A composite fermentation microbial agent for improving equol production, a fermentation liquid and application thereof

Abstract

The application discloses a kind of complex fermentation inoculant for improving equol production, fermentation liquor and application thereof, belong to microbial fermentation technical field.The complex fermentation inoculant of the application, streptococcus thermophilus BLCC2-0025 and lactococcus lactis MP1001 have high β-glucosidase activity, and low activity glycoside form is converted into high activity daidzein;Salivary lactobacillus LS1101 and lactobacillus gregius 0010 have the activity of further converting daidzein into equol;Four strains of bacteria are used in combination, which can synergistically improve the content of equol.The fermentation liquor prepared by fermenting pueraria extract with the complex fermentation inoculant of the application can target regulate intestine-ovary axis, significantly improve the reproductive function of POF mice, relieve ovarian oxidative stress and accurately regulate intestinal flora balance, providing a safe and effective preparation and application scheme for non-hormone intervention of premature ovarian failure.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, specifically to a compound fermentation agent, fermentation broth, and their applications for increasing estrol production. Background Technology

[0002] Equol is the final product of soy isoflavones through a stepwise metabolic transformation by specific microorganisms in the intestines of animals and humans. It is also the core active substance with the highest biological activity and most prominent physiological function among all soy isoflavone metabolites. Compared to its precursors daidzein, daidzein, and dihydrodaidzein, equol possesses stronger selective binding capacity to estrogen receptors, antioxidant activity, and biostability. Its antioxidant capacity far exceeds that of daidzein, and its effects on estrogen-related physiological regulation and oxidative stress damage repair are significantly superior to those of natural soy isoflavones. Therefore, it has become a highly promising active ingredient for development in functional foods, pharmaceuticals, health products, skincare, and livestock farming.

[0003] Currently, the preparation methods of equol are mainly divided into two categories: chemical synthesis and microbial synthesis. Chemical synthesis primarily uses daidzein as a raw material, involving multiple steps of catalytic hydrogenation and reduction reactions. This method suffers from drawbacks such as cumbersome process steps, harsh reaction conditions, and high catalyst costs. Microbial synthesis, on the other hand, offers advantages such as mild reaction conditions, strong stereoselectivity, environmental friendliness, and low raw material costs, making it the mainstream research direction for the industrial production of equol.

[0004] Existing microbial fermentation synthesis of equol primarily uses soybeans and soybean processing byproducts as substrates. However, the low total isoflavone content in soybeans (only 0.04%–0.8%) limits further increases in equol production. Recent in-depth studies on the chemical composition, pharmacology, and clinical applications of kudzu root have confirmed that kudzu isoflavones are both pharmacological and important nutritional factors—a newly discovered essential nutrient that the human body cannot synthesize and must obtain from food, offering significant physiological and health benefits. Kudzu root contains 0.4%–15.87% total isoflavones; using it as a fermentation substrate could broaden the raw material supply pathways for equol fermentation production. However, the structure of kudzu isoflavones differs from that of soybean isoflavones, and currently, there is a lack of high-yield strains suitable for kudzu root fermentation to produce equol. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide a compound fermentation agent, fermentation broth and its application for increasing equol production.

[0006] Specifically, the present invention relates to the following technical solutions: In a first aspect, the present invention provides a compound fermentation agent for increasing equol production, which is composed of Streptococcus thermophilus BLCC2-0025, Lactobacillus salivarius LS1101, Pediococcus lactis MP1001 and Lactobacillus gasseri 0010. The thermophilic streptococcus BLCC2-0025 was deposited on March 18, 2015, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2015130. Its classification and nomenclature are: thermophilic streptococcus BLCC2-0025. Streptococcus thermophilus BLCC2-0025.

[0007] The *Lactobacillus salivarius* LS1101 was deposited on November 9, 2020, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2020706. Its classification and nomenclature are: *Lactobacillus salivarius* LS1101. Lactobacillus salivarius LS1101.

[0008] The preservation number of the lactic acid porphyria MP1001 is CCTCC NO: M 2022136, and it is recorded in the patent "A strain of lactic acid porphyria MP1001, its powder and its application", application publication number CN 116814480A.

[0009] The Lactobacillus gastroenteritis 0010 has the accession number CCTCC NO: M 2015340 and is described in the patent "Composition for Assisting Protection of Gastric Mucosa and / or Relieving Gastric Bloating and its Probiotic Preparation", application publication number CN 106420847B.

[0010] Preferably, the compound fermentation agent is composed of Streptococcus thermophilus BLCC2-0025, Lactobacillus salivarius LS1101, Pediococcus lactis MP1001 and Lactobacillus gasseri 0010 in a live count ratio of 1:1:1:1.

[0011] Preferably, the total viable count of the compound fermentation agent is greater than or equal to 10. 8 CFU / mL.

[0012] In a second aspect, the present invention provides the application of the above-mentioned compound fermentation agent in the production of estrol from fermented kudzu root.

[0013] In the compound fermentation agent of this invention, *Streptococcus thermophilus* BLCC2-0025 and *Pediococcus lactis* MP1001 possess high β-glucosidase activity, which can efficiently hydrolyze the glycosidic bonds of puerarin isoflavones, converting the low-activity glycoside form into the high-activity daidzein. *Lactobacillus salivarius* LS1101 and *Lactobacillus gasseri* 0010 have the activity to further convert daidzein into equol. By combining the above four strains, efficient hydrolysis of glycosides in puerarin isoflavones and the directed conversion of daidzein into equol can be achieved, thereby synergistically increasing the equol content in the final product.

[0014] A third aspect of the present invention provides a fermentation broth prepared by the following method: Inoculate the above-mentioned compound fermentation agent into the sterilized kudzu root extract and incubate at 35-40℃ for 20-30 hours.

[0015] Preferably, the method for preparing the kudzu root extract is as follows: add deionized water to kudzu root, soak for 30-60 minutes, decoct and extract, filter, collect the filtrate, concentrate and sterilize.

[0016] In some preferred embodiments, the kudzu root extract is specifically prepared by the following method: Add deionized water to kudzu root, soak for 30-60 minutes, heat to boiling, decoct and extract for 1 hour, filter and collect the filtrate as the first filtrate; add deionized water to the residue again, heat to boiling, decoct and extract for 1 hour, filter and collect the filtrate as the second filtrate. The first and second filtrates were combined, concentrated, and sterilized to prepare kudzu root extract.

[0017] Preferably, the inoculum amount of the compound fermentation agent is 6-10% of the volume of the kudzu root extract.

[0018] The fermentation broth of this invention contains equol at a content greater than 1.5 mg / mL, which can regulate intestinal flora, promote the conversion of phytoestrogens, and improve oxidative stress, thereby achieving safe and effective intervention for premature ovarian failure (POF) and providing a new solution for non-hormonal treatment of POF.

[0019] In a fourth aspect, the present invention provides the use of the above-mentioned fermentation broth in the preparation of a drug for preventing and treating premature ovarian failure.

[0020] In the above applications, the drug for preventing premature ovarian failure can use the above-mentioned fermentation broth as the sole active ingredient; or, the above-mentioned fermentation broth can be used in combination with other active ingredients.

[0021] In some preferred embodiments, other active ingredients may be selected from one or more of the following: wolfberry extract, angelica extract, strawberry powder, and black chokeberry fruit powder.

[0022] In the above applications, the drugs for preventing and treating premature ovarian failure may also contain flavoring ingredients, which may be selected from xylooligosaccharides, erythritol, steviol glycosides, glucose, arabinose, etc.

[0023] In the above applications, the drugs for preventing and treating premature ovarian failure can be prepared into dosage forms such as tablets, granules, powders, capsules, solutions, suspensions, emulsions, and lyophilized powders using conventional methods.

[0024] The beneficial effects of this invention are: (1) Based on the structural characteristics of kudzu isoflavones, this invention combines strains with β-glucosidase production capabilities and strains with equol production capabilities to obtain a compound fermentation agent that increases equol yield. In the compound fermentation agent of this invention, Streptococcus thermophilus BLCC2-0025 and Pediococcus lactis MP1001 have high β-glucosidase production activity, which can efficiently hydrolyze the glycosidic bonds of kudzu isoflavones, converting the low-activity glycoside form into the high-activity daidzein; Lactobacillus salivarius LS1101 and Lactobacillus gasseri 0010 have the activity of further converting daidzein into equol; the combination of the four strains can synergistically convert inactive estrogens (such as puerarin) in kudzu into potent equol.

[0025] (2) The fermentation liquid prepared by fermenting kudzu root extract with the compound fermentation agent of the present invention is rich in phytoestrogens - estrol and also contains probiotics. The two have a synergistic effect and can significantly improve the reproductive function of POF mice, alleviate ovarian oxidative stress and precisely regulate the balance of intestinal flora by targeting and regulating the gut-ovarian axis. It provides a safe and effective preparation and application scheme for non-hormonal intervention of premature ovarian failure. Attached Figure Description

[0026] Figure 1 Preliminary screening results of β-glucosidase-producing strains.

[0027] Figure 2 Results of enzyme activity assay for β-glucosidase-producing strains.

[0028] Figure 3 Morphological diagram of the estrous cycle in normal mice (hematoxylin and eosin HE staining, 1000×).

[0029] Figure 4 : Graph showing the changes in estrous cycle of mice in each group during the modeling period over 7 days. In the graph, P represents proestrus; E represents estrus; M represents metestrus; D represents diaestrus; Z-1, Z-2, Z-3, Z-4, and Z-5 represent the changes in estrous cycle of 5 mice in the normal group over 7 days; M-1, M-2, ..., M-10 represent the changes in estrous cycle of 10 mice in the model group over 7 days.

[0030] Figure 5 The results of serum hormone levels in mice during the modeling period are shown in the figure. In the figure, A is the E2 concentration, B is the LH concentration, and C is the FSH concentration.

[0031] Figure 6 Results of the percentage of interestrus in each group of mice after 4 weeks of intervention. An asterisk indicates a significant difference compared to the MC group. This means P < 0.05. This means P < 0.01. This means P < 0.001.

[0032] Figure 7 Serum hormone levels in mice after 4 weeks of intervention: In the figure, A represents E2 concentration, B represents LH concentration, and C represents FSH concentration. An asterisk (*) indicates a significant difference from the MC group. This means P < 0.05. This means P < 0.01. This means P < 0.001.

[0033] Figure 8 Results of ovarian and uterine index measurements in mice of each group after 4 weeks of intervention. In the figure, A represents the ovarian index, and B represents the uterine index. An asterisk indicates a significant difference from the MC group. This means P < 0.05. This means P < 0.01. This means P < 0.001.

[0034] Figure 9 HE staining results of ovarian tissues of mice in each group after 4 weeks of intervention (40×).

[0035] Figure 10 The total number of ovarian follicles and the follicular atresia rate in each group of mice were measured after 4 weeks of intervention. In the figure, A represents the total number of ovarian follicles, and B represents the ovarian follicular atresia rate. An asterisk indicates a significant difference from the MC group. This means P < 0.05. This means P < 0.01. This means P < 0.001.

[0036] Figure 11 HE staining results of uterine tissue from mice in each group after 4 weeks of intervention (40×).

[0037] Figure 12 : Fluorescent staining results of apoptotic cells in ovarian tissue of mice in each group after 4 weeks of intervention (400×).

[0038] Figure 13 : Results of total cell fluorescence staining in ovarian tissue of mice in each group after 4 weeks of intervention (400×).

[0039] Figure 14 : Results of apoptosis rates in ovarian tissue cells of mice in each group after 4 weeks of intervention. An asterisk indicates a significant difference compared to the MC group. This means P < 0.05. This means P < 0.01. This means P < 0.001.

[0040] Figure 15 The results of antioxidant index measurements in ovarian tissue of mice in each group after 4 weeks of intervention are shown in the figure. In the figure, A represents MDA content in ovarian tissue, and B represents GSH-Px concentration in ovarian tissue. An asterisk indicates a significant difference from the MC group. This means P < 0.05. This means P < 0.01. This means P < 0.001.

[0041] Figure 16 : Results of gut microbiota counts in mice of each group after 4 weeks of intervention. In the figure, A represents the number of intestinal lactobacilli, B represents the number of intestinal bifidobacteria, C represents the number of intestinal Escherichia coli, and D represents the number of intestinal enterococci. An asterisk indicates a significant difference from the MC group. This means P < 0.05. This means P < 0.01. This means P < 0.001. Detailed Implementation

[0042] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0043] To enable those skilled in the art to more clearly understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments. If specific experimental conditions are not specified in the embodiments, they are generally based on conventional conditions or conditions recommended by the reagent company; the reagents, consumables, etc. used in the following embodiments, unless otherwise specified, can be obtained commercially. Wherein: MRS broth medium, purchased from Qingdao Haibo Biotechnology Co., Ltd., product code HB0384-20; MRS agar medium, purchased from Qingdao Haibo Biotechnology Co., Ltd., product code HB0384-51; BBL liquid medium, purchased from Qingdao Haibo Biotechnology Co., Ltd., product code HB8777; eosin methylene blue agar medium, purchased from Qingdao Haibo Biotechnology Co., Ltd., product code HB0107; sodium azide-crystal violet-esculin agar medium, purchased from Qingdao Haibo Biotechnology Co., Ltd., product code HB0133-1; β-glucosidase, purchased from Shanghai Yuanye Biotechnology Co., Ltd., product code S10047; BBL agar medium: Follow the BBL liquid medium formula, add an additional 0.5% agar powder, and sterilize at 121℃ for 15 min.

[0044] 1 M Na2CO3: Accurately weigh 106.0 g of sodium carbonate solid into a small beaker, add an appropriate amount of distilled water (200-300 mL) to dissolve, transfer to a 1 L volumetric flask, and dilute to 1 L with distilled water.

[0045] p-Nitrophenyl-β-D-glucopyranoside (PNPG) was purchased from Fuzhou Feijing Biotechnology Co., Ltd., product code PH9306; p-nitrophenol (PNP) was purchased from Shanghai Maclean Biotechnology Co., Ltd., product code N814723; kudzu root, a commercially available food and medicinal ingredient; puerarin was purchased from Shanghai Yuanye Biotechnology Co., Ltd., product code B20446; daidzein was purchased from Shanghai Yuanye Biotechnology Co., Ltd., product code B20227; estrol was purchased from Shanghai Yuanye Biotechnology Co., Ltd., product code B50771; cisplatin was purchased from Shanghai Maclean Biotechnology Co., Ltd., product code D807330; hematoxylin and eosin (HE) staining kit was purchased from Beijing Solarbio Science & Technology Co., Ltd., product code G1120; mouse estradiol (E2) ELISA kit was purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd., product code m1001962; mouse follicle-stimulating hormone (FSH) ELISA kit... The following reagent kits were purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd. (product code: m1001910); the mouse luteinizing hormone (LH) ELISA kit was purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd. (product code: m1001984); estradiol was purchased from Beijing Solarbio Science & Technology Co., Ltd. (product code: 50-28-2); 4% paraformaldehyde fixative was purchased from Shanghai Beyotime Biotechnology Co., Ltd. (product code: P0099); the TUNEL staining assay kit for apoptosis was purchased from Elabscience (product code: E-CK-A320); the mounting solution containing anti-fluorescence quencher was purchased from Elabscience (product code: E-IR-R119); the MDA kit (TBA method) was purchased from Nanjing Jiancheng Biotechnology Research Institute Co., Ltd. (product code: A003-1-2); and the GSH-Px kit (colorimetric method) was purchased from Nanjing Jiancheng Biotechnology Research Institute Co., Ltd. (product code: A005-1-2).

[0046] Streptococcus thermophilus BLCC2-0025 was deposited on March 18, 2015, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2015130. Its classification and nomenclature are: Streptococcus thermophilus BLCC2-0025. Streptococcus thermophilus BLCC2-0025.

[0047] Lactobacillus salivarius LS1101 was deposited on November 9, 2020, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2020706. Its classification and nomenclature are: Lactobacillus salivarius LS1101 Lactobacillus salivarius LS1101.

[0048] The accession number for *Pediococcus lactis* MP1001 is CCTCC NO: M 2022136, and it is described in the patent "A strain of *Pediococcus lactis* MP1001, its powder and its application", application publication number CN 116814480A. The accession number for *Lactobacillus gastrospermum* 0010 is CCTCC NO: M 2015340, and it is described in the patent "A composition for assisting in the protection of the gastric mucosa and / or relieving bloating and its microecological preparation", application publication number CN 106420847B.

[0049] Example 1: Screening of strains that efficiently convert inactive estrogens in kudzu root into daidzein 1. Qualitative determination of β-glucosidase-producing strains: Thirty-six candidate bacterial strains (Table 1) from the strain resource bank of the Science and Technology Innovation Center of Shandong Phoenix Biotechnology Co., Ltd. were inoculated into 5 mL of MRS broth and cultured at 37°C for 20 h. Four μL of each strain's culture was then inoculated into 96-well plates containing 93 μL of MRS broth as the experimental group. A negative control was set up using 97 μL of MRS broth, and a positive control was set up using a mixture of 93 μL of MRS broth and 4 μL of 200 U / mL standard β-glucosidase solution. The plates were cultured at 37°C for 12 h. Then, 3 μL of 10 mM PNPG substrate was added to each culture system, and the reaction was carried out at 37°C for 40 min. Finally, 100 μL of 1 M Na₂CO₃ was added to each system to terminate the reaction for 5 min, until the color turned yellow and the OD value increased. 405 Determine if enzyme production has occurred. Select strains with absorbance values ​​greater than 0.6 for secondary screening.

[0050] Table 1: Screening strains producing β-glucosidase The results are as follows Figure 1 As shown, except for strains PA-001, PBIB3-001, and PBIB1-010 which lacked effective enzyme production capacity, the remaining 33 strains all possessed a certain ability to produce β-glucosidase. Ten strains with absorbance values ​​greater than 0.6 (Lactobacillus plantarum PBIL1-006, PBIL1-018, PBIL1-016, Lactobacillus rhamnosus PBIL3-001, Lactobacillus delbrueckii PBIL6-008, Lactobacillus reuteri PBIL4-004, Streptococcus thermophilus BLCC2-0025, Lactobacillus salivarius LS1101, Pediococcus lactis MP1001, and PBIP1-002) were selected for quantitative enzyme activity determination.

[0051] 2. Quantitative determination of β-glucosidase-producing strains: Ten strains with absorbance values ​​greater than 0.6 selected in the above experiments were inoculated into MRS broth medium and cultured at 37°C for 24 h. The culture solutions of the strains were then used for quantitative determination of enzyme activity. The specific procedures are as follows: (1) Take 10 25 mL graduated tubes and add 0.4 mL of 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, and 0.8 mM p-nitrophenol solution respectively. Then add 2.0 mL of 1 M Na2CO3 and 9.6 mL of distilled water. Let stand at room temperature for 5 min and then shake well. Use an equal volume of distilled water as a blank control and measure the absorbance A at 390 nm to establish a standard curve.

[0052] (2) Add 200 μL of bacterial culture medium and 200 μL of 1 mM PNPG to each tube, treat at 40℃ for 60 min, then add 2 mL of 1 M Na2CO3 to terminate the reaction, and then add 9.6 mL of distilled water. Measure the absorbance at 390 nm, using an equal volume of distilled water as a blank control. Repeat each sample three times. Express the enzyme production capacity as enzyme activity units (U / mL) and screen for strains with excellent enzyme production capacity.

[0053] The results are as follows Figure 2 As shown, *Lactobacillus reuteri* PBIL4-004, *Streptococcus thermophilus* BLCC2-0025, *Lactobacillus salivarius* LS1101, and *Pediococcus lactis* MP1001 were identified as high-yield enzyme-producing strains. *Pediococcus lactis* MP1001 and *Streptococcus thermophilus* BLCC2-0025, exhibiting the highest enzyme activity, were selected as key strains for the efficient biotransformation of puerarin isoflavones.

[0054] Example 2: Screening of estrol-producing strains 1. Test method: Five candidate strains (Table 2) from the strain resource bank of the Science and Technology Innovation Center of Shandong Phoenix Biotechnology Co., Ltd. were activated, and the number of viable bacteria after activation was the same (1×10⁻⁶). 8 Five strains of bacteria (CFU / mL) were inoculated at an 8% inoculum into sterilized kudzu root extract (the sterilized kudzu root extract was prepared in the same way as in Example 1), and fermented at 37°C for 24 h to obtain fermentation broth.

[0055] Table 2: Screening strains producing estrol A suitable amount of fermentation broth was taken from each group, centrifuged at 8000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm nylon membrane into a sample bottle. The estrol content in the fermentation broth was determined by HPLC, and strains capable of producing estrol were screened.

[0056] The method for determining the estrol content in the fermentation broth is as follows: Standard: Accurately weigh 10 mg of equol standard, dissolve it in 1 mL of methanol to prepare a 10 mg / mL standard stock solution, and use this as a stock solution to dilute with methanol to 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, and 0.0156 mg / mL to obtain the equol standard working solution. After filtering the standard working solution through a 0.22 μm nylon filter membrane, perform HPLC analysis sequentially according to the chromatographic conditions in Table 3, record the peak areas on the chromatograms of each component, and plot a standard curve.

[0057] Table 3: Chromatographic conditions for the determination of equadol 2. Test Results: The results are shown in Table 4. No equol was produced after fermentation of Leuconostoc mesenteroides PBILM-001, PBILM-002 and Bifidobacterium adolescentis PBIB2-001, while the equol content of Lactobacillus salivarius LS1101 and Lactobacillus gasseri 0010 was 0.15 and 0.11 mg / mL, respectively, showing a clear equol synthesis ability, providing specific strains for the preparation of highly active equol.

[0058] Therefore, Lactobacillus salivarius LS1101 and Lactobacillus gasseri 001 were selected as target strains for producing estrol.

[0059] Table 4: Results of equadol content determination for each strain Example 3: Preparation of Compound Fermentation Agent Based on the screening results of Examples 1 and 2, four strains of bacteria, namely Streptococcus thermophilus BLCC2-0025, Lactobacillus salivarius LS1101, Pediococcus lactis MP1001 and Lactobacillus gasseri 0010, were selected as compound fermentation agents to improve equol production.

[0060] Activated Streptococcus thermophilus BLCC2-0025, Lactobacillus salivarius LS1101, Pediococcus lactis MP1001, and Lactobacillus gasseri 0010 were inoculated into MRS broth medium and cultured to obtain Streptococcus thermophilus BLCC2-0025, Lactobacillus salivarius LS1101, Pediococcus lactis MP1001, and Lactobacillus gasseri 0010 bacterial suspensions.

[0061] The bacterial suspensions of *Streptococcus thermophilus* BLCC2-0025, *Lactobacillus salivarius* LS1101, *Pediococcus lactis* MP1001, and *Lactobacillus gasseri* 0010 were centrifuged, and the bacterial pellets were collected. The pellets were then resuspended in physiological saline to obtain bacterial suspensions, and the viable cell count in each suspension was adjusted to 1 × 10⁻⁶. 8 CFU / mL.

[0062] A compound fermentation agent was prepared by mixing a bacterial suspension of Streptococcus thermophilus BLCC2-0025, a bacterial suspension of Lactobacillus salivarius LS1101, a bacterial suspension of Pediococcus lactis MP1001, and a bacterial suspension of Lactobacillus gasseri 0010 in a volume ratio of 1:1:1:1.

[0063] Example 4: Preparation of Fermentation Broth Kudzu root extract was prepared according to the method of Example 1. The compound fermentation agent prepared in Example 3 was inoculated into the sterilized kudzu root extract. The inoculation amount of the compound fermentation agent was 8% of the volume of the kudzu root extract. The mixture was cultured at 37°C for 24 h to obtain the fermentation broth.

[0064] Comparative Example 1: Preparation of Fermentation Broth Kudzu root extract was prepared according to the method in Example 1. Compound fermentation agent A was inoculated into the sterilized kudzu root extract at an inoculation amount of 8% of the volume of kudzu root extract. The mixture was cultured at 37°C for 24 h to obtain fermentation broth A.

[0065] Compound fermentation agent A consists of 1×10⁶ live bacteria. 8 The bacterial suspension of Streptococcus thermophilus BLCC2-0025 with CFU / mL and viable count was 1×10⁻⁶. 8 The bacterial suspension of *Pediococcus lactis* MP1001 (CFU / mL) was prepared by mixing at a volume ratio of 1:1.

[0066] Comparative Example 2: Preparation of Fermentation Broth Kudzu root extract was prepared according to the method in Example 1. Compound fermentation agent B was inoculated into the sterilized kudzu root extract at an inoculation amount of 8% of the volume of kudzu root extract. The mixture was then cultured at 37°C for 24 h to obtain fermentation broth B.

[0067] Compound fermentation agent B consists of 1×10⁻⁶ live bacteria. 8 The bacterial suspension of Lactobacillus salivarius LS1101 with CFU / mL and the viable count was 1×10⁻⁶. 8 The bacterial suspension of Lactobacillus gasseri 0010 at a volume ratio of 1:1 was prepared.

[0068] Experimental Example 1: Evaluation of the Effects of Different Fermentation Processes 1. Test method: The equol content in the fermentation broth prepared in Example 4, Comparative Example 1 and Comparative Example 2, and the kudzu root extract prepared according to the method in Example 1 were determined according to the "method for determining equol content in fermentation broth" described in Example 2.

[0069] 2. Test Results: The results are shown in Table 5. In Comparative Example 1, after fermentation of kudzu root extract by a combination of *Pediococcus lactis* MP1001 and *Streptococcus thermophilus* BLCC2-0025, no equol was detected in the fermentation broth, confirming that these two strains only possess the ability to convert daidzein and do not have the function of further metabolizing daidzein to equol. In Comparative Example 2, although equol was detected in the fermentation broth of kudzu root extract by a combination of *Lactobacillus salivarius* LS1101 and *Lactobacillus gasseri* 0010, the content was far lower than that of the four-strain co-fermentation system (*Pediococcus lactis* MP1001, *Streptococcus thermophilus* BLCC2-0025, *Lactobacillus salivarius* LS1101, and *Lactobacillus gasseri* 0010). The results showed that, compared with the combined fermentation of Pediococcus lactis MP1001 and Streptococcus thermophilus BLCC2-0025, or the combined fermentation of Lactobacillus salivarius LS1101 and Lactobacillus gasseri 0010, the combined fermentation of kudzu root extract by four strains of Pediococcus lactis MP1001, Streptococcus thermophilus BLCC2-0025, Lactobacillus salivarius LS1101 and Lactobacillus gasseri 0010 synergistically increased the estrol content in the fermentation broth.

[0070] Table 5: Results of equadol content determination in each group In summary, this invention, through systematic screening, identified a composite bacterial strain composed of four specific strains and established a one-step co-fermentation process with a fermentation cycle of 24 hours. This process can simultaneously achieve efficient hydrolysis of glycosides in kudzu isoflavones and the directed conversion of daidzein to equol, significantly increasing the content of highly active isoflavone metabolites in the final product. It effectively overcomes the technical shortcomings of incomplete transformation pathways in single-strain formulations and low efficiency in traditional fermentation, providing key process support and a technological foundation for developing efficient, safe, and orally administered probiotic-phytoestrogen synergistic preparations.

[0071] Experimental Example 2: Construction of a cisplatin-induced POF mouse model 1. Test method: Healthy female Kunming mice weighing 37-38 g (purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.) were selected and randomly divided into two groups after 5 days of pre-feeding: normal group (NC) and model group (MC), with 20 mice in the normal group and 60 mice in the model group. The model group was injected intraperitoneally with cisplatin 3 mg / kg body weight for 5 consecutive days, while the normal group was injected with an equal volume of physiological saline.

[0072] 1.1 Condition observation and weight monitoring During the modeling period, the mice's condition was observed daily, and changes in their body weight before and after modeling were recorded.

[0073] 1.2 Observation of the estrous cycle During the modeling period, the estrous cycle changes in mice were observed for 7 consecutive days (starting from day 1 of intraperitoneal injection). The specific procedures were as follows: Sterile cotton swabs were soaked in sterile saline in advance. After soaking, the sterile cotton swabs were inserted into the vagina of mice and the secretions were scraped in one direction. The secretions were placed in a centrifuge tube containing 1 mL of saline. Then, the cotton swabs were stained onto a glass slide according to the HE staining kit. Cell morphology was observed under a microscope at 1000× to determine the estrous cycle of mice, including proestrus (P), estrus (E), metestrus (M), and diaestrus (D).

[0074] 1.3 Hormone Level Detection Two days after the modeling was completed, medication was stopped. Blood was collected from the orbital sinus of 5 mice in the normal group and 15 mice in the modeling group. The blood was allowed to stand at room temperature for 2 hours, then centrifuged at 3500 rpm for 15 minutes at 4°C. The supernatant was collected and temporarily stored at -20°C. Serum E2, FSH, and LH levels were measured according to the kit instructions.

[0075] 2. Test Results: 2.1 Observation of condition and changes in weight Mice in the normal group drank water and ate normally, were in good spirits, and had smooth fur; most mice in the model group injected with cisplatin intraperitoneally showed depression, disheveled fur, and dull fur.

[0076] The changes in mouse body weight before and after modeling are shown in Table 6.

[0077] Table 6: Changes in body weight of mice in each group before and after modeling Note: Different lowercase letters in the same column indicate significant differences (P < 0.05).

[0078] Before modeling, there was no significant difference in body weight between the normal group and the model group; after modeling, the body weight of the model group mice was significantly lower than that of the normal group (P<0.05).

[0079] 2.2 Observation results of the estrous cycle The normal estrous cycle in mice is 4-5 days, with the estrous period lasting approximately 6-12 hours. The determination of the estrous cycle in mice based on vaginal cytology smears from normal mice is as follows: Figure 3 ): (1) Proestrus (P): Numerous nucleated epithelial cells, either singly or in clusters, accompanied by a small number of keratinized cells; (2) Estrous phase (E): Abundant anucleate keratinocytes; (3) Late estrus (M): abundant keratinocytes, nucleated epithelial cells and leukocytes; (4) Interestrus (D): a large number of white blood cells.

[0080] This application uses intraperitoneal injection of cisplatin to establish a mouse model of ovarian dysfunction, and the estrous cycle monitoring results are as follows: Figure 4 As shown, the estrous cycle of mice in the model group was significantly disrupted over 7 consecutive days, with the absence of the estrus phase and most individuals remaining arrested in the late estrus or interestrus phase for a long period of time. In contrast, the normal control group mice were able to complete 1 to 2 complete cycles within 7 days. These results indicate that cisplatin induction alters the estrous cycle in mice.

[0081] 2.3 Hormone level test results The results of the detection of serum E2, FSH, and LH in mice are as follows: Figure 5 As shown, the E2 level in the model group mice was lower than that in the normal group, while the FSH and LH levels were higher, which is consistent with the symptoms of premature ovarian failure.

[0082] The experimental data above confirm that the mouse model of premature ovarian failure has been successfully established.

[0083] Experimental Example 3: Validation of the intervention effect of compound fermentation agent and fermentation broth on POF mice 1. Test method: 1.1 Experimental Grouping: The mice in the model group and the normal group that were successfully modeled in Experiment Example 2 were used as experimental subjects. The mice in the model group were randomly divided into 4 groups, with 10 mice in each group.

[0084] Normal group (NC): Mice in the normal group were given 0.2 mL of physiological saline per mouse daily; this served as a negative control.

[0085] Model group (MC): Mice in the model group were given 0.2 mL of physiological saline per mouse daily; serving as the model control.

[0086] Kudzu root extract group (GT): Mice in the model group were given 0.2 mL of sterilized kudzu root extract per mouse daily; the preparation of sterilized kudzu root extract was the same as in Example 1.

[0087] Compound fermentation agent group (CP): Mice in the model group were given 0.2 mL of compound fermentation agent per mouse daily; the preparation of compound fermentation agent was the same as in Example 3.

[0088] Kudzu root fermentation broth group (GF): Mice in the model group were given 0.2 mL of fermentation broth per mouse daily; the preparation of the fermentation broth was the same as in Example 4.

[0089] All mice were treated by gavage for 4 consecutive weeks, and their weight was recorded weekly.

[0090] 1.2 Observation of the estrous cycle: Estrogenesis was observed in mice during the fourth week of intervention, using the same method as in Experiment 2.

[0091] 1.3 Serum hormone levels: After the experiment, blood was collected from the eyeballs. The blood was allowed to stand at room temperature for 2 hours, then centrifuged at 3500 r / min for 15 minutes at 4°C to separate the serum. The serum E2, FSH and LH levels were detected using an ELISA kit. The specific operating steps were performed according to the instructions in the corresponding kit.

[0092] 1.4 Organ Index: After the mice were sacrificed, both ovaries and uteruses were removed and weighed. The organ index was calculated using the following formula: 1.5 HE and TUNEL staining of ovarian tissue: After the experiment, ovarian and uterine tissues from one side were fixed in 4% paraformaldehyde, routinely embedded in paraffin, and stained with hematoxylin and eosin (HE). Ovarian paraffin sections were dewaxed and hydrated, and apoptosis was detected according to the TUNEL staining kit instructions. The paraffin sections were dewaxed by a gradient washing process with xylene and ethanol, washed multiple times with PBS, and permeabilized with proteinase K, followed by multiple washes with PBS. After labeling and washing, an anti-quenching agent was added, and the sections were mounted with coverslips. Ovarian tissue cells were observed and photographed under a fluorescence microscope to observe morphological changes in mouse ovarian tissue.

[0093] 1.6 Measurement of ovarian oxidative stress levels: After the experiment, the other ovary was taken and frozen at -20°C. The ovarian tissue was homogenized and used for the determination of MDA and GSH-Px levels. The specific operation steps were performed according to the instructions in the corresponding kit.

[0094] 1.7 Gut microbiota analysis: After the experiment, 1g of colon contents from each group of mice was added to a 99 mL Erlenmeyer flask containing glass beads and sterile physiological saline. The mixture was shaken for 20 min until thoroughly mixed, and then serially diluted 10-fold with sterile physiological saline. The pipette tip was changed for each gradient, and each gradient was repeated three times. The gradient dilutions were plated on selective agar plates for Lactobacillus, Bifidobacterium, Escherichia coli, and Enterococcus (MRS agar, BBL agar, eosin methylene blue agar, and sodium azide-crystal violet-esculin agar, respectively). After completion, the plates were incubated at 37°C for 48 hours before counting.

[0095] 2. Test Results: 2.1 Results of the effect on mouse body weight: As shown in Table 7, after four weeks of gavage, the normal group mice grew normally, while the body weight of the model group and each intervention group increased over time, but remained significantly lower than that of the normal group. This may be because estrogen has a significant impact on metabolism, affecting fat distribution and energy metabolism. In the early stages of modeling, ovarian function is impaired, leading to a sharp decrease in estrogen secretion and resulting in weight loss. However, after four weeks of intervention, the body may initiate some compensatory mechanisms, stabilizing estrogen levels in a relatively low but balanced state. At this point, the body's metabolic rate no longer continues to decline, and fat breakdown and synthesis gradually reach a new equilibrium, thus causing the body weight to stop decreasing and even rebound.

[0096] Table 7: Body weight of mice in each group during intervention period Note: Different lowercase letters in the same column indicate significant differences (P < 0.05).

[0097] 2.2 Results of the effect on the estrous cycle in mice: The results are as follows Figure 6 As shown, the proportion of interestrus in the model group mice was significantly higher than that in the normal group. After 4 weeks of treatment, the proportion of interestrus in each intervention group mice was significantly reduced. Compared with the estrous cycle during modeling, the interestrus in each group was significantly shortened after intervention, which shows that it can improve the cisplatin-induced estrous cycle disorder.

[0098] 2.3 Results of the effect on mouse serum hormones: To verify the effects of each intervention group on improving ovarian function in premature ovarian failure model mice, serum E2, FSH, and LH levels were measured. The results are as follows: Figure 7 As shown, compared with the normal group, the model group mice had significantly lower E2 levels and higher LH and FSH levels, indicating that the hormone levels of the mice remained disordered after modeling, with insufficient E2 synthesis suggesting follicular development disorders or luteal insufficiency. After 4 weeks of intervention, the serum hormone levels in each intervention group were improved compared with the model group. Compared with the model group, the GF group showed a 121.15% increase in E2 levels, a 23.84% decrease in LH levels, and a 28.30% decrease in FSH levels, with better improvement than GT and CP, indicating that the synergistic effect of probiotics and phytoestrogens has a good effect on improving hormone disorders caused by premature ovarian failure.

[0099] 2.4 Results of the effects on mouse ovarian index and uterine index: The results are as follows Figure 8As shown, the ovarian and uterine indices of the model group mice were lower than those of the normal group, with the ovarian index being significantly lower than that of the normal group (P<0.001). After 4 weeks of intervention, the ovarian and uterine indices of mice in all intervention groups increased, with the GF group showing the best improvement in both ovarian and uterine indices, indicating that probiotics and phytoestrogens can synergistically improve the ovarian and uterine indices of mice with premature ovarian failure.

[0100] 2.5 Effects on mouse ovarian morphology: HE staining results are as follows Figure 9 As shown, in the NC group, follicles at different developmental stages were visible, with mature follicles located at the edge of the ovary and granulosa cells arranged regularly. In the model group, the number of atretic follicles was significantly increased, and the number of granulosa cells in the follicles was reduced and disordered. In each intervention group, the proportion of atretic follicles was reduced compared to the model group, with mature follicles located at the edge of the ovary and granulosa cells arranged in an orderly manner and in large numbers.

[0101] The results of the total number of follicles and the count of atretic follicles are as follows: Figure 10 As shown, the total number of follicles in the model group was significantly lower than that in the normal group, while the follicular atresia rate was significantly higher (P<0.01), indicating insufficient follicular reserve and reduced ovarian function in the model group. After 4 weeks of intervention, both the total number of follicles and the atresia rate in each group showed improvement. Among them, the total number of follicles in the GF group increased by 38.66% compared with the model group, and the number of atretic follicles decreased by 17.72%, thus reducing follicular atresia and maintaining follicular reserve.

[0102] 2.6 Effects on mouse uterine morphology: HE staining results are as follows Figure 11 As shown, the NC group mice had well-developed endometrium with loose stroma and abundant glands. The MC group mice had poorly developed endometrium and fewer glands. Four weeks after intervention, the poor endometrial development and reduced gland count in all groups of mice were improved.

[0103] 2.7 Effects on mouse ovarian cell apoptosis: Results of TUNEL staining of mouse ovarian tissue cells are as follows: Figure 12 and Figure 13 As shown, ovarian cells in the model group exhibited significant apoptosis compared to the normal group, and apoptosis was improved to varying degrees in each intervention group. The statistical results of apoptosis rate in mouse ovarian tissue cells are shown below. Figure 14 As shown, compared with the model group, the GF group reduced the apoptosis rate by 18.44%, which was better than the GT and CP groups and approached the normal group, indicating that the synergistic effect of probiotics and phytoestrogens can effectively improve ovarian cell apoptosis.

[0104] 2.8 Effects on oxidative stress in mouse ovaries: Malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) are core indicators of oxidative stress. MDA reflects the degree of lipid peroxidation and cell damage, while GSH-Px catalyzes the decomposition of hydrogen peroxide and protects the integrity of cell membrane structure and function. By detecting the levels of these indicators in mouse ovarian tissue and serum, the oxidative stress status and intervention effect in a mouse model of premature ovarian failure were assessed. The results are as follows: Figure 15 As shown, the MDA level in the ovarian tissue of the model group mice was significantly higher than that in the normal group (P<0.01), and the GSH-Px activity was lower than that in the normal group, indicating that oxidative stress had occurred in the ovaries of POF mice. After 4 weeks of intervention, the MDA and GSH-Px levels in all intervention groups were improved, which could effectively inhibit lipid peroxidation in ovarian tissue, reduce the damage of reactive oxygen species to ovarian cell membranes and follicle structures, repair and enhance the antioxidant system function of ovarian tissue, and improve the body's ability to scavenge reactive oxygen species.

[0105] 2.9 Results of the effect on the gut microbiota of mice: The results are as follows Figure 16 As shown, the levels of lactobacilli, bifidobacteria, Escherichia coli, and enterococci in the gut of mice were measured to assess the effects of cisplatin modeling and various interventions on the gut microbiota of POF mice. The levels of lactobacilli and bifidobacteria in the model group were significantly lower than in the normal group, while Escherichia coli and enterococci showed a slight decreasing trend. This indicates that cisplatin modeling creates a broad-spectrum ecological niche vacancy in the mouse gut microbiota, resulting in a significant decrease not only in beneficial bacteria but also in opportunistic pathogens. After intervention, compared with the model group, the levels of lactobacilli and bifidobacteria in all intervention groups were significantly increased, while Escherichia coli and enterococci showed a slight increase. In the CP group, the direct supplementation of lactobacilli and other strains directly led to an increase in lactobacilli abundance, which in turn increased bifidobacteria abundance through gut microbiota interaction. To maintain gut microbiota balance, the increase in beneficial bacteria abundance may lead to a certain degree of increase in harmful bacteria; therefore, the levels of Escherichia coli and enterococci in the CP group were also higher than in other groups. The GF group may have benefited from the supplementation of active ingredients such as estrogen, which upregulated the abundance of beneficial bacteria through the gut-ovarian axis interaction. At the same time, the active ingredients after kudzu fermentation inhibited the growth of harmful bacteria, resulting in a significant increase in the number of lactic acid bacteria and bifidobacteria, while the number of harmful bacteria was lower than that in the CP group.

[0106] In summary, this invention uses a compound fermentation agent composed of *Streptococcus thermophilus* BLCC2-0025, *Lactobacillus salivarius* LS1101, *Pediococcus lactis* MP1001, and *Lactobacillus gasseri* 0010. Fermentation of kudzu root extract with this compound fermentation agent effectively converts inactive estrogens in kudzu root into equol. The prepared fermentation broth contains beneficial bacteria and phytoestrogens, which have a synergistic effect. This synergistic effect can significantly improve reproductive function in POF mice, alleviate ovarian oxidative stress, and precisely regulate intestinal flora balance by targeting and regulating the gut-ovarian axis. This provides a safe and effective formulation and application scheme for non-hormonal intervention in premature ovarian failure.

[0107] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A compound fermentation agent for increasing estrol yield, characterized in that, It is a compound of Streptococcus thermophilus BLCC2-0025, Lactobacillus salivarius LS1101, Pediococcus lactis MP1001 and Lactobacillus gasseri 0010; The preservation number of the thermophilic streptococcus BLCC2-0025 is CCTCC NO: M 2015130, the preservation number of the lactobacillus LS1101 is CCTCC NO: M 2020706, the preservation number of the lactic acid pedunculates MP1001 is CCTCC NO: M 2022136, and the preservation number of the lactobacillus gasseri 0010 is CCTCC NO: M 2015340.

2. The compound fermentation agent according to claim 1, characterized in that, The compound fermentation agent is composed of Streptococcus thermophilus BLCC2-0025, Lactobacillus salivarius LS1101, Pediococcus lactis MP1001 and Lactobacillus gasseri 0010 in a live count ratio of 1:1:1:

1.

3. The compound fermentation agent according to claim 1 or 2, characterized in that, The total viable count of the compound fermentation agent is greater than or equal to 10. 8 CFU / mL.

4. The application of the compound fermentation agent according to claim 1 or 2 in the production of estrol from fermented kudzu root.

5. A fermentation broth, characterized in that, It is prepared by the following method: Inoculate the sterilized kudzu root extract with the compound fermentation agent described in claim 1 or 2, and incubate at 35-40℃ for 20-30 hours.

6. The fermentation broth according to claim 5, characterized in that, The method for preparing the kudzu root extract is as follows: add deionized water to kudzu root, soak for 30-60 minutes, decoct and extract, filter and collect the filtrate, concentrate and sterilize.

7. The fermentation broth according to claim 6, characterized in that, The decoction extraction method is as follows: heat the soaked kudzu root to boiling, decoct and extract for 1 hour, filter and collect the filtrate as the first filtrate; add deionized water to the residue again, heat to boiling, decoct and extract for 1 hour, filter and collect the filtrate as the second filtrate; combine the first filtrate and the second filtrate.

8. The fermentation broth according to claim 5, characterized in that, The inoculum amount of the compound fermentation agent is 6-10% of the volume of kudzu root extract.

9. The use of the fermentation broth according to any one of claims 5-8 in the preparation of a drug for preventing and treating premature ovarian failure.

10. The application according to claim 9, characterized in that, The dosage form of the drug is tablets, granules, powders, capsules, solutions, suspensions, emulsions, or lyophilized powders.

Images