A process for fermenting Ganoderma lucidum with Aspergillus cristatus

Abstract

The invention relates to the technical field of ganoderma lucidum fermentation technology, and particularly discloses a ganoderma lucidum fermentation technology of Aspergillus cristatus, comprising: S1, transferring a bacterial block to a 5% NaCl+MYA solid culture medium in a clean bench, culturing at a constant temperature and humidity of 28 DEG C for 7 days, and repeating this process twice to activate the strain; inoculating the activated bacterial strain onto a 5% NaCl+MYA plate culture medium, and culturing at a constant temperature and humidity of 28 DEG C for 7 days; adding an appropriate amount of sterile water to the Aspergillus cristatus plate cultured for 7 days, scraping spores, taking 1 mL of spore liquid and adding it to a centrifuge tube containing glass beads, shaking to break the ascospores, counting them with a hemocytometer, and diluting them into a spore suspension of corresponding concentration; S2, weighing an appropriate amount of ganoderma lucidum blocks into a fermentation bottle, adding distilled water, sterilizing at 121 DEG C for 20 minutes, cooling to room temperature, inoculating an appropriate amount of spore suspension, and culturing at a constant temperature and humidity of 28 DEG C for 10 days. The *Aspergillus cristatus*-fermented *Ganoderma lucidum* fruiting bodies obtained by this invention exhibit high bioactivity, and the bitterness of *Ganoderma lucidum* is significantly improved.

Description

Technical Field

[0001] This invention patent relates to the field of fermentation Ganoderma lucidum technology, specifically to a process for fermenting Ganoderma lucidum with Aspergillus cristatus. Background Art

[0002] Currently, Ganoderma lucidum production typically uses high-quality Ganoderma lucidum mycelium. Existing technology employs liquid fermentation to cultivate this mycelium. First, the mycelium is inoculated into a liquid culture medium containing nutrients such as glucose, yeast extract, and acidic protease, while controlling pH and temperature to allow for suitable growth and reproduction. The cultivated mycelium is then separated, washed, and dried for subsequent fermentation. During the mycelial culture process, the treated mycelium is inoculated into a fermenter, with appropriate amounts of nutrients such as glucose and yeast extract added, and pH and temperature controlled to allow for growth and reproduction. Controlling multiple conditions during fermentation is crucial to ensure the healthy growth and reproduction of Ganoderma lucidum. During fermentation, the mycelium produces active ingredients such as polysaccharides and triterpenoids, which have beneficial health effects. After fermentation, extraction and purification processes are performed to obtain the final Ganoderma lucidum product. Another method is solid-state fermentation, using the fruiting body of Ganoderma lucidum for fermentation; however, this method and the materials used for fermentation are less commonly used currently.

[0003] Existing fermentation processes for Ganoderma lucidum produce products with poor biological activity, a noticeable bitter taste, and low DPPH scavenging rates. Literature reports that fermenting food-medicine homologous substances with probiotics can simultaneously exert the efficacy of these substances and the function of probiotics in regulating intestinal flora, while also improving the taste and flavor of the product. Therefore, the inventors of this application have researched, optimized, and improved the fermentation process for Ganoderma lucidum, providing a novel Aspergillus cristatus fermentation process. Summary of the Invention

[0004] The purpose of this invention is to solve the technical problems mentioned in the background art and to provide a process for fermenting Ganoderma lucidum with Aspergillus cristatus. This process improves the bioactivity of the fermented Ganoderma lucidum fruiting bodies and reduces the bitterness of the Ganoderma lucidum.

[0005] The above-mentioned objective of the present invention is achieved as follows:

[0006] A process for fermenting Ganoderma lucidum with Aspergillus cristatus, the process comprising the following steps:

[0007] S1. Strain activation and culture:

[0008] The bacterial blocks stored at -80 ℃ were transferred to 5% NaCl+MYA solid medium in a clean bench and cultured at 28 ℃ for 7 days. This process was repeated twice to activate the strain. Then, the activated strain was inoculated onto 5% NaCl+MYA plate medium and cultured at 28 ℃ for 7 days for later use.

[0009] S2, Fermentation process:

[0010] Add an appropriate amount of sterile water to a plate of Aspergillus cristatus that has been cultured for 7 days, scrape off the spores, and add the spore suspension to a centrifuge tube containing glass beads. Shake to break the ascospores, count them with a hemocytometer, and finally dilute to the appropriate concentration of spore suspension for later use.

[0011] Weigh an appropriate amount of Ganoderma lucidum pieces into a fermentation bottle, add an appropriate amount of distilled water, sterilize at 121 ℃ for 20 min, cool to room temperature, inoculate with an appropriate amount of spore suspension, and culture at 28 ℃ under constant temperature and humidity for 10 days.

[0012] Furthermore, the water content of the spore suspension is 80%.

[0013] Furthermore, the inoculation volume of the spore suspension is 500 μL.

[0014] Furthermore, the spore concentration of the spore suspension is 10. 4 pcs / mL.

[0015] Furthermore, the fermented Ganoderma lucidum obtained by the aforementioned process is used in the preparation of products that protect against alcoholic liver damage.

[0016] Furthermore, the product is a health supplement or a medicine.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The fermentation process of Ganoderma lucidum of the present invention improves and optimizes the design of moisture content, fermentation time, fermentation temperature, spore suspension concentration and inoculation amount. The Ganoderma lucidum fruiting bodies fermented by Aspergillus cristatus obtained by the newly designed fermentation process have high biological activity and the bitterness of Ganoderma lucidum is significantly improved.

[0019] 2. The fermented Ganoderma lucidum product prepared by the process of the present invention has significantly improved DPPH scavenging rate and LPO inhibition rate compared with the existing process, and has high antioxidant activity, thus significantly improving the quality of the fermented Ganoderma lucidum product.

[0020] 3. The fermented Ganoderma lucidum product obtained by the process of the present invention can be used in the preparation of products that protect against alcoholic liver damage. Attached Figure Description

[0021] Figure 1 The effects of different single factors on DPPH scavenging rate in the embodiments of the present invention (moisture content (a), fermentation time (b), spore concentration (c), inoculum size (d), temperature (e)).

[0022] Figure 2 These are 3D response surface plots and contour lines showing the effects of interaction terms of different independent variables on DPPH clearance rate in embodiments of the present invention (temperature-moisture content interaction terms (a, b); time-moisture content interaction terms (c, d); inoculum size-moisture content interaction terms (e, f)).

[0023] Figure 3 These are the standard curves for total triterpenes, total polysaccharides, amino acids, alkaloids, and flavonoids in the embodiments of the present invention (total triterpenes (a), total polysaccharides (b), amino acids (c), alkaloids (d), and flavonoids (e)).

[0024] Figure 4 These are the antioxidant activity measurement results (hydroxyl radical scavenging rate (a), DPPH scavenging rate (b), LPO inhibition rate (c)) in the embodiments of the present invention.

[0025] Figure 5 This is a PCA score graph analysis in an embodiment of the present invention;

[0026] Figure 6 This is the OPLS-DA score map in an embodiment of the present invention;

[0027] Figure 7 These are the permutation test results in the embodiments of the present invention;

[0028] Figure 8 This is the enrichment of the top 135 KEGG functional pathways in the embodiments of the present invention;

[0029] Figure 9 This is an analysis of antioxidant activity-related metabolites (hydroxyl radical scavenging rate (a), DPPH scavenging rate (b), LPO inhibition rate (c)) in the embodiments of this invention.

[0030] Figure 10 This is a classification of potential bitter metabolites in Ganoderma lucidum in the embodiments of the present invention;

[0031] Figure 11 These are the bitter differential metabolites that are significantly upregulated in the embodiments of this invention;

[0032] Figure 12 These are bitter differential metabolites that are significantly downregulated in the embodiments of the present invention;

[0033] Figure 13 This is a pathway enrichment analysis of potential bitter metabolites in the embodiments of the present invention;

[0034] Figure 14 This is a pathway enrichment analysis of differential bitter metabolites in the embodiments of the present invention;

[0035] Figure 15 This is the map00941-flavonoid biosynthesis pathway in the embodiments of the present invention;

[0036] Figure 16 This is the map00940-phenylpropane biosynthesis pathway in the embodiments of the present invention;

[0037] Figure 17 This is the map00943-isoflavone biosynthesis pathway in the embodiments of the present invention;

[0038] Figure 18 This is a volcano diagram from an embodiment of the present invention;

[0039] Figure 19 It is the flavoring substance in the embodiments of the present invention;

[0040] Figure 20 This is a comparison result between the template selected by SWISS-MODEL and the target sequence of TAS2R14 in this embodiment of the invention;

[0041] Figure 21 It is a homologous model of TAS2R14 in the embodiments of the present invention;

[0042] Figure 22 This is a Laplace diagram in an embodiment of the present invention;

[0043] Figure 23 This is a visualization of the docking results of the homology model and bitter substances in the embodiments of the present invention (ganoderic acid E(a), isoflavones(b), ganoderic acid A(c), matrine(d), glycyrrhizin(e), quinic acid(f)).

[0044] Figure 24 This describes the effect of ethanol on LX-2 proliferation in the embodiments of the present invention;

[0045] Figure 25 This invention relates to the effects of different sample intervention concentrations on cell viability in the embodiments of the present invention (a: effect of unfermented Ganoderma lucidum aqueous extract on LX-2 cell viability; b: effect of fermented Ganoderma lucidum aqueous extract on LX-2 cell viability; c: effect of unfermented Ganoderma lucidum ethanol extract on LX-2 cell viability; d: effect of fermented Ganoderma lucidum ethanol extract on LX-2 cell viability).

[0046] Figure 26 This describes the effect of different Ganoderma lucidum treatment groups on LDH in LX-2 cells with alcoholic liver injury in the embodiments of the present invention;

[0047] Figure 27This invention relates to the effects of different Ganoderma lucidum treatment groups on the AST and ALT levels in cells of an alcoholic liver injury model.

[0048] Figure 28 The activities of cell antioxidant enzymes in the embodiments of the present invention are (a: the content of reduced glutathione in cells of alcoholic liver injury model in different Ganoderma lucidum treatment groups; b: the activity of catalase in cells of alcoholic liver injury model in different Ganoderma lucidum treatment groups). Detailed Implementation

[0049] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0050] The implementation of the present invention will be described in detail below with reference to specific embodiments.

[0051] The following are preferred embodiments provided by the present invention. Example

[0052] The present invention provides a process for fermenting Ganoderma lucidum with Aspergillus cristatus, which includes the following steps:

[0053] S1. Strain activation and culture: The bacterial blocks stored at -80℃ were transferred to 5% NaCl + MYA solid medium in a clean bench and cultured at 28℃ and constant humidity for 7 days. This process was repeated twice to activate the strain. Then, the activated strain was inoculated onto 5% NaCl + MYA agar plates and cultured at 28℃ and constant humidity for 7 days for later use.

[0054] S2. Fermentation Process: Add an appropriate amount of sterile water to *Aspergillus cristatus* plates that have been cultured for 7 days. Spores are scraped off, and the spore suspension is transferred to a centrifuge tube containing glass beads. Shake to break the ascospore walls, count the spores using a hemocytometer, and finally dilute to the appropriate concentration of spore suspension for later use. Weigh an appropriate amount of *Ganoderma lucidum* blocks into a fermentation flask, add an appropriate amount of distilled water, sterilize at 121 ℃ for 20 min, cool to room temperature, inoculate with an appropriate amount of spore suspension, and culture at 28 ℃ under constant temperature and humidity for 10 days.

[0055] In this embodiment, the water content of the spore suspension was 80%. The inoculum size of the spore suspension was 500 μL. The spore concentration of the spore suspension was 10. 8 pcs / mL. The application of fermented Ganoderma lucidum prepared using this process in the preparation of products that protect against alcoholic liver injury, such as health supplements or pharmaceuticals.

[0056] The following is an experiment of the embodiment of the present invention:

[0057] I. Single-factor experiment:

[0058] Place an appropriate amount of Ganoderma lucidum in a fermentation flask, add 40-120% distilled water at a weight:volume (w / v), sterilize at 121℃ for 20 minutes, cool to room temperature, and inoculate with 10... 4 -10 8 Spore suspensions of 100-900 μL were incubated at 20-36℃ for 6-14 days. The effects of moisture content, fermentation time, spore concentration, inoculum size, and fermentation temperature on DPPH scavenging rate were investigated in that order.

[0059] II. Optimization of Fermentation Process Using Box-Behnken Experimental Design:

[0060] Based on the results of the single-factor experiments above, moisture content (A), temperature (B), time (C), and inoculum size (D) were selected as independent variables, and DPPH scavenging rate was selected as the response value. A functional relationship between the independent variables and the response value was established, and the optimal fermentation process conditions for Aspergillus cristatus fermentation of Ganoderma lucidum fruiting bodies to improve biological activity and reduce bitterness were obtained.

[0061]

[0062] III. Verification of the optimal encapsulation process:

[0063] Based on the optimal gradient determined by the single-factor experiments, and the optimal moisture content, time, temperature, and inoculum quantity process parameters determined by the four-factor, three-level Box-Behnken experiments, three sets of parallel experiments were conducted and the average value was taken.

[0064] IV. Sensory Evaluation:

[0065] Weigh an appropriate amount of Ganoderma lucidum, steep in water at a temperature >95 ℃ for 2 min (Xie Sandu et al., 2021), and filter to remove residue. Referring to previously reported methods (Yu Mingguang, 2020; Zhang Qianhui et al., 2022; Zhang Pu et al., 2021), bitterness was divided into 5 levels, each corresponding to a specific bitterness range. The sensory evaluation panel consisted of 10 healthy sensory evaluators (5 males and 5 females, aged 18-28 years) with regular sleep schedules. The scoring criteria are shown in Table 1.2.

[0066]

[0067] V. Methods for testing physicochemical indicators:

[0068] 1. Ganoderma lucidum polysaccharides (phenol-sulfuric acid process)

[0069] The determination was performed using the phenol-sulfuric acid method.

[0070] 2. Ganoderma triterpenes (glacial acetic acid-vanillin-perchloric acid method)

[0071] Refer to the methods in the 2020 edition of the Chinese Pharmacopoeia and NY / T3676-2020.

[0072] 3. Amino acids, total flavonoids, alkaloids

[0073] The assay was performed using the kit, and the standard curve and sample assay methods were performed according to the kit instructions.

[0074] VI. In vitro antioxidant activity assessment:

[0075] The antioxidant capacity before and after fermentation was evaluated by hydroxyl radical scavenging rate, DPPH scavenging rate, and LPO inhibition rate. The assays were performed using a kit; the assay methods are detailed in the kit instructions.

[0076] VII. Metabolomics Analysis:

[0077] 1. Metabolomics Data Analysis Methods

[0078] The raw data was processed using Analysis (v10.0.707.0) software to generate a three-dimensional data matrix. During data processing, internal standard peaks and known false positive peaks were excluded, and redundancy removal and peak merging were performed. Metabolites were also identified using database searches, primarily public databases such as NIST (2017), Fiehn (2013), and MS-DIAL (2021).

[0079] 2. Screening of differentially expressed bitter metabolites in Ganoderma lucidum based on metabolomics analysis:

[0080] Potential bitter substances were screened using the public bitterness database BitterDB, the Chemical Book website, and relevant literature. Metabolites with a VIP ≥ 1 were selected as candidate differentially expressed metabolites using the OPLS-DA model, and a t-test was further applied to assess their significance. When the difference between candidate differentially expressed metabolites reached or exceeded the preset significance level (…),… p When the value is <0.05, it is identified as a true differential metabolite.

[0081] 3. Verification of bitter substances:

[0082] Bioinformatics methods were used to screen bitter taste receptors, perform homology modeling, molecular docking, and visualize the docking results. By analyzing the interaction results between bitter taste receptors and ligands, it was verified whether the screened potential bitter substances were indeed bitter substances from Ganoderma lucidum.

[0083] VIII. Research on the protective function against alcoholic liver injury

[0084] 1. Sample preparation:

[0085] Weigh appropriate amounts of Ganoderma lucidum powder before and after fermentation, and perform water extraction and alcohol extraction respectively. After filtration and concentration of the extracted filtrate, freeze-dry to obtain lyophilized powder. Dissolve in DMSO, filter through a 0.22 μm microporous membrane, and adjust to the required concentration using incomplete culture medium.

[0086] 2. Cell culture:

[0087] (1) Cell resuscitation

[0088] After removing LX-2 cells from the liquid nitrogen container, rapidly thaw them in a 37°C water bath. Centrifuge and remove the culture medium, then resuspend the cells in preheated culture medium. Seed the cells into cell culture flasks and incubate at 37°C with 5% CO2. Once the cells have reached approximately 80-100% confluence, they can be passaged. Newly revived cells should be passaged once before being used for subsequent experiments.

[0089] (2) Cell passage

[0090] Take a cell culture flask with 80-100% cell growth, remove the culture medium, and wash with PBS to remove the PBS. Add trypsin for digestion, examine under a microscope, and when digestion is complete, add 3 times the amount of trypsin and growth medium to stop digestion. After centrifugation, remove the culture medium and aliquot the cells into new culture dishes at a ratio of 1:2 to 1:4 for further culture.

[0091] (3) Cell cryopreservation

[0092] Take a cell culture flask with cells at 80-100% confluence, remove the culture medium, wash with PBS, remove the PBS, digest with trypsin, and examine under a microscope. Once digestion is complete, add 3 times the volume of complete culture medium to stop the digestion. After centrifugation to remove the culture medium, add an appropriate amount of cryopreservation solution, pipette to form a suspension, and transfer the suspension to cryovials. Label the cryovials with the cell type and record the passage number and date. Finally, place them in a cryopreservation box and freeze at progressively decreasing temperatures.

[0093] 3. Determination of the effective concentration of the model group

[0094] LX-2 cells were seeded in 96-well plates and cultured. Each group had 6 replicates, with a control group and a model group. The control group was cultured in incomplete medium, while the model group was cultured in medium containing a series of gradient concentrations of ethanol. The plates were incubated at 37°C in a 5% CO2 incubator. Cell viability was measured using the CCK-8 assay 24 hours after cell adhesion.

[0095] Cell viability = .

[0096] 4. Determination of the effective concentration in the experimental group

[0097] LX-2 cells in logarithmic growth phase were collected and seeded into 96-well plates. Six replicates were placed in each well, and the plates were incubated at 37 ℃ in a 5% CO2 incubator. After 24 hours of ethanol-based cell culture to model cell adhesion, the medium was replaced with fresh medium. Control and experimental groups were established. The control group was cultured in incomplete medium, while the experimental groups were cultured in medium containing a series of sample concentrations. All cells were incubated at 37 ℃ in a 5% CO2 incubator for 24 hours. Cell viability was determined using the CCK-8 assay.

[0098] 5. Effects of the experimental group on oxidative damage in LX-2 cells

[0099] A control group, a model group, and an experimental group combined with alcohol treatment were set up. After 48 hours of cell culture, the leakage rate of lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), reduced glutathione (GSH) content, and catalase (CAT) activity were measured in each group.

[0100] IX. Research Results:

[0101] 1. Optimization of the fermentation process of Ganoderma lucidum by Aspergillus cristatus and analysis of its metabolomics results

[0102] 1.1 Analysis of Single-Factor Experiment Results

[0103] 1.1.1 Effect of moisture content on DPPH scavenging rate after fermentation

[0104] like Figure 1 The results in section a showed that within the moisture content range of 40-80%, the DPPH removal rate increased with increasing moisture content; however, a decreasing trend appeared after exceeding 80%, and no significant difference was observed between 80%, 100%, and 120%. If the moisture content of the fermentation substrate is too low, it will become too dry, thus inhibiting the growth and reproduction of microorganisms and adversely affecting metabolism; if the moisture content is too high, it will hinder oxygen transport and increase the possibility of contamination by other microorganisms.

[0105] 1.1.2 Effect of fermentation time on DPPH scavenging rate after fermentation

[0106] like Figure 1 As shown in b, when the fermentation time is between 6 and 10 days, the DPPH scavenging rate shows a significant upward trend with increasing time; after 10 days, the upward trend becomes less pronounced, and there is no significant difference between 10, 12, and 14 days. Fermentation time plays a crucial role in the growth and reproduction of microorganisms, and microbial metabolites undergo certain changes with prolonged culture time. In the primary growth stage of microorganisms, the quantity of certain secondary metabolites gradually increases with increasing culture time; after a certain period, these secondary metabolites may gradually degrade.

[0107] 1.1.3 Effect of spore concentration on DPPH scavenging rate after fermentation

[0108] like Figure 1 As shown in c, the DPPH scavenging rates at different gradients within each group increased with increasing spore concentration; however, statistical analysis revealed no significant differences among them. This indicates that spore concentration had no significant effect on the response value within the experimental range.

[0109] 1.1.4 Effect of Temperature on DPPH Scavenging Rate After Fermentation

[0110] like Figure 1 As shown in d, the DPPH scavenging rate showed a significant upward trend with increasing temperature between 20-28 ℃, but a significant downward trend appeared after 28 ℃. The optimal temperature range for Aspergillus cristatus growth is generally 25-30 ℃. Excessively high or low temperatures may lead to changes in the morphological characteristics and metabolic direction of microorganisms, and even the production of toxic substances, thus causing death (Jia Zhihua et al., 2010).

[0111] 1.1.5 Effect of inoculum size on DPPH scavenging rate after fermentation

[0112] according to Figure 1 The results from study e showed that, between 100-500 μL inoculum size, the DPPH clearance rate increased significantly with increasing inoculum size. However, after exceeding 500 μL, the upward trend in DPPH clearance rate became less pronounced, and a significant downward trend emerged. This may be because excessive inoculum size leads to rapid bacterial growth and competition for survival. Excessive inoculum size may also result in insufficient dissolved oxygen, thereby reducing the formation rate of metabolites and introducing excessive metabolic waste, ultimately reducing economic efficiency.

[0113] 1.2 Analysis of Box-Behnken Experimental Design Results:

[0114] Four factors that significantly affected the response value within the experimental range—moisture content (A), temperature (B), time (C), and inoculum size (D)—were selected as independent variables, with DPPH scavenging rate as the response value. A Box-Behnken optimization experiment with four factors and three levels was designed to obtain the optimal fermentation process parameters. The experimental design and results are shown in Table 6, and the results of the regression analysis of variance are shown in Table 7. The regression equations of DPPH scavenging rate on response variables A, B, C, and D are as follows:

[0115] Y=92.11+1.14A+1.16B+0.62C+0.43D+0.69AB+0.27AC+0.49AD+0.54BC+0.52BD+0.48CD-0.81A2 -1.62B 2 -0.86C 2 -1.41D 2 .

[0116] As shown in Table 6, the model has an F-value of 33.17 and P < 0.0001, indicating a lack of fit. p =0.5417>0.05, indicating that the quadratic model used in the experiment reached a highly significant level and the model is reliable. 2 =0.9707 and R 2 Adj The values ​​of 0.9415 are all approximately equal to 1, indicating a high accuracy in predicting the response value. 2 Adj With R 2 Pred The difference between the actual and predicted values ​​is less than 0.2, CV% = 0.44 < 10, and Adeq Precision = 20.209 > 4, indicating a good fit between the actual and predicted values, and that the model has excellent accuracy and repeatability. In summary, this regression equation can be used to optimize the fermentation process conditions of *Aspergillus cristatus* on *Ganoderma lucidum* fruiting bodies and to analyze and predict its DPPH scavenging rate. Only the AC interaction term in the regression equation is insignificant, indicating that all other terms have a significant impact on the DPPH scavenging rate. The order of influence of the experimental factors involved in the optimization on the DPPH scavenging rate is: temperature (B) > moisture content (A) > time (C) > inoculum size (D).

[0117]

[0118]

[0119]

[0120] 1.3 Response surface analysis of interactions between factors:

[0121] Figure 2 Tables a and b show the interaction between moisture content and temperature on DPPH removal rate, indicating that they are the most important factors in the experimental results. Increasing moisture content significantly improved DPPH removal rate, and this effect was further enhanced with increasing temperature. This interaction result highlights the need to consider both factors comprehensively in practical applications to achieve optimal DPPH removal performance. This is consistent with the conclusions drawn from the regression analysis of variance in Table 7.

[0122] 1.4 Analysis of the verification results of the optimal inclusion-based bitterness removal process:

[0123] As shown in Table 8, the optimal fermentation conditions for Ganoderma lucidum fruiting bodies using Aspergillus cristatus and their predicted values ​​were obtained from the model prediction group. After adjusting the optimal process to reflect actual conditions, the actual DPPH scavenging rate showed no significant difference from the predicted value, indicating a high degree of fit between the model and reality. The resulting optimal fermentation process is stable and feasible. Compared to the original values, the DPPH scavenging rate in the treatment group increased by 25.62%.

[0124]

[0125] Note: Different lowercase letters indicate significant differences. p < 0.05).

[0126] 1.5 Analysis of Sensory Evaluation Results:

[0127] The results are shown in Table 9. After fermentation with *Aspergillus cristatus*, the medicinal flavor of *Ganoderma lucidum* was significantly reduced, and it acquired a delicate fungal aroma. Although the bitterness value of *Ganoderma lucidum* before and after fermentation did not differ significantly, the taste was improved to some extent after fermentation, with reduced bitterness and a milder flavor. During fermentation, certain components in *Ganoderma lucidum* may be decomposed or transformed by microorganisms, generating new compounds. Some of these newly generated compounds can neutralize the original bitterness of *Ganoderma lucidum*, making the taste more mellow.

[0128]

[0129] Note: Different lowercase letters indicate significant differences. p < 0.05).

[0130] 1.6 Analysis of the results of the determination of the content of the main active ingredients:

[0131] 1.6.1 Plotting the Standard Curve:

[0132] The standard curves for total triterpenes, total polysaccharides, amino acids, alkaloids, and flavonoids obtained are as follows: Figure 3 As shown.

[0133] 1.6.2 Analysis of the results of the determination of the content of the main active ingredients:

[0134] The contents of the main active components of Ganoderma lucidum, namely polysaccharides, triterpenes, amino acids, alkaloids, and flavonoids, were determined, and the results are shown in Table 10. During the fermentation process, the contents of polysaccharides, amino acids, and total flavonoids decreased significantly. p < 0.05), decreased by 24.98%, 13.46%, and 39.13% respectively, while the alkaloid content increased significantly ( p < 0.05), increased by 24.64%, while the triterpene content did not change significantly before and after fermentation. p> 0.05). The main reason for the decrease in polysaccharides is the consumption of nutrients required for microbial growth during fermentation. For filamentous fungi, secondary metabolites are usually generated after the rapid growth phase (trophic phase), and then begin to be generated in the subsequent production phase or mid-phase. In this case, when the depletion of key nutrients limits its growth, secondary metabolism is initiated, at which point carbon (glucose), nitrogen, or phosphate is consumed, and a phase with low or zero proliferation rate but high yield is entered.

[0135]

[0136] Note: Different lowercase letters indicate significant differences. p < 0.05).

[0137] 1.7 Analysis of Antioxidant Activity Results:

[0138] like Figure 4 As shown, the antioxidant activity of Ganoderma lucidum before and after fermentation showed significant differences. p < 0.05). After fermentation, the antioxidant activity of Ganoderma lucidum was significantly enhanced. The hydroxyl radical scavenging rate, DPPH scavenging rate, and LPO inhibition rate of the aqueous extract increased by 3.69%, 22.50%, and 5.98%, respectively; while those of the hydroxyl radical scavenging rate, DPPH scavenging rate, and LPO inhibition rate of the ethanol extract increased by 2.82%, 8.18%, and 8.42%, respectively. Furthermore, the ethanol extract exhibited higher antioxidant capacity than the aqueous extract, with hydroxyl radical scavenging rate, DPPH scavenging rate, and LPO inhibition rate being 2.82%, 8.18%, and 39.54% higher, respectively. The chosen solvent system and extraction process affect antioxidant activity. Most of the main active components of Ganoderma lucidum, such as triterpenoids, flavonoids, and alkaloids, are soluble in organic solvents but insoluble or poorly soluble in water.

[0139] 2. Analysis of metabolomics results:

[0140] 2.1 Principal Component Analysis (PCA)

[0141] according to Figure 5 Principal component analysis results showed that the results for both pre- and post-fermentation samples of Ganoderma lucidum were within the 95% confidence interval, and there were significant differences between the two groups, indicating that fermentation significantly affected the separation energy. Based on the actual situation, it is further inferred that chemical changes or the formation of biologically active substances may have occurred during fermentation, leading to the significant differences in Ganoderma lucidum before and after fermentation. These changes or the formation of substances may be related to the combined effects of multiple factors such as microbial activity, substrate type, temperature, and time.

[0142] 2.2 Orthogonal Partial Least Significance Discriminant Analysis (OPLS-DA)

[0143] like Figure 6 As shown in the OPLS-DA score plot, all samples were within the 95% confidence interval, and the replicates of each group were close together and clustered, indicating good data reproducibility. Meanwhile, there were significant differences between the two groups, indicating significant changes in related metabolites before and after Ganoderma lucidum fermentation. Figure 7 The permutation test results showed that the Q2 point obtained by the random sorting method was lower than the original R2 point, further indicating that the model did not exhibit overfitting and had good robustness. Furthermore, all samples were within the 95% confidence interval, confirming the reliability of the experimental data. Subsequent analysis using the VIP value of the OPLS-DA model can be used to screen for differentially metabolites before and after Ganoderma lucidum fermentation.

[0144] 2.3 Screening of differentially expressed metabolites related to antioxidant activity

[0145] According to the analysis results of OPLS-DA, VIP > 1.2 and p A total of 468 metabolites with a value < 0.05 were identified, and these 468 metabolites played an important role in the OPLS-DA discrimination results. Correlation analysis of hydroxyl radical scavenging rate, DPPH scavenging rate, and LPO inhibition rate revealed 135 differentially expressed metabolites associated with antioxidant activity, of which 45 were positively correlated and 90 were negatively correlated. KEGG pathway analysis was performed on these 135 differentially expressed metabolites, and the results are as follows: Figure 8 As shown, differential metabolites are mainly enriched in the KEGG pathway, which is involved in the synthesis of secondary metabolites, carbohydrate metabolism, amino acid metabolism, phenylpropane biosynthesis, and flavonoid and isoflavone biosynthesis.

[0146] 2.4 Correlation Analysis of Antioxidant Activity

[0147] A classification analysis of these 135 differential metabolites revealed that they are mainly: (1) phenylpropanoids, which have anti-inflammatory, antioxidant, and anticancer effects; (2) terpenoids, which have anti-inflammatory, antioxidant, antitumor, and antibacterial effects; (3) alkaloids, which have anti-inflammatory, anticancer, and analgesic effects; (4) carbohydrates, which have the effects of regulating lipid metabolism, anti-ketogenic, and liver protection; and (5) polypeptides, which have the effects of regulating immunity, antibacterial, and antioxidant effects.

[0148] Among the metabolites with positive and negative correlations, 20 metabolites with the strongest correlations were selected for plotting, such as... Figure 9 As shown (red - positive correlation, green - negative correlation), the differentially correlated metabolites with hydroxyl radical scavenging rate, DPPH scavenging rate and LPO inhibition rate are mainly: eugenol, quinic acid, 5-hydroxyconiferol, furandiene and isoflavones; the negatively correlated metabolites are: malic acid, neohesperidin, xanthan alcohol, coumarin and coumaroyl.

[0149] 2.5 Study and Analysis of Potential Bitter Metabolites in Ganoderma lucidum Based on Metabolomics Technology

[0150] 2.5.1 Analysis of potential bitter metabolites

[0151] Using LC-MS and GC-MS techniques, 2318 substances were detected and identified in Ganoderma lucidum fruiting bodies before and after fermentation. To reveal the source of bitterness in Ganoderma lucidum, we further employed metabolomics to screen for potential bitter components. The 2318 substances were compared and analyzed with the public bitterness database BitterDB to screen for possible bitter metabolites present in Ganoderma lucidum.

[0152] like Figure 10 As shown, a total of 130 metabolites were screened that may be related to the bitterness of Ganoderma lucidum, including 38 terpenes, 16 glycosides, 15 flavonoids, 12 organic acids, 9 amino acids, 9 polypeptides, 8 alkaloids, 7 coumarins, and 16 other types. Terpenes, glycosides, and flavonoids are the three largest categories of potential bitter metabolites in Ganoderma lucidum. Among the terpenes, there are 20 types of ganoderic acids, accounting for 52.63%, which theoretically verifies that the main bitter substances in Ganoderma lucidum are triterpenoids (Gao et al., 2004; Wang Jinyan et al., 2021). Tsuyoshi et al. (Tsuyoshi et al., 1985) found that, except for ganoderic acid A (4), ganoderic acid D (7) had the strongest bitterness. Most glycosides are soluble in water and ethanol, and can usually be divided into flavonoid glycosides and phenolic glycosides, which are closely related to the strong bitterness exhibited in tea and beer.

[0153] 2.5.2 Analysis of Differential Metabolites of Bitterness

[0154] By comprehensively applying the VIP value of the OPLS-DA model through multivariate statistical analysis and the t-test through univariate statistical analysis, p The VIP value was used to screen for metabolites with significant differences. To ensure the accuracy and reliability of the screening results, the screening criteria were set as VIP value ≥ 1 and T-test. p Values ​​≤ 0.05 and FC ≥ 1. Based on these criteria, a total of 40 metabolites showed significant differences between the control and experimental groups (Table 11). Figure 11 As shown, among the nine significantly upregulated bitter metabolites, alkaloids and glycosides had the highest proportion (22.22%); among the 31 significantly downregulated bitter metabolites, flavonoids had the highest proportion (22.58%). Figure 12 This further verified the results that the alkaloid content was significantly increased and the total flavonoid content was significantly decreased after fermentation.

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[0158] 2.5.3 KEGG Pathway Enrichment Analysis

[0159] Metabolic pathway annotation and enrichment analysis were performed on potential bitter metabolites and differentially bitter metabolites of Ganoderma lucidum. Results are as follows: Figure 13 and Figure 14 As shown, these metabolites are mainly enriched in pathways such as secondary metabolite biosynthesis, amino acid metabolism, phenylpropanoid biosynthesis, and flavonoid biosynthesis. It is speculated that the bitter metabolites in Ganoderma lucidum play a key role in the above-mentioned biological metabolic pathways and have high activity levels. This difference may ultimately affect the presentation of bitterness in Ganoderma lucidum.

[0160] 2.6 Analysis of the metabolic pathway of Aspergillus cristatus fermentation

[0161] 2.6.1 Analysis of the MAP00941-flavonoid biosynthesis pathway

[0162] The results of the determination of the main active ingredients in Ganoderma lucidum showed that total flavonoids decreased the most after fermentation, and flavonoids accounted for the largest proportion of the differentially downregulated bitter metabolites. KEGG pathway analysis showed that the main metabolic pathway for potential bitter metabolites in Ganoderma lucidum is map00941, such as... Figure 15 As shown in red (annotated metabolites), 16 potential bitter metabolites were annotated in the pathway, including naringenin, coumaroyl quinic acid, glycyrrhizin, hesperidin, epicatechin, chlorogenic acid, and epicatechin. Among them, hesperidin and epicatechin were significantly downregulated, while chlorogenic acid was significantly upregulated.

[0163] 2.6.2 Analysis of the map00940-phenylpropane biosynthesis pathway

[0164] Further analysis of the changing trends of potential bitter metabolites in the pathway, Figure 16 (Green - down metabolite, red - up metabolite, blue - both up and down metabolites present) represents the phenylpropane biosynthetic pathway, a precursor process for flavonoid biosynthesis. Flavonoid biosynthesis begins with phenylalanine, undergoing a series of enzymatic reactions to produce the intermediates quinic acid and sinapic acid. Quinic acid further undergoes decomposition and transformation to generate 5-hydroxyconiferyl alcohol, which is significantly upregulated and is an important biosynthetic precursor used to synthesize various compounds, including flavonoids and lignins, and possesses various physiological activities such as antioxidant, anti-inflammatory, and anticancer effects. Quinic acid is also a component of chlorogenic acid, an important bioactive substance with antiviral, hepatoprotective, choleretic, antitumor, and free radical scavenging effects. Sinapic acid is a precursor to sinapic esters, which are directly related to the synthesis of flavonoids, anthocyanins, and lignans.

[0165] 2.6.3 Analysis of the isoflavone biosynthesis pathway of map00943

[0166] The top four pathways involving flavonoid metabolites are: map00941 - flavonoid synthesis, map00942 - anthocyanin synthesis, map00943 - isoflavone synthesis, and map00944 - flavonoid and flavonol synthesis. For these four pathways, map00941 synthesizes upstream substances for the other three pathways, including the node substances Apigenin and Kaempferol in map00944; the node substances Liquiritigenin and Naringenin in map00943; and the node substances Pelargonidin, Cyanidin, and delphinidin in map00942. For example... Figure 17 As shown, the map00943 metabolic pathway annotated six potential bitter metabolites in Ganoderma lucidum: glycyrrhizin, daidzein, coumarin, sophoridine, naringenin, and hydroxyisoflavone naringenin. Among them, glycyrrhizin, daidzein, and sophoridine were significantly downregulated. These potential bitter metabolites may participate in multiple metabolic pathways. After transformation and decomposition during biosynthesis, they may form non-bitter structures, thereby improving the taste of fermented Ganoderma lucidum.

[0167] 2.7 Analysis of flavor-differentiating metabolites

[0168] like Figure 18 As shown in the volcano diagram, after fermentation with Aspergillus cristatus, Ganoderma lucidum produced 468 differentially expressed metabolites, of which 115 were significantly upregulated and 353 were significantly downregulated. 31 bitter metabolites were significantly downregulated. Figure 19 Among the significantly upregulated differential metabolites, 33 were flavor-contributing substances. Figure 19 Among them are 18 organic acids, such as azelaic acid, succinic acid, and ketoglutaric acid; 8 odor substances, such as terpenes and sinapic acid; and 7 flavor peptides, such as side-chain amino acids that impart sweet and sour tastes, like glycine and aspartic acid. Due to the complex function of taste buds, sour and sweet tastes can neutralize some of the bitterness. The improved taste of brewed Ganoderma lucidum after fermentation is not due to a change in a single bitter substance, but rather the result of the combined effects of multiple metabolites and various other factors.

[0169] 3. Verification of bitter substances:

[0170] 3.1 Results of bitter taste receptor screening:

[0171] 3.1.1 Main active substances of Ganoderma lucidum

[0172] Through literature review, representative substances of the five major active ingredients in Ganoderma lucidum were summarized, totaling 49 substances. See Table 12 for a detailed summary.

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[0174] 3.1.2 Analysis of bitter taste receptor screening results

[0175] Bitter X analysis (Table 13) predicted that among the 7 polysaccharide compounds, only fucose and dextran were predicted to be bitter compounds, with dextran having the highest interaction probability at 63.862% and the specific bitter receptor being TAS2R16; among the 11 triterpenoid compounds, except for benzoylpaeoniflorin which was not a bitter compound, isosteviolidin had the highest interaction probability at 83.437% and the universal bitter receptor being TAS2R10; and among the 6 flavonoid compounds, all were bitter compounds, with kaempferol having the highest interaction probability at 77%. 801% of the bitter taste receptors were identified as the universal TAS2R14 and the specific TAS2R39. Of the 6 alkaloids, all were bitter compounds, with decenoic acid exhibiting the highest interaction probability at 74.242%, and its bitter taste receptors being the universal TAS2R14 and the specific TAS2R38. Of the 19 amino acids, 8 were not bitter compounds, with glutamate exhibiting the highest interaction probability at 95.055%, and its bitter taste receptors being the universal TAS2R14 and the specific TAS2R39. TAS2R14 appeared as the matched receptor in all five major categories of bitter substances predicted, therefore, TAS2R14 was selected as the receptor model for subsequent experiments.

[0176] Most of the compounds predicted so far are identified as bitter compounds, but there are not many literature reports that they are bitter compounds in Ganoderma lucidum. The main reason for this may be that their content is insufficient to cause bitterness, or that a single compound may not present a bitter taste, but rather that multiple compounds work together to produce a bitter taste.

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[0178] 3.2 Analysis of Homology Modeling Results

[0179] 3.2.1 Sequence alignment results

[0180] like Figure 20 As shown, the template Q645T2 and T2R14 sequences have a similarity of 83.28% and an overall quality estimate (GMQE) of 0.81 (0-1). This indicates that the constructed homology model is of good quality.

[0181] 3.2.2. Results of Homology Modeling

[0182] like Figure 21As shown, the TAS2R14 model constructed by SWISS-MODEL consists of 8 amino acid helices. SWISS-MODEL uses the TM score to evaluate the modeling quality. The TM score of the TAS2R14 homology model is 0.82±0.05>0.5, indicating that the structure of the model is similar to that of the natural protein. Figure 22 The Laplace plot results showed that 99.3% of the amino acid residues were within the allowed region (95% in the reasonable region, 4.3% in the allowed region, and 0.7% in the abnormal region), which is at a high level. Overall, both assessments indicate that the homology model has high reliability and warrants further experimentation.

[0183] 3.3 Analysis of the Validation Results of Potential Bitter Metabolites in Ganoderma lucidum

[0184] 3.3.1 Molecular docking binding free energy

[0185] The statistical results of binding free energy are shown in Table 14 (terpenes 1-38, glycosides 39-54, flavonoids 55-69, organic acids 70-81, amino acids 82-90, polypeptides 91-99, alkaloids 100-107, coumarins 108-111, others 112-125). Preliminary verification of 130 potential bitter substances was conducted through molecular docking. 125 bitter metabolites successfully docked with the TAS2R14 homology model, and all had binding free energies <-1.2 Kcal / mol. These 125 metabolites can be preliminarily identified as bitter substances in Ganoderma lucidum. As can be seen from the table, the binding energy of terpenes is lower than that of other types, indicating that under the same conditions, terpenes are more likely to bind with TAS2R14, thus exhibiting bitterness. This further verifies that the main bitter substances in Ganoderma lucidum are terpenes.

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[0190] 3.3.2 Visual Analysis of Dating Results

[0191] Six representative bitter metabolites—ganoderic acid E, ganoderic acid A, isoflavones, matrine, glycyrrhizin, and quinic acid—were selected for molecular docking visualization. For example... Figure 23 As shown, ganoderic acid E ( Figure 23 a) and ganoderic acid A ( Figure 23c) Both ganoderic acids bind tightly to amino acid residues THR-233 and THR-236, stabilizing the ligand within the binding pocket. This result suggests that ganoderic acids may preferentially bind to the TAS2R14 receptor via these two amino acid residues. This binding mode may have a significant impact on the bitterness of ganoderic acids. Additionally, isoflavones ( Figure 23 (b) and quinic acid ( Figure 23 f) can all form hydrogen bonds with LEU-240 amino acid residues. Based on the above experimental results, it can be preliminarily inferred that THR, LEU, MET, and ILE amino acids are likely the main binding sites of TAS2R14. These amino acid residues play a key role in the binding of the TAS2R14 receptor to bitter metabolites. This discovery has important guiding significance for subsequent research, helping us to better understand how these metabolites bind to TAS2R14 and produce bitterness.

[0192] 4. Cellular experiment results:

[0193] 4.1 Effect of acetaldehyde on LX-2 cell proliferation

[0194] The effect of different concentrations of ethanol (0.00096, 0.0048, 0.024, 0.12, 0.6, 3, 15, and 75 mmol / L) on the proliferation of LX-2 cells was detected using the CCK-8 assay. After 24 h of cell culture, ethanol promoted cell proliferation. The effect was most pronounced at an ethanol concentration of 3 mmol / L. Figure 24 Therefore, we designed the acetaldehyde concentration for the experiment to be 3 mmol / L.

[0195] 4.2 Effects of different intervention concentrations on cell viability

[0196] Depend on Figure 25 As shown in a, b, c, and d, the survival rate of LX-2 cells was above 90% in both the water and alcohol extracts of Ganoderma lucidum before and after fermentation at 0.25 mg / mL, indicating no toxicity. Furthermore, the cell survival rates of the different samples were similar, suggesting that the concentrations of each sample selected in the experiment did not affect the normal growth of LX-2 cells. Therefore, 0.25 mg / mL was chosen as the subsequent effective concentration for the intervention group.

[0197] 4.3 LDH enzyme activity

[0198] In normal cells, lactate dehydrogenase (LDH) cannot pass through the cell membrane or can only pass through in small amounts and be released extracellularly. Figure 26The LDH leakage rate in the model group was 234.19%, indicating that alcohol-induced modeling damaged the cell membrane, altering its permeability and allowing large amounts of LDH to leak out. The LDH leakage rates in both the unfermented and fermented Ganoderma lucidum treatment groups were significantly reduced (P < 0.05), suggesting that the water and alcohol extracts of Ganoderma lucidum before and after fermentation can mitigate cell membrane damage. Compared to the model group, the different treatment groups showed reductions of 41.78%, 81.02%, 83.55%, and 93.68%, respectively. This indicates that Ganoderma lucidum itself also has a protective effect against liver damage.

[0199] 4.4 Measurement of transaminase levels

[0200] Depend on Figure 27 The results showed that by measuring the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the supernatant of LX-2 cells, the AST and ALT levels in the control group supernatant were 1.94 nmol / min / mL and 2.15 nmol / min / mL, respectively, while those in the model group supernatant were 5.13 nmol / min / mL and 4.86 nmol / min / mL, respectively. Compared with the control group, the release of AST and ALT in the model group increased by 1.64 and 1.26 times, respectively, with significant differences (P < 0.05). This indicates that ethanol treatment causes certain damage to cells, leading to an increase in transaminase release.

[0201] Ganoderma lucidum extracts (unfermented Ganoderma lucidum aqueous extract, fermented Ganoderma lucidum aqueous extract, unfermented Ganoderma lucidum ethanol extract, and fermented Ganoderma lucidum ethanol extract) all showed efficacy against alcohol-induced alcoholic liver injury at an intervention concentration of 0.25 mg / mL, reducing AST release by 34.29%, 50.88%, 52.95%, and 66.42%, respectively, compared to the model group. ALT release was reduced by 17.52%, 29.55%, 44.86%, and 45.96%, respectively, compared to the model group. The fermented Ganoderma lucidum group showed significantly better regulatory effects on AST and ALT in alcohol-injured LX-2 cells than the unfermented Ganoderma lucidum group (P < 0.05), and the ethanol extract was more effective than the aqueous extract.

[0202] 4.5 Cellular antioxidant enzyme activity

[0203] As shown in Figure 28(a), the fermented Ganoderma lucidum alcohol extract showed the best effect in increasing the intracellular reduced glutathione (GSH) content. Compared with the model group, the intervention concentration of 0.25 mg / mL increased the intracellular GSH level by 1.22 times. The GSH levels in the unfermented Ganoderma lucidum water extract, alcohol extract, and fermented Ganoderma lucidum water extract groups increased by 0.66, 1.06, and 1.09 times, respectively, compared with the model group.

[0204] like Figure 28 In the second group, the four Ganoderma lucidum extracts significantly increased the activity of intracellular catalase (CAT) by 0.41, 0.50, 0.55, and 1.02 times, respectively, compared with the model group. Although the four Ganoderma lucidum extract groups could increase the intracellular CAT activity, they could not match the CAT activity of the initial control group. Compared with the control group, the model group showed a significant decrease in both intracellular GSH content and CAT activity.

[0205] Compared with the alcohol-treated model group, the activities of key antioxidant enzymes GSH and CAT in cells were increased after treatment with the four Ganoderma lucidum extract samples, indicating that Ganoderma lucidum can improve the occurrence of cellular oxidative damage, and the fermented Ganoderma lucidum group had a better effect than the unfermented group.

[0206] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An application of Aspergillus cristatus in improving the antioxidant properties and bitterness of Ganoderma lucidum, characterized in that, Includes the following steps: S1. Strain activation and spore suspension preparation: The bacterial blocks stored at -80 ℃ were transferred to 5% NaCl+MYA solid medium in a clean bench and cultured at 28 ℃ for 7 days. This process was repeated twice to activate the strain. Then, the activated bacterial strain was inoculated onto a 5% NaCl+MYA plate medium and cultured at 28 ℃ under constant temperature and humidity for 7 days. Add an appropriate amount of sterile water to a plate of Aspergillus cristatus that has been cultured for 7 days, scrape off the spores, take 1 mL of spore solution and add it to a centrifuge tube containing glass beads, shake to break the ascospores, count them with a hemocytometer, and finally prepare a spore suspension of the corresponding concentration for later use. S2, Fermentation process: Weigh 6-10g of Ganoderma lucidum blocks with a thickness of 2-3 mm into a fermentation bottle, sterilize at 121 ℃ for 20 min, cool to room temperature, add sterile water to adjust the moisture content of Ganoderma lucidum, and inoculate with an appropriate amount of spore suspension. Then, culture at a constant temperature and humidity of 28-36 ℃ for 7-10 days. The spore concentration of the spore suspension is 10. 4 -10 8 pcs / mL; The moisture content of the Ganoderma lucidum is 80%-120%.

2. The application according to claim 1, characterized in that, The inoculation volume of the spore suspension is 500-900 μL.

3. The use of fermented Ganoderma lucidum prepared by the process according to any one of claims 1-2 in the preparation of products that protect against alcoholic liver injury.

4. The application according to claim 3, characterized in that, The product in question is a health supplement or a medicine.

5. Aspergillus cristatus fermented Ganoderma lucidum tablets prepared according to any one of claims 1-2.

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