Application of bifidobacterium animalis subsp. Lactis in male reproductive aging resistance

Abstract

The invention firstly provides a bifidobacterium animalis subsp. Lactis HX-BA7357 with strong exopolysaccharide production capacity, and belongs to the technical field of microorganisms, the preservation number of the bifidobacterium animalis subsp. Lactis HX-BA7357 is CGMCC (China General Microbiological Culture Collection Center) No.32959, and the bifidobacterium animalis subsp. Lactis HX-BA7357 is preserved in China General Microbiological Culture Collection Center on December 6, 2024. In addition, the invention further discloses a preparation method of the exosome of the animal bifidobacterium subsp. Lactis HX-BA7357 and application of the exosome of the animal bifidobacterium subsp. Lactis HX-BA7357. Compared with the yield before optimization, the yield of the exosome preparation method provided by the invention is increased by 48.91%, the yield of exopolysaccharides is increased by 30.48%, and meanwhile, the bifidobacterium animalis subsp. Lactis HX-BA7357 and the exosome thereof provided by the invention have an improvement effect on reproductive function decline caused by aging of male mice, and have a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of probiotics, and specifically relates to an application of animal Bifidobacterium lactis subspecies and its exosomes in male reproductive anti-aging. Background Art

[0002] Aging is the progressive decline in sexual function that occurs with aging, and its mechanisms involve multi-layered biological imbalances. Aging manifests itself in decreased physiological reserve, imbalanced metabolic homeostasis, diminished adaptability, and increased disease susceptibility. These changes are regulated by genetic, environmental, and stochastic factors and are essentially the cumulative effects of dynamic imbalances in complex networks. Similarly, male reproductive aging is the progressive decline in reproductive system function that occurs with aging. Its mechanisms and manifestations involve the interaction of multiple factors at the endocrine, cellular, and molecular levels, manifesting as decreased hormone levels, reduced sperm quality, and decreased sexual function. Exosomes are extracellular vesicles ranging in size from 30 to 200 nm, encapsulated by a lipid bilayer membrane. They carry bioactive substances such as proteins, nucleic acids, lipids, and metabolites, and serve as key mediators of intercellular communication. They are widely distributed in both eukaryotes and prokaryotes. Their inherent biocompatibility, low immunogenicity, and efficient barrier-crossing capabilities hold significant potential for applications in regenerative medicine, tumor therapy, and intervention for neurodegenerative diseases. Escherichia coli and lactic acid bacteria are the most widely used strains of bacterial exosomes. The core advantage of using strains to prepare exosomes is low cost and efficient production. Not only are gene-directed modifications convenient, but they also have natural immune activation properties. Their high stability and tolerance make them more suitable for oral delivery.

[0003] With the advancement of science and technology, the application of exosomes in various human diseases has also begun to increase. The invention patent with publication number CN119144523A discloses an exosome of Enterococcus fulvida and explores the application of the exosome in the treatment of myocarditis. It can significantly reduce the inflammation level of myocardial cells under pathological conditions and maintain myocardial cell homeostasis; the invention patent with publication number CN119552782A discloses an exosome of Lactobacillus plantarum LP35 with antioxidant activity, and its exosomes can have a significant inhibitory effect on ferroptosis. Summary of the Invention

[0004] One of the objects of the present invention is to provide a strain of animal Bifidobacterium lactis subspecies HX-BA7357 with high extracellular polysaccharide production, wherein the animal Bifidobacterium lactis subspecies HX-BA7357 was deposited in the General Microbiology Center of the China Culture Collection Administration on December 6, 2024, with a deposit number of CGMCC No.32959; the complete 16SrDNA sequence of the strain is as shown in SEQ ID No: 1.

[0005] The second object of the present invention is to provide an application of animal Bifidobacterium lactis subspecies HX-BA7357 or a probiotic preparation thereof in the preparation of a medicine or health product for improving male reproductive aging.

[0006] The third object of the present invention is to provide a method for preparing the probiotic preparation.

[0007] The present invention is achieved through the following technical solutions:

[0008] The invention relates to Bifidobacterium animalis subsp. lactis HX-BA7357, whose preservation number is CGMCC No.32959.

[0009] An exosome of Bifidobacterium animalis subsp. lactis.

[0010] A probiotic preparation comprising the bacterial slurry of Bifidobacterium animalis subsp. lactis according to claim 1, the exosomes of Bifidobacterium animalis subsp. lactis according to claim 2, and a freeze-drying protective agent.

[0011] The freeze-drying protective agent comprises 15% skim milk powder, 8% trehalose, 5% sucrose, 3% sodium glutamate, 0.2% ascorbic acid, 0.1% Tween 80 (liquid), and the balance is sterile PBS solution.

[0012] The preparation method of the bacterial mud comprises the following steps:

[0013] Inoculate animal Bifidobacterium lactis subspecies into TYP medium, ferment at 37°C, and centrifuge after the fermentation is completed to obtain the product.

[0014] The inoculation amount of the inoculation is 4%;

[0015] The culture time is 36h;

[0016] The centrifugal force of the centrifugation is 4000g; the centrifugation time is 10 minutes;

[0017] The TPY liquid culture medium includes 10.0 g / L casein peptone, 5.0 g / L phytone, 2.5 g / L yeast extract, 5.0 g / L glucose, 0.5 g / L L-cysteine hydrochloride, 2.0 g / L dipotassium hydrogen phosphate, 0.1 g / L magnesium sulfate, 2.0 g / L sodium chloride, 1.0 mL / L Tween 80 (liquid), 50 mg / L mupirocin lithium, 10.0 g / L lactose, and the balance is water.

[0018] The TPY liquid culture medium also includes 0.3 wt% of apigenin.

[0019] A use of the animal Bifidobacterium lactis subspecies in the preparation of food, medicine or health care products for resisting male reproductive aging. A use of the animal Bifidobacterium lactis subspecies in the preparation of food, medicine or health care products for resisting male reproductive aging.

[0020] A use of the probiotic preparation in preparing food, medicine or health care product for resisting male reproductive aging.

[0021] The beneficial effects of the present invention are:

[0022] 1. The present invention isolated a strain of Bifidobacterium animalis subsp. lactis HX-BA7357 from the feces of newborn infants. Studies have found that the strain has a strong ability to produce exopolysaccharides, and has good acid and bile resistance and antibacterial ability and other beneficial functions; 2. The exosome particle size distribution of the Bifidobacterium animalis subsp. lactis HX-BA7357 described in the present invention is concentrated in the range of 100nm-200nm, and the exosome concentration is 9.2×10 10 / mL;

[0023] 3. The present invention screened apigenin from numerous plant-derived factors as an exogenous substance that promotes exosome secretion in HX-BA7357. Adding 0.3% apigenin to the culture medium promoted the secretion of HX-BA7357 exopolysaccharides and exosomes by 30.48% and 48.91%, respectively, compared to the unadded group. This effect was significantly superior to that of hydrogen peroxide.

[0024] 4. The animal Bifidobacterium lactis subspecies HX-BA7357 and its exosomes described in the present invention have a significant improvement effect on the reproductive aging of male mice caused by D-galactose, and improve the sperm motility and oxidative stress resistance level of mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Transmission electron micrographs of exosomes of Bifidobacterium animalis subsp. lactis HX-BA7357 are shown;

[0026] Figure 2 The nanoparticle size distribution of exosomes of Bifidobacterium animalis subsp. lactis HX-BA7357 is shown;

[0027] Figure 3 Shows the effects of different substances on the growth curve of Bifidobacterium animalis subsp. lactis HX-BA7357;

[0028] Figure 4 The effects of different substances on the exopolysaccharide content of Bifidobacterium animalis subsp. lactis HX-BA7357 are shown;

[0029] Figure 5Shown are the effects of different concentrations of apigenin on the growth of Bifidobacterium animalis subsp. lactis HX-BA7357;

[0030] Figure 6 The effect of different concentrations of apigenin on the exopolysaccharide content of Bifidobacterium animalis subsp. lactis HX-BA7357 is shown;

[0031] Figure 7 Transmission electron micrographs of HX-BA7357 exosomes secreted by the strain after apigenin induction are shown;

[0032] Figure 8 The effect of H2O2 on the growth of Bifidobacterium animalis subsp. lactis HX-BA7357 is shown;

[0033] Figure 9 Transmission electron micrographs of HX-BA7357 exosomes secreted by the strain after H2O2 induction are shown. DETAILED DESCRIPTION

[0034] The present invention is further described below with reference to the accompanying drawings and examples. The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0035] Exosome production in the prior art is primarily based on Gram-negative bacteria, such as Escherichia coli, and some Gram-positive strains, such as Bacillus subtilis. These strains produce high levels of exopolysaccharides, which facilitate exosome secretion. Based on this, the present invention screened a high-exopolysaccharide-producing strain of Bifidobacterium animalis subsp. lactis to investigate its exosome secretion capacity and the ameliorative effects of exosomes on reproductive aging in mice.

[0036] The experimental materials involved in the following examples are as follows:

[0037] Bifidobacterium animalis subsp. lactis HX-BA7357 was isolated from the intestines of healthy infants and stored frozen at -80°C in glycerol tubes. Generally, this bacterium is inoculated onto the surface of a modified TPY solid medium plate and incubated upside down in a 37°C anaerobic incubator for 24 hours to obtain colonies. A single colony is then selected and incubated in modified TPY liquid medium for 18 hours to obtain a fermentation broth. The following culture media / lyoprotectants are sterilized at 121°C for 15 minutes.

[0038] Components of the modified TYP medium: casein peptone 10.0 g / L, phytone peptone 5.0 g / L, yeast extract 2.5 g / L, glucose 5.0 g / L, L-cysteine hydrochloride 0.5 g / L, dipotassium hydrogen phosphate 2.0 g / L, magnesium sulfate 0.1 g / L, sodium chloride 2.0 g / L, Tween 80 (liquid) 1.0 mL / L, mupirocin lithium 50 mg / L, lactose 10.0 g / L, and the balance is water. For solid culture medium, 15.0 g / L of agar is added.

[0039] Lyophilization protective agent components: skim milk powder 15%, trehalose 8%, sucrose 5%, sodium glutamate 3%, ascorbic acid 0.2%, Tween 80 (liquid) 0.1%, and the balance is sterile PBS solution.

[0040] Example 1 Isolation and Identification of Bifidobacterium animalis subspecies lactis HX-BA7357

[0041] Weigh 0.1g of fresh infant feces collected, add 0.9mL of 60% glycerol, vortex for 1-3min, centrifuge at 1000rpm for 5min, and then draw the supernatant into a sterile centrifuge tube. Centrifuge the supernatant at 6000rpm for 10min, discard the supernatant, and resuspend in physiological saline to prepare a fecal suspension. Inoculate the fecal suspension into TPY liquid culture medium at a ratio of 2%, and enrich and culture at 37℃ for 48h. Dilute the fecal suspension after enrichment culture to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 After vortexing and mixing, 100 μL of the dilution was spread onto the surface of modified TPY solid medium and incubated anaerobically at 37°C for 48 hours. Single colonies with regular milky white edges were inoculated onto the surface of TPY solid medium and repeatedly streaked for purification. Pure bacteria were obtained after incubation at 37°C for 24 hours. Two strains of Bifidobacterium were identified in the initial screening and were identified as Bifidobacterium animalis subsp. lactis and Bifidobacterium animalis subsp. animalis. The probiotic activities of the two strains were compared, and the results are shown in Table 1.

[0042] Table 1 Determination of probiotic function of strains

[0043]

[0044] The fermentation supernatants of the two strains were collected, and the polysaccharide content in the supernatants was determined using the phenol-sulfuric acid method. The results are shown in Table 2. Obviously, the exopolysaccharide content of FY96T7 (HX-BA7357) was higher than that of FY108T12, so FY96T7 (HX-BA7357) was selected for exosome extraction in subsequent experiments.

[0045] Table 2 Extracellular polysaccharide production of strains

[0046] strain Extracellular polysaccharide mg / L FY96T7(HX-BA7357) 49.18 FY108T12 28.46

[0047] The strain was sequenced using 16S rDNA universal primers, and the gene sequence of Bifidobacterium animalis subsp. lactis HX-BA7357 was shown in SEQ ID No: 1.

[0048] SEQ ID No: 1

[0049] CTCAGGATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGGGATCCCTGGCAGCTTGCTGTCGGG

[0050] GTGAGAGTGGCGAACGGGTGAGTAATGCGTGACCAACCTGCCCTGTGCACCGGAATAGCTCCTGGAAACG

[0051] GGTGGTAATACCGGATGCTCCGCTCCATCGCATGGTGGGGTGGGAAATGCTTTTTGCGGCATGGGATGGGGT

[0052] CGCGTCCTATCAGCTTGTTGGCGGGGTGATGGCCCACCAAGGCGTTGACGGGTAGCCGGCCTGAGAGGGT

[0053] GACCGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAA

[0054] TGGGCGCAAGCCTGATGCAGCGACGCCGCGTGCGGGATGGAGGCCTTCGGGTTGTAAACCGCTTTTGTTC

[0055] AAGGGCAAGGCACGGTTTCGGCCGTGTTGAGTGGATTGTTCGAATAAGCACCGGCTAACTACGTGCCAGCA

[0056] GCCGCGGTAATACGTAGGGTGCGAGCGTTATCCGGATTTATTGGGCGTAAAGGGCTCGTAGGCGGTTCGTC

[0057] GCGTCCGGTGTGAAAGTCCATCGCCTAACGGTGGATCTGCGCCGGGTACGGGCGGGCTGGAGTGCGGTAG

[0058] GGGAGACTGGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAACACCAATGGCGAAGGCAG

[0059] GTCTCTGGGCCGTCACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGT

[0060] CCACGCCGTAAACGGTGGATGCTGGATGTGGGGCCCTTTCCACGGGTCCCGTGTCGGAGCCAACGCGTTAA

[0061] GCATCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGG

[0062] CGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTGCCGGATCGCCGTG

[0063] GAGACACGGTTTCCCTTCGGGGCCGGTTCACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGT

[0064] TGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCGCATGTTGCCAGCGGGTGATGCCGGGAACTCATGTGG

[0065] GACCGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAGATCATCATGCCCCTTACGTCCAGGGCTTC

[0066] ACGCATGCTACAATGGCCGGTACAACGCGGTGCGACACGGTGACGTGGGGCGGATCGCTGAAAACCGGTC

[0067] TCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGGCGGAGTCGCTAGTAATCGCGGATCAGCAACGC

[0068] CGCGGTGAATGCGTTCCCGGGCCTTGT

[0069] Example 2 Isolation and Identification of Exosomes from Bifidobacterium animalis subsp. lactis HX-BA7357

[0070] 4% Bifidobacterium animalis subsp. lactis HX-BA7357 was inoculated into TPY liquid medium and incubated anaerobically at 37°C for 24 hours. After incubation, the supernatant was collected by centrifugation at 300g for 10 minutes, filtered through a 0.22μm filter, and supplemented with 1mM PMSF and 0.01% Triton X-100. Exosomes were collected from the supernatant using differential centrifugation at 4°C. First, broken bacterial fragments were removed by centrifugation at 2000g for 10 minutes. The supernatant was then centrifuged at 10,000g for 30 minutes to remove the precipitate, and the supernatant was collected again. Finally, the supernatant collected in the previous step was centrifuged at 100,000g for 90 minutes to precipitate the exosomes. The collected exosome pellet was resuspended in sterile PBS and ultrapurified again by centrifugation at 100,000g for 60 minutes. Finally, the exosome pellet was brought to 1mL with sterile PBS to prepare the exosome solution of Bifidobacterium animalis subsp. lactis HX-BA7357. The process is shown in the table below:

[0071] step Centrifugal force (g) Time (min) Collection Low-speed centrifugation 300 10 supernatant Low-speed centrifugation 2000 10 supernatant High-speed centrifugation 10000 30 supernatant Ultracentrifugation (collection) 100000 90 precipitation Ultracentrifugation (purification) 100000 60 precipitation

[0072] Transmission electron microscopy image analysis and nanoparticle tracking analysis were performed to determine its morphology, purity and particle size distribution. Figure 1 and Figure 2 As shown, the purity is high, the particle size is 100nm-200nm, and the exosome concentration is 9.2×10 10 pieces / mL.

[0073] After 24 hours of culture, the strain was shaken and mixed to determine the free radical scavenging ability of Bifidobacterium animalis subsp. lactis HX-BA7357 and its exosomes. The results, shown in Table 3, indicate that both HX-BA7357 and its exosomes possess strong antioxidant capacity, potentially ameliorating aging-related effects caused by oxidative stress.

[0074] Table 3 Antioxidant capacity of HX-BA7357 and its exosomes

[0075]

[0076] Example 3 Apigenin induces exosome secretion of Bifidobacterium animalis subsp. lactis HX-BA7357

[0077] The use of plant-derived factors to promote the secretion of exosomes by bacterial strains has been an emerging direction in biotechnology and medical research in recent years. Plant-derived factors generally refer to plant extracts (such as polyphenols, polysaccharides, terpenoids, etc.) or plant-derived signaling molecules. They have low toxicity and good biocompatibility, making them suitable for use in biomedical fields (such as drug delivery and regenerative medicine), avoiding the side effects that may be caused by chemically synthesized substances. In addition, some plant factors (such as curcumin and resveratrol) have anti-inflammatory or antioxidant properties, which may indirectly enhance the functionality of exosomes by changing the physiological state of the strain.

[0078] Currently, there are not many plant exogenous substances that can stimulate cells to produce exosomes. The commonly used method in research is to use some irritating substances (such as H2O2, EDTA and SDS) during the culture process to promote cell lysis and produce exosomes. This method may change the immunogenicity, drug delivery efficiency or virulence factor activity of exosomes. In the early stage of the present invention, apigenin was screened from curcumin, ginsenosides, apigenin, tea polyphenols and quercetin and found that apigenin can promote the secretion of extracellular polysaccharides of animal Bifidobacterium lactis subspecies HX-BA7357. Therefore, the strain culture intervened with apigenin was used to explore its effect on the secretion of exosomes of HX-BA7357. First, a TPY liquid culture medium containing 0.05% curcumin, ginsenosides, apigenin, tea polyphenols and quercetin was prepared to observe the effect of the culture medium on the growth of the strain and the production of extracellular polysaccharides. The results are as follows. Figure 3 and Figure 4 As shown in the results, apigenin had little effect on the growth of animal Bifidobacterium lactis subsp. HX-BA7357, did not inhibit the growth of the strain, and increased the secretion content of its extracellular polysaccharide. After preliminary screening, TYP culture medium containing 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, and 1.0% apigenin was prepared, and the results were as follows: Figure 5 and Figure 6 As shown in Figure 2, adding 0.3% apigenin to the culture medium can promote the good growth of the strain, and the strain's extracellular polysaccharide production is also increased to 58.17 mg / L, an increase of 30.48% compared to before addition. The exosomes of HX-BA7357 were extracted and measured according to the method in Example 1, and the electron microscopy results are shown in Figure 2. Figure 7 As shown, the exosome concentration was 1.37×10 11 / mL, an increase of 48.91% compared with not using apigenin.

[0079] Comparative Example 1 0.2 mM H2O2 induces exosome secretion of animal Bifidobacterium lactis subsp. HX-BA7357

[0080] Although using H2O2 as an inducer to promote bacterial exosome secretion can increase bacterial vesicle production through the oxidative stress mechanism, the reactive oxygen species produced by oxidative stress during the induction process will increase the permeability of the cell membrane and may directly lyse the bacteria, which will reduce the total production of exosomes and even change the metabolic state of the bacteria, affecting the natural composition and function of the exosomes. The method mentioned in the reference of the present invention adds 0.2mM H2O2 to the culture medium to stimulate the antioxidant activity of the strain and increase the permeability of the cell membrane of the strain. The HX-BA7357 exosomes are extracted and measured according to the method in the above example. The electron microscopy results are shown as follows: Figure 9 As shown, the exosome concentration was 1.19×10 11 / mL, which was 13.14% lower than that of apigenin. In addition, the cell morphology of the strain showed lysis and fragmentation under the stimulation of H2O2, resulting in the outflow of contents, which seriously affected the growth of the strain.

[0081] The above results indicate that the use of 0.3% apigenin to induce HX-BA7357 exosome secretion is better than that of 0.2 mM H2O2.

[0082] Example 4 Preparation of Live Bacterial Powder of Bifidobacterium animalis subspecies lactis HX-BA7357

[0083] This example provides a method for preparing live bacterial powder of animal Bifidobacterium lactis subsp. lactis HX-BA7357. HX-BA7357 was inoculated into a modified TYP medium at a 4% inoculum, fermented at 37°C for 36 h, and centrifuged at 4000 g for 10 min after the fermentation to collect the bacterial sludge. The collected bacterial sludge was placed in a 50 mL centrifuge tube under sterile conditions, a lyophilization protectant was added at a ratio of 1:1, and the powder was lyophilized to obtain a live bacterial count of 1.2 × 10 12 CFU / g.

[0084] The components of the lyoprotectant are as described above.

[0085] Example 5 Effects of Bifidobacterium lactis subsp. lactis HX-BA7357 and its exosomes on the improvement of reproductive aging in male mice (1) Mouse husbandry: 40 8-week-old male C57BL / 6J mice were randomly divided into 4 groups (n=10) after adaptive feeding for one week. (2) Mouse grouping: ① Normal group (Group C): Oral administration of sterile water every day; ② Model group (Group M): Subcutaneous injection of D-galactose 150 mg / kg BW / d was performed on the back of the mice for 21 consecutive days to establish a subacute aging model in mice. After the model was successfully established, the culture was carried out in the same manner as in Group C; ③ Probiotic preparation group (Group BA): Oral administration of HX-BA7357 every day after modeling, with the number of viable bacteria being no less than 1×10 9CFU; ④ HX-BA7357 exosome group (BAE group): After modeling, 200 μL of HX-BA7357 exosome solution was gavage daily. The experimental period lasted 28 days. Signs of successful aging in mice included weight loss, decreased food intake, slowed movement, and dry, dull fur. After the experimental period, the mice were anesthetized with sodium pentobarbital. After semen was removed, blood was collected from the eyeballs, and back fat was removed, and both kidneys were intact.

[0086] (3) Effects of HX-BA7357 and its exosomes on basic indicators of aging mice

[0087] After 28 days of intervention, mice in the M group showed obvious signs of aging, including thinning and dry hair, listlessness, and a significant decrease in food intake. However, mice in the BA and BAE groups showed significant improvement in their physical appearance, with adverse physical signs effectively alleviated.

[0088] Table 4 Effects of HX-BA7357 exosomes on basic indicators of aging mice

[0089] Grouping Weight / g Average food intake / g / d Renal index / mg / g C 32.56 1.46 8.75 M 27.46 1.17 6.12 BA 30.63 1.31 7.34 BAE 31.54 1.38 7.65

[0090] (4) Effects of HX-BA7357 and its exosomes on sperm quality in aging mice

[0091] After the experiment, mice were anesthetized with sodium pentobarbital, and semen samples were obtained from their epididymal tissue. Semen was incubated at 37°C for 15 minutes, diluted 10-fold with saline, and 10 μL was aspirated onto a hemocytometer. Sperm motility and morphology were observed, and sperm density, total sperm motility, and sperm deformity rate were recorded. The results showed that both HX-BA7357 and its exosomes significantly improved sperm status in mice with kidney yang deficiency, increasing sperm motility and reducing sperm deformity rates, thereby improving sperm quality.

[0092] Table 5 Effects of HX-BA7357 and its exosomes on sperm quality in aging mice

[0093] Grouping <![CDATA[Sperm density 10 6 / mL]]> Total sperm motility% Sperm deformity rate% C 3.44 78.96 22.32 M 1.11 44.845 44.46 BA 2.73 60.81 32.38 BAE 2.99 64.309 29.82

[0094] (5) Effects of HX-BA7357 and its exosomes on reproductive function in aging mice

[0095] Oxidative stress is a damaging state caused by an imbalance in the production and clearance of reactive oxygen species in the body. Its mechanisms in male reproductive aging include sperm damage, testicular dysfunction, and weakened antioxidant defenses. Exosomes may intervene in oxidative stress through multiple targets, including antioxidant, anti-inflammatory, and pro-repair, thereby improving reproductive aging caused by oxidative stress. A kit was used to detect the levels of key oxidative stress indicators in mouse kidney samples. The results, shown in Table 6, showed that both HX-BA7357 and its exosomes significantly reduced the levels of inflammatory factors and MDA content in the kidney tissue of aged mice, and significantly increased the activity of antioxidant enzymes in the tissue and the levels of sex hormones in the serum. This alleviated the body's oxidative stress and hyperinflammatory state, improved the decline in reproductive function in mice, and mitigated reproductive aging caused by inflammation and oxidative stress.

[0096] Table 6 Effects of Bifidobacterium animalis subsp. lactis HX-BA7357 and Cistanche deserticola fermentation product on reproductive function in mice with kidney yang deficiency

[0097]

[0098] In summary, the present invention provides a strain of Bifidobacterium animalis subspecies lactis HX-BA7357 isolated and identified from the feces of healthy newborn babies. At the same time, the present invention characterizes and analyzes the exosomes of Bifidobacterium animalis subspecies lactis HX-BA7357. Moreover, the present invention also provides a method for promoting the secretion of exosomes of Bifidobacterium animalis subspecies lactis HX-BA7357. This method can significantly increase the production of exopolysaccharides and exosomes of Bifidobacterium animalis subspecies lactis HX-BA7357 by adding 0.3% apigenin to the culture medium. Finally, the present invention verifies the improvement effect of Bifidobacterium animalis subspecies lactis HX-BA7357 and its exosomes on reproductive dysfunction in male aging mice.

Claims

1. A Bifidobacterium animalis subsp. lactis HX-BA7357, characterized by: Its accession number is CGMCC No.32959.

2. An exosome of Bifidobacterium animalis subsp. lactis as claimed in claim 1.

3. A probiotic preparation, characterized in that: The invention comprises the bacterial mud of animal Bifidobacterium lactis subspecies according to claim 1, the exosomes of animal Bifidobacterium lactis subspecies according to claim 2, and a freeze-drying protective agent.

4. The probiotic preparation according to claim 3, characterized in that The freeze-drying protective agent comprises 15% skim milk powder, 8% trehalose, 5% sucrose, 3% sodium glutamate, 0.2% ascorbic acid, 0.1% Tween 80 (liquid), and the balance is sterile PBS solution.

5. The probiotic preparation according to claim 3, wherein: The preparation method of the bacterial mud comprises the following steps: Inoculate animal Bifidobacterium lactis subspecies into TYP medium, ferment at 37°C, and centrifuge after the fermentation is completed to obtain the product.

6. The probiotic preparation according to claim 3, wherein: The inoculation amount of the inoculation is 4%; The culture time is 36h; The centrifugal force of the centrifugation is 4000g; the centrifugation time is 10 minutes; The TPY liquid culture medium includes 10.0 g / L casein peptone, 5.0 g / L phytone, 2.5 g / L yeast extract, 5.0 g / L glucose, 0.5 g / L L-cysteine hydrochloride, 2.0 g / L dipotassium hydrogen phosphate, 0.1 g / L magnesium sulfate, 2.0 g / L sodium chloride, 1.0 mL / L Tween 80 (liquid), 50 mg / L mupirocin lithium, 10.0 g / L lactose, and the balance is water.

7. The probiotic preparation according to claim 6, wherein: The TPY liquid culture medium also includes 0.3 wt% of apigenin.

8. Use of the animal Bifidobacterium lactis subspecies according to claim 1 in the preparation of food, medicine or health care product for resisting male reproductive aging.

9. Use of the exosomes of animal Bifidobacterium lactis subsp. lactis as claimed in claim 2 in the preparation of food, medicine or health care product for resisting male reproductive aging.

10. Use of the probiotic preparation according to claim 3 in the preparation of food, medicine or health care product for resisting male reproductive aging.

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