Active factor of animal bifidobacterium subspecies JD37 with cell repairing effect
By isolating Bifidobacterium subspecies JD37 from breast milk and extracting its exosomes to prepare a pharmaceutical composition, the shortcomings of existing growth factor preparations and stem cell therapies are overcome, achieving efficient cell repair and multi-pathway regulation. It has diverse applications and is suitable for skin repair and systemic regulation.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HAIKOU BIHUO INVESTMENT CO LTD
- Filing Date
- 2025-12-02
- Publication Date
- 2026-05-12
AI Technical Summary
Existing growth factor preparations have short half-lives and high costs, while stem cell therapy faces ethical controversies and tumorigenic risks. The active ingredients of Bifidobacterium animalis subspecies are unclear, and there is a lack of verification on direct repair mechanisms such as cell migration and autophagy.
Bifidobacterium animalis subspecies JD37 was isolated from the breast milk microbiome, and its secreted exosomes were extracted to prepare a pharmaceutical composition containing exosomes for promoting cell repair, including skin fibroblast migration and reducing inflammatory factors, and applied in the form of lyophilized powder.
Exosomes can significantly promote the migration of skin fibroblasts, reduce inflammatory factors, achieve multi-pathway regulation, and have diverse applications, suitable for local repair, systemic regulation, and targeted delivery.
Smart Images
Figure CN122012304A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial technology, and more specifically, to an active factor of Bifidobacterium animalis subspecies JD37 with cell repair function. Background Technology
[0002] Impaired cell repair is a core pathological mechanism in skin trauma, muscle atrophy, and metabolic diseases. Currently used growth factor preparations (such as EGF and bFGF) suffer from short half-lives and high costs, while stem cell therapy faces ethical controversies and tumorigenic risks. Bifidobacteria, as a recognized safe probiotic, has metabolites proven to have functions such as immune regulation and cholesterol reduction, but research on their direct cell repair effects is still limited.
[0003] In the prior art, CN 118844633 B discloses that *Bifidobacterium animalis* subspecies CP-9 and HN019 can improve the intestinal barrier by competitively inhibiting pathogenic bacterial adhesion; CN 106535908 A discloses that strain LMG P-28149 can reduce adipocyte infiltration by regulating the PPARγ pathway; and CN 120549985 A discloses that strain F1-3-2 improves sarcopenia by downregulating the muscle atrophy marker MuRF-1. However, the active ingredients of these strains have not been clearly isolated, and there is a lack of verification on direct repair mechanisms such as cell migration and autophagy.
[0004] This invention is the first to isolate Bifidobacterium animalis subspecies JD37 from the breast milk microbiome and demonstrates that its secreted exosomes can promote cell repair, providing a new approach for the development of novel cell repair agents. Summary of the Invention
[0005] This invention first provides a subspecies of Bifidobacterium animalis, JD37, wherein the subspecies of Bifidobacterium animalis, JD37, is... Bifidobacterium animalis subsp. lactis JD37 is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC No: M20251293.
[0006] The present invention also provides an active factor of the above-mentioned animal Bifidobacterium subspecies JD37, which includes exosomes with a particle size of 60-80 nm.
[0007] In some embodiments, exosomes are extracted by ultracentrifugation, specifically including: centrifuging the bacterial fermentation broth at 10,000×g to remove cell debris, filtering the supernatant through a 0.22μm filter membrane, and then collecting the exosome precipitate by ultracentrifugation at 100,000×g for 120 min.
[0008] The present invention also provides a pharmaceutical composition comprising the above-mentioned active factors, containing a pharmaceutically acceptable carrier.
[0009] The present invention also provides the use of the pharmaceutical composition described above in the preparation of a cell repair agent, wherein the cell repair includes skin fibroblast migration and skin repair.
[0010] The present invention also provides the use of the pharmaceutical composition described above in the preparation of formulations that reduce inflammatory factors TNF-α and CRP.
[0011] The present invention also provides a freeze-dried powder formulation, which is prepared by mixing the above-mentioned strain with a freeze-drying protectant at a volume ratio of 1:1, pre-freezing at -70°C and then freeze-drying.
[0012] In some embodiments, the freeze-drying protectant includes maltodextrin and sucrose.
[0013] In some embodiments, the mass fraction ratio of the freeze-drying protectant is maltodextrin:sucrose 1:1.
[0014] Compared with the prior art, the present invention has at least the following beneficial effects: (1) Highly efficient cell repair: JD37 exosomes can promote fibroblast migration, which is beneficial to skin repair.
[0015] (2) Multi-pathway regulation: Simultaneously activates autophagy pathway (LC3-II / LC3-I) and inhibits inflammatory factors (TNF-α).
[0016] (3) Diverse application forms: It can be made into injection (local repair), fermented milk (oral systemic regulation) or microcapsules (targeted delivery).
[0017] Preservation Instructions Bifidobacterium animalis subsp. JD37 ( Bifidobacterium animalis subsp. lactis JD37, isolated from healthy breast milk samples, was deposited at the China Center for Type Culture Collection (CCTCC) on June 6, 2025, and classified as: Bifidobacterium animalis subsp. lactis JD37. Bifidobacterium animalis subsp. lactis JD37), with accession number CCTCC NO: M20251293, located at Wuhan University, Wuhan, Hubei, China. Attached Figure Description
[0018] Figure 1 Electron micrograph of JD37 exosomes.
[0019] Figure 2 This is a particle size distribution diagram of JD37 exosomes. Detailed Implementation
[0020] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with specific embodiments.
[0021] Example 1: Isolation and identification of strain JD37 (1) Materials and methods Sample source: Healthy breast milk sample (taken from Beijing Maternal and Child Health Hospital) (2) Separation method: The samples were diluted with PBS and spread on MRS agar plates containing cysteine (0.05%). They were then anaerobically incubated at 37°C for 48 h. White raised colonies were picked and Gram staining confirmed that they were Gram-positive irregular bacilli. (3) Molecular identification: Genomic DNA was extracted, and the 16S rRNA gene was amplified by PCR (using universal primers 27F / 1492R). The sequencing results, as shown in SEQ ID NO.1, show a 99.7% similarity to *Bifidobacterium animalis* subsp. *lactis*. Preservation information: Deposited on June 6, 2025 at the China Center for Type Culture Collection (CCTCC), classified and named as: Bifidobacterium animalis subsp. lactis JD37, accession number: CCTCC NO: M20251293, address: Wuhan University, Wuhan, Hubei, China.
[0022] Example 2: Preparation of exosome active factors (1) Materials and methods Fermentation culture: JD37 was inoculated into MRS medium and anaerobic fermented at 37℃ for 24 h (viable count 1×10⁻⁶). 9 (CFU / mL) (2) Exosome extraction: The fermentation broth was centrifuged at 10,000×g for 20 min to remove the cells; the supernatant was filtered through a 0.22 μm filter membrane and then centrifuged at 100,000×g for 120 min (4℃); the precipitate was resuspended in PBS to obtain the exosome solution.
[0023] (3) Characterization analysis: Transmission electron microscopy was used to observe the morphology of exosomes to visually confirm their structure and integrity; nanoparticle tracking analysis (NTA) was used to detect particle size distribution; Western blot was used to detect the marker proteins CD63 and TSG101.
[0024] (4) Results: Transmission electron microscopy showed that the exosomes exhibited a typical cup-shaped structure with uniform particle size, confirming their morphological integrity. Figure 1 ); obtained exosomes with a particle size mainly distributed in the range of 60-100 nm (Figure 2 Western blot analysis showed positive expression of CD63 and TSG101, consistent with exosome characteristics. These results indicate that JD37 exosomes were successfully extracted and possess good physicochemical properties, laying the foundation for future applications.
[0025] Example 3: Cell Migration and Repair Experiment (1) Materials and Methods Cell model: Human skin fibroblasts (HSF, ATCC CRL-2522), purchased from the American Center for Type Culture Collection, passaged to the 5th-8th generation for experiments.
[0026] (2) Culture medium and grouping: Control group: serum-free DMEM medium (containing 1% penicillin-streptomycin); Experimental group: serum-free DMEM medium containing 50 μg / mL JD37 exosomes (exosomes were pre-sterilized by a 0.22 μm filter membrane).
[0027] (3) Scratch test procedure: HSF cells were used at a rate of 5 × 10 5 Cells were seeded at a density of cells / well in 6-well plates and cultured for 24 h until a dense monolayer formed (cell confluence > 95% observed under a microscope). Using a 200 μL sterile pipette tip, three parallel scratches were made perpendicular to the well markings (1 mm spacing per well). The plates were gently rinsed twice with PBS to remove detached cells. Three replicates were set up for each group, and the scratched areas were photographed under an inverted microscope (10× objective) at 0 h (initial state), 12 h, and 24 h after scratching (the middle scratch in each well was selected, covering approximately 0.5 mm²). ImageJ software was used for analysis: After importing the image, a grayscale threshold (100-255) was set to distinguish between cell-covered areas (black) and scratch areas (white). Calculate the percentage of the scratched area (defined as "wound healing rate" HNv% = [1 - (scratch area / initial scratch area)] × 100%).
[0028] (4) Results At 0 h post-scratching, the HNv% in both groups was 0%; at 12 h, the HNv% in the control group was 28.7±2.1%, while that in the experimental group increased to 49.5±3.4% (p<0.05); at 24 h, the HNv% in the control group reached 35.6±2.7%, while that in the experimental group significantly increased to 78.3±3.1% (p<0.01). The wound healing rate in the experimental group at 24 h was 119.9% higher than that in the control group, indicating that JD37 exosomes can significantly accelerate HSF cell migration and promote skin repair.
[0029] Example 4: Experiment on the Regulation of Inflammatory Factors (1) Materials and Methods Cell model: LPS-induced RAW264.7 macrophages (2) Handling method: LPS group: 1 μg / mL LPS stimulation LPS + exosome group: LPS + 50 μg / mL JD37 exosomes (3) Detection indicators: ELISA was used to detect the levels of TNF-α and CRP in the supernatant; Western blot was used to determine the LC3-II / LC3-I ratio; (0) Results: JD37 exosomes reduced TNF-α and CRP by 38.2% and 29.6%, respectively (Table 1), and increased the LC3-II / LC3-I ratio by 2.1 times, indicating that it inhibits inflammation by activating autophagy.
[0030] Table 1. Changes in inflammatory factor levels (μg / mL)
[0031] Note: * indicates comparison with the LPS group, p<0.01.
[0032] Example 5: Preparation of freeze-dried powder and animal repair experiment (1) Materials and methods Preparation of freeze-dried powder: The bacterial strain and protectant were mixed at a mass ratio of 1:1; pre-frozen at -70℃ for 2 h, then freeze-dried, yielding a viable count of 1.2 × 10⁻⁶. 10 CFU / g; the protective agent contains 8% maltodextrin and 8% sucrose by mass fraction.
[0033] (1) Animal model: full-thickness skin laceration (1 cm in diameter) in SD rats; Handling method: Control group: Saline compresses; Experimental group: 50 mg JD37 lyophilized powder (dissolved in PBS) applied topically; (3) Evaluation indicators: healing rate 7 days after injury, histological analysis; (4) Results: The healing rate of the experimental group was 89.7±3.2%, which was significantly higher than that of the control group (64.5±4.1%) (p<0.01). The tissue sections showed more complete epidermal regeneration.
[0034] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A subspecies of Bifidobacterium animalis, JD37, characterized in that, The animal bifidobacterium subspecies JD37 is... Bifidobacterium animalis subsp. lactis JD37 is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC No: M20251293.
2. The active factor of Bifidobacterium animalis subspecies JD37 as described in claim 1, characterized in that, It contains exosomes with a particle size of 60-80 nm.
3. The active factor as described in claim 2, characterized in that, Exosomes were extracted using ultracentrifugation, specifically by centrifuging the bacterial fermentation broth at 10,000×g to remove cell debris, filtering the supernatant through a 0.22 μm filter membrane, and then collecting the exosome precipitate by ultracentrifugation at 100,000×g for 120 min.
4. A pharmaceutical composition comprising the active ingredient of claims 2-3, characterized in that, It contains a pharmaceutically acceptable carrier.
5. The use of the pharmaceutical composition of claim 4 in the preparation of a cell repair agent, wherein the cell repair includes skin fibroblast migration and skin repair.
6. The use of the pharmaceutical composition of claim 4 in the preparation of formulations that reduce inflammatory factors TNF-α and CRP.
7. A lyophilized powder formulation, characterized in that, It is prepared by mixing the strain described in claim 1 with a freeze-drying protectant at a volume ratio of 1:1, pre-freezing at -70°C and then freeze-drying.
8. The lyophilized powder formulation as described in claim 7, characterized in that, Freeze-drying protectants include maltodextrin and sucrose.
9. The lyophilized powder formulation according to claim 7, characterized in that, The mass fraction ratio of the freeze-drying protectant is maltodextrin to sucrose at 1:1.