Probiotic fermentation compound with cell repairing and cell senescence delaying functions and application of probiotic fermentation compound

Abstract

The invention provides a bifidobacterium animalis subspecies HX-BA21, and the preservation number of the bifidobacterium animalis subspecies HX-BA21 is CGMCC (China General Microbiological Culture Collection Center) NO.32958. The invention discloses bifidobacterium animalis subspecies HX-BA21 containing exosome metabiotics and an exosome-containing fermentation compound obtained by fermenting gastrodia elata through the HX-BA21. The fermentation compound comprises a probiotic protective layer (a core layer), an exosome delivery layer (a shell layer) and a prebiotics sustained release layer (a cocoon layer) from inside to outside. The probiotic protection layer comprises a sodium alginate-milk fat globule membrane compound and animal bifidobacterium subsp.animalis; the exosome delivery layer comprises a gelatin-chitosan compound and a gastrodia elata and probiotic mixed exosome; the prebiotic sustained-release layer comprises an octenyl succinic anhydride starch / polyvinyl alcohol compound and inulin. The compound can start cell repair by improving the cell migration rate, up-regulate DNA helicase and telomere related gene expression, delay premature senescence and delay cell senescence caused by D-galactose by adjusting the oxidative stress level.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms and biomedicine, and specifically to the preparation of exosomes containing probiotics and Gastrodia elata, and in particular to the preparation of a nanofiber fermentation complex containing probiotics and Gastrodia elata exosomes and its application in cell repair. Background Art

[0002] Exosomes, nanosized vesicles (30-150 nm) secreted by cells, carry bioactive molecules such as proteins, nucleic acids, and lipids, playing a central role in intercellular signaling. Recent studies have found that exosomes derived from probiotics and medicinal foods exhibit unique anti-aging potential. Exosomes from probiotics are often postbiotics.

[0003] Current technologies for enhancing exosome stability primarily include chemical modification, freeze-drying, and nanocarrier encapsulation, but these still present significant drawbacks. Chemical modification (such as targeting peptide grafting or lipid insertion) can alter the surface charge of exosomes, leading to decreased membrane fluidity and increased immunogenicity. Traditional freeze-drying processes induce exosome aggregation, requiring long-term storage at -80°C even with the addition of protective agents such as trehalose. Liposome or polymer nanoparticle encapsulation technologies, however, offer less than 30% encapsulation efficiency and suffer from burst release and poor interfacial compatibility, making controlled sustained release difficult to achieve.

[0004] To address these technical bottlenecks, the field urgently needs to develop an exosome delivery system that combines protective and synergistic functions. Existing research suggests that nanofiber materials, due to their high surface area, tunable porosity, and biomimetic extracellular matrix structure, hold significant potential for bioactive substance loading. However, conventional nanofiber membranes rely on a single pore size for physical encapsulation of exosomes, which cannot withstand the enzymatic and oxidative stresses of complex physiological environments. Furthermore, they lack kinetic design for the coordinated release of exosomes from multiple sources, limiting their anti-aging effects.

[0005] Therefore, there is an urgent need for a new exosome delivery system that can not only protect exosomes from the influence of the external environment but also achieve long-term sustained release to improve their practical application effects in cell repair and anti-aging.

[0006] Chinese patent application CN111225659B discloses an anti-aging composition containing extracellular vesicles (EVs) derived from lactic acid bacteria. The composition contains EVs as active ingredients, which are physiologically active compounds derived from lactic acid bacteria. The EVs can be exosome-like vesicles with a diameter of 20 to 200 nm. The composition inhibits the expression of MMP-1 protein, thereby improving skin wrinkles, increasing skin elasticity, inhibiting collagen loss, and preventing UV-induced skin damage.

[0007] Chinese patent application publication number CN118615187A discloses an anti-aging composition, cosmetic preparation, and preparation method containing limonin. The anti-aging composition comprises the following components by weight: 20-25 parts of a first exosome encapsulated with limonin, 50-60 parts of a second exosome encapsulated with probiotic powder, and 20-25 parts of a third exosome encapsulated with a prebiotic. This application discloses an anti-aging composition containing limonin, encapsulating the limonin within the exosomes and synergizing with the probiotic powder and prebiotics to effectively alleviate skin aging. However, the application fails to consider the issue of cellular aging. Summary of the Invention

[0008] The first object of the present invention is to provide a strain of Bifidobacterium animalis subspecies animalis HX-BA21.

[0009] The second object of the present invention is to provide exosomes prepared from Bifidobacterium animalis subspecies animalis HX-BA21.

[0010] The third object of the present invention is to provide a gastrodia elata exosome prepared by fermenting gastrodia elata with Bifidobacterium animalis subspecies HX-BA21.

[0011] The fourth object of the present invention is to provide a silkworm pupa-type nanofiber fermentation complex composed of probiotics and Gastrodia elata exosomes.

[0012] The present invention is achieved through the following technical solutions:

[0013] The present invention provides a strain of Bifidobacterium animalis subsp. animalis HX-BA21, whose Latin name is Bifidobacterium animalis subsp. animalis, which was deposited on December 6, 2024, at the General Microbiology Center of the China Culture Collection Administration, with the depository code being CGMCC. The depository address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, zip code 100101, and the deposit number is CGMCC No. 32958. The bacteria were alive at the time of deposit.

[0014] A probiotic containing exosomes and postbiotics is primarily isolated from the culture supernatant of HX-BA21 via ultracentrifugation. Transmission electron microscopy revealed the presence of HX-BA21 probiotic exosomes, with a high proportion of 62% being 112.5 nm. A Gastrodia elata containing exosomes is primarily obtained by fermenting Gastrodia elata with Bifidobacterium animalis subsp. animalis HX-BA21 and then isolating it using differential centrifugation and density centrifugation. The high proportion of exosomes is 137.5 nm, accounting for 67%.

[0015] A silkworm pupa-like nanofiber fermentation complex is prepared, comprising, from the inside out, a probiotic protective layer (core layer), an exosome delivery layer (shell layer), and a prebiotic sustained-release layer (cocoon layer). The probiotic protective layer comprises a sodium alginate-milk fat globule membrane complex and Bifidobacterium animalis subsp. animalis; the exosome delivery layer comprises a gelatin-chitosan complex and exosomes mixed with Gastrodia elata and probiotics; and the prebiotic sustained-release layer comprises an octenylsuccinic anhydride starch / polyvinyl alcohol complex and inulin.

[0016] The specific process mainly includes:

[0017] Spinning solution preparation

[0018] Nuclear layer (probiotic protective layer)

[0019] S1: Probiotic suspension preparation: Bifidobacterium animalis subsp. animalis HX-BA21 was inoculated at a 2% (v / v) inoculum into MRS liquid medium and incubated at 37°C for 20 hours for two consecutive generations to activate the strain. The culture was centrifuged at 6000 rpm for 5 minutes at 4°C to collect the cells. The cells were washed twice with sterile phosphate-buffered saline (PBS) and then resuspended in PBS to obtain a probiotic suspension with a viable count of 1 to 5 billion CFU / mL.

[0020] S2: Preparation of protective layer spinning solution: Weigh 8g of sodium alginate and 2g of milk fat globule membrane, add 125mL of deionized water to prepare a mixed solution with a concentration of 8% (w / v), stir at 95°C for 2h, and after the above solution is cooled to room temperature, add 2.0% of the total mass of the solution prepared by S1 and continue stirring for 30min to ensure that the bacterial solution is evenly dispersed to obtain a probiotic protective layer (nuclear layer).

[0021] Shell layer (exosome delivery layer)

[0022] S3: Preparation of exosome mixed solution: The probiotic exosomes prepared according to Example 1 and Example 2 were mixed with the Gastrodia elata exosomes at a volume ratio of 1:2 to obtain an exosome mixed solution.

[0023] S4: Preparation of delivery layer spinning solution: 7 g of gelatin and 3 g of chitosan were weighed and dissolved in 50 mL of deionized water and 50 mL of 1% (w / v) acetic acid solution, respectively. The two solutions were stirred at 150 r / min at room temperature for 30 min to form a gelatin-chitosan composite solution. 1%-3% exosome mixture was added to the composite solution and stirred evenly to obtain the shell layer (exosome delivery layer).

[0024] Cocoon layer (prebiotic sustained-release layer)

[0025] S5: Weigh 5 g of octenylsuccinic anhydride (OSA) starch and dissolve it in 30 mL of deionized water, and 5 g of polyvinyl alcohol (PVA) and dissolve it in 30 mL of deionized water. Mix the two and stir at 200 r / min and 95°C for 3 h until completely dissolved. After the above solution is cooled to room temperature, 2.0% (w / v) inulin is added and stirred continuously to obtain a cocoon layer (prebiotic sustained-release layer).

[0026] S6: adding the core layer spinning solution, the shell layer spinning solution and the cocoon layer spinning solution into a three-axis electrospinning device in a ratio of 1:1:1 for spinning, and forming a three-level embedding structure through three-axis electrospinning.

[0027] Finally, freeze-dry and you’re done.

[0028] The nanofiber fermentation composite according to claim 4, wherein the process parameters of the triaxial electrospinning are as follows:

[0029] The core layer spinning solution was spun through a needle with an inner diameter of 0.4 mm at a flow rate of 0.3 mL / h and a voltage of 10 kV;

[0030] The shell-layer spinning solution was spun through a 0.5 mm inner diameter needle at a flow rate of 0.5 mL / h and a voltage of 15 kV;

[0031] The cocoon layer spinning solution was spun through a needle with an inner diameter of 0.6 mm at a flow rate of 0.8 mL / h and a voltage of 20 kV;

[0032] The receiving device is a rotating drum covered with aluminum foil, the receiving distance is 9 cm, the spinning environment temperature is 20° C., and the humidity is 30%.

[0033] Freeze-drying and crushing treatment: The nanofiber membrane was pre-frozen at -80℃ for 2h and then transferred to a freeze dryer. The resulting freeze-dried membrane was gently ground in a sterile mortar and passed through an 80-mesh sieve to obtain a powder with a moisture content of ≤3% and a particle size distribution of D50 ≤ 50μm. Scanning electron microscopy observation (magnification 10000×) showed that the fiber structure integrity rate was ≥90%.

[0034] The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:

[0035] 1) Probiotics and Gastrodia elata exosomes enhance cell viability and activate cell repair by increasing cell migration rate. The fermentation complex (PG group) showed a more significant effect, with cell viability superior to that of the PQQ group, with a cell migration rate as high as 89.28%. 2) Probiotics, Gastrodia elata exosomes, and fermentation complex alleviated D-galactose-induced oxidative stress damage by reducing the proportion of SA-β-gal-positive cells, decreasing intracellular ROS fluorescence intensity, and increasing mitochondrial membrane potential, thereby delaying fibroblast aging.

[0036] 3) Probiotics, Gastrodia elata exosomes, and fermentation complexes can upregulate the expression of DNA helicases (WRN and BLM); by increasing the mRNA levels of telomere-related genes (TERT and TRF1), they promote the replication of telomeric DNA and thus delay the occurrence of premature aging.

[0037] 4) Fermentation supernatant and composite exosomes can maintain redox homeostasis and delay D-galactose-induced aging by increasing organ coefficient, serum SOD activity, GSH-Px, SOD and CAT activities in the liver and kidney, and reducing serum and liver MDA levels.

[0038] 8. Furthermore, the probiotics, Gastrodia elata exosomes, and fermentation complex have at least one of the following properties

[0039] Antioxidant

[0040] Cell Repair

[0041] Improve telomere replication

[0042] Increase DNA helicase expression

[0043] Delaying fibroblast senescence

[0044] Delaying aging caused by D-galactose BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 An electron micrograph of HX-BA21 exosomes is shown;

[0046] Figure 2 shows the particle size analysis of HX-BA21 exosomes;

[0047] Figure 3 An electron micrograph of Gastrodia elata exosomes is shown;

[0048] Figure 4 Particle size analysis of Gastrodia elata exosomes is shown. DETAILED DESCRIPTION

[0049] Example 1 Probiotic Performance and Exosome Extraction

[0050] ① Screening of amylase-producing strains

[0051] After the strain is activated, it is spread on an LB plate to obtain a single colony. A single colony is picked by toothpick inoculation and inoculated onto a starch hydrolysis medium. After aerobic culture at 37°C for 3 to 7 days, 1 to 2 drops of iodine solution are added to the surface of the medium. A transparent hydrolysis zone appears around the colony with the ability to produce amylase, and the size of the hydrolysis zone is measured.

[0052] ② Screening of cellulase-producing strains

[0053] The method for obtaining single colonies is the same as above. The colonies are inoculated onto sodium carboxymethyl cellulose plates by spot inoculation and cultured aerobically at 37°C for 24 hours. Transparent hydrolysis zones appear around the colonies that produce cellulase, and the sizes of the hydrolysis zones are measured.

[0054] HX-BA21 has excellent ability to produce amylase and cellulase, with Bifidobacterium animalis BB-12 as the control group. The specific performance is as follows:

[0055] Table 1 Comparison of enzyme production performance of different strains

[0056] strain Amylase production capacity Cellulase production capacity HX-BA21 15.25±1.25 16.28±1.21 BB-12 7.25±0.58 8.57±0.86

[0057] ③Isolation and identification of probiotic exosomes

[0058] Exosomes were isolated from the culture supernatant of HX-BA21 cells by ultracentrifugation.

[0059] Culture and collection: Bifidobacterium animalis subsp. animalis HX-BA21 was activated twice and inoculated into 1 L of MRS medium at a 2% inoculum volume.

[0060] Purification: The fermentation broth in the logarithmic growth phase was centrifuged at 7,000 g for 20 minutes, and the resulting supernatant was collected. The supernatant was then centrifuged at 20,000 g for 30 minutes and filtered through a 0.22 μm microporous filter. The filtered supernatant was centrifuged at 30,000 g for 1–2 hours at 4°C, discarded, and the precipitate suspended in PBS. The presence of exosomes was confirmed by transmission electron microscopy, and the size range of the exosomes was analyzed using nanoparticle tracking.

[0061] like Figure 1 As shown in the figure, transmission electron microscopy showed that HX-BA21 probiotic exosomes existed, and the particle size was 112.5 nm, accounting for a high proportion of 62%.

[0062] Based on the above results, the present invention identified the strain HX-BA21.

[0063] Example 2

[0064] The source of strain HX-BA21 was feces of a two-month-old infant.

[0065] The strain HX-BA21 was identified as Bifidobacterium animalis subsp. animalis.

[0066] The 16S sequence of the Bifidobacterium animalis subspecies HX-BA21 is as follows:

[0067] CTCAGGATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGGGATCCCTGGCA

[0068] GCTTGCTGTCGGGGTGAGAGTGGCGAACGGGTGAGTAATGCGTGACCAACCTGCCCT

[0069] GTGCACCGGAATAGCTCCTGGAAACGGGTGGTAATACCGGATGCTCCGCTCCATCGCAT

[0070] GGTGGGGTGGGAAATGCTTTTGCGGCATGGGATGGGGTCGCGTCCTATCAGCTTGTTG

[0071] GCGGGGTGATGGCCCACCAAGGCGTTGACGGGTAGCCGGCCTGAGAGGGTGACCGGC

[0072] CACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTG

[0073] CACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGCGGGATGGAGGCCTTCGGGT

[0074] TGTAAACCGCTTTTGTTCAAGGGCAAGGCACGGTTTCGGCCGTGTTGAGTGGATTGTT

[0075] CGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCG

[0076] TTATCCGGATTTATTGGGCGTAAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGTGTGAAA

[0077] GTCCATCGCCTAACGGTGGATCTGCGCCGGGTACGGGCGGGCTGGAGTGCGGTAGGGG

[0078] AGACTGGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAACACCAATGG

[0079] CGAAGGCAGGTCTCTGGGCCGTCACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGA

[0080] ACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGATGCTGGATGTGGGGCC

[0081] CTTTCCACGGGTCCCGTGTCGGAGCCAACGCGTTAAGCATCCCGCCTGGGGAGTACGG

[0082] CCGCAAGGCTAAAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGC

[0083] GGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTGCCGGATCGCC

[0084] GTGGAGACACGGTTTCCCTTCGGGGCCGGTTCACAGGTGGTGCATGGTCGTCGTCAGC

[0085] TCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCGCATGTTG

[0086] CCAGCGGGTGATGCCGGGAACTCATGTGGGACCGCCGGGGTCAACTCGGAGGAAGGT

[0087] GGGGATGACGTCAGATCATCATGCCCCTTACGTCCAGGGCTTCACGCATGCTACAATGG

[0088] ​​​​​​​​​​​​S1: 1000 g of Gastrodia elata from Xiaocaoba, Yiliang, Zhaotong, Yunnan was selected, washed and dried, and then ultrafinely ground (particle size < 50 μm), added with PBS buffer, and low-temperature extracted at 4°C for 12 h. The juice was pre-treated by juicing and wall-breaking to obtain wall-broken Gastrodia elata juice.

[0093] S2: Gastrodia elata was fermented using Bifidobacterium animalis subsp. animalis HX-BA21 (inoculation size was 3%, viable count was 1-5 billion CFU / mL) at a temperature of 37° C. for 24-48 h and a constant pH of 5.9-7.0.

[0094] S3: Exosomes were isolated by differential centrifugation at 700 g for 15 minutes, 3500 g for 15 minutes, and 12,000 g for 30 minutes. The supernatant was collected after gradient centrifugation and filtered through a 0.22 μm microporous filter. The filtered supernatant was placed in a fixed-angle rotor and centrifuged at 800,000 g for 1-2 hours. The supernatant was discarded and the pellet was suspended in PBS.

[0095] S4: The suspended liquid was further extracted by density centrifugation. The liquid was placed in 30%, 50%, and 70% sucrose solutions and centrifuged at 150,000 g for 2 hours. The middle layer of the 30-50% sucrose solution was collected, and an equal amount of PBS was added to wash away the sucrose. The solution was then centrifuged at 15,000 g for 1 hour. The precipitate was collected and resuspended in 1 mL of PBS to obtain the Gastrodia elata exosome solution.

[0096] S5: Transmission electron microscopy was used to characterize the presence of exosomes, and nanoparticle tracking was used to analyze the size range of exosomes.

[0097] Through transmission electron microscopy, Gastrodia elata exosomes were found to exist, with a particle size of 137.5 nm accounting for a high proportion of 67%.

[0098] The main components of probiotic exosomes include proteins, miRNA, mRNA and other nucleic acids and short-chain fatty acids, while Gastrodia elata exosomes are of plant origin, and most of their components are secondary metabolites (gastrodin, Gastrodia elata polysaccharides, etc.). Probiotic exosomes can clear extracellular ROS, and Gastrodia elata exosomes (especially gastrodin) can inhibit the excessive production of intracellular mitochondrial ROS. The two form a double extracellular-intracellular barrier. The combination of probiotics and Gastrodia elata exosomes may reduce the damage of oxidative stress to mitochondrial membrane potential, synergistically maintain the homeostasis of mitochondrial function, and thus repair cells and delay cell aging.

[0099] Due to the lack of stability and short half-life of exosomes, and the limited drug loading capacity of natural exosomes, it is difficult to meet the treatment needs. Therefore, the method of Example 4 is adopted to enhance the stability and sustained release effect of exosomes, providing physical protection and controlled release. The shell of the silkworm chrysalis fiber can encapsulate the exosomes, and the porous structure of the fiber allows the exosomes to be gradually released through diffusion or material degradation, prolonging the duration of action.

[0100] Example 4 Preparation of Silkworm Pupa Nanofiber Fermentation Composite

[0101] Spinning solution preparation

[0102] Core layer spinning solution (probiotic protective layer)

[0103] S1: Probiotic suspension preparation: Bifidobacterium animalis subsp. animalis HX-BA21 was inoculated at a 2% (v / v) inoculum into MRS liquid medium and incubated at 37°C for 20 hours for two consecutive generations to activate the strain. The culture was centrifuged at 6000 rpm for 5 minutes at 4°C to collect the cells. The cells were washed twice with sterile phosphate-buffered saline (PBS) and then resuspended in PBS to obtain a probiotic suspension with a viable count of 1 to 5 billion CFU / mL.

[0104] S2: Preparation of protective layer spinning solution: Weigh 8g of sodium alginate and 2g of milk fat globule membrane, add 125mL of deionized water to prepare a mixed solution with a concentration of 8% (w / v), stir at 95°C for 2h, and after the above solution is cooled to room temperature, add 2.0% of the total mass of the solution prepared by S1 and continue stirring for 30min to ensure that the bacterial solution is evenly dispersed to obtain a probiotic protective layer (nuclear layer).

[0105] Shell spinning solution (exosome delivery layer)

[0106] S3: Preparation of exosome mixed solution: The probiotic exosomes prepared according to Example 1 and Example 2 were mixed with the Gastrodia elata exosomes at a volume ratio of 1:2 to obtain an exosome mixed solution.

[0107] S4: Preparation of delivery layer spinning solution: 7 g of gelatin and 3 g of chitosan were weighed and dissolved in 50 mL of deionized water and 50 mL of 1% (w / v) acetic acid solution, respectively. The two solutions were stirred at 150 r / min at room temperature for 30 min to form a gelatin-chitosan composite solution. 1%-3% exosome mixture was added to the composite solution and stirred evenly to obtain the shell layer (exosome delivery layer).

[0108] Cocoon layer spinning solution (prebiotic sustained-release layer)

[0109] S5: Weigh 5 g of octenylsuccinic anhydride (OSA) starch and dissolve it in 30 mL of deionized water, and 5 g of polyvinyl alcohol (PVA) and dissolve it in 30 mL of deionized water. Mix the two and stir at 200 r / min and 95°C for 3 h until completely dissolved. After the above solution is cooled to room temperature, 2.0% (w / v) inulin is added and stirred continuously to obtain a cocoon layer (prebiotic sustained-release layer).

[0110] S6: The core layer spinning solution, shell layer spinning solution, and cocoon layer spinning solution were added to a triaxial electrospinning apparatus in a ratio of 1:1:1 for spinning. The triaxial electrospinning apparatus was as follows: the core layer spinning solution was spun through a 0.4 mm inner diameter needle at a flow rate of 0.3 mL / h and a voltage of 10 kV; the shell layer spinning solution was spun through a 0.5 mm inner diameter needle at a flow rate of 0.5 mL / h and a voltage of 15 kV; and the cocoon layer spinning solution was spun through a 0.6 mm inner diameter needle at a flow rate of 0.8 mL / h and a voltage of 20 kV. The receiving device was a rotating drum covered with aluminum foil with a receiving distance of 9 cm. The spinning environment temperature was 20°C and the humidity was 30%.

[0111] The nanofiber membrane collection and spinning process lasted for 6 hours, and the resulting nanofiber membrane was a silkworm pupa-like nanofiber membrane with a three-level encapsulation of probiotics (core)-exosomes (shell)-prebiotics (cocoon). The membrane thickness uniformity error was ≤5%, and the fiber diameter distribution was within the range of 100-300nm. Freeze-drying and pulverization treatment: The nanofiber membrane was pre-frozen at -80℃ for 2 hours and transferred to a freeze dryer. The resulting freeze-dried membrane was gently ground in a sterile mortar and passed through an 80-mesh sieve to obtain a powder with a moisture content of ≤3% and a particle size distribution D50 ≤50μm. Scanning electron microscopy (magnification 10,000×) showed a fiber structure integrity rate of ≥90%.

[0112] Example 5

[0113] Cell viability and wound repair assays were used to evaluate the effects of exosomes on fibroblasts.

[0114] ① Use the CCK-8 kit to assess cell proliferation. When the cell density reaches 5*105cell / m, prepare sample solutions with different concentration gradients, ranging from 30 to 100μg / mL. Add three replicates of each concentration to a 96-well plate. After incubation for 24 hours at 37°C, 5% CO2, and 90% humidity, calculate cell viability according to the CCK-8 kit method = [(experimental well - blank well) / (negative control well - blank well)] × 100%

[0115] Experimental wells (culture medium containing cells, CCK-8, and 3 groups of exosomes)

[0116] Negative control wells (containing cell culture medium, CCK-8, and no test substance)

[0117] Positive control wells (culture medium containing cells, CCK-8, positive control sample (pyrroloquinoline quinone disodium salt, PQQ group, 30 μg / mL)

[0118] Blank wells (medium without cells, test substances, or CCK-8)

[0119] ② A cell scratch assay was used to observe cell migration. Wild-type (WT) mouse embryonic fibroblasts (MEFs) were cultured in DMEM medium supplemented with 10% fetal bovine serum, 1% penicillin, and streptomycin until the logarithmic growth phase. When the cell density reached 5 × 105 cells / m, a scratch was made on the culture surface using a sterile cell scraper. The cells were gently rinsed with PBS to remove dead cells and impurities. The standard cell culture medium was replaced with serum-free medium to inhibit cell proliferation and promote migration. Immediately after the scratch, 30 μg / mL of probiotic exosomes (P group), Gastrodia elata exosomes (G group), and fermentation complex (PG group) were added. A control group received no drug treatment, and a positive control group received 30 μg / mL of PQQ. The initial scratch was observed and recorded under a microscope. The scratch was photographed 24 hours later. The cell migration rate was calculated by measuring the change in scratch width over time. The width (μm) and area (μm2) of the scratch area were measured using Image J software. The cell migration rate at 24 h was calculated by (0 h scratch area-24 h scratch area) / 0 h scratch area.

[0120] Table 2 Changes in cell proliferation and migration rates in different groups

[0121] Group Cell viability Cell migration rate (%) control group 100％ 59.28±0.58 Group P 108％ 72.52±2.59 Group G 115％ 75.89±3.24 PG Group 121％ 89.28±2.65 PQQ group 114％ 85.12±3.89

[0122] It can be seen that probiotics and Gastrodia elata exosomes have the effect of improving cell viability and can activate cell repair function by increasing cell migration rate. The effect of the complex (PG group) is more significant, with cell viability better than that of the PQQ group, and the cell migration rate is as high as 89.28%. When galactose (D-Gal) accumulates in large quantities in cells, it can induce changes in cell metabolism, produce a large amount of reactive oxygen species, cause oxidative stress in cells, and lead to cell aging. Therefore, in this example, D-galactose was used to act on fibroblasts to establish a cell aging model.

[0123] Example 6

[0124] To establish a D-galactose (D-Gal)-induced premature aging model: Fibroblasts were seeded in a 96-well plate and cultured in a cell culture incubator for 24 hours. The original cell culture medium was discarded, and the fibroblasts were pretreated with exosome culture medium for 24 hours. The exosome culture medium was discarded, the cells were washed once with PBS buffer, and the mixed conditioned medium containing exosomes and D-Gal was replaced and cultured for another 48 hours.

[0125] Exosome culture medium: Dilute to 30 μg / mL using complete culture medium. D-galactose conditioned medium: Dilute to 20 mg / mL using complete culture medium.

[0126] Exosomes + D-Gal mixed conditioned medium: Exosomes were diluted to 30 μg / mL using D-galactose medium to obtain mixed conditioned medium.

[0127] ①SA-β-gal activity index determination:

[0128] Cell inoculation and pretreatment: Skin fibroblasts were passaged and inoculated into 6-well plates and cultured for 24 hours. The groups were set as follows:

[0129] Modeling group: D-galactose group: cells were pretreated with 20 mg / mL D-galactose for 48 h;

[0130] Experimental groups: The corresponding exosomes (30 μg / mL) + D-Gal mixed culture medium was used for 48 h, including probiotic exosomes (P group), Gastrodia elata exosomes (G group) and nanofiber complex (PG group);

[0131] Negative control group: complete medium, no treatment, pretreatment of cells for 48 h;

[0132] Positive drug group: cells were treated with 30 μg / mL PQQ+D-Gal mixture for 48 h.

[0133] Remove the 6-well plate from the cell culture incubator and discard the original cell culture medium. Wash once with PBS buffer, add 1 mL of β-galactosidase staining fixative and let it stand at room temperature for 15 minutes. Then wash three times with PBS buffer for 3 minutes each time, 1 mL per well. Add 1 mL of cell senescence β-galactosidase (SA-β-gal) staining working solution to each well and stain at 37°C for 10 hours. Discard the staining working solution and add 1 mL of PBS buffer to each well. Observe and photograph the staining results under an optical microscope, look for senescent cells that show blue staining, and calculate the proportion of senescent cells (number of blue cells / total number of cells).

[0134] ② ROS content determination

[0135] Intracellular ROS levels were measured using the Beyotime Reactive Oxygen Species Reagent Kit and analyzed using a high-content imaging system. The DAPI channel was used to excite the Hochest 33342 nuclear dye, and the DCFH-DA channel was used to excite the oxidative stress dye.

[0136] ③Mitochondrial membrane potential (MMP)

[0137] Mitochondrial membrane potential was measured using the Beyotime Red mitochondrial fluorescent probe. Treated cells were placed on a high-content imaging system and scanned. Channels matching the fluorescent probe's specifications were selected, with 15 fields of view selected per well. A 20× objective lens with a 0.45mm aperture was used. Focus mode was selected, and data acquisition began after the image was clear. Image acquisition conditions: Hochest 33342 nuclear dye was excited by the DAPI channel, and the mitochondrial dye was excited by the Mito-Tracker Red CMXRos channel. Analysis was performed using a high-content imaging system.

[0138] Table 3 Quantitative analysis of SA-β-gal positive staining cells in fibroblasts

[0139]

[0140] Detecting SA-β-gal activity can be used to determine cell aging. Under normal physiological conditions, intracellular β-galactosidase functions under acidic conditions. When cells age, the permeability of the lysosomal membrane increases, allowing the activity of β-galactosidase to be detected at pH 6.0. The results showed that the proportion of SA-β-gal positively stained cells in the modeling group was significantly higher than that in the control group. However, pretreatment with exosomes before D-Gal treatment significantly reduced the proportion of SA-β-gal positive cells. This means that exosomes can delay fibroblast aging.

[0141] As ROS production and accumulation increase, senescent cells proliferate. Excessive production and accumulation of reactive oxygen species (ROS) disrupt the cellular antioxidant defense system, leading to oxidative stress and oxidative damage. D-Gal treatment induced cellular oxidative stress, resulting in elevated intracellular ROS levels. Pretreatment of cells with exosomes before D-Gal induction showed a decrease in intracellular ROS fluorescence intensity. The Gastrodia elata exosome and nanofiber complex group showed significantly better efficacy than the positive control group (PQQ group), indicating that exosomes can alleviate D-galactose-induced oxidative stress damage.

[0142] Mitochondria are essential for aerobic respiration and oxidative phosphorylation. Stable mitochondrial membrane potential is crucial for maintaining normal cellular function and delaying aging. Changes in mitochondrial membrane potential influence cellular energy metabolism, redox status, apoptosis, and calcium homeostasis through multiple pathways, thereby playing a crucial role in cellular and organismal aging. Mitochondrial membrane potential was significantly decreased in the model group. However, it gradually increased after mitochondrial intervention, indicating that exosomes significantly ameliorate the D-Gal-induced decrease in mitochondrial membrane potential.

[0143] With age, the activity of DNA helicase may decrease, resulting in the occurrence of telomeres during DNA replication. The main function of telomeres is to protect the ends of chromosomes, prevent fusion and degradation between chromosomes, and maintain chromosome stability. During cell division, telomeres gradually shorten due to end replication problems of DNA replication. When telomeres shorten to a certain extent, cells will enter a state of senescence or apoptosis, which is called the "telomere hypothesis." Furthermore, using embryonic fibroblasts (MEFs) of Werner progeria syndrome mice as a model, we observed whether probiotic exosomes, Gastrodia elata exosomes, and complexes would affect the expression of DNA helicase and telomere-related genes after intervening in progeria cells to affect aging.

[0144] Example 7

[0145] After modeling, 5 × 104 cells were plated per well in a six-well plate, and 30 μg / mL of the corresponding exosomes were added. After 48 hours, the cells in the six-well plate were collected, counted, and plated again at 5 × 104 cells per well, and exosomes were added at the same time. Similarly, the cells were passaged once every 48 hours for a total of 5 passages. The intervention was performed according to the grouping of Example 6, and the intervention time was 10 days. The cells of the last passage were collected, RNA was extracted, and the RNA was reverse transcribed into cDNA. Subsequently, the successfully reverse transcribed cDNA was subjected to fluorescent quantitative PCR reaction with primers for Blm, WRN, TERT, TRF1, and GAPDH. After the reaction procedure, the Ct value was obtained. Using GAPDH as the internal reference, the expression levels of Blm, Recql4, Mcm7, Parp1, and Terf1 mRNA were calculated and analyzed using 2-ΔΔCt.

[0146] Table 4 Primer sequences

[0147]

[0148] DNA helicase-related genes: WRN and BLM are core members of the RecQ family, directly linked to progeria, and commercially available inhibitors are available. The WRN gene, associated with Werner syndrome (progeria), possesses helicase and exonuclease activities and is involved in telomere maintenance.

[0149] Telomere-related genes:

[0150] TERT (telomerase reverse transcriptase) is responsible for adding telomeric repeat sequences to the ends of chromosomes, slowing telomere shortening.

[0151] TRF1, a member of the Shelterin complex, primarily binds to telomeric double-stranded DNA, protecting chromosome ends from activation of the DNA damage response (DDR).

[0152] Table 5 Expression of DNA helicase and telomere-related genes in different groups

[0153]

[0154]

[0155] The above data show that after 48 hours of exosome treatment, the mRNA levels of DNA helicase (WRN, BLM) and telomere-related genes (TERT, TRF1) were upregulated, and were better than those in the positive control group (PQQ). In summary, exosomes can upregulate the expression of DNA helicase and promote DNA replication, especially the replication of telomeric DNA. Among them, the PG group has a better effect. In order to improve the application range of Gastrodia elata-probiotic exosomes, a comparative verification experiment was carried out according to the fermentation supernatant prepared in Example 2 and Example 3.

[0156] Example 8 Animal Verification

[0157] Establishment of D-galactose-induced aging model and animal grouping

[0158] An aging model was established by intraperitoneal injection of D-galactose for 8 weeks. Mice were weighed and divided into 5 groups of 10 each. The aging model was established by intraperitoneal injection of D-galactose, and the treatment group was given exosomes by oral gavage. The experimental period lasted 8 weeks. After the end of the experimental period, blood was collected from the orbital cavity, and the serum was aliquoted and stored at -80°C until further use. The heart, liver, spleen, lungs, kidneys, and thymus were dissected and weighed. Some liver and kidney tissues were fixed in formalin, and others were aliquoted and stored at -80°C until further use.

[0159] Fermentation supernatant: The fermentation supernatants prepared according to Example 2 and Example 3 were mixed at a ratio of 1:1 and freeze-dried.

[0160] Table 6 Animal grouping

[0161] Animal Grouping D-Galactose Drug name and dosage Negative control group - 0.5 mL normal saline Model Group + 0.5 mL normal saline Fermentation supernatant group + 250 mg / kg / day PG Group + 20 μg / mouse Positive control group (PQQ) + 5 mg / kg / day

[0162] Index evaluation: Evaluation of organ coefficient, antioxidant enzyme content SOD, CAT, GSH-Px, MDA detection method refers to the enzyme-linked immunosorbent assay (ELISA) kit instructions for determination

[0163] Table 7 Effects of different groups on the main organ indexes of aging mice

[0164]

[0165] Organ indices decline with aging, with the thymus index often used as a marker for evaluating the success of aging animal models. Compared to the negative control group, injection of D-galactose led to a significant decrease in the indices of major organs in mice. However, administration of fermentation supernatant, PG exosomes, and PQQ significantly improved organ indices, demonstrating that the fermentation supernatant is effective in protecting organs, with a particularly strong protective effect on the liver.

[0166] Increased oxidative stress is a characteristic of aging. In order to study the protective effect of the dominant group on D-galactose-treated mice, ELISA was used to further detect the levels of antioxidant enzymes SOD, CAT, GSH-Px and MDA in mouse serum, liver and kidney tissues, as shown in the table below.

[0167] Table 8 Effects of different groups on antioxidant enzymes in mouse serum, liver and kidney tissues

[0168]

[0169]

[0170] SOD is an important antioxidant enzyme that scavenges superoxide free radicals in the body, protecting cells from oxidative damage. GSH-Px (glutathione peroxidase) scavenges hydrogen peroxide and lipid peroxides in the body, protecting cells from oxidative damage. CAT is an enzyme that breaks down hydrogen peroxide, preventing it from damaging cells. MDA (malondialdehyde) is a product of lipid peroxidation, and its level can reflect the level of oxidative stress in the body.

[0171] Compared with the negative control group, the SOD activity in the serum of mice in the model group was significantly decreased, while the serum MDA level was significantly increased. However, compared with the model group, the SOD activity in the fermentation supernatant and PG groups was significantly increased, while the MDA level was significantly decreased. This study suggests that the fermentation supernatant and PG exosomes can slow the aging process by regulating redox homeostasis, with the PG group showing the strongest effect.

[0172] Regarding antioxidant enzyme activity in the liver, compared with the negative control group, the model group mice showed significantly decreased SOD, CAT, and GSH-Px activities in the liver and kidneys, and significantly increased MDA levels. Compared with the model group, treatment with fermentation supernatant, PG exosomes, and PQQ significantly increased GSH-Px, SOD, and CAT activities in the liver and kidneys, and significantly decreased MDA levels. There was no significant difference between treatment with fermentation supernatant and PQQ, but PG exosomes showed a stronger antioxidant capacity. All three can regulate the levels of antioxidant enzymes in liver and kidney tissues, thereby delaying D-galactose-induced aging.

Claims

1. A Bifidobacterium animalis subsp. animalis HX-BA21, characterized by: Its accession number is CGMCC No.32958.

2. A probiotic containing exosome postbiotics, characterized by: The method is obtained by fermenting the Bifidobacterium animalis subspecies HX-BA21 according to claim 1.

3. A Gastrodia elata containing exosomes, characterized by: The method is obtained by fermenting Gastrodia elata with the animal Bifidobacterium animalis subspecies HX-BA21 as claimed in claim 1.

4. A fermentation complex, characterized in that: The method comprises the exosome-containing postbiotic composition according to claim 2 and claim 3.

5. A fermentation complex preparation, characterized in that: It includes three-level embedding structure; The tertiary embedding structure consists of a core composed of probiotics, a shell composed of the fermentation complex as claimed in claim 4, and a cocoon composed of prebiotics.

6. The method for preparing the fermentation complex preparation according to claim 5, wherein: The core layer spinning solution, shell layer spinning solution and cocoon layer spinning solution are spun in a ratio of 1:1:1 and freeze-dried to obtain; The method for preparing the core layer spinning solution comprises the following steps: Bifidobacterium animalis subsp. animalis HX-BA21 was inoculated into MRS liquid culture medium to activate the bacteria, and then the bacteria were collected; then the bacteria were resuspended in PBS to obtain a probiotic suspension; Sodium alginate and milk fat globule membrane are mixed, water is added to prepare a mixed solution, and the mixed solution is then mixed with a probiotic suspension to obtain; The method for preparing the shell spinning solution comprises the following steps: The probiotic exosomes are mixed with the Gastrodia elata exosomes to obtain an exosome mixture. Dissolve gelatin and chitosan in water and acetic acid solution respectively, then mix to form a gelatin-chitosan composite solution, and add the exosome mixture to the composite solution to obtain; The preparation method of the cocoon layer spinning solution comprises the following steps: Dissolve octenyl succinic anhydride starch in water, dissolve polyvinyl alcohol in water, mix the two, add inulin and continue stirring to obtain the product.

7. The method for preparing the fermentation complex preparation according to claim 6, characterized in that: The process parameters of the triaxial electrospinning are as follows: The core layer spinning solution was spun through a needle with an inner diameter of 0.4 mm at a flow rate of 0.3 mL / h and a voltage of 10 kV; The shell spinning solution was spun through a needle with an inner diameter of 0.5 mm at a flow rate of 0.5 mL / h and a voltage of 15 kV; The cocoon layer spinning solution was spun through a needle with an inner diameter of 0.6 mm at a flow rate of 0.8 mL / h and a voltage of 20 kV; The spinning environment temperature is 20°C and the humidity is 30%; The inoculation amount of the Bifidobacterium animalis subspecies HX-BA21 is 2% (v / v); The activation temperature is 37°C and the activation time is 20h; The solution used in the probiotic suspension is PBS solution; The number of viable bacteria in the probiotic suspension is 1-5 billion CFU / mL; The weight ratio of the sodium alginate to the milk fat globule membrane is 4:1; The concentration of the mixed solution is 8% (w / v); The added amount of the probiotic suspension is 2 wt % of the mixed solution; The volume ratio of the probiotic exosomes to the Gastrodia elata exosomes is 1:2; The weight ratio of gelatin to chitosan is 7:3; The amount of the exosome mixture added to the composite solution is 1-3 wt %; The weight ratio of the octenyl succinic anhydride starch to the polyvinyl alcohol is 1:1; The added amount of the inulin is 2.0% (w / v) of the mixed solution of octenyl succinic anhydride starch and polyvinyl alcohol.

8. The use of probiotics according to claim 2, characterized in that: Used in the preparation of food or medicine for improving cell migration rate; or Used in the preparation of food or medicine for reducing the proportion of SA-β-gal positive cells; or Used in the preparation of food or medicine that reduces the fluorescence intensity of intracellular ROS; or Used in the preparation of food or medicine for increasing mitochondrial membrane potential; or Used in the preparation of food or medicine that alleviates oxidative stress damage caused by D-galactose; or Used in the preparation of food or medicine for delaying fibroblast aging; or Used in the preparation of food or medicine for upregulating the expression of DNA helicase; or Used in the preparation of foods or medicines that increase the mRNA expression level of telomere-related genes; or The invention is used for preparing food or medicine for promoting the replication of telomere DNA.

9. The use of Gastrodia elata exosomes according to claim 3, characterized in that: Used in the preparation of food or medicine for improving cell migration rate; or Used in the preparation of food or medicine for reducing the proportion of SA-β-gal positive cells; or Used in the preparation of food or medicine that reduces the fluorescence intensity of intracellular ROS; or Used in the preparation of food or medicine for increasing mitochondrial membrane potential; or Used in the preparation of food or medicine that alleviates oxidative stress damage caused by D-galactose; or Used in the preparation of food or medicine for delaying fibroblast aging; or Used in the preparation of food or medicine for upregulating the expression of DNA helicase; or Used in the preparation of foods or medicines that increase the mRNA expression level of telomere-related genes; or The invention is used for preparing food or medicine for promoting the replication of telomere DNA.

10. The use of the fermentation complex preparation according to claim 4, characterized in that: Used in the preparation of food or medicine for improving cell migration rate; or Used in the preparation of food or medicine for reducing the proportion of SA-β-gal positive cells; or Used in the preparation of food or medicine that reduces the fluorescence intensity of intracellular ROS; or Used in the preparation of food or medicine for increasing mitochondrial membrane potential; or Used in the preparation of food or medicine that alleviates oxidative stress damage caused by D-galactose; or Used in the preparation of food or medicine for delaying fibroblast aging; or Used in the preparation of food or medicine for upregulating the expression of DNA helicase; or Used in the preparation of foods or medicines that increase the mRNA expression level of telomere-related genes; or Used in the preparation of foods or medicines that promote the replication of telomeric DNA; or Used in the preparation of food or medicine for increasing serum SOD activity; or Used in the preparation of foods or medicines for increasing the activities of GSH-Px, SOD and CAT in the liver and kidneys; or For use in the preparation of foods or drugs that reduce serum and liver MDA levels; or The invention can be used for preparing food or medicine for delaying aging caused by D-galactose.

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