Ganoderma lucidum and traditional Chinese medicine bidirectional fermentation product, preparation method and application thereof

Abstract

The present application relates to the technical field of microbial fermentation, in particular to a ganoderma lucidum-traditional Chinese medicine bidirectional fermentation product, a preparation method and application thereof, using a PDA culture plate or a PDB culture medium to obtain a fermentation seed, crushing traditional Chinese medicine, passing through an 80-mesh screen to obtain traditional Chinese medicine powder, inoculating ganoderma lucidum liquid obtained by expansion of the fermentation seed into a liquid or solid culture medium to perform fermentation culture, adding the traditional Chinese medicine powder during the culture, and obtaining the ganoderma lucidum-traditional Chinese medicine bidirectional fermentation product after the fermentation is completed. The ganoderma lucidum-traditional Chinese medicine bidirectional fermentation product solves the problem of how to develop a fermentation food with the functions of reducing blood sugar and helping weight loss for obese, hyperglycemic and even diabetic people by using ganoderma lucidum and traditional Chinese medicine.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, specifically to a two-way fermentation product of Ganoderma lucidum and traditional Chinese medicine, its preparation method, and its application. Background Technology

[0002] Diabetes mellitus is a metabolic disease caused by insufficient insulin secretion or impaired insulin action, leading to persistently elevated blood sugar levels. It is mainly divided into type I (autoimmune destruction of pancreatic beta cells) and type II (insulin resistance combined with secretion defects). Its core causes involve the combined effects of genetic susceptibility, unhealthy lifestyle, obesity, and environmental factors. Diabetic patients suffer long-term physical and psychological burdens, including the need for repeated blood glucose testing, insulin injections, slow wound healing, retinopathy, and peripheral neuropathy.

[0003] Studies have shown that Ganoderma lucidum mycelium and spores contain a variety of active ingredients, including triterpenoids, polysaccharides, proteins, polypeptides, and steroids, with a wide range of pharmacological effects, including enhancing immunity, improving hypoxia tolerance, and lowering blood sugar. Lotus leaf, hawthorn, rehmannia root, polygonatum rhizome, mulberry leaf, monk fruit, notoginseng, astragalus root, and kudzu root all fall under the category of "medicine and food homology" or can be used in health foods approved by the state. Ancient texts explicitly or implicitly point to symptoms related to modern glucose metabolism disorders, such as "diabetes," "dry mouth due to deficiency," and "deficiency of both qi and yin." However, due to shortcomings such as insufficient standardization and slow onset of action, they have not been further developed.

[0004] In view of the above-mentioned defects, the inventors of this invention have finally obtained this invention after a long period of research and practice. Summary of the Invention

[0005] The purpose of this invention is to solve the problem of how to develop functional fermented foods and medicines that can lower blood sugar and help with weight loss for people with obesity, high blood sugar, or even diabetes by utilizing Ganoderma lucidum and traditional Chinese medicine. The invention provides a Ganoderma lucidum-traditional Chinese medicine bidirectional fermentation product, its preparation method, and its application.

[0006] To achieve the above objectives, this invention discloses a method for preparing a two-way fermentation product of Ganoderma lucidum and traditional Chinese medicine, comprising the following steps:

[0007] S1, use PDA culture plates or PDB medium to obtain fermentation seeds;

[0008] S2, pulverize the Chinese medicine and pass it through an 80-mesh sieve to obtain Chinese medicine powder;

[0009] S3 involves inoculating the Ganoderma lucidum mycelium solution obtained from the fermentation of the seed culture into a liquid or solid culture medium for fermentation culture. During the culture, traditional Chinese medicine powder is added. After the fermentation is completed, a two-way fermentation product of Ganoderma lucidum and traditional Chinese medicine is obtained.

[0010] In step S1, the Ganoderma lucidum strain is any one of Ganoderma lucidum, Ganoderma sinense, Ganoderma pine and fir, Ganoderma truncatum, Ganoderma sclerophylla, Ganoderma lucidum var. truncatum, Ganoderma lucidum antler, Ganoderma lucidum var. truncatum, and Ganoderma lucidum var. truncatum.

[0011] In step S2, the Chinese medicine is any one of the following: lotus leaf, hawthorn, rehmannia root, polygonatum rhizome, mulberry leaf, monk fruit, notoginseng, astragalus root, and kudzu root.

[0012] In step S1, a PDA culture plate is inverted at 28°C for 7-10 days to obtain fermentation seeds.

[0013] In step S1, the fermentation seed is obtained by culturing in PDB medium at 28°C with shaking for 5-7 days.

[0014] In step S3, the liquid culture medium contains the following components: corn flour 10-20 g / L, soybean meal 10-20 g / L, peanut meal 10-20 g / L, wheat bran 10-20 g / L, and potassium dihydrogen phosphate 3-6 g / L, sterilized at 121°C for 30 min.

[0015] In step S3, the solid culture medium contains the following components: 200-400 g / L corn flour, 200-400 g / L wheat bran, 200-600 g / L peanut shells, 100-200 g / L traditional Chinese medicine powder, 3-5 g / L potassium dihydrogen phosphate, and a moisture content of 40-50%; it is then packaged into mushroom bags, each containing 150-300 g, and sterilized at 121℃ for 30 min.

[0016] In step S3, the fermentation culture is a solid-state fermentation, as follows: the inoculated bags are placed in a constant temperature and humidity environment of 20-25℃ and 60%-70% relative humidity, and then statically cultured for 7-21 days.

[0017] In step S3, the fermentation culture is liquid fermentation, specifically as follows: the temperature of the fermentation tank is controlled at 25 ~ 30℃, the air flow rate is 100 ~ 300 L / h, and the stirring speed is gradually increased from 0 ~ 300 rpm as the viscosity of the fermentation mash increases.

[0018] In step S3, after culturing for 3 to 7 days, add 20 to 30 g of Chinese herbal powder and culture for another 1 to 3 days to complete the fermentation.

[0019] This invention also discloses the Ganoderma lucidum-traditional Chinese medicine bidirectional fermentation product obtained by the above preparation method, and the application of this Ganoderma lucidum-traditional Chinese medicine bidirectional fermentation product in the preparation of functional fermented foods that lower blood sugar and help with weight loss.

[0020] Compared with existing technologies, the beneficial effects of this invention are as follows: the two-way fermentation technology of this invention is simple to operate and highly controllable; the fermentation products have the function of controlling blood sugar and weight. It is expected to reduce insulin use, alleviate the suffering of diabetic patients, and improve their quality of life. Attached Figure Description

[0021] Figure 1 Volcano plot for differentially expressed genes;

[0022] Figure 2 For GO enrichment histogram;

[0023] Figure 3 A bar chart showing the enrichment results for the KEGG Pathway;

[0024] Figure 4 The effect of Ganoderma lucidum-Siraitia grosvenorii bidirectional fermentation on OGTT in T2DM mice is shown in the figure. a is the blood glucose fluctuation curve and b is the area under the blood glucose curve, which reflects the overall blood glucose level of an individual over a certain period of time.

[0025] Figure 5 The effect of Ganoderma lucidum-Siraitia grosvenorii dual fermentation product on ITT in T2DM mice is shown in the figure. In the figure, a is the blood glucose fluctuation curve and b is the area under the blood glucose curve, which reflects the overall blood glucose level of an individual over a certain period of time.

[0026] Figure 6 Effects of Ganoderma lucidum-Siraitia grosvenorii fermentation on HOMA-IR in T2DM mice;

[0027] Figure 7 The effects of Ganoderma lucidum-Siraitia grosvenorii fermentation on four lipid parameters in T2DM mice were investigated. a, b, c, and d represent the measured values ​​of total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in different groups of mice, respectively.

[0028] Figure 8 The expression folds of three key genes in the PI3K / Akt signaling pathway were determined by q-PCR, where a is the PI3K gene, b is the Akt gene, and c is the GSK-3β gene.

[0029] Figure 9 Effects of Ganoderma lucidum-Siraitia grosvenorii fermentation on pancreatic islet morphology and structure in T2DM mice;

[0030] Figure 10 The effects of Ganoderma lucidum-Siraitia grosvenorii dual fermentation product on liver morphology and structure in T2DM mice;

[0031] Figure 11 The effects of Ganoderma lucidum-Siraitia grosvenorii fermentation on the morphology and structure of kidneys in T2DM mice. Detailed Implementation

[0032] The above-mentioned and other technical features and advantages of the present invention will be described in more detail below with reference to the accompanying drawings.

[0033] Example 1

[0034] This embodiment obtains the Ganoderma lucidum-Siraitia grosvenorii dual fermentation product through the following steps:

[0035] (1) Preparation of Ganoderma lucidum strain

[0036] Ganoderma lucidum was inoculated into PDB medium and cultured in a secondary scale-up manner, with each inoculation amount being 2%.

[0037] (2) Preparation of liquid culture medium

[0038] The culture medium contains the following ingredients (per liter): 20 g corn flour, 20 g soybean meal, 30 g peanut meal, 20 g wheat bran, and 5 g potassium dihydrogen phosphate. It is sterilized at 121°C for 30 min.

[0039] (3) Processing of monk fruit

[0040] Take a quantity of monk fruit, crush it using a grinder, and pass it through an 80-mesh sieve for later use.

[0041] (4) Constant temperature fermentation

[0042] The fermentation temperature was controlled at 30℃, and the air flow rate and stirring speed are shown in Table 1. At the 96th hour of fermentation, a sterile sample was taken to determine the reducing sugar content in the fermentation broth. When the reducing sugar content dropped to below 30% of the initial value, monk fruit powder was added. Fermentation continued for another 48 hours until the wet weight of the mycelium no longer increased significantly and the pH value stabilized between 4.5 and 5.5, at which point fermentation was complete. This yielded the Ganoderma lucidum-monk fruit dual-fermentation product.

[0043] Table 1 Fermentation Condition Control

[0044]

[0045] Feeding diabetic model mice with this bidirectional fermentation product for five weeks showed that the diabetic symptoms of the mice were improved and the blood glucose level tended to be normal.

[0046] Example 2

[0047] Functional validation of bidirectional fermentation products:

[0048] 1. Transcriptomics analysis:

[0049] After adding monk fruit powder and continuing fermentation for 24 hours, samples were taken, centrifuged at 4°C to remove the supernatant precipitate, washed 2-3 times with phosphate-buffered saline (PBS), and then frozen in liquid nitrogen for 0.5 hours before being sent for transcriptome sequencing. The changes in the metabolic pathways of Ganoderma lucidum after the addition of monk fruit were analyzed to infer product changes. Sequencing quality is shown in Table 2.

[0050] Table 2 Overall Statistics of Sequences

[0051]

[0052] (1) Analysis of differentially expressed genes (DEGs) between groups

[0053] The study set the screening criteria as log2FoldChange > 1 and p-value < 0.05, such as Figure 1 As shown, a total of 1276 differentially expressed genes were detected. Among them, 592 genes were significantly upregulated and 684 genes were significantly downregulated in the Ganoderma lucidum-Siraitia grosvenorii bidirectional fermentation group compared with the Ganoderma lucidum group.

[0054] (2) GO annotation of differentially expressed genes (DEGs) between transcriptomes

[0055] The annotation system is mainly divided into three categories: biological processes, molecular functions, and cellular components. Using a p-value < 0.05 as the criterion for significant enrichment, the top ten GO terms with the highest enrichment level and lowest p-value in each category were selected to complete the results display. The results are as follows: Figure 2 As shown.

[0056] The bidirectional fermentation group showed significant enrichment of mitosis-related functional proteins, and the corresponding proteins in the mitochondrial matrix also exhibited high significance, indicating that the bidirectional fermentation conditions of Ganoderma lucidum and Luo Han Guo (monk fruit) are conducive to the development and proliferation of Ganoderma lucidum strains. The significant enrichment of 3-hydroxyacyl-CoA dehydrogenase indicates active fatty acid catabolism in the system, suggesting a high energy demand and metabolically active state. The protein content of genes ORF429 and UCE2 was also significantly increased, possibly helping P450 enzymes to locate their corresponding targets on the membrane. P450 enzymes are often associated with fungal secondary metabolism, thus it is speculated that the addition of Luo Han Guo powder can enhance the secondary metabolism of Ganoderma lucidum.

[0057] (3) KEGG annotation of differentially expressed genes (DEGs) between transcriptomes

[0058] KEGG (Kyoto Encyclopedia of Genes and Genomes), as a mainstream public database for pathways, performs pathway significance enrichment analysis on differentially expressed genes, which can clarify the core biochemical metabolic and signal transduction pathways they participate in. Based on the KEGG enrichment analysis data of differentially expressed genes, this study screened 30 pathways with the lowest p-values ​​and the most significant enrichment effects. The results are as follows: Figure 3 As shown.

[0059] In the glycolysis / gluconeogenesis pathway, the expression of the key gene HK2 was significantly upregulated, directly leading to enhanced glucose metabolism in Ganoderma lucidum. In the p53 signaling pathway, Cyclin B gene expression was upregulated, while Apaf-1 gene expression was downregulated. Upregulation of Cyclin B may prolong cell division, while downregulation of Apaf-1 inhibits apoptosis and prolongs mycelial survival. This also provides further evidence that the addition of monk fruit powder can enhance the secondary metabolism of Ganoderma lucidum. It is speculated that the addition of monk fruit powder leads to the generation of more Ganoderma lucidum triterpenoids and polysaccharides, which further regulate carbohydrate synthesis by targeting and promoting phosphorylation of the PI3K / Akt pathway, thereby intervening in diabetes.

[0060] 2. Animal experiments

[0061] Male SPF-grade C57BL / 6J mice aged 5–6 weeks were selected and acclimatized for two weeks. They were allowed free access to food and water throughout the period. The rearing environment was kept at a constant temperature of 24±2℃ and a relative humidity of 60%, using a day-night cycle of 12 hours of light and 12 hours of darkness.

[0062] (1) Establishment of type II diabetic mouse model

[0063] After a week of acclimatization feeding with a basal diet, mice were randomly divided into a normal group (N) and a model group (C) based on their body weight. The normal group continued to be fed a regular basal diet, while the model group was fed a high-fat diet composed of 40% basal diet, 32% lard, 6.5% soybean oil, 9.4% sucrose, 25.8% casein, and 16.15% maltodextrin, with appropriate amounts of minerals, vitamins, and gelatin. After four weeks of continuous feeding, the mice were subjected to overnight fasting, followed by three consecutive days of injection of streptozotocin (STZ) at a dose of 60 mg / kg to establish a diabetic animal model. Fasting blood glucose levels were measured 72 hours after modeling. If the value exceeded 11.0 mmol / L and remained stable in subsequent fasting blood glucose tests, the model was considered to be effectively established. The modeling results are shown in Table 3.

[0064] Table 3 Results of type II diabetic mouse modeling

[0065]

[0066] Mice that successfully developed the model were randomly assigned to four groups: the model group (C group), the metformin group (MET group), the low-dose unfermented Ganoderma lucidum-Siraitia grosvenorii (GSL group), the high-dose unfermented Ganoderma lucidum-Siraitia grosvenorii (GSH group), the low-dose bi-fermented Ganoderma lucidum-Siraitia grosvenorii (GSFL group), and the high-dose bi-fermented Ganoderma lucidum-Siraitia grosvenorii (GSFH group). All groups were fed a high-fat diet throughout the experiment and maintained free access to food and water. The experiment involved daily gavage administration. Groups N and C received the same volume of distilled water; the MET group received 150 mg / kg of metformin solution; the GSL and GSH groups were treated with unfermented Ganoderma lucidum-Siraitia grosvenorii culture medium; and the GSFL and GSFH groups were fed with bi-fermented Ganoderma lucidum-Siraitia grosvenorii culture medium. The dose gradient was set at a low dose of 6 mL / kg and a high dose of 12 mL / kg, both administered once daily via gavage.

[0067] (2) Measurement of fasting blood glucose (FBG) in mice

[0068] During the experimental period, fasting blood glucose levels in mice were measured at fixed times each week to evaluate the effect of the Ganoderma lucidum-Siraitia grosvenorii dual fermentation product on improving fasting blood glucose levels in mice. The results are shown in Table 4.

[0069] Table 4 Fasting blood glucose levels in mice (x±s, n=6)

[0070]

[0071] Compared with the blank N group, the blood glucose levels of mice in the model control C group showed a significantly elevated level (p < 0.01). Compared with group C, the MET and GSFL groups showed a significant decrease after 5 weeks of gavage (p < 0.05), while the GSFH group showed a highly significant decrease (p < 0.01). Data calculations showed that the blood glucose reduction rates in the MET, GSFL, and GSFH groups were 20.56%, 50.4%, and 20.05%, respectively. This confirms that the Ganoderma lucidum-Siraitia grosvenorii dual fermentation product can effectively regulate the body's glucose metabolism and effectively improve hyperglycemia symptoms in mice.

[0072] (3) Oral glucose tolerance test (OGTT) in mice

[0073] One week before the end of the experiment, the mice were fasted overnight and then uniformly administered a 1 g / kg glucose solution via gavage. Blood glucose data were recorded at five time points (0 min, 30 min, 60 min, 90 min, and 120 min) after oral glucose administration. Blood glucose trend curves were plotted based on the results, and the area under the curve (AUG) was calculated using the corresponding formula. The results are shown below. Figure 4 As shown.

[0074] Compared to group N, group C mice showed a significant increase in blood glucose levels, peaking at 30 minutes after intervention and gradually declining, but remaining significantly higher than the lowest blood glucose level in the control group even at 120 minutes, indicating impaired pancreatic function and a substantial decrease in blood glucose regulation in the model mice. Compared to the model group, the MET and GSFL groups showed similar trends in blood glucose changes, with significant decreases in blood glucose levels at all time points. The integrals of their curves are shown below. Figure 4 As shown in b), the area under the curve (AUC) of the GSFL and MET groups was significantly lower than that of group C. Therefore, the results indicate that the Ganoderma lucidum-Siraitia grosvenorii dual fermentation product has a regulatory effect on postprandial blood glucose levels in diabetic mice.

[0075] (4) Insulin tolerance test (ITT) in mice

[0076] Three days before the end of the experiment, ITT (intracytoplasmic insulin tolerance) was measured. Mice were fasted overnight, and all subjects were administered insulin intraperitoneally at a dose of 0.75 U / kg. Blood glucose data were collected at 0, 30, 60, 90, and 120 minutes after injection. Dynamic change curves were plotted based on the results, and the corresponding AUG (autoimmune glucose tolerance) values ​​were calculated using a formula to analyze the regulatory effects of different drug interventions on insulin tolerance in mice. The results are as follows: Figure 5 As shown.

[0077] Compared to group N, group C showed lower insulin sensitivity after insulin injection, while the MET, GSFL, and GSFH groups showed some recovery. The integral results of the curves are as follows: Figure 8 As shown in b), compared with group C, the areas under the curves of the MET, GSFL, and GSFH groups were significantly lower than those of group C. Therefore, the results indicate that the Ganoderma lucidum-Siraitia grosvenorii dual fermentation product can improve insulin utilization and insulin sensitivity in diabetic mice.

[0078] (5) Measurement of mouse serum insulin resistance index

[0079] After all experimental procedures were completed, the mice were fasted overnight. Blood samples were obtained via retroorbital vein sampling and placed in polystyrene tubes pre-filled with anticoagulant. The samples were allowed to stand at room temperature for one hour, followed by centrifugation at 4000 rpm for 15 minutes. The resulting serum was used for lipid-related marker detection. Insulin levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit, strictly following the instructions. The HOMA-IR insulin resistance index was calculated using the corresponding formula to investigate the regulatory effects of various drugs on insulin sensitivity and pancreatic islet cell secretion function in mice. The results are as follows: Figure 6 As shown.

[0080] Compared with group N, group C mice showed a significantly higher HOMA-IR than the control group, indicating that group C mice developed insulin resistance and had higher blood glucose levels. Compared with group C, the MET group, GSFL group, and group M showed significantly lower HOMA-IR values, indicating that the mice's insulin regulation ability was restored and insulin resistance was reduced. Therefore, the results show that the Ganoderma lucidum-Siraitia grosvenorii dual fermentation product can improve insulin sensitivity in diabetic mice.

[0081] (6) Measurement of hyperlipoproteinemia in mice

[0082] Serum and lipid samples were obtained in the same manner as in the previous section. Enzyme-linked immunosorbent assay (ELISA) kits were used to detect triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDC-C), and low-density lipoprotein cholesterol (LDC-C) in mouse blood lipids, in order to analyze the regulatory effects of different drug interventions on lipid metabolism in mice. The results are as follows: Figure 7 As shown.

[0083] Compared with group N, group C showed a significant increase in TC and TG levels, exceeding normal values. The GSFL and GSFH groups showed some recovery, with TC and TG levels remaining within the normal range. HDL-C and LDL-C levels in all groups were within the normal range, indicating no adverse effects. Therefore, the Ganoderma lucidum-Siraitia grosvenorii dual-fermentation product can improve glucose and lipid metabolism in T2DM mice, restoring TC and TG levels to normal and reducing the risk of diabetic complications.

[0084] (7) Real-time quantitative PCR (q-PCR)

[0085] Using q-PCR technology, the expression differences of key genes in the PI3K / Akt signaling pathway in the liver tissues of different groups of mice were detected at the gene level. The gene sequences of key proteins were obtained by searching the NCBI website, and upstream and downstream primers for the target genes were designed based on these sequences, as shown in Table 5.

[0086] Table 5. q-PCR primer design sequences

[0087]

[0088] Weigh 20 mg of liver tissue into a pre-treatment grinding tube and grind it thoroughly using a grinder until the tissue is completely pulverized. Centrifuge the sample at 4°C and 12,000 rpm for 10 min and collect the supernatant. Add 100 μL of chloroform substitute to the system, mix vigorously by inverting for 15 s, let stand for 3 min, and then centrifuge again at low temperature. Collect the supernatant, add 550 μL of isopropanol, mix well, and incubate at -20°C for 15 min. Centrifuge again at low temperature and high speed for 10 min; the white precipitate at the bottom of the tube is the total RNA. Discard the supernatant, add 1 mL of 75% ethanol, invert and rinse the precipitate, centrifuge at 4°C and 12,000 rpm for 5 min, and repeat the rinsing process twice. After completely removing residual ethanol, place the centrifuge tube in a clean bench and air dry for 3-5 min. Finally, add 15 μL of RNA lysis buffer to fully dissolve the precipitate, completing the RNA extraction.

[0089] The reverse transcription reaction was further carried out using the standard operating procedure of the reverse transcription kit, and DNA removal from the samples was performed simultaneously. The system is shown in Table 6. After thoroughly mixing all reaction reagents, the sample was placed in the PCR instrument and the preset program was started. The reaction was sequentially set to 25℃ for 5 min, 42℃ for 30 min, and 85℃ for 5 s. After the program was completed, the sample was stored at 4℃ for later use.

[0090] Table 6. Configuration of the reverse transcription reaction system

[0091]

[0092] Finally, 0.1 mL PCR reaction plates were selected for system preparation, and the specific component ratios were performed according to Table 7. Each reverse transcription product was used in triplicate. After sample loading, the plates were sealed using a PCR-specific sealing film and a sealing device, and then centrifuged using a microplate centrifuge.

[0093] Table 7 q-PCR reaction system configuration

[0094]

[0095] Sample amplification was performed using a real-time PCR instrument. The amplification program first set a pre-denaturation treatment at 95℃ for 30 s, followed by a denaturation step at 95℃ for 15 s, and then annealing and extension at 60℃ for 30 s. This entire cycle was repeated 40 times. The melting curve detection range was set from 65℃ to 95℃, and fluorescence signals were collected simultaneously for every 0.5℃ temperature increase. The results are shown below. Figure 8 As shown.

[0096] (8) Histopathological sections of experimental mice

[0097] After all experimental mice were sacrificed, their pancreas, liver, and kidneys were dissected and pathological sections were prepared. This part of the experiment was conducted by Wuhan Saiwei Biotechnology Co., Ltd., and the results are as follows: Figure 9-11 As shown.

[0098] like Figure 9 As shown, the N group's pancreas surface was covered by a thin, loose connective tissue capsule. The pancreas contained numerous lobules separated by fine connective tissue septa. The pancreatic acini were branching tubular alveolar glands, with abundant, clearly defined acinar cells; the apex was eosinophilic, and the base basophilic. The acini were tightly clustered to form lobules. The islets were clusters of endocrine cells distributed among the acini; the islets were abundant, regularly shaped, and clearly defined. Compared to the N group, the model control group C showed more acinar hypertrophy (green arrows), an increased eosinophilic area at the apex, and a small amount of connective tissue hyperplasia in the stroma (brown arrows). The islets were irregularly shaped, and acinar invasion was observed. Compared to group C, the MAT, GSFL, and GSFH groups showed symptom relief, with only a small amount of acinar vacuolar degeneration (yellow arrows), and a small amount of acinar hypertrophy without obvious necrosis or inflammatory cell infiltration. Therefore, the results indicate that the Ganoderma lucidum-Siraitia grosvenorii dual-fermentation product can improve pancreatic islet damage in diabetic mice and alleviate diabetes.

[0099] like Figure 10 As shown, the liver capsule of mice in group N was a dense connective tissue with uniform thickness and abundant elastic fibers. The liver lobules were centered on the central vein, and hepatocytes and sinusoids were radially and regularly distributed. The hepatocytes were plump and round, the liver plates were neatly arranged, and there was no dilation or compression of the sinusoids. The portal area structure was normal, and there were no pathological abnormalities. Compared with group N, mice in group C showed obvious pathological damage to the liver: a large number of hepatocytes underwent fatty degeneration (yellow arrows), and the cytoplasm showed irregularly sized round vacuoles; accompanied by significant hepatocyte edema (blue arrows), ballooning degeneration, swollen and enlarged cells, with the nucleus centered and the cytoplasm loose and reticular. Punctate hepatocyte necrosis (purple arrows) was visible (nuclear condensation and fragmentation, cytoplasm red staining), a small number of lymphocytes and granulocytes showed focal infiltration (green arrows), and a small amount of connective tissue hyperplasia was present in the portal area and around the sinusoids (brown arrows). Compared to the model group, the liver pathological symptoms in mice in the MET, GSFL, and GSFH groups were significantly alleviated, with only a small amount of mild hepatocyte steatosis, smaller cytoplasmic vacuoles, significantly reduced hepatocyte edema, improved cytoplasmic looseness, and only sporadic lymphocyte infiltration in the portal areas. In conclusion, the Ganoderma lucidum-Siraitia grosvenorii dual-fermentation product can effectively repair liver damage in T2DM mice, promote glycogen synthesis, and improve insulin resistance.

[0100] like Figure 11As shown, the outer capsule of the kidneys in group N was a dense connective tissue of uniform thickness, with clear and distinct layers between the renal cortex and medulla. Glomeruli were evenly distributed within the cortex, with balanced cell numbers and matrix content. Tubular epithelial cells were plump and regular, with orderly brush borders, and no abnormal lesions were observed in the medulla. No proliferation of renal interstitial connective tissue was observed, and no inflammatory cell infiltration was noted; the overall kidney structure was intact and healthy. Compared to the normal group, mice in group C showed typical pathological damage in their kidneys, with extensive proliferation of glomerular mesangial matrix (orange arrows), resulting in widening of the mesangial area and blurred and disordered capillary structure. Extensive tubular epithelial edema was also observed (blue arrows), with enlarged cells, loose and lightly stained cytoplasm, and multiple vacuolar degeneration of the tubular epithelium (yellow arrows). Round microvacuoles formed in the cytoplasm, and some cell nuclei showed chromatin margination. Localized connective tissue proliferation was observed in the renal interstitium (brown arrows), and a small amount of focal infiltration of lymphocytes and granulocytes was observed around blood vessels (green arrows). Compared to the model group, the MET, GSFL, and GSFH intervention groups all showed effective improvement in renal pathological damage, significantly reduced glomerular mesangial matrix proliferation, and markedly improved renal tubular swelling and cytoplasmic loosening, with only a small amount of mild epithelial vacuolar degeneration remaining. No abnormal proliferation or significant inflammatory cell infiltration was observed in the renal interstitium of the intervention groups. These pathological observations confirm that the Ganoderma lucidum-traditional Chinese medicine dual-fermentation product can effectively repair renal pathological damage and improve renal function in T2DM mice.

[0101] 3. Isolation and purification of Ganoderma lucidum triterpenes

[0102] The fermentation mash was collected and ultrasonically disrupted. After filtering out cell debris, Ganoderma triterpenes were extracted with 60% ethanol. HPLC analysis revealed that the ganoderic acids were ganoderic acid C and ganoderic acid C2, with concentrations of 2.3 mg / L and 44.1 mg / L, respectively.

[0103] Example 3

[0104] This embodiment obtains the Ganoderma lucidum-Siraitia grosvenorii bidirectional fermentation product through the following steps:

[0105] (1) Preparation of Ganoderma lucidum strain

[0106] Ganoderma lucidum was inoculated into PDB medium and cultured in stages, with each inoculation amount being 1%.

[0107] (2) Processing of monk fruit

[0108] Take a quantity of monk fruit, crush it using a grinder, and pass it through an 80-mesh sieve for later use.

[0109] (3) Preparation of solid culture medium

[0110] The culture medium contains the following ingredients (per kilogram): 600 g corn cobs, 100 g soybean meal, 20 g glucose, 250 g wheat bran, 20 g traditional Chinese medicine powder, 10 g potassium dihydrogen phosphate, and has a water content of 58-62%.

[0111] (4) Constant temperature fermentation

[0112] The fermentation temperature was controlled at 24℃, the air humidity at 65%, and the fermentation time was 18 days.

[0113] After fermentation, the fermentation product had no off-odor and a unique floral and fruity aroma. After being crushed and dried, it was mixed into standard mouse feed at a ratio of 15% by mass and fed to mice. Not only was the condition of their fur improved, but their blood glucose levels also showed significant changes, exhibiting the same trend as the liquid two-way fermentation product.

[0114] Example 4

[0115] This embodiment obtains the Ganoderma lucidum-Siraitia grosvenorii bidirectional fermentation product through the following steps:

[0116] (1) Preparation of Ganoderma lucidum strain

[0117] Ganoderma lucidum was inoculated into PDB medium and cultured in stages, with each inoculation amount being 1%.

[0118] (2) Traditional Chinese medicine treatment

[0119] Take a quantity of monk fruit, crush it using a grinder, and pass it through an 80-mesh sieve for later use.

[0120] (3) Preparation of liquid culture medium

[0121] The culture medium contains the following ingredients (per liter): 20 g corn flour, 20 g soybean meal, 30 g peanut meal, 20 g wheat bran, and 5 g potassium dihydrogen phosphate. It is sterilized at 121°C for 30 min.

[0122] (4) Constant temperature fermentation

[0123] The fermentation temperature was controlled at 30℃, and the airflow rate and stirring speed were adjusted according to the viscosity of the fermentation broth. At the 96th hour of fermentation, a sterile sample was taken to determine the reducing sugar content in the fermentation broth. When the reducing sugar content dropped to below 30% of the initial value, monk fruit powder was added. Fermentation continued for another 48 hours until the wet weight of the mycelium no longer increased significantly and the pH value stabilized between 4.5 and 5.5, at which point fermentation was complete. This yielded the Ganoderma lucidum-monk fruit dual-fermentation product.

[0124] After fermentation, the fermentation product had no off-odor and a unique floral and fruity aroma. Feeding mice with the product for five weeks not only improved their fur and mental state, but also showed significant changes in blood glucose levels, exhibiting the same trend as in Example 2.

[0125] Example 5

[0126] This embodiment obtains the Ganoderma lucidum-Astragalus bidirectional fermentation product through the following steps:

[0127] (1) Preparation of Ganoderma lucidum strain

[0128] Ganoderma lucidum was inoculated into PDB medium and cultured in stages, with each inoculation amount being 1%.

[0129] (2) Traditional Chinese medicine treatment

[0130] Take a quantity of Astragalus membranaceus, crush it using a grinder, and pass it through an 80-mesh sieve for later use.

[0131] (3) Preparation of liquid culture medium

[0132] The culture medium contains the following ingredients (per liter): 20 g corn flour, 20 g soybean meal, 30 g peanut meal, 20 g wheat bran, and 5 g potassium dihydrogen phosphate. It is sterilized at 121°C for 30 min.

[0133] (4) Constant temperature fermentation

[0134] The fermentation temperature was controlled at 30℃, and the airflow rate and stirring speed were adjusted according to the viscosity of the fermentation broth. At the 96th hour of fermentation, a sterile sample was taken to determine the reducing sugar content in the fermentation broth. Astragalus powder was added when the reducing sugar content dropped below 30% of the initial value. Fermentation continued for another 48 hours until the wet weight of the mycelium no longer increased significantly and the pH value stabilized between 4.5 and 5.5, at which point fermentation was complete. This yielded the Ganoderma lucidum-Astragalus bidirectional fermentation product.

[0135] After fermentation, the fermentation product had no off-odor and a unique herbal aroma, and the fermentation liquid was significantly viscous. Its total polysaccharide content was 130% higher than the control fermentation liquid without astragalus. Feeding mice with this product for five weeks significantly reduced their blood glucose levels.

[0136] Example 6

[0137] This embodiment obtains the Ganoderma lucidum-Pueraria lobata bidirectional fermentation product through the following steps:

[0138] (1) Preparation of Ganoderma lucidum strain

[0139] Ganoderma lucidum was inoculated into PDB medium and cultured in stages, with each inoculation amount being 1%.

[0140] (2) Traditional Chinese medicine treatment

[0141] Take a quantity of kudzu root, crush it using a grinder, and pass it through an 80-mesh sieve for later use.

[0142] (3) Preparation of liquid culture medium

[0143] The culture medium contains the following ingredients (per liter): 20 g corn flour, 20 g soybean meal, 30 g peanut meal, 20 g wheat bran, and 5 g potassium dihydrogen phosphate. It is sterilized at 121°C for 30 min.

[0144] (4) Constant temperature fermentation

[0145] The fermentation temperature was controlled at 30℃, and the airflow rate and stirring speed were adjusted according to the viscosity of the fermentation broth. At the 96th hour of fermentation, a sterile sample was taken to determine the reducing sugar content in the fermentation broth. When the reducing sugar content dropped to below 30% of the initial value, kudzu root powder was added. Fermentation continued for another 48 hours until the wet weight of the mycelium no longer increased significantly and the pH value stabilized between 4.5 and 5.5, at which point fermentation was complete. This yielded the Ganoderma lucidum-kudzu root dual-fermentation product.

[0146] After fermentation, the fermentation product had no off-odor and a unique herbal aroma. Its total triterpenoid content was 28% higher than that of the control fermentation broth without kudzu root.

[0147] Example 7

[0148] This embodiment obtains the Ganoderma lucidum-mulberry leaf bidirectional fermentation product through the following steps:

[0149] (1) Preparation of Ganoderma lucidum strain

[0150] Ganoderma lucidum was inoculated into PDB medium and cultured in stages, with each inoculation amount being 1%.

[0151] (2) Traditional Chinese medicine treatment

[0152] Take a quantity of dried mulberry leaves, crush them using a pulverizer, and pass them through an 80-mesh sieve for later use.

[0153] (3) Preparation of liquid culture medium

[0154] The culture medium contains the following ingredients (per liter): 20 g corn flour, 20 g soybean meal, 30 g peanut meal, 20 g wheat bran, and 5 g potassium dihydrogen phosphate. It is sterilized at 121°C for 30 min.

[0155] (4) Constant temperature fermentation

[0156] The fermentation temperature was controlled at 30℃, and the airflow rate and stirring speed were adjusted according to the viscosity of the fermentation broth. At the 96th hour of fermentation, a sterile sample was taken to determine the reducing sugar content in the fermentation broth. When the reducing sugar content dropped to below 30% of the initial value, mulberry leaf powder was added. Fermentation continued for another 48 hours until the wet weight of the mycelium no longer increased significantly and the pH value stabilized between 4.5 and 5.5, at which point fermentation was complete. This yielded the Ganoderma lucidum-mulberry leaf dual-fermentation product.

[0157] After fermentation, the fermentation product had no off-odor and a unique herbal aroma. Its total ganoderic acid content was 42% higher than that of the control fermentation liquid without mulberry leaves.

[0158] Other co-fermented products of traditional Chinese medicine can positively affect the secondary metabolism of Ganoderma lucidum. The content of active ingredients in the two-way fermentation products is shown in Table 8.

[0159] Table 8 Comparison of active ingredient content in bidirectional fermentation products of different Chinese medicinal materials

[0160]

[0161] Comparative Example 1

[0162] This comparative example obtained the Ganoderma lucidum-Siraitia grosvenorii bidirectional fermentation product through the following steps:

[0163] (1) Preparation of Ganoderma lucidum strain

[0164] Ganoderma lucidum was inoculated into PDB medium and cultured in stages, with each inoculation amount being 1%.

[0165] (2) Preparation of liquid culture medium

[0166] The culture medium contains the following ingredients (per liter): 20 g corn flour, 20 g soybean meal, 20 g glucose, 20 g wheat bran, and 3 g potassium dihydrogen phosphate. Sterilize at 121°C for 30 min.

[0167] (3) Processing of monk fruit

[0168] Take a quantity of monk fruit, crush it using a grinder, and pass it through an 80-mesh sieve for later use.

[0169] (4) Constant temperature fermentation

[0170] The fermentation temperature was controlled at 30℃, and the air flow rate and stirring speed were as shown in Table 8. The herbal powder was added at the 96th hour of fermentation, and fermentation continued for another 48 hours to complete the process. This yielded the Ganoderma lucidum-Siraitia grosvenorii dual-fermentation product.

[0171] Table 9 Fermentation Condition Control

[0172]

[0173] The fermentation product has a floral and fruity aroma, but no characteristic taste of traditional Chinese medicine. Subsequent feeding to mice only resulted in changes in their fur; blood glucose levels remained largely unchanged. The presumed reason is that high ventilation and a high carbon source led to vigorous bacterial growth and metabolism, resulting in insufficient accumulation of active ingredients.

[0174] Comparative Example 2

[0175] This comparative example obtained the Ganoderma lucidum-Siraitia grosvenorii bidirectional fermentation product through the following steps:

[0176] (1) Preparation of Ganoderma lucidum strain

[0177] Ganoderma lucidum was inoculated into PDB medium and cultured in stages, with each inoculation amount being 1%.

[0178] (2) Preparation of liquid culture medium

[0179] The culture medium contains the following ingredients (per liter): 10 g corn flour, 30 g soybean meal, 10 g peanut meal, 20 g wheat bran, and 3 g potassium dihydrogen phosphate. It is sterilized at 121°C for 30 min.

[0180] (3) Processing of monk fruit

[0181] Take a quantity of monk fruit, crush it using a grinder, and pass it through an 80-mesh sieve for later use.

[0182] (4) Constant temperature fermentation

[0183] The fermentation temperature was controlled at 25℃, and the air flow rate and stirring speed were as shown in Table 10. The herbal powder was added at the 72nd hour of fermentation, and fermentation continued for another 24 hours to complete the process. This yielded the Ganoderma lucidum-Siraitia grosvenorii dual-fermentation product.

[0184] Table 10 Fermentation Condition Control

[0185]

[0186] The fermentation product had a fig aroma and also retained the characteristic flavor of traditional Chinese medicine. Subsequent feeding of mice showed no significant changes in blood glucose levels. The presumed reason is that the low aeration and high nitrogen source led to an imbalance in secondary metabolic flux, resulting in the failure of the bidirectional fermentation.

[0187] Comparative Example 3

[0188] This comparative example obtained the Ganoderma lucidum-Siraitia grosvenorii bidirectional fermentation product through the following steps:

[0189] (1) Preparation of Ganoderma lucidum strain

[0190] Ganoderma lucidum was inoculated into PDB medium and cultured in stages, with each inoculation amount being 1%.

[0191] (2) Processing of monk fruit

[0192] Take a quantity of monk fruit, crush it using a grinder, and pass it through an 80-mesh sieve for later use.

[0193] (3) Preparation of solid culture medium

[0194] The culture medium contains the following ingredients (per kilogram): 600 g corn cobs, 100 g soybean meal, 20 g glucose, 250 g wheat bran, 20 g traditional Chinese medicine powder, 10 g potassium dihydrogen phosphate, and has a water content of 58-62%.

[0195] (4) Constant temperature fermentation

[0196] The fermentation temperature was controlled at 28℃, the air humidity at 70%, and the fermentation time was 18 days.

[0197] After fermentation, the fermentation product had no off-odor, a yellowish surface, and a significant amount of water accumulation at the base. After being pulverized and dried, it was mixed into standard mouse feed at a ratio of 15% by mass and fed to mice. The results showed that this batch of fermentation product did not have a blood sugar-lowering effect. This was because the excessively high core temperature and rapid metabolism during solid-state fermentation led to mycelial aging and a decrease in the accumulation of active ingredients.

[0198] Comparative Example 4

[0199] This comparative example obtained the Ganoderma lucidum-Siraitia grosvenorii bidirectional fermentation product through the following steps:

[0200] (1) Preparation of Ganoderma lucidum strain

[0201] Ganoderma lucidum was inoculated into PDB medium and cultured in stages, with each inoculation amount being 1%.

[0202] (2) Traditional Chinese medicine treatment

[0203] Take a quantity of monk fruit, crush it using a grinder, and pass it through an 80-mesh sieve for later use.

[0204] (3) Preparation of solid culture medium

[0205] The culture medium contains the following ingredients (per kilogram): 400 g corn cobs, 300 g soybean meal, 10 g glucose, 250 g wheat bran, and 40 g traditional Chinese medicine powder, with a moisture content of 58-62%.

[0206] (4) Constant temperature fermentation

[0207] Control the fermentation temperature at 20℃, the air humidity at 70%, and the fermentation time at 18 days.

[0208] After fermentation, the culture medium was contaminated with Penicillium and Aspergillus niger. This was because the core temperature was too low during solid-state fermentation, allowing other microorganisms to become the dominant species.

[0209] The above description is merely a preferred embodiment of the present invention and is illustrative rather than restrictive. Those skilled in the art will understand that many changes, modifications, and even equivalents can be made within the spirit and scope defined by the claims of the present invention, all of which will fall within the protection scope of the present invention.

Claims

1. A method for preparing a Ganoderma lucidum-traditional Chinese medicine bidirectional fermentation product, characterized in that, Includes the following steps: S1. Ganoderma lucidum strains are cultured using PDA culture plates or PDB culture medium to obtain fermentation seeds; S2, pulverize the Chinese medicine and pass it through an 80-mesh sieve to obtain Chinese medicine powder; S3 involves inoculating the Ganoderma lucidum mycelium solution obtained from the fermentation of the seed culture into a liquid or solid culture medium for fermentation culture. During the culture, traditional Chinese medicine powder is added. After the fermentation is completed, a two-way fermentation product of Ganoderma lucidum and traditional Chinese medicine is obtained.

2. The method for preparing a Ganoderma lucidum-traditional Chinese medicine bidirectional fermentation product as described in claim 1, characterized in that, In step S1, the Ganoderma lucidum strain is any one of Ganoderma lucidum, Ganoderma sinense, Ganoderma pine and fir, Ganoderma truncatum, Ganoderma sclerophylla, Ganoderma lucidum var. truncatum, Ganoderma lucidum antler, Ganoderma lucidum var. truncatum, and Ganoderma lucidum var. truncatum. In step S2, the Chinese medicine is any one of the following: lotus leaf, hawthorn, rehmannia root, polygonatum rhizome, mulberry leaf, monk fruit, notoginseng, astragalus root, and kudzu root.

3. The method for preparing a Ganoderma lucidum-traditional Chinese medicine bidirectional fermentation product as described in claim 1, characterized in that, In step S1, fermentation seeds are obtained by inverting a PDA culture plate at 28°C for 7-10 days; and by shaking a PDB culture medium at 28°C for 5-7 days.

4. The method for preparing a Ganoderma lucidum-traditional Chinese medicine bidirectional fermentation product as described in claim 1, characterized in that, In step S3, the liquid culture medium contains the following components: corn flour 10-20 g / L, soybean meal 10-20 g / L, peanut meal 10-20 g / L, wheat bran 10-20 g / L, and potassium dihydrogen phosphate 3-6 g / L, sterilized at 121℃ for 30 min.

5. The method for preparing a Ganoderma lucidum-traditional Chinese medicine bidirectional fermentation product as described in claim 1, characterized in that, In step S3, the solid culture medium contains the following components: 200-400 g / L corn cob, 200-400 g / L wheat bran, 200-600 g / L peanut shell, 100-200 g / L traditional Chinese medicine powder, 3-5 g / L potassium dihydrogen phosphate, and a water content of 40-50%; it is then packaged into mushroom bags, each containing 150-300 g, and sterilized at 121℃ for 30 min.

6. The method for preparing a Ganoderma lucidum-traditional Chinese medicine bidirectional fermentation product as described in claim 1, characterized in that, In step S3, the fermentation culture is a solid-state fermentation, as follows: the inoculated bags are placed in a constant temperature and humidity environment of 20-25℃ and 60%-70% relative humidity, and then statically cultured for 7-21 days.

7. The method for preparing a Ganoderma lucidum-traditional Chinese medicine bidirectional fermentation product as described in claim 1, characterized in that, In step S3, the fermentation culture is liquid fermentation, specifically as follows: the temperature of the fermentation tank is controlled at 25 ~ 30℃, the air flow rate is 100 ~ 300L / h, and the stirring speed is gradually increased from 0 ~ 300 rpm as the viscosity of the fermentation mash increases.

8. The method for preparing a Ganoderma lucidum-traditional Chinese medicine bidirectional fermentation product as described in claim 1, characterized in that, In step S3, after culturing for 3 to 7 days, add 20 to 30 g of Chinese herbal powder and culture for another 1 to 3 days to complete the fermentation.

9. A Ganoderma lucidum-traditional Chinese medicine bidirectional fermentation product prepared by the preparation method according to any one of claims 1 to 8.

10. The application of the Ganoderma lucidum-traditional Chinese medicine bidirectional fermentation product as described in claim 9 in the development of functional fermented foods and drugs that lower blood sugar and aid weight loss.

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