Fermentation preparation method and application of ganoderma lucidum with full-effect components

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to a fermentation preparation method and application of ganoderma lucidum with full-effect components. The method comprises the following steps: sequentially carrying out strain pretreatment, two-stage fermentation regulation, graded extraction, eutecticum construction and composite preparation: treating a ganoderma lucidum strain by adopting a domestication culture medium containing corn steep liquor and Tween-80 and an activation culture medium containing beta-cyclodextrin; two-stage fermentation is formed through aerobic proliferation and anaerobic induction, and methyl jasmonate is added in the anaerobic stage and is irradiated by blue light; carrying out supercritical COextraction on fat-soluble components, carrying out water extraction on water-soluble components, and carrying out ultrafiltration and enrichment on small molecule peptides to finish graded extraction; the ganoderic acid DM and the lycium barbarum polysaccharide are prepared into eutecticum, and then the eutecticum is compounded with the water-soluble component and the small molecule peptide in proportion. The strain adaptability and mycelium biomass can be improved, the ratio of ganoderic acid DM is directionally increased, and the solubility and absorption efficiency of fat-soluble components are improved.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a method for preparing Ganoderma lucidum with all-purpose components through fermentation and its application. Background Technology

[0002] Reishi mushroom, a traditional fungus with both medicinal and health-promoting value, contains active substances such as reishi polysaccharides, reishi triterpenes (especially the specific component ganoderic acid DM), and small molecule peptides. These substances have irreplaceable application potential in areas such as immune regulation, anti-oxidation, and skin barrier repair, making it one of the core raw materials in the pharmaceutical, cosmetic, and health food industries. Currently, the industrial preparation of reishi's effective components mainly relies on two technical routes: fruiting body cultivation and extraction, and liquid fermentation. Liquid fermentation, due to its advantages such as short production cycle, strong environmental controllability, and ease of large-scale scaling, is gradually becoming the mainstream technology in the industry. However, existing liquid fermentation and subsequent processing technologies still face several key technical bottlenecks, severely restricting the development and application of highly active and comprehensive reishi products.

[0003] The primary problem is that the strains are not adapted to plant-derived fermentation substrates. Certain high-yielding strains of Ganoderma lucidum naturally have difficulty absorbing plant-derived nutrients efficiently, and traditional culture media lack the "domestication-activation" synergistic design, resulting in low strain survival rate and insufficient mycelial biomass, which restricts the synthesis of active ingredients from the source.

[0004] Secondly, the precision of fermentation metabolism regulation is low. The synthesis of secondary metabolites of Ganoderma lucidum (such as ganoderic acid DM) requires the "conversion from primary metabolism to secondary metabolism," which is difficult to meet by traditional single aerobic or anaerobic culture modes: aerobic environment easily leads to excessive mycelial proliferation and inhibits secondary metabolism, resulting in low total triterpenoids; anaerobic environment, due to the lack of exogenous signal induction, can increase the total amount of triterpenoids, but the proportion of specific components is low, and the activity of key synthetic enzymes cannot be effectively activated, making it difficult to meet the standards for product purity and specificity.

[0005] Therefore, there is an urgent need for a Ganoderma lucidum fermentation preparation technology that can solve the above-mentioned technical bottlenecks. Summary of the Invention

[0006] To address the aforementioned shortcomings in the existing technology, this invention provides a method for preparing Ganoderma lucidum with all-purpose components through fermentation and its application, thereby solving the problems mentioned in the background technology.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: A method for preparing Ganoderma lucidum with all-purpose components by fermentation includes the following steps: A. Pretreatment of the strain: Ganoderma lucidum strain CGMCC 5.1816 was inoculated into an acclimatization medium containing corn steep liquor and Tween-80, and cultured with shaking at 25-28℃ for 5-7 days to obtain an adapted strain; then it was transferred to an activation medium containing wheat bran extract and β-cyclodextrin, and cultured statically at 27-29℃ for 40-50 hours to obtain activated mycelial moss; B, Two-stage fermentation control: B1, Aerobic stage: Inoculate the activated mycelium with a 12%-18% inoculation amount into a fermentation medium containing Ganoderma lucidum residue hydrolysate and wolfberry powder, and culture at 28-30℃ and 170-190r / min for 4-6 days; B2, Anaerobic Induction Stage: Add 0.08-0.12% methyl jasmonate to the fermentation system, replace with nitrogen until the oxygen content is ≤0.2%, and irradiate with 460nm, 10-20μmol / m² / s blue light for the first 6 hours of the anaerobic induction stage, and then let it ferment in the dark at 24-26℃ for 18-22 days. C. Fractional Extraction: The fermentation broth is centrifuged at 3500-4500 r / min for 12-18 minutes to separate the mycelium from the supernatant. The solid residue remaining after supercritical CO2 extraction of the mycelium is the residue, which is then extracted with water to obtain water-soluble components. The water-soluble components mainly contain water-soluble polysaccharides. The supernatant is filtered through an 8-12 kDa ultrafiltration membrane to obtain small molecule peptide components. The supercritical CO2 extract is a lipid-soluble component. D. Construction of co-crystal: Ganoderma lucidum acid DM in the fat-soluble component and Lycium barbarum polysaccharide in the water-soluble component are co-dissolved at a mass ratio of 1:1-1:3, the mass ratio corresponding to a molar ratio of 1:1.0-1:3.2; the mixture is dissolved in an ethanol-water system and spray-dried to obtain a co-crystal with a particle size D90 < 200 nm; E. Composite preparation: The co-crystal is mixed with the water-soluble component and the small molecule peptide component in a ratio of 1:4-6:2-4 to obtain the Ganoderma lucidum product with all-effect components.

[0008] Further, in step A, the acclimatization culture medium contains 1.0%-2.0% corn steep liquor, 1.0%-1.5% glucose, 0.01%-0.03% Tween-80, and 0.05%-0.1% MgSO4, with a shaking speed of 120-150 r / min; the activation culture medium contains 2%-4% wheat bran extract, 0.3%-0.7% yeast extract, and 0.2%-0.4% β-cyclodextrin.

[0009] Further, in step B1, the fermentation culture medium also contains 2%-4% rice bran and 0.03%-0.07% complex mineral elements; the preparation method of the Ganoderma lucidum residue hydrolysate is to mix Ganoderma lucidum residue and water at a ratio of 1:7-9, add 700-900U / g cellulase and 400-600U / g xylanase, enzymatically hydrolyze at 50-60℃ for 5-7 hours, and then filter to obtain the supernatant.

[0010] Furthermore, in step B2, the light source of the blue light is an LED cold light source; during static fermentation, the ORP is maintained at -200mV by monitoring the redox potential.

[0011] Furthermore, in step C, the supercritical CO2 extraction pressure is 28-32 MPa and the temperature is 43-47℃; the water extraction conditions are that the residue is boiled with water for 2-3 hours, and the filtrate is taken as the water-soluble component after filtration.

[0012] Further, in step D, the volume ratio of the ethanol-water system is 6:4-8:2; the inlet temperature of the spray dryer is 170-190℃ and the outlet temperature is 70-90℃; the eutectic is an amorphous solid dispersion, and no characteristic crystal peaks are detected by X-ray powder diffraction.

[0013] A Ganoderma lucidum product with all active components, prepared by the above method, comprises a co-crystal from step D, a water-soluble polysaccharide from step C, and a small molecule peptide component; the particle size D90 of the co-crystal is 100-200 nm; the content of ganoderic acid DM in the co-crystal is 1.8%-5.0%, and the mass ratio of ganoderic acid DM to total triterpenes is 45%-70%; the water solubility of the co-crystal is 30-60 mg / mL; the water-soluble polysaccharide is 26%-40% based on β-glucan; and the proportion of <5 kDa components in the small molecule peptide is 80%-95%.

[0014] Furthermore, the Ganoderma lucidum products of the whole-effect components showed characteristic peaks when detected by high performance liquid chromatography, with a retention time of 8.2±0.1 min corresponding to ganoderic acid DM and 12.5±0.1 min corresponding to lycopene polysaccharide glycosides.

[0015] The present invention also provides the application of the above-described method for preparing Ganoderma lucidum fermentation with all-purpose components in the extraction of effective components from Ganoderma lucidum.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. Pretreatment of strains enhances their adaptability and biomass to plant-derived substrates through specialized culture medium combinations; two-stage fermentation utilizes the synergistic effects of low oxygen, blue light, and signal induction to directionally regulate secondary metabolism and improve the synthesis efficiency of specific active ingredients; graded extraction and co-crystallization achieve efficient separation of multiple active ingredients while solving the problems of poor solubility and difficult absorption of lipid-soluble components, laying a solid foundation for subsequent high-value utilization.

[0017] 2. By scientifically combining co-crystals, water-soluble components, and small molecule peptides, a system with synergistic effects of multiple active ingredients is formed, achieving all-around efficacy; and by utilizing Ganoderma lucidum residue to achieve the recycling of fermentation waste, the cost of raw materials is reduced. Attached Figure Description

[0018] Figure 1 This is a flowchart of a method for preparing Ganoderma lucidum fermentation with all-purpose components according to the present invention; Figure 2 This is a flowchart of the two-stage fermentation control method of the present invention. Detailed Implementation

[0019] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0020] The accompanying drawings are for illustrative purposes only and are schematic diagrams, not actual images. They should not be construed as limiting the scope of this application. To better illustrate the embodiments of the present invention, some parts in the drawings may be omitted, enlarged, or reduced, and do not represent the actual dimensions of the product. It is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings.

[0021] In the accompanying drawings of the embodiments of the present invention, the same or similar reference numerals correspond to the same or similar components. In the description of the present invention, it should be understood that if terms such as "upper," "lower," "left," "right," "inner," and "outer" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, they are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, the terms used to describe positional relationships in the drawings are only for illustrative purposes and should not be construed as limiting the present application. For those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.

[0022] In the description of this invention, unless otherwise explicitly specified and limited, the term "connection" or similar designation indicating a connection between components should be interpreted broadly. For example, it can refer to a fixed connection, a detachable connection, or an integral part; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium; it can refer to the internal communication between two components or the interaction between two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0023] like Figure 1-2 As shown, this invention provides a method for preparing Ganoderma lucidum with all active components through fermentation. The method involves strain pretreatment, two-stage fermentation regulation, graded extraction, co-crystal construction, and compound preparation to obtain a Ganoderma lucidum product with highly active ingredients. The following detailed description includes specific examples and comparative experiments.

[0024] Specifically, a method for preparing Ganoderma lucidum with all-purpose components by fermentation includes the following steps: A. Pretreatment of the strain: Ganoderma lucidum strain CGMCC 5.1816 was inoculated into an acclimatization medium containing corn steep liquor and Tween-80, and cultured with shaking at 25-28℃ for 5-7 days to obtain an adapted strain; then it was transferred to an activation medium containing wheat bran extract and β-cyclodextrin, and cultured statically at 27-29℃ for 40-50 hours to obtain activated mycelial moss; B, Two-stage fermentation control: B1, Aerobic stage: Inoculate the activated mycelium with a 12%-18% inoculation amount into a fermentation medium containing Ganoderma lucidum residue hydrolysate and wolfberry powder, and culture at 28-30℃ and 170-190r / min for 4-6 days; B2, Anaerobic Induction Stage: Add 0.08-0.12% methyl jasmonate to the fermentation system, replace with nitrogen until the oxygen content is ≤0.2%, and irradiate with 460nm, 10-20μmol / m² / s blue light for the first 6 hours of the anaerobic induction stage, and then let it ferment in the dark at 24-26℃ for 18-22 days. C. Fractional Extraction: The fermentation broth is centrifuged at 3500-4500 r / min for 12-18 minutes to separate the mycelium from the supernatant. The solid residue remaining after supercritical CO2 extraction of the mycelium is the residue, which is then extracted with water to obtain water-soluble components. The water-soluble components mainly contain water-soluble polysaccharides. The supernatant is filtered through an 8-12 kDa ultrafiltration membrane to obtain small molecule peptide components. The supercritical CO2 extract is a lipid-soluble component. D. Construction of co-crystal: Ganoderma lucidum acid DM in the fat-soluble component and Lycium barbarum polysaccharide in the water-soluble component are co-dissolved at a mass ratio of 1:1-1:3, the mass ratio corresponding to a molar ratio of 1:1.0-1:3.2; the mixture is dissolved in an ethanol-water system and spray-dried to obtain a co-crystal with a particle size D90 < 200 nm; E. Composite preparation: The co-crystal is mixed with the water-soluble component and the small molecule peptide component in a ratio of 1:4-6:2-4 to obtain the Ganoderma lucidum product with all-effect components.

[0025] In this embodiment, the strain of the present invention was purchased from the China General Microbiological Culture Collection Center, with the number CGMCC5.1816, but is not limited to this number. In step A, the acclimatization culture medium contains 1.0%-2.0% corn steep liquor, 1.0%-1.5% glucose, 0.01%-0.03% Tween-80, and 0.05%-0.1% MgSO4, with a shaking speed of 120-150 r / min; the activation culture medium contains 2%-4% wheat bran extract, 0.3%-0.7% yeast extract, and 0.2%-0.4% β-cyclodextrin.

[0026] In this embodiment, in step B1, the fermentation culture medium also contains 2%-4% rice bran and 0.03%-0.07% complex mineral elements; the preparation method of the Ganoderma lucidum residue hydrolysate is to mix Ganoderma lucidum residue and water at a ratio of 1:7-9, add 700-900 U / g cellulase and 400-600 U / g xylanase, enzymatically hydrolyze at 50-60℃ for 5-7 hours, and then filter to obtain the supernatant of the fermentation broth by centrifugation.

[0027] In this embodiment, in step B2, the light source of the blue light is an LED cold light source; during static fermentation, the ORP is maintained at -200mV by monitoring the redox potential.

[0028] In this embodiment, in step C, the supercritical CO2 extraction pressure is 28-32 MPa and the temperature is 43-47℃; the water extraction conditions are that the residue is boiled with water for 2-3 hours, and the filtrate is taken as the water-soluble component after filtration.

[0029] In this embodiment, in step D, the volume ratio of the ethanol-water system is 6:4-8:2; the spray drying inlet temperature is 170-190℃ and the outlet temperature is 70-90℃; the eutectic is an amorphous solid dispersion, and no characteristic crystal peaks are detected by X-ray powder diffraction.

[0030] A Ganoderma lucidum product with all active components, prepared by the above method, comprises a co-crystal from step D, a water-soluble polysaccharide from step C, and a small molecule peptide component; the particle size D90 of the co-crystal is 100-200 nm; the content of ganoderic acid DM in the co-crystal is 1.8%-5.0%, and the mass ratio of ganoderic acid DM to total triterpenes is 45%-70%; the water solubility of the co-crystal is 30-60 mg / mL; the water-soluble polysaccharide is 26%-40% based on β-glucan; and the proportion of <5 kDa components in the small molecule peptide is 80%-95%.

[0031] Among them, the Ganoderma lucidum products of the whole-effect components have characteristic peaks as detected by high performance liquid chromatography. The retention time of 8.2±0.1 min corresponds to ganoderic acid DM and 12.5±0.1 min corresponds to lycopene polysaccharide.

[0032] The present invention also provides the application of the above-described method for preparing Ganoderma lucidum fermentation with all-purpose components in the extraction of effective components from Ganoderma lucidum.

[0033] As a preferred embodiment of the present invention, the fermentation preparation steps of the Ganoderma lucidum with the full-effect components are as follows: Step A: Strain pretreatment The Ganoderma lucidum strain CGMCC 5.1816 was inoculated into an acclimatization medium containing 1.5% corn steep liquor, 1.2% glucose, 0.02% Tween-80, and 0.08% MgSO4. The culture was carried out at 26°C and 130 r / min for 6 days to obtain an adapted strain. The strain was then transferred to an activation medium containing 3% wheat bran extract, 0.5% yeast extract, and 0.3% β-cyclodextrin. The culture was carried out at 28°C for 45 hours to obtain an activated mycelial colony.

[0034] Step B: Two-stage fermentation regulation B1 Aerobic Stage: The activated mycelium was inoculated into the fermentation medium at an inoculation rate of 15%. The medium contained Ganoderma lucidum mycelium residue hydrolysate (mycelium residue and water were mixed at a ratio of 1:8, with the addition of 800 U / g cellulase and 500 U / g xylanase, and enzymatic hydrolysis at 55℃ for 6 hours and then filtered), 2% wolfberry powder, 3% rice bran, and 0.05% compound mineral elements. The medium was cultured at 29℃ and 180 r / min for 5 days. B2 anaerobic induction stage: 0.1% methyl jasmonate was added to the system, and nitrogen was used to replace the oxygen content to ≤0.2%. For the first 6 hours, the system was irradiated with blue light from an LED cold light source (460nm, 15μmol / m² / s), and then fermented in the dark at 25℃ for 20 days. During this period, the ORP was maintained at ≤-200mV by monitoring the redox potential.

[0035] Step C: Graded Extraction The fermentation broth was centrifuged at 3800 r / min for 15 minutes to separate the mycelium from the supernatant. The mycelium was extracted by supercritical CO2 extraction (pressure 30 MPa, temperature 45℃) to obtain the lipid-soluble component. The remaining residue was boiled with water for 2 hours and filtered to obtain the water-soluble polysaccharide component. The supernatant was filtered through a 10 kDa ultrafiltration membrane to obtain the small molecule peptide component.

[0036] Step D: Eutectic Construction The fat-soluble component ganoderic acid DM and the water-soluble component lycine polysaccharide were co-dissolved in an ethanol-water system (volume ratio 7:3) at a mass ratio of 1:2 (molar ratio 1:2.1). The mixture was then spray-dried (inlet temperature 180℃, outlet temperature 80℃) to obtain an amorphous eutectic crystal with a particle size of D90=150nm. X-ray powder diffraction analysis showed no characteristic crystal peaks.

[0037] Step E: Composite Preparation The co-crystal was mixed with water-soluble components and small molecule peptide components in a mass ratio of 1:5:3 to obtain the Ganoderma lucidum product with all-effect components.

[0038] The present invention will now be described in detail with reference to specific embodiments.

[0039] Example 1: For the strain pretreatment in step A, fresh mycelial growth (approximately 1 cm² in area) at the edge of the slant culture tube of Ganoderma lucidum strain CGMCC 5.1816 was picked up with a sterile inoculation loop and inoculated into a 250 mL Erlenmeyer flask containing 100 mL of acclimatization medium, which contained 1.5% corn steep liquor, 1.2% glucose, 0.02% Tween-80, and 0.08% MgSO4.

[0040] Place the Erlenmeyer flask in a constant temperature shaker, set the temperature to 26℃ and the shaking speed to 150r / min, and continue to incubate for 6 days. Observe the turbidity of the bacterial solution every day during the incubation period, and take a sample on the 6th day for subsequent testing.

[0041] Take 5 mL of the acclimatized bacterial culture and transfer it to a 250 mL Erlenmeyer flask containing 100 mL of activation medium. Place the flask in a 28°C incubator and incubate for 48 hours to form activated bacterial colonies. The activation medium contains 3% wheat bran extract, 0.5% yeast extract, and 0.3% β-cyclodextrin.

[0042] The indicators for detection include strain survival rate, activated mycelial biomass, and mycelial integrity.

[0043] The survival rate of the strains was determined using the "gradient dilution-plate counting method": the acclimated bacterial culture was serially diluted with sterile water to 10-1. -5 10 -6 10 -7 Take 0.1 mL of each diluted solution and spread it on a PDA plate. After incubating at 28°C for 48 hours, count the single colonies and calculate the survival rate using the formula "Survival rate = (Number of single colonies after incubation ÷ Number of theoretical colonies after inoculation) × 100%".

[0044] To activate the mycelial biomass, the activated mycelial biomass was separated by filtering with qualitative filter paper, dried in a 60℃ forced-air drying oven to constant weight, and the dry weight was calculated (unit: g / L).

[0045] Hyphae breakage rate: Activated mycelial moss was prepared into an aqueous slide, and 20 fields of view were randomly observed under a microscope. The proportion of broken hyphae to total hyphae was counted (breakage was defined as the appearance of obvious gaps or fragmentation in hyphae).

[0046] Comparative Example 1-1: The only difference from Example 1 is that the acclimatization medium does not contain Tween-80, while the other conditions are the same.

[0047] Comparative Examples 1-2: The difference from Example 1 is that the acclimatization medium was replaced with traditional PDA medium, which contains 20% potato juice, 2% sucrose and 2% agar, while the other conditions are the same.

[0048] Comparative Examples 1-3: The only difference from Example 1 is that the activation medium does not contain β-cyclodextrin (i.e., the activation medium contains 3% wheat bran extract and 0.5% yeast extract), while the other conditions are the same.

[0049] Results analysis: Effect of Tween-80 on strain survival rate: The survival rate of strain 1 (containing Tween-80) was 92%, while that of Comparative Example 1-1 (without Tween-80) was 78%, representing an increase of 14 percentage points. As a nonionic surfactant, Tween-80 can enhance the cell membrane permeability of Ganoderma lucidum strains, promoting their absorption and utilization of plant-derived nutrients such as corn steep liquor in the acclimatization medium, significantly improving the survival ability of the strains before fermentation, and laying the foundation for subsequent high-yield fermentation.

[0050] Example 1, using the culture medium adapted to this invention, achieved an activated mycelial biomass of 3.8 g / L; Comparative Examples 1-2, using conventional PDA culture medium, showed a biomass of only 2.3 g / L. The culture medium of this invention, through the combination of "corn steep liquor + Tween-80 + β-cyclodextrin," provides a suitable carbon and nitrogen source while the inclusion effect of β-cyclodextrin protects the mycelium, increasing biomass by approximately 65% ​​compared to conventional culture media, ensuring sufficient cell mass for subsequent fermentation and metabolism.

[0051] The hyphal breakage rate of Example 1 (containing β-cyclodextrin) was only 8%, while that of Comparative Examples 1-3 (without β-cyclodextrin) reached 22%, and the biomass (2.5 g / L) was significantly lower than that of Example 1 (3.8 g / L). β-cyclodextrin forms a protective film by encapsulating active ingredients (such as terpene precursors) on the surface of the hyphae, reducing mechanical damage and autolysis of the hyphae during cultivation, maintaining hyphal integrity, and thus ensuring biomass and subsequent metabolic activity. Comparative Examples 1-3, lacking β-cyclodextrin, showed easily broken hyphae and decreased biomass, demonstrating the necessity of "activating the medium containing 0.2%-0.4% β-cyclodextrin."

[0052] The biomass and mycelial integrity of Comparative Example 1-1 (without Tween-80), Comparative Example 1-2 (conventional culture medium), and Comparative Example 1-3 (without β-cyclodextrin) were significantly inferior to those of Example 1. This indicates that the combination of "corn steep liquor + Tween-80 (acclimation culture medium)" and "wheat bran extract + β-cyclodextrin (activation culture medium)" is the key to solving the problem of Ganoderma lucidum strains' adaptability to plant-derived fermentation substrates, and provides a prerequisite for high-yield production of active ingredients in subsequent two-stage fermentation regulation.

[0053] Example 2: Take 15 mL of bacterial suspension (15% inoculum) from the activated bacterial culture of Example 1 using a sterile pipette and transfer it to a 500 mL Erlenmeyer flask containing 100 mL of fermentation medium.

[0054] Fermentation medium formula: 5% Ganoderma lucidum residue hydrolysate, 1.0% wolfberry powder, 3% rice bran, 0.05% compound mineral elements (containing Fe²⁺, Zn²⁺, Mn²⁺, mass ratio 1:1:1), natural pH.

[0055] Preparation of Ganoderma lucidum mycelium residue hydrolysate: Ganoderma lucidum mycelium residue and water are mixed at a ratio of 1:8, and 800 U / g of cellulase and 500 U / g of xylanase are added. The mixture is enzymatically hydrolyzed in a constant temperature water bath at 55℃ for 6 hours. The supernatant of the fermentation broth is obtained by filtration through 4 layers of gauze and centrifugation. The supernatant is then sterilized at 121℃ for 15 minutes for later use.

[0056] B1 Aerobic stage: Place the Erlenmeyer flask in a constant temperature shaker, set the temperature to 29℃ and the shaking speed to 180r / min, and incubate for 5 days; take samples daily to measure the dry weight of mycelium, and enter the anaerobic stage when the dry weight of mycelium reaches 12.5g / L on the 5th day.

[0057] B2 anaerobic induction phase: First, add 0.1% methyl jasmonate (by volume) to the fermentation system and stir magnetically for 10 minutes to disperse it evenly. Secondly, the top space of the Erlenmeyer flask was replaced with nitrogen three times (nitrogen was introduced for 30 seconds each time and then the gas was exhausted) to make the oxygen content of the system ≤0.2% (measured by a portable dissolved oxygen meter). Next, a 460nm LED cold light source was used to irradiate the plants for the first 6 hours at intensities of 10μmol / m² / s (Example 2-1), 15μmol / m² / s (Example 2-2), and 20μmol / m² / s (Example 2-3), respectively. The plants were then moved to a 25°C constant temperature incubator and allowed to ferment in the dark for 20 days. Finally, during the settling period, the ORP value was monitored daily using an oxidation-reduction potential meter, and nitrogen was added to maintain ORP ≤ -200mV.

[0058] The indicators tested included the content of ganoderic acid DM, the proportion of total triterpenes, and the activity of CYP450 enzyme.

[0059] The content of ganoderic acid DM and the proportion of total triterpenes were determined as follows: After fermentation, 10 mL of fermentation broth was taken and centrifuged at 4000 r / min for 15 minutes. The mycelium was extracted three times with methanol using ultrasonic extraction (30 minutes each time). The extracts were combined and detected by high performance liquid chromatography (HPLC): C18 column (250 mm × 4.6 mm, 5 μm), mobile phase methanol-0.1% phosphoric acid water (75:25), flow rate 1.0 mL / min, detection wavelength 254 nm, and the content was calculated by external standard method.

[0060] CYP450 enzyme activity: Take 0.5g of fresh mycelium, add 5mL of phosphate buffer (0.1mol / L, pH7.0), grind in an ice bath, centrifuge at 12000r / min for 20 minutes at 4℃, collect the supernatant, and determine the enzyme activity (unit: U / mg protein) using a kit.

[0061] Comparative Example 2-1: The difference from Example 2 is that there was no blue light irradiation during the anaerobic induction stage, while the other conditions were the same.

[0062] Comparative Example 2-2: The difference in Example 2 is that nitrogen replacement was not performed during the anaerobic induction stage (oxygen content was maintained at 8%-10%), and only 15 μmol / m² / s blue light was used for irradiation, while the other conditions were the same.

[0063] Results analysis: Regulation of synthesis efficiency by blue light intensity: In Example 2-2 (blue light intensity 15 μmol / m² / s), the content of ganoderic acid DM (2.3%), the proportion of total triterpenes (52%), and the CYP450 enzyme activity (162 U / mg) were the highest among all groups. Compared with Example 2-1 (10 μmol / m² / s), low-intensity blue light could not fully activate the key enzyme (CYP450) for triterpenoid synthesis; compared with Example 2-3 (20 μmol / m² / s), high-intensity blue light induced a cellular stress response, which in turn inhibited enzyme activity. This indicates that 15 μmol / m² / s is the optimal intensity for blue light induction, which not only meets the range limit of "10-20 μmol / m² / s", but also experimentally verifies that there is a parameter value within this range that maximizes the synthesis efficiency.

[0064] Synergistic effect of anaerobic environment and blue light: The CYP450 enzyme activity (162 U / mg) in Example 2-2 (anaerobic + blue light) was significantly higher than that in Comparative Example 2-1 (anaerobic only, 95 U / mg) and Comparative Example 2-2 (aerobic + blue light, 102 U / mg). Anaerobic conditions promote the shift of Ganoderma lucidum cells from primary metabolism to secondary metabolism (triterpenoid synthesis belongs to secondary metabolism), while blue light, as an exogenous signaling molecule, further upregulates the expression of key genes in the triterpenoid synthesis pathway. The synergistic effect of the two increases the proportion of ganoderic acid DM in total triterpenoids from 39% in Comparative Example 2-1 to 52%, solving the technical pain point of "high total amount of triterpenoids but low proportion of specific components (ganoderic acid DM)" in traditional fermentation.

[0065] Verification of the necessity of blue light-assisted anaerobic fermentation regulation: The yield of ganoderic acid DM in Comparative Example 2-1 (without blue light) was only 1.3%, and in Comparative Example 2-2 (aerobic + blue light) it was only 1.5%, both far lower than the 2.3% in Example 2-2. This directly proves that both an anaerobic environment is needed to initiate secondary metabolism and specific intensity of blue light is needed to precisely regulate the synthesis of specific components, providing a high-activity and high-purity raw material basis for the subsequent "construction of ganoderic acid DM-lycine polysaccharide cocrystal".

[0066] Example 3: The fermentation broth and mycelium mixture of Example 2-2 (blue light 15μmol / m² / s) were used as raw materials for fractional extraction.

[0067] Step C: Pour the fermentation mixture into a 500mL centrifuge bottle and centrifuge at 4000r / min for 15 minutes to separate the upper supernatant and the lower mycelium. Collect the lower mycelium and load it into a supercritical CO2 extraction vessel. Set the extraction pressure to 30MPa, temperature to 45℃, and CO2 flow rate to 20L / h. Extract for 2 hours and collect the effluent from the bottom of the vessel, which is the lipid-soluble component (mainly containing ganoderic acid DM). After extraction, the remaining mycelial residue was transferred to a 250mL Erlenmeyer flask, and deionized water was added at a solid-liquid ratio of 1:10. After boiling, the mixture was kept at a gentle boil for 2.5 hours. The mixture was filtered through four layers of gauze, and the filtrate was collected. This filtrate is the water-soluble component (mainly containing Lycium barbarum polysaccharides and β-glucan). Take the supernatant from centrifugation and filter it through an 8-12 kDa ultrafiltration membrane (operating pressure 0.2 MPa, temperature 30℃). Collect the permeate, which is the small molecule peptide component.

[0068] Step D: Separate and purify ganoderic acid DM from the lipid-soluble component, and separate and purify Lycium barbarum polysaccharides from the water-soluble component. Set up 3 groups of experiments according to the following mass ratios: Example 3-1: Ganoderic acid DM: Lycium barbarum polysaccharide = 1:1 (corresponding to a molar ratio of 1:1.0, calculated based on a molecular weight of 516.6 for ganoderic acid DM and an average molecular weight of 450 for Lycium barbarum polysaccharide). Example 3-2: Ganoderma lucidum acid DM: Lycium barbarum polysaccharide = 1:2 (corresponding molar ratio 1:1.2); Example 3-3: Ganoderma lucidum acid DM: Lycium barbarum polysaccharide = 1:3 (corresponding molar ratio 1:3.2); Add the mixture of each group into the ethanol-water system (volume ratio 7:3) and stir magnetically for 30 minutes until completely dissolved; then transfer it to a spray dryer, set the inlet temperature to 180℃, the outlet temperature to 80℃, and the feed rate to 15mL / min, and collect the dried product, which is the ganoderic acid DM-lycine polysaccharide cocrystal.

[0069] The indicators include: Eutectic ratio: Detected using X-ray powder diffraction (XRD, 2θ scanning range 5°-80°). The absence of characteristic crystalline peaks indicates an amorphous eutectic. Combined with HPLC determination of free ganoderic acid (DM) content, the ratio is calculated using the following formula: .

[0070] Particle size D90: Take 0.1g of eutectic crystal, ultrasonically disperse it in deionized water for 5 minutes, measure the particle size distribution using a laser particle size analyzer, and read the D90 value; Water solubility: Take 0.5g of cocrystal, add 10mL of deionized water, shake at 37℃ for 24 hours, centrifuge at 4000r / min for 10 minutes, take the supernatant and measure the concentration of ganoderic acid DM by HPLC, which is the water solubility; 24-hour transdermal absorption rate: A Franz diffusion cell was used with porcine skin as the transdermal medium. The receiving cell contained phosphate buffer solution at pH 7.4, with a cocrystal concentration of 1 mg / mL. The mixture was stirred at 37°C for 24 hours. The content of ganoderic acid (DM) in the receiving cell was measured and calculated using the following formula: .

[0071] Comparative Example 3-1: The only difference from Examples 3-2 is that ganoderic acid DM and wolfberry polysaccharide are directly physically mixed at a mass ratio of 1:2 without undergoing ethanol-water co-solution and spray drying; the rest of the operations are the same. Comparative Example 3-2: The difference from Example 3-2 is that the co-solvent is replaced with pure ethanol (without aqueous solution), while the rest of the operation is the same; Comparative Example 3-3: The difference from Examples 3-2 is that the ratio of ganoderic acid DM: wolfberry polysaccharide is 1:4 (exceeding the ratio range of 1:1-1:3), while the rest of the operation is the same.

[0072] Results analysis: Verification of the optimal cocrystallization ratio: Example 3-2 (1:2 mass ratio) showed the highest cocrystallization rate (92%), water solubility (42 mg / mL), and transdermal absorption rate (22%) among all groups. Compared with Example 3-1 (1:1), the amount of Lycium barbarum polysaccharides was insufficient, failing to completely form a hydrogen bond network with ganoderic acid DM, resulting in a lower cocrystallization rate. Compared with Example 3-3 (1:3), excessive Lycium barbarum polysaccharides led to intermolecular aggregation, which in turn reduced solubility and transdermal efficiency. The 1:2 ratio, which limits the range to 1:1-1:3 and achieves the best balance between hydrogen bond matching and molecular dispersion, is the optimal parameter for cocrystallization construction.

[0073] Technical advantages of co-crystallization process: Comparative Example 3-1 (physical mixing) lacks a co-crystallization structure. Due to its high lipid solubility, ganoderic acid DM has a water solubility of only 8 mg / mL and a transdermal absorption rate of only 8%. In contrast, Example 3-2, through co-crystallization, forms an amorphous solid dispersion (no characteristic peaks in XRD), with a 4.2-fold increase in solubility and a 1.75-fold increase in transdermal absorption rate compared to physical mixing. This demonstrates that the co-crystallization process can overcome the dissolution bottleneck of lipid-soluble components, solving the technical pain point of traditional Ganoderma lucidum extracts being "highly active but poorly absorbed."

[0074] Necessity of Solvent System and Proportion Range: Comparative Example 3-2 (pure ethanol solvent) lacked water-mediated hydrogen bonding, resulting in a eutectic rate of only 60% and a 33% decrease in solubility compared to Example 3-2. Comparative Example 3-3 (1:4 ratio) exceeded the specified range, with excessive Lycium barbarum polysaccharides leading to molecular aggregation, significantly reducing both eutectic rate and solubility. This further illustrates that the "ethanol-water (7:3) solvent system" and the "1:1-1:3 ratio range" are key to the efficient construction of the eutectic. Both work together to ensure the physicochemical properties and application effects of the eutectic, providing a core functional carrier for subsequent composite preparation (step E) and skin / oral applications.

[0075] Example 4: Raw material quantitative preparation: Take the DM-lycine cocrystal of ganoderic acid from Examples 3-2 (cocrystal ratio 1:2), the water-soluble component (containing β-glucan) extracted from Example 3, and the small molecule peptide component. First, determine the basic content of each raw material through pretreatment to ensure that the proportion of the overall effective component can be accurately calculated after compounding. Cocrystal: Take 10g of cocrystal from Example 3-2 and analyze it by HPLC: the content of ganoderic acid DM is 2.3% (that is, 10g of cocrystal contains 0.23g of ganoderic acid DM), the proportion of ganoderic acid DM in the total triterpenes is 52%, and the particle size D90=150nm; Water-soluble components: The water extract filtrate from Example 3 was concentrated by rotary evaporation to a solid content of 20% (100 mL of concentrate contains 20 g of solids). The β-glucan content was determined by the phenol-sulfuric acid method to be 32% (i.e., 20 g of solids contains 6.4 g of β-glucan). Small molecule peptide components: The ultrafiltration permeate from Example 3 was freeze-dried to obtain peptide powder. The total peptide content was determined to be 44% by the Coomassie Brilliant Blue method. The content of <5kDa components was determined by the GPC method (mobile phase 0.1mol / L NaCl, flow rate 0.8mL / min): 88% (i.e., 10g of peptide powder contains 3.87g of <5kDa peptides).

[0076] Composite preparation: Three parallel experiments were set up according to the mass ratio of "cocrystal: water-soluble component: small molecule peptide component = 1:4-6:2-4", with each group repeated three times and the average value taken. Example 4-1: 10g of cocrystal (containing 0.23g of ganoderic acid DM) + 40g of water-soluble component (take 200mL of concentrate, containing 12.8g of β-glucan) + 20g of small molecule peptide component (containing 8.8g of <5kDa peptide) were added to 50mL of deionized water, and the mixture was magnetically stirred at 30℃ for 30 minutes, and emulsified in a homogenizer at 8000r / min for 10 minutes to obtain 70g of the total effective component mixture; Example 4-2 (Core Validation Group): 10g cocrystal (containing 0.23g ganoderic acid DM) + 50g water-soluble component (take 250mL concentrate, containing 16.0g β-glucan) + 30g small molecule peptide component (containing 13.2g <5kDa peptide), the operation is the same as in Example 4-1, to obtain 90g of total effective component mixture; Example 4-3: 10g of cocrystal (containing 0.23g of ganoderic acid DM) + 60g of water-soluble component (take 300mL of concentrate, containing 19.2g of β-glucan) + 40g of small molecule peptide component (containing 17.6g of <5kDa peptide), the operation is the same as in Example 4-1, to obtain 110g of all-purpose component mixture.

[0077] Full-effect component content detection: Content of ganoderic acid DM and proportion of total triterpenes: Take 1g of the total effective component mixture, add 10mL of 80% methanol, extract 3 times by ultrasonication at 350W (30 minutes each time), centrifuge at 4000r / min for 15 minutes, combine the supernatants and make up to 25mL, and detect by HPLC (same as in Example 2: C18 column 250mm×4.6mm, 5μm; mobile phase methanol-0.1% phosphoric acid water 75:25; flow rate 1.0mL / min; detection wavelength 254nm), and calculate the content and proportion by external standard method.

[0078] Water-soluble polysaccharide (β-glucan) content: Take 1g of the total effective component mixture, add 20mL of deionized water and boil for 2 hours. After filtration, take 1mL of the filtrate, add 1mL of 5% phenol solution and 5mL of concentrated sulfuric acid, and incubate in a water bath at 40℃ for 20 minutes. Measure the absorbance at 490nm using a UV spectrophotometer. Plot a standard curve using dextran standards and calculate the β-glucan ratio.

[0079] Small molecule peptide content and percentage of <5kDa: Total peptide content: Take 1g of the total effective component mixture, add water to dissolve to 10mL, take 1mL and add 5mL of Coomassie brilliant blue reagent, measure the absorbance at 595nm, and calculate the total peptide content using bovine serum albumin as a standard. <5kDa percentage: Pass the above peptide solution through a 5kDa ultrafiltration membrane (operating pressure 0.2MPa, 30℃), collect the permeate and measure the peptide content, and calculate according to "<5kDa percentage = (permeate peptide content / total peptide content) × 100%".

[0080] Eutectic grain size D90: Take 0.1g of the total effective component mixture, add 10mL of deionized water, ultrasonically disperse at 300W for 5 minutes, measure the particle size distribution using a laser particle size analyzer (wet method), and read the D90 value.

[0081] Comparative Example 4-1 (Deviating from the composite ratio): 10g of co-crystal + 30g of water-soluble component + 10g of small molecule peptide component (ratio 1:3:1, exceeding the range of 1:4-6:2-4), the rest of the operation is the same as in Example 4-2; Comparative Example 4-2 (Defective Eutectic): Only 50g of water-soluble component + 30g of small molecule peptide component, no co-crystal, the rest of the operation is the same as in Example 4-2; Comparative Example 4-3 (Traditional Extraction Method): Take 100g of regular Ganoderma lucidum fruiting body, reflux extract with 70% ethanol for 2 hours (80℃), add 10 times the amount of water to the residue and boil for 2 hours, combine the extracts and concentrate to 100g, test the content of each component according to the same method, and compare with the total effective components of the present invention.

[0082] Results Analysis: The content of all active ingredients fully complies with the limits: The test results of Example 4-2 (core group) accurately matched the index limits. The content of ganoderic acid DM was 0.26%, which, although lower than the 2.3% of the co-crystal raw material due to compound dilution, was still within the reasonable range of "1.8%-5.0%". The total triterpenoid content was 52%, meeting the requirement of 45%-70%. The water-soluble polysaccharide (β-glucan) content was 17.8%, which met the dilution ratio of 26%-40%. Since the solid content in the whole effective component is about 50%, the actual β-glucan content in the solid content reached 35.6%, which fully met the standard. The proportion of small molecule peptides <5kDa was 88.0% (80%-95% range), and the co-crystal D90 = 155nm (100-200nm range), proving that the whole effective product prepared by the method of the present invention fully meets the quality standards.

[0083] The key impact of compound ratio on content synergy: The component contents of Example 4-2 (1:5:3) showed the best balance. Compared with Example 4-1 (1:4:2): the proportion of water-soluble components and small molecule peptides is slightly lower, and the content of ganoderic acid DM is slightly higher due to less dilution (0.33% vs 0.26%), but the total content of small molecule peptides is insufficient (25.1% vs 29.3%), resulting in a decrease in synergistic activity. Compared with Example 4-3 (1:6:4): the excessive water-soluble components further diluted ganoderic acid DM to 0.21%, which still met the standard, but the concentration of fat-soluble active ingredients decreased, affecting the application effect. In Comparative Example 4-1 (deviating from the ratio 1:3:1), due to insufficient water-soluble components, the cocrystals agglomerated (D90=220nm, exceeding the 100-200nm range), and the proportion of ganoderic acid DM dropped to 45% (close to the lower limit), proving that the composite ratio of "1:4-6:2-4" is the core to ensure uniform dispersion and content compliance of each component. Deviating from it will destroy the stability of the overall effective components.

[0084] The present invention has significant advantages in the content of all-purpose components: Compared with traditional extraction methods (Comparative Examples 4-3): the content of ganoderic acid DM (0.26% vs 0.09%) increased by 1.9 times, and the proportion of total triterpenes (52% vs 28%) increased by 85.7%, solving the pain points of "low content and poor specificity of fat-soluble active ingredients" in traditional methods; the content of water-soluble polysaccharides (17.8% vs 8.5%) increased by 1.1 times, the total content of small molecule peptides (29.3% vs 3.2%) increased by 8.2 times, and the proportion of <5kDa (88% vs 45%) doubled. This proves that the present invention achieves the simultaneous enrichment of three types of effective components of Ganoderma lucidum, namely "fat-soluble + water-soluble + peptides", through the whole process of "fermentation regulation-graded extraction-compound preparation", truly achieving the "all-round" effect.

[0085] The eutectic is the core active carrier of the all-around effective components: Comparative Example 4-2 (without co-crystal) had 0 ganoderic acid DM content. Although it had a high content of water-soluble polysaccharides and small molecule peptides, it could not achieve the comprehensive effect of "antioxidation-repair" due to the lack of synergistic effect of fat-soluble components. This further proves that the co-crystal construction in step D is an indispensable link of the all-effect components, and together with water-soluble components and small molecule peptides, it constitutes a "three-in-one" active system.

[0086] Example 5: Multi-component content analysis of the overall effective component Operating steps: Sample preparation: Take the all-purpose components of Example 4-2 (compound ratio 1:5:3), freeze-dry them, pulverize them and pass them through an 80-mesh sieve for later use.

[0087] Detection methods: Ganoderma lucidum polysaccharides: phenol-sulfuric acid method (using dextran as standard, detection wavelength 490nm); Ganoderma lucidum triterpenes: vanillin-glacial acetic acid method (using oleanolic acid as standard, detection wavelength 548nm); Total amino acids and free amino acids: automated amino acid analyzer (hydrolysis conditions: 6mol / L HCl, 110℃ hydrolysis for 24h; free amino acids are directly detected after water dissolution and filtration). Control group: Simultaneous detection of control ratios 4-3 (traditional Ganoderma lucidum fruiting body extract) Results analysis: The Ganoderma lucidum polysaccharide content in Example 4-2 (8.2%) was increased by 134% compared to the traditional method (3.5%). Cause Analysis: The fermentation medium in Example 2 used "Ganoderma lucidum mycelium residue hydrolysate + wolfberry powder". After enzymatic hydrolysis by cellulase (800 U / g) and xylanase (500 U / g), the mycelium residue released a large amount of polysaccharide precursors. Simultaneously, the two-stage fermentation process, regulating "aerobic proliferation (29℃, 180 r / min for 5 days) + anaerobic secondary metabolism (oxygen content ≤0.2%, ORP ≤-200 mV)," optimized the polysaccharide synthesis pathway of the Ganoderma lucidum strain, significantly increasing the accumulation of polysaccharides in the mycelium. Step C, combining "supercritical CO2 extraction (lipid-soluble components) + water extraction (water-soluble polysaccharides)," avoided polysaccharide degradation in organic solvents and fully recovered water-soluble polysaccharides from the mycelial residue through water extraction, achieving efficient polysaccharide enrichment.

[0088] The total triterpenoid content (4.5%) in Example 4-2 was increased by 114% compared to the conventional method (2.1%). Analysis of the cause: The synergistic effect of "anaerobic environment (oxygen content ≤0.2%, ORP ≤-200mV) + 15μmol / m² / s blue light irradiation" in Example 2 is key - the anaerobic environment forces Ganoderma lucidum cells to shift from primary metabolism to triterpenoid secondary metabolism; blue light of a specific intensity acts as an exogenous signal, further activating the key enzyme CYP450 for triterpenoid synthesis (enzyme activity reached 162U / mg in Example 2, which is 1.7 times that of the traditional method), greatly improving the efficiency of triterpenoid synthesis; supercritical CO2 extraction (pressure 30MPa, temperature 45℃) specifically and efficiently extracts fat-soluble triterpenoids, ensuring the yield of total triterpenoids.

[0089] The total amino acid content of Example 4-2 (12.8%) is nearly three times that of the traditional method (4.3%), and the proportion of essential amino acids (42.5%) is also higher than that of the traditional method (28.7%). Analysis of causes: The fermentation medium of "goji berry powder + rice bran" provides abundant amino acid precursors (goji berry powder contains a variety of free amino acids, and rice bran is rich in essential amino acids such as lysine and leucine); the separation process of "8-12kDa ultrafiltration membrane" in step C not only efficiently enriches small molecule peptides (reaching 29.3% in Example 4-2, compared to only 3.2% in the traditional method), but also retains and concentrates free amino acids (especially essential amino acids with small molecular weights that easily pass through the membrane), which significantly enhances the synergistic effect of amino acid nutrition and activity of the whole component.

[0090] Combined with the verified results of ganoderic acid DM (0.26% vs 0.09%) and β-glucan (17.8% vs 8.5%) in Example 4, this multi-component analysis further proves that: through the whole-chain innovation of “strain pretreatment (Example 1) → two-stage fermentation regulation (Example 2) → graded extraction (Example 3) → co-crystal construction (Example 3) → compound preparation (Example 4)”, this invention achieves the simultaneous and efficient enrichment of four major categories of active ingredients: polysaccharides (Ganoderma lucidum polysaccharides, β-glucan), triterpenes (total triterpenes, ganoderic acid DM), peptides (small molecule peptides), and amino acids (total amino acids, essential amino acids), truly achieving the technical goal of “all-effect components”.

[0091] The above are merely embodiments of the present invention. The circuits, electronic components, and modules involved are all prior art, fully achievable by those skilled in the art, and require no further explanation. The scope of protection in this application does not involve improvements to the software and methods. Commonly known structures and characteristics in the solutions are not described in detail here. Those skilled in the art are aware of all common technical knowledge in the field prior to the application date or priority date, are aware of all prior art in that field, and have the ability to apply conventional experimental methods prior to that date. Those skilled in the art can, under the guidance of this application, improve and implement this solution in combination with their own capabilities. Some typical known structures or methods should not be obstacles for those skilled in the art to implement this application. It should be noted that those skilled in the art can make several modifications and improvements without departing from the structure of the present invention. These should also be considered within the scope of protection of the present invention, and will not affect the effectiveness of the implementation of the present invention or the practicality of the patent.

Claims

1. A method for preparing Ganoderma lucidum with all-purpose components by fermentation, characterized in that: Includes the following steps: A. Pretreatment of the strain: Ganoderma lucidum strain CGMCC 5.1816 was inoculated into an acclimatization medium containing corn steep liquor and Tween-80, and cultured with shaking at 25-28℃ for 5-7 days to obtain an adapted strain; then it was transferred to an activation medium containing wheat bran extract and β-cyclodextrin, and cultured statically at 27-29℃ for 40-50 hours to obtain activated mycelial moss; B, Two-stage fermentation control: B1, Aerobic stage: Inoculate the activated mycelium with a 12%-18% inoculation amount into a fermentation medium containing Ganoderma lucidum residue hydrolysate and wolfberry powder, and culture at 28-30℃ and 170-190r / min for 4-6 days; B2, Anaerobic Induction Stage: Add 0.08-0.12% methyl jasmonate to the fermentation system, replace with nitrogen until the oxygen content is ≤0.2%, and irradiate with 460nm, 10-20μmol / m² / s blue light for the first 6 hours of the anaerobic induction stage, and then let it ferment in the dark at 24-26℃ for 18-22 days. C. Fractional Extraction: The fermentation broth is centrifuged at 3500-4500 r / min for 12-18 minutes to separate the mycelium from the supernatant. The solid residue remaining after supercritical CO2 extraction of the mycelium is the residue, which is then extracted with water to obtain water-soluble components. The water-soluble components mainly contain water-soluble polysaccharides. The supernatant is filtered through an 8-12 kDa ultrafiltration membrane to obtain small molecule peptide components. The supercritical CO2 extract is a lipid-soluble component. D. Construction of co-crystal: Ganoderma lucidum acid DM in the fat-soluble component and Lycium barbarum polysaccharide in the water-soluble component are co-dissolved at a mass ratio of 1:1-1:3, the mass ratio corresponding to a molar ratio of 1:1.0-1:3.2; the mixture is dissolved in an ethanol-water system and spray-dried to obtain a co-crystal with a particle size D90 < 200 nm; E. Composite preparation: The co-crystal is mixed with the water-soluble component and the small molecule peptide component in a ratio of 1:4-6:2-4 to obtain the Ganoderma lucidum product with all-effect components.

2. The method for preparing Ganoderma lucidum fermentation of the all-purpose components as described in claim 1, characterized in that: In step A, the acclimatization culture medium contains 1.0%-2.0% corn steep liquor, 1.0%-1.5% glucose, 0.01%-0.03% Tween-80, and 0.05%-0.1% MgSO4, with a shaking speed of 120-150 r / min; the activation culture medium contains 2%-4% wheat bran extract, 0.3%-0.7% yeast extract, and 0.2%-0.4% β-cyclodextrin.

3. The method for preparing Ganoderma lucidum fermentation of the all-purpose components as described in claim 1, characterized in that: In step B1, the fermentation culture medium also contains 2%-4% rice bran and 0.03%-0.07% complex mineral elements; the preparation method of the Ganoderma lucidum residue hydrolysate is to mix Ganoderma lucidum residue and water at a ratio of 1:7-9, add 700-900U / g cellulase and 400-600U / g xylanase, enzymatically hydrolyze at 50-60℃ for 5-7 hours, and then filter to obtain the supernatant of the fermentation broth by centrifugation.

4. The method for preparing Ganoderma lucidum fermentation of the all-purpose components as described in claim 1, characterized in that: In step B2, the blue light source is an LED cold light source; during static fermentation, the ORP is maintained at -200mV by monitoring the redox potential.

5. The method for preparing Ganoderma lucidum fermentation of the all-purpose components as described in claim 1, characterized in that: In step C, the supercritical CO2 extraction pressure is 28-32 MPa and the temperature is 43-47℃; the water extraction conditions are: the residue is boiled with water for 2-3 hours, and the filtrate is taken as the water-soluble component after filtration.

6. The method for preparing Ganoderma lucidum fermentation of the all-purpose components as described in claim 1, characterized in that: In step D, the volume ratio of the ethanol-water system is 6:4-8:2; the spray drying inlet temperature is 170-190℃ and the outlet temperature is 70-90℃; the eutectic is an amorphous solid dispersion, and no characteristic crystal peaks are detected by X-ray powder diffraction.

7. A Ganoderma lucidum product with all-around active ingredients, prepared by the method according to any one of claims 1-6, characterized in that, The product comprises the co-crystal from step D, the water-soluble polysaccharide from step C, and small molecule peptide components; the particle size D90 of the co-crystal is 100-200 nm; the content of ganoderic acid DM in the co-crystal is 1.8%-5.0%, and the mass ratio of ganoderic acid DM to total triterpenes is 45%-70%; the water solubility of the co-crystal is 30-60 mg / mL; the water-soluble polysaccharide is 26%-40% based on β-glucan; and the proportion of <5 kDa components in the small molecule peptides is 80%-95%.

8. The Ganoderma lucidum product with the complete active ingredients as described in claim 7, characterized in that, The Ganoderma lucidum products of the total effective components have characteristic peaks as detected by high performance liquid chromatography. The retention time of 8.2±0.1 min corresponds to ganoderic acid DM, and 12.5±0.1 min corresponds to lycopene polysaccharide.

9. The application of the Ganoderma lucidum fermentation preparation method of the all-purpose components as described in any one of claims 1 to 6 in the extraction of effective components from Ganoderma lucidum.

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