Black soldier fly oil grease freeze-dried liposome and preparation method thereof

Abstract

The application discloses black soldier fly oil freeze-dried liposomes and a preparation method thereof. The preparation method of the black soldier fly oil freeze-dried liposomes comprises the following steps: dissolving phospholipid, sterol, black soldier fly oil and a surfactant in an organic solvent to form a film, hydrating the film with a buffer solution, and preparing nano-liposomes through pulse ice bath ultrasonic treatment; adding a freeze-drying protective agent, uniformly mixing, pre-freezing, and freeze-drying, so as to obtain the black soldier fly oil freeze-dried liposomes. The stability of the black soldier fly oil is remarkably improved through the freeze-drying process, long-term storage of the liposomes at room temperature is realized, and a new strategy is provided for the development of a functional oil delivery system.

Description

Technical Field

[0001] This invention relates to the field of liposomes, specifically to a freeze-dried liposome of black soldier fly larvae and its preparation method. Background Technology

[0002] Black soldier flies, as a resource-rich insect, have attracted widespread attention in recent years in the fields of feed, food, and resource utilization due to their advantages such as short growth cycle, high reproductive capacity, and efficient conversion of organic waste. Black soldier fly larvae are rich in nutrients such as protein, lipids, amino acids, minerals, and vitamins, and also contain bioactive compounds such as fatty acids, antimicrobial peptides, and chitosan, exhibiting significant antibacterial, antiviral, and antioxidant capabilities, making them of significant application potential in the food, feed, and bioenergy sectors.

[0003] Black soldier fly larvae oil (BSFL O) has a balanced composition of saturated and unsaturated fatty acids. Lauric acid (C12:0) is the main saturated fatty acid, exhibiting significant antibacterial activity. Medium-chain fatty acids are easily digested and absorbed, contributing to the oil's high nutritional and functional value. Among the unsaturated fatty acids, oleic acid (C18:1, n-9) and linoleic acid (C18:2, n-6) are dominant. Oleic acid is known as a "healthy fatty acid," effectively improving blood lipid profiles and reducing the risk of cardiovascular disease; linoleic acid is a precursor to the synthesis of arachidonic acid and other eicosanoic acid-like substances, participating in important physiological processes such as regulating inflammatory responses and promoting vasodilation. This unique fatty acid composition makes black soldier fly oil a promising candidate for applications in nutritional health products and functional food development.

[0004] Although black soldier fly oil has diverse functions and wide applications, it is rich in polyunsaturated fatty acids, which are easily affected by factors such as light, heat, and oxygen, resulting in oxidative rancidity and unpleasant odor. In addition, its poor water solubility and low bioavailability severely restrict its application in the food, pharmaceutical, and cosmetic fields.

[0005] Liposomes, as classic bilayer nanocarriers, can effectively encapsulate hydrophobic active ingredients and improve their bioavailability. However, conventional liquid liposomes are prone to phospholipid hydrolysis, oxidation, vesicle fusion, and active ingredient leakage during storage, resulting in a short shelf life. If traditional freeze-drying processes are used directly, ice crystal puncture, membrane phase transition, or lack of effective protection mechanisms often lead to the collapse of freeze-dried products, a sharp increase in particle size after reconstitution, and a precipitous drop in encapsulation efficiency.

[0006] In conclusion, it is necessary to develop a method for preparing freeze-dried liposomes of black soldier fly oil with well-defined process parameters, a suitable protection system, and strong structural reversibility, in order to achieve high stability, high reconstitution rate, and industrial mass production, and to overcome the shortcomings of existing technologies. Summary of the Invention

[0007] This invention aims to overcome the technical defects of existing black soldier fly larvae oil liposomes, such as short storage period, easy structural damage during freeze-drying, and poor reconstitution performance. It provides a black soldier fly larvae oil freeze-dried liposome with high encapsulation efficiency, good stability, uniform particle size, complete freeze-drying formation, and highly restored nanostructure after reconstitution. It can be stored at room temperature. The invention also provides a method for preparing the black soldier fly larvae oil freeze-dried liposome, which solves the existing problems in current applications and has good application prospects.

[0008] The first aspect of this invention provides a method for preparing freeze-dried liposomes of black soldier fly larvae, comprising the following steps: (1) Phospholipids, sterol membrane stabilizers, black soldier fly oil and nonionic surfactants are dissolved in anhydrous pharmaceutical grade volatile organic solvent, mixed and the organic solvent is removed under reduced pressure to form a lipid membrane; buffer solution is added to the lipid membrane for hydration, and then ultrasonic treatment is performed to obtain nanoliposomes loaded with black soldier fly oil. (2) Add a freeze-drying protectant to the nanoliposomes, mix them evenly, and then perform pre-freezing and freeze-drying in sequence to obtain black soldier fly oil freeze-dried liposomes; The freeze-drying protectant is β-lactose; The amount of the freeze-drying protectant added is 8%~15% w / v of the aqueous phase volume.

[0009] Furthermore, in step (1), The phospholipid is one or more of soybean lecithin, egg yolk lecithin, or hydrogenated soybean lecithin; The sterol membrane stabilizer is one of cholesterol, β-sitosterol, or stigmasterol; The nonionic surfactant is one or more of the following: self-polysorbates, poloxamers, PEGylated phospholipids, or bile salts; The organic solvent is anhydrous ethanol or isopropanol; The buffer solution is PBS buffer or HEPES buffer, with a pH of 6.5-7.5.

[0010] Preferably, the phospholipid is soybean lecithin; The sterol membrane stabilizer is cholesterol; The nonionic surfactant is self-polysorbate 80; The organic solvent is anhydrous ethanol; The buffer solution is PBS buffer with a pH of 7.0.

[0011] 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 5:1; The mass ratio of the phospholipid to the black soldier fly oil is 10:1 to 14:1; 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 6.0~8.0; the hydration temperature is 40~60℃, and the concentration of the hydration medium is 0.034~0.06mol / L. In step (3), the ultrasonic power is 100~300 W; the ultrasonic time is 5~25 min; the working time is 0~99 s, and the pause time is 0~99 s.

[0012] Further, in step (2), the freeze-drying protectant is preferably sucrose or β-lactose; the amount of freeze-drying protectant added is 10% w / v of the volume of the aqueous phase.

[0013] Further, in step (2), the pre-freezing temperature is -30℃ to -80℃, preferably -30℃ or -80℃.

[0014] The pre-freezing time is 6 to 24 hours, preferably 12 hours.

[0015] The drying time is 6 to 36 hours, preferably 24 hours.

[0016] The second objective of this invention is to provide a method for preparing a black soldier fly larvae oil liposome dispersion: adding a reconstitution medium to the freeze-dried black soldier fly larvae oil liposomes, vortexing for 2-6 min, and performing a reconstitution treatment; the reconstitution medium is PBS buffer, physiological saline, or deionized water, preferably PBS buffer.

[0017] The third objective of this invention is to provide a freeze-dried liposome of black soldier fly larvae oil, which is prepared by the aforementioned method for preparing freeze-dried liposomes of black soldier fly larvae oil. The freeze-dried liposomes have an average particle size of 135.1~145.1 nm, a polydispersity index (PDI) of 0.337~0.479, a black soldier fly larvae oil encapsulation rate of >81%, and a particle size change rate of 9.58%~17.89% after reconstitution.

[0018] The application of the freeze-dried liposomes of black soldier fly oil provided by this invention in the preparation of functional foods, feed growth promoters, transdermal cosmetic carriers, or fat-soluble nutrient delivery formulations.

[0019] Compared with the prior art, the advantages of the present invention are as follows: (1) This invention uses soybean lecithin and cholesterol as composite membrane materials. Through a reasonable ratio, black soldier fly oil is encapsulated in liposomes, which solves the problems of poor water solubility, low bioavailability and unpleasant odor of black soldier fly oil. (2) Phospholipid bilayer physically isolates oxygen, and freeze-drying thoroughly dehydrates the oil, effectively inhibiting the oxidation and rancidity of polyunsaturated fatty acids, further improving the stability of black soldier fly oil and enabling its storage at room temperature.

[0020] (3) The range of each parameter is orthogonally optimized, which has a good production tolerance. The prepared black soldier fly oil freeze-dried liposomes are evenly distributed, the system is stable and the encapsulation rate is high. After adding freeze-drying protectant, it has high temperature storage effect, good stability and extremely high application value, and can seamlessly meet the downstream processing needs of food, feed, cosmetics and pharmaceutical preparations. Attached Figure Description

[0021] Figure 1 This is a particle size distribution diagram for Example 5.

[0022] Figure 2 This is a Zeta potential distribution diagram for Example 5.

[0023] Figure 3 This is a TEM image of Example 5.

[0024] Figure 4 The image shown is the infrared spectrum of Example 5. Detailed Implementation

[0025] 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.

[0026] 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, polysorbate 80, Tween 80, D-mannitol, sucrose, glucose, maltose, β-lactose and D-trehalose were all 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.

[0027] The amounts of each component added in the embodiments of the present invention are all in parts by weight.

[0028] Example 1: A method for preparing freeze-dried liposomes of black soldier fly larvae oil, comprising the following steps: S1: In this embodiment, the mass ratio of soybean lecithin, cholesterol, black soldier fly oil, and polysorbate 80 is 8.1:1.7:0.6:1.6. Soybean lecithin, cholesterol, black soldier fly oil, and polysorbate 80 are accurately weighed into anhydrous ethanol and ultrasonicated for 15 min to ensure thorough mixing. Subsequently, the solution is subjected to rotary evaporation at 40°C for 30 min to remove the anhydrous ethanol from the mixture, yielding a lipid membrane. S2: 10 mL of PBS buffer with pH 7.0 and concentration of 0.05 mol / L was added to the lipid membrane for hydration and washing. The membrane was then subjected to sonication in an ice bath for 10 min with a sonication power of 300 W and a sonication interval of 60 s / 5 s to obtain black soldier fly larvae oil nanoliposomes. S3: Add 10% (w / v, based on the volume of black soldier fly larvae oil nanoliposomes) of sucrose, vortex to mix, and pre-freeze at -80℃ for 12 h. Then transfer to a vacuum freeze dryer for 24 h to obtain freeze-dried black soldier fly larvae oil liposomes. Add PBS buffer and vortex until the freeze-dried liposomes are completely reconstituted for later use.

[0029] Example 2: A method for preparing freeze-dried liposomes of black soldier fly larvae oil, comprising the following steps: S1: In this embodiment, the mass ratio of soybean lecithin, cholesterol, black soldier fly oil, and polysorbate 80 is 8.1:1.7:0.6:1.6. Soybean lecithin, cholesterol, black soldier fly oil, and polysorbate 80 are accurately weighed into anhydrous ethanol and ultrasonicated for 15 min to ensure thorough mixing. Subsequently, the solution is subjected to rotary evaporation at 40°C for 30 min to remove the anhydrous ethanol from the mixture, yielding a lipid membrane. S2: 10 mL of PBS buffer with pH 7.0 and concentration of 0.05 mol / L was added to the lipid membrane for hydration and washing. The membrane was then subjected to sonication in an ice bath for 10 min with a sonication power of 300 W and a sonication interval of 60 s / 5 s to obtain black soldier fly larvae oil nanoliposomes. S3: Add 10% (w / v, based on the volume of black soldier fly oil nanoliposomes) of sucrose, vortex mix, and pre-freeze in an ultra-low temperature freezer for 12 h at -30℃. Then transfer to a vacuum freeze dryer for freeze-drying for 24 h to obtain black soldier fly oil lyophilized liposomes. After adding PBS buffer, vortex until the black soldier fly lyophilized liposomes are completely reconstituted and ready for use.

[0030] Example 3: A method for preparing freeze-dried liposomes of black soldier fly larvae oil, comprising the following steps: S1: In this embodiment, the mass ratio of soybean lecithin, cholesterol, black soldier fly oil, and polysorbate 80 is 8.1:1.7:0.6:1.6. Soybean lecithin, cholesterol, black soldier fly oil, and polysorbate 80 are accurately weighed into anhydrous ethanol and ultrasonicated for 15 min to ensure thorough mixing. Subsequently, the solution is subjected to rotary evaporation at 40°C for 30 min to remove the anhydrous ethanol from the mixture, yielding a lipid membrane. S2: 10 mL of PBS buffer with pH 7.0 and concentration of 0.05 mol / L was added to the lipid membrane for hydration and washing. The membrane was then subjected to sonication in an ice bath for 10 min with a sonication power of 300 W and a sonication interval of 60 s / 5 s to obtain black soldier fly larvae oil nanoliposomes. S3: Add 10% (w / v, based on the volume of black soldier fly larvae oil nanoliposomes) of sucrose, vortex mix, and pre-freeze at -80℃ for 18 h. Then transfer to a vacuum freeze dryer for 24 h to obtain freeze-dried black soldier fly larvae oil liposomes. Add PBS buffer and vortex until the freeze-dried liposomes are completely reconstituted for later use.

[0031] Example 4: A method for preparing freeze-dried liposomes of black soldier fly larvae oil, comprising the following steps: S1: In this embodiment, the mass ratio of soybean lecithin, cholesterol, black soldier fly oil, and polysorbate 80 is 8.1:1.7:0.6:1.6. Soybean lecithin, cholesterol, black soldier fly oil, and polysorbate 80 are accurately weighed into anhydrous ethanol and ultrasonicated for 15 min to ensure thorough mixing. Subsequently, the solution is subjected to rotary evaporation at 40°C for 30 min to remove the anhydrous ethanol from the mixture, yielding a lipid membrane. S2: 10 mL of PBS buffer with pH 7.0 and concentration of 0.05 mol / L was added to the lipid membrane for hydration and washing. The membrane was then subjected to sonication in an ice bath for 10 min with a sonication power of 300 W and a sonication interval of 60 s / 5 s to obtain black soldier fly larvae oil nanoliposomes. S3: Add 10% (w / v, based on the volume of black soldier fly oil nanoliposomes) of sucrose, vortex to mix, and pre-freeze at -80℃ for 12 h. Then transfer to a vacuum freeze dryer for 18 h to obtain black soldier fly oil lyophilized liposomes. Add PBS buffer and vortex until the lyophilized liposomes are completely reconstituted for later use.

[0032] Example 5: A method for preparing freeze-dried liposomes of black soldier fly larvae oil, comprising the following steps: S1: In this embodiment, the mass ratio of soybean lecithin, cholesterol, black soldier fly oil, and polysorbate 80 is 8.1:1.7:0.6:1.6. Soybean lecithin, cholesterol, black soldier fly oil, and polysorbate 80 are accurately weighed into anhydrous ethanol and ultrasonicated for 15 min to ensure thorough mixing. Subsequently, the solution is subjected to rotary evaporation at 40°C for 30 min to remove the anhydrous ethanol from the mixture, yielding a lipid membrane. S2: 10 mL of PBS buffer with pH 7.0 and concentration of 0.05 mol / L was added to the lipid membrane for hydration and washing. The membrane was then subjected to sonication in an ice bath for 10 min with a sonication power of 300 W and a sonication interval of 60 s / 5 s to obtain black soldier fly larvae oil nanoliposomes. S3: Add 10% (w / v, based on the volume of black soldier fly larvae oil nanoliposomes) of β-lactose, vortex to mix, and pre-freeze at -80℃ for 12 h. Then transfer to a vacuum freeze dryer for 24 h to obtain lyophilized black soldier fly larvae oil liposomes. After adding PBS buffer, vortex until the lyophilized black soldier fly larvae are completely reconstituted and set aside for later use.

[0033] Example 6: A method for preparing freeze-dried liposomes of black soldier fly larvae oil, comprising the following steps: S1: In this embodiment, the mass ratio of soybean lecithin, cholesterol, black soldier fly oil, and polysorbate 80 is 8.1:1.7:0.6:1.6. Soybean lecithin, cholesterol, black soldier fly oil, and polysorbate 80 are accurately weighed into anhydrous ethanol and ultrasonicated for 15 min to ensure thorough mixing. Subsequently, the solution is subjected to rotary evaporation at 40°C for 30 min to remove the anhydrous ethanol from the mixture, yielding a lipid membrane. S2: 10 mL of PBS buffer with pH 7.0 and concentration of 0.05 mol / L was added to the lipid membrane for hydration and washing. The membrane was then subjected to sonication in an ice bath for 10 min with a sonication power of 300 W and a sonication interval of 60 s / 5 s to obtain black soldier fly larvae oil nanoliposomes. S3: Add 8% (w / v, based on the volume of black soldier fly larvae oil nanoliposomes) of β-lactose, vortex to mix, and pre-freeze at -80℃ for 12 h. Then transfer to a vacuum freeze dryer for 24 h to obtain lyophilized black soldier fly larvae oil liposomes. After adding PBS buffer, vortex until the lyophilized black soldier fly larvae are completely reconstituted and set aside for later use.

[0034] Example 7: A method for preparing freeze-dried liposomes of black soldier fly larvae oil, comprising the following steps: S1: In this embodiment, the mass ratio of soybean lecithin, cholesterol, black soldier fly oil, and polysorbate 80 is 8.1:1.7:0.6:1.6. Soybean lecithin, cholesterol, black soldier fly oil, and polysorbate 80 are accurately weighed into anhydrous ethanol and ultrasonicated for 15 min to ensure thorough mixing. Subsequently, the solution is subjected to rotary evaporation at 40°C for 30 min to remove the anhydrous ethanol from the mixture, yielding a lipid membrane. S2: 10 mL of PBS buffer with pH 7.0 and concentration of 0.05 mol / L was added to the lipid membrane for hydration and washing. The membrane was then subjected to sonication in an ice bath for 10 min with a sonication power of 300 W and a sonication interval of 60 s / 5 s to obtain black soldier fly larvae oil nanoliposomes. S3: Add 10% β-lactose, vortex mix, and pre-freeze in an ultra-low temperature freezer for 12 h at -80℃. Then transfer to a vacuum freeze dryer for freeze-drying for 24 h to obtain black soldier fly larvae oil freeze-dried liposomes. After adding physiological saline, vortex until the black soldier fly larvae freeze-dried liposomes are completely reconstituted and set aside for later use.

[0035] Comparative Example 1: A method for preparing freeze-dried liposomes of black soldier fly larvae, comprising the following steps: S1: In this embodiment, the mass ratio of soybean lecithin, cholesterol, black soldier fly oil, and polysorbate 80 is 8.1:1.7:0.6:1.6. Soybean lecithin, cholesterol, black soldier fly oil, and polysorbate 80 are accurately weighed into anhydrous ethanol and ultrasonicated for 15 min to ensure thorough mixing. Subsequently, the solution is subjected to rotary evaporation at 40°C for 30 min to remove the anhydrous ethanol from the mixture, yielding a lipid membrane. S2: 10 mL of PBS buffer with pH 7.0 and concentration of 0.05 mol / L was added to the lipid membrane for hydration and washing. The membrane was then subjected to sonication in an ice bath for 10 min with a sonication power of 300 W and a sonication interval of 60 s / 5 s to obtain black soldier fly larvae oil nanoliposomes. S3: Add 20% (w / v, based on the volume of black soldier fly larvae oil nanoliposomes) of β-lactose, vortex to mix, and pre-freeze at -80℃ for 12 h. Then transfer to a vacuum freeze dryer for 24 h to obtain lyophilized black soldier fly larvae oil liposomes. Add PBS buffer and vortex until the lyophilized black soldier fly larvae liposomes are completely reconstituted for later use.

[0036] Comparative Example 2: A method for preparing freeze-dried liposomes of black soldier fly larvae, comprising the following steps: S1: In this embodiment, the mass ratio of soybean lecithin, cholesterol, black soldier fly oil, and polysorbate 80 is 8.1:1.7:0.6:1.6. Soybean lecithin, cholesterol, black soldier fly oil, and polysorbate 80 are accurately weighed into anhydrous ethanol and ultrasonicated for 15 min to ensure thorough mixing. Subsequently, the solution is subjected to rotary evaporation at 40°C for 30 min to remove the anhydrous ethanol from the mixture, yielding a lipid membrane. S2: 10 mL of PBS buffer with pH 7.0 and concentration of 0.05 mol / L was added to the lipid membrane for hydration and washing. The membrane was then subjected to sonication in an ice bath for 10 min with a sonication power of 300 W and a sonication interval of 60 s / 5 s to obtain black soldier fly larvae oil nanoliposomes. S3: Add 10% (w / v, based on the volume of black soldier fly larvae oil nanoliposomes) of β-lactose, vortex to mix, and pre-freeze at -80℃ for 12 h. Then transfer to a vacuum freeze dryer for 3 h to obtain lyophilized black soldier fly larvae oil liposomes. Add PBS buffer and vortex until the lyophilized black soldier fly larvae liposomes are completely reconstituted for later use.

[0037] Comparative Example 3: A method for preparing freeze-dried liposomes of black soldier fly larvae, comprising the following steps: S1: In this embodiment, the mass ratio of soybean lecithin, cholesterol, black soldier fly oil, and polysorbate 80 is 8.1:1.7:0.6:1.6. Soybean lecithin, cholesterol, black soldier fly oil, and polysorbate 80 are accurately weighed into anhydrous ethanol and ultrasonicated for 15 min to ensure thorough mixing. Subsequently, the solution is subjected to rotary evaporation at 40°C for 30 min to remove the anhydrous ethanol from the mixture, yielding a lipid membrane. S2: 10 mL of PBS buffer with pH 7.0 and concentration of 0.05 mol / L was added to the lipid membrane for hydration and washing. The membrane was then subjected to sonication in an ice bath for 10 min with a sonication power of 300 W and a sonication interval of 60 s / 5 s to obtain black soldier fly larvae oil nanoliposomes. S3: Add 10% (w / v, based on the volume of black soldier fly larvae oil nanoliposomes) of β-lactose, vortex to mix, and pre-freeze at -80℃ for 3 h. Then transfer to a vacuum freeze dryer for 24 h to obtain lyophilized black soldier fly larvae oil liposomes. After adding PBS buffer, vortex until the lyophilized black soldier fly larvae liposomes are completely reconstituted and set aside for later use.

[0038] Comparative Example 4: A method for preparing liposomes, comprising the following steps: S1: In this embodiment, the mass ratio of soybean lecithin, cholesterol, and polysorbate 80 is 8.1:1.7:1.6. Soybean lecithin, cholesterol, and polysorbate 80 are accurately weighed into anhydrous ethanol and ultrasonicated for 15 min to ensure thorough mixing. Subsequently, the solution is subjected to rotary evaporation at 40°C for 30 min to remove the anhydrous ethanol from the mixture, yielding a lipid membrane. S2: Measure 10 mL of PBS buffer with pH 7.0 and concentration of 0.05 mol / L and add it to the lipid membrane. Perform hydration washing on the membrane, and then sonicate it with an ice bath probe for 10 min. The sonication power is 300 W, and the sonication is performed for 60 s with a 5 s interval to obtain black soldier fly oil nanoliposomes.

[0039] Comparative Example 5: A method for preparing black soldier fly larvae lipid liposomes, comprising the following steps: S1: In this embodiment, the mass ratio of soybean lecithin, cholesterol, black soldier fly oil, and polysorbate 80 is 8.1:1.7:0.6:1.6. Soybean lecithin, cholesterol, black soldier fly oil, and polysorbate 80 are accurately weighed into anhydrous ethanol and ultrasonicated for 15 min to ensure thorough mixing. Subsequently, the solution is subjected to rotary evaporation at 40°C for 30 min to remove the anhydrous ethanol from the mixture, yielding a lipid membrane. S2: Measure 10 mL of PBS buffer with pH 7.0 and concentration of 0.05 mol / L and add it to the lipid membrane. Perform hydration washing on the membrane, and then sonicate it with an ice bath probe for 10 min. The sonication power is 300 W, and the sonication is performed for 60 s with a 5 s interval to obtain black soldier fly oil nanoliposomes.

[0040] Comparative Example 6: 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.

[0041] S2: Measure a certain amount of pH 8.0, 0.05 mol / L PBS buffer and add it to the lipid membrane. Place the membrane in a water bath and shake manually for 5-10 min to detach the lipid membrane, obtaining a preliminary crude liposome suspension. Transfer the crude liposome suspension to a glass bottle and sonicate it with an ice bath probe (300 W, 10 min, 60 s on, 5 s off) to obtain nanoliposomes.

[0042] 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.

[0043] Table 1 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 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.

[0044] S2: Measure a certain amount of pH 8.0, 0.05 mol / L PBS buffer and add it to the lipid membrane. Place the membrane in a water bath and shake manually for 5-10 min to detach the lipid membrane, obtaining a preliminary crude liposome suspension. Transfer the crude liposome suspension to a glass bottle and sonicate it with an ice bath probe (300 W, 10 min, 60 s on, 5 s off) to obtain nanoliposomes.

[0045] 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.

[0046] Table 2 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 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 the membrane in a water bath and shake manually for 5-10 min to detach the lipid membrane, obtaining a preliminary crude liposome suspension. Transfer the crude liposome suspension to a glass bottle and sonicate it with an ice bath probe (300 W, 10 min, 60 s on, 5 s off) to obtain nanoliposomes.

[0048] 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.

[0049] Table 3 Comparative Example 9: 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.

[0050] S2: Measure a certain amount of pH 8.0, 0.05 mol / L PBS buffer and add it to the lipid membrane. Place the membrane in a water bath and shake manually for 5-10 min to detach the lipid membrane, obtaining a preliminary crude liposome suspension. Transfer the crude liposome suspension to a glass bottle and sonicate it with an ice bath probe (300 W, 10 min, 60 s on, 5 s off) to obtain nanoliposomes.

[0051] 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%.

[0052] Table 4 Comparative Example 10: 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.

[0053] S2: Measure a certain amount of pH 7.0, 0.05 mol / L PBS buffer and add it to the lipid membrane. Place the membrane in a water bath and shake manually for 5-10 min to detach the lipid membrane, obtaining a preliminary crude liposome suspension. Transfer the crude liposome suspension to a glass bottle and sonicate it with an ice bath probe (300 W, 10 min, 60 s on, 5 s off) to obtain nanoliposomes.

[0054] 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℃.

[0055] Table 5 Comparative Example 11: 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.

[0056] S2: Set the pH of the PBS buffer to 6.0, 6.5, 7.0, 7.5, and 8.0. Measure a certain amount of PBS buffer and add it to the lipid membrane. Place the membrane in a water bath and shake manually for 5-10 minutes to detach the lipid membrane, obtaining a preliminary crude liposome suspension. Transfer the crude liposome suspension to a glass bottle and sonicate it with an ice bath probe (300 W, 10 minutes, 60 seconds on, 5 seconds off) to obtain nanoliposomes.

[0057] 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.

[0058] Table 6 Comparative Example 12: 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.

[0059] S2: A certain amount of pH 7.0, 0.05 mol / L PBS buffer was added to the lipid membrane, and the membrane was placed in a water bath and manually shaken for 5-10 min to detach the lipid membrane, thus obtaining a preliminary crude liposome suspension. The crude liposome suspension was transferred to a glass bottle, and the ultrasonic power was set to 100, 150, 200, 250, and 300 W, with an ultrasonic time of 10 min, intermittent operation for 60 s, and a pause for 5 s. The nanoliposomes were obtained by ultrasonication with an ice bath probe.

[0060] 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.

[0061] Table 7 Comparative Example 13: 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: A certain amount of pH 7.0, 0.05 mol / L PBS buffer was added to the lipid membrane, and the membrane was placed in a water bath and manually shaken for 5-10 min to detach the lipid membrane, thus obtaining a preliminary crude liposome suspension. The crude liposome suspension was transferred to a glass bottle, and the sonication time was set to 5, 10, 15, 20, and 25 min, with a sonication power of 300 W, intermittent operation for 60 s, and a 5 s pause. The nanoliposomes were obtained by sonication with an ice bath probe.

[0063] 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.

[0064] Table 8 Comparative Example 14: 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.

[0065] S2: Measure a certain amount of pH 7.0, 0.05 mol / L PBS buffer and add it to the lipid membrane. Place the membrane in a water bath and shake manually for 5-10 min to detach the lipid membrane, obtaining a preliminary crude liposome suspension. Transfer the crude liposome suspension to a glass bottle and sonicate it with an ice bath probe (300 W, 10 min, 60 s on, 5 s off) to obtain nanoliposomes.

[0066] Comparative Example 15: 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.

[0067] S2: Measure a certain amount of pH 7.0, 0.05 mol / L PBS buffer and add it to the lipid membrane. Place the membrane in a water bath and shake manually for 5-10 min to detach the lipid membrane, obtaining a preliminary crude liposome suspension. Transfer the crude liposome suspension to a glass bottle and sonicate it with an ice bath probe (300 W, 10 min, 60 s on, 5 s off) to obtain nanoliposomes.

[0068] Comparative Example 16: 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.

[0069] S2: Measure a certain amount of pH 7.0, 0.05 mol / L PBS buffer and add it to the lipid membrane. Place the membrane in a water bath and shake manually for 5-10 min to detach the lipid membrane, obtaining a preliminary crude liposome suspension. Transfer the crude liposome suspension to a glass bottle and sonicate it with an ice bath probe (300 W, 10 min, 60 s on, 5 s off) to obtain nanoliposomes.

[0070] Comparative Example 17: 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.

[0071] S2: Measure a certain amount of pH 7.0, 0.05 mol / L PBS buffer and add it to the lipid membrane. Place the membrane in a water bath and shake manually for 5-10 min to detach the lipid membrane, obtaining a preliminary crude liposome suspension. Transfer the crude liposome suspension to a glass bottle and sonicate it with an ice bath probe (300 W, 10 min, 60 s on, 5 s off) to obtain nanoliposomes.

[0072] Comparative Example 18: 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.

[0073] S2: Measure a certain amount of pH 7.0, 0.05 mol / L PBS buffer and add it to the lipid membrane. Place the membrane in a water bath and shake manually for 5-10 min to detach the lipid membrane, obtaining a preliminary crude liposome suspension. Transfer the crude liposome suspension to a glass bottle and sonicate it with an ice bath probe (300 W, 10 min, 60 s on, 5 s off) to obtain nanoliposomes.

[0074] Comparative Example 19: 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.

[0075] S2: Measure a certain amount of pH 7.0, 0.05 mol / L PBS buffer and add it to the lipid membrane. Place the membrane in a water bath and shake manually for 5-10 min to detach the lipid membrane, obtaining a preliminary crude liposome suspension. Transfer the crude liposome suspension to a glass bottle and sonicate it with an ice bath probe (50W, 5 min, 60 s on, 5 s off) to obtain nanoliposomes.

[0076] Comparative Example 20: 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.

[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 crude liposome suspension.

[0078] Comparative Example 21: 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.

[0079] 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 the PBS buffer. Add the mixture to the lipid membrane and place it in a water bath. Shake manually for 5-10 minutes to detach the lipid membrane, obtaining a preliminary crude liposome suspension. Transfer the crude liposome suspension to a glass bottle and sonicate with an ice bath probe (300 W, 10 minutes, 60 seconds on, 5 seconds off) to obtain nanoliposomes.

[0080] Comparative Example 22: 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.

[0081] S2: A certain amount of pH 7.0, 0.05 mol / L PBS buffer was added to the lipid membrane, and the membrane was placed in a water bath and manually shaken for 5-10 min to detach the lipid membrane, thus obtaining a preliminary crude liposome suspension. The crude liposome suspension was transferred to a glass bottle, and black soldier fly oil was added at a mass ratio of soybean lecithin to black soldier fly oil of 10:1. The mixture was then sonicated with an ice bath probe (300 W, 10 min, 60 s on, 5 s off) to obtain nanoliposomes.

[0082] This application evaluates the appearance of Examples 1-6 and Comparative Examples 1-3 before reconstitution and their redispersibility after reconstitution. Specific steps: Freeze-dried black soldier fly larvae oil nanoliposome powder was taken and observed and recorded under natural light, including its color, morphology, and surface condition (whether it collapsed, shrank, or cracked). Subsequently, PBS buffer was added to the freeze-dried powder, and the mixture was vortexed for 30 seconds. The reconstitution rate, solution clarity, and whether a uniform opalescence was observed and recorded to evaluate its redispersibility.

[0083] Please refer to Table 9. The appearance and redispersibility of Examples 1-7 are significantly better than those of Comparative Examples 1-3, indicating that preparation methods outside the range of formulation and preparation process set in this application cannot form lyophilized liposomes well. In Comparative Example 1, the amount of protective agent was too high, resulting in excessively high system viscosity and difficulty in reconstitution; in Comparative Example 2, the freeze-drying time was too short, leading to insufficient drying time, high residual moisture content, and sample structural collapse; in Comparative Example 3, the pre-freezing time was insufficient, resulting in incomplete freezing of the system, inadequate vitrification, and unstable liposome structure.

[0084] Table 9 Then, this application used a Malvern laser particle size analyzer (nano-ZS90) and a UV spectrophotometer (U-T6A) to determine the particle size distribution, zeta potential, and encapsulation efficiency of the freeze-dried black soldier fly oil liposomes obtained in Examples 1-7 and Comparative Examples 1-22. Specific steps: The reconstituted liposomes were diluted 50-fold, and the particle size, PDI, and zeta potential were measured at 25°C and a scattering angle of 90°. 2 mL of n-hexane was added to the liposomes, 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 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, collect the supernatant n-hexane extract, repeat the extraction until the aqueous phase is clear, combine the n-hexane extracts and make up to volume, measure the absorbance at 231 nm (A1), and calculate the encapsulated black soldier fly oil content (C1). Calculate the encapsulation efficiency using the following formula: In the formula: C0 is the free black soldier fly oil content, and C1 is the encapsulated black soldier fly oil content.

[0085] Please see Figure 1 and Figure 2 In Example 5, the average particle size of the freeze-dried liposomes of black soldier fly oil was 145.1 nm, the PDI was 0.337, and the Zeta potential was -46.8 mV.

[0086] Please refer to Table 10. The freeze-dried liposomes from black soldier fly oil in Examples 1-7 are significantly better than those in Comparative Examples 1, 2, and 3. In Comparative Example 1, the preservative content in the freeze-dried liposomes was too high. The high osmotic pressure of the preservative caused deformation or rupture of the liposome membrane. Furthermore, excessive preservative itself may crystallize or form complexes, resulting in a decrease in encapsulation efficiency, a slight increase in particle size, and uneven distribution. In Comparative Example 2, the freeze-drying time was too short, resulting in a high residual moisture content and poor liposome stability. In Comparative Example 3, the pre-freezing time was insufficient, the system was not completely frozen, and the liposome structure was unstable, leading to an increase in average particle size and PDI, and a decrease in Zeta potential and encapsulation efficiency. Compared to Comparative Examples 4 and 5, although the average particle size and PDI of Examples 1-7 increased slightly, the Zeta potential increased significantly, indicating that the liposomes after freeze-drying have superior stability. In summary, compared to Comparative Examples 1-3, Examples 1-7 show reduced average particle size and PDI, increased Zeta potential and encapsulation efficiency, and a more stable and uniformly distributed system. Compared to Comparative Examples 4-5, the stability is enhanced. Furthermore, the method provided in Example 5 of this invention offers better encapsulation, significantly improving the bioactivity and bioavailability of black soldier fly oil.

[0087] The black soldier fly larvae oil nanoliposomes in Example 5 were superior to those in Comparative Examples 14-22. In Comparative Example 14, the soybean lecithin concentration exceeded the range defined in the claims of this application. Excessive phospholipids led to increased system viscosity, disordered molecular arrangement, and the formation of multi-compartmental or heterogeneous vesicles, manifested as increased particle size and PDI, and decreased encapsulation efficiency. In Comparative Example 15, the mass ratio of soybean lecithin to black soldier fly larvae oil exceeded the range defined in the claims of this application, resulting in a significant decrease in encapsulation efficiency. This indicated 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 16, 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 17 and 18, the polysorbate 80 content of the liposomes was outside the range defined in the claims of 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 17 decreased significantly, while that in Comparative Example 18 increased sharply. Comparative Example 19 exceeded the upper temperature limit of rotary evaporation. Compared with Example 5, 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 20, 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 21, polysorbate 80 was added in step S2. The surfactant interfered with the self-assembly of liposomes during hydration, leading to a significant extension of hydration time. Furthermore, the particle size, PDI, and encapsulation efficiency were all inferior to those of Example 5. Therefore, this method is not within the scope of protection of the claims in this application. Compared to Comparative Example 22, Example 5 showed a 20.4 nm increase in particle size, a decrease in PDI, and an increase in Zeta potential, indicating that Example 5 successfully encapsulated black soldier fly oil, and the encapsulated nanoliposomes exhibited superior colloidal stability. Compared to Comparative Examples 14-22, Example 5, 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 5 of this invention offers smaller particle size and PDI, as well as better encapsulation effect, maximizing the bioavailability of black soldier fly oil.

[0088] Table 10 Then, the microstructure of Example 5 was determined using a high-resolution transmission electron microscope (H-7650). Specific steps: The reconstituted liposomes were diluted a certain factor and mixed with 2% phosphotungstic acid at a 1:1 volume ratio. After standing at room temperature for 2-3 minutes, 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 minutes before imaging observation.

[0089] Please refer to Figure 3 The reconstituted liposomes exhibited a spherical or elliptical vesicle structure, with uniform size, clear boundaries, and no obvious aggregation or fusion. The lyophilized liposomes maintained their intact bilayer structure, and light-colored regions were visible within the vesicles, indicating that black soldier fly larvae oil was successfully embedded in the liposome core. Furthermore, the average particle size measured by TEM showed good agreement with the hydrodynamic diameter measured by dynamic light scattering (DLS), verifying the reliability of the particle size data.

[0090] Then, this application conducted stability analysis on Example 5 and Comparative Example 5. Three batches of lyophilized black soldier fly larvae oil nanoliposome powder were taken and stored in sealed containers at 4 ℃ (refrigerated), 25 ℃ (room temperature), and 60 ℃, respectively. Samples were taken at 0, 7, 15, and 30 days, and reconstituted with an equal volume of PBS buffer to examine their appearance, particle size distribution, redispersibility, zeta potential, and encapsulation efficiency.

[0091] Please refer to Table 11. In Example 5, after storage at 4℃ and 25℃ for 30 days, the liposomes maintained a white, loose powder appearance, were easily reconstituted, and showed minimal changes in average particle size and encapsulation efficiency after reconstitution. This indicates that the freeze-dried liposomes of black soldier fly oil can be stored under refrigeration and at room temperature with good stability. After storage at 60℃ for 30 days, the encapsulation efficiency decreased significantly, and the average particle size increased considerably. This may be due to high-temperature-induced phospholipid oxidation and hydrolysis, as well as membrane structure damage. However, the encapsulation efficiency remained at 66.47%, and the average particle size only increased slightly. Furthermore, reconstitution was easy, and no phase separation or precipitation occurred. In Comparative Example 5, the nanoliposomes stored at 4°C for 30 days showed almost no change in average particle size and encapsulation efficiency, exhibiting good stability. However, with increasing temperature, the average particle size and encapsulation efficiency of the nanoliposomes were increasingly affected by temperature. Especially after 30 days of storage at 60°C, the encapsulation efficiency of the nanoliposomes was only 44.25%, indicating a decrease in encapsulation efficiency. This makes the unencapsulated oil more susceptible to oxidation. Therefore, nanoliposomes that have not undergone freeze-drying treatment cannot be stored at room temperature or high temperatures. The above results demonstrate that the black soldier fly larvae oil freeze-dried liposomes in Example 5 of this invention possess excellent storage stability and achieve room temperature and high temperature storage of black soldier fly larvae oil nanoliposomes.

[0092] Table 11 Then, this application performed Fourier transform infrared spectroscopy analysis on Example 5, Comparative Examples 4 and 5, and β-lactose. Specific steps: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] -1 Within the wavenumber range, spectra of black soldier fly oil nanoliposomes, blank liposomes, freeze-dried black soldier fly oil liposomes, and β-lactose were acquired. For the determination, the samples were mixed with dry potassium bromide powder and pressed into transparent thin sheets before measurement.

[0093] Please see Figure 4 3600~3200 cm -1 Within the wavenumber range, β-lactose exhibits a broad and strong characteristic absorption peak, attributed to the stretching vibrations of numerous hydroxyl groups in its molecular structure. The three liposome samples also show broad absorption bands in this region, mainly due to the hydrogen bonding of hydrated hydroxyl groups in the polar lipid heads and residual water in the system. In Example 5, the absorption peak of the freeze-dried liposomes from black soldier fly oil was broader and slightly more intense than that before freeze-drying, indicating that the freeze-drying process altered the hydration state of the liposomes and enhanced intermolecular hydrogen bonding. The absorption peak was observed in the 1640–1630 cm⁻¹ range. -1 At this location, absorption peaks were observed in all three liposome samples from Example 5 and Comparative Examples 4 and 5, attributed to the C=O stretching vibration of the phosphatidyl chain, while β-lactose showed weaker absorption at this point. The absorption was observed in the 1200–900 cm⁻¹ region. -1 At this region, β-lactose exhibits multiple sharp absorption peaks, belonging to the characteristic absorption region of carbohydrates. The absorption in this region of the three liposome samples is relatively smooth, showing a significant difference in peak shape compared to β-lactose, indicating that the characteristic peaks of β-lactose overlap with the strong absorption of liposomes. Furthermore, the infrared spectral profiles of the three liposome samples are highly similar, indicating that the loading of black soldier fly oil and the addition of lyophilization protectants did not significantly alter the basic molecular structure of the liposomes, and the chemical environment of the hydrophobic segments of the lipid bilayer remained stable.

[0094] 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 technology 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 freeze-dried liposomes of black soldier fly larvae oil, characterized in that, Includes the following steps: (1) Phospholipids, sterol membrane stabilizers, black soldier fly oil and nonionic surfactants are dissolved in anhydrous pharmaceutical grade volatile organic solvent, mixed and the organic solvent is removed under reduced pressure to form a lipid membrane; buffer solution is added to the lipid membrane for hydration, and then ultrasonic treatment is performed to obtain nanoliposomes loaded with black soldier fly oil. (2) Add a freeze-drying protectant to the nanoliposomes, mix them evenly, and then perform pre-freezing and freeze-drying in sequence to obtain black soldier fly oil freeze-dried liposomes; The freeze-drying protectant is β-lactose; The amount of the freeze-drying protectant added is 8%~15% w / v of the aqueous phase volume.

2. The method for preparing freeze-dried liposomes of black soldier fly larvae oil according to claim 1, characterized in that, In step (1), The phospholipid is one or more of soybean lecithin, egg yolk lecithin, or hydrogenated soybean lecithin; The sterol membrane stabilizer is one of cholesterol, β-sitosterol, or stigmasterol; The nonionic surfactant is one or more of the following: self-polysorbates, poloxamers, PEGylated phospholipids, or bile salts; The organic solvent is anhydrous ethanol or isopropanol; The buffer solution is PBS buffer or HEPES buffer, with a pH of 6.5~7.

5.

3. The method for preparing freeze-dried liposomes of black soldier fly larvae oil according to claim 1, characterized in that, 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 5:1; The mass ratio of the phospholipid to the black soldier fly oil is 10:1 to 14:1; 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 6.0 to 8.0; the hydration temperature is 40 to 60°C, and the concentration of the hydration medium is 0.034 to 0.06 mol / L. In step (3), the ultrasonic power is 100~300 W; the ultrasonic time is 5~25 min; the working time is 0~99 s, and the pause time is 0~99 s.

4. The method for preparing freeze-dried liposomes of black soldier fly larvae oil according to claim 1, characterized in that, In step (2), the pre-freezing temperature is -30℃ to -80℃; the pre-freezing time is 6 to 24 h; and the drying time is 6 to 36 h.

5. The method for preparing freeze-dried liposomes of black soldier fly larvae oil according to claim 1, characterized in that, In step (3), the pre-freezing temperature is -30℃ or -80℃.

6. A method for preparing a black soldier fly larvae lipid liposome dispersion, characterized in that, The reconstitution medium is added to the freeze-dried liposomes of black soldier fly larvae according to any one of claims 1 to 5, and then vortexed to achieve reconstitution; the reconstitution medium is PBS buffer, physiological saline or deionized water.

7. A freeze-dried liposome of black soldier fly larvae, characterized in that, The lyophilized liposomes were prepared by the method described in any one of claims 1 to 5. The average particle size of the lyophilized liposomes was 135.1 to 145.1 nm, the polydispersity index (PDI) was 0.337 to 0.479, the encapsulation efficiency of black soldier fly oil was >81%, and the particle size change rate after reconstitution was 9.58% to 17.89%.

8. The application of the freeze-dried liposomes of black soldier fly oil as described in claim 7 in the preparation of functional foods, feed growth promoters, transdermal cosmetic carriers, or fat-soluble nutrient delivery formulations.

Images