Black soldier fly larvae oil nanoliposomes and their preparation method

Abstract

This invention discloses a black soldier fly larvae oil nanoliposome and its preparation method, belonging to the field of nanodelivery system preparation technology. This invention employs a thin-film dispersion-hydration ultrasonic method to prepare black soldier fly larvae oil-encapsulated nanoliposomes. This invention can effectively improve the solubility, stability, and antioxidant properties of black soldier fly larvae oil, thereby enhancing its bioavailability and providing a new avenue for the application of black soldier fly larvae oil in the food, feed, and pharmaceutical fields.

Description

Technical Field

[0001] This invention relates to the field of liposomes, and more specifically to a nanoliposome loaded with oil from black soldier fly larvae and its preparation method. Background Technology

[0002] Black soldier fly larvae, as insects of significant resource value, demonstrate remarkable advantages in the field of waste resource utilization. Their larvae have a short growth cycle, a wide diet, and can efficiently convert various organic wastes into their own biomass, possessing dual ecological value in environmental remediation and resource recycling. Furthermore, black soldier fly larvae have a high oil content (30%–35%) and protein content (40%–44%), making them a high-quality renewable biological resource with broad application prospects in biodiesel production, high-protein feed development, and functional food raw materials.

[0003] Black soldier fly larvae oil is primarily composed of unsaturated fatty acids. Its core components include lauric acid (40-50%), which exhibits significant antibacterial, antiviral, antifungal, and anticancer effects; palmitic acid (7-15%), a fundamental component of cell membrane lipids; oleic acid, a major component of animal fats, possessing anti-inflammatory, antioxidant, blood sugar regulating, and cardiovascular disease-improving effects; and linoleic acid, one of the most abundant polyunsaturated fatty acids in nature, and an important raw material for the production of paints, coatings, epoxy resins, and other chemical products. Therefore, black soldier fly larvae oil has rich nutritional value and broad application prospects.

[0004] Although the oil from black soldier fly larvae is highly functional and widely used, it still has some problems, such as poor stability, poor water solubility, unpleasant odor, and low bioavailability.

[0005] Traditional liposome technology faces multiple technical bottlenecks when encapsulating black soldier fly larvae oil: (1) Insect oils have short fatty acid chains and special polarity, which easily leads to excessive fluidity of phospholipid membranes and a significant increase in leakage rate during storage. (2) Conventional high-temperature rotary evaporation (>60℃) and continuous ultrasound are prone to causing oxidation of unsaturated fatty acids and hydrolysis of phospholipids, resulting in low retention of active ingredients; (3) Single surfactants or membrane conditioners are difficult to balance “high loading” and “colloidal stability”, often resulting in problems such as wide particle size distribution (PDI>0.3), easy flocculation or phase separation.

[0006] In conclusion, it is necessary to develop a new technical solution to address the shortcomings of existing technologies. Summary of the Invention

[0007] To achieve the above objectives, this invention provides a method for preparing black soldier fly larvae oil nanoliposomes. This method utilizes the synergistic effect of a specific ratio of phospholipids, sterol membrane stabilizers, and nonionic surfactants, combined with low-temperature reduced-pressure rotary evaporation and intermittent ultrasonic processes, to successfully prepare black soldier fly larvae oil nanoliposomes with uniform particle size, high zeta potential, and excellent stability. These black soldier fly larvae oil nanoliposomes can improve solubility, protect black soldier fly oil from oxidation due to external environmental factors, and effectively improve the bioavailability of black soldier fly oil.

[0008] The first objective of this invention is to provide a method for preparing black soldier fly larvae oil nanoliposomes, comprising the following steps: (1) Dissolve black soldier fly oil, phospholipids, sterol film stabilizers and nonionic surfactants in pharmaceutical grade volatile organic solvent, sonicate until clear, and then remove the solvent by rotary evaporation under reduced pressure to form a uniform lipid film on the inner wall of the container. (2) Hydrate the lipid film by adding a physiologically compatible isotonic buffer system to obtain a crude liposome suspension; (3) The crude liposome suspension is subjected to ultrasonic treatment to obtain black soldier fly oil nanoliposomes.

[0009] Furthermore, the phospholipid is soybean lecithin, preferably soybean lecithin with a phosphatidylcholine content of ≥50%.

[0010] Furthermore, the sterol membrane stabilizer is cholesterol.

[0011] Furthermore, the nonionic surfactant is polysorbate 80.

[0012] Furthermore, the pharmaceutical-grade volatile organic solvent is anhydrous ethanol.

[0013] Furthermore, the concentration of the phospholipids mentioned in step (1) is 8~10 mg / mL; The mass ratio of the phospholipid to the sterol stabilizer is 4:1 to 6:1; The mass ratio of the phospholipid to the black soldier fly oil is 10:1 to 20:1; The amount of the nonionic surfactant added is 15% to 20% of the phospholipid; The temperature of the vacuum rotary evaporation is 40℃~60℃; In step (2), the buffer system is one of phosphate-buffered saline (PBS), Tris-HCl buffer, or HEPES buffer; the pH of the buffer system is 6.5 to 8.0; the hydration temperature is 40℃ to 60℃, and the concentration of the hydration medium is 0.034 mol / L to 0.06 mol / L. The ultrasonic power in step (3) is 250 W ~ 300 W; the ultrasonic time is 5 min ~ 10 min; the working time is 0 s ~ 99 s, and the pause time is 0 s ~ 99 s.

[0014] The second objective of this invention is to provide a black soldier fly larvae oil nanoliposome, which is prepared by the aforementioned method for preparing black soldier fly larvae oil nanoliposomes. The nanoliposomes have an average particle size of 133.1~138.3 nm, a polydispersity index (PDI) of 0.165~0.211, and a black soldier fly larvae oil encapsulation efficiency of 81.79~83.55%.

[0015] Furthermore, in the method for preparing black soldier fly larvae oil nanoliposomes, the concentration of phospholipid is 8 mg / mL, the mass ratio of phospholipid to sterol stabilizer is 4:1, the mass ratio of phospholipid to black soldier fly larvae oil is 10:1, the amount of nonionic surfactant added is 20% of the phospholipid, the temperature of the reduced pressure rotary evaporation is 40℃, the buffer system is 0.05 mol / L PBS buffer, the pH value of the buffer system is 7.0, the ultrasonic power is 300 W, the ultrasonic time is 10 min, the operation is 60 s, and the pause is 5 s.

[0016] Furthermore, the nanoliposomes have an average particle size of 133.6 nm, a polydispersity index (PDI) of 0.197, and a black soldier fly larvae oil encapsulation rate of 83.55%.

[0017] The black soldier fly larvae oil nanoliposomes provided by this invention have applications in the preparation of functional foods, animal feed additives, cosmetic active ingredient delivery carriers, or antibacterial and anti-inflammatory drug formulations. Compared with the prior art, the beneficial effects of this invention are as follows: (1) Component synergistic stabilization mechanism: A specific ratio of sterol membrane stabilizers (preferably cholesterol) are embedded in the hydrophobic region of the phospholipid bilayer, which significantly reduces membrane permeability and inhibits the leakage of black soldier fly oil; nonionic surfactants form a steric hindrance layer on the vesicle surface, which effectively prevents the aggregation and fusion of nanoparticles. The two work together to overcome the defects of "membrane too rigid and easy to break" or "membrane too soft and easy to leak" caused by a single stabilizer, and achieve a dynamic balance between drug loading and colloidal stability.

[0018] (2) Mild process for preservation design: Low-temperature vacuum rotary evaporation at 40~60℃ avoids the oxidation and rancidity of heat-sensitive medium-chain fatty acids in black soldier fly oil; intermittent ultrasonication in ice bath effectively disperses the heat of cavitation, and the system temperature rise is <8℃, so as to maximize the preservation of the structure and biological efficacy of active ingredients such as lauric acid while achieving nano-sized products.

[0019] (3) High loading capacity and long-term stability: The optimized window of phospholipid: oil = 4:1~20:1 takes into account both the encapsulation space and membrane mechanical strength. The resulting liposomes have a PDI of <0.225, a particle size change rate of <1.78% after 30 days of storage at 4℃ / 25℃, and an encapsulation efficiency of more than 67.01%, which meets the requirements of industrial scale-up and end-use shelf life.

[0020] (4) Strong application compatibility: Nano-sizing significantly improves the aqueous dispersibility and transmembrane absorption efficiency of black soldier fly oil, effectively masking unpleasant odors. It can be directly embedded in water-based formulations and is suitable for functional food emulsification systems, feed premixes, transdermal delivery and veterinary antibacterial agents, with significant commercial conversion value.

[0021] The black soldier fly larvae oil nanoliposomes provided by this invention are prepared by encapsulating black soldier fly larvae oil using a thin-film dispersion-hydration ultrasonic method. These black soldier fly larvae oil nanoliposomes have the advantages of uniform particle size, high zeta potential, and excellent stability (storage stability and temperature stability). They can improve solubility, protect black soldier fly larvae oil from oxidation due to external environmental factors, and effectively improve the bioavailability of black soldier fly larvae oil. Attached Figure Description

[0022] Figure 1 This is a particle size distribution diagram for Example 9.

[0023] Figure 2 The particle size distribution diagrams for Examples 1-4 are as follows: (a) Example 1, (b) Example 2, (c) Example 3, and (d) Example 4.

[0024] Figure 3 Particle size distribution diagrams for Examples 5-8: (a) Example 5, (b) Example 6, (c) Example 7, (d) Example 8.

[0025] Figure 4 Zeta potential diagrams for Examples 1-4: (a) Example 1, (b) Example 2, (c) Example 3, (d) Example 4.

[0026] Figure 5 Zeta potential diagrams for Examples 5-8: (a) Example 5, (b) Example 6, (c) Example 7, (d) Example 8.

[0027] Figure 6 This is a graph showing the particle size variation in the storage stability of Example 9.

[0028] Figure 7 This is a graph showing the PDI variation in storage stability for Example 9.

[0029] Figure 8 This is a graph showing the change in Zeta potential for storage stability in Example 9.

[0030] Figure 9 This is a graph showing the change in encapsulation efficiency in the storage stability of Example 9.

[0031] Figure 10 This is a pH change graph showing the storage stability of Example 9.

[0032] Figure 11 This is a TEM image of black soldier fly larvae oil nanoliposomes.

[0033] Figure 12 The curves show the DPPH free radical scavenging rates of black soldier fly oil nanoliposomes and black soldier fly oil. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise stated, the raw materials, reactions, and post-processing methods appearing in the embodiments are all commercially available raw materials and technical methods well known to those skilled in the art.

[0035] The embodiments and comparative examples of the present invention use the following raw materials: Black soldier fly oil was purchased from Probio Biotechnology (Hubei) Co., Ltd. Soybean lecithin and polysorbate 80 were both purchased from Shanghai Maclean Biochemical Technology Co., Ltd. Anhydrous ethanol was purchased from Sinopharm Chemical Reagent Co., Ltd. Cholesterol, purchased from Shandong Keyuan Biochemical Technology Co., Ltd. PBS buffer was purchased from Shanghai Yuanye Co., Ltd. Example 1:

[0036] A method for preparing black soldier fly larvae oil nanoliposomes includes the following steps: S1: With a fixed soybean lecithin to cholesterol ratio of 4:1 and a soybean lecithin to black soldier fly oil ratio of 20:1, and with polysorbate 80 added at 20% of the soybean lecithin content, soybean lecithin concentrations of 2, 4, 6, 8, and 10 mg / mL were set. Soybean lecithin, cholesterol, polysorbate 80, and black soldier fly oil were accurately weighed into anhydrous ethanol according to the ratios and sonicated for 15 min to ensure complete dissolution. Subsequently, the solution was transferred to a 250 mL round-bottom flask, and anhydrous ethanol was removed from the mixed solution by rotary evaporation (water bath 40 ℃, 30 min, vacuum < 5 mbar) until a uniform, transparent lipid film formed on the flask wall.

[0037] S2: Measure a certain amount of pH 8.0, 0.05 mol / L PBS buffer and add it to the lipid membrane. Place it in a water bath and shake it manually for 5-10 minutes to detach the lipid membrane and obtain a preliminary crude liposome suspension.

[0038] S3: Transfer the crude liposome suspension to a glass bottle and sonicate it with an ice bath probe (300 W, 10 min, 60 s working, 5 s pause) to obtain nanoliposomes.

[0039] The encapsulation efficiency of black soldier fly larvae oil nanoliposomes was determined, as shown in Table 1. Lower concentrations resulted in a significant decrease in encapsulation efficiency, which may be due to insufficient lipid content, which fails to effectively encapsulate bioactive components. The optimal concentration of soybean lecithin for encapsulation efficiency was 8 mg / mL.

[0040] Table 1 Example 2:

[0041] A method for preparing black soldier fly larvae oil nanoliposomes includes the following steps: S1: With a fixed soybean lecithin concentration of 8 mg / mL, a soybean lecithin to black soldier fly oil mass ratio of 20:1, and polysorbate 80 added at 20% of the soybean lecithin mass, the soybean lecithin to cholesterol mass ratios were set at 2:1, 4:1, 6:1, 8:1, and 10:1. Soybean lecithin, cholesterol, polysorbate 80, and black soldier fly oil were accurately weighed into anhydrous ethanol and sonicated for 15 min until completely dissolved. The solution was then transferred to a 250 mL round-bottom flask, and the anhydrous ethanol was removed from the mixture by rotary evaporation (water bath 40 ℃, 30 min, vacuum < 5 mbar) until a uniform, transparent lipid film formed on the flask wall.

[0042] S2: Measure a certain amount of pH 8.0, 0.05 mol / L PBS buffer and add it to the lipid membrane. Place it in a water bath and shake it manually for 5-10 minutes to detach the lipid membrane and obtain a preliminary crude liposome suspension.

[0043] S3: Transfer the crude liposome suspension to a glass bottle and sonicate it with an ice bath probe (300 W, 10 min, 60 s working, 5 s pause) to obtain nanoliposomes.

[0044] The encapsulation efficiency of black soldier fly larvae oil nanoliposomes was determined, as shown in Table 2. When the ratio exceeded 4:1, the cholesterol ratio was too low to effectively stabilize the liposome bilayer structure, leading to reduced membrane fluidity and easy vesicle fusion or deformation, thus decreasing the encapsulation efficiency. The optimal soybean lecithin to cholesterol mass ratio was 4:1.

[0045] Table 2 Example 3:

[0046] A method for preparing black soldier fly larvae oil nanoliposomes includes the following steps: S1: With a fixed soybean lecithin concentration of 8 mg / mL, a soybean lecithin to cholesterol mass ratio of 4:1, and polysorbate 80 added at 20% of the soybean lecithin mass, the soybean lecithin to black soldier fly oil mass ratios were set at 20:1, 10:1, 20:3, 5:1, and 4:1. Soybean lecithin, cholesterol, polysorbate 80, and black soldier fly oil were accurately weighed into anhydrous ethanol and sonicated for 15 min until completely dissolved. The solution was then transferred to a 250 mL round-bottom flask, and the anhydrous ethanol was removed from the mixture by rotary evaporation (water bath 40 ℃, 30 min, vacuum < 5 mbar) until a uniform, transparent lipid film formed on the flask wall.

[0047] S2: Measure a certain amount of pH 8.0, 0.05 mol / L PBS buffer and add it to the lipid membrane. Place it in a water bath and shake it manually for 5-10 minutes to detach the lipid membrane and obtain a preliminary crude liposome suspension.

[0048] S3: Transfer the crude liposome suspension to a glass bottle and sonicate it with an ice bath probe (300 W, 10 min, 60 s working, 5 s pause) to obtain nanoliposomes.

[0049] The encapsulation efficiency of black soldier fly oil nanoliposomes was determined, as shown in Table 3. Excess oil exceeded the encapsulation capacity of the lipid bilayer, leading to phase separation and an increase in unencapsulated oil, thus reducing the encapsulation efficiency. The optimal mass ratio of soybean lecithin to black soldier fly oil for encapsulation efficiency was 10:1.

[0050] Table 3 Example 4:

[0051] A method for preparing black soldier fly larvae oil nanoliposomes includes the following steps: S1: With a fixed soybean lecithin concentration of 8 mg / mL, a soybean lecithin to cholesterol mass ratio of 4:1, and a soybean lecithin to black soldier fly oil mass ratio of 10:1, and Tween 80 addition amounts of 10, 15, 20, 25, and 30% (w / w), soybean lecithin, cholesterol, polysorbate 80, and black soldier fly oil were accurately weighed into anhydrous ethanol and sonicated for 15 min until completely dissolved. Subsequently, the solution was transferred to a 250 mL round-bottom flask, and anhydrous ethanol was removed from the mixed solution by rotary evaporation (water bath 40 ℃, 30 min, vacuum < 5 mbar) until a uniform, transparent lipid film formed on the flask wall.

[0052] S2: Measure a certain amount of pH 8.0, 0.05 mol / L PBS buffer and add it to the lipid membrane. Place it in a water bath and shake it manually for 5-10 minutes to detach the lipid membrane and obtain a preliminary crude liposome suspension.

[0053] S3: Transfer the crude liposome suspension to a glass bottle and sonicate it with an ice bath probe (300 W, 10 min, 60 s working, 5 s pause) to obtain nanoliposomes.

[0054] The encapsulation efficiency of black soldier fly larvae oil nanoliposomes was determined, as shown in Table 4. When the polysorbate 80 content was too high, the curvature of the small vesicles was too high, and the volume of the hydrophobic core was limited, which was not conducive to the stable embedding of hydrophobic oils. In addition, the excessive insertion of surfactants damaged the integrity of the membrane structure, leading to leakage of contents. The optimal polysorbate 80 addition amount was 20%.

[0055] Table 4 Example 5:

[0056] A method for preparing black soldier fly larvae oil nanoliposomes includes the following steps: S1: With a fixed soybean lecithin concentration of 8 mg / mL, a soybean lecithin to cholesterol mass ratio of 4:1, a soybean lecithin to black soldier fly oil mass ratio of 10:1, and polysorbate 80 added at 20% of the soybean lecithin content, soybean lecithin, cholesterol, polysorbate 80, and black soldier fly oil were accurately weighed into anhydrous ethanol and sonicated for 15 min to ensure complete dissolution. Subsequently, rotary evaporation temperatures were set at 40, 45, 50, 55, and 60 °C. The solutions were transferred to 250 mL round-bottom flasks, and rotary evaporation was used to remove anhydrous ethanol from the mixed solutions (water bath 40 °C, 30 min, vacuum < 5 mbar) until a uniform, transparent lipid film formed on the flask wall.

[0057] S2: Measure a certain amount of pH 7.0, 0.05 mol / L PBS buffer and add it to the lipid membrane. Place it in a water bath and shake it manually for 5-10 minutes to detach the lipid membrane and obtain a preliminary crude liposome suspension.

[0058] S3: Transfer the crude liposome suspension to a glass bottle and sonicate it with an ice bath probe (300 W, 10 min, 60 s working, 5 s pause) to obtain nanoliposomes.

[0059] The encapsulation efficiency of black soldier fly larvae oil nanoliposomes was determined, and the results are shown in Table 5. At higher temperatures, the thermal motion of phospholipid molecules intensifies, membrane fluidity increases, and collisions and fusion between vesicles intensify, leading to a decrease in encapsulation efficiency. The optimal rotary evaporation temperature for encapsulation efficiency was 40℃.

[0060] Table 5 Example 6:

[0061] A method for preparing black soldier fly larvae oil nanoliposomes includes the following steps: S1: With a fixed soybean lecithin concentration of 8 mg / mL, a soybean lecithin to cholesterol mass ratio of 4:1, a soybean lecithin to black soldier fly oil mass ratio of 10:1, and polysorbate 80 added at 20% of the soybean lecithin content, accurately weigh soybean lecithin, cholesterol, polysorbate 80, and black soldier fly oil into anhydrous ethanol and sonicate for 15 min to completely dissolve them. Then, transfer the solution to a 250 mL round-bottom flask and remove the anhydrous ethanol from the mixture by rotary evaporation (water bath 40 ℃, 30 min, vacuum < 5 mbar) until a uniform, transparent lipid film forms on the flask wall.

[0062] S2: Set the pH of the PBS buffer to 6.0, 6.5, 7.0, 7.5 and 8.0 respectively. Measure a certain amount of PBS buffer and add it to the lipid membrane. Place it in a water bath and shake it manually for 5 to 10 minutes to detach the lipid membrane and obtain a preliminary crude liposome suspension.

[0063] S3: Transfer the crude liposome suspension to a glass bottle and sonicate it with an ice bath probe (300 W, 10 min, 60 s working, 5 s pause) to obtain nanoliposomes.

[0064] The encapsulation efficiency of black soldier fly larvae oil nanoliposomes was determined, and the results are shown in Table 6. The optimal pH of the PBS buffer for encapsulation efficiency was 7.0.

[0065] Table 6 Example 7:

[0066] A method for preparing black soldier fly larvae oil nanoliposomes includes the following steps: S1: With a fixed soybean lecithin concentration of 8 mg / mL, a soybean lecithin to cholesterol mass ratio of 4:1, a soybean lecithin to black soldier fly oil mass ratio of 10:1, and polysorbate 80 added at 20% of the soybean lecithin content, accurately weigh a specific proportion of soybean lecithin, cholesterol, polysorbate 80, and black soldier fly oil into anhydrous ethanol and sonicate for 15 min to completely dissolve them. Then, transfer the solution to a 250 mL round-bottom flask and remove the anhydrous ethanol from the mixture by rotary evaporation (water bath 40℃, 30 min, vacuum < 5 mbar) until a uniform, transparent lipid film forms on the flask wall.

[0067] S2: Measure a certain amount of pH 7.0, 0.05 mol / L PBS buffer and add it to the lipid membrane. Place it in a water bath and shake it manually for 5-10 minutes to detach the lipid membrane and obtain a preliminary crude liposome suspension.

[0068] S3: Transfer the crude liposome suspension to a glass bottle, set the ultrasonic power to 100, 150, 200, 250, and 300 W, the ultrasonic time to 10 min, with 60 s intermittent operation and 5 s pause, and obtain nanoliposomes by ultrasonication with an ice bath probe.

[0069] The encapsulation efficiency of black soldier fly larvae oil nanoliposomes was determined, as shown in Table 7. With increasing ultrasonic power, the cavitation effect intensified, resulting in more intense shear forces. This facilitated the formation of a nanoscale dispersed phase between phospholipids and black soldier fly larvae oil, increasing the oil-water interface area and promoting the orderly self-assembly of phospholipid molecules on the oil droplet surface to form a complete bilayer vesicle structure, thereby improving the encapsulation efficiency. The optimal ultrasonic power for encapsulation efficiency was 300 W.

[0070] Table 7 Example 8:

[0071] A method for preparing black soldier fly larvae oil nanoliposomes includes the following steps: S1: With a fixed soybean lecithin concentration of 8 mg / mL, a soybean lecithin to cholesterol mass ratio of 4:1, a soybean lecithin to black soldier fly oil mass ratio of 10:1, and polysorbate 80 added at 20% of the soybean lecithin content, accurately weigh soybean lecithin, cholesterol, polysorbate 80, and black soldier fly oil into anhydrous ethanol and sonicate for 15 min to completely dissolve them. Then, transfer the solution to a 250 mL round-bottom flask and remove the anhydrous ethanol from the mixture by rotary evaporation (water bath 40 ℃, 30 min, vacuum < 5 mbar) until a uniform, transparent lipid film forms on the flask wall.

[0072] S2: Measure a certain amount of pH 7.0, 0.05 mol / L PBS buffer and add it to the lipid membrane. Place it in a water bath and shake it manually for 5-10 minutes to detach the lipid membrane and obtain a preliminary crude liposome suspension.

[0073] S3: Transfer the crude liposome suspension to a glass bottle, set the sonication time to 5, 10, 15, 20 and 25 min respectively, the sonication power to 300 W, intermittent operation for 60 s, pause for 5 s, and obtain nanoliposomes by sonication with an ice bath probe.

[0074] The encapsulation efficiency of black soldier fly larvae oil nanoliposomes was determined, as shown in Table 8. Prolonged sonication time may lead to cumulative thermal effects and continuous mechanical stress, disrupting vesicle integrity and the phospholipid bilayer structure, resulting in a decrease in encapsulation efficiency. The optimal sonication time for encapsulation efficiency was 10 min.

[0075] Table 8 Example 9:

[0076] A method for preparing black soldier fly larvae oil nanoliposomes includes the following steps: S1: With a fixed soybean lecithin concentration of 8 mg / mL, a soybean lecithin to cholesterol mass ratio of 4:1, a soybean lecithin to black soldier fly oil mass ratio of 10:1, and polysorbate 80 added at 20% of the soybean lecithin content, accurately weigh soybean lecithin, cholesterol, polysorbate 80, and black soldier fly oil into anhydrous ethanol and sonicate for 15 min to completely dissolve them. Then, transfer the solution to a 250 mL round-bottom flask and remove the anhydrous ethanol from the mixture by rotary evaporation (water bath 40 ℃, 30 min, vacuum < 5 mbar) until a uniform, transparent lipid film forms on the flask wall.

[0077] S2: Measure a certain amount of pH 7.0, 0.05 mol / L PBS buffer and add it to the lipid membrane. Place it in a water bath and shake it manually for 5-10 minutes to detach the lipid membrane and obtain a preliminary crude liposome suspension.

[0078] S3: Transfer the crude liposome suspension to a glass bottle and sonicate it with an ice bath probe (300 W, 10 min, 60 s working, 5 s pause) to obtain nanoliposomes. Example 10:

[0079] A method for preparing black soldier fly larvae oil nanoliposomes includes the following steps: S1: With a fixed soybean lecithin concentration of 8 mg / mL, a soybean lecithin to cholesterol mass ratio of 4.76:1, a soybean lecithin to black soldier fly oil mass ratio of 13.5:1, and polysorbate 80 added at 19.75% of the soybean lecithin content, soybean lecithin, cholesterol, polysorbate 80, and black soldier fly oil were accurately weighed into anhydrous ethanol and sonicated for 15 min until completely dissolved. The solution was then transferred to a 250 mL round-bottom flask, and anhydrous ethanol was removed from the mixture by rotary evaporation (water bath 40 ℃, 30 min, vacuum < 5 mbar) until a uniform, transparent lipid film formed on the flask wall.

[0080] S2: Measure a certain amount of pH 7.0, 0.05 mol / L PBS buffer and add it to the lipid membrane. Place it in a water bath and shake it manually for 5-10 minutes to detach the lipid membrane and obtain a preliminary crude liposome suspension.

[0081] S3: Transfer the crude liposome suspension to a glass bottle and sonicate it with an ice bath probe (300 W, 10 min, 60 s working, 5 s pause) to obtain nanoliposomes.

[0082] Comparative Example 1: A method for preparing black soldier fly larvae oil nanoliposomes includes the following steps: S1: Soy lecithin concentration 20 mg / mL, soy lecithin to cholesterol mass ratio 4:1, soy lecithin to black soldier fly oil mass ratio 10:1, polysorbate 80 added at 20% of the soy lecithin content. Soy lecithin, cholesterol, polysorbate 80, and black soldier fly oil were accurately weighed into anhydrous ethanol and sonicated for 15 min to completely dissolve. The solution was then transferred to a 250 mL round-bottom flask, and the anhydrous ethanol was removed from the mixture by rotary evaporation (water bath 40℃, 30 min, vacuum < 5 mbar) until a uniform, transparent lipid film formed on the flask wall.

[0083] S2: Measure a certain amount of pH 7.0, 0.05 mol / L PBS buffer and add it to the lipid membrane. Place it in a water bath and shake it manually for 5-10 minutes to detach the lipid membrane and obtain a preliminary crude liposome suspension.

[0084] S3: Transfer the crude liposome suspension to a glass bottle and sonicate it with an ice bath probe (300 W, 10 min, 60 s working, 5 s pause) to obtain nanoliposomes.

[0085] Comparative Example 2: A method for preparing black soldier fly larvae oil nanoliposomes includes the following steps: S1: With a fixed soybean lecithin concentration of 8 mg / mL, a soybean lecithin to cholesterol mass ratio of 3:1, a soybean lecithin to black soldier fly oil mass ratio of 10:1, and polysorbate 80 added at 20% of the soybean lecithin content, accurately weigh soybean lecithin, cholesterol, polysorbate 80, and black soldier fly oil into anhydrous ethanol and sonicate for 15 min to completely dissolve them. Then, transfer the solution to a 250 mL round-bottom flask and remove the anhydrous ethanol from the mixture by rotary evaporation (water bath 40 ℃, 30 min, vacuum < 5 mbar) until a uniform, transparent lipid film forms on the flask wall.

[0086] S2: Measure a certain amount of pH 7.0, 0.05 mol / L PBS buffer and add it to the lipid membrane. Place it in a water bath and shake it manually for 5-10 minutes to detach the lipid membrane and obtain a preliminary crude liposome suspension.

[0087] S3: Transfer the crude liposome suspension to a glass bottle and sonicate it with an ice bath probe (300 W, 10 min, 60 s working, 5 s pause) to obtain nanoliposomes.

[0088] Comparative Example 3: No Cholesterol Added A method for preparing black soldier fly larvae oil nanoliposomes includes the following steps: S1: With a fixed soybean lecithin concentration of 8 mg / mL, a soybean lecithin to black soldier fly oil mass ratio of 10:1, and polysorbate 80 added at 20% of the soybean lecithin content, accurately weigh soybean lecithin, polysorbate 80, and black soldier fly oil into anhydrous ethanol and sonicate for 15 min to completely dissolve them. Then, transfer the solution to a 250 mL round-bottom flask and remove the anhydrous ethanol from the mixture by rotary evaporation (water bath 40 ℃, 30 min, vacuum < 5 mbar) until a uniform, transparent lipid film forms on the flask wall.

[0089] S2: Measure a certain amount of pH 7.0, 0.05 mol / L PBS buffer and add it to the lipid membrane. Place it in a water bath and shake it manually for 5-10 minutes to detach the lipid membrane and obtain a preliminary crude liposome suspension.

[0090] S3: Transfer the crude liposome suspension to a glass bottle and sonicate it with an ice bath probe (300 W, 10 min, 60 s working, 5 s pause) to obtain nanoliposomes.

[0091] Comparative Example 4: A method for preparing black soldier fly larvae oil nanoliposomes includes the following steps: S1: With a fixed soybean lecithin concentration of 8 mg / mL, a soybean lecithin to cholesterol mass ratio of 4:1, a soybean lecithin to black soldier fly oil mass ratio of 10:1, and polysorbate 80 added at 50% of the soybean lecithin content, accurately weigh soybean lecithin, cholesterol, polysorbate 80, and black soldier fly oil into anhydrous ethanol and sonicate for 15 min to completely dissolve them. Then, transfer the solution to a 250 mL round-bottom flask and remove the anhydrous ethanol from the mixture by rotary evaporation (water bath 40 ℃, 30 min, vacuum < 5 mbar) until a uniform, transparent lipid film forms on the flask wall.

[0092] S2: Measure a certain amount of pH 7.0, 0.05 mol / L PBS buffer and add it to the lipid membrane. Place it in a water bath and shake it manually for 5-10 minutes to detach the lipid membrane and obtain a preliminary crude liposome suspension.

[0093] S3: Transfer the crude liposome suspension to a glass bottle and sonicate it with an ice bath probe (300 W, 10 min, 60 s working, 5 s pause) to obtain nanoliposomes.

[0094] Comparative Example 5: 80g without Tween A method for preparing black soldier fly larvae oil nanoliposomes includes the following steps: S1: With a fixed soybean lecithin concentration of 8 mg / mL, a soybean lecithin to cholesterol mass ratio of 4:1, and a soybean lecithin to black soldier fly oil mass ratio of 10:1, accurately weigh soybean lecithin, cholesterol, and black soldier fly oil into anhydrous ethanol according to the specified ratios. Sonicate for 15 min to ensure complete dissolution. Then, transfer the solution to a 250 mL round-bottom flask and remove the anhydrous ethanol from the mixture by rotary evaporation (water bath 40℃, 30 min, vacuum < 5 mbar) until a uniform, transparent lipid film forms on the flask wall.

[0095] S2: Measure a certain amount of pH 7.0, 0.05 mol / L PBS buffer and add it to the lipid membrane. Place it in a water bath and shake it manually for 5-10 minutes to detach the lipid membrane and obtain a preliminary crude liposome suspension.

[0096] S3: Transfer the crude liposome suspension to a glass bottle and sonicate it with an ice bath probe (300 W, 10 min, 60 s working, 5 s pause) to obtain nanoliposomes.

[0097] Comparative Example 6: A method for preparing black soldier fly larvae oil nanoliposomes includes the following steps: S1: With a fixed soybean lecithin concentration of 8 mg / mL, a soybean lecithin to cholesterol mass ratio of 4:1, a soybean lecithin to black soldier fly oil mass ratio of 10:1, and polysorbate 80 added at 20% of the soybean lecithin content, accurately weigh soybean lecithin, cholesterol, polysorbate 80, and black soldier fly oil into anhydrous ethanol and sonicate for 15 min to completely dissolve them. Then, transfer the solution to a 250 mL round-bottom flask and remove the anhydrous ethanol from the mixture by rotary evaporation (water bath 70℃, 30 min, vacuum < 5 mbar) until a uniform, transparent lipid film forms on the flask wall.

[0098] S2: Measure a certain amount of pH 7.0, 0.05 mol / L PBS buffer and add it to the lipid membrane. Place it in a water bath and shake it manually for 5-10 minutes to detach the lipid membrane and obtain a preliminary crude liposome suspension.

[0099] S3: Transfer the crude liposome suspension to a glass bottle and sonicate it with an ice bath probe (50W, 5 min, 60 s working, 5 s pause) to obtain nanoliposomes.

[0100] Comparative Example 7: A method for preparing black soldier fly larvae oil nanoliposomes includes the following steps: S1: With a fixed soybean lecithin concentration of 8 mg / mL, a soybean lecithin to cholesterol mass ratio of 4:1, a soybean lecithin to black soldier fly oil mass ratio of 10:1, and polysorbate 80 added at 20% of the soybean lecithin content, accurately weigh a specific proportion of soybean lecithin, cholesterol, polysorbate 80, and black soldier fly oil into anhydrous ethanol and sonicate for 15 min to completely dissolve them. Then, transfer the solution to a 250 mL round-bottom flask and remove the anhydrous ethanol from the mixture by rotary evaporation (water bath 35℃, 30 min, vacuum < 5 mbar) until a uniform, transparent lipid film forms on the flask wall.

[0101] S2: Measure a certain amount of pH 7.0, 0.05 mol / L PBS buffer and add it to the lipid membrane. Place it in a water bath and shake it manually for 5-10 minutes to detach the lipid membrane and obtain a crude liposome suspension.

[0102] Comparative Example 8: A method for preparing black soldier fly larvae oil nanoliposomes includes the following steps: S1: With a fixed soybean lecithin concentration of 8 mg / mL, a soybean lecithin to cholesterol mass ratio of 4:1, and a soybean lecithin to black soldier fly oil mass ratio of 10:1, accurately weigh soybean lecithin, cholesterol, and black soldier fly oil into anhydrous ethanol and sonicate for 15 min to ensure complete dissolution. Then, transfer the solution to a 250 mL round-bottom flask and remove the anhydrous ethanol from the mixture by rotary evaporation (water bath 40 ℃, 30 min, vacuum < 5 mbar) until a uniform, transparent lipid film forms on the flask wall.

[0103] S2: Measure a certain amount of pH 7.0, 0.05 mol / L PBS buffer, add polysorbate 80 at 20% of the amount of soybean lecithin, dissolve polysorbate 80 in PBS buffer, add the mixture to the lipid membrane, place in a water bath and shake manually for 5-10 min to detach the lipid membrane, and initially obtain a crude liposome suspension.

[0104] S3: Transfer the crude liposome suspension to a glass bottle and sonicate it with an ice bath probe (300 W, 10 min, 60 s working, 5 s pause) to obtain nanoliposomes.

[0105] Comparative Example 9: No oils added A method for preparing black soldier fly larvae oil nanoliposomes includes the following steps: S1: With a fixed concentration of 8 mg / mL for soy lecithin and a soy lecithin to cholesterol mass ratio of 4:1, and polysorbate 80 added at 20% of the soy lecithin content, accurately weigh the soy lecithin, cholesterol, and polysorbate 80 into anhydrous ethanol and sonicate for 15 min until completely dissolved. Then, transfer the solution to a 250 mL round-bottom flask and remove the anhydrous ethanol from the mixture by rotary evaporation (water bath 40 ℃, 30 min, vacuum < 5 mbar) until a uniform, transparent lipid film forms on the flask wall.

[0106] S2: Measure a certain amount of pH 7.0, 0.05 mol / L PBS buffer and add it to the lipid membrane. Place it in a water bath and shake it manually for 5-10 minutes to detach the lipid membrane and obtain a preliminary crude liposome suspension.

[0107] S3: Transfer the crude liposome suspension to a glass bottle, add black soldier fly oil at a mass ratio of soybean lecithin to black soldier fly oil of 10:1, and sonicate with an ice bath probe (300 W, 10 min, 60 s working, 5 s pause) to obtain nanoliposomes.

[0108] This application uses a Malvern laser particle size analyzer (nano-ZS90) to determine the average particle size, polydispersity index (PDI), and zeta potential of the black soldier fly larvae oil nanoliposomes obtained in Examples 1-9 and Comparative Examples 1-9. The testing procedures include: diluting the nanoliposomes 50 times, using the instrument at a testing temperature of 25°C and a scattering angle of 90°, and performing three parallel measurements on each sample to determine the influence of different factors on the particle size distribution of the black soldier fly larvae oil nanoliposomes.

[0109] Please see Figures 1-3 The polydispersity index (PDI) is an important indicator for evaluating the uniformity of particle size distribution in colloidal systems. A smaller PDI value indicates a narrower particle size distribution range, more uniform size, and better dispersibility. Examples 1-8 all feature nanoliposomes with small average particle size and PDI, indicating good dispersibility. Meanwhile, the particle size distribution diagram of Example 9 shows that the nanoliposomes exhibit a unimodal distribution and a relatively concentrated particle size distribution, with an average particle size of 133.6 nm and a PDI of 0.197, indicating that these nanoliposomes have a small average particle size and low PDI.

[0110] Please see Figures 4-5 Zeta potential is an important parameter characterizing the surface charge density and electrostatic stability of colloidal particles. A larger Zeta potential can inhibit the aggregation of particles in solution, so the Zeta potential can predict the stability of colloidal solutions to some extent. The nanoliposomes formed in Examples 1-8 all have high Zeta potentials, indicating that they have good stability.

[0111] Then, the encapsulation efficiency of Examples 9 and Comparative Examples 1-9 was determined using a UV spectrophotometer (U-T6A). Specific steps: An appropriate amount of black soldier fly larvae oil nanoliposomes was placed in a centrifuge tube, 2 mL of n-hexane was added, vortexed for 1 min, centrifuged at 4000 r / min for 15 min, and the upper n-hexane layer was collected. This extraction was repeated twice, and the n-hexane layers were combined and brought to a final volume of 10 mL. Using n-hexane as a blank, the absorbance at 231 nm (A0) was measured, and the free black soldier fly larvae oil content (C0) was calculated. Anhydrous methanol was added to the lower precipitate after the above extraction, vortexed for 30 s, and ultrasonically demulsified for 10 min. Add 2 mL of n-hexane, centrifuge at 4000 r / min for 15 min, take the upper n-hexane layer, repeat the extraction until the aqueous phase is clear, combine the n-hexane layers and make up to volume, measure the absorbance at 231 nm (A1), calculate the encapsulated black soldier fly oil content (C1), and calculate the encapsulation efficiency using the following formula: Please refer to Table 9. The black soldier fly oil nanoliposomes in Example 9 are superior to those in Comparative Examples 1-9. In Comparative Example 1, the concentration of soybean lecithin exceeded the range defined in this application. Excessive phospholipids led to increased system viscosity and disordered molecular arrangement, forming multi-compartmental or heterogeneous vesicles, manifested as increased particle size and PDI, and decreased encapsulation efficiency. In Comparative Example 2, the mass ratio of soybean lecithin to black soldier fly oil exceeded the range defined in this application, resulting in a significant decrease in encapsulation efficiency. This indicates that the excess oil exceeded the encapsulation capacity of the lipid bilayer, leading to phase separation and an increase in unencapsulated oil. In Comparative Example 3, no cholesterol was added, which could not effectively stabilize the liposome bilayer structure, resulting in reduced membrane fluidity. After sonication, the nanoliposome particles were easily dispersed, and the encapsulated black soldier fly oil leaked out, resulting in a significant decrease in particle size and encapsulation efficiency. In Comparative Examples 4 and 5, the polysorbate 80 content of the liposomes was outside the range defined in this application. Polysorbate 80, as a nonionic surfactant, can reduce interfacial tension and increase bilayer fluidity, thereby promoting the formation of smaller particles. Therefore, the particle size of the liposomes in Comparative Example 4 decreased significantly, while that in Comparative Example 5 increased sharply. Comparative Example 6 broke through the upper limit of rotary evaporation temperature. Compared with Example 9, its average particle size increased significantly, and its encapsulation efficiency decreased significantly, indicating that the low-temperature rotary evaporation process used in this invention maximizes the preservation of the biological efficacy of black soldier fly oil while achieving nano-sized particles. In Comparative Example 7, no probe sonication was performed, and the liposomes failed to be effectively broken down and dispersed. The resulting nanoliposomes exhibited obvious opalescence or turbidity, with a relatively large average particle size and PDI, and relatively low Zeta potential and encapsulation efficiency. In Comparative Example 8, polysorbate 80 was added in step S2. The surfactant interfered with the self-assembly of liposomes during hydration, resulting in a significant extension of hydration time. Furthermore, the particle size, PDI, and encapsulation efficiency were all inferior to those of Example 9. Therefore, this method is not within the scope of protection of this application. Compared to Comparative Example 9, Example 9 showed a 20.4 nm increase in particle size, a decrease in PDI, and an increase in Zeta potential, indicating that Example 9 successfully encapsulated black soldier fly oil, and the encapsulated nanoliposomes exhibited superior colloidal stability. Compared to Comparative Examples 1-9, Example 9, overall, showed a decrease in average particle size and PDI, an increase in potential, and a more stable and uniformly distributed system. The method provided in Example 9 of this invention offers smaller particle size and PDI, as well as better encapsulation effect, maximizing the bioavailability of black soldier fly oil.

[0112] Table 9 Then, this application conducted storage stability and temperature stability tests on Examples 1-9 and Comparative Examples 1-9, and conducted ionic strength stability tests on Example 9.

[0113] Storage stability: The prepared black soldier fly larvae oil nanoliposomes were sealed and stored at 4℃, 25℃, and 37℃ for 30 days. Samples were taken on days 0, 7, 15, and 30 to observe changes in appearance (layering, turbidity, precipitation, etc.) and to evaluate them. Changes in average particle size, PDI, Zeta potential, pH value, and encapsulation efficiency in Example 9 were measured using a Malvern laser particle size analyzer (nano-ZS90), a pH meter, and a UV spectrophotometer (U-T6A).

[0114] Temperature stability: Take 5.0 mL of nanoliposome sample and place it in a stoppered heat-resistant glass beaker. Heat it in a water bath at 25℃, 50℃, 70℃ and 90℃ for 10 min respectively, and observe the appearance changes (layering, turbidity, precipitation, etc.).

[0115] Ionic strength stability: NaCl or MgCl2 stock solutions were added to the nanoliposome samples to achieve final concentrations of 0 (control), 0.1% (w / v), 0.2% (w / v), 0.3% (w / v), 0.4% (w / v), and 0.5% (w / v), respectively. After magnetic stirring at 25 °C for 10 min, particle size, PDI, and zeta potential were measured.

[0116] Please see Figure 6 , Figure 7 and Figure 8 In Example 9, the average particle size stored at 4°C, 25°C, and 37°C remained within the nanometer range, showing slight fluctuations but no significant increase. Compared to 4°C, the liposomes stored at 25°C and 37°C had larger particle sizes, which is attributed to increased membrane fluidity and vesicle fusion due to the higher temperature. The sample stored at 4°C maintained the smallest and most stable particle size (approximately 120 nm), indicating that low temperature effectively inhibits aggregation and fusion, maintaining the initial particle size distribution and colloidal homogeneity. Figure 7 It can be seen that all PDI values ​​remained below 0.3, indicating good particle size uniformity during storage. Figure 8 As can be seen, the Zeta potential remains negative, stable between -41.6 and -45.0 mV, indicating good colloidal stability and anti-aggregation ability. Therefore, under the experimental conditions, 4℃ is the optimal storage temperature.

[0117] Please see Figure 9 and Figure 10 Both encapsulation efficiency and pH value gradually decreased during storage, with the rate of decrease accelerating at higher temperatures. This decrease is attributed to leakage of contents due to bilayer membrane structure disruption and vesicle fusion. Simultaneously, the decrease in pH value may accelerate the hydrolysis of ester bonds linking fatty acids to the phospholipid glycerol backbone.

[0118] Please refer to Table 10. The stability of Examples 1-8 is significantly better than that of Comparative Examples 1-9, indicating that preparation methods outside the range of formulations and preparation processes set in this application cannot effectively form nanoliposomes. The material formulations used in Comparative Examples 1-2 are not within the scope of this application, and the preparation process parameters used in Comparative Example 2 are also not within the scope of this application. This results in the black soldier fly larvae oil not being well encapsulated, making it prone to aggregation, fusion, leakage, and other phenomena, leading to reduced stability.

[0119] Table 10 Referring to Table 11, across the entire ion concentration range (0–0.5%, w / v), in Example 9, the particle size and PDI increased slightly with increasing ion concentration, but remained within a reasonable range, indicating that the liposome structure was not significantly disturbed. However, due to the electrostatic shielding effect of counterions on the surface charge, the absolute value of the Zeta potential decreased with increasing ion concentration. Overall, these data demonstrate that nanoliposomes can coexist stably with metal ions, maintaining structural integrity within the experimental concentration range.

[0120] Table 11 Then, Example 9 was characterized using transmission electron microscopy (TEM) with negative staining: the nanoliposome suspension was appropriately diluted with deionized water and mixed with 2% (w / v) phosphotungstic acid at a 1:1 volume ratio. After standing at room temperature for 2-3 min, a drop of the mixture was placed on a clean copper grid, excess liquid was absorbed with filter paper, and the mixture was air-dried at room temperature for 5 min before imaging observation.

[0121] Please see Figure 11 The vesicles are spherical or nearly spherical with smooth, clear edges and no visible aggregation, indicating good morphological integrity. A clear bilayer membrane structure can be observed, consistent with the typical liposome structure.

[0122] Then, this application tested the DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) concentration of Example 9 and black soldier fly oil, using ascorbic acid (vitamin C (VC)) as a positive control. Example 9, black soldier fly oil, and ascorbic acid were diluted to the same concentration. 7.9 mg of DPPH was dissolved in 100 mL of anhydrous ethanol to prepare a 0.2 mmol / L DPPH solution. The DPPH solution was mixed with an equal volume of Example 9 and black soldier fly oil, and reacted at room temperature in the dark for 30 min. The absorbance at 517 nm was measured and denoted as A. 样品 The absorbance of the mixture of DPPH solution and anhydrous ethanol was used as the control group (A). 对照 The absorbance of the blank group (A) was measured after the sample was mixed with anhydrous ethanol.空白 All measurements were repeated three times. DPPH radical scavenging rate was calculated using formula 3-2: In the formula: A 样品 A represents the absorbance of the reaction system. 空白 A represents the background absorbance of the sample. 对照 The initial absorbance of DPPH.

[0123] Please see Figure 12 As concentration increases, the scavenging rate correspondingly increases. Compared with unencapsulated black soldier fly oil, nano-encapsulation significantly improved DPPH free radical scavenging activity. This may be due to the weak antioxidant activity of phospholipid molecules themselves, and the fact that nano-encapsulation improves the dispersibility of the oil, making the antioxidant components in the core material more readily react with DPPH free radicals. Specifically, at the same concentration, the scavenging rate of liposomes reached 68%, while that of free oil was only 55%. Therefore, nano-encapsulation effectively enhances the antioxidant activity of black soldier fly oil, thereby improving its bioavailability.

[0124] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing black soldier fly larvae oil nanoliposomes, characterized in that, Includes the following steps: (1) Dissolve black soldier fly oil, phospholipids, sterol film stabilizers and nonionic surfactants in pharmaceutical grade volatile organic solvent, sonicate until clear, and then remove the solvent by rotary evaporation under reduced pressure to form a uniform lipid film on the inner wall of the container. (2) Hydrate the lipid film by adding a physiologically compatible isotonic buffer system to obtain a crude liposome suspension; (3) The crude liposome suspension is subjected to ultrasonic treatment to obtain black soldier fly oil nanoliposomes; The concentration of phospholipids mentioned in step (1) is 8~10 mg / mL; The mass ratio of the phospholipid to the sterol stabilizer is 4:1 to 6:1; The mass ratio of the phospholipid to the black soldier fly oil is 10:1 to 20:1; The amount of the nonionic surfactant added is 15% to 20% of the phospholipid; The temperature of the vacuum rotary evaporation is 40℃~60℃; In step (2), the buffer system is one of phosphate-buffered saline (PBS), Tris-HCl buffer, or HEPES buffer; the pH of the buffer system is 6.5 to 8.0; the hydration temperature is 40℃ to 60℃, and the concentration of the hydration medium is 0.034 mol / L to 0.06 mol / L. The ultrasonic power in step (3) is 250 W ~ 300 W; the ultrasonic time is 5 min ~ 10 min; the working time is 0 s ~ 99 s, and the pause time is 0 s ~ 99 s.

2. The preparation method according to claim 1, characterized in that, The phospholipid is soybean lecithin; the sterol film stabilizer is cholesterol; the nonionic surfactant is polysorbate 80; and the pharmaceutical-grade volatile organic solvent is anhydrous ethanol.

3. The preparation method according to claim 1, characterized in that, The concentration of the phospholipid is 8 mg / mL, the mass ratio of the phospholipid to the sterol stabilizer is 4:1, the mass ratio of the phospholipid to the black soldier fly oil is 10:1, the amount of nonionic surfactant added is 20% of the phospholipid, the temperature of the reduced pressure rotary evaporation is 40℃, the buffer system is 0.05 mol / L PBS buffer, the pH value of the buffer system is 7.0, the ultrasonic power is 300 W, the ultrasonic time is 10 min, the working time is 60 s, and the pause time is 5 s.

4. A black soldier fly larvae oil nanoliposome, prepared by the method for preparing black soldier fly larvae oil nanoliposomes according to any one of claims 1-3, wherein the average particle size of the nanoliposomes is 133.1~138.3 nm, the polydispersity index (PDI) is 0.165~0.211, and the black soldier fly larvae oil encapsulation efficiency is 81.79~83.55%.

5. The application of the black soldier fly larvae oil nanoliposomes as described in claim 4 in the preparation of functional foods, animal feed additives, cosmetic active ingredient delivery carriers, or antibacterial and anti-inflammatory drug formulations.

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