KR20080032809A - Cell lysis composition of bacterial and yeast cell wall and method for preparing the same - Google Patents

Abstract

A method for preparing a cell lysis composition of bacterial and yeast cell wall is provided to improve yield of cell wall, and selectively employ antigen substances and immune-enhancing substances in the cell wall by using biochemical and chemical treatment, so that the composition is useful as vaccine antigen, heterologous delivery system, immune enhancer, vaccine assistant and complex feed additive. A method for preparing a cell lysis composition of bacterial and yeast cell wall comprises the steps of: culturing gram positive bacteria, gram negative bacteria and yeast; treating the cultured gram positive bacteria, gram negative bacteria and yeast biochemically and chemically by using transparent outer membrane extraction, TNT(Tris-HCl, sodium chloride, Triton X-114) method, NaOH-EDTA(sodium hydroxide-(ethylene dinitrilo) tetraacetic acid), TMC(Tris-HCl, magnesium chloride, calcium chloride) method or TET(Tris-HCl, EDTA, Triton X-100) method; and extracting useful ingredients of cell wall from the treated gram positive bacteria, gram negative bacteria and yeast, wherein the gram negative bacteria include Escherichia coli 055 and Bordetella pertussis; the gram positive bacteria include Staphylococcus aureus Cowan 1 and Erysipelothrix rhusiopathiae T2; and the yeast is Saccharomyces cerevisiae or Candida albicans. Further, the useful ingredients of cell wall are selected from a group consisting of a transparent cell outer membrane, a cell wall and bacteriolysant.

Description

CELL LYSIS COMPOSITION OF BACTERIAL AND YEAST CELL WALL AND METHOD FOR PREPARING THE SAME

1 is a photograph of a transparent cell membrane of Gram-positive bacteria Staphylococcus aureus Cowan I obtained by using a transparent extracellular membrane extraction method.

Figure 2 is a photograph of the translucent cell membrane of Gram-positive bacteria Erysipelothrix rhusiopathiae T2 obtained by using the DMSO method.

Figure 3 is a photograph of the translucent extracellular membrane of Gram-negative bacteria Escherichia coli (Escherichia coli O55) obtained by using the alkaline lysis method.

4 is a photograph of the transparent extracellular membrane of Bodetella pertussis, a Gram-negative bacterium obtained using the TNT method.

Figure 5 is a photograph of the transparent cell membrane of Saccharomyces cerevisiae yeast bacteria obtained by using the Bacterial ghost method.

Figure 6 is a photograph of the clear extracellular membrane of Saccharomyces cerevisiae obtained by using the TNT method.

7 is a photograph of the extracellular wall of Candida albicans yeast obtained by using the TNT method.

8 is a photograph of the cell wall of Escherichia coli O55 obtained using the NaOH method.

9 is a photograph of the cell wall of Escherichia coli O55 obtained using the TNT method.

10 is a photograph of the cell wall of Escherichia coli O55 obtained using the TMC method.

Figure 11 is a photograph of the cell wall of Bodetella pertussis obtained by using the clear cell membrane extraction method.

12 is a photograph of a cell wall without contents of Staphylococcus aureus Cowan I, a Gram-positive bacterium obtained by TMC method.

FIG. 13 is a photograph of a clear extracellular membrane of Saccharomyces cerevisiae obtained using the TMC method. FIG.

Figure 14 is a photograph of the cell wall of granulocyte form of Staphylococcus aureus Cowan I obtained by using the TNT method.

FIG. 15 is a photograph of granulocyte-shaped cell walls of P. aeruginosa (Erysipelothrix rhusiopathiae T2) obtained using the TNT method.

Figure 16 is a photograph of the cell wall of granulocyte form of Escherichia coli (Escherichia coli O55) obtained by using the TET method.

Figure 17 is a photograph of the granulocyte-like cell wall of Escherichia coli (Escherichia coli O55) obtained by using the clear cell membrane extraction method.

18 is a photograph of granulocyte-shaped cell walls of Saccharomyces cerevisiae obtained using the TET method.

19 is a photograph of cell walls of granulocyte form of Candida albicans obtained using the TET method.

20 is a photograph of the lysate of Escherichia coli O55 obtained using the NaOH-EDTA extraction method.

Figure 21 is a photograph of the lysate of Escherichia coli (Escherichia coli O55) obtained by using the TET method.

Figure 22 is a photograph of the lysate of the Erysipelothrix rhusiopathiae T2 obtained by using the TET method.

The present invention relates to a cell wall lysate composition of bacteria and yeast and a method for producing the same, and more particularly, bacteria obtained by biochemical and chemical treatment of gram-negative bacteria, gram-positive bacteria and yeast, which are rich in microbial specific antigens and immunopotentiators. The present invention relates to a cell wall lysate composition of bacteria and yeast, including a cell wall component of yeast, which can be used as a vaccine antigen, a heterologous antigen carrier, an adjuvant, a vaccine adjuvant and a compound feed additive, and a method for producing the same.

Gram-negative bacteria, Gram-positive bacteria and yeast cell walls are known to have various forms of antigenic substances and immunopotentiators. Currently, homogenizers are used to obtain cell walls of bacteria and yeasts that contain antigenic substances and immunopotentiators. Alternatively, a method of extracting the cell wall after physically destroying the cell wall using a cell pressure by pressure is known, but the yield of the cell wall obtained by the method is very small and only experimental production is possible, Not only are they unsuitable for production, but the complete destruction of the cell wall is known to make it difficult to selectively use the useful components present in the cell wall. In addition, a lysis method using bacteriophage is also known, but it is limited to only some Gram-negative bacteria, making it difficult to mass-produce, and also because of the high specificity of bacterial phage to microorganisms, it is difficult to put it to practical use. In addition to this, the alkaline lysis method is used, but it is difficult to apply to yeast, there is a problem that can not obtain a variety of cell walls according to the intended use.

As a result of the intensive efforts to solve the above problems, the present inventors have developed a biochemical and chemical method capable of extracting and lysing various cell walls suitable for a purpose from gram-negative bacteria, gram-positive bacteria, and yeast, By obtaining useful transparent extracellular membranes, cell walls and lytic materials, the present invention has been completed.

The present invention has been proposed to solve the problems of the prior art as described above, the present invention is a bacterium and yeast obtained by biochemical and chemical treatment of gram-negative bacteria, gram-positive bacteria and yeast rich in microbial specific antigens and immune enhancing substances To provide a cell wall lysis composition of bacteria and yeast, including cell wall components of, and can be used as vaccine antigens, heterologous antigen carriers, immunopotentiators, vaccine adjuvant and multi-feed additives.

In order to achieve the above object, the first aspect of the present invention provides a bacterium and yeast comprising a cell wall component obtained by biochemically and chemically treating gram-negative bacteria, gram-positive bacteria, and yeast, which are rich in microbial specific antigens and immune enhancing substances. As a cell wall lysis composition, the cell wall active ingredient provides a cell wall lysis composition of bacteria and yeast, characterized in that it is used as a vaccine antigen, a heterologous antigen carrier, an immunopotentiator, a vaccine adjuvant and a compound feed additive.

In the present invention, the Gram-negative bacteria is preferably selected from the group consisting of Escherichia coli O55, Bodetella pertussis, etc., and the Gram-positive bacteria are Staphylococcus aureus Cowan I, Don alone It is preferably selected from the group consisting of bacteria (Erysipelothrix rhusiopathiae T2) and the like. In the present invention, the yeast is preferably selected from the group consisting of Saccharomyces cerevisiae, Candida albicans and the like.

In addition, the cell wall component included as an active ingredient in the composition of the present invention is preferably selected from the group consisting of a transparent extracellular membrane, a cell wall and a lytic material.

Thus, the lytic composition of the present invention can be used for the preparation of antigen vaccines, vaccine backing agents, antigen carriers, immunopotentiators, feed additives and negative formulations.

The pharmaceutical compositions of the present invention can be used to prepare various forms of formulations according to conventional methods. In the preparation of the formulation, it is preferable to mix or dilute the cell wall component according to the invention as an active ingredient with the carrier or to enclose it in a carrier in the form of a container. When the carrier is used as a diluent, it may be a solid, semisolid or liquid substance which acts as a vehicle, excipient or medium for the active ingredient. Thus, the formulations may be in the form of tablets, pills, powders, assays, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile injectables, sterile powders and the like. The formulation may further comprise fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like. The lytic compositions according to the invention may be formulated using methods well known in the art to provide rapid, sustained or delayed release of the active ingredient after administration. As an active ingredient, the cell wall component of the present invention may be divided or divided once a day in an amount of 0.1 to 1000 mg / kg (body weight), preferably 1 to 100 mg / kg (body weight) per day for animals including humans. Administration can be via oral or parenteral routes.

In order to achieve the above object, the present invention provides a cell wall lysis composition comprising gram-negative bacteria, gram-positive bacteria and yeast transparent and translucent cell walls as an active ingredient, which comprises culturing gram-negative bacteria, gram-positive bacteria and yeast. step; Biochemically and chemically treating the cultured Gram-negative bacteria, Gram-positive bacteria and yeast; And it provides a method for producing a cell wall lytic composition of bacteria and yeast comprising the step of extracting the cell wall active ingredient from the biochemical and chemically treated Gram-negative bacteria, Gram-positive bacteria and yeast.

In the method for preparing a cell wall lysate composition according to the present invention, in the biochemical and chemical treatment step, the transparent extracellular membrane extraction method, TNT (Tris-HCl, Sodium chloride, Triton X-114) method and NaOH-EDTA (Sodium Hydroxide- ( Ethylenedinitrilo) Tetraacetic Acid lysis method is used, and the cell wall active ingredient thus obtained is preferably a transparent or translucent cell wall. In addition, in the biochemical and chemical treatment step, the clear cell membrane extraction method, TNT (Tris-HCl, Sodium chloride, Triton X-114) method, TMC (Tris-HCl, Magnesium chloride, Calcium chloride) method and TET (Tris-HCl) , EDTA, Triton X-100) method is used, the cell wall active ingredient obtained is preferably a pure cell wall without cell contents, NaOH-EDTA (Sodium Hydroxide- (Ethylenedinitrilo) Tetraacetic Acid) lysis and TET (Tris- HCl, EDTA, Triton X-100) method is also used, and the cell wall active ingredient thus obtained is preferably a transparent or translucent cell wall.

The cell wall lysate composition of the bacteria and yeast prepared according to the method of the present invention is rich in microbial specific antigens and immunopotentiators, and the transparent extracellular membrane thus obtained is useful as a vaccine antigen and heterologous antigen delivery system without a vaccine support agent. The cell wall may be usefully used as an adjuvant and vaccine adjuvant, and the lysate may be usefully used as a compound feed additive.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Separation of Staphylococcus Membranes by Transparent Extracellular Membrane Extraction

After Gram-positive Staphylococcus aureus Cowan I was incubated in 500 ml of Brain Heart Infusion (BHI) broth at 28 ° C. for 18 hours, 500 ml of 10% magnesium sulfate was added to the resulting culture, followed by an additional 18 hours. Incubated with Then, as a transparent extracellular membrane extraction method, two-fold amount of distilled water was added to promote lysis to obtain a transparent cell membrane (see FIG. 1).

Example 2 Separation of Membrane Membranes by DMSO Method

After culturing the Gram-positive bacteria Erysipelothrix rhusiopathiae T2 in 500 ml medium prepared by adding 0.1% Tween 80 to BHI (Brain Heart Infusion) broth for 18 hours at 37 ° C, 1 ml of sodium hydroxide and 1 ml of DMSO per 10 ml were added and incubated for 7 days. Translucent cell membranes were obtained from the culture (see FIG. 2).

Example 3 Extracellular Membrane Separation of Escherichia Coli by Alkali Lysis Method

E. coli (Escherichia coli O55) was cultured in TSA (Tryptic Soy Agar) broth for 24 hours at 37 ° C, sterile distilled water was added to collect the cultured bacteria, and 0.25M NaOH was added to the collected bacteria. Incubated at room temperature for 7 days to obtain a clear cell membrane (see FIG. 3).

Example 4 Extracellular Membrane Separation of Escherichia Coli by Alkali Lysis Method

The gram-negative bacteria Bodetella pertussis was cultured and collected in the same manner as in Example 3, and then 50 mM Tris-HCl, 0.15 M sodium chloride and Triton X-114 were added to the collected bacteria, and then 7 days at room temperature. Incubated to obtain a clear cell membrane (see FIG. 4).

Example 5 Extracellular Membrane Separation of Saccharomyces

Saccharomyces cerevisiae, a yeast bacterium, was extracted in the same manner as the transparent extracellular membrane extraction method of Example 1 and the TNT method, the alkaline lysis method of Example 4, to extract the transparent extracellular membrane (FIGS. 5 and 6). Reference).

Example 6 Separation of Extracellular Membrane of Candida

Candida albicans yeast was carried out in the same manner as in the TNT method of Example 5 to extract the transparent extracellular wall (see Fig. 7)

Example 7 Separation of Cell Walls Without Cell Contents

The Gram-negative bacterium Escherichia coli O55 was incubated for 24 hours at 37 ° C. in TSA (Tryptic Soy Agar) broth, sterile distilled water was added to collect the cultured bacteria, and 0.25M to 2M NaOH was collected. After addition, the cells were cultured for 7 days at room temperature to obtain a cell wall free of cell contents (see FIG. 8). The collected bacteria were also treated by TNT method and TMC (10 mM Tris-HCl, 20 mM magnesium chloride and 1 mM calcium chloride) method. To obtain cell walls without cell contents, respectively (see FIGS. 9 and 10).

In addition, Bodetella pertussis was treated with clear cell membrane extraction to obtain a cell wall without cell contents (see FIG. 11), and treated with Gram-positive bacteria Erysipelothrix rhusiopathiae T2 by TMC method. Saccharomyces cerevisiae (Saccharomyces cerevisiae) was treated by the TNT method to obtain cell walls without contents, respectively (see FIGS. 12 and 13).

Example 8 Separation of Extracellular Wall in Granular Sphere Form

Gram-positive bacteria Staphylococcus aureus Cowan I and Erysipelothrix rhusiopathiae T2 were treated with TNT method to obtain extracellular wall of granulocyte form (see FIGS. 14 and 15), and Escherichia coli (Escherichia coli) O55) was treated with TET (50 mM Tris-HCl, 25 mM EDTA, 1% Triton X-100) and clear extracellular membrane extraction to obtain extracellular granules in the form of granulocytes (see FIG. 16 and FIG. 17). Saccharomyces cerevisiae and Candida albicans were treated by TET to obtain granular globular extracellular wall, respectively (see FIGS. 18 and 19).

Example 9 Separation of Cell Lysis Material by Lysis Treatment

Gram-negative bacterium Escherichia coli (Escherichia coli O55) was treated with 1M NaOH-EDTA extraction and TET to easily lyse the cells to obtain cell lysates (see FIGS. 20 and 21). Cell lysates were obtained by lysing Erysipelothrix rhusiopathiae T2) by TET method to completely lyse the cells (see FIG. 22).

As described above, the cell wall lysate composition of bacteria and yeast according to the present invention is a variety of microorganism-specific antigens and immunopotentiators, such as TNT (Tris-NaCl-Triton X-114) method, DMSO method, etc. Biochemical and chemical methods can be used to obtain a variety of transparent extracellular membranes, cell walls and lytic materials in large quantities. The transparent extracellular membrane obtained by the method of the present invention is a vaccine antigen and heterologous antigen delivery system without a vaccine supplement. The cell wall may be usefully used as an adjuvant and vaccine adjuvant, and the lysate may be usefully used as a compound feed additive.

Claims (9)

As a cell wall lytic composition of bacteria and yeast comprising cell wall components obtained by biochemically and chemically treating gram-negative bacteria, gram-positive bacteria and yeast, which are rich in microbial specific antigens and immunopotentiators, as an active ingredient, The cell wall active ingredient is a cell wall lysis composition of bacteria and yeast, characterized in that it is used as a vaccine antigen, a heterologous antigen carrier, an immune enhancer, a vaccine adjuvant and a compound feed additive. The method of claim 1, The Gram-negative bacteria E. coli (Escherichia coli O55), Bodetella pertussis (Bordetella pertussis), etc., characterized in that the cell wall lysis composition of bacteria and yeast. The method of claim 1, The Gram-positive bacteria is a cell wall lytic composition of bacteria and yeast, characterized in that selected from the group consisting of Staphylococcus aureus Cowan I, Erysipelothrix rhusiopathiae T2. The method of claim 1, The yeast cell wall lysis composition of bacteria and yeast, characterized in that selected from the group consisting of Saccharomyces cerevisiae, Candida albicans and the like. The method of claim 1, The cell wall active ingredient is a cell wall lytic composition of bacteria and yeast, characterized in that selected from the group consisting of a transparent extracellular membrane, a cell wall and a lytic material. As a method for producing a cell wall lysate composition comprising gram-negative bacteria, gram-positive bacteria and yeast as transparent, translucent cell walls, Culturing Gram-negative bacteria, Gram-positive bacteria and yeast; Biochemically and chemically treating the cultured Gram-negative bacteria, Gram-positive bacteria and yeast; And Extracting the cell wall active ingredient from the biochemically and chemically treated Gram-negative bacteria, Gram-positive bacteria and yeast. The method of claim 6, The biochemical and chemical treatment steps were performed using a clear cell membrane extraction method, TNT (Tris-HCl, Sodium chloride, Triton X-114) method and NaOH-EDTA (Sodium Hydroxide- (Ethylenedinitrilo) Tetraacetic Acid) lysis method, The active ingredient is a method for producing a cell wall lysate composition of bacteria and yeast, characterized in that the transparent or translucent cell wall. The method of claim 6, The biochemical and chemical treatment steps include transparent extracellular membrane extraction, TNT (Tris-HCl, Sodium chloride, Triton X-114) method, TMC (Tris-HCl, Magnesium chloride, Calcium chloride) method and TET (Tris-HCl, EDTA). , Triton X-100) method, wherein the obtained cell wall active ingredient is a cell wall lysis composition of bacteria and yeast, characterized in that the pure cell wall without cell contents. The method of claim 6, The biochemical and chemical treatment steps are performed using NaOH-EDTA (Sodium Hydroxide- (Ethylenedinitrilo) Tetraacetic Acid) lysis and TET (Tris-HCl, EDTA, Triton X-100) method, and the cell wall active ingredient obtained is transparent or translucent. A method for producing a cell wall lysate composition of bacteria and yeast, characterized in that it is a cell wall.

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