Application of animal bifidobacterium lactis BGI-L99 in prevention and treatment of hormone imbalance and aging related diseases

Abstract

The application provides application of animal Bifidobacterium lactis BGI-L99 in prevention and treatment of hormone imbalance and aging related diseases, and belongs to the technical field of biotechnology. The aforementioned animal Bifidobacterium lactis BGI-L99 is preserved in Guangdong Microbial Culture Collection Center (GDMCC) on March 31, 2026, and the preservation number is GDMCC No. 68017. Products prepared from the animal Bifidobacterium lactis BGI-L99 in the examples of the application can effectively prevent and treat sex hormone imbalance related diseases and delay aging related degenerative diseases, including relieving polycystic ovary syndrome and osteoporosis, specifically including improving ovarian function, endocrine disorder, depression, metabolic disorder, inflammatory reaction, abnormal intestinal metabolic product, bone microstructure degradation, imbalance between bone formation and bone resorption and the like.

Description

Technical Field

[0001] This application belongs to the field of biotechnology and relates to Bifidobacterium animalis subsp. lactis BGI-L99 and its application in the prevention and treatment of diseases related to sex hormone imbalance and in delaying age-related degenerative diseases. Background Technology

[0002] Sex hormones are core signaling molecules that regulate multiple physiological functions in the human body, including reproductive development, bone metabolism homeostasis, glucose and lipid metabolism, immune regulation, and neuroendocrine function. Within the body, they primarily function through the hypothalamic-pituitary-gonadal axis (HPG axis), forming a sophisticated pathway for synthesis, metabolism, and negative feedback regulation. The dynamic balance of sex hormone concentration and ratio is the core endocrine basis for maintaining homeostasis and slowing the aging process. With age, the function of the HPG axis progressively declines. Simultaneously, exposure to endocrine disruptors in the environment, unhealthy lifestyles, metabolic disorders, surgical interventions (such as oophorectomy), and ovarian damage can easily lead to an imbalance in sex hormone secretion and metabolic pathways, primarily manifested as two core pathological phenotypes: a relative dominance of androgens and a relative deficiency of estrogen. Numerous studies have confirmed that sex hormone imbalance is not only a direct cause of female reproductive system diseases but also a key upstream mechanism driving multidimensional aging of the skeletal, metabolic, cardiovascular, and nervous systems, and is one of the common pathological bases of degenerative diseases prevalent in middle-aged and elderly populations.

[0003] Polycystic ovary syndrome (PCOS) is the most common reproductive endocrine and metabolic disorder in women of reproductive age, and it is also the most representative phenotype of androgen dominance. Its core pathological features are hyperandrogenemia, ovulation disorders, and polycystic ovarian changes. It is often accompanied by short-term and long-term complications such as insulin resistance, abdominal obesity, infertility, and endometrial lesions. Hyperandrogenemia is the core link throughout the development of the disease. Excessive androgens disrupt the negative feedback regulation of the HPG axis, inhibit follicle development and ovulation, and exacerbate insulin resistance, creating a vicious cycle. Furthermore, as women of reproductive age age and ovarian function decline, postmenopausal women have a significantly higher risk of developing estrogen deficiency-related osteoporosis and cardiovascular disease than healthy women. Current clinical treatment for PCOS is mainly symptomatic intervention, with core regimens including oral short-acting contraceptives, anti-androgens, and insulin sensitizers. However, these treatments have limitations, including only addressing the symptoms and not the root cause, significant adverse reactions, limited applicability to certain populations, and a lack of comprehensive intervention capabilities throughout the entire reproductive cycle.

[0004] Postmenopausal osteoporosis (PMOP) is the most representative disease phenotype of estrogen deficiency and the most common degenerative bone disease in middle-aged and elderly women. Its core pathogenesis involves ovarian failure due to natural menopause or iatrogenic factors, resulting in absolute estrogen deficiency. This disrupts bone metabolic homeostasis, leading to increased osteoclast activity and suppressed osteoblast function, ultimately causing bone loss, bone microstructure damage, and an increased risk of fractures. More than half of survivors are unable to live independently, and estrogen deficiency also triggers multi-system aging phenotypes such as reproductive system atrophy and glucose and lipid metabolism disorders. Currently, the mainstream clinical interventions are hormone replacement therapy (HRT) and bisphosphonates, but these have drawbacks such as high safety risks, strict usage restrictions, and limited therapeutic effects.

[0005] More importantly, there are still significant technological gaps in the current technology. No single treatment plan has yet been found that can simultaneously exert a significant therapeutic effect on two completely opposite pathological states: androgen dominance and estrogen deficiency. Most existing related treatment plans can only achieve mild improvement in a single pathological direction and cannot achieve precise bidirectional regulation of sex hormone balance. Furthermore, they cannot simultaneously cover the two major indications of reproductive endocrine diseases and bone degenerative diseases, making it difficult to meet the core clinical needs for safe, multi-target, and full-cycle intervention in sex hormone imbalance and related age-related diseases. Summary of the Invention

[0006] The embodiments of this application aim to at least partially solve one of the technical problems in the related art. To this end, the embodiments of this application provide *Bifidobacterium animalis* subsp. *lactobacter* BGI-L99 and its applications. Products prepared based on *Bifidobacterium animalis* subsp. *lactobacter* BGI-L99 can effectively prevent and treat diseases related to sex hormone imbalance and delay age-related degenerative changes.

[0007] Firstly, this application provides a strain of Bifidobacterium lactis subsp. (Bifidobacterium animalis) Bifidobacterium animalis subsp. lactis The animal Bifidobacterium lactis subspecies BGI-L99 has the accession number GDMCC No. 68017.

[0008] The *Bifidobacterium lactis* subsp. BGI-L99 provided in this application embodiment is classified and named as follows: Bifidobacterium animalis subsp. lactis The gene was deposited on March 31, 2026, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), Guangzhou, China, with accession number GDMCC No. 68017. In an exemplary embodiment, the 16S rDNA sequence of the *Bifidobacterium animalis* subsp. *lactospirum* BGI-L99 is shown in SEQ ID NO: 1.

[0009] (SEQ ID NO: 1).

[0010] Secondly, embodiments of this application provide the application of Bifidobacterium animalis subsp. Lactobacillus BGI-L99 in the preparation of products for the prevention and treatment of diseases related to sex hormone imbalance and for delaying age-related degenerative diseases.

[0011] In an exemplary embodiment, the product includes at least one of the following: microecological preparations, pharmaceuticals, health foods, functional beverages, probiotic products, or foods for special medical purposes.

[0012] In some examples of this application, the dosage form of the microecological preparation includes powder, tablet, capsule, granule or liquid preparation.

[0013] In some examples of this application, the number of live bacteria in the microecological preparation is not less than 1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g.

[0014] In some examples of this application, the formulation further includes food- or pharmaceutically acceptable excipients. Preferably, the excipients include any one or a combination of at least two of the following: carriers, wetting agents, protective agents, fillers, solubilizers, disintegrants, osmotic pressure regulators, coating materials, pH adjusters, antioxidants, or buffers.

[0015] In some examples of this application, the sex hormone imbalance-related diseases and age-related degenerative diseases include at least one of ovarian dysfunction, endocrine disorders, depression, metabolic disorders, inflammatory responses, abnormal intestinal metabolites, bone microstructure degeneration, and imbalance between bone formation and bone resorption.

[0016] Thirdly, embodiments of this application provide a culture comprising: live cells, inactivated cells, metabolites, fermentation broth, or bacterial suspension of Bifidobacterium animalis subsp. Lactobacillus BGI-L99 as in any example of the first aspect.

[0017] Fourthly, embodiments of this application provide a microbial agent or preparation comprising: live cells, inactivated cells, metabolites, fermentation broth, or bacterial suspension of Bifidobacterium animalis subsp. Lactobacillus BGI-L99 as described in any example of the first aspect.

[0018] Fifthly, embodiments of this application provide a composition comprising: live cells, inactivated cells, metabolites, fermentation broth, or bacterial suspension of Bifidobacterium animalis subsp. Lactobacillus BGI-L99 as described in any example of the first aspect.

[0019] In some examples of this application, the compositions include: food compositions and pharmaceutical compositions.

[0020] In some examples of this application, the composition further includes at least one of probiotics, prebiotics, and vitamins.

[0021] Sixthly, embodiments of this application provide the application of Bifidobacterium animalis subsp. lactis BGI-L99 in the preparation of a microbial preparation for alleviating estrous cycle disorders caused by sex hormone imbalance.

[0022] Seventhly, embodiments of this application provide the application of Bifidobacterium animalis subsp. lactis BGI-L99 in the preparation of a microbial preparation for alleviating ovarian morphological abnormalities caused by sex hormone imbalance.

[0023] Eighthly, embodiments of this application provide the use of Bifidobacterium animalis subsp. lactis BGI-L99 in the preparation of microbial preparations for alleviating endocrine disorders caused by sex hormone imbalance.

[0024] Ninthly, embodiments of this application provide the use of Bifidobacterium animalis subsp. lactis BGI-L99 in the preparation of a microbial preparation for alleviating depression caused by sex hormone imbalance.

[0025] In a tenth aspect, embodiments of this application provide the use of Bifidobacterium animalis subsp. lactis BGI-L99 in the preparation of microbial preparations for alleviating metabolic disorders caused by sex hormone imbalance.

[0026] Eleventhly, embodiments of this application provide the application of Bifidobacterium animalis subsp. lactis BGI-L99 in the preparation of microbial preparations for alleviating inflammatory responses caused by sex hormone imbalance.

[0027] In a twelfth aspect, embodiments of this application provide the use of Bifidobacterium animalis subsp. lactis BGI-L99 in the preparation of a microbial preparation for alleviating abnormal intestinal metabolites caused by sex hormone imbalance.

[0028] In a thirteenth aspect, embodiments of this application provide the use of Bifidobacterium animalis subsp. lactis BGI-L99 in the preparation of a microbial preparation for alleviating bone microstructural degeneration caused by sex hormone imbalance.

[0029] In a fourteenth aspect, embodiments of this application provide the use of Bifidobacterium animalis subsp. lactis BGI-L99 in the preparation of a microbial preparation for alleviating the imbalance between bone formation and bone resorption caused by sex hormone imbalance.

[0030] The Bifidobacterium lactis subsp. BGI-L99 of this application embodiment has the following beneficial effects: 1) Using Bifidobacterium lactis subsp. BGI-L99 from the embodiments of this application to intervene in mice with polycystic ovary syndrome can effectively alleviate the estrous cycle disorder in mice; 2) Using Bifidobacterium lactis subsp. BGI-L99 from the embodiments of this application to intervene in mice with polycystic ovary syndrome can effectively improve the abnormal ovarian morphology of mice, specifically manifested as: a decrease in the number of abnormal cystic follicles in the ovaries and an increase in the number of corpora lutea; 3) Using Bifidobacterium lactis subsp. BGI-L99 from the embodiments of this application to intervene in mice with polycystic ovary syndrome can effectively improve the secretion of sex hormones in mice, specifically manifested as: decreased levels of luteinizing hormone and testosterone, and increased levels of estradiol; 4) Using Bifidobacterium lactis subsp. BGI-L99 from the embodiments of this application to intervene in polycystic ovary syndrome mice can effectively improve the depressive-like behavior of the mice; 5) Using Bifidobacterium lactis subsp. BGI-L99 from the embodiments of this application to intervene in mice with polycystic ovary syndrome can effectively improve the metabolic disorders in mice, specifically manifested as: reduced weight gain in mice, and reduced total cholesterol, triglycerides, and low-density lipoprotein cholesterol; 6) Using Bifidobacterium lactis subsp. BGI-L99 from the embodiments of this application to intervene in mice with polycystic ovary syndrome can effectively improve the intestinal and systemic inflammatory responses in mice, specifically manifested as: significantly increased levels of IL-10 and IL-22, and significantly decreased levels of IL-6, IL-1β and TNF-α; 7) Using Bifidobacterium lactis subsp. BGI-L99 from the embodiments of this application to intervene in mice with polycystic ovary syndrome can effectively improve the abnormality of short-chain fatty acids in the intestinal metabolites of mice, specifically manifested as: significantly increased levels of acetic acid, butyric acid, isobutyric acid and valerate; in addition, the expression level of short-chain fatty acid receptor GPR43 mRNA in the intestine of mice was significantly increased after dietary intervention with Bifidobacterium lactis subsp. BGI-L99. 8) Using Bifidobacterium lactis subsp. BGI-L99 from the embodiments of this application to intervene in mice with polycystic ovary syndrome can effectively improve the abnormal bile acid metabolism products in the intestines of mice, specifically manifested as: significantly increased levels of bile acids chenodeoxycholic acid (CDCA) and tauroursodeoxycholic acid (TUDCA) in feces. 9) Using Bifidobacterium lactis subsp. BGI-L99 from the embodiments of this application to intervene in osteoporotic mice can effectively improve the bone microstructure of mice, specifically by increasing bone volume fraction (BV / TV), number of trabeculae (Tb.N) and trabecular thickness (Tb.Th), and reducing trabecular separation (Tb.Sp). 10) Using Bifidobacterium lactis subsp. BGI-L99 from the embodiments of this application to intervene in osteoporotic mice can effectively improve the balance between bone formation and bone resorption in mice. Specifically, it can increase mineralization deposition rate (MAR), bone formation rate (BFR / BS), osteocalcin (OCN), and the level of type I collagen amino-terminal elongated peptide (PINP), while decreasing the levels of type I collagen cross-linked terminal peptide (CTX) and tartrate-resistant acid phosphatase (TRACP). 11) Using Bifidobacterium lactis subsp. BGI-L99 from the embodiments of this application to intervene in osteoporotic mice can effectively improve the systemic inflammatory response in mice, specifically manifested as: a significant increase in the level of the anti-inflammatory factor IL-10 and a significant decrease in the level of the pro-inflammatory factor TNF-α. In summary, the Bifidobacterium animalis subsp. Lactobacillus BGI-L99 of the present application, compared with existing drug intervention strategies, can be applied to the food, health product and pharmaceutical fields, and has broad industrial prospects and public health value. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 A schematic diagram illustrating the effect of Bifidobacterium lactis subsp. BGI-L99 on the estrous cycle of mice, provided in one embodiment of this application. Figure 2 A schematic diagram showing the effect of Bifidobacterium lactis subsp. BGI-L99 on the number of cystic follicles and corpora lutea in mice, provided in one embodiment of this application; and a schematic diagram showing the HE staining results of mouse ovarian tissue sections. Figure 3 This is a schematic diagram illustrating the effect of Bifidobacterium lactis subsp. BGI-L99 on serum sex hormones in mice, provided in one embodiment of this application. Figure 4 This is a schematic diagram illustrating the effect of Bifidobacterium lactis subsp. BGI-L99 on cytokine levels in mouse serum and intestinal tissue, as provided in one embodiment of this application. Figure 5 This is a schematic diagram showing the effect of Bifidobacterium lactis subsp. BGI-L99 on various metabolic indicators in mice, according to one embodiment of this application. Figure 6 A schematic diagram illustrating the effect of Bifidobacterium lactis subsp. BGI-L99 on the level of short-chain fatty acids (SCFAs) in mouse feces, provided in one embodiment of this application. Figure 7 This is a schematic diagram showing the effect of Bifidobacterium animalis subsp. lactis BGI-L99 on bile acid levels in mouse feces according to one embodiment of this application. Figure 8 A schematic diagram illustrating the effect of Bifidobacterium lactis subsp. BGI-L99 on depressive-like behavior in mice, provided in one embodiment of this application. Figure 9 A schematic diagram illustrating the effects of Bifidobacterium lactis subsp. BGI-L99 on bone microstructure and biochemical indicators in osteoporotic mice, provided in one embodiment of this application. Figure 10 A schematic diagram illustrating the effect of Bifidobacterium lactis subsp. BGI-L99 on inflammation in osteoporotic mice, provided in one embodiment of this application. In the polycystic ovary syndrome (PCOS) mouse model, the horizontal axis in each graph represents: Con represents the normal control group, PCOS represents the PCOS model group, and PCOS+B. animalis represents the intervention group with Bifidobacterium lactis subsp. BGI-L99. In the osteoporosis mouse experiment, normal represents the sham-operated control group, OVX represents the ovariectomized osteoporosis model group, and B. animalis represents the intervention group with Bifidobacterium lactis subsp. BGI-L99. Detailed Implementation

[0033] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0034] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this application are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of this application described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion; for example, a process, method, system, product, or server that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or devices.

[0035] In the embodiments of this application, those skilled in the art should understand that the nucleic acids mentioned in the specification and claims actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases in this specification and claims, the other complementary strand is actually disclosed as well. In addition, the nucleic acid sequences in this application include DNA or RNA forms; disclosing one means that the other is also disclosed.

[0036] In the embodiments of this application, "relief" refers to the attainment of desired pharmacological and / or physiological effects. These effects may be preventative in terms of complete or partial prevention of the disease or its symptoms, and / or therapeutic in terms of partial or complete cure of the disease and / or adverse effects caused by the disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of symptoms in individuals susceptible to disease but not yet diagnosed with the disease; (b) suppression of disease, such as inhibiting disease progression; or (c) relief of disease, such as reducing symptoms associated with the disease. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, relieve, improve, reduce, or suppress the individual's disease, including but not limited to administration of a drug containing the compositions described herein to an individual in need.

[0037] In this document, "pharmaceutical acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components of the formulation and / or the mammals to which it is treated. Preferably, "pharmaceutical acceptable" as used herein means approved by a federal regulatory agency or national government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, particularly in humans.

[0038] In this document, the term "pharmaceutically acceptable carrier" includes any solvent, drug stabilizer, or combination thereof, which are known to those skilled in the art. Except in cases where any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is covered.

[0039] In this document, the term "pharmaceuticalally acceptable excipient" can include sugars, including monosaccharides or polysaccharides such as lactose, sucrose, mannitol, and sorbitol; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and methyl cellulose; calcium phosphates, such as dicalcium phosphate and tricalcium phosphate; sodium sulfate; calcium sulfate; polyvinylpyrrolidone; polyvinyl alcohol; stearic acid; alkaline earth metal stearates, such as magnesium stearate and calcium stearate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, and corn oil; nonionic, cationic, and anionic surfactants; ethylene glycol polymers; fatty alcohols; and hydrolyzed cereal solids, as well as other nontoxic and compatible fillers, binders, disintegrants, buffers, preservatives, antioxidants, lubricants, colorants, and other excipients commonly used in pharmaceutical formulations.

[0040] In the embodiments of this application, the preparation for relieving polycystic ovary syndrome can be used by oral administration or other means.

[0041] Unless otherwise specified, the techniques or conditions described in the following examples were performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0042] The following examples establish a PCOS disease model by continuous injection of dehydroepiandrosterone (DHEA) to investigate the effects of gavage administration of Bifidobacterium lactis subsp. BGI-L99 on behavioral indicators, endocrine function, estrous cycle, systemic inflammation, and microbial metabolites in PCOS mice. Furthermore, an osteoporosis disease model is established by ovariectomy to investigate the effects of gavage administration of Bifidobacterium lactis subsp. BGI-L99 on bone metabolism and systemic inflammation in osteoporotic mice.

[0043] Example 1: Identification of Bifidobacterium animalis subsp. Lactobacillus BGI-L99 This embodiment describes the use of Bifidobacterium lactis subsp. animalis (Bifidobacterium lactis) Bifidobacterium animalis subsp. lactis BGI-L99 was isolated, cultured, and identified. Specifically, this included: 1. Sample collection The isolated sample was obtained from the feces of an adolescent female. The feces were collected into a sterile sample tube and brought back to the laboratory for isolation and culture within 1 hour.

[0044] 2. Isolation and purification of BGI-L99 Freshly collected samples were immediately transferred to an anaerobic chamber (Bactron, Shellab, USA; gas ratio 90% N2, 5% CO2, and 5% H2). 1 g of sample was added to 1 mL of sterile PBS (phosphate-buffered saline), thoroughly vortexed, and then serially diluted and spread. MPYG medium was used. The detailed formulation of MPYG liquid medium is as follows: 8.00 g tryptone, 2.00 g soybean peptone, 1.00 g polypeptone, 1.00 g casein, 10.00 g yeast extract, 5.00 g beef extract, 5.00 g glucose, 0.50 g cellobiose, 0.50 g maltose, 0.50 g soluble starch, 2.00 g K2HPO4, 5.00 g sodium acetate, and Tween 80. 0.50 mL of glycerol, 40.00 mL of salt solution, 10.00 mL of trace element solution, 10.00 mL of vitamin solution, and 10.00 mL of heme solution are added. For anaerobic conditions, the following should be added: 0.50 g of cysteine, 0.25 g of Na₂S, 1.00 mg of resazurin, and ddH₂O to a final volume of 1 L. Adjust the pH to 6.8-7.0 and sterilize at 121℃ for 20 min. For solid culture media, add 1.5% agar. Spread the culture plates and incubate anaerobicly at 37℃ for 4 days. After incubation, pick single colonies for streak plating to obtain pure cultures of each strain.

[0045] 3. Preservation of microbial strains After 16S rRNA sequencing of the cultured bacterial culture confirmed that it was Bifidobacterium animalis subsp. lactis BGI-L99, 400 μL of 40% glycerol and 400 μL of the bacterial culture were transferred into glycerol tubes and stored at -80℃.

[0046] 4. Identification of 16S rRNA Take 1 mL of cultured bacterial suspension in an anaerobic incubator and transfer it to a 1.5 mL centrifuge tube. Centrifuge at 10000 rpm for 5 min at room temperature, retain the bacterial cells, and resuspend them in 1×PBS. Use a 25 μL amplification system: 10×Buffer 2.50 μL, dNTP 2.00 μL, primer 341F 0.50 μL, primer 926R 0.50 μL, Taq enzyme 0.30 μL, bacterial suspension 1.00 μL, ddH2O 18.20 μL. Set the amplification program as follows: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 1 min 30 s, 10 cycles; 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 1 min 30 s, 25 cycles; 72℃ extension for 10 min, store at 4℃. PCR products were sent to Beijing Liuhe BGI Genomics Co., Ltd. for Sanger sequencing. The sequencing results were quality controlled using Sequence Scanner v1.0 to remove low-quality sequences. Sequences meeting quality standards were then aligned using Ezbiocloud (https: / / www.ezbiocloud.net / identify). The bacterium with the highest 16S rRNA homology to BGI-L99 in the database was *Bifidobacterium animalis* subsp. *lactamella*. Bifidobacterium animalis subsp. lactis The similarity to DSM 10140 was 99.63%. This indicates that BGI-L99 belongs to the subspecies of Bifidobacterium animalis (Lactobacillus lactis). The 16S rDNA sequencing results of BGI-L99 are shown in SEQ ID NO: 1.

[0047] The sequence of primer 341F is shown in SEQ ID NO: 2; CCTACGGGAGGCAGCAG (SEQ ID NO: 2); The sequence of primer 926R is shown in SEQ ID NO: 3; CCGTCAATTCCTTTRAGTTT (SEQ ID NO: 3).

[0048] Example 2: Effects of Bifidobacterium animalis subsp. lactis BGI-L99 on symptoms of polycystic ovary syndrome in mice. This embodiment constructs a mouse model of polycystic ovary syndrome (PCOS) and uses methods such as vaginal smear, ovarian H&E staining, and ELISA to systematically evaluate the effects of Bifidobacterium lactis subsp. BGI-L99 on the estrous cycle, ovarian morphology, and hormone secretion phenotypes in PCOS mice.

[0049] Experimental methods: (1) Animal model establishment and drug administration: ① Thirty female mice were randomly divided into three groups: control group (Con), model group (PCOS), and Bifidobacterium lactis subsp. BGI-L99 intervention group (PCOS+B. animalis); ② Mice in the PCOS group and PCOS+B. animalis group were subcutaneously injected daily with dehydroepiandrosterone (DHEA, 60 mg / kg) dissolved in sesame oil for 21 consecutive days to induce the PCOS model; the Con group was injected with an equal volume of sesame oil solvent at the same time; ③ After 21 days of model establishment, the intervention phase began. The PCOS+B. animalis group was administered 1×10 mg / kg of sesame oil by gavage daily. 9 CFU BGI-L99 bacterial suspension was administered for 21 days; Con group and PCOS group were simultaneously gavaged with an equal volume of PBS; ④ After all interventions were completed, mice were sacrificed and relevant tissues and samples were collected for subsequent analysis. (2) Vaginal smear: A cotton swab dipped in physiological saline was gently inserted into the vagina to collect vaginal cells. The cells were then evenly smeared onto a glass slide, air-dried, and stained with Wright-Gymsa compound stain. The main cell types in the mouse vaginal smears were analyzed under a microscope for 10 consecutive days, and the estrous cycle stage was determined based on the epithelial cell type (Table 1).

[0050] (3) H&E staining of ovarian tissue: First, paraffin sections were prepared and pretreated for staining. After euthanizing the mice, the ovarian tissues of each group of mice were fixed in 4% paraformaldehyde (v / v) solution, then embedded in paraffin and cut into 5 μm thick sections. The sections were dried in a 37 ℃ incubator, dewaxed with xylene for 20 min, and then rehydrated with 100%, 90%, 80%, and 70% graded ethanol for 5 min each. Finally, the sections were washed 5 times with pH 7.4 PBS for 4 min each time. After pretreatment, H&E staining was performed. The tissue was stained with hematoxylin for 5 min, rinsed with tap water, differentiated with 1% hydrochloric acid alcohol for 15 s, rinsed with tap water again, stained with 0.5% eosin for 3 min, washed 5 times with PBS (4 min each time), dehydrated with 70%, 80%, 90%, and 100% graded ethanol, and then cleared with xylene for 20 min. Finally, the slides were mounted with neutral resin and dried, and then observed under an optical microscope to observe changes in ovarian morphology.

[0051] (4) Serum hormone level determination: The levels of LH, FSH, T, E2, IL-10, and IL-22 in mouse serum were detected using an ELISA kit according to the instructions. A standard curve was established using standards of known concentrations to calculate the levels of the corresponding indicators in serum. All procedures were performed in accordance with the manufacturer's instructions.

[0052] Table 1. Characteristics of epithelial cell types and relative numbers in vaginal smears from different stages of the estrous cycle in mice.

[0053] Note: 0 indicates none, + indicates few, ++ indicates moderate, and +++ indicates many. Experimental results: (1) Intervention with Bifidobacterium animalis subsp. Lactobacillus BGI-L99 can effectively alleviate estrus cycle disorders in PCOS mice: such as Figure 1 As shown in Figure A, compared with the control group, the proportions of proestrus (P) and metestrus / diestrus (M / D) in the PCOS model group mice were significantly reduced. P< 0.01), the proportion of estrus (E) increased significantly ( P< 0.05). The estrous cycle of mice in the Con group was normal ( Figure 1 B), PCOS-like mice induced by subcutaneous DHEA injection exhibit estrous cycle disorder, often remaining in estrus or experiencing cycle loss. Figure 1 C). After intervention with Bifidobacterium animalis subsp. lactis BGI-L99, the estrous cycle disorder in PCOS+B. animalis group mice was improved (Figure 1A), and the proportion of proestrus and metestrus / interestrus was significantly increased ( P< 0.05), the proportion of the estrus period decreased significantly ( P< 0.01), which indicates a normal estrous cycle ( Figure 1 D). The above results indicate that intervention with Bifidobacterium lactis subsp. BGI-L99 can effectively alleviate estrous cycle disorders in PCOS mice.

[0054] (2) Bifidobacterium animalis subsp. lactis BGI-L99 can effectively improve ovarian morphological abnormalities and restore ovarian function in PCOS mice: compared with the control group, the number of ovarian follicles in the PCOS model group mice increased ( P< 0.05) Figure 2 A), decreased number of corpora lutea ( P< 0.01) Figure 2 B). For example... Figure 2 As shown in Figure C, the ovaries of Con mice contained follicles and corpora lutea at various developmental stages, but no cystic follicles were observed. In contrast, the ovaries of the PCOS model group mice had multiple cystic follicles and a reduced number of corpora lutea. Intervention with Bifidobacterium lactis subsp. BGI-L99 reduced the number of cystic follicles in the ovaries of PCOS mice. P< 0.05), increased the formation of the corpus luteum ( P< 0.01) Figure 2(AE). The above results indicate that supplementation with Bifidobacterium lactis subsp. BGI-L99 can effectively improve ovarian morphological abnormalities and restore ovarian function in PCOS mice.

[0055] (3) Intervention with Bifidobacterium animalis subsp. Lactobacillus BGI-L99 can effectively improve abnormal sex hormone levels and alleviate endocrine disorders in PCOS mice: Hyperandrogenemia is one of the important diagnostic criteria for PCOS, as shown in Table 2 and Figure 3 As shown in AD, compared with the control group mice, the serum levels of sex hormone LH and androgen T in the PCOS group mice were significantly increased ( P< 0.01), FSH levels showed no significant change (p=0.14), while estrogen E2 levels decreased significantly ( P< 0.05). However, after intervention with the probiotic *Bifidobacterium animalis* subsp. *lactamase* BGI-L99, compared with PCOS mice, the LH and T levels in the PCOS+B. animalis group were significantly reduced ( P< 0.05), E2 level significantly increased ( P< 0.05). Simultaneously, the expression of genes related to sex hormone synthesis and secretion was measured. Compared with the Con group mice, the expression level of LH hormone receptor LHr in the hypothalamus of the PCOS group mice was significantly increased ( ). P< 0.05) Figure 3 E), the expression level of estrogen receptor Erα was significantly reduced ( P< 0.01) Figure 3 F), the expression levels of CYP17A1 and CYP19A1, genes related to sex hormone synthesis in the ovary, were significantly increased (F). P< 0.01) Figure 3 These results indicate that DHEA-induced endocrine disorders and abnormal sex hormone synthesis and secretion occur in PCOS mice. However, supplementation with the probiotic Bifidobacterium animalis subsp. lactis BGI-L99 improved the abnormal sex hormone secretion. The expression level of LHr in the hypothalamus of PCOS+B. animalis group mice was significantly reduced (GH). P< 0.01), Erα expression level significantly increased ( P< 0.01), ovarian CYP17A1 and CYP19A1 expression was significantly decreased ( P< 0.01). The above results indicate that intervention with Bifidobacterium lactis subsp. BGI-L99 can effectively improve abnormal sex hormone levels and alleviate endocrine disorders in PCOS mice.

[0056] Table 2. Hormone levels in mice

[0057] Note: Compared to the PCOS group, * indicates P <0.05, ** indicates P <0.01.

[0058] Example 3: Effects of Bifidobacterium animalis subsp. lactis BGI-L99 on inflammation in mice with polycystic ovary syndrome In this embodiment, the levels of IL-10 and IL-22 in mouse serum were detected by ELISA, and the expression of inflammatory genes was detected by RT-qPCR to explore the effect of BGI-L99 on inflammation in mice with polycystic ovary syndrome.

[0059] Test method: (1) The ELISA detection procedure is the same as in Example 2; (2) RT-qPCR detection: The steps include RNA extraction, reverse transcription, and RT-qPCR. Weigh 50-70 mg of tissue sample on ice and place it in a 1.5 mL RNase-free centrifuge tube. Add 1 mL of BIOZOL reagent to the centrifuge tube and extract mRNA according to the specific operation method and steps provided in the kit. Use a nucleic acid quantification instrument to measure the absorbance value (260nm / 280nm) and concentration of the extracted mRNA sample. Select samples with a ratio between 1.8 and 2.0 for subsequent operations, calculate the concentration of the mRNA sample, and dilute it to the same concentration as required. Then prepare a reverse transcription system and reverse transcribe the mRNA into cDNA using a PCR amplification instrument. Continue to dilute the reverse transcribed sample to the required concentration and prepare a PCR reaction system. Quantitative PCR is performed using a quantitative PCR instrument QuantStudio®5. The endogenous control gene is GAPDH. All reactions are performed in eight-tube sets. Record the threshold (Ct) and use 2 The relative expression levels of relevant genes were calculated using the ΔΔCT method. The PCR primers used in the experiment were synthesized by Shanghai Bioengineering Co., Ltd., and their sequences are shown in Table 3. Table 3 RT-qPCR Primer Sequences

[0060] Experimental results: Intervention with Bifidobacterium lactis subsp. BGI-L99 improved intestinal and systemic inflammatory responses in PCOS mice: serum IL-10 and IL-22 levels were as follows: Figure 4 As shown in Figure AB, compared with the control group, the serum levels of IL-10 and IL-22 in PCOS group mice were significantly reduced ( P<0.01), while intervention with Bifidobacterium lactis subsp. BGI-L99 significantly increased the serum IL-22 level in PCOS mice ( P< 0.01). Further studies examined the gene expression levels of pro-inflammatory factors IL-6, IL-1β, TNF-α, and anti-inflammatory factor IL-10 in the intestines of mice in each group. The results are as follows: Figure 4 As shown in CF, compared with the control group, the PCOS group mice had higher levels of IL-6 ( ) in their intestines. P< 0.01), IL-1β P< 0.05) and TNF-α P< The expression levels of IL-6, IL-1β, and TNF-α in the intestines of PCOS mice were significantly increased by 0.05%. Intervention with Bifidobacterium lactis subsp. BGI-L99 significantly reduced the expression levels of IL-6, IL-1β, and TNF-α in the intestines of PCOS mice. P< 0.05), while intervention with Bifidobacterium lactis subsp. BGI-L99 significantly increased the expression level of IL-10 in the intestine of PCOS mice ( P< 0.05). These results indicate that intervention with Bifidobacterium lactis subsp. BGI-L99 significantly improved the level of intestinal and systemic inflammatory responses in PCOS mice.

[0061] Example 4: Effects of Bifidobacterium animalis subsp. lactis BGI-L99 on metabolic indicators in mice with polycystic ovary syndrome In this embodiment, a fully automated biochemical analyzer was used to detect serum lipid levels, and targeted metabolomics was used to quantify intestinal short-chain fatty acids and bile acids.

[0062] Experimental methods (1) Blood lipid level measurement: According to the instructions for use of the fully automated biochemical analyzer, the levels of TC, TG and LDL-C in serum were measured; (2) Detection of short-chain fatty acids: SCFAs in mouse feces were detected using GC-MS. A 200 mg fecal sample was placed in a pre-centrifuged tube and 1 mL of MilliQ water was added and mixed thoroughly. The mixture was vortexed for 10 min at room temperature to ensure uniform dispersion. 150 μL of 50% sulfuric acid (w / w) was added and vortexed again. 1600 μL of diethyl ether was added to the centrifuge tube, and the mixture was incubated for 30 min, which aided in the extraction of the fatty components. After incubation, the mixture was centrifuged for 5 min (8000 g, 4°C) to separate the organic phase (upper liquid) and aqueous phase (lower liquid) from the mixture. The upper liquid contained fats and diethyl ether and required further processing. The upper liquid was transferred to a 0.22 µm filter membrane for filtration to remove particulate matter and residues. The filtered liquid was then transferred to a clean gas chromatograph (GC) vial, to which diethyl ether was added as an internal standard. This internal standard serves as a quantitative reference in GC analysis, aiding in the accurate measurement of component concentrations. The concentrations of SCFAs were analyzed using a Shimadzu GC-2014C gas chromatograph equipped with a 30 m × 0.25 µm × 0.25 mm DB-FFAP capillary column. Different concentrations of acetate, propionate, butyrate, isobutyrate, valerate, and isovalerate were used to prepare mixed standards and establish standard curves. Shimadzu GC data processing software was used to integrate the chromatographic peaks. (3) Bile acid detection: Bile acids were quantitatively analyzed using an Eksigent ultrafluid chromatography-100 system and an AB 5600 TripleTOF system (AB SCIEX). An XBridge Peptide BEH C18 column (100 mm × 2.1 mm id; 1.7 μm; Waters Corp) was used, and separation was performed at 0.4 mL / min at 40 °C. The mobile phase consisted of a mixture of 0.1% formic acid, 10 mM ammonium acetate (A), and 0.1% formic acid, 80% methanol, and 20% acetonitrile (B). The gradient flow rate was set as follows: initially 35% B (0.5 min), linearly increasing to 60% B over the next 2.5 min, linearly increasing to 80% B over the next 7 min, linearly increasing to 90% B over the next 6 min, and linearly decreasing to 35% B over the next 4.5 min, maintaining this composition for 2.5 min. Each injection volume was 5 μl. The m / z range for TOF MS scanning was set to 200–800 Da, and the ion scan range for automated tandem mass spectrometry acquisition was 50–800 Da. The raw data were processed using Peak View 1.2 and Multi Quant 2.1 software, and analyzed based on the m / z values ​​and sample retention times.

[0063] Experimental results: (1) Intervention with Bifidobacterium animalis subsp. Lactobacillus BGI-L99 can improve DHEA-induced abnormal glucose and lipid metabolism in PCOS mice: such as Figure 5 As shown in AB, compared with the control group, PCOS mice had a higher body weight at the experimental endpoint. P< 0.05), after intervention with Bifidobacterium lactis subsp. BGI-L99, the body weight of mice in the PCOS+B. animalis group decreased ( P< 0.05). Meanwhile, regarding changes in mouse body weight during the experiment, the PCOS group mice showed significantly higher weight than the Con group ( P< 0.01), the PCOS+B. animalis group mice were significantly lower than the PCOS group ( P< 0.01) Figure 5 C). Furthermore, fasting blood glucose levels in mice were measured, and it was found that intervention with Bifidobacterium animalis subsp. lactis BGI-L99 reduced blood glucose levels in the PCOS+B. animalis group ( P< 0.01) Figure 5 D). Serum total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) levels were measured using a fully automated biochemical analyzer. The results are shown in Table 4. Figure 5 As shown in EG, higher levels of TC, TG, and LDL-C were observed in DHEA-induced PCOS mice, while these levels were significantly reduced after intervention with Bifidobacterium lactis subsp. BGI-L99. P< 0.05). The above results indicate that intervention with Bifidobacterium lactis subsp. BGI-L99 can improve DHEA-induced abnormal glucose and lipid metabolism in PCOS mice and has the potential to alleviate PCOS metabolic disorders.

[0064] Table 4. Mean blood lipid levels in mice (mmol / L)

[0065] Note: Compared to the PCOS group, * indicates P <0.05, ** indicates P <0.01.

[0066] (2) Intervention with Bifidobacterium lactis subsp. BGI-L99 increased the level of SCFAs in PCOS mice: e.g. Figure 6 As shown in AE, the levels of acetic acid, propionic acid, butyric acid, and isobutyric acid in the feces of PCOS group mice were significantly decreased. P< 0.05), while dietary intervention with Bifidobacterium lactis subsp. BGI-L99 significantly increased acetic acid ( ) in the feces of PCOS mice. P< 0.05), butyric acid ( P< 0.05), isobutyric acid ( P< 0.01) and valeric acid ( P< The content was 0.01%. Furthermore, dietary intervention with Bifidobacterium lactis subsp. BGI-L99 significantly increased the expression level of the short-chain fatty acid receptor GPR43 in the intestine of PCOS mice. P< 0.01) Figure 6 F). The above results indicate that intervention with Bifidobacterium lactis subsp. BGI-L99 reversed the abnormal decrease in SCFAs in PCOS mice and helped restore normal gut microbiota metabolism.

[0067] (3) Intervention with Bifidobacterium animalis subsp. Lactobacillus BGI-L99 improved bile acid levels in PCOS mice: The content of bile acids in mouse feces was detected, including chenodeoxycholic acid (CDCA), cholic acid (CA), glycochenodeoxycholic acid (GCDCA), tauroursodeoxycholic acid (TUDCA), ursodeoxycholic acid (UDCA), glycocholic acid (GCA), deoxycholic acid (DCA), and lithocholic acid (LCA). See Table 5 and Figure 7 As shown by AI, compared with the control group, the feces of PCOS group mice contained CDCA (… P< 0.05), UDCA ( P<0.05) and TUDCA ( P< The level of TBA was significantly reduced (0.01), and the serum TBA content was significantly reduced ( P< 0.05). However, after dietary intervention with Bifidobacterium animalis subsp. lactis BGI-L99, the levels of LCA, CDCA, and TUDCA in the feces of mice in the B. animalis+PCOS group were significantly increased ( P< (0.01). These results indicate that intervention with Bifidobacterium animalis subsp. lactis BGI-L99 improved bile acid metabolism disorder in PCOS mice.

[0068] Table 5. Bile acid levels in mice (μg / g)

[0069] Note: Compared to the PCOS group, * indicates P <0.05, ** indicates P <0.01.

[0070] Example 5: Effects of Bifidobacterium animalis subsp. lactis BGI-L99 on depressive-like behavior in mice with polycystic ovary syndrome This embodiment uses behavioral experiments to identify the effect of BGI-L99 on depressive-like behavior in mice with polycystic ovary syndrome.

[0071] Experimental methods: (1) Open field test: The open field test is a classic test for emotion-related behavioral testing. The test is conducted in a quiet environment. The mouse open field test box is 50 cm*50 cm*40 cm in size. Before the test, the mice are allowed to adapt to the test environment for 5 minutes. Then, each time the test is conducted, the mice are placed in the center of the test box from the same position and direction. The test is started. The Supermaze animal behavior analysis software is used to automatically track the movement trajectory of the target mouse, identify the animal's center of gravity, record the animal's movement trajectory, and analyze behavioral indicators such as movement distance and movement speed. The test time is 5 minutes.

[0072] (2) Tail suspension test: The tail suspension test uses the time it takes for a mouse to remain still after being suspended by its tail and unable to escape, and then give up struggling, to reflect its depressive state. During the test, the mouse's tail was fixed to a support with non-adhesive tape and the video was recorded for 6 minutes. The first 2 minutes were the adaptation time, and the mouse's still time in the last 4 minutes was recorded.

[0073] Experimental results: Bifidobacterium animalis subsp. lactis BGI-L99 improved DHEA-induced depressive-like behavior and alleviated depressive symptoms in DHEA-induced PCOS mice. Figure 8As shown in Figure A, compared with the Con group, the PCOS group mice had a significantly longer resting time in the tail suspension test. P< 0.01), while compared with the PCOS group mice, the resting time of mice in the tail suspension test was significantly reduced after intervention with Bifidobacterium lactis subsp. BGI-L99 (0.01). P< 0.01). Meanwhile, the total distance traveled by mice in the open field test can reflect depressive-like behavior. The experiment found that the total distance traveled by mice in the PCOS group was significantly shorter than that in the Con group ( ). P< 0.05), while the total distance traveled by mice supplemented with Bifidobacterium lactis subsp. BGI-L99 was significantly longer than that in the PCOS intervention group ( P< 0.05) Figure 8 B). These results indicate that supplementation with Bifidobacterium animalis subsp. lactis BGI-L99 improves DHEA-induced depressive-like behavior and alleviates depressive symptoms in DHEA-induced PCOS mice.

[0074] Example 6: This example illustrates the effect of Bifidobacterium lactis subsp. BGI-L99 on the bone microstructure of mice.

[0075] Micro-CT scanning and three-dimensional reconstruction analysis were used to identify the intervention effect of BGI-L99 on ovariectomized osteoporosis mice from the perspective of bone microstructure.

[0076] Experimental methods: (1) Animal model establishment and drug administration: ① Eight-week-old female C57BL / 6J mice were randomly divided into three groups: sham-operated group (Normal), model group (OVX), and animal bifidobacterium lactis subsp. BGI-L99 intervention group (B. animalis); ② Bilateral ovariectomy was performed on mice in the OVX and BGI-L99 groups, while only the adipose tissue around the ovaries was removed in the Normal group; ③ The mice were routinely fed for 4 to 12 weeks after surgery to induce a bone loss model; ④ The intervention phase lasted for 30 days. The BGI-L99 group was given bacterial suspension by gavage once every 1 day (15 times in total), while the Normal and OVX groups were given an equal volume of PBS; ⑤ After the intervention, the mice were sacrificed, the right femur was dissected, soft tissue was removed, and the mice were fixed in 4% paraformaldehyde solution for Micro-CT detection.

[0077] (2) Micro-CT detection method: The distal metaphysis of the mouse femur was scanned using a high-resolution Micro-CT system. After the scan, three-dimensional reconstruction was performed, and the cancellous bone region at the same height below the growth plate was selected as the region of interest (ROI). Bone microstructure parameters were calculated using analysis software, mainly including: bone volume fraction (BV / TV), number of trabeculae (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp).

[0078] (3) Experimental Results: The changes in bone mass in each group of mice were visually assessed by Micro-CT imaging analysis. As shown in Figure 9, the three-dimensional reconstructed images showed that, compared with the Normal group, the trabecular bone structure in the distal femur of mice in the OVX group was sparse and fractured, and the bone microstructure was severely damaged. After intervention with Bifidobacterium lactis subsp. BGI-L99, the trabecular bone structure of mice was significantly denser and more connected than that in the OVX group. Quantitative analysis of bone microstructure parameters further confirmed the above results (Table 6). Compared with the Normal group, the bone volume fraction (BV / TV), trabecular bone number (Tb.N), and trabecular bone thickness (Tb.Th) of the OVX group were significantly reduced (P<0.01), while the trabecular bone separation (Tb.Sp) was significantly increased (P<0.01), indicating that the osteoporosis model was successfully constructed. Compared with the OVX group, the BGI-L99 group showed a significant increase in BV / TV, Tb.N, and Tb.Th (P<0.01), and a significant decrease in Tb.Sp (P<0.01). These results indicate that Bifidobacterium animalis subsp. lactis BGI-L99 can effectively inhibit the degradation of bone trabecular microstructure caused by estrogen deficiency and reduce bone loss.

[0079] Table 6. Mouse bone microstructure parameters

[0080] Note: Compared with the OVX group, * indicates P<0.05, and ** indicates P<0.01.

[0081] Example 7: Effects of Bifidobacterium animalis subsp. lactis BGI-L99 on bone metabolism in osteoporotic mice The regulatory effect of BGI-L99 on the balance between bone formation and bone resorption in mice was identified by detecting serum bone metabolism markers and analyzing calcein fluorescence labeling in bone tissue.

[0082] Experimental methods: (1) Detection of serum bone metabolism markers: After the mice were sacrificed at the end of the experiment, serum was collected and bone resorption markers were detected by ELISA kit: type I collagen cross-linked terminal peptide (CTX) and tartrate-resistant acid phosphatase (TRACP); and bone formation markers: osteocalcin (OCN) and type I collagen amino-terminal elongated peptide (PINP).

[0083] (2) Measurement of bone dynamic parameters: On day 10 and day 3 before sacrifice, mice were injected intraperitoneally with calcein (20 mg / kg) for double fluorescent labeling. After sacrifice, the left femur was harvested, and undecalcified bone sections were prepared. Fluorescent bands on the bone surface were observed using a laser confocal microscope, and the mineralization deposition rate (MAR, μm / d) and bone formation rate (BFR / BS, μm3 / μm2 / d) were calculated.

[0084] Experimental results: (1) Bone dynamics analysis (calcein labeling) The rate of bone mineralization deposition directly reflects the strength of osteogenic activity. As shown in Table 7 and Figure 9, laser confocal microscopy revealed two clear, continuous, and widely spaced green fluorescent bands on the bone surface of mice in the Normal group, representing the normal bone mineralization deposition process. The spacing between the fluorescent bands in the OVX group was significantly narrower, and some areas were even blurred or merged, indicating that new bone mineralization was inhibited. After intervention with Bifidobacterium animalis subsp. BGI-L99, the spacing between the fluorescent bands in the B. animalis group was significantly wider than that in the OVX group, the clarity of the bands was restored, and the morphology was close to that of the Normal group. Quantitative analysis of the above images showed that compared with the Normal group, the mineralization deposition rate (MAR) and bone formation rate (BFR / BS) of mice in the OVX group were significantly reduced (P<0.001), indicating that estrogen deficiency led to the inhibition of new bone formation. Supplementation with Bifidobacterium animalis subsp. Lactobacillus BGI-L99 significantly improved the MAR and BFR / BS in the B. animalis group (P<0.001), indicating that BGI-L99 greatly enhanced the mineralization and formation capacity of bone tissue.

[0085] (2) Serum bone metabolism marker analysis: ELISA results further confirmed the regulatory effect of BGI-L99 on bone turnover balance (Table 7 and...). Figure 9In the OVX group, serum levels of bone resorption markers (CTX and TRACP) were significantly higher than those in the Normal group (P<0.001), indicating increased osteoclast activity and accelerated bone loss. After BGI-L99 intervention, CTX and TRACP levels significantly decreased (P<0.001), demonstrating the strain's potent inhibitory effect on bone resorption. Serum levels of bone formation markers (OCN and PINP) in the OVX group were significantly lower (P<0.001). After BGI-L99 intervention, OCN and PINP levels significantly rebounded (P<0.001), recovering to levels close to those in the Normal group. In conclusion, *Bifidobacterium animalis* subsp. *lactamase* BGI-L99 can significantly promote bone formation (increasing MAR, BFR / BS, PINP, and OCN) and potently inhibit bone resorption (decreasing CTX and TRACP) through bidirectional regulation, thereby improving bone quality and alleviating osteoporosis.

[0086] Table 7. Mouse bone dynamics and serum bone metabolism indicators

[0087] Note: * indicates comparison with the OVX group. P <0.05, ** indicates P <0.01, *** indicates P <0.001.

[0088] Example 8: Effects of Bifidobacterium animalis subsp. lactis BGI-L99 on inflammation in osteoporotic mice The expression levels of pro-inflammatory and anti-inflammatory factors in mouse serum were detected by ELISA to explore the immunomodulatory mechanism of BGI-L99 in alleviating bone injury.

[0089] Experimental methods: After the experiment, mice were euthanized, and blood was collected from their eyeballs. Serum was then separated by centrifugation. Following the instructions of the ELISA kit, the concentrations of the anti-inflammatory factor IL-10 and the pro-inflammatory factor TNF-α in the serum of each group of mice were quantitatively analyzed. The specific levels of each indicator were calculated using a standard curve.

[0090] Experimental results: The occurrence of osteoporosis is closely related to immune system disorders, particularly an imbalance between pro-inflammatory and anti-inflammatory factors. ELISA test results are shown in Table 8. Figure 10Compared with the Normal group, mice in the OVX model group showed significant abnormal secretion of inflammatory factors, with a highly significant decrease in the level of the anti-inflammatory factor IL-10 (P<0.001) and a significant increase in the level of the pro-inflammatory factor TNF-α (P<0.05), suggesting that ovariectomy led to drastic changes in the body's immune microenvironment. After intervention with Bifidobacterium animalis subsp. lactis BGI-L99, the immune status of mice was significantly improved: compared with the OVX group, the serum IL-10 level in the B. animalis group mice significantly increased (P<0.001), enhancing the body's anti-inflammatory protective ability; at the same time, the TNF-α level significantly decreased (P<0.05), returning to near-normal levels, effectively blocking the potential erosion of bone tissue by inflammation. In conclusion, Bifidobacterium animalis subsp. lactis BGI-L99 can play a protective role against osteoporosis by reshaping immune balance, particularly by increasing the expression of the anti-inflammatory factor IL-10 and inhibiting the expression of the key pro-inflammatory factor TNF-α, thereby improving the microenvironment disorder caused by ovariectomy.

[0091] Table 8 Serum levels of inflammatory factors in mice

[0092] Note: * indicates comparison with the OVX group. P <0.05, ** indicates P <0.01, *** indicates P <0.001.

[0093] Example 9: Food composition containing Bifidobacterium animalis subsp. lactis BGI-L99 Table 9. Raw Material Proportions for Food Compositions

[0094] Mix milk and sugar according to the formula ratio in Table 9, stir until fully combined, preheat, homogenize under 20 MPa pressure, sterilize at about 90℃ for 5-10 minutes, cool to 40-43℃, mix in vitamin C as a protective agent, and inoculate with 1-100×10 6 cfu / g Bifidobacterium animalis subsp. lactis BGI-L99, which is made containing Bifidobacterium animalis subsp. lactis BGI-L99 food composition.

[0095] Example 10: Pharmaceutical composition containing Bifidobacterium animalis subsp. Lactobacillus BGI-L99 Table 10. Raw Material Proportions for Pharmaceutical Compositions

[0096] According to the formula ratio in Table 10, microcrystalline cellulose, magnesium stearate, silicon dioxide, Bifidobacterium animalis subsp. lactis BGI-L99 is mixed thoroughly and then filled into pharmaceutical gelatin empty capsules to produce a product containing... Bifidobacterium animalis subsp. lactis Pharmaceutical composition of BGI-L99.

[0097] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0098] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application without departing from the principles and spirit of this application.

Claims

1. A strain of Bifidobacterium animalis subsp. lactis ( Bifidobacterium animalis subsp. lactis BGI-L99, characterized in that, The accession number is GDMCC No. 68017.

2. The Bifidobacterium animalis subsp. Lactobacillus BGI-L99 according to claim 1, characterized in that, Its 16S rDNA sequence is shown in SEQ ID NO:

1.

3. The use of Bifidobacterium lactis subsp. BGI-L99 as described in claim 1 or 2 in the preparation of products for the prevention and treatment of diseases related to sex hormone imbalance and for delaying age-related degenerative diseases.

4. The application according to claim 3, characterized in that, The products include at least one of the following: microecological preparations, drugs, health foods, functional beverages, probiotic products, or foods for special medical purposes.

5. The application according to claim 4, characterized in that, The dosage forms of the microecological preparations include powders, tablets, capsules, granules, or liquid preparations; Preferably, the number of live bacteria in the microecological preparation is not less than 1×10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g; Preferably, the microecological preparation further includes food- or pharmaceutically acceptable excipients; the excipients include at least one of a carrier, wetting agent, protective agent, filler, solubilizer, disintegrant, osmotic pressure regulator, coating material, pH adjuster, antioxidant, or buffer.

6. The application according to any one of claims 3-5, characterized in that, The aforementioned sex hormone imbalance-related diseases and age-related degenerative diseases include at least one of the following: ovarian dysfunction, endocrine disorders, depression, metabolic disorders, inflammatory responses, abnormal intestinal metabolites, bone microstructure degeneration, and imbalance between bone formation and bone resorption.

7. A culture, characterized in that, The culture includes: Live cells, inactivated cells, metabolites, fermentation broth, or bacterial suspension of Bifidobacterium lactis subsp. BGI-L99 as described in claim 1 or 2.

8. A microbial agent or preparation, characterized in that, The microbial agent or preparation include: Live cells, inactivated cells, metabolites, fermentation broth, or bacterial suspension of Bifidobacterium lactis subsp. BGI-L99 as described in claim 1 or 2.

9. A composition, characterized in that, include: Live cells, inactivated cells, metabolites, fermentation broth, or bacterial suspension of Bifidobacterium lactis subsp. BGI-L99 as described in claim 1 or 2.

10. The composition according to claim 9, characterized in that, The composition includes: a food composition and a pharmaceutical composition; Optionally, the composition further includes at least one of probiotics, prebiotics, and vitamins.

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