A notoginseng fermented liquid, a fermentation method and application thereof

Abstract

The present application relates to the technical field of bioengineering, and particularly relates to a fermented liquid of Gomphrena yatschekii, a fermentation method and application thereof.The present application provides a fermentation method of Gomphrena yatschekii, comprising the following steps: (1) mixing Gomphrena yatschekii with mixed enzymes for enzymolysis, and sterilizing to obtain an enzymolysis liquid of Gomphrena yatschekii; (2) mixing the enzymolysis liquid of Gomphrena yatschekii with a compound microbial agent for fermentation, and filtering and sterilizing after fermentation to obtain a fermented liquid of Gomphrena yatschekii; the compound microbial agent is Lactobacillus plantarum LZU-J-TSL6 and Lactobacillus plantarum LZU-J-QA25.After fermentation by the method, the effective components in Gomphrena yatschekii are fully dissolved, the drug activity is improved and increased, the body can rapidly absorb the drug, inflammation and oxidative stress can be regulated, and gastric ulcer can be resisted.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a fermentation broth of Codonopsis pilosula, a fermentation method, and its application. Background Technology

[0002] Codonopsis pilosula, a medicinal and edible herb, is a high-quality medicinal material from Zhouqu County, Gannan Prefecture. The industry is large, with high output and good effects. By giving full play to its advantages as both a food and a medicine, the development of Gannan's industry has been guaranteed to a certain extent. In addition, with the top-level design to popularize knowledge such as "prevention of disease", "Chinese medicine food" will be recognized by more and more people, and the market potential is huge.

[0003] Secondly, Codonopsis pilosula, as a major medicinal herb, has a long history and is widely used in both food and medicine. It is rich in various polysaccharides, saponins, alkaloids, trace elements, selenium, vitamin B1, vitamin B2, and contains more than 20 trace elements, 12 of which are essential nutrients for the human body. Modern pharmacological research shows that alkynes, flavonoids, and sugars are the main active ingredients of Codonopsis pilosula, and the content of ginsenosides and polysaccharides determines the quality of the herb, exhibiting various pharmacological effects. For decades, it has been studied by numerous scholars both domestically and internationally. However, the limited dosage forms and routes of administration in traditional Chinese medicine result in slow onset of action, making standardization and regulation difficult. Therefore, finding the optimal processing methods for traditional Chinese medicine to maximize its medicinal and nutritional benefits is of paramount importance for the future. Summary of the Invention

[0004] The purpose of this invention is to provide a fermentation liquid of Codonopsis pilosula, a fermentation method, and its application.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a fermentation method for Codonopsis pilosula, comprising the following steps:

[0007] (1) Codonopsis pilosula was mixed with a mixed enzyme for enzymatic hydrolysis, sterilized and filtered to obtain Codonopsis pilosula hydrolysate;

[0008] (2) The enzyme hydrolysate of Codonopsis pilosula was mixed with a compound microbial agent for fermentation, and then filtered and sterilized to obtain the fermentation liquid of Codonopsis pilosula; the compound microbial agent was Lactobacillus plantarum LZU-J-TSL6 and Lactobacillus plantarum LZU-J-QA25.

[0009] Preferably, the mixed enzyme in step (1) is cellulase and pectinase in a mass ratio of 2-1:1-2.

[0010] Preferably, the enzymatic hydrolysis in step (1) is carried out at a temperature of 55-65°C for 80-100 min.

[0011] Preferably, the Codonopsis pilosula in step (1) is a Codonopsis pilosula homogenate; the preparation method of the Codonopsis pilosula homogenate is: mix Codonopsis pilosula with water at a mass ratio of 1:13-17 and then pulverize to obtain Codonopsis pilosula homogenate.

[0012] Preferably, the mass ratio of the rhizome in step (1) to the mixed enzyme is 100:0.1 to 0.3.

[0013] Preferably, the fermentation time in step (2) is 20 to 28 hours, and the fermentation temperature is 34 to 38°C.

[0014] Preferably, in step (2), the mass ratio of the Codonopsis pilosula enzymatic hydrolysate to the compound microbial agent is 100:2-4; the mass ratio of Lactobacillus plantarum LZU-J-TSL6 to Lactobacillus plantarum LZU-J-QA25 is 2-4:1; and the bacterial count of the compound microbial agent is 7-9 × 10⁻⁶. 8 per mL.

[0015] The present invention also provides a fermentation broth of Codonopsis pilosula prepared by the aforementioned fermentation method.

[0016] The present invention also provides the application of the aforementioned Codonopsis pilosula fermentation broth in the preparation of antioxidant agents.

[0017] The present invention also provides the application of the aforementioned Codonopsis pilosula fermentation liquid in the preparation of drugs for treating gastric mucosal damage.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] In the extraction of active ingredients from medicinal plants, the diffusion of these ingredients into the extraction medium must overcome the dual resistance of the cell wall and the intercellular matrix. Plant cell walls are dense structures composed of substances such as cellulose, hemicellulose, pectin, and lignin. By selecting appropriate enzymes to act on the medicinal plant material, cellulase and pectinase can disrupt the dense structure of the cell wall, thereby facilitating the dissolution of the active ingredients. Choosing suitable enzymes can effectively dissolve the target substance while controlling the dissolution of non-target substances, improving dissolution efficiency and creating favorable conditions for subsequent purification of the extract.

[0020] This invention uses a mixture of cellulase and pectinase to extract Codonopsis pilosula. The compound enzyme-assisted extraction conditions are mild, the extraction time is short, the energy consumption is low, the cost is low, and it helps to maintain the original efficacy of the active ingredients.

[0021] Traditional Chinese medicine (TCM) fermentation technology has a long history and is an important method of processing TCM. It utilizes the growth and metabolism of microorganisms to ferment TCM under appropriate conditions (temperature, oxygen levels, moisture, etc.). During fermentation, microorganisms decompose and transform macromolecular components such as sugars, fats, proteins, and cellulose in the TCM, while simultaneously degrading cell wall components, promoting the dissolution or transformation of active ingredients, and altering its original properties. This process can more significantly moderate the medicinal properties and improve efficacy compared to general physical or chemical processing methods. TCM fermented with probiotics has the effects of increasing the content of active ingredients, enhancing efficacy, expanding indications, and reducing toxicity.

[0022] Codonopsis pilosula contains polysaccharides, flavonoids, and polyacetylene compounds, exhibiting significant antibacterial, antitumor, anti-inflammatory, and antioxidant effects. After fermentation using the method of this invention, the active ingredients in Codonopsis pilosula are fully dissolved, improving and enhancing drug activity. After rapid absorption by the body, it can regulate inflammation and oxidative stress, and resist gastric ulcers. Attached Figure Description

[0023] Figure 1 The effects of different conditions on the yield of Codonopsis pilosula glycosides in Example 1: (A) dosage of compound enzyme; (B) enzymatic hydrolysis time; (C) enzymatic hydrolysis temperature; (D) material-to-liquid ratio (n=3);

[0024] Figure 2 The effect of the interaction on the yield of codonopsis glycosides in Example 1;

[0025] Figure 3 Example 2 shows the growth of different strains in Codonopsis pilosula enzyme extract;

[0026] Figure 4 This is an example of the growth of different proportions of strains in the enzyme extract of Codonopsis pilosula in Example 2;

[0027] Figure 5 The effects of different conditions on the growth of the strain in Example 2: (A) inoculum size; (B) fermentation temperature; (C) fermentation time (n=3);

[0028] Figure 6 Changes in active ingredients of Codonopsis pilosula enzymatic extract before and after fermentation in Example 4: (A) Glucose standard curve; (B) Rutin standard curve; (C) Codonopsis pilosula glycoside standard curve; (D) Total polysaccharide content; (E) Total flavonoid content; (F) Codonopsis pilosula glycoside content; (G) pH; (H) Total acid content (n=3);

[0029] Figure 7 The changes in antioxidant activity of Codonopsis pilosula enzymatic extract before and after fermentation in Example 4: (A) ABTS free radical scavenging ability; (B) DPPH free radical scavenging ability; (C) OH free radical scavenging ability (n=3);

[0030] Figure 8 Example 5 PCA score chart;

[0031] Figure 9 Example 5: PLS-DA score chart;

[0032] Figure 10 This is the overall sample hierarchical clustering tree for Example 5;

[0033] Figure 11 The heatmap shows the differential metabolites in Example 5.

[0034] Figure 12 Ion diagrams of Codonopsis pilosula enzyme extract and Codonopsis pilosula fermentation broth from Example 5: Top of the diagram shows positive ions; bottom of the diagram shows negative ions.

[0035] Figure 13 Dendrogram and multivariate statistical analysis diagram of nonvolatile components before and after fermentation in Example 5; (A) PCA score diagram, (B) PLS-DA score diagram, (C) OPLS-DA score diagram, (D) differential metabolite volcano diagram;

[0036] Figure 14 This is a heatmap of differential metabolite clustering before and after fermentation in Example 5;

[0037] Figure 15 The changes in body weight of rats in each group during modeling and drug administration in Example 6;

[0038] Figure 16 The gastric mucosal ulceration status of rats in each group in Example 6;

[0039] Figure 17 HE staining of gastric mucosal sections from each group of rats in Example 6 (×40);

[0040] Figure 18 The antioxidant capacity of gastric tissue in Example 6 was determined by: (A) glutathione peroxidase; (B) malondialdehyde; (C) superoxide dismutase; and (D) catalase.

[0041] Compared with the blank group, ###P≤0.001; compared with the model group, *P≤0.05, **P≤0.01; compared with the unfermented Codonopsis pilosula liquid, △P≤0.05, △△P≤0.01.

[0042] Figure 19 Serum inflammatory factor expression in Example 6: (A) Tumor necrosis factor-α; (B) Interleukin-1β; (C) Interleukin-10; (D) Myeloperoxidase;

[0043] Compared with the control group, ##P≤0.01, ###P≤0.001; compared with the model group, *P≤0.05, **P≤0.01, ***P≤0.01; compared with the unfermented Codonopsis pilosula liquid, △P≤0.05, △△P≤0.01.

[0044] Figure 20 Example 6: Gastric mucosal protective factors: (A) prostaglandin E2; (B) nitric oxide;

[0045] Compared with the control group, ##P≤0.01; compared with the model group, *P≤0.05, **P≤0.01; compared with the unfermented Codonopsis pilosula liquid, △P≤0.05, △△P≤0.01. Detailed Implementation

[0046] Example 1

[0047] 1.1 Experimental Apparatus and Reagents

[0048] Table 1 Instruments and Reagents

[0049]

[0050] 1.2 Enzymatic Extraction

[0051] This invention uses a mixture of cellulase and pectinase to extract Codonopsis pilosula.

[0052] 1.3 Pretreatment of Codonopsis pilosula and Determination of Codonopsis pilosula Glycosides

[0053] 1.3.1 Pre-treatment of Codonopsis pilosula medicinal material

[0054] The Codonopsis pilosula herb was pulverized, and 10.0g of the herb was accurately weighed. An appropriate amount of distilled water was added, and the mixture was blended using a high-speed blender. A certain amount of compound enzymes (cellulase + pectinase) were then added, and the mixture was enzymatically hydrolyzed at a specific time and temperature. After hydrolysis, the enzyme activity was inactivated at 90℃ for 60 minutes, and the mixture was set aside for later use.

[0055] 1.3.2 Determination method of codonopsis glycosides

[0056] 1) Chromatographic conditions: C18 column, mobile phase acetonitrile:water = 25:75, flow rate 1mL / min, column temperature 30℃, detection wavelength 267nm, injection volume 10μL.

[0057] 2) Preparation of Codonopsis pilosula reference standard: Accurately weigh an appropriate amount of Codonopsis pilosula reference standard, add methanol to dissolve and dilute it to prepare a reference standard stock solution of 0.2048 mg / mL.

[0058] 3) Preparation of test sample: Take the prepared Codonopsis pilosula fermentation broth and filter it through a 0.22 micropore membrane to obtain the test sample.

[0059] 1.4 Single-factor experiment on enzymatic extraction of Codonopsis pilosula

[0060] 1.4.1 Dosage of compound enzyme

[0061] Accurately weigh 5.0g of Codonopsis pilosula medicinal material into 5 portions, add 15 times the amount of purified water to each portion, and enzymatically hydrolyze for 1.5h at 60℃. The dosage of the compound enzyme is 0.10%, 0.15%, 0.20%, 0.25%, and 0.30%, respectively, with the yield of codonopsis glycosides as the indicator. After enzymatic hydrolysis, the enzyme activity is inactivated at 90℃ for 60min, and the mixture is cooled to room temperature. The mixture is then processed according to the method in section "1.3.1". The content of codonopsis glycosides is then determined by liquid chromatography, and the yield is calculated.

[0062] 1.4.2 Enzymatic hydrolysis time

[0063] Five portions of Codonopsis pilosula were accurately weighed and divided into five parts. Each part was then mixed with 15 times the amount of purified water. The mixture was kept at 60°C with a compound enzyme dosage of 0.20%. The effect of enzymatic hydrolysis time (0.5, 1, 1.5, 2, and 2.5 h) on the enzymatic hydrolysis process was investigated, with the yield of codonopsis pilosula as the indicator. After the enzymatic hydrolysis was completed, the enzyme activity was inactivated at 90°C for 60 min. The mixture was then cooled to room temperature and processed according to the method described in section "1.3.1". The content of codonopsis pilosula was then determined by liquid chromatography, and the yield was calculated.

[0064] 1.4.3 Enzymatic hydrolysis temperature

[0065] Five portions of Codonopsis pilosula were accurately weighed and divided into five parts. Each part was then added to 15 times the volume of purified water. The enzymatic hydrolysis was carried out at 60℃ with a compound enzyme dosage of 0.20% for 1.5 hours. The effect of enzymatic hydrolysis temperature (40, 50, 60, 70, and 80℃) on the enzymatic hydrolysis process was investigated, with the yield of codonopsis pilosula as the indicator. After the enzymatic hydrolysis was completed, the enzyme activity was inactivated at 90℃ for 20 minutes. The mixture was then cooled to room temperature and processed according to the method in section "1.3.1". The content of codonopsis pilosula was then determined by liquid chromatography, and the yield was calculated.

[0066] 1.4.4 Material-to-liquid ratio

[0067] Five portions of Codonopsis pilosula were accurately weighed and hydrolyzed at 60℃ with a compound enzyme dosage of 0.20% for 1.5 hours. The effect of the material-to-liquid ratio (1:5, 1:10, 1:15, 1:20, 1:25) on the hydrolysis process was investigated, with the yield of codonopsis glycosides as the indicator. After hydrolysis, the enzyme activity was inactivated at 90℃ for 60 minutes, and the mixture was cooled to room temperature. The mixture was then processed according to the method in section "1.3.1". The content of codonopsis glycosides was then determined by liquid chromatography, and the yield was calculated.

[0068] 1.5 Response Surface Experiment of Enzymatic Extraction of Codonopsis pilosula

[0069] Based on the results of the single-factor experiments, the amount of compound enzyme (A), enzymatic hydrolysis time (B), enzymatic hydrolysis temperature (C) and material-liquid ratio (D) were used as influencing factors, and the yield of Codonopsis pilosula glycosides was used as the response value. A central composite design of 4 factors and 3 levels was used. The design is shown in Table 1-2, and the results are shown in Table 1-3.

[0070] Table 24 Factor 3 Level Table

[0071]

[0072] Table 3 Experimental Results

[0073]

[0074]

[0075] 1.6 Results and Analysis

[0076] 1.6.1 Results and Analysis of Single-Factor Experiments on Enzymatic Extraction of Codonopsis pilosula

[0077] Figure 1 It can be seen that when the amount of compound enzyme is 0.10% to 0.20%, the yield of codonopsis glycosides increases with the increase of enzyme concentration, while when the enzyme concentration is 0.20% to 0.30%, the yield of codonopsis glycosides decreases. Therefore, the amount of compound enzyme used in this invention is 0.20%. Figure 1 It can be seen that the yield increases with the increase of enzymatic hydrolysis time from 0.5 to 1.5 h, and the yield of codonopsis glycosides decreases after 1.5 h. Therefore, the enzymatic hydrolysis time of this invention is 1.5 h. Figure 1 It can be seen that the yield of codonopsis glycosides increases with the increase of enzymatic hydrolysis temperature between 40 and 60°C, and the yield is the largest at 60°C. The extraction rate decreases when the temperature is higher than 60°C. Therefore, the enzymatic hydrolysis temperature of this invention is 60°C. Figure 1 It can be seen that the yield of codonopsis glycoside is the highest when the material-to-liquid ratio is 1:15. When the ratio is further increased, the yield of codonopsis glycoside decreases. Therefore, the material-to-liquid ratio of the present invention is 1:15.

[0078] 1.6.2 Results and Analysis of Response Surface Experiments for Enzymatic Extraction of Codonopsis pilosula

[0079] The experimental results in Table 3 were fitted using Design-Expert 12 software, and the fitted model formula was obtained as follows:

[0080] Y=90.994+0.4575*A+0.3142*B+0.7442*C-0.1992*D-2.06*AB-0.9350*AC+0.2850

[0081] *AD+0.1175*BC+1.03*BD-0.5650*CD-3.61*A2 -4.27*B 2 -1.89*C 2 -5.17*D 2

[0082] Analysis of variance was performed on the established response surface regression model for the yield of codonopsis glycosides, and the results are shown in Table 4. The p-value of the model was 0.0012 < 0.05, reaching a significant level, and the error of lack of fit was 0.1533 > 0.1, indicating that the model lack of fit was not significant. The established quadratic model can be used to analyze and predict the yield of codonopsis glycosides with high reliability. The significance analysis of the regression model coefficients showed that the linear terms B and D, and the quadratic term B... 2 D 2 The significance levels of the coefficients were all less than 0.05, indicating a significant impact on the yield of codonopsis glycosides. This suggests that enzymatic hydrolysis time and the material-to-liquid ratio are important influencing factors in the enzymatic extraction process of Codonopsis pilosula. The response surface of the interaction between the factors is shown below. Figure 2 As shown in the figure. The steeper the slope of the response surface plot, the greater the impact of the change in conditions on the yield of codonopsis glycosides. Figure 2 The yield of codonopsis glycosides increased slowly at first as the enzymatic hydrolysis time and the material-to-liquid ratio decreased, then dropped sharply after reaching a maximum, forming a relatively steep slope. This indicates that the enzymatic hydrolysis time and the material-to-liquid ratio have a significant impact on the yield of codonopsis glycosides, which is consistent with the results of the analysis of variance in Table 4.

[0083] Based on the quadratic model of the corresponding surface analysis, the optimal enzymatic hydrolysis conditions for extracting Codonopsis pilosula were obtained: compound enzyme dosage: 0.201%, hydrolysis time: 1.514 h, hydrolysis temperature: 60.346℃, and material-to-liquid ratio: 14.868 g / ml. Under these conditions, the yield of codonopsis glycosides was 91.980%. Under these optimized conditions, combined with actual conditions, the optimal enzymatic extraction conditions for Codonopsis pilosula were confirmed as: compound enzyme dosage: 0.2%, hydrolysis time: 1.5 h, hydrolysis temperature: 60℃, and material-to-liquid ratio: 15 g / ml. Experiments conducted under these conditions yielded a codonopsis glycoside yield of 92.060%, which is close to the model prediction.

[0084] Table 4. Analysis of Variance for Response Surface Regression Model

[0085]

[0086]

[0087] This invention determined the necessary conditions for enzymatic extraction of Codonopsis pilosula through single-factor experiments, and then further optimized the extraction process using response surface methodology to obtain the optimal extraction process: 0.20% compound enzyme dosage, 1.5 h enzymatic hydrolysis time, 60℃ enzymatic hydrolysis temperature, and a solid-liquid ratio of 1:15 (g / ml). Codonopsis pilosula, a major medicinal and edible herb, has cell walls mainly composed of cellulose, hemicellulose, and pectin. The compound enzymes used in the experiment—cellulase and pectinase—can loosen and rupture its cell walls, thereby increasing the dissolution of active ingredients and improving the yield of codonopsis glycosides. Therefore, the extraction of traditional Chinese medicine using compound enzymes is an important development direction and warrants further research.

[0088] Example 2

[0089] 1. Materials and Methods

[0090] 1.1 Instruments and Reagents

[0091] Table 5 Instruments and Reagents

[0092]

[0093]

[0094] 1.2 Experimental medicinal materials and strains

[0095] Codonopsis pilosula (Wen Dang Shen): Identified by Chief Pharmacist Ni Lin of the Gansu Provincial Institute for Drug Control as the dried root of Codonopsis pilosula, a plant in the Campanulaceae family. The Codonopsis pilosula herb is pulverized and prepared for use.

[0096] Strains: *Lactobacillus plantarum* LZU-J-Q25 (GDMCC No: 63278), *Lactobacillus plantarum* LZU-J-QA85 (GDMCC No: 61192), *Lactobacillus plantarum* LZU-J-QA21 (GDMCC No: 63277), *Lactobacillus plantarum* LZU-J-TSL6, and *Lactobacillus plantarum* LZU-S-ZCJ. The positive control strain *Lactobacillus rhamnosus* LGG (BNCC-185356) was purchased from Beijing Bena Innovation Biotechnology Research Institute.

[0097] 1.3 Activation of fermentation strains and preparation of Codonopsis pilosula enzyme extract

[0098] Strain activation: Weigh 66.2g of MRS agar, add 1000mL of distilled water, stir until completely dissolved, and autoclave (121℃, 20min) for later use. After the culture medium cools to a suitable temperature, pour plates. Take the glycerol-preserved bacterial solution, streak the plate, and incubate it upside down in an incubator at 37℃ for 18h for later use. Add 9.8mL of sterile MRS liquid culture medium to a sterile 10ml test tube, pick a single colony into the test tube, mix well, and incubate at 37℃ for 24h to obtain the first-generation bacterial solution. Then, take 200μL of the first-generation bacterial solution and inoculate it into 9.8mL of MRS liquid culture medium, mix well, and incubate at 37℃ for 18-20h to obtain the bacterial solution used for fermentation. The initial concentration of the bacterial strain used for fermentation is controlled at 10 using plating counting. 10 CFU / mL.

[0099] Preparation of Codonopsis pilosula enzymatic extract: Codonopsis pilosula was extracted according to the optimal enzymatic extraction process obtained in Example 1. An appropriate amount of Codonopsis pilosula powder was weighed into a beaker, and fifteen times the volume of purified water was added. The mixture was then broken down using a cell wall disruptor. 0.20% of a compound enzyme (equal masses of cellulase and pectinase) was added, and the mixture was stirred electromagnetically at 60℃ for 1.5 hours. The supernatant was then inactivated at 90℃ for 60 minutes and set aside for later use.

[0100] 2.2 Single-factor experiments to explore optimal fermentation parameters

[0101] 2.2.1. Screening of fermentation strains

[0102] Take eight 10mL test tubes and add 9.8mL of Codonopsis pilosula enzyme extract. Inoculate LZU-J-QA25, LZU-J-QA85, LZU-J-QA21, LZU-J-TSL6, LZU-S-ZCJ, LGG (positive control), MRS, and LGG-inactivated bacterial suspension (SJ, negative control) at a 2% inoculation rate. Place in a constant temperature incubator and incubate at 37℃. Take samples every 6 hours to measure the OD of the fermentation broth. 600 The optimal fermentation strain was determined by screening out the fermentation values. Two strains with good fermentation effects were selected and mixed in different volume ratios (1:3, 1:1, 3:1) to ferment the Codonopsis pilosula enzyme extract. The fermentation effect of the strains after different mixing ratios was observed, and the optimal mixing ratio of strains for fermentation was selected.

[0103] 2.2.2 Fermentation single-factor conditions

[0104] After screening to obtain the strains required for fermentation, the optimal inoculum size, fermentation time, and fermentation temperature of the fermentation broth of Codonopsis pilosula were explored, using the number of viable bacteria in the fermentation broth as an indicator.

[0105] (1) Inoculation amount: Add Codonopsis pilosula enzyme extract to sterile test tubes, inoculate with bacterial solutions of different proportions (1%, 2%, 3%, 4%, 5%), incubate at 37℃ for 24h, plate the fermentation broth and calculate the number of viable bacteria to determine the optimal inoculation amount A.

[0106] (2) Fermentation time: Add Codonopsis pilosula enzyme extract to sterilized test tubes, inoculate the bacterial solution according to the optimal inoculum A, ferment at 36℃ for different times (6h, 12h, 18h, 24h, 30h, 36h), plate the fermentation liquid and calculate the number of viable bacteria to determine the optimal fermentation time B.

[0107] (3) Fermentation temperature: Add Codonopsis pilosula enzyme extract to sterilized test tubes, inoculate the bacterial solution according to the optimal inoculum A, ferment for B hours at different temperatures (34℃, 35℃, 36℃, 37℃, 38℃, 39℃), plate the fermentation liquid to calculate the number of viable bacteria, and determine the optimal fermentation temperature C.

[0108] 3. Orthogonal Experiments Optimize the Fermentation Process of Codonopsis pilosula

[0109] Based on the results of the single-factor experiments, a three-factor, three-level orthogonal analysis experiment was designed with inoculum size, fermentation time, and fermentation temperature as independent variables and viable cell count as dependent variable. The experimental design is shown in Table 6.

[0110] Table 63: Orthogonal Experiment Table with 3 Factors and 3 Levels

[0111]

[0112] Conclusion: After fermentation, the OD values ​​of the fermentation broths of LZU-J-QA25, LZU-J-QA85, LZU-J-QA21, LZU-J-TSL6, LZU-S-ZCJ, LGG, MRS, and SJ were determined. 600 The result is as follows Figure 3 As shown. Throughout the growth cycle, except for the positive group, all other strains showed a vigorous growth trend. However, after 24 hours, strains LZU-J-TSL6 and LZU-J-QA25 were significantly superior to the other strains. Therefore, these two strains were combined with Codonopsis pilosula enzyme extract in a certain proportion for fermentation to screen for dominant combined strains of Codonopsis pilosula. From Figure 4 It can be seen that the growth was best when LZU-J-TSL6:LZU-J-QA25 = 3:1. Compared with single-strain fermentation, the co-fermentation of the two strains showed better growth from the beginning, followed by OD... 600 The value continued to increase and remained higher than that of single-strain fermentation broth. Therefore, LZU-J-TSL6:LZU-J-QA25 = 3:1 was determined as the optimal strain for fermenting Codonopsis pilosula enzyme extract.

[0113] 4.2 Results and Analysis of Single-Factor Exploration

[0114] In the single-factor exploration, this invention adopted the univariate method, keeping the other two variables constant. First, the inoculum size of the fermented Codonopsis pilosula enzyme extract was determined. Then, temperature and time were explored, ultimately determining the optimal conditions for probiotic fermentation of the Codonopsis pilosula enzyme extract: an inoculum size of 3%, a fermentation temperature of 36℃, and a fermentation time of 24 hours. The results are shown in [Figure number missing]. Figure 5 .

[0115] 4.3 Results and Analysis of Orthogonal Experiment Optimization of Fermentation Process for Codonopsis pilosula

[0116] Based on the experimental results, a three-factor, three-level orthogonal experimental array was designed. Using the number of viable bacteria in the fermentation broth as the indicator, the influence of single factors on the fermentation results (R-value) was analyzed. Table 7 shows that the factor with the greatest impact on fermentation was the inoculum size, followed by fermentation time, and finally fermentation temperature. The optimal combination for the experiment of fermenting the *Codonopsis pilosula* enzyme extract was determined by the K-value to be A2B2C2, i.e., the optimal fermentation conditions were an inoculum size of 3%, a fermentation temperature of 36℃, and a fermentation time of 24 h. The results of the analysis of variance are shown in Table 8, indicating that each factor had a significant impact on the number of viable bacteria in the fermentation broth.

[0117] Table 7 Analysis of Orthogonal Experimental Design Results

[0118]

[0119] Table 8. Experimental Variance Analysis Table

[0120]

[0121]

[0122] Note: P < 0.01 is represented by **.

[0123] Conclusion: The performance of the bacterial strain used in fermentation determines the level of microbial fermentation. Optimal fermentation conditions can increase the number of viable bacteria in the product and fully utilize the advantages of probiotics. This example, based on dominant strains isolated in the laboratory, explored single-strain fermentation, combined strain fermentation, and the ratio of combined strains to screen out the best strains and ratios for the growth of Codonopsis pilosula enzyme extract. Experimental results showed that the combined strains were suitable for the growth of Codonopsis pilosula enzyme extract. Through screening at different ratios, the optimal ratio of LZU-J-TSL6:LZU-J-QA25 = 3:1 for fermenting the Codonopsis pilosula enzyme extract was determined to be the best for bacterial growth. Furthermore, the optimal inoculum size for fermentation was determined to be 3%, the fermentation temperature to be 36℃, and the fermentation time to be 24 hours.

[0124] Example 3

[0125] A fermentation method for Codonopsis pilosula, the steps of which are as follows:

[0126] (1) Mix Codonopsis pilosula and water at a mass ratio of 1:15 and then break the cell wall with a cell wall breaker to obtain Codonopsis pilosula homogenate;

[0127] (2) Codonopsis pilosula was mixed with mixed enzymes (cellulase and pectinase in a mass ratio of 1:1) at a mass ratio of 100:0.2, enzymatically hydrolyzed at 60℃ for 90 min, sterilized at 121℃ for 15 min, and filtered to obtain Codonopsis pilosula enzymatic hydrolysate.

[0128] (3) The enzymatic hydrolysate of Codonopsis pilosula was mixed with a compound microbial agent (Lactobacillus plantarum LZU-J-TSL6 and Lactobacillus plantarum LZU-J-QA25) at a mass ratio of 3:1; the bacterial count was 8×10⁻⁶. 8 Mix (each 100 sachets / mL) at a mass ratio of 100:3, ferment at 36℃ for 24 hours, filter out the residue, and sterilize at 90℃ for 20 minutes to obtain the fermented broth of Codonopsis pilosula.

[0129] Example 4

[0130] The changes in the content of polysaccharides, flavonoids, and codonopsis glycosides of Codonopsis pilosula before and after fermentation were measured. The changes in pH and total acid before and after fermentation were also measured. Subsequently, DPPH, ABTS, and hydroxyl radical scavenging rate were measured to investigate the changes in the in vitro antioxidant activity of Codonopsis pilosula before and after fermentation.

[0131] Table 9 Materials and Instruments

[0132]

[0133]

[0134] 1 Experimental Methods

[0135] 1.1 Determination of total polysaccharides

[0136] 1) Preparation of sulfuric acid-anthrone solution: Accurately weigh 0.1g of anthrone, add 100mL of sulfuric acid to a brown bottle to dissolve and shake well to obtain the solution.

[0137] 2) Preparation of glucose standard solution: Accurately weigh anhydrous glucose, dissolve it in purified water and shake well to prepare a glucose standard solution with a concentration of 0.12 mg / mL.

[0138] 3) Construction of the standard curve: Measure 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mL of glucose standard solution into test tubes, respectively, add water to a final volume of 2.0 mL, and quickly add 6 mL of sulfuric acid-anthrone solution. Shake well. After standing at room temperature for 15 min, cool in an ice-water bath for 15 min, and then measure the OD. 625 Value. Plot concentration-OD. 625 Standard curve of values.

[0139] 4) Preparation of the test sample: Take 5 mL each of the Codonopsis pilosula enzyme extract and fermentation broth, slowly add 50 mL of ethanol, let stand overnight at 4℃, and centrifuge at 5000 r / min for 5 min to obtain the total polysaccharide. Redissolve the total polysaccharide in hot water and bring the volume to 50 mL, then cool. Shake well and take an appropriate amount of the solution for centrifugation. Take 3 mL of the supernatant and add water to bring the volume to 25 mL, which is the sample solution. Accurately measure 2 mL of the sample solution into a test tube, and proceed as described above, "quickly add 6 mL of sulfuric acid-anthrone solution and shake well," to determine the OD. 625 The anhydrous glucose content in the enzyme extract and fermentation broth of Codonopsis pilosula was calculated based on the standard curve.

[0140] 1.2 Determination of total flavonoids

[0141] The total flavonoid content in the samples was determined by the aluminum trichloride (AlCl3) colorimetric method.

[0142] 1) Preparation of AlCl3 solution: Accurately weigh AlCl3, dissolve it in purified water, and prepare a 0.1 mol / L AlCl3 solution.

[0143] 2) Accurately weigh anhydrous sodium acetate and glacial acetic acid, dissolve them in purified water, and prepare 0.2 mol / L sodium acetate solution and 0.2 mol / L acetic acid solution; then prepare a sodium acetate-acetic acid buffer solution with pH 5.2.

[0144] 3) Preparation of rutin reference standard: Take 10 mg of rutin standard and dilute to 50 mL with methanol to prepare a 0.2 mg / mL rutin reference standard solution.

[0145] 4) Construction of the standard curve: Measure 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mL of rutin standard solution into test tubes, add 2 mL of AlCl3 solution and 1 mL of buffer solution, and bring the volume to 10 mL with methanol. Incubate in a 40℃ water bath for 10 min for color development, and measure the OD. 421 Value. Plot concentration-OD. 421 Standard curve of values.

[0146] 5) Preparation of the test sample: Accurately measure 0.2 mL of the sample solution into a test tube, and proceed as described above, starting with "Add 2 mL of AlCl3 solution". Measure the OD. 421 The rutin content in the Codonopsis pilosula enzyme extract and Codonopsis pilosula fermentation broth was calculated based on the standard curve.

[0147] 1.3 Determination of Codonopsis glycosides

[0148] 1) Chromatographic conditions: Waters C18 column, acetonitrile:water = 25:75 mobile phase, flow rate 1 mL / min, column temperature 30℃, detection wavelength 267 nm, injection volume 10 μL.

[0149] 2) Preparation of Codonopsis pilosula reference standard: Accurately weigh an appropriate amount of Codonopsis pilosula reference standard, dissolve and dilute it in methanol to prepare a reference standard stock solution of 0.2048 mg / mL.

[0150] 3) Construction of the standard curve: Accurately measure an appropriate amount of codonopsis glycoside reference standard stock solution to prepare codonopsis glycoside reference standard solutions with mass concentrations of 10, 20, 50, 100, 150, and 200 μg / mL, respectively. Inject 10 μL of each solution for HPLC analysis. Plot the standard curve with peak area (Y) on the ordinate and codonopsis glycoside mass concentration (X) on the abscissa.

[0151] 4) Preparation of test sample: Accurately measure 20 mL of Codonopsis pilosula fermentation broth sample, place it in an evaporating dish and evaporate to dryness, dissolve it in methanol to 5 mL, shake well, and filter it through a 0.22 microfiltration membrane to obtain the test sample.

[0152] 1.4 pH measurement: Take appropriate amounts of pH 2.0, pH 4.0 and pH 7.0 calibration solutions to calibrate the pH meter. Then take appropriate amounts of Codonopsis pilosula enzyme extract and fermentation broth, and measure the pH three times consecutively with the pH meter. Record the readings and take the average value.

[0153] 1.5 Determination of total acid: The enzyme extract and fermentation broth of Codonopsis pilosula were sonicated for 10 min to remove CO2 from the system. 30 mL of the test liquid was taken, 3 drops of phenolphthalein reagent were added, and titrated with 0.1 mol / L standard NaOH titrant. The titration endpoint was reached when the solution turned red and did not fade for 30 seconds. The titration volume was recorded. The titration was performed 3 times and the average value was taken. The total acid content was calculated based on the volume of titrant consumed.

[0154] 1.6 Determination of antioxidant activity of Codonopsis pilosula enzymatic extract before and after in vitro fermentation

[0155] 1) Preparation of DPPH solution: Accurately weigh 2.5 mg of DPPH and place it in a 25.0 mL volumetric flask. Prepare a DPPH solution with ethanol to a concentration of 0.1 mg / mL. Store in a refrigerator at 4°C in the dark.

[0156] 2) Preparation of ABTS solution: Accurately weigh 2.0 mM ABTS standard and 3.5 mM potassium persulfate, dissolve them separately in ultrapure water, mix them in equal volumes, and react in a dark environment for 24 h to form an ABTS+ solution. Store the solution at 4°C in a refrigerator, protected from light, for later use. Dilute the solution to achieve an absorbance of 0.70 ± 0.02 at 734 nm during the experiment.

[0157] 3) Preparation of test solution: Take 10 mL of Codonopsis pilosula enzyme extract and fermentation broth respectively, centrifuge at 3000 rpm for 10 min, and take the supernatant for later use.

[0158] 4) Preparation of positive control solution: Accurately weigh approximately 50.0 mg of vitamin C reference standard and place it in a 100 mL volumetric flask. Prepare a stock solution of the reference standard with methanol to a concentration of 0.5 mg / mL. Store the stock solution at 4°C in a refrigerator protected from light for later use. Before the assay, dilute the stock solution with methanol sequentially to prepare vitamin C reference solutions with mass concentrations of 0.05, 0.04, 0.03, 0.02, and 0.01 mg / mL.

[0159] 5) DPPH free radical scavenging rate determination: Dilute the test solution to concentrations of 6, 8, 10, 12, 14, 16, 18, and 20 mg / mL. Accurately pipette 60 μL of the test solution and 100 μL of the DPPH reference solution into a 96-well plate, mix well, and react at room temperature for 15 min. Measure the absorbance at 517 nm. Perform the determination in triplicate and take the average value. Using vitamin C as a positive control, calculate the DPPH free radical scavenging rate: DPPH free radical scavenging rate = [1 - (A1 - A2) / A0] × 100%; A0 is the absorbance of the control group, A1 is the absorbance of the sample group, and A2 is the absorbance of the blank sample group.

[0160] 6) ABTS free radical scavenging rate determination: Transfer the test solution of each component and dilute it to concentrations of 8, 10, 12, 14, 16, 18 and 20 mg / mL. Accurately transfer 80 μL and 100 μL of ABTS solution to each concentration. + The solutions were placed together in a 96-well plate, mixed well, and reacted at room temperature for 15 min. The absorbance was then measured at 734 nm. The measurements were performed in triplicate, and the average value was taken. Vitamin C was used as a positive control. ABTS + The formula for calculating the free radical scavenging rate is the same as the formula for calculating the DPPH free radical scavenging rate.

[0161] 7) Hydroxyl radical scavenging rate determination: The determination was performed according to the instructions of the hydroxyl radical scavenging kit.

[0162] 1.7 Data Processing

[0163] The comparisons between the two groups involved in the experiments in this chapter were performed using the t-test, with P ≤ 0.05 considered statistically significant. GraphpadPrism 9 software was used for graphing and analysis.

[0164] 2. Conclusion:

[0165] 2.1 Changes in active ingredients of Codonopsis pilosula enzymatic extract before and after fermentation

[0166] Changes in active ingredients of Codonopsis pilosula enzymatic extract before and after fermentation are shown in the figure. Figure 6 The polysaccharide content of *Codonopsis pilosula* enzyme extract before and after fermentation was determined using the sulfuric acid-anthrone method. The values ​​of glucose concentration and OD were plotted on the x-axis. 625Using the ordinate as the ordinate, a standard curve is plotted, yielding the linear regression equation: Y = 0.5857X + 0.1578, R0. 2 =0.9965, indicating a good linear relationship. Figure 6 A). The total polysaccharide content in the fermentation broth of Codonopsis pilosula was significantly lower than that in the enzyme extract. Figure 6 D) This indicates that during fermentation, polysaccharides are used by bacteria as a carbon source for growth, thus reducing their content.

[0167] The total flavonoid content in the system was determined using the AlCl3 method. The concentration of rutin standard solution was plotted as the x-axis, and OD... 421 Using the ordinate as the vertical axis, a standard curve is constructed, yielding the standard curve's linear regression equation: Y = 0.4233X - 0.0034, R0 2 =0.9966, indicating a good linear relationship. Figure 6 B). The total flavonoid content in the fermentation broth of Codonopsis pilosula was significantly increased compared to that in the enzymatic extract. Figure 6 E).

[0168] The content of ginsenosides in the enzyme extract of Codonopsis pilosula before and after fermentation was determined by high performance liquid chromatography (HPLC). A standard curve was plotted with ginsenoside concentration on the x-axis and peak area on the y-axis, and the equation of the standard curve was obtained as: Y = 5750X - 3691, R 2 =0.9969, indicating a good linear relationship. Figure 6 C). The content of codonopsis glycosides in the fermentation broth of Codonopsis pilosula was significantly higher than that in the enzyme extract. Figure 6 F).

[0169] The pH of the Codonopsis pilosula fermentation broth before and after enzyme extraction was measured using a pH meter. The results showed that the pH of the fermentation broth was significantly lower than that of the enzyme extract. Figure 6 G). Using phenolphthalein as an indicator, acid-base titration was performed on the enzyme extract and fermentation broth of Codonopsis pilosula. The results showed that the total acid content in the fermentation broth was significantly higher than that in the enzyme extract, which may be due to the accumulation of organic acid metabolites produced by Lactobacillus plantarum during fermentation.

[0170] 2.2 In vitro antioxidant activity of Codonopsis pilosula fermentation broth

[0171] from Figure 7 The results show that the fermentation broth of Codonopsis pilosula exhibits good antioxidant activity. Furthermore, compared to the enzyme extract of Codonopsis pilosula, the fermentation broth significantly improves the scavenging ability of ABTS, DPPH, and OH free radicals. Figure 7 AC).

[0172] Conclusion: This study investigated the changes in chemical composition and antioxidant activity of *Codonopsis pilosula* enzymatic extract before and after fermentation. The results showed that after fermentation, the polysaccharide content significantly decreased compared to before fermentation, while the flavonoid and codonopsis glycoside content significantly increased. The reduced polysaccharide content provided the carbon and nitrogen sources and nutrients needed for bacterial growth during fermentation. The increased flavonoid and codonopsis glycoside content indicated that large molecules were broken down into smaller molecules during fermentation, leading to their increased content. The pH value of the fermentation broth was lower than before fermentation, and the total acid content was significantly higher, possibly due to the accumulation of acidic metabolites produced during bacterial growth. Polysaccharides and flavonoids are the active components of *Codonopsis pilosula*, and also the main active components for combating oxidative stress, exhibiting anti-inflammatory, immunomodulatory, and metabolic effects. Therefore, the antioxidant activity of the *Codonopsis pilosula* enzymatic extract before and after fermentation was measured. The results showed that the scavenging capacity of ABTS, DPPH, and OH free radicals was significantly improved after fermentation, indicating that metabolites may have been transformed during fermentation, or substances related to antioxidant activity may have been produced.

[0173] Example 5

[0174] Changes in volatile components of Codonopsis pilosula enzymatic extract before and after fermentation

[0175] Volatile compounds in the samples were analyzed using gas chromatography-mass spectrometry (GC-SPME-GC-MS). GC-MS analysis was performed using an Agilent 7890B GC-MS system (Agilent, Santa Clara, CA, USA) equipped with a LEGO Pegasus BT mass-selective detector. A DB-WAS capillary column (30 m × 0.25 mm × 0.25 μm) was used. The helium carrier gas flow rate was 1.0 mL / min. 1 mL of sample was accurately weighed and placed in a 20 mL headspace vial, sealed, and incubated at 40 °C for 5 minutes. The temperature was then increased to 220 °C at a rate of 5 °C / min, followed by an increase to 250 °C at a rate of 20 °C / min, and maintained for 2.5 minutes. For mass spectrometry, the parameters were configured as follows: electron impact mode ionization 70 eV, full scan mode range 20–400 AMU. The compounds were identified by comparison with the NIST 2017 mass spectrometry library.

[0176] Results: The enzyme extract and fermentation broth samples of *Codonopsis pilosula* showed an aggregation trend within groups and a separation trend between groups, indicating a significant difference between the enzyme extract and fermentation samples. (PCR score graph) Figure 14The results showed that the samples of Codonopsis pilosula enzyme extract before and after fermentation were significantly different, with all samples falling within the 95% confidence interval. A total of 108 differentially expressed metabolites were identified, of which 80 were upregulated and 28 were downregulated. These 108 differentially expressed metabolites were screened according to the criterion of VIP > 1 and P < 0.05. The top 52 metabolites with significant changes were selected for cluster heatmap analysis, and the results are as follows: Figures 8-11 As shown, the abundance of 11 volatile compounds in the FCR was significantly lower than that in the NFCR, while the abundance of 23 volatile compounds was significantly higher than that in the NFCR, especially acid compounds. This indicates that fermentation of Codonopsis pilosula broth by Lactobacillus plantarum LZU-J-TSL and LZU-J-Q25 can increase the acid content in the Codonopsis pilosula broth, thereby extending its shelf life. The main compounds whose NFCR decreased after fermentation include acetone, 2,3-pentanedione, hexaldehyde, n-pentanoic acid, ethyl acetate, and 3-methylbutanal. n-pentanoic acid is a short-chain fatty acid and a bacterial metabolite. The main compounds that are elevated include ethanol, hexanoic acid, sorbic acid, propionic acid, nonanoic acid, acetic acid, 4-hexen-1-ol(4e)-acetate, alpha-terpineol, alpha-ionene, 2-acetylpyrrole, and 2(5h)-furanone. Alpha-ionene, 4-hexen-1-ol, (4e)-((4E)-4-hexen-1-ol), and furanone have a strong fruity and jam-like aroma. Alpha-terpineol can block the expression of NF-κB in tumor cell growth and has broad-spectrum antitumor activity. In summary, fermentation of Codonopsis pilosula by Lactobacillus plantarum LZU-J-TSL and LZU-J-Q25 can significantly alter its volatile components.

[0177] Whether in positive or negative ion mode, the effective substances in the Codonopsis pilosula enzyme extract and fermentation broth can be completely eluted in a short time, with good separation effect. As clearly shown in the figure, the types and quantities of chemical components in the Codonopsis pilosula enzyme extract and fermentation broth changed, and the content of compounds before and after fermentation also changed significantly. This indicates that fermentation consumes and utilizes the original substances while simultaneously generating new substances. Figure 12 FCR and NFCR are separated in the PCA scoring plot. Figure 13 A) All samples were within the 95% confidence interval, indicating a significant difference in the chemical composition of FCR and NFCR. The OPLS-DA score plot showed a clear separation between FCR and NFCR, which also confirmed the results of the PCA score plot. Figure 13 B). The R²Y value is 0.99 and the q² value is 0.1, indicating that the original model is reliable and there is no overfitting. Figure 13 C). The scatter plot size represents the VIP value of the OPLS-DA model; the larger the scatter plot, the larger the VIP value. The scatter plot color represents the final screening result: red indicates a significant upregulation of metabolites, blue indicates a significant downregulation of metabolites, and gray indicates no significant difference. Figure 13 D).

[0178] A statistical test was conducted using a pre-set threshold (VIP > 1 and P < 0.05) to screen for 220 compounds, including alkaloids, phenylpropanoids, organic acids, phenols, flavonoids, terpenes, vitamins, and amino acid derivatives. Cluster analysis was performed on the top 50 compounds showing significant changes, and the results are as follows: Figure 14As shown. Compared with NFCR, FCR showed a decrease in 24 potential biomarkers, such as Allose, Gamma-Linolenic acid, 2,6-Dimethylaniline, Maltol, 13-L-Hydroperoxylinoleic acid, Nonadecanoic acid, 3,4-Dihydroxyphthalate, Adenosine, Cytidine, and 4-Acetylbutyrate; in addition, 26 potential biomarkers, including 6-Phospho-beta-D-glucosyl-(1,4)-D-glucose, Gardenoside, Erucic acid, L-Valine, 5-Hydroxyindoleacetic acid, Aucubin, Pyridoxal phosphate, L-Histidine, Serotonin, D-Glucuronic Acid, 6-Hydroxymelatonin, L-Galactose, L-Phenylalanine, and Guanosine, increased. Among them, 6-Phospho-beta-D-glucosyl-(1,4)-D-glucose is a reducing monosaccharide with immune activity and can be used as an antioxidant in food; Gardenoside has laxative, analgesic, choleretic, anti-inflammatory, and soft tissue injury treatment effects, as well as inhibiting gastric juice secretion and reducing pancreatic amylase; L-Valine and L-Phenylalanine are natural essential amino acids for the human body. Figure 14 Fermentation significantly increased their content, laying the foundation for further research on FCR.

[0179] Example 6

[0180] 1. Experimental grouping, modeling, drug administration and experimental procedure

[0181] Sixty-four male SD rats were acclimatized for 7 days. After 7 days, the rats were randomly divided into 8 groups (n=8 per group) according to their body weight: blank control group (Group C), model group (Group M), omeprazole positive control group (Group A), low, medium, and high dose groups of Codonopsis pilosula fermentation broth (FCR-L, M, and H groups), unfermented Codonopsis pilosula group (NFCR group), and probiotic group (Group PB). The treatment of each group is shown in Table 10. After fasting for 24 hours but not water, all groups except the normal control group were administered anhydrous ethanol by gavage at a dose of 1.2 mL / 200 g. Within 5 minutes of successful gavage, rats showed symptoms such as generalized paralysis, mental stress, and rapid breathing and heartbeat. After fasting and water restriction for 4 hours, rats showed symptoms of generalized paralysis after 4 hours, indicating that the acute gastric mucosal injury model was successfully established. After successful modeling and routine feeding for 1 day, each drug-treated group was continuously administered the corresponding test drug for 15 days, while the normal group and model group were given an equal volume (1 mL / 200 g) of physiological saline. After modeling, rats in the positive group were treated with omeprazole (1.26 mL / d). Rats in the low, medium, and high dose groups of fermented Codonopsis pilosula were all treated with different doses of fermentation broth after modeling. According to the Chinese Pharmacopoeia, the recommended daily dose range for Codonopsis pilosula is 9-30 g / day. Based on traditional experience and relevant literature, and referring to the equivalent dose calculated by body surface area conversion between humans and rats, the low, medium, and high doses of Codonopsis pilosula fermentation broth prepared per rat (based on 200 g) were 2.835 mL, 4.725 mL, and 9.45 mL, respectively. The Codonopsis pilosula enzyme extract group and the probiotic group were given the same dose as the medium dose group of Codonopsis pilosula fermentation broth.

[0182] Table 10 Experimental Groups and Drug Administration

[0183]

[0184]

[0185] ① Codonopsis pilosula (Dang Shen) dosage: Humans: 9-30g / 60kg / day; Rats: 0.945-3.15g / kg / day. The feed-to-liquid ratio is 1:15. Based on a rat weight of 200g, the intake is 2.835-9.45mL / day. ② Omeprazole solution preparation: Dissolve 120mg of omeprazole in 40mL of distilled water and shake thoroughly to obtain a 3mg / mL omeprazole solution. Humans: 20mg / 60kg / day; Rats: 0.00033mg / kg / day. Based on a rat weight of 200g, the intake of the 3mg / mL omeprazole solution is 1.26mL / day.

[0186] 2 Evaluation Indicators

[0187] (1) Changes in body weight: Observe the rats' food intake, activity level, mental state, etc., and record the rats' body weight (once every 3 days).

[0188] (2) Morphological observation and index determination of gastric tissue: After 15 days of oral administration of the drug to rats in each group, they were fasted for 24 hours but allowed free access to water. The stomach was then removed by laparotomy, cut along the greater curvature, rinsed with ice-cold saline to remove contents, and laid flat for observation and photographic recording. The rinsed gastric tissue was gently wiped clean of blood vessels from the gastric mucosa with a cotton ball, and the ulcer index was assessed after unfolding. The ulcer index (ulcer area) was calculated based on the size and number of gastric mucosal bleeding and ulcer foci. The following criteria were used: 1 point for punctate ulcers, 2 points for linear ulcers <1mm in length, 3 points for 1-2mm, 4 points for 2-4mm, 5 points for lesion length >4mm, and doubled for erosion width >2mm. All scores were summed to obtain the total lesion score for each rat. The average score for each group was calculated as the ulcer index for that group. Gastric ulcer inhibition rate / % = [(Gastric ulcer index of model group rats - Gastric ulcer index of drug-treated group rats) / Gastric ulcer index of model group rats] × 100%.

[0189] (3) Pathological morphological observation of gastric tissue: After sacrificing rats, gastric tissue was taken and fixed in 10% formalin overnight. After dehydration, clearing, paraffin embedding, sectioning, spreading, scooping, baking, dewaxing and rehydration, the sections were sectioned and stained with hematoxylin-eosin (HE staining). Histological images were taken under a fluorescence microscope with 40x magnification.

[0190] Antioxidant properties of rat gastric tissue, serum inflammatory factors, and gastric mucosal protective factor activity assays: Antioxidant properties: Rat gastric tissue was homogenized into a 10% homogenate using physiological saline, centrifuged at 3000 r / min for 10 min, and the supernatant was collected for later use. According to the kit instructions, the activities or contents of oxidative stress factors superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GSH-PX), and catalase (CAT) in gastric tissue were detected. Serum inflammatory factors: According to the kit instructions, the expression levels of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and interleukin-10 (IL-10) inflammatory cytokines in the serum of SD rats in each group were detected, as well as the activity of myeloperoxidase (MPO). Gastric mucosal protective factors: According to the kit instructions, the expression level of prostaglandin E2 (PEG2) cytokine in the serum of SD rats in each group was measured; the activity of nitric oxide (NO) in the supernatant of SD rat gastric tissue homogenate was also measured.

[0191] Results: During the modeling period, except for the control group, the body weight of rats in all other groups decreased significantly; after administration, the body weight of rats in all groups gradually increased, so gavage with anhydrous ethanol will cause weight loss in rats. After administration, the gastric mucosal damage was repaired to some extent, the rats' food intake gradually recovered, and their body weight gradually increased. Figure 15 ).

[0192] After the experiment, the gastric tissue of each group of SD rats was dissected to observe the effect of FCR on the gastric mucosa of SD rats with experimental gastric ulcers. The results are as follows: Figure 16 As shown, the gastric mucosa of rats in the blank control group was intact, smooth, and light pink, with mucosal folds and no congestion or bleeding. In the model group, the gastric mucosa of rats showed multiple irregular, elongated bleeding streaks accompanied by numerous bleeding points, appearing deep red, with severe mucosal ulceration and erosion. Rats using omeprazole solution as a positive control showed reduced gastric ulcer severity. Rats using FCR solution as a preventative drug showed a reduction in gastric ulcer area, possibly in a dose-dependent manner.

[0193] The results of ulcer index and ulcer inhibition rate are shown in Table 11. Compared with the blank control group (Group C), the ulcer index of the model group (Group M) rats increased significantly, reaching 44.26±1.02, while the ulcer index of the model group (Group M) and the positive control group (Group A) decreased significantly to 4.13±0.89. The ulcer index of the Codonopsis pilosula enzyme extract group, the probiotic group, and the low, medium, and high dose groups of Codonopsis pilosula fermentation liquid were significantly reduced. Among them, the high dose group of Codonopsis pilosula fermentation liquid had the lowest ulcer index, reaching 6.75±0.62, and the highest gastric ulcer inhibition rate, reaching 69.67±3.01, almost close to that of the positive control group. The results indicate that FCR can effectively improve the bleeding status of the gastric mucosa in ethanol-induced acute gastric ulcer SD rats and effectively protect the gastric mucosa from ethanol damage. Meanwhile, compared with the NFCR group, the FCR group rats had a lower ulcer index and a higher ulcer inhibition rate, indicating that FCR had a better protective effect on the gastric mucosa of rats with gastric ulcers than the NFCR group. Figure 17 ).

[0194] Table 11 Gastric ulcer index and inhibition rate

[0195]

[0196]

[0197] In the KB group, the gastric mucosa of rats was intact and smooth, with regular gland arrangement, no shedding of mucosal epithelial cells, no congestion points or inflammatory cells, and no edema. In the M group, the gastric mucosa epithelium and gland arrangement were significantly damaged, with extensive epithelial cell shedding, disordered umbrella-shaped gland arrangement, and obvious congestion and swelling. In the A control group, the gastric mucosa showed mild damage, with no deep mucosal damage observed. Oral administration of FCR-L / M / H rats improved the shedding of epithelial cells and hemorrhagic damage to the gastric mucosa, and the gland arrangement gradually became more regular. The FCR-H group showed the least damage, closest to that of the KB group. These results indicate that FCR has a good therapeutic effect on the gastric tissue of SD rats with gastric ulcers, and the therapeutic effect of high-dose FCR is close to that of omeprazole solution.

[0198] Compared to the blank group (Group C), the levels of GSH-PX, SOD, and CAT were decreased, while the level of MDA was increased in the model group (Group M). Compared to the model group (Group M), the levels of GSH-PX, SOD, and CAT were increased, while the level of MDA was decreased in all other groups. Compared to the Codonopsis pilosula enzyme extract group (NFCR group), the levels of all components in the low, medium, and high dose groups of Codonopsis pilosula fermentation broth (FCR-L, M, H) were superior to those in the NFCR group. The results indicate that FCR can inhibit the oxidative stress response in SD rats with gastric ulcers and significantly improve the body's antioxidant capacity. Figure 18 ).

[0199] Compared to the blank group (Group C), anhydrous ethanol increased the levels of TNF-α, IL-1β, and MPO, while decreasing the level of IL-10. Compared to the model group (Group M), the ranitidine group (Group A) and the low, medium, and high dose groups of Codonopsis pilosula fermentation broth (FCR-L, M, and H groups) all decreased the levels of TNF-α and IL-1β to varying degrees, and increased the level of IL-10. Furthermore, only FCR-H could inhibit anhydrous ethanol-induced serum MPO. The results indicate that FCR has a strong protective effect against anhydrous ethanol-induced gastric injury. Figure 19 ).

[0200] Compared to the blank group (C group), the levels of PEG2 in the serum and NO in the gastric tissue of rats in the model group (M group) were significantly reduced. Compared to the M group, the levels of PEG2 and NO in the A group, FCR group, NFCR group, and PB group were significantly increased, indicating that FCR has an effective and good protective effect on the gastric mucosa of SD rats with gastric ulcers. Figure 20 ).

[0201] The above description is only a preferred embodiment of the present invention.

Claims

1. A fermentation method for Codonopsis pilosula, characterized in that, Includes the following steps: (1) Codonopsis pilosula is mixed with a mixed enzyme for enzymatic hydrolysis, sterilized and filtered to obtain Codonopsis pilosula hydrolysate; the mixed enzyme is cellulase and pectinase, with a mass ratio of 2~1:1~2; (2) The enzyme hydrolysate of Codonopsis pilosula was mixed with a compound microbial agent for fermentation, and then filtered and sterilized to obtain the fermentation liquid of Codonopsis pilosula; the compound microbial agent is Lactobacillus plantarum LZU-J-TSL6 and Lactobacillus plantarum LZU-J-QA25; The mass ratio of the *Codonopsis pilosula* involved in the mixed enzyme in step (1) is 100:0.1~0.3; In step (2), the mass ratio of the Codonopsis pilosula enzymatic hydrolysate to the compound microbial agent is 100:2~4; the mass ratio of Lactobacillus plantarum LZU-J-TSL6 to Lactobacillus plantarum LZU-J-QA25 is 2~4:1; and the bacterial count of the compound microbial agent is 7~9×10⁻⁶. 8 per mL.

2. The fermentation method of Codonopsis pilosula according to claim 1, characterized in that, The enzymatic hydrolysis in step (1) is carried out at a temperature of 55~65℃ for 80~100 min.

3. The fermentation method of Codonopsis pilosula according to claim 1, characterized in that, The Codonopsis pilosula mentioned in step (1) is a Codonopsis pilosula homogenate; the preparation method of the Codonopsis pilosula homogenate is: mix Codonopsis pilosula with water at a mass ratio of 1:13~17 and then crush it to obtain Codonopsis pilosula homogenate.

4. The fermentation method of Codonopsis pilosula according to claim 1, characterized in that, The fermentation time in step (2) is 20~28h, and the fermentation temperature is 34~38℃.

5. The fermented liquid of Codonopsis pilosula prepared by the fermentation method of Codonopsis pilosula according to any one of claims 1 to 4.

6. The application of the Codonopsis pilosula fermentation broth according to claim 5 in the preparation of antioxidant agents.

7. The use of the Codonopsis pilosula fermentation liquid according to claim 5 in the preparation of a drug for treating gastric mucosal injury.

Images