7 in chicken farming, characterized in that, The functional feed additives are added to the chicken's staple food, and the chicken is irradiated with blue light with a wavelength of 400-480nm for 5-30 minutes every day.

Images