Process for fermenting lucid ganoderma by aspergillus cristatus

Abstract

The invention relates to the technical field of ganoderma lucidum fermentation processes, and particularly discloses an aspergillus cristatus ganoderma lucidum fermentation process which comprises the following steps: S1, transferring a fungus block into a 5% NaCl + MYA solid culture medium in a super clean bench, culturing for 7 days under the condition of constant temperature and constant humidity of 28 DEG C, and repeating the process twice to activate a strain; inoculating the activated strain to a 5% NaCl + MYA plate culture medium, and culturing for 7 days under the condition of constant temperature and constant humidity of 28 DEG C; the method comprises the following steps: adding a proper amount of sterile water into an aspergillus cristatus flat plate cultured for 7 days, scraping spores, adding 1mL of spore liquid into a centrifugal tube filled with glass beads, shaking to break wall of ascospores, counting by using a blood cell counting plate, and diluting into spore suspension with corresponding concentration; s2, weighing a proper amount of ganoderma lucidum blocks, putting the ganoderma lucidum blocks into a fermentation bottle, adding distilled water, sterilizing for 20 minutes at 121 DEG C, cooling to room temperature, inoculating a proper amount of spore suspension, and culturing for 10 days at constant temperature and humidity at 28 DEG C; the aspergillus cristatus fermented ganoderma lucidum sporocarp obtained by the method is high in biological activity, and the bitter taste of ganoderma lucidum is obviously improved.

Description

Technical Field

[0001] The patent of this invention relates to the technical field of fermentation technology of Ganoderma lucidum, specifically, to a process of fermentation of Ganoderma lucidum by Aspergillus cristatus. Background Art

[0002] At present, the production of Ganoderma lucidum usually uses high-quality Ganoderma lucidum mycelium. In the prior art, there is a method of cultivating Ganoderma lucidum mycelium by liquid fermentation. First, the Ganoderma lucidum mycelium is inoculated into a liquid culture medium containing nutrients such as glucose, yeast extract, acid protease, etc., and the pH value and temperature are controlled at the same time to make it grow and reproduce under suitable conditions. Then the cultured Ganoderma lucidum mycelium is separated, washed, dried, etc. for subsequent fermentation production. During the strain culture process, the treated Ganoderma lucidum mycelium is inoculated into a fermentation tank, and appropriate amounts of nutrients such as glucose, yeast extract, etc. are added, and the pH value and temperature are controlled to make it grow and reproduce under suitable conditions. Among them, for the control of fermentation conditions, multiple conditions need to be controlled to ensure the growth and reproduction of Ganoderma lucidum. During the fermentation process, Ganoderma lucidum mycelium will produce some active ingredients, such as polysaccharides, triterpenoid compounds, etc., which have a good health care effect on the human body. After the fermentation is completed, extraction, purification, etc. are carried out to finally obtain the Ganoderma lucidum product. Another method is to use solid fermentation to ferment through Ganoderma lucidum fruiting bodies. At present, less fermentation is carried out using this method and this material.

[0003] The ganoderma products obtained by the existing fermentation process of ganoderma have poor biological activity, obvious bitterness, and low DPPH clearance rate. According to literature reports, the use of probiotics to ferment medicinal and edible substances can simultaneously exert the efficacy of medicinal and edible substances and the function of probiotics in regulating intestinal flora, and can also improve the taste of medicinal and edible substances and enhance the flavor of the product. To this end, the inventors of this application have optimized and improved the fermentation process of ganoderma through research, and provided a new process for fermenting ganoderma with Aspergillus cristatus. Summary of the invention

[0004] The purpose of the present invention is to solve the technical problems raised in the above background technology and provide a process for fermenting Ganoderma lucidum with Aspergillus cristatus. The biological activity of Ganoderma lucidum fruiting bodies fermented with Aspergillus cristatus is improved and the bitterness of Ganoderma lucidum is improved.

[0005] The above-mentioned purpose of the present invention is achieved like this:

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

[0007] S1. Strain activation and cultivation:

[0008] The bacterial blocks stored at -80°C were transferred to 5% NaCl+MYA solid culture medium in a clean bench, and cultured at 28°C constant temperature and humidity for 7 days, and this process was repeated twice to activate the strains; then, the activated strains were inoculated on 5% NaCl+MYA plate culture medium, and cultured at 28°C constant temperature and humidity for 7 days for use;

[0009] S2. Fermentation process:

[0010] Add an appropriate amount of sterile water to the Aspergillus cristatus plate that has been cultured for 7 days, scrape the spores, absorb the spore liquid and add it to a centrifuge tube with glass beads, shake it to break the ascospores, count them with a hemocytometer, and finally dilute them into a spore suspension of corresponding concentration for use;

[0011] Weigh an appropriate amount of Ganoderma lucidum blocks into a fermentation bottle, add an appropriate amount of distilled water, sterilize at 121°C for 20 min, cool to room temperature, inoculate an appropriate amount of spore suspension, and culture at a constant temperature and humidity of 28°C 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] Further, the spore concentration of the spore suspension is 10 4 pcs / mL.

[0015] Furthermore, the fermented Ganoderma lucidum obtained by the process is used in the preparation of products for protecting alcoholic liver damage.

[0016] Furthermore, the product is a health product 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 moisture content, fermentation time, fermentation temperature, concentration of spore suspension and inoculation amount. The biological activity of the Ganoderma lucidum fruiting body fermented by Aspergillus cristatus obtained by the newly designed fermentation process of Ganoderma lucidum is high, and the bitterness of Ganoderma lucidum is significantly improved;

[0019] 2. Compared with the existing process, the fermented Ganoderma lucidum product prepared by the process of the present invention has significantly improved DPPH clearance rate and LPO inhibition rate, high antioxidant activity, and significantly improved 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 for protecting alcoholic liver damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the effect of different single factors on DPPH clearance in the examples of the present invention (water content (a), fermentation time (b), spore concentration (c), inoculation amount (d), temperature (e));

[0022] Figure 2 3D response surface diagram and contour lines of the interaction terms of different independent variables in the embodiment of the present invention affecting the DPPH clearance rate (temperature, moisture content interaction term (ab); time, moisture content interaction term (cd); inoculum size, moisture content interaction term (ef); time, temperature interaction term (gh); inoculum size, temperature interaction term (ij); inoculum size, time interaction term (kl));

[0023] Figure 3 is the standard curve of total triterpenes, total polysaccharides, amino acids, alkaloids and flavonoids in the examples of the present invention (total triterpenes (a), total polysaccharides (b), amino acids (c), alkaloids (d), flavonoids (e));

[0024] Figure 4 are the antioxidant activity assay results (hydroxyl radical scavenging rate (a), DPPH scavenging rate (b), LPO inhibition rate (c)) in the examples of the present invention;

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

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

[0027] Figure 7 is the permutation test result in the embodiment of the present invention;

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

[0029] Fig. 9 is the analysis of metabolites related to antioxidant activity in the embodiments of the present invention (hydroxyl radical scavenging rate (a), DPPH scavenging rate (b), LPO inhibition rate (c));

[0030] Fig.10 It is the classification of potential bitter metabolites in Ganoderma lucidum in the embodiments of the present invention;

[0031] Fig.11 is a bitter differential metabolite significantly upregulated in the embodiments of the present invention;

[0032] Fig.12 It is a bitter differential metabolite significantly downregulated in the embodiments of the present invention;

[0033] Fig.13is the pathway enrichment analysis of potential bitter metabolites in the embodiments of the present invention;

[0034] Fig.14 is the pathway enrichment analysis of differential bitter metabolites in the embodiments of the present invention;

[0035] Fig.15 is the map00941-flavonoid biosynthetic pathway in the embodiments of the present invention;

[0036] Fig.16 is the map00940-phenylpropanoid biosynthetic pathway in the embodiments of the present invention;

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

[0038] Fig.18 is a volcano map in an embodiment of the present invention;

[0039] Fig.19 is the flavoring substance in the embodiment of the present invention;

[0040] Fig. 20 is the comparison result between the template selected by SWISS-MODEL and the target sequence of TAS2R14 in the embodiment of the present invention;

[0041] Fig.21 is the homology model of TAS2R14 in the embodiment of the present invention;

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

[0043] Fig.23 It is the visualization of the docking results of the homology model-bitter substances in the embodiment of the present invention (ganoderic acid E (a), isoflavones (b), ganoderic acid A (c), matrine (d), liquiritin (e), quinic acid (f));

[0044] Fig.24 is the effect of ethanol on LX-2 proliferation in the embodiment of the present invention;

[0045] Fig.25 The effects of different sample intervention concentrations on cell viability in the examples of the present invention (a: the effect of unfermented Ganoderma lucidum water extract on LX-2 cell viability; b: the effect of fermented Ganoderma lucidum water extract on LX-2 cell viability; c: the effect of unfermented Ganoderma lucidum alcohol extract on LX-2 cell viability; d: the effect of fermented Ganoderma lucidum alcohol extract on LX-2 cell viability);

[0046] Fig.26 The effects of different Ganoderma lucidum treatment groups on LDH in LX-2 cells with alcoholic liver damage in the present invention;

[0047] Fig. 27 The effects of different Ganoderma lucidum treatment groups on the AST and ALT levels in alcoholic liver injury model cells in the embodiments of the present invention;

[0048] Fig.28 It is the cellular antioxidant enzyme activity in the embodiment of the present invention (a: the reduced glutathione content in the cells of the alcoholic liver injury model in different Ganoderma lucidum treatment groups; b: the catalase activity in the cells of the alcoholic liver injury model in different Ganoderma lucidum treatment groups). DETAILED DESCRIPTION

[0049] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with 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 is described in detail below in conjunction with specific embodiments.

[0051] The following are preferred embodiments of the present invention.

[0052] Example:

[0053] The solution of the embodiment of the present invention provides a process for fermenting Ganoderma lucidum by Aspergillus cristatus, which process comprises the following steps:

[0054] S1. Activation and cultivation of strains: The bacterial blocks stored at -80°C were transferred to 5% NaCl+MYA solid culture medium in a clean bench, and cultured at 28°C constant temperature and humidity for 7 days. This process was repeated twice to activate the strains. Then, the activated strains were inoculated onto 5% NaCl+MYA plate culture medium, and cultured at 28°C constant temperature and humidity for 7 days for use.

[0055] S2. Fermentation process: Add appropriate amount of sterile water to the Aspergillus cristatus plate that has been cultured for 7 days, scrape the spores, absorb the spore liquid and add it to the centrifuge tube with glass beads, shake to break the ascospores, count them with a hemocytometer, and finally dilute them into a spore suspension of corresponding concentration for later use. Weigh an appropriate amount of Ganoderma lucidum block into a fermentation bottle, add an appropriate amount of distilled water, sterilize at 121℃ for 20 minutes, cool to room temperature, inoculate an appropriate amount of spore suspension, and culture at 28℃ for 10 days.

[0056] In this embodiment, the water content of the spore suspension is 80%. The inoculation volume of the spore suspension is 500 μL. The spore concentration of the spore suspension is 10 8 pcs / mL. The fermented Ganoderma lucidum prepared by the process is used in the preparation of a product for protecting alcoholic liver damage, and the product is a health product or a medicine.

[0057] The following is an experiment of the embodiment of the present invention:

[0058] 1. Single factor test:

[0059] Take an appropriate amount of Ganoderma lucidum in a fermentation bottle, add 40-120% distilled water in a mass: volume (w / v) ratio, sterilize at 121℃ for 20 minutes, cool to room temperature, and inoculate for 10 4 -10 8 The spore suspension of 100-900 μL was cultured in an incubator at 20-36°C for 6-14 days. The effects of moisture content, fermentation time, spore concentration, inoculation amount and fermentation temperature on DPPH removal rate were investigated in sequence.

[0060] 2. Box-Behnken experimental design to optimize fermentation process:

[0061] Based on the above single factor test results, moisture content (A), temperature (B), time (C) and inoculation size (D) were selected as independent variables, DPPH clearance rate was used as response value, and the functional relationship between independent variables and response values ​​was established to optimize the optimal fermentation process conditions for improving the biological activity and bitterness of Ganoderma lucidum fruiting bodies fermented by Aspergillus cristatus.

[0062] Table 1.1 Box-Behnken test design factor levels

[0063]

[0064] 3. Verification of the best inclusion process:

[0065] According to the optimal gradient determined by the above single factor experiment, the optimal process parameters of moisture content, time, temperature and inoculation amount were determined by the four-factor three-level Box-Behnken test. Three groups of parallel experiments were carried out and the average value was taken.

[0066] 4. Sensory evaluation:

[0067] Weigh an appropriate amount of Ganoderma lucidum, brew with water temperature > 95℃ for 2 minutes (Xie Sandu et al., 2021), and filter to remove the residue. Referring to the reported methods (Yu Mingguang, 2020; Zhang Qianhui et al., 2022; Zhang Pu et al., 2021), the bitterness is divided into 5 levels, each level corresponds to a certain bitterness range. The sensory evaluation team consists of 10 healthy and normal sensory evaluation personnel (5 men and 5 women, aged 18-28 years old). The scoring criteria are shown in Table 1.2.

[0068] Table 1.2 Sensory evaluation score sheet

[0069]

[0070] V. Physical and chemical index testing methods:

[0071] 1. Ganoderma lucidum polysaccharide (phenol-sulfuric acid method)

[0072] The determination was carried out using the phenol-sulfuric acid method.

[0073] 2. Ganoderma lucidum triterpenoids (glacial acetic acid-vanillin-perchloric acid method)

[0074] Refer to the methods of the 2020 edition of the Chinese Pharmacopoeia and NY / T3676-2020.

[0075] 3. Amino acids, total flavonoids, alkaloids

[0076] The kit was used for determination, and the standard curve and sample determination methods were carried out according to the kit instructions.

[0077] 6. Evaluation of in vitro antioxidant activity:

[0078] The antioxidant capacity before and after fermentation was evaluated by hydroxyl radical scavenging rate, DPPH scavenging rate and LPO inhibition rate. The test kit was used for determination. The determination method is shown in the kit manual.

[0079] 7. Metabolomics analysis:

[0080] 1. Metabolomics data analysis methods

[0081] The raw data were 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. At the same time, metabolites were searched and identified, mainly using public databases such as NIST (2017), Fiehn (2013), and MS-DIAL (2021).

[0082] 2. Screening of Ganoderma lucidum bitter metabolites based on metabolomics analysis:

[0083] Potential bitter substances were screened through the public bitter database BitterDB, Chemical Book website and related literature. The OPLS-DA model was used to screen metabolites with VIP≥1 as candidate differential metabolites, and the t-test was further used to evaluate their significant differences. When the difference of the candidate differential metabolites reached or exceeded the preset significance level (p < 0.05), they were confirmed as true differential metabolites.

[0084] 3. Bitter substance verification:

[0085] Bioinformatics methods are used for bitter receptor screening, homology modeling, molecular docking and visualization analysis of docking results. By analyzing the interaction results between bitter receptors and ligands, it is verified whether the potential bitter substances screened out are the bitter substances of Ganoderma lucidum.

[0086] 8. Study on the protective function against alcoholic liver damage

[0087] 1. Sample preparation:

[0088] Weigh an appropriate amount of Ganoderma lucidum before and after fermentation, crush it, and extract it with water and alcohol respectively. After the filtrate after extraction is filtered and concentrated, freeze-dried to obtain freeze-dried powder. After dissolving it with DMSO, pass it through a 0.22μm microporous filter membrane, and use incomplete culture medium to make the volume to the required concentration.

[0089] 2. Cell culture:

[0090] (1) Cell recovery

[0091] Take out the LX-2 cells from the liquid nitrogen tank and thaw them quickly in a 37℃ constant temperature water bath. Remove the culture medium after centrifugation and resuspend the cells in preheated culture medium. Inoculate them in a cell culture bottle and culture them in a constant temperature incubator at 37℃ and 5% CO2. Passage can be performed when the cells are about 80-100% full. The newly recovered cells can be used for subsequent related experiments only after one pass.

[0092] (2) Cell passaging

[0093] Take the cell culture flask with 80-100% growth, remove the culture medium, aspirate PBS for washing and remove PBS. Add trypsin for digestion and examine under a microscope. When the digestion requirement is reached, add 3 times the growth medium of trypsin to stop digestion. Remove the culture medium after centrifugation and divide into new culture dishes at a ratio of 1:2-1:4 for continued culture.

[0094] (3) Cell cryopreservation

[0095] Take a cell culture flask with 80-100% growth, remove the culture medium, wash with PBS, remove PBS, digest with trypsin, and examine under a microscope. When the digestion requirements are met, add 3 times complete culture medium to terminate the digestion. After centrifugation to remove the culture medium, add an appropriate amount of freezing solution, blow it into a suspension, and transfer the suspension to a cryopreservation tube. Label the cryopreservation tube with the cell type, record the passage number and date. Finally, place it in a cryopreservation box and freeze it step by step.

[0096] 3. Determination of the effective concentration of the model group

[0097] LX-2 cells were inoculated in 96-well plates for culture. There were 6 replicates in each group, and control and model groups were set up. The control group was cultured with incomplete culture medium, and the model group was added with ethanol culture medium containing a series of gradient concentrations, and cultured in a 37°C, 5% CO2 incubator. After observing the cell attachment and growth for 24 hours, the cell viability was determined by CCK-8 method.

[0098]

[0099] 4. Determination of the effective concentration of the test group

[0100] LX-2 cells in the logarithmic growth phase were collected and inoculated in 96-well plates for culture. Six replicate wells in each group were placed in a 37°C, 5% CO2 incubator for culture. After 24 hours of ethanol modeling culture, the cells adhered to the wall and grew, and then fresh culture medium was replaced. A control group and an experimental group were set up. The control group was cultured with incomplete culture medium, and the experimental group was added with sample culture medium containing a series of gradient concentrations, respectively, and placed in a 37°C, 5% CO2 incubator for culture for 24 hours. The cell viability was determined by CCK-8 method.

[0101] 5. Effects of the experimental group on oxidative damage in LX-2 cells

[0102] A control group, a model group, and a test group and an alcohol combined treatment group were set up. After 48 hours of cell culture in each group, the lactate dehydrogenase (LDH) leakage rate, alanine aminotransferase (ALT), aspartate aminotransferase (AST), reduced glutathione (GSH) content and catalase (CAT) activity were measured.

[0103] IX. Research Results:

[0104] 1. Optimization of the fermentation process of Ganoderma lucidum by Aspergillus cristatum and analysis of its metabolomics results

[0105] 1.1 Analysis of single factor test results

[0106] 1.1.1 Effect of moisture content on DPPH clearance rate after fermentation

[0107] like Figure 1 The results of a in the figure show that within the range of 40-80% moisture content, the DPPH removal rate increases with the increase of moisture content; however, after exceeding 80%, a downward trend appears, and no significant difference is observed between 80%, 100% and 120%. If the moisture content of the fermentation substrate is too low, it will cause it to be too dry, thereby inhibiting the growth and reproduction of microorganisms and having an adverse effect on metabolism; if the moisture content is too high, it will not only hinder oxygen transmission, but also increase the possibility of contamination by bacteria.

[0108] 1.1.2 Effect of fermentation time on DPPH clearance rate after fermentation

[0109] like Figure 1As shown in b, when the fermentation days were between 6 and 10 days, the DPPH clearance rate showed a clear upward trend with the increase of time; after 10 days, the upward trend became less obvious, and there was no significant difference between 10, 12 and 14 days. Fermentation time plays a very important role in the growth and reproduction of microorganisms. With the extension of culture time, microbial metabolites will undergo certain changes. In the primary growth stage of microorganisms, the amount of a certain secondary metabolite will gradually increase with the increase of culture time; after a certain period of time, this secondary metabolite may gradually degrade.

[0110] 1.1.3 Effect of spore concentration on DPPH clearance after fermentation

[0111] like Figure 1 As shown in Figure c, as the spore concentration increased, the DPPH clearance rates of different gradients in each group showed an upward trend, but statistical analysis found no significant difference between them. This indicates that the spore concentration has no significant effect on the response value within the scope of this experiment.

[0112] 1.1.4 Effect of temperature on DPPH clearance rate after fermentation

[0113] like Figure 1 As shown in d, between 20-28°C, as the temperature rises, the DPPH clearance rate shows a clear upward trend; and after 28°C, there is a clear downward trend. Generally, the most suitable temperature range for the growth of Aspergillus cristatus is 25-30°C. Too high or too low temperature may cause changes in microbial morphological characteristics and metabolic direction, and even produce toxic substances, leading to death (Jia Zhihua et al., 2010).

[0114] 1.1.5 Effect of inoculum size on DPPH clearance rate after fermentation

[0115] according to Figure 1 The results of e in Figure 2 show that when the inoculum size is between 100-500 μL, the DPPH clearance rate shows a significant upward trend with the increase of the inoculum size. However, after exceeding 500 μL, the upward trend of the DPPH clearance rate becomes less obvious, and a significant downward trend appears. This may be due to the excessive inoculum size, which leads to rapid growth of the bacterial count and competition for survival. Excessive inoculum size may lead to insufficient dissolved oxygen, thereby reducing the generation rate of metabolites and introducing too much metabolic waste, thereby reducing economic benefits.

[0116] 1.2. Analysis of Box-Behnken test design results:

[0117] The four factors of moisture content (A), temperature (B), time (C) and inoculation amount (D) that have a significant effect on the response value within the test range were selected as independent variables, DPPH clearance rate was the response value, and a 4-factor 3-level Box-Behnken optimization test was designed to obtain the optimal fermentation process parameters. The experimental design and results are shown in Table 6, and the experimental regression variance analysis results are shown in Table 7. The regression equation of DPPH clearance rate for response variables A, B, C, and D is as follows:

[0118] Y=92.11+1.14A+1.16B+0.62C+0.43D+0.69AB+0.27AC+0.49AD+0.54BC+0.52BD+0.48CD-0.81A 2 -1.62B 2 -0.86C 2 -1.41D 2 .

[0119] As shown in Table 6, the model F value = 33.17 and P < 0.0001, the lack of fit term p = 0.5417> 0.05, indicating that the quadratic model used in the experiment reached an extremely significant level and the model was reliable. 2 =0.9707 and R 2 Adj =0.9415 are both approximately equal to 1, indicating that the accuracy of the predicted response value is high. 2 Adj With R 2 Pred The difference is less than 0.2, CV%=0.44<10 and Adeq Precision=20.209>4, indicating that the fit between the actual value and the predicted value is good, and the model has excellent accuracy and repeatability. In summary, it is shown that the regression equation can be used to optimize the process conditions of Aspergillus cristatus fermentation of Ganoderma lucidum fruiting bodies and analyze and predict its DPPH clearance rate. Only the AC interaction term in the regression equation is an insignificant factor, indicating that except for the AC interaction term, the other terms have a significant effect on the DPPH clearance rate; the order of influence of the experimental factors involved in the optimization on the DPPH clearance rate is: temperature (B)> moisture content (A)> time (C)> inoculation amount (D).

[0120] Table 6 Box-Behnken experimental design and results (n=3)

[0121]

[0122]

[0123] Table 7 Box-Behnken test regression analysis of variance

[0124]

[0125]

[0126] 1.3 Response surface analysis of interactions between factors:

[0127] Figure 2 Figures a and b show the interaction between water content and temperature on DPPH removal, and the results show that they are the most important factors in the actual experiment. Increasing water content significantly increased DPPH removal, and this effect was further enhanced as the temperature increased. This interactive result emphasizes that in practical applications, these two factors need to be considered comprehensively to achieve the best DPPH removal effect. In addition, Figure 2 Figures g and h also show that the interaction between temperature and time also has an important influence on DPPH clearance. Therefore, during the experiment, it is necessary not only to control the temperature, but also to reasonably set the experimental time to ensure that the DPPH clearance rate is stable and effective. According to the contour density, it was found that compared with other influencing factors, temperature has a more significant effect on DPPH clearance. This is consistent with the conclusion drawn from the regression variance analysis in Table 7.

[0128] 1.4. Analysis of the verification results of the best inclusion and debittering process:

[0129] As shown in Table 8, the optimized optimal process conditions and predicted values ​​of Aspergillus cristatus fermentation of Ganoderma lucidum fruiting bodies are the model prediction group values. Combined with the actual situation, the optimal process is adjusted to the actual value group data. The actual value of DPPH clearance obtained is not significantly different from the predicted value, indicating that the model has a high degree of fit with the actual situation, and the optimal fermentation process obtained is stable and feasible. Compared with the original value, the DPPH clearance rate of the treatment group increased by 25.62%.

[0130] Table 8 Verification results (n=3)

[0131]

[0132] Note: Different lowercase letters indicate significant differences (p<0.05).

[0133] 1.5. Analysis of sensory evaluation results:

[0134] The results are shown in Table 9. After fermentation by Aspergillus cristatus, the medicinal taste of Ganoderma lucidum was significantly weakened, and it had a light and elegant fungus aroma. Although the bitterness value of Ganoderma lucidum before and after fermentation was not significantly different, the taste after fermentation was improved to a certain extent, the bitterness was reduced, and the taste was softer. During the fermentation process, some components in Ganoderma lucidum may be decomposed or transformed by microorganisms to generate some new compounds. Some of these newly produced compounds can neutralize the original bitterness of Ganoderma lucidum, making the taste softer.

[0135] Table 9 Sensory evaluation of Ganoderma lucidum before and after fermentation

[0136]

[0137] Note: Different lowercase letters indicate significant differences (p<0.05).

[0138] 1.6. Analysis of the results of determination of the content of main active ingredients:

[0139] 1.6.1、Drawing of standard curve:

[0140] The obtained standard curves for total triterpenes, total polysaccharides, amino acids, alkaloids and flavonoids are shown in Figure 3 shown.

[0141] 1.6.2 Analysis of the results of determination of the content of main active ingredients:

[0142] The contents of polysaccharides, triterpenes, amino acids, alkaloids and flavonoids, the main active ingredients of Ganoderma lucidum, were determined. 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), decreasing by 24.98%, 13.46% and 39.13% respectively, while the content of alkaloids increased significantly (p<0.05), increasing by 24.64%, and the content of triterpenes 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 the fermentation process. For filamentous fungi, secondary metabolites usually begin to be produced in the subsequent production stage or mid-stage after the rapid growth stage (trophic stage). In this case, when the depletion of key nutrients limits its growth, secondary metabolism will start, at which time carbon (glucose), nitrogen or phosphate is consumed, and enters a stage with low or zero proliferation rate but high yield.

[0143] Table 10 Physical and chemical index determination results

[0144]

[0145] Note: Different lowercase letters indicate significant differences (p<0.05).

[0146] 1.7. Analysis of antioxidant activity results:

[0147] like Figure 4As shown in the results, 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, among which the hydroxyl radical scavenging rate, DPPH scavenging rate and LPO inhibition rate of the water extract increased by 3.69%, 22.50% and 5.98%, respectively; the hydroxyl radical scavenging rate, DPPH scavenging rate and LPO inhibition rate of the alcohol extract increased by 2.82%, 8.18% and 8.42%, respectively. In addition, the alcohol extract showed higher antioxidant capacity than the water extract, and the hydroxyl radical scavenging rate, DPPH scavenging rate and LPO inhibition rate of the alcohol extract were 2.82%, 8.18% and 39.54% higher than those of the water extract, respectively. The selected solvent system and extraction process will affect the antioxidant activity. Most of the main active ingredients of Ganoderma lucidum, such as triterpenes, flavonoids and alkaloids, are soluble in organic solvents and insoluble or difficult to dissolve in water.

[0148] 2. Metabolomics results analysis:

[0149] 2.1. Principal Component Analysis (PCA)

[0150] according to Figure 5 The results of principal component analysis showed that the samples before and after fermentation of Ganoderma lucidum were within the 95% confidence interval, and there were significant differences between the two groups of samples, indicating that fermentation had a significant effect on separation. Combined with the actual situation, it is further inferred that chemical changes may have occurred or biologically active substances were generated during the fermentation process, resulting in significant differences in Ganoderma lucidum before and after fermentation. These changes or the generation of substances may be related to the combined effects of multiple factors such as microbial action, substrate type, temperature and time.

[0151] 2.2 Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA)

[0152] like Figure 6 As shown in the OPLS-DA score graph, all samples are within the 95% confidence interval, and the repeated points of each group of samples are close and clustered together, indicating that the data has good repeatability. At the same time, there are obvious differences between the two groups of samples, indicating that the related metabolites before and after Ganoderma lucidum fermentation have significant changes. Figure 7 The permutation test results show that the Q2 point obtained by the random sorting method is lower than the original R2 point, which further shows that the model does not have overfitting and has good robustness. All samples are within the 95% confidence interval, and the experimental data are true and reliable. The differential metabolites before and after fermentation of Ganoderma lucidum can be screened based on the VIP value analysis of the OPLS-DA model.

[0153] 2.3 Screening of differential metabolites related to antioxidant activity

[0154] According to the analysis results of OPLS-DA, there are 468 metabolites with VIP>1.2 and p<0.05, which play an important role in the discrimination results of OPLS-DA. Combined with the association analysis of hydroxyl radical scavenging rate, DPPH scavenging rate and LPO inhibition rate, there are 135 differential metabolites related to antioxidant activity, of which 45 are positively correlated and 90 are negatively correlated. KEGG functional pathway analysis was performed on these 135 differential metabolites, and the results are as follows Figure 8 As shown, the differential metabolites were mainly enriched in KEGG pathways related to the synthesis of secondary metabolites, carbohydrate metabolism, amino acid metabolism, phenylpropanoid biosynthesis, and flavonoid and isoflavone biosynthesis.

[0155] 2.4 Correlation analysis of antioxidant activity

[0156] The 135 differential metabolites were classified and analyzed, and they were mainly divided into: (1) phenylpropanoids, which have anti-inflammatory, antioxidant, and anticancer effects; (2) terpenes, which have anti-inflammatory, antioxidant, anti-tumor, and antibacterial effects; (3) alkaloids, which have anti-inflammatory, anticancer, and analgesic effects; (4) carbohydrates, which have the effects of regulating fat metabolism, anti-ketosis, and liver protection; (5) peptides, which have the effects of regulating immunity, antibacterial, and antioxidant effects.

[0157] Among the positively and negatively correlated metabolites, 20 metabolites with the strongest correlation were selected for plotting, such as Fig. 9 As shown (red - positive correlation, green - negative correlation), the differential metabolites positively correlated with hydroxyl radical scavenging rate, DPPH scavenging rate and LPO inhibition rate are mainly: syringaresinol, quinic acid, 5-hydroxyconiferyl alcohol, furandiene and isoflavones; the negatively correlated metabolites are: malic acid, neohesperidin, xanthanol, coumarin and coumaroyl.

[0158] 2.5 Research and analysis of potential bitter metabolites in Ganoderma lucidum based on metabolomics technology

[0159] 2.5.1 Analysis of potential bitter metabolites

[0160] By using LC-MS and GC-MS techniques, 2318 substances were detected and identified before and after the fermentation of Ganoderma lucidum fruiting bodies. In order to reveal the source of the bitter taste of Ganoderma lucidum, we further used metabolomics methods to screen for potential bitter components. The 2318 substances were compared and analyzed with the public bitter database BitterDB to screen out the bitter metabolites that may exist in Ganoderma lucidum.

[0161] like Fig.10As shown in the results, a total of 130 metabolites were screened out that may be related to the bitter taste of Ganoderma lucidum, including 38 terpenes, 16 glycosides, 15 flavonoids, 12 organic acids, 9 amino acids, 9 peptides, 8 alkaloids, 7 coumarins and 16 other types. Terpenes, glycosides and flavonoids are the three largest categories of potential bitter metabolites in Ganoderma lucidum. There are 20 ganoderic acids in terpenoids, accounting for 52.63%, which theoretically verifies that the main bitter substances in Ganoderma lucidum are triterpenoid compounds (Gao et al., 2004; Wang Jinyan et al., 2021). Tsuyoshi et al. (Tsuyoshi et al., 1985) found that, in addition to ganoderic acid A (4), ganoderic acid D (7) has 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 presented in tea and beer.

[0162] 2.5.2 Analysis of Bitter Metabolites

[0163] By comprehensively using the VIP value of the multivariate statistical analysis OPLS-DA model and the univariate statistical analysis T test p value, the metabolites with significant differences were screened out. In order to ensure the accuracy and reliability of the screening results, the screening criteria were set as VIP value ≥ 1, T test p value ≤ 0.05, and FC ≥ 1. According to these criteria, a total of 40 metabolites showed significant differences between the control group and the experimental group (Table 11). Fig.11 As shown in Figure 2, among the 9 significantly upregulated bitter metabolites, alkaloids and glycosides accounted for the highest proportion (22.22%); among the 31 significantly downregulated bitter metabolites, flavonoids accounted for the highest proportion (22.58%) ( Fig.12 ). The results further verified that the alkaloid content increased significantly and the total flavonoid content decreased significantly after fermentation.

[0164] Table 11 Differential bitter metabolites

[0165]

[0166]

[0167]

[0168] 2.5.3 KEGG pathway enrichment analysis

[0169] Metabolic pathway annotation and enrichment analysis were performed on the potential bitter metabolites and differential bitter metabolites of Ganoderma lucidum. Fig.13 and Fig.14As shown in the results, 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 a higher activity level. This difference may ultimately affect the presentation of the bitter taste of Ganoderma lucidum.

[0170] 2.6 Analysis of fermentation metabolic pathways of Aspergillus cristatus

[0171] 2.6.1. MAP00941-Analysis of flavonoid biosynthesis pathway

[0172] The results of the determination of the content of the main active ingredients of Ganoderma lucidum showed that the total flavonoids decreased the most after fermentation, and the flavonoids accounted for the largest proportion of the significantly downregulated differential bitter metabolites. The results of KEGG pathway analysis showed that the main metabolic pathway of Ganoderma lucidum potential bitter metabolites was map00941, such as Fig.15 As shown in the figure (red - annotated metabolites), there are 16 potential bitter metabolites annotated by the pathway, such as naringenin, coumaroylquinic acid, licorice root, hesperidin, epicatechin, chlorogenic acid and epicatechin, among which hesperidin and epicatechin were significantly downregulated, and chlorogenic acid was significantly upregulated.

[0173] 2.6.2. MAP00940-phenylpropanoid biosynthesis pathway analysis

[0174] Further analysis of the changing trends of potential bitter metabolites in the pathways. Fig.16 (Green - dowm metabolites, red - up metabolites, blue - up + dowm metabolites coexist) is the phenylpropanoid biosynthesis pathway, which is a precursor process for flavonoid biosynthesis. The biosynthesis of flavonoids begins with phenylalanine, and a series of enzymatic reactions generate intermediate products quinic acid and sinapinic acid. Quinic acid is further decomposed and transformed to generate 5-hydroxyconiferyl alcohol, which is significantly upregulated and is an important biosynthetic precursor. It can be used to synthesize a variety of compounds including flavonoids and lignin, and has multiple physiological activities such as antioxidant, anti-inflammatory and anti-cancer. Quinic acid is also a component of chlorogenic acid, which is an important biologically active substance with antiviral, liver-protecting and choleretic, anti-tumor and free radical scavenging effects. Sinapic acid is a precursor of sinapic acid esters, which are directly related to the synthesis of flavonoids, anthocyanins, lignans and other substances.

[0175] 2.6.3. Analysis of isoflavone biosynthesis pathway in map00943

[0176] The top four pathways involving flavonoid metabolites are: map00941-flavonoid synthesis, map00942-anthocyanidin synthesis, map00943-isoflavone synthesis and map00944-flavonoid and flavonol synthesis. For these four pathways, map00941 synthesizes the upstream substances of the other three pathways, including the node substances Apigenin (Apigenin) and Kaempferol (Kaempferol) of map00944; the node substances Liquiritigenin (Liquiritigenin) and Naringenin (Naringenin) of map00943; the node substances Pelargonidin (Pelargonidin), Cyanidin (Cyanidin) and delphinidin (Delphinium pigment) of map00942. Fig.17 As shown in the figure, six potential bitter metabolites of Ganoderma lucidum were annotated on the map00943 metabolic pathway: liquiritigenin, daidzein, coumarol, acaciatin, naringenin, and hydroxyisoflavone naringenin, among which liquiritigenin, daidzein, and acaciatin were significantly downregulated. Potential bitter metabolites may participate in multiple metabolic pathways, and after transformation and decomposition during the biosynthesis process, they may form structures without bitterness, thereby improving the taste of fermented Ganoderma lucidum.

[0177] 2.7 Analysis of flavor metabolites

[0178] like Fig.18 As shown in the volcano plot, there are 468 differential metabolites after Ganoderma lucidum is fermented by Aspergillus cristatus, of which 115 are significantly upregulated and 353 are significantly downregulated. There are 31 bitter metabolites that are significantly downregulated ( Fig.19 Among the significantly upregulated metabolites, 33 substances exhibited flavor ( Fig.19 ). Among them, there are 18 organic acids, such as azelaic acid, succinic acid, ketoglutaric acid, etc.; 8 odor substances, such as terpenes, sinapinic acid, etc.; 7 flavor peptides, such as side chain amino acids that present sweet and sour tastes, such as glycine and aspartic acid. Due to the complex role of taste buds, sourness and sweetness can neutralize part of the bitterness. The reason why the taste of brewed Ganoderma lucidum after fermentation has improved is not caused by the change of a single bitter substance, but the result of the combined action and multi-faceted influence of multiple metabolites.

[0179] 3. Bitter substance verification:

[0180] 3.1. Bitter taste receptor screening results:

[0181] 3.1.1. Main active substances in Ganoderma lucidum

[0182] Through literature research, we summarized the representative substances of the five major active ingredients in Ganoderma lucidum, totaling 49. See Table 12 for a detailed summary.

[0183] Table 12 Summary of main active substances in Ganoderma lucidum

[0184]

[0185] 3.1.2 Analysis of bitter taste receptor screening results

[0186] According to the prediction of Bitter X analysis (Table 13), among the 7 polysaccharide compounds, only fucose and glucan were predicted to be bitter compounds, and the highest interaction probability was glucan (63.862%), and the bitter receptor was the specific TAS2R16; among the 11 triterpenes, except for benzoylpeonidin, which was not a bitter compound, the highest interaction probability was isosteviol (83.437%), and the bitter receptor was the universal TAS2R10; among the 6 flavonoids, all were bitter compounds, and the highest interaction probability was kaempferol (77. 801%, the bitter receptors are universal TAS2R14 and specific TAS2R39; 6 alkaloids, all of which are bitter compounds, the highest probability of interaction is decenoylcholine 74.242%, the bitter receptors are universal TAS2R14 and specific T2R38; 19 amino acids, 8 of which are not bitter compounds, the highest probability of interaction is glutamate 95.055%, the bitter receptors are universal TAS2R14 and specific TAS2R39. In the prediction results of the five major categories of bitter substances, TAS2R14 appears as a matching receptor, so TAS2R14 is selected as the receptor model for subsequent experiments.

[0187] 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 possible main reason is that their content is not enough to cause bitterness. It is also possible that when a single compound exists, it does not exhibit bitterness, but multiple compounds work together to produce bitterness.

[0188] Table 13 Prediction results

[0189]

[0190] 3.2 Analysis of homology modeling results

[0191] 3.2.1 Sequence alignment results

[0192] like Fig. 20 As shown, the sequence similarity between template Q645T2 and T2R14 is 83.28%, and the overall quality estimate (GMQE) is 0.81 (0-1), indicating that the constructed homology model is of good quality.

[0193] 3.2.2 Homology modeling results

[0194] like Fig.21As shown, the TAS2R14 model constructed by SWISS-MODEL consists of 8 amino acid helices. SWISS-MODEL uses 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 the natural protein. Fig. 22 The pull-through plot results showed that 99.3% of the amino acid residues were in the allowed region (95% in the reasonable region, 4.3% in the allowed region, and 0.7% in the abnormal region), which was at a high level. Overall, both evaluations showed that the homology model had a high reliability and was worthy of further experiments.

[0195] 3.3 Analysis of the validation results of potential bitter metabolites of Ganoderma lucidum

[0196] 3.3.1. Molecular docking binding free energy

[0197] 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, peptides 91-99, alkaloids 100-107, coumarins 108-111, and others 112-125). The 130 potential bitter substances were preliminarily verified by molecular docking, and 125 bitter metabolites were successfully docked with the TAS2R14 homology model, and the binding free energy was <-1.2Kcal / mol. These 125 metabolites can be preliminarily identified as Ganoderma lucidum bitter substances. As can be seen from the table, the binding energy of terpenoid substances is lower than that of other types, indicating that under the same conditions, terpenoid substances are more likely to bind to TAS2R14, thus presenting a bitter taste. It also further verifies that the main bitter substance of Ganoderma lucidum is terpenoid compounds.

[0198] Table 14 Binding free energy of homology model-bitter substance docking conformation

[0199]

[0200]

[0201]

[0202]

[0203] 3.3.2 Visual analysis of docking results

[0204] Six representative bitter metabolites were selected: ganoderic acid E, ganoderic acid A, isoflavones, matrine, liquiritin and quinic acid for visualization of molecular docking results. Fig.23 As shown, ganoderic acid E ( Fig.23 a) and ganoderic acid A( Fig.23c) can bind tightly to THR-233 and THR-236 amino acid residues, and this binding keeps the ligand stable in the binding pocket. This result shows that ganoderic acid may be more inclined to bind to the TAS2R14 receptor through these two amino acid residues. This binding mode may have an important influence on the bitter taste of ganoderic acid. In addition, isoflavones ( Fig.23 b) and quinic acid ( Fig.23 f) can form hydrogen bonds with the LEU-240 amino acid residue. Through the above experimental results, it can be preliminarily inferred that THR, LEU, MET and ILE amino acids may be the main binding sites of TAS2R14. These amino acid residues play a key role in the binding process between the TAS2R14 receptor and bitter metabolites. This discovery has important guiding significance for subsequent research and can help us understand more deeply how these metabolites bind to TAS2R14 and produce bitterness.

[0205] 4. Cell experiment results:

[0206] 4.1 Effects of acetaldehyde on LX-2 cell proliferation

[0207] The CCK-8 method was used to detect the effects of different concentrations of ethanol (0.00096, 0.0048, 0.024, 0.12, 0.6, 3, 15, 75 mmol / L) on the proliferation of LX-2 cells. After 24 hours of cell culture, ethanol promoted cell proliferation. When the ethanol concentration was 3 mmol / L, the effect on the proliferation of LX-2 cells was most obvious ( Fig.24 ). So we designed the acetaldehyde experimental concentration to be 3mmol / L.

[0208] 4.2 Effects of different intervention concentrations on cell viability

[0209] Depend on Fig.25 From a, b, c, and d, we can see that the survival rate of LX-2 cells in the water extract and alcohol extract treatment groups of Ganoderma lucidum before and after fermentation was above 90% at 0.25 mg / mL, showing no toxic effect, and the cell survival rates of each test sample were similar, indicating that the concentrations of each sample selected in the test would not affect the normal growth of LX-2 cells. Therefore, 0.25 mg / mL was used as the subsequent action concentration of the intervention group.

[0210] 4.3 LDH enzyme activity

[0211] In normal cells, lactate dehydrogenase (LDH) cannot pass through the cell membrane or can only pass through a small amount of it and be released outside the cell. Fig.26It can be seen that the LDH leakage rate of the model group was 234.19%, indicating that alcohol modeling caused damage to the cell membrane, the permeability of the cell membrane changed, and LDH leaked out of the cell through the damaged cell membrane. The LDH leakage rate of the unfermented Ganoderma lucidum treatment group and the fermented Ganoderma lucidum treatment group was significantly reduced (P < 0.05), indicating that the water extract and alcohol extract of Ganoderma lucidum before and after fermentation can reduce the damage to the cell membrane. The different treatment groups decreased by 41.78%, 81.02%, 83.55%, and 93.68% respectively compared with the model group. This shows that Ganoderma lucidum itself also has the effect of protecting liver damage.

[0212] 4.4 Determination of transaminase levels

[0213] Depend on Fig. 27 It can be seen that by measuring the content of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the supernatant of LX-2 cells, the content of AST and ALT in the supernatant culture fluid of the control group was 1.94nmol / min / mL and 2.15nmol / min / mL, respectively, and the content of AST and ALT in the supernatant culture fluid of the model group was 5.13nmol / min / mL and 4.86nmol / 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, and the results were significantly different (P < 0.05), indicating that ethanol can cause certain damage to cells and increase the release of transaminases.

[0214] Ganoderma lucidum extracts (unfermented Ganoderma lucidum water extract, fermented Ganoderma lucidum water extract, unfermented Ganoderma lucidum alcohol extract, fermented Ganoderma lucidum alcohol extract) had an effect on alcohol-induced alcoholic liver injury at an intervention concentration of 0.25 mg / mL, reducing the release of AST by 34.29%, 50.88%, 52.95%, and 66.42% compared with the model group, respectively. The release of ALT was reduced by 17.52%, 29.55%, 44.86%, and 45.96% compared with the model group, respectively. The fermented Ganoderma lucidum group had a significantly better regulatory effect on AST and ALT in alcohol-damaged LX-2 cells than the unfermented Ganoderma lucidum (P < 0.05), and the alcohol extract had a better effect than the water extract.

[0215] 4.5. Cellular antioxidant enzyme activities

[0216] like Fig.28 As shown in (a), the fermented Ganoderma lucidum alcohol extract has the best effect on increasing the intracellular reduced glutathione (GSH) content. Compared with the model group, the intervention concentration of 0.25 mg / mL can increase the intracellular GSH level by 1.22 times. The cellular GSH levels of 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.

[0217] like Fig.28 b. The four kinds of Ganoderma lucidum extracts intervened in cells, and the activity of catalase (CAT) in cells increased significantly by 0.41, 0.50, 0.55, and 1.02 times compared with the model group. Although the four kinds of Ganoderma lucidum extracts can increase the activity of CAT in cells, they cannot be close to the CAT activity of the initial control. Compared with the control group, the intracellular GSH content and CAT activity of the model group decreased significantly.

[0218] Compared with the model group treated with alcohol, the activities of key antioxidant enzymes such as GSH and CAT in cells treated with the four Ganoderma lucidum extract samples increased, indicating that Ganoderma lucidum can improve the occurrence of cellular oxidative damage, and the effect of the fermented Ganoderma lucidum group is better than that of the unfermented group.

[0219] The above are only 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 in the protection scope of the present invention.

Claims

1. A process for fermenting Ganoderma lucidum with Aspergillus cristatus, characterized in that: The process comprises the following steps: S1. Strain activation and starter culture preparation: The bacterial blocks stored at -80°C were transferred to 5% NaCl+MYA solid culture medium in a clean bench, and cultured at 28°C constant temperature and humidity for 7 days. This process was repeated twice to activate the strains. Then, the activated strains were inoculated on 5% NaCl+MYA plate culture medium and cultured at 28°C constant temperature and humidity for 7 days. Add an appropriate amount of sterile water to the Aspergillus cristatus plate that has been cultured for 7 days, scrape the spores, take 1 mL of spore liquid and add it to a centrifuge tube containing glass beads, shake to break the ascospores, count them with a hemocytometer, and finally prepare a starter culture of corresponding concentration for use; S2. Fermentation process: Weigh 6-10g of 2-3mm thick Ganoderma lucidum blocks into a fermentation bottle, sterilize at 121°C for 20min, cool to room temperature, add sterile water to adjust the moisture content of Ganoderma lucidum, inoculate an appropriate amount of fermentation agent, and then culture at a constant temperature and humidity of 28-36°C for 7-10d.

2. The process for fermenting Ganoderma lucidum with Aspergillus cristatus according to claim 1, characterized in that: The spore concentration of the spore suspension is 10 4 -10 8 pcs / mL.

3. The process for fermenting Ganoderma lucidum with Aspergillus cristatus according to claim 1, characterized in that: The water content of the ganoderma lucidum is 80%-120%.

4. The process for fermenting Ganoderma lucidum with Aspergillus cristatus according to claim 1, characterized in that: The inoculation volume of the fermentation agent is 500-900 μL.

5. The process for fermenting Ganoderma lucidum with Aspergillus cristatus according to claim 1, characterized in that: The fermentation temperature is 28-36°C.

6. The process for fermenting Ganoderma lucidum with Aspergillus cristatus according to claim 1, characterized in that: The fermentation days are 7-10 days.

7. The process for fermenting Ganoderma lucidum by Aspergillus cristatus according to any one of claims 1 to 6, characterized in that: The fermented ganoderma lucidum prepared by the process is used in preparing products for protecting alcoholic liver damage.

8. The process for fermenting Ganoderma lucidum with Aspergillus cristatus according to claim 7, characterized in that: The product is a health product or a medicine.

9. The Ganoderma lucidum tablets fermented by Aspergillus cristatus prepared according to the method of claims 1-6.

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