Method for increasing yield of extracellular vesicles of lactobacillus bacteria
By adding galactooligosaccharides to a selective culture medium for Lactobacillus and then separating and purifying them, the problem of obtaining plant-derived extracellular vesicles during lactic acid bacteria fermentation was solved, enabling efficient production of Lactobacillus extracellular vesicles and facilitating commercial application.
Patent Information
- Application Number
- CN202511692546.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-18
- Publication Date
- 2026-01-20
AI Technical Summary
In existing technologies, it is inconvenient to obtain edible plant-derived extracellular vesicles during lactic acid bacteria fermentation, making it difficult to achieve large-scale production of lactobacillus extracellular vesicles.
Galacto-oligosaccharides are added to the selective culture medium of Lactobacillus, Lactobacillus bacteria are inoculated and cultured, and extracellular vesicles are obtained by differential or ultracentrifugation and purification, avoiding the addition of edible plant-derived extracellular vesicles.
It effectively promotes the secretion of extracellular vesicles of lactobacilli, increases yield, facilitates commercial production, and is simple and easy to implement.
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Figure CN121362664A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of microbial fermentation engineering, and particularly relates to a method for improving the yield of extracellular vesicles of lactobacillus. BACKGROUND
[0002] Galactooligosaccharides are well-known prebiotics for selectively stimulating the growth of lactobacilli and bifidobacteria. Supplementing galactooligosaccharides can promote the colonization and proliferation of lactobacilli in the gastrointestinal tract, thereby improving the intestinal barrier function and immune development. Galactooligosaccharides enhance the competitive exclusion of intestinal pathogens, thereby reducing the inflammatory response. Galactooligosaccharides have been widely used to improve intestinal function and health.
[0003] Lactobacilli, as an important member of the intestinal microbiota, can participate in the physiological and pathological regulation of the brain by regulating physiological processes such as intestinal barrier function, immune response, and neurotransmitter metabolism. Lactobacilli have a wide range of health benefits for the host, such as immune regulation and intestinal barrier promotion. Targeted modification of the intestinal microbiota by supplementing probiotics has become a promising option for the treatment of intestinal diseases. Probiotics derived from healthy individuals have been widely proven to maintain the intestinal epithelial barrier and improve the intestinal flora structure and regulate immunity. Lactobacilli belong to gram-positive bacteria, grow and reproduce quickly, have low culture cost, and simple growth conditions, which are conducive to the industrialized production of extracellular vesicles.
[0004] Extracellular vesicles are a class of lipid bilayer vesicles secreted by cells into the body, which can be produced by both eukaryotes and prokaryotes. According to the particle size, extracellular vesicles can be divided into exosomes (30nm~150nm), microvesicles (100nm~1000nm), and apoptotic bodies released by dying cells (50nm~5000nm). As an important medium for communication between organisms, extracellular vesicles have a natural lipid bilayer membrane structure and carry biological molecules such as proteins and nucleic acids, have good biocompatibility and low immunogenicity, and thus exhibit great application potential in the fields of disease diagnosis and treatment. The influence of bacterial secreted extracellular vesicles on the host is mediated by the transport and delivery of effector molecules. Extracellular vesicles from probiotics are beneficial to regulate host immunity and intestinal barrier integrity. Lactobacillus johnsonii extracellular vesicles have a significant effect on relieving intestinal inflammation and diarrhea diseases, which is of great significance to the aquaculture industry. However, the mechanism of improving the yield of Lactobacillus johnsonii extracellular vesicles is still unclear.
[0005] At present, the method for improving the yield of lactobacillus extracellular vesicles is to add plant-derived extracellular vesicles, that is, to add edible plant-derived extracellular vesicles in the fermentation process of lactobacillus. The edible plant-derived extracellular vesicles required by this method are not easy to obtain, which is not conducive to the large-scale production of lactobacillus extracellular vesicles. SUMMARY
[0006] In order to solve the problem that the edible plant source extracellular vesicles need to be added in the fermentation process of lactic acid bacteria in the prior art, and the edible plant source extracellular vesicles required to be added are not convenient to obtain and are not conducive to large-scale production of lactobacillus extracellular vesicles, the application provides a method for improving the yield of lactobacillus extracellular vesicles.
[0007] The application provides a method for improving the yield of lactobacillus extracellular vesicles, which comprises the following steps: Galactooligosaccharides are added in a lactobacillus selective culture medium, and lactobacillus bacteria are inoculated and cultured; after the culture, the bacterial liquid is separated and purified to obtain bacterial extracellular vesicles.
[0008] The concentration of the galactooligosaccharides in the lactobacillus selective culture medium is 30 g / L to 50 g / L.
[0009] The method provided by the application is to add galactooligosaccharides in a lactobacillus selective culture medium for fermentation, inoculate lactobacillus bacteria and then culture; after the culture, the bacterial liquid is separated and purified to obtain bacterial extracellular vesicles. The method provided by the application does not need to add extracellular vesicles of edible plant sources in the fermentation process of lactic acid bacteria, and only needs to add galactooligosaccharides in the fermentation process. The galactooligosaccharides are convenient to obtain and facilitate commercial production of lactobacillus extracellular vesicles.
[0010] Further, the concentration of the galactooligosaccharides in the lactobacillus selective culture medium is 40 g / L.
[0011] Further, the lactobacillus bacteria are selected from at least one of lactobacillus johnsonii, lactobacillus reuteri, lactobacillus mucosae, lactobacillus amylovorus and lactobacillus animalis.
[0012] Further, the lactobacillus selective culture medium is an MRS culture medium, and the MRS culture medium is prepared from materials with the following final concentrations: Peptone 9 g / L to 11 g / L, beef infusion powder 7 g / L to 9 g / L, yeast infusion powder 3 g / L to 5 g / L, glucose 19 g / L to 21 g / L, dimethyl bicarbonate 1 g / L to 3 g / L, dimethyl bicarbonate 1 g / L to 3 g / L, sodium acetate 4 g / L to 6 g / L, magnesium sulfate 0.1 g / L to 0.3 g / L, manganese sulfate 0.03 g / L to 0.05 g / L, and Tween 80 0.1 g / L to 2 g / L, and the solvent is water.
[0013] Further, the culture conditions are 36°C to 38°C and 11 hours to 12 hours.
[0014] Further, the inoculation amount of the lactobacillus bacteria is 2 x 108 cfu / mL~8×10 8 cfu / mL.
[0015] Further, the separation process is: after centrifugation of the cultured bacterial liquid, the bacterial extracellular vesicles are obtained.
[0016] Further, the centrifugation method is a differential centrifugation method or an ultracentrifugation method.
[0017] Further, the ultracentrifugation method has the following conditions: Centrifugation at 7000g-8000g for 28-32 minutes, centrifugation at 10000g-11000g for 43-47 minutes.
[0018] Further, the purification process is as follows: After centrifugation of the cultured bacterial liquid, the supernatant is collected, the supernatant is filtered through a 0.21-0.23 μm filter membrane, and the filtrate is collected; the filtrate is centrifuged at 120000g-150000g for 118-122 minutes, the supernatant is discarded, and the precipitate is resuspended to obtain the bacterial extracellular vesicles.
[0019] Compared with the prior art, the present application has the following beneficial effects: 1. The present application provides a method for improving the yield of Lactobacillus extracellular vesicles. The method provided by the present application is to add oligogalactose to the Lactobacillus selective medium for fermentation, and then inoculate Lactobacillus bacteria for culture; after separation and purification of the cultured bacterial liquid, bacterial extracellular vesicles are obtained, wherein the added oligogalactose in the fermentation process can effectively promote the secretion of Lactobacillus extracellular vesicles, and the method provided by the present application does not need to add extracellular vesicles from edible plant sources in the fermentation process of lactic acid bacteria, only needs to add oligogalactose in the fermentation process, and oligogalactose is convenient to obtain, which is convenient for commercial production of Lactobacillus extracellular vesicles.
[0020] 2. The method provided by the present application also extracts Lactobacillus extracellular vesicles by ultracentrifugation, which can effectively remove bacteria and more completely retain the morphology and quantity of extracellular vesicles.
[0021] 3. The present application provides a method for improving the yield of Lactobacillus extracellular vesicles, which adds oligogalactose to the culture of Lactobacillus johnsonii (Lactobacillus selective medium) to effectively improve the yield of Lactobacillus johnsonii extracellular vesicles. The present application adds oligogalactose to the culture of Lactobacillus johnsonii, which can effectively improve the production of Lactobacillus johnsonii extracellular vesicles. This method can be used for large-scale production of Lactobacillus johnsonii extracellular vesicles in factories, and has important value in the field of producing Lactobacillus johnsonii extracellular vesicles. BRIEF DESCRIPTION OF DRAWINGS
[0022] Figure 1 The addition of galactooligosaccharides in the present application significantly increases the number of Lactobacillus johnsonii.
[0023] Figure 2 The addition of galactooligosaccharides in the present application significantly increases the number of particles of extracellular vesicles of Lactobacillus johnsonii.
[0024] Figure 3 The addition of galactooligosaccharides in the present application significantly increases the number of particles of extracellular vesicles of single bacteria of Lactobacillus johnsonii. DETAILED DESCRIPTION
[0025] The present application will be described in detail below in conjunction with the accompanying drawings and specific examples, but should not be understood as limiting the present application. If not specifically stated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, if not specifically stated, can be obtained from commercial channels.
[0026] Example 1: Preliminary screening of Lactobacillus johnsonii A strain with the function of relieving pig intestinal inflammation and diarrhea was screened out from porcine-derived Lactobacillus johnsonii isolated from normal piglet feces through in vivo and in vitro experiments, and the Lactobacillus johnsonii (Lactobacillus johnsonii) selected by the present application was used for experiments. Lactobacillus johnsonii ).
[0027] Among them, the method for isolating porcine-derived Lactobacillus johnsonii from normal piglet feces and the porcine-derived Lactobacillus johnsonii obtained by isolation are described in detail in the following literature: “Tao S, Fan J, Li J, Wu Z, Yao Y, Wang Z, Wu Y, Liu X, Xiao Y, Wei H. Extracellular vesicles derived from Lactobacillus johnsonii promote gut barrier homeostasis by enhancing M2 macrophage polarization. J Adv Res. 2025 Mar;69:545-563. doi: 10.1016 / j.jare.2024.03.011.”.
[0028] The Lactobacillus johnsonii (Lactobacillus johnsonii) selected by the present application is used for experiments. Lactobacillus johnsoniiSee document: Extracellular vesicles derived from Lactobacillus johnsonii promote gut barrier homeostasis by enhancing M2 macrophage polarization, i.e. the strain in the document L.john (H9). If needed, the public can be provided by the College of Animal Science and Technology, Huazhong Agricultural University.
[0029] Example 2: Cultivation and counting of Lactobacillus johnsonii (1) 0.3 mL of Lactobacillus johnsonii was inoculated into an anaerobic culture tube containing 15 mL of Lactobacillus selective medium as a control group, also known as a blank control group; at the same time, a test group, i.e. a 4% oligogalactose group, was set up. The specific operation of the 4% oligogalactose group was as follows:
[0030] In the 4% oligogalactose group, the most important thing was to add sterile oligogalactose to the culture medium of the control group, and the addition amount was 4% of the volume of the Lactobacillus selective medium, i.e. 0.6 g of oligogalactose was added to 15 mL of MRS culture medium.
[0031] The two groups of culture tanks treated according to the above requirements were cultured at 37°C for 12 hours.
[0032] Among them, the Lactobacillus selective culture medium is MRS culture medium.
[0033] The MRS culture medium is made of the following materials with final concentrations: 10.0 g / L of proteose peptone, 8.0 g / L of beef extract powder, 4.0 g / L of yeast extract powder, 20.0 g / L of glucose, 2.0 g / L of dimethyl bicarbonate, 2.0 g / L of dimethyl bicarbonate, 5.0 g / L of sodium acetate, 0.2 g / L of magnesium sulfate, 0.04 g / L of manganese sulfate, and 1.0 g / L of Tween 80, and distilled water is added to 1 L, and autoclaved at 118°C for 15 min.
[0034] (2) Dilution and spread plate count: After 12 hours of culture in (1), 0.1 mL of bacterial solution was taken with a 1 mL sterile syringe into 0.9 mL of sterile PBS, diluted 10 times, and the cycle was repeated. According to the gradient dilution method, 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 and 10 7 times were sequentially diluted. 10 6 and 10 7The bacterial liquid was diluted 10 times with MRS agar medium plate, and then plated respectively. After anaerobic culture at 37°C for 12 hours, the number of colonies was counted.
[0035] The sterile PBS (product number BL302A) was purchased from Biosharp Company, and the pH was 7.4.
[0036] MRS agar medium (product number: HB0384-51): 10.0 g / L of proteose peptone, 10.0 g / L of beef extract powder, 5.0 g / L of yeast extract powder, 20.0 g / L of glucose, 2.0 g / L of dimethyl bicarbonate, 2.0 g / L of dimethyl ammonium citrate, 5.0 g / L of sodium acetate, 0.1 g / L of magnesium sulfate, 0.05 g / L of manganese sulfate, 15.0 g / L of agar, 1.0 g / L of Tween 80, and distilled water was added to 1 L, and then autoclaved at 121°C for 15 min. After cooling to about 45°C, the MRS agar medium plate was poured, and then stored for use.
[0037] Example 3: Production of Lactobacillus johnsonii extracellular vesicles 1. Preparation of Lactobacillus johnsonii extracellular vesicles: (1) The bacterial liquid of the two groups (control group and 4% galactooligosaccharide group) cultured in Example 2 was separated by ultracentrifugation to obtain Lactobacillus johnsonii extracellular vesicles of the two groups (control group and 4% galactooligosaccharide group). The Lactobacillus johnsonii extracellular vesicles obtained from the control group were Lactobacillus johnsonii extracellular vesicles of the control group, and the Lactobacillus johnsonii extracellular vesicles obtained from the 4% galactooligosaccharide group were Lactobacillus johnsonii extracellular vesicles of the 4% galactooligosaccharide group.
[0038] The Lactobacillus johnsonii extracellular vesicles are bacterial extracellular vesicles.
[0039] The method for separating Lactobacillus johnsonii extracellular vesicles by ultracentrifugation is as follows: The bacterial liquid of the two groups (control group and 4% galactooligosaccharide group) cultured in Example 2 was centrifuged at 8000 g for 30 minutes and 10000 g for 45 minutes at 4°C, and the supernatant was filtered with a 0.22 μm filter. Then, the supernatant was centrifuged at 120000 g for 120 minutes, and the supernatant was discarded and poured into sterile PBS. The vesicle precipitate was resuspended in sterile PBS after repeated centrifugation, and Lactobacillus johnsonii extracellular vesicles of the two groups (control group and 4% galactooligosaccharide group) were obtained.
[0040] (2) Extracellular vesicle quantification: The Lactobacillus johnsonii vesicles were quantitatively analyzed using a NS300 nanoparticle tracking analyzer, and the specific method was as follows: The purified extracellular vesicles of Lactobacillus johnsonii in the above group (control group and 4% oligogalactose group) were adjusted to a suitable concentration, and then 500 muL was loaded into a NS300 nanoparticle tracking analyzer for detection. Five different fields of each sample were detected, and the acquired data were analyzed by NTA3.2 software. The instrument detection parameter settings are as follows: Camera Level is 16, Time of Video Records is 60 s, Detect Threshold is 7, and other parameters are set to default.
[0041] The results are shown as Figures 1 to 3
[0042] Compared with the blank control group, the number of Lactobacillus johnsonii in the 4% oligogalactose group was increased, but the difference was not significant Figure 1 Compared with the blank control group, the number of Lactobacillus johnsonii in the 4% oligogalactose group was increased, but the difference was not significant
[0043] Figure 2 Compared with the blank control group, the number of Lactobacillus johnsonii in the 4% oligogalactose group was increased, but the difference was not significant
[0044] Compared with the blank control group, the number of Lactobacillus johnsonii in the 4% oligogalactose group was increased, but the difference was not significant Figure 3
[0045] In summary, the added oligogalactose can significantly promote the secretion of Lactobacillus johnsonii extracellular vesicles, providing a new scheme for industrial large-scale production of Lactobacillus extracellular vesicles.
[0046] The method provided by the present application is to add oligogalactose in the Lactobacillus selective culture medium for fermentation, and then inoculate Lactobacillus bacteria for culture; after the culture, the bacterial liquid is separated and purified to obtain bacterial extracellular vesicles, wherein the added oligogalactose in the fermentation process can effectively promote the secretion of Lactobacillus bacterial extracellular vesicles, and the method provided by the present application does not need to add extracellular vesicles of edible plant sources in the fermentation process of lactic acid bacteria, only needs to add oligogalactose in the fermentation process, and the oligogalactose is convenient to obtain, and the commercial production of Lactobacillus extracellular vesicles is facilitated.
[0047] It should be noted that when numerical ranges are involved in the present application, both endpoints of each numerical range and any number between the two endpoints can be selected, and in order to prevent repetition, the preferred embodiments are described in the present application.
[0048] Although the preferred embodiments of the present application have been described, those skilled in the art can make further changes and modifications to the embodiments once they know the basic creative concept, and these changes and modifications all fall within the scope of all changes and modifications of the present application.
Claims
1. A method of increasing the yield of extracellular vesicles of bacteria of the genus Lactobacillus, characterized in that, The method comprises the following steps: adding galactooligosaccharides to a lactobacillus selective culture medium, inoculating lactobacillus bacteria and then culturing; and separating and purifying the cultured bacterial liquid to obtain bacterial extracellular vesicles. The concentration of the galactooligosaccharides in the lactobacillus selective culture medium is 30 g / L to 50 g / L.
2. The method of claim 1, wherein, The lactobacillus bacteria are selected from at least one of Lactobacillus johnsonii, Lactobacillus reuteri, Lactobacillus mucosae, Lactobacillus amylovorus and Lactobacillus animalis.
3. The method of claim 1, wherein, The lactobacillus selective culture medium is an MRS culture medium, which is prepared from the following materials with final concentrations: peptone 9 g / L to 11 g / L, beef infusion powder 7 g / L to 9 g / L, yeast infusion powder 3 g / L to 5 g / L, glucose 19 g / L to 21 g / L, dimethyl bicarbonate 1 g / L to 3 g / L, dimethyl bicarbonate 1 g / L to 3 g / L, sodium acetate 4 g / L to 6 g / L, magnesium sulfate 0.1 g / L to 0.3 g / L, manganese sulfate 0.03 g / L to 0.05 g / L, and Tween 80 0.1 g / L to 2 g / L, and the solvent is water.
4. The method of claim 1, wherein, The culture conditions are 36°C to 38°C and 11 hours to 12 hours.
5. The method of claim 1, wherein, The inoculation amount of the Lactobacillus bacteria is 2 x 10 8 cfu / mL ~ 8 x 10 8 cfu / mL.
6. The method of claim 1, wherein, The separation process is centrifuging the cultured bacterial liquid to obtain the bacterial extracellular vesicles.
7. The method of claim 6, wherein, The centrifugation method is a differential centrifugation method or an ultracentrifugation method.
8. The method of claim 7, wherein, The ultracentrifugation method has the following conditions: centrifugation at 7000 g to 8000 g for 28 minutes to 32 minutes, and centrifugation at 10000 g to 11000 g for 43 minutes to 47 minutes.
9. The method of claim 6, wherein, The purification process is as follows: centrifuging the cultured bacterial liquid, collecting the supernatant, filtering the supernatant through a 0.21 μm to 0.23 μm filter membrane, collecting the filtrate, centrifuging the filtrate at 120000 g to 150000 g for 118 minutes to 122 minutes, discarding the supernatant, collecting the precipitate and resuspending it to obtain the bacterial extracellular vesicles.