Extraction method of extracellular vesicles of animal bifidobacterium

Abstract

The invention relates to a method for extracting extracellular vesicles of animal bifidobacterium, which comprises the following steps: 1) sequentially carrying out low-speed centrifugal treatment and high-speed centrifugal treatment on bacterial suspension of animal bifidobacterium, and taking supernate; 2) performing ultrafiltration centrifugal treatment on the supernate, and taking filtrate; (3) carrying out ultra-high-speed centrifugal treatment on the filtrate, and taking supernate; and (4) carrying out ultrafiltration treatment on the supernate, wherein filtrate contains the extracellular vesicles of the bifidobacterium animalis. The yield of the extracellular vesicles extracted by the method is high, and the extracellular vesicles can enter various organs of animal bodies through blood.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and in particular to an extraction method of extracellular vesicles of animal bifidobacterium. BACKGROUND

[0002] Membrane vesicles (MVs) of lactic acid bacteria (such as animal bifidobacterium) are vesicular small bodies released by lactic acid bacteria (such as animal bifidobacterium) into its environment. The structure is a lipid bilayer wrapping the components in the cell, and is a nanoscale active substance with a diameter of 20-400 nm, which can shuttle and transfer information between cells. MVs are natural carriers of lactic acid bacteria (such as animal bifidobacterium) molecules, including peptidoglycan, lipid, protein, nucleic acid, etc. As a kind of cell secretion, it has been found that MVs can be transported to various organs of the body through the blood, and can also reach the brain of the host through the blood-brain barrier. At present, in the medical field, MVs are an important treatment material carrier. However, there is still a lack of research on the extraction method of MVs, especially the method for improving the yield and protecting the basic characteristics of MVs.

[0003] Therefore, it is of long-term significance to provide a preparation method without chemical substance stimulation, which can improve the yield of MVs and protect the basic characteristics of MVs. SUMMARY

[0004] The present application provides an extraction method of extracellular vesicles of animal bifidobacterium. The yield of the extracted extracellular vesicles is high, which can enter various organs of the animal body through the blood, and can enter the mouse brain through the blood-brain barrier, and has a wide application prospect in the medical field.

[0005] To this end, in the first aspect of the present application, the present application provides a method for extracting extracellular vesicles of animal bifidobacterium, comprising:

[0006] 1) sequentially performing low-speed centrifugal treatment and high-speed centrifugal treatment on a bacterial suspension of animal bifidobacterium, and taking the supernatant;

[0007] 2) performing ultrafiltration centrifugal treatment on the supernatant, and taking the filtrate;

[0008] 3) performing ultra-high-speed centrifugal treatment on the filtrate, and taking the supernatant;

[0009] 4) performing ultrafiltration treatment on the supernatant, and the filtrate contains the extracellular vesicles of animal bifidobacterium.

[0010] In some embodiments, the pore size of the ultrafiltration centrifugal treatment is 100 KDa.

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

[0012] In some embodiments, the ultrafiltration centrifugation is performed for a time of 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 embodiments, the ultrafiltration centrifugation is performed using an ultrafiltration centrifuge tube (e.g., an Amicon Ultra-15 ultrafiltration centrifuge tube).

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

[0015] In some embodiments, the high speed centrifugation is performed at a speed of 10000-12000 g (e.g., 10000 g, 10200 g, 10400 g, 10600 g, 10800 g, 11000 g, 11200 g, 11400 g, 11600 g, 11800 g, or 12000 g, preferably 11000 g) for 50-70 min (e.g., 50 min, 55 min, 60 min, 65 min, or 70 min, preferably 60 min).

[0016] In some embodiments, the ultracentrifugation is performed twice, the first ultracentrifugation is performed at 110000-130000g (such as 112000g, 114000g, 116000g, 118000g, 120000g, 122000g, 124000g, 126000g, 128000g or 130000g, preferably 120000g) for 80-100min (such as 80min, 85min, 90min, 95min or 100min, preferably 90min), and the second ultracentrifugation is performed at 110000-130000g (such as 112000g, 114000g, 116000g, 118000g, 120000g, 122000g, 124000g, 126000g, 128000g or 130000g, preferably 120000g) for 80-100min (such as 80min, 85min, 90min, 95min or 100min, preferably 90min).

[0017] In some embodiments, the ultrafiltration is performed using an ultrafiltration membrane.

[0018] In some embodiments, the pore size of the ultrafiltration is 0.22um.

[0019] In some embodiments, the low speed centrifugation is performed at a temperature of 4℃.

[0020] In some embodiments, the high speed centrifugation is performed at a temperature of 4℃.

[0021] In some embodiments, the ultracentrifugation is performed at a temperature of 4℃.

[0022] In some embodiments, in step 4), after the supernatant is subjected to ultrafiltration, further comprising: diluting the filtrate with PBS buffer, and the PBS dilution solution contains the animal Bifidobacterium extracellular vesicles.

[0023] In some embodiments, the animal Bifidobacterium is Bifidobacterium animalis lactis BL-99, and the preservation number is CGMCC No.15650. The Bifidobacterium animalis lactis BL-99 was preserved in the China General Microbiological Culture Collection Center (CGMCC) on April 26, 2018, and the classification and naming is Bifidobacterium lactis, and the preservation number is CGMCC No.15650. The specific information can be found in CN110964657B.

[0024] In some embodiments, the bacterial suspension of animal Bifidobacterium is obtained by pre-fermentation of the animal Bifidobacterium. Wherein, the fermentation can use the fermentation conditions conventional in the art, for example, 37℃ culture in MRS liquid medium.

[0025] In the second aspect of the present application, the present application provides an extracellular vesicle of animal Bifidobacterium, which is extracted by the method of the first aspect.

[0026] In some embodiments, the particle size of the extracellular vesicle of animal Bifidobacterium is 50-100 nm (such as 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 the third aspect of the present application, the present application provides a modified extracellular vesicle of animal Bifidobacterium, which is modified from a natural extracellular vesicle of animal Bifidobacterium, wherein the natural extracellular vesicle of animal Bifidobacterium is extracted by the method of the first aspect or as described in the second aspect.

[0028] In the fourth aspect of the present application, the present application provides 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, wherein the extracellular vesicle of animal Bifidobacterium is extracted by the method of the first aspect or as described in the second aspect.

[0029] In the fifth aspect of the present application, the present application provides a composition comprising the extracellular vesicle of animal Bifidobacterium extracted by the method of the first aspect, or comprising the extracellular vesicle of animal Bifidobacterium as described in the second aspect, or comprising the modified extracellular vesicle of animal Bifidobacterium as described in the third aspect, or comprising the lysate and / or cracked product of the extracellular vesicle of animal Bifidobacterium as described in the fourth aspect.

[0030] In the sixth aspect of the present application, the present application provides the use of an extracellular vesicle of animal Bifidobacterium as a drug carrier or the use of an extracellular vesicle of animal Bifidobacterium in the preparation of a drug carrier, wherein the extracellular vesicle of animal Bifidobacterium is natural or modified, wherein the natural extracellular vesicle of animal Bifidobacterium is extracted by the method of the first aspect or as described in the second aspect, and the modified extracellular vesicle of animal Bifidobacterium is as described in the third aspect.

[0031] Beneficial effects

[0032] 1. The present application discloses an extracellular vesicle derived from animal Bifidobacterium subsp. lactis BL-99 and its extraction process and method.

[0033] 2. The application provides the animal Bifidobacterium lactis BL-99 extracellular vesicle extraction method, through the low-speed centrifugation, high-speed centrifugation, ultrafiltration centrifugation, ultracentrifugation and ultrafiltration under certain conditions are combined, the number of extracted extracellular vesicles is more, the protein concentration is high, and the dispersibility is good.

[0034] 3. The application finds that the animal Bifidobacterium BL-99 extracellular vesicle can enter various organs of the animal body through blood.

[0035] 4. The application finds that the animal Bifidobacterium BL-99 extracellular vesicle can enter the brain of a mouse through the blood-brain barrier. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 It is a BL-99 bacterial source vesicle extraction and separation method schematic diagram in the embodiment one of the application.

[0037] Figure 2 It is a BL-99-MVs protein concentration comparison chart extracted by different methods in the embodiment one of the application.

[0038] Figure 3 It is a BL-99-MVs particle size chart extracted by different methods in the embodiment one of the application.

[0039] Figure 4 It is a BL-99-MVs morphological structure chart under a transmission electron microscope extracted by different methods in the embodiment one of the application.

[0040] Figure 5 It is a BL-99-MVs relative quantity comparison chart extracted by different methods in the embodiment one of the application.

[0041] Figure 6 It is a mouse fluorescence display chart after the mouse is gavaged with fluorescently labeled BL-99-MVs for 2h in the embodiment three of the application.

[0042] Figure 7 It is a mouse brain-intestine fluorescence display chart after the mouse is gavaged with fluorescently labeled BL-99-MVs for 2h in the embodiment three of the application. DETAILED DESCRIPTION

[0043] Embodiments of the present application will be described in detail below with reference to examples and drawings, but those skilled in the art will understand that the following examples and drawings are only used to illustrate the present application, and are not limited to the scope of the present application. According to the following detailed description of the drawings and preferred embodiments, various purposes and advantages of the present application will become apparent to those skilled in the art. Unless otherwise specified, each raw material and reagent can be commercially available. Among them, the animal Bifidobacterium lactis BL-99 was deposited in the China General Microbiological Culture Collection Center (CGMCC) on April 26, 2018 (address: No. 1, Beichen West Road, Yard 3, Beijing Chaoyang District, Institute of Microbiology of Chinese Academy of Sciences, China), and the classification name is Bifidobacterium lactis; the preservation number is CGMCC No. 15650. For example, see CN110964657B for specific information.

[0044] Example One:

[0045] In this embodiment, an extracellular vesicle of animal Bifidobacterium lactis BL-99 is prepared, and the protein concentrations of two preparation methods are compared. As shown in Figure 1 , compared with method two, method one is the same as method two in all steps and operations except that it does not use an ultrafiltration centrifuge tube (pore size 100KDa, Amicon Ultra-15) for ultrafiltration centrifugation (5000g, 10min).

[0046] Using animal Bifidobacterium lactis BL-99 for fermentation (37℃ culture in MRS liquid medium), 1L of bacterial suspension was obtained.

[0047] Method one: under the condition of 4℃, the bacterial suspension of animal Bifidobacterium was sequentially subjected to low speed centrifugation (first time, 3600g, 30min; second time, 5000g, 30min) and high speed centrifugation (11000g, 60min), and the supernatant was extracted after centrifugation. The supernatant was subjected to two times of ultra-high speed centrifugation (120000g, 90min) at 4℃, and the supernatant was extracted. The supernatant was subjected to ultrafiltration using an ultrafiltration membrane (0.22um), and the filtrate was diluted with PBS buffer, and the PBS dilution liquid was the extracellular vesicle of BL-99.

[0048] 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.

[0049] The protein concentration was determined according to GB 5009.5-2016, "National Food Safety Standard - Determination of Protein in Food". The test results are as follows: Figure 2 As shown.

[0050] 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.

[0051] Example Two:

[0052] Particle size analysis was performed on the BL-99-MVs extracted using 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 were added to a particle size distribution dish for particle size analysis, and the solution was gradually diluted until the particle size no longer changed, which was taken as the final result. The analytical results are as follows: Figure 3 As shown.

[0053] Depend on Figure 3 The results show that 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.

[0054] The morphological structures of BL-99-MVs extracted by methods one and two as described in Example 1 are shown in the transmission electron microscope images. Figure 4 As shown. The method for morphological detection by transmission electron microscopy is as follows: 20 μL of extracellular vesicles of Bifidobacterium animalis BL-99 is placed on a 300-mesh copper grid, allowed to dry naturally, 20 μL of phosphotungstic acid is added for negative staining, allowed to dry naturally, and then the sample is analyzed by the instrument.

[0055] Depend on Figure 4The results show that the BL-99-MVs structures extracted by methods one and two are generally spherical, but the particle size differs significantly.

[0056] 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.

[0057] 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 cancertherapeutics and diagnostics[J]. Bioeng Transl Med, 2017, 2(1):70.). The results are as follows: Figure 5 As shown in Table 1.

[0058] Figure 5 As shown in Table 1, the number of extracellular vesicles extracted by method 2 is approximately 1.6 times that extracted by method 1 ("**" indicates p<0.01). This demonstrates that the number of extracellular vesicles extracted using method 2 is significantly higher than that extracted using method 1.

[0059] Table 1: Comparison of the number of extracellular vesicles extracted by different methods

[0060]

[0061] Example Three:

[0062] 100 μL of BL99-MVs (200 ng / μL) extracted using Method 2 in Example 1 was co-cultured with 10 μL of Dir dye (100 μM). The 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 as follows... Figure 6 As shown.

[0063] Depend on Figure 6 Fluorescence results showed strong fluorescence in the stomach, intestines, liver, and kidneys of mice 2 hours after gavage, proving that BL-99-MVs entered different organs of the body through the digestive tract.

[0064] Further detailed analysis of individual organs in mice yielded the following results: Figure 7 As shown.

[0065] Depend on Figure 7The fluorescence results can find that there are fluorescence in the brain, kidney, spleen, liver, heart of mice, which proves that the BL-99-MVs can enter different organs of the mouse body through the digestive tract and blood vessels, and at the same time can enter the brain through the blood-brain barrier.

[0066] It is to be understood that the application described herein is not limited to particular methodologies, protocols or reagents, as such can vary. The discussion and examples provided herein are presented to describe particular embodiments presented and are not intended to limit the scope of the application, which is limited only 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 modified from 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 lysate of an extracellular vesicle of animal Bifidobacterium, which is obtained by lysing or lysing 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 lysate 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.

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