Exosome-coated turmeric fermentation product as well as preparation method and application thereof

Abstract

The invention discloses an exosome coated turmeric fermentation product and a preparation method and application thereof, the preparation method comprises the following steps: adding turmeric root powder into a culture medium, inoculating lactobacillus for fermentation, centrifugally collecting supernate, filtering and drying to obtain turmeric fermentation product dry powder; preparing plant source or animal source exosome freeze-dried powder; respectively adding the exosome freeze-dried powder and the turmeric fermentation product dry powder into a PBS buffer solution, redissolving, and mixing to obtain a mixed solution; the mixed solution is subjected to electroporation and purification under the conditions that the voltage is 300-500 V, the capacitance is 150-250 [mu] F, and the number of pulses is 1-3, free components are removed, and the curcuma longa fermentation product nano-vesicles wrapped by the exosome are obtained. The prepared exosome-coated turmeric fermentation product can be used as a cosmetic raw material for inhibiting inflammation, regulating skin micro-ecology and promoting collagen regeneration, and the preparation method improves the curcumin encapsulation efficiency, reduces the particle size of nano-vesicles and ensures the uniformity at the same time.

Description

Technical Field

[0001] This invention belongs to the technical field of skin care products, and particularly relates to an exosome-encapsulated turmeric fermentation product, its preparation method, and its application. Background Technology

[0002] Curcumin is a polyphenolic compound extracted from the rhizome of turmeric. It possesses remarkable anti-inflammatory, antioxidant, and antibacterial bioactivities, showing great potential for application in skin care such as acne treatment, acne scar repair, and anti-photoaging. However, its near-insoluble nature in water and extremely low bioavailability significantly limit its utilization. Current technologies generally employ nanocarriers (such as liposomes and polymer nanoparticles) to encapsulate curcumin, but this faces significant challenges: the highly hydrophobic curcumin crystals are difficult to load efficiently and stably into hydrophilic nanocarriers, easily leading to leakage and crystallization, resulting in low encapsulation efficiency, uneven particle size, and poor stability.

[0003] Exosomes are nanoscale vesicles actively secreted by cells. They possess excellent biocompatibility, low immunogenicity, and natural targeting properties, making them ideal endogenous drug delivery carriers. Plant and animal exosomes themselves are also rich in various bioactive substances (such as nucleic acids, proteins, and lipids), which can participate in intercellular communication and regulate the biological functions of recipient cells, such as inhibiting inflammation and promoting tissue repair.

[0004] Hydrophobic molecules such as curcumin are difficult to disperse in aqueous buffer solutions and tend to aggregate. Currently, using exosomes as carriers to encapsulate such substances, like curcumin, remains a technical challenge. This is because curcumin cannot effectively penetrate the phospholipid bilayer, and crystal precipitation can damage the exosome membrane structure, leading to loading failure and exosome damage. Some studies have attempted to pretreat curcumin, such as using organic solvents for solubilization, but residual solvents can compromise the integrity of exosomes and introduce safety risks. Another approach is to use crude turmeric extract, but its complex hydrophobic composition also presents insurmountable difficulties for exosome encapsulation. Summary of the Invention

[0005] The purpose of this invention is to solve at least one problem in the prior art by proposing an exosome-encapsulated turmeric fermentation product, its preparation method, and its application.

[0006] To achieve the above objectives, this invention proposes a method for preparing turmeric fermentation products encapsulated in exosomes, comprising the following steps: Turmeric root powder was added to a culture medium, inoculated with lactobacillus, and fermented at 35℃-42℃. The supernatant was collected by centrifugation, filtered, and dried to obtain turmeric fermentation product dry powder. Exosomes are extracted, purified, and freeze-dried from animal or plant cell culture supernatants or tissues to obtain exosome freeze-dried powder. The exosome lyophilized powder and the turmeric fermentation product powder were respectively added to PBS buffer for reconstitution and then mixed to obtain a mixture. The mixture was subjected to electroporation at a voltage of 300V-500V, a capacitance of 150μF-250μF, and a pulse frequency of 1-3 times, followed by an ice bath for 10-30 minutes, so that exosomes encapsulated the turmeric fermentation product. After purification and removal of free components, exosome-encapsulated turmeric fermentation product nanovesicles were obtained.

[0007] Electroporation is performed in an electroporator with an electrode spacing of 2mm-4mm. During the electroporation process, controlled electrical pulses are applied to form transient, nanoscale hydrophilic channels on the exosome phospholipid bilayer. This allows water-soluble turmeric fermentation products to diffuse into the exosome via electroosmosis and concentration gradient. By optimizing electroporation parameters (such as voltage, capacitance, and number of pulses), the channels can be ensured to close rapidly after the pulse ends, maximizing the integrity and function of the exosome membrane and preventing leakage of contents or permanent damage to the membrane structure. After electroporation, an ice bath treatment can maximize the recovery of the exosome membrane structure.

[0008] In the process of preparing turmeric fermentation product dry powder, the drying method is either spray drying or freeze drying. Spray drying is preferred as it is more economical. The spray drying process is carried out in a spray dryer with the inlet temperature set at 150℃-180℃ and the outlet temperature at 80℃-90℃.

[0009] The obtained exosome-encapsulated turmeric fermentation product nanovesicles can be stored for a short period of time at 3℃-6℃, or 1%-8% by mass of trehalose can be added as a freeze-drying protectant, and after pre-freezing, freeze-drying is carried out at -50℃ and 0.1mbar vacuum for 24-48 hours to obtain freeze-dried powder that is easy to transport and store.

[0010] Short-chain fatty acids (such as lactic acid and acetic acid) produced during the fermentation of turmeric root powder can enhance the anti-inflammatory effects of curcumin; oligosaccharides produced during fermentation act as prebiotics, promoting the growth of beneficial bacteria in the skin and further balancing the microecology. Exosome encapsulation further enhances the stability, transdermal permeability, and targeting of these components, and introduces the exosomes' own repair and regeneration functions. Because the phospholipid bilayer structure of exosomes effectively isolates turmeric fermentation products from light, oxygen, and enzyme degradation, it prolongs the retention time of activity. The surface of exosomes contains proteins such as CD47 and integrins, which can prevent rapid clearance by the mononuclear-macrophage system and achieve active targeting by binding to receptors on specific cells (such as fibroblasts and keratinocytes) through surface ligands.

[0011] As an optional implementation, the lactobacillus is Lactobacillus plantarum or Lactobacillus rhamnosus, the inoculation amount of lactobacillus is 1%-5%, the fermentation time after inoculation is 48h-72h, and the fermentation is carried out until the pH of the fermentation broth is 3.5-6.5.

[0012] As an optional implementation, the specific steps for centrifuging and collecting the supernatant are as follows: centrifuge the fermentation product after fermentation treatment at a temperature of 3℃-5℃ and a rotation speed of 8000rpm-12000rpm for 15min-30min, and collect the supernatant.

[0013] As an optional implementation, the turmeric root powder is added to the culture medium at a mass concentration of 3%-8%, and the culture medium contains a carbon source, a nitrogen source, and inorganic salts. The nitrogen source includes, but is not limited to, peptone and yeast extract, and the nitrogen source accounts for 0.5%-2.5% of the mass concentration of the culture medium. The carbon source includes, but is not limited to, glucose and sucrose, and the carbon source accounts for 1%-5% of the mass concentration of the culture medium.

[0014] As an optional implementation, the mass ratio of the exosome freeze-dried powder to the turmeric fermentation product powder is (5:1) to (1:5).

[0015] As an optional implementation, the pH of the PBS buffer is 6-7.5, the exosome lyophilized powder is reconstituted with PBS buffer to form an exosome solution with a concentration of 1 mg / ml-50 mg / ml, and the turmeric fermentation product powder is reconstituted with PBS buffer to form a turmeric fermentation product solution with a concentration of 1 mg / ml-100 mg / ml. The mixture is prepared by mixing the exosome solution and the turmeric fermentation product solution at a mass ratio of (1:2) to (5:1).

[0016] As an optional implementation, the exosome lyophilized powder is a plant-derived exosome lyophilized powder. The preparation steps of the plant-derived exosome lyophilized powder are as follows: extracting plant-derived exosomes from the supernatant of plant cell suspension culture, obtaining an exosome suspension by differential ultracentrifugation, multi-stage membrane filtration, or tangential flow filtration, filtering for sterilization, and then freeze-drying to obtain plant exosome lyophilized powder. The plant cells in the plant cell suspension culture supernatant are taken from grape stem cells or Chlorella vulgaris.

[0017] As an optional implementation, the exosome lyophilized powder is an animal-derived exosome lyophilized powder, wherein the animal-derived exosomes are extracted from the culture supernatant of human mesenchymal stem cells or human dermal fibroblasts.

[0018] The present invention also proposes an exosome-encapsulated turmeric fermentation product prepared according to the above-described method for preparing exosome-encapsulated turmeric fermentation product, wherein the average particle size of the exosome-encapsulated turmeric fermentation product is 50nm-200nm and is in the form of nanovesicles.

[0019] This invention also proposes the use of the above-mentioned exosome-encapsulated turmeric fermentation product as a cosmetic ingredient for inhibiting inflammation, regulating skin microecology, and promoting collagen regeneration.

[0020] The beneficial effects of this invention are as follows: This invention, by mixing turmeric fermentation products with exosomes and then processing them using electroporation, cleverly transforms hydrophobic raw materials that cannot be directly and efficiently encapsulated by exosomes into an ideal form that is easily encapsulated, thus successfully solving the problem of exosome loading of turmeric active ingredients and improving the encapsulation efficiency. The combination of exosome extraction and electroporation technology ensures highly uniform nanovesicle size, excellent batch-to-batch consistency, and significantly improved storage stability. The entire preparation process is mild, maximizing the protection of the integrity of exosomes and the bioactivity of turmeric fermentation products. Synergistically, turmeric fermentation products not only provide a means for exosome encapsulation of curcumin, but also, through the synergistic effect of prebiotics and post-biotics produced by the fermentation products themselves with the active ingredients of turmeric, enhance anti-inflammatory, antibacterial, and microecological regulation effects, exhibiting excellent repair and anti-inflammatory properties. During the preparation process, the efficacy focus of the final product can be precisely customized by selecting exosomes from different sources (such as MSC exosomes with anti-inflammatory effects or plant exosomes with antioxidant properties), meeting diverse market demands. The entire preparation process does not require the use of harmful organic solvents, the raw materials are natural and safe, and it can be mass-produced, possessing extremely high transformation and application value.

[0021] The features and advantages of the present invention will be described in detail through embodiments and in conjunction with the accompanying drawings. Attached Figure Description

[0022] Figure 1 This is a schematic diagram showing the particle size analysis results of the turmeric fermentation product nanovesicles encapsulated in exosomes according to Example 1 of the present invention.

[0023] Figure 2 This is a schematic diagram showing the particle size analysis results of the turmeric fermentation product nanovesicles encapsulated in exosomes in Example 2 of the present invention.

[0024] Figure 3 This is a schematic diagram comparing the proliferation efficiency of HaCaT cells using different compositions from embodiments of the present invention.

[0025] Figure 4 This is a schematic diagram comparing the expression levels of inflammatory factor mRNA in HaCaT cells induced by Propionibacterium acnes using different compositions from embodiments of the present invention. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments.

[0027] Example 1 This embodiment proposes a method for preparing turmeric fermentation products encapsulated in exosomes, which specifically includes the following steps: S01. Preparation of turmeric fermentation product: Take 50g of turmeric root powder, add it to 1L of MRS medium containing 3% glucose and 1.5% yeast extract, sterilize at 121℃ for 20 minutes, cool to 37℃, inoculate with 5% (v / v) Lactobacillus plantarum (CGMCC No. 1.555) bacterial suspension, and anaerobic ferment at 37℃ for 60 hours until the pH of the fermentation broth is 4.2. After fermentation, centrifuge the fermentation broth at 4℃ and 10000rpm for 20 minutes, take the supernatant, filter it with a 0.22μm filter membrane, and spray dry the filtrate (set the inlet temperature to 170℃ and the outlet temperature to 85℃ during spray drying) to obtain turmeric fermentation product dry powder; S02. Preparation of MSC exosome lyophilized powder: After human mesenchymal stem cells (MSCs) were cultured to 80% confluence, the culture medium was replaced with exosome-free serum and cultured for another 24-48 hours at 37℃ and 5% CO2. The conditioned medium of human mesenchymal stem cells (MSCs) was collected, and after differential centrifugation (centrifugation conditions: 2000×g 30min, 10000×g 45min), exosome precipitate was obtained by ultracentrifugation at 120000×g for 20 minutes. The precipitate was resuspended in PBS, purified by size exclusion chromatography (qEVoriginal, Izon Science), and the exosome peak was collected. The solution was filtered through a 0.22μm filter membrane and then freeze-dried to obtain MSC exosome lyophilized powder. S03. Preparation of a mixed solution of exosomes and turmeric fermentation product: Take 10 mg of lyophilized MSC exosome powder prepared in step S02 above and 50 mg of turmeric fermentation product powder prepared in step S01 above, and respectively add 1 ml of pre-cooled PBS buffer (pH 6-7.5) to obtain exosome solution and turmeric fermentation product solution. Mix the turmeric fermentation product solution and exosome solution evenly and incubate at room temperature for 10 minutes to obtain a mixed solution. S04. Preparation of turmeric fermentation product encapsulated in human mesenchymal stem cell exosomes: The mixture obtained in step S03 was transferred to an electroporation cup (electrode spacing of 4 mm) of an electroporator. Single-pulse electroporation was performed at 400 V and 200 μF. After electroporation, the sample was placed on ice for 20 minutes (to allow the exosome membrane structure to recover). The eluent was collected and purified by centrifugation at 4000 × g for 30 minutes using a 100 kDa ultrafiltration centrifuge tube. The sample was resuspended in PBS to obtain a vesicle suspension of turmeric fermentation product encapsulated in human mesenchymal stem cell exosomes, denoted as Cur-Ferm@MSC-Exo nanovesicle suspension.

[0028] The Cur-Ferm@MSC-Exo nanovesicle suspension of this embodiment was analyzed and detected using a nano-Coultre analyzer. The specific detection results are as follows: Figure 1 As shown, by Figure 1 It can be seen that the average particle size of the encapsulated particles in this Cur-Ferm@MSC-Exo nanovesicle suspension is 64 nm, D10 is 57 nm, D90 is 72 nm, the particle size is uniform, and the particle concentration is 2.94 × 10⁻⁶. 11 particles / ml.

[0029] This embodiment also proposes an anti-aging face cream composed of the following raw materials in percentage by weight: 0.2%-0.7% of the above-mentioned Cur-Ferm@MSC-Exo nanovesicle suspension, 0.3%-1% of ergothioneine, 0.8%-1.5% of vitamin E acetate, 0.2%-0.6% of bisabolol, 3%-5% of shea butter, 4%-8% of hydrogenated polyisobutylene, 1%-3% of glyceryl stearate, 3%-5% of 1,3-propanediol, 0.3%-1% of p-hydroxyacetophenone, and the balance being deionized water. The face cream containing the above-mentioned Cur-Ferm@MSC-Exo nanovesicle suspension, through the effects of human mesenchymal stem cell exosomes stimulating collagen regeneration and promoting cell proliferation, combined with the anti-inflammatory aging and skin microecological balancing effects of fermented turmeric products, can achieve a comprehensive conditioning effect on aging skin.

[0030] Example 2 This embodiment provides a method for preparing turmeric fermentation products encapsulated in exosomes, which specifically includes the following steps: S01. Preparation of turmeric fermentation product: Take 50g of turmeric root powder, add it to 1L of MRS medium containing 3% glucose and 1.5% yeast extract, sterilize at 115℃ for 25 minutes, cool to 37℃, inoculate with 5% (v / v) Lactobacillus plantarum (CGMCC No. 1.555) bacterial suspension, and anaerobic ferment at 37℃ for 60 hours until the pH of the fermentation broth is 4.2. After fermentation, centrifuge the fermentation broth at 4℃ and 10000rpm for 20 minutes, take the supernatant, filter it with a 0.22μm filter membrane, and spray dry the filtrate (set the inlet temperature to 170℃ and the outlet temperature to 85℃ during spray drying) to obtain turmeric fermentation product dry powder; S02. Preparation of Chlorella exosome lyophilized powder: Chlorella was cultured, and the culture supernatant was collected. The supernatant was filtered sequentially through 0.45 μm and 0.22 μm pore size filter membranes. The solution was concentrated and replaced using a tangential flow filtration system (TFF, 100 kDa membrane pack) (PBS buffer was used for replacement). The concentrated solution was purified by size exclusion chromatography column (qEVoriginal, Izon Science). The exosome peaks were collected and freeze-dried to obtain light green Chlorella exosome lyophilized powder. S03. Preparation of a mixed solution of exosomes and turmeric fermentation product: Take 10 mg of Chlorella exosome lyophilized powder obtained in step S02 above and 40 mg of turmeric fermentation product powder obtained in step S01 of this embodiment, and respectively use 1 ml of pre-cooled PBS buffer (pH of PBS buffer is 6-7.5) to obtain exosome solution and turmeric fermentation product solution. Mix the turmeric fermentation product solution and exosome solution evenly and incubate at room temperature for 10 minutes to obtain a mixed solution; S04. Preparation of turmeric fermentation products encapsulated in human mesenchymal stem cell exosomes: The mixture obtained in step S03 was transferred to an electroporation cup (electrode spacing of 3 mm) of an electroporator. Single-pulse electroporation was performed at 400 V and 200 μF. After electroporation, the sample was placed on ice for 20 minutes (to allow the exosome membrane structure to recover). The effluent was collected and purified by centrifugation at 4000 × g for 30 minutes using a 100 kDa ultrafiltration centrifuge tube. The sample was resuspended in PBS to obtain a vesicle suspension of turmeric fermentation products encapsulated in Chlorella exosomes, denoted as Cur-Ferm@Chlorella-Exo nanovesicle suspension.

[0031] The Cur-Ferm@Chlorella-Exo nanovesicle suspension of this embodiment was analyzed and detected using a nano-Coultre analyzer. The specific detection results are as follows: Figure 2 As shown, by Figure 2It can be seen that the average particle size of the encapsulated particles in this Cur-Ferm@Chlorella-Exo nanovesicle suspension is 59 nm, D10 is 56 nm, D90 is 63 nm, the particle size is uniform, and the particle concentration is 8.92 × 10⁻⁶. 12 particles / ml.

[0032] This embodiment also proposes an acne-removing essence, which is composed of the following raw materials in percentage by weight: 0.3%-1% of the above-mentioned Cur-Ferm@Chlorella-Exo nanovesicle suspension, 1%-3% of Sophora flavescens root extract, 0.3%-0.7% of salicylic acid, 1.5%-3% of niacinamide, 2.5%-4% of oat β-glucan, 4%-8% of glycerin, 0.5%-1.5% of panthenol, 0.3%-1% of p-hydroxyacetophenone, and the balance being deionized water. The essence containing the above-mentioned Cur-Ferm@Chlorella-Exo nanovesicle suspension can quickly inhibit inflammation, kill Propionibacterium acnes, promote the repair of damaged skin, and prevent the formation of acne scars.

[0033] Example 3 This embodiment provides a method for preparing turmeric fermentation products encapsulated in exosomes, which specifically includes the following steps: S01. Preparation of turmeric fermentation product: Take 40g of turmeric root powder, add it to 1L of MRS medium containing 4% sucrose and 1% peptone, sterilize at 110℃ for 30 minutes, cool to room temperature, inoculate with 3% (v / v) Lactobacillus rhamnosus (L. rhamnosusATCC 53103) bacterial suspension, and anaerobic ferment at 38℃ for 72 hours until the pH of the fermentation broth is 3.5. After fermentation, centrifuge the fermentation broth at 5℃ and 12000rpm for 15 minutes, take the supernatant, filter it through a 0.25μm filter membrane, freeze-dry the filtrate to obtain turmeric fermentation product dry powder; S02. Preparation of MSC exosome lyophilized powder: After human mesenchymal stem cells (MSCs) were cultured to 80% confluence, the culture medium was replaced with exosome-free serum and cultured for another 24-48 hours at 37℃ and 5% CO2. The conditioned medium of human mesenchymal stem cells (MSCs) was collected, and after differential centrifugation (centrifugation conditions: 3000×g 20min, 12000×g 30min), exosome precipitate was obtained by ultracentrifugation at 150000×g for 10 minutes. The precipitate was resuspended in PBS, purified by size exclusion chromatography (qEVoriginal, Izon Science), and the exosome peak was collected. The solution was filtered through a 0.25μm filter membrane and then freeze-dried to obtain MSC exosome lyophilized powder. S03. Preparation of a mixed solution of exosomes and turmeric fermentation product: Take 20 mg of lyophilized MSC exosome powder prepared in step S02 above and 10 mg of turmeric fermentation product powder prepared in step S01 above, and respectively add 1 ml of pre-cooled PBS buffer (pH 6-7.5) to obtain exosome solution and turmeric fermentation product solution. Mix the turmeric fermentation product solution and exosome solution evenly and incubate at room temperature for 15 minutes to obtain a mixed solution. S04. Preparation of turmeric fermentation product encapsulated in human mesenchymal stem cell exosomes: The mixture obtained in step S03 was transferred to an electroporation cup (electrode spacing of 2 mm) of an electroporator. Three pulse electroporations were performed at 300 V and 150 μF. After electroporation, the sample was placed on ice for 25 minutes (to allow the exosome membrane structure to recover). The eluent was collected and purified by centrifugation at 4000 × g for 25 minutes using a 100 kDa ultrafiltration centrifuge tube. The sample was resuspended in PBS to obtain a vesicle suspension of turmeric fermentation product encapsulated in human mesenchymal stem cell exosomes, denoted as Cur-Ferm@MSC-Exo nanovesicle suspension.

[0034] Example 4 This embodiment provides a method for preparing turmeric fermentation products encapsulated in exosomes, which specifically includes the following steps: S01. Preparation of turmeric fermentation product: Take 30g of turmeric root powder, add it to 1L of MRS medium containing 5% sucrose and 2% yeast extract, sterilize at 121℃ for 20 minutes, cool to room temperature, inoculate with 2% (v / v) Lactobacillus rhamnosus (L. rhamnosusATCC 53103) bacterial suspension, and anaerobic ferment at 40℃ for 50 hours until the pH of the fermentation broth is 6. After fermentation, centrifuge the fermentation broth at 3℃ and 8000rpm for 30 minutes, take the supernatant, filter it with a filter membrane with a pore size of 0.22μm, freeze dry the filtrate to obtain turmeric fermentation product dry powder; S02. Preparation of Chlorella exosome lyophilized powder: Chlorella was cultured, and the culture supernatant was collected. The supernatant was filtered sequentially through 0.5 μm and 0.2 μm pore size membranes. The solution was concentrated and replaced using a tangential flow filtration system (TFF, 100 kDa membrane pack) (PBS buffer was used for replacement). The concentrate was purified by size exclusion chromatography column (qEVoriginal, Izon Science). The exosome peaks were collected and freeze-dried to obtain light green Chlorella exosome lyophilized powder. S03. Preparation of a mixed solution of exosomes and turmeric fermentation product: Take 10 mg of Chlorella exosome lyophilized powder obtained in step S02 above and 10 mg of turmeric fermentation product powder obtained in step S01 of this embodiment, and respectively use 1 ml of pre-cooled PBS buffer (pH 6-7.5) to obtain exosome solution and turmeric fermentation product solution. Mix the turmeric fermentation product solution and exosome solution evenly and incubate at room temperature for 20 minutes to obtain a mixed solution; S04. Preparation of turmeric fermentation products encapsulated in human mesenchymal stem cell exosomes: The mixture obtained in step S03 was transferred to an electroporation cup (electrode spacing of 3 mm) of an electroporator. Two pulsed electroporations were performed at 500 V and 250 μF. After electroporation, the sample was placed on ice for 30 minutes (to allow the exosome membrane structure to recover). The effluent was collected and purified by centrifugation at 5000 × g for 20 minutes using a 100 kDa ultrafiltration centrifuge tube. The suspension was resuspended in PBS to obtain a vesicle suspension of turmeric fermentation products encapsulated in Chlorella exosomes, denoted as Cur-Ferm@Chlorella-Exo nanovesicle suspension.

[0035] Comparative Example This comparative example provides a method for preparing exosome-encapsulated curcumin products. The exosomes are MSC exosomes. The preparation process of the exosomes is the same as step S02 of Example 1. During preparation, 50 mg of curcumin standard (purity >95%) and 10 mg of lyophilized MSC exosome powder are dissolved in 1 mL of PBS buffer and then mixed. The mixture is then transferred to the electroporation cup of the electroporator for electroporation. The electroporation parameters are the same as those in step S03 of Example 1. After centrifugation and filtration, the exosome-encapsulated curcumin products are obtained.

[0036] During the preparation of the curcumin-encapsulated product by exosomes, it was found that due to the extreme difficulty in dissolving curcumin, a large number of visible orange suspended particles were present in the mixture of curcumin, exosomes and PBS buffer. After electroporation treatment, a large number of flocculent precipitates appeared in the mixture.

[0037] The exosome-encapsulated curcumin product of this comparative example was analyzed and detected using a nano-coulter analyzer. The average particle size detected by NTA was 572 nm, and most of the exosomes were destroyed. This comparative example, compared with Example 1, demonstrates that directly encapsulating hydrophobic curcumin crystals with exosomes is almost infeasible. However, the present invention, which uses fermentation followed by exosome encapsulation, can reduce the particle size, improve the encapsulation efficiency, and ensure the integrity of the exosome membrane structure.

[0038] Tests on the effects of different electroporation parameters on the performance of exosome-encapsulated turmeric fermentation product nanovesicle suspensions: The turmeric fermentation product prepared according to step S01 of Example 1 and the MSC exosome lyophilized powder prepared according to step S02 were mixed in a ratio of 1:2 and reconstituted in PBS buffer. The mixture was then transferred to the electroporation cup of the electroporator for electroporation. The mixture was divided into 11 equal groups, and each group was divided into 3 equal parts. Each group of mixtures was processed according to the electroporation parameters in Table 1. After processing, centrifugation and filtration were performed to obtain the turmeric fermentation product nanovesicle suspension encapsulated by exosomes. The average particle size (nm) and particle density (particles / mL) of the turmeric fermentation product nanovesicle suspension encapsulated by exosomes in each group were measured using a nanocoulter analyzer (NTA). The average value of each group was taken. The specific test results are shown in Table 1 below.

[0039] Table 1. Comparison of performance parameters of turmeric fermentation product nanovesicle suspensions encapsulated in exosomes under different electroporation parameters.

[0040] Based on the data analysis in Table 1, 400V, 250μF, and single pulse are the optimal electroporation conditions. These conditions ensure high particle density and vesicle integrity while maintaining efficient material exchange between the inside and outside of the membrane, thus improving encapsulation efficiency.

[0041] It's important to note that electroporation involves polarizing the phospholipid bilayer of nanovesicles under an external high-voltage electric field. When the transmembrane potential exceeds a critical value, the phospholipid molecules rearrange, forming hydrophilic transient channels. The size, number, and lifetime of these channels are precisely controlled by the electroporation parameters. Increasing the voltage increases the transmembrane potential applied to the phospholipid bilayer, resulting in more and larger channels, which facilitates the entry of active substances. However, excessively high voltages create too many and too large channels, exceeding the membrane's self-repair capacity, leading to channel failure and permanent membrane damage. This results in a significant decrease in particle density and an increase in average particle size. Therefore, it is crucial to select an appropriate voltage that precisely reaches the membrane's self-repair critical value, maximizing encapsulation efficiency while minimizing damage to the vesicles.

[0042] Capacitance is directly related to energy; the larger the capacitance, the longer the pulse discharge time, and the higher the total energy transferred to the sample. A longer pulse time allows the formed pores to remain open for a longer period. This provides a longer time window for turmeric fermentation products to enter the exosomes via electroosmosis and diffusion. Therefore, at low capacitance, a short pulse time results in rapid pore closure, less encapsulation, smaller particle size, and the most intact vesicles. Conversely, at high capacitance, an excessively long pulse time leads to prolonged pore opening and overloading of active ingredients, which may alter the internal and external osmotic pressures of the vesicles, making them prone to fusion or rupture, thus significantly reducing the particle count.

[0043] Increasing the number of pulses has a cumulative effect on the membrane structure of exosomes, creating more new channels. Excessive pulse count can severely damage the membrane integrity, causing vesicles to aggregate and fuse, resulting in a significant increase in average particle size and a substantial decrease in particle density.

[0044] Regarding the repair efficacy test of turmeric fermentation products encapsulated in exosomes: Human immortalized keratinocyte cell line (HaCaT) was used. Cells were divided into 5 equal groups. One group served as a blank control group without treatment. The other four groups were treated with equal amounts of 0.5% turmeric fermentation products from Example 1, 0.5% MSC exosomes from Example 1, 0.5% a physical mixture of turmeric fermentation products and MSC exosomes, and 0.5% Cur-Ferm@MSC-Exo nanovesicles from Example 1, respectively. These were designated as the 0.5% turmeric fermentation product group, the 0.5% MSC exosome group, the 0.5% physical mixture group, and the 0.5% Cur-Ferm@MSC-Exo nanovesicle group. After 24 hours of intervention, the supernatant of each group was collected, and the optical density at 450 nm was measured using a microplate reader. Cell viability was calculated. The test results are as follows: Figure 3 As shown. By Figure 3 It can be seen that, compared with the blank control group, the cell proliferation rate of the Cur-Ferm@MSC-Exo nanovesicle group reached 125.89%, which was significantly higher than that of other groups. This confirms that the turmeric fermentation products and MSC exosomes in Cur-Ferm@MSC-Exo nanovesicles have a synergistic repair effect, and the cell proliferation rate of electroporation treatment is higher than that of simple physical mixing treatment.

[0045] The anti-inflammatory efficacy of turmeric fermentation products encapsulated in exosomes was tested: Human immortalized keratinocyte cell line (HaCaT) was used, and cells were divided into 6 equal groups. One group served as a blank control. The other four groups were treated with *Propionibacterium acnes* induction (as a model control), with equal amounts of 0.5% turmeric fermentation product (Example 2), 0.5% *Chlorella* exosomes (Example 2), 0.5% a physical mixture of turmeric fermentation product and *Chlorella* exosomes (Example 2), and 0.5% *Cur-Ferm@Chlorella-Exo* nanovesicles (Example 2), respectively. These were designated as the 0.5% turmeric fermentation product group, the 0.5% *Chlorella* exosome group, the 0.5% physical mixture group, and the 0.5% *Cur-Ferm@Chlorella-Exo* nanovesicle group. After 24 hours of intervention, RNA was extracted from cells in each group, reverse transcribed into cDNA, and TNF-α mRNA and IL-6 levels were detected using RT-qPCR. mRNA expression levels, specific test results are as follows Figure 4 As shown. By Figure 4It was found that, compared with the model control group, the inhibition rates of TNF-α mRNA and IL-6 mRNA in the Cur-Ferm@Chlorella-Exo nanovesicle group reached 43.81% and 63.47%, respectively, which were significantly higher than those in other groups at the same concentration. This indicates that the turmeric fermentation product and Chlorella exosomes treated with electroporation have a synergistic anti-inflammatory effect, and the effect is better than that of simple physical mixing.

[0046] The above embodiments are illustrative of the present invention and are not intended to limit the present invention. Any simple modifications to the present invention are within the scope of protection of the present invention.

Claims

1. A method for preparing turmeric fermentation products encapsulated in exosomes, characterized in that: It includes the following steps: Turmeric root powder was added to a culture medium, inoculated with lactobacillus, and fermented at 35℃-42℃. The supernatant was collected by centrifugation, filtered, and dried to obtain turmeric fermentation product dry powder. Exosomes are extracted, purified, and freeze-dried from animal or plant cell culture supernatants or tissues to obtain exosome freeze-dried powder. The exosome lyophilized powder and the turmeric fermentation product powder were respectively added to PBS buffer for reconstitution and then mixed to obtain a mixture. The mixture was subjected to electroporation at a voltage of 300V-500V, a capacitance of 150μF-250μF, and a pulse frequency of 1-3 times, followed by an ice bath for 10-30 minutes, so that exosomes encapsulated the turmeric fermentation product. After purification and removal of free components, exosome-encapsulated turmeric fermentation product nanovesicles were obtained.

2. The method for preparing turmeric fermentation products encapsulated in exosomes as described in claim 1, characterized in that: The lactobacillus is either Lactobacillus plantarum or Lactobacillus rhamnosus. The inoculation amount of lactobacillus is 1%-5%, and the fermentation time after inoculation is 48h-72h until the pH of the fermentation broth is 3.5-6.

5.

3. The method for preparing turmeric fermentation products encapsulated in exosomes as described in claim 1, characterized in that: The specific steps for collecting the supernatant by centrifugation are as follows: centrifuge the fermentation product after fermentation treatment at a temperature of 3℃-5℃ and a rotation speed of 8000rpm-12000rpm for 15min-30min, and collect the supernatant.

4. The method for preparing turmeric fermentation products encapsulated in exosomes as described in claim 1, characterized in that: The turmeric root powder is added to the culture medium at a mass concentration of 3%-8%, and the culture medium contains carbon source, nitrogen source and inorganic salt.

5. The method for preparing turmeric fermentation products encapsulated in exosomes as described in claim 1, characterized in that: The mass ratio of the exosome freeze-dried powder to the turmeric fermentation product powder is (5:1) to (1:5).

6. The method for preparing turmeric fermentation products encapsulated in exosomes as described in claim 1, characterized in that: The pH of the PBS buffer is 6-7.

5. The exosome lyophilized powder is reconstituted with PBS buffer to form an exosome solution with a concentration of 1 mg / ml-50 mg / ml. The turmeric fermentation product powder is reconstituted with PBS buffer to form a turmeric fermentation product solution with a concentration of 1 mg / ml-100 mg / ml. The mixture is prepared by mixing the exosome solution and the turmeric fermentation product solution at a mass ratio of (1:2) to (5:1).

7. The method for preparing turmeric fermentation product encapsulated in exosomes as described in claim 1, characterized in that: The exosome lyophilized powder is a plant-derived exosome lyophilized powder. The preparation steps of the plant-derived exosome lyophilized powder are as follows: extracting plant-derived exosomes from the supernatant of plant cell suspension culture, obtaining an exosome suspension by differential ultracentrifugation, multi-stage membrane filtration, or tangential flow filtration, filtering for sterilization, and then freeze-drying to obtain plant exosome lyophilized powder. The plant cells in the plant cell suspension culture supernatant are taken from grape stem cells or Chlorella vulgaris.

8. The method for preparing turmeric fermentation product encapsulated in exosomes as described in claim 1, characterized in that: The exosome lyophilized powder is an animal-derived exosome lyophilized powder, and the animal-derived exosomes in the animal-derived exosome lyophilized powder are extracted from the culture supernatant of human mesenchymal stem cells or human dermal fibroblasts.

9. A method for preparing exosome-encapsulated turmeric fermentation product according to any one of claims 1 to 8, characterized in that: The exosomes encapsulating the turmeric fermentation products have an average particle size of 50nm-200nm and are in the form of nanovesicles.

10. An application of the turmeric fermentation product encapsulated in exosomes as described in claim 9 as a cosmetic ingredient for inhibiting inflammation, regulating skin microecology, and promoting collagen regeneration.

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