A fermentation process for increasing the content of ganoderma lucidum polysaccharide, and a fermentation product and application thereof

Abstract

The present application relates to Ganoderma lucidum polysaccharide fermentation technical field, disclose a kind of fermentation process for improving the content of ganoderma polysaccharide and its fermentation product and application, including the following steps: using gradient mixing method, gradually mix ganoderma lucidum powder and aspergillus niger according to certain proportion, static culture fermentation in incubator, obtain aspergillus niger ganoderma fermentation product.Add sterile water and glucose to aspergillus niger ganoderma fermentation product, obtain mixed solution, after stirring evenly, inoculate rhamnose lactose casein bacillus liquid and lactobacillus plantarum liquid in sterile clean bench, after mixing evenly, transfer to sterile fermentation bag, constant temperature vibration fermentation obtains mixed fermentation liquor, mixed fermentation liquor freeze-drying is obtained, and mixed fermentation freeze-dried powder is obtained.The fermentation process of the present application effectively improves the content of main active substance in ganoderma fermentation product and its antioxidant activity, anti-inflammatory activity and bioavailability, effectively improves the symptoms of mouse ulcerative colitis;For exploring the application of fermented ganoderma in the development of functional food and drug provides a theoretical basis.

Description

Technical Field

[0001] This invention relates to the field of Ganoderma lucidum polysaccharide fermentation technology, specifically to a fermentation process for increasing the content of Ganoderma lucidum polysaccharides, its fermentation products, and their applications. Background Technology

[0002] Ganoderma lucidum polysaccharides are one of the core bioactive components of Ganoderma lucidum, possessing rich pharmacological effects and application value. They are particularly prominent in regulating immunity, anti-oxidation, anti-inflammation, and anti-tumor effects, and have a significant effect on improving ulcerative colitis. They can alleviate colonic pathological damage and regulate the level of inflammatory cytokines, providing a highly effective and low-toxicity natural intervention for this refractory disease. There are 5 to 10 million patients with ulcerative colitis worldwide, clinically manifested as bloody stools, diarrhea, and abdominal pain, sometimes even lifelong. Western medicine treatment often results in many adverse reactions. Ganoderma lucidum polysaccharides, as a natural product, have unique advantages in nutritional health care and disease intervention. Furthermore, fully utilizing their biological efficacy is of great significance for increasing the added value of Ganoderma lucidum products and promoting the high-quality development of the Ganoderma lucidum industry.

[0003] In existing technologies, to improve the polysaccharide content of Ganoderma lucidum, the main focus is on optimizing fermentation processes, screening strains, and processing raw materials, resulting in several technical approaches: First, probiotic fermentation technology is used, screening suitable fermentation strains, including *Lactobacillus rhamnosus* and *Lactobacillus plantarum*, to utilize probiotic fermentation to degrade macromolecules in Ganoderma lucidum raw materials, releasing effective components without consuming polysaccharides in the raw materials, thereby improving the yield and purification of Ganoderma lucidum polysaccharides. For example, fermentation of Ganoderma lucidum powder with *Lactobacillus rhamnosus* can significantly increase the polysaccharide content of Ganoderma lucidum. Second, strains such as *Aspergillus* are selected for fermentation to enhance the bioavailability of nutrients in Ganoderma lucidum raw materials, indirectly promoting the release and enrichment of Ganoderma lucidum polysaccharides. Third, fermentation-related parameters are optimized by controlling fermentation temperature, time, and nutrient substrate to provide a suitable environment for strain growth and Ganoderma lucidum polysaccharide synthesis, thus helping to improve polysaccharide content. Fourth, raw material pretreatment methods are improved to break down the cell wall structure of Ganoderma lucidum, promote the dissolution of Ganoderma lucidum polysaccharides, and further improve the extraction and content levels.

[0004] However, existing technologies for increasing Ganoderma lucidum polysaccharide content still have the following technical problems, which are difficult to meet the actual application needs: (1) Insufficient compatibility of fermentation strains. Existing technologies mostly use single strains for fermentation, without fully exploring the advantages of synergistic fermentation of different strains. The compatibility between strains and Ganoderma lucidum raw materials has not been precisely optimized, resulting in a limited increase in Ganoderma lucidum polysaccharide content and difficulty in simultaneously enhancing its core effects such as anti-inflammatory and immune regulation; (2) Imperfect fermentation process. Existing fermentation parameters lack systematic optimization, and there is insufficient regulation of the correlation between nutrient matrix metabolism, strain growth and Ganoderma lucidum polysaccharide synthesis during fermentation, resulting in unstable Ganoderma lucidum polysaccharide content in fermentation products. In addition, some processes have the problems of cumbersome operation and high cost; (3) The bioavailability of polysaccharides has not been improved simultaneously. Existing fermentation processes mostly focus on increasing polysaccharide content, without fully solving the problem that Ganoderma lucidum polysaccharide macromolecules are difficult to be directly absorbed and utilized by the human body, which limits the full play of its biological efficacy; (4) Severe homogenization of Ganoderma lucidum products. Existing processes have not been optimized in combination with specific application scenarios such as ulcerative colitis, and the functional characteristics of fermentation products are not prominent, making it difficult to meet the market demand for precision nutrition and health care and disease intervention. In summary, existing technologies cannot achieve a synergistic improvement in the content, bioavailability, and efficacy of Ganoderma lucidum polysaccharides. There is an urgent need for an efficient and stable fermentation process to overcome the current technological bottlenecks and provide support for the widespread application of Ganoderma lucidum polysaccharides. Summary of the Invention

[0005] The present invention aims to provide a fermentation process for increasing the polysaccharide content of Ganoderma lucidum, as well as its fermentation products and applications, in order to solve the technical problems of low polysaccharide content and insignificant active efficacy of the active ingredients in existing Ganoderma lucidum fermentation products.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: a fermentation process for increasing the polysaccharide content of Ganoderma lucidum, comprising the following steps: Step 1, primary fermentation of Aspergillus niger: Ganoderma lucidum powder and Aspergillus niger are gradually mixed using a gradient mixing method and then statically fermented in an incubator to obtain Aspergillus niger Ganoderma lucidum fermentation product. Step 2: Preparation of Lactobacillus rhamnosus and Lactobacillus plantarum bacterial cultures: Lactobacillus rhamnosus and Lactobacillus plantarum were activated and cultured separately to obtain Lactobacillus rhamnosus bacterial cultures and Lactobacillus plantarum bacterial cultures; Step 3: Re-fermentation of Lactobacillus rhamnosus and Lactobacillus plantarum: Add sterile water and glucose to the Aspergillus niger Ganoderma lucidum fermentation product to obtain a mixed solution. After stirring evenly, inoculate Lactobacillus rhamnosus and Lactobacillus plantarum bacterial solutions in a sterile ultra-clean bench. After mixing evenly, transfer to a sterile fermentation bag and ferment at a constant temperature with shaking to obtain a mixed fermentation broth. Freeze-dry the mixed fermentation broth to obtain a mixed fermented freeze-dried powder.

[0007] Preferably, as an improvement, in step one, the amount of Aspergillus niger added is 0.3 to 0.7‰ of the mass of Ganoderma lucidum powder; and the amount of sterile water added is 30 to 40% of the mass of Ganoderma lucidum powder.

[0008] Preferably, as an improvement, in step one, the culture is carried out by static culture in an incubator at 30°C for 24-48 hours.

[0009] Preferably, as an improvement, in step two, the bacterial concentration in the *Lactobacillus rhamnosus* and *Lactobacillus plantarum* bacterial solutions is 1 × 10⁻⁶. 8 ~1.0×10 9 CFU / mL.

[0010] Preferably, as an improvement, in step three, the amount of sterile water added is such that the ratio of Aspergillus niger ferment to sterile water is 1:5~10, and the amount of glucose added is such that the mass concentration of glucose in the mixture is 1~5%.

[0011] Preferably, as an improvement, in step three, the amount of *Lactobacillus rhamnosus* bacterial solution added is such that the mass concentration of *Lactobacillus rhamnosus* bacterial solution in the mixture is 3-5%, and the amount of *Lactobacillus plantarum* bacterial solution added is such that the mass concentration of *Lactobacillus plantarum* bacterial solution in the mixture is 3-5%.

[0012] Preferably, as an improvement, this solution also provides a Ganoderma lucidum fermentation product, including the mixed fermented freeze-dried powder obtained by the above fermentation process.

[0013] Preferably, as an improvement, the crude polysaccharide content in the mixed fermented freeze-dried powder is 1.98 ± 0.03 g / 100g, the total triterpenoid content is 18.93 ± 1.87 g / 100g, and the total flavonoid content is 3.55 ± 0.23%.

[0014] Preferably, as an improvement, this solution also provides an application of Ganoderma lucidum fermentation products, including using the above-mentioned mixed fermented freeze-dried powder to prepare drugs for treating inflammation.

[0015] Preferably, as an improvement, this solution also provides an application of Ganoderma lucidum fermentation products, including using the above-mentioned mixed fermented freeze-dried powder to prepare a drug for treating ulcerative colitis.

[0016] The principles and advantages of this solution are as follows: This study first fermented Ganoderma lucidum powder using Aspergillus niger, then fermented the powder using Lactobacillus rhamnosus and Lactobacillus plantarum. The changes in major active substances such as crude polysaccharides, total triterpenes, flavonoids, polyphenols, proteins, and amino acids, as well as their antioxidant, anti-inflammatory, and bioavailability, were analyzed before and after fermentation. The study also aimed to clarify the mechanism and effect of fermentation in alleviating ulcerative colitis in mice. Results showed that the content of active substances in fermented Ganoderma lucidum was significantly increased, especially the contents of crude polysaccharides, total triterpenes, and total flavonoids, which increased by 50%, 42%, and 106%, respectively. In vitro antioxidant experiments showed that the DPPH free radical scavenging rate and ABTS cation scavenging rate of fermented Ganoderma lucidum powder were significantly higher than those of unfermented Ganoderma lucidum powder and the blank control fermented Ganoderma lucidum powder (P < 0.05). Animal experiments showed that compared with the model group, the weight loss trend in mice in the fermented Ganoderma lucidum powder group was alleviated, the DAI score was significantly reduced, and the colon length was significantly increased (P < 0.05). Furthermore, colon pathological sections showed reduced inflammatory cell infiltration and improved goblet cell count. In mice, serum NGAL levels were increased, and the levels of interleukin (IL)-1β, IL18, and interferon-γ in colon tissue were significantly decreased, while the level of the anti-inflammatory factor IL-4 was significantly increased (P<0.05). NLRP3 and ASC protein expression were downregulated (P<0.05). The beneficial gut microbiota, Lachnospiraceae_NK4A136_group, was the dominant genus. Therefore, fermented Ganoderma lucidum powder may alleviate colitis symptoms by regulating inflammatory factor levels and inhibiting NLRP3 protein expression through gut microbiota. This study explores the fermentation and value-added transformation application of Ganoderma lucidum fruiting bodies, providing a theoretical basis for the application of fermented Ganoderma lucidum in the development of functional foods and pharmaceuticals. Attached Figure Description

[0017] Figure 1 The graph shows the effect of different inoculum amounts on the fermentation results of *Lactobacillus rhamnosus* during the fermentation process screening of the present invention.

[0018] Figure 2 The figure shows the effect of different glucose addition amounts on fermentation in the fermentation process screening of Lactobacillus rhamnosus in this invention.

[0019] Figure 3 The figure shows the effect of different fermentation times on fermentation of Lactobacillus rhamnosus in the fermentation process screening of the present invention.

[0020] Figure 4 The graph shows the change in crude polysaccharide content in the fermentation products obtained from the fermentation of Lactobacillus rhamnosus in the fermentation process screening of the present invention.

[0021] Figure 5 The graph shows the change in total triterpenoid content in the fermentation products obtained from the fermentation of Lactobacillus rhamnosus in the fermentation process screening of the present invention.

[0022] Figure 6 The graph shows the change in polyphenol content in the fermentation products obtained from the fermentation of Lactobacillus rhamnosus in the fermentation process screening 1 of this invention.

[0023] Figure 7 The graph shows the change in total flavonoid content in the fermentation products obtained from the fermentation of Lactobacillus rhamnosus in the fermentation process screening of the present invention.

[0024] Figure 8 This is a graph showing the change in protein content in the fermentation products obtained from the fermentation of *Lactobacillus rhamnosus* in the fermentation process screening method 1 of this invention.

[0025] Figure 9 The purpose of this invention is to screen the DPPH free radical scavenging rate of the fermentation products obtained from the fermentation of Lactobacillus rhamnosus in the fermentation process 1 of this invention.

[0026] Figure 10 The ABTS cation scavenging rate of the fermentation product obtained from the fermentation of Lactobacillus rhamnosus in the fermentation process of the present invention was screened.

[0027] Figure 11 To screen the Fe content of the fermentation product obtained from the fermentation of *Lactobacillus rhamnosus* in fermentation process 1 of this invention. 3+ Reduction rate.

[0028] Figure 12 The image shows the cell inhibition rate (left) and NO inhibition rate (right) of the fermentation products obtained from the mixed fermentation of Ganoderma lucidum powder by Aspergillus niger, Lactobacillus rhamnosus, and Lactobacillus plantarum in this invention.

[0029] Figure 13 The fermentation process of this invention screened five experimental groups to observe changes in body weight (A) and DAI score (B) in mice over seven days.

[0030] Figure 14 The fermentation process of this invention screened the colon length changes and pathological tissue sections of 5 experimental groups.

[0031] Figure 15 This study compares the effects of five different experimental groups on the levels of inflammatory factors NGAL, IFN-γ, IL-4, and IL-1β, as well as the activities of MDA and SOD, in mice using the fermentation process of this invention.

[0032] Figure 16 This study compares the immunoblotting results of NLRP3 (A) and ASC (B) proteins in five experimental groups selected for screening using the fermentation process of this invention.

[0033] Figure 17 This is a comparison of the gut microbiota analysis results of five experimental groups in the fermentation process screening of this invention. Detailed Implementation

[0034] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0035] Overview of the Plan This method provides a fermentation process to increase the polysaccharide content of Ganoderma lucidum, including the following steps: Weigh 15 g of Ganoderma lucidum fruiting body powder into an Erlenmeyer flask for sterilization; weigh 0.0075 g of Aspergillus niger (i.e., 0.3-0.7‰ of Aspergillus niger added to the Ganoderma lucidum powder); gradually dilute and mix well; add 6 mL of sterile water; and incubate statically in a 30℃ incubator for 24-48 h. Add sterile water and 1-5% glucose at a material-to-liquid ratio of 1:5, stir well, and then inoculate with a concentration of 1.0×10⁻⁶ in a sterile laminar flow hood. 9 CFU / mL of Lactobacillus rhamnosus and Lactobacillus plantarum (with Lactobacillus rhamnosus and Lactobacillus plantarum added at 3-5% each) were added to the blank control group. After mixing evenly, the mixture was transferred to a sterile fermentation bag and placed in a 37 ℃ intelligent constant temperature shaker for 48 h of fermentation. After fermentation, the mixture was freeze-dried for later use.

[0036] Fermentation process screening 1: Lactobacillus rhamnosus fermentation Weigh 5 g of Ganoderma lucidum powder and place it in an Erlenmeyer flask. Autoclave at 121℃ for 15 min. After cooling, add sterile water at a material-to-liquid ratio of 1:5, and inoculate with activated Lactobacillus rhamnosus (total bacterial count 1.7 × 10⁻⁶). 9 (CFU / mL, activated inoculum amount 4%) was placed in a fermentation bag for fermentation. The inoculum amount, glucose addition amount, and fermentation time were selected as single factors, and the crude polysaccharide content in the fermentation product was used as the experimental index to conduct an orthogonal experiment.

[0037] After freeze-drying, the crude polysaccharide content was determined by spectrophotometry according to NY / T 1676-2023 Determination of Crude Polysaccharides in Edible Fungi. The determination was repeated three times, with the original Ganoderma lucidum powder and Ganoderma lucidum powder without added fungi used as control groups.

[0038] 1. Single-factor experimental design (1) Single-factor test of inoculum quantity Activated bacterial cultures at concentrations of 3%, 5%, and 7% were inoculated into fermentation bags with 5% glucose added, and incubated at 37°C for 48 hours. After fermentation, the fermentation broth was freeze-dried, and the polysaccharide content was determined. This process was repeated three times.

[0039] The results are as follows Figure 1As shown, the inoculum quantity is a core parameter for regulating the metabolic activity of the microorganisms and the efficiency of substrate utilization during fermentation. Too low an inoculum quantity leads to low fermentation efficiency, while too high an inoculum quantity results in insufficient substrate and the secretion of harmful substances. For example... Figure 1 As shown, the polysaccharide content in fermented Ganoderma lucidum powder is highest when the inoculum content is 5%, therefore, 5% inoculum content is the optimal inoculum content.

[0040] (2) Single-factor experiment on glucose addition amount Add 1%, 3%, and 5% glucose respectively, with an inoculum size of 5%, and inoculate into fermentation bags. Incubate at 37℃ for 48 hours. After fermentation, freeze-dry the fermentation broth, determine the polysaccharide content, and repeat three times.

[0041] The results are as follows Figure 2 As shown, glucose, as a key carbon source in the fermentation process, directly affects the metabolic activity of *Lactobacillus rhamnosus* and the synthesis efficiency of *Ganoderma lucidum* polysaccharides. Too little glucose may lead to insufficient carbon source and limited cell growth; too much may result in the accumulation of metabolic byproducts or osmotic pressure inhibition. Figure 2 As shown, the polysaccharide content in fermented Ganoderma lucidum powder is highest when the glucose addition is 5%, therefore, 5% glucose addition is the optimal amount.

[0042] (3) Single-factor experiment on fermentation time Take 5% of the activated bacterial culture, add 5% glucose, and inoculate it into fermentation bags. Incubate at 37℃ for 24 h, 36 h, and 48 h in a constant temperature shaker. After fermentation, freeze-dry the fermentation broth and determine the polysaccharide content. Repeat the process three times.

[0043] like Figure 3 As shown, the polysaccharide content of Ganoderma lucidum powder fermented for 48 hours was the highest, possibly because the microorganisms accelerated their growth and reproduction, entering the logarithmic growth phase, which increased the polysaccharide content. Therefore, a fermentation time of 48 hours is considered the optimal fermentation time.

[0044] 2. Orthogonal experimental design As shown in Table 1, orthogonal experiments were conducted based on the results of single-factor experiments. The optimal process conditions for fermenting Ganoderma lucidum powder with Lactobacillus rhamnosus were screened using the polysaccharide content of fermented Ganoderma lucidum powder as an indicator.

[0045] Table 1. Experimental conditions for single factors

[0046] Based on the results of the single-factor experiment, the inoculum quantity, glucose addition amount, and fermentation time were selected using a three-factor, three-level (L3) method. 3 An orthogonal experiment was designed, and the experimental results are shown in Table 2.

[0047] Table 2 Results of Orthogonal Experiments

[0048] Table 2 shows that different fermentation conditions affect the crude polysaccharide content of Ganoderma lucidum powder. Range analysis revealed that the amount of glucose added had the greatest impact, with the third level showing the largest value, indicating that a glucose addition of 5% yielded the best fermentation results. This was followed by the inoculum size and fermentation time, with the second and third levels showing the best results, respectively. Orthogonal experimental results showed that the order of influence of fermentation factors on crude polysaccharide content was: glucose addition > inoculum size > fermentation time. The optimal fermentation conditions within the experimental range were: 5% glucose addition, 5% inoculum size, and 48 h fermentation time. To verify the reproducibility of the experiment, three replicate experiments were conducted under the optimal conditions, with an average crude polysaccharide content of 1.84 ± 0.10 g / 100g. The results indicate that the experimental design was reasonable and feasible, and the data were reliable, demonstrating that the optimized conditions for fermenting Ganoderma lucidum powder with *Lactobacillus rhamnosus* were accurate and reliable.

[0049] 3. Changes in the main active substances of Ganoderma lucidum powder fermented with Lactobacillus rhamnosus The following results were obtained from fermenting Ganoderma lucidum powder according to the above-mentioned "5% glucose addition, 5% inoculum, and 48 h fermentation time": crude polysaccharide (referring to NY / T 1676-2023 Determination of Crude Polysaccharide in Edible Fungi - Spectrophotometric Method), total triterpenes (referring to NY / T3676-2020 Determination of Total Triterpenes in Ganoderma lucidum - Spectrophotometric Method), polyphenols (referring to GB / T 44349-2024 Determination of Total Polyphenols in Bee Pollen - Folin-Ciocalteu Reagent Colorimetric Method), total flavonoids (referring to NY / T 3903-2021 Determination of Flavonoids in Lycium barbarum), and protein (referring to GB 5009.5-2025 National Food Safety Standard - Determination of Protein in Food), and other major substances were tested as follows: Figures 4-8 As shown in the figure. The experimental groups in the figure are Lactobacillus rhamnosus fermented Ganoderma lucidum powder (…). Lactobacillus rhamnosus Fermented Ganoderma Lucidum Blank Fermented Ganoderma Lucidum Powder (F-GL) Ganoderma Lucidum B-GL) and original Ganoderma lucidum powder ( Ganoderma Lucidum , GL).

[0050] Ganoderma lucidum polysaccharides are mainly distributed in mycelium and fruiting bodies, and possess antioxidant, immunomodulatory, anti-neurodegenerative, and anti-diabetic activities. Figure 4 The crude polysaccharide content in F-GL was 1.84 ± 0.10 g / 100g, significantly higher than that in B-GL (P < 0.05). There was no significant difference between GL and B-GL (P > 0.05). This demonstrates that *Lactobacillus rhamnosus* fermentation enhances the crude polysaccharide content of *Ganoderma lucidum*.

[0051] The content of triterpenes in Ganoderma lucidum is of great significance in evaluating its quality. Total triterpenes are one of the important active components of Ganoderma lucidum, possessing antibacterial, anti-inflammatory, anti-tumor, immune-regulating, anti-aging, cardiovascular disease prevention and treatment, liver-protective, and kidney-protective effects. Figure 5 The total triterpenoid content in F-GL was 0.51 ± 0.07 g / 100g, significantly higher than that in B-GL (P < 0.05). There was no significant difference between GL and B-GL (P > 0.05). This indicates that the total triterpenoid content of Ganoderma lucidum powder significantly increased after fermentation with *Lactobacillus rhamnosus*, suggesting that *Lactobacillus rhamnosus* fermentation has an enhancing effect on the total triterpenoid content of Ganoderma lucidum.

[0052] Ganoderma lucidum polyphenols are important active components among the triterpenoids of Ganoderma lucidum, possessing anti-aging, antioxidant, anti-tumor, and immunomodulatory effects. Figure 6 The polyphenol content in F-GL was 12.1 ± 0.72 mg / g, significantly higher than that in B-GL (P < 0.05). There was no significant difference between GL and B-GL (P > 0.05). This demonstrates that *Lactobacillus rhamnosus* fermentation enhances the polyphenol content of Ganoderma lucidum.

[0053] Ganoderma lucidum flavonoids typically possess antioxidant and anti-inflammatory properties and exhibit specific flavonoid chemical structures. Figure 7 The total flavonoid content in F-GL was 3.43 ± 0.62 mg / g, significantly higher than that in B-GL (P < 0.05). There was no significant difference between GL and B-GL (P > 0.05). This demonstrates that *Lactobacillus rhamnosus* fermentation enhances the total flavonoid content of *Ganoderma lucidum*.

[0054] Ganoderma lucidum protein is an essential substance for the life activities of Ganoderma lucidum and possesses unique immunogenicity, giving it significant effects in immune regulation and anti-tumor treatment. It is also one of the important active substances in Ganoderma lucidum. Figure 8 The protein content of F-GL was 8.88 ± 0.00 g / 100g, significantly higher than that of B-GL (P < 0.05). There was no significant difference between GL and B-GL (P > 0.05). This demonstrates that *Lactobacillus rhamnosus* fermentation enhances the protein content of *Ganoderma lucidum*.

[0055] 4. Effects of Lactobacillus rhamnosus fermentation on the antioxidant activity of Ganoderma lucidum powder The antioxidant activity test results of the product obtained by fermenting Ganoderma lucidum powder according to the above-mentioned "glucose addition 5%, inoculum 5%, fermentation time 48 h" are as follows: Figures 9-11 As shown.

[0056] (1) Analysis of DPPH free radical scavenging rate results The scavenging rate of fermentation products against DPPH free radicals was determined using the following steps: 0.5 g of GL, F-GL, and B-GL samples were dissolved in hot water at 37℃ and soaked for 10 min, then centrifuged (5000 r / min, 10 min) to obtain a clear solution. 2 mL of the above liquid and 2 mL of DPPH reagent were taken, vortexed, and reacted in the dark for 30 min. The absorbance value (λ=517 nm) was then measured and recorded as A1. Several other test tubes were taken and labeled A0 and A2, respectively. The sample in A1 was replaced with anhydrous ethanol, and the A value was measured, which became A0. Then, DPPH was replaced with anhydrous ethanol, and the A value was measured, which became A2. The above operation was repeated three times. The scavenging rate of different Ganoderma lucidum powders against DPPH free radicals was calculated using the following formula.

[0057]

[0058] The results are as follows Figure 9 As shown, F-GL exhibited the strongest DPPH free radical scavenging ability, significantly higher than GL and B-GL (P < 0.05). The DPPH free radical scavenging rate increased from 86% before fermentation to 92% after fermentation. Therefore, Lactobacillus rhamnosus fermentation can significantly improve the DPPH free radical scavenging rate of Ganoderma lucidum powder, demonstrating stronger antioxidant capacity.

[0059] (2) Analysis of ABTS cation scavenging rate results The scavenging rate of ABTS cations by the fermentation products was determined using the following steps: A 7.4 mmol / L ABTS aqueous solution and a 2.6 mmol / L K₂S₂O₈ aqueous solution were prepared, and equal volumes of both were mixed thoroughly. The mixture was then incubated at 25°C in the dark for 16 h to allow for complete reaction. The solution was diluted with phosphate buffer to achieve an absorbance value (λ=734 nm) of 0.70. 40 μL of GL, F-GL, and B-GL samples, and 160 μL of ABTS were added to each well of a 96-well plate, mixed thoroughly, and incubated at 25°C in the dark for 20 min. The absorbance value (A0) was measured. Distilled water was used as a blank control (A1) instead of the sample solution, and its absorbance was measured using the same method. The experiment was repeated three times, and the average value was taken.

[0060]

[0061] The results are as follows Figure 10 As shown, F-GL exhibited the strongest scavenging ability, significantly higher than GL and B-GL (P < 0.05). The ABTS cation scavenging rate increased from 68% before fermentation to 87% after fermentation, indicating that Lactobacillus rhamnosus fermentation can significantly improve the ABTS free radical scavenging rate of Ganoderma lucidum powder, further enhancing the antioxidant capacity of Ganoderma lucidum powder.

[0062] (3) Fe 3+ Analysis of reduction rate results The following steps were used to detect the effect of fermentation products on Fe. 3+ Reduction rate: Mix 100 μL of sample solution with 100 μL of pH 6.6 phosphate buffer solution and 100 μL of 1% potassium ferricyanide solution, react in a 50℃ water bath for 20 min, cool, add 100 μL of 10% trichloroacetic acid solution, and centrifuge (3000 r / min, 10 min). Take 90 μL of the supernatant, add 100 μL of distilled water and 10 μL of 0.1% ferric chloride, let stand for 10 min, and measure the absorbance at 700 nm, recorded as A1. Simultaneously, replace the potassium ferricyanide solution with 100 μL of distilled water as a blank control, recorded as A0. Repeat three times, and take the average value.

[0063]

[0064] The results are as follows Figure 11 As shown, the Fe of F-GL 3+ It exhibits the strongest reduction rate, significantly higher than GL and B-GL. Fe 3+ The reduction capacity increased from 76% before fermentation to 82% after fermentation, indicating that its electron donor capacity was enhanced, suggesting that fermentation with Lactobacillus rhamnosus can improve the antioxidant capacity of Ganoderma lucidum powder.

[0065] Fermentation process screening 2: Lactobacillus plantarum fermentation Weigh 5 g of Ganoderma lucidum powder and place it in an Erlenmeyer flask. Autoclave at 121℃ for 15 min. After cooling, add sterile water at a material-to-liquid ratio of 1:5, and inoculate with activated Lactobacillus plantarum (total bacterial count 1.75 × 10⁻⁶). 9 The inoculum was 4% (CFU / mL) and placed in a fermentation bag for fermentation. After freeze-drying, the content of the main active substances and antioxidant activity were determined according to the aforementioned method. The experiment was repeated three times, and the results are shown in Table 3.

[0066] Table 3 Results of Single-Factor Experiments on Lactobacillus Plantarum Fermented Ganoderma Lucidum Powder

[0067] Experimental data show that when the amount of Lactobacillus plantarum added is 5%, the content of effective components in the fermentation product is better, and the antioxidant activity is the strongest.

[0068] Fermentation process screening 3: Lactobacillus bulgaricus fermentation Weigh 5 g of Ganoderma lucidum powder and place it in an Erlenmeyer flask. Sterilize it at 121℃ for 15 min. After cooling, add sterile water at a material-to-liquid ratio of 1:5. Inoculate with 4% activated Lactobacillus delbrueckii subsp. bulgaricus and add 5% glucose. Place the flask in a fermentation bag and incubate at 37℃ for 48 h. After freeze-drying, determine the content of the main active substances and antioxidant activity according to the aforementioned method. Repeat three times. The results are shown in Table 4.

[0069] Table 4 Results of Single-Factor Experiments on Lactobacillus bulgaricus Fermented Ganoderma Lucidum Powder

[0070] Experimental data show that the product obtained by fermenting Ganoderma lucidum powder with Lactobacillus bulgaricus has low antioxidant activity.

[0071] Fermentation process screening 4: Aspergillus niger fermentation Weigh 15 g of Ganoderma lucidum fruiting body powder into an Erlenmeyer flask for sterilization. Weigh 0.0075 g of Aspergillus niger, gradually dilute and mix well, then add 6 mL of sterile water. Incubate at 30℃ for 48 h and 72 h. After freeze-drying, determine the content of the main active substances and antioxidant activity according to the aforementioned method. Repeat three times. The results are shown in Table 5.

[0072] Table 5 Results of single-factor experiments on Ganoderma lucidum powder fermented with Aspergillus niger.

[0073] Experimental data showed that the content of active ingredients in the product fermented by Aspergillus niger for 48 h was not significantly different from that fermented for 72 h, and the ABTS cation scavenging effect was better. Therefore, a fermentation time of 48 h was selected.

[0074] Fermentation process screening 5: Optimization of mixed-culture fermentation process 1. Optimization of the fermentation process of Ganoderma lucidum powder using a mixture of Lactobacillus plantarum and Lactobacillus rhamnosus Lactobacillus plantarum and Lactobacillus rhamnosus ATCC5310 were inoculated into MRS liquid medium and cultured at 37 ℃ for 24 h. This process was repeated three times to obtain the fermentation seed liquid. 15 g of Ganoderma lucidum fruiting body powder was weighed into a sterilized Erlenmeyer flask. Sterile water and 5% glucose were added at a material-to-liquid ratio of 1:5 and stirred thoroughly. Different proportions of Lactobacillus rhamnosus and Lactobacillus plantarum were then inoculated into the flask in a sterile laminar flow hood. An equal volume of sterile water was added to the blank control group. After thorough mixing, the mixture was transferred to a sterile fermentation bag and fermented in a 37 ℃ intelligent thermostatic shaker for 48 h. After fermentation, the mixture was freeze-dried, and the content of the main active substances and antioxidant activity were determined using the aforementioned method. This process was repeated three times.

[0075] (1) The results of the changes in the main active ingredients and antioxidant activity of mixed fermented Ganoderma lucidum powder are shown in Table 6.

[0076] Table 6. Changes in the main active ingredients and antioxidant activities of Ganoderma lucidum powder fermented with Lactobacillus rhamnosus and Lactobacillus plantarum.

[0077] 2. Optimization of the fermentation process for Ganoderma lucidum powder using a mixture of Aspergillus niger, Lactobacillus plantarum, and Lactobacillus rhamnosus. Weigh 15 g of Ganoderma lucidum fruiting body powder into an Erlenmeyer flask and sterilize. Weigh 0.0075 g of Aspergillus niger, gradually dilute and mix well, then add 6 mL of sterile water and incubate at 30℃ for 48 h. Add sterile water and 5% glucose at a material-to-liquid ratio of 1:5, stir well, and then inoculate with a concentration of 1.0 × 10⁻⁶ in a sterile laminar flow hood. 9 CFU / mL of Lactobacillus rhamnosus and Lactobacillus plantarum were added to the blank control group, and an equal volume of sterile water was added. After mixing evenly, the mixture was transferred to a sterile fermentation bag and placed in a 37 ℃ intelligent constant temperature shaker for 48 h of fermentation. After fermentation, the mixture was freeze-dried for later use. Each group had three replicates, and the content of the main active substances and antioxidant activity were determined according to the aforementioned method.

[0078] (1) The results of the changes in the main active ingredients and antioxidant activity of mixed fermented Ganoderma lucidum powder are shown in Tables 7 and 8, respectively.

[0079] Table 7. Crude polysaccharide content from mixed fermentation of Lactobacillus bulgaricus with Lactobacillus rhamnosus and Lactobacillus plantarum, respectively.

[0080] Table 8. Changes in the main active ingredients and antioxidant activities of Ganoderma lucidum powder fermented with mixed strains of Aspergillus niger, Lactobacillus rhamnosus, and Lactobacillus plantarum.

[0081] Table 8 shows that the contents of crude polysaccharides, total triterpenes, protein, polyphenols, and total flavonoids in F-GL were significantly higher than those in B-GL and GL (P < 0.05). Compared with GL, the crude polysaccharide content increased by 50%, the total triterpenes content increased by 42%, and the total flavonoid content increased by 106%. The DPPH free radical scavenging capacity and ABTS cation scavenging rate were also significantly improved (P < 0.05), indicating that the combined fermentation of *Aspergillus niger* with *Lactobacillus rhamnosus* and *Lactobacillus plantarum* can enhance the antioxidant capacity of Ganoderma lucidum powder. The contents of polysaccharides, triterpenes, polyphenols, flavonoids, and protein in Ganoderma lucidum are important active components and key indicators for evaluating its quality. These active components give Ganoderma lucidum its anti-inflammatory, antibacterial, immune-regulating, and cardiovascular disease-preventing effects. *Lactobacillus rhamnosus* can synthesize lactic acid bacteria extracellular polysaccharides (such as β-glucan), directly increasing the total polysaccharide content. During fermentation, *Lactobacillus rhamnosus* may secrete cellulase and hemicellulase, which degrade the cell wall of *Ganoderma lucidum*, making it easier for active ingredients such as polysaccharides, triterpenes, and flavonoids to dissolve. Microbial metabolites may also regulate the secondary metabolic pathways of *Ganoderma lucidum*, thereby promoting the synthesis of polysaccharides, triterpenes, and flavonoids. The enhanced antioxidant capacity may be related to the increase in total triterpenes, flavonoids, and other antioxidant components after fermentation. These components can effectively scavenge free radicals, thereby enhancing the antioxidant capacity of *Ganoderma lucidum*. Research by Sun Yuwei et al. shows that many lactic acid bacteria in fermented foods can exert antioxidant activity in animal bodies by increasing polyphenol content, enhancing metal ion chelating activity, scavenging free radicals, and regulating enzyme activity, thus improving the antioxidant capacity of fermented substances. Triterpenoids and flavonoids can scavenge free radicals in the body through multiple mechanisms and are known antioxidants that can reduce oxidative stress damage to cells. Therefore, the increase in the content of these components after fermentation directly leads to the enhancement of the antioxidant capacity of *Ganoderma lucidum*.

[0082] (2) Analysis of amino acid content results Reishi mushroom is rich in 8 essential amino acids and 10 non-essential amino acids, characterized by its high variety, high content, and high activity, exhibiting antibacterial, antitumor, and antiviral effects. Table 9 shows that the serine (Ser) content in F-GL was significantly higher than that in B-GL and GL (P < 0.05), possibly related to the sulfur metabolism activity and protease release of microorganisms. The methionine (Met) content in fermented reishi powder was also significantly increased (P < 0.05). As an essential amino acid, increased Met content enhances the protein nutritional value of reishi powder, possessing certain antioxidant properties, immune-boosting effects, and lipid balance regulation. The contents of aspartic acid (Asp), leucine (Leu), and lysine (Lys) also increased to some extent, but the contents of Glu, Ala, Ile, His, Arg, Pro, GL, and B-GL did not change significantly (P > 0.05), possibly due to structural stability or limitations of the microbial metabolic pathways under experimental conditions.

[0083] Table 9. Analysis of amino acid content in Ganoderma lucidum powder products fermented by mixed strains of Aspergillus niger, Lactobacillus rhamnosus, and Lactobacillus plantarum.

[0084] Experimental data show that mixed fermentation with Aspergillus niger, Lactobacillus rhamnosus, and Lactobacillus plantarum is beneficial for increasing the content of active ingredients in Ganoderma lucidum and enhancing its antioxidant capacity.

[0085] (3) Evaluation of the safety and anti-inflammatory activity of fermentation products obtained from Ganoderma lucidum powder by mixed fermentation of Aspergillus niger, Lactobacillus rhamnosus and Lactobacillus plantarum. The experimental steps are as follows: 1) Cytotoxic activity assay: RAW264.7 cells were cultured in DMEM medium containing 10% FBS at 37°C and 5% CO2 using standard methods. After seeding and culturing the cells in 96-well plates for 24 h, 100 μL of fermentation broth at concentrations of 0.1 g / mL, 0.05 g / mL, 0.025 g / mL, 0.0125 g / mL, and 0.00625 g / mL were added, and the cells were cultured for another 48 h at 37°C and 5% CO2. Subsequently, 15 μL of 5 mg / mL MTT solution was added to each well, and the reaction was carried out at 37°C for 4 h. After the reaction, the supernatant was aspirated, and 100 μL of DMSO was added to each well to fully dissolve the precipitate. Finally, the absorbance of each well was measured at 490 nm using a microplate reader, and each experiment was performed in triplicate. Based on the measured data, the cell growth inhibition rate (I) was calculated using formula (1) to assess the cytotoxic effect of the compound.

[0086] I = (1 - A1 / A0) × 100% (1) In the formula, A0 and A1 are the absorbance of the blank group and the experimental group, respectively.

[0087] 2) The Griess method was used to test the compound's activity in inhibiting NO production. RAW264.7 cells were routinely cultured in DMEM medium containing 10% FBS at 37°C in a 5% CO2 incubator. After culturing for 24 h in 96-well plates, 50 μL of the test compound solution (compound concentrations of 0.1 g / mL, 0.05 g / mL, 0.025 g / mL, 0.0125 g / mL, and 0.00625 g / mL) was added, and the cells were cultured for another 1 h. Then, 50 μL (1 mg / mL or 1.5 mg / mL) of LPS was added, and the cells were placed in a 37°C, 5% CO2 incubator for 24 h to allow the cells to adhere. 100 μL of the solution was then transferred to a new 96-well plate, and 100 μL (40 mg / mL) of Griess reagent was added to each well. After mixing, the absorbance (A) of each well was measured at 540 nm using a microplate reader. Each experiment was repeated in triplicate. The NO inhibition rate (I) was calculated according to formula (2).

[0088] I=(A2-A1) / (A2-A0)×100%(2) In the formula, A0, A1, and A2 are the absorbance of the blank control group (50 μL culture medium + 50 μL culture medium), the drug group (50 μL compound + 50 μL LPS), and the LPS-induced group (50 μL culture medium + 50 μL LPS), respectively.

[0089] The results are as follows Figure 12 As shown, when the concentrations of GL (the original Ganoderma lucidum powder control group) and F-GL (the mixed fermentation experimental group of Aspergillus niger, Lactobacillus rhamnosus, and Lactobacillus plantarum) were 10 mg / mL, their effects on cell viability were not significant, and cells grew normally. When the concentration of Ganoderma lucidum powder exceeded 10 mg / mL, the cell inhibition rate increased with increasing concentration. When the concentration reached 20 mg / mL, the inhibition rate exceeded 50%, exhibiting certain cytotoxicity. The inhibitory effects of GL and F-GL on LPS-induced NO production in mouse monocyte / macrophage RAW 264.7 cells were tested using the Griess method. When their concentrations reached 10 mg / mL, both GL and F-GL showed certain anti-inflammatory activity, and their safety was good within this concentration range.

[0090] (4) Effects of fermentation products obtained from mixed fermentation of Ganoderma lucidum powder by Aspergillus niger, Lactobacillus rhamnosus and Lactobacillus plantarum on acute colitis in mice. The experimental steps include the following: Forty-eight C57BL / 6 mice were randomly divided into six groups of eight each: a normal control group (NC), a model group (Model), a positive control group (PC, mesalazine, 130 mg / (kg mb·d), a Ganoderma lucidum fermentation group (F-GL, 758 mg / kg / bw), a Ganoderma lucidum blank fermentation group (B-GL), and a Ganoderma lucidum powder group (GL, 758 mg / kg / bw). Mice in all groups were acclimatized for one week. Except for the NC group, the other groups were allowed free access to 3% DSS solution for seven consecutive days to induce a colitis model. Simultaneously, the PC, F-GL, B-GL, and GL groups were treated with the corresponding doses of the drug via gavage (at 9:00 AM daily). The NC and Model groups were given an equal volume of saline solution in the same manner. Body weight and disease activity index were recorded during the modeling period. DAI (Digital Intestinal Acid) score; After the experiment, mice were fasted for 12 hours but allowed free access to water. Blood was collected from each mouse by enucleation. After standing for 2 hours, the blood was centrifuged at 3000 r / min for 20 min, and the serum was collected and stored at -80℃ for later use. Mice were euthanized by cervical dislocation. The colonic segment from the anus to the end of the cecum was weighed and its length was measured. The colonic tissue was rinsed with pre-cooled physiological saline, dried with filter paper, and weighed. The colonic tissue was divided into two parts. One part was fixed with 10% neutral formaldehyde, and the other part was aliquoted into cryovials and stored at -80℃. The colonic contents were aseptically collected for intestinal flora determination.

[0091] (4-1) Evaluation of mouse body weight changes and activity index During the experiment, mice in the NC group were in good condition, with normal stools and no loose stools or bleeding around the anus; mice in the Model group were lethargic, emaciated, sluggish, with rough fur, and severe loose stools and bleeding around the anus; mice in the PC and F-GL groups were in better condition, with milder loose stools and bleeding. Results are as follows: Figure 13As shown, the body weight of mice in the NC group showed a gradual upward trend. Compared with the NC group, the body weight of mice in the Model group, PC group, B-GL group, and GL group all decreased significantly. The Model group and PC group showed extremely significant differences (P<0.001). The body weight loss trend of mice in the F-GL group was slower (P<0.05), and their DAI scores were significantly lower than those in the Model group (P<0.05). Therefore, Ganoderma lucidum powder fermented by Aspergillus niger, Lactobacillus rhamnoides, and Lactobacillus plantarum has a significant effect on alleviating body weight loss in mice with colitis.

[0092] (4-2) Colon length and pathological changes in colon Results of changes in colon length as follows Figure 14 As shown. Figure 14 A and Figure 14 As shown in B, compared with the NC group, the colon length in the Model group was significantly reduced (P<0.0001); after intervention with fermented Ganoderma lucidum powder and positive control drug, the colon length recovered to the level of the NC group. Figure 14 HE staining of the colon in mice showed that the colonic structure of mice in the NC group was intact and clear, with glands and epithelial cells arranged neatly. In the Model group, the colonic structure was damaged, with severe erosion and ulceration, moderate inflammatory cell infiltration and mucosal edema, and mild intestinal gland dilation. Compared with the Model group, the colonic damage in the PC group was slightly reduced, while the colonic damage and inflammatory infiltration in the B-GL and GL groups were significantly reduced. The colonic structure in the F-GL group was clear and intact, with reduced inflammatory infiltration, glandular damage, and mucosal edema. In conclusion, Ganoderma lucidum powder fermented with Aspergillus niger, Lactobacillus rhamnoides, and Lactobacillus plantarum can alleviate the pathological changes in the colon induced by DSS, such as erosion, edema, and inflammatory cell infiltration, thereby alleviating and treating colitis.

[0093] The results of the determination of inflammatory factor content and oxidative stress level in mouse colon tissue are as follows: Figure 15 As shown. By Figure 15 A~ Figure 15 As shown in Figure D, the levels of pro-inflammatory factors NGAL, IL-1β, and IFN-γ were significantly increased in the Model group (P<0.05), while the level of anti-inflammatory factor IL-4 was significantly decreased (P<0.01). Compared with the Model group and the B-GL group, the levels of pro-inflammatory factors NGAL, IL-1β, and IFN-γ in the colonic tissue of the F-GL group and the PC group were significantly decreased (P<0.05), while the level of anti-inflammatory factor IL-4 was significantly increased (P<0.05). Figure 15 E~ Figure 15F showed that the MDA content in the Model group was significantly increased compared to the NC group (P<0.05), while the MDA content in the PC group and F-GL group decreased by 26.75% and 27.56%, respectively. The SOD activity in the Model group was significantly decreased by 20% compared to the NC group (P<0.05). After intervention with fermented Ganoderma lucidum powder, the SOD activity in the PC group and F-GL group significantly increased by 24.17% and 24.27% compared to the Model group (P<0.05), returning to the NC group level. In normal intestinal mucosa, immune factors are balanced between pro-inflammatory and anti-inflammatory factors. When colitis occurs, this balance is disrupted. Inflammatory cytokine interactions, immune cell infiltration, epithelial cell damage, and intestinal barrier disruption also lead to the secretion of IFN-γ, resulting in intestinal mucosal inflammation. Simultaneously, the increase in IL-1β is considered related to the pathogenesis of colitis, and IL-4 plays an important role in the colitis model. Oxidative stress is a key pathogenic mechanism of ulcerative colitis. When DSS (diethylsaturated saline) acts on the mouse colon, it stimulates an oxidative stress response, producing a large number of harmful free radicals and other oxidation products, such as NO and MDA. These oxidation products damage colonic tissue and mediate the development of colitis. Studies have shown that in a DSS-induced mouse colitis model, papaya polysaccharide upregulates SOD activity and reduces MDA expression in mouse colonic tissue, effectively improving oxidative stress damage and protecting colonic tissue function. Sea buckthorn polysaccharide can reduce MDA concentration in the colon and plasma of colitis mice and enhance the activity of the antioxidant enzyme SOD, exerting an antioxidant effect. Ellagic acid (50 and 100 mg / kg) for 7 consecutive days can reduce ROS levels and the release of the pro-inflammatory factor IL-1β in the colonic tissue of ulcerative colitis model mice, indicating that ellagic acid can improve DSS-induced ulcerative colitis in mice by reducing oxidative stress generation. Simultaneously, ellagic acid increases SOD activity and reduces MDA content in ulcerative colitis mice. Our findings revealed that fermented Ganoderma lucidum powder significantly reduced the levels of pro-inflammatory cytokines IL-1β and IFN-γ in the colon of colitis-affected mice, increased the level of the anti-inflammatory factor IL-4, significantly reduced MDA content in colitis-affected mouse tissues (P < 0.05), increased SOD activity, and alleviated DSS-induced oxidative stress. This indicates that fermented Ganoderma lucidum powder can repair colonic tissue damage by inhibiting oxidative stress and inflammatory factor levels in the colonic tissue of colitis-affected mice.

[0094] Western blot results for proteins are as follows Figure 16 As shown, NLRP3, a member of the NOD-like receptor family, plays a crucial role in colitis. Upon activation of the NLRP3 inflammasome, the NLRP3 protein recruits ACS (acute colitis syndromes). The recruited ACS further activates Caspase-1, promoting the maturation and secretion of factors such as IL-1β and IL-18, thus inducing an inflammatory response. Figure 16It was observed that, compared with the NC group, the protein expression levels of NLRP3 and ASC in the colonic tissue of the Model group mice were significantly increased (P < 0.05), indicating that DSS can activate the NLRP3 inflammasome in mouse colonic tissue, inducing an inflammatory response. Compared with the Model group, the protein expression levels of NLRP3 and ASC in the colonic tissue of the PC and F-GL groups mice were significantly downregulated (P < 0.05). Studies have shown that Hericium erinaceus fruiting body polysaccharides can regulate the NLRP3 pathway, inhibit the expression of inflammatory factors, and have a good delaying effect on colitis. Ellagic acid can inhibit the activation of the NLRP3 inflammasome, reduce the release of pro-inflammatory factors IL-1β and IL-6, and block inflammatory signal transduction. Codonopsis pilosula polysaccharides can regulate intestinal immune response, inhibit excessive activation of the NLRP3 signaling pathway, and alleviate colonic tissue damage. Our study found that the IL-1β content and NLRP3 and ASC protein expression levels in the colonic tissue of Model group mice were significantly increased. After Ganoderma lucidum powder intervention, IL-1β was significantly decreased, and the protein expression levels of NLRP3 and ASC were significantly downregulated (P < 0.05). These results indicate that intervention with Ganoderma lucidum powder co-fermented by Aspergillus niger, Lactobacillus rhamnosus, and Lactobacillus plantarum can inhibit the activation of NLRP3 inflammasomes in the colon of DSS-induced colitis mice.

[0095] Gut microbiota analysis results as follows Figure 17As shown. Gut microbiota dysbiosis has long been considered a key factor in the pathogenesis of colitis. In this study, fermented Ganoderma lucidum powder could improve the changes in gut microbiota induced by DSS in mice. At the phylum level, the F-GL group intervention significantly increased the abundance of Bacteroidota, which, as the dominant gut bacteria, ferments polysaccharides and produces short-chain fatty acids. Pseudomonadota decreased; Proteobacteria (such as Enterobacteriaceae) are often increased in the gut of patients with inflammatory bowel disease, and the decrease in abundance may alleviate mucosal inflammation. At the genus level, fermented Ganoderma lucidum powder increased the relative abundance of Psychrobacter in colitis mice, reaching the level of the NC group; it decreased the number of Escherichia-Shigella, which is beneficial to gut health. LeFSe results indicate that Lachnospiraceae_NK4A136_group, as the dominant bacteria in the fermented Ganoderma lucidum powder group, is one of the major genera present in the mouse gut and is a potential beneficial bacterium. Lachnospiraceae_NK4A136_group can produce SCFAs through the fermentation of dietary polysaccharides, which are negatively correlated with various metabolic diseases and chronic inflammation. Although the direct protective mechanism of Lachnospiraceae_NK4A136_group in the occurrence and progression of colitis is unclear, recent reports suggest its potential anti-colitis activity. In summary, DSS induction adversely affects the composition of the gut microbiota in mice, leading to gut microbiota dysbiosis. Fermented Ganoderma lucidum powder intervention significantly increases the abundance of beneficial bacteria and improves gut health.

[0096] In summary, this method utilizes *Aspergillus niger*, *Lactobacillus rhamnosus*, and *Lactobacillus plantarum* to ferment Ganoderma lucidum powder, yielding the optimal fermentation process: *Aspergillus niger* 0.5‰ is added to the Ganoderma lucidum powder, fermented at 30℃ for 48 h, followed by 5% addition of *Lactobacillus rhamnosus*, 5% of *Lactobacillus plantarum*, and 5% of glucose, with fermentation continuing at 37℃ for another 48 h. This fermentation process significantly increased the content of active substances in Ganoderma lucidum (P < 0.05), with crude polysaccharide content at 1.98 ± 0.03 g / 100g, total triterpenoid content at 18.93 ± 1.87 g / 100g, and total flavonoid content at 3.55 ± 0.23%, representing increases of 50%, 42%, and 106%, respectively. In vitro antioxidant experiments showed that the DPPH free radical scavenging rate and ABTS cation scavenging rate of fermented Ganoderma lucidum powder were significantly higher than those of unfermented Ganoderma lucidum powder and the blank control fermented Ganoderma lucidum powder (P < 0.05). The mixed-culture fermented Ganoderma lucidum powder exhibited a significant protective effect against DSS-induced colitis in mice, improving pathological changes in colonic mucosa, reducing inflammatory cell infiltration, improving goblet cell function, and decreasing the NAGL content in the serum of colitis-affected mice. Its mechanism involves the regulation of inflammatory factors, intestinal barrier repair, and gut microbiota balance. Primarily, it exerted its colonic protective effect by reducing the expression levels of pro-inflammatory cytokines IL-1β and IL-18 and increasing IL-4 expression in the colon of colitis-affected mice; inhibiting oxidative stress in the colonic tissue of colitis-affected mice and repairing tissue damage; increasing gut microbiota diversity and the abundance of beneficial bacteria; and inhibiting the activation of the NLRP3 inflammasome in the colon of colitis-affected mice. This study provides a scientific basis for the functional development of fermented Ganoderma lucidum powder.

[0097] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A fermentation process for increasing the polysaccharide content of Ganoderma lucidum, characterized in that: Includes the following steps: Step 1, primary fermentation of Aspergillus niger: Ganoderma lucidum powder and Aspergillus niger are gradually mixed and placed in an incubator for static fermentation to obtain Aspergillus niger Ganoderma lucidum fermentation product; Step 2: Preparation of Lactobacillus rhamnosus and Lactobacillus plantarum bacterial cultures: Lactobacillus rhamnosus and Lactobacillus plantarum were activated and cultured separately to obtain Lactobacillus rhamnosus bacterial cultures and Lactobacillus plantarum bacterial cultures; Step 3: Re-fermentation of Lactobacillus rhamnosus and Lactobacillus plantarum: Add sterile water and glucose to the Aspergillus niger Ganoderma lucidum fermentation product to obtain a mixed solution. After stirring evenly, inoculate Lactobacillus rhamnosus and Lactobacillus plantarum bacterial solutions in a sterile ultra-clean bench. After mixing evenly, transfer to a sterile fermentation bag and ferment at a constant temperature with shaking to obtain a mixed fermentation broth. Freeze-dry the mixed fermentation broth to obtain a mixed fermented freeze-dried powder.

2. The fermentation process for increasing the polysaccharide content of Ganoderma lucidum according to claim 1, characterized in that: In step one, the amount of Aspergillus niger added is 0.3 to 0.7‰ of the mass of Ganoderma lucidum powder; the amount of sterile water added is 30 to 40% of the mass of Ganoderma lucidum powder.

3. The fermentation process for increasing the polysaccharide content of Ganoderma lucidum according to claim 2, characterized in that: In step one, the culture is carried out by static incubation in a 30°C incubator for 24-48 hours.

4. The fermentation process for increasing the polysaccharide content of Ganoderma lucidum according to claim 3, characterized in that: In step two, the bacterial concentration in the *Lactobacillus rhamnosus* and *Lactobacillus plantarum* bacterial solutions is 1 × 10⁻⁶. 8 ~1.0×10 9 CFU / mL.

5. The fermentation process for increasing the polysaccharide content of Ganoderma lucidum according to claim 4, characterized in that: In step three, the amount of sterile water added is such that the ratio of Aspergillus niger ferment to sterile water is 1:5~10, and the amount of glucose added is such that the mass concentration of glucose in the mixture is 1~5%.

6. The fermentation process for increasing the polysaccharide content of Ganoderma lucidum according to claim 5, characterized in that: In step three, the amount of Lactobacillus rhamnosus bacterial solution added is such that the mass concentration of Lactobacillus rhamnosus bacterial solution in the mixture is 3-5%, and the amount of Lactobacillus plantarum bacterial solution added is such that the mass concentration of Lactobacillus plantarum bacterial solution in the mixture is 3-5%.

7. A Ganoderma lucidum fermentation product, characterized in that: Includes the mixed fermented freeze-dried powder obtained by the fermentation process described in any one of claims 1 to 6.

8. A Ganoderma lucidum fermentation product according to claim 7, characterized in that: The mixed fermented freeze-dried powder contained 1.98 ± 0.03 g / 100g of crude polysaccharide, 18.93 ± 1.87 g / 100g of total triterpenes, and 3.55 ± 0.23% of total flavonoids.

9. An application of a Ganoderma lucidum fermentation product, characterized in that: This includes using the mixed fermented freeze-dried powder of claim 8 to prepare a drug for treating inflammation.

10. An application of a Ganoderma lucidum fermentation product, characterized in that: This includes using the mixed fermented lyophilized powder of claim 8 to prepare a medicament for treating ulcerative colitis.

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