Pediococcus pentosaceus A2 for preventing or improving polycystic ovary syndrome, bacterial agent thereof and application thereof

Abstract

Embodiments of the present invention relate to the field of microorganisms, and specifically relate to Lactiplantibacillus pentosus A2, a bacterial agent and its application for preventing or improving polycystic ovary syndrome. The preservation number of Lactiplantibacillus pentosus A2 is CGMCC NO. 27253. Lactiplantibacillus pentosus A2 of the present invention can correct irregular estrous cycles, improve ovarian polycystic lesions, sex hormone disorders, and abnormal blood lipid and blood glucose metabolism, can effectively reduce the LPS level in serum and oxidative stress damage of ovarian tissue, is a product capable of repairing ovarian function and improving sex hormone secretion, and can be used for preventing, alleviating or treating PCOS.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and particularly to Lactiplantibacillus pentosus A2, a bacterial agent for preventing or improving polycystic ovary syndrome, and its application. Background Art

[0002] Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disease in women of reproductive age, with an incidence rate of about 8% - 13%. It is mainly related to hormonal imbalance, chronic low-grade inflammation, insulin resistance, and high androgen levels. Some studies also suggest that excessive oxidative stress levels in follicular fluid and serum of PCOS patients are one of the potential causes of PCOS. The clinical symptoms of PCOS mainly include hyperandrogenemia, ovulatory dysfunction, insulin resistance, dyslipidemia, chronic inflammation, obesity, etc. So far, there is no recognized best treatment plan for PCOS, and common clinical intervention methods mainly include dietary intervention, and drug treatments with clomiphene and gonadotropin.

[0003] Probiotics are a class of live microbial agents beneficial to the health of the host. Some studies have found that probiotics can regulate the structure of the host intestinal flora and have the effect of improving abnormal sex hormone levels and glucose and lipid metabolism disorders. Therefore, the prevention and treatment effects of probiotics and their related products are currently a research hotspot at home and abroad. Therefore, developing a food-derived product that can comprehensively improve PCOS is of great significance to human health.

[0004] Lactiplantibacillus pentosus is an important lactic acid bacterium widely present in fermented foods, with special antibacterial and probiotic properties. For example, Lactiplantibacillus pentosus AUP2202 (CN116656523A) isolated from naturally fermented chili peppers in Changsha has anti-inflammatory and antioxidant properties. Its DPPH free radical scavenging ability in vitro is 57.42 ± 10.02%, and it can effectively reduce the release of pro-inflammatory factors (IL-6, IL-1β, TNF-α) in macrophages of inflamed mice. The oyster fermented sauce prepared by fermenting Lactiplantibacillus pentosus 7-K from natural oyster fermentation broth has strong antioxidant ability (CN117551584A). The secondary laver fermentation broth sugar metabolism regulator prepared by using Lactiplantibacillus pentosus OUC-por(WH)-W#Pro#Bs14-XYX screened from rotten laver can effectively reduce insulin resistance in normal rats, increase insulin sensitivity, and long-term drinking is more helpful for blood glucose control (CN 116790434A). However, there is currently no report on whether Lactiplantibacillus pentosus can improve ovarian lesions and the expression levels of ovarian-related oxidative stress factors in PCOS rats, and there is still a large space for the demand of food-derived probiotics to improve PCOS.

[0005] The information disclosed in this background art section is only intended to increase the understanding of the overall background of the present invention, and should not be regarded as an admission or any form of suggestion that this information constitutes prior art already known to those of ordinary skill in the art. Summary of the Invention

[0006] Object of the Invention

[0007] The purpose of the present invention is to provide a Lactiplantibacillus pentosus A2, a bacterial agent and its application for preventing or improving polycystic ovary syndrome.

[0008] The Lactiplantibacillus pentosus A2 of the present invention was screened from pickled bamboo shoot fermentation broth, and its preservation number is CGMCC NO.27253, and it has good potential antioxidant, lipid-lowering and blood sugar-lowering abilities. Confirmed by in vivo animal experiments, when Lactiplantibacillus pentosus A2 is ingested at 10 9 cfu / mL or more can significantly improve the reproductive cycle disorder, ovarian polycystic lesions, abnormal testosterone level, abnormal gonadotropin (LH, FSH) levels, abnormal lipid metabolism, abnormal mRNA expression of colon connexin and oxidative stress-related factors in the ovary induced by letrozole, can improve the symptoms and pathophysiological state of PCOS induced by letrozole, and can be used for preventing, alleviating or treating PCOS.

[0009] Solution

[0010] To achieve the object of the present invention, the present invention adopts the following technical solutions.

[0011] In a first aspect, the present invention provides Lactiplantibacillus pentosus A2 for preventing or improving polycystic ovary syndrome. Lactiplantibacillus pentosus A2 was deposited with the China General Microbiological Culture Collection Center on May 4, 2023, and the deposit number is CGMCC NO. 27253.

[0012] In a second aspect, there is provided a bacterium agent comprising Lactiplantibacillus pentosus A2 as described in the first aspect, and further comprising a pharmaceutically acceptable carrier.

[0013] Further, in the bacterium agent, the viable count of Lactiplantibacillus pentosus A2 is 0.01 - 100×10 9 cfu, optionally 0.1 - 100×10 9 cfu, optionally 1 - 100×10 9 cfu, optionally 1 - 10×10 9 cfu / d.

[0014] Further, it further comprises other probiotics in addition to Lactiplantibacillus pentosus A2.

[0015] Further, the bacterium agent is an oral dosage form. Optionally, the oral dosage form is selected from: solution, suspension, emulsion, powder, lozenge, pill, syrup, buccal tablet, tablet and capsule.

[0016] In a third aspect, there is provided an application of Lactiplantibacillus pentosus A2 described in the first aspect and the bacterium agent described in the second aspect in the preparation of a medicament for the following uses:

[0017] 1) Preventing, alleviating or improving polycystic ovary syndrome; optionally, the polycystic ovary syndrome includes ovarian lesions caused by polycystic ovary syndrome and abnormal expression of oxidative stress-related factors in the ovary. Optionally, the oxidative stress-related factors in the ovary include Sod1, Sod2, Cat and Mgst1;

[0018] 2) Improving abnormal blood lipid metabolism; optionally, the abnormal blood lipid metabolism includes high expression of triglyceride, total cholesterol, low-density lipoprotein and high-density lipoprotein;

[0019] 3) Improving hyperglycemia;

[0020] 4) Antioxidation;

[0021] 5) Improve abnormal gonadotropin levels; optionally, the gonadotropin includes one or more of estradiol, testosterone, luteinizing hormone, follicle-stimulating hormone, progesterone;

[0022] 6) Improve the levels of LPS in rat serum and the mRNA levels of tight junction proteins in the colon.

[0023] Beneficial Effects

[0024] The Lactiplantibacillus pentosus A2 (deposit number CGMCC NO. 27253) of the present invention was screened from the pickled bamboo shoot fermentation broth. Through the letrozole-induced PCOS rat model, it was confirmed that the daily intake of Lactiplantibacillus pentosus A2 at 10 9 cfu / mL and above can significantly improve the reproductive cycle disorder, ovarian polycystic lesions, abnormal testosterone levels, abnormal gonadotropin (LH, FSH) levels, abnormal blood lipid metabolism, and abnormal mRNA expression of oxidative stress-related factors in the ovaries of letrozole-induced PCOS rats. By increasing the mRNA expression of tight junction proteins in the colon and oxidative stress-related factors in the ovaries, it can effectively reduce the LPS level in serum and oxidative stress damage in ovarian tissue. It is a product that can repair ovarian function and improve sex hormone secretion, and can be used for the prevention, alleviation or treatment of PCOS. It is expected to be developed and applied in the pharmaceutical industry, which can meet the specific needs of consumers to the greatest extent and has broad market prospects. Brief Description of the Drawings

[0025] One or more embodiments are exemplarily illustrated by the pictures in the corresponding drawings. These exemplary illustrations do not constitute a limitation on the embodiments. The special word "exemplary" here means "serving as an example, embodiment or illustrative". Any embodiment illustrated as "exemplary" here does not have to be interpreted as superior to or better than other embodiments.

[0026] Figure 1 They are the top 20 species in the NR functional classification of the gene function annotation of strain A2 in Example 2; among them, the abscissa represents the species ID, and the ordinate represents the number of annotated genes.

[0027] Figure 2 They are the in vitro lipid-lowering ability results of each bacterium in Example 3, where * represents a significant difference from strain A2 (P<0.05). A2 is Lactiplantibacillus pentosus A2; 299253 is Lactiplantibacillus plantarum 299253; 17938 is Lactobacillus gasseri 17938; LGG is Lactobacillus rhamnosus LGG.

[0028] Figure 3Results of the ABTS radical scavenging ability of each bacterium in Example 3, where * represents a significant difference from strain A2 (P<0.05). A2 is Lactiplantibacillus pentosus A2; 299253 is Lactiplantibacillus plantarum 299253; LGG is Lactobacillus rhamnosus LGG; 17938 is Lactobacillus gasseri 17938.

[0029] Figure 4 Results of the inhibitory effect of each bacterium on α-amylase in Example 3, where * represents a significant difference from strain A2 (P<0.05). A2 is Lactiplantibacillus pentosus A2; 299253 is Lactiplantibacillus plantarum 299253; 17938 is Lactobacillus gasseri 17938; LGG is Lactobacillus rhamnosus LGG.

[0030] Figure 5 Results of the effect of Lactiplantibacillus pentosus A2 in Test Example 1 on the estrous cycle of PCOS rats; among them, Control is the Control (normal) group, PCOS is the PCOS group, and A2 is the PCOS + Lactiplantibacillus pentosus A2 bacterial group. Figure 5 On the left are vaginal exfoliated cell smears (Papanicolaou staining) of rats in each group; on the right: changes in the estrous cycle of rats in each group.

[0031] Figure 6 Results of the effect of Lactiplantibacillus pentosus A2 in Test Example 2 on ovarian tissue lesions in PCOS rats; among them, C is the Control (normal) group, P is the PCOS group, and A2 is the PCOS + Lactiplantibacillus pentosus A2 bacterial group.

[0032] Figure 7 Results of the effect of Lactiplantibacillus pentosus A2 in Test Example 3 on blood lipid metabolism in PCOS rats; #: significant difference compared with group C; *: significant difference compared with group P. A: triglyceride (TG); B: total cholesterol (TC); D: low-density lipoprotein (LDL-C); E: high-density lipoprotein (HDL-C). C is the Control (normal) group, P is the PCOS group, and A2 is the PCOS + Lactiplantibacillus pentosus A2 bacterial group.

[0033] Figure 8 Results of the effect of Lactiplantibacillus pentosus A2 in Test Example 3 on serum sex hormone levels in PCOS rats; #: significant difference compared with group C; *: significant difference compared with group P; A: testosterone (T); B: estradiol (E2); C: luteinizing hormone (LH); D: LH / FSH; E: progesterone (PROG). C is the Control (normal) group, P is the PCOS group, and A2 is the PCOS + Lactiplantibacillus pentosus A2 bacterial group.

[0034] Figure 9It is the result of the effect of Lactiplantibacillus pentosus A2 on insulin resistance in PCOS rats in Test Example 3; among them, #: significant difference compared with group C; *: significant difference compared with group P; A: fasting insulin (FINS); B: insulin resistance index HOMA-IR. C is the Control (normal) group, P is the PCOS group, and A2 is the PCOS + Lactiplantibacillus pentosus A2 bacteria group.

[0035] Figure 10 It is the result of the effect of Lactiplantibacillus pentosus A2 on the mRNA levels of ovarian oxidative stress-related factors in PCOS rats in Test Example 4, where: #: significant difference compared with group C; *: significant difference compared with group P. A-D: mRNA expressions of Sod1, Sod2, Cat, and Mgst1. C is the Control (normal) group, P is the PCOS group, and A2 is the PCOS + Lactiplantibacillus pentosus A2 bacteria group.

[0036] Figure 11 It is the result of the effect of Lactiplantibacillus pentosus A2 on the serum LPS level and the mRNA expression of colonic tight junction proteins in PCOS rats in Test Example 5, where: #: significant difference compared with the Control group (P < 0.05); *: significant difference compared with the PCOS group (P < 0.05). A: lipopolysaccharide (LPS); B: colonic tight junction protein Zo1; C: Occludin; D: Claudin7. C is the Control (normal) group, P is the PCOS group, and A2 is the PCOS + Lactiplantibacillus pentosus A2 bacteria group. Detailed implementation manners

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention. Unless otherwise clearly stated, in the entire specification and claims, the term "including" or its variations such as "comprising" or "including" will be understood to include the stated elements or components, without excluding other elements or other components.

[0038] In addition, to better illustrate the present invention, numerous specific details are given in the following detailed implementation manners. Those skilled in the art should understand that the present invention can also be implemented without some specific details. In some embodiments, details of raw materials, components, methods, means, etc. well-known to those skilled in the art are not described in detail to highlight the gist of the present invention.

[0039] In the following examples, all raw materials used are commercially available.

[0040] Lactiplantibacillus pentosus A2 of the present invention was screened from pickled bamboo shoots, classified and named as Lactiplantibacillus pentosus A2, preservation date: May 4, 2023; preservation unit: China General Microbiological Culture Collection Center; preservation address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China; preservation number: CGMCC NO. 27253.

[0041] Example 1: Isolation and Obtaining of Bacillus A2

[0042] Take the pickled bamboo shoot fermentation sample and rinse it with sterilized PBS. After collecting the rinse solution and performing gradient dilution with PBS, coat it on MRS medium containing 1% CaCO3, and culture it in a 37°C incubator for 48 h. Pick out the single colonies with clear zones, round, milky white or creamy yellow, and good growth for microscopic examination and marking. Select the rod-shaped and Gram-positive bacteria strains for further liquid culture, and screen out the rod-shaped Bacillus A2 (preservation number: CGMCC NO. 27253) from numerous strains.

[0043] Example 2: Identification of Bacillus A2

[0044] Use the Illumina PE150 (350bp small fragment library) sequencing platform to perform bacterial framework sequencing analysis on strain A2, and annotate the sequencing results with the NR (Non-Redundant Protein Database) database.

[0045] The framework sequencing and NR annotation results of Bacillus A2 are as Figure 1 shown. The results show that 86.6% of the coding genes conform to the genus Lactiplantibacillus pentosus. Combining with the morphological characteristics of Bacillus A2, A2 is thus identified as Lactiplantibacillus pentosus and named Lactiplantibacillus pentosus A2.

[0046] Example 3: Safety Evaluation of Lactiplantibacillus pentosus A2

[0047] The excessive use of antibiotics in the pharmaceutical industry can lead to enhanced drug resistance of pathogenic strains, and the intestinal flora in the human body is prone to disorder and imbalance.

[0048] The antibiotic resistance is determined by the following method:

[0049] The isolated and purified Lactiplantibacillus pentosus strain A2 was inoculated into MRS liquid medium at an inoculum size of 2%, cultured in an incubator at 37 °C for 24 h, activated to the second generation, diluted with sterile normal saline to an OD600 of 0.80, mixed with MRS solid medium in a ratio of 1:15 and poured into petri dishes. After solidification, antibiotic susceptibility test discs and blank discs (blank control) were placed equidistantly, cultured at 37 °C for 24 h, observed, and the diameter of the inhibition zone was measured with a vernier caliper. The antibiotic sensitivity characteristics of the test strains were judged according to Table 1 and compared with the commercial strain Lactobacillus gasseri 17938. The results are shown in Table 2.

[0050] Table 1. Drug content of antibiotic susceptibility test discs and resistance judgment criteria

[0051]

[0052]

[0053] Table 2. Results of antibiotic sensitivity characteristics (diameter of inhibition zone / x±sd mm)

[0054]

[0055] As can be seen from the results in Table 2, both Lactiplantibacillus pentosus A2 and the control strain Lactiplantibacillus plantarum 299253 are sensitive to penicillin, streptomycin, tetracycline and chloramphenicol; Lactiplantibacillus pentosus A2 is resistant to neomycin, gentamicin and kanamycin; 299253 is sensitive to norfloxacin and intermediate to novobiocin, while A2 is sensitive to novobiocin and intermediate to norfloxacin, indicating that Lactiplantibacillus pentosus A2 can be used as an edible strain for commercial application.

[0056] Example 3: Probiotic effects of Lactiplantibacillus pentosus A2

[0057] After activation, Lactiplantibacillus pentosus A2 from pickled bamboo shoots was tested for probiotic effects, including TG and cholesterol degradation rate determination, ABTS radical scavenging ability, and potential hypoglycemic effect, together with the commercial strains Lactobacillus gasseri 17938, Lactiplantibacillus plantarum 299253, and Lactobacillus rhamnosus LGG. Specifically:

[0058] Example 3.1 Hypolipidemic Effect

[0059] After activation, Lactiplantibacillus pentosus A2, Lactobacillus gasseri 17938, Lactiplantibacillus plantarum 299253, and Lactobacillus rhamnosus LGG were used to determine the TG and cholesterol degradation rates. The specific measurement methods are as follows:

[0060] (1) Triglyceride-lowering ability

[0061] Dilute the activated bacterial liquid with distilled water to OD600 = 0.4, inoculate it at 2% into MRS medium containing 2.958 mM triglyceride (TG), culture at 37 °C for 24 h, centrifuge at 1000 r / min for 10 min, take the upper-layer fermentation broth, add cholesterol detection working solution, react at 37 °C for 10 min, and then measure the absorbance at 500 nm, denoted as A1; use distilled water and standard TG (5.596 mM) to replace the fermentation broth, denoted as A0 and A2 respectively.

[0062] Calculate the residual TG content in the sample tube after 24 h of fermentation according to formula (2):

[0063]

[0064] A0 —— Absorbance of the distilled water tube

[0065] A1 —— Absorbance of the sample tube

[0066] A2 —— Absorbance of the standard TG tube

[0067] C 标准 —— Concentration of TG in the standard tube

[0068] Calculate the TG degradation rate of each bacterium in 24 h according to formula (3):

[0069]

[0070] (2) Cholesterol-lowering ability

[0071] Dilute the activated bacterial liquid with distilled water to OD600 = 0.6, inoculate it at 2% into MRS medium containing 2.586 mM cholesterol, culture at 37 °C for 24 h, centrifuge at 1000 r / min for 10 min, take the upper-layer fermentation broth, add cholesterol detection working solution, react at 37 °C for 10 min, and then measure the absorbance at 500 nm, denoted as A1; use distilled water and standard cholesterol (5.596 mM) to replace the fermentation broth, denoted as A0 and A2 respectively.

[0072] Calculate the residual cholesterol content in the sample tube after 24 h of fermentation according to formula (4):

[0073]

[0074] A0 —— Absorbance of the distilled water tube

[0075] A1 —— Absorbance of the sample tube

[0076] A2 —— Absorbance of the standard cholesterol tube

[0077] C 标准 —— Concentration of cholesterol in the standard tube

[0078] Calculate the cholesterol degradation rate of each strain at 24 h according to formula (5):

[0079]

[0080] The results of the cholesterol and TG degradation abilities of Lactiplantibacillus pentosus A2 and different commercial strains are as follows Figure 3 . The results show that Lactiplantibacillus pentosus A2 has better cholesterol and triglyceride clearance effects, which are significantly higher than those of the commercial strains Lactiplantibacillus plantarum 299253 and Lactobacillus gasseri 17938 (p < 0.05), and there is no statistical difference from Lactobacillus rhamnosus LGG.

[0081] Example 3.2 Antioxidant Effect

[0082] Lactiplantibacillus pentosus A2 from pickled bamboo shoots was activated with the commercial strains Lactobacillus gasseri 17938, Lactiplantibacillus plantarum 299253, and Lactobacillus rhamnosus LGG, inoculated (inoculation amount of 3-5%) into MRs medium, cultured at 37 °C for 24 h, centrifuged at 10000 r / min for 10 min, and the supernatant was diluted 50 times with PBS as the sample to be tested. The ABTS radical scavenging ability was measured according to the following method:

[0083] (1) ABTS ·+ Stock solution preparation: Mix 7 mM ABTS and 2.45 mM potassium persulfate in equal volumes, and place them in the dark at room temperature for 12-16 h to prepare the ABTS ·+ stock solution;

[0084] (2) ABTS ·+ Working solution preparation: Dilute the ABTS + stock solution with 0.1 M PBS at pH 7.4 to make A 734 = 0.70 ± 0.02;

[0085] (3) ABTS ·+ Radical scavenging rate detection: Take 0.1 mL of the sample to be tested and mix it with 3.9 mL of the ABTS ·+ working solution, and measure A 734 after a 6-min dark reaction in a 30 °C water bath. At the same time, use ultrapure water as the blank control group, and set 3 parallels for each group of samples.

[0086] (4) The ABTS radical scavenging ability of probiotics is expressed by the inhibition rate (%), and its calculation formula is:

[0087]

[0088] In the formula: A S is the absorbance value of the sample; A C is the absorbance value of the blank control group.

[0089] The results of the antioxidant capacity of Lactiplantibacillus pentosus A2 are as follows Figure 2 , and the results show that the fermentation broth of Lactiplantibacillus pentosus A2 has a better ABTS free radical scavenging effect, which is significantly higher than that of the commercial strain Lactobacillus rhamnosus GG, and there is no significant difference from Lactiplantibacillus plantarum 299253 and Lactobacillus gasseri 17938.

[0090] Example 3.3 Potential Hypoglycemic Effect

[0091] α-Amylase inhibitors are a class of substances with good hypoglycemic effects and no obvious toxic and side effects. With the in-depth study of probiotics, the hypoglycemic ability of probiotics has attracted more and more attention.

[0092] After activation, Lactiplantibacillus pentosus A2, Lactobacillus gasseri 33985, Lactobacillus gasseri 17938, Lactiplantibacillus plantarum 299253, and Lactobacillus rhamnosus GG were diluted with distilled water to OD 600 = 0.2, inoculated into MRs at 5%, incubated at 37 °C for 12 h, centrifuged at 4 °C and 10,000 r / min for 10 min, and the supernatant was diluted 10 times with PBS as the sample to be tested for the determination of α-amylase inhibitory activity. The specific determination method is as follows:

[0093] In a 0.6 mL reaction system, PBS buffer, the sample to be tested, and α-amylase solution (1 mg / mL) were added in sequence, mixed evenly, incubated in a water bath at 37 °C for 10 min, then 1.5% soluble starch solution preheated at 37 °C was added, mixed thoroughly, reacted in a water bath at 37 °C for 5 min, 1 mL DNS solution was added, mixed evenly, reacted in a boiling water bath for 5 min and then quickly cooled to room temperature, diluted 10 times and allowed to stand for 30 min, and the absorbance was measured at 540 nm. According to formula (6), the inhibition rate of α-amylase of each sample can be calculated.

[0094]

[0095] Among them: A is the sample group, containing the sample solution and α-amylase solution;

[0096] B is the sample blank group, containing the sample solution without α-amylase solution;

[0097] C is the control group, without the sample solution but containing α-amylase solution;

[0098] D is the blank group, without the sample solution and without α-amylase solution.

[0099] The results of the in vitro hypoglycemic ability determination of Lactiplantibacillus pentosus A2 and three commercial strains are as follows Figure 4, The results showed that Lactiplantibacillus pentosus A2 had excellent α-amylase inhibitory activity, which was similar to Lactiplantibacillus plantarum 299253 and significantly higher than that of Lactobacillus rhamnosus LGG and Lactobacillus gasseri 17938.

[0100] In summary, compared with other strains (such as Lactobacillus rhamnosus LGG), Lactiplantibacillus pentosus A2 had better ABTS radical scavenging ability and potential hypoglycemic ability, and also had good lipid-lowering ability.

[0101] Example 4 Effect of Lactiplantibacillus pentosus A2 on rats with polycystic ovary syndrome

[0102] Thirty healthy female SD rats at 6 weeks of age with a body weight of 213.75 ± 11.23 g were randomly divided into the following 3 groups, with 10 rats in each group:

[0103] Group C: Control group, intragastrically administered 1 mL of 1% sodium carboxymethylcellulose (CMC) solution during the experiment.

[0104] Group P: PCOS model group, induced with letrozole (1 mg / kg / d, dissolved in 1% sodium carboxymethylcellulose solution) for 28 consecutive days to establish a PCOS model. Vaginal smears were taken from the 17th day to the 26th day. When the estrous cycle of the rats changed regularly, the model was considered successfully established.

[0105] Group A2: PCOS + Lactiplantibacillus pentosus A2 intervention group, induced with letrozole (1 mg / kg / d, dissolved in 1% sodium carboxymethylcellulose solution) for 28 consecutive days to establish a PCOS model. At the same time, on the first day of administration (letrozole), Lactiplantibacillus pentosus (the viable cell count was 1×10 9 CFU / d) was intragastrically administered for intervention, once a day for 28 consecutive days;

[0106] Observe the effects on the estrous cycle, blood glucose, blood lipid-related indicators, serum sex hormone detection, gene expression levels of rat ovaries and oxidative stress-related factors of the rats.

[0107] Test Example 1 Detection of the estrous cycle of rats

[0108] A total of 28 days of intervention was carried out. Vaginal smears were taken for 10 days from the 17th day to the 26th day. The changes in the estrous cycle of the rats were observed by the Papanicolaou staining method: mainly flat epithelial cells in the proestrus; mainly keratinized cells in the estrus; the proportions of the three types of cells, namely white blood cells, keratinized cells, and flat epithelial cells, were quite equal in the late estrus; mainly white blood cells in the diestrus.

[0109] The results were as Figure 5As shown, the results indicated that in the normal control (Control) group, the estrous cycles of rats cycled through proestrus, estrus, metestrus, and diestrus in sequence, showing a regular estrous cycle. However, the estrous cycles in the PCOS group showed irregular changes, mostly staying in diestrus. During the intervention with Lactiplantibacillus pentosus A2, the irregular estrous cycles were significantly improved. From the 3rd day of vaginal smear, the estrous cycles resumed regular changes, indicating that Lactiplantibacillus pentosus A2 could correct the irregular estrous cycles in PCOS rats.

[0110] Observation on histological morphology of ovarian tissues in rats of Test Example 2

[0111] One side of the ovaries of the above-mentioned rats sacrificed under anesthesia was placed in 4% (volume fraction) paraformaldehyde for fixation, paraffin embedding, and H&E staining. After sealing with neutral resin, the histological morphology of ovarian tissues in rats of each group was observed under a microscope.

[0112] The results were as Figure 6 , and the results showed that in the ovaries of rats in group C, there were multiple corpora lutea neatly arranged with complete morphology and a relatively thick granulosa cell layer. While in the ovaries of rats in the PCOS group, the volume of corpora lutea was extremely small, the granulosa cell layer was thinned, and cystic dilated follicles and atretic follicles were mostly seen. Compared with the PCOS group, the number of corpora lutea in the ovaries of rats in the Lactiplantibacillus pentosus A2 intervention group increased, cystic dilated follicles were rarely seen, and the granulosa cell layer was relatively thick, indicating that the intervention with Lactiplantibacillus pentosus A2 could effectively improve ovarian lesions in PCOS rats.

[0113] Detection of blood glucose, blood lipid-related indicators, and serum sex hormones in Test Example 3

[0114] After the intervention ended, the rats were fasted for 12 h, and tail vein blood was taken to detect fasting plasma glucose (FPG). The rats were sacrificed by CO2 anesthesia, and about 5 mL of abdominal aortic blood was taken, allowed to stand and centrifuged to separate the serum. According to the instructions of the ELISA kit, triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), testosterone (T), estradiol (E2), luteinizing hormone (LH), follicle-stimulating hormone (FSH) in the serum of rats were detected, the LH / FSH ratio was calculated, and the levels of progesterone (PROG) and fasting insulin (FINS) were calculated. The insulin resistance index (HOMA-IR) was calculated according to formula (7).

[0115]

[0116] The results were as Figures 7 - 9 .

[0117] Effect of Lactiplantibacillus pentosus A2 on Blood Lipid Metabolism in PCOS Rats

[0118] Figure 7The results showed that compared with the normal group (Group C), the levels of TC, TG, and LDL-C in the serum of rats in the PCOS group were significantly increased (P<0.05), while the level of HDL-C was significantly decreased (P<0.05). Compared with the PCOS group, the levels of TC, TG, and LDL-C in the serum of rats in the A2 intervention group were significantly decreased (P<0.05), while the level of HDL-C was significantly increased (P<0.05). This indicated that the intervention of Lactiplantibacillus pentosus A2 could improve the disorder of blood lipid metabolism in PCOS rats.

[0119] Effect of Lactiplantibacillus pentosus A2 on Serum Sex Hormone Levels in PCOS Rats

[0120] Figure 8 The results showed that compared with the normal (C) group, the levels of T, LH, and LH / FSH in the serum of rats in the PCOS group were significantly increased (P<0.05), while the levels of E2 and PROG were significantly decreased (P<0.05). Compared with the PCOS group, the levels of T, LH, and LH / FSH in the serum of rats in the A2 intervention group were significantly decreased (P<0.05), and the levels of serum E2 and PROG were significantly increased (P<0.05). This indicated that the intervention of Lactiplantibacillus pentosus A2 could improve the disorder of sex hormones in PCOS rats.

[0121] Effect of Lactiplantibacillus pentosus A2 on Insulin Resistance in PCOS Rats

[0122] Figure 9 The results showed that compared with the normal (C) group, the levels of FINS and HOMA-IR in the serum of rats in the PCOS group were significantly increased (P<0.05). Compared with the normal group and the probiotic intervention group, the fasting blood glucose of rats in the PCOS group was slightly increased, but there was no significant difference among the groups (P>0.05). Compared with the PCOS group, the level of HOMA-IR in the serum of rats in the A2 group was significantly decreased (P<0.05), and FINS also decreased, but there was no statistical significance (P>0.05). This indicated that the intervention of Lactiplantibacillus pentosus A2 could alleviate insulin resistance in PCOS rats.

[0123] Test Example 4 Detection of Gene Expression Levels of Oxidative Stress-Related Factors in Ovaries

[0124] Take the ovaries of the rats euthanized under anesthesia above, and use reverse transcription real-time fluorescence quantitative PCR (RT-qPCR) to detect the mRNA expression of oxidative stress-related factors Sod1, Sod2, Cat, and Mgst1 in the rat ovaries, and observe the effect of Lactiplantibacillus pentosus A2 on ovarian oxidative stress injury in PCOS rats. The method is as follows: Use Trizol reagent to extract the total RNA of colon tissue and ovarian tissue at a ratio of 10:1, configure the reverse transcription reaction system according to the reverse transcription fluorescence quantitative kit instructions, and reverse transcribe the RNA into cDNA; then use the cDNA obtained by reverse transcription as a template, and configure a 10 μL reaction system according to the SYBR Green quantitative PCR kit instructions. After mixing well, place it in a QuantStudioTM 6Flex PCR instrument to detect the mRNA expression of the target gene. The RT-qPCR reaction conditions are: 95 °C for 15 min, 1 cycle; 95 °C for 10 s, 60 °C for 32 s, 40 cycles. The detection of related genes uses Gapdh as a reference, and the 2-ΔΔCt method is used to calculate the relative mRNA content. The primer sequences of Sod1, Sod2, Cat, and Mgst1 are synthesized by a biological company referring to the literature (Zhou YY, Wu YQ, Chong CJ, Zhong SM, Wang ZX, Qin XH, Liu ZQ, Liu JY, Song JL. Irpex lacteus polysaccharide exhibits therapeutic potential for ovarian fibrosis in PCOS rats via the TGF-β1 / smad pathway. Heliyon. 2023;9(8):e18741. doi:10.1016 / j.heliyon.2023.e18741). The results are as Figure 10 .

[0125] Figure 10 The results showed that compared with the normal group, the mRNA expression levels of Sod1, Sod2, Cat, and Mgst1 in the ovarian tissues of rats in the P group were significantly decreased (P < 0.05). Compared with the PCOS group, the mRNA expression levels of Sod1, Cat, and Mgst1 in rats in the Lactiplantibacillus pentosus A2 group were significantly increased (P < 0.05), and Sod2 also increased, indicating that the intervention of Lactiplantibacillus pentosus A2 can effectively improve the mRNA expression levels of oxidative stress-related factors Sod1, Sod2, Cat, and Mgst1 in the ovaries of PCOS rats and inhibit the oxidative stress injury of the ovaries in PCOS rats.

[0126] Test Example 5 Effects of Lactiplantibacillus pentosus A2 on Serum LPS Level and mRNA of Colonic Tight Junction Proteins in PCOS Rats

[0127] Take the colon of the rats euthanized by the above anesthesia, and use real-time fluorescence quantitative PCR method to detect the mRNA expression of tight junction proteins Zo1, Occludin and Claudin7 in the rat colon. Taking Gapdh as a reference, the specific method refers to the detection of ovarian oxidative stress factors in Test Example 4. The primers for Zo1, Occludin and Claudin7 are shown in Table 3. At the same time, use a lipopolysaccharide (LPS) assay kit to detect the LPS level in the serum.

[0128] Table 3. Primer sequences of Zo1, Occludin and Claudin7

[0129]

[0130]

[0131] The detection results are as Figure 11 shown. Compared with the normal group, the LPS level in the serum of PCOS rats was significantly increased (P<0.05), and the levels of tight junction proteins Zo1, Occludin and Claudin7 in the colon were significantly decreased (P<0.05); compared with the PCOS group, the LPS levels in the serum of rats in the A2 intervention group were significantly decreased (P<0.05), and the levels of tight junction proteins Zo1, Occludin and Claudin7 in the colon were significantly increased (P<0.05), indicating that A2 intervention can effectively reduce the LPS level in the serum of PCOS rats, probably by increasing the mRNA expression of tight junction proteins in the colon.

[0132] The present invention uses Lactiplantibacillus pentosus A2 (deposit number CGMCC NO.27253) with good probiotic effects screened from pickled bamboo shoots. Through a PCOS rat model induced by letrozole, it is confirmed that A2 can correct irregular estrous cycles, improve ovarian polycystic lesions, sex hormone disorders and abnormal blood lipid and blood glucose metabolism, and by increasing the mRNA expression of tight junction proteins in the colon and oxidative stress-related factors in the ovary, effectively reduce the LPS level in the serum and oxidative stress damage in the ovarian tissue. It is a product that can repair ovarian function and improve sex hormone secretion, and can be used for the prevention, alleviation or treatment of PCOS. And it is expected to be developed and applied in the pharmaceutical industry.

[0133] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Lactiplantibacillus pentosus ( Lactiplantibacillus pentosus ) A2 for preventing or improving polycystic ovary syndrome. Lactiplantibacillus pentosus A2 was deposited at the China General Microbiological Culture Collection Center on May 4, 2023, with the deposit number CGMCC NO. 27253.

2. A bacterial agent containing Lactiplantibacillus pentosus A2 as described in claim 1, characterized in that, It also includes a pharmaceutically acceptable carrier.

3. The microbial agent according to claim 2, wherein In the said microbial agent, the viable count of Lactiplantibacillus pentosus A2 is 0.01 to 100×10 9 cfu.

4. The microbial agent according to claim 2, characterized in that, In the said microbial agent, the viable count of Lactiplantibacillus pentosus A2 is 0.1 to 100×10 9 cfu.

5. The microbial agent according to claim 2, wherein In the said microbial agent, the viable count of Lactiplantibacillus pentosus A2 is 1 to 100×10 9 cfu.

6. The microbial agent according to claim 2, wherein In the said microbial agent, the viable count of Lactiplantibacillus pentosus A2 is 1 to 10×10 9 cfu.

7. The microbial agent according to any one of claims 2 to 6, characterized in that, It also includes other probiotics except Lactiplantibacillus pentosus A2.

8. The microbial agent according to any one of claims 2 to 6, characterized in that The bacterial agent is an oral dosage form.

9. The microbial agent according to claim 8, wherein The oral dosage form is selected from: solution, suspension, emulsion, powder, lozenge, pill, syrup, tablet and capsule.

10. The microbial agent according to claim 8, characterized in that, The oral dosage form is a buccal lozenge.

11. Use of Lactiplantibacillus pentosus A2 as claimed in claim 1 and the bacterial agent as claimed in any one of claims 2 to 10 in the preparation of a medicament for preventing, alleviating or improving polycystic ovary syndrome.

12. The application according to claim 11, wherein The polycystic ovary syndrome includes ovarian lesions caused by polycystic ovary syndrome and abnormal expression of oxidative stress-related factors in the ovary.

13. The application according to claim 11, wherein The oxidative stress-related factors in the ovary include Sod1, Sod2, Cat and Mgst1.

14. Use of Lactiplantibacillus pentosus A2 as claimed in claim 1 and the bacterial agent as claimed in any one of claims 2 to 10 in the preparation of a medicament for improving abnormal blood lipid metabolism, and the abnormal blood lipid metabolism includes high expression of triglyceride, total cholesterol, low density lipoprotein and high density lipoprotein.

15. Use of Lactiplantibacillus pentosus A2 as claimed in claim 1 and the bacterial agent as claimed in any one of claims 2 to 10 in the preparation of a medicament for antioxidant.

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