Exosomes derived from beer fermentation liquid and preparation method and application thereof

Abstract

The present invention belongs to the field of biological fermentation technology, and relates to an exosome derived from beer fermentation broth, and a preparation method and application thereof. The present invention provides an exosome derived from beer fermentation broth, and uses beer fermentation broth as a raw material to extract exosomes to obtain exosomes derived from beer fermentation broth. The present invention utilizes beer puree to enrich exosomes, which has the characteristics of low cost, easy acquisition, low toxicity, high yield, high biocompatibility and safety. It can be used as a functional exosome and a drug delivery carrier for the treatment of tissue damage repair and neurodegenerative diseases, etc., and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological fermentation, and in particular relates to exosomes derived from beer fermentation liquid, and a preparation method and application thereof. Background Art

[0002] Exosomes are mainly extracellular vesicles with a lipid bilayer structure secreted by cells, with a diameter of about 40 to 100 nm. Exosomes are widely present in various body fluids, such as blood, urine, cerebrospinal fluid, milk, amniotic fluid and ascites. Exosomes contain active ingredients such as lipids, proteins, DNA, mRNA and siRNA, and participate in many physiological and pathological processes in the body. In addition to their own activity, exosomes have advantages such as higher biocompatibility, outstanding structural stability, high loading capacity and low immunogenicity than traditional drug carriers, and have great potential as a new drug delivery system. In recent years, research on exosome delivery of drugs has gradually increased. Some small molecule chemical drugs and gene drugs have been successfully loaded into exosomes, showing good effects in the treatment of neurological diseases, especially brain diseases and tumors.

[0003] A large amount of evidence shows that exosomes can not only load traditional chemical drugs, but also load protein and nucleic acid drugs to achieve high efficiency, targeting and sustained release of drug delivery, suggesting that it can be used as a natural and efficient drug delivery carrier. At present, the application of exosomes is in its infancy, and most studies are mainly concentrated in the laboratory stage. Exosomes are mainly derived from tumor cells (such as HeLa cells), 293T (immortalized human embryonic kidney cells), red blood cells, mesenchymal stem cells (MSC), iPSC cells and immune cells. However, the industrial production of cells still faces many problems, including: 1. It requires a lot of equipment and technical support, and the cost is high; 2. The use of a large number of cells and biomaterials poses a biosafety risk; 3. The production technology and process are complex, the production efficiency is low, and the quality control is difficult. Therefore, how to achieve large-scale production of cell-derived exosomes is the key bottleneck problem that currently restricts the application of exosomes. Summary of the invention

[0004] The purpose of the present invention is to provide an exosome derived from beer fermentation broth and a preparation method and application thereof. The exosomes of the present invention are derived from beer fermentation broth, and can achieve large-scale production of exosomes.

[0005] The present invention provides exosomes derived from beer fermentation broth. The beer fermentation broth is used as a raw material to extract the exosomes, thereby obtaining the exosomes derived from the beer fermentation broth.

[0006] Preferably, the beer fermentation liquid comprises beer puree.

[0007] The present invention also provides a method for preparing exosomes according to the above technical solution, comprising the following steps:

[0008] Performing a first filtration on the beer to obtain a first filtrate;

[0009] Performing a first centrifugation on the first filtrate and taking a first supernatant;

[0010] Perform a second centrifugation on the first supernatant to obtain a second supernatant;

[0011] The second supernatant is subjected to a third centrifugation to obtain a third supernatant;

[0012] The third supernatant is subjected to a fourth centrifugation to obtain a precipitate;

[0013] resuspending the precipitate and performing a second filtration to obtain a second filtrate;

[0014] The second filtrate is subjected to a fifth centrifugation to obtain a precipitate to obtain exosomes derived from the beer fermentation broth.

[0015] Preferably, the mesh size of the first filtration and the second filtration is 0.22 μm.

[0016] Preferably, the centrifugation and filtration operations are performed at 4°C.

[0017] Preferably, the first centrifugation condition is 1000g, 10 min; the second centrifugation condition is 2000g, 20 min; the third centrifugation condition is 10000g, 40 min; the fourth centrifugation and the fifth centrifugation condition are 100000g, 70 min, respectively.

[0018] Preferably, the resuspending comprises eluting with PBS.

[0019] The present invention also provides the use of the exosomes described in the above technical solution or the exosomes prepared by the preparation method described in the above technical solution in the preparation of drugs for repairing tissue damage and / or drugs for neurodegenerative diseases.

[0020] Preferably, the tissue damage includes but is not limited to ocular surface tissue damage.

[0021] Preferably, the neurodegenerative disease includes but is not limited to retinal neurodegenerative disease.

[0022] The present invention provides exosomes derived from beer fermentation broth. The exosomes of the present invention are derived from beer, have low preparation cost, are easy to obtain, and have high biocompatibility and safety. The present invention uses exosomes derived from beer puree to find that they can effectively promote the repair of corneal epithelial damage and intervene in retinal degenerative lesions. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0024] Figure 1 This is a transmission electron microscopy image of exosomes (B-exo) derived from beer puree provided in Example 1 of the present invention; wherein A is a particle size image, and B is a transmission electron microscopy image of exosomes;

[0025] Figure 2 This is a graph showing the biocompatibility results of B-exo with different cells provided in Example 2 of the present invention;

[0026] Figure 3 This is a diagram showing the therapeutic effect of B-exo on a mouse epithelial repair model provided in Example 3 of the present invention; wherein A is a macroscopic image of the mouse cornea, and B is a statistical diagram of the corneal epithelial repair rate after 24 hours of intervention;

[0027] Figure 4 This is a diagram showing the therapeutic effect of B-exo on a retinal degenerative disease model provided in Example 4 of the present invention; wherein A is a staining diagram of rat retinal frozen sections, and B is a diagram showing the changes in relative retinal thickness between the normal group and the experimental group. DETAILED DESCRIPTION

[0028] The present invention provides an exosome derived from beer fermentation broth, and the exosome is extracted from the beer fermentation broth using the beer fermentation broth as a raw material to obtain the exosome derived from the beer fermentation broth. In the present invention, the beer fermentation broth preferably includes beer puree. The present invention uses ultracentrifugation to separate and obtain exosomes derived from beer puree. The beer puree exosomes extracted by the present invention have a typical cup-shaped structure. In the present invention, the particle size of the exosome derived from the beer fermentation broth is preferably 30 to 150 nm. Beer is brewed by adding hops and fermenting with yeast using malt as the main raw material. Production raw materials: malt, hops, yeast, water. Its brewing can be roughly divided into the main processes of malting, malt crushing, wort preparation, wort fermentation, beer filling, etc. The process is fully mature and has achieved standardization and process. In view of this, with the help of the standardized process of beer production, the present invention uses its fermentation broth to separate and prepare exosomes, which can extract active ingredients from beer fermentation broth, and can also solve the problem of standardized source of exosomes and realize large-scale batch production. Exosomes derived from beer puree have good biocompatibility, low cost and low toxicity. The present invention preliminarily finds that using exosomes derived from beer puree, it can effectively promote the repair of corneal epithelial damage and intervene in retinal degenerative diseases.

[0029] The present invention also provides a method for preparing exosomes according to the above technical solution, comprising the following steps:

[0030] Performing a first filtration on the beer to obtain a first filtrate;

[0031] Performing a first centrifugation on the first filtrate and taking a first supernatant;

[0032] Perform a second centrifugation on the first supernatant to obtain a second supernatant;

[0033] The second supernatant is subjected to a third centrifugation to obtain a third supernatant;

[0034] The third supernatant is subjected to a fourth centrifugation to obtain a precipitate;

[0035] resuspending the precipitate and performing a second filtration to obtain a second filtrate;

[0036] The second filtrate is subjected to a fifth centrifugation to obtain a precipitate to obtain exosomes derived from the beer fermentation broth.

[0037] The beer is subjected to a first filtration to obtain a first filtrate. In the present invention, the beer is preferably a beer fermentation broth. In the present invention, the beer fermentation broth is preferably a fresh beer puree. The present invention does not specifically limit the method of the first filtration, and a conventional filter can be used for filtration. In the present invention, the mesh size of the first filtration is preferably 0.22 μm. In the present invention, the function of the first filtration is to remove the original larger particle impurities in the beer.

[0038] The first filtrate is subjected to a first centrifugation to obtain a first supernatant. In the present invention, the first centrifugation is preferably performed at 4°C. In the short term, at 4°C, the properties of the exosomes are more stable. In the present invention, the conditions for the first centrifugation are preferably 900-1200g, 10-20min; more preferably 1000g, 10min. In the present invention, the role of the first centrifugation in the present invention is to remove larger impurity fragments in the supernatant.

[0039] After obtaining the first supernatant, the present invention performs a second centrifugation on the first supernatant to obtain a second supernatant. In the present invention, the second centrifugation is preferably performed at 4°C. In the present invention, the conditions of the second centrifugation are preferably 1500-2500g, 10-30min; more preferably 2000g, 20min. In the present invention, the role of the second centrifugation in the present invention is to remove small pieces of impurities.

[0040] After obtaining the second supernatant, the present invention performs a third centrifugation on the second supernatant to obtain a third supernatant. In the present invention, the third centrifugation is preferably performed at 4°C. In the present invention, the conditions of the third centrifugation are preferably 8000-11000g, 40-70min; more preferably 10000g, 40min. In the present invention, the role of the third centrifugation in the present invention is to remove macromolecular protein impurities.

[0041] After obtaining the third supernatant, the present invention performs a fourth centrifugation on the third supernatant to obtain a precipitate. In the present invention, the fourth centrifugation is preferably performed at 4°C. In the present invention, the conditions of the fourth centrifugation are preferably 90,000 to 110,000 g, 60 to 120 min; more preferably 100,000 g, 70 min. In the present invention, the role of the fourth centrifugation in the present invention is to remove non-exosome extracellular vesicles.

[0042] After obtaining the precipitate, the present invention resuspends the precipitate and performs a second filtration to obtain a second filtrate. The present invention does not specifically limit the method of the second filtration, and a conventional filter can be used for filtration. In the present invention, the mesh size of the second filtration is preferably 0.22 μm. In the present invention, the function of the second filtration is to remove fungi, bacteria, etc. in the filtrate. In the present invention, the resuspension includes eluting with PBS.

[0043] After obtaining the second filtrate, the present invention performs a fifth centrifugation on the second filtrate, takes a precipitate, and obtains exosomes derived from beer fermentation broth. In the present invention, the fifth centrifugation is preferably performed at 4°C. In the present invention, the conditions of the fifth centrifugation are preferably 90000-110000g, 60-120min; more preferably 100000g, 70min. In the present invention, the role of the fifth centrifugation in the present invention is to wash and purify the exosome particles.

[0044] The beer puree exosomes extracted by the preparation method of the present invention have a typical cup-shaped structure, and the particle size is preferably 30 to 150 nm.

[0045] The present invention also provides the use of the exosomes described in the above technical solution or the exosomes prepared by the preparation method described in the above technical solution in the preparation of drugs for repairing tissue damage and / or drugs for neurodegenerative diseases. In the present invention, the tissue damage includes but is not limited to ocular surface tissue damage. In the present invention, the neurodegenerative diseases include but are not limited to retinal neurodegenerative diseases. The present invention utilizes beer pulp to enrich exosomes, which has the characteristics of low cost, easy acquisition, low toxicity, high yield, high biocompatibility and safety. It can be used as functional exosomes and drug delivery carriers for the treatment of tissue damage repair and neurodegenerative diseases, etc., and has broad application prospects.

[0046] In order to further illustrate the present invention, the exosomes derived from beer fermentation broth and the preparation method and application thereof provided by the present invention are described in detail below in combination with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0047] In the embodiment of the present invention, the beer puree is directly purchased from a beer direct sales outlet. The present invention uses beer of different brands, including Tsingtao beer puree, Taishan beer puree, Snow beer puree and Tsingtao beer.

[0048] Example 1

[0049] Extraction and identification of exosomes from beer puree (B-exo)

[0050] Fresh beer purees of three different brands, namely, Tsingtao beer puree, Taishan beer puree and Snow beer puree, were filtered using a filter having a mesh size of 0.22 μm;

[0051] The supernatant was collected by centrifugation twice at 4°C: the first time was 1000 g for 10 min; the second time was 2000 g for 20 min;

[0052] The supernatant was centrifuged at 10,000 g for 40 min at 4°C and the precipitate was collected by centrifugation at 100,000 g for 70 min.

[0053] The precipitate obtained in the previous step was eluted with PBS and filtered with a 0.22 μm filter;

[0054] At 4°C, the filtered supernatant was centrifuged at 100,000 g for 70 min to obtain the precipitate to obtain beer exosomes.

[0055] The B-exo ( Figure 1 , Tsingtao beer puree: Figure 1 A and B in the figure; Taishan beer puree: C and D in the figure; Snow beer puree: Figure 1 E and F), the results show that the average particle size of B-exo prepared in this example is 50-150nm ( Figure 1 A, C, E, B-exo particle size detection), with a typical cup-shaped structure of exosomes ( Figure 1 B, D, and F in the figure are transmission electron microscopy images of B-exo).

[0056] Example 2

[0057] Taking B-exo derived from Tsingtao beer puree as an example, its biocompatibility test on different cells was tested

[0058] The B-exo protein content was quantitatively detected using BCA protein, and the exosomes were diluted to 200 μg / ml with PBS buffer and placed on ice for later use;

[0059] Use red fluorescent marker dye (PKH26) working solution to incubate with B-exo, mix well, and let stand for 10 minutes; then add 5-10 ml PBS and mix well, centrifuge at 100000g for 1-1.5 hours to remove excess fluorescent dye, and resuspend the bottom precipitate with PBS;

[0060] Corneal epithelial cells, corneal stromal cells, and corneal endothelial cells were cultured to 80% confluence;

[0061] The labeled exosomes were incubated with corneal epithelial, stromal, and endothelial cells for 4 h, respectively, and then washed with PBS;

[0062] The biocompatibility of exosomes with different cells was observed using an inverted fluorescence microscope. B-exo labeled with red fluorescent dye (PKH26) of exosomes were co-incubated with corneal epithelial cells, stromal cells and endothelial cells, and then stained and photographed. DAPI (blue) cell nucleus. Figure 2 As shown, red-labeled exosomes are enriched around the cell nucleus, suggesting that B-exo has good biocompatibility with epithelial cells, stromal cells, and endothelial cells.

[0063] Example 3

[0064] Taking B-exo derived from Tsingtao beer puree as an example, its application in the treatment of ocular epithelial damage

[0065] The B-exo protein content was quantitatively detected using BCA protein, and the exosomes were diluted to 200 μg / ml with PBS buffer and placed on ice for later use;

[0066] C57BL / 6 mice were used to scrape the central cornea and construct a corneal epithelial injury and repair model (lesion diameter was 2.5 mm). Sterile PBS buffer was used as the control group, and B-exo solution was used as the experimental group for eye drops. 10ul was dropped into the eyes each time, with an interval of 10 minutes. The corneal epithelial healing of the mice was observed 24h and 48h after injury, and the injury repair rate was calculated.

[0067] The statistical results show that ( Figure 3 , Figure 3The results of the B-exo test on the repair function of corneal epithelial damage on the ocular surface are shown; Figure A shows the corneal transparency of corneal epithelial treated with B-exo and PBS at 24h and 48h; Figure B shows the statistical analysis of the corneal epithelial damage area after 24h under different treatment conditions). The repair speed of corneal epithelium in the B-exo group was significantly faster than that in the PBS buffer group. This suggests that B-exo has the effect of promoting the repair of corneal epithelial damage.

[0068] Example 4

[0069] Taking B-exo derived from Tsingtao beer puree as an example, its application in the treatment of retinal degenerative diseases

[0070] The B-exo protein content was quantitatively detected using BCA protein, and the exosomes were diluted to 100 μg / ml with PBS buffer and placed on ice for later use;

[0071] PBS and B-exo (10 μl each) were injected subconjunctivally into both eyes of 3-week-old RCS (Royal College of Surgeon rat) rats. Two weeks later, the eyeballs of the rats were collected and fixed, and frozen sections were made for staining.

[0072] The statistical results show that ( Figure 4 , Figure 4 The results of the test on the intervention effect of B-exo on retinal degeneration are shown; Figure A shows the staining pictures of the retina of RCS rats 2 weeks after treatment with B-exo and PBS, DAPI (blue), cell nuclei; Figure B shows the statistical analysis results of retinal thickness under different treatment conditions for 2 weeks). The thickness of RCS retina in the B-exo group was significantly higher than that in the PBS buffer group, indicating that B-exo has the effect of inhibiting retinal degeneration and protecting retinal cells.

[0073] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of exosomes derived from beer fermentation broth in the preparation of a drug for treating corneal epithelial damage and / or retinal neurodegenerative diseases; the beer fermentation broth includes beer puree; The method for preparing exosomes comprises the following steps: Performing a first filtration on the beer fermentation liquid to obtain a first filtrate; Performing a first centrifugation on the first filtrate and taking a first supernatant; Perform a second centrifugation on the first supernatant to obtain a second supernatant; The second supernatant is subjected to a third centrifugation to obtain a third supernatant; The third supernatant is subjected to a fourth centrifugation to obtain a precipitate; resuspending the precipitate and performing a second filtration to obtain a second filtrate; The second filtrate is subjected to a fifth centrifugation to obtain a precipitate to obtain exosomes derived from the beer fermentation broth; The mesh size of the first filtration and the second filtration is 0.22 μm; The centrifugation and filtration operations are performed at 4°C; The conditions for the first centrifugation are 1000 g, 10 min; the conditions for the second centrifugation are 2000 g, 20 min; the conditions for the third centrifugation are 10000 g, 40 min; the conditions for the fourth and fifth centrifugation are 100000 g, 70 min.

2. The use according to claim 1, characterized in that: The resuspension includes elution with PBS.

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