Preparation method and application of composite multi-dimensional nanoemulsion

Abstract

The application belongs to the technical field of feed additives, and discloses a preparation method and application of a compound multi-vitamin nanoemulsion, and specifically discloses a compound multi-vitamin nanoemulsion, which comprises a vitamin supplement, an emulsifier, a traditional Chinese medicine immunity enhancer, an amino acid supplement and an antioxidant; the vitamin supplement comprises fat-soluble vitamins and water-soluble vitamins, and the traditional Chinese medicine immunity enhancer comprises an extract of Astragalus membranaceus and / or an extract of Ganoderma lucidum. The application provides a nano compound multi-vitamin with an anti-stress and growth-promoting effect, and the multi-vitamin is combined with Astragalus membranaceus and Ganoderma lucidum to enhance the anti-stress effect and the growth-promoting effect.

Description

Technical Field

[0001] This invention belongs to the field of feed additive technology, specifically relating to a method for preparing and applying a composite multidimensional nanoemulsion. Background Technology

[0002] Vitamins are essential nutrients for animal growth and development, playing a vital role in the breeding process, including maintaining growth, development, and stress resistance. In recent years, the scale of broiler farming in my country has been continuously expanding, gradually transitioning from traditional scattered small-scale farming to large-scale farming. Broilers are characterized by rapid growth and development and vigorous metabolism, resulting in high nutritional requirements. Animals are prone to metabolic disorders when subjected to heat stress, cold stress, and immune stress. Vitamin levels in feed can also decrease due to factors such as mixing, transportation, or storage, leading to insufficient vitamin intake. In all these situations, additional vitamin supplementation is necessary.

[0003] Retinol maintains dark vision and color perception, preserves mucosal integrity and prevents epithelial cell keratinization, promotes and maintains normal bone growth, regulates immune cell proliferation, and stimulates antibody production. Cholecalciferol regulates cell growth, promotes cell regeneration, and enhances calcium and phosphorus absorption and bone development. L-Ascorbic acid helps maintain adequate levels of glucocorticoid secretion, increases energy supply while avoiding immunosuppression, and can also alleviate hypothyroidism. As an important antioxidant in extracellular fluid, it also enhances the body's ability to scavenge oxygen free radicals in tissues, protecting biological membranes from oxidative damage. Tocopherol can maintain relatively stable levels of cortisol, thyroxine, and creatine phosphokinase in serum, reducing metabolic disorders caused by decreased hormone or enzyme levels. As a major lipid-soluble antioxidant on cell membranes, it can protect cell membrane integrity by reducing the production of oxygen free radicals. B vitamins can maintain the integrity of epithelial tissues such as the digestive tract mucosa, maintain gastrointestinal function, digestive juice secretion, and appetite. They are closely related to the metabolism of sugars, proteins, and fats. When the body is under stress, they can promote collagen synthesis, enhance the toughness and strength of blood vessel walls, and reduce stress damage. Due to the harsh breeding environment and the complexity of diseases, relying solely on vitamin supplementation for stress resistance and growth promotion has certain limitations.

[0004] Against the backdrop of antibiotic bans, traditional Chinese medicine (TCM) additives have gained attention due to their advantages of abundant active ingredients and low toxicity, leading to widespread research and application. Several TCM herbs have been proven to possess anti-stress functions: Astragalus membranaceus (Huangqi), sweet in taste and slightly warm in nature, contains polysaccharides that tonify Qi. These polysaccharides can enhance animal immunity, antioxidant capacity, anti-stress ability, and antiviral capabilities. They also improve intestinal structure and promote feed intake and growth, and are currently widely used in animal production as feed additives and immune enhancers. Reishi mushroom (Ganoderma lucidum), a saprophytic fungus, has calming and soothing effects. Its cells contain cellulase systems, allowing for the extraction of crude polysaccharides from TCM herbs by utilizing monosaccharides and fiber. Reishi can also transform or modify certain components of TCM herbs, producing new substances. The combination of Astragalus membranaceus and Reishi mushroom possesses calming and anti-stress effects.

[0005] Chinese invention patent CN201410597086.0 discloses a composite nanoemulsion for livestock and poultry, which has achieved clinical effects in promoting the growth of weaned piglets and improving feed conversion rate. Chinese invention patent CN201910292927 discloses granules of Astragalus polysaccharide ethyl acetate extract, which has achieved clinical effects in combating stress in crucian carp, restoring low feed intake caused by copper sulfate-induced stress and inhibiting trichlorfon-induced rollover in fish. Chinese invention patent CN102919547A discloses a traditional Chinese medicine liquid multivitamin preparation, which utilizes saffron, Rhodiola rosea, lentinan, and multiembryoprotein protease fused with multivitamins to protect the animal liver. Nevertheless, there are few patents related to the anti-stress and growth-promoting effects of multivitamins on broilers, and no reports describe the anti-stress and growth-promoting activities of traditional Chinese medicine composite multivitamin preparations. Therefore, developing a traditional Chinese medicine composite multivitamin nanoemulsion for anti-stress and growth-promoting effects is of great significance for improving the clinical growth performance of broilers.

[0006] Because of the complex composition of traditional Chinese medicine, multidimensional nanoemulsions are prone to instability. Under low-temperature conditions, they are susceptible to demulsification, precipitation, and gelation, which can affect the absorption of fat-soluble vitamins in animals. Unstable multidimensional nanoemulsions are also prone to problems such as adhering to the walls and clogging pipelines during use, causing inconvenience to product use.

[0007] Studies show that adding antifreeze can improve the antifreeze ability of a solution, thereby enhancing its stability. Commonly used antifreeze includes sugar-based antifreeze, chemical solvent antifreeze, and salt-based antifreeze. Sugar-based antifreeze mainly includes gum-based antifreeze, modified starch-based antifreeze, and small-molecule sugar antifreeze. Their molecular structure contains multiple hydroxyl groups, giving them strong hydrophilicity and allowing them to interact with free water in food. Chemical solvent antifreeze mainly includes propylene glycol, glycerol, and dimethyl sulfoxide (DMSO). These substances themselves can act as antifreeze, lowering the freezing point of the solution. Salt-based antifreeze mainly includes sodium chloride, phosphates, tripolyphosphates, and hexametaphosphates, among other complex phosphate compounds. When livestock and poultry experience diarrhea, stress, or other causes leading to electrolyte imbalances, supplementing with salt-containing substances can alleviate water loss and thus relieve stress.

[0008] Achieving cryoprotection with a single antifreeze agent requires a large dosage and can lead to toxicity. Therefore, it is worthwhile to explore the combined use of antifreeze agents with different mechanisms of action. This approach aims to enhance cryoprotection while reducing the dosage of any single antifreeze agent in the system. Studies have shown that salts, as antifreeze agents, exhibit a significant synergistic effect with sugars. This can reduce the loss of thawed juices and total extruded juices in prepared meatballs, improve their cohesiveness, elasticity, and adhesiveness, and enhance their color, thereby improving the cryoprotection effect of the food. Consequently, these two agents are often used in combination. Chinese invention patent CN201210250809.0 discloses a composite antifreeze solution. By combining DMEM culture medium, dimethyl sulfoxide (DMSO), glycerol, and trehalose, it enables the pretreatment of fresh human amniotic membranes without the need for programmed pre-cooling before direct storage in liquid nitrogen, effectively maintaining the amniotic membrane's viability for a long period. However, the DMSO used in this antifreeze has safety issues. Therefore, it is necessary to develop a composite antifreeze agent containing polysaccharides, salts, and chemical solvents to enhance the stability of the nanosolution and to supplement polysaccharides and electrolytes. Summary of the Invention

[0009] The first objective of this invention is to provide a composite multidimensional nanoemulsion.

[0010] The second objective of this invention is to provide a method for preparing the composite multidimensional nanoemulsion of the first aspect of this invention.

[0011] The third objective of this invention is to provide the application of the composite multidimensional nanoemulsion of the first aspect of this invention in livestock and poultry farming.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0013] Current intensive and large-scale animal husbandry models, due to the large number of animals and high density, result in frequent stress on animals due to the interactions between the environment and individuals, and between individuals themselves. This leads to decreased animal resistance and production performance, which is one of the core problems that needs to be addressed in production. To solve these problems, this invention develops a nanoemulsion solution combining multiple vitamins and traditional Chinese medicine, which improves growth performance by enhancing the stress resistance of the entire population. The combined use of multivitamins and Astragalus and Ganoderma lucidum polysaccharides enhances the anti-stress and growth-promoting effects.

[0014] In a first aspect, the present invention provides a composite multidimensional nanoemulsion comprising a vitamin supplement, an emulsifier, a traditional Chinese medicine immune enhancer, an amino acid supplement, and an antioxidant; wherein the vitamin supplement comprises fat-soluble vitamins and water-soluble vitamins, and the traditional Chinese medicine immune enhancer comprises Astragalus membranaceus extract and / or Ganoderma lucidum extract.

[0015] In some embodiments of the present invention, the Astragalus extract or Ganoderma extract is an aqueous extract of Astragalus or Ganoderma.

[0016] In some embodiments of the present invention, the preparation method of the Astragalus extract or Ganoderma extract includes the following steps: Astragalus and Ganoderma are soaked in water at 45-85°C for 30-40 min; boiled for 50-70 min, and the solid and liquid are separated to obtain filtrate and residue; the residue is boiled with water for 50-70 min, and the solid and liquid are separated to obtain filtrate; the filtrates obtained in the two steps are combined and concentrated to obtain Astragalus extract or Ganoderma extract.

[0017] In some embodiments of the present invention, the preparation method of the Astragalus extract or Ganoderma extract includes the following steps: Astragalus and Ganoderma are soaked in water at 45-55°C for 30-40 min; boiled for 50-70 min, and the solid and liquid are separated to obtain filtrate and residue; the residue is boiled with water for 50-70 min, and the solid and liquid are separated to obtain filtrate; the filtrates obtained in the two steps are combined and concentrated to obtain Astragalus extract or Ganoderma extract.

[0018] The extraction methods of Astragalus polysaccharides and Ganoderma lucidum obtained by the present invention can improve the yield of crude polysaccharides.

[0019] In some embodiments of the present invention, the concentration is to concentrate the medicinal material (i.e., Astragalus membranaceus or Ganoderma lucidum) to the liquid (i.e., filtrate) to a mass ratio of (1-2):1.

[0020] In some embodiments of the present invention, the fat-soluble vitamin includes at least one of retinol, cholecalciferol, and tocopherol.

[0021] The fat-soluble vitamin raw materials of this invention are all liquid raw materials, which avoids the precipitation problem of other excipients in liquid preparations, making addition more convenient and the preparation process simpler.

[0022] In some embodiments of the present invention, the water-soluble vitamin includes at least one of thiamine nitrate, pyridoxine, and L-ascorbic acid.

[0023] In some embodiments of the present invention, the amino acid supplement includes at least one of lysine, hydroxymethionine, and threonine.

[0024] In some preferred embodiments of the present invention, the amino acid supplement is hydroxymethionine.

[0025] In some embodiments of the present invention, the antioxidant includes at least one of propyl gallate, tert-butylhydroquinone, 2,6-di-tert-butyl-4-methylphenol, butyl hydroxyanisole, sodium metabisulfite, sodium sulfite, and L-cysteine.

[0026] In some embodiments of the present invention, the emulsifier includes at least one of polysorbate and poloxamer.

[0027] In some embodiments of the present invention, the composite multidimensional nanoemulsion further includes an ion supplement and an antifreeze agent.

[0028] Traditional Chinese medicine and multidimensional formulations have complex components, which can easily cause instability in nanosystems. Under low-temperature conditions, this can lead to demulsification, precipitation, and gelation, resulting in issues such as residue buildup and pipe blockage during use, affecting the user experience. To improve the antifreeze effect, this invention further adds antifreeze agents and ion protectants (i.e., ion supplements) to the composite multidimensional nanoemulsion. The combined use of Astragalus and Ganoderma lucidum polysaccharides, ion protectants, and chemical antifreeze agents enhances the antifreeze effect, improves solution stability, and promotes absorption by the body.

[0029] In some embodiments of the present invention, the ion supplement includes at least one of sodium chloride, amino acid chelated calcium, L-threonate calcium, calcium bicarbonate, calcium gluconate and calcium lactate.

[0030] In some preferred embodiments of the present invention, the ion supplement is sodium chloride.

[0031] In some embodiments of the present invention, the antifreeze solvent is at least one of propylene glycol, glycerol, and polyethylene glycol.

[0032] In some preferred embodiments of the present invention, the antifreeze solvent is propylene glycol.

[0033] In some embodiments of the present invention, the composite multidimensional nanoemulsion comprises the following components in parts by weight: 100-150 parts vitamin supplement, 130-160 parts emulsifier, 100-200 parts traditional Chinese medicine immune enhancer, 60-80 parts amino acid supplement, 5-15 parts antioxidant, 60-80 parts ion supplement and 120-160 parts antifreeze agent.

[0034] In some preferred embodiments of the present invention, the composite multidimensional nanoemulsion comprises the following components in parts by weight: 125-150 parts vitamin supplement, 150-160 parts emulsifier, 100-200 parts traditional Chinese medicine immune enhancer, 75-80 parts amino acid supplement, 10-15 parts antioxidant, 60-75 parts ion supplement and 150-160 parts antifreeze agent.

[0035] In a second aspect, the present invention provides a method for preparing the composite multidimensional nanoemulsion of the first aspect of the present invention, comprising the following steps:

[0036] (1) Mix fat-soluble vitamins and emulsifiers and stir at 10,000 rpm to 15,000 rpm for 20 min to 30 min to obtain a fat-soluble solution;

[0037] (2) Dissolve the amino acid supplement, ion supplement, water-soluble vitamins and antioxidants in water to obtain a water-soluble solution;

[0038] (3) Add the lipid-soluble solution from step (1) to the water-soluble solution from step (2), stir until the solution is clear, and obtain the O / W type composite multidimensional nanoemulsion intermediate solution;

[0039] (4) Mix the Chinese medicine immune enhancer with the O / W type composite multidimensional nanoemulsion intermediate solution, add an antifreeze agent, and stir to obtain the composite multidimensional nanoemulsion.

[0040] In some embodiments of the present invention, the antifreeze is added while stirring to prevent excessively high local concentrations.

[0041] In some embodiments of the present invention, the pH of the composite multidimensional nanoemulsion solution is adjusted to 4.0-5.5 after adding an antifreeze and stirring, and then water is added to bring the volume to 1 L.

[0042] In a third aspect, the present invention provides the application of the composite multidimensional nanoemulsion of the first aspect of the present invention in livestock and poultry farming.

[0043] In some embodiments of the present invention, the composite multidimensional nanoemulsion is added to the drinking water or diet of livestock and poultry.

[0044] The beneficial effects of this invention are:

[0045] This invention provides a nanocomposite multidimensional agent with anti-stress and growth-promoting effects. The multidimensional agent is used in combination with Astragalus membranaceus and Ganoderma lucidum to enhance the anti-stress and growth-promoting effects.

[0046] Further, the combined use of Astragalus polysaccharides, multivitamins, ion protectants, and chemical antifreeze agents enhances the antifreeze effect, improves solution stability, reduces freezing, gelation, precipitation, and exudation caused by low winter temperatures, improves sample adaptability, and promotes absorption by the body.

[0047] This invention uses nanoemulsion technology to prepare composite multidimensional nanoemulsions, which can solve the problem of blocked water lines in clinical settings. The composite multidimensional emulsion formulation has better water solubility than the powder formulation and does not precipitate.

[0048] This invention combines the physiological characteristics of broilers with the feed ratio in winter, and can add rich minerals, various vitamins and trace elements to resist stress and promote animal growth, so that broilers can still maintain a good feed intake in winter. Attached Figure Description

[0049] Figure 1 The images show the state of the samples after preparation in Examples 3 and 5.

[0050] Figure 2 To investigate the effects of different groups of immune stress on the expression of reactive oxygen species (ROS) in chicken serum. Detailed Implementation

[0051] The present invention will be further described in detail below through specific embodiments.

[0052] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0053] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0054] Example 1

[0055] A composite multidimensional nanoemulsion suitable for anti-stress and growth-promoting effects is made from the following components: 150 g of Astragalus membranaceus aqueous extract, 4.11 g of retinol (10,000 IU / g), 2 g of cholecalciferol (1,000,000 IU / g), 5 g of tocopherol (98%), 150 g of emulsifier mixture (75 g of polysorbate-20 + 75 g of poloxamer 407), 75 g of amino acid supplement (50 g of hydroxymethionine + 25 g of lysine), 75 g of ion supplement (50 g of sodium chloride + 25 g of calcium gluconate), 125 g of water-soluble vitamin (50 g of thiamine nitrate + 25 g of pyridoxine + 50 g of L-ascorbic acid), 10 g of antioxidant (5 g of propyl gallate + 5 g of L-cysteine), 150 g of antifreeze propylene glycol, and 253.89 g of deionized water;

[0056] The preparation method of the Astragalus aqueous extract includes the following steps: 100 g of Astragalus is dried, pulverized, and passed through a 40-mesh sieve. It is then soaked in 10 times its volume of water (50±5℃) for 30 min, followed by boiling and extraction for 1 h. The extract is filtered to obtain the filtrate and residue. Eight times its volume of water is added to the residue, and the extract is boiled and extracted for 1 h. The filtrate is then filtered. The two filtrates are combined, centrifuged, and concentrated under reduced pressure until the mass ratio of the medicinal material to the extract is 1.5:1, thus obtaining the Astragalus aqueous extract.

[0057] The preparation method of the above-mentioned composite multidimensional nanoemulsion includes the following steps:

[0058] (1) Take the amount of fat-soluble vitamins in the formula: retinol, cholecalciferol, tocopherol and emulsifier mixture, and shear it at high speed for 25 minutes at 12000 rpm using a homogenizer to obtain a fat-soluble solution for later use;

[0059] (2) Take 30% deionized water for later use, add amino acid supplement, ion supplement, water-soluble vitamin and antioxidant and use 1500KW ultrasonic to dissolve to obtain water-soluble solution for later use;

[0060] (3) Add the lipid-soluble solution obtained in step (1) to the water-soluble solution obtained in step (2) and shear at high speed for 45 min until the solution is clear to obtain the O / W type composite multidimensional nanoemulsion intermediate solution;

[0061] (4) Add the water extract of Astragalus membranaceus to the intermediate solution obtained in step (3) while stirring. Then add the antifreeze propylene glycol and stir. Adjust the pH of the solution to 4.0-5.5 using pH adjuster (0.1 mol / L dilute hydrochloric acid). Finally, add water to make up to 1 L and filter through a plate filter membrane to obtain the composite multidimensional nanoemulsion.

[0062] Example 2

[0063] A composite multidimensional nanoemulsion suitable for anti-stress and growth-promoting effects is made from the following ingredients: 150 g of Ganoderma lucidum water extract, 4.11 g of retinol (1.7 million IU / g), 2 g of cholecalciferol (1 million IU / g), 5 g of tocopherol (98%), 150 g of emulsifier mixture (75 g of polysorbate-20 + 75 g of poloxamer 407), 75 g of amino acid supplement (50 g of hydroxymethionine + 25 g of lysine), 75 g of ion supplement (50 g of sodium chloride + 25 g of calcium gluconate), 125 g of water-soluble vitamin (50 g of thiamine nitrate + 25 g of pyridoxine + 50 g of L-ascorbic acid), 10 g of antioxidant (5 g of propyl gallate + 5 g of L-cysteine), 150 g of antifreeze propylene glycol, and 253.89 g of deionized water;

[0064] The preparation method of the Ganoderma lucidum water extract includes the following steps: 100 g of Ganoderma lucidum is dried, pulverized, and passed through a 40-mesh sieve. It is then soaked in 10 times its volume of water (50±5℃) for 30 min, followed by boiling and extraction for 1 h. The mixture is filtered to obtain the filtrate and residue. Eight times its volume of water is added to the residue, and the mixture is boiled and extracted for 1 h. The filtrate is then filtered. The two filtrates are combined, centrifuged, and concentrated under reduced pressure until the mass ratio of the medicinal material to the liquid is 1.5:1, thus obtaining the Ganoderma lucidum water extract.

[0065] The preparation method of the above-mentioned composite multidimensional nanoemulsion includes the following steps:

[0066] (1) Take the amount of fat-soluble vitamins in the formula: retinol, cholecalciferol, tocopherol and emulsifier mixture, and shear it at high speed for 25 minutes at 12000 rpm using a homogenizer to obtain a fat-soluble solution for later use;

[0067] (2) Take 30% deionized water for later use, add amino acid supplement, ion supplement, water-soluble vitamin and antioxidant and use 1500KW ultrasonic to dissolve to obtain water-soluble solution for later use;

[0068] (3) Add the lipid-soluble solution obtained in step (1) to the water-soluble solution obtained in step (2) and shear at high speed for 45 min until the solution is clear to obtain the O / W type composite multidimensional nanoemulsion intermediate solution;

[0069] (4) Add the Ganoderma lucidum water extract to the intermediate solution obtained in step (3) while stirring. Then add the antifreeze propylene glycol and stir. Adjust the pH of the solution to 4.0-5.5 using pH adjuster (0.1 mol / L dilute hydrochloric acid). Finally, add water to make up to 1 L and filter through a plate filter membrane to obtain the composite multidimensional nanoemulsion.

[0070] Example 3

[0071] A composite multidimensional nanoemulsion suitable for anti-stress and growth-promoting effects is made from the following components: 150 g of Astragalus membranaceus and Ganoderma lucidum water extract, 4.11 g of retinol (1.7 million IU / g), 2 g of cholecalciferol (1 million IU / g), 5 g of tocopherol (98%), 150 g of emulsifier mixture (75 g of polysorbate-20 + 75 g of poloxamer 407), 75 g of amino acid supplement (50 g of hydroxymethionine + 25 g of lysine), 75 g of ion supplement (50 g of sodium chloride + 25 g of calcium gluconate), 125 g of water-soluble vitamin (50 g of thiamine nitrate + 25 g of pyridoxine + 50 g of L-ascorbic acid), 10 g of antioxidant (5 g of propyl gallate + 5 g of L-cysteine), 150 g of antifreeze propylene glycol, and 253.89 g of deionized water;

[0072] The preparation method of the Astragalus and Ganoderma lucidum water extract includes the following steps: 50 g each of Astragalus and Ganoderma lucidum are dried, pulverized, and passed through a 40-mesh sieve. They are soaked in 10 times their volume of water (50±5℃) for 30 min, then boiled for 1 h, and filtered to obtain the filtrate and residue. Eight times their volume of water is added to the residue, and the mixture is boiled for 1 h, and filtered again to obtain the filtrate. The two filtrates are combined, centrifuged, and concentrated under reduced pressure until the mass ratio of medicinal material to liquid is 1.5:1, thus obtaining the Astragalus and Ganoderma lucidum water extract.

[0073] The preparation method of the above-mentioned composite multidimensional nanoemulsion includes the following steps:

[0074] (1) Take the amount of fat-soluble vitamins in the formula: retinol, cholecalciferol, tocopherol and emulsifier mixture, and shear it at high speed for 25 minutes at 12000 rpm using a homogenizer to obtain a fat-soluble solution for later use;

[0075] (2) Take 30% deionized water for later use, add amino acid supplement, ion supplement, water-soluble vitamin and antioxidant and use 1500KW ultrasonic to dissolve to obtain water-soluble solution for later use;

[0076] (3) Add the lipid-soluble solution obtained in step (1) to the water-soluble solution obtained in step (2) and shear at high speed for 45 min until the solution is clear to obtain the O / W type composite multidimensional nanoemulsion intermediate solution;

[0077] (4) Add the water extract of Astragalus membranaceus and Ganoderma lucidum to the intermediate solution obtained in step (3) while stirring to obtain a mixed solution of traditional Chinese medicine nanoparticles. Then add the antifreeze agent propylene glycol and stir. Adjust the pH of the solution to 4.0-5.5 using pH adjuster (0.1 mol / L dilute hydrochloric acid). Finally, add water to make up to 1 L and filter through a plate filter membrane to obtain the composite multidimensional nanoemulsion.

[0078] Example 4

[0079] A composite multidimensional nanoemulsion suitable for anti-stress and growth-promoting effects is made from the following components: 150 g of Astragalus membranaceus and Ganoderma lucidum water extract, 4.11 g of retinol (1.7 million IU / g), 2 g of cholecalciferol (1 million IU / g), 5 g of tocopherol (98%), 150 g of emulsifier mixture (75 g of polysorbate-20 + 75 g of poloxamer 407), 75 g of amino acid supplement (50 g of hydroxymethionine + 25 g of lysine), 75 g of ion supplement (50 g of sodium chloride + 25 g of calcium gluconate), 125 g of water-soluble vitamin (50 g of thiamine nitrate + 25 g of pyridoxine + 50 g of L-ascorbic acid), 10 g of antioxidant (5 g of propyl gallate + 5 g of L-cysteine), 150 g of antifreeze propylene glycol, and 253.89 g of deionized water;

[0080] The preparation method of the Astragalus and Ganoderma lucidum water extract includes the following steps: 50 g each of Astragalus and Ganoderma lucidum are dried, pulverized, and passed through a 40-mesh sieve. They are soaked in 10 times their volume of water (80±5℃) for 30 min, then boiled for 1 h, and filtered to obtain the filtrate and residue. Eight times their volume of water is added to the residue, and the mixture is boiled for 1 h, and filtered again to obtain the filtrate. The two filtrates are combined, centrifuged, and concentrated under reduced pressure until the mass ratio of medicinal material to liquid is 1.5:1, thus obtaining the Astragalus and Ganoderma lucidum water extract.

[0081] The preparation method of the above-mentioned composite multidimensional nanoemulsion includes the following steps:

[0082] (1) Take the amount of fat-soluble vitamins in the formula: retinol, cholecalciferol, tocopherol and emulsifier mixture, and shear it at high speed for 25 minutes at 12000 rpm using a homogenizer to obtain a fat-soluble solution for later use;

[0083] (2) Take 30% deionized water for later use, add amino acid supplement, ion supplement, water-soluble vitamin and antioxidant and use 1500KW ultrasonic to dissolve to obtain water-soluble solution for later use;

[0084] (3) Add the lipid-soluble solution obtained in step (1) to the water-soluble solution obtained in step (2) and shear at high speed for 45 min until the solution is clear to obtain the O / W type composite multidimensional nanoemulsion intermediate solution;

[0085] (4) Add the water extract of Astragalus membranaceus and Ganoderma lucidum to the intermediate solution obtained in step (3) while stirring. Then add the antifreeze agent propylene glycol and stir. Adjust the pH of the solution to 4.0-5.5 using pH adjuster (0.1 mol / L dilute hydrochloric acid). Finally, add water to make up to 1 L and filter through a plate filter membrane to obtain the composite multidimensional nanoemulsion.

[0086] Example 5

[0087] A composite multidimensional nanoemulsion suitable for anti-stress and growth-promoting effects is made from the following components: 150 g of Astragalus membranaceus and Ganoderma lucidum water extract, 4.11 g of retinol (1.7 million IU / g), 2 g of cholecalciferol (1 million IU / g), 5 g of tocopherol (98%), 150 g of emulsifier mixture (75 g of polysorbate-20 + 75 g of poloxamer 407), 75 g of amino acid supplement (50 g of hydroxymethionine + 25 g of lysine), 75 g of ion supplement (50 g of sodium chloride + 25 g of calcium gluconate), 125 g of water-soluble vitamin (50 g of thiamine nitrate + 25 g of pyridoxine + 50 g of L-ascorbic acid), 10 g of antioxidant (5 g of propyl gallate + 5 g of L-cysteine), 150 g of antifreeze propylene glycol, and 253.89 g of deionized water;

[0088] The preparation method of the Astragalus and Ganoderma lucidum water extract includes the following steps: 50 g each of Astragalus and Ganoderma lucidum are dried, pulverized, and passed through a 40-mesh sieve. They are soaked in 10 times their volume of water (50±5℃) for 30 min, then boiled for 1 h, and filtered to obtain the filtrate and residue. Eight times their volume of water is added to the residue, and the mixture is boiled for 1 h, and filtered again to obtain the filtrate. The two filtrates are combined, centrifuged, and concentrated under reduced pressure until the mass ratio of medicinal material to liquid is 1.5:1, thus obtaining the Astragalus and Ganoderma lucidum water extract.

[0089] The preparation method of the above-mentioned composite multidimensional nanoemulsion includes the following steps:

[0090] (1) Take the amount of fat-soluble vitamins in the formula: retinol, cholecalciferol, tocopherol, antifreeze propylene glycol and emulsifier mixture, and shear it at high speed with a homogenizer for 25 min at 12000 rpm to obtain a fat-soluble solution for later use;

[0091] (2) Take 30% deionized water for later use, add amino acid supplement, ion supplement, water-soluble vitamin and antioxidant and use 1500KW ultrasonic to dissolve to obtain water-soluble solution for later use;

[0092] (3) Add the lipid-soluble solution obtained in step (1) to the water-soluble solution obtained in step (2) and shear at high speed for 45 min until the solution is clear to obtain the O / W type composite multidimensional nanoemulsion intermediate solution;

[0093] (4) Add the water extract of Astragalus membranaceus and Ganoderma lucidum to the intermediate solution obtained in step (3) while stirring. Adjust the pH of the solution to 4.0-5.5 using pH adjuster (0.1 mol / L dilute hydrochloric acid). Finally, add water to make up to 1 L and filter through a plate filter membrane to obtain the composite multidimensional nanoemulsion.

[0094] Example 6

[0095] A composite multidimensional nanoemulsion suitable for anti-stress and growth-promoting effects is made from the following components: retinol (1.7 million IU / g) 4.11 g, cholecalciferol (1 million IU / g) 2 g, tocopherol (98%) 5 g, emulsifier mixture (polysorbate-20 75 g + poloxamer 407 75 g) 150 g, amino acid supplement (hydroxymethionine 50 g + lysine 25 g) 75 g, water-soluble vitamin (thiamine nitrate 50 g + pyridoxine 25 g + L-ascorbic acid 50 g) 125 g, antioxidant (propyl gallate 5 g + L-cysteine ​​5 g) 10 g, and deionized water 628.89 g;

[0096] The preparation method of the above-mentioned composite multidimensional nanoemulsion includes the following steps:

[0097] (1) Take the amount of fat-soluble vitamins in the formula: retinol, cholecalciferol, tocopherol and emulsifier mixture, and shear it at high speed for 25 minutes at 12000 rpm using a homogenizer to obtain a fat-soluble solution for later use;

[0098] (2) Take 30% deionized water for later use, add amino acid supplements, water-soluble vitamins and antioxidants and dissolve them using 1500KW ultrasonication to obtain a water-soluble solution for later use;

[0099] (3) Add the lipid-soluble solution obtained in step (1) to the water-soluble solution obtained in step (2) and shear at high speed for 45 min until the solution is clear to obtain the intermediate solution of O / W type composite multidimensional nanoemulsion solution; adjust the pH of the solution to 4.0 to 5.5 using pH adjuster (0.1 mol / L dilute hydrochloric acid), and finally add water to make up to 1 L. Filter through plate filter membrane to obtain composite multidimensional nanoemulsion.

[0100] Example 7

[0101] A composite multidimensional nanoemulsion suitable for anti-stress and growth-promoting effects is made from the following ingredients: 150 g of Astragalus membranaceus and Ganoderma lucidum water extract, 4.11 g of retinol (1.7 million IU / g), 2 g of cholecalciferol (1 million IU / g), 5 g of tocopherol (98%), 150 g of emulsifier mixture (75 g of polysorbate-20 + 75 g of poloxamer 407), 75 g of amino acid supplement (50 g of hydroxymethionine + 25 g of lysine), 125 g of water-soluble vitamin (50 g of thiamine nitrate + 25 g of pyridoxine + 50 g of L-ascorbic acid), 10 g of antioxidant (5 g of propyl gallate + 5 g of L-cysteine), and 478.89 g of deionized water;

[0102] The preparation method of the Astragalus and Ganoderma lucidum water extract includes the following steps: 50 g each of Astragalus and Ganoderma lucidum are dried, pulverized, and passed through a 40-mesh sieve. They are soaked in 10 times their volume of water (50±5℃) for 30 min, then boiled for 1 h, and filtered to obtain the filtrate and residue. Eight times their volume of water is added to the residue, and the mixture is boiled for 1 h, and filtered again to obtain the filtrate. The two filtrates are combined, centrifuged, and concentrated under reduced pressure until the mass ratio of medicinal material to liquid is 1.5:1, thus obtaining the Astragalus and Ganoderma lucidum water extract.

[0103] The preparation method of the above-mentioned composite multidimensional nanoemulsion includes the following steps:

[0104] (1) Take the amount of fat-soluble vitamins in the formula: retinol, cholecalciferol, tocopherol and emulsifier mixture, and shear it at high speed for 25 minutes at 12000 rpm using a homogenizer to obtain a fat-soluble solution for later use;

[0105] (2) Take 30% deionized water for later use, add amino acid supplements, water-soluble vitamins and antioxidants and dissolve them using 1500KW ultrasonication to obtain a water-soluble solution for later use;

[0106] (3) Add the lipid-soluble solution obtained in step (1) to the water-soluble solution obtained in step (2) and shear at high speed for 45 min until the solution is clear to obtain the O / W type composite multidimensional nanoemulsion intermediate solution;

[0107] (4) Add the water extract of Astragalus membranaceus and Ganoderma lucidum to the intermediate solution obtained in step (3) while stirring. Adjust the pH of the solution to 4.0-5.5 using pH adjuster (0.1 mol / L dilute hydrochloric acid). Finally, add water to make up to 1 L and filter through a plate filter membrane to obtain the composite multidimensional nanoemulsion.

[0108] Example 8

[0109] A composite multidimensional nanoemulsion suitable for anti-stress and growth-promoting effects is made from the following ingredients: 150 g of Astragalus membranaceus and Ganoderma lucidum water extract, 4.11 g of retinol (1.7 million IU / g), 2 g of cholecalciferol (1 million IU / g), 5 g of tocopherol (98%), 150 g of emulsifier mixture (75 g of polysorbate-20 + 75 g of poloxamer 407), 75 g of amino acid supplement (50 g of hydroxymethionine + 25 g of lysine), 75 g of ion supplement (50 g of sodium chloride + 25 g of calcium gluconate), 125 g of water-soluble vitamin (50 g of thiamine nitrate + 25 g of pyridoxine + 50 g of L-ascorbic acid), 10 g of antioxidant (5 g of propyl gallate + 5 g of L-cysteine), and 403.89 g of deionized water;

[0110] The preparation method of the Astragalus and Ganoderma lucidum water extract includes the following steps: 50 g each of Astragalus and Ganoderma lucidum are dried, pulverized, and passed through a 40-mesh sieve. They are soaked in 10 times their volume of water (50±5℃) for 30 min, then boiled for 1 h, and filtered to obtain the filtrate and residue. Eight times their volume of water is added to the residue, and the mixture is boiled for 1 h, and filtered again to obtain the filtrate. The two filtrates are combined, centrifuged, and concentrated under reduced pressure until the mass ratio of medicinal material to liquid is 1.5:1, thus obtaining the Astragalus and Ganoderma lucidum water extract.

[0111] The preparation method of the above-mentioned composite multidimensional nanoemulsion includes the following steps:

[0112] (1) Take the amount of fat-soluble vitamins in the formula: retinol, cholecalciferol, tocopherol and emulsifier mixture, and shear it at high speed for 25 minutes at 12000 rpm using a homogenizer to obtain a fat-soluble solution for later use;

[0113] (2) Take 30% deionized water for later use, add amino acid supplement, ion supplement, water-soluble vitamin and antioxidant and use 1500KW ultrasonic to dissolve to obtain water-soluble solution for later use;

[0114] (3) Add the lipid-soluble solution obtained in step (1) to the water-soluble solution obtained in step (2) and shear at high speed for 45 min until the solution is clear to obtain the O / W type composite multidimensional nanoemulsion intermediate solution;

[0115] (4) Add the water extract of Astragalus membranaceus and Ganoderma lucidum to the intermediate solution obtained in step (3) while stirring. Adjust the pH of the solution to 4.0-5.5 using pH adjuster (0.1 mol / L dilute hydrochloric acid). Finally, add water to make up to 1 L and filter through a plate filter membrane to obtain the composite multidimensional nanoemulsion.

[0116] Effect Example

[0117] 1. Determination of crude polysaccharide content in Astragalus membranaceus extract and Ganoderma lucidum extract

[0118] The crude polysaccharide content in Astragalus extract, Ganoderma lucidum extract, and Astragalus-Ganoderma lucidum extract in Examples 1-3 was determined by ultraviolet spectrophotometry to investigate the effect of different extraction conditions on the crude polysaccharide content in Astragalus extract and Ganoderma lucidum extract.

[0119] The results are shown in Table 1. Among the four extractions, the highest crude polysaccharide content (109.78 mg / mL) was obtained from the extraction of Astragalus membranaceus and Ganoderma lucidum after soaking at 50±5℃ for 30 min. Comparison of Examples 3 and 4 shows that the crude polysaccharide content is closely related to the temperature during extraction. Therefore, extracting Astragalus membranaceus and Ganoderma lucidum at a suitable temperature (50±5℃) can improve the extraction efficiency of crude polysaccharides.

[0120] Table 1. Crude polysaccharide content under different extraction methods

[0121]

[0122] 2. The effect of the order of addition of antifreeze propylene glycol on the appearance of the sample.

[0123] The composite multidimensional nanoemulsions of Examples 3 and 5 were left to stand and their appearance was observed. The results are shown in Table 2 and... Figure 1 As shown, adding propylene glycol as an antifreeze agent in the final step of preparing the composite multidimensional nanoemulsion (Example 3) is superior to adding it to a lipid-soluble solution (Example 5) in that the composite multidimensional nanoemulsion can remain clear, indicating that adding propylene glycol as an antifreeze agent in the final step of preparing the composite multidimensional nanoemulsion can improve the stability of the composite multidimensional nanoemulsion.

[0124] Table 2. Effect of the order of propylene glycol addition on the appearance of the samples.

[0125]

[0126] 3. Freeze-resistance test

[0127] The composite multidimensional nanoemulsions of Examples 3 and 6-8 were placed in a refrigerator at -20°C for antifreeze testing, and the flowability of the samples was observed at different times.

[0128] The results are shown in Table 3. The antifreeze properties of the composite multidimensional nanoemulsion in Example 3 are significantly better than those in Examples 6-8, indicating that the combination of polysaccharide (Astragalus membranaceus and Ganoderma lucidum water extract) + ion supplement + propylene glycol can enhance the antifreeze properties of the solution.

[0129] Table 3. Effects of different antifreeze combinations on the antifreeze properties of the samples.

[0130]

[0131] 4. Clinical application efficacy experiment

[0132] 4.1 Test Materials

[0133] Blank group: Ordinary drinking water;

[0134] Drug control group (i.e., competitor group): Commercially available compound nanoemulsion (purchased from a veterinary drug store, product standard number: Q / 371102SHN051-2019);

[0135] Trivalent inactivated vaccine: Purchased from a veterinary drug store, approval number: Veterinary Drug Production Permit No. 150252161;

[0136] Self-developed group: Composite multidimensional nanoemulsions of Examples 3, 7, and 8;

[0137] Reactive oxygen species detection kit (Yisheng Biotechnology, catalog number: 50101ES01).

[0138] 4.2 Test Methods

[0139] 4.2.1 Experimental grouping and drug administration

[0140] Sixty hundred healthy 27-day-old broilers of similar weight were randomly divided into six groups of 100 each. The blank control group received normal drinking water without any additives or vaccinations. The vaccine group received normal drinking water without any additives and was vaccinated after four days of free access to water. The drug control group and the vaccines from Examples 3, 7, and 8 were diluted at a ratio of 1 mL / L and added to the chickens' drinking water. After four days of free access to water, each group was vaccinated with a trivalent inactivated vaccine against Newcastle disease, infectious bronchitis, and avian influenza (H9 subtype) according to the immunization schedule (Table 4). Body weight, feed intake, and survival rate were recorded three days after administration (week 1) and ten days after administration (week 2). Average weight gain, average feed intake, and feed conversion ratio were calculated for each stage.

[0141] Table 4 Grouping and Administration of Experimental Animals

[0142]

[0143] 4.2.2 Measurement Indicators

[0144] (1) Production performance indicators: average weekly weight gain and feed conversion ratio

[0145] Survival rate = [(Total number of chickens in the group - Number of dead chickens in the group / Total number of chickens in the group)] × 100%;

[0146] (One week) Feed conversion ratio = Average weekly weight gain / Average weekly feed intake;

[0147] Average feed intake = Average weekly feed intake / Total number of chickens in the group;

[0148] (2) Biochemical indicators: Determination of reactive oxygen species (ROS) in chicken serum

[0149] Ten days after medication (second week), venous blood was randomly collected from the wings of four chickens in each group. The blood was centrifuged at 2000 rpm for 5 minutes, and the serum was collected for later use. The reactive oxygen species (ROS) assay was performed according to the instructions of the ROS detection kit.

[0150] 4.3 Test Results

[0151] 4.3.1 Production performance measurement

[0152] As shown in Table 5, the addition of the composite multidimensional nanoemulsion prepared in Example 3 of this invention to the drinking water, compared with the blank control group, increased the weekly weight gain of broilers by 21.4 g and reduced the weekly feed conversion ratio by 0.27 after 3 days of administration; after 10 days of administration, the average weekly weight gain during the growth period increased by 44 g and reduced the weekly feed conversion ratio by 0.17. P All values ​​were <0.05, indicating significant differences. Compared with commercially available compound nanoemulsions, after 3 days of administration, it increased the weekly weight gain of broilers by 18.74 g and decreased the weekly feed conversion ratio by 0.27; after 10 days of administration, the average weekly body weight during the growth period increased by 42.36 g and decreased the weekly feed conversion ratio by 0.15. P The values ​​were all >0.05, indicating no significant difference. This suggests that the composite multidimensional nanoemulsion of Example 3 can achieve significant growth promotion and cost reduction effects, and has a certain improvement on the production performance of broilers. Compared with conventional compound nanoemulsions, it can achieve better growth promotion effects and better production economy.

[0153] Table 5. Average feed intake, weight gain, and feed conversion ratio after drug administration in different groups.

[0154]

[0155] Note: 'a' indicates that the weekly weight gain of the sample group was significantly different from that of the control group, p < 0.05.

[0156] As shown in Table 6, no chickens died in the control group during the monitoring period, while chickens in the vaccine group experienced varying degrees of mortality 3 days and 10 days after vaccination. This indicates that immunization-induced stress has a certain impact on the survival rate of the flock. Compared with commercially available compound nanoemulsions, the application of the composite multidimensional nanoemulsions in Examples 3 and 8 increased the survival rate by 2%. The above results indicate that the composite multidimensional nanoemulsions in Examples 3 and 7-8 can achieve significant anti-immune stress effects, effectively protect against stress-induced mortality after vaccination, and improve animal survival rates.

[0157] Table 6. Effects of immune stress on survival rate in different groups.

[0158]

[0159] 4.3.2 Determination of Biochemical Indicators

[0160] ROS levels are important signals of normal cellular physiological function and cell damage caused by environmental factors. ROS levels increase sharply when animals are under stress. (See Table 7 and...) Figure 2The data showed that the expression level of reactive oxygen species (ROS) in the blank control group was 173.75 ng / mL, while that in the vaccine group was 189.96 ng / mL. Compared with the blank control group, the expression level of ROS in the vaccine group increased by 10.98%. Compared with the vaccine group, the composite multidimensional nanoemulsion provided by this invention (Examples 3, 7, and 8) could reduce the expression level of ROS by 15.58%, 12.36%, and 14.80%, respectively. P All values ​​were <0.05, indicating significant differences; compared with the vaccine group, the drug control group showed an 8.53% reduction in reactive oxygen species (ROS) expression. P The values ​​were all >0.05, indicating no significant difference. This demonstrates that the composite multidimensional nanoemulsion provided by this invention can achieve significant antioxidant stress effects, effectively reduce the expression of reactive oxygen species (ROS), and protect the cellular redox balance from being disrupted.

[0161] Table 7. Effects of different groups of immune stress on the expression levels of reactive oxygen species (ROS) in chicken serum.

[0162]

[0163] Note: The different letters on the shoulder insignia indicate significant differences. P <0.05); the difference was not significant if the shoulder insignia had the same letter or no letter marking. P >0.05).

[0164] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A composite multidimensional nanoemulsion, comprising the following components in parts by weight: 100-150 parts vitamin supplement, 130-160 parts emulsifier, 100-200 parts traditional Chinese medicine immune enhancer, 60-80 parts amino acid supplement, 5-15 parts antioxidant, 60-80 parts ion supplement, and 120-160 parts antifreeze agent, wherein the vitamin supplement includes fat-soluble vitamins and water-soluble vitamins, and the traditional Chinese medicine immune enhancer includes Astragalus membranaceus extract and Ganoderma lucidum extract; The ion supplement is sodium chloride and calcium gluconate; The antifreeze agent is propylene glycol; The preparation method of the Astragalus extract or Ganoderma extract includes the following steps: Astragalus and Ganoderma lucidum are soaked in water at 45-85℃ for 30-40 minutes; boiled for 50-70 minutes, and the solid and liquid are separated to obtain filtrate and residue; the residue is boiled in water for 50-70 minutes, and the solid and liquid are separated to obtain filtrate; the filtrates obtained from the two processes are combined and concentrated to obtain Astragalus extract or Ganoderma lucidum extract. The preparation method of the composite multidimensional nanoemulsion includes the following steps: (1) Mix fat-soluble vitamins and emulsifiers and stir at 10,000 rpm to 15,000 rpm for 20 min to 30 min to obtain a fat-soluble solution; (2) Dissolve the amino acid supplement, ion supplement, water-soluble vitamins and antioxidants in water to obtain a water-soluble solution; (3) Add the lipid-soluble solution from step (1) to the water-soluble solution from step (2), stir until the solution is clear, and obtain the O / W type composite multidimensional nanoemulsion intermediate solution; (4) Mix the Chinese medicine immune enhancer with the O / W type composite multidimensional nanoemulsion intermediate solution, add an antifreeze agent, and stir to obtain the composite multidimensional nanoemulsion.

2. The composite multidimensional nanoemulsion according to claim 1, characterized in that, The amino acid supplement includes at least one of lysine, hydroxymethionine, and threonine.

3. The composite multidimensional nanoemulsion according to claim 1 or 2, characterized in that, The antioxidants include propyl gallate and L-cysteine; and / or, the emulsifiers include polysorbate and poloxamer.

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