Probiotic fermentation complex with cell repair and cell aging delay and application thereof

Abstract

The application provides an animal bifidobacterium animalis subsp animalis HX-BA21, which has a preservation number of CGMCC NO.32958. The application discloses an animal bifidobacterium animalis subsp animalis HX-BA21 containing exosome postbiotic and a fermented complex containing exosomes obtained by fermenting Gastrodia elata with HX-BA21, which comprises, from inside to outside, a probiotic protective layer (nuclear layer), an exosome delivery layer (shell layer) and a prebiotic sustained-release layer (cocoon layer). The probiotic protective layer comprises sodium alginate-milk fat globule membrane complex and animal bifidobacterium animalis subsp animalis; the exosome delivery layer comprises gelatin-chitosan complex and Gastrodia elata mixed exosomes with probiotics; and the prebiotic sustained-release layer comprises octenyl succinic anhydride starch / polyvinyl alcohol complex and inulin. The complex can improve cell migration rate to start cell repair, up-regulate DNA helicase and telomere-related gene expression, delay premature aging, and delay cell aging caused by D-galactose by regulating the level of oxidative stress.

Description

Technical Field

[0001] This invention relates to the fields of microbiology and biomedicine, specifically to the preparation of probiotics and Gastrodia elata exosomes, and more particularly to the preparation of a fermentation complex of nanofibers containing probiotics and Gastrodia elata exosomes and its application in cell repair. Background Technology

[0002] Exosomes, as nanoscale vesicles (30-150 nm) secreted by cells, carry bioactive molecules such as proteins, nucleic acids, and lipids, playing a central role in intercellular signal transduction. Recent studies have found that probiotics and exosomes derived from food and medicine exhibit unique anti-aging potential, with probiotic exosomes generally serving as their metatrophs.

[0003] Currently, technologies to improve exosome stability mainly include chemical modification, freeze-drying, and nanocarrier encapsulation, but significant drawbacks remain. Chemical modification (such as targeted peptide grafting or lipid insertion) may alter the surface charge of exosomes, leading to decreased membrane fluidity and increased immunogenicity; traditional freeze-drying processes cause exosome aggregation, requiring long-term storage at -80°C even with the addition of protective agents such as trehalose; while liposome or polymer nanoparticle encapsulation technologies have encapsulation rates of less than 30%, and suffer from burst release effects and poor interfacial compatibility, making it difficult to achieve controlled sustained release.

[0004] To address the aforementioned technological bottlenecks, there is an urgent need in this field to develop an exosome delivery system that combines protective and functional synergy. Existing research indicates that nanofiber materials, due to their high specific surface area, tunable porosity, and biomimetic extracellular matrix structure, have significant potential in the field of loading bioactive substances. However, traditional nanofiber membranes often rely on single-pore structures for the physical encapsulation of exosomes, which cannot withstand the enzymatic and oxidative stresses in complex physiological environments, and lack kinetic design for the synergistic release of multi-source exosomes, thus limiting their anti-aging effects.

[0005] Therefore, there is an urgent need for a novel exosome delivery system that can protect exosomes from external environmental influences and achieve long-term sustained release, so as to improve their practical application in cell repair and anti-aging.

[0006] Chinese patent application CN111225659B discloses an anti-aging composition comprising extracellular vesicles derived from lactic acid bacteria. This composition includes extracellular vesicles as an active ingredient, which are physiologically active complexes derived from lactic acid bacteria. The extracellular vesicles can be exosome-like vesicles with a diameter of 20 nm to 200 nm. Furthermore, this composition inhibits the expression of MMP-1 protein, thereby providing benefits such as improving skin wrinkles, increasing skin elasticity, inhibiting collagen loss, and preventing skin damage caused by ultraviolet radiation.

[0007] Chinese patent application CN118615187A discloses an anti-aging composition containing limonene, a cosmetic formulation, and a preparation method. The anti-aging composition containing limonene comprises the following components in parts by weight: 20-25 parts of a first exosome encapsulating limonene, 50-60 parts of a second exosome encapsulating probiotic powder, and 20-25 parts of a third exosome encapsulating prebiotics. This application presents an anti-aging composition containing limonene, which encapsulates limonene in exosomes and works synergistically with probiotic powder and prebiotics to effectively alleviate skin aging problems. However, it does not consider the issue from the perspective of cellular aging. Summary of the Invention

[0008] The first objective of this invention is to provide a strain of Bifidobacterium animalis subspecies HX-BA21.

[0009] The second objective of this invention is to provide an exosome prepared from Bifidobacterium animalis subspecies HX-BA21.

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

[0011] The fourth objective of this invention is to provide a silkworm pupa-like nanofiber fermentation complex composed of probiotics and gastrodia elata exosomes.

[0012] This invention is achieved through the following technical solution:

[0013] This invention provides a strain of *Bifidobacterium animalis* subsp. *animalis*, which was deposited on December 6, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Chinese Academy of Sciences, 100101, China. The accession number is CGMCC No. 32958. The bacteria were viable at the time of deposit.

[0014] A probiotic containing exosomes and metabiotics was mainly isolated from the supernatant of HX-BA21 culture using ultracentrifugation. Transmission electron microscopy revealed the presence of exosomes from HX-BA21 probiotics, with a high proportion (62%) having a particle size of 112.5 nm. A Gastrodia elata containing exosomes was mainly obtained by fermenting Gastrodia elata with Bifidobacterium animalis subspecies HX-BA21, followed by differential centrifugation and density centrifugation. A high proportion (67%) of the exosomes had a particle size of 137.5 nm.

[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 contains a sodium alginate-milk fat globule membrane complex and *Bifidobacterium animalis* subsp. *animal*; the exosome delivery layer contains a gelatin-chitosan complex and a mixture of *Gastrodia elata* and probiotic exosomes; the prebiotic sustained-release layer contains an octenyl succinic anhydride starch / polyvinyl alcohol complex and inulin.

[0016] The specific processes mainly include:

[0017] Preparation of spinning solution

[0018] Core layer (probiotic protective layer)

[0019] S1: Preparation of probiotic suspension: Bifidobacterium animalis subspecies HX-BA21 was inoculated into MRS liquid medium at an inoculum of 2% (v / v) and then cultured at 37°C for 20 h. This was repeated for two generations to activate the strain. The culture was centrifuged at 4°C and 6000 rpm for 5 min, and the bacterial cells were collected. The bacterial 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 10-50 billion CFU / mL.

[0020] S2: Preparation of protective layer spinning solution: Weigh 8g sodium alginate and 2g milk fat globule membrane, add 125mL of deionized water to prepare a mixed solution with a concentration of 8% (w / v), stir at 95℃ for 2h, and after the solution is cooled to room temperature, add 2.0% of the bacterial suspension prepared in S1 and continue stirring for 30min to ensure uniform dispersion of the bacterial solution, and obtain the probiotic protective layer (core layer).

[0021] Shell (Exosome Delivery Layer)

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

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

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

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

[0026] S6: The core spinning solution, shell spinning solution and cocoon spinning solution are added to the triaxial electrospinning device in a 1:1:1 ratio for spinning, and a three-stage encapsulation structure is formed through triaxial electrospinning.

[0027] Finally, freeze-dry to obtain the final product.

[0028] According to claim 4, the nanofiber fermentation complex is characterized in that the process parameters of the triaxial electrospinning are as follows:

[0029] The core 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 is 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.

[0031] The cocoon spinning solution is 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 9cm, and the spinning environment temperature is 20℃ and the humidity is 30%.

[0033] Freeze-drying and pulverization: The nanofiber membrane was pre-frozen at -80℃ for 2 hours 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 powder. The powder had a moisture content of ≤3% and a particle size distribution D50 ≤50μm. The fiber structure integrity was observed by scanning electron microscopy (magnification 10000×) to be ≥90%.

[0034] The technical solutions provided in this application have the following advantages compared with the prior art:

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

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

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

[0038] 8. Further, the probiotics, gastrodia 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 D-galactose-induced aging Attached Figure Description

[0045] Figure 1 Electron micrographs of HX-BA21 exosomes are shown;

[0046] Figure 2 The particle size analysis of HX-BA21 exosomes is shown;

[0047] Figure 3 Electron micrographs of Gastrodia elata exosomes are shown;

[0048] Figure 4 The particle size analysis of Gastrodia elata exosomes is shown. Detailed Implementation

[0049] Example 1: Probiotic Performance and Exosome Extraction

[0050] ① Screening of amylase-producing strains

[0051] After the strain was activated, it was spread on LB plates and cultured to obtain single colonies. Single colonies were picked up by toothpick inoculation and inoculated onto starch hydrolysis medium. After aerobic culture at 37°C for 3 to 7 days, 1 to 2 drops of iodine solution were added to the surface of the medium. A transparent hydrolysis zone appeared around the colonies with the ability to produce amylase, and the size of the hydrolysis zone was measured.

[0052] ② Screening of cellulase-producing strains

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

[0054] HX-BA21 exhibits excellent amylase and cellulase production capabilities, with Bifidobacterium animalis BB-12 serving as the control group. Specific performance characteristics are as follows:

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

[0056] HX-BA21 15.25±1.25 16.28±1.21 BB-12 7.25±0.58 8.57±0.86

[0057] ③ Isolation and identification of probiotic exosomes

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

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

[0060] Purification: The fermentation broth in the logarithmic growth phase was centrifuged at 7000g for 20 min, and the supernatant was collected. The supernatant was then centrifuged at 20000g for 30 min and filtered through a 0.22 μm microporous filter. The filtered supernatant was centrifuged at 30000g for 1-2 h at 4℃, the supernatant was discarded, and the precipitate was resuspended in PBS. The presence of exosomes was qualitatively determined using transmission electron microscopy, and the particle size range of the exosomes was analyzed using nanoparticle tracking.

[0061] like Figure 1 As shown, HX-BA21 probiotic exosomes were present by transmission electron microscopy, and the proportion of particles with a diameter of 112.5 nm was relatively high, accounting for 62%.

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

[0063] Example 2

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

[0065] After identification, strain HX-BA21 was found to be a subspecies of Bifidobacterium animalis.

[0066] The 16S sequence of the animal subspecies of Bifidobacterium animalis 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] CCGGTACAACGCGGTGCGACACGGTGACGTGGGGCGGATCGCTGAAAACCGGTCTCA

[0089] GTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGGCGGAGTCGCTAGTAATCGCGGA

[0090] TCAGCAACGCCGCGGTGAATGCGTTCCCGGG.

[0091] Example 3 Preparation of Gastrodia elata exosomes

[0092] S1: 1000g of Gastrodia elata from Xiaocaoba, Yiliang County, Zhaotong City, Yunnan Province was washed, dried, and then ultra-finely pulverized (particle size <50μm). PBS buffer was added, and the mixture was extracted at 4℃ for 12h. Pretreatment was performed by juicing to break the cell wall, resulting in broken cell wall Gastrodia elata juice.

[0093] S2: Gastrodia elata was fermented using Bifidobacterium animalis subspecies HX-BA21 (inoculation amount of 3%, viable count of 10-50 billion CFU / mL) at a temperature of 37℃ for 24-48 hours and a constant pH of 5.9-7.0.

[0094] S3: Exosomes were separated using differential centrifugation. The centrifugation sequence was: 700g for 15 min, 3500g for 15 min, and 12000g for 30 min. After gradient centrifugation, the supernatant was collected and filtered using a 0.22μm microporous filter. The filtered supernatant was placed in a fixed-angle rotor and centrifuged at 800000g for 1-2 h. The supernatant was discarded, and the precipitate was resuspended using PBS.

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

[0096] S5: The presence of exosomes was determined using transmission electron microscopy, and the particle size range of exosomes was analyzed using nanoparticle tracking.

[0097] Transmission electron microscopy revealed the presence of exosomes from Gastrodia elata, with a high proportion (67%) having a particle size of 137.5 nm.

[0098] Probiotic exosomes mainly consist of proteins, miRNAs, mRNAs, and other nucleic acids, as well as short-chain fatty acids. Gastrodia elata exosomes, being plant-derived, are mostly composed of secondary metabolites (gastrodin, gastrodin polysaccharides, etc.). Probiotic exosomes can scavenge extracellular ROS, while Gastrodia elata exosomes (especially gastrodin) can inhibit excessive ROS production in intracellular mitochondria. The two form an extracellular-intracellular dual barrier. The combination of probiotics and Gastrodia elata exosomes may reduce the damage to mitochondrial membrane potential caused by oxidative stress, synergistically maintain mitochondrial functional homeostasis, thereby repairing cells and delaying cell aging.

[0099] Due to insufficient stability, short half-life, and limited drug loading capacity of natural exosomes, they are insufficient to meet therapeutic needs. Therefore, the method described in Example 4 is adopted to enhance the stability and sustained-release effect of exosomes, providing physical protection and controlled release. In this method, the shell of the silkworm-like fiber can encapsulate the exosomes, and the porous structure of the fiber allows the exosomes to be released gradually through diffusion or material degradation, thus prolonging the duration of action.

[0100] Example 4: Preparation of silkworm pupa-type nanofiber fermentation complex

[0101] Preparation of spinning solution

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

[0103] S1: Preparation of probiotic suspension: Bifidobacterium animalis subspecies HX-BA21 was inoculated into MRS liquid medium at an inoculum of 2% (v / v) and then cultured at 37°C for 20 h. This was repeated for two generations to activate the strain. The culture was centrifuged at 4°C and 6000 rpm for 5 min, and the bacterial cells were collected. The bacterial 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 10-50 billion CFU / mL.

[0104] S2: Preparation of protective layer spinning solution: Weigh 8g sodium alginate and 2g milk fat globule membrane, add 125mL of deionized water to prepare a mixed solution with a concentration of 8% (w / v), stir at 95℃ for 2h, and after the solution is cooled to room temperature, add 2.0% of the bacterial suspension prepared in S1 and continue stirring for 30min to ensure uniform dispersion of the bacterial solution, and obtain the probiotic protective layer (core layer).

[0105] Shell spinning solution (exosome delivery layer)

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

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

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

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

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

[0111] The nanofiber membrane collection and spinning process lasted for 6 hours, resulting in a silkworm pupa-like nanofiber membrane with a three-stage encapsulation of probiotics (nucleus), exosomes (shell), and prebiotics (cocoon). The membrane thickness uniformity error was ≤5%, and the fiber diameter distribution was within the range of 100-300 nm. Freeze-drying and pulverization: The nanofiber membrane was pre-frozen at -80℃ for 2 hours, 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 powder. The powder had a moisture content ≤3%, a particle size distribution D50 ≤ 50 μm, and scanning electron microscopy (10000× magnification) showed a fiber structure integrity rate ≥90%.

[0112] Example 5

[0113] The effects of exosomes on fibroblasts were assessed using cell viability and cell scratch repair assays.

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

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

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

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

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

[0119] ② Cell migration was observed using a cell scratch assay. Wild-type (WT) mouse embryonic fibroblasts (MEFs) were selected as the experimental subjects and cultured in DMEM medium containing 10% fetal bovine serum, 1% penicillin, and streptomycin until the logarithmic growth phase. When the cell density reached 5*10⁵ cells / m², scratches were created on the culture surface using a sterile cell scraper. Cells were gently rinsed with PBS to remove dead cells and impurities. The regular cell culture medium was replaced with serum-free medium to inhibit cell proliferation and promote migration. Immediately after scratching, 30 μg / mL of probiotic exosomes (P group), Gastrodia elata exosomes (G group), and fermentation complex (PG group) were added. The control group received no treatment, and the positive control group received 30 μg / mL of PQQ. The state of the scratches at the beginning was observed and recorded under a microscope. The scratches were photographed after 24 hours. The cell migration rate was calculated by measuring the change in scratch width over time. The width (μm) and area (μm²) of the scratched region were measured using ImageJ software. The cell migration rate at 24 hours was calculated by dividing the area of ​​the scratch at 0 hours by the area of ​​the scratch at 24 hours.

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

[0121] 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 is evident that probiotics and Gastrodia elata exosomes can enhance cell viability and initiate cell repair functions by increasing cell migration rate. The complex (PG group) showed a more significant effect, exhibiting superior cell viability compared to the PQQ group, with a cell migration rate as high as 89.28%. When galactose (D-Gal) accumulates in large quantities in cells, it can induce changes in cell metabolism, producing a large amount of reactive oxygen species, leading to oxidative stress and cellular senescence. Therefore, in this embodiment, D-galactose was used to act on fibroblasts to establish a cell senescence model.

[0123] Example 6

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

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

[0126] Mixed conditioned medium of exosomes + D-Gal: The mixed conditioned medium was obtained by diluting exosomes to 30 μg / mL with D-galactose medium.

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

[0128] Cell seeding and pretreatment: Skin fibroblasts were passaged and seeded in 6-well plates for 24 h. Groups were set as follows:

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

[0130] Experimental groups: exosomes (30 μg / mL) were cultured in D-Gal mixed medium for 48 h, namely probiotic exosomes (P group), gastrodia exosomes (G group) and nanofiber complex (PG group);

[0131] Negative control group: Complete culture medium, no treatment, cells pretreated for 48 hours;

[0132] Positive drug group: Cells were treated with a mixture of 30 μg / mL PQQ and D-Gal 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 incubate at room temperature for 15 minutes. Then wash three times with PBS buffer, 1 mL per well for 3 minutes each time. 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, identify 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 determined using the Beyotime reactive oxygen species (ROS) kit. Measurements were performed using a high-content imaging system. Acquisition conditions: DAPI channel excited Hochest 33342 nuclear dye, DCFH-DA channel excited oxidative stress dye, and analysis was performed using the high-content imaging system.

[0136] ③ Mitochondrial membrane potential (MMP)

[0137] Mitochondrial membrane potential was measured using Beyotime's mitochondrial red fluorescent probe. Treated cells were placed under a high-content imaging system for plate scanning. Channels matching the fluorescent probe parameters were selected, with 15 fields of view per well. The objective lens was 20× / 0.45mm aperture, and the focusing mode was selected. Data acquisition began after the images were clear. Image acquisition conditions: Hochest 33342 nuclear dye was excited by the DAPI channel, and mitochondrial dye was excited by the Mito-Tracker Red CMXRos channel. Analysis was performed using the high-content imaging system.

[0138] Table 3. Quantitative analysis of SA-β-gal-positive fibroblasts.

[0139]

[0140] Detecting SA-β-gal activity can be used to assess cellular senescence. Under normal physiological conditions, intracellular β-galactosidase functions under acidic conditions. However, during cellular senescence, lysosomal membrane permeability increases, allowing β-galactosidase activity to be detected at pH 6.0. Results showed that the proportion of SA-β-gal-positive cells in the model group was significantly higher than in the control group. However, pretreatment with exosomes before D-Gal treatment significantly reduced the proportion of SA-β-gal-positive cells. This suggests that exosomes can delay fibroblast senescence.

[0141] With the increase in the production and accumulation of ROS, senescent cells proliferate. Excessive production and accumulation of reactive oxygen species (ROS) can 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 with exosomes before D-Gal induction showed a decrease in intracellular ROS fluorescence intensity. The Gastrodia elata exosome and nanofiber complex group was significantly better than the positive control group (PQQ group), indicating that exosomes can alleviate D-galactose-induced oxidative stress damage.

[0142] Mitochondria are essential for cellular aerobic respiration and oxidative phosphorylation, and the stability of mitochondrial membrane potential is crucial for maintaining normal cellular function and delaying aging. Changes in mitochondrial membrane potential affect cellular energy metabolism, redox state, apoptosis, and calcium homeostasis through multiple pathways, thus playing a significant role in cellular and organismal aging. In the model group, mitochondrial membrane potential was significantly decreased; however, after mitochondrial intervention, mitochondrial membrane potential gradually increased, indicating that exosomes have a significant ameliorative effect on the D-Gal-induced decrease in mitochondrial membrane potential.

[0143] As we age, the activity of DNA helicase may decline, leading to problems during DNA replication. Telomeres' main function is to protect chromosome ends, preventing fusion and degradation between chromosomes and maintaining chromosome stability. During cell division, telomeres gradually shorten due to problems with DNA end replication. When telomeres shorten to a certain extent, the cell enters a state of senescence or apoptosis; this is known as the "telomere hypothesis." Furthermore, using embryonic fibroblasts (MEFs) from Werner progeria mice as a model, this study observed whether the intervention of probiotic exosomes, Gastrodia elata exosomes, and complexes in prematurely aging cells affected the expression of DNA helicase and telomere-related genes, thus influencing aging.

[0144] Example 7

[0145] After modeling, fibroblasts were seeded at a rate of 5 × 10⁴ cells per well into six-well plates, with 30 μg / mL of the corresponding exosomes added simultaneously. After 48 hours, the cells in the six-well plates were collected, counted, and seeded again at 5 × 10⁴ cells / well, with exosomes added simultaneously. This process was repeated, with cells passaged every 48 hours for a total of 5 passages. The intervention was performed according to the grouping in Example 6, with an intervention period of 10 days. Cells from the last passage were collected, and their RNA was extracted. The RNA was reverse transcribed into cDNA. Subsequently, the successfully reverse-transcribed cDNA and primers for Blm, WRN, TERT, TRF1, and GAPDH were used for quantitative real-time PCR. After the reaction program was completed, Ct values ​​were obtained. Using GAPDH as an internal control, 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 associated with progeria, and commercially available inhibitors are available. Among them, the WRN gene is associated with Werner syndrome (progeria), possesses helicase and exonuclease activities, and participates in telomere maintenance.

[0149] Telomere-related genes:

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

[0151] TRF1, a member of the Shelterin complex, primarily binds to telomeric double-stranded DNA, protecting chromosome ends from activation by 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 expression levels of DNA helicases (WRN, BLM) and telomere-related genes (TERT, TRF1) were upregulated, and this was superior to the positive control group (PQQ). In summary, exosomes can upregulate the expression of DNA helicases and promote DNA replication, especially telomere DNA replication. The PG group showed the best effect. To broaden the application scope of Gastrodia elata-probiotic exosomes, comparative verification experiments were conducted using fermentation supernatants prepared according to Examples 2 and 3.

[0156] Example 8 Animal Verification

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

[0158] An aging model was induced by intraperitoneal injection of D-galactose for 8 weeks. Mice were weighed and divided into 5 groups of 10 mice each. The aging model was established by intraperitoneal injection of D-galactose, while the treatment group received exosomes via gavage. The experimental period was 8 weeks. After the experimental period, blood was collected from the orbital sinus, and the serum was aliquoted and stored at -80℃ for later use. The heart, liver, spleen, lungs, kidneys, and thymus were dissected and weighed. Some liver and kidney tissues were fixed in formalin, and the rest were aliquoted and stored at -80℃ for later use.

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

[0160] Table 6 Animal Grouping

[0161] negative control group - 0.5 mL physiological saline Model group + 0.5 mL physiological saline Fermentation supernatant group + 250mg / kg / d PG Group + 20μg / each Positive control group (PQQ) + 5mg / kg / d

[0162] Evaluation of indicators: The levels of organ coefficients, antioxidant enzymes (SOD, CAT, GSH-Px, MDA) were assessed. Detection methods were performed according to the instructions for the enzyme-linked immunosorbent assay (ELISA) kit.

[0163] Table 7. Effects of different groups on the major organ indices of aging mice

[0164]

[0165] As age increases, organ coefficients decline, with the thymus index generally serving as a marker for assessing the success of aging animal models. Compared to the negative control group, injection of D-galactose led to a significant decrease in the major organ coefficients in mice. However, administration of fermentation supernatant, PG exosomes, and PQQ significantly improved organ coefficients, indicating that fermentation supernatant also has a certain effect and can effectively protect organs, with particularly good protective effects on the liver.

[0166] Increased oxidative stress is a characteristic of aging. To investigate the protective effect of the dominant group on D-galactose-treated mice, the levels of antioxidant enzymes SOD, CAT, GSH-Px and MDA in mouse serum, liver and kidney tissues were further detected by ELISA, 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] Superoxide dismutase (SOD) is an important antioxidant enzyme that can scavenge superoxide free radicals in the body and protect cells from oxidative damage. Glutathione peroxidase (GSH-Px) can scavenge hydrogen peroxide and lipid peroxides in the body, protecting cells from oxidative damage. Catalytic acid (CAT) is an enzyme that can break down hydrogen peroxide, preventing hydrogen peroxide damage to cells; malondialdehyde (MDA) is a product of lipid peroxidation, and its content can reflect the level of oxidative stress in the body.

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

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

Claims

1. A fermentation complex formulation, characterized in that: Including a three-level embedding structure; The three-level encapsulation structure consists of a core composed of probiotics, a shell composed of fermentation complexes, and a cocoon composed of prebiotics. The preparation method of the fermentation complex formulation includes the following steps: The core spinning solution, shell spinning solution, and cocoon spinning solution are added to a triaxial electrospinning device in a 1:1:1 ratio for spinning. A three-stage encapsulation structure is formed by triaxial electrospinning and then freeze-dried to obtain the desired product. The preparation method of the core spinning solution includes the following steps: Bifidobacterium animalis subsp. animalis HX-BA21 with accession number CGMCC No. 32958 was inoculated into MRS liquid medium to activate the bacterial strain, and the bacterial cells were collected; then the bacterial cells were resuspended in PBS to obtain a probiotic suspension. Sodium alginate and milk fat globule membrane were mixed and water was added to prepare a mixed solution. Then the mixed solution was mixed with a probiotic suspension to obtain a protective layer spinning solution. The preparation method of the shell spinning solution includes the following steps: Probiotic exosomes were mixed with Gastrodia elata exosomes to obtain an exosome mixture. Gelatin and chitosan are dissolved separately in water and acetic acid solutions, then mixed to form a gelatin-chitosan composite solution. Exosome mixture is then added to the composite solution to obtain the final product. The preparation method of the cocoon spinning solution includes the following steps: Octenyl succinic anhydride starch is dissolved in water, polyvinyl alcohol is dissolved in water, and then the two are mixed together and inulin is added while stirring continuously to obtain the product. The method for preparing the probiotic exosomes includes the following steps: Bifidobacterium animalis subspecies HX-BA21 was activated twice and inoculated into 1L of MRS medium at a 2% inoculum. The fermentation broth in the logarithmic growth phase was centrifuged at 7000g for 20min, and the supernatant was collected. The supernatant was then centrifuged at 20000g for 30min and filtered through a 0.22μm microporous filter. The filtered supernatant was centrifuged at 30000g at 4℃ for 1-2h, the supernatant was discarded, and the precipitate was resuspended in PBS to obtain the final product. The method for preparing the Gastrodia elata exosomes includes the following steps: 1000g of Gastrodia elata was washed, dried, and then ultra-finely pulverized to a particle size of <50μm. PBS buffer was added, and the mixture was extracted at 4℃ for 12h. Pretreatment was carried out by juicing to break the cell wall, and the broken cell wall Gastrodia elata juice was obtained. Fermentation of broken-cell wall Gastrodia elata juice was carried out using Bifidobacterium animalis subspecies HX-BA21 at a temperature of 37℃ for 24-48 hours, with a constant pH of 5.9-7.

0. The inoculum size of Bifidobacterium animalis subspecies HX-BA21 was 3%, and the viable bacterial count in the bacterial solution was 10-50 billion CFU / mL. Exosomes were separated using differential centrifugation. The centrifugation sequence was: 700g for 15 min, 3500g for 15 min, and 12000g for 30 min. After gradient centrifugation, the supernatant was collected and filtered using a 0.22μm microporous filter. The filtered supernatant was then placed in a fixed-angle rotor and centrifuged at 800000g for 1-2 h. The supernatant was discarded, and the precipitate was resuspended using PBS. The suspended liquid was placed in 30%, 50%, and 70% sucrose solutions and centrifuged at 150,000g 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 mixture was then centrifuged at 15,000g for 1 hour, and the precipitate was collected. The precipitate was resuspended in 1 mL of PBS to obtain the Gastrodia elata exosome solution.

2. The fermentation complex formulation according to claim 1, characterized in that: The process parameters for the triaxial electrospinning are as follows: The core 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 is 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 spinning solution is 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 was 20℃ and the humidity was 30%. The inoculation amount of the animal subspecies of Bifidobacterium animalis HX-BA21 was 2% (v / v); The activation temperature was 37°C, and the activation time was 20 hours. The probiotic suspension was prepared using a PBS solution. The number of live bacteria in the probiotic suspension is 10-50 billion CFU / mL; The weight ratio of sodium alginate to milk fat globule membrane is 4:1; The concentration of the mixed solution is 8% (w / v); The amount of probiotic suspension added is 2 wt% of the mixed solution; The volume ratio of the probiotic exosomes to the gastrodia exosomes is 1:2; The weight ratio of gelatin to chitosan is 7:3; The amount of exosome mixture added to the composite solution is 1-3 wt%. The weight ratio of the octenyl succinic anhydride starch to polyvinyl alcohol is 1:1; The amount of inulin added is 2.0% (w / v) of the mixed solution of octenyl succinic anhydride starch and polyvinyl alcohol.

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