A method for preparing and applying self-assembled artificial oil from nanoparticles

Abstract

This invention discloses a method for preparing and applying a self-assembled artificial oil body from nanoparticles, comprising: 1) preparing nanoparticles by covalently binding plant oil body caleosin and / or plant oil body oleosin with phenolic acid small molecule compounds; 2) preparing artificial lipid droplets using plant phospholipids and plant oils; 3) mixing the nanoparticles from step 1) and the artificial lipid droplets from step 2) uniformly, ultrasonicating; centrifuging, and collecting the upper layer to obtain the self-assembled artificial oil body; by mixing curcumin or other active substances into the oil, the self-assembled artificial oil body, under gastric juice conditions, exhibits strong interaction due to its negatively charged surface, which can protect curcumin from damage and reduce curcumin leakage. Free fatty acids and monoglycerides generated in intestinal juice combine with bile salts and phospholipids to form mixed micelles, exhibiting higher solubilizing ability for curcumin.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a method for preparing a self-assembled artificial oil body from nanoparticles and its application as a carrier in food, cosmetics and pharmaceuticals. Background Technology

[0002] Oil bodies, a common organelle in higher plants, are typically spherical or oval in shape, ranging from 0.5 to 2.5 μm in diameter, and are mainly composed of triacylglycerols (TAGs), phospholipids (PLs), and surface proteins. The TAGs are enclosed by a phospholipid monolayer membrane. Within the membrane, the two acyl groups of the PL molecule interact inward with the hydrophobic TAGs in the matrix, while the hydrophilic PL head groups are exposed in the cytoplasm. The surface proteins of the oil bodies include various proteins such as oleosin, caleosin, and steroleosin. These proteins share similar structural characteristics, forming hydrophobic proline knots inserted into the TAGs, with N- and C-terminal hydrophilic amino acids embedded on the oil body surface. Based on these unique structural features, artificial lipid droplets were prepared by combining triacylglycerols and phospholipids, and nanoparticles were prepared by combining oil body membrane proteins and phenolic acid small molecule compounds. These nanoparticles were then mixed in a specific ratio and self-assembled into artificial oil bodies. The size, structure, and thermal stability of artificial oil bodies can be controlled by altering the proportions of their components and the types of proteins. Artificial oil bodies have simple compositions and clear structures, making them applicable in various fields such as food, feed, cosmetics, medical aesthetics, pharmaceuticals, and chemicals.

[0003] Phenolic acids are active substances derived from vegetables, fruits, and other species, possessing broad-spectrum bactericidal effects and strong antioxidant properties, such as gallic acid, protocatechuic acid, and rosmarinic acid. Artificial oil bodies, which are self-assembled from nanoparticles composed of small molecule phenolic acids, exhibit antioxidant and bactericidal effects. In food, this can reduce the use of preservatives and improve safety. In feed production, emulsifiers containing such artificial oil bodies can be used as fish feed additives.

[0004] Artificial oils, as emulsifiers, are widely used in the food and feed industries, such as in butter, ice cream, vinaigrette, salad dressing, pudding, fruit juice, coatings, fish products, pet food, and livestock feed. In food processing, using artificial oils as emulsifiers not only effectively increases the content of unsaturated fatty acids and regulates the ratio of saturated to unsaturated fatty acids, but more importantly, it can carry hydrophilic or hydrophobic proteins or metabolites, such as fat-soluble vitamin E, coenzyme Q10, curcumin, and allicin, making it one of the healthiest and most economical emulsifiers. Using artificial oil emulsifiers in fruit juice can enrich its nutritional value and prevent the sedimentation of solid components. Butter made with artificial oils contains a small amount of saturated fatty acids, making it healthier than traditional butter; artificial oils can also replace milk solids, reducing the cost of ice cream production.

[0005] Artificial oils have promising applications in the cosmetics industry, such as creams, lotions, cosmetics, hair care products, and bath products containing various plant oils, such as soaps, detergents, and cleaning agents. For example, in cosmetics, artificial oils can be added as emulsifiers or used to load active ingredients as functional components. In toothpaste products with special functions, oils can also serve as transport carriers for ingredients such as flavoring agents, fluorides, silica, chelating agents, and sweeteners.

[0006] In pharmaceutical formulations, artificial oils can serve as therapeutic, diagnostic, and transport carriers. Utilizing artificial oils as drug delivery carriers, self-assembled artificial oils can target and deliver hydrophilic and hydrophobic drugs, offering excellent encapsulation and sustained-release effects. They can also be fabricated into nanomaterials for precise targeting of ligands, achieving targeted therapeutic effects. In industrial applications, artificial oil emulsifiers can be used in lubricants, paints, coatings, inks, varnishes, films, paper sizes, latexes, etc. Summary of the Invention

[0007] The purpose of this invention is to solve the above-mentioned problems by providing a method for preparing and applying a self-assembled artificial oil body made of nanoparticles.

[0008] A nanoparticle self-assembled artificial oil body is prepared by the following method, comprising:

[0009] 1) Nanoparticles are prepared by covalently binding plant oleosin and / or plant oleosin with small molecule phenolic acid compounds.

[0010] a. Add caleosin and / or oleosin, a caleosin protein from plant oil bodies, to phosphate buffer to prepare a caleosin and / or oleosin protein phosphate buffer solution.

[0011] b. Add small molecule phenolic acid compounds, stir, and incubate;

[0012] c. Adjust the pH of the solution from step b to 7.0; dialyze to remove free phenolic acid molecules, yielding nanoparticles;

[0013] 2) Preparation of artificial lipid droplets

[0014] a. Add plant phospholipids and plant oils to anhydrous ethanol solution and mix thoroughly to obtain a mixture;

[0015] b. Add the mixture to the Tween 80 solution and stir; after the ethanol has completely evaporated, quickly cool to room temperature to obtain artificial lipid droplets; 3) Utilize nanoparticles and artificial lipid droplets to self-assemble artificial oil bodies;

[0016] a. Mix the nanoparticles described in step 1) and the artificial lipid droplets described in step 2) evenly, and then treat with ultrasound;

[0017] b. Centrifuge, centrifuge, take the upper layer, and obtain the self-assembled artificial oil body;

[0018] The phenolic acid small molecule compound mentioned in step 1) is rosmarinic acid (RoSA), gallic acid, catechin, or quercetin;

[0019] The plant phospholipid mentioned in step 2) is soybean lecithin;

[0020] The plants mentioned in step 1) are safflower, soybean, peanut, rapeseed, sesame, castor bean, sunflower, perilla seed, or tiger nut;

[0021] The phenolic acid small molecule compound mentioned is rosmarinic acid (RoSA);

[0022] The plant oil mentioned in step 2) is safflower oil;

[0023] The protein mentioned in step 1) is an artificially expressed protein;

[0024] The concentration of the protein phosphate buffer solution mentioned in step 1) is 3%;

[0025] In step 2), the mass ratio of plant phospholipids to plant oils is 10:1.

[0026] Step 2) involves mixing the plant oils and bioactive substances, followed by mixing them with plant phospholipids;

[0027] The bioactive substance mentioned is curcumin.

[0028] Step 2) involves adjusting the pH of the protein phosphate buffer solution to 9.0 before adding phenolic acid small molecule compounds.

[0029] Sequence Listing 1 is SEQ ID NO 1: Safflower Caleosin nucleotide sequence

[0030] Sequence listing 2 is SEQ ID NO 2: Safflower oleosin nucleotide sequence

[0031] This invention provides a method for preparing and applying a self-assembled artificial oil body from nanoparticles, comprising: 1) preparing nanoparticles by covalently binding plant oil body caleosin and / or plant oil body oleosin with phenolic acid small molecule compounds; 2) preparing artificial lipid droplets using plant phospholipids and plant oils; 3) mixing the nanoparticles from step 1) and the artificial lipid droplets from step 2) uniformly, ultrasonicating; centrifuging, and collecting the upper layer to obtain the self-assembled artificial oil body; the self-assembled artificial oil body prepared by mixing curcumin into the oil exhibits strong interaction with the negatively charged surface of the artificial oil body under gastric juice conditions, protecting curcumin from damage and reducing curcumin leakage. Free fatty acids and monoglycerides generated in intestinal juice combine with bile salts and phospholipids to form mixed micelles, which have a higher solubilizing capacity for curcumin. Attached Figure Description

[0032] Figure 1 It is a map of prokaryotic expression vectors;

[0033] Figure 2 This involves SDS-PAGE analysis of the target protein's induced expression.

[0034] Figure 3 The target protein was purified by affinity chromatography and analyzed by SDS-PAGE.

[0035] Figure 4 This is an image of the artificial oil body.

[0036] Figure 5 It is an in vitro digestion SDS-PAGE analysis. Detailed Implementation

[0037] Example 1: Construction of safflower oil body caleosin and safflower oil body oleosin vectors and preparation of engineered bacteria

[0038] (1) Cloning and vector construction of safflower caleosin and safflower oleosin genes

[0039] Using cDNA from mature safflower seeds as a template, the target caleosin and oleosin genes (sequence listings 1 and 2) were cloned using PCR. The two genes were then ligated into cloning vectors. The recombinant cloning vectors were transformed into *E. coli* host strain DH5α. Single colonies were picked from plating for identification. Positive colonies were expanded and cultured, and recombinant plasmids were extracted. The target genes were obtained by restriction enzyme digestion. Then, gene recombination technology was used to construct the target genes into a prokaryotic expression vector (see appendix). Figure 1The recombinant prokaryotic expression vector was obtained and transformed into Escherichia coli BL21(DE3) to prepare engineered Escherichia coli BL21(DE3) expressing safflower caleosin protein and Escherichia coli BL21(DE3) expressing safflower oleosin protein.

[0040] (2) Induction and purification of safflower caleosin and safflower oleosin proteins

[0041] Recombinant engineered bacteria expressing safflower caleosin and safflower oleosin proteins were inoculated into LB medium and cultured at 37°C and 200 rpm in a shaker until the bacterial OD600nm reached 0.6. Then, different concentrations (0.1–1.0 mM) of IPTG were added for further culture. The bacterial cells were collected by centrifugation, resuspended in PBS, and sonicated. The supernatant and precipitate were collected separately and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). (See attached image) Figure 2 Coomassie Brilliant Blue R-250 staining was performed, and the electrophoresis results were analyzed using ImageJ software to determine the proportions of calcium oleosin and oleosin in the total protein, thus determining the optimal IPTG concentration to be 0.5 mM. After induction for 2-6 hours, the optimal induction time was found to be 5 hours. The recombinant bacteria were cultured under optimal conditions (IPTG concentration 0.5 mM, 37℃, induction for 5 hours), centrifuged, and the cells were harvested, lysed, and sonicated to collect the supernatant. The supernatant was filtered through a 0.45 μm filter membrane. The column was then equilibrated with NI-NAT and 5 column volumes of equilibration buffer, followed by 5 column volumes of wash buffer (containing 20 mM imidazole). Finally, calcium oleosin and oleosin were eluted with elution buffer, and the results were analyzed by SDS-PAGE electrophoresis (see attached). Figure 3 The purity of safflower caleosin and safflower oleosin proteins was determined by HPLC, and the purity of both proteins was ≥90%.

[0042] Example 2: Artificial Oil Body Self-Assembly Process

[0043] (1) Safflower caleosin protein and safflower oleosin protein were covalently bound to phenolic acid small molecule compounds to prepare nanoparticles.

[0044] 3g of caleosin protein and 3g of oleosin protein were separately prepared into 3% caleosin and oleosin protein solutions by placing them in 100ml of sodium phosphate buffer. The solutions were continuously stirred and incubated overnight at 4°C to allow for complete protein hydration. The pH of both caleosin and oleosin protein solutions was adjusted to 9.0, and small molecule phenolic compounds (rosmarinic acid RoSA, gallic acid, catechin, and quercetin) were added and incubated for 12 hours to induce covalent coupling. Then, 0.5... The pH of the solution was adjusted to 7.0 using hydrochloric acid to obtain covalent complexes of different types of phenolic acid small molecules. The covalent complexes were dialyzed in distilled water at 4°C to remove free phenolic acid small molecules (rosmarinic acid RoSA, gallic acid, catechin, quercetin), and then freeze-dried to obtain nanoparticles. The binding rate was measured to find that the nanoparticles obtained by binding safflower calciumoside protein and safflower oleosin protein with rosmarinic acid had binding rates of 95% and 94%, respectively, which were higher than the binding rates of other plant calciumoside protein and oleosin protein (see Table 1). To further determine the optimal concentration of rosmarinic acid, different concentrations (0 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, 150 μM, 200 μM, 250 μM, 300 μM) of rosmarinic acid RoSA were added to two protein solutions and incubated for 12 h to induce covalent coupling. The pH of the solutions was then adjusted to 7.0 using 0.5 M hydrochloric acid to obtain covalent complexes of different concentrations of calciumosin-RosA and oleosin-RosA. These covalent complexes were dialyzed in distilled water at 4 °C to remove free rosmarinic acid RoSA, and then freeze-dried to obtain nanoparticles. The binding rates of safflower calciumosin protein with different concentrations of rosmarinic acid RoSA and the binding rates of safflower oleosin protein solution with different concentrations of rosmarinic acid RoSA were determined, and the optimal binding rate of 3% safflower calciumosin protein with 200 μM rosmarinic acid RoSA was determined. Rosmarinic acid (RoSA) showed the highest binding rate and reached saturation, thus it was identified as nanoparticle 1 (safflower caleosin-RosA). 3% safflower oleosin protein showed the highest binding rate with 250 μM rosmarinic acid (RoSA) and reached saturation, thus it was identified as nanoparticle 2 (safflower oleosin-RosA). The binding rates of both nanoparticle 1 and nanoparticle 2 reached over 94%.

[0045] Binding ratio(%)=(Totalphenolic acid(mg)-phenolic acidindialysate(mg)) / ((Totalphenolic acid(mg))) x 100% ①

[0046] mg of quercetin / mg of protein=(Totalphenolic acid(mg)X Binding ratio%)x100% / (Amountofprotein (mg)) ②

[0047] Table 1 Comparison of the binding rates of safflower oil-related proteins to small phenolic acid compounds.

[0048]

[0049] (2) Preparation of artificial lipid droplets

[0050] Phospholipids (soybean lecithin) and oils (safflower oil, olive oil, sesame oil) were mixed at a mass ratio of 10:1 and heated to dissolve in anhydrous ethanol solution. The mixture was stirred for 30 seconds using a magnetic stirrer to ensure thorough mixing. The mixture was then slowly added to a 0.2% Tween 80 solution and stirred on a magnetic stirrer for 3-4 hours, during which time the ethanol was completely evaporated. The mixture was then rapidly cooled to room temperature in an ice-water bath to obtain artificial lipid droplets of soybean lecithin and oils (safflower oil, olive oil, sesame oil).

[0051] (3) Self-assembly of artificial oil bodies using nanoparticles and artificial lipid droplets

[0052] Dissolve 300 mg of nanoparticle 1 (safflower caleosin-RosA) and 300 mg of nanoparticle 2 (safflower oleosin-RosA) prepared in (1) in phosphate buffer. Add 62.5 mg of artificial lipid droplets (safflower oil, olive oil, sesame oil) prepared in (2) to nanoparticle 1 solution and nanoparticle 2 solution respectively. Mix them thoroughly to obtain mixture 1 and mixture 2. Place the above mixtures on ice and sonicate them for 20 s respectively. Sonicate three times, centrifuge, and the upper layer is artificial oil. Artificial oil bodies can be prepared from sesame oil, olive oil, and safflower oil. Particle size analysis revealed that the artificial oil bodies prepared from olive oil had particle sizes of 1473 nm (nanoparticle 1) and 1584 nm (nanoparticle 2), while those prepared from sesame oil had particle sizes of 1754 nm (nanoparticle 1) and 1842 nm (nanoparticle 2), and those prepared from safflower oil had particle sizes of 819 nm (nanoparticle 1) and 896 nm (nanoparticle 2) (see Table 2). Microstructural observation showed that the artificial oil bodies prepared from safflower oil were uniformly dispersed and had uniform particle sizes. Therefore, it was determined that artificial lipid droplets could be constructed using safflower oil and soybean lecithin, and then self-assembled with nanoparticles 1 and 2 to form artificial oil bodies. Specifically, artificial lipid droplets-safflower caleosin-RosA self-assembled to form artificial oil body 1, and artificial lipid droplets-safflower oleosin-RosA self-assembled to form artificial oil body 2 (see Table 2). Figure 4 ).

[0053] Table 2. Self-assembly of artificial oil bodies by nanoparticles and artificial lipid droplets

[0054]

[0055] Example 3: Optimization of Self-Assembly Conditions for Artificial Oil Bodies

[0056] (1) pH value: Set different pH values ​​(pH 4.5~pH 9.5) of 0.01M phosphate buffer solution, dissolve nanoparticles 1 and 2 in phosphate buffer solution of different pH values ​​respectively, and follow the operation steps of Example 3 (3) above to obtain artificial oil body 1 and artificial oil body 2 under different pH conditions. The particle size and potential detection showed that the particle size of artificial oil body 1 and artificial oil body 2 prepared under pH 7.5 conditions was the smallest.

[0057] (2) Ultrasonic intensity: Nanoparticles 1 and 2 were dissolved in pH 7.5 phosphate buffer solution and the operation steps of Example 3 (3) were followed. The mixture was placed on ice and ultrasonically oscillated. The ultrasonic power was 100W~450W. Artificial oil body 1 and artificial oil body 2 were prepared. The ultrasonic intensity was determined to be 300W by detecting the particle size and potential of artificial oil body 1 and artificial oil body 2.

[0058] (3) Ultrasonic time: Nanoparticles 1 and 2 were dissolved in pH 7.5 phosphate buffer solution, and the operation steps of Example 3 (3) were followed. The mixture was placed on ice and ultrasonically oscillated at an ultrasonic intensity of 300 W for 10 s to 60 s to prepare artificial oil bodies 1 and 2. The ultrasonic time was determined to be 20 s by measuring the particle size and potential of artificial oil bodies 1 and 2. The optimal conditions for the self-assembly of artificial oil bodies were determined to be pH 7.5, ultrasonic intensity of 300 W, and ultrasonic time of 20 s.

[0059] Example 4: Property Testing of Artificial Oil Bodies

[0060] The properties of artificial oil bodies 1 and 2, prepared under conditions of pH=7.5, ultrasonic intensity of 300W, and ultrasonic time of 20s, were analyzed. The ζ-potential of artificial oil bodies 1 and 2 was measured using a Malvern Zetasizer potentiometer, and the particle size of artificial oil bodies 1 and 2 was measured using a Malvern Mastersizer laser particle size analyzer. All tests were conducted at room temperature. The results of three measurements showed that the particle size of artificial oil body 1 was below 600nm, and the particle size of artificial oil body 2 was between 650-950nm. Thermal analysis of the thermogravimetric analysis of both self-assembled artificial oil bodies 1 and 2 showed that the self-assembled artificial oil bodies were more stable than natural oil bodies. FTIR spectroscopy analysis showed that both self-assembled artificial oil bodies 1 and 2 had increased phenolic hydroxyl groups. Rheological measurements of the artificial oil bodies indicated that, within the tested shear rate range, both self-assembled artificial oil bodies 1 and 2 exhibited significant shear-thinning behavior.

[0061] Example 5: Emulsifying properties of artificial oils

[0062] Self-assembled artificial oil body 1, artificial oil body 2, and natural oil body were placed in vials and left at room temperature for 48 hours. After standing, the oil bodies separated into two layers: an upper emulsion layer and a lower whey layer. The emulsion stability index showed that the artificial oil body was more stable than the natural oil body, and artificial oil body 1 was more stable than artificial oil body 2. The artificial and natural oil bodies were diluted with 0.1 wt% sodium dodecyl sulfate aqueous solution, and their absorbance was measured using a spectrophotometer. The emulsion stability index (ESI) was calculated according to the formula, and the results showed that the artificial oil body had better emulsifying performance than the natural oil body (see Table 3). The self-assembled artificial oil body showed significantly improved emulsifying properties, making it more suitable for the application of artificial oil emulsifiers in food, feed, pharmaceuticals, cosmetics, and industrial products, such as butter, ice cream, juice, fish food, pet food, and livestock feed.

[0063] The formula for calculating the emulsion separation index (CI) is: CI / % = HS / HE × 100 (Note: HS is the height of the whey layer; HE is the total height of the emulsion in the bottle).

[0064] Emulsion stability calculation formula: ESI (min) = A0 X 10 / (Ag - Ao) (Note: A0 is the absorbance at 0 min, N=100 is the dilution factor, θ is the oil phase fraction, L is the tube thickness (1 cm), C is the protein concentration (g / mL), and A10 is the absorbance at 10 min)

[0065]

[0066] Example 6: Encapsulation efficiency and release rate of self-assembled artificial oil body 3, using curcumin as an example.

[0067] Artificial oil body 3: Artificial oil body 1 loaded with curcumin

[0068] Different concentrations (0 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, 500 μg / ml, 600 μg / ml) of curcumin were added to safflower oil and allowed to fully dissolve. This solution was then mixed with soybean lecithin in an ethanol solution and heated in a water bath until completely dissolved. The mixture was stirred with a magnetic stirrer for 30 seconds to ensure complete mixing, and then slowly added to a 0.2% Tween 80 solution. The mixture was stirred with a magnetic stirrer for 3–4 hours to allow the ethanol to completely evaporate. The mixture was then rapidly cooled in an ice-water bath to obtain curcumin-loaded artificial lipid droplets. These droplets were mixed with the prepared nanoparticles (1) to form artificial oil bodies containing different curcumin concentrations. The mixture was placed on ice, sonicated, centrifuged, and the curcumin-loaded artificial oil bodies (3) were collected. HPLC analysis of the prepared artificial oil bodies (3) confirmed that the concentration of curcumin added to the safflower oil was 500 μg / mL.

[0069] Encapsulation efficiency testing: Encapsulation efficiency = C1 / C0 × 100%, where C1 is the concentration of curcumin encapsulated in the emulsion gel (μg / mL); C0 is the total concentration of curcumin in the sample (μg / mL). The encapsulation efficiency is between 52% and 95%.

[0070] Drug loading = Curcumin content of the encapsulated oil / mass of the artificial oil body, with a drug loading of 0.25~2.5%.

[0071] Artificial oil containing curcumin was placed in a dialysis bag and immersed in PBS solution. An equal amount of solution was taken out at intervals and an equal amount of dialysis medium was added. The curcumin content was determined by HPLC and the drug release curve was plotted. The results showed that the release rate of 500 μg / mL curcumin added to safflower oil was 70% after 48 hours.

[0072] Example 8: Evaluation of the antioxidant capacity of curcumin-loaded artificial oil body 3

[0073] Artificial oil body 3: Artificial oil body 1 loaded with curcumin was tested for its DPPH free radical scavenging activity and ABTS. •+ Free radical scavenging activity assay to evaluate in vitro antioxidant capacity.

[0074] First, 100 μL of artificial oil 3 was mixed with an equal volume of DPPH stock solution (0.1 mM in 95% methanol) and incubated in the dark at room temperature for 30 min. The absorbance of the mixture was measured at 517 nm (PBS was used as a blank reference), and the DPPH free radical scavenging activity was calculated. Formula: DPPH free radical scavenging rate (%) = [1 (A 样本 -A 空白 )] 100

[0075] An equal volume of 7.4 mM ABTS stock solution was mixed with 2.45 mM potassium persulfate, and the mixture was left to stand in the dark for 12-16 hours to obtain ABTS. -• + Solution. Freshly prepared ABTS -•+ The solution was then diluted with PBS, achieving an absorbance of 0.70 ± 0.02 at 734 nm. 10 μL of the artificial oil dispersion was mixed with 90 μL of diluted ABTS. -•+ The solutions were mixed and reacted in the dark for 6 min; the absorbance of the mixture was measured at 734 nm, and the ABTS was calculated. -•+ Free radical scavenging activity. Formula: ABTS •+ Free radical scavenging rate (%) = [1 (A 样本 -A 空白 100%

[0076] The artificial oil body 3 loaded with curcumin has certain DPPH and ABTS•+ free radical scavenging activities (see Table 4).

[0077] Table 4. Scavenging rates of DPPH and ABTS•+ free radicals

[0078]

[0079] Example 9: Evaluation of the digestibility of curcumin-loaded artificial oil body 3

[0080] Artificial oil body 3: Artificial oil body 1 loaded with curcumin was mixed with SSF containing 5 mg / mL α-amylase, artificial oil body 3 loaded with curcumin, and PBS. The pH was adjusted to 6.8, and the mixture was incubated at 37°C with continuous shaking at 100 r / min for 10 min. Artificial oil body 3 loaded with curcumin was then mixed with simulated gastric juice. The pH was adjusted to 2.0, and the mixture was incubated at 37°C with continuous shaking at 100 r / min for 1 h. (The simulated gastric juice formulation consisted of pepsin (2500 U / mg), 0.7% HCl, and 2 mg / mL NaCl.) The pH of the gastric sample was adjusted to 7.0, and the sample was mixed with intestinal juice. Under constant stirring, the sample was mixed with bile salt solution, and pancreatin (250 U / mg) was added. The mixture was incubated at 37°C with continuous shaking at 100 r / min for 2 h (during this process, the pH of the system was maintained at 7.0 by adding NaOH solution). SDS-PAGE was performed on samples at each digestion stage (10 min, 30 min, 60 min, and 120 min). Figure 5 The particle size, potential, and free fatty acid content were determined. It was found that under gastric fluid conditions, negatively charged artificial oil bodies exhibited strong interactions, protecting curcumin from damage and reducing its leakage. Free fatty acids and monoglycerides generated in intestinal fluid combined with bile salts and phospholipids to form mixed micelles, exhibiting a higher solubilizing capacity for curcumin.

[0081] Example 10: Evaluation of the delivery effect of curcumin-loaded artificial oil body 3

[0082] Artificial oil body 3: Artificial oil body 1 loaded with curcumin

[0083] Mice were administered curcumin-loaded artificial oil 3 via gavage. Six-week-old male BALB / c mice (SPF grade, approximately 20g) were selected and allowed to acclimatize for one week before further experiments. The experiment consisted of a blank control group, a negative control group, and an experimental group (loaded curcumin-loaded artificial oil 3). After 1-7 days of environmental feeding, the experimental groups were administered curcumin-loaded artificial oil 3 daily via gavage (low dose: 10 mg / kg; medium dose: 50 mg / kg; high dose: 300 mg / kg). The negative control group was administered the same amount of artificial oil daily via gavage, and the blank control group was administered the same amount of drinking water daily. This gavage was continued for 14 consecutive days. Mouse weight, fur, feces, food and water intake, and mental status were monitored daily. After gavage, intestinal contents were collected for 16S DNA sequencing to analyze changes in the gut microbiota. The results showed that, compared with oral curcumin, the 50 mg / kg curcumin-loaded artificial oil body 3 in the medium-dose experimental group improved the oral bioavailability and bioaccessibility of curcumin (see Table 5), and had no significant damage to the small intestine, liver, and kidneys. The small particle size of the curcumin-loaded artificial oil body not only protected curcumin from degradation by the special environment of the gastrointestinal tract, but also increased transmembrane absorption by the intestinal epithelium, thereby regulating the richness and distribution frequency of the intestinal flora.

[0084] Table 5 Comparison of bioavailability of curcumin-loaded artificial oil body 3

[0085]

[0086] Other applications of self-assembled artificial oil bodies: In the food processing industry, using oil bodies as emulsifiers has unparalleled advantages over other emulsifiers. Because oil bodies are rich in unsaturated fatty acids and vitamin E, they are the healthiest and most economical emulsifiers. Oil bodies can be used in the cosmetics industry, such as creams, lotions, cosmetics, hair care products, and bath products, such as soaps, detergents, and cleaning agents. While using oil bodies as emulsifiers, some functional ingredients can be added to personal care products. Industrially, oil-based emulsifiers can be used in lubricants, paints, coatings, inks, varnishes, films, paper sizes, latex, etc.

Claims

1. A nanoparticle self-assembled artificial oil body, prepared by the following method, comprising: 1) Nanoparticles were prepared by covalently binding plant oleosin and / or plant oleosin with small phenolic acid compounds. a. Add caleosin and / or oleosin, a caleosin protein from plant oil bodies, to phosphate buffer to prepare a caleosin and / or oleosin protein phosphate buffer solution; b. Add small molecule phenolic acid compounds, stir, and incubate; c. Adjust the pH of the solution from step b to 7.0; dialyze to remove free phenolic acid molecules to obtain nanoparticles; 2) Preparation of artificial lipid droplets a. Add plant phospholipids and plant oils to anhydrous ethanol solution and mix thoroughly to obtain a mixture; b. Add the mixture to the Tween 80 solution and stir; after the ethanol has completely evaporated, quickly cool to room temperature to obtain artificial lipid droplets; 3) Utilizing nanoparticles and artificial lipid droplets to self-assemble artificial oil bodies; a. Mix the nanoparticles described in step 1) and the artificial lipid droplets described in step 2) evenly, and then treat with ultrasound; b. Centrifuge, centrifuge, take the upper layer, and obtain the self-assembled artificial oil body; The plant mentioned in step 1) is safflower; The phenolic acid small molecule compound mentioned is rosmarinic acid (RoSA); The plant oil mentioned in step 2) is safflower oil.

2. The nanoparticle self-assembled artificial oil body according to claim 1, characterized in that: The plant phospholipid mentioned in step 2) is soybean lecithin.

3. The nanoparticle self-assembled artificial oil body according to claim 1, characterized in that: The protein in question is an artificially expressed protein.

4. The nanoparticle self-assembled artificial oil body according to claim 3, characterized in that: The concentration of the protein phosphate buffer solution mentioned in step 1) is 3%.

5. A nanoparticle self-assembled artificial oil body according to claim 1, 2, 3 or 4, characterized in that: Step 2) The mass ratio of plant phospholipids to plant oils is 10:

1.

6. The nanoparticle self-assembled artificial oil body according to claim 5, characterized in that: Step 2) involves mixing the plant oils and bioactive substances, and then mixing them with plant phospholipids.

7. The nanoparticle self-assembled artificial oil body according to claim 6, characterized in that: The bioactive substance mentioned is curcumin.

8. The nanoparticle self-assembled artificial oil body according to claim 7, characterized in that: Step 2) involves adjusting the pH of the protein phosphate buffer solution to 9.0 before adding small molecule phenolic acid compounds.

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