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

Title of Invention: METHOD FOR PRODUCING MEMBRANE VESICLES OF GRAM-POSITIVE BACTERIA AND USE OF MEMBRANE VESICLES THEREOF Technical Field

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

[0002] Membrane vesicles (hereinafter, referred to as “MVs”) are vesicles produced by bacteria. Although the intrinsic purposes of MV production by bacteria are diverse, including the mutual transmission of physiologically active substances between bacteria, and the like, attempts to artificially produce MVs for use in other industrial applications are being conducted in the field of Gram negative bacteria.

[0003] For example, Patent Literature 1 provides a method for producing a vaccine against a specific disease using MVs obtained from Gram-negative bacteria. Citation List Patent Literature

[0004] Patent Literature 1: WO 2001 / 091788 A Summary of Invention Technical Problem

[0005] In contrast, for Gram-positive bacteria, a method for producing MVs has not yet been established, and even the industrial applications of MVs derived from Gram-positive bacteria are also unknown, leaving room for further research and development.

[0006] Accordingly, an object of the present invention is to establish a method for producing MVs from a Gram-positive bacterium and to find a novel use for such MVs. Solution to Problem

[0007] As a result of intensive studies, the present inventors have established a production method for mass-producing MVs of a Gram-positive bacterium. Surprisingly, the present inventors have also found a use of the MVs as an antiinflammatory agent. That is, a method for producing MVs of a Gram-positive bacterium of the present invention and examples of a use of the MVs are as follows.

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

[0009] According to the present invention, it is possible to provide the method for producing MVs of a Gram-positive bacterium, and the novel use thereof. Brief Description of Drawings

[0010] [Fig. 1] Fig. 1 is a flowchart illustrating a method for producing MVs. [Fig. 2] Fig. 2 illustrates SEM images of MVs obtained from Gram-positive bacteria or Gram-negative bacteria. [Fig. 3] Fig. 3 is a bar graph illustrating a comparison of the yields of MVs per bacterial cell weight between Gram-negative bacteria and Gram-positive bacteria when MVs are artificially produced. [Fig. 4] Fig. 4 is a graph illustrating the relationship between the culture time (horizontal axis) of Gram-positive bacteria, and the turbidity (OD) and pH (vertical axis). [Fig. 5a] Fig. 5(a) is a bar graph illustrating the MV yield (in terms of protein amount) of Gram-positive bacteria (Lp0132) for: sMVs produced from a culture supernatant (left); dMVs artificially produced from bacterial cells without pH adjustment (middle); and dMVs artificially produced from bacterial cells with the pH adjusted to 7 (right). [Fig. 5b] Fig. 5(b) is a bar graph illustrating the particle number ratio ( / control) of dMVs artificially produced from bacterial cells adjusted to pH 5, 6, 7, 8, 9, and 10. [Fig. 6a] Fig. 6(a) is a bar graph illustrating the MV yield of Gram-positive bacteria (YIT 0209) for: dMVs artificially produced from bacterial cells without pH adjustment (left); and dMVs artificially produced from bacterial cells with pH adjustment (right). [Fig. 6b] Fig. 6(b) is a bar graph illustrating the MV yield of Gram-positive bacteria (YIT 4014) for: dMVs artificially produced from bacterial cells without pH adjustment (left); and dMVs artificially produced from bacterial cells with pH adjustment (right). [Fig. 7a] Fig. 7(a) is a bar graph illustrating the MV yield of Gram-positive bacteria (Lp0132) for: dMVs produced using deoxycholic acid (DOC) as a surfactant (left); and dMVs produced using sodium cholate (CA) as a surfactant (right). The vertical axis of Fig. 7(a) indicates the number of MV particles (x 109 particles / ml). [Fig. 7b] Fig. 7(b) is a bar graph illustrating the MV yield of Gram-positive bacteria (Lp0132) for: dMVs produced using deoxycholic acid (DOC) as a surfactant (left); and dMVs produced using sodium cholate (CA) as a surfactant (right). The vertical axis of Fig. 7(b) indicates the MV yield (pg) per 50 mL of culture broth (in terms of protein amount). [Fig. 8] Fig. 8 is a bar graph illustrating a comparison of IL-10-inducing activity (anti-inflammatory effect) for heat-killed bacterial cells (HKB) (left), sLpMVs (middle), and dLpMVs (right) from Gram-positive bacteria (Lp0132), at each concentration. [Fig. 9] Fig. 9 is a bar graph illustrating the amount of RNA encapsulated within sMVs (left) and dMVs (right) of Gram-positive bacteria. Description of Embodiments

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

[0012] The present inventors have established a method for producing MVs from Gram-positive bacteria, the method including: a step a of culturing the Gram-positive bacteria; a step b of adjusting a pH of a culture medium upon initiation of the culturing and / or during the culturing in the step a; and a step c of adding a surfactant to bacterial cells after culturing. By including the above-described steps a to c, the MVs of the Gram-positive bacteria can be artificially mass-produced.

[0013] In addition, the present inventors have found that Gram-positive bacterial MVs are useful for use as an anti inflammatory agent because, for example, the MVs induce the production of IL-10.

[0014] Here, the pH adjustment in the step b is preferably carried out to a pH of 5 to 9 from the perspective of increasing the MV yield.

[0015] A Gram-positive bacterium is preferably a lactic acid bacterium or a bifidobacterium from the perspective of pharmaceutical use.

[0016] In addition, the surfactant is, for example, a nonionic or anionic surfactant having a steroid skeleton. From the perspective of pharmaceutical use, the surfactant is preferably a bile acid. Specifically, the bile acid is more preferably cholic acid or deoxycholic acid from the perspective of reproducibility, and is still more preferably deoxycholic acid from the perspective of increasing the MV yield.

[0017] In addition, the surfactant may include a salt thereof as long as the effect of the invention is not impaired. As for candidates of the surfactant presented in the following paragraphs, the salts thereof may be included as well.  The candidate of the salts may be an inorganic salt or an organic salt.

[0018] In addition, based on Examples described below, the content of RNA encapsulated within the artificially produced MVs (dMVs) was determined, and as a result, it was found to be 10% or less of the content of RNA encapsulated within the naturally occurring MVs (sMVs). For MVs derived from Gram-positive bacteria to be applied to uses such as anti-inflammatory agents, it is effective to use MVs in which the RNA content is 10% or less of the RNA content of naturally occurring MVs.

[0019] In the present specification, the phrase “MVs are artificially produced” refers to artificially producing MVs by promoting self-assembly after destruction of cell surface layers of bacterial cells with a surfactant. Therefore, the production method is different from that of naturally-derived MVs spontaneously generated (naturally occurring) by living bacteria.

[0020] Here, in the present specification, the MVs spontaneously generated (naturally occurring) by bacteria are referred to as sMVs, as the MVs are collected from a supernatant of a culture broth. In addition, the artificially produced MVs are referred to as dMVs, as a detergent used for surface layer destruction and selfassembly is employed.

[0021] As will be described in detail later, the present inventors have obtained a finding in the course of research and development that Gram-positive bacteria, as compared to Gram-negative bacteria, result in a negligible yield of MVs when the MVs are artificially produced from a culture broth without pH adjustment. As a result of intensive studies, the present inventors have unexpectedly found that the MV yield is increased by adjusting the pH upon initiation of the culturing or during the culturing process of Gram positive bacteria.

[0022] (1. Gram-Positive Bacteria) Gram-positive bacteria are bacteria that are stained deep blue or purple by Gram staining. A Gram-positive bacterium used in the present invention is preferably a lactic acid bacterium or a bifidobacterium from the perspective of pharmaceutical use.

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

[0024] Examples of the bacteria belonging to the genus Lactobacillus include Lactiplantibacillus plantarum, Lacticaseibacillus paracasei (L. casei), Lactobacillus mali, Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Lactobacillus helveticus.

[0025] Examples of the bacteria belonging to the genus Streptococcus include Streptococcus thermophilus.

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

[0027] Examples of the bacteria belonging to the genus Enterococcus include Enterococcus faecalis.

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

[0029] Among these, Lactiplantibacillus plantarum, Lacticaseibacillus paracasei (L. casei), or Bifidobacteriumbreve is more preferable. Note that, in recent years, lactic acid bacteria belonging to the genus Lactobacillus have been reclassified. That is, for the lactic acid bacteria that conventionally belonged to the genus Lactobacillus, the genus has been subdivided, and the genus names of some bacterial species have 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 the present specification, descriptions will be made using the new classification nomenclature after the reclassification. In addition, for example, among the lactic acid bacteria that were classified into the genus Lactobacillus in the old classification, those that can be newly classified as Lactiplantibacillus or Lacticaseibacillus are included in Lactiplantibacillus or Lacticaseibacillus of the present application.

[0031] (2. Membrane Vesicles) Membrane vesicles (MVs) are vesicles produced by bacteria, and each has a particle size of 20 to 500 nm (with a median size of about 100 nm).

[0032] As described above, in the present invention, the inventors have established manufacturing methods for sMVs and dMVs as MVs derived from Gram-positive bacteria.  Here, as also illustrated in Examples, confirmation of whether MVs have been produced can be carried out by analysis such as SEM imaging or a nanoparticle tracking analysis. However, as for the distinction between sMVs and dMVs, it is impossible and highly impractical to directly identify the structures of both MVs using an electron microscope or the like.

[0033] Note that, based on Examples described below, the content of RNA encapsulated within the artificially produced MVs (dMVs) was determined, and as a result, it was found to be 10% or less of the content of RNA encapsulated within the naturally occurring MVs (sMVs). For MVs derived from Gram-positive bacteria to be applied to uses such as anti-inflammatory agents, it is effective that the RNA content of the membrane vesicles (MVs) is 10% or less of the RNA content of naturally occurring MVs. For RNA measurement, for example, a commercially available measurement kit such as QubitTM RNA BR Assay Kit (Invitrogen) is used.

[0034] (3. Method for Producing MVs) Fig. 1 is a flowchart illustrating a method for producing MVs. The outline of the method for producing MVs will be described with reference to Fig. 1. Note that, since the production methods for sMVs and dMVs branch after step S1003, for convenience, steps will be referred to as a step Ss in the case of sMVs and a step Sd in the case of dMVs.

[0035] When the operation is started, in step S1001, culturing of the bacteria is performed. Preferred bacterial types are as described above.

[0036] In a case of producing dMVs, it is preferable that the pH of a culture medium is adjusted within a predetermined range from the perspective of increasing the MV yield.

[0037] From the perspective of the optimal pH for the lactic acid bacteria or the bifidobacteria, the pH is preferably pH 5 or higher as a lower limit.

[0038] From the perspective of the optimal pH for the lactic acid bacteria or the bifidobacteria, the pH is preferably pH 9 or lower as an upper limit.

[0039] From the perspective of the optimal growth temperature, the culture temperature is preferably 30°C or higher, and more preferably 33°C or higher, as a lower limit.

[0040] As an upper limit, the culture temperature is preferably 45°C or lower, and more preferably 43°C or lower, from the perspective of the optimal growth temperature.

[0041] In a case where the culture temperature is lower than the lower limit, the bacterial cells do not grow appropriately, and in a case where the culture temperature is higher than the upper limit, there is a risk that the bacterial cells may be destroyed due to protein denaturation.

[0042] The culture time is not particularly limited, but from the perspective of increasing the MV yield, 10 hours or longer is preferable, and 24 hours or longer is more preferable, as a lower limit.

[0043] As an upper limit, the culture time is preferably 96 hours or shorter, and more preferably 48 hours or shorter, from the perspective of increasing the MV yield.

[0044] In a case where the culture time is shorter than the lower limit, the constituent materials of MVs in the bacteria are not sufficiently produced, and in a case where the culture time is longer than the upper limit, it is not preferable from the perspective of productivity.

[0045] Examples of a culture method include agitation culture, static culture, shaking culture, and neutralization culture.

[0046] Next, in step S1002, the culture broth is centrifuged to separate the culture broth into a supernatant and bacterial cells. Here, in a case of extracting sMVs that have been spontaneously produced by bacteria, the culture supernatant of step S1002 is obtained in step Ss1003.

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

[0048] On the other hand, in a case of producing artificially prepared dMVs, the bacterial cell fraction is obtained in step Sd1003 through step S1002.

[0049] Next, the bacterial cell fraction is suspended in a buffer solution (step Sd1004), and a predetermined detergent is added (step Sd1005) to destroy cell walls of the bacteria, the solution is then agitated (step Sd1006) to promote self-assembly, and after a predetermined time has elapsed, the solution is filtered (step Sd1007) and finally ultracentrifuged (step Sd1008) to obtain dMVs.

[0050] Here, the detergent is not particularly limited as long as it is capable of producing dMVs, and examples thereof include a nonionic or anionic detergent, bile acids having an action of lysing bacterial cell walls, or secondary bile acids produced as metabolites through the metabolism of bile acids by the bacteria. In addition, the detergent may include a salt thereof as long as the effect of the invention is not impaired.

[0051] Examples of the bile acid include cholic acid, glycocholic acid, taurocholic acid, chenodeoxycholic acid, hyocholic acid, and 5alpha-cyprinol.

[0052] Examples of the secondary bile acid include deoxycholic acid, lithocholic acid, hyodeoxycholic acid, and ursodeoxycholic acid.

[0053] Among these, cholic acid or deoxycholic acid is preferable from the perspective of reproducibility in producing artificial MVs, and deoxycholic acid is more preferable from the perspective of increasing the MV yield.

[0054] The concentration of the detergent added is preferably within a range of 0.1% to 5%. In a case where the concentration is less than the lower limit, the bacterial cell walls cannot be lysed, and in a case where the concentration is more than the upper limit, there is a risk that the high concentration of the detergent may adversely affect the self-assembly.

[0055] In addition, the time required for self-assembly (agitation time) after adding the detergent is preferably within a range of 15 minutes to 1 hour. In a case where the time is shorter than the lower limit, self-assembly is insufficient, and the time is longer than the upper limit, it is not preferable from the perspective of productivity.

[0056] In addition, the agitation speed is preferably 100 to 200 min-1. In a case where the agitation speed is lower than the lower limit, self-assembly is insufficient, and in a case where the agitation speed is higher than the upper limit, it is not preferable from the perspective of productivity.

[0057] The MVs obtained through the above-described steps may be stored after being subjected to treatments such as concentration, dilution, freezing, drying, and powdering.

[0058] In addition, in a case where the MVs are formulated for pharmaceutical use or the like, dosage forms may be selected from, for example, tablets, capsules, granules, sugar-coated tablets, pills, fine granules, powders, dusting powders, sustained-release preparations, suspensions, emulsions, syrups, lyophilized preparations, and liquids.

[0059] In addition, the above-described formulation may use the MVs alone, or may use the MVs in combination with a pharmaceutically acceptable carrier.

[0060] In addition, as necessary, excipients, stabilizers, preservatives, wetting agents, emulsifiers, lubricants, sweeteners, coloring agents, flavoring agents, buffering agents, antioxidants, pH adjusters, and the like may be appropriately added. Examples

[0061] (Examples) The present invention will be described in detail based on the following examples, but the present invention is not limited to these examples. In addition, the abbreviations mainly used in the following examples are described in the table below. CA: Cholic acid DOC: Deoxycholic acid AI12060: Alistipes indistinctus YIT 12060 AiMVs: Generic term for MV from AI12060 sAiMVs: MVs isolated from the culture supernatant of AI12060 dAiMVs: MVs artificially produced from the bacterial cells of AI12060 using DOC Lp0132: Lactiplantibacillus plantarum YIT 0132 LpMVs: Generic term for MVs from Lp0132 sLpMVs: MVs isolated from the culture supernatant of Lp0132 dLpMVs: MVs artificially produced from the bacterial cells of Lp0132 using DOC YIT 0209: Lacticaseibacillus paracasei YIT 0209 YIT 4014: Bifidobacterium breve YIT 4014 Note that, Lp0132 (Lactiplantibacillus plantarum YIT 0132) has been internationally deposited as Lactobacillus plantarum YIT 0132 (Accession No. FERM BP-11349) on February 24, 2010, with the NITE Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (NITE-NPMD) (#122, 2-5-8 Kazusa-kamatari, Kisarazu-shi, Chiba 292-0818, Japan).

[0062] (Example 1) <Bacteria> Cryopreserved AI12060, Lp0132, YIT 0209, and YIT 4014 were used. Here, it has been known that AI12060 is a Gramnegative bacteria, and Lp0132, YIT 0209, and YIT 4014 are Gram-positive bacteria.

[0063] AI12060 and YIT 4014 were cultured in GAM (1% glucose) medium under anaerobic conditions, and Lp0132 and YIT 0209 were cultured in MRS medium under aerobic conditions, with all strains being subjected to subculture and static culture at 37°C for 48 to 96 hours.

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

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

[0066] Next, a second Buffer (10% DOC solution) was prepared. A 10% (w / v) DOC solution was prepared by dissolving sodium DOC (FUJIFILM Wako Pure Chemical Corporation) in the first buffer, and the resultant solution was designated as the second buffer.

[0067] <Detergent (DOC) Treatment> The bacterial culture broth was centrifuged (8,000 x g, 20 min, 4°C), and the culture supernatant was removed. The collected bacterial cells were washed with PBS and resuspended in the first buffer to 1 / 10 of the volume of the culture broth.

[0068] After the bacterial pellet was completely dissolved, the second buffer was added to adjust the final concentration of DOC to 0.5%, and the mixture was stirred using a stirrer at room temperature (100 to 200 min-1, 30 min).

[0069] (Example 2) <Isolation of sMVs> For sAiMVs, ultracentrifugation was employed. The bacterial culture broth was centrifuged (8,000 x g, 20 min, 4°C), and the supernatant was collected. The collected culture supernatant was passed through a PES syringe filter having a pore size of 0.45 pm (Millipore), and then subjected to ultracentrifugation (P70AT rotor, 200,000 x g, 2 h, 4°C) to pelletize the MVs, which were then resuspended in PBS. Thereafter, a washing operation was performed three times using an ultrafiltration device (Millipore) with a molecular weight cut-off of 100 kDa to obtain sAiMVs. On the other hand, in a case of sLpMVs or sMVs from YIT 0209 or YIT 4014, density gradient ultracentrifugation was employed. The bacterial culture broth was centrifuged (8,000 x 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 PES syringe filter having a pore size of 0.45 pm (Millipore), and then concentrated to approximately 1 mL of the MV-containing culture supernatant by ultrafiltration (Millipore) with a molecular weight cut-off of 100 kDa. OptiPrep (60% iodixanol) (Sigma-Aldrich) was used for the preparation of the density gradient.

[0071] The OptiPrep solution was diluted with MQ water, and 2 mL each of 25% to 40% iodixanol solutions were layered in 5% increments, followed by layering 1 mL of the concentrated supernatant on the top layer.

[0072] Furthermore, ultracentrifugation (P40ST rotor, 200,000 x g, 3 h, 4°C) was performed, and 1 mL was collected using a pipette to include the target band, thereby obtaining an isolated sMV solution derived from the culture supernatant.

[0073] <Isolation of dMVs> For dAiMVs, ultracentrifugation was employed. The solution after the DOC treatment was centrifuged (8,000 x g, 20 min, 4°C), and the supernatant was collected. The collected culture supernatant was passed through a PES syringe filter having a pore size of 0.45 pm (Millipore), and then subjected to ultracentrifugation (P70AT rotor, 200,000 x g, 2 h, 4°C) to pelletize the MVs, which were then resuspended in PBS. Thereafter, a washing operation was performed three times using an ultrafiltration device (Millipore) with a molecular weight cut-off of 100 kDa to obtain dAiMVs.

[0074] On the other hand, in a case of dLpMV or dMVs from YIT 0209 or YIT 4014, density gradient ultracentrifugation was employed. The solution after the DOC treatment was centrifuged (12,000 x g, 30 min to 1 h, 4°C), and the supernatant was collected. The collected culture supernatant was passed through a PES syringe filter having a pore size of 0.45 pm (Millipore), and then concentrated to approximately 1 mL by an ultrafiltration device (Millipore) with a molecular weight cut-off of 100 kDa.

[0075] OptiPrep (60% iodixanol, Sigma-Aldrich) was used for the preparation of the density gradient. The OptiPrep solution was diluted with MQ water, and 1 mL each of 10 to 40% iodixanol solutions were layered in 5% increments, followed by layering 1 mL of the concentrated supernatant on the top layer. Ultracentrifugation (P40ST rotor, 200,000 x g, 3 h, 4°C) was performed, and 1 mL was collected using a pipette to include the target band, thereby obtaining respective dMVs.

[0076] <Quantification of MVs> The protein concentration of the MVs was quantified using the Pierce (registered trademark) BCA Protein Assay Kit (manufactured by Thermo Fisher Scientific Inc.) according to the prescribed protocol.

[0077] <SEM Observation of MVs> In a case of MVs isolated by ultracentrifugation, the MVs were washed with MQ water and then subjected to freeze drying. The dried MV powder was coated using an auto fine coater (JEC-3000FC) and observed with a benchtop scanning electron microscope (JCM-6000).

[0078] On the other hand, in a case of MVs isolated by density gradient ultracentrifugation, 1% BSA was passed through an ultrafiltration device (Millipore) to block the membrane, followed by washing three times with MQ water to remove excess BSA on the membrane. Thereafter, the isolated MVs in the OptiPrep solution were washed and replaced with MQ water. Freeze-drying was performed, and the resulting MV powder was coated using an auto fine coater (JEC-3000FC) and observed with a benchtop scanning electron microscope (JCM-6000).

[0079] Fig. 2 illustrates SEM images of dMVs obtained from Gram-positive bacteria or Gram-negative bacteria. Fig. 2(A) is a SEM image of dAiMVs, Fig. 2(B) is a SEM image of dLpMVs, Fig. 2(C) is a SEM image of dMVs of YIT 0209, and Fig. 2(D) is a SEM image of dMVs of YIT 4014.

[0080] In all of the SEM images, spherical structures considered to be MVs could be observed. From these results, it was found that artificial MVs could be prepared from both Gram-negative and Gram-positive bacteria using DOC, and the MVs (dMVs) could be obtained not only from the supernatant (sMVs) but also from the bacterial cells, in a single cultivation.

[0081] In addition, since all DOC treatments were performed by stirring with a stirrer at room temperature for 30 min, it has been found that the dMVs could be homogenized by stirring at room temperature for at least about 30 min.

[0082] (Example 3) <Examination for Increasing Yield of dMVs> On the other hand, it has been also found that the production of dMVs from Gram-positive bacteria involves the following challenges. Fig. 3 is a bar graph illustrating a comparison of the yields of MVs per bacterial cell weight between Gram-negative bacteria (left) and Gram-positive bacteria (right) when MVs are artificially produced. On the horizontal axis of Fig. 3, the Gram-negative bacteria (left) indicates AI12060, and the Gram-positive bacteria (right) indicates Lp0132. The vertical axis indicates the MV yield (pg) (in terms of protein amount) per bacterial cell weight (mg).

[0083] From Fig. 3, it has been found that the MV yield per bacterial cell weight for dMVs from Gram-positive bacteria was significantly lower than that of dMVs from Gram negative bacteria. Therefore, in order to put dMVs from Gram-positive bacteria into practical use for prescribed applications, it was necessary to produce artificial MVs more efficiently.

[0084] The inventors examined the DOC treatment conditions, such as the DOC concentration and the stirring time during DOC addition, but the dLpMV yield did not increase (data not shown). Therefore, as a further examination, it was examined whether the sensitivity to DOC could be improved by controlling the state of the bacteria.

[0085] Fig. 4 is a graph illustrating the relationship between the culture time (horizontal axis) of Gram-positive bacteria (Lp0132), and the turbidity (OD) and pH (vertical axis). The culture time (h) is illustrated on the horizontal axis of Fig. 4, and the relative value of turbidity (OD) and the pH value are illustrated on the vertical axis. The numerical values on the vertical axis are pH. In the graph, the solid line indicates the turbidity (OD), and the dotted line indicates the pH.

[0086] From Fig. 4, in the culture without pH adjustment, the pH of the culture broth began to decrease from 5 hours after the start of cultivation and reached a low-pH environment (around pH 4) at 10 hours after the start of cultivation. Therefore, at 8 hours after the start of cultivation, i.e., prior to the middle stage of the logarithmic growth phase and before entering a low-pH environment, the pH of the culture broth was adjusted once to 7, thereby preventing prolonged cultivation under the low-pH environment.

[0087] <pH Adjustment of Lp0132> Based on the above-described points, an investigation was conducted to assess whether or not pH adjustment increases the yield of dMVs in Lp0132, Gram-positive bacteria. Fig. 5(a) illustrates the result of dMV preparation using DOC from Lp0132 bacterial cells whose pH was adjusted to 7 after 8 hours of cultivation. In addition, the pH was adjusted to 5, 6, 7, 8, 9, and 10 to examine the extent to which the effect of pH adjustment extends. The results are illustrated in Fig. 5(b).

[0088] Fig. 5(a) is a bar graph illustrating the MV yield of the Gram-positive bacteria (Lp0132) for: sMVs (left) produced from a culture supernatant; dMVs (middle) artificially produced from a culture broth without pH adjustment; and dMVs (right) artificially produced from a culture broth with pH adjusted to 7. The vertical axis indicates the MV yield (pg) (in terms of protein amount) per 50 mL of the culture broth. Fig. 5(b) is a bar graph illustrating the MV yield of dMVs artificially produced from culture broths of the Gram-positive bacteria (Lp0132), whose pH was adjusted to 5, 6, 7, 8, 9, and 10. The vertical axis indicates the particle number ratio ( / control) of MVs. The control refers to the number of dMV particles artificially produced from the culture broth without pH adjustment.

[0089] From Fig. 5(a), it has been found that the yield from the bacterial cells whose pH was adjusted during cultivation is significantly higher than that from the bacterial cells without pH adjustment. From Fig. 5(b), it has been found that the effect is particularly effective within the pH range of 5 to 9.

[0090] <pH Adjustment of YIT 0209 and YIT 4014> Similarly, the effect of pH adjustment on the yield of dMVs was examined in YIT 0209 and YIT 4014. Fig. 6 illustrates the results of dMV preparation using DOC from bacterial cells of YIT 0209 and YIT 4014, whose pH was adjusted to 7 at 8 hours after the start of cultivation.

[0091] Fig. 6(a) and 6(b) are bar graphs illustrating the MV yields of Gram-positive bacteria (YIT 0209 and YIT 4014): dMVs (left) artificially produced from a culture broth without pH adjustment; and dMVs (right) artificially produced from a culture broth with pH adjusted to 7. Fig. 6(a) illustrates the number of dMV particles per milliliter on the vertical axis for YIT 0209, with the left side of the horizontal axis representing dMVs derived from bacterial cells without pH adjustment, and the right side representing those derived from bacterial cells whose pH was adjusted to 7. In addition, Fig. 6(b) illustrates the number of dMV particles per milliliter on the vertical axis for YIT 4014, with the left side of the horizontal axis representing dMVs derived from bacterial cells without pH adjustment, and the right side representing those derived from bacterial cells whose pH was adjusted to 7.

[0092] From Fig. 6(a) and 6(b), it has been found that, for Gram-positive bacteria other than Lp0132, bacterial cells subjected to pH adjustment during cultivation also exhibited a substantially higher yield compared to those without pH adjustment.

[0093] Previous studies have provided findings that Grampositive bacteria develop thickened cell walls when cultured under the low-pH environment, and it was therefore expected that such thickening would reinforce the cell walls to reduce sensitivity to DOC. Therefore, it has been considered that the pH adjustment led to the thinning of the cell walls of the Gram-positive bacteria during cultivation, which moderately promoted the self-assembly of MVs, thereby resulting in an increased yield of dMVs.

[0094] Based on the above results, it was found that in the method for producing membrane vesicles (MVs) of Grampositive bacteria, mass production of dMVs can be achieved by: in the culturing step a, adjusting the pH of the culture medium upon initiation of the culturing and during the culturing in the culturing step a (step b); and adding a surfactant to bacterial cells after the culturing (step c).

[0095] (Example 4) <Examination of Detergent> In the present example, a detergent used for producing dMVs was examined with cholic acid (CA) as a primary bile acid.

[0096] dMVs from Lp0132, Gram-positive bacteria, were isolated in the same manner as in Examples 1 and 2, except that sodium cholate (FUJIFILM Wako Pure Chemical Corporation) was used as the detergent. The results are illustrated in Fig. 7.

[0097] Fig. 7(a) and 7(b) are bar graphs illustrating the MV yields of Gram-positive bacteria (Lp0132): dMVs (left) produced using DOC as a detergent; and dMVs (right) produced using sodium cholate as a detergent. The vertical axis of (a) indicates the number of MV particles (x 109 particles / ml). The vertical axis of (b) indicates the MV yield (pg) (in terms of protein amount) per 50 mL of the culture broth.

[0098] From Fig. 7(a) and 7(b), it has been found that dMVs can be produced even when CA, which is a primary bile acid, is used as the detergent. In addition, from the perspective of the dMV yield, the use of DOC provided favorable results.

[0099] (Example 5) In the present example, an exploration of applications of dMVs from Gram-positive bacteria, for which mass production had been achieved, was conducted.

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

[0101] RAW264.7 was subcultured at 37°C under an environment of 5% CO2 using a DMEM medium containing 10% FBS and 100 pg / mL streptomycin (10% FBS / DMEM).

[0102] <Examination of IL-10-Inducing Activity> HKB or LpMVs derived from Lp0132 (at final concentrations of 1 pg / ml, 5 pg / ml, and 10 pg / ml), each suspended in a 10% DMEM medium, were seeded together with RAW264.7 (2.0 x 105 cells / well) into a 96-well plate, and cultured for 24 hours at 37°C under a 5% CO2 atmosphere in an incubator.

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

[0104] The capture antibody was diluted with PBS to a predetermined concentration, added to a 96-well plate for ELISA at 100 pL per well, and incubated overnight at 4°C. Each well was washed with PBS containing 0.05% (w / v) of Tween20 (washing solution), and thereafter, 100 pL of PBS containing 1% (w / v) BSA (BSA / PBS) was added to each well for blocking, which was carried out at 37°C for 90 minutes.

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

[0106] After washing three times with a washing solution, 50 pL of 1-Step (registered trademark) Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific Inc.) was added to each well, and the reaction was allowed to proceed at room temperature for 5 minutes.

[0107] After confirming the color development, 50 pL of 2M H2SO4 was added to each well to stop the reaction, and the absorbances were measured at 450 nm and 595 nm with a microplate reader.

[0108] <Differences in Properties between sLpMV and dLpMV in Lp0132> The activity of LpMV was evaluated based on the ability to induce IL-10 in the macrophage cell line RAW264.7. Fig. 8 is a bar graph illustrating a comparison of IL-10 inducing activity for HKB (left), sLpMVs (middle), and dLpMVs (right) from Gram-positive bacteria (Lp0132), at each concentration.

[0109] The IL-10-inducing activity on the vertical axis of Fig. 8 represents the amount of IL-10 produced by RAW264.7 treated with either HKB or LpMVs, which is expressed as the signal intensity per volume of the culture medium. The amount of IL-10 produced is determined by collecting the culture supernatant after cultivation and measuring the amount of IL-10 in the supernatant by ELISA. The horizontal axis in Fig. 8 indicates, from left to right, the addition amounts (10 pg / mL, 5 pg / mL, and 1 pg / mL) of heat-killed bacterial cells (HKB), and sLpMV, and dLpMV, all derived from Lp0132.

[0110] HKB, sLpMVs, and dLpMVs from Lp0132 were added to RAW264.7 at final concentrations of 1 pg / ml, 5 pg / ml and 10 pg / ml, respectively, and the amount of IL-10 produced was measured; as a result, IL-10 production was not observed with the sLpMV treatment, whereas IL-10 production similar to that with the HKB treatment was confirmed with the dLpMV treatment.

[0111] From this, it was found that sLpMVs do not inherit the IL-10-inducing activity present in HKB, whereas dLpMVs retain that activity. The IL-10-inducing activity of dLpMVs was also higher than that of HKB.

[0112] A mechanism in which Gram-positive bacteria produce MVs from cell membranes and release the MVs outside the bacterial cells by the MVs passing through the cell walls is widely accepted. When bacteria spontaneously produce MVs, it is considered that the incorporation of cell wall components is minimized.

[0113] On the other hand, in a case where destruction was carried out with a detergent, it is assumed that cell membranes to which cell wall components are attached serve as materials for the self-assembly of MVs. Therefore, it was considered that the membrane components of MVs from both cases differed, leading to the presence or absence of IL-10-inducing activity.

[0114] From the above, it has been suggested that dMVs of Gram-positive bacteria can be utilized as an active ingredient of an anti-inflammatory agent or an active ingredient of an agent with IL-10-inducing activity.

[0115] (Example 6) <Characteristics of dMVs from Gram-Positive Bacteria> As illustrated in Fig. 2, whether or not MVs have been produced can be confirmed by SEM images or the like. However, as for the distinction between sMVs and dMVs, it is impossible and highly impractical to directly identify the structures of both MVs using an electron microscope or the like.

[0116] Therefore, a method was attempted to distinguish between sMVs and dMVs by measuring the amount of RNA encapsulated within the MVs. Fig. 9 is a bar graph illustrating the amount of RNA encapsulated within sMVs (left) and dMVs (right) of Gram-positive bacteria. The vertical axis in Fig. 9 represents a value obtained by measuring the amount of RNA in MVs from Gram-positive bacteria using Qubit (registered trademark) RNA BR Assay Kit (Invitrogen). The horizontal axis indicates sMVs and dMVs from left to right.

[0117] In Fig. 9, the content of RNA encapsulated within the artificially produced MVs (dMVs) was determined, and as a result, it was found to be 10% or less of the content of RNA encapsulated within the naturally occurring MVs (sMVs). For MVs derived from Gram-positive bacteria to be applied to uses such as anti-inflammatory agents, it is effective to use dMVs in which the RNA content of the membrane vesicles is 10% or less of that of naturally occurring MVs.

Claims

1. A method for producing membrane vesicles (MVs) of a Gram-positive bacterium, the method comprising:a step a of culturing the Gram-positive bacterium;a step b of adjusting a pH of a culture medium uponinitiation of the culturing and / or during the culturing in the step a; anda step c of adding a surfactant to bacterial cellsafter the culturing.

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

3. The production method according to claim 1,wherein the Gram-positive bacterium is a lactic acidbacterium or a bifidobacterium.

4. The production method according to any one ofclaims 1 to 3, wherein the surfactant is a nonionic oranionic surfactant having a steroid skeleton.

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

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

7. Membrane vesicles (MVs) derived from a Grampositive bacterium, wherein an RNA content of the MVs is10% or less of an RNA content of naturally occurring MVs.

8. An anti-inflammatory agent comprising, as an active ingredient, the Gram-positive bacterial MVs according to claim 7 .

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