Lycium barbarum exosome composite beverage as well as preparation method and application thereof

Abstract

The invention relates to the technical field of drinks, in particular to a Chinese wolfberry exosome composite drink and a preparation method and application thereof, the Chinese wolfberry exosome composite drink with good sensory properties is prepared from Chinese wolfberry exosomes and Chinese wolfberry puree for the first time, and the defects that an existing Chinese wolfberry puree product loses active substances and is low in bioavailability can be overcome. Cell experiments are combined to verify that the compound beverage has good effects of reducing the oxidative stress level and delaying skeletal muscle cell senescence, and it is indirectly proved that the fructus lycii exosome compound beverage has good application prospects. Compared with a Chinese wolfberry fruit puree product, the Chinese wolfberry fruit exosome composite beverage contains a large amount of Chinese wolfberry fruit exosomes, so that the defect of loss of bioactive substances such as partial protein and miRNA can be overcome, and the Chinese wolfberry fruit exosome composite beverage has better and higher bioavailability and can play a better role in resisting myotube atrophy and skeletal muscle cell senescence.

Description

Technical Field

[0001] The present application relates to the field of beverage technology, and in particular to a wolfberry exosome composite beverage and a preparation method and application thereof. Background Art

[0002] Aging is a universal physiological phenomenon, which is manifested by the gradual decline of the body's ability to adapt to the living environment and the responsiveness of related organ functions. Changes in the expression levels of aging markers can be observed in this process. Aging not only causes serious damage to the physical and mental health of individuals, but also increases the burden on families and society.

[0003] During the aging process of various organs in the human body, skeletal muscle tissue is often accompanied by pathological phenotypes mainly characterized by muscle atrophy, which are mainly manifested by reduced myotube diameter, increased expression of aging markers, oxidative damage to skeletal muscle cells, and impaired normal physiological functions of skeletal muscle. Aging-related muscle atrophy not only increases the risk of locomotor system diseases and the incidence of chronic metabolic diseases such as diabetes, but also affects the quality of life of the elderly and further increases the burden on the medical system and society. As the aging problem becomes increasingly severe, it is very important to explore an effective and feasible anti-skeletal muscle aging program or auxiliary means.

[0004] As a traditional Chinese herbal medicine with the characteristics of "medicine and food homology", wolfberry (Lycium barbarum berry) is rich in wolfberry polysaccharides, polyphenols, betaine and other bioactive ingredients, and has anti-inflammatory, antioxidant, anti-aging, growth-promoting effects. Previous studies have shown that wolfberry extract can play an anti-aging role through mechanisms such as anti-oxidative damage.

[0005] Exosomes are membrane vesicles with a particle size of 30nm-150nm that are peeled off from the cell membrane or actively secreted by the cell. They can be produced by microorganisms, mammals, plants and human cells, and have the functions of promoting intercellular communication and regulating physiological and pathological processes. Plant-derived exosomes are mainly isolated and extracted from fresh plants. They have the same bioactive components (proteins, lipids, carbohydrates, phytochemicals, miRNA, etc.) as the original plants. Due to their beneficial effects and tissue specificity, they have been used in the medical field.

[0006] In addition to phytochemicals such as Lycium barbarum polysaccharides and polyphenols, Lycium barbarum exosomes also carry bioactive substances such as miRNA, which are more easily absorbed by the human body. At the same time, 1 kg of fresh Lycium barbarum fruit can extract 1×10 12-13The granular wolfberry exosomes can retain the nutrients of fresh fruit and can be stored at -80℃ for half a year. However, although wolfberry exosomes have good application prospects, due to their small particles and limited loading of bioactive substances, they are easily degraded under normal storage conditions (4℃ or -20℃), so it is difficult to apply them to the market as a product that can be stored for a short period of time. In addition, wolfberry exosomes are colorless and tasteless, and their sensory properties are not as good as those of wolfberry homogenate.

[0007] At present, the existing wolfberry puree products extracted by the Not From Concentrate (NFC) process on the market are made from fresh wolfberries through low-temperature pressing. However, this product has defects such as inconsistent raw material standards (since wolfberry NFC juice does not have high requirements for the source of raw materials, it usually contains different varieties and easily adulterated moldy or stale fruits) and process limitations, which will lead to the loss of bioactive substances such as plant exosomes and miRNA, affecting the overall antioxidant, anti-inflammatory and / or anti-aging capabilities of the beverage. These defects are often overlooked, and there is no process or method that can effectively solve this defect. Summary of the invention

[0008] The purpose of the present application is to overcome the deficiencies of the above-mentioned prior art and to provide a wolfberry exosome composite beverage and a preparation method and application thereof.

[0009] To achieve the above purpose, the technical solution adopted by this application is:

[0010] The present application provides a wolfberry exosome compound beverage, the wolfberry exosome compound beverage comprising wolfberry exosomes and wolfberry puree;

[0011] The wolfberry puree comprises fresh wolfberry fruits, antioxidants and water.

[0012] As a traditional Chinese medicinal material with a long history of both medicine and food, wolfberry contains a variety of bioactive substances. However, wolfberry puree products have the defect of losing some bioactive substances (especially wolfberry exosomes), which affects the efficacy of wolfberry.

[0013] Exosomes have proven pharmacological activity, good biocompatibility, specific tissue targeting and good drug loading capacity, and are considered to be a promising therapeutic active substance and drug delivery carrier. Compared with exosomes from mammals, plant-derived exosomes have attracted extensive attention in recent years due to their advantages such as easy separation, high yield, wide sources and obvious activity. The morphology and composition of plant exosomes are similar to those of animal extracellular vesicles, and they have similar functions to the source plants, playing an important role in non-cell autonomous manner and even in information exchange between species.

[0014] After extensive research and experiments, the inventors of this application found that compared with wolfberry fruit or wolfberry puree, the exosomes derived from wolfberry may contain bioactive substances similar to those in fresh wolfberry fruit (such as wolfberry polysaccharides, betaine, etc.), and have the same antioxidant and anti-aging effects. The bioactive ingredients such as proteins and miRNA carried by wolfberry exosomes can be directly endocytosed into cells, which has the function of promoting intercellular communication and regulating the physiological functions of the body, and is conducive to giving full play to the physiological functions such as antioxidant and anti-aging; it also has the advantages of high target organ efficacy concentration and high bioavailability, which can make up for the defects of the loss of active substances and low bioavailability of existing wolfberry puree products.

[0015] In addition, this application attempts for the first time to mix wolfberry exosomes with wolfberry puree to prepare a wolfberry exosome composite beverage with good sensory properties (named Qi Neng), which can fully utilize / give full play to the effective ingredients in wolfberry, and also greatly slow down the degradation rate of wolfberry exosomes, stably preserve wolfberry exosomes in a short period of time, and retain the nutritional components of wolfberry fruit. Therefore, the wolfberry exosome composite beverage of this application can comprehensively or superimpose the advantages of wolfberry puree and wolfberry exosomes. The composite beverage has potential good antioxidant damage and can inhibit the expression of skeletal muscle aging markers, etc., and is also better than the existing wolfberry puree products on the market.

[0016] In summary, "Qi Neng" has excellent application prospects in anti-aging and anti-oxidation. It can be used as a healthy drink or health food to resist skeletal muscle aging or sports fatigue, and has a wide range of application scenarios and channels.

[0017] As a preferred embodiment of the wolfberry exosome composite beverage described in the present application, the number of vesicles added per milliliter of wolfberry puree is 5×10 10 ~1×10 11 of wolfberry exosomes.

[0018] The present application determines the number of vesicles in the wolfberry exosome suspension by NTA detection.

[0019] In the technical solution of the present application, it was found that wolfberry exosomes contain miRNA similar to that of wolfberry fruit. Wolfberry exosomes have the effect of resisting skeletal muscle aging. Because they are derived from wolfberry, a species with both medicinal and edible properties, they have the potential to be developed into a compound beverage.

[0020] Experiments have found that the content of wolfberry exosomes can directly affect the biological function of wolfberry puree in the body. The number of vesicles added to wolfberry puree is 5×10 10 ~1×10 11The wolfberry exosomes can alleviate the cytotoxicity caused by H2O2, and can also have a synergistic effect. The various components interact and promote each other, which can improve the bioavailability of the effective ingredients of wolfberry, thereby enhancing the product's antioxidant and anti-aging effects, and solving the problem of loss of active substances in oral wolfberry puree and low bioavailability.

[0021] This application uses wolfberry puree and wolfberry exosomes to prepare a composite beverage with good sensory properties and anti-skeletal muscle cell aging effects, providing new ideas and strategies for anti-aging research for the population.

[0022] The inventors explored the dosage of wolfberry exosomes. If the dosage of wolfberry exosomes is too small, the synergistic effect will be less than the effect brought by the appropriate addition amount. If the dosage of wolfberry exosomes is too large, antagonism may occur, which will also lead to a decrease in the effect of improving the bioavailability of wolfberry's effective ingredients, and a decrease in the antioxidant and anti-aging effects of the product. This shows that the above-mentioned raw materials have a more ideal synergistic effect when they are within the scope of this application.

[0023] As a preferred embodiment of the wolfberry exosome composite beverage described in the present application, the number of vesicles added per milliliter of wolfberry puree is 5×10 10 of wolfberry exosomes.

[0024] When the dosage of wolfberry exosomes in the present application is within the above range, the cytotoxic effect caused by H2O2 can be better alleviated, the synergistic effect is more obvious, the bioavailability of the effective ingredients of wolfberry can be better improved, and the loss of active substances in oral wolfberry puree can be reduced, thereby better improving the antioxidant and anti-aging effects of the product.

[0025] As a preferred embodiment of the wolfberry exosome composite beverage described in the present application, the particle size range of the wolfberry exosomes in the wolfberry exosome composite beverage is 50nm to 110nm.

[0026] As a preferred embodiment of the wolfberry exosome compound beverage described in the present application, the pH value of the wolfberry exosome compound beverage is 4.0-4.5, the pH value of the wolfberry exosomes is 7.2-7.7, and the pH value of the wolfberry puree is 3.9-4.4.

[0027] In the present application, wolfberry exosomes are a colorless transparent solution with no obvious odor and a pH range of 7.2 to 7.7; wolfberry puree is an orange transparent solution with no obvious foreign matter visible to the naked eye and an odor similar to that of wolfberry fruit, with a pH range of 3.9 to 4.4.

[0028] The wolfberry exosome compound beverage prepared in the present application is an orange transparent solution, which also has no obvious foreign matter visible to the naked eye and has a smell similar to that of wolfberry puree, with a pH range of 4.0 to 4.5.

[0029] In some specific implementations, the antioxidant includes vitamin C and citric acid.

[0030] The above-mentioned types of antioxidants are used to inhibit bacteria in the wolfberry exosome compound beverage, and also have the effects of protecting color and improving flavor.

[0031] As a preferred embodiment of the wolfberry exosome composite beverage described in the present application, the method for preparing the wolfberry exosomes comprises the following steps:

[0032] 1) Wash and crush fresh wolfberries, first centrifuge them at a low speed to remove cells and cell debris, take the supernatant, and then centrifuge the supernatant at an ultra-high speed to take the precipitate;

[0033] 2) After the precipitate is resuspended, it is added to a density gradient sucrose solution and centrifuged, and the solution between the 30% and 45% density layers is taken, the solution is resuspended and centrifuged, and the precipitate is taken, and it is resuspended and filtered again to obtain wolfberry exosomes.

[0034] In some specific embodiments, the method for preparing the density gradient sucrose solution comprises the following steps:

[0035] Density gradient sucrose solution was prepared from sucrose and deionized water in proportion (sucrose / total solution volume = 80 g / L, 300 g / L, 450 g / L, 600 g / L) to prepare 8%, 30%, 45%, 60% sucrose solution. After high pressure sterilization (121°C, 30 min), the solution was added to the centrifuge tube in density layers (8%, 30%, 45%, 60% from top to bottom).

[0036] Experiments have shown that the liquid extracted between two visible layers (the density layers are between the 8% layer and the 30% layer, and between the 30% layer and the 45% layer), and vesicles are found in both layers after NTA detection. The vesicle concentration in the layered solution with a density layer of 8% to 30% is lower than that in the layered solution with a density layer of 30% to 45%.

[0037] The present application extracts wolfberry exosomes from fresh wolfberries through the sucrose gradient-ultracentrifugation method to obtain wolfberry exosomes with better quality. The obtained wolfberry exosomes are mixed with wolfberry puree to make up for the low bioavailability of wolfberry puree. The resulting wolfberry exosome composite beverage has better anti-aging and antioxidant properties, can be used as a health drink or health food against skeletal muscle aging or sports fatigue, and has a wide range of application scenarios and approaches.

[0038] In some specific embodiments, the fresh wolfberry is fresh Ningqi No. 7 wolfberry, and can also be other types of wolfberry, not limited to the above types.

[0039] As a preferred embodiment of the wolfberry exosome composite beverage described in the present application, in the step 1), the low-speed centrifugation conditions are: first centrifugation at 1000×g to 3000×g for 10 to 20 minutes, and then centrifugation at 9000×g to 11000×g for 40 to 60 minutes;

[0040] Ultra-high-speed centrifugation conditions are: centrifugation at 150,000 × g to 170,000 × g for 80 to 100 min;

[0041] The centrifugal condition in step 2) is 150000×g to 170000×g for 80 to 100 min.

[0042] As a preferred embodiment of the wolfberry exosome composite beverage described in the present application, the sucrose concentration range corresponding to the layered solution with a density layer of 30% to 45% in the step 2) is 25 to 50%.

[0043] In some specific embodiments, the sucrose concentration range of the layered solution with a density layer of 30% is 25-30%, and the sucrose concentration range of the layered solution with a density layer of 45% is 45-50%.

[0044] As a preferred embodiment of the wolfberry exosome composite beverage described in the present application, the wolfberry exosomes are stored at a temperature of 2 to 5°C.

[0045] This application compares the degradation of wolfberry exosomes under different storage conditions and finds that under the above-mentioned temperature conditions, after mixing wolfberry exosomes with existing raw pulp products, a wolfberry exosome composite beverage with better sensory properties can be prepared. The degradation rate of wolfberry exosomes is also greatly slowed down, and the vesicles in wolfberry exosomes do not decrease significantly, thereby retaining the nutritional components of wolfberry fruit.

[0046] As a preferred embodiment of the wolfberry exosome composite beverage described in the present application, the wolfberry puree includes 95.0-99.9% by mass of fresh wolfberry fruit, 0.03-0.15% by mass of vitamin C and 0.05-0.1% by mass of citric acid, and the remainder is water.

[0047] It should be noted that in the present application, the preparation method of the wolfberry puree is not limited, and the wolfberry puree can be prepared by mixing the raw materials evenly.

[0048] Among them, the representative wolfberry puree is the "Keyikang" wolfberry puree, which includes 99% by mass of fresh wolfberry fruit, 0.05% by mass of vitamin C and 0.05% by mass of citric acid, and the remainder is water.

[0049] The present application also provides a method for preparing the wolfberry exosome composite beverage, which is prepared by centrifuging and filtering wolfberry pulp at a low speed and then mixing it with wolfberry exosomes;

[0050] The number of vesicles added per ml of wolfberry puree is 5×10 10 ~1×10 11 of wolfberry exosomes.

[0051] Preferably, the number of vesicles added per ml of wolfberry puree is 5×10 10 of wolfberry exosomes.

[0052] As a preferred embodiment of the method for preparing the wolfberry exosome composite beverage described in the present application, the low-speed centrifugation is: centrifugation at 2000×g for 20 min.

[0053] The present application also provides the use of the above-mentioned wolfberry exosome composite beverage in the preparation of health products.

[0054] As a preferred embodiment of the application described in the present application, the health care products include health care products for resisting skeletal muscle aging or fatigue.

[0055] This application is the first to formulate wolfberry exosomes and wolfberry puree into a wolfberry exosome composite beverage "Qi Neng". "Qi Neng" combines the advantages of wolfberry homogenate and wolfberry exosomes. While delaying the degradation rate of wolfberry exosomes, it has good antioxidant and anti-aging (skeletal muscle atrophy) effects.

[0056] TEM and NTA tests confirmed that the content of exosomes in wolfberry puree was low due to long-term storage or process loss, while the "Qi Neng" configured after classified storage contained more wolfberry exosomes, and under the storage condition of 4°C, about 2 / 3 of the wolfberry exosomes in "Qi Neng" could still be preserved after 7 days.

[0057] Through miRNA sequencing and QPCR experiments, it was preliminarily inferred that although wolfberry exosomes are small in size, they still retain a considerable amount of miRNA; at the same time, there are differences in the type, distribution and content of miRNA retained in wolfberry homogenate and that in wolfberry exosomes. The type and distribution of miRNA in "Qi Neng" combines the characteristics of wolfberry homogenate and extracted wolfberry exosomes.

[0058] Through cell experiments, it was further found that "Qi Neng" has a good antioxidant effect and has better functions of improving cell activity, inhibiting the expression of aging markers, alleviating the reduction of myotube diameter, increasing ATP levels and inhibiting cell apoptosis than wolfberry puree. At the same time, compared with wolfberry homogenate, the miRNA in "Qi Neng" is more easily absorbed by cells. It can be preliminarily inferred that "Qi Neng" can improve the performance of wolfberry puree products and play a better role in resisting skeletal muscle cell aging.

[0059] Compared with the prior art, this application has the following beneficial effects:

[0060] The present application provides a wolfberry exosome compound beverage and its preparation method and application. The present application uses wolfberry exosomes to improve wolfberry puree products, and prepares wolfberry exosome compound beverages with good sensory properties and better anti-skeletal muscle cell aging effects. At the same time, NTA analysis verified that the degradation rate of wolfberry exosomes in the wolfberry exosome compound beverage was delayed, and further combined with miRNA sequencing technology and cell experiments proved that the wolfberry exosome compound beverage has a richer variety of information substances / relative content and good antioxidant effects. Compared with the existing wolfberry puree products on the market, it can better inhibit the expression of skeletal muscle cell aging markers and alleviate muscle cell aging. Compared with traditional anti-aging technologies or solutions, the wolfberry exosome compound beverage "Qi Neng" prepared by wolfberry exosomes and wolfberry puree in the present application has the advantages of high safety, easy access to raw materials, economical and affordable, and portable and easy to operate, providing an effective and feasible new idea for anti-aging research and / or practical applications for the population. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 The figure is a diagram of the vesicle concentration in the layered solution with a density layer of 30-45% (the density layers from top to bottom are 8%, 30%, 45%, 60%);

[0062] Figure 2 This is a graph showing the concentration of vesicles in a layered solution with a density layer of 8% to 30%;

[0063] Figure 3 This is a flow chart for constructing the wolfberry exosome composite beverage prepared in Example 4;

[0064] Figure 4 The sensory properties of wolfberry exosomes, wolfberry puree and the wolfberry exosome composite beverage prepared in Example 4;

[0065] Figure 5 TEM test results of wolfberry exosomes, wolfberry puree and wolfberry exosome composite beverage prepared in Example 4 (Note: G represents wolfberry puree; D represents wolfberry exosomes; GD represents wolfberry exosome composite beverage);

[0066] Figure 6The NTA test results and exosome content analysis chart of wolfberry exosomes, wolfberry puree and wolfberry exosome compound beverage (Note: GD represents wolfberry exosome compound beverage; D-7d represents exosomes after 7 days of storage; GD-7d represents wolfberry exosome compound beverage after 7 days of storage. After ANOVA analysis, multiple comparisons were performed by Tukey's test, and the results are indicated by "*". "ns" means P>0.05, "*" means P<0.05, "**" means P<0.01, "***" means P<0.001, and "****" means P<0.0001);

[0067] Figure 7 This is the result graph of the percentage of remaining vesicles after wolfberry exosomes and wolfberry exosome compound beverage were stored at different temperatures for 7 days (D represents wolfberry exosomes; GD represents wolfberry exosome compound beverage);

[0068] Figure 8 The results of cell viability detection (CCK8) of different groups in Experimental Example 2 (wherein, C represents PBS intervention in the control group; G represents wolfberry puree intervention; D represents wolfberry exosome intervention; GD represents wolfberry exosome compound beverage intervention; H represents H2O2 intervention; HD represents H2O2 and wolfberry exosome co-intervention; HG represents H2O2 and wolfberry puree co-intervention; HGD1 represents H2O2 and wolfberry exosome compound beverage prepared in Comparative Example 1 co-intervention; HGD2, H2O2 and wolfberry exosome compound beverage prepared in Example 6 co-intervention; HGD3, H2O2 and wolfberry exosome compound beverage prepared in Example 4 co-intervention; HGD4, H2O2 and wolfberry exosome compound beverage prepared in Example 5 co-intervention; HGD5, H2O2 and wolfberry exosome compound beverage prepared in Comparative Example 2 co-intervention; HGD represents wolfberry exosome compound beverage prepared in Example 4. After ANOVA analysis, by Multiple comparisons were performed by test, and the results are indicated by "*". "ns" indicates P>0.05, "*" indicates P<0.05, "**" indicates P<0.01, "***" indicates P<0.001, and "****" indicates P<0.0001);

[0069] Fig. 9The results of cell viability detection (CCK8) of different groups in Experimental Example 2 (wherein, C represents PBS intervention in the control group; H represents H2O2 intervention; HG1 represents H2O2 and wolfberry puree prepared in Example 4 co-intervention; HG2 represents H2O2 and wolfberry puree treated in Comparative Example 3 co-intervention; HG3 represents H2O2 and wolfberry puree treated in Comparative Example 4 co-intervention; HGD1 represents H2O2 and wolfberry exosome composite beverage prepared in Example 4 co-intervention; HGD2, H2O2 and wolfberry exosome composite beverage prepared in Comparative Example 3 co-intervention; HGD3, H2O2 and wolfberry exosome composite beverage prepared in Comparative Example 4 co-intervention. After ANOVA analysis, by Multiple comparisons were performed by test, and the results are indicated by "*". "ns" indicates P>0.05, "*" indicates P<0.05, "**" indicates P<0.01, "***" indicates P<0.001, and "****" indicates P<0.0001);

[0070] Fig.10 The results of myotube diameter detection after different groups of intervention in Experimental Example 3 (wherein, C represents the control group PBS intervention; H represents H2O2 intervention; HD represents H2O2 and wolfberry exosomes co-intervention; HG represents H2O2 and wolfberry puree co-intervention; HGD represents H2O2 and wolfberry exosomes composite beverage prepared in Example 4 co-intervention. After ANOVA analysis, Multiple comparisons were performed by test, and the results are indicated by "*". "ns" indicates P>0.05, "*" indicates P<0.05, "**" indicates P<0.01, "***" indicates P<0.001, and "****" indicates P<0.0001);

[0071] Fig.11 The results of the detection of β-galactosidase (aging marker) expression levels after intervention in different groups in Experimental Example 3 (wherein, the percentage of β-galactosidase staining area to the total area of ​​the visual field is used as the experimental result for comparison. C represents the control group PBS intervention; H represents H2O2 intervention; HD represents H2O2 and wolfberry exosomes co-intervention; HG represents H2O2 and wolfberry puree co-intervention; HGD represents H2O2 and wolfberry exosomes composite beverage prepared in Example 4 co-intervention. After ANOVA analysis, Multiple comparisons were performed by test, and the results are indicated by "*". "ns" indicates P>0.05, "*" indicates P<0.05, "**" indicates P<0.01, "***" indicates P<0.001, and "****" indicates P<0.0001);

[0072] Fig.12The results of oxidative stress index detection after intervention in different groups in Experimental Example 3 (a: ROS content detection results (the percentage of ROS staining area to the total area of ​​the visual field is used as the experimental result for comparison); b: GSH-Px content detection results; c: Superoxide dismutase (SOD) content detection results; d: Lactate dehydrogenase (LDH) content detection results, wherein C represents the control group PBS intervention; H represents H2O2 intervention; HD represents H2O2 and wolfberry exosomes co-intervention; HG represents H2O2 and wolfberry puree co-intervention; HGD represents H2O2 and wolfberry exosomes composite beverage prepared in Example 4 co-intervention. After ANOVA analysis, by Multiple comparisons were performed by t-test. "ns" means P>0.05, "*" means P<0.05, "**" means P<0.01, "***" means P<0.001, and "****" means P<0.0001. "#" means the t-test result of this group and group H is P<0.05);

[0073] Fig.13 The results of mitochondrial related index detection after intervention in different groups in Experimental Example 3 (a: JC-1 monomer detection result; Figure 8 b: JC-1 polymer test results; c: JC-1 polymer to monomer ratio; d: ATP content test results; C represents the control group PBS intervention; H represents H2O2 intervention; HD represents H2O2 and wolfberry exosomes co-intervention; HG represents H2O2 and wolfberry puree co-intervention; HGD represents H2O2 and wolfberry exosomes composite beverage prepared in Example 4 co-intervention. After ANOVA analysis, Multiple comparisons were performed by t-test. "ns" means P>0.05, "*" means P<0.05, "**" means P<0.01, "***" means P<0.001, and "****" means P<0.0001. "#" means that the t-test result of this group and group H is P<0.05. ).

[0074] Fig.14 Figure 2 is the result of miRNA sequencing and RNA extraction experiment of different groups in Experimental Example 4 (a: miRNA sequencing result; b: RNA extraction result; G represents wolfberry puree; D represents wolfberry exosomes; GD represents wolfberry exosome compound beverage prepared in Example 4. After ANOVA analysis, Multiple comparisons were performed by test, and the results are indicated by "*". "ns" indicates P>0.05, "*" indicates P<0.05, "**" indicates P<0.01, "***" indicates P<0.001, and "****" indicates P<0.0001);

[0075] Fig.15 The Q-PCR experimental results of plant-derived miRNA after intervention in different groups in Experimental Example 4 (a, b, c: miRNA detection results in wolfberry puree, wolfberry exosomes and "Qi Neng"; d, e, f: miRNA detection results in C2C12 cells; wherein, G represents wolfberry puree; D represents wolfberry exosomes; GD represents the wolfberry exosome compound beverage prepared in Example 4; C represents the control group PBS intervention; H represents H2O2 intervention; HD represents the joint intervention of H2O2 and wolfberry exosomes; HG represents the joint intervention of H2O2 and wolfberry puree; HGD represents the joint intervention of H2O2 and the wolfberry exosome compound beverage prepared in Example 4. After ANOVA analysis, multiple comparisons were performed by Tukey's test, and the results are indicated by "*". "ns" indicates P>0.05, "*" indicates P<0.05, "**" indicates P<0.01, "***" indicates P<0.001, and "****" indicates P<0.0001);

[0076] Fig.16 The results of the Q-PCR experiment on muscle atrophy and mitochondria after intervention in different groups in Experimental Example 4 (C represents the control group PBS intervention; H represents H2O2 intervention; HD represents H2O2 and wolfberry exosomes intervention; HG represents H2O2 and wolfberry puree intervention; HGD represents H2O2 and wolfberry exosomes composite beverage prepared in Example 4 intervention. After ANOVA analysis, Multiple comparisons were performed by test, and the results are indicated by "*". "ns" indicates P>0.05, "*" indicates P<0.05, "**" indicates P<0.01, "***" indicates P<0.001, and "****" indicates P<0.0001). DETAILED DESCRIPTION

[0077] In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application. In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0078] In this application, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are deemed to be continuous and include the minimum and maximum values ​​of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0079] In the following examples and comparative examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified, and the components and raw materials used in each parallel experiment are of the same type.

[0080] The wolfberry puree used in the embodiments and comparative examples is "Keyikang" wolfberry puree, which includes 99% by mass of fresh wolfberry fruit, 0.05% by mass of vitamin C and 0.05% by mass of citric acid, and the remainder is water.

[0081] MISEV-2023 standard: Welsh, Joshua A et al. "Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches." Journal of extracellular vesicles vol.13,2(2024):e12404.doi:10.1002 / jev2.12404.

[0082] In the following examples and comparative examples:

[0083] The density gradient sucrose solution was prepared from sucrose and deionized water in proportion (sucrose / total solution volume = 80 g / L, 300 g / L, 450 g / L, 600 g / L) to prepare sucrose solutions with concentrations of 8%, 30%, 45%, and 60%. After high-pressure sterilization (121°C, 30 min), the solution was added to the centrifuge tube in density layers (8%, 30%, 45%, and 60% from top to bottom), thereby dividing the solution into layered solutions with density layers of 8% to 30%, 30% to 45%, and 45% to 60%, respectively.

[0084] Example 1: Preparation of Lycium barbarum exosomes

[0085] This embodiment provides a method for preparing wolfberry exosomes, comprising the following steps:

[0086] 1) Wash and crush fresh wolfberries, first centrifuge at low speed (first at 1000 × g for 10 min, then at 2000 × g for 20 min, and finally at 10,000 × g for 60 min) to remove cells and cell debris, take the supernatant, and then centrifuge the supernatant at ultra-high speed (150,000 × g for 90 min) to enrich the exosomes in the precipitate, and take the precipitate;

[0087] 2) resuspending the precipitate obtained in step 1) and adding it to a density gradient sucrose solution for centrifugation (using 150,000 × g for 90 min), taking the solution between the 30% layer and the 45% layer (density layer is 30% to 45%), resuspending the solution and centrifuging it (using 150,000 × g for 90 min), taking the precipitate, resuspending and filtering it again, obtaining wolfberry exosomes, and storing it at 4°C.

[0088] Example 2: Preparation of Lycium barbarum exosomes

[0089] Compared with Example 1, step 2) is different in that a solution with a density layer between 8% and 30% is taken (the density layer is 8% to 30%), and the other parameters are the same as those in Example 1.

[0090] Example 3: Preparation of Lycium barbarum exosomes

[0091] Compared with Example 1, step 2) is different in that a solution with a density layer between 45% and 60% is taken, and the other parameters are the same as those in Example 1.

[0092] Through experiments, it can be seen that the present application extracted liquids between two layers (density layers of 8% to 30% and 30% to 45%) (i.e., Examples 1-2), and both contained vesicles after NTA detection, but the vesicle concentration in the layered solution with a density layer of 8% to 30% was much lower than that in the layered solution with a density layer of 30% to 45%, such as Figure 1-Figure 2 Therefore, the present application selected the wolfberry exosomes prepared in Example 1 (a layered solution with a density layer of 30% to 45%) to conduct an experiment on preparing a wolfberry exosome composite beverage.

[0093] Example 4: A wolfberry exosome composite beverage and a preparation method thereof

[0094] This embodiment provides a method for preparing a wolfberry exosome composite beverage, comprising the following steps:

[0095] 1. Centrifuge the wolfberry puree (2000×g, 20 min), discard the precipitate, and take the supernatant. Then filter (using a 0.22 μm filter) the supernatant to remove residual fruit fragments.

[0096] 2. Add wolfberry exosomes to the filtered wolfberry puree (pH value is 3.9-4.4) (the number of vesicles added per ml of wolfberry puree is 5×10 10 The wolfberry exosomes, the particle size of which is distributed between 50nm and 150nm (determined by the results of nanoparticle tracking analysis), are prepared to obtain a wolfberry exosome compound beverage ("Qi Neng") and stored at 4°C. The specific operation process is as follows Figure 3 shown.

[0097] Example 5: A wolfberry exosome composite beverage and a preparation method thereof

[0098] Compared with Example 4, the only difference is that the number of vesicles added per milliliter of wolfberry puree is 1×10 11 The parameters in the preparation method of wolfberry exosomes are the same as those in Example 4.

[0099] Example 6: A wolfberry exosome composite beverage and its preparation method

[0100] Compared with Example 4, the only difference is that the number of vesicles added per ml of wolfberry puree is 2.5×10 10 The parameters in the preparation method of wolfberry exosomes are the same as those in Example 4.

[0101] Comparative Example 1: A wolfberry exosome composite beverage and a preparation method thereof

[0102] Compared with Example 4, the difference in Comparative Example 1 is that the number of vesicles added per milliliter of wolfberry puree is 0.5×10 10 The parameters in the preparation method of wolfberry exosomes are the same as those in Example 4.

[0103] Comparative Example 2: A wolfberry exosome composite beverage and a preparation method thereof

[0104] Compared with Example 4, the difference of Comparative Example 2 is that the number of vesicles added per milliliter of wolfberry puree is 4×10 11 The parameters in the preparation method of wolfberry exosomes are the same as those in Example 4.

[0105] Comparative Example 3: A wolfberry exosome composite beverage and a preparation method thereof

[0106] Compared with Example 4, the only difference is that step 2 is different:

[0107] 2. The pH value of the filtered wolfberry pulp was adjusted with phosphate from 4.2 to 5, and then wolfberry exosomes were added to the treated wolfberry pulp (the number of vesicles added per ml of wolfberry pulp was 5×10 10 The remaining parameters in the preparation method of the wolfberry exosome composite beverage are the same as those in Example 4.

[0108] Comparative Example 4: A wolfberry exosome composite beverage and a preparation method thereof

[0109] Compared with Example 4, the only difference is that step 2 is different:

[0110] The pH value of the filtered wolfberry puree was adjusted with phosphate from 4.2 to 5.5, and then wolfberry exosomes were added to the treated wolfberry puree (the number of vesicles added per ml of wolfberry puree was 5×10 10 The remaining parameters in the preparation method of the wolfberry exosome composite beverage are the same as those in Example 4.

[0111] Experimental Example 1: Detection and Identification of Sensory Properties of Lycium barbarum Exosomes Composite Drink

[0112] This experiment tested the sensory properties of the wolfberry exosomes prepared in Example 1 and the wolfberry puree and wolfberry exosome composite beverage prepared in Example 4;

[0113] The prepared wolfberry exosome compound beverage is an orange transparent solution, with no obvious foreign matter visible to the naked eye and a smell similar to that of wolfberry puree, with a pH range of 4.0-4.5; wolfberry exosomes are a colorless transparent solution with no obvious odor, and a pH range of 7.2-7.7; wolfberry puree is an orange transparent solution with no obvious foreign matter visible to the naked eye and a smell similar to that of wolfberry fruit, with a pH range of 3.9-4.4 (such as Figure 4 shown).

[0114] Through transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA), it can be observed that the suspension of wolfberry exosomes contains a large number of regularly shaped cup-disc-shaped vesicles with a particle size distribution between 50nm and 150nm. The morphology and particle size meet the relevant standards for plant exosomes in the MISEV-2023 standard.

[0115] Although there are a small number of vesicles in the wolfberry homogenate, their morphology is irregular and the particle (vesicle) size is small, concentrated between 40nm-80nm. Similar to the wolfberry exosome suspension, the wolfberry exosome compound beverage can be observed by TEM to contain a large number of vesicles with regular shapes, and the particle size is evenly distributed between 50nm-100nm, which is closer to the vesicle morphology of the wolfberry exosome suspension. The results are as follows Figure 5 shown.

[0116] The wolfberry exosomes (prepared in Example 1) stored at -80°C were taken out and packaged, and then stored in refrigerators at 4°C and -20°C respectively;

[0117] After wolfberry puree and wolfberry exosomes were prepared into "Qineng" (prepared in Example 4), they were also packaged and stored in 4°C and -20°C refrigerators respectively. After 7 days, all the above samples and wolfberry exosomes were tested by NTA.

[0118] like Figure 6-Figure 7 After the wolfberry exosomes were stored at -20°C for 7 days, the vesicle concentration decreased significantly (the remaining exosome concentration was about 1 / 5); the vesicle concentration stored at 4°C also decreased significantly (the remaining exosome concentration was about 3 / 5), but was higher than the sample stored at -20°C.

[0119] The vesicle concentration of the samples of "Qi Neng" stored at 4°C (the remaining exosome concentration was about 7 / 10) and -20°C (the remaining exosome concentration was about 1 / 2) also decreased, but the degree of decrease was smaller.

[0120] The above results suggest that the content of wolfberry exosomes in wolfberry puree products is low; the vesicle concentration of wolfberry exosomes will decrease when stored at 4°C, and the degree of decrease will be more obvious after freeze-thaw; but in the present application, after the wolfberry exosomes are stored in wolfberry puree to make "Qi Neng", the wolfberry exosomes are relatively stable, and the vesicle concentration decreases more slowly when stored at 4°C and -20°C, and the storage effect at 4°C is better.

[0121] like Figure 6 Through the two-sample t-test, it was found that the newly prepared samples of wolfberry exosomes and "Qineng" and the samples stored for 7 days were higher than those of wolfberry puree (P<0.05).

[0122] Experimental Example 2: Intervention Experiment of Different Lycium Barbarum Exosome Compound Drinks

[0123] In this experiment, H2O2 was used to induce oxidative damage in C2C12 mouse myoblasts, and wolfberry puree, wolfberry exosome compound beverage (Examples 4 to 6 and Comparative Examples 1 to 4) and wolfberry exosomes (prepared in Example 1) were used for cell co-culture intervention. The volume ratio of the intervention substances (wolfberry puree, wolfberry exosome compound beverage and wolfberry exosomes) to GIBCO's DMEM high-glucose medium (from Thermo Fisher Scientific) was 1:19.

[0124] The CCK8 test results showed that Figure 8 As shown, wolfberry exosomes can improve cell activity and alleviate the cytotoxicity caused by H2O2, while wolfberry puree of the same concentration has no obvious effect. The wolfberry exosome composite beverage prepared in Example 4 can also significantly improve cell activity and alleviate the cytotoxicity caused by H2O2.

[0125] The wolfberry exosome composite beverages prepared in Examples 5 to 6 can also alleviate the cytotoxicity caused by H2O2. The wolfberry exosome composite beverages prepared in Comparative Examples 1 to 2 have no obvious effect in alleviating the cytotoxicity caused by H2O2. Therefore, this application chooses to add 5×10 vesicles per milliliter of wolfberry puree. 10 ~1×10 11 The wolfberry exosomes (determined by the results of nanoparticle tracking analysis) were used for subsequent experiments.

[0126] In this experiment, H2O2 was used to induce oxidative damage in C2C12 mouse myoblasts, and wolfberry puree with different pH values ​​(wolfberry puree prepared in Example 4, wolfberry puree treated in Comparative Examples 3-4) and wolfberry exosome composite beverage (wolfberry exosome composite beverage prepared in Example 4 and Comparative Examples 3-4) were used for cell co-culture intervention. The volume ratio of the intervention substance (wolfberry puree and wolfberry exosome composite beverage) to the culture medium was 1:19.

[0127] The CCK8 test results showed that Fig. 9 As shown, the wolfberry exosome composite beverage prepared in Example 4 and Comparative Example 3 can significantly improve cell activity and alleviate the cytotoxicity caused by H2O2; the wolfberry puree treated with Example 4 and Comparative Example 3 has no obvious effect; the wolfberry puree treated with Comparative Example 4 and the wolfberry exosome composite beverage prepared in Comparative Example 4 have certain toxicity to cells.

[0128] Through the above CCK8 experiment, the wolfberry exosome composite beverages prepared in Examples 4 to 6 have the effect of alleviating the cytotoxicity caused by H2O2, indicating that the number of vesicles added per milliliter of wolfberry puree is 1×10 10 ~1×10 11 The wolfberry exosomes can alleviate the cytotoxicity caused by H2O2. Therefore, in the subsequent experiments, the best ratio was selected (the number of vesicles added per ml of wolfberry puree was 5×10 10 of wolfberry exosomes).

[0129] Experimental Example 3: Antioxidant and anti-aging effects of wolfberry exosomes complex beverage

[0130] After the C2C12 cells differentiated into myotubes, H2O2 was used to induce cellular oxidative stress and myotube atrophy, and wolfberry puree, the wolfberry exosome composite drink prepared in Example 4, and wolfberry exosomes were used for intervention, respectively. After 24 hours, the required indicators were detected using the corresponding biochemical kits.

[0131] Compared with wolfberry puree, the wolfberry exosome compound beverage and wolfberry exosomes of the present application have more obvious anti-skeletal muscle atrophy effect (reference Fig.10 ) and inhibited the expression level of β-galactosidase, a marker of aging (reference Fig.11). At the same time, the wolfberry exosome compound beverage of the present application combines the anti-oxidative stress effect of wolfberry puree, which can reduce the content of reactive oxygen species (ROS) in muscle cells (reference Fig.12 ), increase the content of glutathione peroxidase (GSH-Px) (reference Fig.13 ), thereby reducing the level of oxidative stress induced by H2O2. In addition, "Qi Neng" also combines the advantages of wolfberry exosomes, and can more effectively alleviate the H2O2-induced decrease in mitochondrial membrane potential and the decrease in ATP level / content compared to wolfberry puree. The decrease in mitochondrial membrane potential is a landmark event in the early stage of cell apoptosis. When the mitochondrial membrane potential is high, JC-1 aggregates in the matrix of mitochondria to form aggregates; when the mitochondrial membrane potential is low, JC-1 cannot aggregate in the matrix of mitochondria, and JC-1 is a monomer at this time. The decrease in cell membrane potential can be easily detected by the transition of JC-1 from aggregates to monomers, and this transition can be used as a detection indicator for early cell apoptosis.

[0132] Experimental Example 4: RNA sequencing and real-time fluorescence quantification (Q-PCR) detection of wolfberry exosome compound beverage

[0133] The miRNA sequencing and Q-PCR experiments were performed on the wolfberry exosome compound beverage, wolfberry puree and wolfberry exosome prepared in Example 4, and the results showed that there were significant differences in the types, distribution and content of miRNA among the three ( Fig.14 ). The miRNA in wolfberry homogenate mainly comes from cell debris. Although wolfberry exosomes do not contain cell debris and are small in size, they still contain a certain amount of miRNA, among which miR166d, miR167a, and miR168a are at higher levels.

[0134] Further Q-PCR detection of miR166d, miR167a, and miR168a in the wolfberry exosome compound beverage (GD), wolfberry puree (G), and wolfberry exosome (D) prepared in Example 4 revealed that miR166d, miR167a, and miR168a were all found in wolfberry puree (G) at the highest levels, but the difference was not statistically significant. After H2O2 and wolfberry exosome compound beverage (HGD) and wolfberry puree (HG) intervened in C2C12 cells, Q-PCR detection of miR166d, miR167a, and miR168a in cells revealed that the three miRNAs increased after intervention, and the relative levels in C2C12 cells after "Qi Neng" intervention were the highest ( Fig.15The above results suggest that although there is little difference in the content of miRNA between the wolfberry exosome compound drink and the wolfberry puree, the miRNA in the wolfberry exosome compound drink is more easily absorbed by cells.

[0135] After H2O2 induced myotube atrophy, compared with wolfberry homogenate (HG), wolfberry exosome compound beverage reduced the relative content of miRNA of muscle atrophy-related muscular atrophy box F gene (Atrogin1) and mitochondrial autophagy-related ubiquitin protein ligase gene (Parkin) in C2C12 cells (HGD), while increased the relative content of miRNA of nuclear respiratory factor 1 (Nrf1) involved in maintaining mitochondrial homeostasis ( Fig.16 ). The experimental results show that the wolfberry exosome compound drink has the function of resisting myotube atrophy and maintaining mitochondrial homeostasis, and although the wolfberry homogenate has a certain improvement effect, the effect is not as good as the wolfberry exosome compound drink of the present application.

[0136] The above key results show that the present application can successfully prepare "Qi Neng" with good sensory properties, that is, the wolfberry exosome compound beverage of the present application, which combines the advantages of wolfberry exosomes and wolfberry homogenate, in which the wolfberry exosomes are more stable than those stored in PBS solution, and the content of bioactive substances (especially miRNA) in the cell model intervened by "Qi Neng" is higher than that in the cell model intervened by wolfberry homogenate. At the same time, while retaining the antioxidant effect similar to that of wolfberry puree, "Qi Neng" also has better effects of improving muscle cell activity, inhibiting the expression of aging markers, inhibiting myotube atrophy, maintaining mitochondrial homeostasis, and increasing ATP levels and inhibiting cell apoptosis, further improving / making up for the shortcomings of wolfberry puree, and effectively exerting the biological activity effect of anti-aging of skeletal muscle cells.

[0137] This application is the first to use wolfberry exosomes and wolfberry puree to prepare a wolfberry exosome composite beverage "Qi Neng" with good sensory properties, and combined with cell experiments to verify that "Qi Neng" has good effects in reducing oxidative stress levels and delaying skeletal muscle cell aging, indirectly proving that "Qi Neng" has good application prospects.

[0138] The ingredients in "Qi Neng" are similar to those of its source plant, wolfberry, but it is superior to fresh wolfberry in terms of storage and taste and flavor. The degradation rate of wolfberry exosomes is significantly lower than that of wolfberry exosomes stored in PBS.

[0139] Compared with the wolfberry puree products on the market, the large amount of wolfberry exosomes contained in "Qi Neng" can make up for the loss of some of its bioactive substances such as proteins and miRNAs, have better and higher bioavailability, and can play a better role in resisting myotube atrophy and skeletal muscle cell aging. At the same time, compared with existing anti-aging solutions or reagents, the raw materials used in this application are all wolfberries, which are both medicinal and edible, and have the advantages of being economical, safe, and easy to obtain.

[0140] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. A wolfberry exosome compound beverage, It is characterized in that The wolfberry exosome compound beverage comprises wolfberry exosomes and wolfberry puree; The wolfberry puree comprises fresh wolfberry fruits, antioxidants and water.

2. The wolfberry exosome composite beverage according to claim 1, It is characterized in that The number of vesicles added per ml of wolfberry puree is 5×10 10 ~1×10 11 of wolfberry exosomes.

3. The wolfberry exosome composite beverage according to claim 1, It is characterized in that The number of vesicles added per ml of wolfberry puree is 5×10 10 ~1×10 11 of wolfberry exosomes.

4. The wolfberry exosome composite beverage according to claim 1, It is characterized in that The particle size range of the wolfberry exosomes in the wolfberry exosome composite beverage is 50nm to 150nm.

5. The wolfberry exosome composite beverage according to claim 1, It is characterized in that The pH value of the wolfberry exosome compound beverage is 4.0-4.5, the pH value of the wolfberry exosome is 7.2-7.7, and the pH value of the wolfberry puree is 3.9-4.

4.

6. The wolfberry exosome composite beverage according to claim 1, It is characterized in that The antioxidants include vitamin C and citric acid.

7. The wolfberry exosome composite beverage according to claim 1, It is characterized in that The method for preparing wolfberry exosomes comprises the following steps: 1) Wash and crush fresh wolfberries, first centrifuge them at a low speed to remove cells and cell debris, take the supernatant, and then centrifuge the supernatant at an ultra-high speed to take the precipitate; 2) After the precipitate is resuspended, it is added to a density gradient sucrose solution and centrifuged, and the solution between the 30% and 45% density layers is taken, the solution is resuspended and centrifuged, and the precipitate is taken, and it is resuspended and filtered again to obtain wolfberry exosomes.

8. The wolfberry exosome composite beverage according to claim 7, It is characterized in that In the step 1), the low-speed centrifugation conditions are: first centrifugation at 1000×g to 3000×g for 10 to 20 min, and then centrifugation at 9000×g to 11000×g for 40 to 60 min; Ultra-high-speed centrifugation conditions are: centrifugation at 150,000 × g to 170,000 × g for 80 to 100 min; The centrifugal condition in step 2) is 150000×g to 170000×g for 80 to 100 min.

9. The wolfberry exosome composite beverage according to claim 7, It is characterized in that In the step 2), the sucrose concentration range of the solution with density stratification between 30% stratification and 45% stratification is 30-45%.

10. The wolfberry exosome composite beverage according to claim 7, It is characterized in that The wolfberry exosomes are stored at a temperature of 2 to 5°C.

11. The wolfberry exosome composite beverage according to claim 1, It is characterized in that The wolfberry puree comprises 95.0-99.9% by mass of fresh wolfberry fruit, 0.03-0.15% by mass of vitamin C and 0.05-0.1% by mass of citric acid, and the remainder is water.

12. The method for preparing the wolfberry exosome composite beverage according to any one of claims 1 to 11, It is characterized in that The wolfberry puree is centrifuged and filtered at low speed, and then mixed with wolfberry exosomes to obtain the product; The number of vesicles added per ml of wolfberry puree is 5×10 10 ~1×10 11 of wolfberry exosomes.

13. The method for preparing the wolfberry exosome composite beverage according to claim 12, It is characterized in that The low-speed centrifugation is: centrifugation at 2000-3000×g for 20-30 min.

14. Use of the wolfberry exosome composite beverage according to any one of claims 1 to 12 in the preparation of health products.

15. The use according to claim 14, It is characterized in that The health care products include health care products for resisting skeletal muscle aging or fatigue.

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