Exosome production method

Abstract

Provided is an exosome production method, comprising the following steps: providing a Ganoderma culture solution containing Ganoderma mycelium, and providing an algal culture solution containing a chlorophyte alga; placing the algal culture solution and the Ganoderma culture solution in a container and co-culturing, to obtain a mixed culture solution; and extracting exosomes from the mixed culture solution. By allowing the Ganoderma mycelium and the chlorophyte alga to interact under co-culturing conditions, exosome yield is increased, and the particle size of the exosomes tends to be uniform.

Description

Exosome production methods Technical Field

[0001] The present disclosure relates to a method for producing exosomes. Background Art

[0002] Exosomes are nanosized vesicles that differentiate from the cell membrane and are released into the cellular environment. They range in size from 30 to 150 nanometers and are a type of extracellular vesicle (EV). Exosomes are composed of a lipid bilayer similar to the cell membrane, as well as active biomolecules such as nucleic acids and proteins. Their composition and function depend on the cell in which they originate.

[0003] Research on animal exosomes indicates that the properties of the exosomes secreted by the animals themselves are significantly correlated. For example, exosomes from mesenchymal stem cells can have similar effects to those of stem cells. The effects of animal exosomes have been extensively studied and proven, particularly those derived from humans. However, animal exosomes are highly sensitive to their environment, making their use difficult beyond direct in vivo administration (injection). This significantly limits their potential applications.

[0004] In recent years, plant exosomes have been shown to possess similar therapeutic benefits to those of animal exosomes. Therefore, plant-derived exosomes will be a key focus of future pharmaceutical research and development. Medicinal plants hold the greatest potential for development, as they have already been proven to be of medicinal use. Therefore, obtaining more and better exosomes has become a primary focus.

[0005] Therefore, pharmaceutical companies have invested a lot of resources in developing new drug carriers, hoping to deliver small molecules or biologics more efficiently and accurately. Exosomes have long been considered the best candidates for drug carriers due to their low immunogenicity, good biocompatibility, bioactivity, low cell rejection, and nano-size that targets the microenvironment. In addition to the above advantages, exosomes also have other significant characteristics, such as high affinity, easy phagocytosis by target cells, easy degradation and release of drugs in cells, and the ability to avoid being consumed by the immune system.

[0006] Because exosomes are cell-autonomously secreted substances and readily enter surrounding cells, they are readily available in large quantities from liquid sources, such as various plant sap or plant (cell) culture solutions. Currently, traditional Chinese medicine (TCM) (traditional medicinal plants) is often consumed by extracting the plant itself / fermentation broth, filtering / concentrating it, or directly (drying and grinding it into powder). This process can disrupt the structure of the exosomes, reducing their stability and quantity, and thus losing their inherent properties.

[0007] Lingzhi, on the other hand, is a highly valued traditional medicinal plant, a highly effective and widely accepted remedy in traditional Chinese medicine. The growth of fungi like Lingzhi can generally be categorized into the following stages: hyphae → mycelium → fruiting body → sporophyte. The hyphae are similar to the aerial tentacles of a root, or what might be considered "growing roots." The root, once mature, becomes the mycelium. The fruiting body, on the other hand, is the familiar mushroom-like structure. While in the mycelium, Lingzhi increases its number entirely through division and replication. These hyphae, like plant roots, specialize in nutrient absorption and storage. As Lingzhi gradually develops into a stipe, cap, and finally a full-fledged mushroom body, spores are formed, producing numerous complex metabolites. Therefore, in terms of composition, the fruiting body contains far more diverse components than the mycelium. It is generally believed that the full efficacy of Ganoderma lucidum comes from the fruiting body, a woody structure that develops from the mycelium. For human use, the fruiting body is the only one that possesses its full physiological benefits. Currently, commercial production relies on culturing Ganoderma lucidum spores to the fruiting body stage to obtain its medicinal components (such as secondary metabolites). However, obtaining exosomes from Ganoderma lucidum requires culturing the mycelium stage. Currently, the production of exosomes from Ganoderma lucidum mycelium is not widely accepted due to low yields, long cultivation times, and a cumbersome process. Furthermore, the overall efficacy and value of the resulting product are inferior to those obtained from cultivating intact fruiting bodies.

[0008] Furthermore, in addition to solid fruiting body culture and dynamic mycelium culture, Ganoderma lucidum culture also has a static mycelium culture method. The growth pattern and physiological products of static mycelium culture are between the fruiting body and the mycelium, so it also has some of the characteristics (effects) of the fruiting body. Therefore, in the prior art, in order to increase the yield of exosomes, the method adopted is to culture Ganoderma lucidum in the form of individual static mycelium culture in order to secrete exosomes with pharmacological properties. However, in fact, the method of simply using static mycelium culture alone cannot produce a sufficient yield of exosomes, and the particle size of the produced exosomes is very dispersed, and the greater the difference in the particle size of the exosomes, the more types of exosomes obtained and the more chaotic, which means that exosomes with uniform particle size with common pharmacological properties cannot be obtained (in theory, exosomes with consistent particle size belong to the same type of exosomes and will produce the same pharmacological effect). Therefore, how to increase the yield of exosomes with pharmacological development prospects during Ganoderma lucidum mycelium culture is the problem to be overcome by this application.

[0009] Therefore, it is necessary to propose solutions to the problems in the prior art. Technical issues

[0010] In conventional techniques, the yield of Ganoderma lucidum exosomes is low under single culture conditions, the culture process is difficult, and the particle size of the produced exosomes is dispersed, making it impossible to obtain exosomes of uniform particle size with common pharmacological properties. Technical Solutions

[0011] The present disclosure aims to provide a method for producing exosomes by co-culturing Ganoderma lucidum and algae of the genus Chlorella to effectively increase the yield and particle size uniformity of exosomes while overcoming the disadvantages of the cumbersome cultivation process.

[0012] To address the aforementioned issues, the present disclosure provides a method for producing exosomes, comprising the following steps: providing a Ganoderma lucidum culture solution containing mycelium of Ganoderma lucidum and providing an algae culture solution containing algae of the genus Chlorophytum; placing the algae culture solution and the Ganoderma lucidum culture solution in a container and co-culturing them to obtain a mixed culture solution; and extracting exosomes from the mixed culture solution.

[0013] In one embodiment, the method further comprises pre-culturing the Ganoderma lucidum, wherein the pre-culturing comprises the following steps:

[0014] Steps: obtaining the mycelium of Ganoderma lucidum, and planting the mycelium in a solid culture medium dish under a sterile environment;

[0015] The method comprises the steps of taking out a portion of mycelium, placing the portion in a liquid culture medium dish and culturing the portion until the mycelium forms the mycelium cake. When the area of ​​the mycelium cake formed by the mycelium reaches 70% to 90% of the liquid surface area of ​​the liquid culture medium dish, the Ganoderma lucidum culture solution containing the mycelium cake is obtained.

[0016] In one embodiment, the method further comprises the following steps:

[0017] Step: mixing the green algae culture solution and the ganoderma culture solution to obtain the mixed culture solution;

[0018] Step: shaking the mixed culture solution, wherein the shaking prevents the algae from settling in the mixed culture solution or remaining still at the bottom, and maintains the mycelium cake suspended on the surface of the mixed culture solution.

[0019] In one embodiment, when a container of the mixed culture medium is placed flat, the frequency of the shaking is lower than 50 rpm.

[0020] In one embodiment, when a container containing the mixed culture medium is tilted, the frequency of the oscillation is lower than 100 rpm.

[0021] In one embodiment, the mixed culture solution is prevented from infiltrating the surface of the mycelium cake during the shaking.

[0022] In one embodiment, the method further comprises centrifuging the mixed culture solution.

[0023] In one embodiment, the algae is Chlorella vulgaris.

[0024] In one embodiment, the culture medium of the Ganoderma lucidum is pre-cultured first, and then the culture medium of the algae is pre-cultured. Beneficial effects

[0025] Based on the above, the present disclosure aims to provide a method for producing exosomes. After the mycelia of Ganoderma lucidum aggregate on the surface of a mixed culture solution to form a mycelial cake, Ganoderma lucidum is co-cultured with algae of the genus Chlorophyta. The culture conditions are controlled so that the algae do not settle in the mixed culture solution or remain stationary at the bottom of the container, and the mycelial cake is maintained suspended on the surface of the mixed culture solution. This method can effectively increase the yield of exosomes from the Ganoderma lucidum mycelium and make the exosomes more uniform in size. Furthermore, a high yield of exosomes with pharmacological efficacy can be obtained using a simple culture method. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG1 is a schematic diagram of the process for producing exosomes disclosed herein.

[0027] FIG2 is a schematic diagram of the exosome concentration and particle size distribution when Ganoderma lucidum mycelium is statically cultured alone in the prior art.

[0028] FIG3 is a schematic diagram of the concentration and particle size distribution of exosomes when Ganoderma lucidum and Chlorella vulgaris are co-cultured in one embodiment of the present disclosure.

[0029] FIG4 is a schematic diagram of the exosome concentration and particle size distribution when Ganoderma lucidum and Chlorella pyrenoidosa are co-cultured in another embodiment of the present disclosure.

[0030] FIG5 shows schematic diagrams of exosome concentrations when green algae is cultured alone, when Ganoderma lucidum is cultured alone statically, and when Ganoderma lucidum is co-cultured with Chlorella vulgaris and Chlorella pyrenoidosa, respectively. Modes for Carrying Out the Invention

[0031] The following descriptions of the embodiments, with reference to the accompanying drawings, illustrate specific embodiments in which the present disclosure may be implemented. The present disclosure may be embodied in a variety of different forms and should not be construed as limited to the embodiments described herein. The embodiments described do not limit the scope of the invention as defined by the claims.

[0032] The term "exosomes" as used herein refers to cell-derived double-membrane vesicles produced by cells through exocytosis and released into various body fluids, interstitial fluids, or culture medium. Within their vesicles are substances such as messenger RNA (mRNA), microRNA (miRNA), and / or proteins. The detailed composition of these substances varies depending on the source cell and the cell's state at the time of secretion. As mentioned above, exosomes are currently hypothesized to play an important role in intercellular signaling pathways.

[0033] The term "Ganoderma" used herein is also known as Lingzhi grass, Wannianrong, Fucao, Mu Lingzhi, Shenzhi, Zhicao, Xiancao, Ruicao, etc. In a broad sense, Lingzhi includes species of the Ganodermaceae family and its closely related families and genera, while in a narrow sense, it refers to a specific species that is widely cultivated. It belongs to the Basidiomycota, Hymenomycetes, Aphyllaes, Ganodermataceae, and Ganoderma genus. Lingzhi is a white rot fungus that mostly grows on broad-leaved trees and grows well in a hot and humid environment. The genus Ganoderma includes many species. In the embodiments of the present disclosure, Ganoderma lucidium is used as a representative to conduct the following experiments, but the present disclosure should not be limited to this species.

[0034] The term "green algae" as used herein refers to the phylum Chlorophyta, which includes many species.

[0035] The term "Chlorella" as used herein refers to the genus Chlorella, a common single-celled algae belonging to the family Chlorophyceae of the class Chlorophyceae. It is a genus of single-celled green algae that lives on the surface of water. The genus Chlorella includes many species. In the examples of this disclosure, Chlorella vulgaris and Chlorella pyrenoidosa were used as representatives for the following experiments. However, this disclosure should not be limited to these species.

[0036] It is currently generally believed that the full efficacy of Ganoderma lucidum comes from the fruiting bodies that develop from the mycelium. Commercial use involves culturing Ganoderma lucidum spores alone to the fruiting body stage to obtain its medicinal components. However, Ganoderma lucidum exosomes also possess medicinal properties. To obtain Ganoderma lucidum exosomes, the process must be performed at the mycelial stage, a complex process that results in a low yield. In the prior art, to increase exosome production, Ganoderma lucidum is cultured in a static mycelial culture system in the hope of secreting exosomes with medicinal properties. However, static mycelial culture alone cannot produce sufficient exosomes. Therefore, in the present disclosure, a co-culture stimulant (e.g., algae) that can stimulate Ganoderma lucidum to secrete exosomes is added to the Ganoderma lucidum mycelium during the culture process. This co-culture stimulant encourages the Ganoderma lucidum mycelium to produce more exosomes during the static mycelial culture, resulting in exosomes of uniform particle size with common medicinal properties.

[0037] The present disclosure provides a method for producing exosomes, which is primarily used to increase the yield and size of exosomes produced by Ganoderma lucidum mycelium. The exosome production method according to one embodiment of the present disclosure will be described in detail below with reference to FIG1 . Please refer to FIG1 , which is a schematic flow diagram of the exosome production method according to the present disclosure. The exosome production method disclosed herein comprises the following steps:

[0038] Step S01: providing a Ganoderma lucidum culture solution containing a mycelium cake of Ganoderma lucidum and providing an algae culture solution containing an algae of the genus Chlorophytum;

[0039] Step S02: placing the algae culture solution and the Ganoderma lucidum culture solution in a container for co-culturing to obtain a mixed culture solution; and

[0040] Step S03: extracting exosomes from the mixed culture medium.

[0041] Step S01 further includes pre-culturing the algae, and the pre-culturing includes the following steps:

[0042] Step S011A: obtaining the algae, and culturing the algae in a first culture medium;

[0043] Step S012A: shaking the culture solution at a frequency between 150 rpm and 250 rpm;

[0044] Step S013A: taking the first culture solution and coating a culture medium;

[0045] Step S014A: culturing under the culture conditions of step S012A until a single bacterial colony appears in the culture medium; and

[0046] Step S015A: Take a single colony and culture it in a second culture medium until the density of the green algae reaches 6*10 6 -1*10 7 cells / ml to obtain the algae culture solution.

[0047] In addition, step S01 further includes pre-culturing the Ganoderma lucidum, and the pre-culturing includes the following steps:

[0048] Step S011B: obtaining the mycelium of the Ganoderma lucidum, and planting the mycelium in a solid culture medium dish under a sterile environment; and

[0049] Step S012B: Take out a portion of the mycelium, place it in a liquid culture medium culture dish and culture it statically until the mycelium forms the mycelium cake. When the area of ​​the mycelium cake formed by the mycelium reaches 70-90% of the liquid surface area of ​​the liquid culture medium culture dish, the mycelium cake and culture liquid of the Ganoderma lucidum are obtained.

[0050] In the present disclosure, the algae is Chlorella vulgaris, but is not limited thereto.

[0051] It should be noted that while it takes 15 to 21 days for Ganoderma lucidum to grow from spores to mycelium cakes, algae only takes 7 to 14 days to grow from spawn to algae. Because Ganoderma lucidum grows more slowly, pre-culturing Ganoderma lucidum can be performed before pre-culturing algae. Therefore, in step S01, the Ganoderma lucidum culture medium is pre-cultured until mycelium cakes are formed, followed by pre-culturing the algae culture medium.

[0052] Step S02 further includes the following steps:

[0053] S021: mixing the green algae culture solution and the ganoderma lucidum culture solution to obtain the mixed culture solution;

[0054] S022: shaking the mixed culture solution, so that the algae do not settle in the mixed culture solution or remain still at the bottom, but maintain the mycelium of the Ganoderma lucidum aggregated on the surface of the mixed culture solution to form the mycelium cake.

[0055] In one embodiment, if the culture medium is not shaken during cultivation, the algae will settle to the bottom of the container and cease growth. However, if the frequency of the shake is too high, the growth of the Ganoderma mycelium cake will be affected, preventing the formation of a mycelium cake. Therefore, controlling the shake frequency is crucial. An appropriate shake frequency can prevent the algae from settling in the mixed culture solution or becoming stationary at the bottom of the container and cessation of growth, while also maintaining the Ganoderma mycelium in a state of forming a mycelium cake on the surface of the mixed culture solution.

[0056] In one embodiment, when the container of the mixed culture medium is placed horizontally, the frequency of the shaking is lower than 50 rpm.

[0057] In another embodiment, when a container of the mixed culture medium is tilted, the frequency of the shaking is lower than 100 rpm.

[0058] It should be noted that when the container is tilted, that is, when it is simultaneously tilted and shaken, the mycelium cake formed on the surface of the mixed culture solution is less susceptible to damage from the shaking during the shaking process. In other words, the Ganoderma mycelium stored in a tilted container has a higher tolerance to shaking or vibration. Therefore, the oscillation frequency of the tilted container is higher than when the container is horizontal. In one embodiment, during the shaking process, the mixed culture solution should be prevented from infiltrating the surface of the mycelium cake. In other words, the mixed culture solution should not contact the surface of the mycelium cake. More specifically, during the cultivation process, the algae are suspended in the mixed culture solution, while the Ganoderma mycelium is suspended above the surface of the mixed culture solution, forming a ring-shaped mycelium cake. The roots of the mycelium absorb the components of the culture solution. Therefore, only the roots of the mycelium need to contact the mixed culture solution; the rest of the mycelium does not need to contact the mixed culture solution, preventing the mixed culture solution from infiltrating the surface of the mycelium cake and damaging it.

[0059] In one embodiment, the Ganoderma lucidum and algae are co-cultured for 14 to 21 days, preferably 14 days, but not limited thereto. The mixed culture medium is then removed and centrifuged to remove impurities such as cell debris and large vesicles, thereby extracting the exosomes.

[0060] In one embodiment, exosomes are extracted from the mixed culture medium by column extraction, ultracentrifugation, PEG precipitation, affinity magnetic beads, and antibody separation.

[0061] Hereinafter, the present disclosure will be described in detail through examples. However, the following examples are for illustrative purposes only, and the scope of the present disclosure is not limited by the following examples.

[0062] Example

[0063] Green algae (Chlorella) pre-cultivation, comprising the following steps:

[0064] 1.1) Obtain a strain of green algae and culture it in a first culture medium. The first culture medium is Walne medium, whose composition per liter includes 1.3 grams of FeCl3·6H2O, 0.36 grams of MnCl2·4H2O, 33.6 grams of H3BO3, 45 grams of Na2EDTA, 20 grams of NaH2PO4·2H2O, 100 grams of NaNO3, and 1 ml of a trace metal solution. The trace metal solution has a formula of 2.1 grams of ZnCl2, 2.0 grams of CoCl2·6H2O, and 0.9 grams of (NH4)6Mo7O per liter. 24 ·4H2O and 2.0 g of CuSO4·5H2O.

[0065] 1.2) The culture conditions are set as room temperature, 8-12 hours of illumination, and 2000-4000 lux, preferably a mixture of red and blue light, but not limited thereto. The first culture solution is shaken at a frequency of 150 rpm to 250 rpm.

[0066] 1.3) After culturing for 8-12 days, the first culture solution is plated on a culture medium containing 1.5% agar powder.

[0067] 1.4) Cultivate under the culture conditions of step 1.2) until a single colony appears in the culture medium.

[0068] 1.5) Take a single colony and culture it in a second culture medium for 7 to 14 days until the density of the green algae reaches 6*10 6 -1*10 7 Cell number / ml, and obtain green algae culture solution.

[0069] Ganoderma lucidum pre-cultivation, including the following steps:

[0070] 1.1) Obtain the mycelia of Ganoderma lucidum and, under a sterile environment, inoculate the mycelia onto solid culture medium (PDA) culture dishes. The solid culture medium comprises 200 g potato extract, 20 g glucose extract, and 15 g agar powder per liter.

[0071] 1.2) Set the culture conditions as a culture temperature of 26-30°C and a culture time of 15-21 days.

[0072] 1.3) Remove a portion of mycelium and place it in a liquid culture medium dish for static culture. Set the light source to sunlight and the illumination time to the daylight time.

[0073] 1.4) Cultivating until the mycelia form the mycelial cake, and when an area of ​​the mycelial cake formed by the mycelia reaches 70%-90% of the liquid surface area of ​​the liquid culture medium culture dish, obtaining the mycelial cake and culture solution of Ganoderma lucidum.

[0074] Green algae and Ganoderma lucidum co-cultivation

[0075] After obtaining the green algae culture solution and the ganoderma lucidum culture solution respectively, the green algae culture solution and the ganoderma lucidum culture solution are mixed in a container to obtain the mixed culture solution. In the present embodiment, the mixing is an equal volume mixing, but it is not limited thereto. The mixed culture solution is then shaken. It should be noted that since algae need to be shaken during the growth process to avoid algae settling at the bottom of the container and stopping growth, and ganoderma lucidum cannot be shaken excessively during the cultivation process to avoid damaging the mycelium cake, it is necessary to control the frequency of the shaking within a certain range. Such shaking will not cause the algae to settle in the mixed culture solution or stop growing by being stationary at the bottom of the container, and will not damage the mycelium cake on the liquid surface of the mixed culture solution. In short, during the co-cultivation process, it is necessary to avoid the green algae being in a static state, so the green algae activity will be maintained by shaking or vibrating the container. Preferably, the frequency of the shaking or vibration is in the range of 20rpm to 100rpm, and more preferably, the frequency of the shaking or vibration is 40rpm. In various embodiments, when the container is shaken or oscillated in a flat position, the frequency of the oscillation is less than 50 rpm; and when the container is shaken or oscillated in a tilted position, the frequency of the oscillation is less than 100 rpm. In other words, compared to Ganoderma mycelium stored in a flat container, Ganoderma mycelium stored in a tilted container has a higher tolerance to shaking or oscillation because, when the container is tilted and oscillated, the mycelium cake in the container is less likely to be damaged by the shaking process. It should also be noted that during the oscillation process, the mixed culture solution must not infiltrate the surface of the Ganoderma mycelium cake to prevent the infiltration of the mixed culture solution and damage to the mycelium cake.

[0076] In this example, the culture conditions were set to 8-12 hours of daily illumination, preferably 10 hours per day. The light intensity was set to 1000 to 2000 lux, preferably 1500 lux, and a mixed red and blue light source, but not limited thereto. The culture was continued for 14 days to obtain a mixed culture solution containing exosomes.

[0077] Mixed culture fluid sample processing

[0078] After the co-cultivation process is complete, the mixed culture medium is removed. In this embodiment, the mixed culture medium has a volume of 5 ml and is stored at a temperature between 4°C and -80°C. Processing of the mixed culture medium includes the following steps: Step 1.1) Centrifugation at 300g for 10 minutes at 4°C to remove cells. Step 1.2) Centrifugation at 2000g for 20 minutes at 4°C to remove cell debris and impurities. Step 1.3) Finally, centrifugation at 15,000g for 30 minutes at 4°C to remove large cellular vesicles, and exosomes can be extracted. For example, exosomes can be extracted from the mixed culture medium using column extraction, ultracentrifugation, PEG precipitation, affinity magnetic beads, and antibody separation. More specifically, the process includes step 1.4) adding the sample to the top of the column, mixing, fractionating, and storing at 4°C. Preferably, the microcolumn extraction method is used in this embodiment. Depending on the size of the column, exosomes ranging from 35 nm to 1000 nm can be obtained. Finally, the obtained exosomes can be analyzed for NTA particle size concentration.

[0079] Particle size and concentration analysis results: In this embodiment, the particle size and concentration of the obtained exosomes were analyzed using the following instruments and conditions, and the results are shown in Table 1 below.

[0080] Instrument: Nanoparticle Tracking Analyzer (NTA)

[0081] Malvern Panalytical

[0082] Model: NanoSight NS300

[0083] Lens type: sCMOS

[0084] Light source type: blue light 488

[0085] Temperature: 23.7℃-24.9℃

[0086] Mixed culture medium viscosity: 0.9 cP

[0087] Table 1

[0088] Please refer to Figure 2, which is a schematic diagram of the exosome concentration and particle size distribution when Ganoderma mycelium is statically cultured alone, according to the prior art, and the particle size / concentration analysis data shown in Table 1 above. Figure 2 is a comparative example corresponding to sample number FDS-05 in Table 1 above. Ganoderma mycelium was statically cultured alone using the prior art as the comparative example (FDS-05), and the exosome size distribution was observed. The culture results show that the exosome particles secreted by Ganoderma mycelium statically cultured alone are quite diverse in size, with the maximum particle size significantly different from the average particle size, indicating a high degree of particle size dispersion. A higher degree of particle size dispersion indicates that too many different types of exosomes are present, preventing the production of exosomes with uniform particle sizes (theoretically, exosomes with uniform particle sizes belong to the same type and produce the same pharmacological effects). Therefore, the above culture results show that under the conditions of Ganoderma lucidum culture alone, the points indicating the distribution intensity of exosomes fall in a relatively scattered area, indicating that the particle size of the produced exosomes varies greatly. In other words, the exosome particle size is not uniform, which may lead to the instability of the pharmacological efficacy of the obtained exosomes. It can also be seen that the points indicating the distribution intensity of exosomes are relatively loose, indicating a low concentration of the produced exosomes.

[0089] Please refer to Figure 3, which is a schematic diagram of the exosome concentration and particle size distribution when Ganoderma lucidum and Chlorella vulgaris are co-cultured in one embodiment of the present disclosure. Figure 3 is Example 1 corresponding to sample number FDS-03 in Table 1 above. In this example, Ganoderma lucidum mycelium cakes were co-cultured with Chlorella vulgaris using the above-described culture method, and the exosome size distribution was observed. It can be seen that under the co-culture conditions, the exosome size distribution differs from the size distribution of exosomes obtained from Ganoderma lucidum cultured alone in the prior art. More specifically, compared to the comparative example, the difference between the maximum particle size and the average particle size is lower, indicating that the exosome particle size dispersion is lower and the exosome particle size is similar, indicating that the exosomes obtained belong to the same type of exosomes and will produce the same pharmacological effects. Conversely, as shown in the Comparative Example, the difference between the maximum particle size and the average particle size is large, indicating a greater degree of size dispersion among the exosome particles, indicating that the sample contains a greater number of different exosome types. The experimental results above demonstrate that when Ganoderma lucidum mycelium is co-cultured with Chlorella, the size of the secreted exosomes is relatively concentrated within a certain range, as evidenced by the relatively concentrated distribution of points. Furthermore, the exosome concentration obtained under the co-culture conditions is significantly higher than that in the Comparative Example, as evidenced by the denser distribution of points. Co-culturing Ganoderma lucidum with Chlorella results in more consistent exosome size, further contributing to the stability of the exosomes' pharmacological effects.

[0090] Please refer to Figure 4, which is a schematic diagram of the exosome concentration and particle size distribution when Ganoderma lucidum is co-cultured with Chlorella pyrenoidosa in another embodiment of the present disclosure. Figure 4 is Example 2 corresponding to sample number FDS-04 in Table 1 above. In this example, Ganoderma lucidum mycelial cake was co-cultured with Chlorella pyrenoidosa using the above-described culture method, and the exosome size distribution was observed. It can be seen that under the co-culture conditions, the exosome size distribution differs from the size distribution of exosomes obtained from Ganoderma lucidum cultured alone in the prior art. More specifically, compared to the comparative example, the difference between the maximum particle size and the average particle size is lower, indicating that the exosome particle size dispersion is lower and the exosome particle size is similar, indicating that the exosomes obtained belong to the same type of exosomes and will produce the same pharmacological effect. Conversely, as shown in the Comparative Example, the difference between the maximum particle size and the average particle size is large, indicating a greater degree of size dispersion among the exosome particles, indicating that the sample contains a greater number of different exosome types. The experimental results above demonstrate that when Ganoderma lucidum mycelium is co-cultured with Chlorella pyrenoidosa, the size of the secreted exosomes is relatively concentrated within a certain range, as evidenced by the relatively concentrated distribution of points. Furthermore, the exosome concentration obtained under the co-culture conditions is significantly higher than that in the Comparative Example, as evidenced by the denser distribution of points. Co-culturing Ganoderma lucidum with Chlorella pyrenoidosa results in more consistent exosome size, further contributing to the stability of the exosomes' pharmacological effects.

[0091] Please refer to Figure 5, which shows the exosome concentrations when green algae is cultured alone, when Ganoderma lucidum is cultured statically alone, and when Ganoderma lucidum is co-cultured with Chlorella vulgaris and Chlorella pyrenoidosa, respectively. As can be seen from the figure, when Chlorella is cultured alone, if the unit concentration of Chlorella exosomes is set at 10 particles / ml, then when Ganoderma lucidum is cultured statically alone, the unit concentration of Ganoderma lucidum exosomes is 17 particles / ml. However, when Ganoderma lucidum is co-cultured with Chlorella vulgaris, the unit concentration of Ganoderma lucidum exosomes is approximately 94 particles / ml, and when Ganoderma lucidum is co-cultured with Chlorella pyrenoidosa, the unit concentration of Ganoderma lucidum exosomes exceeds 600 particles / ml. This shows that co-culturing Ganoderma lucidum with green algae does significantly increase exosome production.

[0092] The present disclosure provides a method for producing exosomes, wherein a co-culture medium (i.e., algae) that can stimulate Ganoderma lucidum to secrete exosomes is added to the Ganoderma lucidum mycelium during the culturing process. The co-culture medium allows the Ganoderma lucidum mycelium to produce more exosomes during static mycelial culture and improves the uniformity of the exosome particle size. More specifically, after the Ganoderma lucidum mycelium aggregates on the surface of a mixed culture solution to form a mycelial cake, the Ganoderma lucidum is co-cultured with algae from the genus Chlorella. The culture conditions are controlled to prevent the algae from settling in the mixed culture solution or remaining stationary at the bottom of the container, while maintaining the mycelial cake suspended on the surface of the mixed culture solution. This effectively increases the exosome yield of the Ganoderma lucidum mycelium and makes the size of the exosomes secreted by the Ganoderma lucidum mycelium more uniform, producing exosomes with pharmacological stability and achieving a high yield of pharmacologically effective exosomes using a relatively simple culture method.

[0093] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this disclosure belongs. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used to practice this disclosure. Of course, this disclosure is in no way limited to the methods and materials described.

[0094] The above embodiments are merely illustrative of the principles and effects of this disclosure. Their purpose is to enable those skilled in the art to understand and implement the present disclosure. They are not intended to limit this disclosure. Therefore, those skilled in the art may make equivalent modifications, alterations, and variations to the above embodiments without departing from the spirit of this disclosure. The scope of rights of this disclosure shall be as set forth in the patent claims described below.

Claims

1. A method for producing exosomes, comprising the following steps: Step S01: Provide a Ganoderma lucidum culture solution containing a mycelial cake of Ganoderma lucidum and provide an algal culture solution containing algae of the genus Chlorella; Step S02: Co-culture the algal culture solution and the Ganoderma lucidum culture solution in a container to obtain a mixed culture solution; and Step S03: Extract exosomes from the mixed culture solution.

2. The production method according to claim 1, wherein Step S01 further comprises pre-culturing the algae, and the pre-culture comprises the following steps: Step S011A: Obtain the algae and culture the algae in a first culture solution; Step S012A: Shake the culture solution, and the shaking frequency is between 150 rpm and 250 rpm; Step S013A: Take the first culture solution and plate it on a culture medium; Step S014A: Culture under the culture conditions of Step S012A until a single colony appears in the culture medium; Step S015A: Pick a single colony and culture it using a second culture medium until a density of the green algae is 6*10 6 -1*10 7 cells / mL to obtain the algal culture solution.

3. The production method according to claim 1, wherein Step S01 further comprises pre-culturing the Ganoderma lucidum, and the pre-culture comprises the following steps: Step S011B: Obtain the mycelium of Ganoderma lucidum and seed the mycelium in a solid culture medium petri dish in a sterile environment; Step S012B: Take out a part of the mycelium and place it in a liquid culture medium petri dish for static culture until the mycelium forms the state of the mycelial cake. When the area of the mycelial cake formed by the mycelium reaches 70%-90% of the liquid surface area of the liquid culture medium petri dish, the Ganoderma lucidum culture solution with the mycelial cake is obtained.

4. The production method according to claim 1, wherein Step S02 further comprises the following steps: Step S021: Mix the Chlorella culture solution and the Ganoderma lucidum culture solution to obtain the mixed culture solution; Step S022: Shake the mixed culture solution, and the shaking prevents the algae from precipitating in the mixed culture solution or staying still at the bottom, and maintains the state of the mycelial cake suspended on the liquid surface of the mixed culture solution.

5. The production method according to claim 4, wherein, In Step S022, when the container of the mixed culture solution is placed flat, the shaking frequency is lower than 50 rpm.

6. The production method according to claim 1, wherein, In Step S022, when the container of the mixed culture solution is placed obliquely, the shaking frequency is lower than 100 rpm.

7. The production method according to claim 4, wherein, In Step S022, prevent the mixed culture solution from wetting the surface of the mycelial cake during the shaking.

8. The production method according to claim 1, wherein Step S03 further comprises performing a centrifugation procedure on the mixed culture solution.

9. The production method according to claim 1, wherein the algae is Chlorella vulgaris.

10. In the production method according to claim 1, in Step S01, first pre-culture the culture solution of Ganoderma lucidum, and then pre-culture the culture solution of the algae.

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