Traditional Chinese medicine fermentation composition for ovarian cell repair and preparation method thereof

Abstract

The invention relates to the field of biological fermentation, and discloses a traditional Chinese medicine fermentation composition for ovarian cell repair and a preparation method thereof. The traditional Chinese medicine fermented composition is prepared from traditional Chinese medicine raw materials through microbial liquid fermentation, the traditional Chinese medicine raw materials comprise lily, angelica sinensis, sea-buckthorn, raspberry and fructus lycii in a mass ratio of (1-2): (1-2): (1-2): (1-2): (1-2). The traditional Chinese medicine fermented composition is obtained by mixing lily, angelica sinensis, sea-buckthorn, raspberry and fructus lycii as traditional Chinese medicine raw materials according to a specific proportion and performing biological liquid fermentation, and the traditional Chinese medicine fermented composition has extremely high antioxidant activity and an excellent ovarian cell repairing function.

Description

Technical Field

[0001] The present invention relates to the field of biological fermentation, and particularly to a traditional Chinese medicine fermentation composition for ovarian cell repair and a preparation method thereof. Background Art

[0002] Free radicals, also known as "free radicals", refer to molecules or groups with unpaired electrons formed by the cleavage of covalent bonds in compounds under the influence of external factors. In the human body, free radicals are often harmful compounds produced during physiological metabolic reactions, with strong oxidizing properties, capable of damaging tissue cells and triggering aging and the occurrence of various diseases.

[0003] Traditional Chinese medicine believes that ovarian function decline is mostly related to syndromes such as "insufficient kidney essence", "deficiency of qi and blood", and "liver depression and spleen weakness". Traditional Chinese medicines such as Liuwei Dihuang Pills and Bazhen Decoction regulate endocrine and improve the ovarian microenvironment through functions such as tonifying the kidney and filling essence, replenishing qi and nourishing blood. However, traditional Chinese medicine decoctions or pills have limitations such as low ingredient absorption rate and slow onset of action, while fermentation technology can break through this bottleneck.

[0004] Through the enzymatic hydrolysis of microorganisms, traditional Chinese medicine fermentation can decompose macromolecular substances (such as polysaccharides and proteins) into smaller molecular active ingredients (such as oligosaccharides and polypeptides) that are more easily absorbed, and at the same time generate new metabolites (such as vitamins and organic acids), enhancing the medicinal effect. By screening specific strains (such as lactic acid bacteria and Saccharomyces cerevisiae) and formulating them with traditional Chinese medicine compounds, solid-state or liquid fermentation is carried out. For example, fermented traditional Chinese medicine can improve antioxidant, anti-inflammatory, and immunomodulatory abilities, which has potential significance for improving ovarian oxidative stress and promoting follicle development. Fermented traditional Chinese medicine not only increases active ingredients. For example, flavonoids and saponins can promote the expression of angiogenesis factors (such as VEGF), relieve ovarian ischemia problems, and provide nutritional support for follicle development; it can also balance estrogen and progesterone levels and delay premature ovarian failure by mimicking phytoestrogens (such as soy isoflavones) or regulating gonadotropin secretion.

[0005] Traditional Chinese medicine fermentation technology has gradually transitioned from traditional fermentation methods to modern fermentation methods. Modern traditional Chinese medicine fermentation technology draws on modern technologies such as pharmaceutical engineering technology and biological engineering technology to expand traditional fermentation methods, applying probiotics to traditional Chinese medicine fermentation to achieve the biological transformation of traditional Chinese medicine by microorganisms. Among them, multi-stage fermentation utilizes the metabolic characteristics of different microorganisms or the same microorganisms under different conditions to optimize the fermentation process by gradually adjusting environmental factors (such as temperature, pH, and oxygen concentration). Each stage in multiple stages targets specific metabolites, which can significantly improve the selectivity and yield of target products.

[0006] In summary, it has positive significance to utilize multi-stage fermentation technology to develop safe and effective traditional Chinese medicine fermentation products for ovarian repair. Summary of the Invention

[0007] To solve the above technical problems, the present invention provides a traditional Chinese medicine fermentation composition for ovarian cell repair and a preparation method thereof. The traditional Chinese medicine fermentation composition of the present invention is obtained by mixing lily, angelica, seabuckthorn, raspberry and wolfberry fruit as traditional Chinese medicine raw materials in a specific ratio and through biological liquid fermentation. The traditional Chinese medicine fermentation composition has extremely high antioxidant activity and excellent ovarian cell repair function.

[0008] The specific technical solution of the present invention is as follows: In the first aspect, the present invention provides a traditional Chinese medicine fermentation composition for ovarian cell repair, which is obtained by microbial liquid fermentation of traditional Chinese medicine raw materials; the traditional Chinese medicine raw materials include lily, angelica, seabuckthorn, raspberry and wolfberry fruit in a mass ratio of (1-2):(1-2):(1-2):(1-2):(1-2).

[0009] The present invention discovers that when a specific combination of traditional Chinese medicine raw materials of lily, angelica, seabuckthorn, raspberry and wolfberry fruit is selected and mixed and fermented in a specific ratio, the obtained fermentation product has extremely high antioxidant activity and excellent ovarian cell repair function.

[0010] Preferably, the mass ratio of lily, angelica, seabuckthorn, raspberry and wolfberry fruit is 1:1:2:2:1 or 1:1:2:2:2 or 1:1:2:1:2 or 2:1:2:1:2 or 2∶1∶2:2:2; most preferably, the mass ratio of lily, angelica, seabuckthorn, raspberry and wolfberry fruit is 1:1:2:2:2.

[0011] Preferably, the fermentation inoculant for the microbial liquid fermentation is selected from one of the following combinations: (1) Saccharomyces cerevisiae ML-002, Lactobacillus paracasei ML-001 and Lactobacillus johnsonii 021; or (2) Saccharomyces cerevisiae ML-002, Lactobacillus paracasei ML-001 and Lactobacillus casei ML-003.

[0012] Among them: Saccharomyces cerevisiae ML-002 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on January 16, 2025, with the deposit number CGMCC No. 33435, and the microbial taxonomic name is Saccharomyces cerevisiae; Lactiplantibacillus paracasei ML-001 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on January 16, 2025, with the deposit number CGMCC No. 33434, and the microbial taxonomic name is Lactiplantibacillus paracasei; Lactobacillus johnsonii 021 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on July 24, 2002, with the deposit number CGMCC No. 1.3221, and the microbial taxonomic name is Lactobacillus johnsonii. Lactobacillus casei ML-003 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on January 16, 2025, with the deposit number CGMCC No. 33436, and the microbial taxonomic name is Lactobacillus casei.

[0013] Through previous research, the research team of the present invention found that the above-mentioned strains have good activity in fermenting traditional Chinese medicines, and have excellent free radical scavenging activity after fermenting the above-mentioned traditional Chinese medicine raw material composition. Compared with other reported traditional Chinese medicine fermentation products, its characteristic is that even under the condition of a lower initial strain inoculation amount, a fermentation broth with higher antioxidant activity can still be obtained. The present invention further found that the fermentation activity of the above-mentioned strains is very stable. The traditional Chinese medicine fermentation products prepared by multi-stage fermentation still maintain a high level of antioxidant activity after being stored under different temperatures and lower pH conditions, indicating that they can maintain their efficacy in the human gastrointestinal environment. In addition, the traditional Chinese medicine products obtained by multi-stage fermentation of such strains still have a very high antioxidant activity during the long shelf life at room temperature, and there is currently no research on the antioxidant level of ovarian cells of the above-mentioned strain combination in multi-stage fermentation products.

[0014] In a second aspect, the present invention provides a method for preparing a traditional Chinese medicine fermentation composition, which comprises the following steps: 1) Prepare the traditional Chinese medicine raw materials into a sterilized and cooled liquid material.

[0015] 2) Inoculate Saccharomyces cerevisiae for primary fermentation, then inoculate Lactiplantibacillus paracasei for secondary fermentation, and finally inoculate Lactobacillus johnsonii or Lactobacillus casei for tertiary fermentation to obtain a traditional Chinese medicine base material.

[0016] 3) Centrifuge the traditional Chinese medicine base material and filter and sterilize the supernatant.

[0017] The present invention has found that when the above-mentioned traditional Chinese medicine raw material composition is fermented by a three-stage fermentation process, the antioxidant activity of the obtained traditional Chinese medicine fermentation product after fermentation is higher, and the efficacy of the product can also be guaranteed during a long shelf life.

[0018] The inoculation sequence of multi-stage fermentation is designed based on the pH gradient. In the first-stage fermentation, the pH drops to 4.0 - 4.5 to avoid premature acidification from inhibiting the activity of yeast. In the second-stage fermentation, Lactobacillus paracasei becomes the dominant strain in a weakly acidic environment, and at this time the pH drops to 3.5 - 4.0. In the third-stage fermentation, Lactobacillus johnsonii can further produce acid (pH = 3.0 - 3.5) and generate specific metabolites in a strongly acidic environment. This sequence is adapted through the pH gradient to ensure that each strain functions under optimal conditions and obtain a fermentation product with the best efficacy.

[0019] Preferably, for the first-stage fermentation: the temperature is 28 - 37°C, the fermentation lasts for 4 - 7 days, and the inoculation amount is 2.5×10 5 -1×10 6 CFU / mL; for the second-stage fermentation: the temperature is 28 - 45°C, the fermentation lasts for 1 - 6 days, and the inoculation amount is 2.5×10 5 -1×10 6 CFU / mL; for the third-stage fermentation: the temperature is 28 - 45°C, the fermentation lasts for 5 - 10 days, and the inoculation amount is 2.5×10 5 -1×10 6 CFU / mL.

[0020] The present invention has found that during the three-stage fermentation process, the inoculation timing of each strain has a significant impact on the fermentation effect. The inoculation timing of multi-stage fermentation needs to consider the pH change and the metabolic characteristics of the microbial community during the fermentation process. The first-stage fermentation for 4 - 7 days ensures that Saccharomyces cerevisiae fully degrades macromolecules such as polysaccharides and slowly reduces the pH to 4.0 - 4.5 to avoid premature acidification from inhibiting yeast. The second-stage fermentation for 1 - 6 days inoculates Lactobacillus paracasei after the pH drops to the appropriate range to quickly produce acid (pH = 3.5 - 4.0). The third-stage fermentation for 5 - 10 days inoculates Lactobacillus johnsonii under strongly acidic conditions to further metabolize complex carbon sources, deeply acidify (pH = 3.0 - 3.5) and generate antibacterial substances to ensure complete fermentation and product stability. The time design matches the metabolic rate and pH adaptability of each strain to achieve efficient and stable multi-stage fermentation.

[0021] Preferably, stirring is carried out during the fermentation process, and the stirring speed is 150 - 250 rpm.

[0022] The present invention discovers that during the three-stage fermentation process, the stirring speed has a significant impact on the fermentation effect. The selection of the stirring speed is based on the growth characteristics, metabolic requirements, and fermentation products of microorganisms. For Saccharomyces cerevisiae, this speed provides an appropriate amount of dissolved oxygen to support initial proliferation and metabolism, while avoiding cell damage and by-product accumulation caused by too high a speed. For lactic acid bacteria, 150 - 250 rpm can maintain a low dissolved oxygen environment, promote strain metabolism, and ensure uniform distribution of nutrients. In multi-stage fermentation, selecting this speed can simplify the operation, support a smooth transition and pH control, while reducing energy consumption. In addition, a moderate speed can also optimize the composition of metabolites and is applicable to fermentations of different scales.

[0023] Preferably, in step 2), the viable count of the Chinese medicine base material ≥ 1×10 9 CFU / mL.

[0024] Preferably, it further includes: 4) Filling and sterilization: filling and sterilizing the liquid material obtained in step 3) to obtain the finished product.

[0025] More preferably, step 4) specifically includes: filling and sealing the liquid material obtained in step 3), pasteurizing and cooling; or subjecting it to UHT sterilization and hot filling or aseptic cold filling into a packaging container made of PET material or composite paper material and sealing it to obtain the Chinese medicine fermentation composition.

[0026] Compared with the prior art, the beneficial effects of the present invention are: (1) The Chinese medicine fermentation composition of the present invention is obtained by mixing lily, angelica, seabuckthorn, raspberry, and wolfberry as Chinese medicine raw materials in a specific ratio and subjecting them to biological liquid fermentation. This Chinese medicine fermentation composition has extremely high antioxidant activity and excellent ovarian cell repair function. And the fermentation activity of these strains is very stable. The Chinese medicine fermentation product prepared by multi-stage fermentation still maintains a high level of antioxidant activity after being stored under different temperatures and at a relatively low pH, and can maintain its efficacy in the human gastrointestinal environment.

[0027] (2) The fermentation inoculant of the present invention has excellent fermentation performance, a fast fermentation speed, can significantly shorten the production cycle, and is very suitable for Chinese medicine fermentation. Especially in the process of synthesizing flavonoids and polyphenolic compounds, these strains can not only achieve efficient conversion but also effectively increase the yield of the final product. In addition, the flavonoids and polyphenols produced by these strains have extremely high antioxidant activity. Description of the Drawings

[0028] Figure 1 Shows the changes in antioxidant activity and saponins of different formulations of ML-001 fermentation.

[0029] Figure 2 Schematic diagram of the multi-stage fermentation process.

[0030] Figure 3 The antioxidant activity changes of strain ML-001 at different initial inoculation amounts and different fermentation times.

[0031] Figure 4 Graphs showing the changes in DPPH scavenging rate and hydroxyl radical scavenging rate during the fermentation of traditional Chinese medicine for ovarian repair at different rotation speeds.

[0032] Figure 5 The antioxidant activity changes during the secondary fermentation for 14 days at different transfer days.

[0033] Figure 6 The antioxidant activity changes during the tertiary fermentation for 16 days at different transfer days.

[0034] Figure 7 Results of ROS generation and scavenging. COV434 cells were pretreated with FJZY and AST, then treated with hydrogen peroxide, and the ROS level was detected using the DCFH-D1 kit.

[0035] Figure 8 Results of ROS generation and scavenging. COV434 cells were pretreated with FJZY and AST, then treated with hydrogen peroxide, and the ROS level was detected using the DCFH-D1 kit.

[0036] Figure 9 Results of cell apoptosis. COV434 cells were pretreated with FJZY and AST, then treated with hydrogen peroxide, and the apoptosis level of cells in each experimental group was detected using a cell apoptosis detection kit. Detailed implementation manners

[0037] The present invention will be further described below in conjunction with the embodiments.

[0038] General embodiment A traditional Chinese medicine fermentation composition for ovarian cell repair, which is obtained by microbial liquid fermentation of traditional Chinese medicine raw materials; the traditional Chinese medicine raw materials include lily, angelica, seabuckthorn, raspberry and wolfberry fruit in a mass ratio of (1-2):(1-2):(1-2):(1-2):(1-2).

[0039] Preferably, the mass ratio of lily, angelica, seabuckthorn, raspberry and wolfberry fruit is 1:1:2:2:1 or 1:1:2:2:2 or 1:1:2:1:2 or 2:1:2:1:2 or 2:1:2:2:2; most preferably, the mass ratio of lily, angelica, seabuckthorn, raspberry and wolfberry fruit is 1:1:2:2:2.

[0040] Preferably, the fermentation inoculant for the microbial liquid fermentation is selected from one of the following combinations: (1) Saccharomyces cerevisiae ML-002, Lactiplantibacillus paraplantarum ML-001 and Lactobacillus johnsonii 021; or (2) Saccharomyces cerevisiae ML-002, Lactiplantibacillus paraplantarum ML-001 and Lactobacillus casei ML-003. Among them: Saccharomyces cerevisiae ML-002 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on January 16, 2025, with the deposit number CGMCC No. 33435, and the microbial taxonomic name is Saccharomyces cerevisiae; Lactiplantibacillus paraplantarum ML-001 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on January 16, 2025, with the deposit number CGMCC No. 33434, and the microbial taxonomic name is Lactiplantibacillus paracasei; Lactobacillus johnsonii 021 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on July 24, 2002, with the deposit number CGMCC No. 1.3221, and the microbial taxonomic name is Lactobacillus johnsonii.

[0041] Lactobacillus casei ML-003 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on January 16, 2025, with the deposit number CGMCC No. 33436, and the microbial taxonomic name is Lactobacillus casei.

[0042] A preparation method of a traditional Chinese medicine fermentation composition, which comprises the following steps: 1) Prepare the traditional Chinese medicine raw materials into a sterilized and cooled liquid material.

[0043] 2) Inoculate Saccharomyces cerevisiae for primary fermentation, then inoculate Lactiplantibacillus paraplantarum for secondary fermentation, and finally inoculate Lactobacillus johnsonii or Lactobacillus casei for tertiary fermentation to obtain a traditional Chinese medicine base material.

[0044] 3) Centrifuge the traditional Chinese medicine base material and filter and sterilize the supernatant.

[0045] Preferably, for primary fermentation: the temperature is 28 - 37 °C, the fermentation time is 4 - 7 days, and the inoculation amount is 2.5×10 5 -1×10 6 CFU / mL; for secondary fermentation: the temperature is 28 - 45 °C, the fermentation time is 1 - 6 days, and the inoculation amount is 2.5×10 5 -1×10 6 CFU / mL; for tertiary fermentation: the temperature is 28 - 45 °C, the fermentation time is 5 - 10 days, and the inoculation amount is 2.5×10 5 -1×106 CFU / mL.

[0046] Preferably, stirring is carried out during the fermentation process, and the stirring speed is 150 - 250 rpm.

[0047] Preferably, in step 2), the viable bacteria count in the traditional Chinese medicine base material ≥ 1×10 9 CFU / mL.

[0048] Preferably, it further includes: 4) filling and sterilization: filling and sterilizing the liquid material obtained in step 3) to obtain the finished product.

[0049] More preferably, step 4) specifically includes: filling and sealing the liquid material obtained in step 3), pasteurizing and cooling; or subjecting it to UHT sterilization and hot filling or aseptic cold filling into a packaging container made of PET material or composite paper material and sealing to obtain the traditional Chinese medicine fermentation composition. Specific examples Example 1: Optimization of the traditional Chinese medicine raw material formula Traditional Chinese medicine fermentation is a process of transforming traditional Chinese medicine into a new form through the action of microorganisms. This process not only has an important impact on the effective components of the medicine, but also enhances its biological activity. Fermentation can increase the content of components such as flavonoids, polyphenols, and saponins in traditional Chinese medicine, and ultimately helps to enhance its antioxidant activity. The fermentation characteristics of traditional Chinese medicine compatibility show its complexity and uniqueness. The present invention uses advanced pharmacological research tools to optimize a combination of medicinal materials with efficacy for female ovarian health as the basis of the test formula, and optimizes the formula using the following test steps.

[0051] Meanwhile, the present invention selects strain ML - 001 to ferment different formulas as shown in the following table. The traditional Chinese medicine is pulverized and passed through a 40 - mesh sieve, the traditional Chinese medicine raw materials are configured according to the ratio and mixed evenly with 2.5 times the mass of water. After sterilization at 115°C for 20 min, the medicinal powder base material is obtained. After cooling, it is inoculated with ML - 001 at an inoculation amount of 5×10 5 CFU / mL, fermented in a sealed manner at 37°C for 15 days, and the changes in the content of key active components such as antioxidant activity and saponins during the fermentation process are carefully monitored, as Figure 1 shown. This research aims to deeply understand the dynamic changes and mutual relationships of these active components during the fermentation process, so as to provide important data support and scientific basis for further application development and theoretical research, and determine the fermentation formula components. Name Formulation ratio (mass ratio) Formulation 1 Lily: Angelica sinensis: Sea buckthorn: Raspberry: Lycium barbarum = 1:1:2:2:1 Formulation 2 Lily: Angelica sinensis: Sea buckthorn: Raspberry: Lycium barbarum = 1:1:2:2:2 Formulation 3 Lily: Angelica sinensis: Sea buckthorn: Raspberry: Lycium barbarum = 1:1:2:1:2 Formulation 4 Lily: Angelica sinensis: Sea buckthorn: Raspberry: Lycium barbarum = 2:1:2:1:2 Formulation 5 Lily: Angelica sinensis: Sea buckthorn: Raspberry: Lycium barbarum = 2:1:2:2:2

[0052] As Figure 1As shown in the figure, the initial antioxidant and saponin contents of Formula Five are the highest, but there is no obvious improvement after fermentation by the strain; while for Formula Two, the saponin and antioxidant contents reach their highest values on the 15th day and the 12th day respectively after fermentation; the antioxidant change trend of Formula One is similar to that of Formula Two, reaching the peak on the 12th day and then being lower than the initial fermentation level; for Formula Three, the antioxidant activity and saponin content do not increase after fermentation; for Formula Four, the saponin and antioxidant activities increase significantly in the early stage of fermentation, but perform poorly during long-term fermentation. Therefore, the performance of Formula Two is the best, with a relatively high antioxidant activity after fermentation, an increase of 62.5%; at the same time, the saponin content increases after the 12th day.

[0053] Example 2: Contents of Various Components in Multistage Fermentation The present invention explores the comparison of various chemical components and antioxidant activities under different fermentation methods. It is mainly divided into two fermentation methods: "single-strain fermentation" and "multistage fermentation", and analyzes their performance in terms of pH value, polysaccharide, flavonoid, polyphenol, saponin and antioxidant activity.

[0054] In the above-mentioned powder base material with the best effect (Lily: Angelica sinensis: Sea buckthorn: Raspberry: Lycium barbarum = 1:1:2:2:2), single-strain fermentation and multistage fermentation are carried out respectively. Under the condition of a fermentation temperature of 37°C, inoculate single-strain bacteria at an inoculation amount of 5×10 5 CFU / mL. Special attention should be paid that the products of secondary fermentation and tertiary fermentation need to be sterilized at 115°C for 15 minutes and then inoculated again; the secondary fermentation is inoculated on the 10th day of fermentation, and the tertiary fermentation is inoculated on the 5th day and the 10th day of fermentation respectively. After 20 days of fermentation, relevant components are detected, and the detection results are shown in the following table. Within a certain culture time, as the fermentation time prolongs, the inhibitory antioxidant activity effect of the fermentation broth also increases, which indicates that the antioxidant substances in the fermentation broth are produced during the culture fermentation process. The results show that the pH value of single-strain fermentation is higher and the fermentation acidity is weaker, while the pH value of multistage fermentation is at an intermediate level. This indicates that different fermentation methods have a significant impact on the acidity and alkalinity of the environment. In addition, lactic acid bacteria fermentation shows a low utilization rate of polysaccharides, while yeast fermentation performs better in terms of flavonoids and antioxidant ability. Multistage fermentation shows the diversity of components and the overall performance is better than that of single-strain fermentation. Yeast fermentation performs the best in terms of antioxidant ability, indicating that it may be more effective in enhancing the efficacy of traditional Chinese medicine health products.

[0055] Example 3: Preparation of Traditional Chinese Medicine Fermentation Product for Ovarian Repair A preparation method of a traditional Chinese medicine fermentation product for ovarian repair, as Figure 2 shown, includes the following steps: Fermentation broth preparation: After accurately weighing the traditional Chinese medicine powder of the required formula, mix it according to the mass ratio (lily: angelica: seabuckthorn: raspberry: wolfberry = 1:1:2:2:2), and gradually add an appropriate amount of water until the water content of the whole mixture reaches 65 wt%. Ensure thorough stirring during this process to enable the powder to uniformly absorb water, thereby obtaining an ideal wet state and preparing for subsequent processing and fermentation.

[0056] 2) Sterilization: Perform high-pressure sterilization on the fermentation broth obtained in step 1), then sterilize at 115 °C for 30 minutes, and cool to 42 °C to obtain a sterile cooled feed liquid.

[0057] 3) Inoculation and fermentation: Add 5×10 5 CFU / mL of Saccharomyces cerevisiae ML-002 to the sterilized and cooled feed liquid obtained in step 2), stir and mix evenly, place it in a constant-temperature incubator, and ferment at 30 °C at a rotation speed of 220 rpm for 5 days. The viable cell count requirement is ≥1×10 9 CFU / mL. On the 5th day, add 5×10 5 CFU / mL of Lactobacillus paracasei ML-001, place it in a constant-temperature incubator, and ferment at 37 °C at a rotation speed of 220 rpm for 4 days. Finally, inoculate Lactobacillus casei ML-003 at an inoculation amount of 5×10 5 CFU / mL on the 10th day of fermentation, and continue to culture at 37 °C at a rotation speed of 220 rpm for 11 days to obtain a traditional Chinese medicine base material. The antioxidant activity of this traditional Chinese medicine base material is up to 96.30%, the saponin content reaches 14.33 mg / mL, the polysaccharide content is 10.47 mg / mL, the polyphenol content is 25.63 mg / mL, and the flavonoid content is 22.08 mg / mL.

[0058] 4) The medicinal residues and impurities therein. Centrifuge the traditional Chinese medicine base material. During centrifugation, the solid part in the traditional Chinese medicine base material will precipitate to the bottom of the centrifuge tube due to the action of centrifugal force, while the liquid part will form the upper crude extraction medicinal liquid. Subsequently, carefully pour or separate the supernatant, and collect the obtained crude extraction medicinal liquid for subsequent analysis and application to ensure the concentration and purity of the active ingredients in the medicinal liquid.

[0059] 5) Filling and sterilization: Fill the centrifuged feed liquid in step 4) into a packaging container made of HDPE material and seal it, and perform pasteurization at 87 °C for 18 min and then cool it to obtain a traditional Chinese medicine oral liquid for ovarian repair with a long shelf life at room temperature.

[0060] Example 4: Antioxidant and free radical scavenging rates of traditional Chinese medicine fermentation broth under different inoculation amounts and stirring speeds. The inoculation amount is a key factor in the traditional Chinese medicine fermentation process. Reasonably optimizing the inoculation amount can significantly improve the fermentation effect of medicinal materials and the quality of the final product. Therefore, in actual operation, it is necessary to determine the appropriate inoculation amount according to specific traditional Chinese medicinal materials and target products to obtain the best fermentation effect. Inoculate the multi-stage fermentation strain with an inoculation amount of 0 - 1×10 6 CFU / mL, and the fermentation process parameters are the same as in Example 4. Detect the changes in its antioxidant activity over time as Figure 3 shown. The antioxidant activity first increases and then decreases with the increase of time. The increase of the traditional Chinese medicine base material without inoculated bacteria is not obvious, indicating that the fermentation process plays a decisive role in the metabolic transformation of antioxidant components. The antioxidant activity of the fermentation broth with an inoculation amount of 1×10 6 CFU / mL initially increases rapidly, but on the 6th day of fermentation, it is lower than that with inoculation amounts of 5×10 5 CFU / mL and 7.5×10 5 CFU / mL. And the fermentation broth with an inoculation amount of 5×10 5 CFU / mL reaches the highest value on the 9th day, far higher than other values. And although the antioxidant activity in the later fermentation decreases, it still remains at a high level. On the 20th day, it is equal to the antioxidant activity of the fermentation broth with an inoculation amount of 7.5×10 5 CFU / mL.

[0061] The stirring speed plays a crucial role in the fermentation effect and the quality of the final product during the traditional Chinese medicine fermentation process. Appropriate stirring speed can effectively increase the oxygen solubility in the culture medium, promote the growth and metabolism of aerobic microorganisms, accelerate the fermentation rate, and at the same time evenly distribute nutrients and metabolites to avoid precipitation. However, too low a stirring speed may lead to uneven nutrient distribution and affect the activity of microorganisms, while too high a speed may cause excessive bubbles and local overheating, reduce the oxygen transfer efficiency, and even damage the structure of microorganisms. Different stirring speeds also affect the choice of metabolic pathways, change the production ratio of active ingredients in medicinal materials, and ultimately affect the antioxidant activity and sensory characteristics. Therefore, in this example, the hydroxyl radical content and DPPH scavenging rate are detected at stirring speeds of 150 rpm, 180 rpm, 200 rpm, 220 rpm, and 240 rpm respectively. The test results are shown in Figure 4 . The hydroxyl radical scavenging rate reaches the maximum value of 97.25% at a stirring speed of 180 rpm, while the DPPH scavenging rate reaches the maximum value of 96.21% at a stirring speed of 200 rpm. Through the above speed optimization of this traditional Chinese medicine fermentation broth, the DPPH scavenging rate > 93%, and the hydroxyl radical scavenging rate > 94%.

[0062] Example 5: Changes in the components of three-stage fermentation at different transfer days Multistage fermentation can significantly improve the yield and fermentation efficiency by dividing the fermentation process into multiple stages. At different stages, optimizing various parameters such as temperature, pH, and oxygen supply can specifically meet the needs of microorganisms while reducing the inhibitory effect of metabolites on microbial growth. In addition, multistage fermentation also allows for the selection of different microorganisms or strains, promoting the production of diverse products and enhancing economic benefits and resource utilization efficiency. This flexible control and optimization make multistage fermentation of great application value in modern biotechnology. In this example, the antioxidant activities of the liquid medicines in secondary fermentation and tertiary fermentation were respectively detected under the inoculation condition of 5×10 5 CFU / mL. The strain ML-002 was inoculated in the primary fermentation and cultured at a rotation speed of 220 rpm and a temperature of 30°C. The secondary fermentation was inoculated on the 4th, 5th, and 6th days of fermentation respectively, and the strain ML-001 was selected. The subsequent culture was carried out at a rotation speed of 220 rpm and a temperature of 37°C. The changes in its antioxidant activity are shown in Figure 5 . We found that it was best to transfer the secondary fermentation strain on the 5th day of the primary fermentation. Its antioxidant activity continued to increase and reached a peak on the 8th day, but then the antioxidant activity decreased on the 10th day. Therefore, we inoculated ML-003 for tertiary fermentation on the 8th, 9th, and 10th days, and the culture conditions were the same as those of the secondary fermentation. The change in antioxidant activity over time is as shown in Figure 6 .

[0063] After the fermentation process ended, we conducted a detailed component analysis on the obtained fermentation products, focusing on analyzing the contents of bioactive components such as polyphenols, flavonoids, saponins, and polysaccharides as shown in the following table. At the same time, we also measured the pH value of the fermentation broth to evaluate its acid-base characteristics. In the secondary fermentation stage: on the 4th day, the pH was 3.28. At this time, the contents of polysaccharides, flavonoids, polyphenols, and saponins were relatively high. As the number of transfer days increased, especially on the 5th and 6th days, the pH decreased after fermentation ended, and the contents of polysaccharides and flavonoids also decreased significantly, indicating that these components may have been consumed or transformed during the fermentation process. In the tertiary fermentation stage: from the 8th day to the 10th day, the pH gradually rose to 4.08, indicating the change in the fermentation environment over time. The contents of polysaccharides and flavonoids reached 16.00 mg / mL and 15.00 mg / mL on the 8th day and then gradually decreased. When the tertiary strain was transferred on the 10th day, the flavonoid content recovered to 22.08 mg / mL after fermentation ended, which may be related to the metabolic patterns of different microorganisms.

[0064] Based on the above quantitative analysis of various components and parameters, the secondary strain was transferred on the 5th day and the tertiary strain was transferred on the 10th day to achieve the best effects of various components of the fermentation. The results are shown in the following table.

[0065] Example 6: Experiment on the repair of oxidative damage to human ovarian cells In this example, COV434 human ovarian granulosa cells were used as the research object to study the protective effect of the traditional Chinese medicine fermentation broth obtained under the optimal conditions of Example 4 on H2O2-induced damage to ovarian granulosa cells.

[0066] (1) COV434 cells were cultured in DMEM and MEM supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin respectively. The cells were maintained at 37 °C in a humidified atmosphere with 5% CO2. The COV434 cells were placed in a 96-well plate with a cell density of 8000 cells / well. FJZY and AST were incubated with COV434 cells for 24 hours at drug concentrations of 40 ng / mL, 80 ng / mL and 4 μg / mL, 8 μg / mL respectively. After washing three times with PBS, 1 mmol H2O2 was added and the cells were treated for 4 h. The standard CCK-8 assay was used to preliminarily evaluate the function of FJZY and AST in relieving oxidative stress.

[0067] After treatment with 1 mmol hydrogen peroxide for 4 h, as Figure 7 shown, COV434 cells showed significantly increased cytotoxicity under oxidative stress. Pretreatment with low concentrations of FJZY and AST significantly alleviated the cytotoxicity caused by oxidative stress, and was more significant under high-concentration treatment. Compared with the control group, the cell viability increased by 37% and 44% respectively. It should be noted that the high-concentration FJZY group (80 ng / mL) showed a cell activity protection function comparable to that of the high-concentration AST group (8 μg / mL).

[0068] (2) COV434 cells were cultured in DMEM and MEM supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin respectively. The cells were maintained at 37 °C in a humidified atmosphere with 5% CO2. The COV434 cells were placed in a 12-well plate with a cell density of 6W cells / well. FJZY and AST were incubated with COV434 cells for 24 hours at drug concentrations of 40 ng / mL, 80 ng / mL and 4 μg / mL, 8 μg / mL respectively. After washing three times with PBS, 1 mmol H2O2 was added and the cells were treated for 4 h. The DCFH-DA kit was used to evaluate the free radical scavenging ability of FJZY and AST.

[0069] After treatment with 1 mmol hydrogen peroxide for 4 h, as Figure 8As shown, COV434 cells were subjected to oxidative stress, showing a significantly increased level of reactive oxygen species. Pretreatment with low concentrations of FJZY and AST could scavenge the ROS generated by oxidative stress, and it was more significant in the high-concentration groups, reducing by 73% and 84% respectively compared with the group treated with H2O2 alone. Notably, the high-concentration FJZY group (80 ng / mL) showed an equivalent level of in vitro reactive oxygen species scavenging as the high-concentration AST group (8 μg / mL).

[0070] (3) COV434 cells were cultured in DMEM and MEM supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin respectively. The cells were maintained at 37 °C in a humidified environment with 5% CO2. COV434 cells were placed in a 6-well plate, maintaining a cell density of 300,000 cells / well. FJZY and AST were incubated with COV434 cells for 24 hours, and the drug concentrations were 40 ng / mL, 80 ng / mL and 4 μg / mL, 8 μg / mL respectively. After washing three times with PBS, 1 mmol H2O2 was added and the cells were treated for 4 h. A cell apoptosis detection kit was used to evaluate the free radical scavenging ability of FJZY and AST.

[0071] Treated with 1 mmol hydrogen peroxide for 4 h, as Figure 9 shown, COV434 cells were subjected to oxidative stress, showing a significantly increased cytotoxicity. Pretreatment with low concentrations of FJZY and AST could reduce the cytotoxicity caused by oxidative stress, and it was positively correlated with the dosage used. When using high concentrations of FJZY and AST, the cell viability increased by 32% and 34% respectively compared with the control group (H2O2). Notably, the high-concentration FJZY group (80 ng / mL) showed an equivalent cell repair ability as the high-concentration AST group (8 μg / mL).

[0072] The above experiments showed that the present invention obtained a composition capable of repairing ovarian cells through traditional Chinese medicine fermentation, and the effective concentrations for scavenging oxygen free radicals and repairing ovarian cells were much lower than those of the currently commercially available antioxidants of the same kind that can be used for ovarian cell repair.

Claims

1. A traditional Chinese medicine fermentation composition for ovarian cell repair, characterized in that: It is obtained by microbial liquid fermentation of traditional Chinese medicine raw materials; the traditional Chinese medicine raw materials include lily, angelica, seabuckthorn, raspberry and wolfberry fruit with a mass ratio of (1-2):(1-2):(1-2):(1-2):(1-2).

2. The traditional Chinese medicine fermentation composition according to claim 1, wherein: The mass ratio of the lily, angelica, seabuckthorn, raspberry and wolfberry fruit is 1:1:2:2:1 or 1:1:2:2:2 or 1:1:2:1:2 or 2:1:2:1:2 or 2:1:2:2:

2.

3. The traditional Chinese medicine fermentation composition according to claim 2, characterized in that: The mass ratio of the lily, angelica, seabuckthorn, raspberry and wolfberry fruit is 1:1:2:2:

2.

4. The traditional Chinese medicine fermentation composition according to any one of claims 1-3, wherein: The fermentation inoculum for the microbial liquid fermentation is selected from one of the following combinations: (1) Saccharomyces cerevisiae ML-002, Lactiplantibacillus paracasei ML-001 and Lactobacillus johnsonii 021; or (2) Saccharomyces cerevisiae ML-002, Lactiplantibacillus paracasei ML-001 and Lactobacillus casei ML-003; Wherein: Saccharomyces cerevisiae ML-002 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on January 16, 2025, with the deposit number CGMCC No. 33435, and the microbial taxonomic name is Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) Lactobacillus paracasei ML-001, was deposited at the China General Microbiological Culture Collection Center on January 16, 2025, with the deposit number CGMCC No. 33434, and the microbial taxonomic name is Lactobacillus paracasei ( Lacticaseibacillus paracasei ); Lactobacillus johnsonii 021, with the preservation number of CGMCC No.1.3221; Lactobacillus casei ML-003, which was deposited at the China General Microbiological Culture Collection Center on January 16, 2025, with the deposit number CGMCC No. 33436, and the microbial taxonomic name is Lactobacillus casei ( Lactobacillus casei ).

5. A preparation method of the traditional Chinese medicine fermentation composition according to any one of claims 1-4, characterized in that Including: 1) Prepare the traditional Chinese medicine raw materials into a sterilized and cooled liquid material; 2) Inoculate Saccharomyces cerevisiae for primary fermentation, then inoculate Lactiplantibacillus paracasei for secondary fermentation, and finally inoculate Lactobacillus johnsonii or Lactobacillus casei for tertiary fermentation to obtain a traditional Chinese medicine base material; 3) Centrifuge the traditional Chinese medicine base material and filter and sterilize the supernatant.

6. The preparation method according to claim 5, characterized in that: Primary fermentation: temperature 28 - 37°C, fermentation for 4 - 7 days, inoculum size 2.5×10 5 -1×10 6 CFU / mL; Secondary fermentation: temperature 28 - 45°C, fermentation for 1 - 6 days, inoculum size 2.5×10 5 -1×10 6 CFU / mL; Tertiary fermentation: temperature 28 - 45 °C, fermentation for 5 - 10 days, inoculum size 2.5×10 5 -1×10 6 CFU / mL.

7. The preparation method according to claim 6, characterized in that: Stirring is carried out during the fermentation process, and the stirring speed is 150-250 rpm.

8. The preparation method according to claim 5, characterized in that: In step 2), the viable count of the Chinese medicine base material ≥ 1×10 9 CFU / mL.

9. The preparation method according to claim 5, characterized in that: It also includes: 4) Filling and sterilization: Fill and sterilize the liquid material obtained in step 3) to obtain the finished product.

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