Extracellular vesicle of bifidobacterium animalis, extraction method therefor, and use thereof in treatment and / or prevention of nerve injury

Abstract

Provided are an extracellular vesicle of Bifidobacterium animalis, an extraction method therefor, and use of the extracellular vesicle of Bifidobacterium animalis in the treatment and / or prevention of nerve injury. The extraction method comprises: 1) subjecting a bacterial suspension of Bifidobacterium animalis to a low-speed centrifugation treatment and a high-speed centrifugation treatment in sequence, and collecting a supernatant; 2) subjecting the supernatant to an ultrafiltration centrifugation treatment, and collecting a filtrate; 3) subjecting the filtrate to an ultra-high-speed centrifugation treatment, and collecting a supernatant; and 4) subjecting the supernatant to an ultrafiltration treatment, wherein the filtrate contains the extracellular vesicle of Bifidobacterium animalis.

Description

Extracellular vesicles of Bifidobacterium animalis, their extraction method, and their application in the treatment and / or prevention of nerve damage. Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an extracellular vesicle of Bifidobacterium animalis, its extraction method, and its application in the treatment and / or prevention of nerve damage. Background Technology

[0002] Extracellular vesicles (MVs) of lactic acid bacteria (such as Bifidobacterium animalis) are vesicular bodies released into their environment. Their structure consists of a lipid bilayer encapsulating cellular components. These are nanoscale active substances with a diameter of 20-400 nm, capable of shuttling between cells and transmitting information. MVs are natural carriers of lactic acid bacteria (such as Bifidobacterium animalis) molecules, including peptidoglycans, lipids, proteins, and nucleic acids. As a cellular secretion substance, studies have found that MVs can be transported throughout the body via the bloodstream and can also cross the blood-brain barrier to reach the host's brain. Currently, MVs are an important therapeutic carrier in the medical field. However, research on extraction methods for MVs, especially methods to increase yield and preserve basic properties, is still lacking. Therefore, providing a preparation method that is free of chemical irritants and can simultaneously increase MV yield and preserve its basic properties is of long-term significance.

[0003] Inflammatory neurological injury refers to inflammation of nerve tissue. There are many causes of this inflammation, including infection, traumatic brain injury, toxic metabolites from mental illness, or autoimmune disorders. This injury can further lead to a range of neurological diseases, including neurodegenerative diseases such as Parkinson's and Alzheimer's. In the central nervous system (CNS), including the brain and spinal cord, microglia are resident innate immune cells that respond to signals. The CNS is a typical site of immune privilege because peripheral immune cells are usually blocked by the blood-brain barrier (BBB). When the BBB weakens, microglia produce reactive oxygen species (ROS) and release signals that invoke an inflammatory response from peripheral immune cells. Therefore, exploring the effects of novel active ingredients on brain nerves is crucial for developing novel treatments for neuroinflammation. Summary of the Invention

[0004] This invention provides a method for extracting extracellular vesicles from Bifidobacterium animalis. The method yields high-yield extracellular vesicles that can enter various organs of the animal body via the bloodstream and cross the blood-brain barrier to enter the mouse brain, showing broad application prospects in the medical field. Furthermore, this invention also reveals that the extracellular vesicles of this Bifidobacterium animalis have a role in alleviating nerve damage.

[0005] Therefore, in a first aspect, the present invention provides a method for extracting extracellular vesicles of Bifidobacterium animalis, comprising:

[0006] 1) The bacterial suspension of Bifidobacterium animalis was subjected to low-speed centrifugation and high-speed centrifugation in sequence, and the supernatant was collected.

[0007] 2) Perform ultrafiltration and centrifugation on the supernatant, and collect the filtrate;

[0008] 3) Centrifuge the filtrate at ultra-high speed and collect the supernatant;

[0009] 4) The supernatant is subjected to ultrafiltration, and the filtrate contains the extracellular vesicles of Bifidobacterium animalis.

[0010] In some implementations, the pore size of the ultrafiltration centrifugation process is 100 kDa.

[0011] In some embodiments, the ultrafiltration centrifugation is carried out at a rotation speed of 4000-6000g (e.g., 4000g, 4200g, 4400g, 4600g, 4800g, 5000g, 5200g, 5400g, 5600g, 5800g or 6000g, preferably 5000g).

[0012] In some embodiments, the ultrafiltration centrifugation treatment time is 5-15 min (e.g., 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min or 15 min), preferably 10 min.

[0013] In some implementations, the ultrafiltration centrifugation process is performed using ultrafiltration centrifuge tubes (such as Amicon Ultra-15 ultrafiltration centrifuge tubes).

[0014] In some embodiments, the low-speed centrifugation is performed in two steps. The first low-speed centrifugation is carried out at 3500-4000g (e.g., 3500g, 3600g, 3700g, 3800g, 3900g or 4000g, preferably 3600g) for 20-40 minutes (e.g., 20min, 25min, 30min, 35min or 40min, preferably 30min). The second low-speed centrifugation is carried out at 5000-5500g (e.g., 5000g, 5100g, 5200g, 5300g, 5400g or 5500g, preferably 5000g) for 20-40 minutes (e.g., 20min, 25min, 30min, 35min or 40min, preferably 30min).

[0015] In some embodiments, the high-speed centrifugation is carried out at 10,000-12,000g (e.g., 10,000g, 10,200g, 10,400g, 10,600g, 10,800g, 11,000g, 11,200g, 11,400g, 11,600g, 11,800g or 12,000g, preferably 11,000g) for 50-70 minutes (e.g., 50 minutes, 55 minutes, 60 minutes, 65 minutes or 70 minutes, preferably 60 minutes).

[0016] In some embodiments, the ultracentrifugation process is performed in two stages. The first ultracentrifugation is carried out at 110,000-130,000 g (e.g., 112,000 g, 114,000 g, 116,000 g, 118,000 g, 120,000 g, 122,000 g, 124,000 g, 126,000 g, 128,000 g, or 130,000 g, preferably 120,000 g) for 80-100 min (e.g., 80 min, 85 min, 90 min, 95 min, or 100 min). The second ultra-high speed centrifugation is carried out at 110,000-130,000g (e.g., 112,000g, 114,000g, 116,000g, 118,000g, 120,000g, 122,000g, 124,000g, 126,000g, 128,000g or 130,000g, preferably 120,000g) for 80-100 minutes (e.g., 80 minutes, 85 minutes, 90 minutes, 95 minutes or 100 minutes, preferably 90 minutes).

[0017] In some implementations, the ultrafiltration process is achieved using an ultrafiltration membrane.

[0018] In some implementations, the pore size of the ultrafiltration process is 0.22 μm.

[0019] In some embodiments, the low-speed centrifugation is carried out at a temperature of 4°C.

[0020] In some implementations, the high-speed centrifugation process is carried out at a temperature of 4°C.

[0021] In some implementations, the ultra-high-speed centrifugation process is carried out at a temperature of 4°C.

[0022] In some implementations, step 4) further includes ultrafiltration of the supernatant followed by diluting the filtrate with PBS buffer, wherein the PBS buffer contains the extracellular vesicles of the animal Bifidobacterium.

[0023] In some embodiments, the *Bifidobacterium animalis* is *Bifidobacterium lactis* subspecies BL-99, with accession number CGMCC No. 15650. *Bifidobacterium lactis* subspecies BL-99 was deposited on April 26, 2018, at the China General Microbiological Culture Collection Center (CGMCC) (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), and is classified as *Bifidobacterium lactis*; accession number CGMCC No. 15650. For further details, see CN110964657B.

[0024] In some embodiments, the bacterial suspension of *Bifidobacterium animalis* is obtained by pre-fermenting the *Bifidobacterium animalis*. The fermentation can be performed using conventional fermentation conditions in the art, such as incubation at 37°C in MRS liquid medium.

[0025] In a second aspect of the invention, the invention provides extracellular vesicles of Bifidobacterium animalis, which are extracted by the method of the first aspect.

[0026] In some embodiments, the extracellular vesicles of the animal Bifidobacterium have a particle size of 50-100 nm (e.g., 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm or 100 nm, such as 85-90 nm).

[0027] In a third aspect of the invention, the invention provides modified Bifidobacterium animalis extracellular vesicles, which are obtained by modifying natural Bifidobacterium animalis extracellular vesicles, said natural Bifidobacterium animalis extracellular vesicles being extracted by the method of the first aspect or as described in the second aspect.

[0028] In a fourth aspect of the invention, the invention provides a lysate or lysate of extracellular vesicles of Bifidobacterium animalis, obtained by dissolving or lysing the extracellular vesicles of Bifidobacterium animalis, wherein the extracellular vesicles of Bifidobacterium animalis are extracted by the method of the first aspect or as described in the second aspect.

[0029] In a fifth aspect of the invention, the invention provides a composition comprising extracellular vesicles of Bifidobacterium animalis extracted by the method of the first aspect, or comprising extracellular vesicles of Bifidobacterium animalis of the second aspect, or comprising modified extracellular vesicles of Bifidobacterium animalis of the third aspect, or comprising a lysate and / or a lysate of extracellular vesicles of Bifidobacterium animalis of the fourth aspect.

[0030] In a sixth aspect of the invention, the invention provides the use of Bifidobacterium animalis extracellular vesicles as a drug carrier or the use of Bifidobacterium animalis extracellular vesicles to prepare a drug carrier, wherein the Bifidobacterium animalis extracellular vesicles are natural or modified, wherein the natural Bifidobacterium animalis extracellular vesicles are obtained by extraction by the method of the first aspect or as described in the second aspect, and the modified Bifidobacterium animalis extracellular vesicles are as described in the third aspect.

[0031] In a seventh aspect, the present invention provides the use of extracellular vesicles of *Bifidobacterium animalis* subsp. *lactamase* BL-99 in the preparation of pharmaceuticals, common foods, health foods, food additives, and / or dietary supplements, wherein the pharmaceuticals are used to treat and / or prevent nerve damage, and the common foods, health foods, food additives, and / or dietary supplements are used to alleviate nerve damage, wherein the *Bifidobacterium animalis* subsp. *lactamase* BL-99 has the accession number CGMCC No. 15650. In some embodiments, the extracellular vesicles of *Bifidobacterium animalis* subsp. *lactamase* BL-99 are obtained by extraction using the method of the first aspect or as described in the second aspect.

[0032] In an eighth aspect of the invention, the invention provides the use of a composition in the preparation of a pharmaceutical, a common food, a health food, a food additive, and / or a dietary supplement, said composition comprising extracellular vesicles of *Bifidobacterium lactis* subsp. *animal* BL-99, said pharmaceutical for treating and / or preventing nerve damage, said common food, health food, food additive, and / or dietary supplement for alleviating nerve damage, said *Bifidobacterium lactis* subsp. *animal* BL-99 having the accession number CGMCC No. 15650. In some embodiments, said *Bifidobacterium lactis* subsp. *animal* BL-99 extracellular vesicles are obtained by extraction using the method of the first aspect or as described in the second aspect.

[0033] In a ninth aspect of the invention, the invention provides the use of a metabolite of *Bifidobacterium lactis* subsp. 99 in the preparation of pharmaceuticals, common foods, health foods, food additives, and / or dietary supplements, wherein the *Bifidobacterium lactis* subsp. 99 metabolite comprises *Bifidobacterium lactis* subsp. 99 extracellular vesicles, the pharmaceuticals are used to treat and / or prevent nerve damage, the common foods, health foods, food additives, and / or dietary supplements are used to alleviate nerve damage, and the *Bifidobacterium lactis* subsp. 99 has the accession number CGMCC No. 15650. In some embodiments, the *Bifidobacterium lactis* subsp. 99 extracellular vesicles are obtained by the method of the first aspect or as described in the second aspect.

[0034] In a tenth aspect of the invention, the present invention provides the use of a solution or lysate of extracellular vesicles of *Bifidobacterium lactis* subsp. *lactamase* BL-99 in the preparation of pharmaceuticals, common foods, health foods, food additives, and / or dietary supplements. The solution or lysate is obtained by dissolving or lysing the extracellular vesicles of *Bifidobacterium lactis* subsp. *lactamase* BL-99. The pharmaceuticals are used to treat and / or prevent nerve damage, and the common foods, health foods, food additives, and / or dietary supplements are used to alleviate nerve damage. The *Bifidobacterium lactis* subsp. *lactamase* BL-99 has the accession number CGMCC No. 15650. In some embodiments, the extracellular vesicles of *Bifidobacterium lactis* subsp. *lactamase* BL-99 are obtained by extraction using the method of the first aspect or as described in the second aspect.

[0035] In an eleventh aspect of the present invention, the present invention provides extracellular vesicles of *Bifidobacterium lactis* subsp. laciliforme, metabolites of *Bifidobacterium lactis* subsp. laciliforme BL-99, solutions or lysates of extracellular vesicles of *Bifidobacterium lactis* subsp. laciliforme BL-99, or compositions thereof, for the treatment and / or prevention of nerve injury or the relief of nerve injury. The composition or the metabolites of *Bifidobacterium lactis* subsp. laciliforme BL-99 contain extracellular vesicles of *Bifidobacterium lactis* subsp. laciliforme BL-99 extracellular vesicles are obtained by dissolving or lysing the extracellular vesicles of *Bifidobacterium lactis* subsp. laciliforme BL-99 has the accession number CGMCC No. 15650. In some embodiments, the extracellular vesicles of *Bifidobacterium lactis* subsp. laciliforme BL-99 are obtained by extraction using the method of the first aspect or as described in the second aspect.

[0036] In a twelfth aspect of the invention, the invention provides a method for treating and / or preventing or alleviating nerve injury, comprising administering to a subject in need an effective amount of *Bifidobacterium lactis* subsp. lacryma-jobi BL-99 extracellular vesicles, *Bifidobacterium lactis* subsp. lacryma-jobi BL-99 metabolites, a solution or lysate of *Bifidobacterium lactis* subsp. lacryma-jobi BL-99 extracellular vesicles, or a composition thereof, wherein the composition or the *Bifidobacterium lactis* subsp. lacryma-jobi BL-99 metabolites comprise *Bifidobacterium lactis* subsp. lacryma-jobi BL-99 extracellular vesicles, and the solution or lysate of the *Bifidobacterium lactis* subsp. lacryma-jobi BL-99 is obtained by dissolving or lysing the *Bifidobacterium lactis* subsp. lacryma-jobi BL-99, the accession number of which is CGMCC No. 15650. In some embodiments, the *Bifidobacterium lactis* subsp. lacryma-jobi BL-99 extracellular vesicles are obtained by the method of the first aspect or as described in the second aspect.

[0037] In a thirteenth aspect of the invention, a method for inhibiting / alleviating inflammation of nerve cells is provided, comprising administering to nerve cells an effective amount of *Bifidobacterium lactis* subsp. lacryma-jobi BL-99 extracellular vesicles, *Bifidobacterium lactis* subsp. lacryma-jobi BL-99 metabolites, a solution or lysate of *Bifidobacterium lactis* subsp. lacryma-jobi BL-99 extracellular vesicles, or a composition thereof, wherein the composition or the *Bifidobacterium lactis* subsp. lacryma-jobi BL-99 metabolites comprise *Bifidobacterium lactis* subsp. lacryma-jobi BL-99 extracellular vesicles, and the solution or lysate of the *Bifidobacterium lactis* subsp. lacryma-jobi BL-99 is obtained by dissolving or lysing the *Bifidobacterium lactis* subsp. lacryma-jobi BL-99, the preservation number of which is CGMCC No. 15650. In some embodiments, the *Bifidobacterium lactis* subsp. lacryma-jobi BL-99 extracellular vesicles are obtained by extraction using the method of the first aspect or as described in the second aspect.

[0038] In some embodiments, the dosage of the extracellular vesicles of Bifidobacterium animalis subsp. lactis BL-99 is 50-400 ng / μL, such as 50 ng / μL, 100 ng / μL, 200 ng / μL or 400 ng / μL, preferably 200 ng / μL.

[0039] In some implementations, the nerve injury is an inflammatory nerve injury.

[0040] In some implementations, the inflammatory nerve injury is LPS-induced inflammatory nerve injury.

[0041] The term "treatment" generally refers to achieving a desired pharmacological and / or physiological effect. This effect can be preventative, based on the complete or partial prevention of the disease or its symptoms; and / or therapeutic, based on the partial or complete stabilization or cure of the disease and / or side effects resulting from the disease. As used herein, "treatment" encompasses any treatment of a patient's disease, including: (a) prevention of disease or symptoms occurring in a patient who is susceptible to the disease or its symptoms but has not yet been diagnosed with the disease; (b) suppression of the symptoms of the disease, i.e., prevention of its progression; or (c) relief of the symptoms of the disease, i.e., causing the disease or its symptoms to regress.

[0042] The term "subject" refers to a vertebrate. In some embodiments, vertebrate refers to a mammal. Mammals include, but are not limited to, livestock (such as cattle), pets (such as cats, dogs, and horses), primates, mice, and rats. In some embodiments, mammal refers to a human.

[0043] The term "effective amount" refers to the amount that effectively achieves the desired therapeutic or preventative effect at the necessary dose and time. The "therapeutic effective amount" of the substance / molecule of this invention can vary depending on factors such as an individual's disease state, age, sex, weight, and the substance / molecule's ability to elicit the desired response in the individual. Therapeutic effective amount also encompasses the amount in which the beneficial therapeutic effect of the substance / molecule outweighs any toxic or harmful consequences. "Preventative effective amount" refers to the amount that effectively achieves the desired preventative effect at the necessary dose and time. Typically, but not necessarily, the preventative effective amount will be lower than the therapeutic effective amount because the preventative dose is administered to the subject before or in the early stages of the disease. Beneficial effects

[0044] 1. This invention clarifies an extracellular vesicle derived from Bifidobacterium lactis subsp. BL-99 and its extraction process and method.

[0045] 2. The extracellular vesicle extraction method of Bifidobacterium lactis subsp. BL-99 provided by the present invention combines low-speed centrifugation, high-speed centrifugation, ultrafiltration centrifugation, ultra-high-speed centrifugation and ultrafiltration under certain conditions, resulting in a large number of extracted extracellular vesicles with high protein concentration and good dispersibility.

[0046] 3. This invention has discovered that extracellular vesicles of Bifidobacterium animalis BL-99 can enter various organs of an animal through the bloodstream.

[0047] 4. This invention has discovered that extracellular vesicles of Bifidobacterium animalis BL-99 can cross the blood-brain barrier and enter the mouse brain.

[0048] 5. This invention has discovered that extracellular vesicles of Bifidobacterium lactis subsp. BL-99 have the effect of relieving nerve damage (such as inflammatory nerve damage).

[0049] 6. The extracellular vesicles of Bifidobacterium lactis subsp. BL-99 provided by this invention can inhibit neuroinflammation and alleviate nerve damage (such as inflammatory nerve damage) by regulating the levels of growth factors, anti-inflammatory factors and pro-inflammatory factors.

[0050] 7. The extracellular vesicles of Bifidobacterium lactis subsp. BL-99 provided by this invention can inhibit nerve inflammation and alleviate nerve damage (such as inflammatory nerve damage) by reducing the production of reactive oxygen species.

[0051] 8. Animal experiments have confirmed that the extracellular vesicles of Bifidobacterium lactis subsp. BL-99 provided by this invention can effectively alleviate brain nerve damage (such as inflammatory nerve damage). Attached Figure Description

[0052] Figure 1 is a schematic diagram of the BL-99 bacterial vesicle extraction and separation method in Embodiment 1 of the present invention.

[0053] Figure 2 is a comparison of the protein concentrations of BL-99-MVs extracted by different methods in Example 1 of the present invention.

[0054] Figure 3 shows the particle size distribution of BL-99-MVs extracted by different methods in Embodiment 1 of the present invention.

[0055] Figure 4 shows the morphological structure of BL-99-MVs extracted by different methods in Embodiment 1 of the present invention under a transmission electron microscope.

[0056] Figure 5 is a comparison of the relative quantities of BL-99-MVs extracted by different methods in Embodiment 1 of the present invention.

[0057] Figure 6 shows the fluorescence display of mice 2 hours after gavage administration of fluorescently labeled BL-99-MVs in Example 3 of the present invention.

[0058] Figure 7 shows the fluorescence display of mouse brain and intestine 2 hours after gavage administration of fluorescently labeled BL-99-MVs in Example 3 of the present invention.

[0059] Figure 8 shows the effect of different concentrations of Bifidobacterium animalis BL-99 extracellular vesicles on the survival rate of LPS-induced inflammatory HT22 cells in Example 4 of the present invention; "*" indicates p<0.05; "**" indicates p<0.01; "***" indicates p<0.001.

[0060] Figure 9 shows the effect of 200 ng / μl concentration of Bifidobacterium animalis BL-99 extracellular vesicles on growth factors in HT22 cells in Example 5 of the present invention; "*" indicates p<0.05; "**" indicates p<0.01; "***" indicates p<0.001.

[0061] Figure 10 shows the effect of 200 ng / μl concentration of Bifidobacterium animalis BL-99 extracellular vesicles on inflammatory factors in HT22 cells in Example 5 of the present invention; "*" indicates p<0.05; "**" indicates p<0.01; "***" indicates p<0.001.

[0062] Figure 11 shows the detection results of reactive oxygen species (ROS) in HT22 cells by extracellular vesicles of Bifidobacterium animalis BL-99 at a concentration of 200 ng / μl in Example 6 of the present invention; "*" means p<0.05; "**" means p<0.01; "***" means p<0.001.

[0063] Figure 12 shows the intervention status of the Ctrl group, LPS-encephalitis group, and MV group in Embodiment 7 of the present invention.

[0064] Figure 13 shows typical water maze trajectories of mice in each group in Example 7 of the present invention.

[0065] Figure 14 shows typical novel object recognition trajectories of mice in each group in Example 7 of the present invention. Detailed Implementation

[0066] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings. However, those skilled in the art will understand that the following embodiments and drawings are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the accompanying drawings and preferred embodiments. Unless otherwise specified, all raw materials and reagents are commercially available. Specifically, *Bifidobacterium lactis* subsp. BL-99 was deposited on April 26, 2018, at the China General Microbiological Culture Collection Center (CGMCC) (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), classified and named as *Bifidobacterium lactis*; accession number CGMCC No. 15650. For example, see CN110964657B for further information.

[0067] Example 1:

[0068] This embodiment prepared extracellular vesicles of Bifidobacterium lactis subsp. BL-99 and compared the protein concentrations of two preparation methods. As shown in Figure 1, compared with method two, method one is exactly the same as method two except that it does not use ultrafiltration centrifuge tubes (100 kDa pore size, Amicon Ultra-15) for ultrafiltration centrifugation (5000 g, 10 min).

[0069] Fermentation was performed using Bifidobacterium animalis subsp. lactis BL-99 (cultured in MRS liquid medium at 37°C) to obtain 1 L of bacterial suspension.

[0070] Method 1: At 4°C, the bacterial suspension of *Bifidobacterium animalis* was subjected to sequential low-speed centrifugation (first time, 3600g, 30min; second time, 5000g, 30min) and high-speed centrifugation (11000g, 60min). The supernatant was then extracted. The supernatant was further subjected to two ultracentrifugations at 4°C (120000g, 90min) to extract the supernatant. The supernatant was then ultrafiltered using a 0.22µm ultrafiltration membrane, and the filtrate was diluted with PBS buffer. The PBS buffer contained the extracellular vesicles of BL-99.

[0071] Method 2: At 4°C, the bacterial suspension of *Bifidobacterium animalis* was sequentially centrifuged at low speed (3600g, 30min; 5000g, 30min) and high speed (11000g, 60min), and the supernatant was extracted after centrifugation. The supernatant was then ultrafiltered and centrifuged (5000g, 10min) using an ultrafiltration centrifuge tube (100kDa pore size, Amicon Ultra-15) to obtain a relatively pure supernatant. The supernatant obtained after ultrafiltration was then subjected to two ultra-high speed centrifugations at 4°C (120000g, 90min) to extract the supernatant. The supernatant was then ultrafiltered using an ultrafiltration membrane (0.22µm), and the filtrate was diluted with PBS buffer. The PBS dilution contained the extracellular vesicles of BL-99.

[0072] The protein concentration was determined according to GB 5009.5-2016, "National Food Safety Standard - Determination of Protein in Food". The test results are shown in Figure 2.

[0073] The test results show that the concentration of BL-99-MVs protein extracted by method 1 is 1.29 ug / ul; the concentration of BL-99-MVs protein extracted by method 2 is 1.36 ug / ul. It can be seen that the protein concentration extracted by method 2 is significantly higher than that extracted by method 1.

[0074] Example 2:

[0075] Particle size analysis was performed on the BL-99-MVs extracted by methods one and two as described in Example 1. The particle size analysis was performed using a particle size analyzer. The particle size distribution detection method was as follows: 700 μL of extracellular vesicles of 20 μg / mL Bifidobacterium animalis BL-99 was added to a particle size distribution dish for particle size analysis, and the mixture was gradually diluted until the particle size no longer changed, which was taken as the final result. The analysis results are shown in Figure 3.

[0076] As shown in Figure 3, the BL-99-MVs extracted by Method 1 have a particle size concentrated at 141.9 nm, while the BL-99-MVs extracted by Method 2 have a particle size concentrated at 87.11 nm.

[0077] Figure 4 shows the morphological structure of BL-99-MVs extracted by methods one and two described in Example 1 under a transmission electron microscope. The morphological detection method for transmission electron microscopy is as follows: 20 μL of extracellular vesicles of Bifidobacterium animalis BL-99 was placed on a 300-mesh copper grid, allowed to dry naturally, and then 20 μL of phosphotungstic acid was added for negative staining. After drying naturally, the sample was then analyzed by the microscope.

[0078] As shown in Figure 4, the BL-99-MVs structures extracted by Method 1 and Method 2 are generally spherical, but the particle size differences are obvious.

[0079] Based on transmission electron microscopy images and particle size analysis, it was found that the BL-99-MVs extracted by method two had smaller particle size and better dispersibility compared with method one.

[0080] The number and size of BL-99-MVs extracted by methods one and two described in Example 1 were determined by flow cytometry (Reference: Inamdar S, Nitiyanandan R, Rege K. Emerging applications of exosomes in cancer therapeutics and diagnostics[J]. Bioeng Transl Med, 2017, 2(1):70.). The results are shown in Figure 5 and Table 1.

[0081] Figure 5 and Table 1 show that the number of extracellular vesicles extracted by method 2 is about 1.6 times that extracted by method 1 ("**" means p<0.01). It can be seen that the number of extracellular vesicles extracted by method 2 is significantly higher than that extracted by method 1.

[0082] Table 1: Comparison of the number of extracellular vesicles extracted by different methods

[0083] Example 3:

[0084] 100 μL of BL99-MVs (200 ng / μL) extracted by method 2 in Example 1 was co-cultured with 10 μL of Dir dye (100 μM). BL-99-MVs were fluorescently labeled, and after incubation at 37°C for 30 min, mice were administered the solution via gavage. The fluorescence detection results (using the IVIS Spectrum small animal in vivo tracer system) are shown in Figure 6.

[0085] As shown in Figure 6, strong fluorescence was observed in the stomach, intestine, liver, and kidney of the mice 2 hours after gavage, proving that BL-99-MVs entered different organs of the body through the digestive tract.

[0086] Further detailed analysis of individual organs in mice yielded results, as shown in Figure 7.

[0087] As shown in Figure 7, fluorescence was observed in the brain, kidneys, spleen, liver, and heart of the mice, proving that BL-99-MVs can enter different organs of the mouse body through the digestive tract and blood vessels, and can also cross the blood-brain barrier to enter the brain.

[0088] Example 4:

[0089] This embodiment tested the effect of extracellular vesicles of Bifidobacterium animalis BL-99 extracted by method 2 in embodiment 1 on the relative survival rate of nerve cells HT22.

[0090] Mouse hippocampal neurons (HT22, purchased from iCell (Bioscience Inc., Shanghai, China)) and normal epithelial cell lines (purchased from iCell (Bioscience Inc., Shanghai, China)) were revived and passaged at least three times. They were then seeded into well plates and cultured adherently for 12 hours, followed by starvation for 12 hours. At 24 hours of culture, different concentrations of BL-99 extracellular vesicles were added to the BL-99 vesicle + LPS-induced inflammation group. After 36 hours of culture, both the LPS-induced inflammation group and the BL-99 vesicle + LPS-induced inflammation group were treated with LPS (specific method can be found in CN102807557B) to induce inflammation in mouse hippocampal neurons (HT22). Culture continued for 60 hours. Samples were collected and measured after LPS induction treatment.

[0091] Blank group (N): HT22 cells from the hippocampus of normal mice

[0092] LPS-induced inflammation group: LPS-induced neuroinflammation in mouse hippocampal neurons HT22 at 36 h.

[0093] Low-dose BL-99 vesicles + LPS-induced inflammation group: BL-99 extracellular vesicles (50 ng / μL) were added at 24 h, and LPS-induced neuroinflammation in mouse hippocampal neurons HT22 was induced at 36 h.

[0094] Medium-dose BL-99 vesicles + LPS-induced inflammation group: BL-99 extracellular vesicles (100ng / μL) were added at 24h, and LPS induced neuroinflammation in mouse hippocampal neurons HT22 at 36h.

[0095] High-dose BL-99 vesicles + LPS-induced inflammation group: BL-99 extracellular vesicles (200 ng / μL) were added at 24 h, and LPS-induced neuroinflammation in mouse hippocampal neurons HT22 was induced at 36 h.

[0096] Higher dose BL-99 vesicles + LPS-induced inflammation group: BL-99 extracellular vesicles (400ng / μL) were added at 24h, and LPS-induced neuroinflammation of mouse hippocampal neurons HT22 was induced at 36h.

[0097] The methods for detecting total cell count and dead cell count were based on GB 4789.35-2016, "National Food Safety Standard: Microbiological Examination of Food - Examination of Lactic Acid Bacteria". The experimental results are shown in Figure 8.

[0098] The results showed that the relative survival rate of HT22 cells with LPS-induced inflammatory damage was significantly reduced. The addition of low, medium, high and higher doses of BL-99 extracellular vesicles could significantly improve the relative survival rate of HT22 cells with inflammatory damage, and the survival rate was highest at a dose of 200 ng / μL.

[0099] Example 5:

[0100] This embodiment tested the effect of extracellular vesicles of Bifidobacterium animalis BL-99 extracted by method 2 in embodiment 1 on inflammatory factors of HT-22 cells.

[0101] Nerve injury has many causes, but neuroinflammation is considered a major cause. Mouse hippocampal neurons (HT22, purchased from iCell (Bioscience Inc., Shanghai, China)) and normal epithelial cell lines (purchased from iCell (Bioscience Inc., Shanghai, China)) were revived and passaged at least three times, then seeded into well plates and cultured adherently for 12 hours, followed by starvation for 12 hours. At 24 hours of culture, BL-99 extracellular vesicles were added to the BL-99 vesicle + LPS-induced inflammation group. After 36 hours of culture, LPS-induced inflammation was induced in both the LPS-induced inflammation group and the BL-99 vesicle + LPS-induced inflammation group (specific method can be found in CN102807557B) to induce inflammation in mouse hippocampal neurons (HT22). Samples were collected at 60 hours of culture. The expression levels of Ki67, IL-10, TNF-α, and IL-17 were detected using an ELISA kit.

[0102] Blank group (N): HT22 cells from the hippocampus of normal mice

[0103] LPS-induced inflammation group: LPS-induced neuroinflammation in mouse hippocampal neurons HT22 at 36 h.

[0104] BL-99 vesicles + LPS-induced inflammation group: BL-99 extracellular vesicles (200 ng / μL) were added at 24 h, and LPS-induced neuroinflammation in mouse hippocampal neurons HT22 was induced at 36 h.

[0105] Figure 9 shows that, compared with the blank group, the expression of cell growth factor Ki67 in the LPS-induced group was significantly reduced; compared with the LPS-induced neuroinflammation group, BL-99-MVs can significantly increase the level of cell growth factor Ki67 in cells after LPS-induced inflammation.

[0106] Figure 10 shows that, compared with the control group, the expression of anti-inflammatory factor IL-10 in cells induced by LPS was significantly reduced, while the expression of pro-inflammatory factors TNF-α and IL-17 was significantly increased. Compared with the LPS-induced neuroinflammation group, BL-99-MVs can significantly increase the level of anti-inflammatory factor IL-10 in cells after LPS-induced inflammation, while significantly reducing the levels of pro-inflammatory factors TNF-α and IL-17.

[0107] Experimental results show that BL-99-MVs can inhibit neuroinflammation by regulating the levels of growth factors, anti-inflammatory factors, and pro-inflammatory factors.

[0108] Example 6:

[0109] This embodiment tested the effect of extracellular vesicles of Bifidobacterium animalis BL-99 extracted by method 2 in embodiment 1 on the antioxidant activity of HT-22 cells.

[0110] Reactive oxygen species (ROS) are important signaling molecules that regulate metabolism and inflammatory responses. Under normal circumstances, they are maintained at low levels in the body, but their accumulation may lead to neurodegeneration and neuroinflammation.

[0111] Mouse hippocampal neurons (HT22, purchased from iCell (Bioscience Inc., Shanghai, China)) and normal epithelial cell lines (purchased from iCell (Bioscience Inc., Shanghai, China)) were revived and passaged at least three times. They were then seeded into well plates and cultured adherently for 12 hours, followed by starvation for 12 hours. At 24 hours of culture, BL-99 extracellular vesicles were added to the BL-99 vesicle + LPS-induced inflammation group. After 36 hours of culture, both the LPS-induced inflammation group and the BL-99 vesicle + LPS-induced inflammation group were treated with LPS (specific method can be found in CN102807557B) to induce inflammation in mouse hippocampal neurons (HT22). At 60 hours of culture, cells were collected, and samples were analyzed using a reactive oxygen species (ROS) detection kit.

[0112] Blank group (N): HT22 cells from the hippocampus of normal mice

[0113] LPS-induced inflammation group: LPS-induced neuroinflammation in mouse hippocampal neurons HT22 at 36 h.

[0114] BL-99 vesicles + LPS-induced inflammation group: BL-99 extracellular vesicles (200 ng / μL) were added at 24 h, and LPS-induced neuroinflammation in mouse hippocampal neurons HT22 was induced at 36 h.

[0115] Figure 11 shows that, compared with the control group, the LPS-induced inflammation group showed a significant increase in ROS production; compared with the LPS-induced neuroinflammation group, BL-99-MVs significantly reduced intracellular ROS (reactive oxygen species) production. These results indicate that BL-99-MVs can inhibit neuronal damage by reducing the production of reactive oxygen species.

[0116] Example 7:

[0117] This embodiment tested the alleviating effect of extracellular vesicles of Bifidobacterium animalis BL-99 extracted by method 2 in embodiment 1 on neuroinflammatory damage in the brain of mice (Balb / c mice).

[0118] Mice were divided into the Ctrl group, LPS-encephalitis group, and MV group, and the corresponding interventions are shown in Figure 12.

[0119] The specific dosage for each group is as follows:

[0120] Blank control group (Ctrl group): administered normal saline by gavage (3 mg / kg);

[0121] Model group (LPS-encephalitis group): administered normal saline by gavage (3 mg / kg), followed by LPS injection (1 mg / kg) on ​​days 28-30;

[0122] BL99-MV group (BL-99 vesicles + LPS-encephalitis group): BL99-MV (200ul, 3mg / kg) was administered by gavage daily, and LPS (1mg / kg) was injected every 28-30 days.

[0123] The experimental results are shown in Figures 13 and 14.

[0124] Figure 13: The movement trajectories of the mice show that, compared with the normal control group, the model group mice traversed the platform significantly less often and their movement within the platform quadrant was also reduced; the BL99-MV group mice showed a significant increase in the number of platform traversals and increased activity within the target quadrant. The results indicate that BL99-MV intervention significantly improved the movement trajectories of the mice, increasing both the number of platform traversals and activity within the platform quadrant.

[0125] Figure 14: The activity trajectories of the mice show that, compared with the normal control group, the model group mice were unwilling to explore new things and showed virtually no movement in the quadrant where the platform was located; although the BL99-MV group mice also did not show high performance in exploring new things, their activity in the target quadrant increased significantly. The results indicate that after BL99-MV intervention, the mice's activity in the target quadrant increased significantly.

[0126] In summary, BL99-MV intervention can effectively alleviate neuroinflammatory damage in the brain of mice.

[0127] It should be understood that the invention described herein is not limited to specific methodologies, experimental protocols, or reagents, as these can vary. The discussions and examples provided herein are for illustrative purposes only and are not intended to limit the scope of the invention, which is defined solely by the claims.

Claims

1. A method for extracting extracellular vesicles of Bifidobacterium animalis, comprising: 1) sequentially subjecting a bacterial suspension of Bifidobacterium animalis to low-speed centrifugation and high-speed centrifugation, and taking the supernatant; 2) subjecting the supernatant to ultrafiltration centrifugation, and taking the filtrate; 3) subjecting the filtrate to ultracentrifugation, and taking the supernatant; 4) subjecting the supernatant to ultrafiltration, and the filtrate contains the extracellular vesicles of Bifidobacterium animalis.

2. The method of claim 1, wherein, The method further has one or more technical features selected from (i)-(iv) below: (i) the pore size of the ultrafiltration centrifugation is 100 KDa; (ii) the ultrafiltration centrifugation is performed at a speed of 4000-6000 g (preferably 5000 g); (iii) the time of the ultrafiltration centrifugation is 5-15 min, preferably 10 min; (iv) the ultrafiltration centrifugation is achieved by using an ultrafiltration centrifuge tube (such as an Amicon Ultra-15 ultrafiltration centrifuge tube).

3. The method according to any one of claims 1-2, wherein, The method further has one or more technical features selected from (i)-(v) below: (i) the low-speed centrifugation is performed twice, the first low-speed centrifugation is performed at 3500-4000 g (preferably 3600 g) for 20-40 min (preferably 30 min), and the second low-speed centrifugation is performed at 5000-5500 g (preferably 5000 g) for 20-40 min (preferably 30 min); (ii) the high-speed centrifugation is performed at 10000-12000 g (preferably 11000 g) for 50-70 min (preferably 60 min); (iii) the ultracentrifugation is performed twice, the first ultracentrifugation is performed at 110000-130000 g (preferably 120000 g) for 80-100 min (preferably 90 min), and the second ultracentrifugation is performed at 110000-130000 g (preferably 120000 g) for 80-100 min (preferably 90 min); (iv) the ultrafiltration is achieved by using an ultrafiltration membrane; (v) the pore size of the ultrafiltration is 0.22 um.

4. The method according to any one of claims 1 to 3, wherein, The method further has one or more technical features selected from (i)-(iv) below: (i) the low-speed centrifugation is performed at a temperature of 4°C; (ii) the high-speed centrifugation is performed at a temperature of 4°C; (iii) the ultracentrifugation is performed at a temperature of 4°C; (iv) after the ultrafiltration of the supernatant in step 4), further comprising: diluting the filtrate with PBS buffer, and the PBS diluent contains the extracellular vesicles of Bifidobacterium animalis.

5. The method according to any one of claims 1 to 4, wherein, The method further has one or more technical features selected from (i)-(ii) below: (i) the Bifidobacterium animalis is Bifidobacterium animalis subsp. lactis BL-99, and the accession number is CGMCC No. 15650; (ii) the bacterial suspension of the animal Bifidobacterium is obtained by pre-fermentation of the animal Bifidobacterium.

6. An extracellular vesicle of animal Bifidobacterium, which is extracted by the method of any one of claims 1-5; Preferably, the particle size of the extracellular vesicle of animal Bifidobacterium is 50-100 nm.

7. A modified extracellular vesicle of animal Bifidobacterium, which is obtained by modification of a native extracellular vesicle of animal Bifidobacterium, the native extracellular vesicle of animal Bifidobacterium being extracted by the method of any one of claims 1-5 or as defined in claim 6.

8. A lysate or a cracked product of an extracellular vesicle of animal Bifidobacterium, which is obtained by lysing or cracking the extracellular vesicle of animal Bifidobacterium, the extracellular vesicle of animal Bifidobacterium being extracted by the method of any one of claims 1-5 or as defined in claim 6.

9. A composition comprising the extracellular vesicle of animal Bifidobacterium extracted by the method of any one of claims 1-5, or comprising the extracellular vesicle of animal Bifidobacterium as defined in claim 6, or comprising the modified extracellular vesicle of animal Bifidobacterium as defined in claim 7, or comprising the lysate and / or cracked product of the extracellular vesicle of animal Bifidobacterium as defined in claim 8.

10. Use of an extracellular vesicle of Bifidobacterium animalis as a drug carrier or for the preparation of a drug carrier, said extracellular vesicle of Bifidobacterium animalis being native or modified, wherein, The native extracellular vesicle of animal Bifidobacterium is extracted by the method of any one of claims 1-5 or as defined in claim 6, and the modified extracellular vesicle of animal Bifidobacterium is as defined in claim 7.

11. Use of an extracellular vesicle of animal Bifidobacterium lactis BL-99 in the preparation of a medicament for treating and / or preventing nerve injury, and / or a common food, a health food, a food additive and / or a dietary supplement for relieving nerve injury, the deposit number of the animal Bifidobacterium lactis BL-99 being CGMCC No. 15650; Preferably, the extracellular vesicle of animal Bifidobacterium lactis BL-99 is extracted by the method of any one of claims 1-5 or as defined in claim 6.

12. Use of a composition comprising an extracellular vesicle of animal Bifidobacterium lactis BL-99 in the preparation of a medicament for treating and / or preventing nerve injury, and / or a common food, a health food, a food additive and / or a dietary supplement for relieving nerve injury, the deposit number of the animal Bifidobacterium lactis BL-99 being CGMCC No. 15650; Preferably, the extracellular vesicle of animal Bifidobacterium lactis BL-99 is extracted by the method of any one of claims 1-5 or as defined in claim 6.

13. Use of metabolites of Bifidobacterium animalis lactis BL-99 comprising extracellular vesicles of Bifidobacterium animalis lactis BL-99 in the preparation of a medicament for the treatment and / or prevention of nerve injury, a common food, a health food, a food additive and / or a dietary supplement for alleviating nerve injury, the Bifidobacterium animalis lactis BL-99 having the accession number CGMCC No. 15650; preferably the extracellular vesicles of Bifidobacterium animalis lactis BL-99 are obtained by the method of any one of claims 1-5 or as defined in claim 6.

14. Use of a lysate or a lysate of extracellular vesicles of Bifidobacterium animalis lactis BL-99 obtained by lysing or lysing extracellular vesicles of Bifidobacterium animalis lactis BL-99 in the preparation of a medicament for the treatment and / or prevention of nerve injury, a common food, a health food, a food additive and / or a dietary supplement for alleviating nerve injury, the Bifidobacterium animalis lactis BL-99 having the accession number CGMCC No. 15650; preferably the extracellular vesicles of Bifidobacterium animalis lactis BL-99 are obtained by the method of any one of claims 1-5 or as defined in claim 6. The composition or the metabolites of Bifidobacterium animalis lactis BL-99 comprise extracellular vesicles of Bifidobacterium animalis lactis BL-99, a lysate or a lysate of extracellular vesicles of Bifidobacterium animalis lactis BL-99 obtained by lysing or lysing extracellular vesicles of Bifidobacterium animalis lactis BL-99, the Bifidobacterium animalis lactis BL-99 having the accession number CGMCC No. 15650; preferably the extracellular vesicles of Bifidobacterium animalis lactis BL-99 are obtained by the method of any one of claims 1-5 or as defined in claim 6. The composition or the metabolites of Bifidobacterium animalis lactis BL-99 comprise extracellular vesicles of Bifidobacterium animalis lactis BL-99, a lysate or a lysate of extracellular vesicles of Bifidobacterium animalis lactis BL-99 obtained by lysing or lysing extracellular vesicles of Bifidobacterium animalis lactis BL-99, the Bifidobacterium animalis lactis BL-99 having the accession number CGMCC No. 15650; preferably the extracellular vesicles of Bifidobacterium animalis lactis BL-99 are obtained by the method of any one of claims 1-5 or as defined in claim 6.

15. A method of treating and / or preventing or alleviating a neurological injury, comprising administering to a subject in need thereof an effective amount of an extracellular vesicle of Bifidobacterium animalis lactis BL-99, a metabolite of Bifidobacterium animalis lactis BL-99, a lysate or a fraction of an extracellular vesicle of Bifidobacterium animalis lactis BL-99, or a composition thereof, wherein, The composition or the metabolites of Bifidobacterium animalis lactis BL-99 comprise extracellular vesicles of Bifidobacterium animalis lactis BL-99, a lysate or a lysate of extracellular vesicles of Bifidobacterium animalis lactis BL-99 obtained by lysing or lysing extracellular vesicles of Bifidobacterium animalis lactis BL-99, the Bifidobacterium animalis lactis BL-99 having the accession number CGMCC No. 15650; preferably the extracellular vesicles of Bifidobacterium animalis lactis BL-99 are obtained by the method of any one of claims 1-5 or as defined in claim 6. The composition or the metabolites of Bifidobacterium animalis lactis BL-99 comprise extracellular vesicles of Bifidobacterium animalis lactis BL-99, a lysate or a lysate of extracellular vesicles of Bifidobacterium animalis lactis BL-99 obtained by lysing or lysing extracellular vesicles of Bifidobacterium animalis lactis BL-99, the Bifidobacterium animalis lactis BL-99 having the accession number CGMCC No. 15650; preferably the extracellular vesicles of Bifidobacterium animalis lactis BL-99 are obtained by the method of any one of claims 1-5 or as defined in claim 6.

16. A method of inhibiting / relieving inflammation of a neural cell, comprising administering to the neural cell an effective amount of Bifidobacterium animalis lactis BL-99 extracellular vesicles, Bifidobacterium animalis lactis BL-99 metabolites, a lysate or a lysate of Bifidobacterium animalis lactis BL-99 extracellular vesicles, or a composition thereof, wherein, ​ ​ 17. The use according to any one of claims 11 to 14 or the method according to claim 15 or 16, wherein, The administration dose of the extracellular vesicles of Bifidobacterium animalis ssp. lactis BL-99 is 50-400 ng / μL, such as 50 ng / μL, 100 ng / μL, 200 ng / μL or 400 ng / μL, preferably 200 ng / μL.

18. The use according to any one of claims 11 to 14 or the method according to claim 15, wherein, The nerve injury is an inflammatory nerve injury.

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