Donkey milk exosome preparation, preparation method and application

Abstract

The invention provides a donkey milk exosome preparation, a preparation method and application, and belongs to the technical field of life sciences, the donkey milk exosome preparation comprises a donkey milk exosome and a complex microbial inoculant; wherein the complex microbial inoculant is prepared from clostridium butyricum, bacillus subtilis and enterococcus faecium. The scheme that the donkey milk exosome and the complex microbial inoculant are compounded for use is created for the first time, and the donkey milk exosome and the complex microbial inoculant can achieve synergistic interaction, so that inflammatory factors can be effectively reduced, the number of goblet cells is increased, and the intestinal mucosa is repaired; meanwhile, by utilizing the natural anti-inflammatory property of the donkey milk exosome and the micro-ecological regulation of the complex microbial inoculant, the side effects of chemical drugs are avoided, and an integrated scheme of'intestinal barrier repair-immune regulation 'is provided.

Description

Technical Field

[0001] This invention belongs to the field of life science technology, specifically to a donkey milk exosome preparation, its preparation method, and its application. Background Technology

[0002] Inflammatory bowel disease (IBD) is a group of chronic, relapsing, and nonspecific inflammatory bowel diseases affecting millions of people worldwide. It is characterized by an imbalance between enteritis and immune response, primarily including ulcerative colitis and Crohn's disease. It is a complex intestinal disease influenced by genetic, environmental, and microbial factors. With the accelerating pace of life and increasing stress levels, coupled with environmental factors such as exposure to antibiotics and processed foods, the incidence of IBD has increased dramatically. However, the lack of effective drugs makes research into the prevention and treatment of this difficult-to-cure chronic inflammatory bowel disease particularly important, and it remains a significant challenge to human health.

[0003] As research into IBD deepens and the advocacy for dietary therapy grows, researchers are beginning to explore the potential of certain foods to alleviate IBD symptoms. Milk, as a natural product, contains bioactive components called milk-derived exosomes (MEVs), which are nanoscale extracellular vesicles. These exosomes not only serve as drug carriers but also play important physiological roles in animal metabolism and immunity. While bovine milk exosomes can regulate gut health, innate immunity, and prevent or treat inflammatory diseases, their low concentration and limited production hinder large-scale application.

[0004] Therefore, a new treatment for enteritis that can be applied on a large scale and is highly effective is needed.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a donkey milk exosome preparation, its preparation method, and its application. This exosome preparation has a synergistic therapeutic effect on dextran sulfate sodium-induced colitis, thus effectively solving the problem of insufficient efficacy of single exosome treatment for enteritis. Furthermore, the preparation method of the donkey milk exosome preparation can, on the one hand, overcome the bottleneck of large-scale application of donkey milk exosomes, and on the other hand, improve the stability of donkey milk exosomes. This provides a safe and efficient new strategy for the treatment of intestinal diseases.

[0007] To achieve the above-mentioned objectives of this invention, the following technical solution is adopted:

[0008] A donkey milk exosome preparation, comprising: donkey milk exosomes and a compound bacterial agent;

[0009] The compound microbial agent is composed of Clostridium butyricum, Bacillus subtilis and Enterococcus faecalis.

[0010] Preferably, the ratio of viable bacteria among Clostridium butyricum, Bacillus subtilis and Enterococcus faecalis is 1:10:1.

[0011] Preferably, the viable counts of Clostridium acidophilum, Bacillus subtilis, and Enterococcus faecalis are all 1×10⁻⁶. 9 cfu / g, the dosage of donkey milk exosomes is 1×10⁻⁶. 11 particles / kg.

[0012] Preferably, the *Clostridium butyricum* has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M2021016; the *Bacillus subtilis* has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M2021018; and the *Enterococcus faecium* has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M2021017.

[0013] Preferably, the donkey milk exosomes are either donkey colostrum exosomes or donkey normal milk exosomes.

[0014] Preferably, the particle size of the colostrum exosomes is 79.8±0.59 nm; the particle size of the normal milk exosomes is 82.6±0.93 nm.

[0015] This invention also provides a method for preparing a donkey milk exosome preparation, comprising the following steps:

[0016] S1. Centrifuge donkey milk at 4℃ and 2000-4000×g for 10-15 minutes, then filter to remove impurities and obtain whey.

[0017] S2. Add rennet to the whey, incubate in a water bath at 36℃-38℃ for 40min-50min, and then centrifuge at 11000-14000×g at 4℃ for 1h-2h.

[0018] S3. Filter the filtrate obtained by centrifugation, centrifuge at 60000-80000×g for 1-2 hours, and then ultracentrifuge at 130000-140000×g for 1-2 hours to obtain a precipitate. Resuspend the precipitate in PBS and filter to obtain donkey milk exosomes.

[0019] S4. Mix the donkey milk exosomes with Clostridium butyricum, Bacillus subtilis and Enterococcus faecalis until homogeneous to obtain the final product.

[0020] Preferably, in step S1, centrifugation is performed at 3000×g for 15 min;

[0021] Preferably, in step S2, the water bath is carried out at 37°C for 45 min, followed by centrifugation at 12000×g at 4°C for 1 h.

[0022] Preferably, in step S3, after centrifugation at 70000×g for 1 hour, the mixture is ultracentrifuged at 135000×g for 1.5 hours.

[0023] This invention also provides the application of a donkey milk exosome preparation in the preparation of a drug for treating intestinal diseases.

[0024] Preferably, the intestinal disease is inflammatory bowel disease.

[0025] Preferably, the inflammatory bowel disease is either ulcerative colitis or Crohn's disease.

[0026] Preferably, the donkey milk exosome preparation is used to reduce serum IL-6 and IL-1β levels.

[0027] Alternatively, it can increase the number of goblet cells in the colon and the thickness of the mucus layer;

[0028] Alternatively, it can improve colonic mucosal damage.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] This innovative approach combines donkey milk exosomes with a compound probiotic agent, achieving a synergistic effect that effectively reduces inflammatory factors and increases goblet cell count, thereby repairing the intestinal mucosa. Simultaneously, by leveraging the natural anti-inflammatory properties of donkey milk exosomes and the microecological regulation of the compound probiotic agent, the side effects of chemical drugs are avoided, providing an integrated solution for "intestinal barrier repair and immune regulation." Attached Figure Description

[0031] Figure 1 Transmission electron micrograph of colostrum exosomes;

[0032] Figure 2 Transmission electron micrograph of normal mammary exosomes;

[0033] Figure 3 : Particle size distribution of exosomes obtained from colostrum preparation;

[0034] Figure 4 : Particle size distribution of exosomes prepared from normal emulsion;

[0035] Figure 5Western blot analysis of donkey milk exosome marker proteins CD81, CD9, and TSG101 and negative marker protein Calnexin; among them, TM, MM, and CM are donkey colostrum exosomes; the rest are donkey normal milk exosomes;

[0036] Figure 6 Box plot of the main dimensions of donkey colostrum exosomes and donkey normal milk exosomes, where DC represents donkey colostrum exosomes and DM represents donkey normal milk exosomes;

[0037] Figure 7 Box plots of protein concentrations in donkey colostrum exosomes and donkey normal milk exosomes, where DC represents donkey colostrum exosomes and DM represents donkey normal milk exosomes;

[0038] Figure 8 Morphological and staining observations of the alleviating effect of donkey milk exosomes on DSS-induced colitis in mice;

[0039] Figure 9 The effect of donkey milk exosomes on DSS-induced colonic DAI scores in mice was investigated. The DSS+DK EVs group was the donkey milk exosome group, and the DSS+DK EVs+Pro group was the donkey milk exosome compound bacterial agent group.

[0040] Figure 10 The effect of donkey milk exosomes on DSS-induced serum IL-6 in mice was investigated. The DSS+DK EVs group was the donkey milk exosome group, and the DSS+DK EVs+Pro group was the donkey milk exosome compound bacterial agent group.

[0041] Figure 11 The effect of donkey milk exosomes on DSS-induced serum IL-1β in mice was investigated. Among them, DSS+DK EVs was the donkey milk exosome group, and DSS+DK EVs+Pro was the donkey milk exosome compound bacterial agent group. Detailed Implementation

[0042] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0043] Example 1

[0044] A. Preparation of donkey milk exosomes

[0045] Preparation of impurity-free donkey whey: Fresh donkey milk or donkey milk samples stored at -80℃ were wrapped with sealing film and placed in a preheated 37℃ water bath for rapid melting, which took 20-30 minutes. The melted donkey milk was centrifuged at 3000×g for 15 minutes at 4℃, and the whey was filtered through gauze into a new 50ml centrifuge tube. This process was repeated once to remove impurities and cell debris from the donkey milk. Rennet was added to the obtained whey at a ratio of 0.02g / 100ml, and the mixture was incubated in a 37℃ water bath for 45 minutes, then centrifuged at 12000×g for 1 hour at 4℃. The whey was then filtered through a 0.22µm filter into an ultracentrifuge tube to remove milk fat, casein, and residual rennet.

[0046] Ultracentrifugation extraction of donkey milk exosomes: After treatment, the whey was ultracentrifuged at 70,000 × g (Beckman) at 4°C for 1 h. The supernatant was collected, and larger vesicles and other proteins were discarded. The ultracentrifuge tubes were thoroughly rinsed with ultrapure water. The whey was then ultracentrifuged again at 135,000 × g at 4°C for 1.5 h, and the supernatant was discarded. The precipitate was separated into two ultracentrifuge tubes by rinsing with 1 ml of pre-cooled 4°C 1×PBS. Both tubes were balanced with an appropriate amount of pre-cooled 4°C 1×PBS and ultracentrifuged at 135,000 × g at 4°C for 1.5 h. The precipitate was then dissolved in 1 ml of pre-cooled 4°C 1×PBS and filtered through a 0.22 μm filter into a sterile, enzyme-free 1.5 ml centrifuge tube. 10 μL was used for protein concentration detection by BSA, and the remainder was stored at -80°C.

[0047] B. Identification of donkey milk exosomes

[0048] Protein concentration determination method: The protein concentration of exosomes was quantitatively extracted using the BCA protein assay kit (Bio-Rad, USA). This data can be used to calculate the ratio of donkey milk exosome particle number to protein content. Transmission electron microscopy (TEM) morphology observation: Exosome morphology was observed using a transmission electron microscope (TEM; HT-7700, Hitachi, Japan). Milk exosomes were precipitated using a copper grid, and then 10 μL of uranium acetate was added to the copper grid for 1 min. The precipitate was absorbed with filter paper, dried at room temperature for several minutes, and then imaged under 80 kV. TEM analysis showed that the exosomes were cup-shaped, round or nearly round, membranous vesicles with a visible double-membrane structure. The central region contained a relatively concentrated low-electron-density component with clear boundaries, and the average diameter was approximately 80 nm (experimental results are shown in the figure). Figure 1-2 As shown, where Figure 1 Transmission electron micrograph of exosomes prepared from colostrum; Figure 2 Transmission electron micrograph of exosomes prepared from normal milk.

[0049] C. Particle concentration and particle size distribution determination

[0050] Particle concentration and size distribution were determined using a nanoflow cytometer (NanoFCM, N30E, China). Particle size distribution analysis showed that donkey milk exosomes were distributed in the 50-150 nm range, with a peak size within this range. There was a difference between colostrum and normal milk exosomes; colostrum exosomes were significantly smaller (79.8 ± 0.59 nm) compared to normal milk exosomes (82.6 ± 0.93 nm, p < 0.05) (experimental results are shown in the figure). Figure 3-4 As shown, where Figure 3 The particle size distribution of exosomes prepared from colostrum; Figure 4 (Particle size distribution of exosomes prepared from normal emulsion).

[0051] D. Marker protein analysis

[0052] Western blot analysis was performed using antibodies against Calnexin (Cell Signaling Technology, Danvers, MA, USA), CD9 (Cell Signaling Technology, Danvers, MA, USA), CD81 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Flot1 (BD Transduction Lab, Alphen aan den Rijn, Netherlands), and TSG101 (Novus Biologicals, Littleton, CO, USA) to analyze specific exosome markers such as CD9, CD81, Flot1, and TSG101. The procedure was as follows: A 12% SDS-PAGE gel was prepared according to the protein size of the sample to be tested; after assembling the electrophoresis apparatus, an appropriate amount of running buffer was added. The protein sample was removed, boiled in a constant-temperature metal bath for 3–5 minutes, and centrifuged; the protein sample and protein marker were added to the wells of the electrophoresis gel in the required order using a pipette or sample needle. Cover the tank, turn on the power, and run the gel at 80V until the sample exits the stacking gel. Then, switch to 100V and run the gel until the bromophenol blue reaches the bottom of the gel. After electrophoresis, remove the gel, trim its size, and cut off the edges and excess material. Mark the lower left corner. Based on the trimmed gel size, cut a PVDF membrane of the corresponding size, mark the lower left corner, activate it in a suitable amount of methanol for 20 seconds, and then soak it in pre-cooled transfer buffer. Take 8 sheets of filter paper and cut them into 8cm × 10cm pieces. Soak them in pre-cooled transfer buffer as well. Prepare the transfer "sandwich" in the following order: sponge-filter paper-electrophoresis gel-membrane-filter paper-sponge. Add the transfer buffer to the tank, assemble the transfer apparatus, add floating ice, and bury the apparatus in an ice bath or place it in a refrigerator. Turn on the power and run the transfer at 300mA. The transfer time depends on the size of the protein to be detected; generally, 1 kDa = 1 min. After transfer, remove the PVDF membrane and immerse it in 5% skim milk TBST with the protein side facing up for 1 hour. Primary antibody blocking: Cut the milk-blocked membrane as needed and immerse it in a prepared primary antibody solution (antibody dilution ratio 1:1000) and incubate overnight at 4°C. Recover the primary antibody and wash the membrane with 20 mL TBST at room temperature for 10 minutes, repeating three times. Select the secondary antibody based on the primary antibody, prepare a 1:5000 solution in 5% skim milk TBST, and immerse the membrane in the secondary antibody solution for approximately 1 hour at room temperature. Recover the secondary antibody and wash the membrane with 20 mL TBST at room temperature for 10 minutes, repeating three times. Remove the PVDF membrane, drain the water, and lay it flat on plastic wrap with the protein side facing up. Add an equal volume of ECL A / B solution to the membrane and react in the dark for 5 minutes. Clamp the membrane and transfer it into a sealing film, ensuring the protein side remains facing up throughout.The membrane was placed in the imaging system, the parameters were set, and exposure began; the brightness and contrast were adjusted, and the image was saved. Western blotting confirmed the presence of exosome marker proteins in the extracted donkey milk exosomes. Figure 5 As shown, the results for milk-derived exosome marker proteins CD9, CD81, and TSG101 were positive, while Calnexin expression was negative.

[0053] from Figures 6-7 It can be seen that the protein concentration of colostrum exosomes is significantly higher than that of normal milk exosomes. Based on the data in Table 1, the average ratio of donkey milk exosome particle number to protein content is 1 × 10⁻⁶. 9 particles / μg.

[0054] Table 1: Donkey milk exosome particle size, protein content, and particle concentration

[0055]

[0056] Experimental Example 1: Effect of donkey milk exosome preparations on sodium dextran sulfate-induced acute colitis

[0057] Donkey milk exosomes + probiotics compound preparation includes: 1×10 11 particles / kg exosomes, 1×10⁻⁶ deposited on January 5, 2021 9 cfu / g Clostridium butyricum Cb-01 (inventor-screwed and preserved by the inventor, deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M2021016), 1×10 10 CFU / g of Bacillus subtilis DK-03 (inventor-screwed and preserved at the China Center for Type Culture Collection, accession number CCTCC NO: M2021018) and 1×10 9 CFU / g of *Enterococcus faecium* DK-01 (inventor-strained and preserved at the China Center for Type Culture Collection, accession number CCTCC NO: M2021017). 1×10⁻⁶ 11 Donkey milk exosomes particles / kg and 1×10 9 cfu / g Clostridium butyricum, 1×10 10 CFU / g of Bacillus subtilis and 1×10 9 A mixture of CFU / g Enterococcus faecalis was used to obtain a donkey milk exosome preparation.

[0058] Healthy male C57BL / 6N mice were selected and acclimatized for one week. They were then randomly divided into 5 treatment groups, with 8 mice in each group. The experimental groups were as follows: control group, DSS enteritis group (4% (W / V)), donkey milk exosomes (1×10¹¹ particles / kg / day) + DSS group, and donkey milk exosomes + compound bacterial agent + DSS group.

[0059] On day 30 of feeding with donkey milk exosomes, acute enteritis was induced by DSS (Digital Substances Sedimentation), with the mice perfused with 100 mL of water containing 4% DSS for 7 consecutive days. Body weight was recorded weekly, and the DAI (Digital Inflammation and Intestinal Acidity) index was assessed. At the end of the experiment, blood was collected, and intestinal and colonic tissue samples were collected after dissection. A portion was fixed in formaldehyde for histological observation and analysis (including HE staining, Alcian blue staining, and PAS staining). Colonic mucosal morphology and histopathological scores, intestinal mucus thickness, goblet cell count, levels of colonic mucosal inflammatory response-related proteins and inflammatory factors, and molecular signaling pathways were measured to investigate the effects of donkey milk exosomes on colonic tissue damage and inflammatory responses in mice.

[0060] Test results:

[0061] Experimental results are as follows Figure 8-9 As shown, compared with the DSS-induced mouse colitis model group, the donkey milk exosome treatment group and the exosome compound bacterial agent significantly increased the length of the mouse colon, improved the integrity of the mouse colonic mucosa, and increased the number of goblet cells; donkey milk exosomes also significantly reduced the DAI score of the mouse colon and significantly improved the intestinal health. Figure 10-11 As shown, the combination of donkey milk exosomes and probiotics significantly reduced serum inflammatory factors IL-6 and IL-1β in mice, alleviating inflammatory symptoms and significantly improving acute colitis in mice. Meanwhile, Figure 8-11 It can be seen that the combined treatment of exosomes and compound bacteria preparations is more effective than that of exosomes alone, indicating that exosomes and compound bacteria preparations have a synergistic effect and are more effective in treating enteritis.

[0062] In summary, compared with the acute colitis group, the donkey milk exosome administration group, especially the group treated with donkey milk exosomes combined with a compound probiotic preparation, showed significantly reduced damage to the intestinal mucosa and villi, a significant increase in the number of intestinal goblet cells, a significant decrease in the DAI score, and a significant reduction in serum inflammatory factor levels. Furthermore, the combined treatment of exosomes and the compound probiotic preparation demonstrated superior therapeutic effects compared to the exosome-only treatment group, indicating a synergistic effect between exosomes and the compound probiotic preparation, resulting in a more effective treatment for enteritis.

[0063] The donkey milk exosome preparation process of this invention is simple, has good stability and high safety. The prepared donkey milk exosomes can be used to relieve or treat acute enteritis, providing a new strategy for the treatment of intestinal diseases.

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A donkey milk exosome preparation, characterized in that, include: Donkey milk exosomes and compound bacterial agents; The compound microbial agent is composed of Clostridium butyricum, Bacillus subtilis and Enterococcus faecalis.

2. The donkey milk exosome preparation according to claim 1, characterized in that, The ratio of viable bacteria among Clostridium butyricum, Bacillus subtilis, and Enterococcus faecalis is 1:10:

1.

3. The donkey milk exosome preparation according to claim 2, characterized in that, The *Clostridium butyricum* has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M2021016; the *Bacillus subtilis* has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M2021018; and the *Enterococcus faecium* has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M2021017.

4. The donkey milk exosome preparation according to claim 1, characterized in that, The donkey milk exosomes are either donkey colostrum exosomes or donkey normal milk exosomes.

5. The donkey milk exosome preparation according to claim 4, characterized in that, The particle size of the colostrum exosomes was 79.8 ± 0.59 nm; the particle size of the normal milk exosomes was 82.6 ± 0.93 nm.

6. A method for preparing a donkey milk exosome preparation according to any one of claims 1-5, characterized in that, Includes the following steps: S1. Centrifuge donkey milk at 4℃ and 2000-4000×g for 10-15 minutes, then filter to remove impurities and obtain whey. S2. Add rennet to the whey, incubate in a water bath at 36℃-38℃ for 40min-50min, and then centrifuge at 11000-14000×g at 4℃ for 1h-2h. S3. Filter the filtrate obtained by centrifugation, centrifuge at 60000-80000×g for 1-2 hours, and then ultracentrifuge at 130000-140000×g for 1-2 hours to obtain a precipitate. Resuspend the precipitate in PBS and filter to obtain donkey milk exosomes. S4. Mix the donkey milk exosomes with Clostridium butyricum, Bacillus subtilis and Enterococcus faecalis until homogeneous to obtain the final product.

7. The use of a donkey milk exosome preparation as described in claim 1 in the preparation of a medicament for treating intestinal diseases.

8. The use of the donkey milk exosome preparation according to claim 7 in the preparation of a drug for treating intestinal diseases, characterized in that, The intestinal disease mentioned is inflammatory bowel disease.

9. The application of the donkey milk exosome preparation according to claim 8 in the preparation of a drug for treating intestinal diseases, characterized in that, The inflammatory bowel disease mentioned is either ulcerative colitis or Crohn's disease.

10. The use of the donkey milk exosome preparation according to claim 8 in the preparation of a drug for treating intestinal diseases, characterized in that, The donkey milk exosome preparation is used to reduce serum IL-6 and IL-1β levels. Alternatively, it can increase the number of goblet cells in the colon and the thickness of the mucus layer; Alternatively, it can improve damage to the colonic mucosa.

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