Method for fermenting astragalus membranaceus by lactobacillus plantarum

Abstract

The invention belongs to the field of microbial fermentation, and particularly discloses a method for fermenting astragalus membranaceus by lactobacillus plantarum. The method comprises the following steps: S1, crushing and sterilizing astragalus membranaceus; s2, inoculating lactobacillus plantarum LP-301; and S3, carrying out fermentation culture. After astragalus membranaceus is fermented by the lactobacillus plantarum LP-301, the contents of substances such as flavonoids, quinones and terpenes in a fermentation product are remarkably increased, so that the lactobacillus plantarum LP-301 can be used for preparing antioxidant products or anti-inflammatory products and has a relatively high application value.

Description

Technical Field

[0001] The invention belongs to the field of microbial fermentation, and particularly relates to a method for fermenting astragalus with Lactobacillus plantarum and application thereof. Background Art

[0002] Astragalus, also known as cotton-shaped astragalus and cotton-shaped astragalus, belongs to the genus Astragalus in the Leguminosae family. It is a perennial, deep-rooted medicinal plant. The Chinese Pharmacopoeia lists astragalus as the dried root of Astragalus mongolica and Astragalus membranaceus. Due to its sweet and warm nature, astragalus has the effects of tonifying the middle qi and replenishing qi, detoxifying and promoting urination, promoting fluid production and nourishing blood, and healing sores and promoting tissue regeneration. It is clinically used to treat qi deficiency and fatigue, spontaneous sweating due to superficial deficiency, edema due to qi deficiency, poor appetite and loose stools, qi deficiency, persistent carbuncle ulcers, chronic diarrhea and prolapse of the rectum, and hematochezia and metrorrhagia. It is the most widely used staple Chinese medicinal herb. Astragalus is a food and medicine with both medicinal and nutritional value, and its value as a food is also outstanding. It is not only rich in nutrients, such as calcium, iron, zinc, selenium, and other trace elements, but can also provide health benefits through various dietary methods.

[0003] The active ingredients of Astragalus are mainly saponins, polysaccharides and flavonoids, which have anti-fatigue and anti-tumor effects, and also have certain protective effects on organs such as the heart, lungs, brain, gastrointestinal tract, liver and kidneys, and the nervous system. In 2022, the total consumption of Astragalus in China reached 75,400 tons, and 476 Astragalus health foods were registered with the State Administration for Market Regulation (Li Mingze, Li Guofeng, Huang Yulong, et al., Research Progress on the Physiological Functions of Astragalus Active Ingredients and Their Application in Food [J]. Food and Fermentation Industries, 2024, 50(13): 398-407.). Based on the biological activity of Astragalus in the fields of anti-inflammatory, anti-aging, and hypoglycemic, a diversified functional product system has been successfully developed, covering innovative forms such as fermented health drinks, herbal tea bags, and compound juices. In order to accelerate the exploration of the value of the entire industrial chain of Astragalus resources, it is of far-reaching significance to conduct more comprehensive and extensive research.

[0004] Fermentation involves the decomposition, synthesis, and transformation of organic matter through biological transformation, utilizing the metabolism of organisms such as microorganisms or cells under specific environmental conditions. The large amounts of active enzymes produced during fermentation significantly enhance drug absorption, allowing the active ingredients to be rapidly absorbed and utilized by tissues and cells. This achieves the therapeutic goals of curing illnesses, strengthening the body, and regulating immunity, maximizing the medicinal value of natural Chinese medicines. Under fermentation, the active ingredients of traditional Chinese medicines undergo extensive biotransformation, breaking down macromolecules into small active substances that can be directly absorbed through the intestinal barrier, significantly improving their bioavailability (Wang Zichao, Zheng Yi, Dai Yibo, Yang Rongchao, Zhao Renyong, Sun Gangchun, Zhou Wenwen, Feng Shouai, Feng Yingjie, Li Na, Yang Jinchu, Zhang Huiru, An Lemei. Effect of probiotic fermentation on the extraction rate and bioactivity of plant-based polysaccharides: A review [J]. Innovative Food Science & Emerging Technologies, 2024, 98: 103863.). Therefore, utilizing microbial fermentation for traditional Chinese medicine can reduce costs, improve efficacy, and provide a new avenue for developing health products such as medicinal and edible products. The creation and development of fermented traditional Chinese medicine in my country dates back thousands of years, with the earliest fermented traditional Chinese medicines being qu-based, including red koji, liushenqu, jianqu, and pinellia ternata koji. Currently, there are already patented medicines made through the fermentation of traditional Chinese medicines, including Shenqu, Jianshenqu, Banxiaqu, Caiyunqu, Chenxiangqu, Hongqu, Dandouchi, Baiyaojian, and Pienzaihuang. However, due to various limitations, such as environmental conditions and technical requirements, traditional fermentation techniques are unable to effectively decompose the active ingredients of traditional Chinese herbal medicines.

[0005] Traditional Chinese medicine fermentation often uses fungi as the fermenting agent. In recent years, research on bacterial fermentation has gradually gained momentum, with probiotics becoming an ideal choice due to their unique advantages. Probiotics are beneficial live bacteria and their metabolites that can modulate the host's intestinal flora and enhance health. Lactobacillus plantarum, an anaerobic or facultative anaerobic bacterium commonly found in fermented dairy products, is widely used in the fermentation of foods such as soy milk and yogurt and has promising applications in healthcare. It contributes to improved nutrition, enriches food diversity, and possesses a variety of beneficial effects. It plays an important role in intestinal health, metabolic disorders, and brain health, improving gastrointestinal function, regulating immunity, and alleviating alcoholic liver damage. In recent years, the application of Lactobacillus plantarum fermentation in food, feed silage and plant material fermentation has become increasingly common, and has attracted widespread attention from researchers at home and abroad (Borjihan Qinggele, Yang Zhan, Liu Kailonng, Yang Chengcong, Zhang Xiaoyuan, Yao Guoqiang. Comparative genomics and metabolite analysis of Lactiplantibacillus plantarum strains in dairy and vegetable fermentation environments [J]. Food Bioscience, 2025, 65: 106058.).

[0006] From the perspective of intestinal flora metabolism, traditional Chinese medicine (TCM) and probiotics interact: when orally administered, TCM can be metabolized and transformed by probiotics through the intestines; at the same time, TCM ingredients can also regulate the growth and activity of probiotics. The absorption, distribution, transformation, and excretion of drugs in the digestive tract require lipid-soluble diffusion. Most glycoside compounds, due to their sugar structure, have low lipid solubility and are difficult to penetrate the biomembrane barrier to reach their target sites. However, the human intestinal microbial community can secrete specific glycoside hydrolases that precisely sever glycosidic bonds through catalytic reactions, promoting the dissociation of glycosides from bound glycosides and converting them into free aglycones.

[0007] Astragalus membranaceus is a classic tonic in Traditional Chinese Medicine, documented in numerous ancient texts. Its chemical composition, pharmacological effects, processing techniques, and extraction and isolation are currently under extensive research both domestically and internationally. With advances in modern Chinese medicine fermentation technology, research on fermented Astragalus membranaceus is increasing, with a particular focus on strain screening, process optimization, fermentation models, efficacy evaluation, and component evolution. Astragalus membranaceus has diverse active ingredients, and fermentation can facilitate their release, thereby increasing their utilization. Zhou Qingmin et al. found that the contents of saponins, polysaccharides, flavonoids, nutrients and trace elements in fermented Astragalus stems and leaves were higher than those of the active ingredients and nutrients in Astragalus stems and leaves directly measured (Li Junxiang, Ma Yingchun, Li Xiaofeng, Wang Yafei, Huo Zeqi, Lin Yang, Li Jiaru, Yang Hui, Zhang Zhiming, Yang Pingrong, Zhang Chunjiang. Fermented Astragalus and its metabolites regulate inflammatory status and gutmicrobiota to repair intestinal barrier damage in dextran sulfate sodium-induced ulcerative colitis [J]. Frontiers in Nutrition, 2022, 9: 1035912.).

[0008] Astragalus membranaceus, a traditional Chinese medicinal herb used for both medicinal and edible purposes, is not only a classic herb commonly used in TCM clinics, but also a popular folk food. Not only is it natural, additive-free, green, and safe, it also has the health-promoting effect of delaying skin aging, making it an ideal choice that combines health-promoting value with ease of consumption. Although the consumption of astragalus membranaceus is a tradition among the Chinese people, there are currently few fermented astragalus membranaceus products on the market, and foods using fermented astragalus membranaceus freeze-dried powder as a raw material are even rarer. In response to market demands and technological innovations, scholars have gradually focused their development efforts on health supplements and biological preparations with specific efficacy. Therefore, the research and development of innovative fermented astragalus membranaceus foods with health benefits is of great value and significance in promoting the industrialization of astragalus membranaceus. Summary of the Invention

[0009] The present invention screens and uses Lactobacillus plantarum LP-301 to ferment astragalus, studies its optimal fermentation conditions through single-factor experiments and response surface experiments; studies the active ingredient composition and in vitro antioxidant activity of the fermented astragalus; and studies the inflammation-relieving effect of the fermented astragalus, thereby ultimately completing the present invention.

[0010] The present invention provides a method for fermenting astragalus by using Lactobacillus plantarum, which comprises the following steps: S1: crushing and sterilizing the Astragalus membranaceus; S2: inoculating Lactobacillus plantarum LP-301; the Lactobacillus plantarum LP-301 has a preservation number of CGMCC NO. 25279; and S3: performing fermentation culture.

[0011] Specifically, in S1, the astragalus is crushed with an ultrafine grinder and passed through a 120-180 mesh sieve to obtain astragalus powder.

[0012] Preferably, in S1, the astragalus is crushed with an ultrafine grinder and passed through a 140-160 mesh sieve (e.g., a 150 mesh sieve) to obtain astragalus powder.

[0013] In a specific embodiment, the astragalus powder is weighed in S1, thoroughly mixed with water at a material-to-liquid ratio of 1:20-40, and sterilized at high temperature.

[0014] Preferably, the astragalus powder is weighed in S1, thoroughly mixed with water at a material-to-liquid ratio of 1:28-32, and sterilized at a high temperature of 121° C. for 20 minutes.

[0015] Specifically, in S2, after being sterilized and cooled to room temperature, 1×10 7 - 1×10 9 CFU / mL of Lactobacillus plantarum LP-301.

[0016] Preferably, in S2, after being sterilized and cooled to room temperature, 0.9-1.1×10 8 CFU / mL of Lactobacillus plantarum LP-301.

[0017] Specifically, in S3, the cells were placed in a constant temperature incubator at 35-39°C for 24-64 h.

[0018] More specifically, in S3, the cells were placed in a 37°C constant temperature incubator for 46-50 h of constant temperature culture.

[0019] The present invention provides a fermentation product obtained by the method, which is the obtained fermentation liquid, or is obtained by drying or freeze-drying the obtained fermentation liquid.

[0020] The present invention further provides the use of the fermentation product in preparing antioxidant products or anti-inflammatory products.

[0021] A total of 1,716 metabolites were detected using the UPLC-MS / MS detection platform and relevant databases. Compared with unfermented broth, 713 substances were upregulated in the fermented Astragalus lyophilized powder, including 5 terpenes, 9 flavonoids, 3 alkaloids, 2 other compounds, and 1 quinone. Compared with unfermented broth, 144 substances were upregulated in the fermented Astragalus lyophilized powder, while 20 substances were significantly downregulated, including 11 flavonoids, 4 phenolic acids, 3 other compounds, 1 lignan and coumarin, and 1 quinone.

[0022] The data analysis results showed that after Lactobacillus plantarum LP-301 fermented Astragalus, the contents of substances such as astragaloside IV, formononetin, dragon's blood C, quercetin, isorhamnetin, apigenin, dioscorea ginseng, luteolin, rhein, ursolic acid, and betulinic acid increased significantly, indicating that fermentation significantly increased the content of flavonoids, quinones, terpenes and other substances.

[0023] When the concentration was 1.0 mg / mL, the DPPH radical scavenging rates of the unfermented Astragalus group, fermented Astragalus liquid group, and fermented Astragalus liquid freeze-dried powder group all reached the maximum, which were 58.54%, 95.68%, and 74.34%, respectively, and the Vc of the control group was 95.69%; the superoxide anion radical scavenging rates were 81.36%, 82.38%, and 71.56%, respectively, and the Vc of the control group was 85.34%; the hydroxyl radical scavenging rates were 48.24%, 52.27%, and 62.39%, respectively, and the Vc of the control group was 82.26%; the total reducing capacity was 0.217, 0.256, and 0.361, respectively, and the Vc of the control group was 1.145.

[0024] Compared with the model group (M group), treatment with fermented astragalus reduced liver and kidney weights in mice and ameliorated pathological changes such as inflammatory infiltration and cell necrosis in the liver and kidneys. Serum MDA levels were significantly reduced. Analysis of differential metabolites in mouse serum revealed that both fermented astragalus and astragaloside IV had a moderate effect on LPS-induced intestinal microbial disturbances.

[0025] Probiotic fermentation can significantly promote the release of effective ingredients in Astragalus, greatly enhance its pharmacological activity, and may produce new active substances, opening up new paths for improving the efficacy of Astragalus, which plays an important role in broadening the application of Astragalus. The present invention uses response surface methodology to optimize the process of fermenting Astragalus with Lactobacillus plantarum LP-301, and evaluates the in vitro antioxidant activity and inflammation-relieving effect of fermented Astragalus. The study uses metabolomics technology to systematically analyze the active ingredients of fermented Astragalus, expounds on the changes in its components, and provides a solid theoretical basis for the subsequent development of fermented Astragalus-related products.

[0026] Biomaterial deposit information: Lactobacillus plantarum LP-301 was deposited on July 11, 2022, at the General Microbiology Center of China Culture Collection Administration (CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China) with the deposit number CGMCC NO. 25279 and the classification name Lactobacillus plantarum. Lactobacillus plantarum . BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Demonstrate the effect of material-liquid ratio on the total flavonoids content of fermented Astragalus.

[0028] Figure 2 The effect of material-liquid ratio on the total polysaccharide content of fermented Astragalus was demonstrated.

[0029] Figure 3 To demonstrate the effect of fermentation time on the total flavonoids content of fermented Astragalus.

[0030] Figure 4 To demonstrate the effect of fermentation time on the total polysaccharide content of fermented Astragalus.

[0031] Figure 5 To demonstrate the effect of fermentation temperature on the total flavonoids content of fermented Astragalus.

[0032] Figure 6 To demonstrate the effect of fermentation temperature on the total polysaccharide content of fermented Astragalus.

[0033] Figure 7 The effect of inoculation amount on the total flavonoids content of fermented Astragalus was demonstrated.

[0034] Figure 8 The effect of inoculation amount on the total polysaccharide content of fermented Astragalus was demonstrated.

[0035] Figure 9 To demonstrate the effect of sieve aperture on the total flavonoids content of fermented Astragalus.

[0036] Figure 10 Demonstrate the effect of sieve mesh size on the total polysaccharide content of fermented Astragalus.

[0037] Figure 11 shows the effect of the interaction between fermentation time (A) and fermentation temperature (B) on the total flavonoids content.

[0038] Figure 12 Showing the effect of the interaction between fermentation time (A) and material-liquid ratio (C) on the total flavonoids content.

[0039] Figure 13 Showing the effect of the interaction between fermentation temperature (B) and solid-liquid ratio (C) on the total flavonoids content.

[0040] Figure 14Showing the effect of the interaction between fermentation time (A) and fermentation temperature (B) on the total polysaccharide content.

[0041] Figure 15 Showing the effect of the interaction between fermentation time (A) and material-to-liquid ratio (C) on the total polysaccharide content.

[0042] Figure 16 Showing the effect of the interaction between fermentation temperature (B) and solid-liquid ratio (C) on the total polysaccharide content.

[0043] Figure 17 Display DPPH free radical scavenging rate.

[0044] Figure 18 Demonstrates superoxide anion radical scavenging rate.

[0045] Figure 19 Demonstrates hydroxyl radical scavenging rate.

[0046] Figure 20 Demonstrate total reduction capability.

[0047] Figure 21 Displays a circular diagram of metabolite class composition.

[0048] Figure 22 PCA score plots showing the mass spectrometry data of each group of samples and quality control samples. (a) Unfermented Astragalus group, (b) Fermented Astragalus liquid group, and (c) Fermented Astragalus liquid freeze-dried powder group (QC) quality control samples. DETAILED DESCRIPTION

[0049] 1 Materials and Methods 1.1 Experimental Materials Astragalus membranaceus from the Babai Village Base of the Inner Mongolia Academy of Science and Technology. Lactobacillus plantarum LP-301, accession number CGMCC No. 25279, was isolated from kimchi in Ganxi Town, Pujiang County, Sichuan Province in 2008 by the Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education, Inner Mongolia Agricultural University. Angel Diamond Yogurt Starter, Angel Yeast Co., Ltd. Lactobacillus plantarum and Lactobacillus bulgaricus, Shaanxi Xingda Biotechnology Co., Ltd.

[0050] 1.2 Experimental Methods 1.2.1 Screening of Astragalus Fermentation Strain Astragalus root was washed three times, drained, and dried in a forced-air drying oven until constant weight. The root was then removed and pulverized using an ultrafine grinder. The powder was passed through a 150-mesh sieve to obtain the powder, which was then stored in a cool, dry place until ready for use. Five portions of 3.0 g of Astragalus powder were accurately weighed and fermented with five bacterial strains: Angel Diamond Yogurt Starter, Lactobacillus plantarum, Lactobacillus bulgaricus, Lactobacillus plantarum LP-301, and a mixed culture (Lactobacillus plantarum:Lactobacillus bulgaricus = 1:1). The mixture was thoroughly mixed in a 200 mL Erlenmeyer flask at a solid-liquid ratio of 1:30, sterilized, cooled to room temperature, inoculated with the appropriate bacterial strain, and incubated in a 37°C incubator for 48 hours. The fermentation performance of each starter was analyzed using total flavonoid and total polysaccharide content as indicators.

[0051] 1.2.2 Optimization of Astragalus Fermentation Process by Lactobacillus plantarum LP-301 1) Fermentation process Weigh 3 g of astragalus powder (passed through a 150-mesh sieve), mix thoroughly with distilled water at a solid-liquid ratio of 1:30, sterilize, cool to room temperature, inoculate with Lactobacillus plantarum LP-301, and place in a 37°C constant temperature incubator for 48 h to obtain fermented astragalus liquid.

[0052] 2) Fermentation condition single factor experiment This study used total flavonoid content and total polysaccharide content as indicators, and conducted single-factor experimental analysis to analyze the effects of fermentation time, material-liquid ratio, fermentation temperature, inoculation amount, and sieve aperture on the total flavonoid content and total polysaccharide content of fermented astragalus. The Box-Behnken response surface experimental design was used to systematically explore the key influencing factors in the fermentation process of astragalus, and accurately optimize the process parameters to determine the optimal conditions for improving the fermentation effect.

[0053] (1) Effect of material-liquid ratio on the total flavonoids and total polysaccharide contents of fermented Astragalus 3 g of Astragalus powder was accurately weighed and mixed with water according to different material-liquid ratios. The mixture was then inoculated with Lactobacillus plantarum LP-301, with a fixed fermentation time of 48 h, a fermentation temperature of 37 °C, and an inoculation volume of 1*10 8 CFU / mL, with different solid-liquid ratios including 1:20, 1:30, 1:40, 1:50, and 1:60 g / mL, to study their effects on the total flavonoids content and total polysaccharide content of fermented Astragalus.

[0054] (2) Effect of fermentation time on the total flavonoids and polysaccharide contents of fermented Astragalus The material-liquid ratio was fixed at 1:30 g / mL, the fermentation temperature was 37°C, and the inoculation amount was 1*10 8 CFU / mL, and the fermentation time (24, 36, 48, 60, and 72 h) was changed to study its effects on the total flavonoids and total polysaccharide contents of fermented Astragalus.

[0055] (3) Effect of fermentation temperature on the total flavonoids and total polysaccharide contents of fermented Astragalus The fixed fermentation time was 48 h, the material-liquid ratio was 1:30 g / mL, and the inoculation amount was 1*10 8 CFU / mL, and the fermentation temperature (28, 31, 34, 37, 40℃) was changed to study its effect on the total flavonoids content and total polysaccharide content of fermented Astragalus.

[0056] (4) Effect of inoculation amount on the total flavonoids and total polysaccharide contents of fermented Astragalus The fermentation time was fixed at 48 h, the fermentation temperature was 37 °C, the material-liquid ratio was 1:30 g / mL, and the inoculum amount (1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 CFU / mL) to study its effects on the total flavonoids and total polysaccharide contents of fermented Astragalus.

[0057] (5) Effect of sieve aperture on total flavonoids and total polysaccharide content in fermented Astragalus The fixed fermentation time was 48 h, the fermentation temperature was 37 °C, the material-liquid ratio was 1:30 g / mL, and the inoculum size was 1×10 8 CFU / mL, and the sieve aperture (60, 80, 100, 150, 200 mesh) was changed to study its effect on the total flavonoids content and total polysaccharide content of fermented Astragalus.

[0058] 3) Response surface experimental design Based on the results of the single-factor experiment, three key factors with significant influencing factors were screened: fermentation time (A), fermentation temperature (B), and material-to-liquid ratio (C). A three-factor, three-level response surface optimization experiment was constructed using Design-Expert 13 software, following the Box-Behnken experimental design principle, to evaluate the total flavonoid and total polysaccharide contents of fermented Astragalus. The specific experimental factors and their level codes are detailed in Table 1.

[0059] Table 1 Response surface experimental factor level design

[0060] 1.2.3 Sample metabolome analysis 1) Liquid sample extraction process (1) Thaw the sample until there is no ice and vortex for 10 seconds to mix.

[0061] (2) Take 200 μL of the sample and add it to the corresponding numbered 1.5 mL centrifuge tube. Add 200 μL of 70% methanol containing internal standard extract. The internal standard extract is prepared by dissolving 1 mg of the standard in 1 mL of 70% methanol to prepare a 1000 μg / mL standard stock solution. The 1000 μg / mL stock solution is then diluted with 70% methanol to prepare a 250 μg / mL internal standard solution.

[0062] (3) Vortex for 15 min, 12000 rpm, 4°C, and centrifuge for 3 min.

[0063] (4) Take the supernatant, filter it through a 0.22 μm microporous filter membrane, and transfer it to a sample injection bottle for testing.

[0064] 2) Solid sample extraction process (1) The samples were placed in a freeze dryer and vacuum-dried for 63 h.

[0065] (2) Grind into powder using a grinder (30 Hz, 1.5 min).

[0066] (3) Weigh 50 mg of sample and add 1200 μL of 70% methanol internal standard extract pre-cooled at -20°C. The internal standard extract is prepared by dissolving 1 mg of standard in 1 mL of 70% methanol to prepare a 1000 μg / mL standard stock solution. The 1000 μg / mL stock solution is diluted with 70% methanol to prepare a 250 μg / mL internal standard solution.

[0067] (4) Vortex once every 30 minutes, each time for 30 seconds, for a total of 6 times.

[0068] (5) After centrifugation at 12,000 rpm for 3 min, the supernatant was collected, filtered through a 0.22 μm microporous filter membrane, and transferred to a sample injection bottle for testing and UPLC-MS / MS analysis.

[0069] 3) Metabolomics testing instruments and conditions Data acquisition was mainly completed with the help of an ultra-performance liquid chromatography (UPLC) coupled with tandem mass spectrometry (MS / MS) instrument system.

[0070] 4) Ultra-high performance liquid chromatography conditions (1) Chromatographic column: AgilentSB-C18 1.8 µm, 2.1 mm * 100 mm.

[0071] (2) Mobile phase: A is ultrapure water containing 0.1% formic acid, and B is acetonitrile containing 0.1% formic acid.

[0072] (3) Elution gradient: Phase B accounts for 5% at 0.00 min, increases linearly to 95% within 9.00 min and maintains for 1 min, then decreases to 5% between 10.00 and 11.10 min, and then equilibrates at 5% for 14 min.

[0073] (4) Flow rate: 0.35 mL / min; column temperature: 40°C; injection volume: 2 μL.

[0074] 5) Mass spectrometry conditions (1) Electrospray ionization (ESI) temperature: 500°C; ion spray voltage (IS): 5500 V (positive ion mode) / -4500 V (negative ion mode).

[0075] (2) The ion source gas I (GSI), gas II (GSII), and curtain gas (CUR) were set to 50, 60, and 25 psi, respectively, and the collision-induced ionization parameter was set to high.

[0076] (3) The QQQ scan used the MRM mode and the collision gas (nitrogen) was set to medium.

[0077] (4) By optimizing the declustering potential (DP) and collision energy (CE), the DP and CE of each MRM transition were completed. A specific set of MRM transitions was monitored in each period, depending on the metabolites eluting in each period.

[0078] 1.2.4 In vitro antioxidant activity study methods

[0079] 1) DPPH free radical scavenging ability determination method Prepare 0.2mmol / L DPPH solution immediately before use. Take 1mL of sample solution with concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0mg / mL and the same concentration of vitamin C positive control solution and place them in 5mL centrifuge tubes. Add 1mL of DPPH anhydrous ethanol solution to each, mix well, and let it stand at room temperature in the dark for 30 minutes. Measure the absorbance A at 517nm. i .

[0080]

[0081] Where: Y is the clearance rate, %; A i is the absorbance of the sample; A j is the absorbance without adding DPPH; A0 is the absorbance of the sample without adding DPPH.

[0082] 2) Determination of superoxide anion free radical scavenging ability Place 0.1 mL of sample solutions at concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL, and a vitamin C positive control solution of the same concentration, into separate 15 mL centrifuge tubes. Add 4.5 mL of Tris-HCl buffer (pH 8.2, 0.05 mol / L), mix thoroughly, and incubate in a 25°C water bath for 20 min. Add 0.4 mL of preheated pyrogallol (2.5 mmol / L), vortex mix, and incubate in a 25°C water bath for 20 min. Immediately terminate the reaction by adding 8 mol / L hydrochloric acid. Measure the absorbance at 300 nm.

[0083]

[0084] Where: Y is the clearance rate, %; A0 is the absorbance value after distilled water and pyrogallol are mixed; A1 is the absorbance value after pyrogallol is mixed with the sample to be tested.

[0085] 3) Hydroxyl radical (·OH) scavenging ability determination method Take 1.0 mL of sample solutions at concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL and a vitamin C positive control solution in the same concentration gradient. Add 2 mL each of salicylic acid solution (6 mmol / L), ferrous sulfate solution, and hydrogen peroxide solution (6 mmol / L) in that order. Vortex mix thoroughly, let stand for 10 minutes, and measure the absorbance at a wavelength of 510 nm, denoted as A1. Use the blank solution A0 for comparison. The hydroxyl radical scavenging rate is calculated as follows:

[0086] In the formula: Y is the clearance rate, %; A0 is the absorbance value after distilled water and salicylic acid are mixed; A1 is the absorbance value after salicylic acid and the sample to be tested are mixed; A2 is the absorbance value after distilled water and the sample to be tested are mixed.

[0087] 4) Total reducing capacity determination method 1.0 mL of sample solutions at concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL, along with a vitamin C positive control solution in the same concentration gradient, were placed in separate 50 mL centrifuge tubes. 1.0 mL of 0.2 mol / L, pH 6.6 phosphate buffer and 1% potassium ferricyanide solution were added, respectively, and the mixture was thoroughly mixed. The mixture was incubated in a 60°C water bath for 15 minutes. After cooling to room temperature, 1.0 mL of 10% trichloroacetic acid solution was added, and the mixture was centrifuged at 4000 rpm for 10 minutes at 4°C. 2 mL of the supernatant was transferred to a 10 mL test tube, followed by 0.4 mL of 0.1% ferric chloride solution and 2.0 mL of distilled water. The mixture was vortexed and allowed to stand for 10 minutes. The absorbance (A1) at 700 nm was measured, and the absorbance (A2) of the blank solution was determined.

[0088]

[0089] Where: A1 is the absorbance of the sample solution; A2 is the absorbance of the blank solution.

[0090] 1.2.5 Alleviating effect of fermented Astragalus freeze-dried powder on LPS-induced inflammation (1) Experimental groups After one week of adaptive feeding, 30 female mice and 20 male mice were randomly divided into five groups: blank group (NC group), model group (M group), astragaloside IV group (AS group), low-dose fermented astragalus group (FAL group), and high-dose fermented astragalus group (FAH group). After the acclimation period, mice in each group received preventive medication for 21 days. On the 22nd day, mice in the M, FAL, FAH, and AS groups were intraperitoneally injected with 0.5 mg / mL LPS (0.2 mL / mouse) to establish an acute inflammation model. Six hours after modeling, the mice were sacrificed, and samples were collected and stored as required for subsequent experiments.

[0091] Table 2 Experimental groups and treatments

[0092] (2) Serum sample collection After modeling, blood was collected from the eyeball and placed in a 1.5 mL centrifuge tube. The tube was incubated at 4°C overnight until stratification occurred. The tube was centrifuged at 3000 rpm for 5 minutes, and the supernatant was transferred to a clean centrifuge tube. The tube was centrifuged at 12000 rpm for 10 minutes at 4°C. The serum was collected and transferred to a cryovial and stored at -80°C until testing.

[0093] (3) Tissue sample collection Mice were killed by cervical dislocation, sprayed with 75% alcohol for disinfection, and placed on a clean operating table. The abdominal cavity was quickly opened and the liver and kidney were removed.

[0094] (4) Relative organ weight (organ coefficient) The liver and kidneys were weighed using an analytical balance, and the relative organ weights were calculated according to the relative organ weight formula: relative organ weight = organ weight / body weight × 100.

[0095] (5) Organizational structure observation Fresh liver and kidney were rinsed with PBS buffer, dried with filter paper, and fixed in 4% paraformaldehyde at 4°C for at least 24 hours. Fixed tissues were then dehydrated, cleared, paraffin-impregnated, paraffin-embedded, and trimmed, and 4 μm sections were prepared. The sections were flattened on 40°C warm water, removed from the slides, and then oven-dried at 60°C to melt the wax. The sections were then removed and allowed to stand at room temperature until ready for use. After staining with hematoxylin-eosin, the sections were mounted with neutral resin and examined under a light microscope at 20x magnification.

[0096] The method for making paraffin sections is as follows: ① Dehydration: Cut the tissue into 3x3x2 mm pieces and place them in 70% alcohol overnight.

[0097] Dehydrate according to the following steps: 80% alcohol for 2 h → 85% alcohol for 1 h → 90% alcohol for 1 h → anhydrous ethanol I for 1 h → anhydrous ethanol II for 30 min.

[0098] ② Tissue transparency: xylene for 3 minutes.

[0099] ③ Wax immersion: Paraffin I for 1 h, Paraffin II for 30 min.

[0100] ④Embedding: Use a tissue embedding machine.

[0101] ⑤ Wax block trimming: trim the wax block to make it trapezoidal to ensure a smooth tissue section.

[0102] ⑥ Sectioning: Fix the trimmed wax block on the microtome with a fixed thickness of 5 μm.

[0103] ⑦ Patching: Adjust the temperature of the water bath to 37°C. Spread the stain flat and attach it to a glass slide. Then place it in an oven to prevent the stain from falling off. Set the temperature to 50°C and set aside.

[0104] The sections were stained with HE in this order: ① Baking: Baking at 60℃ for 2h; Dewaxing: Xylene I and Xylene II, 10 min each.

[0105] ②Anhydrous ethanol → 95% alcohol → 90% alcohol → 85% alcohol → 80% alcohol → 70% alcohol, 4 min each.

[0106] ③ Distilled water for 2 min → Hematoxylin staining for 3 min → Tap water for 5 min → 0.5% hydrochloric acid alcohol for 30 s → Distilled water for 30 s → 0.5% ammonia water for 30 s → Distilled water for 30 s.

[0107] ④70%, 85%, and 95% alcohol for 3 min each → Eosin staining solution for 1.5 min → 95% alcohol for 1 min → 100% ethanol I and II for 3 min each.

[0108] ⑤ Xylene I and II for 7 min each.

[0109] ⑥Seal the slides with neutral gum and collect images using a microscope.

[0110] (6) Detection of malondialdehyde (MDA) content in mouse serum Serum MDA levels were determined using an enzyme-linked immunosorbent assay (ELSA). Serum samples were returned to room temperature and assayed according to the kit instructions. Samples were added to a 96-well plate and the results were read using a microplate reader. Three replicates were performed for each sample group, and the mean optical density was used as the statistical indicator.

[0111] (7) Non-targeted metabolomics analysis of mouse serum Sample pretreatment: Take 100 μL of serum sample and place it in an EP tube. Add the same volume of 80% methanol aqueous solution. Take an equal volume of sample from the experimental sample and mix it as the QC sample. Vortex and shake, let it stand in an ice bath for 5 minutes, and centrifuge it at 15,000g and 4°C for 20 minutes. Take a certain amount of supernatant and dilute it with mass spectrometry grade water to a methanol content of 53%. Centrifuge it under the same conditions, collect the supernatant, and inject it into LC-MS for analysis.

[0112] Chromatographic conditions: Select Hypersil Gold column (C18) for the chromatographic column, control the column temperature at 40°C, the flow rate at 0.2 mL / min, select mobile phase A: 0.1% formic acid, mobile phase B: methanol, and set the chromatographic gradient elution program as shown in the table below.

[0113] Table 3 Chromatographic gradient elution program

[0114] Mass spectrometry conditions: Select a scan range of 100-1500; MS / MS secondary scans are data-dependent scans; ESI source settings are shown in the table below.

[0115] Table 4 ESI source settings

[0116] Data processing: The data was processed based on the Linux operating system (CentOS version 6.6) and R and Python.

[0117] (8) Mouse feces 16S second generation amplicon analysis Fecal DNA Extraction: Mouse fecal samples were collected and stored in sterile cryovials, snap-frozen in liquid nitrogen, and then transferred to a -80°C freezer for testing. Genomic DNA was extracted using the CTAB method.

[0118] PCR products were obtained using primers in the 16SV4 region (515F and 806R) for bacterial diversity, primers in the 18SV4 region (528F and 706R) for eukaryotic microbial diversity, and primers in the ITS1 region (ITS5-1737F and ITS2-2043R) for fungal diversity. In addition, amplified regions included 16SV3-V4, 16SV4-V5, and 16SV5-V7; archaeal 16SV4-V5 and 16SV8; and 18SV9 and the ITS2 region. All PCR mixtures were added with 15 μL Phusion High-Fidelity PCR MasterMix, 0.2 μM primers, and 10 ng genomic DNA template. The first denaturation was performed at 98°C for 1 minute, followed by 30 cycles at 98°C (10 seconds), 50°C (30 seconds), and 72°C (30 seconds). Finally, the mixture was kept at 72°C for 5 minutes. Samples were mixed, purified, libraries were constructed, and the DNA was loaded onto the instrument. PCR products were purified using magnetic beads and mixed in equal amounts based on product concentration. The thoroughly mixed PCR products were then detected and the target bands recovered. After library construction, quantification was performed using Qubit and qPCR, and qualified libraries were sequenced.

[0119] Bioinformatics Analysis: The raw tag data were truncated and filtered to remove duplicate chimeric sequences to obtain the final valid tags. Based on the valid data, the sequences were clustered into operational taxonomic units (otu) with a default identity of 97% using Uparse software. Species annotation was performed using the Mothur method and the SILVA138 SSU rRNA database. Alpha diversity was described using the Chao1 and Shannon indices, and beta diversity was described using principal coordinate analysis (PCoA). Species with significant differences between groups were compared using LEFSe analysis.

[0120] 2 Results and Analysis 2.1 Optimization of Astragalus Fermentation Process 1) Determination of Astragalus Fermentation Strain During the fermentation process of Astragalus membranaceus, selecting the appropriate bacterial strain is crucial for enhancing the fermentation efficiency and active ingredient release. Different bacterial strains can significantly influence the fermentation results. The appropriate bacterial strain not only effectively accelerates the fermentation rate of Astragalus membranaceus but also achieves a higher active ingredient content and bioavailability.

[0121] Table 5 Total flavonoids and total polysaccharide contents of Astragalus fermented by different strains

[0122] Note: a, b, c, d represent P The difference was significant at the <0.05 level.

[0123] Results showed significant differences in the total flavonoid and polysaccharide contents of Astragalus membranaceus fermented by five different strains. The total flavonoid content of unfermented Astragalus membranaceus was 1.54 mg / g, and the total polysaccharide content was 177 mg / g. Lactobacillus plantarum LP-301 fermented Astragalus membranaceus with the highest total flavonoid and polysaccharide contents, while the contents of Astragalus membranaceus fermented by the other four strains were significantly lower than those of LP-301. Astragalus membranaceus is a plant material, and most of its nutrients and active ingredients, such as flavonoids and polysaccharides, are intracellular and encapsulated by cell walls, making them difficult to bioavailable. During fermentation, microorganisms use Astragalus membranaceus as a nutrient for growth, reproduction, and metabolism. The cellulase, ligninase, and pectinase produced by these microorganisms digest and degrade the cell wall, causing cell wall damage and releasing intracellular lysates. This increases the content of flavonoids and polysaccharides in the fermentation broth, thereby enhancing their bioavailability. When Astragalus is fermented with different starter cultures, the higher the content of total flavonoids and total polysaccharides in the fermentation broth, the higher its biological activity may be. Therefore, Lactobacillus plantarum LP-301 was selected as the fermentation strain.

[0124] 2) Determination of the material-liquid ratio of Astragalus fermentation The material-to-liquid ratio, which refers to the mass-to-volume ratio of astragalus to solvent, is a key factor influencing fermentation performance. Different material-to-liquid ratios result in varying fermentation results, alter the types and levels of metabolites in the fermentation broth, and affect the viscosity and concentration of the solution. Therefore, determining the optimal material-to-liquid ratio is crucial for fermenting astragalus.

[0125] like Figure 1 、 Figure 2As shown, the material-liquid ratio has a significant impact on Astragalus fermentation. Excessively low moisture content dries the substrate, reducing nutrient dissolution and inhibiting microbial growth. With increasing moisture content, the total flavonoid and total polysaccharide contents initially increase and then decrease. At a material-liquid ratio of 1:30 g / mL, the total flavonoid content reaches a peak of 2.93 mg / g, significantly higher than at other levels. At a material-liquid ratio of 1:40 g / mL, the total polysaccharide content reaches a peak of 410.1 mg / g, but there is no significant difference compared to the material-liquid ratio of 1:30 g / mL. This may be because the microbial fermentation capacity has reached its maximum at this point, and the release of dissolved substances in the Astragalus has reached its peak. However, increasing the material-liquid ratio reduces the concentration of these substances, resulting in no significant increase in total polysaccharide content. As the moisture content continues to increase, the total flavonoid and total polysaccharide contents show a decreasing trend. Increasing moisture content reduces the bacterial metabolic rate, inhibits normal bacterial growth and reproduction, and dilutes the metabolic products in the fermentation broth, reducing their concentration and hindering the microbial decomposition of organic matter. From the perspective of fermentation effect and cost saving, the solid-liquid ratio of fermented Astragalus was determined to be 1:30 g / mL.

[0126] 3) Determination of Astragalus Fermentation Time Fermentation time is an important factor affecting the fermentation results. If the fermentation time is insufficient, the growth and reproduction of the microflora will be low, which may lead to incomplete fermentation of Astragalus and insufficient release of dissolved substances. On the contrary, if the fermentation time is too long, some macromolecular substances such as sugars in the system that provide energy for the growth and reproduction of the microflora may be decomposed and utilized by microorganisms in the late fermentation stage, reducing the content of effective substances and active ingredients, and affecting the content of total flavonoids and total polysaccharides. The results of the single-factor experiment on Astragalus fermentation time are shown in Figure 3 、 Figure 4 .

[0127] from Figure 3 and Figure 4 It can be seen that the total flavonoid and total polysaccharide contents in the fermentation broth increased with fermentation time, reaching their peaks at 48 hours, with total flavonoid content reaching 3.28 mg / g and total polysaccharide content reaching 470.46 mg / g. When the fermentation time exceeded 48 hours, the total flavonoid and total polysaccharide contents showed a downward trend. This may be due to the fact that in the early stages of fermentation, the bacterial strain is actively growing and metabolizing, and the fermentation broth is nutrient-rich, leading to rapid and extensive fermentation of Astragalus membranaceus, releasing large amounts of intracellular flavonoids and polysaccharides into the fermentation broth. In the later stages of fermentation, the accumulation of metabolites inhibits the growth of Lactobacillus plantarum LP-301. Furthermore, due to the decrease in nutrient content, some flavonoids and polysaccharides may be decomposed and utilized by the microorganisms, resulting in a downward trend in the total flavonoid and total polysaccharide contents in the fermentation broth. Therefore, a fermentation time of 48 hours was selected.

[0128] 4) Determination of Astragalus Fermentation Temperature Fermentation temperature is an important factor affecting the fermentation process. Both too low and too high temperatures will affect the growth of bacteria and their metabolic rate. When the fermentation temperature is too low, the growth rate and metabolic rate of the bacteria are slow, and the fermentation rate of Astragalus is slow; when the fermentation temperature is too high, it will cause some bacteria to have a faster metabolic rate, a decrease in vitality or even death, and delay the fermentation process. The results of the single factor experiment on Astragalus fermentation temperature are shown in Figure 5 、 Figure 6 .

[0129] from Figure 5 、 Figure 6 As can be seen, fermentation temperature significantly affects the total flavonoid and polysaccharide contents in the Astragalus fermentation product, with both low and high temperatures being unsuitable for their accumulation. This may be because low temperatures restrict microbial metabolism, slowing the metabolic rate and leading to reduced fermentation efficiency, while high temperatures inhibit enzyme activity, limiting microbial growth and metabolism. When the temperature is below 37°C, the total flavonoid and polysaccharide contents gradually increase with increasing temperature. Above 37°C, however, the total flavonoid and polysaccharide contents decrease significantly. These results indicate that the optimal temperature for Astragalus fermentation by Lactobacillus plantarum LP-301 is 37°C.

[0130] 5) Determination of the inoculum amount for fermented Astragalus The inoculation amount directly determines the number of bacteria at the beginning of fermentation, thus affecting the fermentation speed. When the inoculation amount is small, the initial number of bacteria is low and the bacteria proliferates slowly, resulting in a longer fermentation cycle. As the inoculation amount continues to increase, the initial number of bacteria continues to increase and the fermentation speed accelerates. The results of the single factor experiment on the inoculation amount of Astragalus fermentation agent are shown in Figure 7 、 Figure 8 .

[0131] like Figure 7 、 Figure 8 As shown in the figure, the inoculum amount is the number of bacteria at the beginning of fermentation, which determines the fermentation speed. When the inoculum amount is small, the initial number of bacteria is low and the bacteria proliferates slowly, resulting in a slow fermentation speed and low total flavonoids and total polysaccharide contents in the fermentation liquid. As the inoculum amount continues to increase, the fermentation speed gradually increases. When the inoculum amount is 10 8 CFU / mL, the fermentation speed is the fastest; as the inoculation amount continues to increase, the total flavonoid content and total polysaccharide content show a downward trend. The reason for this is: the inoculation amount is less than 10 8 CFU / mL, with the increase of inoculation amount, the number of Lactobacillus plantarum LP-301 in the fermentation broth continued to increase, nutrients were sufficient, and the fermentation speed gradually increased; when the inoculation amount was greater than 10 8 CFU / mL, the number of microorganisms in the fermentation broth is too large, the oxygen demand increases, and the nutrients are insufficient, resulting in a decrease in the fermentation rate. The results showed that the optimal inoculation amount of Lactobacillus plantarum LP-301 is 10 8 CFU / mL.

[0132] 6) Determination of sieve aperture The particle size of Astragalus powder varies with the sieve aperture, and the difficulty of decomposition and utilization by fermentation microorganisms varies. The results of the single factor experiment on the effect of sieve aperture on the fermentation degree of Astragalus are shown in Figure 9 、 Figure 10 .

[0133] As shown in the figure, the mesh size has little effect on the fermentation degree of Astragalus. However, as the mesh size decreases, the fermentation degree of Astragalus increases. Based on the experimental results, a 150-mesh sieve is the optimal choice for fermenting Astragalus and is beneficial for improving the quality of subsequent products.

[0134] 2.2 Response surface optimization experiment 1) Response surface model fitting and variance analysis of experimental data The response surface optimization method uses scientifically designed experiments to obtain data and constructs a functional relationship between factors and responses using a multivariate quadratic regression equation. This method can systematically examine the impact of multiple factors on the response variable during experimental parameter optimization, analyze the effects of each factor level, and visually demonstrate the interactions between factors through response surface graphs. Regression analysis can identify significant associations between independent and dependent variables, assess the reliability of the response surface regression model, and determine whether the approximate model can be used for subsequent optimization designs.

[0135] Table 6 Response surface experiment results

[0136] The F value of the model equation for the total flavonoids content of fermented Astragalus was 57.56, the regression model P<0.01 (extremely significant), and the lack of fit P =0.9547 (not significant), and R²=0.9867, RAdj²=0.9695, indicating that the equation is highly reliable. The F value measures the influence of the variable on the response value; the larger the value, the higher the contribution. Variance analysis showed that the order of influence of the linear terms was material-liquid ratio (C) > fermentation time (B) > fermentation temperature (A); the interaction terms were AB>AC>BC; and the quadratic terms were B²>C²>A². The significance test showed that the quadratic terms B² and C² had a very significant effect on the total flavonoid content of fermented Astragalus membranaceus ( P <0.01); the linear term C, the interaction term AB and the quadratic term A² also had a significant effect on its content ( P <0.05).

[0137] Table 7 Response surface regression model analysis of total flavonoids content

[0138] Note: “*” indicates significant difference. P <0.05; "**" indicates extremely significant difference. P<0.01.

[0139] The F value of the model equation for the total polysaccharide content of fermented Astragalus was 126.18, the regression model P < 0.01 (extremely significant), and the lack of fit P =0.0807 (not significant), and R²=0.9939, RAdj²=0.9860, indicating that the equation is highly reliable. The F value measures the influence of the variable on the response value. The larger the value, the higher the contribution. The variance analysis shows that the order of influence of the linear term is fermentation temperature (A)>fermentation time (B)>material-liquid ratio (C); the interaction term is AB>AC>BC; the quadratic term is A 2 >C 2 >B 2 The significance test shows that the quadratic term A 2 、B 2 、C 2 All of them have a very significant effect on the total polysaccharide content of fermented Astragalus ( P <0.01); the linear terms A, B and the interaction terms AB, AC all had significant effects on its content ( P <0.05).

[0140] Table 8 Response surface regression model analysis of total polysaccharide content

[0141] Among them, R 2 =0.9939, R Adj 2 =0.9860, R Pre 2 =0.9211CV%=4.06 2) Interaction analysis Using DesignExpert 13 software, we designed 3D response plots and contour plots for the interactions between three factors and three levels. We analyzed the effects of each factor on the total flavonoid and total polysaccharide content of fermented astragalus, as well as the strength of the interactions between the factors. The results are shown in the figure below.

[0142] Depend on Figure 11 The 3D response graph shows a steep surface, with the slope at point B being steeper than that at point A. This indicates that fermentation temperature has a greater impact on the total flavonoid content of fermented astragalus than fermentation time. The edges of the AB contour lines tend to be elliptical, with rapid color changes and tight contour lines, indicating a significant interaction between fermentation temperature and fermentation time.

[0143] Depend on Figure 12 It can be seen that although the response graph surface is relatively steep, and the slope at C is steeper than that at A, that is, the effect of the solid-liquid ratio on the total flavonoids content of fermented astragalus is greater than the fermentation time; but the edges of the AC contour lines tend to be circular, the contour lines are sparse, and the color changes slowly, indicating that the interaction between the solid-liquid ratio and the fermentation time is not significant.

[0144] Depend on Figure 13 It can be seen that the graph surface is steep, but the contour lines are nearly circular, and the color gradient is slow and sparse, indicating that the BC interaction is weak. This shows that the interaction between fermentation temperature and material-liquid ratio is not significant.

[0145] Depend on Figure 14 It can be seen that the graph surface is relatively steep, and the slope at A is steeper than that at B, that is, the effect of fermentation time on the total polysaccharide content of fermented Astragalus is greater than that of fermentation temperature. The edges of the AB contour lines tend to be elliptical, the color changes rapidly, and the contour lines are relatively close, indicating that the interaction between the material-liquid ratio and the fermentation time is significant.

[0146] Depend on Figure 15 It can be seen that the graph surface is steeper, and the slope at A is steeper than that at C, that is, the effect of fermentation time on the total polysaccharide content of fermented Astragalus is greater than the material-liquid ratio. The edges of the AC contour lines tend to be elliptical, the color changes relatively quickly, and the contour lines are relatively close, indicating that the interaction between the material-liquid ratio and fermentation time is significant.

[0147] Depend on Figure 16 It can be seen that the graph surface is steep, but the contour lines are nearly circular, and the color gradient is slow and sparse, indicating that the BC interaction is weak. This shows that the interaction between fermentation temperature and material-liquid ratio is not significant.

[0148] Response surface analysis revealed that the total flavonoids and total polysaccharides contents of fermented Astragalus reached a maximum value, corresponding to the optimal combination of solid-liquid ratio, fermentation time, and fermentation temperature. Software predictions revealed the optimal fermentation conditions to be a solid-liquid ratio of 1:27.50 g / mL, a fermentation temperature of 36.73°C, and a fermentation time of 47.96 h. Under these conditions, the theoretical total flavonoid content of Astragalus was 5.20 mg / g, and the theoretical total polysaccharide content was 467.20 mg / g.

[0149] 3) Verification experiment Three batches of samples were fermented to verify the optimal fermentation conditions optimized by the response surface analysis. To facilitate experimental operation, the optimized fermentation conditions were modified to: fermentation time at 37°C, solid-liquid ratio of 1:28 g / mL, and fermentation time of 48 h. Other conditions (inoculation amount and sieve aperture) remained unchanged. Total flavonoids and total polysaccharide contents were calculated.

[0150] The results of the validation experiment showed that the total flavonoids content and total polysaccharide content of fermented Astragalus were consistent with the statistical analysis results, indicating that the response surface analysis results were correct and the fermentation conditions were determined to be used for Astragalus fermentation.

[0151] 2.3 Comparison of active ingredient content in Astragalus before and after fermentation Astragalus polysaccharides, flavonoids, and saponins are the main active ingredients in Astragalus, possessing excellent antiviral properties and enhancing immunity. However, most of its active ingredients are present in cells. After fermentation, various metabolic and biochemical reactions occur, causing significant changes in the components of Astragalus. This increases the content of active ingredients, particularly total polysaccharides and flavonoids.

[0152] Table 9 Effect of fermentation on the active ingredients in Astragalus

[0153] Table 9 Comparison of the content of active ingredients in Astragalus before and after fermentation. Lactobacillus plantarum is a commonly used bacterial agent for fermenting traditional Chinese medicine, which helps the traditional Chinese medicine produce more active substances after fermentation. Wang Shizhong inoculated Astragalus with Pleurotus ostreatus to ferment it, tested the content of the main active ingredients in the fermentation broth, and compared it with the corresponding active ingredients in the Astragalus decoction. It was found that fermentation increased the content of Astragalus polysaccharides and total flavonoids. The results of this study are similar to those of the previous study. The total flavonoid content and total polysaccharide content in Astragalus after fermentation were significantly higher than those before fermentation, increasing by 237.66% and 163.36%, respectively. This indicates that fermentation promotes the release of active ingredients in Astragalus.

[0154] 2.4 Results of in vitro antioxidant effects of fermented Astragalus 1) DPPH free radical scavenging rate The results of DPPH free radical scavenging experiment are as follows Figure 17 As shown, the experimental results of DPPH free radical scavenging rate of unfermented astragalus, fermented astragalus liquid, and fermented astragalus liquid freeze-dried powder are shown.

[0155] The results showed that samples of varying concentrations all had a certain scavenging effect on DPPH free radicals, and the scavenging rate increased in a dose-response relationship with increasing concentration. The order of DPPH scavenging capacity among the different samples was: Vc positive control > fermented Astragalus liquid > fermented Astragalus liquid freeze-dried powder > unfermented Astragalus solution. When the solution concentration reached 1.0 mg / mL, the free radical scavenging rate reached its maximum, and the DPPH free radical scavenging rate of the Vc control group was essentially the same as that of the fermented liquid group, and significantly higher than that of the other groups ( P<0.05). The DPPH free radical scavenging capacity of the Vc control group was 95.69%, that of the fermented Astragalus liquid was 95.68%, that of the fermented Astragalus liquid freeze-dried powder was 74.34%, and that of the unfermented Astragalus liquid was 58.54%. The experimental results showed that at concentrations of 0.6 to 1.0 mg / mL, the DPPH free radical scavenging capacity of the fermented Astragalus liquid was essentially the same as that of the same Vc concentration, indicating a strong DPPH free radical scavenging capacity, significantly higher than that of the unfermented Astragalus liquid group. Therefore, fermentation significantly increases the content of substances with strong antioxidant capacity in Astragalus. The results showed that the scavenging rate of the fermented Astragalus liquid freeze-dried powder was lower than that of the fermented Astragalus liquid, possibly due to the loss of some antioxidant substances with DPPH free radical scavenging capacity during the freeze-drying process.

[0156] 2) Superoxide anion free radical scavenging rate Figure 18 The results of the superoxide anion free radical scavenging test of unfermented Astragalus, fermented Astragalus liquid, and fermented Astragalus liquid freeze-dried powder were shown. Among them, fermented Astragalus liquid freeze-dried powder, fermented Astragalus liquid, unfermented Astragalus solution and Vc positive control group all had certain O2 - The scavenging capacity ranked in the order: vitamin C positive control > fermented Astragalus liquid > unfermented Astragalus liquid > fermented Astragalus liquid freeze-dried powder. With increasing mass concentration, the free radical scavenging rates of all sample groups showed an upward trend. At a concentration of 1.0 mg / mL, all four sample groups achieved the highest scavenging rates. The superoxide anion free radical scavenging rate of the vitamin C control group was slightly higher than that of the other groups, at 85.34%, 82.38% for the fermented Astragalus liquid, 71.56% for the fermented Astragalus liquid freeze-dried powder, and 81.36% for the unfermented Astragalus liquid. The experimental results showed that at a mass concentration of 1.0 mg / mL, the superoxide anion free radical scavenging capacity of the fermented Astragalus liquid was no significantly different from that of the same concentration of vitamin C, indicating that its superoxide anion free radical scavenging capacity is very strong and significantly higher than that of the fermented Astragalus liquid freeze-dried powder. Therefore, fermentation significantly increases the content of substances with strong antioxidant capacity in Astragalus. The results showed that the clearance rate of the freeze-dried powder of fermented astragalus liquid was lower than that of the fermented astragalus liquid, which may be caused by the loss of some antioxidant substances with superoxide anion free radical scavenging ability during the freeze-drying process.

[0157] 3) Hydroxyl radical scavenging rate Figure 19The results of the hydroxyl radical scavenging experiments for unfermented Astragalus membranaceus, fermented Astragalus membranaceus liquid, and fermented Astragalus membranaceus liquid freeze-dried powder are shown. As shown in the figure, the fermented Astragalus membranaceus liquid freeze-dried powder, fermented Astragalus membranaceus liquid, unfermented Astragalus membranaceus liquid, and the vitamin C positive control group all have a certain degree of hydroxyl radical scavenging ability. The order of ·OH scavenging ability is: vitamin C positive control > fermented Astragalus membranaceus liquid freeze-dried powder > fermented Astragalus membranaceus liquid > unfermented Astragalus membranaceus liquid. With increasing concentration, the ·OH scavenging ability of each group of samples increases positively. At a mass concentration of 1.0 mg / mL, the ·OH scavenging ability of the fermented Astragalus membranaceus liquid freeze-dried powder, fermented Astragalus membranaceus liquid, unfermented Astragalus membranaceus liquid, and the vitamin C positive control group reaches its highest ·OH scavenging ability. At this concentration, the ·OH scavenging rate is 82.26% for the positive control group, 62.39% for the fermented Astragalus membranaceus liquid freeze-dried powder, 52.27% for the fermented Astragalus membranaceus liquid, and 48.24% for the unfermented Astragalus membranaceus liquid. These results indicate that fermentation enhances the hydroxyl radical scavenging ability of Astragalus membranaceus, likely due to the generation and release of more active substances with hydroxyl radical scavenging abilities during the fermentation process.

[0158] 4) Total reduction capacity The reducing power is positively correlated with the antioxidant activity. 3+ Reduction to Fe 2+ The absorbance at 700 nm is positively correlated with the reducing ability. Figure 20 These are the total reducing capacity test results of unfermented Astragalus, fermented Astragalus liquid, and fermented Astragalus liquid freeze-dried powder.

[0159] As shown in the figure, each group of samples had a certain degree of iron ion reducing ability. The order of total reducing ability was: Vc positive control > fermented Astragalus liquid freeze-dried powder > fermented Astragalus liquid > unfermented Astragalus liquid. As the mass concentration increased, the iron ion reducing ability gradually increased, and the iron ion reducing ability of each group of samples reached its maximum at a mass concentration of 1.0 mg / mL. The total reducing ability of the Vc control group was significantly higher than that of the other groups ( P <0.05). This indicates that the iron ion reducing ability of Astragalus membranaceus is not high, and fermentation has no significant effect on its iron ion reducing ability.

[0160] 2.5 Metabolite Detection Results of Fermented Astragalus 1) Secondary metabolite composition of the sample The metabolite composition of biological samples is sample-specific, and different samples contain different metabolite types and proportions; the metabolite composition will also change during different treatments or biological processes. The metabolite composition of the three samples in this study - fermented astragalus liquid, fermented astragalus liquid freeze-dried powder and unfermented astragalus is shown in Figure 21 .

[0161] Based on the UPLC-MS / MS detection platform and the self-built database MWDB (metware database), this study detected a total of nine major categories of secondary metabolites, namely flavonoids, alkaloids, phenolic acids, terpenes, lignans and coumarins, quinolines, steroids, tannins and others.

[0162] A total of 1,716 secondary metabolites were detected, including 529 flavonoids, 260 alkaloids, 245 phenolic acids, 232 terpenes, 138 lignans and coumarins, 51 quinones, 7 tannins, 3 steroids and 250 other secondary metabolites.

[0163] Most of these secondary metabolites possess strong biological activity. For example, flavonoids, present in many traditional Chinese medicinal herbs, possess a variety of biological activities, including anti-tumor, immunomodulatory, lipid-lowering, and antioxidant effects. Alkaloids, extracts from common Chinese herbal medicines, can prevent and improve diabetes by influencing the intestinal flora. Phenolic acids play an important role in antibacterial, anti-inflammatory, and antioxidant activities. Terpenoids possess anti-inflammatory and antibacterial properties and can regulate the secretion of the intestinal hormone glucagon-like peptide-1 (GLP-1), affecting appetite and energy metabolism, thereby effectively improving obesity. Numerous previous studies have shown that these metabolites act on multiple target proteins and have therapeutic effects on a variety of diseases, including tumors or cancers, neurological diseases, circulatory diseases, hematological diseases, psychiatric disorders, and immune disorders.

[0164] 2) Principal component analysis of test results Principal component analysis (PCA) was used to examine the differences in metabolites among three sample groups: fermented astragalus liquid, lyophilized fermented astragalus liquid, and unfermented astragalus. Figure 22 shows PC1 representing the first principal component, PC2 the second principal component, and PC3 the third principal component. Quality control (QC) samples are prepared by mixing samples and are used to test the reproducibility of samples processed using the same method. They are also used to evaluate the stability of the mass spectrometry system. If QC samples are closely clustered, this indicates good experimental stability and reproducibility.

[0165] The metabolite composition in the figure determines the position of the sample. Samples in the same or similar positions have similar metabolite compositions, and the farther apart they are, the greater the difference in their metabolites. The experimental results show that the quality control samples are in similar positions, indicating good repeatability of the experiment. Principal component analysis (PCA) showed that the PC1 score was 59%, the PC2 score was 16.94%, the PC3 score was 6.95%, and the total PCA score was 82.89%. The scatter plots corresponding to the three groups of samples showed a relatively close clustering within the group, indicating that the repeatability within the group was relatively good, the sample data was very similar, and there was a high degree of parallelism between the samples; on the other hand, there was good discrimination between the groups, indicating that the secondary metabolites in the three groups of samples were quite different. This shows that the metabolites underwent significant changes during the fermentation and freeze-drying processes of Astragalus membranaceus, and the secondary metabolites produced were very different.

[0166] 3) Analysis of differential metabolites in samples (1) Heat map of differential metabolites The results showed that compared with the unfermented astragalus sample, most of the differential secondary metabolites showed an up-regulation trend after astragalus fermentation; after the fermented astragalus liquid was freeze-dried, most of the differential metabolites were significantly up-regulated, which were displayed in red on the graph. Among them, the flavonoids with significantly increased content include taxifolin (dihydroquercetin), genistein (genistein), citronellol (dihydrokaempferol), isocarpon, apigenin, glycyrrhizin, naringenin chalcone, formononetin, medipterone, kaempferol, biochanin A, naringenin, daidzein, isoliquiritigenin, etc., followed by phenolic acids such as salicin, gallic acid, 2,3-dihydroxybenzoic acid, 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, and protocatechuic acid, and other types such as resveratrol, vanillin, 4-hydroxy-3-methoxybenzaldehyde, p-coumarol, 4-hydroxybenzaldehyde, salicylaldehyde, as well as lignans and coumarins such as coumestrol. The main reason may be that after fermentation of Astragalus with Lactobacillus plantarum, the enzymes produced by the probiotics can degrade the plant cell wall, significantly increasing the intercellular spaces and allowing the precipitation of Astragalus's active ingredients and metabolites, leading to an increase in the content of most metabolites. After fermentation and freeze-dried fermentation broth, the levels of small amounts of flavonoids such as 2-phenylacetamide, epiafrucatechin, afrucatechin, naringenin-7-O-glucoside (cherrin), hesperetin-7-O-glucoside, isobarbitoside (phlorizin chalcone), schizonepetaside, formononetin-7-O-glucoside (formononetin), biochanin A-7-O-glucoside (Indian dalbergia glycoside), and daidzein-7-O-glucoside (daidzin) were downregulated, as were phenolic acids such as chlorogenic acid (3-O-caffeoylquinic acid), cinnamic acid, α-hydroxycinnamic acid, 2-hydroxycinnamic acid, 3-hydroxycinnamic acid, and phenylglyoxylic acid. A possible explanation is that microbial growth requires nutrients, and some metabolites in the fermentation broth are consumed by the microorganisms as nutrients, leading to a decrease in their content. The hydroxyl groups on flavonoid glycosides can typically react with one or more caffeic acids, fatty acids, and ferulic acid to form mono- or polyacyl acylated flavonoid glycosides. This may also account for the significant decrease in the content of some phenolic acid compounds, such as gallic acid and 4-hydroxybenzoic acid.

[0167] (2) Differential metabolite analysis Volcano plots were constructed to display differential metabolites based on relative content fold change (FC) values ​​≥2 or ≤0.5. As shown in the figure, a total of 418 differential metabolites were identified between the fermented broth and the unfermented broth, of which 122 were upregulated and 296 were downregulated. A total of 875 differential metabolites were identified between the fermented Astragalus freeze-dried powder and the unfermented broth, of which 682 were upregulated and 193 were downregulated. A total of 718 differential metabolites were identified between the fermented Astragalus freeze-dried powder and the fermented broth, of which 701 were upregulated and 17 were downregulated. The results indicate that the fermented Astragalus freeze-dried powder had the most upregulated metabolites, indicating that the freeze-drying process had the greatest impact on the metabolites in Astragalus. This is likely due to sample concentration caused by freeze-drying, which significantly increased the metabolite content. The information of the metabolites with the largest up-regulated differences between the fermented Astragalus freeze-dried powder and the unfermented liquid and the metabolites with the largest down-regulated differences between the fermented liquid and the unfermented liquid are shown in Table 10.

[0168] Table 10 Identification results of significantly up-regulated and down-regulated metabolites among differential metabolites ;

[0169] The metabolites with the greatest differences screened out mainly include flavonoids, terpenes, alkaloids, phenolic acids, quinones, lignans and coumarins and others. Modern pharmacological research shows that flavonoids and alkaloids have anti-inflammatory, antibacterial, antiviral, and antioxidant functions; phenolic acids have an inhibitory effect on biofilms but do not have a clearing effect, and their inhibitory effect on biofilms is reflected in both antibacterial and inhibition of glycosyltransferase activity; the functionality and nutritional value of terpenes and their derivatives have been continuously confirmed, and some diterpenes and sesquiterpenes can change the surface characteristics of bacteria and reduce their adhesion ability; quinone compounds are natural active compounds and are the active ingredients of medicinal plants such as rhubarb and salvia miltiorrhiza, with antioxidant and anti-cancer pharmacological activities; lignans, as a natural compound, have the effects of enhancing immunity and cardiac contractility, and also have analgesic, lipid-lowering, enzyme-lowering and other functions, and can promote cardiac activity and respiration, and also affect the excitation and inhibition processes of the cerebral cortex; coumarin can reduce capillary permeability, promote blood circulation and increase blood flow, promote lymphatic return, effectively reduce edema, and have anti-inflammatory effects. For example, 3,9-dihydroxypterocarpon primarily affects the isoflavone biosynthesis pathway and the secondary metabolite biosynthesis pathway. Apigenin and luteolin jointly affect the flavonoid degradation pathway and the isoflavone biosynthesis pathway. Biochanin A primarily affects the phenylpropanoid biosynthesis pathway. Drastic changes in these compounds in fermented astragalus may alter its efficacy and activity, potentially making it a more valuable functional ingredient.

[0170] 4) Analysis of differential metabolic pathways among samples All detected metabolites were annotated using the KEGG database. A total of 249 metabolites were annotated by KEGG, and 149 significantly differentially expressed metabolites were annotated. Among the annotated metabolites, 79 belonged to metabolic pathways, 65 to secondary metabolic pathways, 33 to microbial metabolic pathways in diverse environments, 30 to phenylpropanoid biosynthesis pathway metabolites, 27 to flavonoid biosynthesis pathway metabolites, 21 to isoflavone biosynthesis pathway metabolites, 14 to aminobenzoate degradation pathway metabolites, and 14 to flavonoid and flavonol biosynthesis pathway metabolites.

[0171] 2.6 Alleviating effect of fermented Astragalus freeze-dried powder on LPS-induced inflammation 1) Relative organ weight The results showed that intraperitoneal injection of LPS increased the relative liver weight of mice. Intraperitoneal injection of LPS 21 days after the use of fermented Astragalus freeze-dried powder and Astragalus hepatitis A prophylaxis decreased the relative liver weight of mice compared with the M group, but there were no significant differences. Intraperitoneal injection of LPS significantly increased the relative left kidney weight, relative right kidney weight, and relative kidney weight of mice. The relative left kidney weight, relative right kidney weight, and relative kidney weight of mice in the FAL, FAH, and AS groups all decreased, but not significantly.

[0172] 2) Organizational structure observation To further assess liver and kidney damage in mice, H&E-stained sections of the mice's livers and kidneys were analyzed. H&E staining of the livers and kidneys showed that the NC group had normal and clear liver tissue morphology. Compared with the NC group, the M group had disorganized hepatic cords, large intercellular spaces, and inflammatory infiltration in the portal area. Compared with the M group, the FAL group had improved hepatic cord disorganization and intercellular spaces, but inflammatory infiltration was still visible. Compared with the M group, the FAL and FAH groups had improved hepatic cord disorganization, intercellular spaces, and inflammatory infiltration. Compared with the M group, the AS group had substantially improved hepatic cord disorganization, intercellular spaces, and inflammatory infiltration.

[0173] The renal tissue structure of mice in the NC group was clear, without congestion, edema, or cell necrosis. Compared with the NC group, the tissue structure of mice in the M group underwent damaging changes, with loosely arranged renal tubules and large gaps. Interstitial vascular congestion, cell necrosis, and inflammatory infiltration were observed. Compared with the M group, inflammatory infiltration and cell necrosis were still observed in mice in the FAL group, and the renal tubular gaps were improved. Compared with the M group, interstitial vascular congestion, cell necrosis, and inflammatory infiltration were improved in mice in the FAH and AS groups.

[0174] 3) Detection of MDA content in mouse serum MDA is a byproduct of lipid peroxidation and a widely recognized marker of oxidative stress. The effectiveness of fermented Astragalus freeze-dried powder in preventing acute inflammation was evaluated by measuring MDA levels in mouse serum. The results showed that compared with the NC group, the MDA level in the M group was significantly increased. Compared with the M group, the FAL, FAH, and AS groups all had significantly lower MDA levels in the mouse serum. There was no significant difference in MDA levels in the FAH group compared with the AS group.

[0175] 4) Non-targeted metabolite detection in mouse serum A total of 2,471 differential metabolites were detected in mouse serum samples, classified into 17 categories. Lipids and lipid-like molecules were the most abundant, followed by organic acids and their derivatives. Significant differences in metabolic patterns were observed between the M and NC groups, the FAL and M groups, the FAH and M groups, and the AS and M groups.

[0176] set up p The differences in serum metabolites between the M group and the NC group, the FAL group and the M group, the FAL group and the M group, and the AS group and the M group were obtained by VIP>1. Compared with the NC group, the M group had 136 differential metabolites up-regulated and 189 differential metabolites down-regulated; compared with the M group, the FAL group had 97 differential metabolites up-regulated and 48 differential metabolites down-regulated; compared with the M group, the FAH group had 217 differential metabolites up-regulated and 151 differential metabolites down-regulated; compared with the M group, the AS group had 227 differential metabolites up-regulated and 168 differential metabolites down-regulated.

[0177] There were 36 up-regulated metabolites shared by the NC and M groups, and 34 down-regulated metabolites shared by the NC and M groups, and 36 down-regulated metabolites shared by the FAH and M groups. Metabolic pathway enrichment analysis was performed on these 70 disordered differential metabolites. The results showed that the disordered differential metabolites were enriched in 20 metabolic pathways, including arginine and proline metabolism, central carbon metabolism in cancer, and aminoacyl-tRNA biosynthesis.

[0178] 5) Mouse feces 16S second-generation amplicon analysis (1) OTU cluster analysis Operational taxonomic units (OTUs) are formed by clustering different sequences using distance metrics. A total of 1478 OTUs were obtained from the N, M, FAL, FAH, and AS groups. Of these, 345 OTUs were shared between the NC and M groups; 326 OTUs were shared between the M and FAL groups, and 346 OTUs were shared between the NC and FAL groups; 388 OTUs were shared between the M and FAH groups, and 355 OTUs were shared between the NC and FAH groups; 429 OTUs were shared between the M and AS groups, and 399 OTUs were shared between the NC and AS groups.

[0179] (2) α-diversity analysis α-Diversity measures the richness and diversity of microbial communities within a group. Chao 1 reflects richness, while Shannon's index reflects uniformity. After LPS treatment, the richness of samples in the M group was generally lower than that in the NC group, while the uniformity of samples in the M group was generally higher than that in the NC group. This indicates that LPS reduces the abundance of the mouse intestinal microbiota, but the species within the intestine are relatively uniform. The richness and uniformity of the FAL, FAH, and AS groups were generally higher than those in the M group, indicating that fermented Astragalus and Astragalus hepatitis A have a certain effect on improving the LPS-induced intestinal microbial disturbance. The Good Coverage Index of each group was greater than 0.99, indicating that the data set was sufficient and the sequence analysis results were reliable.

[0180] (3) β-diversity analysis β-diversity analysis showed that the M group and the NC group could be well distinguished, indicating that there were differences in the diversity of intestinal flora of mice in the M and NC groups; the distances between most samples in the FAL, FAH, and AS groups and most samples in the NC groups were relatively close, indicating that the diversity of intestinal flora of mice in the FAL, FAH, AS, and NC groups was similar, and fermented astragalus and astragalus hepatitis A had a certain preventive effect on intestinal flora disorders induced by LPS.

[0181] (4) Effect of fermented Astragalus freeze-dried powder on the richness of intestinal flora in mice Selection of the top 15 most abundant species at the phylum level in each group revealed that Firmicutes and Bacteroidota were the most abundant bacterial groups in the mouse intestinal microbiota, accounting for over 75% in each group. Patescibacteria, Desulfobacterota, Proteobacteria, and Actinobacteriota accounted for the majority of the remaining bacterial groups. Treatment with LPS, fermented Astragalus freeze-dried powder, and astragaloside IV significantly altered the intestinal microbiota structure of mice. Compared with the NC group, the abundance of Firmicutes and Desulfobacteria increased in the M group, while the abundance of Bacteroidetes and Proteobacteria decreased, and the ratio of Firmicutes to Bacteroidetes increased. Compared with the model group, the abundance of Firmicutes and Desulfobacteria decreased to varying degrees in the FAL and FAH groups, while the abundance of Bacteroidetes and Proteobacteria increased to varying degrees, and the ratio of Firmicutes to Bacteroidetes decreased to varying degrees. This indicates that fermented astragalus and astragaloside IV effectively maintain the homeostasis of intestinal flora in mice.

[0182] The top 15 species with the highest abundance at the genus level in each group were selected. The results showed that compared with the NC group, the abundance of the Muribaculaceae and Rikenellaceae families in the M group decreased, while the abundance of the Bacteroidaceae and Lachnospiraceae families increased; compared with the M group, the abundance of the Muribaculaceae and Rikenellaceae families in the FAL, FAH and AS groups increased, while the abundance of the Bacteroidaceae and Lachnospiraceae families decreased.

[0183] The top 15 species with the highest abundance at the genus level within each group were selected for analysis. The results showed that the abundance of Desulfovibrio and Helicobacter increased in the M group compared with the NC group. Desulfovibrio is a strict anaerobe that has been linked to primary bacteremia and intra-abdominal infection. Helicobacter promotes inflammation by inducing immune tolerance, contributing to the pathogenesis of inflammatory bowel disease. Compared with the model group, the abundance of Desulfovibrio and Helicobacter decreased in the FAL, FAH, and AS groups.

Claims

1. A method for fermenting Astragalus with Lactobacillus plantarum, characterized in that: The steps include: S1 crushes and sterilizes the Astragalus root; S2 is inoculated with Lactobacillus plantarum LP-301; the deposit number of the Lactobacillus plantarum LP-301 is CGMCC NO.25279; S3 is used for fermentation culture.

2. The method according to claim 1, wherein In S1, the astragalus is crushed with an ultrafine grinder and passed through a 120-180 mesh sieve to obtain astragalus powder; preferably, the astragalus is passed through a 140-160 mesh sieve to obtain astragalus powder.

3. The method according to claim 1, wherein Weigh the Astragalus powder in S2, mix it thoroughly with water at a solid-liquid ratio of 1:20-40, and sterilize it at high temperature.

4. The method according to claim 3, wherein Astragalus powder was weighed in S1, mixed thoroughly with water at a material-to-liquid ratio of 1:28-32, and sterilized at 121°C for 20 minutes.

5. The method according to any one of claims 1 to 4, characterized in that In S2, after sterilization and cooling to room temperature, 1×107-1×109 CFU / mL of Lactobacillus plantarum LP-301 was inoculated.

6. The method according to claim 5, wherein In S2, after sterilization and cooling to room temperature, 0.9-1.1×108 CFU / mL of Lactobacillus plantarum LP-301 was inoculated.

7. The method according to any one of claims 1 to 4, characterized in that S3 was placed in a 35-39°C constant temperature incubator for 24-64 hours of constant temperature culture.

8. The method according to claim 7, wherein In S3, place in a 37°C constant temperature incubator for 46-50 hours.

9. The fermentation product obtained by the method according to any one of claims 1 to 8, characterized in that The fermentation liquid is obtained, or the fermentation liquid is obtained by drying or freeze-drying.

10. Use of the fermentation product according to claim 9 in the preparation of antioxidant products or anti-inflammatory products.

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