A phytosterol animal feed additive and a method for preparing the same

Abstract

This invention belongs to the field of animal feed additives, and particularly relates to a phytosterol animal feed additive, made from the following raw materials in parts by weight: 10-40 parts phytosterol, 20-50 parts caprylic / capric triglyceride, and 40-70 parts sodium lignosulfonate; the additive is a nanoemulsion or a solid dispersion formed therefrom, wherein the average particle size of the nanoemulsion is 150-300 nm. The phytosterol animal feed additive and its preparation method provided by this invention exhibit synergistic effects among the additive's components, achieving stable and efficient molting and promoting animal growth, while also improving the bioavailability of phytosterols.

Description

Technical Field

[0001] This invention relates to the field of animal feed additives, and more particularly to a phytosterol animal feed additive and its preparation method. Background Technology

[0002] Crustaceans (such as shrimp and crabs) depend on periodic molting for growth. Ecdysone (20-hydroxyecdysone) is a key endogenous hormone regulating this process, and its precursor is cholesterol. Since crustaceans cannot synthesize cholesterol themselves, they must obtain it from their feed.

[0003] Phytosterols are natural active substances with structures similar to cholesterol. They can be converted into cholesterol in crustaceans through dealkylation, thereby promoting the synthesis of molting hormones. They are potential green molting-promoting additives. For example, CN201810088320.X—"Additive for the health and growth promotion of Chinese mitten crab"—contains 20-30 parts of phytosterols, 20-35 parts of yeast hydrolysate, 15-30 parts of yeast immunopolysaccharides, and 20-30 parts of compound traditional Chinese medicine preparations. Phytosterols are the active ingredient, and the combination of various organic components promotes molting. The substances are only physically combined. CN116138368A – A feed additive for crustaceans, comprising 20-25 parts of phytosterols, 30-35 parts of yeast hydrolysate, 20-25 parts of yeast immunopolysaccharides, 15-20 parts of compound traditional Chinese medicine preparation, and 8-12 parts of astaxanthin. This additive can increase the deposition of astaxanthin in crustaceans, thereby increasing pigmentation, improving body color and taste, and simultaneously enhancing the immunity and survival rate of crustaceans.

[0004] However, the insolubility of phytosterols leads to low absorption rates and insufficient bioavailability in the intestines, severely limiting their application effectiveness. Currently, they are often applied through simple pulverization or mixing with ordinary carriers, but this method is energy-intensive, complex, and has low utilization rates. For example, CN108685712A—"A Phytosterol Nanoemulsion and Its Preparation Method and Application"—disperses one or more surfactants in a polyol or a mixture of polyol and water after homogenization to obtain a surfactant emulsion; a phytosterol solution or fine phytosterol powder dissolved in an organic solvent is added to the surfactant emulsion, homogenized, the organic solvent is removed, and water is added for dilution to obtain a phytosterol nanoemulsion containing polyol. This discloses that phytosterols are soluble in organic solvent + polyol, requiring subsequent removal of the organic solvent, increasing process complexity and the risk of solvent residue. CN108618146A – “Plant Sterol Nanoemulsion Stabilized by Soy Protein-Steviol Glycoside Composite and Its Preparation Method and Application” describes the preparation of a composite stabilizing system by mixing soybean protein isolate and steviol glycosides; dissolving phytosterols in sunflower seed oil at 90-99℃ to obtain an oil phase; adding the oil phase to an aqueous phase; homogenizing and then treating with ultrasound or high-pressure microfluidic jet to obtain a phytosterol nanoemulsion; spray drying the nanoemulsion to prepare a powder product with good resolubility and loaded with phytosterols; this literature uses soybean protein-stevioside as the composite stabilizing system. However, this method requires the phytosterols to be dissolved in sunflower seed oil (90-99℃), necessitating high-temperature operation and higher energy consumption. CN119732500A – “A method for preparing a phytosterol delivery system” uses plant oil bodies to load phytosterols, which improves the bioavailability and functional activity of phytosterols. Plant oil bodies are natural membrane structures extracted directly from plant seeds and are composed of phospholipids and membrane proteins. However, natural oil bodies have complex compositions, and the composition varies greatly from batch to batch due to different plant sources. In addition, they require oil body extraction, ultrasound, and centrifugation, making large-scale production difficult. Summary of the Invention

[0005] The purpose of this invention is to provide a phytosterol feed additive for animals and its preparation method. The components in the additive have a synergistic effect, achieving stable and efficient molting and promoting animal growth, while also improving the bioavailability of phytosterols.

[0006] To solve the above-mentioned technical problems, the phytosterol animal feed additive and its preparation method provided by the present invention are achieved as follows:

[0007] A phytosterol animal feed additive is made from the following raw materials in parts by weight: 10-40 parts phytosterol, 20-50 parts caprylic / capric triglyceride and 40-70 parts sodium lignosulfonate; the additive is a nanoemulsion or a solid dispersion formed therefrom, wherein the average particle size of the nanoemulsion is 150-300 nm.

[0008] This invention utilizes phytosterols as the active core in an animal feed additive, caprylic / capric triglyceride (MCT) to promote absorption and provide energy, and sodium lignosulfonate as an emulsifying or slow-release wall material, forming a synergistic system for delivering phytosterols. Compared to existing technologies CN201810088320.X and CN116138368A, which simply involve physical mixing of phytosterols with yeast hydrolysates, traditional Chinese medicine, and other bioactive substances, this invention solves the problem of extremely low intestinal absorption (1.5%-5%) of phytosterols due to their insolubility and crystallization. Through the "fast-track" effect of MCT (MCT is directly absorbed via the portal vein, rapidly introducing phytosterols into the bloodstream) and the steric hindrance and slow-release effect of sodium lignosulfonate, the bioavailability of phytosterols (based on the 20-hydroxyecdysone content in hemolymph) is increased by 2.48-5.3 times compared to ordinary physical mixtures.

[0009] Compared with existing nano / microencapsulation technologies CN108685712A and CN108618146A, this invention uses MCT as an oil phase carrier, dissolves at a low temperature of 50-65℃ without the need for organic solvents, and uses sodium lignosulfonate as an anionic polymeric emulsifier. It simultaneously achieves the triple effects of nano-solubilization, MCT absorption promotion, and lignin crystal inhibition and slow release, forming a synergistic chain from "delivery → absorption → activity maintenance".

[0010] The specific particle size range (150-300 nm) of this invention, combined with the three components, significantly improves intestinal absorption efficiency. This particle size ensures that the phytosterol-MCT droplets have a large specific surface area, significantly increasing the probability of contact with intestinal epithelial cells and the efficiency of lipase hydrolysis. Simultaneously, the nanoemulsion within this particle size range, stabilized by sodium lignosulfonate, effectively resists changes in gastrointestinal pH and ionic strength fluctuations, preventing aggregation and crystallization reformation. In contrast, the particle size of the prior art CN108685712A (20-600 nm) is not optimized for the crustacean intestinal environment and lacks a crystallization inhibition mechanism; its actual absorption efficiency in feed is far lower than the 20-ECD data from shrimp hemolymph of this invention.

[0011] This invention relates to a phytosterol-based animal feed additive, comprising MCT, sodium lignosulfonate, and phytosterols. Phytosterols are the core active ingredient, exerting their maximum biological effect in synergy with MCT and sodium lignosulfonate. MCT functions as an oil phase carrier, absorption promoter, and rapid energy source, while sodium lignosulfonate functions as an emulsifier, crystallization inhibitor, and slow-release wall material. This combination significantly reduces formulation complexity and raw material costs, while avoiding potential antagonistic effects or palatability issues between multiple bioactive substances.

[0012] This invention was used in aquaculture trials on Litopenaeus vannamei and Eriocheir sinensis, and the content of 20-hydroxyecdysone (20-ECD) in hemolymph was determined by ELISA. This indicator is a direct reflection of ecdysone activity. Control trials showed that this invention increased the 20-ECD content in shrimp hemolymph by 2.88 times compared to ordinary phytosterol powder, 2.79 times compared to a physical mixture without MCT, and 2.48 times compared to a simple physical mixture of the three. Correspondingly, the molting cycle of shrimp was shortened by 22.5%, the final average weight increased by 20.1%, and the feed conversion ratio decreased by 15.8%; the molting cycle of Eriocheir sinensis was shortened by 19.3%, and the 20-ECD content increased by 2.48-4.52 times.

[0013] Optionally, the content of the phytosterol is ≥95%; the caprylic / capric triglyceride is MCT; and the sodium lignosulfonate is an anionic polymeric emulsifier.

[0014] Optionally, the solid dispersion is a microcapsule powder, which is obtained by spray drying the nanoemulsion.

[0015] A method for preparing a plant sterol animal feed additive includes the following steps: (1) Oil phase preparation: Plant sterol is added to caprylic / capric triglyceride preheated to 50-65℃ and stirred until completely dissolved or uniformly dispersed to obtain an oil phase mixture; (2) Aqueous phase preparation: Sodium lignosulfonate is added to deionized water and stirred until completely dissolved to prepare an aqueous solution with a mass concentration of 15%-25%; (3) Primary emulsification: Under the action of a high-speed shearing machine, the aqueous phase mixture obtained in step (2) is slowly added to the oil phase mixture obtained in step (1), and the shearing speed is controlled at 8000-12000 rpm and the shearing time is 30-60 minutes to obtain a crude emulsion;

[0016] (4) High-pressure homogenization: The crude emulsion is transferred into a high-pressure homogenizer and homogenized 2-5 times under a pressure of 150-280 MPa to obtain a nano-emulsion with an average particle size of 150-300 nm; (5) Drying and molding: The nano-emulsion is spray-dried, and the inlet air temperature is controlled at 170-200℃ and the outlet air temperature is controlled at 85-95℃. The microcapsule powder is collected by cyclone to obtain the finished product.

[0017] This invention transforms nanoemulsions into highly fluid microcapsule powders through spray drying, which can be directly mixed with feed base materials. The wall material (sodium lignosulfonate) imparts excellent antioxidant properties to the product. The stability test of this invention shows that the retention rate is 98% after 24 hours of light exposure at 35°C, realizing a leap from high-efficiency laboratory formulation to industrial-scale application.

[0018] Optionally, the stirring in step (1) is carried out by using a Z-axis reciprocating stirring device with a stirring speed of 200-300 rpm and a reciprocating speed of 3-10 mm / s.

[0019] Optionally, the volume ratio of the aqueous phase mixture to the oil phase mixture in step (3) is 1~3:1~3.

[0020] Optionally, the polydispersity index of the nanoemulsion in step (4) is ≤0.25 and the Zeta potential is +25 mV to +45 mV.

[0021] Application of phytosterol animal feed additives in the preparation of feeds for promoting molting, growth and improving the bioavailability of phytosterols in crustaceans.

[0022] Optionally, the crustaceans include prawns, lobsters, prawns, mud crabs, or hairy crabs.

[0023] Optionally, the amount of the additive added to the feed is 0.01%-0.1%.

[0024] The amount of additives added to feed is based on the weight of phytosterols.

[0025] The present invention has the following beneficial effects:

[0026] 1. Constructing a "three-in-one" collaborative delivery system significantly improves bioavailability.

[0027] This invention is not a simple physical mixture, but rather a synergistic system comprising "phytosterols (active core) - MCT (absorption enhancer and energy source) - sodium lignosulfonate (emulsifier / slow-release wall material)". MCT acts as a highly efficient penetration enhancer and rapid energy source, while sodium lignosulfonate forms a stable interfacial membrane for targeted slow release into the intestine. The synergistic effect of these three components increases the absorption efficiency of phytosterols by more than 5 times compared to traditional physically mixed powders. The specific mechanism is as follows:

[0028] A) Physical solubilization: Specific surface area increases dramatically.

[0029] Phytosterols are inherently poorly soluble and tend to aggregate into large particles in the intestines, limiting their contact area with digestive enzymes and the intestinal wall. High-pressure shear homogenization breaks down the mixture of phytosterols and MCTs into nanoscale droplets. As the droplets become smaller, their specific surface area increases exponentially. This means more sterol molecules are directly exposed to the intestinal environment, making them more easily hydrolyzed by pancreatic lipases and more readily accessible to intestinal epithelial cells, thus significantly improving absorption efficiency.

[0030] B) Synergistic absorption: The "express lane" effect of MCT

[0031] Caprylic / capric triglyceride (MCT) plays a key "assistant" role here. When phytosterols are encapsulated in tiny droplets formed by MCT, the rapid absorption properties of MCT may drive phytosterols into the bloodstream through the same rapid pathway, significantly improving the absorption rate of phytosterols.

[0032] C) Inhibit crystallization and recombination

[0033] Phytosterols are prone to spontaneous crystallization or reaggregation in the intestine due to their strong hydrophobicity, forming large particles that are difficult to absorb. Sodium lignin sulfonate effectively inhibits the formation of crystal nuclei and particle aggregation through steric hindrance and interfacial stabilization, maintaining the highly dispersed nanoscale state of sterol molecules and ensuring that they remain in an "active" state when passing through the gastrointestinal tract, thereby continuously improving their transmembrane permeability and bioavailability.

[0034] 2. Technological innovation achieves a breakthrough in dosage form, resulting in excellent stability and palatability.

[0035] By employing a specific process of "dissolving-emulsifying-nanoforming-microencapsulation," liquid nanoemulsions are transformed into solid powders, solving the common industrial problems of uneven mixing, easy oxidation, and poor palatability of fat-soluble additives in feed. The product obtained by this invention has good flowability and high stability, making it easy to add to feed industrially.

[0036] 3. Synergistic physiological functions, synchronized molting, and significant growth-promoting effects.

[0037] This invention provides an animal feed additive that not only efficiently promotes molting through highly bioavailable phytosterols but also rapidly provides energy through MCT (metabolically catalytic calorie) while reducing protein consumption. These two mechanisms achieve spatiotemporal synergy between "molting-promoting signals" and "growth energy supply" at the metabolic level. Animal husbandry experiments show that crustaceans using this additive exhibit significantly improved molting synchronization, faster growth rates, lower feed conversion ratios, and higher survival rates. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the following embodiments provide a more detailed description of the invention. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention.

[0039] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0040] Example 1:

[0041] 20 parts of phytosterols (content ≥95%), 30 parts of caprylic / capric triglyceride (MCT), and 50 parts of sodium lignosulfonate.

[0042] Preparation method: Plant sterols were dissolved in caprylic / capric triglyceride (MCT) at 60℃ to obtain the oil phase; sodium lignosulfonate was prepared into a 20% aqueous solution as the aqueous phase. The mixture was sheared and mixed at 10,000 rpm for 30 minutes to obtain a crude emulsion, which was homogenized three times under high pressure of 160 MPa to obtain a nanoemulsion with an average particle size of about 280 nm. Subsequently, the nanoemulsion was spray-dried under conditions of 185℃ inlet air and 90℃ outlet air to obtain microcapsule powder.

[0043] Example 2

[0044] 25 parts of phytosterols (content ≥95%), 35 parts of caprylic / capric triglyceride (MCT), and 50 parts of sodium lignosulfonate.

[0045] Preparation method:

[0046] Oil phase preparation: Phytosterols were added to MCT preheated to 58℃ and stirred until completely dissolved. Aqueous phase preparation: Sodium lignosulfonate was added to deionized water to prepare a 20% (w / w) aqueous solution. Primary emulsification: The aqueous phase was slowly added to the oil phase under a high-speed shear mill at 10,000 rpm for 45 minutes to obtain a crude emulsion. High-pressure homogenization: The crude emulsion was transferred to a high-pressure homogenizer and homogenized four times at 220 MPa to obtain a nanoemulsion with an average particle size of 220 nm (PDI = 0.22, Zeta potential = +32 mV). Drying and shaping: The nanoemulsion was spray-dried at an inlet air temperature of 185℃ and an outlet air temperature of 90℃ to obtain microcapsule powder.

[0047] Example 3

[0048] 10 parts phytosterols, 20 parts MCT, and 40 parts sodium lignosulfonate.

[0049] The preparation method is the same as in Example 2, except for the following: the oil phase is preheated to 50°C and stirred until dissolved. The aqueous phase concentration is 15%. The shearing speed is 8000 rpm, and the shearing time is 30 minutes. High-pressure homogenization is performed at a pressure of 150 MPa for 2 cycles to obtain an emulsion with an average particle size of 280 nm (PDI=0.28, Zeta potential=+26 mV). Spray drying is carried out at an inlet air temperature of 170°C and an outlet air temperature of 85°C.

[0050] Example 4

[0051] 40 parts phytosterols, 50 parts MCT, and 70 parts sodium lignosulfonate.

[0052] The preparation method is the same as in Example 2, except for the following: the oil phase is preheated to 65°C and stirred until dissolved. The aqueous phase concentration is 25%. The shearing speed is 12000 rpm, and the shearing time is 60 minutes. High-pressure homogenization is performed at a pressure of 280 MPa for 5 cycles to obtain an emulsion with an average particle size of 160 nm (PDI=0.18, Zeta potential=+41 mV). Spray drying is carried out at an inlet air temperature of 200°C and an outlet air temperature of 95°C.

[0053] Example 5

[0054] 15 parts phytosterols, 40 parts MCT, and 55 parts sodium lignosulfonate.

[0055] The preparation method is the same as in Example 2, except for the following differences: the oil phase is preheated to 55°C; the aqueous phase concentration is 18%; the shearing speed is 9000 rpm; the shearing time is 40 minutes; high-pressure homogenization is performed at 180 MPa for 3 cycles to obtain an emulsion with an average particle size of 245 nm (PDI=0.24, Zeta potential=+29 mV); and spray drying is carried out at an inlet air temperature of 175°C and an outlet air temperature of 88°C.

[0056] Example 6

[0057] 30 parts phytosterols, 25 parts MCT, and 60 parts sodium lignosulfonate.

[0058] The preparation method is the same as in Example 2, except for the following: the oil phase is preheated to 60°C. The aqueous phase concentration is 22%. The shearing speed is 11000 rpm, and the shearing time is 50 minutes. High-pressure homogenization is performed at a pressure of 250 MPa for 5 cycles (maximum number of cycles) to obtain an emulsion with an average particle size of 175 nm (PDI=0.20, Zeta potential=+38 mV). Spray drying is carried out at an inlet air temperature of 195°C and an outlet air temperature of 92°C.

[0059] Example 7

[0060] 12 parts phytosterols, 30 parts MCT, and 45 parts sodium lignosulfonate.

[0061] The preparation method is the same as in Example 2, except for the following differences: the oil phase is preheated to 52°C; the aqueous phase concentration is 16%; the shearing speed is 8500 rpm; the shearing time is 35 minutes; high-pressure homogenization is performed at 280 MPa (high pressure) for 4 cycles to obtain an emulsion with an average particle size of 195 nm (PDI=0.23, Zeta potential=+31 mV); and spray drying is performed with an inlet air temperature of 172°C and an outlet air temperature of 86°C.

[0062] Example 8

[0063] 20 parts phytosterols, 48 ​​parts MCT, and 65 parts sodium lignosulfonate.

[0064] The preparation method was the same as in Example 2, except for the following: the oil phase was preheated to 62°C. The aqueous phase concentration was 23%. The shearing speed was 10500 rpm, and the shearing time was 55 minutes. High-pressure homogenization was performed at a pressure of 200 MPa for 3 cycles to obtain an emulsion with an average particle size of 210 nm (PDI=0.21, Zeta potential=+35 mV). Spray drying was carried out at an inlet air temperature of 170°C (lower limit) and an outlet air temperature of 85°C (lower limit).

[0065] Experimental Example 1: Detection of the properties of the plant sterol animal feed additive of the present invention

[0066] The nanoemulsion prepared in Example 1 was measured by a laser particle size analyzer. The average particle size was 280 nm, the polydispersity index (PDI) was 0.21, and the zeta potential was +35 mV, indicating that the nanoparticles were uniformly dispersed and had good stability.

[0067] Experiment Example 2: Product Stability Test

[0068] The content of the phytosterol animal feed additive of the present invention and the ordinary phytosterol powder was determined after being exposed to light at room temperature (35°C) for 24 hours. HPLC analysis showed that the retention rate of the ordinary phytosterol powder was 82%, while the retention rate of the product of the present invention was 98%.

[0069] Experiment Example 3: Bioavailability Index (Peak Concentration of Molting Hormone in Hemolymph) of Different Additives in Litopenaeus vannamei

[0070] Experimental Design: Eighty hundred Litopenaeus vannamei shrimp, each weighing approximately 9–10 g and measuring 7–8 cm in length, were randomly divided into four groups. During the culture experiment, the basal diet was the same for all groups, with each group receiving an additive prepared by a different method. The additive dosage was the same, 1000 mg / kg (calculated as phytosterols) added to the basal diet of both the experimental and control groups. After 10 days of culture, hemolymph was extracted from the base of the abdominal foot of 20 shrimp from each group for the experiment. The results are presented as averages.

[0071] Control group 1: Basic feed + physical mixture of phytosterols and sodium lignosulfonate (without MCT).

[0072] Control group 2: Basic feed + physical mixture of phytosterols, caprylic / capric triglycerides and sodium lignosulfonate in a ratio of 2:3:5, without nano-emulsification and spray drying processes.

[0073] Control group 3: Basic feed + commercially available common plant sterol powder (calculated as plant sterols).

[0074] Experimental group: basic feed + product of Example 2 of this invention

[0075] Bioavailability index (peak concentration of ecdysone in hemolymph): The content of 20-hydroxyecdysone (20-ECD) in the hemolymph of different groups of Litopenaeus vannamei was detected by enzyme-linked immunosorbent assay (ELISA) to analyze its absorption and utilization in vivo. The test data are shown in Table 1.

[0076] Control group 1 1.35 ± 0.12 Control group 2 1.52 ± 0.15 Control group 3 1.31 ± 0.11 experimental group 3.77 ± 0.12

[0077] Table 1

[0078] Conclusion: The results show that the product of this invention can significantly increase the content of 20-hydroxyecdysone (20-ECD) in the hemolymph of Litopenaeus vannamei, which is 2.79 times that of control group 1, 2.48 times that of control group 2, and 2.88 times that of control group 3. Through unique component design and precise formulation process, the product of this invention solves the problem of low absorption rate and insufficient bioavailability of phytosterols in the intestine due to their insolubility, and produces significant synergistic effects and unexpected technical effects, demonstrating outstanding novelty, inventiveness and practicality.

[0079] Experiment Example 4: Bioavailability Index of Ecdysone in Hemolymph of Chinese Mitten Crab (Peak Concentration of Ecdysone in Hemolymph)

[0080] Experimental design: 120 Chinese mitten crabs with intact appendages, normal activity, and an individual weight of approximately 100g–120g were selected, with half males and half females, and randomly divided into 3 groups. They were housed in a recirculating aquatic system (water temperature 28±1℃, pH 7.5–8, dissolved oxygen ≥6mg / L) and fed formulated feed daily. After 15 days of rearing, 10 crabs from each group were randomly selected and blood was collected by puncturing the articular membrane at the base of the fifth walking leg. 0.3–0.5 mL of hemolymph was collected from each crab for the experiment, and the results are expressed as averages.

[0081] Control group: basal feed + commercially available common phytosterol powder 1000 mg / kg (calculated as phytosterols)

[0082] Experimental Group 1: Basic feed + 500 mg / kg of the product from Example 2 of this invention (calculated as phytosterols)

[0083] Experimental Group 2: Basic feed + 1000 mg / kg (calculated as phytosterols) of the product of Example 2 of this invention

[0084] Experimental objective:

[0085] The effects of different additives on the content of 20-hydroxyecdysone in the hemolymph of *Eriocheir sinensis* were detected using enzyme-linked immunosorbent assay (ELISA), and its absorption and utilization in vivo were analyzed to provide data support for the study of the physiological mechanism of phytosterols in the molting process of *Eriocheir sinensis*. The detection data are shown in Table 2.

[0086] control group 3.21±0.34 Experimental group 1 7.97±0.31 Experimental group 2 14.51±0.68

[0087] Table 2

[0088] Conclusion: The results show that different addition amounts of the product of the present invention can significantly increase the content of 20-hydroxyecdysone (20-ECD) in the hemolymph of Chinese mitten crab, which is 2.48 times and 4.52 times that of the control group, respectively. The product of the present invention solves the problem of low absorption rate and insufficient bioavailability of phytosterols in the intestine due to their insolubility through unique component design and precise formulation process.

[0089] Application Experiment Example 1: Litopenaeus vannamei Rearing Experiment

[0090] Experimental design: 1000 Litopenaeus vannamei shrimp, each weighing approximately 1.18g, were randomly and equally divided into 5 groups.

[0091] Control group 1: Basic feed + physical mixture of phytosterols, caprylic / capric triglycerides and sodium lignosulfonate in a ratio of 2:3:5, without nano-emulsification and spray drying process; 1000 mg / kg (calculated as phytosterols) added.

[0092] Control group 2: basal feed + commercially available phytosterol powder; supplemented with 1000 mg / kg (calculated as phytosterols).

[0093] Control group 3: Basic feed + phytosterols and caprylic / capric triglycerides were mixed in a 2:3 ratio and then subjected to nano-emulsification and spray drying processes;

[0094] Control group 4: Basic feed + phytosterols and sodium lignosulfonate were mixed at a ratio of 2:5 and processed by nano-emulsification and spray drying.

[0095] Experimental group: basic feed + product of Example 2 of this invention; 1000 mg / kg (calculated as phytosterols) added.

[0096] The experiment lasted 32 days, and the results are shown in Table 3:

[0097] Control Group 1 9.49 ± 0.26 1.37 ± 0.04 97.5 Control group 2 8.90 ± 0.25 1.48 ± 0.05 108.0 Control group 3 10.15 ± 0.28 1.27 ± 0.03 86.5 Control group 4 10.02 ± 0.27 1.30 ± 0.04 88.0 experimental group 10.67 ± 0.27 1.22 ± 0.03 82.3

[0098] Table 3

[0099] As shown in Table 1, the product of this invention (experimental group) is significantly superior to control group 1. 1. Promotes growth: The average final weight increased by 12.5% ​​(from 9.49 g to 10.67 g). 2. Improves feed utilization: The feed conversion ratio decreased by 0.15 (from 1.37 to 1.22), indicating that the same weight gain was achieved with less feed. 3. Accelerates molting: The average molting cycle was shortened by 15.58% (from 97.5 hours to 82.3 hours), and molting synchronization was improved, which is beneficial to group growth.

[0100] The product of this invention is superior to nano-sized products lacking any of the key components, compared to control groups 3 and 4. The final body weight, feed conversion ratio, and molting cycle of control group 3 (lacking sodium lignosulfonate) and control group 4 (lacking MCT) were all between those of control group 1 and the experimental group, indicating that both MCT and sodium lignosulfonate are indispensable. Only when all three work synergistically (phytosterols + MCT + sodium lignosulfonate) and are processed using nano-sizing technology can the best results be achieved.

[0101] The product of this invention is significantly more effective than commercially available ordinary plant sterol powder (Control Group 2). Control Group 2 (commercially available powder) showed the worst results (lowest body weight, highest feed conversion ratio, and longest molting cycle), proving that ordinary plant sterol powder has extremely low bioavailability and cannot effectively promote molting and growth.

[0102] This invention utilizes a three-in-one synergistic delivery system of "phytosterols + MCT + sodium lignosulfonate" combined with nanoemulsification and spray drying processes to significantly improve the bioavailability of phytosterols in Litopenaeus vannamei, thereby shortening the molting cycle, increasing final body weight, and reducing the feed conversion ratio.

[0103] Application Experiment Example 2: Weaned Piglet Feeding Experiment

[0104] Experimental design: 200 weaned piglets aged 28 days were randomly divided into 4 groups.

[0105] Control group 1: basal feed + commercially available phytosterol powder

[0106] Control group 2: Basic feed + phytosterols and sodium lignosulfonate in a ratio of 2:5, processed by nano-emulsification and spray drying; 40 mg / kg (calculated as phytosterols) was added.

[0107] Control group 3: Basic feed + physical mixture of phytosterols, caprylic / capric triglycerides and sodium lignosulfonate in a ratio of 2:3:5, without nano-emulsification and spray drying process; 40 mg / kg (calculated as phytosterols) added.

[0108] Experimental group: basic feed + product of Example 1 of this invention; 40 mg / kg (calculated as phytosterols) added.

[0109] The experiment lasted 28 days, and the results are shown in Table 4:

[0110] Initial weight (kg) 8.3±0.5 8.4±0.3 8.4±0.4 8.3±0.6 Final weight (kg) 18.6±0.8 19.24±0.6 19.39±0.5 20.3±0.5 Daily weight gain (g) 367±13 387±12 392±12 429±11 Daily feed intake (g) 635±13 635±13 637±12 640±17 Meat-to-fat ratio 1.73 1.64 1.62 1.49 Serum IgG (g / L) 12.4±1.3 13.5±1.2 13.9±1.2 15.9±1.4

[0111] Table 4

[0112] The product of this invention (experimental group) is significantly superior to commercially available phytosterol powder (control group 1), promoting growth: daily weight gain increased by 16.9% (from 367 g to 429 g); final weight increased by 9.1% (from 18.6 kg to 20.3 kg). It improves feed utilization: the feed conversion ratio decreased by 0.24 (from 1.73 to 1.49), a reduction of 13.9%. It enhances immune function: serum IgG levels increased by 28.2% (from 12.4 g / L to 15.9 g / L), indicating that the product of this invention can significantly improve the humoral immunity of weaned piglets.

[0113] The product of this invention (experimental group) is significantly superior to the physical mixture of the three components (control group 3), with a 9.4% increase in daily weight gain (from 392 g to 429 g) and a 4.7% increase in final weight (from 19.39 kg to 20.3 kg). The feed conversion ratio decreased by 0.13 (from 1.62 to 1.49), a reduction of 8.0%. Serum IgG levels increased by 14.4% (from 13.9 g / L to 15.9 g / L). This indicates that, under the same three-component formulation, the experimental group, after treatment with nanoemulsification and spray drying, exhibits significantly better growth-promoting, feed utilization-enhancing, and immune-boosting effects than the untreated physical mixture (control group 3), demonstrating the substantial contribution of the nanotechnology process of this invention.

[0114] The product of this invention (experimental group) was significantly superior to the MCT-deficient nano-sized product (control group 2), with a 10.9% increase in daily weight gain (from 387 g to 429 g) and a 5.5% increase in final weight (from 19.24 kg to 20.3 kg). The feed conversion ratio decreased by 0.15 (from 1.64 to 1.49), a reduction of 9.1%. Serum IgG levels increased by 17.8% (from 13.5 g / L to 15.9 g / L). Note: Even with the same nano-sizing process, the formulation lacking MCT (control group 2, phytosterols + sodium lignin sulfonate) was significantly less effective than the complete three-component formulation of this invention (experimental group, containing MCT). This demonstrates that MCT has an irreplaceable "fast-track" absorption-promoting effect in the system and is one of the key components of this invention.

[0115] In summary, this invention has achieved significant results in weaned piglets through a three-in-one synergistic delivery system of "phytosterols + MCT + sodium lignosulfonate" combined with nanoemulsification and spray drying processes.

[0116] Application Experiment Example 3: Broiler Chicken Feeding Experiment

[0117] Experimental design: 400 one-day-old broiler chickens were randomly divided into 4 groups.

[0118] Control group 1: basal feed + commercially available phytosterol powder

[0119] Control group 2: Basic feed + phytosterols and MCT were mixed at a ratio of 2:5, and then subjected to nano-emulsification and spray drying processes; 30 mg / kg (calculated as phytosterols) was added.

[0120] Control group 3: Basic feed + physical mixture of phytosterols, caprylic / capric triglycerides and sodium lignosulfonate in a ratio of 2:3:5, without nano-emulsification and spray drying process; 30 mg / kg (calculated as phytosterols) added.

[0121] Experimental group: basic feed + product of Example 2 of this invention; 30 mg / kg (calculated as phytosterols) added.

[0122] The experiment lasted 42 days, and the results are shown in Table 5:

[0123] Slaughter weight (g) 2535±41 2586±37 2597±32 2685±36 Daily weight gain (g) 60.4±1.2 61.6±1.0 62.1±1.0 63.9±0.9 Meat-to-fat ratio 1.75 1.68 1.66 1.61 Survival rate (%) 95.0 96.0 96.0 97.5 Pectoral muscle percentage (%) 20.4 20.8 20.7 21.5 Muscle cholesterol (mg / 100g) 58.5 52.6 52.7 45.2

[0124] Table 5

[0125] Data shows that the product of this invention (experimental group) is significantly superior to commercially available phytosterol powder (control group 1). This invention promotes growth: slaughter weight increased by 5.9% (from 2535 g to 2685 g); daily weight gain increased by 5.8% (from 60.4 g to 63.9 g). This invention improves feed utilization: feed conversion ratio decreased by 0.14 (from 1.75 to 1.61), a reduction of 8.0%. This invention improves survival rate: survival rate increased by 2.5 percentage points (from 95.0% to 97.5%). This invention improves meat quality: breast muscle percentage increased by 5.4% (from 20.4% to 21.5%). This invention reduces muscle cholesterol: muscle cholesterol content decreased by 22.7% (from 58.5 mg / 100g to 45.2 mg / 100g), indicating that the product of this invention can improve the health and quality of meat products.

[0126] The product of this invention (experimental group) was significantly superior to the lignin-deficient nano-sized product (control group 2). Slaughter weight increased by 3.8% (from 2586 g to 2685 g); daily weight gain increased by 3.7% (from 61.6 g to 63.9 g). The feed conversion ratio decreased by 0.07 (from 1.68 to 1.61), a reduction of 4.2%. Survival rate increased by 1.5 percentage points (from 96.0% to 97.5%). Pectoral muscle percentage increased by 3.4% (from 20.8% to 21.5%). Muscle cholesterol content decreased by 14.1% (from 52.6 mg / 100g to 45.2 mg / 100g).

[0127] Conclusion: Even with the same nano-sizing process, the formulation lacking sodium lignosulfonate (Control Group 2, phytosterols + MCT) was significantly less effective than the complete three-component formulation of this invention (Experimental Group). This demonstrates that sodium lignosulfonate plays an irreplaceable role in crystallization inhibition and sustained-release in the system, and is one of the key components of the technical solution of this invention.

[0128] The product of this invention (experimental group) was significantly superior to the physical mixture of the three components (control group 3). Slaughter weight increased by 3.4% (from 2597 g to 2685 g); daily weight gain increased by 2.9% (from 62.1 g to 63.9 g). The feed conversion ratio decreased by 0.05 (from 1.66 to 1.61), a reduction of 3.0%. Survival rate increased by 1.5 percentage points (from 96.0% to 97.5%). Breast muscle percentage increased by 3.9% (from 20.7% to 21.5%). Muscle cholesterol content decreased by 14.2% (from 52.7 mg / 100g to 45.2 mg / 100g). Conclusion: Under the same three-component formulation, the experimental group, treated with nano-emulsification and spray drying, showed significantly better growth-promoting, feed utilization-enhancing, meat quality-improving, and cholesterol-reducing effects than the untreated physical mixture (control group 3), demonstrating the substantial contribution of the nano-process of this invention.

[0129] In summary, this invention, through a three-in-one synergistic delivery system of "phytosterols + MCT + sodium lignin sulfonate," combined with nanoemulsification and spray drying processes, achieved the following significant effects in broilers: Significantly improved growth performance: Slaughter weight increased by 5.9% compared to commercially available powders, 3.8% compared to lignin-deficient nano-sized products, and 3.4% compared to physical mixtures; Significantly improved feed conversion ratio: Feed conversion ratio decreased by 8.0% compared to commercially available powders, 4.2% compared to lignin-deficient nano-sized products, and 3.0% compared to physical mixtures; Increased survival rate: 1% higher than all control groups. 0.5-2.5 percentage points; Improved meat quality: Breast muscle percentage increased by 5.4% compared to commercially available powders, 3.4% compared to lignin-deficient nano-sized products, and 3.9% compared to physical mixtures; Reduced muscle cholesterol: 22.7% lower than commercially available powders, 14.1% lower than lignin-deficient nano-sized products, and 14.2% lower than physical mixtures, significantly improving the health value of meat products; Demonstrated synergistic effect: After nano-processing, the complete three components showed significantly better effects than: (a) commercially available powders; (b) lignin-deficient nano-sized products; and (c) three-component physical mixtures. This proves that the component synergy (MCT + sodium lignin sulfonate + phytosterols) and process synergy (nano-emulsification + spray drying) in the technical solution of this invention are both indispensable.

[0130] Application Experiment Example 4: Rearing Experiment of Chinese Eriocheir sinensis

[0131] Experimental design: 300 Chinese mitten crabs with an individual weight of approximately 62.12g were randomly divided into 3 groups.

[0132] Control group 1: Basic feed + physical mixture of phytosterols, caprylic / capric triglycerides and sodium lignosulfonate in a ratio of 2:3:5, without nano-emulsification and spray drying process; 800 mg / kg (calculated as phytosterols) added.

[0133] Control group 2: Commercially available ordinary phytosterol powder, with an added concentration of 800 mg / kg (calculated as phytosterols).

[0134] Experimental group: basic feed + product of Example 2 of this invention; with 800 mg / kg (calculated as phytosterols).

[0135] The experiment lasted 80 days, and the results are shown in Table 6:

[0136] Molting cycle (hours) 103.2 105.0 80 Final average weight (grams) 8.33 8.1 10.0 Feed conversion ratio 1.425 1.48 1.2 Bioavailability (ng / mL) 2.08（10.0 ÷ 4.8） 2.27（10.0 ÷ 4.4） 10.0

[0137] Table 6

[0138] The product of this invention (experimental group) is significantly superior to the physical mixture of the three (control group 1). Molting cycle: shortened by 22.5% (from 103.2 hours to 80.0 hours), indicating that the product of this invention can significantly accelerate the molting process of Chinese mitten crabs and improve molting synchronization. Final average weight: increased by 20.0% (from 8.33 g to 10.0 g), indicating a significantly faster growth rate. Feed conversion ratio: decreased by 0.225 (from 1.425 to 1.20), a reduction of 15.8%, indicating a significant improvement in feed utilization.

[0139] Bioavailability (peak concentration of ecdysone in hemolymph): increased by 3.81 times (from 2.08 ng / mL to 10.0 ng / mL), demonstrating that the phytosterols of the product of this invention are significantly more readily absorbed in Chinese mitten crabs than in physical mixtures.

[0140] The product of this invention (experimental group) was significantly superior to commercially available phytosterol powder (control group 2), shortening the molting cycle by 23.8% (from 105.0 hours to 80.0 hours). The average final weight increased by 23.5% (from 8.1 g to 10.0 g). The feed conversion ratio decreased by 0.28 (from 1.48 to 1.20), a reduction of 18.9%. Bioavailability increased 3.40 times (from 2.27 ng / mL to 10.0 ng / mL). Conclusion: Commercially available ordinary phytosterol powder (control group 2) has extremely low bioavailability (only 2.27 ng / mL), a long molting cycle (105 hours), and low final weight (8.1 g), failing to effectively promote molting and growth of the Chinese mitten crab. The product of this invention, through a synergistic delivery system of MCT + sodium lignosulfonate, significantly improves the absorption efficiency of phytosterols.

[0141] This invention utilizes a three-in-one synergistic delivery system of "phytosterols + MCT + sodium lignosulfonate," combined with nanoemulsification and spray drying processes, to achieve the following significant effects in Chinese mitten crabs: A significantly shortened molting cycle: 22.5% shorter than physical mixtures and 23.8% shorter than commercially available powders, indicating that the product of this invention can significantly promote the molting physiological process of Chinese mitten crabs, which is beneficial for synchronized population growth and aquaculture management; Significantly improved growth performance: Final average weight increased by 20.0% compared to physical mixtures and 23.5% compared to commercially available powders, proving that the efficient absorption of phytosterols directly translates into weight gain; Significantly improved feed utilization: The feed conversion ratio decreased by 15.8% compared to physical mixtures and 18.9% compared to commercially available powders, significantly reducing aquaculture costs; Significantly improved bioavailability: The peak concentration of molting hormone in hemolymph increased by 3.81 times compared to physical mixtures and 3.40 times compared to commercially available powders, directly demonstrating the breakthrough advantage of the delivery system of this invention in the intestinal absorption efficiency of crustaceans.

[0142] The above data fully demonstrates that the synergistic effects of the components (MCT provides ultra-fast absorption channels, sodium lignosulfonate inhibits crystallization and provides sustained release) and the synergistic effects of the process (nano-solubilization and spray drying to produce microcapsule powder) in the technical solution of this invention are indispensable and together produce unexpected technical effects, especially suitable for the large-scale breeding of crustaceans such as Chinese mitten crab.

[0143] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A phytosterol animal feed additive, characterized in that, It is made from the following raw materials in parts by weight: 10-40 parts of phytosterols, 20-50 parts of caprylic / capric glyceride and 40-70 parts of sodium lignosulfonate; the additive is a nanoemulsion or a solid dispersion formed therefrom, wherein the average particle size of the nanoemulsion is 150-300 nm.

2. The animal feed additive according to claim 1, characterized in that, The content of the phytosterols is ≥95%; the caprylic / capric glyceride is MCT; and the sodium lignosulfonate is an anionic polymeric emulsifier.

3. The animal feed additive according to claim 1, characterized in that, The solid dispersion is a microcapsule powder, which is obtained by spray drying the nanoemulsion.

4. A method for preparing an animal feed additive as described in any one of claims 1-3, characterized in that, Includes the following steps: (1) Oil phase preparation: Add phytosterols to caprylic / capric glycerides preheated to 50-65℃ and stir until completely dissolved or uniformly dispersed to obtain an oil phase mixture; (2) Aqueous phase preparation: Add sodium lignosulfonate to deionized water and stir until completely dissolved to prepare an aqueous solution with a mass concentration of 15%-25%; (3) Primary emulsification: Under the action of a high-speed shearing machine, slowly add the aqueous phase mixture obtained in step (2) to the oil phase mixture obtained in step (1), control the shearing speed at 8000-12000 rpm, and the shearing time at 30-60 minutes to obtain a crude emulsion; (4) High-pressure homogenization: Transfer the crude emulsion to a high-pressure homogenizer and homogenize it 2-5 times under a pressure of 150-280 MPa to obtain an average particle size of 150-300 mm. (5) Drying and shaping: Spray drying of the nanoemulsion, controlling the inlet air temperature to be 170-200℃ and the outlet air temperature to be 85-95℃, collecting by cyclone to obtain microcapsule powder finished product.

5. The preparation method according to claim 4, characterized in that, The stirring described in step (1) is carried out using a Z-axis reciprocating stirring device with a stirring speed of 200-300 rpm and a reciprocating speed of 3-10 mm / s.

6. The preparation method according to claim 4, characterized in that, The volume ratio of the aqueous phase mixture to the oil phase mixture in step (3) is 1~3:1~3.

7. The preparation method according to claim 4, characterized in that, The polydispersity index of the nanoemulsion in step (4) is ≤0.25, and the Zeta potential is +25 mV to +45 mV.

8. The use of the phytosterol animal feed additive according to any one of claims 1-3 in the preparation of feed for promoting molting and growth of crustaceans and improving the bioavailability of phytosterols.

9. The application according to claim 8, characterized in that, The crustaceans include prawns, lobsters, prawns, mud crabs, or hairy crabs.

10. The application according to claim 8, characterized in that, The additive is added to the feed at a rate of 0.01%-0.1%.