Preparation method of extracellular vesicles derived from co-fermentation of fingered orange and termitomyces albuminosus, product and application thereof

Abstract

The present application relates to a kind of preparation methods of exocyst of co-fermentation of Goniopora and Termitomyces, and its product and application, the preparation method includes: (1) Goniopora seed liquid is inoculated in the fermentation medium containing raw material, fermentation culture is carried out, and co-fermentation product is obtained;(2) co-fermentation product and polyethylene glycol are mixed, and co-precipitation reaction, centrifugation are carried out, and the exocyst is obtained.The cellulase, pectinase and other secretions of Termitomyces population in the co-fermentation system of the present application can gently partially degrade the cell wall components of Goniopora, promote the vesicles originally retained in the apoplast to be released into the fermentation broth, so that the exocyst with higher purity and less pollution can be directly separated from the fermentation supernatant;Extraction of vesicle from fermentation broth avoids the complex purification burden brought by plant tissue crushing, and the fermentation process itself has the effects of sterilization, reduction of anti-nutritional factors, etc., and is safer.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to a method for preparing extracellular vesicles derived from co-fermentation of finger lime and Tibetan kefir, as well as its products and applications. Background Technology

[0002] Extracting exosomes directly from fresh plant tissues presents significantly different technical challenges compared to extracting exosomes from animal cells. Animal cell exosomes can be collected directly from cell culture supernatants, while plant exosomes, encased in dense plant cell walls, require complete disruption of the cell walls through physical fragmentation or enzymatic hydrolysis for release. However, this drastic cell disruption inevitably leads to the co-release and co-separation of numerous intracellular components (such as chloroplast fragments, mitochondrial fragments, endoplasmic reticulum fragments, soluble proteins, and nucleic acids) and cell wall fragments. These non-vesicle contaminants highly overlap with the target exosomes in particle size and sedimentation behavior, making them difficult to distinguish effectively using conventional differential centrifugation or polymer precipitation methods. This severely reduces the purity of the final product, thereby interfering with the specific attribution and functional analysis of the extract's biological effects. Therefore, exploring a novel extraction strategy that can gently release plant exosomes and reduce interference from intracellular contaminants is of great significance for advancing basic research and applied translation of plant exosomes.

[0003] Currently, the mainstream extraction methods for plant exosomes include ultracentrifugation and polyethylene glycol (PEG) precipitation. While ultracentrifugation is considered the "gold standard" for separation, it is expensive, cumbersome, and time-consuming. Furthermore, the high shear forces during repeated ultracentrifugation can cause irreversible damage to the vesicle structure of exosomes, affecting their bioactivity. PEG precipitation is simple to operate, inexpensive, and suitable for large-scale sample processing. However, existing PEG precipitation methods mostly use single-molecular-weight PEG for precipitation. The principle is to utilize the exclusion effect of PEG to cause exosomes to aggregate and settle. However, this method has poor specificity, and while precipitating exosomes, it easily co-precipitates a large amount of free proteins, nucleic acids, and other non-vesicle impurities, resulting in low purity of the extracted product. In addition, different molecular weights of PEG have different precipitation preferences for vesicles of different particle sizes. Single-molecular-weight PEG cannot achieve broad-spectrum and efficient enrichment of exosomes, limiting its application in the large-scale preparation of plant exosomes.

[0004] Therefore, how to achieve the gentle release and efficient purification of plant exosomes without relying on severe mechanical force and high-cost enzyme preparations is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a method for preparing extracellular vesicles derived from co-fermentation of finger lime and Tibetan kefir, as well as the products and applications thereof.

[0006] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for preparing extracellular vesicles derived from co-fermentation of finger lime and Tibetan kefir, the method comprising: (1) The seed liquid of Tibetan Ganoderma lucidum was inoculated into a fermentation medium containing finger lime raw material and fermented to obtain co-fermentation product; (2) The co-fermentation product and polyethylene glycol are mixed, co-precipitated, and centrifuged to obtain the extracellular vesicles.

[0007] This invention creatively designs a method for preparing extracellular vesicles. In the co-fermentation system of this invention, cellulase and pectinase secreted by the *Ganoderma lucidum* microorganisms can gently and partially degrade the cell wall components of finger lime pulp, promoting the release of vesicles that were originally retained in the apoplast into the fermentation broth. Therefore, extracellular vesicles with higher purity and less contamination can be directly separated from the fermentation supernatant. Furthermore, the vesicles in this co-fermentation system not only include extracellular vesicles released by the plant itself, but also include high-yield extracellular vesicles secreted by *Ganoderma lucidum* microorganisms.

[0008] Finger lime exosomes possess high biocompatibility and stability, while probiotic-derived vesicles carry microbial-specific immunomodulatory and intestinal barrier protective molecules. Both coexist in fermentation products as a "plant-microbe mixed vesicle community," potentially exhibiting synergistic bioactivity. Studies have shown that adding edible plant-derived exosomes not only increases lactic acid bacteria vesicle yield but also significantly enriches probiotic indole metabolites within the vesicles.

[0009] Furthermore, vesicle extraction from fermentation broth is a non-destructive separation strategy, avoiding the complex purification burden caused by plant tissue disruption. In addition, the fermentation process itself has bactericidal and anti-nutritional factor-reducing effects, resulting in higher safety. Internationally, standardized frameworks such as the MISEV guidelines have been proposed, ensuring the rigor and reproducibility of vesicle research. This design can achieve efficient transformation from laboratory-scale to large-scale production by optimizing the fermentation process, reducing industrial preparation costs and meeting the industrialization needs of functional foods and biopharmaceuticals.

[0010] Preferably, the fermentation medium in step (1) comprises: finger lime raw material, extracellular polysaccharide and animal milk.

[0011] This invention adds exogenous extracellular polysaccharides to the fermentation medium. On the one hand, these polysaccharides can serve as an additional carbon source, providing energy for *King Kong Mikojima* during the later stages of fermentation, extending its active metabolic period, and facilitating more thorough decomposition of plant cell walls. On the other hand, the added extracellular polysaccharides may induce or stimulate *King Kong Mikojima* to synthesize more extracellular polysaccharides, forming a positive cycle. Simultaneously, the extracellular polysaccharides can also improve the physical stability of the fermentation broth, preventing plant particles from settling too quickly.

[0012] Preferably, the amount of finger lime ingredient added is 10-30% of the animal milk mass percentage (e.g., 10%, 15%, 20%, 25%, 30%, etc.).

[0013] Preferably, the amount of extracellular polysaccharide added is 0.05-0.5% of the animal milk mass percentage (e.g., 0.05%, 0.1%, 0.15%, 0.2%, 0.3%, 0.4%, 0.5%, etc.).

[0014] Preferably, the Tibetan Ganoderma lucidum seed liquid in step (1) is obtained by a preparation method including the following steps: Tibetan Ganoderma lucidum mycelium particles are inoculated into animal milk culture medium for activation culture, and thus obtained.

[0015] Preferably, the inoculation amount of the Tibetan Lingzhi mushroom granules is 3-6% (for example, it can be 3%, 4%, 5%, 6%, etc.).

[0016] Preferably, the activation culture temperature is 25-30℃ (e.g., 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, etc.), the culture time is 24-48 h (e.g., 24 h, 30 h, 36 h, 42 h, 48 h, etc.), and the initial pH of the culture is 6.5-7.0 (e.g., 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, etc.).

[0017] Preferably, the inoculation amount of the Tibetan Ganoderma lucidum seed liquid in step (1) is 3-5% (for example, it can be 3%, 4%, 5%, etc.).

[0018] Preferably, the fermentation culture temperature in step (1) is 25-30℃ (e.g., 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, etc.), and the time is 36-60 h (e.g., 36 h, 42 h, 48 h, 54 h, 60 h, etc.).

[0019] Preferably, the fermentation process further includes a filtration step.

[0020] Preferably, the amount of polyethylene glycol added in step (2) is 6-14% of the mass percentage of the co-fermentation product (e.g., 6%, 8%, 10%, 12%, 14%, etc.).

[0021] Preferably, the molecular weight of the polydiethanol in step (2) is 500-4000 Da (for example, it can be 500 Da, 1000 Da, 1500 Da, 2000 Da, 2500 Da, 3000 Da, 3500 Da, 4000 Da, etc.).

[0022] Preferably, the polyethylene glycol in step (2) is a combination of polyethylene glycol 2000 and polyethylene glycol 3000.

[0023] Preferably, the temperature of the coprecipitation reaction in step (2) is 0-10℃ (e.g., 0℃, 2℃, 4℃, 6℃, 8℃, 10℃, etc.), and the time is 1.5-3 h (e.g., 1.5 h, 2 h, 2.5 h, 3 h, etc.).

[0024] Preferably, the centrifugation method in step (2) is a three-step centrifugation method, specifically: the reaction product is centrifuged for the first time at 9000-10000 rpm (for example, it can be 9000 rpm, 9200 rpm, 9400 rpm, 9600 rpm, 9800 rpm, 10000 rpm, etc.) to obtain the first precipitate; The first precipitate is resuspended in water and / or phosphate buffer and centrifuged a second time at 5000-6500 rpm (e.g., 5000 rpm, 5500 rpm, 6000 rpm, 6500 rpm, etc.) to obtain the second supernatant. The second supernatant is centrifuged a third time at 11,000-13,000 rpm (e.g., 11,000 rpm, 11,500 rpm, 12,000 rpm, 12,500 rpm, 13,000 rpm, etc.) to obtain the precipitate, which is the extracellular vesicle.

[0025] All other specific point values ​​not listed above within the numerical ranges mentioned above can be selected and are all within the protection scope of this invention. For the sake of brevity, they will not be described in detail here.

[0026] In a second aspect, the present invention provides extracellular vesicles obtained by the preparation method described in the first aspect.

[0027] Thirdly, the present invention provides the use of extracellular vesicles as described in the second aspect in the preparation of cosmetics.

[0028] Compared with the prior art, the present invention has the following beneficial effects: This invention creatively designs a method for preparing extracellular vesicles. In the co-fermentation system of this invention, cellulase and pectinase secreted by the *Ganoderma lucidum* microorganisms can gently and partially degrade the cell wall components of finger lime pulp, promoting the release of vesicles that were originally retained in the apoplast into the fermentation broth. Therefore, extracellular vesicles with higher purity and less contamination can be directly separated from the fermentation supernatant. Furthermore, the vesicles in this co-fermentation system not only include extracellular vesicles released by the plant itself, but also include high-yield extracellular vesicles secreted by *Ganoderma lucidum* microorganisms.

[0029] Finger lime exosomes possess high biocompatibility and stability, while probiotic-derived vesicles carry microbial-specific immunomodulatory and intestinal barrier protective molecules. Both coexist in fermentation products as a "plant-microbe mixed vesicle community," potentially exhibiting synergistic bioactivity. Studies have shown that adding edible plant-derived exosomes not only increases lactic acid bacteria vesicle yield but also significantly enriches probiotic indole metabolites within the vesicles.

[0030] Furthermore, vesicle extraction from fermentation broth is a non-destructive separation strategy, avoiding the complex purification burden caused by plant tissue disruption. In addition, the fermentation process itself has bactericidal and anti-nutritional factor-reducing effects, resulting in higher safety. Internationally, standardized frameworks such as the MISEV guidelines have been proposed, ensuring the rigor and reproducibility of vesicle research. This design can achieve efficient transformation from laboratory-scale to large-scale production by optimizing the fermentation process, reducing industrial preparation costs and meeting the industrialization needs of functional foods and biopharmaceuticals. Attached Figure Description

[0031] Figure 1 Transmission electron microscope image of extracellular vesicles prepared in Example 1 of this invention; Figure 2 The figure shows the particle size distribution of the extracellular vesicles prepared in Example 1 of the present invention. The upper left of the figure is a three-dimensional particle size-intensity distribution map, the upper right of the figure is a microscopic imaging field of view, the lower left of the figure is a particle size-scattering intensity scatter plot, and the lower right of the figure is a particle size distribution histogram. Detailed Implementation

[0032] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0033] The Tibetan Ganoderma lucidum used in this invention is a variety from Tibet (collected from farmers in Linzhi City), the milk is from Mengniu High-tech Dairy Co., Ltd., the extracellular polysaccharide is from Best Pharmaceutical Co., Ltd., the cellulase is from Shanghai Yuanye Biotechnology Co., Ltd., and the pectinase is from Shanghai Yuanye Biotechnology Co., Ltd.

[0034] Example 1 This embodiment provides an extracellular vesicle, prepared by the following method: (1) Activation of strain: 5% of the *Ganoderma lucidum* mycelium was inoculated into pure milk culture medium, the pH was adjusted to 6.6, and the medium was incubated at 28℃ for 48 h to obtain *Ganoderma lucidum* seed liquid (live cell concentration 1×10⁻⁶). 9 (CFU / mL) (2) Fermentation culture: 25% fresh finger lime juice (obtained by squeezing finger lime), 0.3% extracellular polysaccharide and 74.7% pure milk were mixed by mass percentage to prepare a fermentation culture medium with an initial pH of 6.5. The seed liquid of Ganoderma lucidum was inoculated into the fermentation culture medium at an inoculation rate of 5%. Under aseptic conditions, the fermentation culture was carried out at 28°C for 48 h. After filtration, the supernatant was taken to obtain the co-fermentation product. (3) Polyethylene glycol coprecipitation: The co-fermentation product and a combination of PEG 2000 and PEG 3000 accounting for 12% of the mass of the co-fermentation product (the mass ratio of PEG 2000 and PEG 3000 is 1:1) were mixed, vortexed for 2 min, and then allowed to stand at 4℃ for 2 h to coprecipitate, and a mixture was obtained. (4) The mixture is centrifuged in three steps as follows: the mixture is centrifuged at 10,000 rpm for 30 min to obtain the first precipitate; The first precipitate was resuspended in phosphate buffer and centrifuged at 6000 rpm for 10 min to obtain the second supernatant. The second supernatant was centrifuged at 12,000 rpm for 10 min to obtain the precipitate, which is the extracellular vesicle.

[0035] Example 2 This embodiment provides an extracellular vesicle, prepared by the following method: (1) Activation of strain: 5% of the *Ganoderma lucidum* mycelium was inoculated into pure milk culture medium, the pH was adjusted to 6.5, and the medium was incubated at 28℃ for 42 h to obtain *Ganoderma lucidum* seed liquid (live cell concentration 1×10⁻⁶). 9 (CFU / mL) (2) Fermentation culture: 20% fresh finger lime juice (obtained by squeezing finger lime), 0.15% extracellular polysaccharide and 79.85% pure milk were mixed by mass percentage to prepare a fermentation culture medium with an initial pH of 6.5. The seed liquid of Ganoderma lucidum was inoculated into the fermentation culture medium at an inoculation rate of 3%. Under aseptic conditions, the mixture was allowed to ferment at 30°C for 48 h. After filtration, the supernatant was collected to obtain the co-fermentation product. (3) Polyethylene glycol coprecipitation: The co-fermentation product and a combination of PEG 2000 and PEG 3000 accounting for 10% of the mass of the co-fermentation product (the mass ratio of PEG 2000 and PEG 3000 is 2:1) were mixed, vortexed for 2 min, and then allowed to stand at 4℃ for 3 h to coprecipitate, and a mixed solution was obtained. (4) The mixture was centrifuged in three steps as follows: the mixture was centrifuged at 9000 rpm for 30 min to obtain the first precipitate; The first precipitate was resuspended in phosphate buffer and centrifuged at 5500 rpm for 10 min to obtain the second supernatant. The second supernatant was centrifuged at 13,000 rpm for 10 min to obtain the precipitate, which is the extracellular vesicle.

[0036] Example 3 This embodiment provides an extracellular vesicle, prepared by the following method: (1) Activation of strain: 4% of the *Gymnocalycium tigrinum* mycelium was inoculated into milk culture medium, the pH was adjusted to 6.5, and the medium was incubated at 28℃ for 42 h to obtain *Gymnocalycium tigrinum* seed culture (live cell concentration 1×10⁻⁶). 9 (CFU / mL) (2) Fermentation culture: 15% fresh finger lime juice (obtained by squeezing finger lime), 0.4% extracellular polysaccharide and 84.6% milk were mixed by mass percentage to prepare a fermentation culture medium with an initial pH of 6.5. The seed liquid of Ganoderma lucidum was inoculated into the fermentation culture medium at an inoculation rate of 4%. Under aseptic conditions, the mixture was allowed to ferment at 30°C for 48 h. After filtration, the supernatant was collected to obtain the co-fermentation product. (3) Polyethylene glycol coprecipitation: The co-fermentation product and PEG 2000 and PEG 3000 (10% by mass of the co-fermentation product) were mixed (the mass ratio of PEG 2000 and PEG 3000 was 1:2). After vortexing for 2 min, the mixture was allowed to stand at 4℃ for 1.5 h to coprecipitate and obtain a mixture. (4) The mixture was centrifuged in three steps as follows: the mixture was centrifuged at 9000 rpm for 25 min to obtain the first precipitate; The first precipitate was resuspended in phosphate buffer and centrifuged at 6500 rpm for 10 min to obtain the second supernatant. The second supernatant was centrifuged at 11,500 rpm for 15 min to obtain the precipitate, which is the extracellular vesicle.

[0037] Example 4 This embodiment provides an extracellular vesicle. The preparation method differs from that of Example 1 only in that extracellular polysaccharides are not added when preparing the fermentation medium in step (2), while other conditions remain unchanged.

[0038] Example 5 This embodiment provides an extracellular vesicle. The preparation method differs from that of Example 1 only in that the percentage of finger orange juice is changed to 8% when preparing the fermentation medium in step (2). The reduced mass is proportionally allocated to the extracellular polysaccharide and milk, while other conditions remain unchanged.

[0039] Example 6 This embodiment provides an extracellular vesicle. The preparation method differs from that of Example 1 only in that the percentage of finger orange juice is changed to 32% when preparing the fermentation medium in step (2). The increased mass is deducted from the extracellular polysaccharide and milk in proportion, while other conditions remain unchanged.

[0040] Example 7 This embodiment provides an extracellular vesicle. The preparation method differs from that of Example 1 only in that PEG 2000 is not used in step (3), and all the mass reduced by PEG 2000 is allocated to PEG 3000. All other conditions remain unchanged.

[0041] Example 8 This embodiment provides an extracellular vesicle. The preparation method differs from that of Example 1 only in that PEG 3000 is not used in step (3), and all the mass reduced by PEG 3000 is allocated to PEG 2000. All other conditions remain unchanged.

[0042] Example 9 This embodiment provides an extracellular vesicle. The preparation method differs from that of Example 1 only in that step (4) is: centrifuge the mixture at 12000 rpm for 20 min to obtain the precipitate, which is the extracellular vesicle.

[0043] Example 10 This embodiment provides an extracellular vesicle. The preparation method differs from that of Example 1 only in that the total mass of PEG 2000 and PEG 3000 in step (3) is changed to 5% of the mass of the co-fermentation product, while other conditions remain unchanged.

[0044] Example 11 This embodiment provides an extracellular vesicle. The preparation method differs from that of Example 1 only in that the total mass of PEG 2000 and PEG 3000 in step (3) is changed to 15% of the mass of the co-fermentation product, while other conditions remain unchanged.

[0045] Comparative Example 1 This comparative example provides an extracellular vesicle. The preparation method differs from that of Example 1 only in that step (1) is omitted, and the seed liquid of Ganoderma lucidum in step (2) is replaced with a complex composed of cellulase and pectinase in a mass ratio of 1:1. The total inoculation amount of the complex is 5%, and other conditions remain unchanged.

[0046] Comparative Example 2 This comparative example provides an extracellular vesicle. The preparation method differs from that of Example 1 only in that finger lime juice is not added in step (2), and the reduced mass of finger lime juice is proportionally allocated to the extracellular polysaccharide and milk. All other conditions remain unchanged.

[0047] Comparative Example 3 This comparative example provides an extracellular vesicle. The preparation method differs from that of Example 1 only in that step (1) is omitted and the seed liquid of Ganoderma lucidum is not added in step (2). All other conditions remain unchanged.

[0048] Test Example 1 This test case performs basic performance tests on the extracellular vesicles obtained in Examples 1-11 and Comparative Examples 1-3.

[0049] (1) Physical characteristics Transmission electron microscope images of the extracellular vesicles prepared in Example 1 of this invention are as follows: Figure 1 As shown, the particle size distribution is as follows: Figure 2 As shown, the results indicate that the extracellular vesicles prepared by this invention have uniform size, good morphology, and an average particle size of about 125 nm.

[0050] (2) Purity test Take the extracellular vesicles obtained in Examples 1-11 and Comparative Examples 1-3 of this invention, add 1 mL of sterile phosphate buffer (PBS, pH 7.4) to resuspend them, and let them stand at 4°C for 2 h to dissolve them fully, so as to obtain the sample solution to be tested.

[0051] Total protein concentration determination: The total protein concentration of each sample solution was determined using a BCA (diquinoline carboxylic acid) protein quantification kit. The procedure was performed according to the kit instructions, and the absorbance (OD value) of each well was measured at 562 nm using a microplate reader. The total protein concentration (unit: μg / mL) of each sample was calculated based on the BSA standard curve.

[0052] Particle concentration: The particle concentration of each sample solution was determined using a nanoparticle tracking analyzer (NTA). Before detection, the samples were diluted with PBS to a concentration of 1 × 10⁻⁶. 7 (particles / mL), three videos were collected for each sample, the average value was taken, and the total particle concentration of each group of samples was recorded.

[0053] Purity Calculation and Evaluation: Calculate the purity index of each sample using the following formula: Purity (number of particles / protein amount, particles / μg) = Particle concentration (particles / mL) / Protein concentration (μg / mL) The purity index of each embodiment was compared with that of each comparative sample. The higher the purity index, the higher the purity of the vesicle extract. The test results are shown in Table 1. The results showed that the extracellular vesicles prepared by this invention had higher purity and less contamination, indicating that the preparation method of this invention can effectively remove impurities such as free proteins and significantly improve vesicle purity. The extracellular vesicle purity of Examples 4-11 and Comparative Examples 1-2 of this invention was slightly lower than that of Examples 1-3, indicating that each technical feature (addition of extracellular polysaccharides, specific selection and content of finger lime juice, selection of polyethylene glycol, application of three-step centrifugation, etc.) has excellent synergistic effects. The absence or substitution of any of these features would not achieve the purity level of this invention.

[0054] Test Example 2 This test case uses the tumor necrosis factor α (TNF-α) gene expression level evaluation method to test the anti-inflammatory and soothing efficacy of extracellular vesicles obtained in Examples 1-11 and Comparative Examples 1-3.

[0055] (1) Experimental materials Human skin fibroblasts (BJ cell line) were used as the test cell model, and the samples to be tested were extracellular vesicles obtained in Examples 1-11 and Comparative Examples 1-3.

[0056] (2) Cell culture and treatment Take BJ cells in the logarithmic growth phase, at 4 × 10⁻⁶ 5 Cells were seeded at a density of [specific value] in 6-well cell culture plates and cultured routinely at 37°C and 5% CO2. When the cells reached approximately 70% confluence, the original culture medium was discarded and replaced with basal medium containing 10% fetal bovine serum (FBS), and cultured synchronously for 24 hours. Subsequently, the culture medium was replaced with medium containing the sample to be tested, and lipopolysaccharide (LPS) was added simultaneously for stimulation, and the treatment continued for another 24 hours.

[0057] (3) Experimental grouping Positive control group: Cell culture medium was supplemented with 0.2 mg / mL dexamethasone and treated for 24 hours; Blank control group: Cells were cultured in basal medium containing 10% FBS for 24 hours (without LPS or test samples). Model control group: Cells were stimulated with LPS only, without the test sample or dexamethasone. Test sample group: cells were respectively added with extracellular vesicles obtained in Examples 1-11 and Comparative Examples 1-3 at a mass percentage of 1%, and LPS stimulation was added simultaneously.

[0058] (4) Total RNA extraction and reverse transcription After treatment, the cell culture medium was discarded, and 1 mL of TRIzol reagent was added to each well to extract total RNA from the cells according to the kit instructions. After determining the purity and concentration of RNA by UV spectrophotometry, an equal amount of total RNA was taken and reverse transcribed into complementary deoxyribonucleic acid (cDNA) using a reverse transcription kit. The obtained cDNA was stored at -20℃ for later use.

[0059] (5) Real-time quantitative PCR detection Using cDNA as a template, real-time quantitative PCR (qPCR) amplification was performed using TNF-α-specific primers to detect the relative expression level of the TNF-α gene. The results were standardized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. After the amplification reaction, the relative expression level of TNF-α mRNA in each group of cells was calculated using the 2^(-ΔΔCt) method.

[0060] Each sample group had 3 parallel duplicate wells, and the experiment was independently repeated 3 times. The results are shown in Table 2. The results showed that, compared with the blank control group, the TNF-α gene expression level was significantly increased after 24 hours of treatment in the model group, indicating that the model was successfully constructed. After 24 hours of treatment in the positive control group (dexamethasone), the TNF-α gene expression level decreased significantly. The extracellular vesicles of Examples 1-3 of this invention showed better results than the positive control group. Changing the extracellular polysaccharide or finger lime content in the fermentation medium would lead to an increase in the TNF-α gene expression level compared with Example 1. Changing the amount of Tibetan kefir grains added or replacing Tibetan kefir grains would further decrease the effect. Changing the operation steps of the co-precipitation method would also significantly decrease the technical effect of extracellular vesicles.

[0061] Test Example 3 This test case uses the interleukin-6 (IL-6) gene expression level anti-inflammatory evaluation method to test the anti-inflammatory effect of extracellular vesicles obtained in Examples 1-11 and Comparative Examples 1-3.

[0062] (1) Experimental materials Human skin fibroblasts (BJ cell line) were used as the test cell model, and the samples to be tested were extracellular vesicles obtained in Examples 1-11 and Comparative Examples 1-3.

[0063] (2) Cell culture and treatment Take BJ cells in the logarithmic growth phase, and use 4 × 10⁴ cells per well. 5Cells were seeded at a density of 1000 cells / well in 6-well cell culture plates and cultured routinely at 37°C and 5% CO2. When the cells reached approximately 70% confluence, the original culture medium was discarded and replaced with basal medium containing 10% fetal bovine serum (FBS), and cultured synchronously for 24 hours. Subsequently, the culture medium was replaced with media containing different concentrations of the test samples, and lipopolysaccharide (LPS) was added simultaneously for stimulation, and the treatment continued for another 24 hours.

[0064] (3) Experimental grouping Positive control group: Cell culture medium was supplemented with 0.2 mg / mL dexamethasone and treated for 24 hours; Blank control group: Cells were cultured in basal medium containing 10% FBS for 24 hours (without LPS or test samples). Model control group: Cells were stimulated with LPS only (1 μg / mL), without the test sample or dexamethasone. Test sample group: cells were respectively added with extracellular vesicles obtained in Examples 1-11 and Comparative Examples 1-3 at a mass percentage of 1%, and LPS stimulation was added simultaneously.

[0065] (4) Total RNA extraction and reverse transcription After treatment, the cell culture medium was discarded, and 1 mL of TRIzol reagent was added to each well to extract total RNA from the cells according to the kit instructions. After determining the purity and concentration of RNA by UV spectrophotometry, an equal amount of total RNA was taken and reverse transcribed into complementary deoxyribonucleic acid (cDNA) using a reverse transcription kit. The obtained cDNA was stored at -20℃ for later use.

[0066] (5) Real-time quantitative PCR detection Using cDNA as a template, real-time quantitative PCR (qPCR) amplification was performed using interleukin-6 (IL-6) specific primers to detect the relative expression level of the IL-6 gene. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal control gene to standardize the results. After the amplification reaction, the relative expression level of IL-6 mRNA in each group of cells was calculated using the 2^(-ΔΔCt) method.

[0067] (6) Results statistics Each sample group has 3 parallel duplicate wells, and the experiment is independently repeated 3 times.

[0068] The inhibitory effect of extracellular vesicles obtained from different embodiments and comparative examples on LPS-induced inflammatory responses was evaluated by comparing the differences in IL-6 gene expression levels between the test sample groups and the model control group. Lower IL-6 expression levels indicated stronger anti-inflammatory and soothing activity in the test samples. The test results are shown in Table 3. The results showed that, compared with the blank control group, the IL-6 gene expression level was significantly increased after 24 hours of treatment in the model group, indicating that the model was successfully constructed. After 24 hours of treatment in the positive control group (dexamethasone), the IL-6 gene expression level decreased significantly. The extracellular vesicles of Examples 1-3 of this invention showed better results than the positive control group. Changing the extracellular polysaccharide or finger lime content in the fermentation medium would lead to an increase in the IL-6 gene expression level compared with Example 1. Changing the amount of *Ganoderma lucidum* added or replacing *Ganoderma lucidum* would further decrease the effect. Changing the operation steps of the co-precipitation method would also significantly decrease the technical effect of extracellular vesicles.

[0069] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0070] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0071] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A method for preparing extracellular vesicles derived from co-fermentation of finger lime and Tibetan kefir, characterized in that, The preparation method includes: (1) The seed liquid of Tibetan Ganoderma lucidum was inoculated into a fermentation medium containing finger lime raw material and fermented to obtain co-fermentation product; (2) The co-fermentation product and polyethylene glycol are mixed, co-precipitated, and centrifuged to obtain the extracellular vesicles.

2. The preparation method according to claim 1, characterized in that, The fermentation medium formulation in step (1) includes: finger lime raw material, extracellular polysaccharide and animal milk; Preferably, the amount of finger lime ingredient added is 10-30% of the animal milk mass percentage; Preferably, the amount of extracellular polysaccharide added is 0.05-0.5% of the animal milk mass percentage.

3. The preparation method according to claim 1 or 2, characterized in that, The Tibetan Ganoderma lucidum seed liquid in step (1) is obtained by a preparation method including the following steps: Tibetan Ganoderma lucidum mycelium is inoculated into animal milk culture medium for activation culture, and thus obtained.

4. The preparation method according to claim 3, characterized in that, The inoculation amount of the Tibetan Kumquat mycelium is 3-6%; Preferably, the activation culture temperature is 25-30℃, the culture time is 24-48 h, and the initial pH of the culture is 6.5-7.

0.

5. The preparation method according to any one of claims 1-4, characterized in that, The inoculation amount of the Tibetan Ganoderma lucidum seed liquid in step (1) is 3-5%.

6. The preparation method according to any one of claims 1-5, characterized in that, The fermentation culture in step (1) is carried out at a temperature of 25-30℃ for 36-60 h. Preferably, the fermentation process further includes a filtration step.

7. The preparation method according to any one of claims 1-6, characterized in that, The amount of polyethylene glycol added in step (2) is 6-14% of the mass percentage of the co-fermentation product; Preferably, the molecular weight of the polydiethanol in step (2) is 500-4000 Da; Preferably, the polyethylene glycol in step (2) is a combination of polyethylene glycol 2000 and polyethylene glycol 3000; Preferably, the temperature of the coprecipitation reaction in step (2) is 0-10℃ and the time is 1.5-3 h.

8. The preparation method according to any one of claims 1-7, characterized in that, The centrifugation method described in step (2) is a three-step centrifugation method. The specific steps are as follows: the product after reaction is centrifuged for the first time at 9000-10000 rpm to obtain the first precipitate; The first precipitate was resuspended in water and / or phosphate buffer and centrifuged a second time at 5000-6500 rpm to obtain the second supernatant. The second supernatant was centrifuged a third time at 11,000-13,000 rpm, and the resulting precipitate was the extracellular vesicle.

9. Extracellular vesicles obtained by the preparation method according to any one of claims 1-8.

10. The application of the extracellular vesicles according to claim 9 in the preparation of cosmetics.

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