A traditional Chinese medicine fermentation composition for repairing ovarian cells and a preparation method thereof

Abstract

The application relates to the field of biological fermentation, and discloses a traditional Chinese medicine fermentation composition for repairing ovary cells and a preparation method thereof. The traditional Chinese medicine fermentation composition is obtained by subjecting traditional Chinese medicine raw materials to microbial liquid fermentation; the traditional Chinese medicine raw materials include lilies, angelica, sea buckthorn, raspberries and medlar in a mass ratio of (1-2):(1-2):(1-2):(1-2):(1-2). The traditional Chinese medicine fermentation composition is obtained by mixing lilies, angelica, sea buckthorn, raspberries and medlar in a specific proportion and subjecting the mixture to biological liquid fermentation. The traditional Chinese medicine fermentation composition has extremely high antioxidant activity and excellent ovary cell repairing function.

Description

Technical Field

[0001] This invention relates to the field of bio-fermentation, and in particular to a traditional Chinese medicine fermentation composition for ovarian cell repair and its preparation method. Background Technology

[0002] Free radicals, also known as "free radicals," are molecules or groups with unpaired electrons formed when covalent bonds in a compound break under the influence of external factors. In the human body, free radicals are often harmful compounds produced during physiological metabolic reactions. They have strong oxidizing properties, can damage tissue cells, and trigger aging and various diseases.

[0003] Traditional Chinese medicine believes that decreased ovarian function is often related to syndromes such as "kidney essence deficiency," "qi and blood deficiency," and "liver stagnation and spleen deficiency." Traditional Chinese medicines such as Liuwei Dihuang Wan and Bazhen Tang regulate endocrine function and improve the ovarian microenvironment by tonifying the kidneys and replenishing essence, as well as nourishing qi and blood. However, traditional Chinese medicine decoctions or pills have limitations such as low absorption rate and slow onset of action, while fermentation technology can overcome this bottleneck.

[0004] Traditional Chinese medicine (TCM) fermentation utilizes the enzymatic hydrolysis of microorganisms to break down macromolecules (such as polysaccharides and proteins) into more easily absorbed small-molecule active ingredients (such as oligosaccharides and polypeptides), while simultaneously generating new metabolites (such as vitamins and organic acids), thus enhancing efficacy. Specific bacterial strains (such as lactic acid bacteria and brewer's yeast) are screened and combined with TCM formulas for solid-state or liquid fermentation. For example, fermented TCM can enhance its antioxidant, anti-inflammatory, and immunomodulatory capabilities, which has potential significance for improving ovarian oxidative stress and promoting follicle development. Fermented TCM not only increases its active ingredients, such as flavonoids and saponins which can promote the expression of angiogenic factors (such as VEGF), alleviate ovarian ischemia, 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 gonadal hormone secretion.

[0005] Traditional Chinese medicine (TCM) fermentation technology has gradually transitioned from traditional methods to modern ones. Modern TCM fermentation technology expands upon traditional methods by drawing on pharmaceutical engineering and bioengineering techniques, incorporating probiotics to achieve microbial biotransformation of TCM. Multi-stage fermentation utilizes the metabolic characteristics of different microorganisms or the same microorganisms under varying conditions, optimizing the fermentation process by progressively adjusting environmental factors (such as temperature, pH, and oxygen concentration). Each of the multiple stages targets specific metabolites, significantly improving the selectivity and yield of the desired product.

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

[0007] To address the aforementioned technical problems, this invention provides a traditional Chinese medicine fermentation composition for ovarian cell repair and its preparation method. The traditional Chinese medicine fermentation composition of this invention is obtained by mixing lily bulb, angelica root, sea buckthorn, raspberry, and wolfberry as raw materials in a specific ratio and then subjecting them to biological liquid fermentation. This traditional Chinese medicine fermentation composition exhibits extremely high antioxidant activity and excellent ovarian cell repair function.

[0008] The specific technical solution of this invention is as follows:

[0009] In a 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, sea buckthorn, raspberry and wolfberry in a mass ratio of (1-2):(1-2):(1-2):(1-2):(1-2):(1-2)

[0010] This invention discovers that when a specific combination of traditional Chinese medicine ingredients—lily, angelica, sea buckthorn, raspberry, and wolfberry—is selected and fermented in a specific ratio, the resulting fermentation product exhibits extremely high antioxidant activity and excellent ovarian cell repair function.

[0011] Preferably, the mass ratio of lily bulb, angelica root, sea buckthorn, raspberry, and wolfberry 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 bulb, angelica root, sea buckthorn, raspberry, and wolfberry is 1:1:2:2:2.

[0012] Preferably, the fermentation inoculum for the microbial liquid fermentation is selected from one of the following combinations:

[0013] (1) Saccharomyces cerevisiae ML-002, Lactobacillus paracasei ML-001, and Lactobacillus johnsonii O21; or

[0014] (2) Saccharomyces cerevisiae ML-002, Lactobacillus paracasei ML-001 and Lactobacillus casei ML-003.

[0015] Among them: Saccharomyces cerevisiae ML-002 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 16, 2025, with accession number CGMCC No. 33435, and is classified as Saccharomyces cerevisiae; Lactobacillus paracasei ML-001 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 16, 2025, with accession number CGMCC No. 33434, and is classified as Lactobacillus paracasei; Lactobacillus johnsonii 021 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 24, 2002, with accession number CGMCC No. 1.3221, and is classified as Lactobacillus johnsonii. Lactobacillus casei ML-003 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 16, 2025, with accession number CGMCC No. 33436 and microbiological classification name Lactobacillus casei.

[0016] Through previous research, our team discovered that the aforementioned strains possess excellent fermentation activity against traditional Chinese medicine (TCM) and exhibit outstanding free radical scavenging activity after fermenting the aforementioned TCM raw material combinations. Compared with other reported TCM fermentation products, its characteristic is that even with low initial inoculum levels, fermentation broths with high antioxidant activity can still be obtained. This invention further found that the fermentation activity of the aforementioned strains is very stable. TCM fermentation products obtained through multi-stage fermentation maintain high levels of antioxidant activity even after storage at different temperatures and low pH conditions, indicating that they can retain their efficacy in the human gastrointestinal environment. Furthermore, TCM products obtained through multi-stage fermentation of these strains still exhibit high antioxidant activity over a long shelf life at room temperature, while currently, there is no research on the role of the aforementioned strain combinations in participating in the antioxidant levels of ovarian cells in multi-stage fermentation products.

[0017] Secondly, the present invention provides a method for preparing a fermented composition of traditional Chinese medicine, comprising the following steps:

[0018] 1) Prepare the Chinese herbal raw materials into a sterilization and cooling solution.

[0019] 2) Inoculate with Saccharomyces cerevisiae for primary fermentation, then inoculate with Lactobacillus paracasei for secondary fermentation, and finally inoculate with Lactobacillus johnsonii or Lactobacillus casei for tertiary fermentation to obtain the Chinese medicine base material.

[0020] 3) Centrifuge the Chinese herbal medicine base material and filter the supernatant to remove bacteria.

[0021] The present invention has found that when the above-mentioned Chinese herbal raw material composition is fermented using a three-stage fermentation process, the resulting Chinese herbal fermentation product has higher antioxidant activity and can also maintain the efficacy of the product within a long shelf life.

[0022] The multi-stage fermentation inoculation sequence is designed based on a pH gradient. In the first stage of fermentation, the pH drops to 4.0-4.5 to avoid premature acidification that inhibits yeast activity. In the second stage of fermentation, *Lactobacillus paracasei* becomes the dominant species in a slightly acidic environment, at which point the pH drops to 3.5-4.0. In the third stage of fermentation, *Lactobacillus johnsonii* can further produce acid (pH = 3.0-3.5) and generate specific metabolites in a strongly acidic environment. This sequence, adapted through a pH gradient, ensures that each species functions under optimal conditions, resulting in the most effective fermentation product.

[0023] As a preferred method, primary fermentation is carried out at a temperature of 28-37℃ for 4-7 days, with an inoculum size of 2.5 × 10⁻⁶. 5 -1×10 6 CFU / mL; Secondary fermentation: Temperature 28-45℃, fermentation time 1-6 days, inoculum size 2.5×10⁻⁶ 5 -1×10 6 CFU / mL; Three-stage fermentation: temperature 28-45℃, fermentation time 5-10 days, inoculum size 2.5×10⁻⁶ 5 -1×10 6 CFU / mL.

[0024] This invention reveals that the timing of inoculation for each strain during the three-stage fermentation process significantly impacts the fermentation effect. The inoculation timing for multi-stage fermentation needs to consider pH changes and the metabolic characteristics of the microbial community during fermentation. The first-stage fermentation lasts 4-7 days to ensure that the *Saccharomyces cerevisiae* fully degrades polysaccharides and other macromolecules, and slowly lowers the pH to 4.0-4.5, avoiding premature acidification that inhibits the yeast. The second-stage fermentation lasts 1-6 days, after the pH has dropped to a suitable range, inoculating with *Lactobacillus paracasei* to rapidly produce acid (pH = 3.5-4.0). The third-stage fermentation lasts 5-10 days, inoculating with *Lactobacillus johnsonii* under strongly acidic conditions to further metabolize complex carbon sources, deeply acidify (pH = 3.0-3.5), and generate antimicrobial substances, ensuring thorough fermentation and product stability. The timing is designed to match the metabolic rate and pH adaptability of each microbial strain, achieving efficient and stable multi-stage fermentation.

[0025] Preferably, stirring is performed during fermentation at a speed of 150-250 rpm.

[0026] This invention reveals that the stirring speed significantly impacts fermentation efficiency during three-stage fermentation. The selection of stirring speed is based on the growth characteristics, metabolic needs, and fermentation products of the microorganisms. For *Saccharomyces cerevisiae*, this speed provides adequate dissolved oxygen, supporting initial proliferation and metabolism while avoiding cell damage and byproduct accumulation caused by excessively high speeds. For lactic acid bacteria, 150-250 rpm maintains a low-oxygen environment, promoting strain metabolism and ensuring uniform nutrient distribution. In multi-stage fermentation, selecting this speed simplifies operation, supports smooth transitions and pH control, and reduces energy consumption. Furthermore, a moderate speed optimizes metabolic composition and is suitable for fermentations of different scales.

[0027] Preferably, in step 2), the number of live bacteria in the traditional Chinese medicine base material is ≥1×10⁻⁶. 9 CFU / mL.

[0028] As a preferred option, it also includes: 4) filling and sterilization: filling and sterilizing the liquid obtained in step 3) to obtain the finished product.

[0029] Further optimization, step 4) specifically includes: filling and sealing the liquid obtained in step 3), pasteurizing and cooling it; or sterilizing it with UHT and hot filling or aseptic cold filling it into PET or composite paper packaging containers and sealing it to obtain the traditional Chinese medicine fermentation composition.

[0030] Compared with the prior art, the beneficial effects of the present invention are:

[0031] (1) The traditional Chinese medicine fermentation composition of this invention is obtained by mixing lily, angelica, sea buckthorn, raspberry and wolfberry as raw materials in a specific ratio and fermenting them through biological liquid fermentation. This traditional Chinese medicine fermentation composition has extremely high antioxidant activity and excellent ovarian cell repair function. Moreover, the fermentation activity of these strains is very stable. The traditional Chinese medicine fermentation product obtained through multi-stage fermentation can still maintain a high level of antioxidant activity after being stored under different temperatures and low pH conditions, and can maintain its efficacy in the human gastrointestinal environment.

[0032] (2) The fermentation agent of this invention has excellent fermentation performance and a rapid fermentation rate, which can significantly shorten the production cycle. It is very suitable for the fermentation of traditional Chinese medicine, especially in the process of synthesizing flavonoids and polyphenols. 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. Attached Figure Description

[0033] Figure 1 The antioxidant activity and saponin changes of different fermentation formulations of ML-001 were studied.

[0034] Figure 2 This is a schematic diagram of a multi-stage fermentation process.

[0035] Figure 3 The changes in antioxidant activity of strain ML-001 under different initial inoculum amounts and fermentation times are shown.

[0036] Figure 4 The graph shows the changes in DPPH scavenging rate and hydroxyl radical scavenging rate of traditional Chinese medicine for ovarian repair fermentation at different speeds.

[0037] Figure 5 The changes in antioxidant activity after 14 days of fermentation with different transfer days in the secondary fermentation stage.

[0038] Figure 6 The changes in antioxidant activity after 16 days of fermentation with different transfer days in the three-stage fermentation process.

[0039] Figure 7 ROS generation and clearance results were analyzed. COV434 cells were pretreated with FJZY and AST, followed by hydrogen peroxide treatment, and ROS levels were detected using the DCFH-D1 kit.

[0040] Figure 8 ROS generation and clearance results were analyzed. COV434 cells were pretreated with FJZY and AST, followed by hydrogen peroxide treatment, and ROS levels were detected using the DCFH-D1 kit.

[0041] Figure 9 The results showed apoptosis. COV434 cells were pretreated with FJZY and AST, followed by hydrogen peroxide treatment, and the apoptosis level in each experimental group was detected using an apoptosis detection kit. Detailed Implementation

[0042] The present invention will be further described below with reference to embodiments.

[0043] General Implementation Examples

[0044] A traditional Chinese medicine fermentation composition for ovarian cell repair is obtained by microbial liquid fermentation of traditional Chinese medicine raw materials; the traditional Chinese medicine raw materials include lily, angelica, sea buckthorn, raspberry and wolfberry in a mass ratio of (1-2):(1-2):(1-2):(1-2):(1-2):(1-2)

[0045] Preferably, the mass ratio of lily bulb, angelica root, sea buckthorn, raspberry, and wolfberry 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 bulb, angelica root, sea buckthorn, raspberry, and wolfberry is 1:1:2:2:2.

[0046] Preferably, the fermentation inoculum for the microbial liquid fermentation is selected from one of the following combinations:

[0047] (1) Saccharomyces cerevisiae ML-002, Lactobacillus paracasei ML-001, and Lactobacillus johnsonii O21; or (2) Saccharomyces cerevisiae ML-002, Lactobacillus paracasei ML-001, and Lactobacillus casei ML-003. Wherein:

[0048] Saccharomyces cerevisiae ML-002 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 16, 2025, with accession number CGMCC No. 33435, and is classified as Saccharomyces cerevisiae; Lactobacillus paracasei ML-001 was deposited at the same center on January 16, 2025, with accession number CGMCC No. 33434, and is classified as Lactobacillus paracasei.

[0049] Lactobacillus johnsonii 021 was deposited on July 24, 2002, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 1.3221 and is classified as Lactobacillus johnsonii.

[0050] Lactobacillus casei ML-003 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 16, 2025, with accession number CGMCC No. 33436 and microbiological classification name Lactobacillus casei.

[0051] A method for preparing a fermented composition of traditional Chinese medicine, comprising the following steps:

[0052] 1) Prepare the Chinese herbal raw materials into a sterilization and cooling solution.

[0053] 2) Inoculate with Saccharomyces cerevisiae for primary fermentation, then inoculate with Lactobacillus paracasei for secondary fermentation, and finally inoculate with Lactobacillus johnsonii or Lactobacillus casei for tertiary fermentation to obtain the Chinese medicine base material.

[0054] 3) Centrifuge the Chinese herbal medicine base material and filter the supernatant to remove bacteria.

[0055] As a preferred method, primary fermentation is carried out at a temperature of 28-37℃ for 4-7 days, with an inoculum size of 2.5 × 10⁻⁶. 5 -1×10 6 CFU / mL; Secondary fermentation: Temperature 28-45℃, fermentation time 1-6 days, inoculum size 2.5×10⁻⁶ 5 -1×10 6CFU / mL; Three-stage fermentation: temperature 28-45℃, fermentation time 5-10 days, inoculum size 2.5×10⁻⁶ 5 -1×10 6 CFU / mL.

[0056] Preferably, stirring is performed during fermentation at a speed of 150-250 rpm.

[0057] Preferably, in step 2), the number of live bacteria in the traditional Chinese medicine base material is ≥1×10⁻⁶. 9 CFU / mL.

[0058] As a preferred option, it also includes: 4) filling and sterilization: filling and sterilizing the liquid obtained in step 3) to obtain the finished product.

[0059] Further optimization, step 4) specifically includes: filling and sealing the liquid obtained in step 3), pasteurizing and cooling it; or sterilizing it with UHT and hot filling or aseptic cold filling it into PET or composite paper packaging containers and sealing it to obtain the traditional Chinese medicine fermentation composition. Specific Implementation

[0061] Example 1: Optimal Selection of Traditional Chinese Medicine Raw Material Formula

[0062] Fermentation of traditional Chinese medicine (TCM) is a process that transforms medicinal herbs into a new form through microbial action. This process not only significantly affects the effective components of the herbs but also enhances their biological activity. Fermentation can increase the content of flavonoids, polyphenols, saponins, and other components in TCM, ultimately contributing to enhanced antioxidant activity. The fermentation characteristics of TCM formulations exhibit complexity and uniqueness. This invention employs advanced pharmacological research tools, selecting a combination of medicinal herbs effective for female ovarian health as the basis for the experimental formulation, and using the following experimental steps to optimize the formulation.

[0063] Meanwhile, this invention uses strain ML-001 to ferment different formulations, as shown in the table below. The traditional Chinese medicine is pulverized and passed through a 40-mesh sieve. The raw materials are prepared according to a specific ratio, and 2.5 times their weight of water is added and mixed thoroughly. The mixture is then sterilized at 115℃ for 20 minutes to obtain a medicinal powder base. After cooling, it is fermented at 5×10⁻⁶... 5 ML-001 was inoculated at a CFU / mL inoculum and fermented in a sealed environment at 37℃ for 15 days. Detailed monitoring was conducted on changes in antioxidant activity and the content of key active ingredients such as saponins during the fermentation process. Figure 1 As shown. This study aims to gain a deeper understanding of the dynamic changes and interrelationships of these active ingredients during fermentation, thereby providing important data support and scientific basis for further application development and theoretical research, and determining the components of fermentation formulations.

[0064] name Formula ratio (by mass) Formula 1 Lily: Angelica: Sea buckthorn: Raspberry: Goji berry = 1:1:2:2:1 Formula 2 Lily: Angelica: Sea buckthorn: Raspberry: Goji berry = 1:1:2:2:2 Formula 3 Lily: Angelica: Sea buckthorn: Raspberry: Goji berry = 1:1:2:1:2 Formula 4 Lily: Angelica: Sea buckthorn: Raspberry: Goji berry = 2:1:2:1:2 Formula 5 Lily: Angelica: Sea buckthorn: Raspberry: Goji berry = 2:1:2:2:2

[0065] like Figure 1 As shown, Formula 5 initially had the highest antioxidant and saponin content, but this did not significantly increase after fermentation. Formula 2, on the other hand, reached its peak saponin and antioxidant levels on days 12 and 15, respectively, after fermentation. Formula 1's antioxidant activity followed a similar trend to Formula 2, peaking on day 12 and subsequently falling below the initial fermentation level. Formula 3 showed no increase in antioxidant activity or saponin content after fermentation. Formula 4 showed a significant increase in saponin and antioxidant activity in the early stages of fermentation, but its performance was poor over longer fermentation periods. Therefore, Formula 2 performed best, exhibiting high antioxidant activity after fermentation, increasing by 62.5%; simultaneously, its saponin content increased after day 12.

[0066] Example 2: Content of various components in multi-stage fermentation

[0067] This invention investigates and compares the various chemical components and antioxidant activities under different fermentation methods. It mainly focuses on two fermentation methods: "single-strain fermentation" and "multi-stage fermentation," analyzing their performance in terms of pH value, polysaccharides, flavonoids, polyphenols, saponins, and antioxidant activity.

[0068] In the above-mentioned optimal medicinal powder substrate (lily: angelica: sea buckthorn: raspberry: wolfberry = 1:1:2:2:2), single-strain fermentation and multi-stage fermentation were carried out respectively. Under a fermentation temperature of 37℃, the fermentation was carried out at a rate of 5 × 10⁻⁶. 5 Inoculation with single bacteria was performed at a CFU / mL inoculum. Special attention was paid to the fact that products from secondary and tertiary fermentations required sterilization at 115℃ for 15 minutes before re-inoculation. Secondary fermentation was inoculated on day 10, and tertiary fermentation on days 5 and 10, respectively. Components were analyzed after 20 days of fermentation; the results are shown in the table below. Within a certain culture period, the inhibitory effect of the fermentation broth on antioxidant activity increased with prolonged fermentation time, indicating that antioxidants in the fermentation broth were produced during the fermentation process. The results showed that single-strain fermentation had a higher pH and weaker acidity, while multi-stage fermentation had a pH at a moderate level. This indicates that different fermentation methods have a significant impact on the acidity or alkalinity of the environment. Furthermore, lactic acid bacteria fermentation showed low polysaccharide utilization, while yeast fermentation performed better in terms of flavonoids and antioxidant capacity. Multi-stage fermentation showed component diversity and overall performance superior to single-strain fermentation. Yeast fermentation showed the best antioxidant capacity, suggesting it may be more effective in enhancing the efficacy of traditional Chinese medicine health products.

[0069]

[0070] Example 3: Preparation of Fermented Traditional Chinese Medicine Products for Ovarian Repair

[0071] A method for preparing a fermented traditional Chinese medicine product for ovarian repair, such as Figure 2 As shown, it includes the following steps:

[0072] Fermentation broth preparation: Accurately weigh the required herbal powders and mix them according to the mass ratio (lily bulb: angelica: sea buckthorn: raspberry: wolfberry = 1:1:2:2:2). Gradually add an appropriate amount of water until the total water content of the mixture reaches 65 wt%. Ensure thorough stirring during this process to allow the powder to absorb water evenly, thus achieving the ideal moist state and preparing for subsequent processing and fermentation.

[0073] 2) Sterilization: The fermentation broth obtained in step 1) is subjected to high-pressure sterilization, then sterilized at 115°C for 30 minutes, and cooled to 42°C to obtain sterile cooled broth.

[0074] 3) Inoculation and fermentation: Add 5×10 to the sterilized and cooled liquid obtained in step 2). 5 Ingredients: CFU / mL Saccharomyces cerevisiae ML-002; mixing thoroughly; fermentation in a constant temperature incubator at 30℃ and 220 rpm for 5 days; viable cell count ≥ 1 × 10⁻⁶. 9 CFU / mL, added on day 5 at 5×10 5 CFU / mL Lactobacillus paracasei ML-001 was placed in a constant temperature incubator and fermented at 37℃ and 220 rpm for 4 days. Finally, on the 10th day of fermentation, 5×10⁻⁶ CFU / mL Lactobacillus paracasei was added. 5 Lactobacillus casei ML-003 was inoculated at a CFU / mL level and cultured at 37℃ for 220 rpm for 11 days to obtain the traditional Chinese medicine base material. This base material exhibited the highest antioxidant activity of 96.30%, with saponin content reaching 14.33 mg / mL, polysaccharide content 10.47 mg / mL, polyphenol content 25.63 mg / mL, and flavonoid content 22.08 mg / mL.

[0075] 4) Residue and impurities. The herbal base is centrifuged. During centrifugation, the solid portion of the herbal base will settle to the bottom of the centrifuge tube due to centrifugal force, while the liquid portion will form the upper crude extract. Subsequently, the supernatant is carefully poured off or separated, and the obtained crude extract is collected for subsequent analysis and application, ensuring the concentration and purity of the effective components in the extract.

[0076] 5) Filling and sterilization: Fill the centrifuged liquid from step 4) into HDPE packaging containers and seal them. Sterilize at 87℃ for 18 minutes and then cool to obtain a room-temperature, long-shelf-life oral liquid for ovarian repair.

[0077] Example 4: Antioxidant and Free Radical Scavenging Rates of Traditional Chinese Medicine Fermentation Broth under Different Inoculation Amounts and Stirring Speeds. Inoculation amount is a key factor in the fermentation process of traditional Chinese medicine. Optimizing the inoculation amount can significantly improve the fermentation effect and the quality of the final product. Therefore, in practice, it is necessary to determine the appropriate inoculation amount based on the specific traditional Chinese medicine and the target product to obtain the best fermentation effect. (0-1×10⁻⁶) 6 Inoculation with CFU / mL of multi-stage fermentation strains was performed, and the fermentation process parameters were the same as in Example 4. The changes in antioxidant activity over time were then detected. Figure 3 As shown, antioxidant activity initially increased and then decreased over time, while the increase was not significant in the uninoculated herbal substrate, indicating that the fermentation process played a decisive role in the metabolic transformation of antioxidant components. The inoculum size was 1×10⁻⁶. 6 Although the antioxidant activity of the fermentation broth at CFU / mL initially increased rapidly, it fell below the level of the inoculum size of 5×10⁻⁶ on day 6 of fermentation. 5 CFU / mL and 7.5×10 5 CFU / mL, and the inoculum size is 5×10 5 The CFU / mL value reached its highest level on day 9, significantly higher than other values. Although the antioxidant capacity decreased somewhat during the later stages of fermentation, it remained high, reaching a level comparable to an inoculum of 7.5 × 10⁻⁶ on day 20. 5 The antioxidant capacity was the same at CFU / mL.

[0078] Stirring speed plays a crucial role in the fermentation process of traditional Chinese medicine, significantly impacting fermentation efficiency and the quality of the final product. An appropriate stirring speed effectively increases oxygen solubility in the culture medium, promotes the growth and metabolism of aerobic microorganisms, accelerates the fermentation rate, and ensures even distribution of nutrients and metabolites, preventing precipitation. However, excessively low stirring speeds may lead to uneven nutrient distribution, affecting microbial activity, while excessively high speeds may cause excessive bubbles, localized overheating, reduced oxygen transfer efficiency, and even damage to microbial structure. Different stirring speeds also influence the selection of metabolic pathways, altering the proportion of active ingredients in the medicinal materials, ultimately affecting antioxidant activity and sensory properties. Therefore, in this embodiment, the content of hydroxyl radicals and the DPPH scavenging rate were measured at stirring speeds of 150 rpm, 180 rpm, 200 rpm, 220 rpm, and 240 rpm, respectively. The test results are shown below. Figure 4 The hydroxyl radical scavenging rate reached a maximum of 97.25% at 180 rpm, while the DPPH scavenging rate reached a maximum of 96.21% at 200 rpm. Through the above-mentioned speed optimization, this traditional Chinese medicine fermentation broth achieved a DPPH scavenging rate of >93% and a hydroxyl radical scavenging rate of >94%.

[0079] Example 5: Changes in Components During Three-Stage Fermentation with Different Transfer Days

[0080] Multistage fermentation, by dividing the fermentation process into multiple stages, can significantly improve yield and fermentation efficiency. Optimizing parameters such as temperature, pH, and oxygen supply at different stages can specifically meet the needs of microorganisms while reducing the inhibitory effects of metabolites on microbial growth. Furthermore, multistage fermentation allows for the selection of different microorganisms or strains, promoting the production of diversified products and improving economic efficiency and resource utilization. This flexible control and optimization makes multistage fermentation of significant application value in modern biotechnology. This example tested the fermentation process at 5 × 10⁻⁶... 5 The antioxidant activity of secondary and tertiary fermentation broths under CFU / mL inoculation conditions was investigated. Primary fermentation was inoculated with strain ML-002 at 220 rpm and 30°C. Secondary fermentation was carried out on days 4, 5, and 6 using strain ML-001, with subsequent cultures conducted at 220 rpm and 37°C. Changes in antioxidant activity were observed. Figure 5 We found that transferring the secondary fermentation strain on day 5 of the primary fermentation yielded the best results, with its antioxidant activity continuously increasing and peaking on day 8. However, the antioxidant activity then declined on day 10. Therefore, we inoculated ML-003 on days 8, 9, and 10 for tertiary fermentation, using the same culture conditions as the secondary fermentation. The changes in antioxidant activity over time were as follows: Figure 6 As shown.

[0081] After the fermentation process, we conducted detailed component analysis on the obtained fermentation products, focusing on the content of bioactive components such as polyphenols, flavonoids, saponins, and polysaccharides (see table below). We also measured the pH of the fermentation broth to assess its acid-base characteristics. In the secondary fermentation stage: On day 4, the pH was 3.28, at which point the contents of polysaccharides, flavonoids, polyphenols, and saponins were relatively high. With increasing transfer days, especially on days 5 and 6, the pH decreased after fermentation, and the contents of polysaccharides and flavonoids also decreased significantly, indicating that these components may have been consumed or transformed during fermentation. In the tertiary fermentation stage: From day 8 to day 10, the pH gradually increased to 4.08, showing 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 day 8, respectively, and then gradually decreased. However, when the tertiary strain was transferred on day 10, the flavonoid content rebounded to 22.08 mg / mL after fermentation, which may be related to the different metabolic pathways of the microorganisms.

[0082] Based on the quantitative analysis of various components and parameters above, secondary strain transfer was performed on day 5 and tertiary strain transfer was performed on day 10 to achieve the best results for the various components of fermentation. The results are shown in the table below.

[0083]

[0084] Example 6: Experiment on Oxidative Damage Repair in Human Ovarian Cells

[0085] This embodiment uses COV434 human ovarian granulosa cells as the research object to study the protective effect of the traditional Chinese medicine fermentation broth obtained under the optimal conditions in Example 4 on H2O2-induced ovarian granulosa cell damage.

[0086] (1) COV434 cells were cultured in DMEM and MEM supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin, respectively. Cells were maintained at 37°C under humidification with 5% CO2. COV434 cells were placed in 96-well plates at a density of 8000 cells / well. COV434 cells were incubated with FJZY and AST for 24 hours at drug concentrations of 40 ng / mL, 80 ng / mL, 4 μg / mL, and 8 μg / mL, respectively. Cells were washed three times with PBS and treated with 1 mmol H2O2 for 4 hours. The oxidative stress-relieving function of FJZY and AST was preliminarily assessed using the standard CCK-8 assay.

[0087] Treat with 1 mmol of hydrogen peroxide for 4 hours, such as Figure 7 As shown, COV434 cells exhibited significantly increased cytotoxicity under oxidative stress. Pretreatment with low concentrations of FJZY and AST significantly alleviated cytotoxicity caused by oxidative stress, with the effect being more pronounced at high concentrations. Compared to the control group, cell viability was increased by 37% and 44%, respectively. Notably, the high-concentration FJZY group (80 ng / mL) showed comparable cell viability protection to the high-concentration AST group (8 μg / mL).

[0088] (2) COV434 cells were cultured in DMEM and MEM supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin, respectively. Cells were maintained at 37°C under humidification with 5% CO2. COV434 cells were placed in 12-well plates at a density of 6 cells / well. COV434 cells were incubated with FJZY and AST for 24 hours at drug concentrations of 40 ng / mL, 80 ng / mL, 4 μg / mL, and 8 μg / mL, respectively. Cells were washed three times with PBS and treated with 1 mmol H2O2 for 4 hours. The free radical scavenging capacity of FJZY and AST was assessed using the DCFH-DA kit.

[0089] Treat with 1 mmol of hydrogen peroxide for 4 hours, such as Figure 8As shown, COV434 cells exhibited significantly elevated reactive oxygen species (ROS) levels under oxidative stress. Pretreatment with low concentrations of FJZY and AST effectively scavenged ROS generated by oxidative stress, with the effect being more pronounced in the high-concentration groups, reducing ROS by 73% and 84%, respectively, compared to the H2O2-only treatment group. Notably, the high-concentration FJZY group (80 ng / mL) demonstrated comparable in vitro ROS scavenging levels to the high-concentration AST group (8 μg / mL).

[0090] (3) COV434 cells were cultured in DMEM and MEM supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin, respectively. Cells were maintained at 37°C under humidification with 5% CO2. COV434 cells were placed in 6-well plates at a density of 30 cells / well. COV434 cells were incubated with FJZY and AST for 24 hours at drug concentrations of 40 ng / mL, 80 ng / mL, 4 μg / mL, and 8 μg / mL, respectively. Cells were washed three times with PBS and treated with 1 mmol H2O2 for 4 hours. The free radical scavenging capacity of FJZY and AST was assessed using an apoptosis detection kit.

[0091] Treat with 1 mmol of hydrogen peroxide for 4 hours, such as Figure 9 As shown, COV434 cells exhibited significantly increased cytotoxicity under oxidative stress. Pretreatment with low concentrations of FJZY and AST reduced oxidative stress-induced cytotoxicity, showing a positive correlation with the dosage. Using high concentrations of FJZY and AST, cell viability was increased by 32% and 34%, respectively, compared to the control group (H2O2). Notably, the high-concentration FJZY group (80 ng / mL) showed comparable cell repair capacity to the high-concentration AST group (8 μg / mL).

[0092] The above experiments show that the present invention obtains a composition that can be used for ovarian cell repair through the fermentation of traditional Chinese medicine, and its effective concentration for scavenging oxygen free radicals and repairing ovarian cells is much lower than that of similar antioxidants currently available on the market 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 Chinese medicinal materials; the Chinese medicinal materials include lily, angelica, sea buckthorn, raspberry and wolfberry in a mass ratio of (1-2):(1-2):(1-2):(1-2):(1-2); The microbial liquid fermentation includes: 1) Prepare a sterilization and cooling solution from the raw materials of traditional Chinese medicine; 2) Inoculate with *Saccharomyces cerevisiae* for primary fermentation, then inoculate with *Lactobacillus paracasei* for secondary fermentation, and finally inoculate with *Lactobacillus johnsonii* or *Lactobacillus casei* for tertiary fermentation to obtain the traditional Chinese medicine base material; wherein, primary fermentation: temperature 28-37℃, fermentation time 4-7 days, inoculation amount 2.5×10 5 -1×10 6 CFU / mL; Secondary fermentation: Temperature 28-45℃, fermentation time 1-6 days, inoculum size 2.5×10⁻⁶ 5 -1×10 6 CFU / mL; Three-stage fermentation: temperature 28-45℃, fermentation time 5-10 days, inoculum size 2.5×10⁻⁶ 5 -1×10 6 CFU / mL; 3) Centrifuge the Chinese herbal medicine base material and filter the supernatant to remove bacteria; The fermentation inoculum 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 O21; or (2) Saccharomyces cerevisiae ML-002, Lactobacillus paracasei ML-001 and Lactobacillus casei ML-003; in: Saccharomyces cerevisiae ML-002 was deposited on January 16, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33435, and its microbiological classification is Saccharomyces cerevisiae (…). Saccharomyces cerevisiae ); Lactobacillus paracasei ML-001 was deposited on January 16, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33434, and its microbiological classification is Lactobacillus paracasei (…). Lacticaseibacillus paracasei ); Lactobacillus johnsonii 021, with accession number CGMCC No. 1.3221; Lactobacillus casei ML-003 was deposited on January 16, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33436, and its microbiological classification is Lactobacillus casei (…). Lactobacillus casei ).

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

2.

4. The traditional Chinese medicine fermentation composition according to claim 1, characterized in that: Stirring is carried out during fermentation at a speed of 150-250 rpm.

5. The traditional Chinese medicine fermentation composition according to claim 1, characterized in that: In step 2), the number of viable bacteria in the herbal medicine base is ≥1×10⁻⁶. 9 CFU / mL.

6. The traditional Chinese medicine fermentation composition according to claim 1, characterized in that: The microbial liquid fermentation also includes: 4) filling and sterilization: filling and sterilizing the liquid obtained in step 3) to obtain the finished product.

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