Method for preparing radix sophorae tonkinensis by fermenting of melanocarpus albomyces and product and application thereof

Abstract

This invention discloses a method for preparing light-colored fermented soybeans using *Gnaphalium affine* fermentation, along with the product and its applications, belonging to the fields of traditional Chinese medicine processing technology and microbial fermentation technology. This invention provides *Gnaphalium affine* (… Epicoccum nigrum This paper provides a novel application for the preparation of light-colored fermented soybeans and a method for fermenting them. The method includes: inoculating *Achnatherum gracile* into a liquid culture medium for activation and culture; decocting *Artemisia annua* and mulberry leaves in water, separating the decoction and combining it with the residue for later use; soaking black beans in the decoction and residue, followed by sterilization; adding activated bacterial solution to the sterilized black beans, and fermenting at a constant temperature in a sealed container; drying after fermentation to obtain the finished light-colored fermented soybean product. Pharmacological experiments show that the obtained light-colored fermented soybean significantly reduces serum TC, TG, LDL, ALT, and AST levels in MASLD model mice, and increases HDL levels. Its lipid-lowering and liver-protecting activities are significantly superior to those of traditionally fermented light-colored fermented soybeans (P < 0.05), and it can be used to prepare drugs or health products for the prevention and / or treatment of glucose and lipid metabolism-related diseases.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine processing technology and microbial fermentation technology, specifically involving *Gnaphalium affine* (…). Epicoccum nigrum The application of ) in the preparation of light fermented soybeans, and a method for preparing light fermented soybeans by fermentation using a single strain of Acanthopanax niger, the resulting product and its medicinal use in the prevention and / or treatment of diseases related to glucose and lipid metabolism. Background Technology

[0002] Fermented soybean (Douchi) is a fermented product made from the mature seeds of soybean (Glycine max), a legume. It is listed in the Pharmacopoeia of the People's Republic of China and its traditional uses include relieving exterior syndromes, alleviating irritability, and dispersing stagnant heat. Modern pharmacological studies have confirmed that fermented soybean possesses various activities such as lowering lipids, anti-oxidation, regulating intestinal flora, and protecting the liver. It is a typical example of food and medicine sharing the same origin and is widely used in traditional Chinese medicine formulas, health foods, and daily diets.

[0003] Currently, the production of light-colored fermented black soybeans is mainly divided into two modes: traditional natural fermentation and industrially modified fermentation. Traditional light-colored fermented black soybeans rely on natural mixed-culture open fermentation. The fermentation microorganisms include various wild bacteria such as Aspergillus oryzae, Mucor, Bacillus, and lactic acid bacteria. The production process consists of three main steps: soaking black soybeans, steaming, and post-fermentation. The fermentation temperature is 22-30℃, and the natural fermentation cycle is about one month, completely dependent on the environmental temperature and humidity to control the fermentation process. Existing industrially modified methods mostly use multi-strain compound fermentation (a combination of molds and bacteria), shortening the fermentation cycle by artificially inoculating some dominant microorganisms, but rarely using a single strain to complete the entire fermentation process.

[0004] Metabolic dysfunction-associated steatotic liver disease (MASLD, formerly known as non-alcoholic fatty liver disease, NAFLD) encompasses a spectrum of conditions including simple fatty liver, non-alcoholic steatohepatitis (NASH), liver fibrosis, and cirrhosis, ultimately progressing to hepatocellular carcinoma. Furthermore, MASLD is closely associated with type 2 diabetes, cardiovascular disease, and chronic kidney disease, with the associated risk of death being more than twice that of the general population.

[0005] Current treatments have significant limitations: the number of approved drugs is extremely limited, and most are targeted at specific stages of fibrosis. Clinical practice mainly relies on lifestyle interventions, but patient adherence is poor and long-term efficacy is limited; metformin and statins can only improve some metabolic indicators and cannot reverse liver inflammation and fibrosis, and they also have side effects such as gastrointestinal reactions and liver and kidney damage. Therefore, the development of safe, effective, and long-term usable natural drugs is an urgent need in the prevention and treatment of MASLD.

[0006] Fermented soybean (douchi), a valuable food and medicine, possesses active ingredients that exert multi-target regulatory effects on MASLD. Daidzein can activate the AMPK pathway to inhibit hepatic lipid synthesis; isoflavones can scavenge free radicals and reduce oxidative stress; and fermented peptides can regulate gut microbiota and improve insulin resistance. However, current fermentation processes severely limit its pharmacological activity and clinical application, presenting the following technical bottlenecks: (1) The pharmacological activity is extremely unstable: Traditional natural mixed fermentation relies on wild bacteria in the environment, and the composition of the strains is uncontrollable. This results in the content of core active ingredients such as daidzein and total isoflavones in different batches of products being more than 3 times different, and the efficacy fluctuates significantly, which cannot meet the dosage standardization requirements for the prevention and control of MASLD.

[0007] (2) Insufficient conversion of active ingredients: Soy isoflavones mainly exist in the form of glycosides during natural fermentation, resulting in low bioavailability; although multi-strain compound fermentation can partially improve the conversion rate of aglycones, the complex interactions between strains make it difficult to precisely regulate the generation of active ingredients, and the overall lipid-lowering and liver-protecting activity is limited.

[0008] (3) Product safety risks: Open fermentation is prone to the growth of putrefactive bacteria and toxin-producing bacteria, which can lead to product spoilage and mold, posing a food safety risk and limiting its large-scale application in the pharmaceutical and health fields.

[0009] To date, there are no systematic reports, either domestically or internationally, on the precise regulation of active ingredients in fermented soybean (Soybean Paste) through single-strain pure culture fermentation to significantly enhance its efficacy in preventing and treating MASLD. Developing standardized production technologies for fermented soybean that can stably enrich active ingredients and significantly enhance lipid-lowering and liver-protective activities is of great significance for promoting the research and development of natural drugs for MASLD. Summary of the Invention

[0010] The first objective of this invention is to provide *Gnaphalium affine* (… Epicoccum nigrum Its application in the preparation of light fermented soybeans overcomes the shortcomings of existing technologies, such as uncontrollable strains, unstable product quality, high risk of contamination by other microorganisms, complex processes, and long production cycles.

[0011] The second objective of this invention is to provide a method for preparing light-colored fermented soybeans using a single strain of *Gnaphalium affine* through fermentation, thereby achieving standardized and controllable production of light-colored fermented soybeans.

[0012] The third objective of this invention is to provide a light-colored fermented soybean product obtained by the above method, and the application of the light-colored fermented soybean in the preparation of drugs or health products for the prevention and / or treatment of diseases related to glucose and lipid metabolism, providing a new natural medicinal and food resource for the prevention and treatment of diseases such as hyperlipidemia and fatty liver disease related to metabolic dysfunction.

[0013] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides *Gnaphalium affine* (…Epicoccum nigrum The application of *Gnaphalium affine* in the preparation of light-colored fermented soybeans. Preferably, the *Gnaphalium affine* strain has the preservation number CGMCC No. 42974, and was deposited at the China General Microbiological Culture Collection Center on August 19, 2026, with the taxonomic name *Gnaphalium affine*. Epicoccum nigrum .

[0014] Black spores belong to the phylum Ascomycota ( Ascomycota) , phylum Cynomycetes ( Dothideomycetes ), Graminosporales ( Pleosporales ), Acetococcus ( Epicoccum *Pseudomonas nigricans* is a type of filamentous fungus widely found in nature. This invention is the first to discover that using *Pseudomonas nigricans* as a single fermentation strain for closed, constant-temperature fermentation of light-colored fermented soybeans can avoid the contamination by other microorganisms and the destruction of active ingredients caused by environmental fluctuations in traditional open fermentation with mixed microorganisms. This significantly improves the liver-soothing and lipid-lowering activity of light-colored fermented soybeans and enhances batch-to-batch consistency, making the fermentation process controllable and ensuring stable product quality.

[0015] Secondly, the present invention provides a method for preparing light-flavored fermented soybeans by fermentation with a single strain, comprising the following steps: (1) Activation of strain: After inoculating *Gnaphalium affine* on a solid culture medium, the culture was transferred to a liquid culture medium for activation culture to obtain activated bacterial solution; (2) Boil Artemisia annua and mulberry leaves in water, separate the decoction from the dregs, concentrate the decoction and mix it with the dregs to obtain a mixture of decoction and dregs; (3) Soak the black beans in the mixture of medicinal juice and dregs obtained in step (2), sterilize them after soaking, and then cool them. (4) Add the activated bacterial solution obtained in step (1) to the sterilized and cooled black beans, mix well, and then seal and ferment at a constant temperature. (5) After fermentation, remove the artemisia and mulberry leaves, dry them, and obtain the light fermented soybean product.

[0016] Preferably, in step (1), the preservation number of the *Gnaphalium affine* is CGMCC No. 42974; The solid culture medium is PDA culture medium, and the liquid culture medium is PDB culture medium; the activation culture temperature is 24±4℃, preferably 22±2℃, and the activation time is 48-72 hours, preferably 48 hours.

[0017] Preferably, the activated bacterial solution in step (1) has a mycelial mass concentration range of 1.55-3.52 g / L, based on the dry mycelial weight.

[0018] Preferably, in step (2), the mass ratio of Artemisia annua to mulberry leaves is 1:1; the mass ratio of the total mass of Artemisia annua and mulberry leaves to water is 1:10-20, more preferably 1:10.

[0019] The concentration is to be reduced to 1 / 2 to 1 / 10 of the original volume before use, more preferably 1 / 2 to 1 / 3, and even more preferably 1 / 2.

[0020] Preferably, in step (3), the soaking time is 1-15 hours, more preferably 10-12 hours, until the black beans absorb 20%-30% of the water; the sterilization is performed at 110-130℃ for 10-20 minutes, more preferably at 120-121℃ for 15-20 minutes.

[0021] Preferably, in step (4), the black beans are allowed to stand and cool to 25±10℃ before adding the activated bacterial solution, and the mass ratio of the black beans to the activated bacterial solution is 10:1; the temperature of the constant temperature fermentation is 20±4℃, more preferably 20±2℃ (the strain has good cold resistance, and overheating will cause the strain to die), and the time is 7-14 days, more preferably 7 days.

[0022] Preferably, in step (5), the drying method is hot air drying, the drying temperature is 50-60℃, and the material is turned over regularly during the drying process until the fermented soybean granules are completely loose and there are no clumps sticking together; the surface is not transparent and wet, there is no sticky feeling when touched, there is no wet and soft cross-section inside the granules when broken open, and the material does not become damp when piled at room temperature.

[0023] The core innovation of this invention lies in: (1) using a single strain of *Gnaphalium affine* for pure closed fermentation, which fundamentally avoids the problems of contamination by miscellaneous bacteria and uncontrollable microbial communities in traditional open mixed fermentation; (2) by precisely controlling key process parameters such as activation culture temperature, sterilization conditions, inoculation ratio, and fermentation temperature and humidity, the targeted enrichment of active ingredients in light soybean and the batch-to-batch consistency of product quality are achieved; (3) the light soybean prepared by the complete set of controllable fermentation process can specifically regulate the metabolism of sugar and lipids in the body, reduce liver damage, and has stable efficacy. It can be used as a food-medicine homology raw material specifically for the intervention of metabolic liver diseases, improving the lipid-lowering and liver-protecting effects of traditional fermented light soybean and stabilizing the differences in efficacy between batches.

[0024] Thirdly, the present invention provides a light-colored fermented soybean product prepared by the above method. This light-colored fermented soybean product has significantly better lipid-lowering and liver-protecting activities, stable content of active ingredients, good batch-to-batch consistency, and is suitable for standardized large-scale production.

[0025] Preferably, the light-colored fermented soybean contains 0.021-0.0272% daidzein and 0.018-0.027% genistein.

[0026] Fourthly, the present invention provides the use of the above-mentioned light fermented soybean in the preparation of pharmaceuticals or health products for the prevention and / or treatment of diseases related to glucose and lipid metabolism. These diseases include, but are not limited to, hyperlipidemia, metabolic dysfunction-associated fatty liver disease (MASLD), non-alcoholic steatohepatitis (NASH), or diseases related to liver qi stagnation.

[0027] The drug or health product works through at least one of the following mechanisms: downregulating serum and liver total cholesterol (TC) and triglycerides (TG); downregulating serum low-density lipoprotein (LDL), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels, upregulating high-density lipoprotein (HDL) levels, reducing liver lipid accumulation, and alleviating liver steatosis and liver damage.

[0028] Preferably, the dosage form of the drug or health product is an oral preparation, and more preferably a solid preparation, a semi-solid preparation, or a liquid preparation.

[0029] Preferably, the drug or health product further includes a pharmaceutically or health product-acceptable carrier.

[0030] Preferably, the drug or health product is administered to humans.

[0031] Compared with the prior art, the present invention has the following beneficial effects: (1) The strains are controllable and the product quality is stable. Traditional natural mixed-culture fermentation relies on wild microorganisms in the environment, resulting in variations of more than three times in the content of active ingredients between different batches. This invention uses a single strain of *Gnaphalium affine* for closed-system fermentation. The strain is clearly defined and controllable, avoiding contamination by other microorganisms and the impact of environmental fluctuations on the fermentation process. This ensures batch-to-batch consistency in the content of active ingredients and efficacy of the fermented soybean products, meeting the standardized production requirements for pharmaceuticals and health products.

[0032] (2) The fermentation cycle is shortened and the production efficiency is improved. Traditional natural fermentation takes about one month. This invention shortens the fermentation cycle to 7-14 days by precisely controlling the culture temperature, inoculation ratio and sealed constant temperature conditions, which greatly improves production efficiency and reduces production costs.

[0033] (3) Significantly enhanced lipid-lowering and liver-protecting activities Pharmacodynamic experiments showed that the fermented soybeans prepared by the single strain of this invention exhibited significantly better lipid-lowering and hepatoprotective activities than conventionally fermented soybeans in MASLD model mice: they could significantly reduce serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), and alanine aminotransferase (ALT). Aspartate aminotransferase (AST) levels increased, high-density lipoprotein (HDL) levels increased, and hepatic lipid accumulation decreased in a dose-dependent manner. Under the same dosage conditions, the single-strain fermented soybean of this invention significantly improved serum TC, TG, HDL, LDL, ALT, and AST levels compared to the same dosage of conventionally fermented soybean (P < 0.05).

[0034] (4) High safety, suitable for long-term use The *Gnaphalium affine* used in this invention is a non-pathogenic filamentous fungus widely found in nature. The fermentation process is carried out under sealed conditions, effectively preventing the growth of putrefactive bacteria and toxin-producing bacteria that occur in open fermentation, resulting in a high product safety. Lightly fermented soybeans are themselves a food and medicine homology product, suitable for long-term use as a health supplement or medicine to prevent metabolic disorders of sugars and lipids.

[0035] (5) The process is simple and suitable for industrial production. The method of this invention has simple process steps, mild operating conditions, and uses conventional culture media and equipment, making it easy to scale up and promote, and suitable for large-scale industrial production.

[0036] The *Gnaphalium affine* involved in this invention was deposited on August 19, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 42974 and taxonomic name: *Gnaphalium affine*. Epicoccum nigrum. Attached Figure Description

[0037] Figure 1 This is a colony diagram of the strain in PDA medium; Figure 2 This is a picture of the product made from fermented light-colored soybeans by this strain; Figure 3 This is a graph showing the pharmacological activity of this strain of fermented soybeans in MASLD model mice compared to traditionally fermented soybeans. In the graph, A represents serum TC content, B represents serum TG content, C represents serum LDL content, D represents serum HDL content, E represents serum ALT content, F represents serum AST content, G represents liver TC content, and H represents liver TG content (ns in the graph indicates P ≥ 0.05). This indicates that P < 0.05. This indicates that P < 0.01. This indicates that P < 0.001. This indicates that P < 0.0001.

[0038] Figure 4 Comparative results of the molecular networks of chemical components in raw soybeans, naturally fermented light soybeans, and light soybeans fermented by a single strain. Detailed Implementation

[0039] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the present invention in any way. Any modifications or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0040] The processes, conditions, reagents, and experimental methods used in implementing this invention, except as specifically mentioned below, are all common knowledge and general knowledge in the field, and this invention does not have any particular limitations. Experimental methods in the embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0041] Unless otherwise stated, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. However, in the event of any conflict, the specification containing the definitions shall prevail.

[0042] In this invention, the term "fermented soybean" refers to soybean, a legume. Glycine max (L.) Fermented products of mature seeds of Merr. are listed in the Pharmacopoeia of the People's Republic of China and are considered both food and medicine.

[0043] In this invention, the term "metabolic dysfunction-associated fatty liver disease (MASLD)" was originally called non-alcoholic fatty liver disease (NAFLD). It is a clinicopathological syndrome characterized by excessive fat accumulation and steatosis in hepatocytes, and its disease spectrum covers simple fatty liver, non-alcoholic steatohepatitis (NASH), liver fibrosis, cirrhosis and hepatocellular carcinoma.

[0044] In this invention, the term "non-alcoholic steatohepatitis (NASH)" is a progressive subtype of MASLD, characterized by hepatocellular steatosis with intralobular inflammation and ballooning degeneration, which can progress to liver fibrosis and cirrhosis.

[0045] In this invention, the term "glucose and lipid metabolism-related diseases" includes, but is not limited to, hyperlipidemia, metabolic dysfunction-associated fatty liver disease (MASLD), non-alcoholic steatohepatitis (NASH), liver qi stagnation-related diseases, and other diseases with glucose and lipid metabolism disorders as their core pathological basis.

[0046] In this invention, the term "PDA medium" refers to Potato Dextrose Agar, a solid culture medium with potato extract, glucose and agar as the main components, which is widely used for the isolation, purification, morphological observation and preservation of fungi.

[0047] In this invention, the term "PDB medium" refers to Potato Dextrose Broth, a liquid culture medium with potato extract and glucose as the main components, which is widely used for the cultivation and activation of fungi.

[0048] In this invention, the term "prevention" means a method of preventing the onset of a disease and / or its accompanying symptoms or protecting a subject from acquiring a disease.

[0049] In this invention, the term "treatment" includes delaying and stopping the progression of a disease, or eliminating the disease, without requiring 100% inhibition, eradication, and reversal.

[0050] The black globulus used in the embodiments of the present invention ( Epicoccum nigrum The strain was deposited on August 19, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 42974 and taxonomic name: *Gnaphalium affine*. Epicoccum nigrum. This strain was collected as a compound strain during the natural fermentation of black soybeans, artemisia annua, and mulberry leaves according to the pharmacopoeia method to prepare light fermented soybean paste, and was isolated by streaking on PDB medium plates.

[0051] Black beans, artemisia annua, and mulberry leaves are all commercially available products and meet the relevant standards of the Pharmacopoeia of the People's Republic of China. PDB culture medium and reagents are all of analytical grade or biochemical reagent grade.

[0052] Example 1 1. Strain activation: The fermentation strain *Gnaphalium affine* was cultured on PDA medium and then transferred to 10 mL of PDB liquid medium. After mixing, it was placed in a 25℃ biochemical incubator and incubated for 48 h to obtain a qualified activated bacterial solution (concentration of 1.93 g / L based on dry mycelial weight). The colony diagram of this strain in PDA medium is shown below. Figure 1 .

[0053] 2. Preparation of fermentation excipients: Weigh 5 g of Artemisia annua and 5 g of mulberry leaves, add 200 mL of drinking water, bring to a boil over high heat, then simmer over low heat for 45 minutes. Filter to separate the decoction from the dregs, concentrate the decoction to 100 mL, and combine it with the dregs for later use.

[0054] 3. Black bean pretreatment: Take 100 g of dried black beans, add the combined medicinal juice and residue to completely submerge them, soak at room temperature for 12 h until the black beans absorb 20% of the water; after soaking, autoclave at 121℃ for 15 min, and let cool naturally to room temperature.

[0055] 4. Inoculation and fermentation: Add 10 g of activated bacterial solution to sterilized black beans and mix thoroughly; place in a biochemical incubator and ferment at a sealed constant temperature of 20℃ for 7 days.

[0056] 5. Finished Product Drying: After fermentation, remove the artemisia and mulberry leaves, transfer the material to a hot air drying oven, and dry at 55℃ until no sticky residue adheres to the surface of the fermented black beans, yielding 60 g of light-colored fermented black beans. See the attached photograph for its appearance. Figure 2 .

[0057] Example 2 1. Activation of the strain: The fermentation strain *Gnaphalium affine* was cultured on PDA medium and then transferred to 100 mL of PDB liquid medium. After shaking well, it was placed in a biochemical incubator at 22℃ and cultured at a constant temperature for 48 h to obtain a qualified activated bacterial solution (with a mass concentration of 2.19 g / L based on the dry mycelium weight).

[0058] 2. Preparation of fermentation excipients: Weigh 50 g of Artemisia annua and 50 g of mulberry leaves, add 1 L of drinking water, bring to a boil over high heat, then simmer over low heat for 30 minutes. Filter to separate the decoction from the residue, concentrate the decoction to 500 mL, and combine it with the residue for later use.

[0059] 3. Black bean pretreatment: Take 1 kg of dried black beans, add the combined medicinal juice and residue to completely submerge them, soak at room temperature for 11 h until the black beans absorb 25% water; after soaking, autoclave at 120℃ for 20 min, and cool naturally to room temperature.

[0060] 4. Inoculation and fermentation: Add 100 g of activated bacterial solution to sterilized black beans and mix thoroughly; place in a biochemical incubator and ferment at a sealed constant temperature of 20℃ for 7 days.

[0061] 5. Finished product drying: After fermentation, remove the artemisia and mulberry leaves, transfer the material to a hot air drying oven, and dry at 50℃ until there is no sticky residue on the surface of the fermented soybeans, to obtain 689 g of light fermented soybean product.

[0062] Example 3 1. Activation of the strain: The fermentation strain *Gnaphalium affine* was cultured on PDA medium and then transferred to 1 L of PDB liquid medium. After shaking well, it was placed in a 28℃ biochemical incubator and cultured at a constant temperature for 72 h to obtain a qualified activated bacterial solution (with a mass concentration of 3.52 g / L based on the dry mycelium weight).

[0063] 2. Preparation of fermentation excipients: Weigh 500 g of Artemisia annua and 500 g of mulberry leaves, add 10 L of drinking water, bring to a boil over high heat, then simmer over low heat for 60 minutes. Filter to separate the decoction from the dregs, concentrate the decoction to 4 L, and combine it with the dregs for later use.

[0064] 3. Black soybean pretreatment: Take 10 kg of dry black soybeans, add the combined medicinal juice and medicinal dregs obtained above until the black soybeans are completely immersed, and soak at room temperature for 15 h; until the water absorption of the black soybeans reaches 30%, perform autoclaving at 120°C for 15 min, and cool naturally to room temperature.

[0065] 4. Inoculation and fermentation: Add 1 kg of activated bacterial liquid to the sterilized black soybeans, stir thoroughly and mix evenly; place in a biochemical incubator, and perform sealed constant-temperature fermentation at 20°C for 7 d.

[0066] 5. Finished product drying: After the fermentation is completed, remove Artemisia annua and mulberry leaves, transfer the materials to a hot air drying oven, and dry at 60°C until no mucus adheres to the surface of the fermented soybean, to obtain 6.7 kg of finished light-fermented soybean.

[0067] Pharmacodynamic test of Example 4 Test objective: To verify the difference in lipid-lowering and liver-protecting activities between the single-strain fermented light-fermented soybean of the present invention and the traditionally fermented light-fermented soybean in MASLD model mice.

[0068] Experimental animals: C57BL / 6J mice, male, weighing 18-22 g, purchased from SPF (Beijing) Biotechnology Co., Ltd., license number: SCXK (Beijing) 2024-0001.

[0069] Experimental reagents: Total cholesterol (TC) assay kit, triglyceride (TG) assay kit, low-density lipoprotein (LDL) assay kit, high-density lipoprotein (HDL) assay kit, aspartate aminotransferase (AST) assay kit, alanine aminotransferase (ALT) assay kit (Nanjing Jiancheng Bioengineering Institute). The traditionally fermented light-fermented soybean is prepared in accordance with the processing requirements of the Chinese Pharmacopoeia.

[0070] Experimental method: (1) Modeling and grouping: After 7 days of adaptive feeding, the mice were randomly divided into a control group (control), a model group (model), a positive drug group (positive drug fenofibrate, 50 mg / kg), a low-dose traditionally fermented light-fermented soybean group (1820 mg / kg, natural low) and a high-dose group (3640 mg / kg, natural high), a low-dose light-fermented soybean group prepared in Example 3 (1820 mg / kg, single strain low) and a high-dose group (3640 mg / kg, single strain high), with 10 mice in each group. Except that the control group was given ordinary feed, the other groups were given high-fat feed for 8 weeks to establish the MASLD model.

[0071] (2) Drug administration: After successful modeling, each drug administration group was administered the corresponding drug by gavage daily, while the control group and model group were administered an equal volume of distilled water by gavage. Drug administration continued for 4 weeks, during which time each group continued to receive the corresponding feed. After the last administration, the animals were fasted for 12 hours, and blood was collected from the eyeballs to separate serum for biochemical analysis. Serum was used to detect TC, TG, HDL, LDL, ALT, and AST, and liver tissue was used to detect TC and TG. The detection methods were performed according to the instructions of each reagent kit.

[0072] The experimental results are shown in Figure 3 The results showed that, compared with the control group, the serum TC, TG, LDL, ALT, AST and liver TC and TG of the model group mice were significantly increased, and the serum HDL was significantly decreased (P<0.0001), indicating that the MASLD model was successfully established.

[0073] Traditional fermented soybean paste showed a weak regulatory effect on lipid accumulation in both low and high dose groups.

[0074] The low and high dose groups of fermented light soybeans of the present invention can significantly improve the above indicators in a dose-dependent manner. Among them, the high dose group of single strain fermentation is significantly better than the traditional fermented light soybeans of the same dose in improving serum TC, TG, LDL and ALT (P<0.05), and the low dose group of single strain fermentation is significantly better than the traditional fermented light soybeans of the same dose in improving serum HDL and AST (P<0.05).

[0075] Example 5: Determination of the content of daidzein and genistein in lightly fermented soybean. 1. Instruments and reagents High performance liquid chromatograph (equipped with UV detector, column oven, and automatic sample injection system); Daidaidzein reference standard (purity ≥98%), genistein reference standard (purity ≥98%); Acetonitrile was of chromatographic grade, glacial acetic acid and methanol were of analytical grade, and the water used in the experiment was ultrapure water; Lightly fermented soybeans as test samples: Three batches of lightly fermented soybeans prepared according to the method in Example 1, which were naturally fermented according to the pharmacopoeia method.

[0076] 2 Chromatographic conditions The chromatographic column was an Agilent ZORBAX Extend-C18 4.6 mm × 250 mm column with a particle size of 5 μm. The mobile phase was acetonitrile-1% glacial acetic acid solution (25:75). The detection wavelength was 260 nm. The flow rate was 1.0 mL / min. The column temperature was 30 °C.

[0077] 3. Solution preparation 3.1 Preparation of mixed reference solution Accurately weigh 10 mg each of daidzein and genistein reference standards, place them in a 50 mL volumetric flask, dissolve and dilute to the mark with methanol, and shake well. Accurately measure 1 mL, place it in a 10 mL volumetric flask, dilute to the mark with methanol, and shake well to obtain a mixed reference solution containing 20 μg each of daidzein and genistein per mL.

[0078] 3.2 Preparation of test solution Accurately weigh approximately 1 g of light fermented soybean powder (passed through a No. 2 sieve), place it in a stoppered conical flask, accurately add 25 mL of methanol, weigh the flask, heat under reflux for 1 hour, cool, and weigh again. Make up the lost weight with methanol, shake well, filter, and collect the filtrate to obtain the test solution. Prepare three parallel solutions of light fermented soybean test solution and three solutions of traditionally naturally fermented light fermented soybean test solution using the same method.

[0079] 4. Sample content determination Accurately pipette 10 μL of the mixed reference solution and each test solution into the high performance liquid chromatograph, determine the peak area under the above chromatographic conditions, and calculate the content of daidzein and genistein in each sample using the external standard method and standard curve. The results are calculated based on dried products. See Table 1 for details.

[0080] Table 1. Results of content determination in light fermented soybean samples

[0081] Conclusion: Using the total content of daidzein and genistein, the hallmark components of fermented soybean (Dou Chi), as evaluation indicators, stability analysis was conducted on parallel samples of naturally fermented and single-strain fermented Dou Chi. The RSD of the total content in the naturally fermented group was 41.60%, with large fluctuations in the content of active ingredients between batches. The fermentation system had a complex microbial community, poor process controllability, and poor product homogeneity. The RSD of the total content in the Dou Chi group of this invention was only 3.69%, far lower than that of the naturally fermented group. The dispersion of daidzein and genistein content between batches was smaller, and the overall content levels were more consistent. In summary, the single-strain fermentation mode of this invention can effectively avoid the problems of interference from miscellaneous bacteria and uncontrollable fermentation process in natural fermentation, significantly improve the batch stability of active ingredients and the uniformity of product quality in Dou Chi, and is more suitable for standardized and industrialized fermentation production of Dou Chi.

[0082] Example 6: Determination of the transformation of single strain and naturally fermented components 1. Experimental Objective High-resolution MS / MS fragment spectra were obtained by LC-QTOF-MS / MS positive and negative ion AutoMS2 on raw soybeans, naturally fermented light soybeans, and light soybeans fermented by a single strain. These fragment spectra were used to construct molecular networks and compare the chemical composition of the three groups of samples.

[0083] 2. Sample grouping is shown in Table 2. Table 2. Sample information analyzed by LC-QTOF-MS / MS

[0084] The three groups comprised a total of nine independent samples, each of which was processed separately throughout the entire process without merging the samples.

[0085] 3. Sample pretreatment First, each sample was individually pulverized and passed through an 80-mesh sieve to obtain a uniform powder. 1.00 g of the sample powder was accurately weighed and added to 20.0 mL of 70% ethanol (v / v) at a material-to-liquid ratio of 1:20 (g / mL) as the extraction solvent. Ultrasonic extraction was performed twice under ice-water bath conditions, each extraction lasting 60 minutes. After extraction, the supernatants from the two extractions were collected and combined. The combined supernatant was aliquoted into 1.00 mL portions, and the solvent was evaporated to dryness using a vacuum centrifuge until the sample was completely dry. The dried residue was reconstituted with 200 μL of 50% methanol (v / v). After reconstitution, the sample was centrifuged at 13000 rpm for 10 minutes at 4 °C. Finally, the centrifuged supernatant was used for mass spectrometry data acquisition and analysis in both positive and negative ion modes. The HPLC conditions are shown in Table 3, the gradient program in Table 4, the mass spectrometry acquisition parameters in Table 5, and the polarity and ion source settings in Table 6.

[0086] Table 3 Liquid phase conditions

[0087] Table 4 Gradient Procedure

[0088] Table 5 Mass Spectrometry Parameters

[0089] Note: Reference quality calibration is enabled for both positive and negative ion methods; average scan count is 1, detection window is 100 ppm, and minimum height is 1000 counts. AutoMS2 uses the dedicated exclusion lists in their respective method files.

[0090] The results are as follows Figure 4As shown, molecular network analysis reveals that single-strain fermentation significantly restructures the composition of soybean. Molecular family 1 comprises 14 nodes, with 13 nodes exhibiting the highest relative abundance in the single-strain fermentation group (M group) and none showing a dominant node from the raw soybean group (F group), indicating that this molecular family is primarily composed of metabolic features enriched after single-strain fermentation. Molecular family 2 contains dominant nodes from the F, M, and natural fermentation groups (N group), exhibiting a combination of preservative and fermentation-related component accumulation. All 7 nodes of molecular family 3 show the highest abundance in the raw soybean group. The clear separation between the dominant families in raw soybean and single-strain fermentation reflects the targeted reshaping of soybean metabolic composition by single-strain fermentation.

[0091] Further differential analysis of all filtering features revealed 92 exploratory differential features between groups M and F that met the original p < 0.05 and |log2FC| ≥ 1, of which 50 still met q < 0.10 after Benjamini–Hochberg correction. In contrast, only 35 exploratory differential features were obtained between groups N and F, and none of them met q < 0.10. Meanwhile, the median Spearman correlation coefficient within groups M was 0.619, higher than that of group N (0.486), and the mean Euclidean distance within groups M was also lower than that of group N, indicating better reproducibility and consistency of component changes induced by single-strain fermentation.

[0092] Among the 20 representative MS / MS libraries matched metabolites, eight components—γ-glutamylleucine, coumarin, lysine, leucine, emodin methyl ether, phenylalanine, isoleucine, and tyrosine—simultaneously satisfied M / F log2FC ≥ 1 and q < 0.10. γ-glutamylleucine showed the largest increase (log2FC = 3.33), followed by coumarin (2.95), lysine (2.93), and leucine (2.90). Considering the molecular family distribution, the number of differentially expressed features, and the intragroup consistency, single-strain fermentation under these experimental conditions exhibited more concentrated, stable, and reproducible component transformation characteristics.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. *Gnaphalium affine* ( Epicoccum nigrum Application of light fermented soybeans in the preparation of light fermented soybeans.

2. The application according to claim 1, characterized in that, The preservation number of the *Gnaphalium affine* is: CGMCC No. 42974.

3. A method for preparing light-flavored fermented soybeans by fermentation using a single strain, characterized in that, Includes the following steps: (1) Activation of strain: Inoculate *Gnaphalium affine* into liquid culture medium and carry out activation culture to obtain activated bacterial solution; (2) Boil Artemisia annua and mulberry leaves in water, separate the decoction from the dregs, concentrate the decoction and mix it with the dregs to obtain a mixture of decoction and dregs; (3) Soak the black beans in the mixture of medicinal juice and dregs obtained in step (2), sterilize them after soaking, and then cool them. (4) Add the activated bacterial solution obtained in step (1) to the sterilized black beans, mix well, and then seal and ferment at a constant temperature. (5) After fermentation, remove the artemisia and mulberry leaves, dry them, and obtain the finished light fermented soybean product.

4. The method according to claim 3, characterized in that, In step (1), the preservation number of the *Gnaphalium affine* is CGMCC No. 42974; The liquid culture medium is PDB medium; the activation culture temperature is 24±4℃ and the time is 48-72 hours.

5. The method according to claim 3, characterized in that, In step (2), the mass ratio of Artemisia annua to mulberry leaves is 1:1; the mass ratio of the total mass of Artemisia annua and mulberry leaves to water is 1:10-20; and the concentration is to concentrate to 1 / 2 to 1 / 10 of the original volume.

6. The method according to claim 3, characterized in that, In step (3), the soaking time is 1-15 hours, until the black beans absorb 20%-30% water; the sterilization is performed at 110-130℃ for 10-20 minutes.

7. The method according to claim 3, characterized in that, In step (4), the mass ratio of black beans to activated bacterial solution is 10:1; the temperature of constant temperature fermentation is 20±4℃, and the time is 7-14 days; In step (5), the drying method is hot air drying, the drying temperature is 50-60℃, and the soybeans are dried until there is no sticky residue on the surface.

8. A light-colored fermented soybean paste prepared by the method according to any one of claims 3-7.

9. The use of the light fermented soybean as described in claim 8 in the preparation of a medicament or health product for the prevention and / or treatment of diseases related to glucose and lipid metabolism.

10. The application according to claim 9, characterized in that, The glucose and lipid metabolism-related diseases mentioned are hyperlipidemia, fatty liver disease related to metabolic dysfunction, non-alcoholic steatohepatitis, or liver qi stagnation-related diseases.

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