Medicinal and edible leavening and preparation method and application thereof

Abstract

The invention provides a medicinal and edible leavening and a preparation method and application thereof. The preparation method of the medicinal and edible leavening comprises the following steps: mixing codonopsis pilosula, astragalus membranaceus, platycodon grandiflorum and pleurotus tuber-regium to obtain a medicinal and edible mixture, and crushing the medicinal and edible mixture to obtain a medicinal and edible crushed mixture; and mixing the medicinal and edible crushed mixture with water to prepare a medicinal and edible crushed mixed solution, inoculating lactobacillus plantarum into the medicinal and edible crushed mixed solution for fermentation treatment, collecting fermentation liquor after the fermentation treatment is finished, and drying the fermentation liquor to obtain the medicinal and edible fermented product. Experiments show that the medicinal and edible homologous substance provided by the invention has remarkable anti-inflammatory and anti-oxidation effects, has a protective effect on kidney injury caused by oxidative stress and inflammation, relieves the progress of chronic kidney diseases, and provides a new thought for preparing products for relieving and treating chronic kidney diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and in particular to a medicine-food homologous fermentation product and a preparation method and application thereof. Background Art

[0002] In recent years, the incidence of kidney diseases such as chronic kidney disease (CKD) has continued to rise. Currently, the main treatments for CKD include drug therapy and dialysis therapy. Drug therapy mainly delays further deterioration of renal function by controlling underlying diseases such as hypertension and diabetes, but it cannot reverse damaged renal function. Although dialysis therapy can replace some kidney function, its high cost and complexity of treatment make it difficult to significantly improve the patient's quality of life. Therefore, it is particularly important to develop new methods to prevent and alleviate CKD.

[0003] Compared with prescription drugs or traditional medicines, functional foods can not only provide preventive nutritional supplements for healthy people, but also help people with abnormal physiological functions to recover faster. The main difference between functional foods and drugs is that they have higher safety and can be freely taken without a prescription. Studies have shown that oxidative stress and inflammation play a key role in the development of CKD, leading to kidney tissue damage. Therefore, exploring strategies to reduce inflammation and oxidative stress responses is of great significance for the treatment of CKD. Summary of the invention

[0004] The present invention provides a medicinal and edible fermented product and a preparation method thereof that can effectively reduce inflammation and oxidative stress reactions, and is expected to provide a solution for alleviating chronic kidney disease.

[0005] The first aspect of the present invention provides a method for preparing a medicinal and edible fermented product, comprising:

[0006] Mixing Codonopsis pilosula, Astragalus membranaceus, Platycodon grandiflorum and Pleurotus ostreatus to obtain a medicine-food mixture, and crushing the medicine-food mixture to obtain a medicine-food crushed mixture;

[0007] The edible and medicinal crushed mixture is mixed with water to obtain an edible and medicinal crushed mixed liquid, Lactobacillus plantarum is inoculated into the edible and medicinal crushed mixed liquid for fermentation, and after the fermentation is completed, the fermentation liquid is collected and dried to obtain an edible and medicinal fermentation product.

[0008] Codonopsis pilosula is a perennial herbaceous vine of the genus Codonopsis in the Campanulaceae family. It contains a variety of active ingredients, such as polysaccharides and saponins, which have been shown to have immunomodulatory and antioxidant effects, can reduce oxidative stress and inflammatory responses, and alleviate kidney damage. Astragalus is a plant of the genus Astragalus in the leguminous family. Its main active ingredients include astragaloside I and polysaccharides, which help to alleviate CKD-related kidney damage by reducing oxidative stress, improving microcirculation, and enhancing immune function. Platycodon grandiflorum is a perennial herb of the genus Platycodon in the Campanulaceae family. It is rich in saponins and a variety of phenolic compounds, has anti-inflammatory, antioxidant and immunomodulatory effects, and can reduce inflammatory responses by inhibiting the production of inflammatory mediators. Tiger milk mushroom is a wood-decomposing fungus with significant antioxidant and anti-inflammatory properties. It helps protect the kidneys from damage by inhibiting oxidative stress responses and regulating the immune system. The present invention mixes the above four edible and medicinal substances and ferments them using Lactobacillus plantarum, which helps to improve the biological activity, absorption rate and bioavailability of the four edible and medicinal substances, enhance the antioxidant and anti-inflammatory properties of the edible and medicinal substances, and increase the regulatory effect on intestinal microorganisms, showing greater potential in the prevention and treatment of chronic kidney disease.

[0009] In the preparation method as described above, the mass ratio of Codonopsis pilosula, Astragalus membranaceus, Platycodon grandiflorum and Pleurotus ostreatus is (0.8-1.2):(0.8-1.2):(0.8-1.2):(0.8-1.2), which can be appropriately adjusted according to actual needs.

[0010] In the preparation method as described above, the particle size of the edible and medicinal powdered mixture is not higher than 80 μm, and an appropriate particle size is selected according to the process requirements.

[0011] In the preparation method as described above, the mass volume ratio of the edible and medicinal powdered mixture to water is 0.2-0.3.

[0012] In the preparation method as described above, the edible and medicinal powdered mixed solution further comprises skim milk powder and sucrose; the mass of the skim milk powder is 3%-5% of the total volume of the edible and medicinal powdered mixed solution, and the mass of the sucrose is 6%-10% of the total volume of the edible and medicinal powdered mixed solution.

[0013] In the preparation method as described above, the fermentation temperature can be selected to be in the range of 35-40° C., and the fermentation time is 8-14 hours.

[0014] The second aspect of the present invention provides a medicinal and edible fermented product prepared according to the above preparation method.

[0015] The third aspect of the present invention provides the use of the above-mentioned medicinal and edible fermentation product in reducing cellular inflammatory response and / or oxidative stress response.

[0016] A fourth aspect of the present invention provides a method for reducing cellular inflammatory response and / or oxidative stress response, wherein the above-mentioned medicinal and edible fermented product is mixed with cells for culture to reduce the cellular inflammatory response and / or oxidative stress response.

[0017] The fifth aspect of the present invention provides the use of the above-mentioned medicinal and edible fermentation product in the preparation of a product for alleviating chronic nephritis.

[0018] The sixth aspect of the present invention provides a product comprising the above-mentioned medicine-food fermentation product.

[0019] The product as described above is one of food, health care product or medicine. Further, the product is preferably a health care product.

[0020] The present invention prepares a medicine-food fermentation product based on four medicine-food raw materials. Experiments show that the medicine-food fermentation product provided by the present invention has significant anti-inflammatory and antioxidant effects, protects kidney damage caused by oxidative stress and inflammation, alleviates the progression of chronic kidney disease, and provides a new idea for preparing products for alleviating and treating chronic kidney disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The present invention is to use Lactobacillus plantarum to ferment the medicine-food crushed mixed liquid and analyze the content of effective ingredients in the fermentation liquid at different fermentation times; wherein A represents the content analysis result of total polyphenols in the fermentation liquid; B represents the content analysis result of total flavonoids in the fermentation liquid; C represents the content analysis result of total saponins in the fermentation liquid; D represents the content analysis result of total polysaccharides in the fermentation liquid;

[0022] Figure 2 The results of the analysis of the antioxidant capacity of the fermented broth at different fermentation times after fermentation of the mixed liquor of medicinal and edible substances by using plant lactobacillus; wherein A represents the analysis result of the scavenging rate of DPPH free radicals by the fermented broth; B represents the determination result of the scavenging rate of ABTS free radicals by the fermented broth; and C represents the determination result of the ferric ion reducing capacity by the fermented broth;

[0023] Figure 3 The results of the determination of the effect of the medicinal and edible fermentation products on cell viability; A represents the determination result of HK-2 cells, and B represents the determination result of Raw264.7 cells;

[0024] Figure 4 Results of the HK-2 cell viability test of the medicinal and edible fermentation products;

[0025] Figure 5The effect of food-medicine fermentation products on the reactive oxygen content of HK-2 cells under oxidative stress; A is the result of flow cytometry detection, and B is the statistical result of the reactive oxygen content of HK-2 cells;

[0026] Figure 6 The effects of the food-medicine fermentation product on SOD, GSH-Px and MDA of HK-2 cells induced by H2O2; A is the effect on MDA, B is the effect on GSH-Px, and C is the effect on SOD;

[0027] Figure 7 The effects of the food-medicine fermentation product on the mRNA expression levels of Caspase-3 gene, MMP-9 gene, JUN gene, HO-1 gene and KIM-1 gene in HK-2 cells; A is the effect on the mRNA expression level of Caspase-3 gene, B is the effect on the mRNA expression level of JUN gene, C is the effect on the mRNA expression level of MMP-9 gene, D is the effect on the mRNA expression level of KIM-1 gene, and E is the effect on the mRNA expression level of HO-1 gene;

[0028] Figure 8 The effect of food and medicine fermentation products on the morphology of Raw264.7 cells;

[0029] Fig. 9 Figure 3 shows the effects of food-medicine fermentation products on the NO content in Raw264.7 cells and the mRNA expression levels of iNOS, IL-6, IL-1β, NF-κB and TNF-α genes; A is the effect on the NO content in Raw264.7 cells, B is the effect on the mRNA expression level of NF-κB gene, C is the effect on the mRNA expression level of iNOS gene, D is the effect on the mRNA expression level of IL-6 gene, E is the effect on the mRNA expression level of IL-1β gene, and F is the effect on the mRNA expression level of TNF-α gene.

[0030] Figure 1-9 Different letters indicate significant differences between the two groups (p < 0.05); ns indicates no significant differences between the two groups. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Example 1. Preparation of food and medicine fermentation product.

[0033] 1. Weigh Codonopsis pilosula, Astragalus membranaceus, Platycodon grandiflorum and Pleurotus eryngii in a mass ratio of 1:1:1:1, and mix to obtain a medicinal and edible mixture. After drying the medicinal and edible mixture including the four raw materials, crush it with an ultrafine grinder, and sieve the crushed mixture with a 75 μm (200 mesh) sieve to obtain a medicinal and edible crushed mixture with a particle size of no more than 75 μm that can pass through the sieve.

[0034] 2. Mix the sieved edible and medicinal powdered mixture with sucrose and skim milk powder, and add water to prepare an edible and medicinal powdered mixture, wherein the mass volume ratio of the edible and medicinal powdered mixture to water is 0.25, the mass of the skim milk powder is 4% of the total volume of the edible and medicinal powdered mixture, and the mass of the sucrose is 8% of the total volume of the edible and medicinal powdered mixture.

[0035] 3. Inoculate Lactobacillus plantarum (strain purchased from China Industrial Microbiological Culture Collection Center, strain number CICC 20764) in the above-mentioned edible and medicinal powdered mixed solution at an inoculation rate of 4%-6%, and perform constant temperature static fermentation.

[0036] Example 2: Effect of fermentation time on the performance of food and medicine fermentation products

[0037] 1. Analysis of the changes of active ingredients during fermentation as the fermentation days increase

[0038] The method provided in Example 1 was used. The fermentation broth was collected after 0, 2, 4, 6, 8, 10, 12, and 14 hours of fermentation, and the fermentation broth was centrifuged at 5000 r / min for 10 minutes to remove the suspended residue in the fermentation broth. The centrifugal supernatant was collected and the content (mg / mL) of total polysaccharides, total flavonoids, total saponins, and total polyphenols in the centrifugal supernatant was detected. Three groups of experiments were set up in parallel, and the experimental data were expressed as mean ± standard deviation. GraphPad Prism 9 software was used for data analysis and visualization, and one-way analysis of variance was used to evaluate statistical significance.

[0039] The total polysaccharides were determined by the phenol-sulfuric acid method, in which polysaccharides were reacted with phenol under strong acidic conditions to produce yellow compounds, and then the absorbance was measured by a spectrophotometer; the total flavonoids were determined by the aluminum ion colorimetric method, in which flavonoid compounds formed a stable complex with aluminum ions, and then the absorbance was measured at a certain wavelength; the total saponins were determined by the vanillin-sulfuric acid method, in which saponins and vanillin reacted under acidic conditions to produce red compounds, and then the absorbance was measured by a spectrophotometer; the total polyphenols were determined by the Folin-phenol reagent method, in which polyphenol compounds reacted with the Folin-phenol reagent to produce blue compounds, and then the absorbance was measured by a spectrophotometer.

[0040] The experimental results are as follows Figure 1 As shown, it can be seen that the fermentation of Lactobacillus plantarum significantly improved the nutritional quality of the edible and medicinal powdered mixture. At different fermentation time points, the total phenol content, total flavonoid content, total polysaccharide content and total saponin content all showed significant increases. Specifically, Figure 1 As shown in Figure A, the total phenol content showed an overall increasing trend during the fermentation process, reaching the highest value at the 14th hour of fermentation, with an increase of 17.6%. Figure 1 As shown in Figure B, as the fermentation time increased, the total flavonoid content first increased and then decreased, reaching the highest value at 10 hours, an increase of 12.7% compared to before fermentation. Figure 1 As shown in Figure C, as the fermentation time increases, the total saponin content increases as a whole, reaching the highest value at 8 hours, with an increase of 45.13%. Figure 1 As shown in D, with the increase of fermentation time, the total polysaccharide content showed a slow upward trend and reached the highest value at 10 hours, an increase of 48.60% compared with before fermentation.

[0041] 2. Antioxidant capacity test of medicinal and edible fermented products obtained at different fermentation times

[0042] The method provided in Example 1 was used to collect the fermentation broth after 0, 2, 4, 6, 8, 10, 12, and 14 hours of fermentation, and the fermentation broth was centrifuged at 5000 r / min for 10 minutes to remove the suspended residue in the fermentation broth, collect the centrifuged supernatant, and detect the antioxidant properties of the fermentation supernatant, including DPPH free radical scavenging rate, iron ion reducing ability (TPTZ method), and ABTS free radical scavenging ability.

[0043] Determination of DPPH free radical scavenging rate: The fermentation broth was centrifuged at 5000 rpm for 10 minutes, and the supernatant was taken. 3.6 mL of 1×10 -4 mol / L DPPH solution, and the absorbance was measured at 517 nm after 30 minutes of reaction. Each sample was measured three times in parallel to calculate the DPPH clearance rate.

[0044] Determination of iron ion reducing ability (TPTZ method): Prepare TPTZ working solution, make Trolox standard solutions with different concentration gradients, mix with TPTZ working solution and measure absorbance at 593nm, and draw a standard curve. Take the sample and mix with TPTZ working solution for 4 minutes and measure absorbance. The iron ion reducing ability is expressed in mmol (Trolox) / L.

[0045] Determination of ABTS free radical scavenging ability: prepare ABTS free radical solution, use Trolox standard solution with different concentration gradients to mix with ABTS solution and measure the absorbance at 734 nm, measure the absorbance after 30 minutes of reaction, and express the ABTS scavenging ability in mmol (Trolox) / L.

[0046] The experimental results are as follows Figure 2 As shown in the figure, it can be seen that the medicinal and edible fermentation broth exhibits significant antioxidant properties. With the extension of fermentation time, the iron ion reduction ability, ABTS free radical scavenging ability and DPPH free radical scavenging ability are significantly improved, which reflects the release of active substances and the enhancement of antioxidant activity during the fermentation process. Figure 2 As shown in Figure A, the DPPH free radical scavenging rate gradually increased during the fermentation process. After 14 hours of fermentation, the scavenging rate increased from 85.8% before fermentation to 95.8%, an increase of 1.12 times. Figure 2 As shown in Figure B, with the increase of fermentation time, the ABTS free radical scavenging capacity showed a slow increase trend. After 14 hours of fermentation, the scavenging capacity increased from 0.92mmol Trolox / L before fermentation to 1.03mmol Trolox / L, an increase of 1.21 times. Figure 2 As shown in C, the iron ion reducing capacity gradually increased with the increase of fermentation time. After 14 hours of fermentation, the iron ion reducing capacity increased from 0.69mmol Trolox / L before fermentation to 0.92mmol Trolox / L, an increase of 1.33 times.

[0047] Example 3: Analysis of the cytotoxicity of the food-drug fermentation product (named LFCAPT)

[0048] HK-2 cells and Raw 264.7 cells were placed in an incubator for 4 hours, and the cell adhesion was observed. When the cells grew to 80% confluence, they were subcultured, the old culture medium was aspirated with a pipette, the cells were rinsed once with PBS, 1 mL of 0.25% trypsin was added for digestion for 120 s, 2 mL of serum-containing culture medium was added to terminate the digestion, the cells were mixed by pipetting, the cell suspension was collected, centrifuged at 1000 r / min for 5 min, 1 mL of new culture medium was added by pipetting to disperse the cells, and the cells were inoculated in a 96-well plate. The remaining cell suspension was inoculated in a new culture flask for continued subculture.

[0049] The food-medicine homologous fermentation product LFCAPT was dissolved in DMEM / F12 medium to prepare a mixed solution with different gradient concentrations (0, 125, 250, 500, 1000, 2000 μg / mL). The food-medicine homologous fermentation product (LFCAPT) was dissolved in DMEM high-glucose medium (containing 10% special fetal bovine serum, 1% antibiotics (penicillin-streptomycin 100×)) to prepare a mixed solution with different gradient concentrations (0, 25, 50, 100, 200, 500, 1000, 2000 μg / mL).

[0050] HK-2 cells and Raw 264.7 cells were cultured at 1×10 5 The cells were inoculated at a concentration of 100 μL / mL in a 96-well plate, with 100 μL per well. After 24 hours of cell growth, a cytotoxicity test was performed. 10 μL of the mixture was added to each well, with 6 replicates per group. After 24 hours of culture, the original culture medium was aspirated, and 100 μL of CCK-8 mixture prepared at a ratio of 1:10 was added to each well. After continuing to culture in the incubator for 20 minutes, the absorbance was detected at 450 nm. The cell viability (%) was calculated according to formula 1.

[0051]

[0052] In formula 1: A b is the absorbance value of the cell group and the LFCAPT group, A0 is the absorbance value of the no-cell group, and A is the absorbance value of untreated cells (without LFCAPT).

[0053] Test results such as Figure 3 As shown, the food-medicine fermentation product LFCAPT has good biosafety for HK-2 cells and RAW264.7 cells at the concentrations studied.

[0054] Example 4: Protective effect of food-drug fermentation product (named LFCAPT) on oxidative stress in HK-2 renal tubular epithelial cells induced by H2O2

[0055] 1. Cell grouping and viability determination

[0056] After being fixed for 24 hours, the HK-2 cells were divided into 6 groups, namely, control group, hydrogen peroxide group, hydrogen peroxide-vitamin C group, hydrogen peroxide-LFCAPT low-dose group, hydrogen peroxide-LFCAPT medium-dose group and hydrogen peroxide-LFCAPT high-dose group; the cells in control group were incubated under normal culture conditions for 12 hours; the cells in hydrogen peroxide group were incubated under normal culture conditions for 9 hours, and then the cells were stimulated with 150 μg / ml H2O2 for 3 hours; the cells in hydrogen peroxide-vitamin C group were incubated under 1 mM vitamin C for 9 hours, and then the cells were stimulated with 150 μg / ml H2O2 for 3 hours; the cells in hydrogen peroxide-LFCAPT low-dose group were incubated under 250 μg / ml LFCAPT for 9 hours, and then the cells were stimulated with 150 μg / ml H2O2 for 3 hours; the cells in hydrogen peroxide-LFCAPT medium-dose group were incubated under 500 μg / ml LFCAPT for 9 hours, and then the cells were stimulated with 150 μg / ml The cells were stimulated with H2O2 for 3 hours; the cells in the hydrogen peroxide-LFCAPT high-dose group were incubated with 1000 μg / ml LFCAPT for 9 hours, and then stimulated with 150 μg / ml H2O2 for 3 hours.

[0057] After the stimulation, the original culture medium was removed, and 100 μL of CCK-8 mixed solution prepared at a ratio of 1:10 was added to each well, and the cells were cultured in the incubator for 20 minutes, and then the absorbance was measured at 450 nm. The cell viability (%) was calculated according to Formula 1.

[0058] Test results such as Figure 4 As shown in the figure, compared with the model group, the pretreatment groups of LFCAPT fermentation products at different concentrations significantly increased the viability of HK-2 cells. In particular, at a concentration of 500 μg / mL, LFCAPT fermentation products increased cell viability to 77.44%, an increase of 27.1% over the model group. The VC control group (1 nM) also significantly increased cell survival rate to 83.92%, an increase of 33.59% over the model group. This indicates that LFCAPT fermentation products have a significant protective effect on HK-2 cells damaged by H2O2.

[0059] 2. Detection of reactive oxygen species (ROS) by flow cytometry

[0060] HK-2 cells in the logarithmic growth phase were taken and digested to make a suspension. The number of cells was counted using a cell counting plate and diluted to 1×10 5 / mL. 1mL of cell suspension and 1mL of serum-containing medium were mixed and inoculated into a 96-well plate. After culturing in the incubator for 24 hours, the supernatant was discarded. The cells were grouped according to the above grouping method and treated in the same way. After the treatment was completed, the supernatant was discarded again. The cells were digested and collected by centrifugation with 1mL of serum-containing medium. Then, ROS in the cells were detected by flow cytometry and using the DCFH-DA fluorescent probe.

[0061] The results are as follows Figure 5 As shown in the figure, compared with the control group, the ROS level in the HK-2 model group increased significantly. The ROS levels in the groups treated with different concentrations of LFCAPT fermentation products did not change significantly, but were significantly lower than those in the model group (P < 0.01). At concentrations of 250, 500, and 1000 μg / mL, the ROS levels decreased by 88.26%, 88.16%, and 87.04%, respectively. The VC control group also significantly reduced the ROS level, indicating that LFCAPT fermentation products can effectively remove H2O2-induced ROS, and its effect is comparable to that of VC.

[0062] 3. Determination of biochemical indicators

[0063] HK-2 cells in the logarithmic growth phase were taken and digested to make a suspension. The number of cells was counted using a cell counting plate and diluted to 1×10 5 / mL. Mix 1mL of cell suspension and 1mL of serum-containing medium and inoculate in a 96-well plate. After culturing in the incubator for 24 hours, aspirate the supernatant. Group the cells according to the above grouping method and perform the same treatment. After the treatment is completed, aspirate the supernatant again. Digest the cells and collect the cells by centrifugation with 1mL of serum-containing medium. Then use SOD, GSH-Px, and MDA commercial kits for determination.

[0064] like Figure 6 As shown in Figure A, after treatment with 150 μg / mL H2O2, the MDA content of HK-2 cells increased significantly to 1.26 nmol / mL. The VC control group significantly reduced the MDA content to 0.26 nmol / mL, and different concentrations of LFCAPT fermentation products also significantly reduced the MDA content, although the difference between different concentrations was not significant. The MDA content of all LFCAPT-treated groups was significantly lower than that of the model group, indicating that LFCAPT fermentation products have the ability to reduce oxidative damage. Figure 6As shown in Figure BC, LFCAPT fermentation products significantly increased the levels of SOD and GSH-Px in HK-2 cells damaged by H2O2. After treatment with 150μg / mL H2O2, the activity of cellular SOD decreased significantly by 27.3%. After adding 250μg / mL LFCAPT fermentation products, the activity of SOD increased significantly to 7.02U / mg prot, and the VC control group also significantly increased the activity of SOD. Similarly, the activity of cellular GSH-Px decreased significantly by 36.5% after H2O2 treatment. The GSH-Px activity of the groups treated with different concentrations of LFCAPT fermentation products was significantly higher than that of the model group, and the GSH-Px activity of the 250μg / mL group increased to 96.42U / mg prot.

[0065] 4. Effects on mRNA expression of oxidative stress cytokines and inflammatory factors

[0066] HK-2 cells in the logarithmic growth phase were taken and digested to make a suspension. The number of cells was counted using a cell counting plate and diluted to 1×10 5 / mL. 1mL of cell suspension and 1mL of serum-containing culture medium were mixed and inoculated into a 96-well plate. After culturing in the incubator for 24 hours, the supernatant was discarded. The cells were grouped according to the above grouping method and treated in the same way. Total RNA in the cells was extracted using the Trizol kit, and the total RNA concentration was measured using an ultra-micro spectrophotometer. cDNA was synthesized using the PrimeScript RT kit, and real-time quantitative polymerase chain reaction (qPCR) was performed using the CFX96 real-time PCR system. The genes detected included the expression levels of Caspase-3 gene, MMP9 gene, JUN gene, HO-1 gene, and KIM-1 gene, and β-actin was the internal reference gene. The primer sequences are shown in Table 1.

[0067] Table 1 Gene primer sequences

[0068]

[0069]

[0070] The results of the test are as follows Figure 7As shown in the figure, it can be seen that after HK-2 cells were co-incubated with H2O2 for 3 hours, the gene expression of oxidative damage genes iNOS, Caspase-3, MMP9, JUN and kidney injury factor KIM-1 was significantly upregulated, and the gene expression of antioxidant gene HO-1 was significantly downregulated. After treatment with 250, 500 and 1000 μg / mL concentrations of LFCAPT fermentation products, and VC control group (1nM), the expression of these oxidative damage genes was significantly reduced, while the expression of antioxidant gene HO-1 was significantly increased. Different concentrations of fermentation products LFCAPT and VC showed significant antioxidant effects. In terms of Caspase-3 gene expression, the highest concentration of LFCAPT fermentation product group (1000 μg / mL) decreased it by about 78.56%, while the VC control group decreased it by 78.00%. Similar significant downward trends were also observed in the expression changes of MMP9 and JUN genes. In contrast, the expression of the antioxidant gene HO-1 was significantly upregulated in the treatment groups, with the 500 μg / mL group increasing by 243.05%. LFCAPT fermentation products significantly regulated the expression of oxidative damage and antioxidant-related genes at different concentrations, reduced the expression of oxidative damage genes, and upregulated the expression of the antioxidant gene HO-1. These results indicate that LFCAPT fermentation products have significant antioxidant effects, demonstrating their potential in alleviating H2O2-induced oxidative stress, and providing a scientific basis for their application in the prevention and treatment of oxidative stress-related diseases.

[0071] Example 5: Protective effect of LFCAPT on inflammation in Raw264.7 cells

[0072] 1. Take Raw264.7 cells in logarithmic growth state, digest the cells and make a suspension. Count the number of cells using a cell counting plate and dilute them to 1×10 5 / mL. Add 1mL of cell suspension and 1mL of serum-containing culture medium to each well, mix well and inoculate in a 6-well plate. After culturing in the incubator for 24 hours, aspirate and discard the supernatant. Raw264.7 cells were grouped and treated as follows. Control group: cells were incubated under normal culture conditions for 24 hours; LPS group: cells were incubated with 1.0 μg / mL LPS in the culture medium for 24 hours to induce inflammatory response in Raw264.7 cells; dexamethasone group: cells were incubated with 1.0 μg / mL LPS and 80 μg / ml dexamethasone in the culture medium for 24 hours; low-concentration fermentation group: cells were incubated with 1.0 μg / mL LPS and 25 μg / ml LFCAPT in the culture medium for 24 hours; medium-concentration fermentation group: cells were incubated with 1.0 μg / mL LPS and 50 μg / ml LFCAPT in the culture medium for 24 hours; high-concentration fermentation group: cells were incubated with 1.0 μg / mL LPS and 100 μg / ml LFCAPT in the culture medium for 24 hours. After the treatment, the supernatant was discarded again. The cells were washed three times with PBS solution and fixed with 4% paraformaldehyde for 10-20 minutes. Then wash the cells 2-3 times with ultrapure water. Then stain with 0.5% crystal violet for 10 minutes. Then wash the stained cells several times with ultrapure water. Finally, observe the stained cells under an inverted microscope.

[0073] The staining results are as follows Figure 8 As shown in the figure, under normal conditions, RAW264.7 cells are oval and smooth and round in shape. However, under LPS induction, these cells showed obvious response characteristics, including an increase in the number of pseudopodia, a dendritic cell morphology, and an increase in intracellular bubbles and particles. Different concentrations of LFCAPT water extract significantly inhibited the response of cells to LPS and did not have a negative impact on cell morphology. When the LFCAPT concentration was 25μg / mL, many cells still showed a dendritic morphology; at concentrations of 50μg / mL and 100μg / mL, most of the cell morphology returned to normal cell morphology.

[0074] 2. The NO content of the cell supernatant of each group was measured using a NO commercial kit, and the expression levels of iNOS gene, IL-6 gene, IL-1β gene, NF-κB gene and TNF-α gene were tested using the same method as in Example 4. The primers used are shown in Table 2.

[0075] Table 2 Nucleotide sequences of primers used

[0076]

[0077] Test results such as Fig. 9As shown, it can be seen that after RAW264.7 cells were co-incubated with LPS for 48 hours, inflammatory mediators (including NO levels and iNOS, NF-кB, IL-6, IL-1β, and TNF-α gene expression) were significantly upregulated, inducing an inflammatory response. After treatment with different concentrations of LFCAPT ferments or 80 μg / mL of dexamethasone, the expression of these inflammatory mediators was significantly reduced, showing an anti-inflammatory effect. Specific results showed that there was no significant difference in the gene expression of NO, IL-1β, NF-кB, and TNF-α under different concentrations of LFCAPT treatment, but all showed anti-inflammatory effects similar to dexamethasone, further confirming the potential of LFCAPT ferments in alleviating LPS-induced inflammation.

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a medicinal and edible fermented product, characterized in that: include: Mixing Codonopsis pilosula, Astragalus membranaceus, Platycodon grandiflorum and Pleurotus ostreatus to obtain a medicine-food mixture, and crushing the medicine-food mixture to obtain a medicine-food crushed mixture; The edible and medicinal crushed mixture is mixed with water to obtain an edible and medicinal crushed mixed liquid, Lactobacillus plantarum is inoculated into the edible and medicinal crushed mixed liquid for fermentation, and after the fermentation is completed, the fermentation liquid is collected and dried to obtain an edible and medicinal fermentation product.

2. The preparation method according to claim 1, characterized in that: The mass ratio of Codonopsis pilosula, Astragalus membranaceus, Platycodon grandiflorum and Pleurotus ostreatus is (0.8-1.2):(0.8-1.2):(0.8-1.2):(0.8-1.2).

3. The preparation method according to claim 1, characterized in that: The mass volume ratio of the edible and medicinal powdered mixture to water is 0.2-0.

3.

4. The preparation method according to claim 1, characterized in that: The edible and medicinal powdered mixed liquid also includes skim milk powder and sucrose; the mass of the skim milk powder is 3%-5% of the total volume of the edible and medicinal powdered mixed liquid, and the mass of the sucrose is 6%-10% of the total volume of the edible and medicinal powdered mixed liquid.

5. The preparation method according to claim 1, characterized in that: The temperature of the fermentation treatment is 35-40° C., and the fermentation time is 8-14 hours.

6. The food-medicine fermented product prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the food-medicine fermented product according to claim 6 in reducing cellular inflammatory response and / or oxidative stress response.

8. A method for reducing cellular inflammatory response and / or oxidative stress response, characterized in that: The food-medicine fermented product according to claim 6 is mixed and cultured with cells to reduce the inflammatory response and / or oxidative stress response of the cells.

9. Use of the medicinal and edible fermented product according to claim 6 in preparing products for relieving chronic nephritis.

10. A product, characterized in that Including the medicine-food homologous fermentation product as described in claim 6.

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