Method for producing membrane vesicle for gram-positive bacteria and use of said membrane vesicle

Abstract

This method for producing a membrane vesicle (MV) for gram-positive bacteria is characterized by comprising: a step a for culturing the gram-positive bacteria; a step b for adjusting the pH of a culture medium at the start of the culturing of step a and / or during the culturing of step a; and a step c for adding a surfactant to a bacteria body after the culturing.

Description

Method for producing membrane vesicles of gram-positive bacteria and use of the membrane vesicles

[0001] The present invention relates to a method for producing membrane vesicles of Gram-positive bacteria and the use of such membrane vesicles.

[0002] Membrane vesicles (hereinafter referred to as "MVs") are small vesicles produced by bacteria. The original purpose of bacterial MV production is to facilitate the mutual transmission of physiologically active substances between bacteria, among other things. However, attempts have been made in the field of Gram-negative bacteria to artificially produce MVs for use in other industrial applications.

[0003] For example, US Pat. No. 5,629,999 provides a method for producing vaccines against specific diseases using MVs obtained from Gram-negative bacteria.

[0004] International Publication No. WO2001 / 091788

[0005] On the other hand, with regard to Gram-positive bacteria, a method for producing MVs has not yet been established, and the industrial uses of MVs from Gram-positive bacteria are unknown to begin with, so there is still room for further research and development.

[0006] Therefore, the present invention aims to establish a method for producing MVs of Gram-positive bacteria and to find new uses for the MVs.

[0007] As a result of extensive research, the present inventors have established a method for mass production of Gram-positive bacterial MVs. Furthermore, they have unexpectedly discovered the use of these MVs as anti-inflammatory agents. Specifically, the method for producing Gram-positive bacterial MVs of the present invention and examples of the use of these MVs are as follows:

[0008] [1] A method for producing membrane vesicles (MVs) of Gram-positive bacteria, comprising: step a) culturing the Gram-positive bacteria; step b) adjusting the pH of the medium at the start of the culture and / or during the culture in step a); and step c) adding a surfactant to the bacterial cells after the culture. [2] The method according to [1] above, wherein the pH of the medium is adjusted to 5 to 9 in step b). [3] The method according to [1] above, wherein the Gram-positive bacteria are lactic acid bacteria or bifidobacteria. [4] The method according to any one of [1] to [3] above, wherein the surfactant is a nonionic or anionic surfactant having a steroid skeleton. [5] The method according to [4] above, wherein the surfactant is a bile acid. [6] The method according to [5] above, wherein the surfactant is cholic acid or deoxycholic acid. [7] Membrane vesicles (MVs) derived from Gram-positive bacteria, characterized in that the RNA content of the MVs is 10% or less of the RNA content of naturally occurring MVs. [8] An anti-inflammatory agent containing the MV derived from the Gram-positive bacterium according to [7] above as an active ingredient.

[0009] According to the present invention, a method for producing MVs of Gram-positive bacteria is provided, and new uses thereof can be provided.

[0010] Figure 1 is a flowchart showing the MV production method. Figure 2 shows SEM images of MVs obtained from Gram-positive and Gram-negative bacteria. Figure 3 is a bar graph comparing the MV yield per cell weight when artificially producing MVs between Gram-negative and Gram-positive bacteria. Figure 4 is a graph showing the relationship between the culture time (horizontal axis) of Gram-positive bacteria and the turbidity (OD) and pH (vertical axis). Figure 5(a) is a bar graph showing the MV yield (converted to protein amount) of Gram-positive bacteria (Lp0132) for sMVs produced from the culture supernatant (left), dMVs artificially produced from cells without pH adjustment (center), and dMVs artificially produced from cells with pH adjusted to 7 (right). Figure 5(b) is a bar graph showing the particle number ratio ( / control) of dMVs artificially produced from cells with pH adjusted to 5, 6, 7, 8, 9, and 10. Figure 6(a) is a bar graph showing the MV yield of dMV artificially produced from cells of a Gram-positive bacterium (YIT 0209) without pH adjustment (left) and from cells with pH adjustment (right). Figure 6(b) is a bar graph showing the MV yield of dMV artificially produced from cells of a Gram-positive bacterium (YIT 4014) without pH adjustment (left) and from cells with pH adjustment (right). Figure 7(a) is a bar graph showing the MV yield of dMV artificially produced from cells of a Gram-positive bacterium (Lp0132) with deoxycholic acid (DOC) as the surfactant (left) and with sodium cholate (CA) as the surfactant (right). The vertical axis of Figure 7(a) represents the number of MV particles (×10 ９ The figure shows the MV yield (particles / ml) of dMV (left) containing deoxycholic acid (DOC) as a surfactant and dMV (right) containing sodium cholate (CA) as a surfactant for Gram-positive bacteria (Lp0132). The vertical axis of Figure 7(b) shows the MV yield (μg) (protein equivalent) per 50 mL of culture medium. Figure 8 is a bar graph comparing the IL-10-inducing activity (anti-inflammatory activity) of heat-killed cells (HKB) (left), sLpMV (center), and dLpMV (right) of Gram-positive bacteria (Lp0132) at different concentrations. Figure 9 is a bar graph showing the amount of RNA encapsulated in sMV (left) and dMV (right) of Gram-positive bacteria.

[0011] The production of membrane vesicles of Gram-positive bacteria and the use of the membrane vesicles of the present invention will be described in detail below. The explanation of the constituent elements described below is an example of one embodiment of the present invention, and the present invention is not limited to these contents.

[0012] The present inventors have established a method for producing MVs of Gram-positive bacteria, which comprises step a) culturing Gram-positive bacteria, step b) adjusting the pH of the medium in step a), and step c) adding a surfactant to the bacterial cells after culturing. By including the above steps a) to c), it is possible to artificially mass-produce MVs of Gram-positive bacteria.

[0013] Furthermore, the present inventors have found that MVs of Gram-positive bacteria are useful as anti-inflammatory agents, for example, because they induce the production of IL-10.

[0014] Here, the pH adjustment in step b is preferably adjusted to pH 5 to 9 from the viewpoint of increasing the yield of MVs.

[0015] Furthermore, from the viewpoint of medical use, the Gram-positive bacteria are preferably lactic acid bacteria or bifidobacteria.

[0016] The surfactant is, for example, a nonionic or anionic surfactant having a steroid skeleton. From the viewpoint of pharmaceutical use, it is preferably a type of bile acid. Specifically, from the viewpoint of reproducibility, the bile acid is more preferably cholic acid or deoxycholic acid, and from the viewpoint of increasing the yield of MVs, it is even more preferably deoxycholic acid.

[0017] The surfactant may also contain its salt as long as it does not impair the effects of the invention. The surfactant candidates shown in the following paragraphs may also contain their salts. The salt candidates may be inorganic salts or organic salts.

[0018] Furthermore, in the Examples described below, the RNA content of artificially produced MVs (dMVs) was determined to be 10% or less of the RNA content of naturally occurring MVs (sMVs). Therefore, it is effective to use membrane vesicles (dMVs) whose RNA content in MVs is 10% or less of the RNA content of naturally occurring MVs as MVs derived from Gram-positive bacteria for applications such as anti-inflammatory agents.

[0019] As used herein, "artificially produced" MVs refers to the artificial production of MVs by disrupting the bacterial cell surface with a surfactant and then promoting self-assembly. Therefore, the production method is different from that of naturally occurring MVs produced spontaneously by living bacteria.

[0020] In this specification, spontaneously produced MVs are called sMVs because they are collected from the culture supernatant, whereas artificially produced MVs are called dMVs because they are produced using detergents for surface disruption and self-assembly.

[0021] As will be explained in detail later, the present inventors discovered during the course of research and development that artificial production of MVs from a culture medium without pH adjustment of Gram-positive bacteria results in a much lower MV yield than that of Gram-negative bacteria. As a result of extensive research, they unexpectedly discovered that adjusting the pH during the start or process of culturing Gram-positive bacteria increases the MV yield.

[0022] (1. Gram-positive bacteria) Gram-positive bacteria are bacteria that stain dark blue or purple by Gram staining. From the viewpoint of pharmaceutical use, the Gram-positive bacteria used in the present invention are preferably lactic acid bacteria or bifidobacteria.

[0023] Examples of lactic acid bacteria or bifidobacteria include, but are not limited to, bacteria of the genus Lactiprantibacillus, Lacticaseibacillus, Lactobacillus, Streptococcus, Lactococcus, Enterococcus, and Bifidobacterium.

[0024] Examples of Lactobacillus bacteria include Lactiprantibacillus plantarum, Lacticaseibacillus paracasei (casei), Lactobacillus mali, Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Lactobacillus helveticus.

[0025] Examples of Streptococcus bacteria include Streptococcus thermophilus.

[0026] Examples of bacteria of the genus Lactococcus include Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris.

[0027] Examples of Enterococcus bacteria include Enterococcus faecalis.

[0028] Examples of the Bifidobacterium bacteria include Bifidobacterium breve, Bifidobacterium bifidum, and Bifidobacterium longum.

[0029] Among them, more preferred is Lactiprantibacillus plantarum, Lacticaseibacillus paracasei (casei) or Bifidobacterium breve.In addition, in recent years, the lactic acid bacteria of the genus Lactobacillus have been reclassified.That is, the lactic acid bacteria that previously belong to the genus Lactobacillus have been subdivided, and the genus name of some of the species has been changed.

[0030] (Regarding reclassification of lactic acid bacteria) Zheng et al., A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae. Int. J. Syst. Evol. Microbiol. 2020 Apr; 70(4):2782-2858 DOI 10.1099 / ijsem.0.004107 Therefore, in this specification, the new classification after reclassification will be indicated. Furthermore, for example, among lactic acid bacteria classified in the genus Lactobacillus in the old classification, those that can be newly classified as Lactiprancibacillus or Lacticaseibacillus are included in the Lactiprancibacillus and Lacticaseibacillus of the present application.

[0031] (2. Membrane Vesicles) Membrane vesicles (MVs) are small vesicles produced by bacteria and have a particle size of 20-500 nm (with a median size of approximately 100 nm).

[0032] As described above, in this invention, the inventors established a method for producing sMVs and dMVs derived from Gram-positive bacteria. As shown in the Examples, the production of MVs can be confirmed by SEM imaging and nanoparticle tracking analysis. However, it is impossible to distinguish between sMVs and dMVs by directly identifying the structures of both MVs using an electron microscope, making this method impractical.

[0033] In the examples described below, the RNA content of artificially produced MVs (dMVs) was determined and found to be 10% or less of the RNA content of naturally occurring MVs (sMVs). For Gram-positive bacterial MVs used in applications such as anti-inflammatory drugs, it is effective to use membrane vesicles (dMVs) whose RNA content is 10% or less of the RNA content of naturally occurring MVs. For RNA measurement, for example, Qubit ＴＭA commercially available measurement kit such as RNA BR Assay Kit (Invitrogen) is used.

[0034] (3. Manufacturing Method of MV) Fig. 1 is a flowchart showing a manufacturing method of MV. An outline of the manufacturing method of MV will be explained using Fig. 1. Note that, since the manufacturing method branches off between sMV and dMV after step S1003, for convenience, the step will be referred to as step Ss for sMV and step Sd for dMV.

[0035] When the operation is started, bacteria are cultured in step S1001. The preferred types of bacteria are as described above.

[0036] When producing dMVs, it is preferable to adjust the pH of the medium to a predetermined range in order to increase the yield of MVs.

[0037] The lower limit of the pH is preferably 5 or higher, from the viewpoint of the optimum pH for lactic acid bacteria or bifidobacteria.

[0038] The upper limit is preferably pH 9 or less, from the viewpoint of the optimum pH for lactic acid bacteria or bifidobacteria.

[0039] The lower limit of the culture temperature is preferably 30°C or higher, more preferably 33°C or higher, from the viewpoint of the optimum growth temperature.

[0040] From the viewpoint of the optimum growth temperature, the upper limit is preferably 45°C or lower, more preferably 43°C or lower.

[0041] If the culture temperature is below the lower limit, the cells will not grow properly, and if the temperature is above the upper limit, the cells may be destroyed due to protein denaturation.

[0042] The culture time is not particularly limited, but from the viewpoint of increasing the yield of MVs, the lower limit is preferably 10 hours or more, and more preferably 24 hours or more.

[0043] From the viewpoint of increasing the yield of MVs, the upper limit is preferably 96 hours or less, and more preferably 48 hours or less.

[0044] If the culture time is below the lower limit, the constituent materials of MVs in the bacteria will not be produced sufficiently, and if it is above the upper limit, it is not preferable from the viewpoint of productivity.

[0045] Examples of the culture method include agitation culture, static culture, shaking culture, and neutral culture.

[0046] Next, in step S1002, the culture solution is centrifuged to separate the supernatant from the bacterial cells. If sMVs spontaneously produced by the bacteria are to be extracted, the culture supernatant from step S1002 is obtained in step Ss1003.

[0047] Furthermore, in step Ss1004, the culture supernatant is filtered, and in step Ss1005, it is ultracentrifuged to obtain sMVs.

[0048] On the other hand, when artificially produced dMVs are produced, in step Sd1003, the bacterial body part is obtained from step S1002.

[0049] Next, the bacterial body is suspended in a buffer solution (step Sd1004), and a predetermined surfactant is added (step Sd1005) to destroy the bacterial cell walls.The solution is then stirred (step Sd1006) to promote self-organization, and after a predetermined time has passed, the solution is filtered (step Sd1007), and finally, ultracentrifugation is performed (step Sd1008) to obtain dMVs.

[0050] The surfactant is not particularly limited as long as it can produce dMVs, but examples thereof include nonionic or anionic surfactants, bile acids that dissolve bacterial cell walls, and secondary bile acids that are produced as metabolites of bile acids by bacteria. The surfactant may also include its salts, provided that the effects of the invention are not impaired.

[0051] Bile acids include cholic acid, glycocholic acid, taurocholic acid, chenodeoxycholic acid, hyocholic acid, 5 alpha-cyprinol, and the like.

[0052] Secondary bile acids include deoxycholic acid, lithocholic acid, hyodeoxycholic acid, and ursodeoxycholic acid.

[0053] Among these, cholic acid or deoxycholic acid is preferred from the viewpoint of reproducibility of artificial MV production, and deoxycholic acid is more preferred from the viewpoint of increasing the yield of MVs.

[0054] The surfactant concentration is preferably in the range of 0.1% to 5%. If the concentration is below the lower limit, the bacterial cell walls cannot be dissolved, and if the concentration is above the upper limit, the high concentration of surfactant may have a negative effect on self-assembly.

[0055] Furthermore, the time required for self-assembly (stirring time) after adding the surfactant is preferably in the range of 15 minutes to 1 hour. If it is below the lower limit, self-assembly is insufficient, and if it is above the upper limit, it is not preferable from the viewpoint of productivity.

[0056] The stirring speed is 100 to 200 min -1 If the value is below the lower limit, the self-organization is insufficient, and if the value is above the upper limit, it is not preferable from the viewpoint of productivity.

[0057] The MVs obtained through the above steps may be stored after being processed, for example, by concentration / dilution, freezing, drying, or powdering.

[0058] When MV is formulated for pharmaceutical use, the dosage form may be selected from the following: tablets, capsules, granules, sugar-coated tablets, pills, fine granules, powders, dusts, sustained-release preparations, suspensions, emulsions, syrups, lyophilized preparations, liquids, etc.

[0059] The above-mentioned preparation may contain MV alone or in combination with a pharmaceutically acceptable carrier.

[0060] If necessary, excipients, stabilizers, preservatives, wetting agents, emulsifiers, lubricants, sweeteners, coloring agents, flavoring agents, buffers, antioxidants, pH adjusters, etc. may be added appropriately.

[0061] EXAMPLES The present invention will be specifically described based on the following examples, but the present invention is not limited to these examples. Abbreviations mainly used in the following examples are explained in the table below. CA Cholic acid DOC Deoxycholic acid AI12060 Alistipes indistinctus YIT 12060 AiMV Generic term for AI12060 MVs sAiMV MVs isolated from the culture supernatant of AI12060 dAiMV MVs artificially produced from AI12060 cells using DOC Lp0132 Lactiplantibacillus plantarum YIT 0132 LpMV Generic term for Lp0132 MVs sLpMV MVs isolated from the culture supernatant of Lp0132 dLpMV MVs artificially produced from Lp0132 cells using DOC YIT 0209 Lacticaseibacillus paracasei YIT 0209 YIT 4014 Bifidobacterium breve YIT 4014 Lp0132 (Lactiplantibacillus plantarum YIT 0132) was deposited internationally as Lactobacillus plantarum YIT 0132 (deposit number FERM BP-11349) on February 24, 2010 at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NITE-NPMD) (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818).

[0062] (Example 1) <Bacteria> AI12060, Lp0132, YIT 0209, and YIT 4014 were used, each of which had been frozen and stored. AI12060 is known to be a Gram-negative bacterium, while Lp0132, YIT 0209, and YIT 4014 are known to be Gram-positive bacteria.

[0063] AI12060 and YIT 4014 were cultured in GAM (1% glucose) medium under anaerobic conditions, while Lp0132 and YIT 0209 were cultured in MRS medium under aerobic conditions. All bacteria were subcultured at 37°C and statically cultured for 48 to 96 hours.

[0064] <Buffer Preparation> First buffer (0.1 M Tris-HCl, 0.01 M EDTA) solution (pH 8.9) was prepared. Trizma® base (Sigma-Aldrich) was dissolved in MQ water to a final concentration of 0.1 M Tris solution, and 0.5 mol / L EDTA solution (pH 8.0) (Nacalai Tesque) was added to a final concentration of 0.01 M.

[0065] After sterilization with a PES syringe filter (Millipore) with a pore size of 0.22 μm, the pH of the solution was adjusted to 8.9 with 2 N HCl solution.

[0066] Next, a second buffer (10% DOC solution) was prepared by dissolving sodium DOC (Fujifilm Wako Pure Chemical Industries, Ltd.) in the first buffer to prepare a 10% (w / v) DOC solution, which was used as the second buffer.

[0067] Treatment with surfactant (DOC) The bacterial cell culture was centrifuged (8,000×g, 20 min, 4° C.) and the culture supernatant was removed. The collected bacterial cells were washed with PBS and suspended in First Buffer in 1 / 10 the volume of the culture medium.

[0068] After the bacterial precipitate has completely disappeared, add Second Buffer so that the final concentration of DOC becomes 0.5%, and stir at room temperature using a stirrer (for 100 to 200 minutes). －１ , 30 min).

[0069] Example 2 Isolation of sMVs Ultracentrifugation was used to isolate sAiMVs. The bacterial culture was centrifuged (8,000 × g, 20 min, 4°C) and the supernatant was collected. The collected culture supernatant was passed through a 0.45 μm PES syringe filter (Millipore) and then ultracentrifuged (P70AT rotor, 200,000 × g, 2 h, 4°C) to pellet the MVs, which were then suspended in PBS. The pellet was then washed three times using an ultrafilter (Millipore) with a molecular weight cutoff of 100 kDa to isolate sAiMVs. Density gradient ultracentrifugation was used to isolate sLpMVs, YIT 0209, or YIT 4014 sMVs. The culture medium containing the bacterial cells was centrifuged (8,000×g, 20 min, 4° C.) to remove the bacterial cells, and the culture supernatant was collected.

[0070] The collected culture supernatant was passed through a 0.45 μm PES syringe filter (Millipore) and then concentrated to approximately 1 mL using ultrafiltration (Millipore) with a molecular weight cutoff of 100 kDa. A density gradient was prepared using OptiPrep (60% iodixanol) (Sigma-Aldrich).

[0071] The OptiPrep solution was diluted with MQ water, and 2 mL of 25% to 40% iodixanol solution was layered at 5% intervals, with 1 mL of the concentrated supernatant layer layered on top.

[0072] Further, the mixture was ultracentrifuged (P40ST rotor, 200,000×g, 3 hours, 4° C.), and 1 mL of the culture supernatant containing the band of interest was collected using a pipette to obtain an isolated sMV solution.

[0073] <Isolation of dMV> For dAiMV, ultracentrifugation was used. The solution after DOC treatment was centrifuged (8,000 × g, 20 min, 4 ° C) and the supernatant was collected. The collected culture supernatant was passed through a 0.45 μm pore size PES syringe filter (Millipore) and then ultracentrifuged (P70AT rotor, 200,000 × g, 2 h, 4 ° C) to pellet the MVs, which were then suspended in PBS. Subsequently, the dAiMVs were obtained by washing three times using an ultrafilter (Millipore) with a molecular weight cutoff of 100 kDa.

[0074] On the other hand, for dLpMV, YIT 0209, or YIT 4014 dMV, density gradient ultracentrifugation was used. The solution after DOC treatment was centrifuged (12,000 × g, 30 min to 1 h, 4°C), and the supernatant was collected. The collected culture supernatant was passed through a 0.45 μm pore size PES syringe filter (Millipore) and then concentrated to approximately 1 mL using an ultrafilter (Millipore) with a molecular weight cutoff of 100 kDa.

[0075] Density gradients were prepared using OptiPrep (60% iodixanol, Sigma-Aldrich). The OptiPrep solution was diluted with MQ water, and 1 mL of 10-40% iodixanol solution was layered at 5% intervals, with 1 mL of the concentrate layer on top. Ultracentrifugation (P40ST rotor, 200,000 × g, 3 h, 4°C) was performed, and 1 mL containing the band of interest was recovered with a pipette to obtain the respective dMVs.

[0076] <Quantification of MVs> Quantification of MV proteins was carried out by a predetermined method using Pierce (registered trademark) BCA Protein Assay Kit (manufactured by Thermo).

[0077] <SEM Observation of MVs> MVs isolated by ultracentrifugation were washed with MQ water and then freeze-dried. The dried MV powder was coated using an Autofine Coater (JEC-3000FC) and observed under a tabletop scanning electron microscope (JCM-6000).

[0078] On the other hand, for MVs isolated by density gradient ultracentrifugation, 1% BSA was passed through an ultrafilter (Millipore) to block the membrane, followed by washing three times with MQ water to remove excess BSA from the membrane. The isolated MVs in the OptiPrep solution were then washed with MQ water. Lyophilization was performed, and the MV powder was coated using an Autofine Coater (JEC-3000FC) and observed under a tabletop scanning microscope (JCM-6000).

[0079] Figure 2 shows SEM images of dMVs obtained from Gram-positive and Gram-negative bacteria: Figure 2(A) shows dAiMV, Figure 2(B) shows dLpMV, Figure 2(C) shows dMV from YIT 0209, and Figure 2(D) shows dMV from YIT 4014.

[0080] Both SEM images confirmed spherical structures that appeared to be MVs. This indicates that it is possible to artificially produce MVs using DOC in both Gram-negative and Gram-positive bacteria, and that MVs (dMVs) can be obtained not only from the supernatant (sMVs) but also from the bacterial cells in a single culture.

[0081] Furthermore, in all DOC treatments, stirring was performed using a stirrer at room temperature for 30 minutes, which revealed that homogenization of dMV is possible if stirring is performed at room temperature for at least about 30 minutes.

[0082] Example 3: <Study to Increase dMV Yield> On the other hand, it was also revealed that the production of dMVs from Gram-positive bacteria involves the following problems. Figure 3 is a bar graph comparing the MV yield per bacterial cell weight when artificially producing MVs between Gram-negative bacteria (left) and Gram-positive bacteria (right). The horizontal axis of Figure 3 represents AI12060 for Gram-negative bacteria (left) and Lp0132 for Gram-positive bacteria (right). The vertical axis represents the amount of MV (μg) (equivalent to protein amount) per bacterial cell weight (mg).

[0083] As shown in Figure 3, the dMV yield per cell weight of Gram-positive bacteria is significantly lower than that of Gram-negative bacteria. Therefore, in order to utilize dMVs from Gram-positive bacteria for specific applications, it was necessary to produce artificial MVs more efficiently.

[0084] The inventors investigated DOC treatment conditions, such as DOC concentration and stirring time during DOC addition, but the yield of dLpMV did not increase (data not shown). Therefore, as a further investigation, they investigated whether it was possible to improve the sensitivity to DOC by controlling the bacterial condition.

[0085] 4 is a graph showing the relationship between the culture time (horizontal axis) of a Gram-positive bacterium (Lp0132) and the turbidity (OD) and pH (vertical axis). The horizontal axis of FIG. 4 shows the culture time (h), and the vertical axis shows the relative value of turbidity (OD) and the pH value. The value on the vertical axis is pH. In the graph, the solid line shows turbidity (OD), and the dotted line shows pH.

[0086] 4, in the case of culture without pH adjustment, the pH of the culture medium began to decrease after 5 hours of culture, and reached a low pH environment (near pH 4) after 10 hours of culture. Therefore, the pH of the culture medium was adjusted to 7 once after 8 hours of culture, just before the middle of the logarithmic growth phase and before the low pH environment was reached, to prevent prolonged culture in a low pH environment.

[0087] <pH Adjustment of Lp0132> Based on the above, we investigated whether pH adjustment would increase the yield of dMVs from the Gram-positive bacterium Lp0132. Figure 5(a) shows the results of dMV production by DOC from Lp0132 cells whose pH was adjusted to 7 after 8 hours of culture. We also examined the extent to which the effect of pH adjustment would be achieved by adjusting the pH to 5, 6, 7, 8, 9, and 10. The results are shown in Figure 5(b).

[0088] Figure 5(a) is a bar graph showing the MV yields of Gram-positive bacteria (Lp0132) for sMVs produced from culture supernatant (left), dMVs artificially produced from culture medium without pH adjustment (center), and dMVs artificially produced from culture medium adjusted to pH 7 (right). The vertical axis shows the MV yield (μg) (protein equivalent) per 50 mL of culture medium. Figure 5(b) is a bar graph showing the MV yields of Gram-positive bacteria (Lp0132) for dMVs artificially produced from culture medium adjusted to pH 5, 6, 7, 8, 9, and 10. The vertical axis shows the MV particle number ratio ( / control). The control is the particle number of dMVs artificially produced from culture medium without pH adjustment.

[0089] Figure 5(a) shows that the yield of the cells cultured with pH adjusted during cultivation is significantly higher than that of cells cultured without pH adjustment. Figure 5(b) shows that the effect is particularly pronounced in the pH range of 5 to 9.

[0090] <pH adjustment of YIT 0209 and YIT 4014> Similarly, we investigated whether adjusting the pH would increase the dMV yield for YIT 0209 and YIT 4014. dMVs were produced by DOC from cells of YIT 0209 and YIT 4014, the pH of which was adjusted to 7 after 8 hours of culture, and the results are shown in Figure 6.

[0091] Figures 6(a) and (b) are bar graphs showing the MV yields of Gram-positive bacteria (YIT 0209 and YIT 4014) artificially produced from culture medium without pH adjustment (left) and artificially produced from culture medium adjusted to pH 7 (right). In Figure 6(a), for YIT 0209, the left horizontal axis shows dMVs obtained from bacterial cells without pH adjustment and the right horizontal axis shows dMVs obtained from culture medium adjusted to pH 7, and the vertical axis shows the number of particles per mL. In Figure 6(b), for YIT 4014, the left horizontal axis shows dMVs obtained from bacterial cells without pH adjustment and the right horizontal axis shows dMVs obtained from culture medium adjusted to pH 7, and the vertical axis shows the number of particles per mL.

[0092] 6(a) and (b) reveal that for Gram-positive bacteria other than Lp0132, the yield of bacterial cells cultured with pH adjustment was significantly higher than that of bacterial cells cultured without pH adjustment.

[0093] Previous studies have shown that the cell walls of Gram-positive bacteria thicken when cultured in a low pH environment, and it was predicted that DOC sensitivity would decrease as the cell walls become thicker and stronger. Therefore, it was thought that adjusting the pH thinned the cell walls of Gram-positive bacteria during culture, moderately promoting MV self-assembly and increasing the yield of dMVs.

[0094] From the above results, it was revealed that in the method for producing membrane vesicles (MVs) of Gram-positive bacteria, mass production of dMVs is possible by adjusting the pH of the medium at the start of culture in step a and during culture (step b), and adding a surfactant to the bacterial cells after culture (step c).

[0095] Example 4 <Study on Surfactants> In this example, cholic acid (CA), a primary bile acid, was used to study surfactants for producing dMV.

[0096] Except for using CA sodium (FUJIFILM Wako Pure Chemical Industries, Ltd.) as the surfactant, dMV of the Gram-positive bacterium Lp0132 was isolated in the same manner as in Examples 1 and 2. The results are shown in Figure 7.

[0097] Figures 7(a) and (b) are bar graphs showing the MV yields of dMVs containing DOC as a surfactant (left) and sodium CA as a surfactant (right) for Gram-positive bacteria (Lp0132). The vertical axis of (a) represents the number of MV particles (×10 ９ The vertical axis of (b) indicates MV yield (μg) (protein equivalent) per 50 mL of culture medium.

[0098] 7(a) and (b) demonstrate that dMV can be produced even when CA, a primary bile acid, is used as the surfactant. Furthermore, from the viewpoint of dMV yield, the use of DOC was preferable.

[0099] Example 5 In this example, applications of dMV of Gram-positive bacteria, which can now be mass-produced, were explored.

[0100] <Cells> The mouse monocytic leukemia macrophage-like cell line RAW264.7 was used. RAW264.7 is known to respond to most TLR ligands and to produce inflammatory cytokines.

[0101] RAW264.7 was cultured in a DMEM medium containing 10% FBS and 100 μg / mL streptomycin (10% FBS / DMEM) under 5% CO ２ Subculture was carried out at 37°C under the following conditions.

[0102] <Examination of IL-10 Inducing Activity> In a 96-well plate, HKB or LpMV of Lp0132 (final concentrations of 1 μg / ml, 5 μg / ml, and 10 μg / ml) suspended in 10% DMEM medium and RAW264.7 (2.0 × 10 ５ cells / well) and incubated in an incubator at 5% CO ２ The cells were cultured at 37°C for 24 hours under the ambient conditions.

[0103] The culture supernatant was collected, and the amount of IL-10 produced was measured by ELISA using Mouse IL-10 Duo Set ELISA (R&D systems).

[0104] The Capture antibody was diluted to a predetermined concentration with PBS, and 100 μL of each was added to a 96-well ELISA plate and incubated overnight at 4° C. After washing each well with PBS containing 0.05% (w / v) Tween 20 (washing solution), 100 μL of PBS containing 1% (w / v) BSA (BSA / PBS) was added to each well, followed by blocking at 37° C. for 90 minutes.

[0105] After blocking, 100 μL of culture supernatant diluted 1000-fold with 1% (w / v) BSA / PBS was added to each well and incubated overnight at 4° C. After incubation, the wells were washed three times, and 100 μL of a detection antibody diluted to a predetermined concentration with 1% (w / v) BSA / PBS was added to each well and incubated at room temperature for 120 minutes. After that, 100 μL of Streptavidin-HRP diluted to a predetermined concentration with 1% (w / v) BSA / PBS was added to each well and incubated in the dark at room temperature for 20 minutes.

[0106] Thereafter, after washing three times with washing solution, 50 μL of 1-Step® Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) was added and reacted at room temperature for 5 minutes.

[0107] After checking the color, 2M H ２ SO ４ The reaction was stopped by adding 50 μL of each of the following, and the absorbance at 450 nm and 595 nm was measured using a microplate reader.

[0108] <Differences in properties between sLpMV and dLpMV in Lp0132> LpMV activity was evaluated by its ability to induce IL-10 in the macrophage cell line RAW264.7. Figure 8 is a bar graph comparing the IL-10-inducing activity of Gram-positive bacteria (Lp0132) at various concentrations against HKB (left), sLpMV (center), and dLpMV (right).

[0109] The IL-10 induction activity on the vertical axis of Figure 8 indicates the amount of IL-10 produced by RAW264.7 upon treatment with HKB or LpMV, expressed as signal intensity per medium. IL-10 production was measured by ELISA after collecting the culture supernatant after culture. The horizontal axis of Figure 8 indicates, from left to right, the amount of heat-killed Lp0132 cells (HKB), sLpMV, and dLpMV added (10 μg / mL, 5 μg / mL, and 1 μg / mL).

[0110] Lp0132 HKB, sLpMV, or dLpMV was added to RAW264.7 at final concentrations of 1 μg / ml, 5 μg / ml, or 10 μg / ml, respectively, and the amount of IL-10 produced was measured. As a result, no IL-10 production was observed with sLpMV treatment, but IL-10 production similar to that seen with HKB treatment was confirmed with dLpMV treatment.

[0111] These results indicate that sLpMV does not inherit the IL-10-inducing activity of HKB, whereas dLpMV retains this activity. Furthermore, the IL-10-inducing activity of dLpMV was higher than that of HKB.

[0112] It is widely recognized that Gram-positive bacteria produce MVs from the cell membrane and then release them outside the cell through the cell wall. When bacteria spontaneously produce MVs, it is thought that few cell wall components are carried over.

[0113] On the other hand, when disrupted with a surfactant, it is assumed that the cell membrane with attached cell wall components is present as a material for MV self-assembly. Therefore, the membrane components of the two MVs are different, which is thought to be the reason for the presence or absence of IL-10 inducing activity.

[0114] From the above, it was suggested that dMV of Gram-positive bacteria could be used as an active ingredient of an anti-inflammatory agent or an active ingredient of an IL-10-inducing agent.

[0115] Example 6 Characteristics of dMVs of Gram-positive bacteria As shown in Figure 2, the production of MVs can be confirmed by SEM images, etc. However, it is impossible to distinguish between sMVs and dMVs directly using an electron microscope, etc., because it is not practical to directly identify the structures of both MVs.

[0116] Therefore, we attempted to distinguish between sMVs and dMVs by measuring the amount of RNA encapsulated in MVs. Figure 9 is a bar graph showing the amount of RNA encapsulated in sMVs (left) and dMVs (right) of Gram-positive bacteria. The vertical axis of Figure 9 shows the amount of RNA encapsulated in MVs of Gram-positive bacteria measured using the Qubit® RNA BR Assay Kit (Invitrogen). The horizontal axis shows sMVs and dMVs from left to right.

[0117] As shown in Figure 9, the RNA content of artificially produced MVs (dMVs) was determined to be 10% or less of that of naturally occurring MVs (sMVs). For Gram-positive bacterial MVs used in applications such as anti-inflammatory drugs, it is effective to use membrane vesicles (dMVs) whose RNA content is 10% or less of that of naturally occurring MVs.

Claims

1. A method for producing membrane vesicles (MVs) of Gram-positive bacteria, comprising: step a) culturing the Gram-positive bacteria; step b) adjusting the pH of the medium at the start of the culturing and / or during the culturing in step a); and step c) adding a surfactant to the bacterial cells after the culturing.

2. The method of claim 1, wherein the pH of the medium is adjusted to 5 to 9 in steps a and / or b.

3. The method according to claim 1, wherein the Gram-positive bacteria are lactic acid bacteria or bifidobacteria.

4. The manufacturing method according to any one of claims 1 to 3, wherein the surfactant is a nonionic or anionic surfactant having a steroid skeleton.

5. The method of claim 4, wherein the surfactant is a bile acid.

6. The method of claim 5, wherein the surfactant is cholic acid or deoxycholic acid.

7. Membrane vesicles (MVs) derived from Gram-positive bacteria, characterized in that the RNA content of the MVs is 10% or less of the RNA content of naturally occurring MVs.

8. An anti-inflammatory agent containing MV derived from Gram-positive bacteria according to claim 7 as an active ingredient.

Images