CN117965402B - Preparation method and application of lactobacillus outer membrane vesicle - Google Patents
Preparation method and application of lactobacillus outer membrane vesicle Download PDFInfo
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- 239000012528 membrane Substances 0.000 title claims abstract description 113
- 241000186660 Lactobacillus Species 0.000 title claims abstract description 37
- 229940039696 lactobacillus Drugs 0.000 title claims abstract description 37
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 239000007788 liquid Substances 0.000 claims abstract description 41
- 239000001963 growth medium Substances 0.000 claims abstract description 25
- 230000001580 bacterial effect Effects 0.000 claims abstract description 24
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- 241000894006 Bacteria Species 0.000 claims description 22
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 16
- 239000000243 solution Substances 0.000 claims description 12
- 235000014655 lactic acid Nutrition 0.000 claims description 9
- 239000004310 lactic acid Substances 0.000 claims description 9
- 239000000872 buffer Substances 0.000 claims description 7
- 239000002609 medium Substances 0.000 claims description 7
- 239000007853 buffer solution Substances 0.000 claims description 5
- 239000011148 porous material Substances 0.000 claims 1
- 238000012258 culturing Methods 0.000 abstract description 9
- 239000003242 anti bacterial agent Substances 0.000 abstract description 7
- 239000012535 impurity Substances 0.000 abstract description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract 1
- 241000194035 Lactococcus lactis Species 0.000 description 45
- 235000014897 Streptococcus lactis Nutrition 0.000 description 44
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- 230000000052 comparative effect Effects 0.000 description 12
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- 229940031000 streptococcus pneumoniae Drugs 0.000 description 10
- 241000194031 Enterococcus faecium Species 0.000 description 8
- 241000186779 Listeria monocytogenes Species 0.000 description 8
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Abstract
The invention discloses a preparation method of lactobacillus outer membrane vesicles, which comprises the following steps: preparing lactobacillus seed liquid; coating the lactobacillus seed liquid on a solid flat-plate culture medium, and continuing culturing; scraping and washing lactobacillus grown in the solid plate culture medium, and collecting bacterial liquid; centrifuging the bacterial liquid, and filtering the obtained supernatant with a microfiltration membrane; intercepting the filtered filtrate by adopting an ultrafiltration tube, and secondarily filtering the obtained intercepted liquid by using a microfiltration membrane to obtain the water-based filter; wherein, the conditions of centrifugation are: the centrifugal temperature is 4 ℃, the centrifugal force is not more than 8000g, and the centrifugal time is 5-30min; the size of the ultrafiltration tube is 30KD; and the application of lactobacillus outer membrane vesicles in the preparation of antibacterial agents. The lactobacillus outer membrane vesicle prepared by the invention has the advantages of less impurities, difficult breaking, good integrity, clean background, high purity and the like.
Description
Technical Field
The present invention relates to the field of biotechnology. More specifically, the invention relates to a preparation method and application of lactobacillus outer membrane vesicles.
Background
Antibiotics play an important role in treating diseases caused by bacterial infection, however, with the wide use of antibiotics, antibiotic resistant bacteria in the environment are gradually increased, and potential threat is caused to human health, so that the search for more natural, safer and less resistant antibacterial agents is of great significance.
Vesicles are natural nanoscale small bodies secreted by bacteria and having a bilayer membrane structure, and play important biological functions in many life events of bacteria, such as intercellular communication, promotion of biofilm formation, participation in immune response, and the like. In recent years, more and more researches indicate that vesicles secreted by bacteria can play an antibacterial role as drug delivery carriers. However, there are few studies on the outer membrane vesicles of lactic acid bacteria, and the studies on the outer membrane vesicles of lactic acid bacteria on the aspect of antibacterial activity are more delicate. In addition, most of the current methods for extracting outer membrane vesicles of bacteria are ultracentrifugation methods, and the outer membrane vesicles separated by the method are easy to break and difficult to detect, so that the number of the extracted outer membrane vesicles is small, and the quality is poor. Therefore, establishing an extraction and purification method capable of rapidly obtaining a large number of lactobacillus outer membrane vesicles is a problem to be solved urgently at present.
Disclosure of Invention
The invention aims to provide a preparation method and application of lactobacillus outer membrane vesicles, so as to at least solve the problems.
To achieve the objects and other advantages and in accordance with the purpose of the invention, there is provided a method for preparing outer membrane vesicles of lactic acid bacteria, comprising: preparing lactobacillus seed liquid; coating the lactobacillus seed liquid on a solid flat-plate culture medium, and continuing to culture; scraping and washing the lactobacillus grown out of the solid plate culture medium, and collecting bacterial liquid; centrifuging the bacterial liquid, and filtering the obtained supernatant with a microfiltration membrane; intercepting the filtered filtrate by adopting an ultrafiltration tube, and secondarily filtering the obtained interception liquid by using a microfiltration membrane to obtain lactobacillus outer membrane vesicles; wherein,
The centrifugation conditions are as follows: the centrifugal temperature is 4 ℃, the centrifugal force is not more than 8000g, and the centrifugal time is 5-30min; the size of the ultrafiltration tube is 30KD.
Preferably, in the preparation method of the lactobacillus outer membrane vesicle, lactobacillus strains are added into an MRS liquid culture medium to be cultured at the temperature of 30-37 ℃ for 12 h.
Preferably, in the preparation method of the lactobacillus outer membrane vesicle, the solid plate culture medium is MRS solid plate culture medium, and the culture time on the solid plate culture medium is 24 hours.
Preferably, in the preparation method of the lactobacillus outer membrane vesicle, 0.01M PBS buffer is adopted to scrape and wash the lactobacillus growing out of the solid plate culture medium.
Preferably, the preparation method of the lactobacillus outer membrane vesicle comprises the following centrifugation conditions: centrifugal temperature 4 ℃, centrifugal force 4000g and centrifugal time 10min.
Preferably, in the preparation method of the lactobacillus outer membrane vesicle, the aperture of the microfiltration membrane is 0.22 μm.
Preferably, the preparation method of the lactobacillus outer membrane vesicle comprises the following steps: centrifugal temperature 4 ℃, centrifugal force 4000g and centrifugal time 10min.
Preferably, in the preparation method of the lactobacillus outer membrane vesicle, the trapped fluid is washed three times with 0.01M PBS buffer solution in an ultrafiltration tube before the secondary filtration, wherein the washing conditions are as follows: centrifugal temperature 4 ℃, centrifugal force 4000g and centrifugal time 10min.
The invention also provides an application of the lactobacillus outer membrane vesicle prepared by the preparation method in preparation of an antibacterial agent.
Preferably, the use of the antimicrobial agent is for inhibiting the growth of enterococcus faecium, listeria monocytogenes and/or streptococcus pneumoniae.
The invention at least comprises the following beneficial effects:
The method effectively solves the problems of easy breakage, difficult detection, and more impurities of vesicles in the traditional extraction method of the lactobacillus outer membrane vesicles by adopting a mode of combining solid flat culture with ultrafiltration interception, and the extracted lactobacillus outer membrane vesicles have the characteristics of less impurities, difficult breakage, good integrity, clean background, high purity and the like.
The outer membrane vesicles extracted by the invention have very good growth inhibition effect on enterococcus faecium, listeria monocytogenes and streptococcus pneumoniae, and provide a technical basis for application of the lactobacillus outer membrane vesicles in antibacterial agents.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is a flow chart of a method for preparing outer membrane vesicles of lactic acid bacteria according to the invention;
FIG. 2 is an electron microscopic view of lactococcus lactis outer membrane vesicles prepared in example 1 of the present invention;
FIG. 3 is an electron microscopic view of lactococcus lactis outer membrane vesicles prepared in comparative example 1 of the present invention;
FIG. 4 is an electron microscopic view of lactococcus lactis outer membrane vesicles prepared in comparative example 2 of the present invention;
FIG. 5 is an electron microscopic image of the outer membrane vesicles of Lactobacillus plantarum prepared in example 2 of the present invention;
FIG. 6 is an electron microscopic image of the outer membrane vesicle of Lactobacillus plantarum prepared in comparative example 3 of the present invention;
FIG. 7 is an electron microscopic image of the outer membrane vesicle of Lactobacillus plantarum prepared in comparative example 4 of the present invention;
FIG. 8 is a graph showing the results of a plate bacteriostasis test of lactococcus lactis outer membrane vesicles prepared in example 1 of the present invention;
FIG. 9 is a graph showing the results of a test of the growth inhibition of enterococcus faecium by the outer membrane vesicles of lactococcus lactis prepared in example 1 of the present invention;
FIG. 10 is a graph showing the results of a test for inhibition of growth of Listeria monocytogenes by lactococcus lactis outer membrane vesicles prepared in example 1 of the present invention;
FIG. 11 is a graph showing the results of a test for growth inhibition of Streptococcus pneumoniae by lactococcus lactis outer membrane vesicles prepared in example 1 of the present invention;
FIG. 12 is a graph showing the results of a test for inhibiting the growth of Bacillus cereus by the lactococcus lactis outer membrane vesicles prepared in example 1 of the present invention.
Detailed Description
The present invention is described in further detail below with reference to examples and drawings to enable those skilled in the art to practice the same and to refer to the description.
It will be understood that terms, such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
The experimental methods described in the following embodiments are conventional methods unless otherwise indicated, and the reagents and materials are commercially available.
Experimental materials:
The lactococcus lactis L-WY410 is classified and named as lactococcus lactis Lactococcus lactis L-WY410, the preservation number is CGMCC No.29830, and the lactococcus lactis L-WY410 is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms at the date of 2024, 01 and 31, and the preservation address is North Star Xiya No. 1 national academy of sciences of China, the Korean area of Beijing.
Example 1:
As shown in fig. 1, the extraction and purification of lactococcus lactis outer membrane vesicles comprises the following steps:
Step one, preparing lactococcus lactis seed liquid.
The lactococcus lactis L-WY410 strain is taken out from a refrigerator at the temperature of minus 80 ℃, added into a 10 mL MRS liquid culture medium, and placed in a 30 ℃ incubator for culturing to 12 h, thus obtaining the lactococcus lactis seed liquid.
And step two, solid flat-plate culture.
And (3) taking 100 mu L of lactococcus lactis seed liquid, coating the lactococcus lactis seed liquid in 10 MRS solid flat-plate culture mediums with the diameters of 6 cm, placing the lactococcus lactis seed liquid in 30 ℃ for continuous culture of 24h, and scraping and washing thalli in the flat-plate by using 0.01M PBS buffer solution to obtain the bacterial liquid.
And thirdly, collecting lactococcus lactis outer membrane vesicles.
And (3) centrifuging the bacterial liquid obtained in the step (II) at 4 ℃ and 4000 g for 10: 10min, and removing most bacterial cells. Collecting supernatant, and filtering the supernatant with 0.22 μm microfiltration membrane to remove most of cell debris and flagella, etc., to obtain mixture containing lactococcus lactis outer membrane vesicle.
And step four, purifying lactococcus lactis outer membrane vesicles.
The filtrate containing lactococcus lactis outer membrane vesicles was trapped by an ultrafiltration tube of 30 KD, concentrated by centrifugation at 4000 g to 1mL, and the trapped fluid was washed three times with 15 mL of 0.01m PBS buffer and finally concentrated to 1mL to obtain outer membrane vesicles. The obtained vesicles were subjected to negative staining analysis by transmission electron microscopy, and the results are shown in fig. 2.
Comparative example 1:
The extraction and purification of lactococcus lactis outer membrane vesicles comprises the following steps:
Step one, preparing lactococcus lactis seed liquid.
The lactococcus lactis L-WY410 strain is taken out from a refrigerator at the temperature of minus 80 ℃, added into a 10 mL MRS liquid culture medium, and placed in a 30 ℃ incubator for culturing to 12 h, thus obtaining the lactococcus lactis seed liquid.
And step two, liquid culture.
The lactococcus lactis seed liquid is transferred into 400 mL MRS liquid culture medium according to 2 percent, and is subjected to stationary culture at 30 ℃ for 24 h, so as to obtain bacterial liquid.
And thirdly, collecting lactococcus lactis outer membrane vesicles.
And (3) centrifuging the bacterial liquid obtained in the step (II) at 4 ℃ and 4000 g for 10: 10min, and removing most bacterial cells. Collecting supernatant, and filtering the supernatant with 0.22 μm microfiltration membrane to remove most of cell debris and flagella, etc., to obtain mixture containing lactococcus lactis outer membrane vesicle.
And step four, purifying lactococcus lactis outer membrane vesicles.
The filtrate containing lactococcus lactis outer membrane vesicles was trapped by an ultrafiltration tube of 30 KD, concentrated by centrifugation at 4000 g to 1mL, and the trapped fluid was washed three times with 15 mL of 0.01m PBS buffer and finally concentrated to 1mL to obtain outer membrane vesicles. The obtained vesicles were subjected to negative staining analysis by transmission electron microscopy, and the results are shown in fig. 3.
Comparative example 2:
The extraction and purification of lactococcus lactis outer membrane vesicles comprises the following steps:
Step one, preparing lactococcus lactis seed liquid.
The lactococcus lactis L-WY410 strain is taken out from a refrigerator at the temperature of minus 80 ℃, added into a 10 mL MRS liquid culture medium, and placed in a 30 ℃ incubator for culturing to 12 h, thus obtaining the lactococcus lactis seed liquid.
And step two, liquid culture.
The lactococcus lactis seed liquid is transferred into 400 mL MRS liquid culture medium according to 2 percent, and is subjected to stationary culture at 30 ℃ for 24 h, so as to obtain bacterial liquid.
And thirdly, collecting lactococcus lactis outer membrane vesicles.
And (3) centrifuging the bacterial liquid obtained in the step (II) at 4 ℃ and 4000 g for 10: 10min, and removing most bacterial cells. Collecting supernatant, and filtering the supernatant with 0.22 μm microfiltration membrane to remove most of cell debris and flagella, etc., to obtain mixture containing lactococcus lactis outer membrane vesicle.
And step four, purifying lactococcus lactis outer membrane vesicles.
The filtrate containing lactococcus lactis outer membrane vesicles was trapped with an ultrafiltration tube of 30 KD, centrifuged at 4 ℃ and 4000 g to 50mL, then centrifuged at 200000 g with an ultracentrifuge at 4h, the supernatant removed and the pellet resuspended with 400 μl of 0.01M PBS buffer to obtain outer membrane vesicles. The obtained vesicles were subjected to negative staining analysis by transmission electron microscopy, and the results are shown in fig. 4.
As can be seen from FIGS. 2,3 and 4, the lactococcus lactis outer membrane vesicles isolated by the method of the invention (example 1) have the characteristics of less impurities, less breakage, good integrity, clean background and high purity compared with outer membrane vesicles extracted by liquid culture (comparative example 1) and liquid culture+ultra-high speed centrifugation (comparative example 2). In addition, the extraction process of the embodiment 1 does not need to adopt ultra-high speed centrifugal equipment, and the used 30KD ultrafiltration tube can be repeatedly used for a plurality of times, so that the method is a low-cost and high-efficiency outer membrane vesicle extraction method.
Example 2:
as shown in fig. 1, the extraction and purification of lactobacillus plantarum outer membrane vesicles comprises the following steps:
step one, preparing lactobacillus plantarum seed solution.
The lactobacillus plantarum strain is taken out from a refrigerator at the temperature of minus 80 ℃ and added into a10 mL MRS liquid culture medium, and is placed in a 37 ℃ incubator for culturing until the temperature reaches 12 h, thus obtaining lactobacillus plantarum seed liquid.
And step two, solid flat-plate culture.
And (3) taking 100 mu L of lactobacillus plantarum seed solution, coating the lactobacillus plantarum seed solution in 10 MRS solid flat-plate culture mediums with the diameters of 6 cm, placing the lactobacillus plantarum seed solution in 37 ℃ for continuous culture of 24h, and scraping and washing thalli in the flat-plate by using 0.01M PBS buffer solution to obtain the bacterial solution.
And thirdly, collecting lactobacillus plantarum outer membrane vesicles.
And (3) centrifuging the bacterial liquid obtained in the step (II) at 4 ℃ and 4000 g for 10: 10min, and removing most bacterial cells. Collecting supernatant, and filtering the supernatant with 0.22 μm microfiltration membrane to remove most of cell debris and flagellum, and obtain mixture containing lactobacillus plantarum outer membrane vesicle.
And step four, purifying lactobacillus plantarum outer membrane vesicles.
The filtrate containing lactobacillus plantarum outer membrane vesicles was trapped by an ultrafiltration tube of 30 KD, concentrated by centrifugation at 4000 g to 1mL, and the trapped fluid was washed three times with 15 mL of 0.01m PBS buffer and finally concentrated to 1mL to obtain outer membrane vesicles. The obtained vesicles were subjected to negative staining analysis by transmission electron microscopy, and the results are shown in fig. 5.
Comparative example 3:
The extraction and purification of the lactobacillus plantarum outer membrane vesicles comprise the following steps:
step one, preparing lactobacillus plantarum seed solution.
The lactobacillus plantarum strain is taken out from a refrigerator at the temperature of minus 80 ℃ and added into a10 mL MRS liquid culture medium, and is placed in a 37 ℃ incubator for culturing until the temperature reaches 12 h, thus obtaining lactobacillus plantarum seed liquid.
And step two, liquid culture.
Inoculating lactobacillus plantarum seed solution into 400 mL MRS liquid culture medium according to 2%, standing and culturing at 37 ℃ for 24: 24 h, and obtaining bacterial solution.
And thirdly, collecting lactobacillus plantarum outer membrane vesicles.
And (3) centrifuging the bacterial liquid obtained in the step (II) at 4 ℃ and 4000 g for 10: 10min, and removing most bacterial cells. Collecting supernatant, and filtering the supernatant with 0.22 μm microfiltration membrane to remove most of cell debris and flagellum, and obtain mixture containing lactobacillus plantarum outer membrane vesicle.
And step four, purifying lactobacillus plantarum outer membrane vesicles.
The filtrate containing lactobacillus plantarum outer membrane vesicles was trapped by an ultrafiltration tube of 30 KD, concentrated by centrifugation at 4000 g to 1mL, and the trapped fluid was washed three times with 15 mL of 0.01m PBS buffer and finally concentrated to 1mL to obtain outer membrane vesicles. The obtained vesicles were subjected to negative staining analysis by transmission electron microscopy, and the results are shown in fig. 6.
Comparative example 4:
The extraction and purification of the lactobacillus plantarum outer membrane vesicles comprise the following steps:
step one, preparing lactobacillus plantarum seed solution.
The lactobacillus plantarum strain is taken out from a refrigerator at the temperature of minus 80 ℃ and added into a10 mL MRS liquid culture medium, and is placed in a 37 ℃ incubator for culturing until the temperature reaches 12 h, thus obtaining lactobacillus plantarum seed liquid.
And step two, liquid culture.
Inoculating lactobacillus plantarum seed solution into 400 mL MRS liquid culture medium according to 2%, standing and culturing at 37 ℃ for 24: 24 h, and obtaining bacterial solution.
And thirdly, collecting lactobacillus plantarum outer membrane vesicles.
And (3) centrifuging the bacterial liquid obtained in the step (II) at 4 ℃ and 4000 g for 10: 10min, and removing most bacterial cells. Collecting supernatant, and filtering the supernatant with 0.22 μm microfiltration membrane to remove most of cell debris and flagellum, and obtain mixture containing lactobacillus plantarum outer membrane vesicle.
And step four, purifying lactobacillus plantarum outer membrane vesicles.
The filtrate containing lactobacillus plantarum outer membrane vesicles is intercepted by an ultrafiltration tube of 30 KD, the temperature is 4 ℃, the centrifugation concentration is 4000 g to 50mL, then an ultracentrifuge is adopted, the centrifugation is performed at 200000 g for 4 h, the supernatant is removed, and the sediment is resuspended by 400 MuL of PBS buffer solution of 0.01M, so that the outer membrane vesicles are obtained. The obtained vesicles were subjected to negative staining analysis by transmission electron microscopy, and the results are shown in fig. 7.
As can be seen from FIGS. 5, 6 and 7, the outer membrane vesicles of Lactobacillus plantarum isolated by the method of the invention (example 2) have the characteristics of less impurities, less breakage, good integrity, clean background and high purity compared with outer membrane vesicles extracted by liquid culture (comparative example 3) and liquid culture+ultra-high speed centrifugation (comparative example 4). In addition, the extraction process of the embodiment 2 does not need to adopt ultra-high speed centrifugal equipment, and the used 30KD ultrafiltration tube can be repeatedly used for a plurality of times, so that the method is a low-cost and high-efficiency outer membrane vesicle extraction method.
Test example 1:
1. plate bacteriostasis test for detecting bacteriostasis effect of purified vesicle
Enterococcus faecium (Enterococcus faeciumKCCM 11197P), listeria monocytogenes (Listeria monocytogenesATCC 19111), bacillus cereus (Bacillus cereus ATCC 14579), streptococcus pneumoniae (Streptococcus pneumoniae R), pseudomonas aeruginosa (Pseudomonas aeruginosaATCC 9027) and Escherichia coli (ESCHERICHIA COLIATCC 8739) were cultured in liquid medium to mid-log phase, then the indicator was added to the non-coagulated agar medium to give final concentration of the indicator at 10 6 CFU/mL, and the mixed medium was poured into 6 cm dishes each having a diameter of about 15 mL. In the ultra clean bench, the dish lid was opened and dried 20 min. The 6mm sterile filter paper sheets are placed in a culture dish, 20 microliters of the concentrated outer membrane vesicle liquid prepared in the example 1 is dripped on the filter paper sheets, after the outer membrane vesicle liquid is completely diffused, the filter paper sheets are placed at a proper culture temperature of indicator bacteria, the culture is carried out for 24 h, and the bacteriostasis phenomenon of the outer membrane vesicles on the indicator bacteria is observed, and the results are shown in figure 8, wherein in figure 8, A is enterococcus faecium, B is listeria monocytogenes, C is streptococcus pneumoniae, D is bacillus cereus, E is escherichia coli and F is pseudomonas aeruginosa.
As can be seen from FIG. 8, the lactococcus lactis outer membrane vesicles have a remarkable inhibitory effect on enterococcus faecium, listeria monocytogenes, streptococcus pneumoniae and Bacillus cereus.
2. Growth inhibition of indicator bacteria by outer membrane vesicles
Enterococcus faecium (Enterococcus faeciumKCCM 11197P), listeria monocytogenes (Listeria monocytogenesATCC 19111), streptococcus pneumoniae (Streptococcus pneumoniae R) and bacillus cereus (Bacillus cereus ATCC 14579) are cultivated in a liquid culture medium to a mid-log phase, then indicator bacteria in a logarithmic growth phase are inoculated in a fresh liquid culture medium according to 2%, the indicator bacteria are randomly divided into two groups, an experimental group and a control group, the outer membrane vesicles prepared in the example 1 are added into the experimental group, the final concentration is 10 9 parts per mL, the outer membrane vesicles are not added into the control group, and then the liquid culture medium is cultivated at a proper cultivation temperature of the indicator bacteria. OD 600 of the indicator bacteria was measured every two hours, and the inhibition of outer membrane vesicles on the indicator bacteria during growth was detected. The experimental results are shown in fig. 9, 10, 11 and 12.
As shown in figures 9-12, compared with a control group, the lactococcus lactis outer membrane vesicle has obvious inhibition effect on the growth of enterococcus faecium, listeria monocytogenes streptococcus pneumoniae and bacillus cereus, which proves that the lactococcus lactis outer membrane vesicle prepared by the invention has very good antibacterial effect and has application prospect as an antibacterial agent.
The number of equipment and the scale of processing described herein are intended to simplify the description of the present invention. Modifications and variations to the methods of preparation and use of the lactococcus lactis outer membrane vesicles of the invention will be apparent to those skilled in the art.
Although embodiments of the present invention have been disclosed above, it is not limited to the details and embodiments shown and described, it is well suited to various fields of use for which the invention would be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the specific details and illustrations shown and described herein, without departing from the general concepts defined in the claims and their equivalents.
Claims (6)
1. The preparation method of the lactobacillus outer membrane vesicle is characterized by comprising the following steps:
Preparing lactobacillus seed liquid;
coating the lactobacillus seed liquid on a solid flat-plate culture medium, and continuing to culture;
scraping and washing the lactobacillus grown out of the solid plate culture medium, and collecting bacterial liquid;
centrifuging the bacterial liquid, wherein the centrifugation conditions are as follows: centrifuging at 4deg.C for 5-30min with centrifugal force of not more than 8000g, and filtering the supernatant with microfiltration membrane;
The filtered filtrate is trapped by an ultrafiltration tube with 30KD, and then the trapped liquid is washed three times by a PBS buffer solution with 0.01M, wherein the conditions of the trapped and washed in the ultrafiltration tube are as follows: centrifuging at 4deg.C for 10min with 4000g, and filtering with microfiltration membrane to obtain lactobacillus outer membrane vesicle.
2. The method for preparing the outer membrane vesicle of lactobacillus according to claim 1, wherein the step of preparing the lactobacillus seed solution is to add lactobacillus strain into MRS liquid culture medium and culture the lactobacillus strain at 30-37 ℃ for 12 h.
3. The method for preparing outer membrane vesicles of lactic acid bacteria according to claim 1 wherein said solid plate medium is MRS solid plate medium and the culture time on said solid plate medium is 24h.
4. The method for preparing outer membrane vesicles of lactic acid bacteria according to claim 1 wherein lactic acid bacteria grown in said solid plate medium are scraped with 0.01M PBS buffer.
5. The method for preparing the outer membrane vesicle of lactic acid bacteria according to claim 1, wherein the centrifugation conditions are as follows: centrifugal temperature 4 ℃, centrifugal force 4000g and centrifugal time 10min.
6. The method for preparing the outer membrane vesicle of lactic acid bacteria according to claim 1, wherein the pore size of the micro-filtration membrane is 0.22 μm.
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