Preparation method and application of active probiotics embedded in bovine milk extracellular vesicles

Abstract

The present invention belongs to the field of biomedical materials and relates to a preparation method and application of active probiotics embedded in milk-derived extracellular vesicles. The probiotics embedded in milk-derived extracellular vesicles include probiotics and functionalized milk-derived extracellular vesicles coated on the surface of the probiotics. The preparation method comprises the following steps: extracting milk-derived extracellular vesicles, mixing and incubating phospholipid-polyethylene glycol-phenylboronic acid with the milk-derived extracellular vesicles according to a mass ratio to enable the phospholipid-polyethylene glycol-phenylboronic acid to be completely connected to the milk-derived extracellular vesicles, thereby obtaining functionalized milk-derived extracellular vesicles with phenylboronic acid groups modified on the surface; fermenting and culturing the probiotics to obtain a probiotic sludge; and mixing and incubating the reconstituted probiotic bacterial solution with the functionalized milk-derived extracellular vesicles according to a volume ratio to prepare a probiotic system embedded in milk-derived extracellular vesicles. The present invention is applicable to the development and utilization of various probiotic oral drugs and provides a possible new development strategy for the future of probiotic delivery systems.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and particularly relates to a preparation method and application of bovine milk extracellular vesicles embedded with active probiotics. Background Art

[0002] The gut microbiota contains a variety of symbiotic bacteria and other microorganisms inhabiting the human gastrointestinal tract, and plays a key role in maintaining human health. Once the homeostasis of the human gut is disrupted, it can lead to complications such as inflammatory bowel disease, colon cancer, and gut-related diseases such as Alzheimer's disease and depression. Probiotics have a variety of positive effects on the host, such as restoring gut homeostasis, regulating the host immune response, producing anti-inflammatory or anti-cancer compounds such as short-chain fatty acids, inhibiting the growth of pathogenic or harmful bacteria, and more importantly, it can regulate the functions of other organs through the microbiome-gut-brain axis, microbiome-gut-lung axis, and microbiome-gut-liver axis. However, strongly acidic gastric juice, various digestive enzymes and bile salts in the small intestine can cause a large number of probiotics to die, resulting in limited colonization and proliferation of probiotics in the gastrointestinal tract, and insufficient probiotic colony levels can also seriously impair their functions. Therefore, in order to prevent the death of probiotics caused by direct ingestion of probiotics, it is necessary to encapsulate the probiotics.

[0003] Extracellular vesicles are natural nanoparticles that play an important role in cell-to-cell communication by transferring RNA, lipids, and proteins from donor cells to recipient cells or binding to receptors on the surface of recipient cells; extracellular vesicles are absorbed after oral administration and accumulate in tissues, crossing the blood-brain barrier; these characteristics make extracellular vesicles have the advantages of being a novel bioactive food compound and delivering drugs to diseased tissues. At the same time, as a biological carrier, extracellular vesicles have obvious advantages compared with traditional synthetic carriers. It has the ability to transport various active biomolecules. Compared with mature liposomes, extracellular vesicles show better performance, including across biological carriers, improving the tissue bioavailability and efficacy of chemotherapeutic drugs, and showing lower toxicity or immunogenicity, thus becoming an effective drug delivery route. Compared with the low yield of extracellular vesicles derived from cell lines, the concentration and product diversity of extracellular vesicles in milk are very high, and the integrity of phospholipid vesicles is stable in vitro for a long time. Summary of the Invention

[0004] The object of the present invention is to solve the above problems existing in the prior art, and a preparation method and application of milk exosome-embedded live probiotics are proposed. The present invention successfully constructs a probiotic embedding system by using milk exosomes. The milk exosomes used in the preparation method have good biocompatibility, can penetrate the blood-brain barrier, accumulate in tissues, improve the survival rate of probiotics, and are applicable to the development and utilization of various probiotic oral drugs, providing a possible new development strategy for the future of the probiotic delivery system; at the same time, the nano-scale milk exosomes embed the micro-scale probiotics, providing a new idea for exosome-targeted bacterial therapy.

[0005] The technical solution of the present invention is as follows:

[0006] A milk exosome-embedded probiotic, comprising probiotics and functionalized milk exosomes coated on the surface of the probiotics; the functionalized milk exosomes are obtained by modifying the surface of milk exosomes with phenylboronic acid groups, and the polysaccharides on the surface of the probiotics have adjacent hydroxyl groups that can covalently bind to phenylboronic acid.

[0007] In the present invention, phospholipid-polyethylene glycol-phenylboronic acid (DSPE-PEG-PBA) is mixed and incubated with milk exosomes, DPP is loaded onto the milk exosomes, the embedding material milk exosomes are functionalized, and the surface of the milk exosomes is modified with phenylboronic acid groups, which covalently bind to the adjacent hydroxyl groups on the surface of the probiotics, so that they can target and bind to the surface of single probiotic cells, thereby achieving the embedding effect.

[0008] Furthermore, the particle size of the milk exosomes is 50-200 nm and is extracted from milk. As a natural nano-carrier, milk exosomes have good biocompatibility. At the same time, milk exosomes can enhance intestinal immunity, repair the intestinal barrier, and regulate the intestinal flora. In this embedding system, milk exosomes can protect probiotics from reaching the intestine and simultaneously synergistically regulate the intestinal flora, having a broader application prospect than other probiotic embedding materials.

[0009] Furthermore, the probiotics are live probiotics, selected from one or more of Bifidobacterium, Akkermansia muciniphila, and Lactobacillus plantarum. Among them, Lactobacillus plantarum is facultatively anaerobic, Bifidobacterium is anaerobic, and Akkermansia muciniphila is strictly anaerobic.

[0010] The present invention also protects a preparation method of milk exosome-embedded probiotics, which is characterized by comprising the following steps:

[0011] (1) Extract milk exosomes, and mix and incubate phospholipid-polyethylene glycol-phenylboronic acid (DPP) with milk exosomes at a mass ratio of 0.5-1.5:1-2 for 18-48 h at room temperature to allow DPP to completely bind to the milk exosomes, obtaining functionalized milk exosomes with phenylboronic acid groups on the surface. Ultracentrifuge to remove the DPP that has not bound to the milk exosomes.

[0012] (2) Ferment the probiotics for 18-48 h, centrifuge to discard the supernatant, wash 2-3 times with PBS buffer, and then centrifuge at high speed to obtain probiotic mud.

[0013] (3) Use the probiotics as the core material, and mix and incubate with the functionalized milk exosomes at a volume ratio of 0.5-1.5:1-2 for 15-45 min at room temperature to allow the phenylboronic acid groups on the surface of the functionalized milk exosomes to covalently bind to the adjacent hydroxyl groups on the surface of the probiotics, preparing a milk exosome-embedded probiotic system.

[0014] In the present invention, milk exosomes are used as a nano-skeleton, and polyethylene glycol polymers are added for assembly to obtain functionalized milk exosomes with phenylboronic acid groups. The reconstituted probiotic solution is mixed and incubated with the functionalized milk exosomes at a volume ratio, so that the phenylboronic acid groups modified on the surface of the milk exosomes undergo a click reaction with the adjacent hydroxyl groups contained in the polysaccharides on the surface of the probiotics. Phenylboronic acid covalently binds to the adjacent hydroxyl groups of the polysaccharides on the surface of the probiotics through the click reaction, enabling the functionalized milk exosomes and probiotics to generate strong covalent bonds, thereby achieving the encapsulation of micron-sized probiotics by nano-sized milk exosomes and protecting the active probiotics in a harsh environment to play their functions in the intestine.

[0015] Further, in step (1), milk exosomes are extracted by ultracentrifugation. The extraction method includes: taking 80-100 mL of fresh raw milk, adding 0.04-0.06 g of rennet and 0.15-0.3 g of CaCl2, quickly mixing evenly and then standing at low temperature for 30-60 min, centrifuging at high speed at low temperature for 30-45 min. After taking the supernatant, ultracentrifuge for 60-90 min, perform ultracentrifugation 2-4 times, suspend with PBS, and centrifuge twice at 4 °C and 5000 r / min using a 100 KDa filter tube to remove small particle precipitates.

[0016] Further, the low temperature is 4 °C, the centrifugal force for the high-speed centrifugation for 30-45 min is 12000-18000×g, and the centrifugal force for the ultracentrifugation is 80000-135000×g. Starting from the second ultracentrifugation, the centrifugal force is set to 120000-140000×g.

[0017] Further, in step (2), when the probiotic is Lactobacillus plantarum, it is cultured in MRS medium for 18 - 24 h;

[0018] When the probiotic is Bifidobacterium, it is anaerobically cultured in MRS for 18 - 24 h;

[0019] When the probiotic is Akkermansia muciniphila, it is anaerobically cultured in BHI medium for 36 - 48 h.

[0020] Further, in step (3), the probiotic mud is redissolved with PBS solution to make the number of bacteria in the probiotic solution reach 10 8 CFU / mL, and then it is mixed with functionalized milk exosomes and incubated at 25 °C for 15 - 45 min to fully bind the phenylboronic acid groups on the surface of the milk exosomes to the adjacent hydroxyl groups on the surface of the probiotics, and then centrifuged at 6000 - 8000 r / min to obtain the probiotic system embedded with milk exosomes.

[0021] The present invention also protects the application of the milk exosome - embedded probiotics described above or the milk exosome - embedded probiotics prepared by the above - described preparation method in the preparation of probiotic oral drugs.

[0022] The present invention also protects a probiotic product, which comprises the milk exosome - embedded probiotics described above or the milk exosome - embedded probiotics prepared by the above - described preparation method.

[0023] Advantages of the present invention:

[0024] The present invention uses functionalized milk exosomes to embed probiotics, enabling the milk exosomes to target and bind to the surface of the probiotics, performing single - cell embedding of the probiotics, endowing them with superior resistance to the harsh gastrointestinal environment, high oral bioavailability, improved adhesion ability and intestinal colonization ability of the probiotics, and also improving the survival rate of the probiotics under gastrointestinal conditions; this active probiotic embedding system provides a possible new development strategy for the probiotic delivery system; meanwhile, it provides a new idea for exosome - targeted bacterial therapy.

[0025] The present invention uses small - molecule natural liposomes to embed active probiotics, which has the advantages of simple process, easy availability of materials, high biocompatibility, etc., realizing the delivery and colonization of probiotics in the digestive tract. At the same time, the milk exosomes can be re - absorbed and utilized by the intestine as prebiotics after reaching the intestine, protecting intestinal health; through experimental verification, this embedding system improves the ability of probiotics to resist the harsh gastrointestinal environment, improves the delivery efficiency of probiotics, and ensures the role of probiotics in regulating the balance of intestinal flora; it can be applied to the development and application field of probiotic products.

[0026] The present invention provides an application of nanoscale extracellular vesicles in the encapsulation of micron-scale probiotics. Milk extracellular vesicles can be used as high-quality drug or probiotic encapsulation and delivery materials, with broad application prospects, providing a new idea for the delivery of probiotics by milk extracellular vesicles. Description of the Drawings

[0027] Figure 1 It is a morphological result diagram and particle size distribution diagram of milk extracellular vesicles under an electron microscope at 100,000X;

[0028] Figure 2 It is a morphological result diagram and particle size distribution diagram of functionalized milk extracellular vesicles under an electron microscope at 100,000X;

[0029] Figure 3 It is a viable count diagram of probiotics after encapsulating probiotics with extracellular vesicles provided in Test Example 3;

[0030] Figure 4 It is an acid tolerance rate diagram of probiotics after encapsulating probiotics with extracellular vesicles provided in Test Example 4;

[0031] Figure 5 It is a bile salt tolerance rate diagram of probiotics after encapsulating probiotics with extracellular vesicles provided in Test Example 5. Detailed Embodiments

[0032] To further understand the present invention, the present invention will be further described in conjunction with the drawings and embodiments. The technical solutions in the embodiments are clearly and completely described; obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the protection scope of the present invention.

[0033] The present invention provides a preparation method for encapsulating probiotics with milk extracellular vesicles, and the milk extracellular vesicle-encapsulated probiotics are prepared, including functionalized milk extracellular vesicles (EV-PBA) with a nanoscale surface-modified phenylboronic acid group, and micron-scale live probiotics encapsulated as the core material. The live probiotics are selected from one or more of Bifidobacterium BB12, Akkermansia muciniphila (AKK), and Lactobacillus plantarum Q7.

[0034] The preparation method provided by the present invention includes the following steps:

[0035] (1) Preparation of functionalized milk exosomes: For freshly produced raw milk purchased on the same day, take 80 - 100 mL, add 0.04 - 0.06 g of rennet and 0.15 - 0.3 g of CaCl₂, quickly mix well and let stand for 30 - 60 min. At 4°C, centrifuge at a high speed of 12000 - 18000×g for 30 - 45 min. After taking the supernatant, centrifuge at an ultra-high speed of 80000 - 135000×g for 60 - 90 min. Starting from the second ultra-high speed centrifugation, set the centrifugal force to 120000 - 140000×g, and after centrifuging 2 - 4 times, suspend the precipitate with PBS and centrifuge twice at 4°C and 5000 r / min using a 100KDa filter tube to remove small particle precipitates, obtaining milk exosomes.

[0036] Mix the fresh milk exosomes with DPP at a mass ratio of 0.5 - 1.5:1 - 2, incubate at 4°C for 18 - 48 h to obtain functionalized milk exosomes with phenylboronic acid groups connected to the surface, and centrifuge at an ultra-high speed of 120000 - 140000×g to remove free polymers.

[0037] (2) Preparation of probiotic bacterial sludge: Ferment a large amount of active probiotics in an Erlenmeyer flask for 18 - 48 h, and centrifuge at 6000 - 8000 r / min to obtain bacterial sludge.

[0038] (3) Preparation of milk exosome-embedded probiotics: Re-dissolve the obtained bacterial sludge with PBS to make the number of bacteria in the probiotic bacterial solution reach 10 8 CFU / mL, and then mix it with the functionalized milk exosomes at a volume ratio of 0.5 - 1.5:1 - 2, and incubate at room temperature for 15 - 45 min to allow the phenylboronic acid groups connected to the surface of the milk exosomes to fully bind to the adjacent hydroxyl groups on the surface of the probiotics.

[0039] Example 1

[0040] This example prepares milk exosome-embedded Lactobacillus plantarum, and its preparation method specifically includes the following steps:

[0041] (1) Preparation of functionalized milk exosomes: For freshly produced raw milk purchased on the same day, take 90 mL, add 0.05 g of rennet and 0.22 g of CaCl₂, quickly mix well and let stand for 45 min. At 4°C, centrifuge at a high speed of 15000×g for 38 min. After taking the supernatant, centrifuge at an ultra-high speed of 110000×g for 75 min. The centrifugal force for the second and third ultra-high speed centrifugations is 130000×g. After centrifuging 3 times, suspend the precipitate with PBS and centrifuge twice at 4°C and 5000 r / min using a 100KDa filter tube to remove small particle precipitates, obtaining milk exosomes.

[0042] Mix fresh milk exosomes with DPP at a mass ratio of 1.0:1.5 and incubate at 4°C for 30 h to obtain functionalized milk exosomes with phenylboronic acid groups attached to the surface. Remove free polymers by ultracentrifugation at 130,000×g.

[0043] (2) Preparation of probiotic bacterial sludge: Culture Lactobacillus plantarum in a conical flask for 21 h and centrifuge at 7000 r / min to obtain bacterial sludge.

[0044] (3) Preparation of milk exosome-embedded probiotics: Resuspend the obtained bacterial sludge with PBS, and then mix the resuspended bacterial liquid with the functionalized milk exosomes at a volume ratio of 1.0:1.5 and incubate at room temperature for 30 min to allow the phenylboronic acid groups attached to the surface of the milk exosomes to fully bind to the adjacent hydroxyl groups on the surface of the probiotics, thus obtaining Lactobacillus plantarum-embedded milk exosomes (Q7@EV-PBA).

[0045] Example 2

[0046] This example prepares a Lactobacillus plantarum-embedded milk exosome, and its preparation method specifically includes the following steps:

[0047] (1) Preparation of functionalized milk exosomes: Take 80 mL of fresh raw milk produced on the same day, add 0.04 g of rennet and 0.15 g of CaCl2, quickly mix well and let stand for 30 min, centrifuge at 12,000×g at 4°C for 30 min, take the supernatant, and then ultracentrifuge at 80,000×g for 60 min. Starting from the second ultracentrifugation, set the centrifugal force to 120,000×g and centrifuge twice. Suspend the precipitate with PBS and centrifuge twice at 5000 r / min at 4°C using a 100KDa filter tube to remove small particle precipitates, obtaining milk exosomes.

[0048] Mix fresh milk exosomes with DPP at a mass ratio of 0.5 - 1.5:1 - 2 and incubate at 4°C for 18 h to obtain functionalized milk exosomes with phenylboronic acid groups attached to the surface. Remove free polymers by ultracentrifugation at 120,000 - 140,000×g.

[0049] (2) Preparation of probiotic bacterial sludge: Culture Lactobacillus plantarum in a conical flask for 18 h and centrifuge at 6000 r / min to obtain bacterial sludge.

[0050] (3) Preparation of milk exosome-embedded probiotics: Resuspend the obtained bacterial sludge with PBS, and then mix the resuspended bacterial liquid with the functionalized milk exosomes at a volume ratio of 0.5:2 and incubate at room temperature for 15 min to allow the phenylboronic acid groups attached to the surface of the milk exosomes to fully bind to the adjacent hydroxyl groups on the surface of the probiotics, thus obtaining Lactobacillus plantarum-embedded milk exosomes (Q7@EV-PBA).

[0051] Example 3

[0052] In this example, a preparation method for embedding Lactobacillus plantarum in milk-derived extracellular vesicles is provided, which specifically includes the following steps:

[0053] (1) Preparation of functionalized milk-derived extracellular vesicles: Take 100 mL of fresh raw milk produced on the same day, add 0.06 g of rennet and 0.3 g of CaCl2, quickly mix well and let stand for 60 min at 4°C, then centrifuge at 18,000×g for 45 min. After taking the supernatant, ultracentrifuge at 135,000×g for 90 min. Starting from the second ultracentrifugation, set the centrifugal force to 140,000×g and centrifuge 4 times. Suspend the precipitate in PBS and centrifuge twice at 5,000 r / min at 4°C using a 100 kDa filter tube to remove small particle precipitates, thus obtaining milk-derived extracellular vesicles.

[0054] Mix the fresh milk-derived extracellular vesicles with DPP at a mass ratio of 1.5:1 and incubate at 4°C for 48 h to obtain functionalized milk-derived extracellular vesicles with phenylboronic acid groups attached to the surface. Ultracentrifuge at 120,000 - 140,000×g to remove free polymers.

[0055] (2) Preparation of probiotic bacterial sludge: Culture Lactobacillus plantarum in an Erlenmeyer flask for 24 h and centrifuge at 8,000 r / min to obtain bacterial sludge.

[0056] (3) Preparation of milk-derived extracellular vesicles embedding probiotics: Re-dissolve the obtained bacterial sludge in PBS, then mix the re-dissolved bacterial liquid with the functionalized milk-derived extracellular vesicles at a volume ratio of 1.5:1 and incubate at room temperature for 45 min to allow the phenylboronic acid groups attached to the surface of the milk-derived extracellular vesicles to fully bind to the adjacent hydroxyl groups on the surface of the probiotics, thus obtaining Lactobacillus plantarum embedded in milk-derived extracellular vesicles (Q7@EV-PBA).

[0057] Example 4

[0058] In this example, a preparation method for embedding Bifidobacterium in milk-derived extracellular vesicles is provided, which specifically includes the following steps:

[0059] (1) Preparation of functionalized milk-derived extracellular vesicles: Take 90 mL of fresh raw milk produced on the same day, add 0.05 g of rennet and 0.22 g of CaCl2, quickly mix well and let stand for 45 min at 4°C, then centrifuge at 15,000×g for 38 min. After taking the supernatant, ultracentrifuge at 110,000×g for 75 min. The centrifugal force for the second and third ultracentrifugations is 130,000×g. Centrifuge 3 times, suspend the precipitate in PBS and centrifuge twice at 5,000 r / min at 4°C using a 100 kDa filter tube to remove small particle precipitates, thus obtaining milk-derived extracellular vesicles.

[0060] Mix fresh milk extracellular vesicles and DPP at a mass ratio of 1:1.5 and incubate at 4 °C for 27 h to obtain functionalized milk extracellular vesicles with phenylboronic acid groups attached to the surface. Ultracentrifuge at 130,000×g to remove free polymers.

[0061] (2) Preparation of probiotic bacterial sludge: Anaerobically culture the Bifidobacterium in an Erlenmeyer flask for 21 h and centrifuge at 7000 r / min to obtain the bacterial sludge.

[0062] (3) Preparation of milk extracellular vesicles embedded with probiotics: Resuspend the obtained bacterial sludge with PBS, and then mix the resuspended bacterial liquid with the functionalized milk extracellular vesicles at a volume ratio of 1:1.5 and incubate at room temperature for 40 min to allow the phenylboronic acid groups attached to the surface of the milk extracellular vesicles to fully bind to the adjacent hydroxyl groups on the surface of the probiotics, thereby preparing milk extracellular vesicles embedded with Bifidobacterium (BB12@EV-PBA).

[0063] Example 5

[0064] This example prepares a milk extracellular vesicle embedded with Akkermansia muciniphila, and its preparation method specifically includes the following steps:

[0065] (1) Preparation of functionalized milk extracellular vesicles: Take 90 mL of freshly produced fresh raw milk purchased, add 0.05 g of rennet and 0.23 g of CaCl2, quickly mix well and let stand for 45 min. Centrifuge at 15,000×g at 4 °C for 38 min. After taking the supernatant, ultracentrifuge at 110,000×g for 75 min. The centrifugal force for the second and third ultracentrifugations is 130,000×g. After centrifuging 3 times, suspend the precipitate with PBS and centrifuge twice at 5000 r / min at 4 °C using a 100KDa filter tube to remove small particle precipitates, thereby obtaining milk extracellular vesicles.

[0066] Mix fresh milk extracellular vesicles and DPP at a mass ratio of 1:1.5 and incubate at 4 °C for 27 h to obtain functionalized milk extracellular vesicles with phenylboronic acid groups attached to the surface. Ultracentrifuge at 130,000×g to remove free polymers.

[0067] (2) Preparation of probiotic bacterial sludge: Anaerobically culture the Akkermansia muciniphila in an Erlenmeyer flask for 42 h and centrifuge at 7000 r / min to obtain the bacterial sludge.

[0068] (3) Preparation of milk extracellular vesicles embedded with probiotics: Resuspend the obtained bacterial sludge with PBS, and then mix the resuspended bacterial liquid with the functionalized milk extracellular vesicles at a volume ratio of 1:1.5 and incubate at room temperature for 38 min to allow the phenylboronic acid groups attached to the surface of the milk extracellular vesicles to fully bind to the adjacent hydroxyl groups on the surface of the probiotics, thereby preparing milk extracellular vesicles embedded with Akkermansia muciniphila (AKK@EV-PBA).

[0069] Test Example 1 Identification of Milk Extracellular Vesicles

[0070] The morphology and particle size distribution of the milk-derived extracellular vesicles extracted in Example 1 were detected using a transmission electron microscope and a particle size analyzer.

[0071] The steps for morphological detection using a transmission electron microscope were as follows: 20 μL of the milk-derived extracellular vesicles were coated on a copper grid covered with a formvar film for 2 min, and the grid was placed on a drop of phosphotungstic acid for 1 min. After drying, it was tested on the machine.

[0072] The steps for particle size distribution detection were as follows: The extracellular vesicles were diluted to a protein concentration of 0.1 mg / mL, and the particle size was detected in a particle size cell.

[0073] The results were as Figure 1 shown. It could be seen from Figure 1 that the milk-derived extracellular vesicles were spherical in shape, had an obvious bilayer structure, and a diameter of about 100 - 200 nm. It could be seen that the obtained milk-derived extracellular vesicles had a uniform particle size distribution, and the average particle size was 142 nm.

[0074] Test Example 2 Identification of Functionalized Milk-Derived Extracellular Vesicles

[0075] The morphology and particle size distribution of the functionalized milk-derived extracellular vesicles prepared in Example 1 were detected using a transmission electron microscope and a particle size analyzer.

[0076] The steps for morphological detection using a transmission electron microscope were as follows: 20 μL of the functionalized milk-derived extracellular vesicles were coated on a copper grid covered with a formvar film for 2 min, and the grid was placed on a drop of phosphotungstic acid for 1 min. After drying, it was tested on the machine.

[0077] The steps for particle size distribution detection were as follows: The extracellular vesicles were diluted to a protein concentration of 0.1 mg / mL, and the particle size was detected in a particle size cell.

[0078] The results were as Figure 2 shown. It could be seen from Figure 2 that the functionalized milk-derived extracellular vesicles were spherical in shape, and the diameter increased to 150 - 200 nm. It could be seen that the obtained milk-derived extracellular vesicles had a uniform particle size distribution, and the average particle size was 190 nm, indicating that the particle size increased after the phenylboronic acid group was connected to the surface of the milk-derived extracellular vesicles.

[0079] Test Example 3 Effect of Probiotics Encapsulated in Functionalized Milk-Derived Extracellular Vesicles on Probiotic Activity

[0080] Test raw materials:

[0081] Lactobacillus plantarum Q7 used in Example 1; Lactobacillus plantarum encapsulated in milk-derived extracellular vesicles prepared in Example 1 (Q7@EV-PBA);

[0082] Bifidobacterium BB12 used in Example 4; Bifidobacterium embedded in milk-derived extracellular vesicles prepared in Example 4 (BB12@EV-PBA);

[0083] Akkermansia muciniphila AKK used in Example 5; Akkermansia muciniphila embedded in milk-derived extracellular vesicles prepared in Example 5 (AKK@EV-PBA);

[0084] Viable cell counts of the above test raw materials were detected. The steps of the detection method are as follows:

[0085] Prepare MRS solid medium and brain heart infusion (BHI) solid medium. Dilute the probiotics (Q7 and BB12) used in Examples 1 and 4 and the encapsulated probiotics (Q7@EV-PBA and BB12@EV-PBA) prepared to concentration gradients of 10 -5 、10 -6 、10 -7 . Take 0.1 mL of each concentration gradient into a sterile petri dish, then pour 15 mL of the sterilized MRS solid medium cooled to 45 - 50 °C, mix well, and incubate at 37 °C after solidification. Dilute the probiotic (AKK) used in Example 5 and the encapsulated probiotic (AKK@EV-PBA) prepared to concentration gradients of 10 -5 、10 -6 、10 -7 . Take 0.1 mL of each concentration gradient into a sterile petri dish, then pour 15 mL of the sterilized BHI solid medium cooled to 45 - 50 °C, mix well, and incubate at 37 °C after solidification. Calculate the viable cell counts after 48 h. The specific results are shown in Figure 3 .

[0086] From Figure 3 it can be concluded that after being encapsulated by functionalized extracellular vesicles, Lactobacillus plantarum, Bifidobacterium, and Akkermansia muciniphila have no significant effect on the survival rate of probiotics, indicating that the functionalized extracellular vesicles have no inhibitory effect on the growth of the three selected strains in the experiment.

[0087] Test Example 4 Acid Resistance Test

[0088] Using the test raw materials of Test Example 3, the acid resistance test was carried out.

[0089] Test method: Adjust PBS to pH 3 with 1 M hydrochloric acid, sterilize at 121 °C. Inoculate each probiotic and the encapsulated probiotic into sterile PBS with pH 3 and incubate at 37 °C for 33 h. Determine the viable cell counts by plate counting method, and the counting results of each group at 0 h are used as the control. The following survival rate formula is used to calculate the acid resistance ability of the strains:

[0090] Survival rate (%) = logCFUN t / logCFUN0 × 100%

[0091] Among them, N0 represents the total number of colonies before the strain treatment; N t represents the total number of colonies after the strain treatment. The specific results are shown in Figure 4 .

[0092] From Figure 4 it can be seen that the survival rates of Lactobacillus plantarum, Bifidobacterium, and Akkermansia muciniphila are significantly increased after being encapsulated by functionalized extracellular vesicles, indicating that the functionalized extracellular vesicles have acid protection ability for probiotics after encapsulating them, and improve the acid tolerance of the strain.

[0093] Test Example 5 Bile Salt Tolerance Test

[0094] Using the test materials in Test Example 3, the bile salt tolerance test was carried out.

[0095] Test method: Add 0.3 g of bile salt to 100 mL of PBS, adjust the pH of the solution to 6.8 - 7.4 and then sterilize; then add the encapsulated probiotics or unencapsulated probiotics into the bile salt solution, culture at 37 °C for 4 h, and determine the viable count by the plate counting method. The intestinal bile salt protection ability of the functionalized extracellular vesicles on probiotics was evaluated by the survival rate of the strain. The following survival rate formula was used to calculate the acid tolerance of the strain;

[0096] Survival rate (%) = logCFUN t / logCFUN0 × 100%

[0097] Among them, N0 represents the total number of colonies before the strain treatment; N t represents the total number of colonies after the strain treatment. The specific results are shown in Figure 5 .

[0098] From Figure 5 it can be seen that the survival rates of Lactobacillus plantarum, Bifidobacterium, and Akkermansia muciniphila are significantly increased after being encapsulated by functionalized extracellular vesicles, indicating that the bile salt tolerance of the probiotics is enhanced after the functionalized extracellular vesicles encapsulate the probiotics.

[0099] Through the above test examples, it can be found that the probiotics encapsulated by the bovine milk extracellular vesicles prepared by the present invention, the extracellular vesicles do not affect the survival rate of the probiotics, and the stability in acidic solution and bile salt solution is much better than that of the unencapsulated probiotics, enabling the probiotics to tolerate the harsh gastrointestinal environment and improving the survival rate of the probiotics reaching the intestine.

[0100] The above description is only a preferred embodiment of the present invention and is not a limitation of the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, modifications, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of probiotics embedded in milk-derived extracellular vesicles, characterized in that, It includes the following steps: (1) Extract milk exosomes, and mix and incubate phospholipid-polyethylene glycol-phenylboronic acid with milk exosomes at a mass ratio of 0.5-1.5:1-2 for 18-48 h at 4°C to completely connect the phospholipid-polyethylene glycol-phenylboronic acid to the milk exosomes, obtaining functionalized milk exosomes with phenylboronic acid groups modified on the surface; (2) Ferment and culture the probiotics for 18-48 h, centrifuge to discard the supernatant, wash 2-3 times with PBS buffer, and then centrifuge at high speed to obtain probiotic mud; (3) Mix and incubate the probiotics with the functionalized milk exosomes at a volume ratio of 0.5-1.5:1-2 for 15-45 min at room temperature to prepare a probiotic system embedded in milk exosomes; The prepared probiotic embedded in milk exosomes includes probiotics and functionalized milk exosomes coated on the surface of the probiotics; the functionalized milk exosomes are obtained by modifying phenylboronic acid groups on the surface of milk exosomes, and the polysaccharides on the surface of the probiotics have adjacent hydroxyl groups that can covalently bind to phenylboronic acid; The probiotics are active probiotics, selected from one or more of Bifidobacterium, Akkermansia muciniphila, and Lactobacillus plantarum.

2. The preparation method according to claim 1, characterized in that, The milk exosomes have a particle size of 50-200 nm and are extracted from milk.

3. The preparation method according to claim 1, characterized in that, In the step (1), the milk exosomes are extracted by the ultracentrifugation method, and the extraction method includes: taking 80-100 mL of fresh raw milk, adding 0.04-0.06 g of rennet and 0.15-0.3 g of CaCl2, quickly mixing and then standing at low temperature for 30-60 min, centrifuging at high speed at low temperature for 30-45 min, taking the supernatant, then ultracentrifuging for 60-90 min, ultracentrifuging 2-4 times, suspending with PBS, and centrifuging twice at 5000 r / min at 4°C using a 100 KDa filter tube to obtain the milk exosomes.

4. The preparation method according to claim 3, characterized in that, The low temperature is 4°C, the centrifugal force for high-speed centrifugation is 12000-18000×g, and the centrifugal force for ultracentrifugation is 80000-135000×g. Starting from the second ultracentrifugation, the centrifugal force is set to 120000-140000×g.

5. The preparation method according to claim 1, characterized in that, In the step (2), when the probiotic is Lactobacillus plantarum, it is cultured in MRS medium for 18-24 h; When the probiotic is Bifidobacterium, it is anaerobically cultured in MRS medium for 18-24 h; When the probiotic is Akkermansia muciniphila, it is anaerobically cultured in BHI medium for 36-48 h.

6. The preparation method according to claim 1, wherein In the step (3), the probiotic bacterial sludge is first redissolved with PBS solution to make the number of bacteria in the probiotic bacterial liquid reach 10 8 CFU / mL, and then mixed with functionalized milk exosomes, incubated at 25 °C for 15 - 45 min to fully bind the phenylboronic acid groups on the surface of milk exosomes to the adjacent hydroxyl groups on the surface of probiotics, and centrifuged at 6000 - 8000 r / min at high speed to obtain the probiotic system embedded with milk exosomes.

7. Use of the probiotic embedded in milk exosomes prepared by the preparation method according to any one of claims 1-6 in the preparation of probiotic oral drugs.

8. A probiotic product, characterized in that, It includes the probiotic embedded in milk exosomes prepared by the preparation method according to any one of claims 1-6.

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