JP6963335B2 - Method for Producing Membrane Vesicle Composition - Google Patents

Description

The present invention relates to a method for producing a membrane vesicle composition. The present invention also relates to a method for producing a pharmaceutical product, a quasi drug, a cosmetic product or a food product, a method for producing a vaccine, and a method for producing an immunity activating agent.

Bacteria and fungi are widely known to produce membrane vesicles (Non-Patent Document 1). There is an example in which membrane vesicles collected from pathogenic bacteria are used in the production of vaccines for diseases such as bacterial meningitis (Non-Patent Document 2). It has also been reported that lactic acid bacteria produce membrane vesicles, and the membrane vesicles have immunomodulatory ability (Non-Patent Document 3).

Brown et al., Nature Reviews Microbiology, 2015, Vol.13, No.10, p620-630 Acevedo et al., Frontiers in Immunology, 2014, Vol. 5, No.121, p1-7 Al-Nedawi et al., The FASEB Journal, 2015, Vol. 29, p684-695

Membrane vesicles produced by bacteria or fungi are usually collected by ultracentrifugation from the culture medium of the bacteria or fungi. However, since the amount of membrane vesicles produced by bacteria or fungi is very small and the amount obtained by the above method is small, a method for producing membrane vesicles more efficiently is required.

An object of the present invention is to provide a method capable of obtaining bacterial and / or fungal membrane vesicles more efficiently.

The inventors of the present application have found that artepirin C has an action of promoting the production of membrane vesicles of bacteria and fungi.

The method for producing a membrane vesicle composition of the present invention includes a step of treating a bacterium and / or a fungus with Artepillin C to cause the bacterium and / or the fungus to produce a membrane vesicle. By the above production method, bacterial and / or fungal membrane vesicles can be efficiently obtained.

In the above production method, the bacterium and / or fungus may be at least one selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria and yeast.

The present invention also provides a method for producing a pharmaceutical product, a quasi drug, a cosmetic product or a food product, which comprises a step of blending the membrane vesicle composition obtained by the above production method. By the above method, pharmaceuticals, quasi-drugs, cosmetics or foods containing useful components produced by bacteria and / or fungi in membrane vesicles can be produced.

The present invention also provides a method for producing a vaccine containing the membrane vesicle composition obtained by the above production method. According to this production method, a vaccine containing a membrane vesicle composition can be produced more efficiently and easily.

The present invention is also a method for producing an immunity activating agent, wherein the immunity activating agent contains the membrane vesicle composition obtained by the above-mentioned production method as an active ingredient, and the above-mentioned bacteria and / or fungi are contained. , Provide a production method having an immunity activating effect.

The present invention also provides a membrane vesicle production promoter for bacteria and / or fungi containing artepirin C as an active ingredient.

According to the production method of the present invention, membrane vesicles of bacteria and / or fungi can be obtained more efficiently.

FIG. 5 is an HS-AFM image showing bacteria before and after treatment with Artepirin C. It is a graph which shows the amount of bacterial membrane vesicle production. 9 is a scanning electron microscope image showing Escherichia coli Nisle 1917 cells after treatment with Artepirin C. 6 is a scanning electron microscope image showing Lactobacillus kunkei JCM 16173 cells after treatment with Artepirin C. 6 is a scanning electron microscope image showing Lactobacillus kunkei BPS402 cells after treatment with Artepirin C. 6 is a scanning electron microscope image showing Bifidobacterium longum BB536 cells after treatment with Artepillin C. 6 is a scanning electron microscope image showing Saccharomyces cerevisiae d3010 cells after treatment with Artepillin C.

Hereinafter, embodiments for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

The method for producing a membrane vesicle composition according to the present embodiment includes a step of treating a bacterium and / or a fungus with Artepillin C to cause the bacterium and / or the fungus to produce a membrane vesicle. Since Artepillin C has an action of promoting the production of membrane vesicles of bacteria and fungi, membrane vesicles can be obtained more efficiently and easily by the production method.

<Bacterial / fungal treatment with Artepillin C>
Treatment of bacteria and / or fungi with Artepillin C can promote membrane vesicle production on the extracorporeal membrane of the bacteria and / or fungi. Treatment of bacteria or fungi with Artepirin C can be specifically carried out, for example, by allowing bacteria and / or fungi and Artepirin C to coexist in the medium. The medium may be, for example, liquid, semi-solid or solid. The treatment of bacteria and / or fungi with Artepirin C can be specifically carried out by adding Artepirin C to, for example, a culture medium or a solid medium containing viable cells. The treatment time of bacteria and / or fungi with Artepillin C may be, for example, 1 minute or more, 5 minutes or more, 10 minutes or more, 15 minutes or more, 20 minutes or more, and 30 minutes or more. The processing time may be, for example, 1 hour or less.

The concentration of artepirin C to be treated with bacteria and / or fungi may be, for example, 1 to 2000 μM, 10 to 1500 μM, 50 to 1200 μM, or 2 to 75 μM in a medium containing bacteria and / or fungi. , 5-50 μM or 7-30 μM. Bacterial treatment with Artepillin C is preferably carried out in liquid.

The artepirin C used in the production method according to the present embodiment may be, for example, one synthesized by a known method or may be derived from a natural raw material such as propolis. As the artepirin C, those obtained by purifying propolis and increasing the concentration of artepirin C in the propolis may be used. As a commercially available product of Artepirin C, for example, Artepirin C (Wako Pure Chemical Industries, Ltd.) or the like can be used.

When treating Artepillin C into bacteria and / or fungi, the concentration of ursolic acid in the medium containing bacteria and / or fungi is preferably 62.5 μM or less, more preferably not containing ursolic acid. It is more preferable that no propolis-derived component other than artepirin C coexists in the medium. As a result, the production of membrane vesicles can be further promoted, and contamination of the membrane vesicles due to lysis of the bacterial cells can be prevented.

The production of membrane vesicles can be confirmed, for example, by observing the cells with a microscope such as a transmission type or operation type electron microscope or an atomic force microscope.

<Membrane vesicle composition>
The membrane vesicle composition obtained by the production method according to the present embodiment may consist only of membrane vesicles produced by bacteria and / or fungi, and may contain a part of the components of the membrane vesicles. It may be a membrane vesicle or one that contains other components in addition to a part of the components of the membrane vesicle. The membrane vesicle composition may be in the form of suspension, paste, powder or the like.

The membrane vesicle composition obtained by the production method of the present embodiment may contain, for example, 1 to 100% by mass of a component derived from the membrane vesicle, and 10% by mass or more of the component derived from the membrane vesicle. , 20% by mass or more, 30% by mass or more, 50% by mass or more, 70% by mass or more, 90% by mass or more, 95% by mass or more, or 99% by mass or more. The membrane vesicle composition preferably contains membrane vesicles that maintain a membrane-covered structure without degradation. Moreover, it is preferable that the membrane vesicle composition does not contain crushed fragments of bacteria and / or fungal cells.

Bacteria and fungi are known to have the ability to produce membrane vesicles in a wide range of species. The bacterium used in the production method according to the present embodiment is not particularly limited as long as it has the ability to produce membrane vesicles. Bacteria may be Gram-negative or Gram-positive. The bacterium may be a coccus, a bacillus, a spiral bacterium, or the like. The bacterium may be any of an aerobic bacterium, an anaerobic bacterium, and a facultative anaerobic bacterium. Bacteria include, for example, meningitis, periodontal disease, Escherichia coli, pyogenic, gonococcus, enteritis vibrio, regionera, erythema, Bordetella pertussis, salmonella, pyrroli, lime (Lime disease boleria), pneumonia. , Staphylococcus aureus, C. It may be Clostridium or the like. The fungus may be, for example, yeast.

When a pathogenic bacterium is used as a bacterium, the membrane vesicles produced by the pathogenic bacterium or a component derived from the membrane vesicle can be used as an immunogen for a vaccine against the pathogenic bacterium by a known method. Therefore, the membrane vesicle composition obtained by the method for producing a membrane vesicle composition according to the present embodiment can be used for producing a vaccine containing the composition. The vaccine may be therapeutic or prophylactic. The vaccine may be for humans and for animals such as fish, birds and mammals. The vaccine may be in powder form or in liquid form. The form of the vaccine is not particularly limited, and may be, for example, uncoated tablets, sugar-coated tablets, granules, powders, tablets, capsules (hard capsules, soft capsules, seamless capsules). When the vaccine is for humans, the administration form is not particularly limited, and for example, it may be ingested by injection, transdermally, orally, or nasally.

It is known that useful bacteria such as lactic acid bacteria and / or useful components secreted by the fungi are also contained in the membrane vesicles produced by the bacteria and / or the fungi. Therefore, membrane vesicles obtained from bacteria and / or fungi can be used as components having the effects of the bacteria and / or fungi. Therefore, a membrane vesicle composition having an immunostimulatory action can be obtained by producing membrane vesicles using, for example, a bacterium or a fungus having an immunostimulatory action. In one embodiment of the present invention, a membrane vesicle composition comprising a step of treating a bacterium and / or a fungus having an immunostimulatory action with Artepillin C to cause the bacterium and / or the fungus to produce a membrane vesicle. A method for producing an immunity activating agent contained as an active ingredient is provided.

The lactic acid bacterium may be, for example, Lactobacillus, Streptococcus, Pediococcus, Leuconostocaceae, Enterococcus, Lactococcus, Bifidobacterium and the like. By using a lactic acid bacterium, a bacterial membrane vesicle having the physiological activity of the lactic acid bacterium can be obtained.

The lactic acid bacterium may be, for example, Lactobacillus kunky, preferably Lactobacillus kunky BPS402 (accession number FERM BP-11439). Lactobacillus kunky BPS402 is known to have an immunostimulatory effect based on a high IgA production promoting effect. Therefore, in the method for producing a bacterial membrane vesicle composition according to the present embodiment, for example, by using Lactobacillus kunky BPS402 as a bacterium, a bacterial membrane vesicle composition having an effect of activating immunity can be obtained. .. In one embodiment, there is provided a method for producing an immunostimulatory agent or an immunoglobulin production promoter such as IgA, which comprises a membrane vesicle composition derived from Lactobacillus kunky BPS402 as an active ingredient. Lactobacillus Kunky BPS402 was established on October 3, 2011 by the National Institute of Technology and Technology Patent Organism Depositary Center (1-1-1, Higashi, Kisarazu City, Japan, Central No. 6, 〒305-8566, now independent. It has been deposited at the National Institute of Technology and Evaluation Patent Organism Depositary (2-5-8 Kazusakamatari, Kisarazu City, Japan, Room 292-0818) as the accession number FERM P-22177. It is possible. The strain is currently being transferred to an international deposit, and the accession number is FERM BP-11439.

As the bacterium and / or fungus used in the method for producing the membrane vesicle composition according to the present embodiment, those prepared by culturing in advance under conditions suitable for each bacterium and / or fungus can be used.

<Membrane vesicle>
The size of the membrane vesicles produced by bacteria and / or fungi in the production method according to the present embodiment may be, for example, 10 to 600 nm, 20 to 400 nm, or 40 to 300 nm. Membrane vesicles have a structure covered by a cell membrane. Membrane vesicles may contain, for example, nucleic acids such as DNA, RNA, proteins, signaling substances, pathogens, immunomodulators, and other physiologically active substances, including bacteria such as lipopolysaccharides, lipoproteins, peptidoglycans, and /. Alternatively, it may contain components derived from the cell membrane or cell wall of the bacterium. Membrane vesicles may be, for example, derived from bacterial and / or fungal cell membranes or outer membranes.

The method for producing a membrane vesicle composition according to the present embodiment may further include a step of isolating the membrane vesicle composition. Membrane vesicles produced on the membranes of bacteria and / or fungi or compositions containing the membrane vesicles can be isolated by known methods. Specifically, for example, the cells are removed from the culture solution containing the cells or the solution in which the medium containing the cells is suspended or dissolved by a centrifuge, and the supernatant is filtered or purified as necessary. Can be obtained through. The supernatant may be further ultracentrifuged to separate and purify the membrane vesicles and other substances. The supernatant may be concentrated and then purified. The membrane vesicle composition obtained by the production method according to the present embodiment may be used together with the bacterial cells without being isolated from the bacterial cells.

<Pharmaceuticals, quasi-drugs, cosmetics, foods>
The membrane vesicle composition obtained by the method for producing a membrane vesicle composition according to the present embodiment can be used in the production of these products as a component of pharmaceuticals, quasi-drugs, cosmetics and / or foods. .. Therefore, one embodiment of the present invention is a method for producing a pharmaceutical product, a quasi-drug, a cosmetic product, and / or a food product, which comprises a step of blending the membrane vesicle composition obtained by the above-mentioned method for producing a membrane vesicle composition. I will provide a. The membrane vesicle composition may be diluted, concentrated, dried, heated or the like, if necessary, and used in the production of the above product.

In particular, when bacteria and / or fungi are useful bacteria or fungi such as lactic acid bacteria, the components derived from the obtained membrane vesicles can be used as components of pharmaceuticals, quasi-drugs, cosmetics and / or foods. , Pharmaceuticals, quasi-drugs, cosmetics and / or foods having the effects of the bacterium and / or the fungus can be produced. For example, by blending a component derived from a bacterium having an immunostimulatory action and / or a membrane vesicle derived from the fungus into a raw material of a drug, a quasi drug, a cosmetic or / or a food, an immunostimulatory drug, It is possible to manufacture quasi-drugs for immunostimulation, cosmetics for immunostimulation, and foods for immunostimulation.

The formulation of the membrane vesicle composition into pharmaceuticals, quasi-drugs, cosmetics or foods is performed, for example, by mixing the membrane vesicle composition prepared in advance with the raw materials of pharmaceuticals, quasi-drugs, cosmetics or foods. Or by blending Artepillin C with bacteria and / or fungi that produce membrane vesicles in pharmaceuticals, quasi-drugs, cosmetics or foods, membrane vesicles in the product. This may be done by producing a composition. Pharmaceuticals, quasi-drugs, cosmetics and foods may contain artepirin C and bacteria and / or fungi that produce membrane vesicles.

The content of the membrane vesicle composition in pharmaceuticals, quasi-drugs, cosmetics and foods may be, for example, 0.001% by mass or more, 0.01% by mass or more, 0.1% by mass or more, It may be 1% by mass or more, 5% by mass or more, or 10% by mass or more. The content of the membrane vesicle composition in pharmaceuticals, non-pharmaceutical products, cosmetics and foods is, for example, 100% by mass or less, 99% by mass or less, 95% by mass or less, 90% by mass or less, 70% by mass or less. It may be 50% by mass or less, 30% by mass or less, 10% by mass or less, 5% by mass or less, and 1% by mass or less.

As a drug or a non-pharmaceutical product, a membrane vesicle or a component derived from the membrane vesicle may be used as it is, including tablets (including uncoated tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, troches, etc.) and capsules. Agents, pills, powders (powder), fine granules, granules, liquids, suspensions, emulsions, syrups, pastes, injections (when used, for infusions such as distilled water or amino acid infusions or electrolyte infusions) (Including the case of blending and preparing as a liquid preparation), etc. may be used. These various preparations can be prepared, for example, by mixing the membrane vesicle composition, which is an active ingredient, with other ingredients, if necessary, and molding them into the above dosage form.

Other ingredients used in pharmaceuticals or quasi-drugs include, for example, pharmaceutically acceptable excipients, binders, lubricants, disintegrants, emulsifiers, surfactants, bases, solubilizers. Agents, suspending agents and the like can be mentioned.

In addition to the membrane vesicle composition, the cosmetics according to the present embodiment include ingredients usually used in cosmetics, such as whitening agents, moisturizers, antioxidants, oily ingredients, ultraviolet absorbers, surfactants, and thickeners. , Alcohols, powder components, coloring materials, aqueous components, water, various skin nutrients and the like can be appropriately blended as needed.

The dosage form of the cosmetic product according to the present embodiment is, for example, a solubilized type, an emulsified type, a powder type, an oil liquid type, a gel type, an ointment type, an aerosol type, a water-oil two-layer system, and a water-oil-powder three-layer. It may be a system or the like. The above cosmetics include, for example, basic cosmetics such as facial cleansers, lotions, milky lotions, creams, gels, essences, beauty liquids, packs, masks, mists, UV preventive cosmetics, foundations, lipsticks, cheeks, eye shadows, eyeliners, and mascara. Make-up cosmetics, wash pigments, massage agents, cleansing agents, after-shave lotions, pre-shave lotions, shaving creams, body soaps, soaps, shampoos, rinses, hair treatments, hair styling products, hair styling products, hair mists, hair It may be foam, hair liquid, hair gel, hair spray, hair restorer, antiperspirant, bathing agent, mouth rinse, oral cosmetics, toothpaste and the like.

The food according to the present embodiment may be a food in which the tertiary function (physical condition adjusting function) of the food is emphasized. Examples of foods in which the tertiary function of foods is emphasized include health foods, foods with functional claims, dietary supplements, supplements, and foods for specified health use.

Foods include minerals, vitamins, flavonoids, quinones, polyphenols, amino acids, nucleic acids, essential fatty acids, refreshing agents, binders, sweeteners, disintegrants, lubricants, colorants, as needed. , Fragrances, stabilizers, preservatives, sustained-release regulators, surfactants, solubilizers, wetting agents and the like may be blended. The food may be in any form such as solid, liquid or paste.

Foods include, for example: soft drinks such as coffee, tea drinks, juices, carbonated drinks, dairy drinks, lactic acid bacteria drinks, yogurt drinks, and liquors such as Japanese liquor, western liquor, fruit liquor and honey liquor. Beverages; Spreads such as custard cream; Pastes such as fruit paste; Western confectionery such as chocolate, donuts, pies, cream puffs, gum, jelly, candy, cookies, cakes and puddings; Japanese confectionery; iced confectionery such as ice cream, iced drinks and sherbets; cooked foods such as curry, beef bowl, miscellaneous dishes, miso juice, soup, meat sauce, pasta, pickles, jam; dressing, sprinkle, flavor seasoning and soup base, etc. Seasoning.

Artepirin C has the effect of promoting the production of membrane vesicles of bacteria and fungi. Therefore, one embodiment of the present invention provides a membrane vesicle production promoter for bacteria and / or fungi containing artepirin C as an active ingredient. As the specific embodiment of artepirin C, bacteria and fungi in the membrane vesicle production promoter, the embodiment in the above-mentioned method for producing a membrane vesicle composition can be applied.

Hereinafter, the present invention will be described in more detail based on examples. However, the present invention is not limited to the following examples.

[Test Example 1]
Artepirin C (refined product from propolis) was dissolved in DMSO to prepare a 25 μM (7.5 μg / mL) solution of Artepirin C. The periodontopathic bacterium Porphyromonas gingivalis ATCC 33277 (obtained from ATCC) was immobilized on a glass slide for HS-AFM (high-speed atomic force microscopy) observation. Artepirin C treatment was performed by dropping an Artepirin C solution onto the immobilized cells. Using an HS-AFM device (manufactured by Olympus Corporation), the behavior of bacterial cells on a nanoscale before and after treatment with Artepirin C was observed. An HS-AFM image before and every 10 minutes (t = 10, 20, 30 minutes) before and after the treatment with Artepirin C is shown in FIG. The size of each image indicates a range of 1520 nm × 1140 nm (xxy).

For bacterial cells 30 minutes after treatment with Artepirin C, three different regions were randomly selected per bacterial cell by HS-AFM observation, and the number of membrane vesicles in each was visually counted, and in each of the three regions. The total number of membrane vesicles was calculated. The total number of membrane vesicles was calculated in the same manner for the three bacterial cells, and the average value per bacterial cell was calculated. The results are shown in Table 1 and FIG. Using Artepillin C, ethanol-extracted propolis 100 μg / mL, and baccarin (refined product from propolis) 100 μM (56.3 μg / mL) dissolved in DMSO, treat with bacteria in the same manner as above to produce membrane vesicles. I counted the amount. As the ethanol-extracted propolis aqueous solution, a propolis raw mass was extracted with ethanol, filtered, and then concentrated to 100 μg / mL. The results are shown in Table 1 and FIG.

The production of membrane vesicles was confirmed by treatment with ethanol-extracted propolis, Artepirin C and Baccarin (Table 1, FIG. 2). In the treatment with Artepillin C, the number of vesicles and the diameter of vesicles tended to increase with the lapse of treatment time (Fig. 1). When converted to 1 μg / mL, the membrane vesicle-producing effect of Artepirin C treatment was calculated to be 18.75 times that of ethanol-extracted propolis treatment.

It is also known that Artepirin C and Baccarin are present in the ethanol-extracted propolis used at concentrations of 9.5% by mass and 3.5% by mass, respectively. Therefore, Artepillin C is present at a concentration of 9.5 μg / mL in 100 μg / mL of an aqueous ethanol-extracted propolis solution. Here, since the amount of membrane vesicles produced per 1 μg / mL of artepirin C calculated from the results of artepirin C alone is 7.5 as shown in Table 1, it is assumed that artepirin C in the ethanol-extracted propolis is artepirin C alone. Treatment with 100 μg / mL of ethanol-extracted propolis should result in the production of at least 71.25 membrane vesicles. However, the effect of membrane vesicle production by the actual treatment with ethanol-extracted propolis was about half of this, indicating the superiority of treatment with Artepirin C alone.

[Test Example 2]
Escherichia coli (Escherichia coli Nistle 1917), lactic acid bacteria (Lactobacillus kunkei JCM 16173, Lactobacillus kunkei BPS402), bifidobacteria (Bifidobacterium longum bleached honey-produced saccharomyces) An experiment was conducted to confirm. The prepared various bacterial cells were suspended in PBS buffer, a solution containing the bacterial cells was added onto the substrate, and the mixture was left at room temperature for 10 to 30 minutes to adhere the bacterial cells on the substrate. After washing the substrate with PBS buffer, each test substance was added dropwise onto the substrate and reacted at room temperature for 2 hours. As the test substance, a solution prepared by dissolving Artepillin C in a 10% aqueous solution of DMSO at a concentration of 100 μM or 1000 μM was used. As a control, a DMSO 10% aqueous solution was used.

The treated cells were fixed with PBS containing 2.5% glutaraldehyde / 2% paraformaldehyde and observed with a scanning electron microscope. The results are shown in FIGS. 3-7. In all bacteria and fungi, the effect of artepirin C on promoting membrane vesicle production was confirmed at concentrations of 100 μM and 1000 μM. With lactic acid bacteria, almost no change in the morphology of the cells due to treatment was observed, and a tendency was observed that vesicles were uniformly formed on the outer membrane. Membrane vesicle-producing activity was observed in Escherichia coli, which is a prokaryotic gram-negative bacterium, lactic acid bacterium and bifidobacteria, which are gram-positive bacteria, and yeast, which is a eukaryote. It was confirmed that the membrane vesicle-inducing effect of Artepillin C is effective against various bacteria and fungi.

Claims (6)

The bacteria and / or fungi treated with Arutepirin C viewing including the step of producing membrane vesicles in the bacterium and / or fungus, in processing bacteria and / or fungi Arutepirin C, the bacteria and / or fungi A method for producing a membrane vesicle composition in which a propolis-derived component other than Artepillin C does not coexist in the containing medium. The production method according to claim 1, wherein the bacterium and / or fungus is at least one selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria and yeast. A method for producing a pharmaceutical product, a quasi drug, a cosmetic product, or a food product, which comprises a step of obtaining a membrane vesicle composition by the production method according to claim 1 or 2 and a step of blending the membrane vesicle composition. A step of obtaining a membrane vesicle composition by the production method according to claim 1 or 2, and a method for producing a vaccine containing the membrane vesicle composition. It is a method for producing an immunostimulatory agent .
請Motomeko includes the step of obtaining a membrane vesicle compositions from bacteria and / or fungi by the method according to 1 or 2, wherein the bacterium and / or fungus all SANYO having immunity activating effect, the immune A production method in which the force activator contains the membrane vesicle composition as an active ingredient. An agent for promoting the production of membrane vesicles of bacteria and / or fungi containing artepirin C as an active ingredient.

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