Bioactive preparation used for skin injury repair and prepared by taking freshwater pearl shell waste as raw material as well as preparation method and application of bioactive preparation

Abstract

The preparation method comprises the following steps: extracting an intershell fluid active substance, extracting extracellular small vesicles as an active substance target, using hyriopsis cumingii pallium for conventional pearl mussel breeding, and preparing the bioactive preparation for skin injury repair from the raw material, so as to obtain the bioactive preparation for skin injury repair. The method comprises the following steps: collecting liquid in a gap between a pearl culture clam shell and an outer mantle, separating and extracting vesicles from the liquid through ultracentrifugation, taking freshwater pearl shell culture by-products as a sustainable source of bioactive substances, grinding the bioactive substances contained in the freshwater pearl shell culture by-products, optimizing components through molecular composition, and preparing the freshwater pearl shell culture by-products. A preparation with a stable bioactive substance structure for repairing skin injury radiated by ultraviolet rays is obtained. The extracellular small vesicles are extracted from pearl shells, the main components of the preparation are biological sources, the safety is high, the absorbability of a nanoscale molecular structure is good, an innovative solution is provided for skin repair, and high value-added conversion of pearl shells is achieved.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and in particular to a bioactive preparation for skin damage repair made from freshwater pearl oyster waste, its preparation method, and its application. Background Technology

[0002] Skin injury repair is a highly programmed biological process, beginning with an inflammatory response to clear necrotic tissue and pathogens. This then progresses to the extracellular matrix (ECM) stage, primarily involving fibroblast proliferation, migration, and the secretion of type III collagen to build granulation tissue. Finally, remodeling is achieved through the orderly replacement of collagen from type III to type I and the re-epithelialization of keratinocytes, restoring the skin's structural and functional integrity. However, in acute or deep injuries, the repair process is often hindered by impaired transitions from the inflammatory to proliferative phases. This impairment is primarily manifested in excessive and persistent inflammatory responses and abnormal activation of matrix metalloproteinases (MMPs), leading to an imbalance in the repair microenvironment and creating an ineffective cycle of "synthesis and degradation" of the extracellular matrix. Simultaneously, exogenous repair factors such as growth factors are easily and rapidly degraded at the wound site, exhibiting low delivery efficiency and short-lived effects. Furthermore, single-component treatment strategies struggle to synergistically regulate the multi-stage and multi-target needs of the repair network.

[0003] To address the aforementioned challenges, this study, based on the principles of resource recycling and sustainable development, innovatively focuses on the intershell fluid—a byproduct of freshwater pearl oysters—as a source of bioactive substances. As benthic organisms, freshwater pearl oysters have developed unique immune and damage repair mechanisms through long-term evolution. The space between their shell and mantle is rich in extracellular vesicles secreted by intrashell stem cells. Proteomics analysis confirmed that these vesicles naturally carry a functionally synergistic molecular network: on the one hand, they are significantly enriched with proteins related to extracellular matrix-receptor interactions and cell adhesion, providing a direct molecular basis for fibroblast attachment, migration, and collagen deposition; on the other hand, they contain proteins involved in regulating key inflammatory signaling pathways such as NF-κB, possessing the potential to actively regulate the immune microenvironment and promote inflammatory homeostasis. These vesicles naturally integrate the dual repair functions of "regulating the inflammatory microenvironment" and "guiding matrix reconstruction." However, despite the clear biological functions and potential application value of these vesicles, the high-value development of the freshwater pearl oyster byproducts from which they originate has not yet begun, and their resource potential has not been fully explored and transformed. Summary of the Invention

[0004] This invention provides a bioactive preparation for skin damage repair made from freshwater pearl oyster waste, its preparation method, and its application. The purpose is to extract highly bioactive substances from freshwater pearl oyster by-products to develop novel skin damage repair preparations and realize high-value-added utilization of shellfish resources.

[0005] This application is achieved through the following technical solution: A bioactive preparation for skin damage repair was developed using freshwater pearl oyster waste as raw material. Active substances were extracted from the intershell fluid, with extracellular vesicles as the target active substances. Using the mantle of the common pearl oyster *Swimmus triangularis*, the liquid between the shell and mantle of the pearl oyster was collected, and vesicles were extracted by ultracentrifugation. This method utilizes freshwater pearl oyster aquaculture byproducts as a sustainable source of bioactive substances. Based on the bioactive substances contained within, the potential biological functions were analyzed. Further optimization of the molecular composition yielded a structurally stable bioactive preparation for repairing skin damage caused by ultraviolet radiation.

[0006] In a preferred embodiment, the extraction of active substances from the intershell fluid specifically includes the following steps: (1) Collecting intershell fluid: Clean the attached substances on the surface of pearl mussels to prevent contamination of the intershell fluid. Then, use a syringe with a long needle, insert the needle parallel to the shell layer, and insert it into the space between the mantle and the upper shell to extract the intershell fluid. (2) Initial impurity removal: The initial impurity removal is performed using a refrigerated high-speed centrifuge. After centrifugation, the supernatant is collected and the bottom sediment is discarded. (3) Remove impurities again: Use a needle filter to slowly push and filter the supernatant obtained in step (2) to prevent the filter membrane structure from being damaged or the extracellular vesicle structure from being damaged, and repeat once; use a needle filter again to slowly push and filter the liquid to obtain the original solution containing extracellular vesicles. (4) Obtaining extracellular vesicles: The original solution obtained in step (3) was centrifuged using a 50 mL ultracentrifuge, and the bottom of the centrifuge tube was slowly blown to resuspend the precipitate and obtain a high concentration of extracellular vesicle active sample. (5) Take a portion of the high-concentration extracellular vesicle active sample obtained in step (4), extract total protein, and perform protein sequencing by high-throughput sequencing to determine the molecular composition of the active material.

[0007] In a preferred embodiment, the length of the long needle in the syringe used in step (1) is 8-10 cm.

[0008] As a preferred embodiment, in step (2), when removing impurities for the first time, the interstitial fluid is first placed in a 50 mL centrifuge tube and a benchtop low-temperature centrifuge is used. The speed is 12000 rpm, the time is 20 min, and the temperature is 4℃. The discarded bottom precipitate is tissue fragments, cells and cell debris, macromolecular proteins and other impurities in the interstitial fluid.

[0009] In a preferred embodiment, when removing impurities again in step (3), a needle filter with a pore size of 0.45 μm and a diameter of 3 cm is used to slowly push and filter the supernatant, and a needle filter with a pore size of 0.22 μm and a diameter of 3 cm is used again to slowly push and filter the liquid.

[0010] As a preferred embodiment, in step (4), the ultracentrifuge parameters are set as follows: 150,000 rpm, 4℃ for 1 h, discard the supernatant, take the precipitate, and slowly blow 1 mL of 0.1% NaCl onto the bottom of the centrifuge tube.

[0011] The preparation method of a bioactive agent for skin damage repair derived from freshwater pearl oyster waste includes the following steps: (a) Sample concentration standardization control: The concentration of the obtained high-concentration extracellular microvesicle active sample protein was determined, and the protein concentration of the solution was controlled at a certain value according to the dilution ratio table; (b) Functional combination: Based on the protein sequencing results, extracellular growth factors bFGF, IGF and EGF were added to activate and enhance the molecular functions of the MAPK and PI3K-AKT-mTOR pathways in the material to promote potential repair functions. (c) Formulation preservation: Glycerol is added to the formulation to ensure the integrity of the membrane structure of extracellular vesicles under repeated freeze-thaw cycles; (d) Biosafety assessment: The endotoxin content of the preparation shall be detected using an endotoxin detection kit, and the measurement result shall be less than 0.125 EU / mL.

[0012] In a preferred embodiment, in step (a), the protein concentration in the solution is controlled at 200 ng / μL using 0.1% NaCl according to the dilution ratio table.

[0013] In a preferred embodiment, 5-10% glycerol is added to the formulation in step (c) to ensure the integrity of the membrane structure of extracellular vesicles under repeated freeze-thaw cycles.

[0014] In a preferred embodiment, the measurement in step (d) showed that the endotoxin content in the formulation was less than 0.125 EU / ml, which met the requirements.

[0015] Beneficial Effects: This invention relates to a bioactive preparation for skin damage repair derived from freshwater pearl oyster waste and its preparation method. Using freshwater pearl oysters with implanted nuclei, which resemble stem cell tissue, as raw materials, extracellular microvesicles are extracted. Based on their molecular composition, a skin repair active preparation is developed. The main components of this preparation are of biological origin, exhibiting high safety and excellent absorption due to its nanoscale molecular structure. The raw materials are derived from aquatic byproducts, resulting in low cost and meeting the requirements of resource recycling and the development of new productive forces. It possesses clear repair efficacy; experiments have confirmed that this preparation can significantly promote skin damage healing. This invention not only provides an innovative solution for skin repair but also realizes the high-value-added transformation of pearl oysters. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the extraction process of liquid and extracellular vesicles between freshwater pearl shells in one embodiment of the present invention.

[0017] Figure 2 This is an electron microscope schematic diagram of the morphology of extracellular vesicles in freshly isolated stem cell tissue in one embodiment of the present invention.

[0018] Figure 3 This is an electron micrograph showing the morphology of extracellular vesicles of stem cell tissue in a formulation according to an embodiment of the present invention.

[0019] Figure 4 This is a schematic diagram of GO enrichment analysis of extracellular small molecule vesicles in the shell fluid in one embodiment of the present invention.

[0020] Figure 5 This is a schematic diagram of the KEGG pathway enrichment analysis of extracellular small molecule vesicles in the shell fluid in one embodiment of the present invention.

[0021] Figure 6 This is a schematic diagram of the skin damage repair technology route and phenotypic results in one embodiment of the present invention.

[0022] Figure 7 This is a schematic diagram of HE and Masson staining results of the skin at the wound site in one embodiment of the present invention.

[0023] Figure 8 This is a schematic diagram illustrating the significance analysis of collagen deposition area in one embodiment of the present invention.

[0024] Figure 9 This is a schematic diagram of the endotoxin detection results in a formulation according to one embodiment of the present invention. Detailed Implementation

[0025] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings: These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes, but the protection scope of the present invention is not limited to the following embodiments. Example 1:

[0026] like Figure 1 As shown, a bioactive preparation for skin damage repair, derived from freshwater pearl oyster waste, and its preparation method, including the extraction of active substances from intershell fluid and the creation of the active preparation, are described. Extraction of active substances from intershell fluid, targeting extracellular vesicles, was performed using the mantle membrane of the common pearl-producing mussel *Triplophysa pulcherrima*. Fluid was collected from the space between the shell and mantle membrane of the pearl-producing mussel, and vesicles were extracted by ultracentrifugation. The specific steps included: (1) Collecting intershell fluid: Clean the attached substances on the surface of pearl mussels to prevent contamination of the intershell fluid. Then, use a syringe with a long needle (8-10 cm in length) to insert the needle parallel to the shell layer into the space between the mantle and the upper shell to extract the intershell fluid. (2) First impurity removal: The first impurity removal was carried out using a refrigerated high-speed centrifuge. First, the interstitial fluid was placed in a 50 mL centrifuge tube and a benchtop low-temperature centrifuge was used. The speed was 12000 rpm, the time was 20 min, and the temperature was 4℃. The supernatant was taken and the bottom precipitate was discarded. The tissue fragments, cells and cell debris, macromolecular proteins and other impurities in the interstitial fluid were removed. (3) Remove impurities again: Use a needle filter with a pore size of 0.45 μm and a diameter of 3 cm to slowly push and filter the supernatant obtained in step (2) to prevent the filter membrane structure from being damaged or the extracellular vesicle structure from being damaged, and repeat once; use a needle filter with a pore size of 0.22 μm and a diameter of 3 cm to slowly push and filter the liquid again to obtain the original solution containing extracellular vesicles; (4) Obtaining extracellular vesicles: The original solution obtained in step (3) was centrifuged using a 50 mL ultracentrifuge. The ultracentrifuge parameters were set as follows: 150,000 rpm, 4℃ for 1 h. The supernatant was discarded, and the precipitate was collected. 1 mL of 0.1% NaCl was slowly blown onto the bottom of the centrifuge tube to resuspend the precipitate and obtain a high concentration of extracellular vesicle activity sample. (5) Take a portion of the high-concentration extracellular vesicle active sample obtained in step (4), extract total protein, and perform protein sequencing by high-throughput sequencing to determine the molecular composition of the active material.

[0027] The creation of active formulations involves using freshwater pearl oyster farming byproducts as a source of bioactive substances, analyzing their potential biological functions, further optimizing their molecular composition, and obtaining formulations with stable bioactive substances for the repair of skin damage caused by ultraviolet radiation. The specific steps include: (a) Sample concentration standardization control: The concentration of the obtained high-concentration extracellular microvesicle active sample protein was determined, and the protein concentration of the solution was controlled at 200 ng / μL using 0.1% NaCl according to the dilution ratio table (see Table 1 below); (b) Functional combination: Based on the protein sequencing results, extracellular growth factors bFGF, IGF and EGF were added to activate and enhance the molecular functions of the MAPK and PI3K-AKT-mTOR pathways in the material to promote potential repair functions. (c) Formulation preservation: Add 5-10% glycerol to the formulation to ensure the integrity of the membrane structure of extracellular vesicles under repeated freeze-thaw conditions; (d) Biosafety evaluation: The endotoxin content of the formulation was detected using an endotoxin detection kit. The measurement result was required to be less than 0.125 EU / mL. The results showed that the endotoxin content in the formulation was less than 0.125 EU / mL. Figure 9 As shown.

[0028] Table 1. Ingredients of Skin Damage Repair Preparations Based on Intermittent Fluid from the Triangular Sail Clam.

[0029] Example 2:

[0030] Electron microscopic observation of extracellular vesicles in the obtained stem cell tissue: like Figure 2 As shown in the figure, the morphology of extracellular microvesicles was systematically characterized by transmission electron microscopy in this embodiment. The microvesicles separated by this method were intact in morphology, with typical saucer-shaped or round structure, clear membrane boundaries, and no obvious rupture or aggregation.

[0031] like Figure 3 As shown, morphological observation of the small vesicles extracted from the formulation after preparation showed that the small vesicles in the formulation still maintained a complete vesicle structure, and their morphological characteristics were basically consistent with those of freshly isolated EVs, indicating that the formulation process did not significantly affect their structural integrity. Example 3:

[0032] Compound formulation of extracellular small molecule vesicle preparations in shell fluid: Proteomics sequencing of the obtained extracellular microvesicles revealed that the vesicles were significantly enriched in a protein network that regulates extracellular matrix remodeling, inflammatory immune homeostasis, and cellular behavior, providing a molecular basis for their repair potential.

[0033] like Figure 4As shown, the significantly enriched ECM-receptor interactions and focal adhesion pathways in KEGG corroborate the findings in the GO entries regarding "cell-matrix adhesion," "calcium ion binding," and "hyaluronic acid binding," collectively revealing its core function in mediating cell-matrix recognition and adhesion. Furthermore, the coexistence of the "proteolysis" process with corresponding "serine-type endopeptidase activity" and "endopeptidase inhibitor activity" indicates its ability to precisely regulate local proteolytic homeostasis.

[0034] like Figure 5 As shown, vesicles exhibit the potential to regulate the immune microenvironment. The rich inflammatory and immune pathways (such as TNF, NF-κB, and Toll-like receptor pathways) revealed in KEGG analysis have partial effector molecular bases at the GO level, including "scavenger receptor activity," "superoxide metabolism," and "peptidoglycan degradation" and "chitin binding" functions, suggesting potential microbial recognition. These characteristics indicate that the vesicles may participate in regulating the inflammatory response and homeostasis at the site of injury.

[0035] This vesicle contains a core signaling network that directly activates the repair-driving pathway. KEGG shows significant enrichment of the PI3K-Akt, mTOR, and MAPK signaling pathways, and extracellular growth factors can activate these pathways, initiating core signaling hubs that regulate cell proliferation, survival, and metabolism. Based on the functional map directly supported by the data, a synergistic strategy of "extracellular vesicle-growth factor" is proposed. This vesicle creates an adhesion-friendly matrix microenvironment, regulates immune homeostasis, and pre-activates key pathways (MAPK and PI3K-Akt-mTOR), creating a "preparatory state" for the efficient action of growth factors. The additional formulation with bFGF, EGF, and IGF-1 is precisely to amplify the specificity and intensity of this basic signal: bFGF and EGF strongly drive the MAPK pathway, promoting fibroblast / angiogenesis and epithelialization, respectively; IGF-1 can maximally activate the PI3K-Akt-mTOR axis, providing continuous anabolic power for repair. The three, together with extracellular microvesicles, complement and synergize in the functions of "microenvironment preparation" and "powerful command execution". Example 4:

[0036] Experiments on the use of the biomaterials prepared in this invention for skin damage repair: To evaluate the in vivo repair efficacy of this formulation, a UV-induced mouse skin injury model was constructed in this embodiment. Figure 6As designed, C57BL / 6 mice were randomly divided into four groups: CON0 (normal light control), CON1 (UV irradiation + saline treatment), SEV+ (UV irradiation + extracellular vesicles), and SEV++ (UV irradiation + growth factor combination treatment). During the experimental period, except for the CON0 group, all other groups received UVA / UVB combined ultraviolet radiation to the back for one hour daily for two consecutive days to establish an acute injury model. Starting after the second irradiation, the corresponding preparation (100 μL) was applied to the injured area of ​​each group of mice once daily for five consecutive days. On day 10 of the experiment, the repair status of the skin tissue in each group of mice was comprehensively evaluated and analyzed.

[0037] like Figure 6 Phenotypic results showed that, compared with the CON0 group (which did not receive UV irradiation), mice in the CON1, SEV+, and SEV++ groups exhibited significant skin damage on their backs, indicating the successful establishment of the UV irradiation model. After the injury intervention, compared with the CON1 group treated with saline, the SEV+ group treated only with extracellular vesicles and the SEV++ group treated with the combined preparation showed significantly smaller wound areas, with no obvious tissue fluid exudation. Tissue proliferation and elevation were observed at the wound edges, suggesting that the skin had entered an active repair phase. Further comparison between the SEV+ and SEV++ groups revealed that mice in the SEV++ group treated with the combined extracellular vesicle and growth factor preparation showed significant new tissue elevation in the center of the wound (as indicated by the arrow), while the SEV+ group treated with only extracellular vesicles, although showing some wound shrinkage, did not exhibit similar significant tissue regeneration in the central area. Example 5:

[0038] HE staining and Masson's trichrome staining were used to assess collagen deposition. like Figure 7 As shown, HE staining results revealed significant differences in the degree of inflammatory infiltration among the groups. The CON1 group showed a large number of diffusely infiltrated inflammatory cells, mainly including neutrophils and lymphocytes (red arrows). In contrast, the SEV++ and SEV+ groups showed significantly reduced inflammatory cell infiltration and newly formed pores (black arrows). The SEV++ group exhibited the lowest degree of inflammatory infiltration among all groups, with only a very small number of scattered inflammatory cells, the highest number of pores, and intact tissue structure.

[0039] like Figure 8As shown, collagen deposition was assessed using Masson's trichrome staining. Only a small amount of sparse blue collagen fibers were observed in the control group tissue. Both the SEV++ and SEV+ groups induced significant collagen deposition, exhibiting a dense and extensive network of blue collagen fibers. Compared to the SEV+ group, the SEV++ group showed the most abundant collagen deposition and a more compact fiber arrangement. Quantitative image analysis revealed that the percentage of collagen area in the SEV++ group was significantly higher than that in the control group (…). p < 0.001), the percentage of collagen area in the SEV+ group was significantly higher than that in the control group, and the amount of collagen deposition in the SEV++ group was significantly higher than that in the SEV+ group ( p < 0.05). Example 6:

[0040] Endotoxin detection in pharmaceutical preparations: like Figure 9 As shown, the endotoxin test kit was used to test the endotoxin content of the preparation obtained in this case. The results showed that the reaction products of the negative control group with an endotoxin content of less than 0.125 EU / mL and the SEV++ group did not form a firm and stable gel, but were in a liquid or shaky soft gel state, and easily slid down the inner wall; the reaction products of the positive control group with an endotoxin concentration of greater than or equal to 0.125 EU / mL formed a firm and complete gel, which was stably attached to the bottom of the tube and did not easily slid down the inner wall.

[0041] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A bioactive preparation for skin damage repair, derived from freshwater pearl oyster waste, characterized in that, Active substances were extracted from the intershell fluid, with extracellular vesicles as the target active substances. Using the mantle of the common pearl oyster, the liquid in the gap between the shell and mantle of the pearl oyster was collected. The vesicles were then separated and extracted by ultracentrifugation. Freshwater pearl oyster aquaculture by-products were used as a sustainable source of bioactive substances. Based on the bioactive substances contained therein, the potential biological functions were analyzed. Furthermore, the composition was optimized through molecular composition to obtain a structurally stable formulation of bioactive substances for the repair of skin damage caused by ultraviolet radiation.

2. The bioactive preparation for skin damage repair derived from freshwater pearl oyster waste according to claim 1, characterized in that, The extraction of active substances from the intershell fluid specifically includes the following steps: (1) Collecting intershell fluid: Clean the attached substances on the surface of pearl mussels to prevent contamination of the intershell fluid. Then, use a syringe with a long needle, insert the needle parallel to the shell layer, and insert it into the space between the mantle and the upper shell to extract the intershell fluid. (2) Initial impurity removal: The initial impurity removal is performed using a refrigerated high-speed centrifuge. After centrifugation, the supernatant is collected and the bottom sediment is discarded. (3) Remove impurities again: Use a needle filter to slowly push and filter the supernatant obtained in step (2) to prevent the filter membrane structure from being damaged or the extracellular vesicle structure from being damaged, and repeat once; use a needle filter again to slowly push and filter the liquid to obtain the original solution containing extracellular vesicles. (4) Obtaining extracellular vesicles: The original solution obtained in step (3) was centrifuged using a 50 mL ultracentrifuge, and the bottom of the centrifuge tube was slowly blown to resuspend the precipitate and obtain a high concentration of extracellular vesicle active sample. (5) Take a portion of the high-concentration extracellular vesicle active sample obtained in step (4), extract total protein, and perform protein sequencing by high-throughput sequencing to determine the molecular composition of the active material.

3. The bioactive preparation for skin damage repair derived from freshwater pearl oyster waste according to claim 2, characterized in that, The syringe in step (1) has a long needle that is 8-10 cm long.

4. The bioactive preparation for skin damage repair derived from freshwater pearl oyster waste according to claim 2, characterized in that, In step (2), when removing impurities for the first time, the interstitial fluid is first placed in a 50 mL centrifuge tube and a benchtop low-temperature centrifuge is used. The speed is 12000 rpm, the time is 20 min, and the temperature is 4℃. The bottom precipitate is discarded as tissue fragments, cells and cell debris, macromolecular proteins and other impurities in the interstitial fluid.

5. The bioactive preparation for skin damage repair derived from freshwater pearl oyster waste according to claim 2, characterized in that, In step (3), when removing impurities again, a needle filter with a pore size of 0.45 μm and a diameter of 3 cm is used to slowly push and filter the supernatant. Then, a needle filter with a pore size of 0.22 μm and a diameter of 3 cm is used again to slowly push and filter the liquid.

6. The bioactive preparation for skin damage repair derived from freshwater pearl oyster waste according to claim 2, characterized in that, In step (4), the ultracentrifuge parameters are set as follows: 150,000 rpm, 4℃ for 1 h, discard the supernatant, take the precipitate, and slowly blow 1 mL of 0.1% NaCl onto the bottom of the centrifuge tube.

7. The method for preparing a bioactive agent for skin damage repair based on freshwater pearl oyster waste as described in claim 1, characterized in that, Specifically, the following steps are included: (a) Sample concentration standardization control: The concentration of the obtained high-concentration extracellular microvesicle active sample protein was determined, and the protein concentration of the solution was controlled at a certain value according to the dilution ratio table; (b) Functional combination: Based on the protein sequencing results, extracellular growth factors bFGF, IGF and EGF were added to activate and enhance the molecular functions of the MAPK and PI3K-AKT-mTOR pathways in the material to promote potential repair functions. (c) Formulation preservation: Glycerol is added to the formulation to ensure the integrity of the membrane structure of extracellular vesicles under repeated freeze-thaw cycles; (d) Biosafety assessment: The endotoxin content of the preparation shall be detected using an endotoxin detection kit, and the measurement result shall be less than 0.125 EU / mL.

8. The method for preparing a bioactive agent for skin damage repair based on freshwater pearl oyster waste as a raw material according to claim 7, characterized in that, In step (a), the protein concentration in the solution is controlled at 200 ng / μL using 0.1% NaCl according to the dilution ratio table.

9. The method for preparing a bioactive agent for skin damage repair based on freshwater pearl oyster waste as a raw material according to claim 7, characterized in that, In step (c), 5-10% glycerol is added to the formulation to ensure the integrity of the membrane structure of extracellular vesicles under repeated freeze-thaw cycles; in step (d), the measurement results show that the endotoxin content in the formulation is less than 0.125 EU / ml, which meets the requirements.

10. The use of the bioactive agent as described in claim 1 in the preparation of a bioactive agent for skin damage repair.

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