A bacterial agent, composition and application thereof for preventing or improving polycystic ovary syndrome

Abstract

The embodiments of the present invention relate to the field of microorganisms, and in particular to a bacterial agent, a composition and an application thereof for preventing or improving polycystic ovary syndrome. The active ingredient of the bacterial agent includes plant lactobacillus (Lactiplantibacillus plantarum) G3, and the deposit number is CGMCC NO.27252. The plant lactobacillus G3 of the present invention, alone or in combination with thermophilic streptococcus and bulgaric lactobacillus delbrueckii, can correct irregular estrous cycles, improve ovarian polycystic lesions, sex hormone disorders and blood lipids, abnormal blood sugar metabolism, and by increasing the mRNA expression of oxidative stress-related factors in the ovary, effectively reduce the oxidative stress damage of ovarian tissue, and can be used to prevent, alleviate or treat PCOS. The effect of plant lactobacillus G3 in combination with thermophilic streptococcus and bulgaric lactobacillus delbrueckii is more significant.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a bacterial agent, a composition and an application 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. Worldwide, its incidence is about 8% to 13%, and the latest prevalence in China in 2020 is about 7.8%. The clinical symptoms of PCOS are mainly hyperandrogenemia, ovulatory dysfunction, insulin resistance, dyslipidemia, chronic inflammation, obesity, etc. Its pathophysiological basis is mainly related to hormone imbalance, chronic low-grade inflammation, insulin resistance and high androgen levels. Some 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. So far, there is no recognized best treatment for PCOS, and common clinical interventions are mainly dietary interventions, clomiphene and gonadotropin drug treatments. Probiotics are a class of live microbial preparations that can improve the balance of the host microecology and are beneficial to the host's health when taken in appropriate amounts. Studies have found that probiotics, as the main component of intestinal flora, have the functions of regulating the host's intestinal flora structure, improving abnormal sex hormone levels and lipid metabolism disorders. Intervention with probiotics and related products may be one of the potential means of clinical treatment of PCOS in the future.

[0003] Probiotics and related products thereof are currently the hotspots of research at home and abroad to the preventive and therapeutic effect of diseases, and probiotics and fermented products thereof are also confirmed in animal experiments to the improvement effect of PCOS. At present, the probiotic strains with the alleviation of polycystic ovary syndrome mainly include Bifidobacterium longum subspecies CCFM1102 (CN111073834 A), plant lactobacillus CCFM1019 (CN113317447A), plant lactobacillus CCFM1202 (CN 114426938A), Lactobacillus reuteri (CN115364126B, CN116355804A, CN116445346B) etc., and probiotic fermented products mainly include: medicine and food homology plant probiotic fermented product (CN114982949A), plant lactobacillus CCFM1019 fermented soy milk (CN113317447A), but the product with breast milk source plant lactobacillus fermented buffalo milk is not reported.

[0004] Therefore, targeted research and development of food-borne products is of great significance to human health.

[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 purpose of the present invention is to provide a bacterial agent, a composition and an application thereof for preventing or improving polycystic ovary syndrome.

[0008] The breast milk-derived Lactobacillus plantarum G3 of the present invention has a deposit number of CGMCC NO.27252 and has good potential antioxidant and lipid-lowering and blood sugar-lowering capabilities. In vivo animal experiments have confirmed that the bacteria has the ability to significantly improve letrozole-induced PCOS symptoms and pathophysiology, including reproductive cycle disorders, ovarian polycystic ovarian lesions, abnormal androgen (testosterone) levels, abnormal gonadotropin (LH / FSH) levels, abnormal blood lipid metabolism, and abnormal mRNA expression of oxidative stress-related factors in the ovaries. It is expected to be developed and applied to the food, health care products or pharmaceutical industries, can meet the specific needs of consumers to the greatest extent, and has broad market prospects.

[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 bacterial agent for preventing or improving polycystic ovary syndrome, the active ingredients of which include Lactobacillus plantarum G3; Lactobacillus plantarum G3 has been deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on May 4, 2023, with the deposit number CGMCCNO.27252.

[0012] Furthermore, it also includes thermophilic streptococcus and / or bulgaric lactobacillus delbrueckii. Optionally, the ratio of plant lactobacillus G3, thermophilic streptococcus and bulgaric lactobacillus delbrueckii is 6 to 10:1, and optionally the ratio is 10:1.

[0013] Further, the inoculation amount of Lactobacillus plantarum G3 is 2-5% by volume of buffalo milk, and the inoculation amount of the combined bacterial agent of Streptococcus thermophilus and Lactobacillus delbrueckii bulgaricus is 0.2-0.5% (w / v);

[0014] In a second aspect, a composition is provided, comprising the bacterial agent as described in the first aspect.

[0015] Furthermore, the composition is a pharmaceutical composition, which comprises the bacterial agent as described in the second aspect, and a pharmaceutically acceptable carrier.

[0016] Furthermore, the pharmaceutical composition is an oral dosage form;

[0017] Optionally, the oral dosage form is selected from the group consisting of solutions, suspensions, emulsions, powders, lozenges, pills, syrups, buccal lozenges, tablets and capsules.

[0018] In a third aspect, a dairy product is provided, which is obtained by fermenting a dairy product with the bacterial agent described in the first aspect or combining it with a dairy product.

[0019] Furthermore, the dairy product is cow's milk.

[0020] Furthermore, the dairy product is selected from milk powder and fermented dairy products.

[0021] In a preferred embodiment, the fermented dairy product is selected from fermented milk.

[0022] Furthermore, in the dairy product, the number of viable bacteria is 0.01 to 100×10 8 cfu / d, optionally 0.1~100×10 8 cfu / d, optionally 1 to 100×10 8 cfu / d, optionally 1 to 10×10 8 cfu / d.

[0023] In a fourth aspect, there is provided a use of the bacterial agent described in the first aspect and the composition described in the second aspect in the preparation of a medicine for the following purposes:

[0024] 1) Prevent, alleviate or improve polycystic ovary syndrome;

[0025] 2) Improve hyperglycemia;

[0026] 3) Antioxidant;

[0027] 4) Improve abnormal blood lipid metabolism;

[0028] 5) Improve abnormal androgen levels;

[0029] 6) Improve abnormal gonadotropin levels.

[0030] Beneficial Effects

[0031] The present invention utilizes plant lactobacillus G3 (preservation number CGMCC NO.27252) with good prebiotic effect screened from breast milk, alone or in combination with thermophilic streptococcus and bulgaric lactobacillus delbrueckii, and through a letrozole-induced PCOS rat model, it is confirmed that G3 alone or in combination with thermophilic streptococcus and bulgaric lactobacillus delbrueckii can correct irregular estrous cycle, improve ovarian polycystic ovarian lesions, sex hormone disorders and blood lipid and blood sugar metabolism abnormalities, and by increasing the mRNA expression of oxidative stress-related factors in the ovary, effectively reduce the oxidative stress damage of serum sex hormones and ovarian tissue, can repair ovarian function and improve sex hormone secretion, and can be used to prevent, alleviate or treat PCOS. The effect of plant lactobacillus G3 in combination with thermophilic streptococcus and bulgaric lactobacillus delbrueckii is more significant. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] 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.

[0033] Figure 1 It is the top 20 species in the NR functional classification of the gene function annotation of the strain G3 in Example 2; wherein the abscissa represents the species ID, and the ordinate represents the number of annotated genes.

[0034] Figure 2 The results of the ABTS free radical scavenging ability of each strain in Example 3, where * represents a significant difference from the G3 strain (P<0.05). G3 is Lactobacillus plantarum G3; 299253 is Lactobacillus plantarum 299253; LGG is Lactobacillus rhamnosus LGG; 17938 is Lactobacillus gasseri 17938; and B5 is Lactobacillus plantarum B5.

[0035] Figure 3 The results of the in vitro lipid-lowering ability of each strain in Example 3, where * represents a significant difference from the G3 strain (P<0.05). G3 is Lactobacillus plantarum G3; 299253 is Lactobacillus plantarum 299253; LGG is Lactobacillus rhamnosus LGG; 17938 is Lactobacillus gasseri 17938; and B5 is Lactobacillus plantarum B5.

[0036] Figure 4 The results of the inhibition of α-amylase by each strain in Example 3 are shown in Table 1. In the results, * indicates a significant difference from the G3 strain (P<0.05). G3 is Lactobacillus plantarum G3; 299253 is Lactobacillus plantarum 299253; LGG is Lactobacillus rhamnosus LGG; 17938 is Lactobacillus gasseri 17938; and B5 is Lactobacillus plantarum B5.

[0037] Figure 5 The results of the effect of fermented milk on the estrous cycle of PCOS rats in Test Example 1; C is the Control (normal) group, P is the PCOS group, G3 is the PCOS+G3 bacteria group, B5 is the PCOS+B5 bacteria group, FM1 is the PCOS+G3 fermented milk group, FM2 is the PCOS+Lactobacillus plantarum G3+yogurt starter (Lactobacillus bulgaricus+Streptococcus thermophilus) fermented milk group, FM3 is the PCOS+yogurt starter fermented milk 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.

[0038] Figure 6 This is the result of the effect of different fermented milks on blood lipid metabolism in PCOS rats in Test Example 2; #: 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 total protein (LDL-C); E: High-density total protein (HDL-C). C is the Control (normal) group, P is the PCOS group, G3 is the PCOS+G3 bacteria group, B5 is the PCOS+B5 bacteria group, FM1 is the PCOS+G3 fermented milk group, FM2 group is the PCOS+Lactobacillus plantarum G3+yogurt starter (Lactobacillus bulgaricus+Streptococcus thermophilus) fermented milk group, and FM3 group is the PCOS+yogurt starter fermented milk group.

[0039] Figure 7 The results of the effects of different fermented milks on the serum sex hormone levels of PCOS rats in test example 2; #: 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. C is the Control (normal) group, P is the PCOS group, G3 is the PCOS+G3 bacteria group, B5 is the PCOS+B5 bacteria group, FM1 is the PCOS+G3 fermented milk group, FM2 is the PCOS+Lactobacillus plantarum G3+yogurt starter (Lactobacillus bulgaricus+Streptococcus thermophilus) fermented milk group, and FM3 is the PCOS+yogurt starter fermented milk group.

[0040] Figure 8The results of the effects of different fermented milks on insulin resistance in PCOS rats in Test Example 2; 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, G3 is the PCOS+G3 bacteria group, B5 is the PCOS+B5 bacteria group, FM1 is the PCOS+G3 fermented milk group, FM2 is the PCOS+Lactobacillus plantarum G3+yogurt starter (Lactobacillus bulgaricus+Streptococcus thermophilus) fermented milk group, and FM3 is the PCOS+yogurt starter fermented milk group.

[0041] Fig. 9 This is the effect of different fermented milks on ovarian tissue lesions in PCOS rats in Test Example 3; wherein, C is the Control (normal) group, P is the PCOS group, G3 is the PCOS+G3 bacteria group, B5 is the PCOS+B5 bacteria group, FM1 is the PCOS+G3 fermented milk group, FM2 group is the PCOS+Lactobacillus plantarum G3+yogurt starter (Lactobacillus bulgaricus+Streptococcus thermophilus) fermented milk group, and FM3 group is the PCOS+yogurt starter fermented milk group.

[0042] Fig.10 The results of test example 4 are the effects of different fermented milks on the mRNA levels of ovarian oxidative stress-related factors in PCOS rats, where: #: significant difference from group C; *: significant difference from group P. AE: mRNA expression of Sod1, Sod2, Cat and Mgst1. C is the Control (normal) group, P is the PCOS group, G3 is the PCOS+G3 bacteria group, B5 is the PCOS+B5 bacteria group, FM1 is the PCOS+G3 fermented milk group, FM2 group is the PCOS+Lactobacillus plantarum G3+yogurt starter (Lactobacillus bulgaricus+Streptococcus thermophilus) fermented milk group, and FM3 group is the PCOS+yogurt starter fermented milk 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 plant lactobacillus G3 of the present invention is screened and obtained from breast milk, and is classified and named as plant lactobacillus (Lactiplantibacillus plantarum) G3, with a preservation date of May 4, 2023; the preservation unit is the General Microbiology Center of the China Microbiological Culture Collection Administration; the preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China; the preservation number is CGMCC NO.27252.

[0047] Example 1: Isolation and acquisition of Bacillus G3

[0048] Fresh breast milk was diluted with sterile PBS and applied on a plate containing 1% CaCO 3 The culture was carried out on MRS medium at 37°C for 48 hours under anaerobism. Single colonies with transparent circles, roundness, milky white or milky yellow color and good growth were picked for microscopic examination. Rod-shaped Bacillus G3 was screened and identified as Gram-positive bacteria. Lactobacillus plantarum B5 (preservation number: CGMCCNO.27251) was also obtained.

[0049] Example 2: Identification of Bacillus G3

[0050] The Illumina PE150 (350bp small fragment library) sequencing platform was used to perform bacterial framework sequencing analysis on G3 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 G3 are shown in Figure 1 As shown, the results showed that 61.8% of the coding genes were consistent with the genus Lactobacillus plantarum. Combined with the morphological characteristics of G3, G3 was identified as Lactobacillus plantarum.

[0052] The genomic characteristics of Lactobacillus plantarum G3 and B5 are shown in Table 1. Both belong to Lactobacillus plantarum, but have different genomic characteristics.

[0053] Table 1 Genomic characteristics

[0054]

[0055] Example 3: Probiotic effects of Lactobacillus plantarum G3

[0056] The screened Lactobacillus plantarum G3 and B5 were activated together with the commercial strains Lactobacillus gasseri 17938, Lactobacillus plantarum 299253, and Lactobacillus rhamnosus LGG, and the probiotic effects were tested, including ABTS free radical scavenging ability, TG and cholesterol degradation rate determination, and potential hypoglycemic effect. Specifically:

[0057] Antioxidant Effect

[0058] Lactobacillus plantarum G3, B5 and commercial strains Lactobacillus gasseri 17938, Lactobacillus plantarum 299253, and Lactobacillus rhamnosus LGG were cremated and inoculated in MRs medium at an inoculum amount of 3-5%, cultured at 37° C. for 24 h, centrifuged at 10000 r / min for 10 min, and the supernatant was diluted 50 times (sample), and the ABTS free radical scavenging ability was determined as follows:

[0059] (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;

[0060] (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;

[0061] (3)ABTS ·+ Free radical scavenging rate determination: Take 0.1mL sample and 3.9mL 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.

[0062] (4) The scavenging ability of probiotic ABTS free radicals is expressed as inhibition rate (%), and its calculation formula is:

[0063]

[0064] Where: A S is the sample absorbance value; A C The absorbance value of the blank control group.

[0065] The results are as follows Figure 2The results showed that the ABTS free radical scavenging ability of Lactobacillus plantarum G3 was significantly higher than that of Lactobacillus plantarum B5, commercial Lactobacillus plantarum 299253, and Lactobacillus rhamnosus LGG, and was equivalent to that of Lactobacillus gasseri 17938.

[0066] Lipid-lowering effect

[0067] Lactobacillus plantarum G3, B5 and commercial strains Lactobacillus gasseri 17938, Lactobacillus plantarum 299253, and Lactobacillus rhamnosus LGG were activated and then the TG and cholesterol degradation rates were determined. The specific determination methods are as follows:

[0068] (1) Triglyceride-lowering ability

[0069] 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 .

[0070] The residual TG content of the sample tube after 24 hours of fermentation was calculated according to formula (2):

[0071]

[0072] A 0 ——Absorbance of distilled water tube

[0073] A 1 ——Absorbance of sample tube

[0074] A 2 ——Absorbance of standard TG tube

[0075] C 标准 ——Concentration of TG in standard tube

[0076] The TG degradation rate of each bacterium for 24 h was calculated according to formula (3):

[0077]

[0078] (2) Cholesterol-lowering ability

[0079] The activated bacterial liquid 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 .

[0080] The residual cholesterol content in the sample tube after 24 hours of fermentation was calculated according to formula (4):

[0081]

[0082] A 0 ——Absorbance of distilled water tube

[0083] A 1 ——Absorbance of sample tube

[0084] A 2 ——Absorbance of standard cholesterol tube

[0085] C 标准 ——Cholesterol concentration in standard tube

[0086] The cholesterol degradation rate of each bacterium in 24 hours was calculated according to formula (5):

[0087]

[0088] The results are as follows Figure 3 The results showed that Lactobacillus plantarum G3 had a better cholesterol-clearing effect, which was significantly higher than the commercial strains Lactobacillus plantarum 299253, Lactobacillus rhamnosus LGG and Lactobacillus gasseri 17938, and was comparable to Lactobacillus plantarum B5.

[0089] Potential hypoglycemic effect

[0090] 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.

[0091] After activation, Lactobacillus plantarum G3, B5, 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, and the supernatant was diluted 10 times (sample) for α-amylase inhibitory activity determination. The specific determination method is as follows:

[0092] PBS buffer, test solution and enzyme 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% soluble starch solution preheated at 37°C was added, mixed well, reacted in a 37°C water bath for 5 min, 1 mL 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).

[0093]

[0094] Wherein: A is a sample group, containing sample solution and α-amylase solution;

[0095] B is the sample blank group, containing the sample solution but not the α-amylase solution;

[0096] C is the control group, which contains α-amylase solution without sample solution;

[0097] D is the blank group, which does not contain sample solution or α-amylase solution.

[0098] The results are as follows Figure 4 The results showed that Lactobacillus plantarum G3 had excellent α-amylase inhibitory activity, which was similar to that of Lactobacillus gasseri 17938, significantly higher than that of Lactobacillus rhamnosus LGG, but significantly lower than that of Lactobacillus plantarum 299253 and Lactobacillus plantarum B5.

[0099] In summary, Lactobacillus plantarum G3 has better ABTS free radical scavenging and lipid-lowering abilities than other strains (such as B5).

[0100] Example 4 Effect of fermented milk on rats with polycystic ovary syndrome

[0101] Preparation of fermented milk :

[0102] The G3 and B5 strains were activated and the concentration was adjusted to 1.25×10 10cfu / mL, and the seed solution was prepared for use. The buffalo milk was homogenized at 175-180 Bar and sterilized at 90-100 ° C, then cooled to 40-45 ° C, inoculated with 2-4% plant lactobacillus G3 and 0.2-0.4% composite bacterial agent (composite bacterial agent of Lactobacillus bulgaricus and Streptococcus thermophilus, purchased from Danisco (China) Co., Ltd.) by volume of the buffalo milk, and after being fully mixed, fermented at 35-43 ° C for 8-10 h until the milk curdled, and the fermented milk (G3+Lactobacillus bulgaricus+Streptococcus thermophilus) was obtained, and the number of viable bacteria in the fermented milk was 3.2×10 9 cfu / mL, and store at 4°C overnight for later use.

[0103] Similarly, 2-4% of the volume of buffalo milk was inoculated with plant lactobacillus G3 and fermented according to the above method to obtain single-bacteria G3 fermented milk, and the number of viable bacteria in the fermented milk was 1.0×10 9 cfu / mL.

[0104] Similarly, 0.2-0.4% of the composite bacterial agent (compounded bacterial agent of Lactobacillus bulgaricus and Streptococcus thermophilus, purchased from Danisco (China) Co., Ltd.) was inoculated based on the volume of buffalo milk and fermented according to the above method to obtain (Lactobacillus bulgaricus + Streptococcus thermophilus) fermented milk, and the number of viable bacteria in the fermented milk was 6.1×10 8 cfu / mL.

[0105] Experimental design

[0106] Seventy healthy female SD rats aged 6 weeks and weighing 213.75±11.23 g (purchased from Hunan Slake Jingda Experimental Animal Co., Ltd. [Production License No.: SCXK(Xiang)2019-0004]) were randomly divided into the following 7 groups, with 10 rats in each group:

[0107] Group C: Control group, normal control, intragastrically administered with 1 mL of 1% sodium carboxymethylcellulose (CMC) solution during the experiment;

[0108] Group P: PCOS group, letrozole (1 mg / kg / d, dissolved in 1% sodium carboxymethylcellulose solution) was administered for 28 consecutive days to induce the PCOS model, and vaginal smears were taken from the 17th to the 26th day. When the estrous cycle of rats changed, the model was considered to be successfully established;

[0109] FM1 group: PCOS + Lactobacillus plantarum G3 fermented milk group, letrozole (1 mg / kg / d, dissolved in 1% sodium carboxymethylcellulose solution) was given for 28 consecutive days to induce PCOS model, and letrozole was given at the same time on the first day at 1×10 8 CFU / d The above G3 fermented milk was given by gavage once a day for 28 consecutive days;

[0110] FM2 group: PCOS + fermented milk (G3 + Lactobacillus bulgaricus + Streptococcus thermophilus) group, letrozole (1 mg / kg / d, dissolved in 1% sodium carboxymethylcellulose solution) was given for 28 consecutive days to induce PCOS model, and letrozole was given at the same time on the first day at 1×10 8 CFU / d was given by intragastric administration of the above fermented milk (G3 + Lactobacillus bulgaricus + Streptococcus thermophilus) once a day for 28 consecutive days;

[0111] FM3 group: PCOS + fermented milk (Lactobacillus bulgaricus + Streptococcus thermophilus) group, letrozole (1 mg / kg / d, dissolved in 1% sodium carboxymethylcellulose solution) was given for 28 consecutive days to induce PCOS model, and letrozole was given at the same time on the first day at 1×10 8 CFU / d, the above-mentioned (Lactobacillus bulgaricus + Streptococcus thermophilus) fermented milk was given by gavage once a day for 28 consecutive days.

[0112] G3 group: PCOS + Lactobacillus plantarum G3 bacterial solution group, letrozole (1 mg / kg / d, dissolved in 1% sodium carboxymethylcellulose solution) was given for 28 consecutive days to induce PCOS model, and letrozole was given at the same time on the first day at 1×10 9 CFU / d, the above-mentioned G3 bacterial solution was given by intragastric gavage once a day for 28 consecutive days.

[0113] Group B5: PCOS + Lactobacillus plantarum B5 bacterial solution group, letrozole (1 mg / kg / d, dissolved in 1% sodium carboxymethylcellulose solution) was given for 28 consecutive days to induce the PCOS model. At the same time, letrozole was given at 1×10 9 CFU / d, the above-mentioned B5 bacterial solution was given by intragastric gavage once a day for 28 consecutive days.

[0114] Test Example 1: Detection of estrus cycle in rats

[0115] 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.

[0116] The results are as follows Figure 5As shown in the figure, the results showed that the rats in group C had a regular estrus cycle with proestrus, estrus, metestrus and diestrus, while the estrus cycle in group P was irregular and mostly stayed in diestrus. The irregular estrus cycle was significantly improved during the fermented milk intervention. Among them, the estrus cycle of group FM1 resumed regular changes on the third day of vaginal smear, and the estrus cycle of groups FM2, FM3, G3 and B5 resumed regular changes from the fourth day of vaginal smear, indicating that fermented milk and bacterial liquid intervention can correct the irregular estrus cycle of PCOS rats more quickly.

[0117] Test Example 2 Blood sugar, blood lipid related indicators, serum sex hormone detection

[0118] 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. The levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL-C), high-density lipoprotein (HDL-C), estradiol (E2), testosterone (T), luteinizing hormone (LH), follicle-stimulating hormone (FSH), progesterone (PROG), and fasting insulin (FINS) in rat serum were detected according to the instructions of the ELISA kit, and the insulin resistance index (HOMA-IR) was calculated according to formula (7).

[0119]

[0120] The results are as follows Figures 6 to 8 .

[0121] Effects of fermented milk on blood lipid metabolism in PCOS rats

[0122] Figure 6 The results showed that compared with the normal group (group C), the TG, TC and LDL-C in the serum of the rats in group P were significantly increased (P<0.05), and the HDL-C was significantly decreased (P<0.05); compared with the P group, the TC, TG and LDL-C in the serum of the rats in the G3, B5 bacterial liquid groups and the 3 fermented milk groups were significantly decreased (P<0.05), and the HDL-C was significantly increased (P<0.05), but the lipid-lowering effects of G3 and B5 bacteria were similar, while the TC-lowering effect of G3 and Lactobacillus bulgaricus and Streptococcus thermophilus synergistically fermented milk (FM2 group) was better than that of G3 single bacteria fermented milk and Lactobacillus bulgaricus and Streptococcus thermophilus fermented milk ( Figure 6 B), indicating that G3, Lactobacillus bulgaricus and Streptococcus thermophilus can synergistically ferment milk, which is more beneficial to improving cholesterol metabolism disorders in PCOS rats.

[0123] Effects of fermented milk on serum sex hormone levels in PCOS rats

[0124] Figure 7 The results showed that compared with the normal (C) group, the serum T, LH and LH / FSH levels of the rats in the P group were significantly increased (P<0.05), and the E2 level was significantly decreased (P<0.05). However, after the fermented milk intervention, the T and LH levels were significantly decreased (P<0.05), and the serum E2 level was significantly increased (P<0.05), but the LH level was still significantly higher than that in the normal group ( Figure 7 C), E2 level ( Figure 7 B) and FM2 group T levels were close to those of the normal group ( Figure 7 A). Relatively speaking, the levels of LH / FSH in the G3, B5, FM1 and FM2 groups were significantly reduced (P<0.05), and those in the FM3 group were reduced, but the difference was not significant compared with the P group (P>0.05). This indicates that fermented milk intervention can improve sex hormone disorders in PCOS rats, and the addition of Lactobacillus plantarum G3 is more conducive to improving serum T levels and LH / FSH in PCOS rats.

[0125] Effects of fermented milk on insulin resistance in PCOS rats

[0126] Figure 8 The results showed that compared with the normal (C) group, the FINS level and HOMA-IR in the serum of the rats in the P group were significantly increased (P<0.05), but after the fermented milk intervention, the FINS level and HOMA-IR were significantly decreased (P<0.05), and there was no significant difference with the normal group level (P>0.05), except that the FINS level in the FM2 group was significantly lower than that in the normal group ( Figure 8 A), indicating that fermented milk intervention can alleviate insulin resistance in PCOS rats, and the addition of G3 may be more beneficial to improving the FINS level in PCOS rats.

[0127] Test Example 3 Histological morphology observation of rat ovary

[0128] 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.

[0129] The results are as follows Fig. 9 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 group P, there were fewer corpora lutea, thinner granulosa cell layer, and more cystic dilated follicles and atretic follicles. Compared with group P, the number of corpora lutea in the ovaries of rats in groups G3, B5 bacteria and fermented milk increased, cystic dilated follicles were rare, and the granulosa cell layer was thicker, especially in group FM2, indicating that the addition of G3, Lactobacillus bulgaricus and Streptococcus thermophilus can more effectively improve the ovarian lesions of rats with PCOS.

[0130] Test Example 4 Detection of gene expression levels of oxidative stress-related factors in ovaries

[0131] The ovaries of the rats anesthetized and killed 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), and the effects of G3, B5 bacteria and fermented milk on ovarian oxidative stress damage in PCOS rats were observed. The method was 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 obtained from references (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) and were commissioned to be synthesized by a biological company. Zhou YY, Wu YQ, ChongCJ, 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 follows Fig.10 .

[0132] Fig.10 The results showed that compared with the normal group, the mRNA expression levels of Sod1, Sod2, Cat and Mgst1 in the ovarian tissue of rats in the P group were significantly decreased (P<0.05). After intervention, the mRNA expression levels of Sod1, Sod2 and Mgst1 in the G3 bacteria and fermented milk groups were significantly increased (P<0.05), while the changes in the B5 bacteria were not significant. For the mRNA expression level of Cat, the G3, FM1 and FM2 groups were significantly increased (P<0.05), while the changes in the FM3 group were not significant (P>0.05). For the B5 bacteria, except for the significant increase in the mRNA expression level of Mgst1, the mRNA expression levels of Sod1, Sod2 and Cat were not significantly affected. In addition, the mRNA expression levels of Cat and Mgst1 in the FM2 group were significantly higher than those in the FM3 group (P<0.05), and the mRNA expression level of Mgst1 in the FM2 group was significantly higher than that in the FM1 group (P<0.05), indicating that fermented milk intervention 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.

[0133] In summary, compared with Lactobacillus plantarum B5, breast milk-derived Lactobacillus plantarum G3 has better lipid-lowering ability and ABTS clearance rate, and can effectively improve the mRNA expression levels of oxidative stress-related factors Sod1, Sod2, Cat and Mgst1; and the composite fermentation bacteria agent (Lactobacillus plantarum G3 + Lactobacillus bulgaricus and Streptococcus thermophilus) is more effective than the single bacteria agent G3, Lactobacillus bulgaricus and Streptococcus thermophilus in improving ovarian lesions in PCOS rats, effectively reducing total cholesterol in blood lipids, improving high-density total protein, especially in improving the mRNA expression levels of oxidative stress-related factors Sod1, Sod2, Cat and Mgst1, and is expected to be developed and applied in the food, health care products or pharmaceutical industries.

[0134] 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 bacterial agent for preventing or improving polycystic ovary syndrome, the active ingredient of which includes Lactobacillus plantarum ( Lactiplantibacillus plantarum ) G3; Lactobacillus plantarum G3 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on May 4, 2023, with the deposit number CGMCC NO.27252.

2. The bacterial agent according to claim 1, characterized in that Also included are Streptococcus thermophilus and / or Lactobacillus delbrueckii bulgaricus.

3. A composition, characterized in that The composition comprises the bacterial agent according to claim 1 or 2.

4. The composition according to claim 3, characterized in that The composition is a pharmaceutical composition, which comprises the bacterial agent according to claim 1 or 2, and a pharmaceutically acceptable carrier.

5. The composition according to claim 4, characterized in that The pharmaceutical composition is in oral dosage form.

6. The composition according to claim 5, characterized in that The oral dosage form is selected from the group consisting of solutions, suspensions, emulsions, powders, lozenges, pills, syrups, tablets and capsules.

7. The composition according to claim 5, characterized in that The oral dosage form is a buccal lozenge.

8. A dairy product, characterized in that The method is obtained by fermenting a dairy product with the bacterial agent according to claim 1 or 2 or combining the dairy product with the bacterial agent.

9. The dairy product according to claim 8, characterized in that The dairy product is cow's milk.

10. The dairy product according to claim 8, characterized in that The dairy product is selected from the group consisting of milk powder and fermented dairy products.

11. The dairy product according to claim 10, characterized in that The fermented dairy product is selected from fermented milk.

12. Use of the bacterial agent according to claim 1 or 2, or the composition according to any one of claims 3 to 7 in preparing a product for the following purposes: 1) Prevent, relieve or improve polycystic ovary syndrome.

13. The use according to claim 12, characterized in that: The product is a medicine.

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