Preparation method of ganoderma lucidum exosome containing medicinal and edible active ingredients

Abstract

The invention relates to the technical field of biotechnology and food science, in particular to a preparation method of ganoderma lucidum exosomes containing medicinal and edible active ingredients, which comprises the following steps: culturing ganoderma lucidum mycelium fermentation liquor; adding the rhizoma polygonati solution into the ganoderma lucidum mycelium fermentation liquor, and carrying out biotransformation on a rhizoma polygonati powder substrate to obtain ganoderma lucidum exosome fermentation liquor; and separating, extracting and purifying the ganoderma lucidum exosome fermentation liquor to obtain the ganoderma lucidum exosome. The ganoderma lucidum exosome is extracted and purified, the medicine and food homology characteristic of the ganoderma lucidum exosome is fully utilized, and a new technical path is provided for development of functional food, health care products and medicines. According to the method, the corresponding exosome can be successfully extracted from the ganoderma lucidum culture solution, and the density of the exosome extracted after the polygonatum sibiricum is added is obviously higher than that of the exosome extracted without the polygonatum sibiricum, that is, after the polygonatum sibiricum is added, the active ingredients of the polygonatum sibiricum permeate into the original ganoderma lucidum exosome, and the ganoderma lucidum exosome has an obvious killing effect on cancer cells and shows a stronger killing effect.

Description

Technical Field

[0001] The present invention relates to the technical field of biotechnology and food science, and in particular to a method for preparing Ganoderma lucidum exosomes containing effective ingredients that are both medicinal and edible. Background Art

[0002] The concept of "medicine and food having the same origin" refers to the fact that certain natural substances can be consumed as food and also possess medicinal value. This concept has a long history in traditional Chinese medicine, embodying the health-preserving philosophy of "medicine and food having the same origin, and medicine and food having the same use." Ganoderma lucidum, as a typical edible and medicinal substance, holds a key position in both traditional medicine and the modern health industry due to its rich bioactive ingredients and wide range of pharmacological effects. Ganoderma lucidum contains a variety of bioactive ingredients, including polysaccharides, triterpenoids, proteins, peptides, and various trace elements. These ingredients endow Ganoderma lucidum with significant immune-modulating, anti-tumor, antioxidant, and metabolic-regulating functions.

[0003] Lingzhi, a traditional Chinese medicinal herb, has been used in my country for over 2,000 years. As early as the Shennong Bencao Jing (Shen Nong's Classic of Materia Medica), it was listed as a top-grade herb, boasting the properties of "lightness, immortality, and longevity." In 2000, the Pharmacopoeia of the People's Republic of China officially added Lingzhi to its list, specifically noting its benefits of "tonifying qi, calming the mind, and relieving cough and asthma." Modern pharmacological research has demonstrated a wide range of pharmacological effects, including anti-tumor, anti-radiation and anti-chemotherapy, anti-myocardial ischemia, anti-hypoxia, free radical scavenging, bidirectional immune regulation, lipid regulation, and anti-HIV-1 and HIV-1 protease activity. These studies not only validate the traditional medicinal value of Lingzhi but also provide scientific evidence for its application in modern medicine and functional foods. With the rapid development of the health industry, Lingzhi, due to its unique medicinal and edible properties, presents enormous market potential.

[0004] Ganoderma triterpenes are one of the main bioactive components of Ganoderma lucidum, exhibiting significant pharmacological activities, including anti-tumor, anti-inflammatory, antioxidant, and immunomodulatory effects. The biosynthesis pathway of Ganoderma triterpenes is complex, and their yield is influenced by multiple factors, including strain characteristics, culture conditions, and medium composition. Therefore, identifying Ganoderma lucidum strains that produce high triterpenes and optimizing culture conditions to maximize triterpenoid yields have been key research areas in the field of Ganoderma lucidum liquid fermentation. In industrial production, utilizing low-cost, widely available culture media to obtain high levels of Ganoderma triterpenes is crucial for achieving large-scale application. For example, utilizing agricultural waste (such as corn stalks and sugarcane bagasse) as culture media not only reduces production costs but also enables resource recycling. Furthermore, the identification and analysis of ganoderic acid components in Ganoderma triterpenes lays the foundation for the subsequent preparation and activity evaluation of high-purity triterpenoid compounds. These studies have not only promoted the development and application of Ganoderma triterpenes but also provided important technical references for the research of other active components of Ganoderma lucidum.

[0005] Exosomes are a type of extracellular vesicles with a diameter of approximately 30-150 nanometers, secreted by cells and widely present in body fluids. Exosomes contain a wealth of bioactive substances, including proteins, nucleic acids (such as mRNA, miRNA), and lipids, and play an important role in intercellular communication, immune regulation, disease diagnosis, and treatment. In recent years, the application of exosomes in the biomedical field has attracted much attention, especially in the development of drug delivery systems and functional foods. Ganoderma lucidum exosomes, as an important active ingredient of Ganoderma lucidum, have received widespread attention in recent years. Studies have shown that Ganoderma lucidum exosomes not only retain the bioactive components of their parent cells, but also have unique biocompatibility and low immunogenicity, making them suitable as carriers of functional ingredients. Ganoderma lucidum, a representative Ganoderma lucidum that can be used as both medicine and food, may have exosomes rich in active substances such as polysaccharides and triterpenes, with potential immune regulation, anti-inflammatory, and antioxidant functions. For example, polysaccharides in Ganoderma lucidum exosomes may exert immunomodulatory effects by activating macrophages and enhancing natural killer cell activity, while triterpenes may exert anti-inflammatory effects by inhibiting the release of inflammatory mediators. Polygonatum sibiricum, a plant with both medicinal and edible properties, contains multiple active ingredients, including polygonatum saponins and polyphenols, exhibiting antioxidant, anti-aging, and immunomodulatory properties. Co-culturing and fermenting Polygonatum sibiricum with Ganoderma lucidum cells not only fully utilizes the medicinal and edible properties of Polygonatum sibiricum but also potentially enhances the bioactivity of Ganoderma lucidum exosomes through synergistic effects. For example, polyphenols in Polygonatum sibiricum may enhance their antioxidant and anti-inflammatory properties by interacting with the polysaccharides and triterpenes in Ganoderma lucidum exosomes. This combination offers new insights into the development of functional foods and drugs, and opens up new research directions for the comprehensive utilization of medicinal and edible substances. Summary of the Invention

[0006] The object of the present invention is to provide a method for preparing Ganoderma lucidum exosomes containing effective ingredients that are both medicinal and edible.

[0007] The present invention achieves its purpose by adopting a method for preparing Ganoderma lucidum exosomes containing active ingredients of medicinal and edible properties, which is characterized by comprising the following steps:

[0008] Step S1: culturing Ganoderma lucidum mycelium fermentation liquid;

[0009] Step S2: adding the polygonatum solution to the Ganoderma lucidum mycelium fermentation liquid obtained in step S1 to biotransform the polygonatum powder substrate to obtain Ganoderma lucidum exosome fermentation liquid;

[0010] Step S3: separating, extracting and purifying the Ganoderma lucidum exosome fermentation broth to obtain Ganoderma lucidum exosomes.

[0011] Preferably, the specific method of step S1 includes:

[0012] Step S11: inoculating high-triterpene-yielding Ganoderma lucidum mycelia into PDA culture medium for slant culture until the Ganoderma lucidum mycelia fill the slant test tube;

[0013] Step S12: Pour sterile water into the inclined test tube covered with Ganoderma mycelium, scrape all the Ganoderma mycelium on the culture medium into the sterile water, and pour the sterile water containing Ganoderma mycelium into the seed culture medium and culture for several days to prepare the first-level seed solution;

[0014] Step S13: The cultured primary seed solution is vibrated to break up the mycelial balls, and the broken mycelia are aspirated and inoculated into a submerged liquid fermentation medium for cultivation for several days to obtain a secondary seed solution;

[0015] Step S14: inoculating the secondary seed liquid into a fermentation medium, fermenting and culturing for several days, and then expanding the culture to obtain a primary Ganoderma lucidum strain;

[0016] Step S15: inoculating the first-level Ganoderma lucidum strain into a fermentation medium for fermentation culture for several days, and then filtering to obtain a Ganoderma lucidum mycelium fermentation liquid.

[0017] Preferably, in step S11, the PDA culture medium comprises: 0.01 g / L-1.0 g / L vitamin B1, 1.0 g / L-5.0 g / L KH2PO4, 0.5 g / L-2.0 g / L MgSO4, 5.0 g / L-30 g / L glucose, 5.0 g / L-30 g / L agar powder, and 100 g / L-300 g / L potato extract;

[0018] In step S12, the seed culture medium includes: 0.01g / L-0.1g / L vitamin B1, 1.0g / L-5.0g / L KH2PO4, 0.1g / L-1g / L MgSO4, 20g / L-50g / L glucose, 1.0g / L-10g / L tryptone, and 1g / L-5g / L yeast extract;

[0019] In step S13, the submerged liquid fermentation medium includes: 0.01g / L-0.1g / L vitamin B1, 1.0g / L-5.0g / L KH2PO4, 0.5g / L-3.0g / L MgSO4, 20g / L-50g / L glucose, 1.0g / L-10g / L tryptone, and 1g / L-5g / L yeast extract.

[0020] Preferably, in step S14, the fermentation medium comprises: 0.01g / L-0.1g / L vitamin B1, 1.0g / L-5.0g / L KH2PO4, 0.5g / L-3.0g / L MgSO4, 20g / L-50g / L glucose, 1.0g / L-10g / L tryptone, 1g / L-5g / L yeast extract,

[0021] The fermentation culture parameters are as follows: culture at 25°C-30°C in a shaker at 110 rpm-130 rpm for 2-5 days, and inoculating the fermentation medium with the secondary seed solution and fermentation medium at a volume ratio of 1:10;

[0022] In step S15, the fermentation medium includes: 0.01g / L-0.1g / L vitamin B1, 1.0g / L-5.0g / L KH2PO4, 0.5g / L-3.0g / L MgSO4, 20g / L-50g / L glucose, 1.0g / L-10g / L tryptone, 1g / L-5g / L yeast extract,

[0023] The fermentation culture parameters are: 25°C-30°C, shaking at 110 rpm-130 rpm, and culture for 2-5 days.

[0024] Preferably, in step S1, the concentration of β-glucosidase in the Ganoderma lucidum mycelium fermentation broth is 10 U / mL-30 U / mL.

[0025] Preferably, the specific method of step S2 includes:

[0026] Step S21: crushing and sieving the naturally dried polygonatum to obtain polygonatum powder;

[0027] Step S22: Mixing polygonatum powder and pure water in a certain proportion and then sterilizing to obtain a sterile polygonatum solution;

[0028] Step S23: uniformly stirring the Ganoderma lucidum mycelium fermentation liquid and the sterile Polygonatum sibiricum solution in a certain ratio under appropriate conditions to perform a conversion reaction;

[0029] Step S24: After the reaction is completed, the Ganoderma lucidum exosome fermentation liquid is collected.

[0030] Preferably, in step S22, the weight-to-volume ratio of polygonatum powder to pure water is 1 g:4 mL;

[0031] In the step S23, the volume ratio of the Ganoderma lucidum mycelium fermentation liquid to the sterile Polygonatum sibiricum solution is 9:1, and the temperature of the bioconversion is 25-30°C.

[0032] Preferably, in step S23, the conversion rate of the macromolecular glycoside glycosides in the polygonatum to the small molecular aglycones needs to be continuously detected, and the reaction termination index is when the conversion rate reaches 75%.

[0033] The conversion rate is calculated as follows: Polygonatum sibiricum saponin aglycone conversion rate = Polygonatum sibiricum saponin aglycone in Example 1 / (Polygonatum sibiricum saponin aglycone in Comparative Example 1 + Polygonatum sibiricum saponin aglycone in Example 1).

[0034] Preferably, the specific method of step S3 includes:

[0035] Step S31: placing the Ganoderma lucidum exosome fermentation liquid into PBS buffer and homogenizing it;

[0036] Step S32: centrifuging the homogenized fermentation broth, gradually increasing the centrifugal speed and time, and finally ultracentrifuging at 4°C;

[0037] Step S33: filtering the Ganoderma lucidum exosome precipitate after centrifugation;

[0038] Step S34: Rinse the Ganoderma lucidum exosome precipitate with PBS buffer, suspend it in PBS buffer or culture medium, and filter it to obtain Ganoderma lucidum exosomes.

[0039] Preferably, in step S32, the specific process of centrifugation is: centrifuging the homogenized fermentation broth at 1000xg-1500xg for 10-20 minutes, 2000xg-3500xg for 20-30 minutes, 4000xg-5000xg for 30-35 minutes, and finally at 100000xg-120000xg at 4°C for 1.0-1.5 hours.

[0040] The present invention has the following advantages and beneficial effects:

[0041] The present invention extracts and purifies Ganoderma lucidum exosomes, fully utilizing their medicinal and edible properties, and provides a new technical path for the development of functional foods, health products and drugs.

[0042] The method of the present invention can successfully extract corresponding exosomes from Ganoderma lucidum culture medium, and the density of the exosomes extracted after adding Polygonatum sibiricum is significantly higher than that of the exosomes extracted without adding Polygonatum sibiricum, that is, after adding Polygonatum sibiricum, its active ingredients penetrate into the original Ganoderma lucidum exosomes.

[0043] The exosomes prepared by the method of the present invention have a significant killing effect on cancer cells, and the exosomes containing the active ingredients of Polygonatum sibiricum have a greater inhibitory effect on the proliferation of cancer cells and show a stronger killing effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a flow chart of a method for preparing Ganoderma lucidum exosomes containing active ingredients that are both medicinal and edible, provided in this application;

[0045] Figure 2 The β-glucosidase activity diagram provided in this application and the saponin content change trend in the Ganoderma lucidum culture medium in Example 1; wherein A is a trend diagram of the change of β-glucosidase over time during the Ganoderma lucidum culture process; B is a trend diagram of the change of polygonatum saponin in the Ganoderma lucidum culture medium over time after the addition of polygonatum;

[0046] Figure 3 The particle size and density diagrams of the exosomes from Ganoderma lucidum mycelium prepared in Comparative Example 1 and Example 1 provided in this application; wherein A is a particle size characterization diagram of the Ganoderma lucidum exosomes prepared in Comparative Example 1; B is a particle size characterization diagram of the Ganoderma lucidum exosomes prepared in Example 1;

[0047] Figure 4 The graphs showing the changes in the electron microscopic morphology of the exosomes from the mycelium of Ganoderma lucidum prepared in Comparative Example 1 and Example 1 provided in this application; wherein A is an electron microscopic characterization graph of the exosomes from Ganoderma lucidum prepared in Comparative Example 1; and B is an electron microscopic characterization graph of the exosomes from Ganoderma lucidum prepared in Example 1;

[0048] Figure 5 The toxicity trend graphs of the freeze-dried Ganoderma lucidum exosome powder prepared in Comparative Example 1 and Example 1 on Hep-G2 cells and CaCo-2 cells provided in this application are shown in Figure A, wherein Figure A is a trend graph showing the inhibition of the supernatant freeze-dried powder prepared in Comparative Example 1 and Example 1 on the growth of Hep-G2 cells; and Figure B is a trend graph showing the inhibition of the supernatant freeze-dried powder prepared in Comparative Example 1 and Example 1 on the growth of CaCo-2 cells.

[0049] Figure 6 This is a volcano plot of the differential expression of the main substances in the exosomes of Ganoderma lucidum mycelium prepared in Comparative Example 1 and Example 1 provided in this application;

[0050] Figure 7 This is a graph showing the changes in the content of the main substances in the exosomes of Ganoderma lucidum mycelium prepared in Comparative Example 1 and Example 1 provided in this application. DETAILED DESCRIPTION

[0051] 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 in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Therefore, it should be clear to those skilled in the art that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present application. In addition, for the sake of clarity and brevity, descriptions of known functions and structures are omitted in the embodiments.

[0052] In addition, the present application may repeat reference numerals and / or letters in different examples. This repetition is for the purpose of simplicity and clarity and does not in itself indicate the relationship between the various embodiments and / or settings discussed.

[0053] It should also be noted that, in this document, the terms "include", "comprises" or any other variations thereof are intended to cover a non-exclusive inclusion.

[0054] Example 1

[0055] Please refer to Figure 1 This embodiment mainly introduces a method for preparing Ganoderma lucidum exosomes containing effective ingredients of medicinal and edible properties, comprising the following steps:

[0056] Step S1, culturing Ganoderma lucidum mycelium fermentation liquid; the specific method includes:

[0057] Step S11: using an inoculating shovel to scoop out a small piece of high-triterpene-yielding Ganoderma lucidum mycelium, placing it in a slant test tube connected to a PDA culture medium, and culturing at 30° C. until the Ganoderma lucidum mycelium fills the test tube;

[0058] PDA medium: vitamin B1 (0.05 g / L), KH2PO4 (3.0 g / L), MgSO4 (1.5 g / L), glucose (20 g / L), agar powder (20 g / L), potato extract (200 g / L).

[0059] Step S12: Pour 10 mL of sterile water into the inclined test tube covered with Ganoderma mycelium, scrape all the Ganoderma mycelium on the PDA medium into the sterile water with an inoculating spatula, and pour the sterile water into a 250 mL conical flask containing 40 mL of seed culture medium; culture at 30° C. and 120 rpm for 5 days to obtain a first-level seed solution;

[0060] Seed culture medium: Seed culture medium: Vitamin B1 (0.05 g / L), KH2PO4 (1.0 g / L), MgSO4 (0.5 g / L), Glucose (35 g / L), Tryptone (5.0 g / L), Yeast extract (2.5 g / L).

[0061] Step S13: The cultured primary seed solution was placed in a 250 mL conical flask containing glass beads, and then the mycelial pellets were broken by shaking. 5 mL of the broken mycelium was aspirated and inoculated into a 250 mL conical flask containing 45 mL of submerged liquid fermentation medium; the culture was carried out at 30° C. and 120 rpm for 2 days to obtain the secondary seed solution;

[0062] Submerged liquid fermentation medium: Submerged liquid fermentation medium: Vitamin B1 (0.05 g / L), KH2PO4 (3.0 g / L), MgSO4 (1.5 g / L), Glucose (35 g / L), Tryptone (5.0 g / L), Yeast extract (2.5 g / L).

[0063] Step S14, inoculating the secondary seed liquid into the fermentation medium at a volume ratio of 1:10, inoculating the fermentation medium, fermenting and culturing for 5 days, and then expanding the culture to obtain a primary Ganoderma lucidum strain;

[0064] Step S15, inoculating the first-level Ganoderma lucidum strain into a fermentation tank filled with fermentation medium, culturing the fermentation tank for 24 to 36 hours, and then filtering to obtain Ganoderma lucidum mycelium fermentation liquid;

[0065] During the fermentation process, the activity of β-glucosidase needs to be detected. The β-glucosidase activity unit (U) is defined as the amount of enzyme required to hydrolyze the substrate to release 1 μmol of pNPG within one minute under the reaction conditions of pH = 5.0 and 50°C.

[0066] pH = 5.0 PC buffer: Weigh 3.689g Na2HPO4:12H2O and 1.01g citric acid and dissolve them in 100ml distilled water, adjust the pH to 5.0 and make up to volume.

[0067] Substrate (pNPG) working solution: Accurately weigh 150.65 mg of pNPG and completely dissolve it in 100 ml of PC buffer (pH = 5.0) for later use.

[0068] β-glucosidase activity determination: Ganoderma mycelium fermentation broth was collected by centrifugation, added with appropriate amount of distilled water and ultrasonically disrupted, centrifuged at 10000xg for 15 min at 4°C, 100 μl of supernatant was mixed with 200 μl of 5 mM pNPG, and kept at 50°C for 30 min. 2 ml of 1 mol / L Na2CO3 was added to terminate the reaction and color was developed, and the absorbance was measured at 400 nm.

[0069] x: measure the pNPG content corresponding to the absorbance value;

[0070] m: weight of Ganoderma lucidum mycelium fermentation liquid;

[0071] Please refer to Figure 2 A. The vertical axis of the figure represents the β-glucosidase concentration (nmol / ml) in the Ganoderma mycelium fermentation broth, and the horizontal axis represents the Ganoderma mycelium fermentation culture duration (days). As shown in the figure, the β-GC (β-glucosidase) content increases continuously over time, with a clear upward trend from day 1 to day 6, and the upward trend reaches a peak between day 6 and day 7. The β-GC content increases from 13.90 nmol / ml to 20.33 nmol / ml, with an average daily increase of 0.919 nmol / ml over the 7 days of culture.

[0072] Adjust the temperature (25°C-30°C), pH value (6.0-7.5), and rotation speed (110rpm-130rpm) in the fermentation tank. Continuously detect the β-glucosidase activity during the culture process. When it reaches the expected concentration, the fermentation reaction is suspended and the next biotransformation reaction is carried out.

[0073] Step S2, biotransforming the polygonatum powder substrate to obtain Ganoderma lucidum exosome fermentation liquid; specifically comprising:

[0074] Step S21, crushing and sieving the naturally dried polygonatum to obtain polygonatum powder;

[0075] Step S22: Mix the polygonatum powder and pure water in a ratio of 1:4 and sterilize with pressurized steam to obtain a sterile polygonatum solution;

[0076] Step S23, placing the Ganoderma lucidum mycelium fermentation liquid and the sterile Polygonatum sibiricum solution in a ratio of 9:1 in a sterile reaction tank, and stirring evenly at 30° C. to carry out a conversion reaction; while stirring the reaction, it is necessary to continuously detect the conversion rate of the macromolecular glycoside glycosides in the Polygonatum sibiricum into small molecular aglycones, and the reaction termination index is when the conversion rate reaches 75%; the conversion rate is calculated as follows: Polygonatum sibiricum saponin aglycone conversion rate = Polygonatum sibiricum saponin aglycone in Example 1 / (Polygonatum sibiricum saponin aglycone in Comparative Example 1 + Polygonatum sibiricum saponin aglycone in Example 1).

[0077] The detection methods of polygonatum saponins include:

[0078] (1) Instruments: High performance liquid chromatography (HPLC): Agilent Technologies 1220 Infinity LC high performance liquid chromatograph, purchased from Agilent Technologies, USA; Agilent HC-C18

[0079] (2) Chromatographic column (4.6 mm × 250 mm, 5 μm),

[0080] (3) Chromatographic acetonitrile (made in the United States) (batch number: 20130318);

[0081] (4) Polygonatum sibiricum aglycone standard (China National Institute for the Control of Pharmaceutical and Biological Products).

[0082] (5) Chromatographic conditions: Acetonitrile (A) and water (B) were used as the mobile phase, with gradient elution. The gradient program was as follows: 0 min: 5% A; 10 min: 10% A; 30 min: 35% A; 40 min: 40% A; 60 min: 5% A. The flow rate was 1 mL / min, the column temperature was 30°C, the detection wavelength was 200 nm, and the injection volume was 20 μL.

[0083] (6) Preparation of control test Accurately weigh an appropriate amount of Polygonatum sibiricum saponin, dissolve it in methanol to prepare a reference solution with a concentration of 0.10, 20 mg / mL, filter it through a 0.45 μm microporous filter membrane, and set aside.

[0084] (7) Sample detection: Add 80% ethanol at a solid-liquid ratio of 1:20, perform ultrasonic extraction twice at 60°C for 50 min, filter, combine the filtrates, evaporate to dryness, add 20 mL of distilled water to dissolve, add (20 mL, 20 mL) of n-butanol to extract twice, take the n-butanol layer, spin dry, add a small amount of methanol to dissolve, place each in a 10 mL volumetric flask, add methanol to make up to volume, filter through a 0.45 μm microporous filter membrane, and take the filtrate as the test sample.

[0085] Step S24: After the reaction is completed, the Ganoderma lucidum exosome fermentation liquid is collected.

[0086] During the fermentation process of Ganoderma lucidum mycelium, the activity of β-glucosidase is detected. When the activity reaches the expected concentration (10U / mL-30U / mL), the mixed solution of Polygonatum sibiricum is added to the fermentation liquid of Ganoderma lucidum mycelium. Through the biotransformation action of β-glucosidase in the fermentation liquid of Ganoderma lucidum mycelium, the macromolecular glycoside glycosides in the effective ingredients of Polygonatum sibiricum are converted into small molecular aglycones.

[0087] Step S3: Separating, extracting and purifying the Ganoderma lucidum exosome fermentation broth to obtain Ganoderma lucidum exosomes. Specifically comprising:

[0088] Step S31, placing the Ganoderma lucidum exosome fermentation liquid into cold phosphate buffered saline (PBS) and homogenizing it in a high-speed blender for 1 minute;

[0089] Step S32, centrifuging the stirred fermentation broth at 1000×g for 10 minutes, 2000×g for 20 minutes, 4000×g for 30 minutes, and finally at 100,000×g for 1.5 hours at 4°C;

[0090] Step S33, filtering and separating the Ganoderma lucidum exosome precipitate;

[0091] Step S34: rinse the Ganoderma lucidum exosome precipitate with PBS, suspend it in PBS or culture medium, and filter it again using a 200 nm Acrodisc filter to obtain Ganoderma lucidum exosomes.

[0092] Please refer to Figure 2 B, The change of saponin content in the culture medium three days after the addition of Polygonatum sibiricum, which was generally maintained at around 3400ug / mL, and gradually increased from the first day to the third day.

[0093] Comparative Example 1

[0094] The difference between Comparative Example 1 and Example 1 is that the Polygonatum sibiricum solution was not added during the culture process, and only the seed culture medium prepared in step S12 and 5 mL of the shaken and broken bacterial solution were added to a 250 mL conical flask and cultured together for 3 days.

[0095] Please refer to Figure 3 A, the vertical axis is the concentration of exosomes from the mycelia of Ganoderma lucidum prepared in Comparative Example 1 (Particles / mL), and the horizontal axis is the sample particle diameter (nm). As can be seen from the figure, the concentration of exosomes from the mycelia of Ganoderma lucidum prepared in Comparative Example 1 is 6.1E+7 Particles / mL, the dilution factor is 500, the original concentration is 3.0E+10 Particles / mL, and the Median (×50) is 149.9 nm.

[0096] Please refer to Figure 3 B. The ordinate of the figure represents the concentration of exosomes from the mycelia of Ganoderma lucidum prepared in Example 1 (Particles / mL), and the abscissa represents the sample particle diameter (nm). As can be seen from the figure, the concentration of exosomes from the mycelia of Ganoderma lucidum prepared in Example 1 is 4.6E+7 Particles / mL. The dilution factor is 20,000, the original concentration is 9.2E+11 Particles / mL, and the median (×50) is 141.4 nm.

[0097] Combine Figure 3 It can be seen that the particle size of the Ganoderma lucidum mycelium exosomes prepared in Comparative Example 1 and Example 1 did not change significantly (front: Median (×50) was 149.9 nm, back: Median (×50) was 141.4 nm. The results showed that the density of the Ganoderma lucidum exosomes prepared in Comparative Example 1 and Example 1 changed significantly, and the density increased from 3.0E+10 Particles / mL in Comparative Example 1 (without the addition of Polygonatum sibiricum) to 9.2E+11 Particles / mL, an increase of 3.06X 100 times (about 300 times).

[0098] Example 2

[0099] Based on Example 1 and Comparative Example 1, this example mainly introduces the effect verification experimental data of Example 1 and Comparative Example 1.

[0100] The detection of Ganoderma lucidum exosomes includes:

[0101] 1. Experimental instruments and reagents

[0102] Table 1 Experimental instruments and reagents

[0103] Instrument name factory model Origin ZetaView_Particle Metrix Particle Metrix PMX-120 Germany 100nm PS beads Thermo Fisher 3100A USA PBS Servicebio G4202-500ML China

[0104] 2. Operation steps

[0105] (1) Wash the sample pool 3 times with pure water;

[0106] (2) After cleaning, calibrate the instrument using a standard (100 nm PS beads, polystyrene microspheres), and perform subsequent testing after calibration.

[0107] (3) Wash the sample pool with 1× PBS;

[0108] (4) The sample was diluted with 1× PBS (the dilution factor is shown in the PDF report) and added to the sample pool. The real-time dynamic image of exosome particles was observed on the computer screen.

[0109] (5) Collect information and issue corresponding test reports;

[0110] (6) After the test, use 1×PBS to clean the sample pool and test the next sample.

[0111] The difference between the substances contained in the exosomes separated after adding Polygonatum sibiricum for biotransformation and those in comparative example 1 can be indirectly reflected by measuring the β-glucosidase activity.

[0112] Please refer to Figure 4 , Figure 4 A is a real-time dynamic image of the exosome particles in the mycelium of Ganoderma lucidum prepared in comparative example 1. Figure 4 B is a real-time dynamic image of the exosome particles in the mycelium of Ganoderma lucidum prepared in Example 1. It can be seen that saucer-shaped exosomes can be detected in the mycelium of Ganoderma lucidum prepared in both Comparative Example 1 and Example 1, with no significant changes. Figure 3 、 Figure 4 According to the characteristics of exosomes, regardless of whether Polygonatum sibiricum is added or not, the corresponding exosomes can be successfully extracted from the Ganoderma lucidum culture medium, and the density of the exosomes extracted after adding Polygonatum sibiricum is significantly higher than that without adding Polygonatum sibiricum. That is, after adding Polygonatum sibiricum, its active ingredients penetrate into the original Ganoderma lucidum exosomes.

[0113] Example 3

[0114] The experiments on the inhibitory effect of the freeze-dried Ganoderma lucidum exosome powder prepared in Example 1 and Comparative Example 1 on cancer cell proliferation included:

[0115] 1. Experimental cells, freeze-dried powder

[0116] (1) The cells selected for the experiment were human liver cancer cells Hep-G2 and human colon adenocarcinoma cells CaCo-2.

[0117] (2) The freeze-dried powders were all derived from the culture medium of Ganoderma lucidum, which was filtered through a 0.22 μm filter and then freeze-dried.

[0118] 2. This experiment uses CCK-8 method to detect the killing effect of the Ganoderma lucidum supernatant culture fluid prepared in Comparative Example 1 and Example 1 on cancer cells

[0119] Please refer to Figure 5 , Figure 5 A. The cell survival rate of the Ganoderma lucidum exosome freeze-dried powder prepared in comparative example 1 and example 1 on Hep-G2 cells, Figure 5 B. Comparative Example 1 and Example 1: Cell viability of the Ganoderma lucidum exosome freeze-dried powders for CaCo-2 cells. The overall trend in the two figures shows that, with increasing concentrations of the two freeze-dried powders, both cell viability levels decreased, regardless of whether Polygonatum sibiricum was added. This indicates that the freeze-dried powders exhibited a certain inhibitory effect on cancer cell proliferation, with this inhibitory effect being more pronounced at high concentrations. This inhibitory effect was more pronounced after the addition of Polygonatum sibiricum compared to the freeze-dried powders without Polygonatum sibiricum. This suggests that the presence of a certain species in the Ganoderma lucidum culture supernatant, namely the two exosomes prepared in Example 1, exhibited a significant killing effect on cancer cells, and that the exosomes containing the active component of Polygonatum sibiricum had a greater inhibitory effect on cancer cell proliferation.

[0120] Example 4

[0121] In this study, liquid chromatography-mass spectrometry (LC-MS) was used to investigate the metabolomics of exosomes.

[0122] 1. Sample Processing

[0123] (1) Accurately weigh 100 ± 5 mg of sample into a 2 mL centrifuge tube and add a 6 mm diameter grinding bead;

[0124] (2) Add 800 μL of extraction solution (methanol:water = 4:1 (v:v)) containing four internal standards (L-2-chlorophenylalanine (0.02 mg / mL) etc.);

[0125] (3) Grind in a frozen tissue grinder for 6 min (-10°C, 50 Hz);

[0126] (4) low-temperature ultrasonic extraction for 30 min (5°C, 40 kHz);

[0127] (5) The sample was placed at -20°C for 30 min; centrifuged for 15 min (13,000 g, 4°C) and the supernatant was transferred to a vial with an internal cannula for analysis.

[0128] (6) In addition, 20 μL of supernatant was taken from each sample and mixed to serve as a quality control sample.

[0129] 2. LC-MS detection

[0130] The instrument platform for this LC-MS analysis is Thermo Fisher Scientific's ultra-high performance liquid chromatography tandem Fourier transform mass spectrometry UHPLC-Q Exactive system.

[0131] C18 column chromatographic conditions: The chromatographic column was ACQUITY UPLC BEH C18 (100 mm × 2.1 mm id, 1.7 μm; Waters, Milford, USA); the mobile phase A was 2% acetonitrile in water (containing 0.1% formic acid), the mobile phase B was acetonitrile (containing 0.1% formic acid), the injection volume was 3 μL, and the column temperature was 40°C.

[0132] Mass spectrometry conditions: The sample was electrospray ionized, and the mass spectrometry signals were collected in positive and negative ion scanning modes. Specific parameters are shown in Table 2 below:

[0133] Table 2 Mass spectrometry parameters

[0134] describe Description parameter Scan range Scan type(m / z) 70-1050 Sheath gas flow rate Sheath gas flow rate(arb) 50 Auxiliary gas flow rate Aux gas flow rate(arb) 13 Heating temperature Heater temp(℃) 450 Capillary temperature Capillary temp(℃) 320 Spray voltage (positive mode) Spray voltage(+)(V) 3500 Spray voltage (negative mode) Spray voltage(-)(V) -3000 Collision energy Normalized collision energy(eV) 20,40,60 Resolution (Full MS) Resolution(Full MS) 70000 Resolution (MS2) Resolution(MS2) 17500 S-Lens voltage S-Lens RF Level 40

[0135] Please refer to Figure 6 and Figure 7 , Figure 6 The figure shows the difference between the exosomes prepared in Example 1 and the exosomes prepared in Comparative Example 1. Figure 7 The figure shows the differential expression of substances contained in the exosomes prepared in Example 1 and the exosomes prepared in Comparative Example 1.

[0136] from Figure 6It can be seen that in the exosomes prepared in Example 1, the contents of Sobetirome, Acorus acid, Zingerone A and various compounds are significantly higher, especially the compound No. 1 in Table 3, whose content difference in the two exosomes is as high as 4838.4615 times, and the second compound Sobetirome, whose difference is as high as 45.9391 times, which shows that in the exosomes prepared in Example 1, the effective ingredients of Polygonatum sibiricum successfully penetrated into the original exosomes, so the content of the corresponding compounds increased accordingly. These substances with significant differences may be related to the pharmacological activities of Polygonatum sibiricum (such as antioxidant, anti-inflammatory, and anti-tumor). For example, Zingerone A and Acorus acid are known to have anti-inflammatory and antioxidant properties, and the increase in their content may enhance the biological activity of exosomes. The addition of Polygonatum sibiricum significantly changed the metabolomic characteristics of Ganoderma lucidum exosomes, especially the content of certain specific substances (such as No. 1-5) increased significantly. These differences may be related to the enhanced anti-cancer activity of exosomes (such as Figure 5 This provides molecular-level evidence for the synergistic effect of Polygonatum sibiricum and Ganoderma lucidum exosomes.

[0137] Combine Figure 6 、 Figure 7 It can be seen that after the addition of Polygonatum sibiricum for biotransformation, the corresponding exosomes contain significant differences in the composition of substances, and the content of some substances varies greatly. Accordingly, the active ingredients in Polygonatum sibiricum that have a killing effect on cancer cells show a stronger killing effect when combined with the active ingredients in Ganoderma lucidum exosomes. Table 3 below shows the names of the substances contained and the differences in their content.

[0138] Table 3 Ratios of the components contained in exosomes in Example 1 and Comparative Example 1

[0139]

[0140]

[0141] In the table, B1 represents Comparative Example 1, B2 represents Example 1, and FC stands for fold change.

[0142] The above description is only a preferred embodiment of the present invention, which certainly cannot be used to limit the scope of rights of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and changes can be made without departing from the principles of the present invention. These improvements and changes are also considered to be within the scope of protection of the present invention.

Claims

1. A method for preparing Ganoderma lucidum exosomes containing active ingredients that are both medicinal and edible, characterized in that: The following steps are involved: Step S1: culturing Ganoderma lucidum mycelium fermentation liquid; Step S2: adding the polygonatum solution to the Ganoderma lucidum mycelium fermentation liquid obtained in step S1 to biotransform the polygonatum powder substrate to obtain Ganoderma lucidum exosome fermentation liquid; Step S3: separating, extracting and purifying the Ganoderma lucidum exosome fermentation broth to obtain Ganoderma lucidum exosomes.

2. The method for preparing Ganoderma lucidum exosomes containing active ingredients of medicinal and edible properties according to claim 1, characterized in that: The specific method of step S1 includes: Step S11: inoculating high-triterpene-yielding Ganoderma lucidum mycelia into PDA culture medium for slant culture until the Ganoderma lucidum mycelia fill the slant test tube; Step S12: Pour sterile water into the inclined test tube covered with Ganoderma mycelium, scrape all the Ganoderma mycelium on the culture medium into the sterile water, and pour the sterile water containing Ganoderma mycelium into the seed culture medium and culture for several days to prepare the first-level seed solution; Step S13: The cultured primary seed solution is vibrated to break up the mycelial balls, and the broken mycelia are aspirated and inoculated into a deep liquid fermentation medium for cultivation for several days to obtain a secondary seed solution; Step S14: inoculating the secondary seed liquid into a fermentation medium, fermenting and culturing for several days, and then expanding the culture to obtain a primary Ganoderma lucidum strain; Step S15: inoculating the first-level Ganoderma lucidum strain into a fermentation medium for fermentation culture for several days, and then filtering to obtain a Ganoderma lucidum mycelium fermentation liquid.

3. The method for preparing Ganoderma lucidum exosomes containing active ingredients of medicinal and edible properties according to claim 2, characterized in that: In step S11, the PDA culture medium includes: 0.01g / L-1.0g / L vitamin B1, 1.0g / L-5.0g / L KH2PO4, 0.5g / L-2.0g / L MgSO4, 5.0g / L-30g / L glucose, 5.0g / L-30g / L agar powder, and 100g / L-300g / L potato extract; In step S12, the seed culture medium includes: 0.01g / L-0.1g / L vitamin B1, 1.0g / L-5.0g / L KH2PO4, 0.1g / L-1g / L MgSO4, 20g / L-50g / L glucose, 1.0g / L-10g / L tryptone, and 1g / L-5g / L yeast extract; In step S13, the submerged liquid fermentation medium includes: 0.01g / L-0.1g / L vitamin B1, 1.0g / L-5.0g / L KH2PO4, 0.5g / L-3.0g / L MgSO4, 20g / L-50g / L glucose, 1.0g / L-10g / L tryptone, and 1g / L-5g / L yeast extract.

4. The method for preparing Ganoderma lucidum exosomes containing active ingredients of medicinal and edible properties according to claim 2, characterized in that: In step S14, the fermentation medium includes: 0.01g / L-0.1g / L vitamin B1, 1.0g / L-5.0g / L KH2PO4, 0.5g / L-3.0g / L MgSO4, 20g / L-50g / L glucose, 1.0g / L-10g / L tryptone, 1g / L-5g / L yeast extract, The fermentation culture parameters are as follows: culture at 25°C-30°C in a shaker at 110 rpm-130 rpm for 2-5 days, and inoculating the fermentation medium with the secondary seed solution and fermentation medium at a volume ratio of 1:10; In step S15, the fermentation medium includes: 0.01g / L-0.1g / L vitamin B1, 1.0g / L-5.0g / L KH2PO4, 0.5g / L-3.0g / L MgSO4, 20g / L-50g / L glucose, 1.0g / L-10g / L tryptone, 1g / L-5g / L yeast extract, The fermentation culture parameters are: 25°C-30°C, shaking at 110 rpm-130 rpm, and culture for 2-5 days.

5. The method for preparing Ganoderma lucidum exosomes containing medicinal and edible active ingredients according to claim 1, characterized in that: In the step S1, the concentration of β-glucosidase in the Ganoderma lucidum mycelium fermentation broth is 10 U / mL-30 U / mL.

6. The method for preparing Ganoderma lucidum exosomes containing active ingredients of medicinal and edible properties according to claim 1, characterized in that: The specific method of step S2 includes: Step S21: crushing and sieving the naturally dried polygonatum to obtain polygonatum powder; Step S22: Mixing polygonatum powder and pure water in a certain proportion and then sterilizing to obtain a sterile polygonatum solution; Step S23: uniformly stirring the Ganoderma lucidum mycelium fermentation liquid and the sterile Polygonatum sibiricum solution in a certain ratio under appropriate conditions to perform a conversion reaction; Step S24: After the reaction is completed, the Ganoderma lucidum exosome fermentation liquid is collected.

7. The method for preparing Ganoderma lucidum exosomes containing active ingredients of medicinal and edible properties according to claim 6, characterized in that: In step S22, the weight-to-volume ratio of polygonatum powder to pure water is 1 g:4 mL; In the step S23, the volume ratio of the Ganoderma lucidum mycelium fermentation liquid to the sterile Polygonatum sibiricum solution is 9:1, and the temperature of the bioconversion is 25-30°C.

8. The method for preparing Ganoderma lucidum exosomes containing active ingredients of medicinal and edible properties according to claim 6, characterized in that: In step S23, the conversion rate of the macromolecular glycoside glycosides in the polygonatum to the micromolecular aglycones needs to be continuously detected, and the reaction termination index is when the conversion rate reaches 75%.

9. The method for preparing Ganoderma lucidum exosomes containing active ingredients of medicinal and edible properties according to claim 1, characterized in that: The specific method of step S3 includes: Step S31: placing the Ganoderma lucidum exosome fermentation liquid into PBS buffer and homogenizing it; Step S32: centrifuging the homogenized fermentation broth, gradually increasing the centrifugal speed and time, and finally ultracentrifuging at 4°C; Step S33: filtering the Ganoderma lucidum exosome precipitate after centrifugation; Step S34: Rinse the Ganoderma lucidum exosome precipitate with PBS buffer, suspend it in PBS buffer or culture medium, and filter it to obtain Ganoderma lucidum exosomes.

10. The method for preparing Ganoderma lucidum exosomes containing active ingredients of medicinal and edible properties according to claim 8, characterized in that: In step S32, the specific process of centrifugation is: centrifuging the homogenized fermentation broth at 1000xg-1500xg for 10-20 minutes, 2000xg-3500xg for 20-30 minutes, 4000xg-5000xg for 30-35 minutes, and finally at 100000xg-120000xg at 4°C for 1.0-1.5 hours.

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