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

Abstract

A method for producing a membrane vesicle (MV) of a gram-positive bacterium, characterized by comprising: a process a in which the gram-positive bacterium is cultured; a process b in which the pH of a culture medium is adjusted at the start of the culture and / or during the culture of the process a; and a process c in which a surfactant is added to the bacterial cells after the culture.

Description

Technical Field

[0001] This invention relates to a method for manufacturing membrane vesicles of Gram-positive bacteria and the use of such membrane vesicles. Background Technology

[0002] Membrane vesicles (MVs) are vesicles produced by bacteria. The original purpose of bacterial MV production includes various purposes such as the exchange of physiologically active substances between bacteria. However, in the field of Gram-negative bacteria, there are attempts to artificially manufacture MVs and utilize them for other industrial applications.

[0003] For example, Patent Document 1 provides a method for manufacturing a vaccine against a specific disease using MV obtained from Gram-negative bacteria.

[0004] Existing technical documents Patent documents Patent Document 1: International Publication No. WO2001 / 091788 Summary of the Invention

[0005] The problem that the invention aims to solve On the other hand, regarding Gram-positive bacteria, the manufacturing method of MV has not yet been established. In addition, the industrial applications of Gram-positive bacteria MV are unclear, leaving room for further research and development.

[0006] Therefore, the object of the present invention is to establish a method for manufacturing MV of Gram-positive bacteria and to discover new uses for the MV.

[0007] Technical solutions for solving the problem The inventors conducted in-depth research and established a method for mass-producing MV of Gram-positive bacteria. Furthermore, they unexpectedly discovered the use of this MV as an anti-inflammatory agent.

[0008] Specifically, the method for manufacturing the Gram-positive bacteria MV of the present invention and the use of the MV are as follows.

[0009] [1] A method for manufacturing membrane vesicles (MV) of Gram-positive bacteria, comprising: Step a, wherein the Gram-positive bacteria are cultured; Step b, wherein the pH of the culture medium is adjusted at the start of and / or during the culture in step a; and Step c, in which a surfactant is added to the bacterial cells after culturing.

[0010] [2] According to the manufacturing method described in [1] above, wherein, In step b, the pH of the culture medium is adjusted to 5-9.

[0011] [3] According to the manufacturing method described in [1] above, wherein, The Gram-positive bacteria are lactic acid bacteria or bifidobacteria.

[0012] [4] The manufacturing method according to any one of [1] to [3] above, wherein, The surfactant is a nonionic or anionic surfactant with a steroid backbone.

[0013] [5] According to the manufacturing method described in [4] above, wherein, The surfactant is bile acid.

[0014] [6] According to the manufacturing method described in [5] above, wherein, The surfactant is cholic acid or deoxycholic acid.

[0015] [7] A MV derived from Gram-positive bacteria, characterized in that, The RNA content of the membrane vesicles (MVs) is less than 10% of the RNA content of naturally occurring MVs.

[0016] [8] An anti-inflammatory agent that uses the above-mentioned [7] MV derived from Gram-positive bacteria as its active ingredient.

[0017] Invention Effects According to the present invention, a method for manufacturing MV of Gram-positive bacteria can be provided, and novel uses thereof can be provided. Attached Figure Description

[0018] Figure 1 This is a flowchart illustrating the method for creating an MV.

[0019] Figure 2 These are SEM images of MV obtained from Gram-positive or Gram-negative bacteria.

[0020] Figure 3 This is a bar chart comparing the yield of MV per unit weight of Gram-negative bacteria with that of Gram-positive bacteria during artificial MV production.

[0021] Figure 4 It is a graph showing the relationship between the culture time (horizontal axis) of Gram-positive bacteria and turbidity (OD) and pH (vertical axis).

[0022] Figure 5(a) is a bar chart showing the MV yield (converted from protein content) of Gram-positive bacteria (Lp0132) produced from culture supernatant (left), artificially produced dMV from culture medium without pH adjustment (center), and artificially produced dMV from culture medium with pH adjusted to 7 (right).

[0023] Figure 5(b) is a bar chart showing the particle number ratio ( / control) of dMV artificially produced from bacteria with pH adjusted to 5, 6, 7, 8, 9, and 10.

[0024] Figure 6(a) is a bar chart showing the MV production of dMV artificially produced by Gram-positive bacteria (YIT 0209) with unpH-adjusted cells (left) and dMV artificially produced by pH-adjusted cells (right).

[0025] Figure 6(b) is a bar chart showing the MV production of dMV artificially produced by Gram-positive bacteria (YIT 4014) with unpH-adjusted cells (left) and dMV artificially produced by pH-adjusted cells (right).

[0026] Figure 7(a) is a bar chart showing the MV yield of Gram-positive bacteria (Lp0132) with deoxycholic acid (DOC) as the surfactant (left) and sodium cholic acid (CA) as the surfactant (right). The vertical axis of Figure 7(a) represents the number of MV particles (×10⁻¹⁰). 9 (particles / ml)

[0027] Figure 7(b) is a bar chart showing the MV yield of Gram-positive bacteria (Lp0132) with deoxycholic acid (DOC) as the surfactant (left) and sodium cholic acid (CA) as the surfactant (right). The vertical axis of Figure 7(b) represents the MV yield (μg) per 50 mL of culture medium (converted from protein mass).

[0028] Figure 8 This is a bar chart comparing the IL-10-induced activity (anti-inflammatory capacity) of heated dead cells (HKB) (left), sLpMV (center), and dLpMV (right) against Gram-positive bacteria (Lp0132) at different concentrations.

[0029] Figure 9 This is a bar chart showing the amount of RNA contained in the sMV (left) and dMV (right) of Gram-positive bacteria. Detailed Implementation

[0030] The following is a detailed description of the manufacture of membrane vesicles of Gram-positive bacteria and the utilization of these membrane vesicles according to the present invention. The description of the constituent elements described below is an example of one embodiment of the present invention and is not limited thereto.

[0031] The inventors have established a method for manufacturing MV of Gram-positive bacteria, comprising step a of culturing Gram-positive bacteria, step b of adjusting the pH of the culture medium in step a, and step c of adding a surfactant to the bacterial cells after culturing. By including the above steps a to c, MV of Gram-positive bacteria can be artificially mass-produced.

[0032] Furthermore, the inventors have discovered that the MV of Gram-positive bacteria is useful as an anti-inflammatory agent because it induces the production of, for example, IL-10.

[0033] From the perspective of increasing MV production, the pH adjustment of process b is preferably set to pH 5-9.

[0034] In addition, from a medical perspective, Gram-positive bacteria are preferably lactic acid bacteria or bifidobacteria.

[0035] Furthermore, the surfactant is, for example, a nonionic or anionic surfactant having a steroid backbone. From a pharmaceutical viewpoint, a bile acid is preferred. Specifically, from a reproducibility viewpoint, the bile acid is more preferably cholic acid or deoxycholic acid, and from the viewpoint of increasing MV production, deoxycholic acid is further preferred.

[0036] Furthermore, to the extent that it does not impair the effects of the invention, the surfactant may contain its salt. Similarly, the salts of the surfactants shown in the following paragraphs may also be included. The salts may be inorganic or organic salts.

[0037] Furthermore, after determining the RNA content encapsulated in the artificially manufactured MV (dMV) according to the examples described later, it was found that the RNA content encapsulated in the MV (sMV) was less than 10%. As a Gram-positive bacteria-derived MV suitable for use as an anti-inflammatory agent, using dMV with an RNA content of less than 10% of the RNA content of the naturally occurring MV is effective.

[0038] In this specification, "artificially manufactured" MV refers to the artificial production of MV by disrupting the cell surface of bacteria with surfactants, thereby promoting self-organization. Therefore, this method differs from the spontaneous (naturally generated) MV produced by living bacteria.

[0039] In this specification, spontaneously generated (naturally produced) bacterial MVs, which are recovered from the supernatant of the culture medium, are referred to as sMVs. Artificially manufactured MVs, which utilize surfactants for surface disruption and self-organization, are referred to as dMVs.

[0040] Although detailed later, the inventors obtained the following insights during the research and development process: compared to Gram-negative bacteria, Gram-positive bacteria produce very little MV when artificially generating MV in culture media with unadjusted pH. Furthermore, in-depth research unexpectedly revealed that MV production increases by adjusting the pH at the beginning or during the culture of Gram-positive bacteria.

[0041] (1. Gram-positive bacteria) Gram-positive bacteria are bacteria that are stained dark blue or purple by Gram staining. From a medical point of view, the Gram-positive bacteria used in this invention are preferably lactic acid bacteria or bifidobacteria.

[0042] Although there are no specific limitations, examples of lactic acid bacteria or bifidobacteria include: *Lactobacillus plantarum*, *Lactobacillus*, *Lactobacillus*, *Streptococcus*, *Lactococcus*, *Enterococcus*, or *Bifidobacterium*.

[0043] Examples of bacteria in the genus *Lactobacillus* include: *Lactiplantibacillus plantarum*, *Lactaseibacillus paracasei*, *Lactobacillus mali*, *Lactobacillus acidophilus*, *Lactobacillus delbrueckii subsp. bulgaricus*, and *Lactobacillus shelveticus*.

[0044] Examples of Streptococcus include Streptococcus thermophilus.

[0045] Among the bacteria of the genus Lactococcus, examples include: Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. lactis fat.

[0046] Among the bacteria of the genus Enterococcus, examples include Enterococcus faecalis.

[0047] Among the bacteria of the genus Bifidobacterium, examples include: Bifidobacterium breve, Bifidobacterium bifidum, and Bifidobacterium longum.

[0048] More preferably, it is *Lactobacillus plantarum*, *Lactobacillus paracasei*, or *Bifidobacterium breve*.

[0049] It should be noted that in recent years, lactic acid bacteria of the genus *Lactobacillus* have been reclassified. That is, the genus of lactic acid bacteria belonging to the existing genus *Lactobacillus* has been subdivided, and in some species, the genus name has been changed.

[0050] (Regarding the reclassification of lactic acid bacteria) According to 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 DOI10.1099 / ijsem.0.004107, this specification indicates the designation of the new classification after reclassification. Additionally, for example, among lactic acid bacteria classified as Lactobacillus in the old classification, those newly classified as *Lactobacillus plantarum* and *Lactobacillus casei* are included in the *Lactobacillus plantarum* and *Lactobacillus casei* of this invention.

[0051] (2. Membrane vesicles) Membrane vesicles (MVs) are vesicles produced by bacteria, with a particle size of 20 nm to 500 nm (center size of about 100 nm).

[0052] As described above, the inventors of this invention have established methods for manufacturing sMV and dMV for MVs derived from Gram-positive bacteria. Here, as shown in the embodiments, it can be confirmed whether MVs have been manufactured using methods such as SEM image analysis and nanoparticle tracking. However, it is impractical to directly determine the structure of sMVs and dMVs using electron microscopy or similar methods.

[0053] It should be noted that after determining the RNA content encapsulated in the artificially manufactured MV (dMV) according to the examples described later, the RNA content encapsulated in the naturally occurring MV (sMV) was found to be less than 10%. As MVs derived from Gram-positive bacteria suitable for applications such as anti-inflammatory agents, using dMVs with an RNA content in the membrane vesicles (MV) that is less than 10% of the RNA content of naturally occurring MVs is effective. In RNA assays, for example, Qubit... TM Commercially available assay kits such as the RNA BR Assay Kit (Invitrogen) are available.

[0054] (3. Methods for creating MV) Figure 1 This is a flowchart illustrating the manufacturing method of MV. (Using...) Figure 1 This section provides an overview of the manufacturing method for MV. It should be noted that, since there are differences in the manufacturing methods for sMV and dMV after step S1003, for convenience, the case of sMV will be referred to as step Ss, and the case of dMV will be referred to as step Sd.

[0055] When the operation begins, bacterial culture is performed in step S1001. The preferred bacterial species are as described above.

[0056] In the case of manufacturing dMV, from the viewpoint of increasing MV yield, it is preferable to adjust the pH of the culture medium to a specified range.

[0057] From the perspective of the optimal pH for lactic acid bacteria or bifidobacteria, the preferred pH is above pH 5 as a lower limit.

[0058] From the perspective of the optimal pH for lactic acid bacteria or bifidobacteria, as an upper limit, pH 9 or below is preferred.

[0059] From the perspective of optimal development temperature, the lower limit of the culture temperature is preferably above 30°C, and more preferably above 33°C.

[0060] From the viewpoint of optimal development temperature, the upper limit is preferably 45°C or below, and more preferably 43°C or below.

[0061] Below the lower limit of the culture temperature, the bacteria cannot develop properly; above the upper limit, the bacteria may be destroyed due to protein modification.

[0062] There is no particular limitation on the cultivation time, but from the viewpoint of increasing MV production, it is preferred to be more than 10 hours, and more preferably more than 24 hours, as a lower limit.

[0063] From the perspective of increasing MV production, the upper limit is preferably 96 hours or less, and more preferably 48 hours or less.

[0064] Below the lower limit of the culture time, the building blocks of MV in bacteria cannot be sufficiently generated; above the upper limit, it is not preferred from a productivity point of view.

[0065] Examples of cultivation methods include: stirred culture, static culture, shaking culture, and neutralization culture.

[0066] Then, in step S1002, the culture medium is centrifuged to separate it into supernatant and bacterial cells. Here, in the case of extracting spontaneously generated sMV from bacteria, the culture supernatant from step S1002 is obtained in step Ss1003.

[0067] Then, in step Ss1004, the culture supernatant is filtered, and in step Ss1005, it is ultracentrifuged to obtain sMV.

[0068] On the other hand, in the case of manufacturing artificially produced dMV, in step Sd1003, the bacterial cell portion is obtained from step S1002.

[0069] Next, the bacterial cells are suspended in a buffer solution (step Sd1004), and a specified surfactant is added (step Sd1005) to disrupt the cell walls of the cells. The solution is then stirred (step Sd1006) to promote self-organization. After a specified time, the solution is filtered (step Sd1007) and finally ultracentrifuged (step Sd1008) to obtain dMV.

[0070] Here, the surfactant is not particularly limited as long as it can produce dMV. Examples include: nonionic or anionic surfactants, bile acids that have the function of dissolving bacterial cell walls, or secondary bile acids generated by bacterial metabolism of bile acids as their metabolites. Salts of surfactants may be included, to the extent that they do not impair the effects of the invention.

[0071] Examples of bile acids include: cholic acid, glycocholic acid, taurocholic acid, chenodeoxycholic acid, porcine cholic acid, and 5α-caryophyllexin.

[0072] Examples of secondary bile acids include: deoxycholic acid, lithocholic acid, porcine deoxycholic acid, and ursodeoxycholic acid.

[0073] From the viewpoint of reproducibility of artificial music video production, cholic acid or deoxycholic acid is preferred, and from the viewpoint of increasing music video production, deoxycholic acid is more preferred.

[0074] The preferred concentration of the surfactant is in the range of 0.1% to 5%. If the concentration is below the lower limit, the bacterial cell wall cannot be dissolved; if the concentration exceeds the upper limit, the high concentration of surfactant may have an adverse effect on self-organization.

[0075] Furthermore, after adding the surfactant, the time required for self-organization (stirring time) is preferably in the range of 15 minutes to 1 hour. If it is below the lower limit, self-organization will be insufficient, and if it exceeds the upper limit, it is not preferred from a productivity point of view.

[0076] In addition, the preferred stirring speed is 100 min. -1 ~200min-1 If the value is below the lower limit, self-organization is insufficient; if it exceeds the upper limit, it is not preferred from a productivity point of view.

[0077] The MV obtained through the above steps can also be processed and stored by methods such as concentration, dilution, freezing, drying, and pulverization.

[0078] In addition, when MV is formulated for pharmaceutical applications, dosage forms such as tablets, capsules, granules, sugar-coated tablets, pills, fine granules, powders, sustained-release preparations, suspensions, emulsions, syrups, freeze-dried agents, and liquids can be selected.

[0079] In addition, the above-mentioned formulations can be used alone or in combination with pharmaceutically permissible carriers.

[0080] In addition, excipients, stabilizers, preservatives, humectants, emulsifiers, lubricants, sweeteners, colorants, flavorings, buffers, antioxidants, pH adjusters, etc., may be added as needed.

[0081] Example (Example) The invention is specifically illustrated by the following examples, but the invention is not limited to these examples.

[0082] In addition, the following table explains the abbreviations mainly used in the following embodiments.

[0083] CA bile acids DOC (deoxycholic acid) AI12060Alistipes indistinctus YIT 12060 The general term for AiMVAI12060's MVs sAiMV is the MV separated from the culture supernatant of AI12060. dAiMV is an artificially manufactured MV from AI12060 cells using DOC. Lp0132 Lactiplantibacillus plantarum YIT 0132 The general term for LpMVLp0132's MV. sLpMV MV separated from the culture supernatant of Lp0132 dLpMV is an artificially produced MV from Lp0132 cells using DOC. YIT 0209Lacticaseibacillus paracasei YIT 0209 YIT 4014 Bifidobacterium breve YIT 4014 It should be noted that Lp0132 (Lactiplantibacillus plantarum YIT 0132) was internationally deposited in Japan on February 24, 2013 at the National Institute for Technology Evaluation Patent Microbial Collection Center (NITE-NPMD) (Room 122, 2-5-8 Kazusa-Kamazore, Kisarazu City, Chiba Prefecture 292-0818) as Lactobacillus plantarum YIT 0132 (accession number FERM BP-11349).

[0084] (Example 1) <bacteria> AI12060, Lp0132, YIT 0209, and YIT 4014 each used cryopreserved bacteria. Here, AI12060 is known to be a Gram-negative bacterium, while Lp0132, YIT 0209, and YIT 4014 are Gram-positive bacteria.

[0085] 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 passaged at 37°C and incubated statically for 48 to 96 hours.

[0086] <Buffer Preparation> Prepare the first buffer solution (0.1 M Tris-HCl, 0.01 M EDTA) (pH 8.9). First, dissolve Trizma (trademark) base (Sigma-Aldrich) in MQ water to a final concentration of 0.1 M Tris solution, and then add 0.5 mol / L EDTA solution (pH 8.0) (NACALAITESQUE) to a final concentration of 0.01 M.

[0087] After sterilization using a 0.22 μm PES needle filter (Millipore), the pH of the solution was adjusted to 8.9 using a 2N HCl solution.

[0088] Next, prepare the second buffer (a 10% DOC solution). Dissolve sodium DOC (Fujifilm and Koichi Pure Chemicals Co., Ltd.) in the first buffer to prepare a 10% (w / v) DOC solution, which will be used as the second buffer.

[0089] <Surfactant (DOC) Treatment> Centrifuge the bacterial culture (8,000×g, 20 min, 4℃) to remove the culture supernatant. Wash the recovered bacterial cells with PBS, then resuspend them in 1 / 10 of the culture medium using the first buffer.

[0090] After the bacterial precipitate has completely disappeared, add the second buffer to achieve a final DOC concentration of 0.5%, and stir with a stirrer at room temperature for 100 min. -1 ~200min -1 (30 minutes).

[0091] (Example 2) <SMV Separation> In the case of sAiMV, ultracentrifugation was used. The bacterial culture was centrifuged (8,000×g, 20 min, 4℃), 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℃) to granulate the MV, which was then resuspended in PBS. Then, the cells were washed three times using an ultrafilter (Millipore) with a molecular weight cutoff of 100 kDa to obtain sAiMV.

[0092] On the other hand, in the case of sLpMV, YIT 0209, or YIT 4014 sMV, density gradient ultracentrifugation is used. The bacterial culture is centrifuged (8,000×g, 20 min, 4℃) to remove the bacterial cells and recover the culture supernatant.

[0093] The recovered 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. The density gradient was prepared using OptiPrep (60% iodixanol) (Sigma-Aldrich).

[0094] Dilute the OptiPrep solution with MQ water, then stack 2 mL of 25%–40% iodixanol solution at 5% intervals, and finally stack 1 mL of the supernatant concentrate on top.

[0095] Then, ultracentrifugation (P40ST rotor, 200,000×g, 3h, 4℃) was performed, and 1 mL was recovered using a pipette in the form of a band containing the target to obtain the sMV solution separated from the culture supernatant.

[0096] <DMV Separation> In the case of dAiMV, ultracentrifugation was used. The DOC-treated solution was centrifuged (8,000×g, 20 min, 4℃), 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℃) to granulate the MV, which was then resuspended in PBS. Then, the MV was obtained by three washing operations using an ultrafilter (Millipore) with a molecular weight cutoff of 100 kDa.

[0097] On the other hand, in the case of dLpMV, YIT 0209, or YIT 4014 dMV, density gradient ultracentrifugation was used. The DOC-treated solution was centrifuged (12,000×g, 30 min–1 h, 4 °C), and the supernatant was recovered. The recovered culture supernatant was passed through a 0.45 μm PES syringe filter (Millipore) and then concentrated through an ultrafilter (Millipore) with a molecular weight cutoff of 100 kDa until approximately 1 mL was obtained.

[0098] 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 stacked at 5% intervals, with a 1 mL concentrate layer on top. The mixture was then ultracentrifuged (P40ST rotor, 200,000 × g, 3 h, 4 °C), and 1 mL was recovered using a pipette as a band containing the target molecules to obtain the respective dMV.

[0099] <Quantitative Analysis of Music Video> Protein quantification of MV was performed using the Pierce (registered trademark) BCA Protein Assay Kit (manufactured by Thermo) and the prescribed method.

[0100] <SEM Observations on MV> After separation by ultracentrifugation, MV was washed with MQ water and then freeze-dried. The dried MV powder was coated using an automatic fine coating machine (JEC-3000FC) and observed using a desktop scanning microscope (JCM-6000).

[0101] On the other hand, in the case of MV separated by density gradient ultracentrifugation, 1% BSA was passed through an ultrafiltration membrane (Millipore), the membrane was sealed, and then washed three times with MQ water to remove the remaining BSA on the membrane. Then, the MV in the separated OptiPrep solution was washed and replaced with MQ water. The solution was then freeze-dried, and MV powder was coated using an automated fine coater (JEC-3000FC) and observed using a desktop scanning microscope (JCM-6000).

[0102] Figure 2 These are SEM images of dMV obtained from Gram-positive or Gram-negative bacteria. Specifically, Figure 2 (A) is the SEM image of dAiMV. Figure 2 (B) is the SEM image of dLpMV. Figure 2 (C) is the SEM image of the dMV of YIT 0209. Figure 2 (D) is the SEM image of the dMV of YIT 4014.

[0103] The spherical structures identified as MVs can be confirmed from any SEM image. This indicates that MVs can be artificially produced from both Gram-negative and Gram-positive bacteria using DOC. MVs (dMVs) can be obtained from the supernatant (sMV) or from the bacterial cells through a single culture.

[0104] In addition, since all DOC processing is carried out by stirring at room temperature for 30 minutes, it can be concluded that if stirring at room temperature for at least 30 minutes, dMV homogenization can be achieved.

[0105] (Example 3) On the other hand, it is also known that the following issues exist in the production of dMV of Gram-positive bacteria. Figure 3 This is a bar chart comparing the yield of MV per unit weight of Gram-negative bacteria (left) and Gram-positive bacteria (right) during the artificial production of MV. Figure 3 The horizontal axis represents Gram-negative bacteria (left) as AI12060 and Gram-positive bacteria (right) as Lp0132. The vertical axis represents the MV (μg) per unit bacterial cell weight (mg) (converted from protein content).

[0106] Depend on Figure 3 It is known that the MV yield per unit weight of Gram-positive bacteria is extremely low compared to that of Gram-negative bacteria. Therefore, in order to practically utilize dMV of Gram-positive bacteria for their intended applications, it is necessary to manufacture artificial MV more efficiently.

[0107] 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 recorded). Therefore, as a further study, they investigated whether the sensitivity to DOC could be improved by controlling the state of the bacteria.

[0108] Figure 4 This is a graph showing the relationship between the culture time (horizontal axis) and turbidity (OD) and pH (vertical axis) of Gram-positive bacteria (Lp0132). Figure 4 The horizontal axis represents the incubation time (h), and the vertical axis represents the relative value of turbidity (OD) and the pH value. The value on the vertical axis is pH. In the graph, the solid line represents turbidity (OD), and the dashed line represents pH.

[0109] according to Figure 4 In culture without pH adjustment, the pH of the culture medium began to decrease after 5 hours of culture, reaching a low pH (around pH 4) environment after 10 hours. Therefore, after 8 hours of culture before the mid-logarithmic growth phase and before reaching the low pH environment, the pH of the culture medium was temporarily adjusted to 7 to prevent long-term culture in a low pH environment.

[0110] <pH Adjustment of Lp0132> Based on the above viewpoints, a study was conducted to investigate whether the production of dMV (drug-dependent methane) from Lp0132, a Gram-positive bacterium, increased due to pH adjustment. Figure 5(a) shows the results of dMV production from Lp0132 cells cultured at pH 7 for 8 hours using DOC (distilled carbon). Furthermore, the effects of pH adjustments at 5, 6, 7, 8, 9, and 10 were investigated. Figure 5(b) shows the results.

[0111] Figure 5(a) is a bar chart showing the MV yield of Gram-positive bacteria (Lp0132) produced from culture supernatant (left), artificially produced dMV from culture medium without pH adjustment (center), and artificially produced dMV from culture medium adjusted to pH 7 (right). The vertical axis represents the MV yield (μg) per 50 mL of culture medium (converted to protein content). Figure 5(b) is a bar chart showing the MV yield of Gram-positive bacteria (Lp0132) produced from culture medium adjusted to pH 5, 6, 7, 8, 9, and 10. The vertical axis represents the MV particle number ratio ( / control group). The control group consists of the number of dMV particles produced from culture medium without pH adjustment.

[0112] As shown in Figure 5(a), the bacterial cells with pH adjustment during cultivation showed a significant increase in yield compared to those without pH adjustment. As shown in Figure 5(b), this was particularly effective in the pH range of 5–9.

[0113] <pH Adjustment of YIT 0209 and YIT 4014> Similarly, the production of dMV was investigated for YIT 0209 and YIT 4014 to determine whether pH adjustment increased the yield. Figure 6 shows the results of dMV production via DOC from cells of YIT 0209 and YIT 4014 that were cultured for 8 hours and then adjusted to pH 7.

[0114] Figures 6(a) and 6(b) are bar charts showing the MV yield of artificially produced dMV (left) from culture medium without pH adjustment and artificially produced dMV (right) from culture medium adjusted to pH 7 for Gram-positive bacteria (YIT 0209 and YIT 4014). In Figure 6(a), for YIT 0209, the horizontal axis represents the bacterial cells without pH adjustment on the left and the dMV at pH 7 on the right, with the vertical axis representing the number of particles per 1 mL. Similarly, in Figure 6(b), for YIT 4014, the horizontal axis represents the bacterial cells without pH adjustment on the left and the dMV at pH 7 on the right, with the vertical axis representing the number of particles per 1 mL.

[0115] As shown in Figures 6(a) and 6(b), for Gram-positive bacteria other than Lp0132, the yield of cells that underwent pH adjustment during culture was significantly increased compared to cells that did not undergo pH adjustment.

[0116] Based on current research, we have gained insights into cell wall thickening in Gram-positive bacteria cultured at low pH. It is expected that as the cell walls become more robust due to this thickening, DOC sensitivity decreases. Therefore, it is believed that by adjusting the pH, the cell walls of cultured Gram-positive bacteria become thinner, and the self-organization of MV is moderately promoted, thereby increasing dMV production.

[0117] Based on the above results, it can be concluded that in the method for manufacturing membrane vesicles (MV) of Gram-positive bacteria, in the culturing step a, the following steps are taken: at the beginning of the culturing in step a, the pH of the culture medium is adjusted during the culturing (step b), and a surfactant is added to the bacterial cells after the culturing (step c). This allows for the mass production of dMV.

[0118] (Example 4) <Research on Surfactants> In this embodiment, a study was conducted on the use of cholic acid (CA) as a primary bile acid for the surfactant used to manufacture dMV.

[0119] Except that the surfactant was sodium CA (Fujifilm and Kojun Pharmaceutical Co., Ltd.), the dMV of Lp0132, a Gram-positive bacterium, was isolated in the same manner as in Examples 1 and 2. The results are shown in Figure 7.

[0120] Figures 7(a) and 7(b) are bar charts showing the MV yield of Gram-positive bacteria (Lp0132) with DOC surfactant (left) and sodium CA surfactant (right). The vertical axis of Figure 7(a) represents the particle number of MV (×10⁻¹⁰). 9 Particles / ml). The vertical axis of Figure 7(b) represents the MV yield (μg) per 50 mL of culture medium (converted from protein mass).

[0121] As shown in Figures 7(a) and 7(b), dMV can still be produced even when the surfactant is set as CA, which is a primary bile acid. Furthermore, from the viewpoint of dMV yield, using DOC is the preferred result.

[0122] (Example 5) In this study, the application of dMV of Gram-positive bacteria that can be mass-produced was explored.

[0123] <cell> The mouse monocytic leukemia macrophage-like cell line RAW264.7 was used. RAW264.7 is known to respond to most TLR ligands, producing inflammatory cytokines, etc.

[0124] RAW264.7 was cultured using DMEM medium (10% FBS / DMEM) containing 10% FBS and 100 μg / mL streptomycin, at 37°C under 5% CO2 conditions.

[0125] <Study on IL-10 induction activity> Lp0132 in HKB, or LpMV (final concentrations of 1 μg / ml, 5 μg / ml, and 10 μg / ml), and RAW264.7 (2.0 × 10⁻⁶) were inoculated into 96-well plates in 10% DMEM medium. 5 The cells / wells were incubated in an incubator at 37°C for 24 hours in a 5% CO2 environment.

[0126] The culture supernatant was recovered, and the amount of IL-10 produced was determined by ELISA using the Mouse IL-10 Duo Set ELISA (R&Dsystems).

[0127] Dilute the Capture antibody to the specified concentration with PBS, add 100 μL to each well of a 96-well ELISA plate, and incubate overnight at 4°C. Wash each well with PBS containing 0.05% (w / v) Tween 20, then add 100 μL of PBS containing 1% (w / v) BSA (BSA / PBS) to each well and block at 37°C for 90 minutes.

[0128] After blocking, 100 μL of culture supernatant diluted 1000-fold with 1% (w / v) BSA / PBS was added to each sample, and the mixture was reacted overnight at 4°C. After the reaction, three washing operations were performed, and 100 μL of detection antibody diluted 1% (w / v) BSA / PBS to the specified concentration was added to each sample. After reacting at room temperature for 120 minutes, 100 μL of streptavidin-labeled horseradish peroxidase (Streptavidin-HRP) diluted 1% (w / v) BSA / PBS to the specified concentration was added to each sample, and the mixture was reacted at room temperature in the dark for 20 minutes.

[0129] Then, after washing three times with the washing solution, add 50 μL of 1-Step (registered trademark) Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific) to each sample and react at room temperature for 5 minutes.

[0130] After confirming color development, add 50 μL of 2M H2SO4 to each sample and stop the reaction. Measure the absorbance at 450 nm and 595 nm using a microplate reader.

[0131] <Differences in the properties of sLpMV and dLpMV in Lp0132> LpMV activity was evaluated by the ability of macrophages to be induced by IL-10 in the RAW264.7 cell line. Figure 8 This is a bar graph comparing the IL-10 induction activity of HKB (left), sLpMV (center), and dLpMV (right) against Gram-positive bacteria (Lp0132) when their concentrations are changed.

[0132] Figure 8 The IL-10 induction activity on the vertical axis represents the amount of IL-10 produced by RAW264.7 when treated with HKB or LpMV, expressed as the signal intensity of each culture medium. IL-10 production was obtained by recovering the culture supernatant after culture and determining the amount of IL-10 in the supernatant by ELISA. Figure 8 The horizontal axis from left to right represents the amount of heat-killed bacterial cells (HKB), sLpMV, and dLpMV added to Lp0132 (10 μg / mL, 5 μg / mL, and 1 μg / mL).

[0133] Lp0132 was added to RAW264.7 at final concentrations of 1 μg / ml, 5 μg / ml, and 10 μg / ml using HKB, sLpMV, or dLpMV, and the amount of IL-10 produced was measured. No IL-10 production was observed in the sLpMV treatment, while the same IL-10 production was confirmed in the dLpMV treatment as in the HKB treatment.

[0134] Therefore, sLpMV did not inherit the IL-10 inducing activity of HKB, while dLpMV retained this activity. Furthermore, the IL-10 inducing activity of dLpMV is higher than that of HKB.

[0135] The mechanism by which Gram-positive bacteria release MV (microbeta) from their cell membranes into the extracellular space via the cell wall after production from the cell membrane is widely understood. It is believed that in cases of spontaneous MV production by bacteria, minimal cell wall components are introduced.

[0136] On the other hand, it is assumed that when disrupted by surfactants, the cell membrane, with its attached cell wall components, exists as a self-organizing material for the MV. Therefore, it is believed that the different composition of the membranes in the two MVs leads to the presence or absence of IL-10-induced activity.

[0137] Based on the above, dMV of Gram-positive bacteria is implied as an effective component of anti-inflammatory agents or an effective component of IL-10 inducing agents.

[0138] (Example 6) <Characteristics of dMV in Gram-positive bacteria> like Figure 2 As shown, SEM images can be used to confirm whether an MV has been manufactured. However, it is impractical to directly determine the structure of sMV and dMV using electron microscopy to differentiate them.

[0139] Therefore, we attempted to differentiate between sMV and dMV by measuring the amount of RNA encapsulated within the MV. Figure 9 This is a bar chart showing the amount of RNA contained in the sMV (left) and dMV (right) of Gram-positive bacteria. Figure 9 The vertical axis represents the value obtained by measuring the amount of RNA in the MV of Gram-positive bacteria using the Qubit (registered trademark) RNA BR Assay Kit (invitrogen). The horizontal axis represents sMV and dMV from left to right.

[0140] exist Figure 9In this study, the RNA content encapsulated in artificially manufactured vesicles (dMV) was determined to be less than 10% of the RNA content encapsulated in naturally occurring vesicles (sMV). For Gram-positive bacteria-derived vesicles suitable for use as anti-inflammatory agents, dMVs with an RNA content in their membrane vesicles (MV) that is less than 10% of the RNA content in naturally occurring MVs are effective. PCT / RO / 134 form

Claims

1. A method for manufacturing membrane vesicles (MV) of Gram-positive bacteria, characterized in that, Include: Step a, wherein the Gram-positive bacteria are cultured; Step b, wherein the pH of the culture medium is adjusted at the start of and / or during the culture in step a; and Step c, in which a surfactant is added to the bacterial cells after culturing.

2. The manufacturing method according to claim 1, wherein, In step a and / or step b, the pH of the culture medium is adjusted to 5-9.

3. The manufacturing 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 with a steroid backbone.

5. The manufacturing method according to claim 4, wherein, The surfactant is bile acid.

6. The manufacturing method according to claim 5, wherein, The surfactant is cholic acid or deoxycholic acid.

7. A membrane vesicle (MV) derived from Gram-positive bacteria, characterized in that, The RNA content of the membrane vesicles (MVs) is less than 10% of the RNA content of naturally occurring MVs.

8. An anti-inflammatory agent, characterized in that, The membrane vesicles (MVs) derived from Gram-positive bacteria as described in claim 7 are used as the active ingredient.

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