Lactobacillus pentosus phytobacterium A2 for preventing or improving polycystic ovarian syndrome, fungicide, dairy product and application of lactobacillus pentosus phytobacterium A2, fungicide and dairy product

Abstract

The embodiment of the invention relates to the field of microorganisms, in particular to lactobacillus pentosus A2 used for preventing or improving polycystic ovarian syndrome, a microbial agent, a dairy product and application of the lactobacillus pentosus A2, the microbial agent and the dairy product. The preservation number of the lactobacillus pentosus A2 is CGMCC (China General Microbiological Culture Collection Center) NO.27253. The lactobacillus pentosus A2 disclosed by the invention can correct irregular estrus cycles, improve ovarian polycystic lesions, sex hormone disorder and abnormal blood fat and blood sugar metabolism, can effectively reduce the LPS level in serum and oxidative stress injury of ovarian tissues, is a product capable of repairing ovarian functions and improving sex hormone secretion, and can be used for preventing, relieving or treating PCOS.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to a Lactiplantibacillus pentosus A2, a bacterial agent, a dairy product and applications thereof for preventing or improving polycystic ovary syndrome. Background Art

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

[0003] Probiotics are a type of live microbial preparation that is beneficial to the host's health. Studies have found that probiotics can regulate the host's intestinal flora structure and improve abnormal sex hormone levels and glucose and lipid metabolism disorders. Therefore, the prevention and treatment of diseases by probiotics and their related products is currently a hot topic of research at home and abroad. Therefore, the development of food-borne products that can comprehensively improve PCOS is of great significance to human health.

[0004] Lactobacillus pentosus is an important lactic acid bacterium widely present in fermented foods, and has special antibacterial and probiotic properties. For example, Lactobacillus pentosus AUP2202 (CN116656523A) isolated from naturally fermented peppers in Changsha has anti-inflammatory and antioxidant properties, and its in vitro DPPH free radical scavenging ability is 57.42±10.02%, which can effectively reduce the release of pro-inflammatory factors (IL-6, IL-1β, TNF-α) in macrophages of inflammatory mice; and the oyster fermented sauce prepared by fermenting Lactobacillus pentosus 7-K derived from natural oyster fermented liquid has strong antioxidant capacity (CN117551584A); the secondary laver fermented liquid sugar metabolism regulator prepared by using Lactobacillus 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 conducive to blood sugar control (CN 116790434A). However, there is no report on whether Lactobacillus pentosus can improve the ovarian lesions and the expression levels of ovarian-related oxidative stress factors in PCOS rats. There is still a lot of room for the demand for food-borne probiotics to improve PCOS.

[0005] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0006] Purpose of the Invention

[0007] The present invention aims to provide a Lactiplantibacillus pentosus A2, a bacterial agent, a dairy product and applications thereof for preventing or improving polycystic ovary syndrome.

[0008] The pentosus lactobacillus A2 of the present invention is screened from the fermentation broth of sour bamboo shoots, and its deposit number is CGMCCNO.27253. It has good potential antioxidant and lipid-lowering and blood sugar-lowering abilities. In vivo animal experiments have confirmed that the pentosus lactobacillus A2 can be taken 10 times a day. 9 cfu / mL and above can significantly improve the reproductive cycle disorders, ovarian polycystic lesions, abnormal testosterone levels, abnormal gonadotropin (LH, FSH) levels, abnormal blood lipid metabolism, abnormal mRNA expression of colicin and oxidative stress-related factors in PCOS rats induced by letrozole, and can improve the symptoms and pathophysiological state of PCOS induced by letrozole, and can be used to prevent, alleviate or treat PCOS.

[0009] Solution

[0010] To achieve the purpose of the present invention, the present invention adopts the following technical scheme.

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

[0012] In a second aspect, a method is provided that includes Lactobacillus pentosus A2 as described in the first aspect, and also includes a food or pharmaceutically acceptable carrier.

[0013] Furthermore, in the bacterial agent, the number of viable bacteria of Lactobacillus pentosus A2 is 0.01 to 100×10 9 cfu, optionally 0.1 to 100×10 9 cfu, optionally 1 to 100×10 9 cfu, optionally 1 to 10×10 9 cfu / d.

[0014] Furthermore, other probiotics except Lactobacillus pentosus A2 are also included.

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

[0016] In a third aspect, a dairy product is provided, which is obtained by fermenting a dairy product with the Lactobacillus pentosus A2 described in the first aspect or the bacterial agent described in the second aspect or by combining the fermentation with a dairy product.

[0017] Furthermore, the dairy product is cow's milk.

[0018] Furthermore, in the dairy product, the number of viable bacteria of Lactobacillus pentosus A2 is 0.01 to 100×10 9 cfu, optionally 0.1 to 100×10 9 cfu, optionally 1 to 100×10 9 cfu, optionally 1 to 10×10 9 cfu / d.

[0019] In a fourth aspect, there is provided a use of the Lactobacillus pentosus A2 described in the first aspect, the bacterial agent described in the second aspect, or the dairy product described in the third aspect in preparing a product for the following purposes:

[0020] 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, and optionally, the oxidative stress-related factors in the ovary include Sod1, Sod2, Cat and Mgst1;

[0021] 2) improving abnormal blood lipid metabolism; optionally, abnormal blood lipid metabolism includes high expression of triglycerides, total cholesterol, low-density lipoprotein, and high-density lipoprotein;

[0022] 3) Improve hyperglycemia;

[0023] 4) Antioxidant;

[0024] 5) Improving abnormal gonadotropin levels; optionally, the gonadotropin includes one or more of estradiol, testosterone, luteinizing hormone, follicle-stimulating hormone, and progesterone;

[0025] 6) Improved serum LPS levels and colon tight junction protein mRNA levels in rats.

[0026] In a feasible embodiment, the product is a food, a health product or a medicine.

[0027] In a preferred embodiment, the dairy product is selected from milk powder and fermented dairy products.

[0028] In a preferred embodiment, the fermented dairy product is selected from fermented milk.

[0029] Beneficial Effects

[0030] The present invention's lactobacillus pentosus A2 (preservation number CGMCC NO.27253) was screened from the fermentation broth of sour bamboo shoots. The PCOS rat model induced by letrozole was used to confirm that lactobacillus pentosus A2 had a daily intake of 10 9 cfu / mL and above can significantly improve the reproductive cycle disorders, 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 PCOS rats induced by letrozole. It can also effectively reduce the LPS level in serum and the oxidative stress damage of ovarian tissue by increasing the mRNA expression of tight junction proteins in the colon and oxidative stress-related factors in the ovaries. It is a product that can repair ovarian function and improve sex hormone secretion, and can be used to prevent, alleviate or treat PCOS. It is expected to be developed and applied in the food, health care products or pharmaceutical industries, which can meet the specific needs of consumers to the greatest extent and has broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] One or more embodiments are exemplarily described by the pictures in the accompanying drawings, and these exemplary descriptions do not constitute limitations on the embodiments. The special word "exemplary" here means "used as an example, embodiment or illustrative". Any embodiment described as "exemplary" here is not necessarily interpreted as being superior or better than other embodiments.

[0032] Figure 1 It is the top 20 species in the NR functional classification of the gene function annotation of strain A2 in Example 2; wherein the abscissa represents the species ID, and the ordinate represents the number of annotated genes.

[0033] Figure 2 The results of the in vitro lipid-lowering ability of each strain in Example 3, where * represents a significant difference from strain A2 (P<0.05). A2 is Lactobacillus pentosus A2; 299253 is Lactobacillus plantarum 299253; 17938 is Lactobacillus gasseri 17938; LGG is Lactobacillus rhamnosus LGG.

[0034] Figure 3 The results of the ABTS free radical scavenging ability of each strain in Example 3, where * represents a significant difference from strain A2 (P<0.05). A2 is Lactobacillus pentosus A2; 299253 is Lactobacillus plantarum 299253; LGG is Lactobacillus rhamnosus LGG; and 17938 is Lactobacillus gasseri 17938.

[0035] Figure 4 The results of the inhibition of α-amylase by each strain in Example 3 are shown in Table 1. * indicates significant difference from strain A2 (P<0.05). A2 is Lactobacillus pentosus A2; 299253 is Lactobacillus plantarum 299253; 17938 is Lactobacillus gasseri 17938; and LGG is Lactobacillus rhamnosus LGG.

[0036] Figure 5 The results are as follows: the effect of Lactobacillus pentosus A2 of Test Example 1 on the estrous cycle of PCOS rats; wherein Control is the Control (normal) group, PCOS is the PCOS group, and A2 is the PCOS+Lactobacillus pentosus A2 bacterial group, Figure 5 The left side is the vaginal exfoliated cell smear (Papanicolaou stain) of rats in each group; the right side: the changes in the estrous cycle of rats in each group.

[0037] Figure 6 This is the effect of test example 2, Lactobacillus pentosus A2, on ovarian tissue lesions in PCOS rats; wherein, C is the Control (normal) group, P is the PCOS group, and A2 is the PCOS+Lactobacillus pentosus A2 bacterial group.

[0038] Figure 7This is the result of the effect of Lactobacillus pentosus A2 on blood lipid metabolism in PCOS rats in test example 3; #: The difference is significant compared with group C; *: The difference is significant 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 group, P is the PCOS group, and A2 is the PCOS+Lactobacillus pentosus A2 group.

[0039] Figure 8 This is the result of the effect of Lactobacillus pentosus A2 on the serum sex hormone levels of PCOS rats in test example 3; #: The difference is significant compared with group C; *: The difference is significant 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 + Lactobacillus pentosus A2 bacteria group.

[0040] Fig. 9 The results of the effect of Lactobacillus pentosus A2 on insulin resistance in PCOS rats in test example 3; where #: 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+Lactobacillus pentosus A2 group.

[0041] Fig.10 The results of test example 4 are the effects of Lactobacillus pentosus A2 on the mRNA levels of ovarian oxidative stress-related factors in PCOS rats, where: #: significant difference from group C; *: significant difference from group P. AD: mRNA expression of Sod1, Sod2, Cat and Mgst1. C is the Control (normal) group, P is the PCOS group, and A2 is the PCOS+Lactobacillus pentosus A2 bacteria group.

[0042] Fig.11 The results of the test example 5 are the effects of Lactobacillus pentosus A2 on serum LPS levels and colon tight junction protein mRNA expression in PCOS rats, 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: Colon tight junction protein Zo1; C: Occludin; D: Claudin7. C is the Control (normal) group, P is the PCOS group, and A2 is the PCOS+Lactobacillus pentosus A2 bacteria group. DETAILED DESCRIPTION

[0043] In order to make the purpose, 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 part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Unless otherwise expressly stated, throughout the 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.

[0044] In addition, in order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In some embodiments, raw materials, elements, methods, means, etc. well known to those skilled in the art are not described in detail, so as to highlight the main purpose of the present invention.

[0045] In the following examples, all the raw materials used are commercially available.

[0046] The pentosus lactobacillus A2 of the present invention is screened and obtained from sour bamboo shoots, and is classified and named as pentosus lactobacillus (Lactiplantibacillus pentosus) A2, with a preservation date of May 4, 2023; the preservation unit is China General Microbiological Culture Collection Management Center; the preservation address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China; the preservation number is CGMCC NO.27253.

[0047] Example 1: Isolation and acquisition of Bacillus A2

[0048] The fermented sour bamboo shoots were washed with sterilized PBS, and the washing solution was collected and graded diluted with PBS, and then applied to a 1% CaCO 3 The strains were cultured on MRS medium at 37°C in an incubator for 48 hours, and single colonies with transparent circles, round, milky white or milky yellow color, and good growth were selected for microscopic examination and marked. The rod-shaped and Gram-positive bacterial strains were selected for further liquid culture, and the rod-shaped Bacillus A2 (deposit number: CGMCC NO.27253) was screened from many strains.

[0049] Example 2: Identification of Bacillus A2

[0050] The Illumina PE150 (350bp small fragment library) sequencing platform was used to perform bacterial framework sequencing analysis on A2 bacteria, and the sequencing results were annotated with the NR (Non-Redundant Protein Database) database.

[0051] The framework sequencing and NR annotation results of Bacillus A2 are shown in Figure 1 As shown, the results showed that 86.6% of the coding genes were consistent with the genus Lactiplantibacillus pentosus. Combined with the morphological characteristics of Bacillus A2, A2 was identified as Lactiplantibacillus pentosus and named Lactiplantibacillus pentosus A2.

[0052] Example 3: Safety evaluation of Lactobacillus pentosus A2

[0053] Excessive use of antibiotics in the pharmaceutical industry can lead to increased drug resistance in pathogenic strains, and the flora in the human intestine is prone to disorder and imbalance. Therefore, obtaining lactic acid bacteria that are sensitive to antibiotics has become one of the important indicators of whether they are safe for use in food.

[0054] Antibiotic resistance was determined using the following method:

[0055] The isolated and purified Lactobacillus pentosus strain A2 was inoculated into MRS liquid culture medium at a 2% inoculation rate, cultured in a constant temperature incubator at 37°C for 24 h, activated to the second generation, diluted with sterile saline to an OD600 of 0.80, mixed with MRS solid culture medium at a ratio of 1:15 and poured into a culture dish. After solidification, drug-sensitive paper and blank paper (blank control) were placed at equal distances, cultured at 37°C for 24 h, and the diameter of the inhibition zone was observed and 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.

[0056] Table 1. Drug content and resistance judgment criteria of drug-sensitive paper sheets

[0057]

[0058] Table 2. Antibiotic sensitivity characteristics results (inhibition zone diameter / x±sd mm)

[0059]

[0060] From the results in Table 2, it can be seen that both Lactobacillus pentosus A2 and the control bacteria commercial Lactobacillus plantarum 299253 are sensitive to penicillin, streptomycin, tetracycline and chloramphenicol; Lactobacillus pentosus A2 is resistant to neomycin, gentamicin and kanamycin; 299253 is sensitive to norfloxacin and is intermediate to novobiocin, but A2 is sensitive to novobiocin and is intermediate to norfloxacin, indicating that Lactobacillus pentosus A2 can be used as an edible strain for commercial application.

[0061] Example 3: Probiotic Effects of Lactobacillus pentosus A2

[0062] After the activation of Lactobacillus pentosus A2 from sour bamboo shoots and commercial strains Lactobacillus gasseri 17938, Lactobacillus plantarum 299253, and Lactobacillus rhamnosus LGG, the probiotic effects were tested, including the determination of TG and cholesterol degradation rates, ABTS free radical scavenging ability, and potential hypoglycemic effect. Specifically:

[0063] Example 3.1 Lipid-lowering effect

[0064] Lactobacillus pentosus A2 and commercial strains Lactobacillus gasseri 17938, Lactobacillus plantarum 299253, and Lactobacillus rhamnosus LGG were activated and the TG and cholesterol degradation rates were determined. The specific determination method is as follows:

[0065] (1) Triglyceride-lowering ability

[0066] The activated bacterial liquid was diluted with distilled water to OD600 = 0.4, and 2% was inoculated into MRS medium containing 2.958mM triglyceride (TG), cultured at 37°C for 24h, centrifuged at 1000r / min for 10min, and the upper fermentation liquid was added to the cholesterol detection working solution. After reacting at 37°C for 10min, the absorbance was measured at 500nm and recorded as A. 1 ; Distilled water and standard TG (5.596mM) were used instead of fermentation broth, respectively denoted as A 0 and A 2 .

[0067] The residual TG content of the sample tube after 24 hours of fermentation was calculated according to formula (2):

[0068]

[0069] A 0 ——Absorbance of distilled water tube

[0070] A 1 ——Absorbance of sample tube

[0071] A 2 ——Absorbance of standard TG tube

[0072] C 标准 ——Concentration of TG in standard tube

[0073] The TG degradation rate of each bacterium for 24 h was calculated according to formula (3):

[0074]

[0075] (2) Cholesterol-lowering ability

[0076] The activated bacterial solution was diluted with distilled water to OD600 = 0.6, and 2% was inoculated into MRS medium containing 2.586mM cholesterol, cultured at 37℃ for 24h, centrifuged at 1000r / min for 10min, the upper fermentation liquid was taken and added to the cholesterol detection working solution, reacted at 37℃ for 10min, and the absorbance was measured at 500nm, recorded as A 1 ; Distilled water and standard cholesterol (5.596mM) were used instead of fermentation broth, respectively denoted as A 0 and A 2 .

[0077] The residual cholesterol content in the sample tube after 24 hours of fermentation was calculated according to formula (4):

[0078]

[0079] A 0 ——Absorbance of distilled water tube

[0080] A 1 ——Absorbance of sample tube

[0081] A 2 ——Absorbance of standard cholesterol tube

[0082] C 标准 ——Cholesterol concentration in standard tube

[0083] The cholesterol degradation rate of each bacterium in 24 hours was calculated according to formula (5):

[0084]

[0085] Results of the cholesterol and TG degradation abilities of Lactobacillus pentosus A2 and different commercial strains are shown in Figure 3 The results showed that Lactobacillus pentosus A2 had a better cholesterol and triglyceride removal effect, which was significantly higher than the commercial strains Lactobacillus plantarum 299253 and Lactobacillus gasseri 17938 (p<0.05), and had no statistical difference with Lactobacillus rhamnosus LGG.

[0086] Example 3.2 Antioxidant effect

[0087] Lactobacillus pentosus A2 from sour bamboo shoots and commercial strains of Lactobacillus gasseri 17938, Lactobacillus plantarum 299253, and Lactobacillus rhamnosus LGG were activated and inoculated (3-5% inoculation amount) in MRs medium, cultured at 37°C for 24h, centrifuged at 10000r / min for 10min, and the supernatant was diluted 50 times with PBS as the test sample, and the ABTS free radical scavenging ability was determined as follows:

[0088] (1)ABTS ·+Preparation of stock solution: Mix 7mM ABTS and 2.45mM potassium persulfate in equal volumes and place at room temperature in the dark for 12-16 hours to prepare ABTS ·+ Stock solution;

[0089] (2)ABTS ·+ Working fluid preparation: ABTS + The stock solution was diluted with 0.1M PBS, pH 7.4 to make A 734 =0.70±0.02;

[0090] (3)ABTS ·+ Free radical scavenging rate detection: Take 0.1mL of the sample to be tested and 3.9mL of ABTS ·+ The working solution was mixed and reacted in a 30℃ water bath for 6 minutes before measuring A 734 At the same time, ultrapure water was used as the blank control group, and three parallel samples were set for each group of samples.

[0091] (4) The scavenging ability of probiotic ABTS free radicals is expressed as inhibition rate (%), and its calculation formula is:

[0092]

[0093] Where: A S is the sample absorbance value; A C The absorbance value of the blank control group.

[0094] The results of antioxidant capacity of Lactobacillus pentosus A2 are as follows Figure 2 The results showed that the fermentation broth of Lactobacillus pentosus A2 had a good ABTS free radical scavenging effect, which was significantly higher than that of the commercial strain Lactobacillus rhamnosus LGG, and had no significant difference from Lactobacillus plantarum 299253 and Lactobacillus gasseri 17938.

[0095] Example 3.3 Potential hypoglycemic effect

[0096] α-Amylase inhibitors are a type of drug that has a good hypoglycemic effect without obvious toxic side effects. With the in-depth research on probiotics, the ability of probiotics to lower blood sugar has received more and more attention.

[0097] After activation, Lactobacillus pentosus A2, Lactobacillus gasseri 33985, Lactobacillus gasseri 17938, Lactobacillus plantarum 299253, and Lactobacillus rhamnosus LGG were diluted with distilled water to OD 600 =0.2, inoculated into MRs at 5%, incubated at 37°C for 12h, centrifuged at 4°C, 10 000r / min for 10min, took the supernatant and diluted it 10 times with PBS as the sample to be tested, and carried out the α-amylase inhibitory activity determination. The specific determination method is as follows:

[0098] PBS buffer, the sample to be tested and α-amylase solution (1 mg / mL) were added to the 0.6 mL reaction system in sequence, mixed evenly, kept in a 37°C water bath for 10 min, then 1.5% of soluble starch solution preheated at 37°C was added, mixed thoroughly, reacted in a 37°C water bath for 5 min, 1 mL of DNS solution was added, mixed evenly, reacted in a boiling water bath for 5 min, then quickly cooled to room temperature, diluted 10 times and allowed to stand for 30 min, and the absorbance was measured at 540 nm. The inhibition rate of α-amylase of each sample can be calculated according to formula (6).

[0099]

[0100] Wherein: A is a sample group, containing sample solution and α-amylase solution;

[0101] B is the sample blank group, containing the sample solution but not the α-amylase solution;

[0102] C is the control group, which contains α-amylase solution without sample solution;

[0103] D is the blank group, which does not contain sample solution or α-amylase solution.

[0104] The results of in vitro glucose-lowering ability test of Lactobacillus pentosus A2 and three commercial strains are shown in Figure 4 The results showed that Lactobacillus pentosus A2 had excellent α-amylase inhibitory activity, which was similar to that of Lactobacillus plantarum 299253 and significantly higher than that of Lactobacillus rhamnosus LGG and Lactobacillus gasseri 17938.

[0105] In summary, compared with other strains (such as Lactobacillus rhamnosus LGG), Lactobacillus pentosus A2 has better ABTS free radical scavenging ability and potential blood sugar lowering ability, and also has good blood lipid lowering ability.

[0106] Example 4 Effect of Lactobacillus pentosus A2 on rats with polycystic ovary syndrome

[0107] Thirty healthy female SD rats aged 6 weeks and weighing 213.75±11.23 g were randomly divided into the following three groups, with 10 rats in each group:

[0108] Group C: Control group, rats were intragastrically administered with 1 mL of 1% sodium carboxymethylcellulose (CMC) solution during the experiment.

[0109] Group P: PCOS model group, letrozole (1 mg / kg / d, dissolved in 1% sodium carboxymethyl cellulose solution) was given for 28 consecutive days to induce the PCOS model, and vaginal smears were taken from the 17th to the 26th day. When the estrus cycle of rats changed, the model was considered to be successfully established.

[0110] Group A2: PCOS + Lactobacillus pentosus intervention group A2, letrozole (1 mg / kg / d, dissolved in 1% sodium carboxymethylcellulose solution) was given for 28 consecutive days to induce the PCOS model, and Lactobacillus pentosus (viable count of 1×10 9 CFU / d) intragastric administration, once a day for 28 consecutive days;

[0111] The effects on the estrus cycle, blood sugar, blood lipid related indicators, serum sex hormone detection, rat ovary and gene expression levels of oxidative stress related factors of rats were observed.

[0112] Test Example 1: Detection of estrus cycle in rats

[0113] The intervention lasted for 28 days in total. Vaginal smears were performed for 10 days from the 17th to the 26th day, and the Papanicolaou staining method was used to observe the changes in the estrus cycle of rats: squamous epithelial cells were the main cells in the proestrus; keratinocytes were the main cells in the estrus; the proportions of leukocytes, keratinocytes, and squamous epithelial cells were equal in the late estrus; and leukocytes were the main cells in the diestrus.

[0114] The results are as follows Figure 5 As shown in the figure, the results showed that the normal control group (Control) rats had a regular estrus cycle with proestrus, estrus, metestrus and diestrus in sequence, while the estrus cycle of the PCOS group showed irregular changes, mostly staying in the diestrus period. During the intervention of Lactobacillus pentosus A2, the irregular estrus cycle was significantly improved, and the estrus cycle resumed regular changes from the third day of vaginal smear, indicating that Lactobacillus pentosus A2 can correct the irregular estrus cycle of PCOS rats.

[0115] Test Example 2 Histological morphology observation of rat ovary

[0116] One side of the ovary of the rats sacrificed by anesthesia was taken and fixed with 4% (volume fraction) paraformaldehyde, embedded in paraffin and stained with H&E. After sealing with neutral resin, the histological morphology of the ovary of each group of rats was observed under a microscope.

[0117] The results are as follows Figure 6 The results showed that there were multiple corpora lutea in the ovaries of rats in group C, which were neatly arranged and complete in shape, and the granulosa cell layer was thicker. In the ovaries of rats in the PCOS group, there were fewer corpora lutea, thinner granulosa cell layer, and more cystic dilated follicles and atretic follicles. Compared with the PCOS group, the number of corpora lutea in the ovaries of rats in the Lactobacillus pentosus A2 intervention group increased, cystic dilated follicles were less common, and the granulosa cell layer was thicker, indicating that Lactobacillus pentosus A2 intervention can effectively improve the ovarian lesions of rats with PCOS.

[0118] Test Example 3 Blood sugar, blood lipid related indicators, serum sex hormone detection

[0119] After the intervention, the rats were fasted for 12 h, and tail vein blood was collected to measure fasting blood glucose (FPG). 2 Rats were anesthetized and killed, and about 5 mL of abdominal aorta blood was collected. The blood was centrifuged to separate serum. According to the instructions of the ELISA kit, triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL-C), high-density lipoprotein (HDL-C), testosterone (T), estradiol (E2), luteinizing hormone (LH), follicle-stimulating hormone (FSH) in rat serum were detected, and the levels of LH / FSH, progesterone (PROG), and fasting insulin (FINS) were calculated. The insulin resistance index (HOMA-IR) was calculated according to formula (7).

[0120]

[0121] The results are as follows Figures 7 to 9 .

[0122] Effects of Lactobacillus pentosus A2 on blood lipid metabolism in PCOS rats

[0123] Figure 7 The results showed that compared with the normal group (group C), the serum TC, TG and LDL-C in the PCOS rats were significantly increased (P<0.05), and the HDL-C was significantly decreased (P<0.05); compared with the PCOS group, the serum TC, TG and LDL-C in the A2 intervention group were significantly decreased (P<0.05), and the HDL-C was significantly increased (P<0.05). This indicates that Lactobacillus pentosus A2 intervention can improve the lipid metabolism disorder in PCOS rats.

[0124] Effects of Lactobacillus pentosus A2 on serum sex hormone levels in PCOS rats

[0125] Figure 8 The results showed that compared with the normal (C) group, the serum T, LH and LH / FSH levels of the PCOS rats were significantly increased (P<0.05), and the E2 and PROG levels were significantly decreased (P<0.05). Compared with the PCOS group, the serum T, LH and LH / FSH levels of the A2 intervention group were significantly decreased (P<0.05), and the serum E2 and PROG levels were significantly increased (P<0.05), indicating that Lactobacillus pentosus A2 intervention can improve the sex hormone disorder of PCOS rats.

[0126] Effects of Lactobacillus pentosus A2 on insulin resistance in PCOS rats

[0127] Fig. 9The results showed that compared with the normal (C) group, the serum FINS level and HOMA-IR of the PCOS rats were significantly increased (P<0.05). Compared with the normal group and the probiotic intervention group, the fasting blood glucose of the PCOS rats was increased, but there was no significant difference among the groups (P>0.05). Compared with the PCOS group, the serum HOMA-IR level of the A2 group rats was significantly decreased (P<0.05), and FINS was also decreased, but there was no statistical significance (P>0.05), indicating that Lactobacillus pentosus A2 intervention can reduce insulin resistance in PCOS rats.

[0128] Test Example 4 Detection of gene expression levels of oxidative stress-related factors in ovaries

[0129] The ovaries of the above-mentioned anesthetized rats were taken, and the mRNA expression of oxidative stress-related factors Sod1, Sod2, Cat and Mgst1 in the rat ovaries was detected by reverse transcription real-time fluorescence quantitative PCR (RT-qPCR), so as to observe the effect of Lactobacillus pentosus A2 on ovarian oxidative stress damage in PCOS rats. The method is as follows: Trizol reagent was used to extract total RNA from colon tissue and ovarian tissue at a ratio of 10:1, and the reverse transcription reaction system was configured according to the instructions of the reverse transcription fluorescence quantitative kit to reverse transcribe RNA into cDNA; then, the cDNA obtained by reverse transcription was used as a template, and a 10μL reaction system was prepared according to the instructions of the SYBR Green quantitative PCR kit, and the mRNA expression of the target gene was detected in the QuantStudioTM 6Flex PCR instrument after being fully mixed. The RT-qPCR reaction conditions were: 95℃15min, 1 cycle; 95℃10s, 60℃32s, 40 cycles. Gapdh was used as a reference for related gene detection, and the relative mRNA content was calculated using the 2-ΔΔCt method. The primer sequences of Sod1, Sod2, Cat and Mgst1 were synthesized by a biological company according to the reference (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). Fig.10 .

[0130] Fig.10The results showed that compared with the normal group, the mRNA expression levels of Sod1, Sod2, Cat and Mgst1 in ovarian tissue of rats in group P were significantly decreased (P<0.05). Compared with the PCOS group, the mRNA expression levels of Sod1, Cat and Mgst1 in rats in group Lactobacillus pentosus A2 were significantly increased (P<0.05), and Sod2 was also increased. This indicates that intervention with Lactobacillus 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 damage of the ovaries of PCOS rats.

[0131] Test Example 5 Effect of Lactobacillus pentosus A2 on serum LPS level and colon tight junction protein mRNA in PCOS rats

[0132] The colon of the above-mentioned rats sacrificed by anesthesia was taken, and the mRNA expression of tight junction proteins Zo1, Occludin and Claudin7 in the rat colon was detected by real-time fluorescence quantitative PCR method, with Gapdh as a reference. The specific method refers to the detection of ovarian oxidative stress factors in Test Example 4, and the primers of Zo1, Occludin and Claudin7 are shown in Table 3. At the same time, the LPS level in serum was detected by Lipopolysaccharide (LPS) assay kit.

[0133] Table 3. Primer sequences of Zo1, Occludin and Claudin7

[0134]

[0135] Test results such as Fig.11 As shown in the data, compared with the normal group, the serum LPS level of rats in the PCOS group was significantly increased (P<0.05), and the levels of colon tight junction proteins Zo1, Occludin and Claudin7 were significantly decreased (P<0.05); compared with the PCOS group, the serum LPS level of rats in the A2 intervention group was significantly decreased (P<0.05), and the levels of colon tight junction proteins Zo1, Occludin and Claudin7 were significantly increased (P<0.05), indicating that A2 intervention can effectively reduce the serum LPS level of PCOS rats, which may be achieved by increasing the mRNA expression of tight junction proteins in the colon.

[0136] The present invention uses Lactobacillus pentosus A2 (preservation number CGMCC NO.27253) screened from sour bamboo shoots with good probiotic effects, and through a letrozole-induced PCOS rat model, it is confirmed that A2 can correct irregular estrous cycles, improve ovarian polycystic lesions, sex hormone disorders, and abnormal blood lipid and blood sugar metabolism, and effectively reduce the LPS level in serum and the oxidative stress damage of ovarian tissue by increasing the mRNA expression of tight junction proteins in the colon and oxidative stress-related factors in the ovary. The product is a product that can repair ovarian function and improve sex hormone secretion, and can be used to prevent, alleviate or treat PCOS. It is expected to be developed and applied to the food, health care products or pharmaceutical industries.

[0137] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

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

2. A bacterial agent comprising the Lactobacillus pentosus A2 according to claim 1, characterized in that: Also included are food or pharmaceutically acceptable carriers.

3. The bacterial agent according to claim 2, characterized in that In the bacterial agent, the number of viable bacteria of Lactobacillus pentosus A2 is 0.01 to 100×10 9 cfu, optionally 0.1 to 100×10 9 cfu, optionally 1 to 100×10 9 cfu, optionally 1 to 10×10 9 cfu / d.

4. The bacterial agent according to claim 2 or 3, characterized in that: Probiotics other than Lactobacillus pentosus A2 are also included.

5. The bacterial agent according to any one of claims 2 to 4, characterized in that: The bacterial agent is an oral dosage form, and optionally, the oral dosage form is selected from: solution, suspension, emulsion, powder, lozenge, pill, syrup, buccal lozenge, tablet and capsule.

6. A dairy product, characterized in that The method is obtained by fermenting a dairy product by using the lactobacillus pentosus A2 described in claim 1 or the bacterial agent described in any one of claims 2 to 5, or by combining the fermented dairy product with the dairy product.

7. The dairy product according to claim 6, characterized in that The dairy product is cow's milk; And / or, in the dairy product, the number of viable bacteria of Lactobacillus pentosus A2 is 0.01 to 100×10 9 cfu, optionally 0.1 to 100×10 9 cfu, optionally 1 to 100×10 9 cfu, optionally 1 to 10×10 9 cfu / d.

8. Use of the Lactobacillus pentosus A2 according to claim 1, the bacterial agent according to any one of claims 2 to 5, or the dairy product according to claim 6 or 7 in preparing a product for the following purposes: 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, and optionally, the oxidative stress-related factors in the ovary include Sod1, Sod2, Cat and Mgst1; 2) improving abnormal blood lipid metabolism; optionally, abnormal blood lipid metabolism includes high expression of triglycerides, total cholesterol, low-density lipoprotein, and high-density lipoprotein; 3) Improve hyperglycemia; 4) Antioxidant; 5) Improving abnormal gonadotropin levels; optionally, the gonadotropin includes one or more of estradiol, testosterone, luteinizing hormone, follicle-stimulating hormone, and progesterone; 6) Improved serum LPS levels and colon tight junction protein mRNA levels in rats.

9. The use according to claim 8, characterized in that: The product is food, health product or medicine.

10. The use according to claim 9, characterized in that: The food is milk powder or fermented dairy product.

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