Hericium erinaceus-ginseng bidirectional solid fermentation product, preparation method thereof, mycoplasm granules and application

Abstract

The invention relates to the field of traditional Chinese medicines, in particular to a hericium erinaceus-ginseng bidirectional solid fermentation product, a preparation method thereof, mycoplasm granules and application. Hericium erinaceus and ginseng are combined to establish a bidirectional solid fermentation system, dynamic changes of active substances in the fermentation process are explored, and a new way is developed for further development of hericium erinaceus and ginseng research. The hericium erinaceus-ginseng bidirectional solid fermentation mycoplasm is prepared into a granule product, the preparation process is optimized, a theoretical basis is provided for health care product development of the bidirectional solid fermentation mycoplasm, the antioxidant activity of the bidirectional solid fermentation mycoplasm and the granule product thereof is researched, and the potential drug efficacy of self-made mycoplasm granules is deeply explored. The protection effect of the hericium erinaceus-ginseng bidirectional solid fermentation mycoplasm and the granule product thereof on ethanol-induced gastric mucosal lesion is researched, a theoretical basis is provided for subsequent further research on hericium erinaceus-ginseng bidirectional solid fermentation experiments, and a new thought is provided for development of traditional Chinese medicine products for treating gastric ulcer.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine, and in particular to a Hericium erinaceus-ginseng bidirectional solid fermentation product, a preparation method thereof, a fungus granule and an application thereof. Background Art

[0002] Hericium erinaceus, also known as Hericium erinaceus, is a highly nutritious fungus used for both medicinal and edible purposes. Rich in carbohydrates, protein, and trace elements, Hericium erinaceus is widely used in Asia, Europe, and the United States due to its diverse active ingredients, including sterols, polyphenols, polysaccharides, and pyrones, for its immune-boosting, digestion-promoting, and anti-tumor properties. Traditional Chinese Medicine considers Hericium erinaceus to be "neutral in nature, sweet in flavor, and beneficial for the five internal organs, aiding digestion, and strengthening the body." Through extensive research and practice, Hericium erinaceus is recognized as a fungus with significant stomach-nourishing properties, and is often used to prevent and alleviate ailments such as gastric ulcers, gastric cancer, and gastritis.

[0003] Ginseng (Panax ginseng C.A. Mey) is an herbaceous plant of the Araliaceae family, known as the "King of Herbs." Ginseng is mostly used as a medicine in its root and is widely distributed. Currently, nearly 200 active substances have been isolated from ginseng, including ginsenosides, ginseng polysaccharides, volatile oils, polyacetylene compounds, and amino acids. The rich active ingredients in ginseng contribute to its diverse physiological activities and medicinal effects. Ginsenosides and ginseng polysaccharides are the most important active ingredients in ginseng, with hypoglycemic, anti-inflammatory, anti-tumor, gastrointestinal regulation, and anti-tumor effects.

[0004] Gastric ulcers are a chronic digestive disease that develops in the lesser curvature of the stomach or between the cardia and pylorus. They are highly prevalent, affecting approximately 5-10% of the global population to varying degrees. Common symptoms of gastric ulcers include abdominal distension, acid reflux, belching, and epigastric pain. Severe cases can include bleeding, perforation, and even cancer, with a cancerous transformation rate as high as 2-3%. The development of gastric ulcers is a complex process, fueled by multiple factors, including the abuse of nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin, Helicobacter pylori infection, and alcohol abuse. As living standards improve, alcohol consumption has become commonplace, and the incidence of gastric mucosal damage-related diseases such as gastric bleeding, gastritis, and gastric ulcers caused by excessive alcohol consumption is increasing year by year. The mechanism by which ethanol induces gastric ulcers is complex. In addition to chemical burns caused by direct contact of ethanol with the gastric mucosa, oxidative stress and inflammation induced by oxygen free radicals (ROS) also contribute to ethanol-induced apoptosis of gastric epithelial cells.

[0005] Bidirectional solid fermentation is a composite fermentation system consisting of one or more Chinese medicinal materials and edible and medicinal fungi. The Chinese medicinal materials serve as the medicinal matrix to provide the fungi with the nutrients required for growth and metabolism. At the same time, the secondary metabolites secreted by the fungi stimulate the transformation and dissolution of bioactive substances in the Chinese medicinal materials.

[0006] Bidirectional solid-state fermentation can enhance the bioactivity of medicinal substrates while providing nutrients for the growth of edible and medicinal fungi. This environmentally friendly and resource-saving approach can significantly promote the recycling of traditional Chinese medicines (TCMs) and facilitate the establishment of a TCM industry chain. It is a key research topic in the development of Traditional Chinese Medicine (TCM). Both Hericium erinaceus and Panax ginseng have been used in bidirectional solid-state fermentation systems, but research on these systems is rare. Summary of the Invention

[0007] In view of this, the present invention provides a Hericium erinaceus-ginseng bidirectional solid fermentation product, a preparation method thereof, mycoplasma granules and applications. Gastric ulcer is a disease that is easy to recur, difficult to cure, and seriously endangers human health. Traditional Chinese medicine is very effective in treating it due to its multiple targets and few adverse reactions. The present invention uses Hericium erinaceus as the fermentation strain and ginseng as the medicinal matrix to carry out bidirectional solid fermentation, monitor the changing trend of the main components in the medicinal matrix, optimize the production process of the bidirectional fermentation product granule product, and explore the protective effect of the bidirectional fermentation product of Hericium erinaceus and ginseng on ethanol-induced gastric mucosal damage, providing a theoretical basis for the development of Hericium erinaceus-ginseng bidirectional fermentation products.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] In a first aspect, the present invention provides the use of bidirectional fermentation of Hericium erinaceus strains and ginseng in any of the following items;

[0010] (I), increasing the content of reducing sugars in the fermentation product; and / or

[0011] (II), increasing the protein content in the fermentation product; and / or

[0012] (III), increasing the ergosterol content in the fermentation product; and / or

[0013] (IV), increasing the content of essential amino acids (EAA) in the fermentation product; and / or

[0014] (V), increasing the content of unsaturated fatty acids (SFAs) in the fermentation product; and / or

[0015] (VI), increasing the crude fat content in the fermentation product; and / or

[0016] (VII), increasing the content of total dietary fiber in the fermentation product; and / or

[0017] (VIII), increasing the ash and / or mineral content; and / or

[0018] (IX) Improve the types and contents of volatile flavor compounds (VFCs) in ginseng;

[0019] The preservation number of the Hericium erinaceus strain is CGMCC No. 22450.

[0020] In some embodiments of the present invention, the types of volatile flavor substances (VFCs) include alcohol VFCs and / or ester VFCs;

[0021] The type and content of the volatile flavor substances (VFCs) in ginseng that are improved include any of the following:

[0022] (i) reducing the content of alcohol VFCs; and / or

[0023] (ii) Increase the content of ester VFCs.

[0024] FAA refers to amino acids that have other special functions in addition to being able to synthesize proteins. FAA is not only a good nutrient, but also plays an important role in flavor. FAA is divided into umami AA (Glu, Asp, Lys), sweet AA (Thr, Ser, Gly, Ala, Leu, Val, Phe) and bitter AA (Tyr, Ile, Leu, Phe, Arg, His) according to different flavoring methods. FAA that cannot be synthesized by the human body and must be supplied through food is called essential amino acids (EAA). The essential amino acids (EAA) include: one or more of Val, Met, Ile, Leu, Phe, Lys, Thr or Trp; in the present invention, the content of flavor FAA is increased, and it can be considered that Hericium erinaceus can regulate the flavor of ginseng fermentation products through FAA metabolism.

[0025] FAs include 15 saturated fatty acids (SFAs) and 20 unsaturated fatty acids (USFAs). The levels of oleic acid, linoleic acid, γ-linolenic acid, dihomo-γ-linolenic acid, and arachidonic acid also increase to varying degrees. Linoleic acid and linolenic acid are two essential FAs for human life. Studies have shown that excessive intake of SFAs can increase serum total cholesterol and the incidence of cardiovascular disease, which is detrimental to human health. However, USFAs such as oleic acid and arachidonic acid have the effects of lowering cholesterol and preventing atherosclerosis. These results indicate that the types and contents of FAs in ginseng undergo changes after fermentation with Hericium erinaceus, and the fermentation product has higher nutritional and pharmacological value.

[0026] Volatile flavor compounds (VFCs) are one of the most important factors influencing the flavor of baked and fermented foods and a key factor in consumer acceptance. Studies have shown that fermentation significantly influences the volatile components and concentrations in foods. However, little is known about the changes in VFCs in ginseng fermented with Hericium erinaceus. Forty-three compounds were detected in UFG, primarily alcohols, accounting for 44.91% of the total volatile flavor compounds; 45 compounds were detected in HEB, primarily acids (44.91%); and 47 compounds were detected in FG, primarily esters (28.12%). The main volatile flavor compounds in UFG were 4-chlorobenzo[1,2,5]thiadiazol-5-ol (14.81%), followed by 4,4,5,8-tetramethyl-4-hydro-1-benzopyran (14.16%) and cineole (8.62%). The three most abundant volatile flavor compounds in HEB are palmitic acid (19.98%), 3,3,5,7-tetramethyl-1-indanone (14.30%), and dimethyl 2-(5-methyl-[1,3]thiothioate-2-methylene)-malonate (6.59%). The most abundant volatile flavor compounds in FG are 3-trifluoromethylbenzoic acid cyclobutyl ester (22.05%), 4-chloro-5-hydroxy-2,1,3-thiadiazole (21.37%), and 2,4,7,8-tetramethyl-3H-1,5-benzodiazepine (18.82%). Some researchers have found that changes in the types and contents of VFCs may be related to FAA degradation and glucose conversion during fermentation. Overall, fermentation significantly affects the types and concentrations of VFCs in ginseng.

[0027] Although minerals are present in relatively low concentrations in the human body, they participate in various metabolic processes, such as protein synthesis, transport, and cell regulation, making them crucial components of the body. HEB has the highest potassium content, while UFG and FG have the highest calcium content. Compared to UFG, FG showed significant increases in K and Ca, by 11.01% and 8.33%, respectively. Studies have reported that the total mineral content of ginseng does not change with the fermentation process. However, the increase in total mineral content observed in this study after fermentation may be related to differences in ginseng variety, microbial diversity, and fermentation environmental conditions.

[0028] Heavy metals generally refer to metals with a specific gravity greater than 5, and their effects on the human body are slow and long-lasting. Once heavy metals enter the human body, they are difficult to metabolize. When excessively accumulated in the body, they can affect the development of cardiovascular and central nervous systems, seriously endangering human health. The heavy metal levels in UFG, HEB, and FG are all far below the limits established by the FAO / WHO Joint Departmental Policy on Essential Medicines and Medicinal Products (1999) (As < 3 mg / kg, Pb < 10 mg / kg, Cd < 0.3 mg / kg, Hg < 0.1 mg / kg). Compared with UFG, the As, Pb, Cd, and Hg levels in FG did not change significantly, indicating that Hericium erinaceus fermentation does not lead to an increase in the heavy metal content in ginseng, and is therefore reliable in terms of safety.

[0029] Protein and crude fat content are used as indicators to evaluate the edible quality of fermented ginseng products. Ash in food refers to the inorganic matter remaining after high-temperature burning, primarily inorganic salts and their oxides. Dietary fiber is a class of substances that cannot be digested and absorbed by the human small intestine, including polysaccharides, oligosaccharides, lignin, cellulose, and hemicellulose. Compared with UFG, the protein and crude fat contents of FG increased by 42.46% and 22.63%, respectively. The ash and total dietary fiber content were in the order HEB > FG > UFG. Compared with UFG, the ash content of FG increased by 33.33% and the total dietary fiber content increased by 24.16%. The increase in ash and total dietary fiber content in FG may be due to the incorporation of nutrients such as inorganic salts by Hericium erinaceus during its growth.

[0030] In a second aspect, the present invention further provides a method for preparing a Hericium erinaceus strain-ginseng bidirectional solid fermentation product, comprising the following steps:

[0031] Step 1: Cultivating a solid culture of Hericium erinaceus strain;

[0032] Step 2: preparing a liquid culture of Hericium erinaceus strain;

[0033] Step 3: Grinding and sieving the ginseng slices to obtain ginseng powder; sterilizing the mixture, mixing it with the liquid strain of Hericium erinaceus, fermenting it with glucose or brown sugar as a carbon source, and separating Hericium erinaceus buds (HEB) and ginseng fungus (FG) in the fermentation system;

[0034] The preservation number of the Hericium erinaceus strain is CGMCC No. 22450.

[0035] In some specific embodiments of the present invention, the preparation of the liquid spawn of Hericium erinaceus in step 2 comprises the following steps:

[0036] Hericium erinaceus was cultured in the primary liquid culture medium and the secondary liquid culture medium in sequence at 26°C and 160 rpm / min in the dark for 7 days.

[0037] The primary liquid culture medium includes 20 g of glucose, 10 g of peptone, 2 g of KH2PO4, 1 g of MgSO4·7H2O, and 1 L of distilled water;

[0038] The secondary liquid culture medium includes 22 g of glucose, 0.2 g of peptone, 0.2 g of yeast powder, 5 g of KH2PO4, 1 g of MgSO4·7H2O, and 1 L of distilled water.

[0039] In some specific embodiments of the present invention, the carbon source in step 3 includes glucose;

[0040] The inoculation amount of the Hericium erinaceus liquid strain is 20% (W / V);

[0041] The fermentation temperature is 26° C., and the fermentation time is 3 to 40 days.

[0042] In some specific embodiments of the present invention, the fermentation time includes 3 days, 12 days, 21 days, 30 days or 40 days.

[0043] In a third aspect, the present invention also provides a Hericium erinaceus-ginseng bidirectional solid fermentation product obtained by the preparation method.

[0044] In a fourth aspect, the present invention also provides a method for preparing mycoplasma granules, comprising taking the ginseng mycoplasma in the Hericium erinaceus-ginseng bidirectional solid fermentation product, extracting with water to obtain an extract, and granulating to obtain the mycoplasma granules.

[0045] In some specific embodiments of the present invention, the solid-liquid ratio of the water extraction is 25:1; and / or

[0046] The water extraction time is 2.5h; and / or

[0047] The water extraction temperature is 60°C; and / or

[0048] The granulation aids include fillers and wetting agents; and / or

[0049] The fillers include starch and sucrose; and / or

[0050] The mass ratio of the extract to the starch is 1:1; and / or

[0051] The mass ratio of the extract to the sucrose is 1:1.4; and / or

[0052] The wetting agent is 75% by volume ethanol, and the amount of the ethanol is 12% (v / v); and / or

[0053] The drying temperature of the plasmid is 50°C.

[0054] In a fifth aspect, the present invention also provides mycoplasma granules prepared by the preparation method.

[0055] In a sixth aspect, the present invention further provides the use of the Hericium erinaceus-ginseng bidirectional solid fermentation product or the fungus granules in any of the following items:

[0056] (I) Improve the antioxidant capacity of cells;

[0057] (II), preparing a drug for improving antioxidant capacity;

[0058] (III) Oxidative repair of ethanol-damaged cells;

[0059] (IV), promoting the growth of ethanol-damaged cells;

[0060] (V) Improve the survival rate of ethanol-damaged cells;

[0061] (VI) improving the migration ability of ethanol-damaged cells;

[0062] (VII) Preparation of a drug for preventing and / or treating ethanol-induced gastric ulcer or a drug for protecting ethanol-induced gastric mucosa.

[0063] In some specific embodiments of the present invention, the antioxidant comprises:

[0064] Increase DPPH free radical scavenging rate;

[0065] Increase OH radical scavenging rate;

[0066] Increase ABTS+ free radical scavenging rate.

[0067] In a seventh aspect, the present invention further provides a medicine comprising the Hericium erinaceus-ginseng bidirectional solid fermentation product or the fungus granules.

[0068] The present invention explores the effects of two carbon sources, glucose and brown sugar, on the metabolites during the fermentation process of Hericium erinaceus, and establishes a Hericium erinaceus-ginseng bidirectional solid fermentation system using Hericium erinaceus as the fermentation strain and ginseng as the medicinal matrix. The dynamic changes of active ingredients during the fermentation process are investigated, the granule extraction and granulation processes are developed and optimized, the antioxidant activity of the fermentation products and mycelium granules is explored, and their repair effect on ethanol-induced gastric ulcer model cells is preliminarily explored.

[0069] The technical solutions of the present invention include: establishing a Hericium erinaceus-ginseng bidirectional solid fermentation system; combining Hericium erinaceus and ginseng to establish a bidirectional solid fermentation system, exploring the dynamic changes of active substances during the fermentation process, and opening up new paths for the further development of Hericium erinaceus and ginseng research. Making Hericium erinaceus-ginseng bidirectional solid fermentation mycelium granules; making Hericium erinaceus-ginseng bidirectional solid fermentation mycelium into a granule product and optimizing its production process, providing a theoretical basis for the development of health products of bidirectional solid fermentation mycelium, and studying the antioxidant activity of Hericium erinaceus-ginseng bidirectional solid fermentation mycelium and its granule products, and deeply exploring the potential drug efficacy of self-made mycelium granules. Exploring the therapeutic effect of bidirectional solid fermentation products and granule products on gastric ulcers; exploring the protective effect of Hericium erinaceus-ginseng bidirectional solid fermentation mycelium and its granule products on ethanol-induced gastric mucosal damage, providing a theoretical basis for further exploring Hericium erinaceus-ginseng bidirectional solid fermentation experiments, and providing new ideas for the development of traditional Chinese medicine products for the treatment of gastric ulcers.

[0070] Glucose and brown sugar were used as carbon sources for liquid culture of Hericium erinaceus mycelium. The total sugar, reducing sugar and polysaccharide contents as well as the cellulase, xylanase and pectinase activities in the fermentation broth of Hericium erinaceus after different fermentation times under different carbon sources were determined by UV spectrophotometry. (2) The total sugar, reducing sugar and protein contents of unfermented ginseng (UFG) and the products of Hericium erinaceus-ginseng bidirectional fermentation, fungus (FG) and fungus buds (HEB) were determined by phenol-sulfuric acid method, DNS and BCA colorimetry at 15, 30 and 40 days of fermentation. The ergosterol content in FG was determined by HPLC; (3) The optimal extraction and granulation process of bidirectional fermentation microbial granules (JZ) was determined by single factor investigation combined with orthogonal experimental method; (4) The antioxidant activities of UFG, FG, HEB, WLX and JZ at different concentrations were determined and compared by DPPH method, ·OH method and ABTS+ method; (5) The effects of each sample on the survival rate of GES-1 cells were detected and compared by CCK-8 method, and the effects of different concentrations of each sample on cell migration rate were compared by cell scratch test.

[0071] By comparing the contents of the main components of the products during the bidirectional solid fermentation process, it was found that the protein content in FG reached the highest on the 30th day, the ergosterol content reached the highest on the 40th day, and the total sugar, reducing sugar and protein contents in HEB all reached the highest on the 40th day; by comparing the component contents in UFG and FG fermented for 40 days, it was found that except for total sugar, the contents of other components increased significantly after 40 days of fermentation (P<0.05).

[0072] FG fermented for 40 days was used as the main raw material for the production of granules and the process was optimized. The optimal extraction conditions were liquid-to-solid ratio of 25:1, extraction time of 2.5h, and extraction temperature of 60℃; the optimal granulation process was extract: sucrose = 1:1.4, ethanol dosage of 12%, ethanol concentration of 75%, and drying temperature of 50℃.

[0073] Comparative experiments showed that HEB had outstanding antioxidant activity. In addition, the scavenging rate of FG against DPPH free radicals in the concentration range of 0.5-3 mg / mL was significantly higher than that of UFG (P<0.05). The scavenging activity of JZ against the three free radicals at a concentration of 3 mg / mL was comparable to that of WLX.

[0074] The experiment found that different concentrations of UFG, FG, HEB, WLX and JZ can significantly improve the cell survival rate of damaged cells, among which FG (600μg / mL) has the most outstanding effect; compared with the ethanol model group, the cell migration rate of damaged cells after 36h treatment with FG (100μg / mL) increased the most, reaching 67.49%, followed by JZ (600μg / mL) at 57.72%.

[0075] The present invention compares the effects of two different carbon sources, glucose and brown sugar, on the metabolites of Hericium erinaceus liquid fermentation, and finds that glucose can promote the secretion of Hericium erinaceus extracellular enzymes, while brown sugar can promote the secretion of Hericium erinaceus polysaccharides. Based on the above results, a Hericium erinaceus-ginseng bidirectional solid fermentation system was preliminarily successfully established. The content of the main components of each product during the fermentation process was compared, and the optimal fermentation time was determined to be 40 days. The granule granulation process was established and optimized with the bidirectional fermentation product mycelium (FG) as the main raw material. The antioxidant activity of the bidirectional fermentation product and the granule product (JZ) was compared, and it was found that the Hericium erinaceus buds (HEB) had outstanding antioxidant activity after fermentation, and the antioxidant activity of the ginseng matrix was significantly improved after fermentation. In vitro cell experiments on the bidirectional fermentation product and the granule product found that ginseng fermentation mycelium can effectively alleviate gastric mucosal damage caused by ethanol.

[0076] Biological Deposit Description

[0077] CCUCM-HE001, taxonomic name: Hericium erinaceus, was deposited on June 10, 2021, at the China General Microbiology Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, under the accession number CGMCC No. 22450. A biological deposit certificate was submitted with the previous patent application. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0079] Figure 1 The technical route of the present invention is shown;

[0080] Figure 2 Shows the growth of Hericium erinaceus-ginseng bidirectional fermentation at 15, 30, and 40 days;

[0081] Figure 3 The FAA content determination spectra in UFG, FG and HEB are shown; wherein, A shows the FAA content determination spectra in UFG; B shows the FAA content determination spectra in FG; C shows the FAA content determination spectra in HEB;

[0082] Figure 4 Spectra of FAs content determination in UFG, FG and HEB are shown; wherein, A shows the spectrum of FAs content determination in UFG; B shows the spectrum of FAs content determination in FG; C shows the spectrum of FAs content determination in HEB;

[0083] Figure 5 Figure 3 shows the changes in nutrient content in FG before and after fermentation; A shows the principal component score plot of bidirectional fermentation metabolites; B shows the heat map of FAA in samples treated with different methods; C shows the PCA plot of FAA content in bidirectional fermentation metabolites; (D) shows the tree-like stacking plot of FAA content in bidirectional fermentation metabolites; (E) shows the violin plot of FAs content in bidirectional fermentation metabolites;

[0084] Figure 6 The figures show the changes in VFCs content in samples before and after fermentation; A shows a bubble chart showing the changes in VFCs content in samples before and after fermentation; B shows an impact chart showing the changes in VFCs content in samples before and after fermentation;

[0085] Figure 7 The results of ergosterol content determination are shown; wherein, (a) shows a spectrum of ergosterol content determination in FG, and (b) shows a line graph of ergosterol content determination in FG;

[0086] Figure 8 The effects of liquid-to-solid ratio on extract yield and extract polysaccharide content are shown;

[0087] Figure 9 The results show the effect of extraction time on the extract yield and polysaccharide content of the extract;

[0088] Figure 10 The effects of extraction temperature on extract yield and polysaccharide content in the extract were shown;

[0089] Figure 11 Showing Hericium erinaceus-ginseng bidirectional fermentation fungus granules;

[0090] Figure 12 The scavenging effects of UFG, FG, HEB, WLX, JZ and Vc on DPPH are shown;

[0091] Note: Compared with the UFG group, the VC group showed significant differences as *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; the WLX group showed significant differences as #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001; the JZ group showed significant differences as ^P<0.05, ^^P<0.01, ^^^P<0.001, ^^^^P<0.0001; the FG group showed significant differences as +P<0.05, ++P<0.01, +++P<0.001, ++++P<0.0001; the HEB group showed significant differences as △P<0.05, △△P<0.01, △△△P<0.001, △△△△P<0.0001;

[0092] Figure 13 The results show the scavenging effects of UFG, FG, HEB, WLX, JZ and Vc on hydroxyl free radicals;

[0093] Figure 14 Shows the effects of UFG, FG, HEB, WLX, JZ and Vc on ABTS + Free radical scavenging effect;

[0094] Figure 15 Shown are the effects of different concentrations of UFG treatment on the activity of GES-1 cells for (a) 12 h (b) 24 h (c) 36 h (d) 48 h (e) 60 h;

[0095] Note: Compared with the UFG concentration of 0 μg / mL, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0096] Figure 16 Figure 3 shows the effect of different concentrations of FG treatment; (a) shows the effect of 12h on the activity of GES-1 cells; (b) shows the effect of 24h on the activity of GES-1 cells; (c) shows the effect of 36h on the activity of GES-1 cells; (d) shows the effect of 48h on the activity of GES-1 cells; (e) shows the effect of 60h on the activity of GES-1 cells;

[0097] Figure 17 Shown are the effects of different HEB concentration treatments on GES-1 cell viability for (a) 12 h (b) 24 h (c) 36 h (d) 48 h (e) 60 h;

[0098] Figure 18Shown are the effects of WLX treatment at different concentrations for (a) 12 h (b) 24 h (c) 36 h (d) 48 h (e) 60 h on the activity of GES-1 cells;

[0099] Figure 19 Shown are the effects of different concentrations of JZ treatment on the activity of GES-1 cells for (a) 12 h (b) 24 h (c) 36 h (d) 48 h (e) 60 h;

[0100] Figure 20 Shown are the effects of UFG, HEB, FG, WLX and JZ on the activity of GES-1 cells damaged by ethanol;

[0101] Note: Different letters in the bar graph indicate significant differences between the groups (P<0.05);

[0102] Figure 21 Figure 3 shows the effects of UFG, HEB, FG, WLX and JZ on the migration ability of GES-1 cells damaged by ethanol; (a) shows the effect of UFG on the migration ability of GES-1 cells damaged by ethanol; (b) shows the effect of HEB and FG on the migration ability of GES-1 cells damaged by ethanol; (c) shows the effect of WLX and JZ on the migration ability of GES-1 cells damaged by ethanol; (d) shows the effect of UFG, HEB, FG, WLX and JZ on the migration rate of GES-1 cells damaged by ethanol;

[0103] Note: Compared with the Model group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; UFGL, M, H represent low, medium, and high concentrations of UFG, respectively; HEB, FG, WLX, and JZ are the same as above. DETAILED DESCRIPTION

[0104] The present invention discloses Hericium erinaceus-ginseng bidirectional solid fermentation product, its preparation method, mycoplasma granules and applications. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters for implementation. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0105] Table 1. English abbreviations

[0106]

[0107]

[0108] Abbreviated term:

[0109] Unfermented Ginseng: UFG

[0110] FG, a product of Hericium erinaceus-ginseng bidirectional fermentation

[0111] Mushroom buds: HEB

[0112] Wei Lexin comparative drug: WLX

[0113] Homemade granules made from fungi (made according to the method of Wei Le Xin granules): JZ.

[0114] This experiment established a Hericium erinaceus-ginseng bidirectional solid fermentation system to monitor the changes in the main active ingredients during the fermentation process, explore the changes in the active substances during the fermentation process, and facilitate the control of fermentation time in subsequent production practices. To explore the application prospects of Hericium erinaceus-ginseng bidirectional fermentation products, this experiment used the bidirectional fermentation products to produce granules and optimize the process. The in vitro antioxidant activity of the bidirectional fermentation products and their products was compared, providing a theoretical basis for the development of granule products of the bidirectional fermentation products. In addition, this experiment established an ethanol-induced GES-1 cell injury model to explore the protective effects of the bidirectional fermentation products and products on gastric ulcer damage, laying the foundation for the screening of the active ingredients and pharmacodynamic mechanism of Hericium erinaceus-ginseng bidirectional fermentation products and products, and providing an experimental basis for the further development and utilization of Hericium erinaceus-ginseng bidirectional fermentation products.

[0115] This study compared the effects of glucose and brown sugar as carbon sources on liquid fermentation of Hericium erinaceus, established a Hericium erinaceus-ginseng bidirectional fermentation system, and compared the main components of the fermentation products. Granules were prepared using bidirectional fermentation bacteria as the main raw material and the process was optimized. The antioxidant activity of the bidirectional fermentation products and granules, as well as their protective effects on a gastric ulcer cell model, were investigated. The results are as follows:

[0116] Study on the main components of Hericium erinaceus-ginseng bidirectional solid-state fermentation products: A Hericium erinaceus-ginseng bidirectional fermentation system was established, and changes in the system's properties during the fermentation process were observed. Dynamic tracking of the changes in the main nutrient contents of unfermented ginseng (UFG), fungus buds (FG), and fungus buds (HEB) was performed. The results showed that the reducing sugar, protein, and ergosterol contents in FG were significantly higher than those in the unfermented ginseng medicinal matrix. A horizontal comparison of the compositional changes in the products during the fermentation process revealed that the protein content in FG reached its highest level on the 30th day of fermentation, while the total sugar, reducing sugar, and protein contents in HEB all reached their highest levels on the 40th day of fermentation. Furthermore, the ergosterol content in FG increased by 41.2% on the 40th day compared to the 30th day of fermentation. Based on comprehensive considerations, 40 days was selected as the fermentation time for Hericium erinaceus-ginseng bidirectional solid-state fermentation, and subsequent experiments were conducted using FG on the 40th day of fermentation.

[0117] Ginseng and Hericium erinaceus are both valuable medicinal and edible Chinese herbal medicines, widely used in health foods and clinical medicine. This study, for the first time, established a Hericium erinaceus-ginseng bidirectional solid-state fermentation system using Hericium erinaceus as the fermentation strain and ginseng as the medicinal matrix. The results comprehensively investigated the dynamic changes in activity and nutrients during the fermentation process.

[0118] This study used ginseng as the medicinal matrix and Hericium erinaceus as the fermentation strain, conducting a bidirectional solid-state fermentation. The study compared changes in the content of ginseng nutrients, including amino acids (EAAs), unsaturated fatty acids (SFAs), volatile flavor compounds (VFCs), minerals, total dietary fiber, ash, total protein, and crude fat, before and after fermentation. The goal was to investigate the transformation patterns of ginseng and Hericium erinaceus nutrients during bidirectional solid-state fermentation, with the goal of developing a novel fermentation method that effectively enhances the nutritional value and taste of ginseng.

[0119] After 30 days of fermentation with Hericium erinaceus, the contents of essential amino acids (EAAs) and unsaturated fatty acids (SFAs) in the fermented microbial medium increased by 68.10% and 19.11%, respectively. Hericium erinaceus fermentation also had varying degrees of effects on the types and contents of volatile flavor compounds (VFCs) in ginseng, as well as other substances (total sugars, reducing sugars, protein, crude fat, minerals, heavy metals, ash, and total dietary fiber). After fermentation, the proportion of alcohol VFCs decreased (44.91% to 1.66%), while the proportion of ester VFCs increased (0.48% to 28.12%). Protein, crude fat, and total dietary fiber contents increased by 42.46%, 22.63%, and 24.16%, respectively. This study provides a theoretical basis for the development and application of ginseng in functional foods and pharmaceuticals.

[0120] Study on the preparation process of Hericium erinaceus-ginseng bidirectional solid fermentation mycelium granules: The extraction process of the 40dFG extract of bidirectional fermentation was optimized with the extract yield and extract polysaccharide content as screening indicators, and the optimal extraction conditions were liquid-to-solid ratio of 25:1, extraction time of 2.5h, and extraction temperature of 60℃. The granulation process was subjected to a single-factor experiment with granulation difficulty, particle morphology and particle formation rate as indicators, and then the comprehensive score was calculated with particle formation rate, bulk density, angle of repose and moisture absorption rate as indicators. An orthogonal experiment was carried out, and the optimal granulation process was obtained as extract: sucrose = 1:1.4, ethanol dosage of 12%, ethanol concentration of 75%, and drying temperature of 50℃.

[0121] Study on the activity of Hericium erinaceus-ginseng bidirectional solid fermentation products and granules: Comparison of the antioxidant activity of bidirectional fermentation products and granule products (JZ) showed that HEB had the highest antioxidant activity. When the HEB concentration was 3 mg / mL, its antioxidant activity against DPPH, ·OH and ABTS was +The scavenging effect of the three free radicals was not significantly different from that of ascorbic acid (Vc) (P>0.05), and when the concentration was 3 mg / mL, the DPPH free radical scavenging rate of FG was significantly higher than that of UFG (P<0.05), increasing by 55.59%. In addition, JZ also has a certain antioxidant effect. When the concentration was 3 mg / mL, it had a significant effect on DPPH and ABTS. + The scavenging rates of hydroxyl radicals and ·OH radicals were 72.57%, 58.60% and 54.80%, respectively.

[0122] Effects of Hericium erinaceus-Ginseng bidirectional solid fermentation products on ethanol-damaged GES-1 cells: Treatment of ethanol-damaged GES-1 cells with Hericium erinaceus-Ginseng bidirectional fermentation products revealed that UFG, FG, HEB, and JZ all exhibited significant repair effects on ethanol-damaged cells. FG exhibited the most prominent protective effect, significantly enhancing cell survival and migration rates in ethanol-damaged cells. When gastric ulcer model cells were treated with 600 μg / mL of FG for 36 hours, cell survival increased by 49.92%, and cell migration reached 57.43%. Furthermore, homemade mycoplasma granules demonstrated excellent cell migration-promoting properties. Treatment with 1600 μg / mL of JZ increased the migration rate of ethanol-damaged cells to 57.72%. This suggests that Hericium erinaceus-Ginseng bidirectional fermentation products and their granular formulations have a certain repair effect on ethanol-induced gastric ulcer damage.

[0123] This study preliminarily explored the product application and product development of Hericium erinaceus-ginseng bidirectional solid fermentation, and conducted a comparative study on the antioxidant activity and anti-gastric ulcer ability of the bidirectional fermentation products and granule products, and obtained relatively ideal results. It is hoped that this study can provide a theoretical basis for the further development and application of Hericium erinaceus-ginseng bidirectional fermentation, and provide new ideas for the relief and treatment of gastric ulcers.

[0124] The Hericium erinaceus-ginseng bidirectional solid fermentation product, its preparation method, mycoplasma granules, and raw materials and reagents used in its application provided by the present invention can all be purchased from the market.

[0125] The present invention will be further described below in conjunction with the embodiments:

[0126] Example 1 Study on the main components of Hericium erinaceus-ginseng bidirectional solid fermentation products

[0127] The use of edible and medicinal fungi and Chinese medicinal materials for bidirectional solid fermentation has many advantages, such as improving efficacy, reducing toxicity, and being green and environmentally friendly, and has become a key research direction for researchers. However, this technology is still in its infancy, and problems such as clarifying the bidirectional fermentation mechanism, controlling the fermentation conditions, and systematically analyzing the fermentation products need to be solved urgently. This chapter compares the changes in the content of chemical components such as total sugar, protein, reducing sugar, and ergosterol in unfermented ginseng (UFG), Hericium erinaceus-ginseng bidirectional fermentation mycelium (FG), and Hericium erinaceus buds (HEB), analyzes the differences in mycelium before and after fermentation, and preliminarily explores the changes in the chemical composition of Hericium erinaceus and ginseng before and after fermentation, providing a theoretical basis for better exploring the nutritional value of mycelium in the future.

[0128] 1 Experimental Materials

[0129] 1.1 Medicinal Materials

[0130] Four-year-old ginseng slices were purchased from Jilin Yisheng Foreign Trade Co., Ltd.

[0131] 1.2 Experimental Reagents

[0132] Table 2. Experimental reagents

[0133]

[0134]

[0135] 1.3 Experimental instruments

[0136] Table 3. Experimental instruments

[0137]

[0138] 2 Experimental methods

[0139] 2.1 Cultivation of Hericium erinaceus solid culture

[0140] The Hericium erinaceus slant culture stored at 4°C was transferred to PDA solid culture medium in a clean bench and cultured in a constant temperature incubator at 28°C for 7 days. After subculturing again, the culture was cultured under the above conditions until the Hericium erinaceus hyphae covered the surface of the culture medium, and then liquid culture could be continued.

[0141] 2.2 Preparation of Hericium erinaceus liquid culture

[0142] The above-mentioned Hericium erinaceus mycelium was inoculated into liquid culture medium (20 g glucose, 10 g peptone, 2 g KH2PO4, 1 g MgSO4·7H2O, and 1 L distilled water), and cultured in a shaking incubator at 140 rpm / min under dark conditions at 28°C until the bottle was evenly covered with light yellow Hericium erinaceus balls, and the fermentation was terminated.

[0143] 2.3 Preparation of Hericium erinaceus-ginseng bidirectional solid fermentation products

[0144] A two-way solid-state fermentation of Hericium erinaceus and Panax ginseng was performed. Four-year-old ginseng slices were crushed and passed through a 16-mesh pharmacopoeia sieve to obtain ginseng powder. 25 g of the ginseng powder was weighed and placed in a 200 mL glass culture bottle with a lid. 10 mL of ddH2O was added, stirred, and sterilized by autoclaving at 121°C for 30 min. The sterilized ginseng matrix was placed in a clean bench to cool, shaken to loosen the matrix, and sterilized under UV light for 30 min. A control group consisted of a ginseng matrix without Hericium erinaceus fermentation broth, while an experimental group consisted of a ginseng matrix with 30% (w / v) Hericium erinaceus fermentation broth. Samples were collected at 15, 30, and 40 days of fermentation to separate the Hericium erinaceus buds (HEB) and Panax ginseng fungus (FG) from the fermentation system. The samples were then dried at 40°C, crushed, and passed through a 50-mesh sieve. The sample powders were sealed and stored in a desiccator.

[0145] 2.4 Determination of main components after fermentation

[0146] 2.4.1 Determination of total sugar content

[0147] Determine the total sugar content in oven-dried samples using the phenol-sulfuric acid method. To create a standard curve, dry anhydrous glucose at 105°C to constant weight and prepare a 0.1 mg / mL standard solution in a volumetric flask. Accurately pipette 0, 200, 400, 600, 800, and 1000 μL of the solution, make up to 1 mL with sterile water, and shake thoroughly. Add 1 mL of 5% phenol and 5 mL of sulfuric acid, mix thoroughly, and boil in a water bath for 5 minutes. Remove and cool to room temperature. Measure the absorbance at 490 nm. Perform the same determination on the sample to be tested, and calculate the total sugar content based on the anhydrous glucose standard curve.

[0148] 2.4.2 Determination of reducing sugar content

[0149] The reducing sugar content in dried samples was determined using a 3,5-dinitrosalicylic acid colorimetric method with slight modifications based on the method described in the literature (Du Yangyang, Bao Yuanyuan, Liu Xiangyu, et al. Effects of Buckwheat Rotation on Enzyme Activities and Microorganisms in the Rhizosphere Soil of Potatoes in Yunnan [J / OL]. China Agricultural Science and Technology Herald, 1-9 [2024-03-14].). To construct a standard curve: Anhydrous glucose was dried at 105°C to constant weight and placed in a volumetric flask to prepare a 0.1 mg / mL standard solution. Accurately pipette 0, 200, 400, 600, 800, and 1000 μL of the standard solution, make up to 1 mL with sterile water, and shake well. Add 2 mL of DNS solution, mix thoroughly, and boil in a water bath for 5 minutes. Remove and cool to room temperature. Add 9 mL of sterile water, mix thoroughly, and measure the absorbance at 540 nm. The test samples were assayed under the same conditions, and the reducing sugar content in the samples was calculated based on the anhydrous glucose standard curve. 2.4.3 Protein content determination

[0150] The total protein content in the oven-dried sample was determined using the Coomassie Brilliant Blue method. Standard curve drawing: 5 mg of bovine serum albumin (BSA) was placed in a volumetric flask and diluted to 100 mL to prepare a protein standard solution with a concentration of 0.05 mg / mL. Accurately draw 0, 200, 400, 600, 800, and 1000 μL of the standard solution, make up to 1 mL with sterile water, and shake evenly. Weigh 100 mg of Coomassie Brilliant Blue G-250, add 50 mL of 95% ethanol to dissolve, then add 100 mL of 85% H3PO4 and dilute to 1000 mL with sterile water. 4 mL of Coomassie Brilliant Blue solution was added to each of the different concentration gradient protein standard solutions, vortexed for 5 minutes, and the absorbance at 595 nm was measured. The test samples were measured under the same conditions, and the protein content in the sample was calculated according to the BSA standard curve.

[0151] 2.5 Data Processing

[0152] Experimental data were analyzed using SPSS 25.0. The data are presented as mean ± standard deviation (mean ± SD). Group differences were compared using one-way analysis of variance (ANOVA) and multiple comparisons between groups using the Tukey HSD test. P < 0.05 was considered statistically significant.

[0153] 3 Experimental results

[0154] 3.1 Bidirectional fermentation growth

[0155] The fermentation of Hericium erinaceus and ginseng on days 15, 30 and 40 is as follows Figure 2 As shown in the figure, on the 15th day of fermentation, the Hericium erinaceus hyphae had essentially covered the entire surface of the ginseng medicinal matrix, forming a radial pattern, with a small amount of hyphae penetrating into the underlying medicinal matrix. By the 30th day, a small number of Hericium erinaceus buds had formed on the upper surface of the medicinal matrix, and the hyphae had almost completely penetrated the entire medicinal matrix. By the 40th day of fermentation, a thick and dense Hericium erinaceus bud structure had formed on the upper surface of the medicinal matrix, and the hyphae had completely penetrated the medicinal matrix.

[0156] 3.2 Determination results of total sugar, reducing sugar and protein content

[0157] According to the methods in 2.4.1, 2.4.2 and 2.4.3, the standard curves of total sugar, reducing sugar and protein were obtained as follows: Y = 5.2817X + 0.1443 (R 2 =0.9997), Y=6.3179X+0.0458(R 2 =0.9990), Y=20.865X+0.5369(R 2=0.9991). The results of total sugar, reducing sugar, and protein content determinations for UFG, HEB, and FG at different fermentation stages are shown in Table 4. As shown in Table 4, the total sugar, reducing sugar, and protein contents in UFG did not change significantly at 15, 30, or 40 days (P>0.05), whereas the contents of each component in FG and HEB showed significant differences during the fermentation process (P<0.05).

[0158] The total sugar content in FG decreased with fermentation time. Compared with fermentation day 15, the total sugar content in FG decreased significantly by 10.13% on day 30 (P < 0.05), and by 35.90% on day 40 (P < 0.05). Compared with UFG of the same period, the total sugar content in FG decreased by 2.39% and 32.79% on days 30 and 40, respectively. Compared with fermentation day 30, the total sugar content in HEB increased significantly (P < 0.05), by 48.38%, on day 40.

[0159] The reducing sugar content in FG did not change significantly at 30 days compared with 15 days (P>0.05). However, the reducing sugar content in FG at 40 days increased significantly compared with both 15 and 30 days, by 40.63% and 63.91%, respectively. The reducing sugar content in HEB was lower than that in UFG and FG during the same period. Furthermore, the reducing sugar content in HEB at 40 days increased significantly (P<0.05), by 9.18%, compared with 30 days (P<0.05).

[0160] Total protein content in FG reached its highest level on day 30 of fermentation, increasing by 66.27% compared to day 15. Total protein content in FG remained unchanged during the later stages of fermentation (P>0.05). Compared to UFG grown at the same time, total protein content in FG increased by 2.74-fold on day 30 of fermentation. Total protein content in HEB significantly increased by 18.47% on day 40 of fermentation compared to day 30 (P<0.05).

[0161] Table 4. Determination results of total sugar, reducing sugar and protein content

[0162]

[0163]

[0164] Note: Different letters in the table indicate significant differences among different fermentation times of UFG, FG and HEB (P<0.05).

[0165] 3.3 Ergosterol content determination results

[0166] The linear regression equation of ergosterol was Y=36.797X-0.7574, R2 =0.9971. The ergosterol spectra of FG at different fermentation times are shown in Figure 2. Figure 7 (a) As shown. The results of the determination of ergosterol content in FG are as follows Figure 7 (b) As shown in the figure, the ergosterol content in FG was 4.95 μg / g on day 15 of fermentation. From day 15 to day 30 of fermentation, the ergosterol content in FG increased rapidly. Compared to day 15, the ergosterol content in FG increased by 99.34 μg / g on day 30 of fermentation. Furthermore, compared to day 30 of fermentation, the ergosterol content in FG increased by 41.2% on day 40 of fermentation.

[0167] 4 Discussions

[0168] Ginseng and Hericium erinaceus are both valuable medicinal and edible Chinese herbs, widely used in health foods and traditional medicines. This study established a Hericium erinaceus-ginseng bidirectional solid-state fermentation system using Hericium erinaceus as the fermentation strain and ginseng as the medicinal matrix. The dynamic changes in activity and nutrients during the fermentation process were comprehensively investigated.

[0169] The experimental results show that, in addition to total sugar content, the contents of other components, such as reducing sugars, protein, and ergosterol, all increased after fermentation compared to UFG. The secretion of various extracellular enzymes (amylase, cellulase, glucosidase, laccase, etc.) by Hericium erinaceus during fermentation may be the primary cause of the changes in sugar content in FG. Ergosterol is the primary active ingredient in Hericium erinaceus. The increase in ergosterol content after fermentation suggests that bidirectional fermentation facilitates the accumulation of active ingredients in the fungus.

[0170] Example 2 Changes in nutrient composition before and after ginseng-hericium erinaceus bidirectional solid fermentation

[0171] 1. Materials and Methods

[0172] Materials and reagents

[0173] Hericium erinaceus was preserved by China General Microorganism Culture Collection (Beijing, China) with the strain number CGMCC No. 22450. Four-year-old ginseng slices were purchased from Jilin Yisheng Foreign Trade Co., Ltd. (Changchun, Jilin, China).

[0174] Fermentation method

[0175] The fermentation process involves two steps. First, the Hericium erinaceus inoculum is prepared. The Hericium erinaceus is inoculated into a primary liquid culture medium (20 g glucose, 10 g peptone, 2 g KH2PO4, 1 g MgSO4·7H2O, and 1 L distilled water) to obtain a primary liquid culture. This primary liquid culture is then inoculated into a secondary liquid culture medium (22 g glucose, 0.2 g peptone, 0.2 g yeast powder, 5 g KH2PO4, 1 g MgSO4·7H2O, and 1 L distilled water, with an inoculum concentration of 20%). The culture is incubated at 26°C, 160 rpm / min, and dark conditions for 7 days to obtain a secondary fermentation broth. The secondary fermentation broth is then inoculated into a sterilized solid ginseng culture medium at a 20% (w / v) inoculum concentration and cultured at 26°C for 3, 12, 21, 30, or 40 days.

[0176] Sample preparation

[0177] The experimental group (FG) was treated with ginseng matrix inoculated with secondary Hericium erinaceus seed culture, while the control group (UFG) was treated with ginseng matrix without secondary Hericium erinaceus seed culture. By day 30 of fermentation, the Hericium erinaceus mycelium had completely filled the entire flask and had formed distinct mycelial buds (HEBs). UFG and FG samples were collected at days 3, 12, 21, 30, and 40, and HEB samples were isolated at days 30 and 40. The samples were dried at 40°C, crushed, passed through a No. 3 sieve, and stored sealed in the dark.

[0178] 1.3.1. Determination of total sugar and reducing sugar content

[0179] 1g of FG and UFG samples with different fermentation times were dissolved in 20mL of distilled water and incubated in a 70℃ water bath for 1h. Filtered through a 0.45μm filter membrane, the filtrate was collected. According to the literature (Wang Huan, Chen Changbao, Zhang Bo, et al. Optimization of the extraction process of polysaccharides from the fruiting body of Agaricus oxyphylla by response surface methodology and its in vitro antioxidant activity [J]. Journal of Mycology, 2019, 38(10): 1681-1688. or Zhao Yanqiao, Wang Shaoping, Wang Yue, et al. Analysis of the in vitro antioxidant and hypoglycemic activity of Perilla leaves [J]. Food Industry Science and Technology, 2024, 45(08): 318-324.), the total sugar content was determined by the phenol-sulfuric acid method. The reference used the 3,5-dinitrosalicylic acid (DNS) colorimetric method to determine reducing sugars.

[0180] 1.4. Nutritional analysis

[0181] 1.4.1. FAA content determination

[0182] FAA content was determined for HEB, FG after 30 days of fermentation, and UFG. An appropriate amount of sample was weighed and 15 mL of 6 mol / L hydrochloric acid solution was added to a hydrolysis tube. The tube was frozen in a cryogen for 5 minutes, then nitrogen was filled and hydrolyzed at 110°C for 24 hours. The sample was then cooled to room temperature. The hydrolyzate was transferred to a 50 mL volumetric flask and dilute to the mark with water. Accurately aspirate 1 mL of the filtrate and dry under reduced pressure at 40°C. Dissolve the solution in 1.0 mL of sodium citrate buffer (pH 2.2), filter through a 0.22 μm filter, and analyze the content using an automatic amino acid analyzer (LA 8080, Hitachi High-Technologies Corporation, Japan).

[0183] 1.4.2.FAs content determination

[0184] Weigh an appropriate amount of the sample to be tested, add 100 mg of pyrogallic acid, 2 mL of 95% ethanol, and 10 mL of hydrochloric acid solution, and hydrolyze in a 70°C water bath for 40 minutes. Cool to room temperature. Add 10 mL of 95% ethanol to the hydrolyzed sample. Rinse the hydrolyzate with 50 mL of a mixture of ether and petroleum ether, and combine the rinse and hydrolyzate. Shake the separatory funnel, collect the ethereal extract, repeat the extraction three times, combine the extracts, evaporate to dryness in a water bath, and dry at 105°C for 2 hours. Add 2 mL of 2% sodium hydroxide-methanol solution to the fat extract, and incubate in a water bath at 85°C for 30 minutes. Add 3 mL of 14% boron trifluoride-methanol solution, and incubate in a water bath at 85°C for 30 minutes. Cool to room temperature. Add 1 mL of n-hexane and extract with vortexing for 2 minutes. After the extract was separated, 0.1 mL of the upper extract was taken and the volume was adjusted to 1 mL with n-hexane. The volume was filtered through a 0.45 μm filter membrane and then determined using a crude fat analyzer (SZF-06A, Shanghai Xinjia, China).

[0185] 1.4.3. Determination of VFCs Content

[0186] 0.2 g of sample was weighed and added to 5 mL of n-hexane. The sample mixture was vortexed for 2 minutes, allowed to stand for extraction for 20 minutes, and then centrifuged at 4000 rpm / min at 4°C for 20 minutes. The supernatant was filtered through a 0.22 μm filter and analyzed using an inductively coupled plasma mass spectrometer (iCAPQ, Thermo, USA). Chromatographic conditions: An HP-INNOWAX column (25 m × 0.20 mm × 0.40 μm) was used, with helium as the carrier gas at a flow rate of 1.0 mL / min. Splitless injection was performed, and the inlet temperature was 240°C. The temperature program was as follows: the column temperature was initially maintained at 100°C for 5 minutes, then increased to 150°C at a rate of 5°C / min, and then to 280°C at a rate of 30°C / min, where it was held for 30 minutes. Mass spectrometry conditions: Agilent 5975B mass spectrometer, ionization mode: EI source, electron energy: 70 eV, filament heating current: 0.35 mA, ion source temperature: 200°C, transfer line temperature: 250°C, scan range: 40-450 m / z.

[0187] 1.4.4. Determination of mineral content

[0188] Weigh an appropriate amount of sample into a polytetrafluoroethylene digestion vessel, add 5 mL of nitric acid, and place in a microwave digester after the reaction is complete. The digestion procedure is to digest at 100°C, 140°C, 160°C, and 180°C for 3 minutes each, followed by digestion at 190°C for 15 minutes. After the digestion vessel temperature cools to below 50°C, rinse the digestion vessel and transfer the rinse solution to a 50 mL volumetric flask and dilute to the mark with water. The reaction conditions for the ICP-MS method are as follows: RF power: 1550 W, pump speed: 40 rpm, spray chamber temperature: 2.7°C, sampling depth: 5 mm, cooling gas flow rate: 14 L / min, auxiliary gas flow rate: 0.8 L / min, and nebulizing gas flow rate: 1.122 L / min.

[0189] 1.4.5. Determination of other nutritional components

[0190] Protein content was determined using the Kjeldahl method. Crude fat and total dietary fiber content were determined using the methods of Yose et al. Ash content was determined using the methods of Juraj et al.

[0191] Data Analysis

[0192] One-way ANOVA was performed using SPSS 20.0 software. Line graphs and bar graphs of changes in active ingredients were drawn using GraphPad Prism 7.00 software. Data were log-transformed and centered (CTR) formatted using SIMCA (14.0) software for principal component analysis (PCA) modeling. Cluster heat maps were drawn using Origin 2022 software. All experiments were repeated three times, and data are presented as means ± SE.

[0193] 2. Results and Discussion

[0194] 2.1. Analysis of changes in nutrient content before fermentation

[0195] Principal component analysis of nutrients in bidirectional fermentation samples

[0196] Principal component analysis (PCA) is an unsupervised pattern recognition method that reduces the dimensionality of complex data to more effectively interpret the main information in the data. PCA pattern was used to identify the differences in nutritional components among UFG, HEB, and FG. The resulting PCA component diagram is shown in Figure 5(A). PC1 and PC2 represent the first and second principal component scores, respectively. The color of the scattered points indicates the experimental grouping samples, all of which are within the 95% confidence interval. The concentration of sample points within each group indicates good reproducibility across treatments.

[0197] 2.1.1. Changes in FAA and FAs

[0198] FAA is closely related to human health, and its content in ginseng shows significant differences under different environments and processing conditions. This study analyzed the FAA content in UFG, HEB and FG, and the amino acid profiles obtained are shown in Figure 2. Figure 3 The changing trends of the FAA contents in the samples during the fermentation process are as follows: Figure 5 (B, C, D) and Table 5. The content of each FAA in HEB was significantly higher than that in UFG and FG. Compared with UFG, the content of Arg decreased in FG, while the content of the remaining 16 FAAs increased. The decrease in Arg content may be due to its conversion to Orn by arginase. Comparing TAA content, HEB > FG > UFG. Hwang et al. believe that the rate of increase in FAA may be positively correlated with the degradation of polyunsaturated acids and other metabolites during fermentation.

[0199] Essential amino acids (EAAs) are amino acids that cannot be synthesized by the human body and must be supplied through food. They include Val, Met, IIe, Leu, Phe, Lys, Thr, and Trp. The proportion of EAA in HEB, FG, and UFG is 30.35%, 39.30%, and 36.01%, respectively. The EAA content in FG increases by 68.10% after fermentation, with Val, Met, IIe, and Phe showing significant increases. The NEAA content in FG increases by 24.54% after fermentation, with Ser, Cys, and His showing significant increases. This suggests that fermentation significantly increases the nutritional content of ginseng.

[0200] FAA is not only a good nutrient, but also plays an important role in flavor. FAA is divided into umami AA (Glu, Asp, Lys), sweet AA (Thr, Ser, Gly, Ala, Leu, Val, Phe) and bitter AA (Tyr, Ile, Leu, Phe, Arg, His) according to different flavoring methods. After fermentation, the umami AA, sweet AA and bitter AA contents in ginseng increased by 31.79%, 71.04% and 26.34%, respectively. Previous studies have shown that lactic acid bacteria can metabolize FAA into characteristic aromas such as fruity and fatty aromas unique to fruits and vegetables during the fermentation process. The increase in the content of flavor FAA in this study suggests that Hericium erinaceus can regulate the flavor of ginseng fermentation products through FAA metabolism.

[0201] Table 5. FAA content in UFG, HEB and FG

[0202]

[0203]

[0204] Note: All values are expressed as Mean ± SE. Tukey's multiple-test was used. Different letters correspond to significant differences (p < 0.05) associated with different treatments. ND indicates not detected according to the detection limit of each substance.

[0205] FAs in UFG, HEB and FG were analyzed, including 15 saturated fatty acids (SFAs) and 20 unsaturated fatty acids (USFAs). Figure 5 E and Table 6, fatty acid profiles are shown in Figure 4 . The content of total fatty acids (TFAs) is FG>MB>UFG. Compared with UFG, the content of FAs in FG increased by 19.11%, the proportion of SFAs increased slightly (17.77%→17.93%), and the content of USFAs increased by 19.11% after fermentation. The contents of oleic acid, linoleic acid, γ-linolenic acid, dihomo-γ-linolenic acid and arachidonic acid also increased to varying degrees. Linoleic acid and linolenic acid are two FAs necessary for human life activities. Studies have shown that excessive intake of SFAs by the human body will cause an increase in total cholesterol in the serum, increase the incidence of cardiovascular disease, and is not conducive to human health. USFAs such as oleic acid and arachidonic acid have the effect of lowering cholesterol and preventing atherosclerosis. The above results show that the types and contents of FAs of ginseng changed after fermentation with Hericium erinaceus, and the fermentation product has higher nutritional and pharmacological value.

[0206] Table 6. FAs content in UFG, HEB and FG

[0207]

[0208]

[0209] Note: All values are expressed as Mean ± SE. Tukey's multiple-test was used. Different letters correspond to significant differences (p < 0.05) associated with different treatments. ND indicates not detected according to the detection limit of each substance.

[0210] 2.1.2. VFCs Changes

[0211] Volatile flavor compounds (VFCs) are one of the most important factors affecting baked and fermented foods and are a key factor in consumer acceptance. Studies have shown that fermentation can have a significant impact on the volatile components and concentrations in foods. However, little is known about the changes in VFCs in ginseng fermented with Hericium erinaceus. Figure 6 As shown in the figure, a total of 43 substances were detected in UFG, mainly alcohol compounds, accounting for 44.91% of the total volatile flavor substances; a total of 45 substances were detected in HEB, mainly acid compounds (44.91%); a total of 47 substances were detected in FG, mainly ester compounds, accounting for 28.12% of the total volatile flavor substances; the main volatile flavor substances in UFG were 4-chlorobenzo[1,2,5]thiadiazol-5-ol (14.81%), followed by 4,4,5,8-tetramethyl-4-hydro-1-benzopyran (14.16%) and cineole (8.62%). The three most abundant volatile flavor compounds in HEB are palmitic acid (19.98%), 3,3,5,7-tetramethyl-1-indanone (14.30%), and dimethyl 2-(5-methyl-[1,3]thiothioate-2-methylene)-malonate (6.59%). The most abundant volatile flavor compounds in FG are 3-trifluoromethylbenzoic acid cyclobutyl ester (22.05%), 4-chloro-5-hydroxy-2,1,3-thiadiazole (21.37%), and 2,4,7,8-tetramethyl-3H-1,5-benzodiazepine (18.82%). Some researchers have found that changes in the types and contents of VFCs may be related to FAA degradation and glucose conversion during fermentation. Overall, fermentation significantly affects the types and concentrations of VFCs in ginseng.

[0212] 2.1.3. Changes in mineral content before and after fermentation

[0213] Although minerals are present in relatively low concentrations in the human body, they participate in various metabolic processes and function in diverse ways, such as protein synthesis, transport, and cell regulation. They are essential components of living organisms. The results in Table 7 show that HEB has the highest K content, while UFG and FG have the highest Ca content. Compared to UFG, FG exhibits significant increases in K and Ca, by 11.01% and 8.33%, respectively. Studies have reported that the total mineral content of ginseng does not change with the fermentation process. However, the increase in total mineral content after fermentation in this study may be related to differences in ginseng variety, microbial diversity, and fermentation environmental conditions.

[0214] Heavy metals generally refer to metals with a specific gravity greater than 5, and their effects on the human body are slow and long-lasting. Once heavy metals enter the human body, they are difficult to metabolize. When excessively accumulated in the body, they can affect the development of cardiovascular and central nervous systems, seriously endangering human health. The heavy metal levels in UFG, HEB, and FG are all far below the limits established by the FAO / WHO Joint Departmental Policy on Essential Medicines and Medicinal Products (1999) (As < 3 mg / kg, Pb < 10 mg / kg, Cd < 0.3 mg / kg, Hg < 0.1 mg / kg). Compared with UFG, the As, Pb, Cd, and Hg levels in FG did not change significantly, indicating that Hericium erinaceus fermentation does not lead to an increase in the heavy metal content in ginseng, and is therefore reliable in terms of safety.

[0215] Table 7. Contents of trace elements and heavy metals in UFG, HEB and FG

[0216]

[0217]

[0218] Note: All values are expressed as Mean ± SE. Tukey's multiple-test was used. Different letters correspond to significant differences (p < 0.05) associated with different treatments. ND indicates not detected according to the detection limit of each substance.

[0219] 2.1.4. Changes in other nutrient contents before and after fermentation

[0220] Table 8 shows the total protein, crude fat, ash, and total dietary fiber contents of UFG, HEB, and FG. Protein and crude fat content are used as indicators for evaluating the edible quality of ginseng fermented products. Ash in food refers to the inorganic matter remaining after high-temperature burning, primarily inorganic salts and their oxides. Dietary fiber is a class of substances that cannot be digested and absorbed by the human small intestine and includes polysaccharides, oligosaccharides, lignin, cellulose, and hemicellulose. Compared with UFG, the protein and crude fat contents of FG increased by 42.46% and 22.63%, respectively. The ash and total dietary fiber content of HEB were higher than those of FG, and higher than those of UFG. Compared with UFG, the ash content of FG increased by 33.33% and the total dietary fiber content increased by 24.16%. The increase in ash and total dietary fiber content in FG may be due to the incorporation of nutrients such as inorganic salts by Hericium erinaceus during its growth.

[0221] Table 8. Content of main components

[0222]

[0223] Note: All values are expressed as Mean ± SE. Tukey's multiple-test was used. Different letters correspond to significant differences (p < 0.05) associated with different treatments. ND indicates not detected according to the detection limit of each substance.

[0224] 3. Conclusion

[0225] Studies have shown that fermentation using medicinal fungi as fermentation strains and traditional Chinese herbal medicines as medicinal matrices can lead to varying degrees of changes in the nutritional composition and content of these herbs. Currently, in-depth and detailed research on fungal fermentation of ginseng is lacking. The results of this study demonstrated that solid-state fermentation of ginseng using Hericium erinaceus as the fermentation strain resulted in significant changes in the content of various components in the fermented products. Compared with untreated ginseng (UFG), the FAA content in FG increased after 30 days of fermentation (5.37 to 7.61 g / 100 g), and the proportion of EAA increased (33.35 to 36.01%). The content of USFAs (USFAs) significantly increased after fermentation (1511.52 to 1800.37 g / 100 g). Furthermore, significant changes were observed in the contents of total sugars, reducing sugars, protein, crude fat, dietary fiber, ash, minerals, and VFCs in ginseng. Based on this, we believe that compared with fresh ginseng, FG fermented with Hericium erinaceus for 30 days has higher medicinal value and research significance. It can be used as a key raw material for developing functional foods for the human body. Research on the biological activity of FG should be carried out to promote the development of health foods and medical products.

[0226] Example 3 Study on the preparation process of Hericium erinaceus-ginseng bidirectional solid fermentation fungal granules

[0227] Traditional Chinese medicine granules, the product of research and evolution of various processing techniques for traditional Chinese medicine slices, are portable and highly effective traditional Chinese medicine preparations. Compared to traditional Chinese medicine decoctions, granules retain the efficacy of the original formula while being convenient to store and carry with you. The dosage can be adjusted according to the severity of symptoms without the need for decoction. The manufacturing process for granules is simpler and more controllable, making them suitable for large-scale factory production. Therefore, in this experiment, granules were selected as the preparation form for the Hericium erinaceus-ginseng bidirectional fermentation microbial culture, and the granule forming process was studied using wet granulation.

[0228] 1 Experimental Materials

[0229] 1.1 Source of raw materials

[0230] Chapter 2 Experimental results of Hericium erinaceus-ginseng bidirectional solid fermentation 40dFG.

[0231] 1.2 Experimental Reagents

[0232] Table 9. Experimental reagents

[0233]

[0234] 1.3 Experimental instruments

[0235] Table 10. Experimental instruments

[0236]

[0237] 2 Experimental methods

[0238] 2.1 Extraction process optimization

[0239] This experiment used a water extraction method to produce the extract. Preliminary investigations revealed that the liquid-to-solid ratio, extraction time, extraction temperature, and number of extractions were the primary factors influencing the extraction effect. The extraction process was optimized using the extract yield and polysaccharide content as indicators.

[0240] 2.1.1 Evaluation index determination

[0241] (1) Determination of extract yield

[0242] Calculate the extract yield (X) according to formula (3-1):

[0243]

[0244] Note: m1: extract mass, mg; m2: Hericium erinaceus-ginseng fungus mass, mg.

[0245] (2) Determination of polysaccharide content in extract

[0246] Accurately weigh an appropriate amount of anhydrous glucose and prepare a glucose solution with purified water to a concentration of 0.1 mg / mL. Add 0, 20, 40, 60, 80, and 100 μL of glucose solution to 1.5 mL centrifuge tubes, respectively, and make up to 100 μL with purified water. Add 100 μL of 5% phenol to each centrifuge tube, followed by 500 μL of sulfuric acid, mix thoroughly, and place in a boiling water bath for 5 minutes. Cool to room temperature and measure the OD value at 490 nm using a microplate reader to create a standard curve. Dissolve an appropriate amount of extract in water to a concentration of 0.0625 mg / mL. Add 100 μL of the extract solution to each centrifuge tube. Measure the OD value as described above and calculate the polysaccharide content in the extract based on the standard curve.

[0247] 2.1.2 Single-factor experiment

[0248] Based on preliminary experiments, 500 mg of FG was precisely weighed and fixed at a liquid-to-solid ratio of 20:1, an extraction temperature of 80°C, and an extraction time of 3 hours. The effects of these factors, including liquid-to-solid ratio, extraction temperature, and extraction time, on extract yield and polysaccharide content were investigated. The fungus was dissolved at liquid-to-solid ratios of 10:1, 15:1, 20:1, 25:1, and 30:1. The solution was placed in a water bath and extracted at 60, 70, 80, 90, and 100°C for 2, 2.5, 3, 3.5, and 4 hours, respectively. The filtrate was filtered and concentrated to an extract relative density of 1.2. The extract was weighed, and the extract yield and polysaccharide content were calculated.

[0249] 2.2 Granulation process optimization

[0250] Starch and sucrose were selected as excipients for Hericium erinaceus-ginseng granules. Preliminary experiments revealed that the excipient ratio, the amount and concentration of the wetting agent ethanol, and the drying temperature significantly impacted the granulation quality. Therefore, granule formation rate was selected as a screening criterion, and these factors were optimized.

[0251] 2.2.1 Evaluation index determination

[0252] (1) Determination of particle forming rate

[0253] The particle forming rate is determined according to the double screening method of the second method in General Chapter 0982 of the Chinese Pharmacopoeia (2020 edition). The dried particles are weighed and passed through a 10-mesh sieve and an 80-mesh sieve in turn. The particles that can pass through the 10-mesh sieve and cannot pass through the 80-mesh sieve are considered qualified particles. The qualified particles are weighed and the particle forming rate is calculated. If the particle forming rate is less than 85%, the particle size of the particles produced by this method is unqualified. The particle qualification rate (X) is calculated according to formula (3-2):

[0254]

[0255] Note: m1: mass of qualified particles, g; m2: mass of particles before sieving, g.

[0256] (2) Determination of particle bulk density

[0257] Take an appropriate amount of qualified particles and weigh them, place them in a dry measuring cylinder, shake the cylinder gently, read the cylinder scale, and calculate the bulk density according to formula (3-3):

[0258]

[0259] Note: X: bulk density of particles, unit is g / mL; m: mass of particles, g; V: middle scale of particle measuring cylinder, mL.

[0260] (3) Determination of particle repose angle

[0261] The fixed funnel method is used to measure the angle of repose. Three triangular funnels are connected in series, the bottom of the bottom funnel is ground flat, and a coordinate paper is placed 1.5 cm horizontally from the bottom of the funnel. The granules after being granulated are poured into the top funnel along the funnel wall until the tip of the cone formed by the granules on the coordinate paper touches the bottom of the funnel. The radius of the cone base (R) is read on the coordinate paper, and the angle of repose is calculated (tanα=H / R). The smaller the angle of repose, the better the fluidity of the granules. It is generally believed that when α≤40°, the granules can meet the fluidity requirements of the production process.

[0262] (4) Determination of particle moisture absorption rate

[0263] The hygroscopicity of the particles was determined according to the Guidelines for Hygroscopicity Tests of Drugs in Appendix 9103, Part IV of the Chinese Pharmacopoeia (2020 Edition). Supersaturated sodium chloride was added to the bottom of a sealed container and allowed to stand for 24 hours, maintaining the relative humidity inside the container at 75%. The granules were placed in the container after granulation and weighed after 24 hours. The hygroscopicity of the particles (X) was calculated according to formula (3-4):

[0264]

[0265] Note: m1: mass of particles before moisture absorption, g; m2: mass of particles after moisture absorption, g.

[0266] (5) Comprehensive score

[0267] References (Wang Xiaoxin, He Ke, Wu Yongmei, et al. Research on the molding process of Shangke Jiedu Granules [J]. Journal of Traditional Chinese Medicine, 2020, 26(14): 17-19.) A comprehensive scoring method with a percentage system was used to evaluate the overall quality of the granules, of which the molding rate accounted for 40 points, the bulk density, angle of repose, and moisture absorption rate each accounted for 20 points, and the comprehensive scoring value = (40×molding rate value) / maximum molding rate + (20×bulk density value) / maximum bulk density + (20×minimum angle of repose) / angle of repose value + (20×minimum moisture absorption rate) / moisture absorption rate value.

[0268] 2.2.2 Single-factor experiment

[0269] Based on preliminary experiments, granules were prepared using a fixed dry extract to sucrose ratio of 1:1.4, a wetting agent ethanol concentration of 75%, an ethanol dosage of 10%, a drying temperature of 60°C, and a drying time of 0.5 h. The effects of excipient dosage, wetting agent dosage and concentration, and drying temperature on granule morphology and yield were investigated. Using a wet granulation method, an appropriate amount of extract was precisely weighed and a soft material was prepared using a dry extract to starch ratio of 1:2, a dry extract to sucrose ratio of 1:1, 1:1.2, 1:1.4, 1:1.6, and 1:1.8, wetting agent ethanol concentrations of 55, 65, 75, 85, and 95%, and ethanol dosages of 6, 8, 10, 12, and 14%. The granules formed a mass when grasped but fell apart when touched. The granules were then passed through a 14-mesh sieve and dried at 40, 50, 60, 70, and 80°C for 0.5 h. The dried granules were sieved through a 10-mesh sieve and then an 80-mesh sieve to evaluate the granule morphology and calculate the granule forming rate.

[0270] 2.2.3 Orthogonal experimental level design

[0271] Based on the results of the single-factor experiment, the mass ratio of dry extract to sucrose, wetting agent ethanol concentration, ethanol dosage, and drying temperature were selected as the factors to be investigated, and a three-factor, three-level orthogonal experiment was established (Table 11). The granule production process was optimized through comprehensive scoring.

[0272] Table 11. Orthogonal experimental factor levels

[0273]

[0274] 2.3 Validation Experiment and Quality Inspection

[0275] Three batches of granules were prepared according to the optimal process conditions, and comprehensive scores were given for each batch, and the following quality checks were performed.

[0276] Property inspection: The prepared granules should be dry, non-sticky, with uniform particle size and color, and without softening or moisture absorption.

[0277] Particle size determination: according to “(1) Determination of particle size ratio”.

[0278] Moisture Determination: Refer to General Chapter 0832, "Determination of Moisture by Drying Method," in the 2020 edition of the Chinese Pharmacopoeia. Take 1g of whole granules and spread them flat on a flat weighing bottle that has been dried to constant weight, with a thickness not exceeding 5mm. Accurately weigh the granules. Remove the cap from the weighing bottle and dry in an oven at 100-105°C for 5 hours. Cover the bottle, place in a desiccator, cool, and then accurately weigh. Granules with a moisture content of no more than 8% are considered acceptable.

[0279] Loss on Drying: Refer to General Chapter 0831 "Loss on Drying Method" in the Chinese Pharmacopoeia (2020 Edition). Take 5g of the granules after granulation and dry them under reduced pressure at 80°C to constant weight. Granules with a weight loss of no more than 2.0% are considered acceptable.

[0280] Solubility test: Refer to General Chapter 0104, "Soluble Granules Test Method," in the 2020 edition of the Chinese Pharmacopoeia. Take 10g of the prepared granules, add 200mL of hot water, and stir. Measure the time required for all granules to dissolve. If all granules do not dissolve within 5 minutes, the granules fail the solubility test.

[0281] 2.4 Data Processing

[0282] The experiment was repeated three times, and the orthogonal experimental data were statistically analyzed using SPSS25.0 software, and the graphs were drawn using GraphPad Prism 8.4.8 software.

[0283] 3 Results and Analysis

[0284] 3.1 Extraction process optimization experimental results

[0285] 3.1.1 Liquid-to-solid ratio screening

[0286] Depend on Figure 8 It can be seen that the use of different liquid-to-material ratios for hot water extraction of Hericium erinaceus-ginseng fungi will have a great impact on the extract yield. Under different liquid-to-material ratios, the change range of the polysaccharide content of the obtained extract is small, and the maximum does not exceed 5%, while the extract yield has obvious changes. Compared with the liquid-to-material ratio of 10:1, the extract yield increased by 15.28% when the liquid-to-material ratio was 25:1; the extract yield when the liquid-to-material ratio was 30:1 decreased by 1.01% compared with the liquid-to-material ratio of 15:1. Figure 8 It can be seen that the peak values of extract yield and polysaccharide content both appear when the liquid-to-solid ratio is 25:1, so 25:1 is selected as the optimal extraction liquid-to-solid ratio.

[0287] 3.1.2 Extraction time screening

[0288] Depend on Figure 9 Extraction time significantly impacted the polysaccharide content of the extract. The lowest polysaccharide content, at 56.49%, was achieved at a 2-hour extraction time. Compared to 2 hours, the polysaccharide content increased by 21.54% at 2.5 hours. The polysaccharide content then decreased with increasing extraction time, reaching its lowest point at 62.82% at 4 hours. The highest extract yield, at 64.33%, was achieved at 2.5 hours, a 2.65% increase compared to 2 hours. The lowest extract yield, at 59.33%, was achieved at 3 hours. Both the extract yield and polysaccharide content reached their highest levels at 2.5 hours, making 2.5 hours the optimal extraction time.

[0289] 3.1.3 Extraction temperature screening

[0290] like Figure 10 As shown, different extraction temperatures significantly affect both extract yield and polysaccharide content. The extract yield decreases with increasing extraction temperature, reaching a maximum of 73.23% at 60°C and a minimum of 67.33% at 100°C. Within the extraction temperature range of 60-90°C, the polysaccharide content of the extract increases with the extraction temperature. The polysaccharide content reaches its highest level at 60°C, reaching 73.23%, while the yield drops to its lowest level at 90°C, reaching 64.72%. Compared to 90°C, the extract yield at 100°C increases by 6.90%. Both the extract yield and polysaccharide content reach their highest levels at 60°C, making 60°C the optimal extraction temperature.

[0291] 3.2 Granulation process optimization experimental results

[0292] 3.2.1 Single-factor experimental results

[0293] (1) Screening of auxiliary material addition amount

[0294] Excipients, also known as fillers, can increase granule production and improve granule formation efficiency and uniformity. In this experiment, starch and sucrose were selected as excipients. A fixed dry extract to starch mass ratio of 1:1 was used to examine the effect of sucrose addition on granulation difficulty, uniformity, and formation efficiency. The results are shown in Table 12. The results show that with increasing sucrose addition, the soft material gradually became easier to sieve, uniformity improved, and the granule formation efficiency initially increased and then decreased. This suggests that moderate increases in sucrose addition can improve granule uniformity and formation efficiency, but excessive sucrose addition can result in looser, more brittle granules, which in turn reduces formation efficiency. When the dry extract to sucrose mass ratio was 1:1 or 1:1.8, the granule formation efficiency was less than 85%, indicating substandard granules. Therefore, dry extract to sucrose mass ratios of 1:1.2, 1:1.4, and 1:1.6 were selected as the three levels for subsequent orthogonal screening.

[0295] Table 12. Screening of excipient addition amounts

[0296]

[0297] (2) Screening of wetting agent dosage

[0298] A wetting agent is a non-sticky liquid that imparts viscosity to raw materials by wetting them. Wetting agents can help soft materials adhere, facilitating granule production. In this experiment, ethanol was used as the wetting agent at a fixed volume fraction of 75%. The effects of ethanol dosage on granulation difficulty, uniformity, and granule formation efficiency were investigated. The results are shown in Table 13. The results show that increasing the wetting agent dosage improves granule uniformity. However, at a wetting agent dosage of 14%, the granules tend to stick to the screen due to the high viscosity of the soft material. At a wetting agent dosage of 6%, the granule formation efficiency was less than 85%, resulting in unqualified granules. At a wetting agent dosage of 12%, the granule formation efficiency reached its highest level, reaching 91.59%. Compared to a wetting agent dosage of 12%, the granule formation efficiency at a wetting agent dosage of 14% decreased by 2.82%. Taking into account production practices and granule quality, 10%, 12%, and 14% were selected as the three levels for subsequent orthogonal screening.

[0299] Table 13. Screening of wetting agent dosage

[0300]

[0301]

[0302] (3) Wetting agent concentration screening

[0303] Ethanol was used as a wetting agent at a fixed wetting agent dosage of 10%. The effects of wetting agent concentration on granulation difficulty, granule morphology, and granule formation efficiency were investigated. The results are shown in Table 14. The results show that granule uniformity and granule formation efficiency decreased with increasing ethanol concentration. When the ethanol concentration exceeded 85%, granule size became uneven, and the granule formation efficiency was less than 85%. When the ethanol concentration was within the range of 55-75%, the soft material easily passed through the sieve, granule morphology was uniform, and granule formation efficiency exceeded 85%, reaching a maximum of 92.72%. Therefore, wetting agent concentrations of 55%, 65%, and 75% were selected as the three levels for subsequent orthogonal screening.

[0304] Table 14. Screening of wetting agent concentrations

[0305]

[0306] (4) Drying temperature screening

[0307] Table 15 shows that the particle formation rate decreases significantly within the 40-80°C drying temperature range. The highest particle formation rate, 93.77%, is achieved at a drying temperature of 40°C. As the drying temperature increases, the formation rate decreases, reaching its lowest point at 80°C, at 82.46%, resulting in unqualified particles. Particles tend to clump easily at drying temperatures between 70 and 80°C, likely due to excessively high drying temperatures and rapid water loss. Considering both particle morphology and formation rate, 40°C, 50°C, and 60°C were selected as the three levels for subsequent orthogonal screening.

[0308] Table 15. Screening of drying temperature

[0309]

[0310] 3.2.2 Orthogonal Experiment Results

[0311] As shown in Table 11, the effects of excipient ratio, wetting agent concentration and dosage, and drying temperature on the comprehensive score of mycoplasma granules were investigated to identify the optimal granulation process. The results are shown in Table 16. Range and variance analyses were performed using SPSS 25.0 software, and the results are shown in Tables 17 and 18. Table 17 shows that, among all factors, the order of influence on the comprehensive score of the granules is A > C > B > D. Specifically, the extract:sucrose ratio has the greatest impact on the comprehensive score, followed by ethanol concentration and ethanol dosage, and drying temperature has the least impact. Table 18 shows that, with the exception of the extract:sucrose ratio, the remaining factors have no significant effect on the comprehensive score. Since the evaluation index selected in this experiment is the comprehensive score of particles, the larger the value, the higher the quality of the particles. Therefore, by comparing the Kavg values in Table 6, the optimal process combination is A2B2C3D2, that is, when the granulation process is extract: sucrose = 1:1.4, the ethanol dosage is 12%, the ethanol concentration is 75%, and the drying temperature is 50°C, the granules score the highest.

[0312] Table 16. Orthogonal experiment results

[0313]

[0314]

[0315] Table 17. Range analysis

[0316]

[0317] Table 18. Analysis of variance

[0318]

[0319] Note: *P<0.05, **P<0.01.

[0320] 3.3 Validation experiments and quality checks

[0321] Three groups of verification experiments were carried out under the optimal granulation conditions of extract: sucrose = 1:1.4, ethanol dosage of 12%, ethanol concentration of 75%, and drying temperature of 50°C. The results are shown in Table 19. The comprehensive score of the obtained granules was improved compared with the highest score of the orthogonal experiment, indicating that the process conditions of the granules screened in this experiment are stable and feasible. The granules prepared under the optimal granulation conditions were quality checked, and the results are shown in Table 20. The three groups of granules prepared under the optimal granulation conditions have been dried, and the granules are yellow-brown, without moisture absorption, softening, agglomeration, deliquescence and other phenomena, meeting the properties requirements of granules. The moisture content, drying loss and solubility of the granules are in compliance with the specifications of the "Chinese Pharmacopoeia" (2020). The Hericium erinaceus-ginseng bidirectional fermentation fungal granules prepared by the above-mentioned optimized process are as follows Figure 11 shown.

[0322] Table 19. Verification experiment results

[0323]

[0324] Table 20. Quality inspection results

[0325]

[0326] 4 Discussions

[0327] As Traditional Chinese Medicine (TCM) is increasingly trusted and chosen by the public, and as formulation technology evolves, TCM granules are emerging as a promising option due to their portability, ease of storage, and the absence of decoction. However, traditional granules are often made directly from crushed TCM slices. While this method is simple and rapid, it also presents challenges such as high material consumption, low solubility, and low human absorption. This study screened a hot water extraction process for ginseng-Hericium erinaceus mycelium, maximizing the extraction of water-soluble active ingredients while ensuring dry extract yield. Dry extract yield is fundamental to the efficacy of TCM granules. This study selected dry extract yield and polysaccharide content as evaluation indicators for extraction process optimization, optimizing the liquid-to-solid ratio, extraction time, and extraction temperature. Ultimately, a liquid-to-solid ratio of 25:1, an extraction time of 2.5 h, and an extraction temperature of 60°C were selected as the optimal extraction conditions for subsequent experiments.

[0328] Dry extracts obtained from traditional Chinese medicines (TCMs) are generally highly hygroscopic and have high viscosity, making granulation difficult. To reduce this difficulty while ensuring the resulting granules have a good forming rate and solubility, it is important to select the appropriate types and amounts of excipients and wetting agents. The appropriate amount of excipients can reduce the viscosity of the dry extract and increase its fluidity, facilitating granule formation and improving content uniformity. During the granule preparation process, a wetting agent is also added to facilitate granulation. Wetting agents themselves are not sticky and primarily serve to wet the extract and excipients. Water and ethanol are commonly used in granulation. Since the extract used in this experiment is made from TCM, which is too viscous when exposed to water, making granulation difficult, ethanol was chosen as the wetting agent. The concentration and amount of the wetting agent, ethanol, are crucial, as they directly affect the hardness of the resulting soft material. In this experiment, the molding rate, bulk density, angle of repose and moisture absorption rate were used as evaluation indicators to comprehensively score the granules. The effects of the ratio of extract to sucrose, the amount and concentration of ethanol, and the drying temperature on the quality of the granules were investigated. The optimal granulation process for the granules was selected as follows: extract to sucrose = 1:1.4, ethanol amount 12%, ethanol concentration 75%, and drying temperature 50℃.

[0329] 5. Summary

[0330] In this experiment, Hericium erinaceus-ginseng fermented mycelium was selected as the raw material, and appropriate amounts of starch, sucrose and other auxiliary materials were added to develop granules, in order to screen out the optimal granulation process for bidirectional fermented mycelium granules. The extract yield and extract polysaccharide content were used to optimize the mycelium water extraction process in a single-factor experiment to obtain the optimal liquid-to-material ratio, time and temperature for extraction. Based on the results of the extraction process optimization, the comprehensive score of the granules was used as the evaluation standard, and the extract: sucrose ratio, ethanol dosage and concentration, and drying temperature were used as influencing factors for an orthogonal experiment to obtain the optimal granulation process for the granules. The granules prepared under these conditions are uniform in size, have a high molding rate, are odorless, and their moisture content, drying loss, and solubility are in compliance with the standards of the "Chinese Pharmacopoeia" (2020). Through the experiments in this chapter, a theoretical basis is provided for the secondary development of bidirectional fermented mycelium, and a technical reference is provided for promoting the deep processing of Hericium erinaceus-ginseng bidirectional fermented products.

[0331] Example 4 Study on the activity of the Hericium erinaceus-ginseng bidirectional solid fermentation microbial products and granules

[0332] Wei Le Xin Granules are a traditional Chinese medicine preparation composed of Hericium erinaceus extract and other substances. They have the effect of nourishing the stomach and regulating the stomach. They are currently used to relieve symptoms such as abdominal pain, acid reflux, and belching in patients with gastric ulcers, reducing physical pain. Numerous studies have shown that the various active ingredients in Hericium erinaceus and ginseng have excellent antioxidant properties. To further explore the bioactivity and drug efficacy of bidirectional fermentation products and products, this experiment aims to compare the antioxidant activity of Hericium erinaceus-ginseng bidirectional fermentation products, Wei Le Xin Granules, and Hericium erinaceus-ginseng bidirectional solid fermentation microbial granules, laying a theoretical foundation for further research and application of fermentation products and microbial granules.

[0333] 1 Experimental Materials

[0334] 1.1 Sample Source

[0335] Hericium erinaceus-ginseng bidirectional solid fermentation fungal granules (JZ) were prepared according to the optimal granulation process screened in Chapter 3.

[0336] 1.2 Experimental Reagents

[0337] Table 21. Experimental reagents

[0338]

[0339] 1.3 Experimental instruments

[0340] Table 22. Experimental instruments

[0341]

[0342] 2 Experimental methods

[0343] 2.1 Sample pretreatment

[0344] Take 5g of UFG, FG and HEB dried samples respectively, dissolve them in sterile water at a solid-liquid ratio of 1:20, shake well and ultrasonicate at a power of 100W for 2h, cool to room temperature, filter, and ultrasonicate the residue again under the above conditions. Combine the filtrate, concentrate the filtrate under reduced pressure to 1 / 4 of the original volume, freeze-dry the concentrate, and seal and dry the freeze-dried powder for storage.

[0345] 2.2 DPPH free radical scavenging rate determination

[0346] DPPH is a relatively stable free radical that appears purple in organic solvents. When it comes into contact with a substance with free radical scavenging ability, the purple color in the solution fades because the DPPH free radical accepts electrons. The DPPH free radical scavenging ability of different substances can be judged according to the absorbance value at the maximum ultraviolet absorption point of the DPPH solution. According to the method in the reference (Zheng Weixiong, Xie Tinghui, Wu Jinneng, et al. Study on the chemical composition and cytotoxic activity of Corydalis umbellata [J]. Shandong Chemical Industry, 2021, 50(21): 27-29.), the DPPH free radical scavenging rate was determined. Appropriate amounts of UFG, FG, HEB freeze-dried samples and WLX, JZ were weighed and dissolved in sterile water to prepare sample solutions with concentrations of 0.5, 1, 1.5, 2, 2.5, and 3 mg / mL. 0.2 mL of sample solution was mixed with an equal volume of 2.6×10 -4 Mix the 10 mol / L DPPH ethanol solution evenly, react in the dark at room temperature for 5 minutes, and measure the absorbance at 517 nm with a microplate reader. Ascorbic acid (Vc) was used as a positive control instead of the sample. The DPPH free radical scavenging rate of each sample was calculated according to formula (4-1):

[0347]

[0348] Note: A X is the sample absorbance, A X0 The absorbance was measured by using anhydrous ethanol instead of DPPH ethanol solution, and A0 was measured by using sterile water instead of the sample.

[0349] 2.3 OH clearance rate determination

[0350] OH radicals react with salicylic acid to form 2,3-dihydroxybenzoic acid, which has a maximum absorption wavelength at 510 nm. Substances with antioxidant properties can inhibit this reaction. The OH radical scavenging ability of the samples was determined according to the method described in the literature (Mohanan A, Nickerson MT, Ghosh S. Oxidative stability of flaxseed oil: effect of hydrophilic, hydrophobic, and intermediate polarity antioxidants [J]. Food Chemistry, 2018, 266, 524-533). Equal volumes of sample solution, 9 mmol / L salicylic acid-ethanol solution, 9 mmol / L FeSO₄, and 9 mmol / L H₂O₂ were mixed and incubated in a 37°C water bath for 30 minutes. After cooling to room temperature, the absorbance at 510 nm was measured. Ascorbic acid (Vc) was used as a positive control in place of the sample. The OH radical scavenging rate of each sample was calculated according to formula (4-2):

[0351]

[0352] Note: A i is the sample absorbance, A j It is the absorbance measured without adding the color developing agent H2O2, and A0 is the absorbance measured when sterile water is used instead of the sample.

[0353] 2.4ABTS + Free radical scavenging rate assay

[0354] ABTS can be oxidized by oxidants to produce turquoise ABTS + , substances with antioxidant capacity can inhibit this oxidation reaction, and the ABTS of the substance can be judged by the color of the product + The free radical scavenging ability is strong or weak. The sample ABTS was determined according to the method in the reference (Yang FR, Chen JL, Ye SH, et al. Characterization of antioxidant activity of exopolysacchrides from endophytic Lysinibacillus sphaericus Ya6 under osmotic stress conditions [J]. Process Biochemistry, 2022, 113: 87-96.) + Free radical scavenging ability. Take 10mL of ABTS ethanol solution with a concentration of 7mmol / L and mix it with an equal volume of K2S2O8 solution with a concentration of 2.4mmol / L, and let it stand at room temperature in the dark for 48h to obtain ABTS mother solution. Take 5mL of ABTS mother solution and dilute it to 100mL with anhydrous ethanol to obtain ABTS working solution. Take 0.2mL of sample solution with different concentrations and mix it with an equal volume of ABTS working solution, let it stand at room temperature in the dark for 6min, and immediately measure the absorbance value at 734nm with an enzyme reader. Ascorbic acid (Vc) is used instead of the sample as a positive control. Calculate the ABTS of each sample according to formula (4-3) + Clearance rate:

[0355]

[0356] Note: A s is the sample absorbance, A d Absorbance was measured by replacing ABTS working solution with anhydrous ethanol, A a The absorbance was measured using sterile water instead of the sample.

[0357] 2.5 Data Processing

[0358] GraphpadPrism 8.4.8 was used to draw the line graph, and SPSS25.0 was used to analyze the data by one-way ANOVA test. P < 0.05 indicated that the difference was statistically significant.

[0359] 3 Experimental results

[0360] 3.1 DPPH free radical scavenging rate determination results

[0361] The scavenging ability of UFG, FG, HEB, WLX and JZ on DPPH free radicals was determined. The results are as follows Figure 12 As shown in the figure, the DPPH free radical scavenging ability of each sample was ranked from high to low as Vc > HEB > FG > WLX > JZ > UFG. The DPPH free radical scavenging ability of HEB was similar to that of Vc, with both maintaining scavenging abilities above 93%. The DPPH free radical scavenging abilities of JZ and UFG increased with increasing concentration, showing a positive correlation between concentration and scavenging rate. Both scavenging rates reached their highest values at 3 mg / mL, reaching 72.67% and 57.98%, respectively. The scavenging rates of FG and WLX did not change significantly with increasing concentration (P < 0.05). Compared with UFG, the DPPH free radical scavenging ability of FG at all concentrations was significantly increased, reaching its highest values at 3 mg / mL, with scavenging rates of 57.98% and 90.21%, respectively. The difference in DPPH free radical scavenging ability between WLX and JZ gradually decreased with increasing concentration, reaching a 36.24% difference at 0.5 mg / mL and a 3.67% difference at 3 mg / mL. All samples had a DPPH free radical scavenging rate greater than 29% within the 0.5-3 mg / mL concentration range.

[0362] 3.2 OH radical scavenging rate determination results

[0363] The scavenging ability of UFG, FG, HEB, WLX and JZ on OH free radicals was determined. The results are as follows Figure 13As shown in the figure. At a sample concentration of 3 mg / mL, the OH radical scavenging rate ranked from high to low as Vc > HEB > UFG > WLX > FG > JZ. The hydroxyl radical scavenging rate of Vc increased significantly in the 0.5-1.5 mg / mL concentration range (P < 0.05), remaining stable at 1.5-3 mg / mL, with a minimum scavenging rate of 70.99% and a maximum of 99.60%. The OH radical scavenging capacity of HEB and FG increased with increasing concentration, showing a positive correlation between concentration and scavenging rate. Both scavenging rates reached their highest values at 3 mg / mL, at 95.36% and 68.66%, respectively. There was no significant difference in the OH radical scavenging effect of FG and UFG across all concentrations (P > 0.05), indicating that fermentation has no significant effect on the OH radical scavenging capacity of the ginseng medicinal matrix (P > 0.05). The scavenging rates of WLX and JZ for ·OH radicals showed little fluctuation with increasing concentration. WLX achieved the highest scavenging rate of 69.97% at 3 mg / mL, while JZ achieved the highest scavenging rate of 65.69% at 1 mg / mL. All samples maintained scavenging rates of >43% for ·OH radicals within the 0.5-3 mg / mL concentration range.

[0364] 3.3ABTS + Free radical scavenging rate determination results

[0365] Determination of the effects of UFG, FG, HEB, WLX and JZ on ABTS + Free radical scavenging ability, the results are as follows Figure 14 As shown. Vc to ABTS + The free radical scavenging ability did not change significantly in the concentration range of 0.5-3 mg / mL (P>0.05), and the scavenging rate remained above 98%. When the concentration of HEB increased from 0.5 mg / mL to 1.5 mg / mL, its scavenging ability to ABTS + The free radical scavenging ability was significantly enhanced (P<0.05), and the scavenging rate increased by 65.31%. When the concentration of HEB was 3 mg / mL, the scavenging rate of ABTS was significantly increased. + The free radical scavenging rate was the highest, at 96.47%, which was close to that of Vc. With the increase of concentration, WLX and JZ had a negative effect on ABTS. + The free radical scavenging rate does not fluctuate much, and the scavenging ability is similar. + The clearance rate remained at 61%-65% in the concentration range of 0.5-3 mg / mL. + The free radical scavenging ability increased with the increase of concentration, and the concentration and scavenging rate showed a positive correlation trend. The scavenging rates of both reached the highest at a concentration of 3 mg / mL, which were 63.11% and 61.02% respectively.

[0366] 4 Discussions

[0367] When the oxidative reaction in the human body exceeds the threshold of its own antioxidant capacity, it will induce oxidative stress, leading to the proliferation of oxygen free radicals, causing cell apoptosis and DNA damage, and inducing the occurrence of various diseases. + Comparison of their free radical scavenging abilities revealed that at a sample concentration of 3 mg / mL, HEB exhibited the strongest inhibitory activity against all three free radicals. This phenomenon may be due to the rich flavonoids and polyphenols in HEB. Furthermore, FG exhibited a significantly higher DPPH free radical scavenging rate than UFG, indicating that the bidirectional fermentation process enhances the antioxidant capacity of the ginseng matrix. This is presumably due to the degradation or conversion of the ginseng matrix into antioxidant substances that were not previously present during the growth of Hericium erinaceus. These substances may possess potent DPPH free radical scavenging activity. At a sample concentration of 3 mg / mL, there was no significant difference in the antioxidant activity of WLX and JZ, and both achieved scavenging rates exceeding 50% for all three free radicals, demonstrating that the Hericium erinaceus-ginseng bidirectional fermentation granules possess considerable antioxidant activity.

[0368] 5. Summary

[0369] Comparative studies of the antioxidant activity and hypoglycemic capacity of Hericium erinaceus-ginseng bidirectional fermentation products and their products with those of Wei Le Xin Granules revealed that the antioxidant activity of the medicinal matrix was significantly increased after fermentation, and the antioxidant activity of the fungal granules was comparable to that of Wei Le Xin Granules. We hope that the preliminary results of this chapter will lay the foundation for further research on Hericium erinaceus-ginseng bidirectional fermentation and for the production, development, and application of these products.

[0370] Example 5 Effects of Hericium erinaceus-Ginseng Bidirectional Solid Fermentation Products and Granule Products on Ethanol-Injured GES-1 Cells

[0371] Excessive drinking in daily life has led to an increasing number of patients with gastric mucosal damage diseases. After drinking a lot of alcohol, the human body synthesizes and secretes a large number of reactive oxygen species (ROS). ROS can attack basic components of cells such as nucleic acids and proteins through oxidative stress reactions inside cells, inducing the production of toxic substances such as oxygen free radicals. Gastric mucosal damage diseases caused by heavy drinking mainly include gastric ulcers, gastritis, gastric bleeding, etc. This chapter uses GES-1 cells as the research object to explore the proliferation and protective effects of ginseng, Hericium erinaceus-ginseng bidirectional fermentation microplasm, microbial buds, and microbial granules on cells, providing a theoretical basis for the development of food and pharmaceutical products derived from bidirectional fermentation products.

[0372] 1 Experimental Materials

[0373] 1.1 Experimental Reagents

[0374] Table 23. Experimental reagents

[0375]

[0376] Fetal bovine serum Zhejiang Tianhang Biotechnology Co., Ltd.

[0377] CCK-8 Detection Kit Shanghai Biyuntian Biotechnology Co., Ltd.

[0378] 1.2 Experimental instruments

[0379] Table 24. Experimental instruments

[0380]

[0381] 2 Experimental methods

[0382] 2.1 Sample pretreatment

[0383] Take 5g of UFG, FG, and HEB powder and dissolve them in sterile water at a liquid-to-solid ratio of 25:1. After oscillation, ultrasonic extraction is performed for 2h and centrifugation is performed at 8000rpm for 10min. The supernatant is collected and concentrated to 1 / 4 of the original volume under reduced pressure in a rotary evaporator. The concentrate is placed at -80℃ overnight and freeze-dried in a vacuum freeze dryer for 48h to obtain UFG, FG, and HEB freeze-dried powders, which are stored in a desiccator.

[0384] 2.5 Effects of drug administration on the survival rate of ethanol-damaged GES-1 cells

[0385] (1) Cell inoculation and culture methods are the same as 2.2;

[0386] (2) The cells were divided into blank group, control group, model group, UFG, FG, HEB low (10 μg / mL), medium (100 μg / mL), high (600 μg / mL) concentration groups, WLX, JZ low (100 μg / mL), medium (400 μg / mL), high (1600 μg / mL) concentration groups, with 6 replicates in each group. The normal group was added with 100 μL of fresh complete medium, and the model group, UFG, FG, HEB, WLX, JZ low, medium, and high concentration groups were all added with 2 mL of complete medium containing 3% anhydrous ethanol and cultured for 5 h;

[0387] (3) After culturing to the corresponding time point, the original culture medium was discarded, and 100 μL of fresh complete culture medium was added to the normal group and model group. 2 mL of drug-containing culture medium of the corresponding concentration was added to the low, medium, and high concentration groups of UFG, FG, HEB, WLX, and JZ, and the cells were cultured to the respective time points: 12 h, 36 h, and 60 h.

[0388] (4) Culture to the corresponding time point, add 10 mL of CCK-8 solution to each well, incubate in the incubator for 2 h, and measure the OD of each well with a microplate reader. 450 .

[0389] 2.6 Effects of drug administration on the migration of ethanol-induced GES-1 cells

[0390] (1) Take a 6-well plate and draw three straight lines on the back with a marker to divide each well into 4 fields of view. Then take cells in the logarithmic growth phase and use the corresponding cell culture medium to prepare 1×10 cells per mL. 5 2 mL of cell suspension was added to a 6-well plate and cultured in a cell culture incubator for 24 h.

[0391] (2) After the cells are tightly connected to form a monolayer, use a 200 μL pipette tip to vertically draw a mark in the center of the 6 wells, aspirate the original culture medium, and then add 1 mL of PBS to each well to wash twice to remove non-adherent cells;

[0392] (3) The cells were divided into blank group, control group, model group, UFG, FG, HEB low (10μg / mL), medium (100μg / mL), high (600μg / mL) concentration groups, WLX, JZ low (100μg / mL), medium (400μg / mL), high (1600μg / mL) concentration groups, with 6 replicates in each group. The normal group was added with 2mL of fresh complete medium, and the model group, UFG, FG, HEB, WLX, JZ low, medium, and high concentration groups were all added with 2mL of complete medium containing 3% anhydrous ethanol and cultured for 5 hours. The wound healing status was recorded by taking pictures, and the wound area was measured using Image J.

[0393] (4) After culturing to the corresponding time point, the original culture medium was discarded, and 2 mL of fresh complete culture medium was added to the normal group and model group. 2 mL of drug-containing culture medium of corresponding concentrations was added to the low, medium, and high concentration groups of UFG, FG, HEB, WLX, and JZ. The healing of the scratch wound was measured at 12 and 24 h after administration, and the wound area was measured using Image J.

[0394] 2.7 Statistical analysis

[0395] Experimental data were statistically analyzed using SPSS 25.0. Data are presented as mean ± standard deviation (mean ± SD), and some results are presented in bar graphs. Group differences were compared using one-way analysis of variance (ANOVA) and multiple comparisons between groups using the Tukey HSD test. P < 0.05 was considered statistically significant.

[0396] 3 Experimental results

[0397] Effect of 3.3UFG on GES-1 cell activity

[0398] After GES-1 cells were treated with complete medium containing UFG at different concentrations and for different time periods, the OD values of each treatment were measured. 450 Value Figure 15 shown.

[0399] After 12 hours of UFG treatment, the OD450 values of cells treated with UFG at concentrations of 2400 μg / mL and 4800 μg / mL were significantly increased compared with the control group (P<0.05), indicating that UFG at concentrations of 2400 μg / mL and 4800 μg / mL significantly promoted the growth of GES-1 cells, with cell viability rates of 130.13% and 177.91%, respectively. Treatment with UFG at concentrations between 10 and 1200 μg / mL for 12 hours had no significant promoting or inhibiting effect on cell growth (P>0.05). The lowest cell viability, at 9600 μg / mL, was 93.25%.

[0400] After 24 hours of UFG treatment, UFG concentrations of 300 μg / mL, 1200 μg / mL, 2400 μg / mL, and 4800 μg / mL significantly promoted cell growth (P<0.05), with cell viability rates of 119.22%, 120.43%, 129.90%, and 128.10%, respectively. Other concentrations of UFG had no significant effect on GES-1 cells, with the lowest cell viability at 9600 μg / mL, at 93.25%.

[0401] After 36 hours of UFG treatment, all concentrations of UFG except 25 μg / mL, 4800 μg / mL, and 9600 μg / mL significantly promoted GES-1 cell growth (P < 0.05). The highest cell survival rate, 124.05%, was achieved at 2400 μg / mL. At 9600 μg / mL, UFG significantly inhibited cell proliferation (P < 0.05), reaching a cell inhibition rate of 38.37%.

[0402] When the cells were treated with UFG for 48 hours, when the UFG concentration was in the range of 10-1200 μg / mL, it had no obvious promoting or inhibiting effect on cell growth (P>0.05). When the UFG concentration was in the range of 2400-9600 μg / mL, it had a significant inhibitory effect on the cells (P<0.05). When the UFG concentration was 9600 μg / mL, the inhibitory effect was the strongest, with an inhibition rate of 33.52%.

[0403] After 60 hours of UFG treatment, UFG at concentrations of 10 μg / mL and 25 μg / mL significantly promoted the growth of GES-1 cells (P<0.05). The highest cell survival rate was achieved at 25 μg / mL, reaching 110.18%. UFG at concentrations of 4800-9600 μg / mL had a significant inhibitory effect on GES-1 cells (P<0.05), with the strongest inhibitory effect at 9600 μg / mL, reaching an inhibition rate of 27.41%.

[0404] Effect of 3.4FG on GES-1 cell activity

[0405] After GES-1 cells were treated with complete medium containing FG at different concentrations and for different time periods, the OD values of each treatment were measured. 450 Value Figure 16 shown.

[0406] After 12 hours of FG treatment, except for 9600μg / mL, all other concentrations of FG had significant promoting effects on GES-1 cells (P<0.05). When the FG concentration was 25μg / mL, the cell survival rate was the highest, at 165.55%.

[0407] After 24 hours of FG treatment, except for FG concentrations of 100 μg / mL and 150 μg / mL, which showed a significant promoting effect on GES-1 cells (P < 0.05), the remaining FG concentrations had no significant promoting or inhibiting effect compared with the control group (P > 0.05). The cell survival rate was highest at 100 μg / mL, reaching 137.70%. The cell inhibition rate was highest at 9600 μg / mL, reaching 13.71%.

[0408] After 36 hours of FG treatment, FG concentrations ranging from 1500 to 4800 μg / mL significantly promoted the growth of GES-1 cells (P<0.05). The highest cell survival rate, at 2400 μg / mL, was 133.56%. After 48 hours of FG treatment, except for a significant inhibitory effect at 9600 μg / mL (P<0.05), other FG concentrations had no significant promoting or inhibiting effects on GES-1 cells (P>0.05).

[0409] After 60 hours of FG treatment, FG concentrations of 50 μg / mL and 100 μg / mL significantly promoted the growth of GES-1 cells (P < 0.05), with cell survival rates of 125.12% and 126.42%, respectively. FG concentrations of 4800 μg / mL and 9600 μg / mL significantly inhibited the growth of GES-1 cells (P < 0.05), with cell inhibition rates of 22.63% and 41.15%, respectively. 3.5 Effect of HEB on GES-1 Cell Activity

[0410] After GES-1 cells were treated with HEB complete medium at different concentrations and for different time periods, the OD values of each treatment were measured. 450 Value Figure 17 As shown. Figure 17 It can be seen that when the HEB concentration was 2400-9600 μg / mL, it had a significant inhibitory effect on GES-1 cells from 12 to 60 hours (P < 0.05). When the HEB concentration was 9600 μg / mL, the cell inhibition rate was the highest, reaching 97.80%, when the GES-1 cells were treated for 60 hours. When the cells were cultured under HEB concentration range of 10-600 μg / mL, HEB had no significant promoting or inhibiting effect on cell growth at 12 and 60 hours (P > 0.05). After the cells were treated with HEB for 24 hours, HEB concentrations within the range of 25-600 μg / mL had a significant promoting effect on cell growth (P < 0.05), among which the cell survival rate was the highest at 600 μg / mL, reaching 125.24%. After 36 hours of HEB treatment, HEB significantly promoted cell growth at concentrations ranging from 10 to 300 μg / mL (P<0.05). The highest cell survival rate, 120.07%, was achieved at 100 μg / mL. After 48 hours of HEB treatment, HEB at a concentration of 100 μg / mL significantly promoted GES-1 cell growth (P<0.05), reaching its highest cell survival rate, 116.10%.

[0411] Effects of 3.6WLX on GES-1 cell viability

[0412] After GES-1 cells were treated with complete medium containing WLX at different concentrations and for different times, the OD values of each treatment were measured. 450 Value Figure 18As shown. After 12 hours of WLX treatment, except for a significant promotion effect at 800 μg / mL (P < 0.05), all other WLX concentrations had no significant promoting or inhibiting effect on GES-1 cell growth (P > 0.05). The highest cell survival rate, reaching 117.63%, was achieved at 800 μg / mL. After 24 hours of WLX treatment, except for a significant inhibition effect at 25,600 μg / mL (P < 0.05), all other WLX concentrations had no significant promoting or inhibiting effect on GES-1 cell growth (P > 0.05). The highest inhibition rate, reaching 23.64%, was achieved at 25,600 μg / mL. After 36 hours of WLX treatment, WLX at concentrations of 800 μg / mL and 1600 μg / mL significantly promoted the growth of GES-1 cells (P < 0.05), with cell viability rates of 112.67% and 113.93%, respectively. The highest inhibition rate, reaching 21.27%, was achieved at a concentration of 25600 μg / mL. After 48 hours of WLX treatment, WLX at a concentration of 25600 μg / mL significantly inhibited the growth of GES-1 cells (P < 0.05), with an inhibition rate of 22.18%. The remaining WLX concentrations had no significant promoting or inhibiting effect on GES-1 cell growth (P > 0.05). After 60 hours of WLX treatment, WLX at concentrations of 100 μg / mL and 400 μg / mL significantly promoted the growth of GES-1 cells (P<0.05), with cell survival rates of 123.1% and 121.03%, respectively. The remaining WLX concentrations had no significant promoting or inhibiting effect on the growth of GES-1 cells (P>0.05).

[0413] Effects of 3.7JZ on GES-1 cell viability

[0414] After GES-1 cells were treated with complete medium containing JZ at different concentrations and for different times, the OD values of each treatment were measured. 450 Value Figure 19 As shown. Figure 19 It can be seen that after treating GES-1 cells with different concentrations of JZ for 12-48 hours, it had no promoting effect on cell growth. When the JZ concentration was 25600μg / mL, the growth of GES-1 cells was inhibited at all time points. After the cells were treated with JZ for 60 hours, JZ concentrations of 1600μg / mL and 6400μg / mL had a significant promoting effect on the growth of GES-1 cells (P<0.05), with cell survival rates of 128.61% and 123.70%, respectively. The remaining JZ concentrations had no significant promoting or inhibiting effect on the growth of GES-1 cells (P>0.05).

[0415] Effects of 3.8UFG, FG, HEB, WLX and JZ on the activity of GES-1 cells damaged by ethanol

[0416] GES-1 cells that had been modeled with 3% ethanol for 5 hours were treated with different concentrations of UFG, HEB, FG, WLX and JZ, and the survival rates of the treated cells were measured. Figure 20 As shown. Figure 20 It can be seen that the Model group at each time point showed significant differences compared with its corresponding Control group, indicating that the modeling was successful at each time point.

[0417] Twelve hours after modeling and administration, the cell survival rates at UFG concentrations of 10 μg / mL and 100 μg / mL were significantly different from those in the model group (P < 0.05), reaching 99.12% and 102.14%, respectively. At a UFG concentration of 600 μg / mL, the cell survival rate was not significantly different from that in the model group. However, the cell survival rates in the low, medium, and high HEB concentration groups were significantly increased compared with those in the model group (P < 0.05), reaching 97.87%, 112.92%, and 110.12%, respectively. The cell survival rates in the low, medium, and high FG concentration groups were significantly increased compared with those in the model group (P < 0.05), with the highest cell survival rate at 600 μg / mL (108.26%). When the WLX concentration was 100 μg / mL, the cell survival rate was not significantly different from that of the model group (P>0.05). However, when the WLX concentration was 400 μg / mL and 1600 μg / mL, the cell survival rate was significantly increased compared with the model group (P<0.05), reaching 101.31% and 100.15%, respectively. There were no significant differences between the low, medium, and high JZ concentration groups and the model group (P>0.05).

[0418] Thirty-six hours after modeling and administration, the cell viability at UFG concentrations of 10 μg / mL and 100 μg / mL was significantly different from that of the model group (P < 0.05), reaching 98.99% and 94.88%, respectively. At a UFG concentration of 600 μg / mL, the cell viability was not significantly different from that of the model group (P > 0.05). The cell viability in the low, medium, and high HEB concentration groups was significantly increased compared with the model group (P < 0.05), reaching 97.76%, 97.38%, and 103.21%, respectively. The cell viability in the low, medium, and high FG concentration groups was significantly increased compared with the model group (P < 0.05), with the highest cell viability at 600 μg / mL (107.82%). When the WLX concentration was 100 μg / mL, the cell survival rate was not significantly different from that of the Model group (P>0.05). When the WLX concentration was 400 μg / mL and 1600 μg / mL, the cell survival rate was significantly increased compared with that of the Model group (94.84% and 91.43%, respectively) (P<0.05). When the JZ concentration was 100 μg / mL, the cell survival rate was significantly increased compared with that of the Model group (P<0.05). When the JZ concentration was 400 μg / mL and 1600 μg / mL, the cell survival rate was not significantly different from that of the Model group (P>0.05).

[0419] 60 hours after modeling and drug administration, the cell survival rates of the UFG, HEB, FG, WLX and JZ groups showed significant differences compared with the Control group (P<0.05). The highest cell survival rate was in the UFG medium concentration group, with a cell survival rate of 112.85%, and HEB was in the high concentration group (113.72%). The FG group had the highest cell survival rate at a concentration of 100 μg / mL, which was 115.97%. The highest cell survival rates in the WLX and JZ groups both appeared in the high concentration group (1600 μg / mL), which were 115.41% and 112.05%, respectively.

[0420] 3.9 Effects of UFG, FG, HEB, WLX and JZ on the migration ability of GES-1 cells damaged by ethanol

[0421] The scratch test was performed on GES-1 cells damaged by ethanol using different treatments. The scratch images obtained under a 4× microscope are shown in the figure below. Figure 21 As shown in (ac), Image J software was used to calculate the scratch area of each group after treatment, and the cell migration rate was calculated. Figure 21 (d) As shown in the results, when ethanol-damaged cells were treated with various drug groups, the cell migration rates were significantly different.

[0422] Twelve hours after administration, the migration rate of cells in the control group was significantly different from that in the model group (P < 0.05), indicating that ethanol significantly affects the migration ability of normal cells. Compared with the model group, the migration rate of cells was significantly increased (P < 0.05) at UFG concentrations of 10 μg / mL (14.31%) and 600 μg / mL (23.01%), increasing by 8.34% and 17.04%, respectively. However, there was no significant change at a UFG concentration of 100 μg / mL (10.62%) (P > 0.05). Compared with the model group, the migration rate of cells was significantly increased (P < 0.05) at HEB concentrations of 10 μg / mL (18.46%) and 100 μg / mL (36.48%), increasing by 12.49% and 25.71%, respectively. There was no significant change at a HEB concentration of 600 μg / mL (6.04%) (P > 0.05). Compared with the Model group, the cell migration rate was significantly increased (P<0.05) when the FG concentration was 10μg / mL (16.56%) and 600μg / mL (30.72%), increasing by 10.59% and 24.75%, respectively, while there was no significant change when the FG concentration was 100μg / mL (10.62%) (P>0.05). Compared with the Model group, the cell migration rates in the WLX and JZ groups showed no significant changes at low concentrations (P>0.05), but increased significantly at medium and high concentrations (P<0.05). The cell migration rates were WLX 100μg / mL (9.20%), WLX 400μg / mL (21.60%), WLX 1600μg / mL (16.99%), JZ 100μg / mL (22.03%), JZ 400μg / mL (30.12%), and JZ 1600μg / mL (39.89%), respectively.

[0423] After 24 hours of administration, compared with the model group, the cell migration rate was significantly increased at UFG concentrations of 10 μg / mL (34.63%) and 600 μg / mL (31.20%) (P < 0.05), increasing by 22.91% and 19.47%, respectively. However, there was no significant change at UFG concentration of 100 μg / mL (15.42%) (P > 0.05). Compared with the model group, the cell migration rate was significantly increased at HEB concentrations of 10 μg / mL (31.22%) and 100 μg / mL (44.52%) (P < 0.05), increasing by 19.50% and 32.80%, respectively. There was no significant change at HEB concentration of 600 μg / mL (8.08%) (P > 0.05). Compared with the Model group, the cell migration rate in the FG group was significantly increased at all concentrations (P < 0.05), with the highest migration rate at 100 μg / mL, reaching 47.77%, an increase of 36.05%. Compared with the Model group, the cell migration rates in the WLX group were significantly increased at low, medium, and high concentrations (P < 0.05), reaching 20.22%, 28.17%, and 24.83%, respectively, representing increases of 8.50%, 16.45%, and 13.11%, respectively. Compared with the Model group, the cell migration rates in the JZ group were significantly increased at low, medium, and high concentrations (P < 0.05), reaching 31.55%, 36.00%, and 48.9%, respectively, representing increases of 19.83%, 24.28%, and 37.18%, respectively.

[0424] After 36 hours of administration, compared with the model group, the cell migration rates in the low, medium, and high concentrations of UFG in the UFG group were significantly increased (P < 0.05), reaching 43.86%, 36.61%, and 36.46%, respectively, and increasing by 26.47%, 19.22%, and 19.07%, respectively. Compared with the model group, the cell migration rates were significantly increased (P < 0.05) at HEB concentrations of 10 μg / mL (40.29%) and 100 μg / mL (54.88%), increasing by 22.90% and 37.49%, respectively. There was no significant change at HEB concentration of 600 μg / mL (14.62%) (P > 0.05). Compared with the Model group, the cell migration rates in the FG group at low, medium, and high concentrations were significantly increased (P < 0.05), reaching 37.49%, 67.49%, and 57.43%, respectively, representing increases of 20.10%, 50.09%, and 40.05%, respectively. Compared with the Model group, the cell migration rates in the WLX group were significantly increased at all concentrations (P < 0.05), with the highest rate at 400 μg / mL, reaching 52.64%, an increase of 35.25%. Compared with the Model group, the cell migration rates in the JZ group at low, medium, and high concentrations were significantly increased (P < 0.05), reaching 52.51%, 48.73%, and 57.72%, respectively, representing increases of 35.12%, 31.34%, and 40.34%, respectively.

[0425] 4 Conclusion

[0426] This study investigated the oxidative repair effects of a bidirectional fermentation product of Hericium erinaceus and Panax ginseng on ethanol-damaged GES-1 cells. Preliminary experiments using different concentrations and durations of ethanol incubation for cell establishment revealed that a 3% ethanol concentration, with a cell inhibition rate of approximately 40% after 5 hours of exposure, was optimal for modeling. Subsequently, the optimal concentrations and durations of UFG, FG, HEB, WLX, and JZ were screened, revealing an optimal concentration range of 10-600 μg / mL for UFG, FG, and HEB, and 100-1600 μg / mL for WLX and JZ. The protective effects of low, medium, and high concentrations of UFG, FG, HEB, WLX, and JZ on ethanol-damaged GES-1 cells were then investigated. Results showed that all treatments exhibited significant cell growth-promoting effects after 60 hours of incubation, with the FG treatment group showing the most pronounced effect.

[0427] Cell migration is a fundamental function of normal cells, a physiological process essential for normal growth and development, and a ubiquitous form of movement in living cells. This study used a scratch wound test to investigate the effects of UFG, FG, HEB, WLX, and JZ on the migration of ethanol-damaged GES-1 cells. The results showed that all treatments exhibited a significant migration-promoting effect, with cells treated with 100 μg / mL FG exhibiting the strongest migration activity.

[0428] Our results further confirmed that Hericium erinaceus-ginseng bi-fermentation products have a strong repair capacity on ethanol-stimulated GES-1 cells. However, the situation of alcohol-induced gastric ulcer is complex and is closely related to oxidative stress, inflammatory response and mitochondrial apoptosis.

[0429] 5. Summary

[0430] By studying the cytotoxicity of Hericium erinaceus-ginseng bidirectional fermentation products on GES-1 cells, as well as their effects on cell proliferation and migration ability of ethanol-damaged GES-1 cells, we explore their physiological activity and pharmacological efficacy. We hope that this can provide a theoretical basis for subsequent bidirectional solid fermentation experiments, deeply explore the potential value of fermentation products, and provide a scientific background for future expansion of experimental directions.

[0431] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Application of bidirectional fermentation of Hericium erinaceus strains and ginseng in any of the following: (I), increasing the content of reducing sugars in the fermentation product; and / or (II), increasing the protein content in the fermentation product; and / or (III), increasing the ergosterol content in the fermentation product; and / or (IV), increasing the content of essential amino acids (EAA) in the fermentation product; and / or (V), increasing the content of unsaturated fatty acids (SFAs) in the fermentation product; and / or (VI), increasing the crude fat content in the fermentation product; and / or (VII), increasing the content of total dietary fiber in the fermentation product; and / or (VIII), increasing the ash and / or mineral content; and / or (IX) Improve the types and contents of volatile flavor compounds (VFCs) in ginseng; The preservation number of the Hericium erinaceus strain is CGMCC No. 22450.

2. The use according to claim 1, characterized in that The types of volatile flavor substances (VFCs) include alcohol VFCs and / or ester VFCs; The type and content of the volatile flavor substances (VFCs) in ginseng that are improved include any of the following: (i) reducing the content of alcohol VFCs; and / or (ii) Increase the content of ester VFCs.

3. A method for preparing a Hericium erinaceus strain-ginseng bidirectional solid fermentation product, characterized in that: The steps include: Step 1: Cultivating a solid culture of Hericium erinaceus strain; Step 2: preparing a liquid culture of Hericium erinaceus strain; Step 3: Grinding and sieving the ginseng slices to obtain ginseng powder; sterilizing the mixture, mixing it with the liquid strain of Hericium erinaceus, fermenting it with glucose or brown sugar as a carbon source, and separating Hericium erinaceus buds (HEB) and ginseng fungus (FG) in the fermentation system; The preservation number of the Hericium erinaceus strain is CGMCC No. 22450.

4. The preparation method according to claim 3, wherein The preparation of Hericium erinaceus liquid spawn in step 2 comprises the following steps: Hericium erinaceus was cultured in the primary liquid culture medium and the secondary liquid culture medium at 26°C and 160 rpm / min in the dark for 7 days. The primary liquid culture medium includes 20 g of glucose, 10 g of peptone, 2 g of KH2PO4, 1 g of MgSO4·7H2O, and 1 L of distilled water; The secondary liquid culture medium includes 22 g glucose, 0.2 g peptone, 0.2 g yeast powder, 5 g KH2PO4, 1 g MgSO4·7H2O, and 1 L distilled water; The carbon source in step 3 includes glucose; The inoculation amount of the Hericium erinaceus liquid strain is 20% (W / V); The fermentation temperature is 26° C., and the fermentation time is 3 to 40 days.

5. The Hericium erinaceus-ginseng bidirectional solid fermentation product obtained by the preparation method according to claim 3 or 4.

6. A method for preparing mycoplasma granules, characterized in that: The ginseng mycelium in the Hericium erinaceus-ginseng bidirectional solid fermentation product according to claim 5 is extracted with water to obtain an extract, and granulated to obtain the mycelium granules.

7. The preparation method according to claim 6, wherein The solid-liquid ratio of the water extraction is 25:1; and / or The water extraction time is 2.5h; and / or The water extraction temperature is 60°C; and / or The granulation aids include fillers and wetting agents; and / or The fillers include starch and sucrose; and / or The mass ratio of the extract to the starch is 1:1; and / or The mass ratio of the extract to the sucrose is 1:1.4; and / or The wetting agent is 75% by volume ethanol, and the amount of the ethanol is 12% (v / v); and / or The drying temperature of the plasmid is 50°C.

8. Mycoplasma granules prepared by the preparation method according to claim 6 or 7.

9. Use of the Hericium erinaceus-ginseng bidirectional solid fermentation product according to claim 5 or the fungus granules according to claim 8 in any of the following items; (I) Improve the antioxidant capacity of cells; (II), preparing a drug for improving antioxidant capacity; (III) Oxidative repair of ethanol-damaged cells; (IV), promoting the growth of ethanol-damaged cells; (V) improve the survival rate of ethanol-damaged cells; (VI) improving the migration ability of ethanol-damaged cells; (VII) preparing a drug for preventing and / or treating ethanol-induced gastric ulcer or preparing a drug for protecting ethanol-induced gastric mucosa; Preferably, the antioxidant comprises: Increase DPPH free radical scavenging rate; Increase OH radical scavenging rate; Increase ABTS+ free radical scavenging rate.

10. A drug, characterized in that It includes the Hericium erinaceus-ginseng bidirectional solid fermentation product as claimed in claim 5 or the fungus granules as claimed in claim 8.

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