A method for preparing ganoderma lucidum exosomes
By using white LED irradiation during Ganoderma lucidum fermentation, mycelium biomass was increased, and the mycelium biomass was separated and prepared in combination with centrifugation, filtration and tangential flow techniques, the problems of low extraction efficiency and insufficient purity of Ganoderma lucidum exosomes were solved, and efficient and economical exosome preparation was achieved.
Patent Information
- Application Number
- CN202411973307.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-30
- Publication Date
- 2025-04-04
AI Technical Summary
In the prior art, the separation and extraction efficiency of Ganoderma lucidum exosomes is low, resulting in low recovery rate and insufficient purity, making it difficult to meet industrial needs.
By irradiating white LED during Ganoderma lucidum fermentation, the conditions are optimized to increase the mycelium biomass of Ganoderma lucidum, thereby increasing the secretion of exosomes, and is isolated and prepared in combination with centrifugation, filtration and tangential flow (TFF) technology to improve the extraction efficiency and purity of exosomes.
It realizes efficient separation and extraction of Ganoderma lucidum exosomes, improves recovery rate and purity, reduces production costs, and has good application prospects and research value.
Smart Images
Figure CN119752646A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology and specifically provides a method for preparing ganoderma lucidum exosomes. Background Art
[0002] Exosomes are small vesicles that are actively secreted by cells outside the cell, and their diameter is usually between 30-150nm. These vesicles contain a variety of molecules, including proteins, RNA and DNA, which can transmit information between cells, thereby affecting the function of receptor cells. Exosomes from different sources have different biological functions, including cell-to-cell communication, immune regulation, antigen presentation, and the occurrence and development of diseases.
[0003] Ganoderma lucidum is a medicinal fungus that contains a variety of natural chemical components, among which the active ingredients are mainly polysaccharides, triterpenes, nucleosides, and sterols. Ganoderma lucidum has a long history of medicinal use, and the research on its active ingredients and pharmacological effects has been extensive and in-depth. Studies have shown that Ganoderma lucidum has certain anti-tumor, anti-inflammatory, liver protection, hypoglycemic, and anti-aging effects. Ganoderma lucidum is most commonly used in traditional medicine or alternative medicine for treatment in powdered or extract form. Combined with the medicinal and edible value of Ganoderma lucidum itself, it is very meaningful to explore new forms of Ganoderma lucidum application. At present, the research, development and application of Ganoderma lucidum exosomes are relatively small.
[0004] Ganoderma lucidum belongs to aerobic fungi. During liquid fermentation, light has an important influence on the growth of Ganoderma lucidum and the biosynthesis of metabolites. Studies have shown that white light LED is most conducive to cell growth, and blue light LED is most conducive to the synthesis of extracellular polysaccharides. At present, the effects of these factors on the secretion of Ganoderma lucidum exosomes have not been reported. Based on these basic studies, a scheme is proposed to increase the secretion of Ganoderma lucidum exosomes by using light.
[0005] At present, most laboratories use ultracentrifugation to separate Ganoderma exosomes, but the recovery rate is low and the time is long, which is difficult to meet the needs of industrialization. Therefore, it is urgent to develop a method for preparing Ganoderma exosomes to reduce costs and obtain Ganoderma exosomes with higher purity and higher yield. Compared with centrifugal concentration devices or ultrafiltration devices, tangential flow (TFF) can obtain higher concentrations in a shorter time. Ganoderma exosomes are separated and prepared by combining centrifugation and TFF to improve the extraction efficiency and purity of Ganoderma exosomes. Further explore new application directions of Ganoderma exosomes and create the feasibility of industrialization and commercialization of Ganoderma exosomes. Summary of the invention
[0006] The purpose of the present invention is to provide a method for preparing exosomes of Ganoderma lucidum. During the fermentation process of Ganoderma lucidum, white light LED irradiation is performed to increase the biomass of Ganoderma lucidum mycelium and the secretion of Ganoderma lucidum exosomes; and high-purity exosomes are separated and prepared by centrifugation, filtration, tangential flow and the like.
[0007] After a lot of experiments, the inventors obtained the following technical solution: a method for preparing Ganoderma lucidum exosomes, the method comprising the following steps:
[0008] 1. Take out the cryopreserved tube containing the Ganoderma lucidum block from the ultra-low temperature refrigerator, thaw it quickly, take out the block with sterile tweezers and put it into a culture dish containing potato dextrose agar (PDA) medium, seal the plate and culture it at 30℃ for 5 days;
[0009] 2. Use a scalpel to cut the outermost layer of the Ganoderma lucidum block and inoculate it on the slope of the test tube containing PDA, and place it in a 30℃ incubator until the Ganoderma lucidum hyphae cover the slope for later use;
[0010] 3. Add an appropriate amount of seed culture medium to the activated slope, gently scrape the mycelium on the slope with a spoon, pour the culture medium back into the conical bottle, place it on a constant temperature shaker, and culture it at 30℃ for 5 days. Shake the culture bottle every day for primary seed culture;
[0011] 4. Transfer 5 mL of the first-level culture medium to a culture bottle containing 45 mL of culture medium, place it on a constant temperature shaker, and culture at 30°C for 2 days. Shake the culture bottle every day for secondary seed culture.
[0012] 5. Transfer 3 mL of the secondary seed culture medium to 47 mL of sterilized fermentation medium, and culture at 30°C and 120 r / min for 9 days with light treatment to obtain Ganoderma lucidum fermentation liquid.
[0013] Further, in step 5, 40-120 μE m -2 s -1 White LED illumination for 2 h.
[0014] 6. Take the Ganoderma lucidum fermentation liquid and place it in a crusher for 1 minute, then filter it through a 100-200 μm cell gauze to recover the Ganoderma lucidum fermentation mycelium;
[0015] 7. The filtrate was centrifuged at 200-400 g for 10 min at 4 °C.
[0016] 8. Remove the supernatant and centrifuge at 500-3500g for 30 min at 4°C.
[0017] 9. Take out the supernatant and filter through 0.45 μm to obtain the filtrate;
[0018] 10. Use TFF technology to concentrate the filtrate and replace the buffer.
[0019] Furthermore, the concentration and buffer exchange parameter ranges of step 10 are as follows: molecular weight cutoff is 500KD, TMP is 3-4psi, back pressure is 2-3psi, inlet flow rate is 80-120mL / min, concentration multiple is 10-20, diafiltration liquid exchange multiple is 6-10 times, and diafiltration buffer is PBS or saline.
[0020] Compared with the prior art, the advantages of the present invention are: 1. The present invention optimizes the conditions through white light LED irradiation, increases the biomass of Ganoderma lucidum mycelium, and effectively promotes the secretion of Ganoderma lucidum exosomes. 2. The method of the present invention avoids the limitations of low yield and high cost of separating Ganoderma lucidum exosomes in existing reports, and adopts a combination of centrifugation and tangential flow to separate and prepare exosomes from Ganoderma lucidum fermentation broth, thereby solving the problems of low recovery rate and insufficient purity in the preparation process of Ganoderma lucidum exosomes. 3. The Ganoderma lucidum exosomes prepared by the present invention showed no potential cytotoxicity in in vitro cell experiments and were able to activate anti-tumor immunity, thus having good application prospects and research value. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 Transmission electron microscopy images of Ganoderma lucidum exosomes, scale bar 100nm in Figure A, scale bar 20nm in Figure B;
[0022] Figure 2 This is the particle size distribution diagram of Ganoderma lucidum exosomes;
[0023] Figure 3 The results of cytotoxicity test of Ganoderma lucidum exosomes at different concentrations;
[0024] Figure 4 This is a comparison of the effects of different concentrations of Ganoderma lucidum exosomes on the maturation of mouse bone marrow-derived dendritic cells (BMDCs). The LPS group was used as a positive control. *p<0.05. DETAILED DESCRIPTION
[0025] The present invention can be more easily understood by the subsequent description of some embodiments of the present invention and the detailed contents of the examples included therein. Before further describing the present invention, it should be understood that the present invention is not limited to the specific embodiments described, because these embodiments are necessarily diverse. It should also be understood that the terms used in this specification are only for the purpose of illustrating specific embodiments, rather than as limitations, because the scope of the present invention will be limited only in the appended claims.
[0026] Example 1 Preparation of Ganoderma lucidum exosomes
[0027] Take out the cryotube containing the Ganoderma lucidum block from the ultra-low temperature freezer, thaw quickly, take out the block with sterile tweezers and put it on a culture dish containing potato dextrose agar (PDA) medium, seal the plate and culture it at 30℃ for 5 days;
[0028] Use a scalpel to cut the outermost layer of the Ganoderma lucidum block and inoculate it on the slope of the test tube containing PDA, and place it in a 30℃ incubator until the Ganoderma lucidum hyphae cover the slope for later use;
[0029] Add an appropriate amount of seed culture medium to the activated slant, gently scrape the mycelium on the slant with a spoon, pour the culture medium back into the conical flask, place it on a constant temperature shaker, and culture it at 30℃ for 5 days. Shake the culture flask every day for primary seed culture;
[0030] 5 mL of the culture medium of the primary culture was transferred to a culture bottle containing 45 mL of culture medium, and placed on a constant temperature shaker at 30°C for 2 days. The culture bottle was shaken every day for secondary seed culture.
[0031] 3 mL of the secondary seed culture was transferred to 47 mL of sterilized fermentation medium and cultured at 30°C and 120 r / min for 9 days;
[0032] 120 μE m per day -2 s -1 After irradiation with white LED light for 2 h, Ganoderma lucidum fermentation liquid was obtained.
[0033] The fermented Ganoderma lucidum liquid was crushed in a crusher for 1 min, and then filtered through a 100 μm cell mesh.
[0034] The filtrate was centrifuged at 400 g for 10 min at 4 °C.
[0035] The supernatant was collected and centrifuged at 3500 g for 30 min at 4 °C.
[0036] Take the supernatant and filter it through 0.45 μm to obtain the filtrate;
[0037] TFF concentration and replacement: TFF technology was used to concentrate the filtrate and replace the buffer to prepare a concentrated solution. The parameters were: molecular weight cutoff 500KD, TMP 4psi, back pressure 3psi, inlet flow rate 120mL / min, concentration multiple 15, diafiltration multiple 10 times, and diafiltration buffer PBS.
[0038] Example 2 Preparation of Ganoderma lucidum exosomes
[0039] Take out the cryotube containing the Ganoderma lucidum block from the ultra-low temperature freezer, thaw quickly, take out the block with sterile tweezers and put it on a culture dish containing potato dextrose agar (PDA) medium, seal the plate and culture it at 30℃ for 5 days;
[0040] Use a scalpel to cut the outermost layer of the Ganoderma lucidum block and inoculate it on the slope of the test tube containing PDA, and place it in a 30℃ incubator until the Ganoderma lucidum hyphae cover the slope for later use;
[0041] Add an appropriate amount of seed culture medium to the activated slant, gently scrape the mycelium on the slant with a spoon, pour the culture medium back into the conical flask, place it on a constant temperature shaker, and culture it at 30℃ for 5 days. Shake the culture flask every day for primary seed culture;
[0042] 5 mL of the culture medium of the primary culture was transferred to a culture bottle containing 45 mL of culture medium, and placed on a constant temperature shaker at 30°C for 2 days. The culture bottle was shaken every day for secondary seed culture.
[0043] 3 mL of the secondary seed culture was transferred to 47 mL of sterilized fermentation medium and cultured at 30°C and 120 r / min for 9 days;
[0044] 40 μE m per day -2 s -1 After irradiation with white LED light for 2 h, Ganoderma lucidum fermentation liquid was obtained.
[0045] The fermented Ganoderma lucidum liquid was crushed in a crusher for 1 min, and then filtered through a 100 μm cell mesh.
[0046] The filtrate was centrifuged at 400 g for 10 min at 4 °C.
[0047] The supernatant was collected and centrifuged at 3500 g for 30 min at 4 °C.
[0048] Take the supernatant and filter it through 0.45 μm to obtain the filtrate;
[0049] TFF concentration and replacement: TFF technology was used to concentrate the filtrate and replace the buffer to prepare a concentrated solution. The parameters were: molecular weight cutoff 500KD, TMP 4psi, back pressure 3psi, inlet flow rate 80mL / min, concentration multiple 15, diafiltration multiple 10 times, and diafiltration buffer PBS.
[0050] Comparative Example 1:
[0051] This comparative example provides a method for preparing Ganoderma lucidum exosomes, which is basically the same as Example 1, except that no illumination is performed during the Ganoderma lucidum shake flask culture process.
[0052] Comparative Example 2:
[0053] This comparative example provides a method for preparing Ganoderma lucidum exosomes, wherein the Ganoderma lucidum fermentation liquid is harvested as in Example 1, except that the exosomes are obtained by ultracentrifugation.
[0054] The fermented liquor of Ganoderma lucidum was crushed in a crusher for 1 min, and then filtered through a 100 μm cell mesh.
[0055] The filtrate was centrifuged at 400 g for 10 min at 4 °C.
[0056] The supernatant was collected and centrifuged at 3500 g for 30 min at 4 °C.
[0057] The supernatant was collected and centrifuged at 10000 g for 10 min at 4 °C.
[0058] Take the supernatant and filter it through 0.45 μm to obtain the filtrate;
[0059] The supernatant was collected and ultracentrifuged at 150,000 g for 2 h at 4 °C.
[0060] The mycelium of Ganoderma lucidum was recovered and dried and weighed, and the mycelium dry weight results are shown in Table 1;
[0061] Table 1 Sample name Example 1 Example 2 Comparative Example 1 Comparative Example 2 Ganoderma lucidum fermentation liquid (g / L) 8.9 8.3 6.3 8.9
[0062] By comparing the dry weight of Ganoderma lucidum mycelium, it was found that the illumination of Ganoderma lucidum fermentation liquid during the cultivation process increased the mycelium biomass, and most of the mycelium obtained were small and medium-sized balls.
[0063] The protein content was detected by BCA method, and the results are shown in Table 2;
[0064] Table 2 Sample name Example 1 Example 2 Comparative Example 1 Comparative Example 2 Ganoderma lucidum fermentation liquid (mg / mL) 3.46 3.24 2.56 3.46 Ganoderma lucidum exosomes (mg / mL) 0.162 0.158 0.123 0.102
[0065] Nanoparticle tracking analysis (NTA) was used to measure the concentration and size of exosome particles, and the results are shown in Table 3 ;
[0066] Table 3 Sample name Example 1 Example 2 Comparative Example 1 Comparative Example 2 Particle number concentration (particles / mL) <![CDATA[2.0X10 11 ]]> <![CDATA[1.9X10 11 ]]> <![CDATA[1.1X10 10 ]]> <![CDATA[4.6X10 9 ]]> Peak particle size (nm) 117.7 117.9 112.0 126.1 Average particle size (nm) 158.7 154.1 128.6 190.1
[0067] The morphology of Ganoderma lucidum exosomes was observed under transmission electron microscopy. Figure 1 As shown, Figure 1 B shows that Ganoderma lucidum exosomes present a typical vesicle structure and the membrane structure is intact.
[0068] Nanoparticle tracking analysis (NTA) was used to measure the concentration and size of Ganoderma lucidum exosome particles. Figure 2 The results show that the number concentration of exosome particles prepared in Example 1 is 2.0X 10 11 (particles / mL), which is significantly better than the samples in the control group.
[0069] According to the results of the Ganoderma lucidum exosome particle concentration determination and protein analysis, the illumination of the Ganoderma lucidum fermentation liquid during the culture process increased the mycelium biomass, reduced the phenomenon of large-scale mycelium aggregation, and was also conducive to the secretion of Ganoderma lucidum exosomes. The recovery rate of Ganoderma lucidum exosomes was further improved by combining centrifugation, filtration and tangential flow.
[0070] Example 4 In vitro experiment of Ganoderma lucidum exosomes
[0071] In vitro cytotoxicity experiment of Ganoderma lucidum exosomes
[0072] Adjust the density of 3T3-L1 mouse embryonic fibroblasts to 1X 10 5 cells / mL, inoculated 100 μL of cell suspension into a 96-well plate, and pre-cultured the plate in an incubator for 24 h (at 37° C., 5% CO 2 ).
[0073] The exosomes of Ganoderma lucidum were diluted with culture medium to the concentrations of 1X 10 8 particles / mL、5X 10 8 particles / mL、1X 10 9 particles / mL were added to a 96-well plate, with 5 replicate wells in each group, and culture was continued for 48 h (at 37° C., 5% CO 2 ).
[0074] Before detection, the culture supernatant was removed, and the cells were washed twice with PBS. 100 μL of culture medium and CCK-8 mixture was added to each well at a ratio of 10:1.
[0075] Incubate the plate in the incubator for 1 hour.
[0076] The absorbance at 450 nm was measured using an enzyme reader.
[0077] The survival rate (%) of 3T3-L1 cells under different concentrations of Ganoderma lucidum exosomes was calculated according to the formula:
[0078]
[0079] A (exosomes): absorbance of wells with cells, CCK-8 solution, and exosome solution
[0080] A (blank): absorbance of wells with culture medium and CCK-8 solution but no cells
[0081] A (0 exosomes): absorbance of the well with cells, CCK-8 solution and no exosome solution
[0082] In vitro cytotoxicity test results Figure 3 As shown, Ganoderma lucidum exosomes at various concentrations had no potential cytotoxicity.
[0083] Experiment on Ganoderma lucidum exosomes promoting the maturation of mouse bone marrow-derived dendritic cells (BMDCs)
[0084] Ganoderma lucidum exosomes were prepared at concentrations of 1X 10 8 particles / mL、5X 10 8 particles / mL、1X10 9 particles / mL, and the exosomes of Ganoderma lucidum at each concentration were mixed with 5X 10 5 BMDCs were incubated for 24 hours, repeated 5 times, and BMDCs were collected. Dead / live staining was first performed, and the cells were blocked after washing 3 times with PBS. After blocking, they were rinsed with PBS twice, and 200μL of cell staining buffer was added, and then 1μL CD11c-FITC (ab95765), CD80-APC (ab25555), and CD86-PeCy7 (ab81995) were added. In addition, single staining tubes and blank cell tubes for the three dyes were set up. After the cells and dyes were mixed evenly, they were stained on ice in the dark for 30 minutes, centrifuged at 400g for 5 minutes to discard the dyes; 1mL of cell staining buffer was added to rinse 3 times, and finally the cells were resuspended with 500μL of cell staining buffer for flow cytometric analysis. At the same time, single staining tubes and blank cell tubes for the three dyes are required. PBS, exosomes, and lipopolysaccharide (LPS, 50ng / mL) promote the maturation of BMDCs. Figure 4 As shown in the results, the exosome group significantly improved the maturation-promoting effect on BMDCs, indicating that Ganoderma exosomes can promote the maturation of dendritic cells in vitro and have the potential to activate immunity.
[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some or all of the technical features therein may be replaced by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. A method for preparing Ganoderma lucidum exosomes, characterized in that: The ganoderma lucidum exosomes are made from ganoderma lucidum fermentation liquid.
2. The method for preparing Ganoderma lucidum exosomes according to claim 1, characterized in that: The ganoderma lucidum strain activation and shaking flask culture adopt potato dextrose agar (PDA) culture medium and culture at 30° C. to obtain ganoderma lucidum fermentation liquid.
3. The method for preparing Ganoderma lucidum exosomes according to claim 2, characterized in that: During the activation and shaking bottle culture of the ganoderma lucidum strains, white light LED irradiation is performed to obtain the ganoderma lucidum fermentation liquid.
4. The method for preparing Ganoderma lucidum exosomes according to claim 3, characterized in that: The light intensity is 40-120 μE m -2 s -1 White LED illumination for 2 h.
5. The method for preparing Ganoderma lucidum exosomes according to claim 5, characterized in that: The steps of separating and extracting Ganoderma lucidum exosomes are as follows: Step 1: taking the Ganoderma lucidum fermentation liquid, placing it in a crusher and crushing it for 1 minute, filtering it through a 100-200 μm cell gauze to recover the Ganoderma lucidum fermentation mycelium; Step 2: Take the filtrate and perform primary centrifugation at 200-400g, 10min, 4°C; Step 3: Remove the supernatant and perform secondary centrifugation at 500-3500g, 30min, 4°C; Step 4: Take out the supernatant and filter it through 0.45 μm to obtain the filtrate; Step 5: Concentrate and buffer exchange the filtrate using tangential flow technology.
6. The method for preparing Ganoderma lucidum exosomes according to claim 6, wherein the tangential flow technical parameter range in step 5 includes: The molecular weight cut-off is 500KD, the TMP is 3-4psi, the back pressure is 2-3psi, the inlet flow rate is 80-120mL / min, the concentration multiple is 10-20, and the filtration and liquid replacement multiple is 6-10 times.
7. The method for preparing Ganoderma lucidum exosomes according to claim 7, wherein the diafiltration buffer is PBS or physiological saline.