Pediococcus pentosaceus NSS0402 and application thereof in improving polycystic ovarian syndrome

Abstract

The invention relates to the technical field of microbial culture, and particularly discloses pediococcus pentosaceus NSS0402 and application thereof to improvement of polycystic ovarian syndrome. The invention relates to pediococcus pentosaceus NSS0402, which is preserved in China Center for Type Culture Collection (CCTCC) on July 1, 2024, the address is Wuhan University, Wuhan, China, the pediococcus pentosaceus NSS0402 is named as pediococcus pentosaceus, and the preservation number is CCTCC NO: M 20241421. The pediococcus pentosaceus NSS0402 can effectively improve the polycystic ovarian syndrome and related metabolic level disorder and sex hormone level abnormality symptoms thereof, and can be applied to preparation of products for preventing, relieving or treating the polycystic ovarian syndrome.

Description

Technical Field

[0001] This application relates to the field of microbial culture technology, and more specifically, to a strain of Pediococcus pentosaceus NSS0402 and its application in improving polycystic ovary syndrome. Background Technology

[0002] Polycystic ovary syndrome (PCOS) is one of the most common endocrine and metabolic disorders among women of reproductive age worldwide, with an incidence rate as high as 6-20%, and this rate is increasing year by year. The main pathological features of this disease include ovulation dysfunction, hyperandrogenemia, and polycystic ovarian changes. This not only leads to external symptoms such as irregular menstruation, hirsutism, and acne, but also severely impacts fertility. Approximately 50-70% of PCOS patients experience ovulation disorders, making it one of the leading causes of female infertility.

[0003] Besides reproductive system abnormalities, PCOS is often accompanied by a series of metabolic problems, such as insulin resistance, obesity, and diabetes. Studies have shown that approximately 50-70% of PCOS patients have varying degrees of insulin resistance, which in turn exacerbates hyperandrogenemia, creating a vicious cycle that increases the risk of long-term complications such as cardiovascular disease and metabolic syndrome, severely impacting patients' quality of life and health prognosis.

[0004] Currently, clinical treatments for PCOS mainly include lifestyle interventions, medication, and assisted reproductive technologies. Lifestyle interventions such as diet control and exercise can improve symptoms to some extent, but patient adherence is poor, making long-term adherence difficult. Commonly used medications include oral contraceptives to regulate menstrual cycles, anti-androgen drugs to improve symptoms such as hirsutism, and insulin sensitizers to alleviate insulin resistance. However, these medications have many limitations. For example, oral contraceptives may cause adverse reactions such as breast tenderness and nausea, and long-term use may affect glucose and lipid metabolism; insulin sensitizers may cause gastrointestinal discomfort and hypoglycemia, and some patients do not respond well to the medication. While assisted reproductive technologies can help some patients achieve their desire for children, they are expensive, have limited success rates, and may cause complications such as ovarian hyperstimulation syndrome.

[0005] In recent years, probiotics, as a class of live microorganisms beneficial to host health, have been widely used to regulate gut microbiota and improve metabolic-related diseases. Existing studies have shown that certain probiotic strains can improve PCOS by improving insulin resistance, reducing inflammatory factor levels, and regulating hormone secretion, providing a new research direction for PCOS treatment. However, the currently discovered probiotic strains still have limitations in their specificity and effectiveness in improving PCOS, failing to meet clinical needs. Therefore, screening for a probiotic strain that can specifically improve PCOS symptoms, with high safety and few side effects, is of great significance for improving the treatment effect of PCOS and improving patients' quality of life.

[0006] Pediococcus pentosaceus, a type of lactic acid bacteria, is widely used in food fermentation and probiotic preparations. It exhibits good human tolerance, can stably colonize the gut, and demonstrates excellent safety and probiotic properties. The inventors, through a systematic process including targeted screening and functional validation, have identified a strain of Pediococcus pentosaceus, NSS0402, aiming to provide a novel and effective approach for the prevention, mitigation, and treatment of PCOS, thus addressing the shortcomings of existing treatment options. Summary of the Invention

[0007] The purpose of this application is to provide a strain of Pediococcus pentosaceus NSS0402 and its application in improving polycystic ovary syndrome (PCOS). The Pediococcus pentosaceus NSS0402 screened in this application has shown excellent performance in improving PCOS, providing a new and effective means for the prevention, relief and treatment of PCOS.

[0008] In the first aspect, this application provides a strain of Pediococcus pentosaceus NSS0402, which adopts the following technical solution: A strain of Pediococcus pentosaceus NSS0402 was deposited on July 1, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, and named Pediococcus pentosaceus NSS0402, with accession number CCTCC NO:M 20241421.

[0009] By adopting the above technical solution, the Pediococcus pentosaceus NSS0402 screened in this application is a probiotic that can specifically improve polycystic ovary syndrome (PCOS). It has high safety and few side effects. On the one hand, it has excellent acid resistance, can adapt to the human intestinal environment and survive stably, and can produce short-chain fatty acids with rich antibacterial activity, which participate in the body's immune response and endocrine regulation, thereby regulating the host's metabolic level and sex hormone secretion. On the other hand, it can significantly improve the pathological symptoms of ovarian tissue in patients with PCOS, including the number of cystic follicles, corpus luteum, and testosterone levels.

[0010] Secondly, this application provides the use of Pediococcus pentosaceus NSS0402 in products for improving polycystic ovary syndrome.

[0011] Optionally, the product may include food, medicine, or health products.

[0012] Optionally, the viable count of Pediococcus pentosaceus NSS0402 in the food, medicine, or health product is ≥1×10⁻⁶. 6 CFU / g.

[0013] Optionally, the Pediococcus pentosaceus NSS0402 in the food, medicine or health product is present in the form of Pediococcus pentosaceus NSS0402 lyophilized powder.

[0014] Optionally, the Pediococcus pentosaceus NSS0402 lyophilized powder is obtained by mixing Pediococcus pentosaceus NSS0402 mycelial sludge with a lyophilization protectant and then freeze-drying it under vacuum, wherein the viable count is ≥1×10⁻⁶. 6 CFU / g.

[0015] Optionally, the mass ratio of the sludge of *Pediococcus pentosaceus* NSS0402 to the freeze-drying protectant is (1-3):1.

[0016] Optionally, the freeze-drying protectant is formulated as follows: 1-2 g / L trehalose, 1-2 g / L resistant dextrin, 1-2 g / L skim milk powder, 0.1-1 g / L lactoferrin, 0.1-0.2 g / L monosodium glutamate, 1 g / L Tween 80 and 1 L water.

[0017] By adopting the above technical solution, Pediococcus pentosaceus is a probiotic. The Pediococcus pentosaceus NSS0402 screened in this application has high safety in products (such as medicines) for the prevention, relief or treatment of polycystic ovary syndrome, and it does not produce drug resistance. It improves the physiological and psychological adverse reactions caused by antibiotics in patients with conventional polycystic ovary syndrome, and can be used for a long time in products for the prevention, relief or treatment of polycystic ovary syndrome in women.

[0018] Thirdly, this application provides the use of Pediococcus pentosaceus NSS0402 or Pediococcus pentosaceus lyophilized powder in the preparation of microecological preparations of γ-aminobutyric acid.

[0019] Optionally, the microecological preparation includes ready-to-eat probiotic powder, probiotic active beverage, and postbiotic probiotic drink.

[0020] Fourthly, this application provides a microecological preparation containing Pediococcus pentosaceus NSS0402, employing the following technical solution: A microecological preparation containing Pediococcus pentosaceus NSS0402, comprising, by weight, 2-5 parts probiotic freeze-dried powder, 10-15 parts maltodextrin, 20-25 parts skim milk powder, 5-8 parts fructooligosaccharides, 5-10 parts emulsified calcium, 1-4 parts black bean powder, 1.5-2.5 parts black date powder, 2.1-2.8 parts hawthorn powder, and 3.6 parts lemon powder, wherein the probiotic freeze-dried powder is Pediococcus pentosaceus NSS0402 freeze-dried powder.

[0021] Optionally, the probiotic freeze-dried powder is a compound probiotic freeze-dried powder, including Pediococcus pentosaceus NSS0402 freeze-dried powder and Lactobacillus reuteri NSL0506 freeze-dried powder.

[0022] By adopting the above technical solution, the inventors discovered that when Pediococcus pentosaceus NSS0402 is combined with Lactobacillus reuteri NSL0506 as a compound probiotic for the prevention, improvement or treatment of polycystic ovary syndrome in women, it has a significantly better effect than single bacterial agents or other compound methods. This indicates that Pediococcus pentosaceus NSS0402 and Lactobacillus reuteri NSL0506 have a synergistic effect in regulating the metabolic level and abnormal sex hormone secretion in patients with polycystic ovary syndrome, and improving the abundance of intestinal flora in patients.

[0023] In summary, this application has the following beneficial effects: 1. The Pediococcus pentosaceus NSS0402 obtained in this application is a probiotic with excellent acid resistance and acid production capacity. It can adapt to the human intestinal environment and survive in the intestine. The short-chain fatty acids with antibacterial activity produced can participate in the body's immune response and endocrine regulation, thereby regulating the host's metabolic level and sex hormone secretion.

[0024] 2. The Pediococcus pentosaceus NSS0402 obtained in this application can specifically improve polycystic ovary syndrome (PCOS), with high safety and few side effects. It can significantly improve the pathological symptoms of ovarian tissue in PCOS patients, including the number of cystic follicles, corpus luteum, and testosterone levels.

[0025] 3. The Pediococcus pentosaceus NSS0402 obtained in this application has high safety when used in products for the prevention, relief or treatment of polycystic ovary syndrome, such as videos, health products and medicines. It does not produce drug resistance, improves the physiological and psychological adverse reactions caused by antibiotic use in patients with conventional polycystic ovary syndrome, and can be used long-term in products for the prevention, relief or treatment of polycystic ovary syndrome in women. Detailed Implementation

[0026] The present application will be further described in detail below with reference to embodiments and comparative examples. Example

[0027] Example 1: Screening, isolation and identification of Pediococcus pentosaceus NSS0402 1. Screening and purification of Pediococcus pentosaceus NSS0402 Sampling location: Pickled vegetable factory in Pingdu City, Qingdao, Shandong Province; The obtained bacterial strain was added to 100 ml of MRS liquid shake flask medium and cultured for 24 h. The culture was then streaked onto MRS solid medium and anaerobically cultured at 37°C for 48 h. Single colonies were then inoculated into MRS liquid medium and anaerobically cultured at 37°C for 24 h. Finally, 0.9% physiological saline was added for 10-fold serial dilutions, diluting the bacterial suspension three times to a final volume of 10. -8 Take 1 mL of bacterial suspension and inoculate it into MRS solid medium, and culture it under anaerobic conditions at 37°C for 48 h. Typical colonies on the plate were picked and re-streaked onto MRS solid medium using the streak method for purification. The purification process was repeated for 3 generations to obtain the purified strain, which was then preserved in 40% glycerol.

[0028] The MRS liquid culture medium formula is as follows: 20g glucose, 10g peptone, 6.5g beef extract, 5g yeast extract, 2g diammonium citrate, 2g dipotassium hydrogen phosphate, 4g anhydrous sodium acetate, 0.58g magnesium sulfate heptahydrate, 0.25g manganese sulfate monohydrate, 1mL Tween 80, 0.5g L-cysteine ​​hydrochloride, and 1L sterile water. For solid culture medium, an additional 18g of agar powder is required.

[0029] 2. Identification of Pediococcus pentosaceus NSS0402 2.1 The purified strain was Gram-stained and observed under a microscope. Morphological observation under an optical microscope revealed that the colony morphology of the strain was 1-2 mm in diameter, round, milky white on the front, smooth, with neat middle and edges, opaque, moist, and easy to pick up. This spherical strain does not produce spores and is Gram-positive after staining, indicating that it is a Gram-positive bacterium. 2.2 Acid Resistance Test After sterilizing PBS buffer (pH=6.8) at 121℃ for 15 min, the purified strain was activated for two generations and then resuspended in PBS buffer at a 4% inoculum to obtain a bacterial suspension. The pH of the bacterial suspension was adjusted to 3 with 37% hydrochloric acid solution and incubated at 37℃. Samples were taken at 0 min, 15 min, 30 min, 45 min, and 60 min to determine the viable count and calculate the survival rate. Survival rate = viable count at sampling time / original viable count × 100%.

[0030] Table 1 Time / min 0 15 30 45 60 Survival rate / % 100 94.2 88.3 80.3 75.4 It can be seen that after culturing at pH 3 for 60 minutes, the survival rate of the strain decreased from 100% to 75.4%, but it still maintained excellent viability, indicating that the strain can grow well in acidic environments and has excellent acid resistance.

[0031] 2.3 Acid production capacity test The acid-producing capacity of the strains was determined by titration. After activation, the strains preserved in glycerol tubes were inoculated into MRS liquid medium at a 2% inoculum and cultured at 37℃ for 24 h. 10 mL of the fermentation broth of each strain was taken and added to 50 mL of sterile water. 2-3 drops of 1 g / L phenolphthalein were added as an indicator, and titration was performed with 0.1 mol / L NaOH standard solution. The titration endpoint was defined as the solution turning pink and not fading after 30 s. Each sample was titrated in triplicate. Using uninoculated MRS liquid culture medium as a blank control, the total acidity (g·L⁻¹) = (V₁ - V₂) × c × 100 × V 0-1 (V1 is the volume of NaOH solution consumed by the sample, mL; V2 is the volume of NaOH solution consumed by the blank control, mL; V0 is the total volume of the diluent, mL; c is the concentration of standard NaOH, mol / L).

[0032] Table 2 Sample Name control group Parallel Sample 1 Parallel Sample 2 Parallel Sample 2 Acidity (g / L) 0.2±0.1 6.8±0.3 7.2±0.4 6.5±0.2 It can be seen that Pediococcus pentosaceus NSS0402 has excellent acid resistance and the ability to produce SCFA (short-chain fatty acids).

[0033] The strain obtained through screening and identification was deposited and named Pediococcus pentosaceus NSS0402. It was deposited at the China Center for Type Culture Collection (CCTCC) on July 1, 2024, at Wuhan University, Wuhan, China, with accession number CCTCC NO:M 20241421.

[0034] Example 2: Detection of physiological and biochemical properties of Pediococcus pentosaceus NSS0402 1. Drug resistance testing The susceptibility of Pediococcus pentosaceus strains to eight antibiotics was determined using the KB method. According to the Clinical Laboratory Standards Institute (CLSI) criteria, the strains were classified as resistant, intermediate, or sensitive. The test results are recorded in Table 3, where S represents sensitive, M represents moderately sensitive, and R represents resistant.

[0035] Table 3 Results of antibiotic susceptibility testing antibiotic penicillin Chloramphenicol Rifampicin tetracycline clindamycin Vancomycin Cefaclor Sensitivity S S S S S S S The overuse of antibiotics in the pharmaceutical industry can lead to increased drug resistance in pathogenic strains, and the gut microbiota in humans is prone to disorder and imbalance. Therefore, obtaining antibiotic-sensitive lactic acid bacteria has become one of the important indicators of whether their application in food is safe.

[0036] Table 3 shows that the sensitivity of *Pediococcus pentosaceus* strain to eight antibiotics was detected by the K=B method. *Pediococcus pentosaceus* NSS0402 was sensitive to all eight antibiotics, indicating that *Pediococcus pentosaceus* NSS0402 does not carry transferable antibiotic resistance genes and has no resistance phenotype. After entering the human body, especially after entering the intestinal environment, it can effectively avoid the potential risk of promoting antibiotic resistance, and the safety risk is low.

[0037] 2. Sugar fermentation experiment The metabolic activity of the strain was determined by sugar fermentation test. Pediococcus pentosaceus NSS0402 was tested to see if it could ferment and produce acid using different types of sugar alcohols listed in Table 4 as substrates. This was to test the nutritional requirements of Pediococcus pentosaceus NSS0402. The culture time was 48 hours. The experimental results are recorded in Table 4, where + indicates positive and fermentation can be carried out using this substrate, and - indicates negative and fermentation cannot be carried out using this substrate.

[0038] Table 4. Results of sugar alcohol fermentation by *Pediococcus pentosacchari* NSS0402 The results of sugar alcohol fermentation in Table 4 show that *Pediococcus pentosaceus* NSS0402 exhibits typical metabolic characteristics of *Pediococcus pentosaceus*, capable of fermenting various sugar alcohols such as D-glucose, D-lactose, D-maltose, D-sucrose, and D-merinobisaccharide. This indicates that *Pediococcus pentosaceus* NSS0402 has a wide and diverse carbon source utilization capacity, high metabolic flexibility, strong environmental adaptability, and strong intestinal colonization potential, laying a solid foundation for the development and application research of microecological preparations containing *Pediococcus pentosaceus* NSS0402.

[0039] Example 3: Safety testing of Pediococcus pentosaceus NSS0402 1. Hemolytic test Hemolytic activity assay of bacterial strains: Hemolysis refers to the rupture of red blood cells and the release of hemoglobin under the influence of hemolytic toxins and other physicochemical factors. The presence or absence of hemolytic activity in the bacterial strain's metabolites is considered an important indicator for evaluating the strain's safety.

[0040] The activated Pediococcus pentosaceus NSS0402 and the quality control strain Escherichia coli were streaked onto Columbia blood agar plates using a sterile inoculation loop and incubated at 37°C for 48 hours. A blank control experiment was set up to observe whether hemolytic zones appeared around the colonies.

[0041] The results showed that no obvious hemolytic zone was formed around Pediococcus pentosaceus NSS0402, indicating that it was a non-toxic γ-hemolytic disease, which also shows that Pediococcus pentosaceus NSS0402 is non-pathogenic and has good safety.

[0042] 2. Acute oral toxicity test in animals Twenty healthy adult SPF-grade KM mice (18-22g each), half male and half female, were selected. The concentration of *Pediococcus pentosaceus* NSS0402 in its growth phase was adjusted to 1×10⁻⁶ using PBS buffer. 8 The mice were administered CFU / mL via gavage once on an empty stomach, with a gavage volume of 0.2 mL / 10 g body weight. Immediately after gavage, the mice were observed for changes in activity, weight, and physical condition. The observation period was 14 days.

[0043] The results showed that the mice had normal activity and appetite during the observation period and gained weight. After the experiment, the surviving mice were dissected and no abnormalities were found in the internal organs such as heart, liver, spleen, lungs, kidneys and intestines. This indicates that Pediococcus pentosaccharis NSS0402 does not have acute toxicity to mice and that Pediococcus pentosaccharis NSS0402 has good safety.

[0044] Example 4: Preparation of Lyophilized Powder of Pediococcus Pentosacchari NSS0402 The preparation method of Pediococcus pentosaceus NSS0402 lyophilized powder includes the following steps: S1: Activated Pediococcus pentosus NSS0402 was streaked into MRS solid medium and anaerobic fermented at 37℃ for 48 h. The microbial suspension was then collected and centrifuged at 8000 r / min for 10 min to collect the centrifuged cells and obtain bacterial sludge. S2: Mix the bacterial sludge with the freeze-drying protectant at a mass ratio of 3:1. The raw material formula of the freeze-drying protectant includes 2 g / L trehalose, 2 g / L resistant dextrin, 2 g / L skim milk powder, 1 g / L lactoferrin, 0.2 g / L monosodium glutamate, 1 g / L Tween 80 and 1 L water to obtain Pediococcus pentosaceus NSS0402 bacterial suspension. S3: Pre-freeze the Pediococcus pentosacchari NSS0402 bacterial suspension at -60℃ for 1 hour in a vacuum freeze dryer, then vacuum to 20Pa, and start gradient sublimation drying at -50℃. After 24 hours, freeze-dry into powder to obtain Pediococcus pentosacchari NSS0402 freeze-dried powder with high viable bacterial count. After dispensing, store in a cold storage at -18℃. S4: Mix the lyophilized powder of Pediococcus pentosaceus NSS0402 with physiological saline (0.85%), and calculate the number of viable bacteria and survival rate using the viable bacteria counting method. The lyophilization survival rate = (total number of viable bacteria per milliliter after lyophilization and rehydration / total number of viable bacteria per milliliter of bacterial solution before lyophilization) × 100%.

[0045] Example 5: Preparation of a probiotic preparation containing Pediococcus pentosaceus NSS0402 The microecological preparation containing Pediococcus pentosaceus NSS0402 comprises, by weight, 2 parts of lyophilized Pediococcus pentosaceus NSS0402 prepared in Example 4, 10 parts of maltodextrin, 20 parts of skim milk powder, 5 parts of fructooligosaccharides, 5 parts of emulsified calcium, 1 part of black bean powder, 1.5 parts of black date powder, 2.1 parts of hawthorn powder and 3 parts of lemon powder; The preparation method of the above-mentioned probiotic preparation containing Pediococcus pentosaceus NSS0402 includes the following steps: First, the raw materials are broken up using a vibrating screen to create uniform, fine particles for easy mixing. Then, an equal or slightly larger amount of excipients (usually a free-flowing carrier such as maltodextrin) is taken and pre-mixed with the probiotic powder in a small mixer. Next, approximately twice the weight of the excipients are added and mixed again. This process is repeated, like a snowball effect, gradually adding more excipients until all are added. This ensures that the probiotic particles are evenly separated by the excipient particles, achieving a uniform microscopic distribution. All the pre-mixed materials are then fed into a large final mixer for a set time to ensure the extreme uniformity of the entire batch. The mixed powder is then directly fed into a fully automatic packaging machine for filling into bags, bottles, or strips. During filling, the packaging containers are vacuum-sealed and filled with nitrogen, a crucial measure to ensure the stability of the active probiotics throughout their shelf life. The lyophilized powder in the probiotic preparation was then diluted using a serial dilution method, and viable cell counts were performed on MRS agar medium at appropriate concentrations. The viable cell count of *Pediococcus pentosaceus* NSS0402 in the probiotic preparation was 4.12 × 10⁻⁶. 8 CFU / g.

[0046] Example 6: Preparation of a probiotic preparation containing Pediococcus pentosaceus NSS0402 The microecological preparation containing Pediococcus pentosaceus NSS0402 differs from that in Example 5 in that the raw materials, by weight, include 4 parts of lyophilized Pediococcus pentosaceus NSS0402 prepared in Example 4, 12.4 parts of maltodextrin, 23 parts of skim milk powder, 7 parts of fructooligosaccharides, 8 parts of emulsified calcium, 3 parts of black bean powder, 2 parts of black date powder, 2.5 parts of hawthorn powder, and 5 parts of lemon powder. All other steps are the same as in Example 5.

[0047] Example 7: Preparation of a probiotic preparation containing Pediococcus pentosaceus NSS0402 The microecological preparation containing Pediococcus pentosaceus NSS0402 differs from that in Example 5 in that the raw materials, by weight, include 6 parts of lyophilized Pediococcus pentosaceus NSS0402 prepared in Example 4, 15 parts of maltodextrin, 25 parts of skim milk powder, 8 parts of fructooligosaccharides, 10 parts of emulsified calcium, 4 parts of black bean powder, 2.5 parts of black date powder, 2.8 parts of hawthorn powder, and 6 parts of lemon powder. All other steps are the same as in Example 5.

[0048] Example 8: Effect of Pediococcus pentosaceus NSS0402 on alleviating polycystic ovary syndrome symptoms in rats. Seventy healthy rats of similar size were purchased and housed under specific pathogen-free conditions, with 12 hours of light and 12 hours of darkness. All rats were acclimatized for one week before the experiment and then randomly divided into 7 groups of 10 rats each.

[0049] The grouping and processing status are as follows: Group 1: Blank control group (CTL); Group 2: Untreated polycystic ovary syndrome model group (MC); Group 3: Polycystic ovary syndrome group treated with only the probiotic preparation containing Pediococcus pentosaccharide NSS0402 from Example 5 (NSS0402); Group 4: Polycystic ovary syndrome group treated with only the probiotic preparation containing Pediococcus pentosaceus NSS0402 from Example 6 (NSS0402); Group 5: Polycystic ovary syndrome group treated with a probiotic preparation containing only Pediococcus pentosaceus NSS0402 from Example 7 (NSS0402); Group 6: Polycystic ovary syndrome group treated with a probiotic preparation containing only Lactobacillus reuteri NSL0506 (NSL0506). The probiotic preparation containing Lactobacillus reuteri NSL0506 differs from that in Example 5 only in the type of probiotic. Group 7: Polycystic ovary syndrome group treated with a combination of probiotic preparations containing Pediococcus pentosaceus NSS0402 and Lactobacillus reuteri NSL0506 from Groups 3 and 6 (NSS0402+NSL0506, with a live bacteria ratio of 3:1 for Pediococcus pentosaceus NSS0402 and Lactobacillus reuteri NSL0506); The intervention dose of the probiotic-containing probiotic preparation in each of the above groups was 1×10⁻⁶. 8 CFU / day, with a 1-week adaptation period before administration.

[0050] 1. Effects of probiotics on polycystic ovary syndrome: Polycystic ovary syndrome symptoms were induced in rats in groups 2-7 by gavage with letrozole (1 mg / kg, 1% carboxymethyl cellulose) for 21 consecutive days. In group 1, rats were gavaged with 1% carboxymethyl cellulose instead of letrozole. All rats were fed normal diets for 30 consecutive days. Body weight was recorded every 3 days, and the results are recorded in Table 5.

[0051] 2. Ovulation function detection: After gavage, the estrous cycle of each group of rats was observed by vaginal smear method, the ovulation rate was calculated, and the results were recorded in Table 6.

[0052] 3. Serum analysis: Rats were fasted overnight and sacrificed on day 31. Blood samples were collected, stored at 20°C for 1 hour, and then centrifuged at 3000g for 15 min to obtain serum.

[0053] 3.1 Ovarian morphology observation: After the rats were sacrificed, the ovaries were removed, weighed, and prepared into pathological sections. HE staining was performed to observe the ovarian morphology.

[0054] The results showed that the ovarian volume of rats in group 2 increased, the number of follicles increased, most of which were atretic follicles, and there was significant stromal hyperplasia; the ovarian volume and weight of rats in groups 3-7 decreased, the follicular development improved, and the number of atretic follicles decreased, indicating that Pediococcus pentosus NSS0402 has a certain improving effect on the ovarian morphology of rats with polycystic ovary syndrome.

[0055] 3.2 Determination of metabolic levels: Total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL C), and low-density lipoprotein cholesterol (LDL C) were measured using a biochemical analyzer (Beckman Coulter, CA), and the results are recorded in Table 7.

[0056] 3.3 Determination of sex hormone levels: Testosterone, luteinizing hormone (LH), estradiol (E2), progesterone, anti-Müllerian hormone (AMH), and gonadotropin-releasing hormone (GnRH) were measured using an enzyme-linked immunosorbent assay kit according to the manufacturer's protocol, and the results are recorded in Table 8.

[0057] Table 5 As shown in Table 5, compared with the blank control group 1, the rats in the intervention group 2 grew rapidly and gained weight quickly after one month. In the probiotic intervention group, the weight gain trend of rats in groups 3-6 slowed down, and the overall growth rate was lower than that of the model group 2.

[0058] In addition, it was unexpectedly found that when Pediococcus pentosaceus NSS0402 and Lactobacillus reuteri NSL0506 were used in combination, the weight loss of rats in the compound probiotic group 7 was more obvious, especially in the 4th week, their average weight tended to approach that of group 1.

[0059] Table 6 Group Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Ovulation rate / % 85 20 40 52 55 49 60 According to the results of the ovulation function test in rats in Table 6, the ovulation rate of rats in the blank control group 1 was 80%, the ovulation rate of rats in the model group 2 was significantly reduced to 20% (P<0.01), and the ovulation rate of rats in the experimental groups 3-7 was increased to 50% (P<0.05). This indicates that Pediococcus pentosaceus NSS0402 can effectively improve the ovulation function of rats with polycystic ovary syndrome.

[0060] Table 7 As shown in Table 7, the results of the metabolic level measurements indicate that, compared with the blank control group 1, the model group 2 had higher levels of triglycerides (TG) and low-density lipoprotein cholesterol (LDL C), and lower levels of total cholesterol (TC) and high-density lipoprotein cholesterol (HDL C), indicating that letrozole successfully induced metabolic disorders in rats.

[0061] However, when probiotics were used for intervention, we found that the TG and LDL C levels of rats treated with Pediococcus pentosaccharide NSS0402 and Lactobacillus reuteri NSL0506 decreased, while the TC and HDL C levels recovered. The effect of Pediococcus pentosaccharide NSS0402 intervention was more obvious in the group. In other words, Pediococcus pentosaccharide NSS0402 has a more significant effect on improving the metabolic disorder symptoms of polycystic ovary syndrome rats than Lactobacillus reuteri.

[0062] In addition, it was found that when the compound probiotic group 7 was treated with a combination of Pediococcus pentosaceus NSS0402 and Lactobacillus reuteri NSL0506, the serum TC, HDL C, TG and LDL C levels of rats were basically restored to normal, and were more similar to those of the CTL group.

[0063] Table 8 Table 8 shows the results of the measurement of sex hormone levels in rats, reflecting the endocrine status of the experimental rats. By analyzing the serum sex hormone concentration, it can be seen that compared with the blank control group 1, the testosterone, LH and GnRH levels of rats in the model group 2 were significantly higher, showing symptoms of polycystic ovary syndrome. At the same time, their progesterone, E2 concentration and AMH levels were lower, reflecting abnormal ovarian function in rats in the model group 2.

[0064] Under the intervention of probiotics, the level of male hormone testosterone in rats in groups 3-6 showed a decreasing trend, while the level of estrogen increased, indicating that the symptoms of polycystic ovary syndrome were improved. It can be seen that Pediococcus pentosaceus NSS0402 has a more significant effect on improving the symptoms of polycystic ovary syndrome than Lactobacillus reuteri.

[0065] In addition, it was found that when the compound probiotic group 7 was treated with a combination of Pediococcus pentosaceus NSS0402 and Lactobacillus reuteri NSL0506, it had a more significant effect on restoring estrogen levels in experimental rats, and the relief effect on polycystic ovary syndrome-related symptoms was more significant.

[0066] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A strain of Pediococcus pentosaceus NSS0402, characterized in that, The Pediococcus pentosaceus NSS0402 strain was deposited on July 1, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, and named Pediococcus pentosaceus NSS0402, with accession number CCTCC NO: M 20241421.

2. The use of Pediococcus pentosaceus NSS0402 as described in claim 1 in products for improving polycystic ovary syndrome.

3. The application according to claim 2, wherein the product includes food, medicine or health products.

4. In the application according to claim 3, the viable count of Pediococcus pentosaceus NSS0402 in the food, medicine, or health product is ≥1×10⁻⁶. 6 CFU / g.

5. In the application according to claim 3, the Pediococcus pentosaceus NSS0402 in the food, medicine or health product exists in the form of Pediococcus pentosaceus NSS0402 lyophilized powder.

6. In the application according to claim 5, the Pediococcus pentosaceus NSS0402 lyophilized powder is obtained by mixing Pediococcus pentosaceus NSS0402 mycelial sludge with a lyophilization protectant and then freeze-drying it under vacuum, wherein the viable count is ≥1×10⁻⁶. 6 CFU / g.

7. A microecological preparation containing Pediococcus pentosaceus NSS0402, characterized in that, By weight, the ingredients include 2-5 parts probiotic freeze-dried powder, 10-15 parts maltodextrin, 20-25 parts skim milk powder, 5-8 parts fructooligosaccharides, 5-10 parts emulsified calcium, 1-4 parts black bean powder, 1.5-2.5 parts black date powder, 2.1-2.8 parts hawthorn powder and 3.6 parts lemon powder, wherein the probiotic freeze-dried powder is Pediococcus pentosaceus NSS0402 freeze-dried powder.

8. The microecological preparation containing Pediococcus pentosaceus NSS0402 according to claim 7, characterized in that, The probiotic freeze-dried powder is a compound probiotic freeze-dried powder, including Pediococcus pentosaceus NSS0402 freeze-dried powder and Lactobacillus reuteri NSL0506 freeze-dried powder.

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