JP7060439B2 - How to recover dead bacteria by sterilization - Google Patents

Description

The present invention relates to a method for recovering dead bacteria by sterilization treatment.

In order to provide food and drink with high safety from a microbiological point of view so as not to cause food poisoning, it is important to sufficiently sterilize the microorganisms contained in the raw materials and products.
As a conventional sterilization method, heat sterilization is generally known. However, when raw materials and products are heated for sterilization, deterioration and heating odor may occur, and nutritional components and flavor may be impaired.

Therefore, electric field sterilization is attracting attention as an alternative sterilization method to heat sterilization.
Electric field sterilization is a method of sterilizing mixed microorganisms by an electrical sterilizing action by sandwiching raw materials and products between electrodes and applying a high AC voltage. The electric field sterilization method can suppress discoloration due to heat, generation of a heated odor, and reduction of nutritional components as compared with the conventional sterilization method using only heating.
Furthermore, the use of electric field sterilization is being considered in order to provide highly safe liquid medicines such as infusions and injections.

In general, electric field sterilization conditions must be set appropriately depending on the type of food and drink and the type of microorganisms mixed in. Then, it is necessary to confirm whether the sterilizing effect is sufficient according to the set electric field sterilization conditions. Furthermore, in order to deepen the knowledge of sterilization and antibacterial treatment, it is important to elucidate how the microorganisms were killed by the bactericidal treatment.

In order to observe the dead bacteria after the sterilization treatment, it is necessary to collect the dead bacteria. As a conventional method for recovering dead bacteria, a method in which a suspension containing the dead bacteria is centrifuged and the dead bacteria are recovered from the separated and settled pellets is common. Further, in Patent Document 1, after collecting and installing a microbial sample on a liquid permeable membrane for preparing a scanning electron microscope (SEM) specimen, at least a fixing step or a washing / fixing step, a washing step, and dehydration are described. A method for treating a microbial sample, in which the step is performed while sucking from the lower part of the liquid permeable membrane, is described.

Japanese Unexamined Patent Publication No. 2008-286591

It is possible to recover live bacteria and dead bacteria killed by heat sterilization by the prior art or the method described in Patent Document 1. However, until now, it has not been reported that dead bacteria killed by electric field sterilization were recovered by the conventional method.

Therefore, an object of the present invention is to provide a method for recovering dead bacteria, which cannot be collected by the conventional method and can recover dead bacteria killed by electric field sterilization or the like.

In view of the above problems, the present inventor has made a diligent study. As a result, it was found that even if the suspension of the dead bacteria killed by the electric field sterilization treatment was centrifuged under the conditions of the conventional method, the dead bacteria killed by the electric field sterilization treatment did not transfer to the pellets. Furthermore, it was found that when the suspension was centrifuged under conditions different from the conventional method, the killed bacteria killed by the electric field sterilization treatment floated in the supernatant. Therefore, the supernatant after centrifugation in which the dead bacteria killed by the electric field sterilization treatment floats is filtered using a membrane filter, the dead bacteria are adsorbed on the membrane filter, and the dead bacteria are fixed on the membrane filter to perform electric field sterilization. It was found that the dead bacteria killed by the treatment can be specifically recovered.
The present invention has been completed based on these findings.

In the present invention, the sterilized bacteria are suspended in a solvent, the suspension is centrifuged, the supernatant after centrifugation is filtered using a membrane filter, and the dead bacteria are adsorbed on the membrane filter to form a membrane filter. The present invention relates to a method for recovering dead bacteria, in which the dead bacteria are fixed and the dead bacteria are collected together with the membrane filter.

Further, in the present invention, the sterilized bacteria are suspended in a solvent, the suspension is centrifuged, the supernatant after centrifugation is filtered using a membrane filter, and the dead bacteria are adsorbed on the membrane filter, and the membrane filter is used. The dead bacteria were fixed in the cell, the dead bacteria were collected together with the membrane filter, the recovered membrane filter was immersed in a critical point drying solution, the membrane filter was frozen, and the frozen membrane filter was dried at the critical point and dried at the critical point. The present invention relates to a method for imaging dead bacteria by subjecting a membrane filter to imaging using an electronic microscope.

According to the present invention, it is possible to recover dead bacteria that have been killed by electric field sterilization or the like, which could not be collected by the conventional method.
Further, in the present invention, the dead bacteria killed by the electric field sterilization treatment or the like are adsorbed and fixed on the membrane filter, and the target dead bacteria are recovered together with the membrane filter. The recovered membrane filter can be subjected to imaging with an electron microscope by performing a predetermined treatment. Therefore, it is possible to easily image the dead bacteria recovered by the present invention.

FIG. 1 (A) is an electron micrograph of the dead Staphylococcus aureus after the electro-sterile treatment, which was recovered by the method for recovering the dead bacteria of the present invention. FIG. 1B is an electron micrograph of another killed Staphylococcus aureus after electrosterilization, which was recovered by the method for recovering killed bacteria of the present invention. FIG. 1C is an electron micrograph of dead Escherichia coli after electrosterilization, which was recovered by the method for recovering dead bacteria of the present invention.

In the method for recovering dead bacteria of the present invention, dead bacteria whose cell structure has been significantly changed (collapsed) due to electric field sterilization or the like are targeted for recovery. Hereinafter, the dead bacteria to be recovered will be described with reference to FIGS. 1 (A) to 1 (C) with reference to the dead bacteria killed by the electric field sterilization treatment as a specific example. However, the present invention is not limited to the dead bacteria that have been killed by the electric field sterilization treatment as long as the dead bacteria have a large change (collapse) in the cell structure.

The killed bacteria after the electric field sterilization treatment have a characteristic shape that is significantly different from the killed bacteria killed by other sterilization methods (for example, heat sterilization and alcohol sterilization) (see FIGS. 1 (A) to 1 (C)). ..
Specifically, the cell surface is pore-shaped with a size of about 0.01 to 0.5 μm and has a protruding structure. In addition, the presence of fold-like protrusions over the entire cell surface is also a characteristic of killed bacteria after electrosterilization. In all of the killed bacteria shown in FIGS. 1 (A) to 1 (C), structures such as these are present throughout the cell surface layer, and the surface is roughened.
Furthermore, most of the killed bacteria after the electric field sterilization treatment are fragmented and pores are present on the cell surface (see FIGS. 1 (A) to 1 (C)). The intracellular substance leaks out of the cell through this pore.
Furthermore, the dead bacteria after the electric field sterilization treatment tend to have a smaller cell diameter than the viable bacteria before the electric field sterilization treatment. For example, the cell diameter of a viable Staphylococcus aureus before the electric field sterilization treatment is about 0.8 to 1.0 μm. On the other hand, the cell diameter of the dead Staphylococcus aureus after the electric field sterilization treatment is about 0.4 to 1.0 μm, and most of them are about 0.4 to 0.5 μm (FIG. 1 (A). )-(C)).

The sterilization conditions for the dead bacteria to be recovered in the present invention are not particularly limited, and can be appropriately set from a conventional method according to the type of food raw material, product, etc., bacterial species, and the like.

The solvent used for preparing the suspension is not particularly limited and can be appropriately selected. For example, Polyoxyethylene Sorbitan Monooleate, Polyoxyethylene sorbitan monolaulate, Polyoxyethylene sorbitan monopalmitate, monopalmitate monostearate. ) And an aqueous solution containing at least one surfactant selected from the group consisting of monostearate monooleate.
Further, it is preferable to appropriately set the concentration of the solvent in order to transfer the killed bacteria to the supernatant after centrifugation. For example, 0.05 to 0.2% by mass is preferable.

Centrifuge the prepared suspension and collect the supernatant. The supernatant contains dead bacteria whose cell shape has changed significantly as described above.
As long as the dead bacteria whose cell shape has changed significantly can be isolated, the conditions for centrifugation are not particularly limited, and the cells can be appropriately set within a range in which the cells are not damaged and collapsed by centrifugation. For example, centrifugation at 7,000 to 15,000 G for 5 to 15 minutes is preferred, 10,000 to 15,000 G for 10 to 15 minutes, and 15,000 G for 15 minutes. This centrifugation condition, in which it is preferable to perform centrifugation while cooling the suspension, is more than the condition in the conventional method (about 10 minutes at 10,000 G) shown in, for example, Japanese Patent Application Laid-Open No. 2008-286591. The degree is large. By adopting such conditions, dead bacteria whose cell shape has changed significantly can be transferred into the supernatant.
In the present invention, the suspension before centrifugation includes dead cells whose cell shape has been significantly changed by electric field sterilization or the like, as well as live bacteria and dead bacteria having a large cell size, cell fragments of microorganisms, and the like. If such a suspension is filtered using a membrane filter described later without centrifuging, clogging easily occurs and the recovery efficiency of dead bacteria is greatly reduced. On the other hand, by centrifuging the suspension before filtering using a membrane filter, many contaminants such as viable cells, dead cells, and microbial cell fragments, which have large cell sizes, can be used as pellets. Can be separated. As a result, filtration using the membrane filter in the subsequent stage can be performed efficiently. Furthermore, by increasing the degree of centrifugation more than the conditions in the conventional method, most of the contaminants such as live bacteria do not remain in the supernatant, and the ability to distinguish between dead bacteria and live bacteria can be improved. Therefore, it is possible to take a clear image of dead bacteria with a small amount of residue.

The supernatant after centrifugation is filtered using a membrane filter, and the dead bacteria in the filtrate are adsorbed on the membrane filter. In the present invention, the pore size of the membrane filter can be appropriately set by comparing the cell sizes before and after the sterilization treatment.
Specifically, the cell diameter of a viable Staphylococcus aureus is about 0.8 to 1.0 μm. On the other hand, when the dead bacteria after the electric field sterilization treatment were observed by SEM, the cell shape was significantly changed, and most of the dead bacteria were about 0.4 to 0.5 μm. Therefore, it is preferable to set the pore size of the membrane filter in consideration that the size of the dead bacterium whose cell shape has changed significantly is about half that of the viable bacterium. Specifically, the pore size of the membrane filter is preferably 0.1 to 0.45 μm.

In the present invention, before filtering using a membrane filter, the dead cells contained in the supernatant whose cell shape has changed significantly are separated from viable cells, dead cells, microbial cell debris, etc., which have a large cell size. In addition, it is preferable to perform dust removal treatment. By performing the dust removal treatment and the filtration using the membrane filter step by step, clogging of the membrane filter can be suppressed and the time required for adsorbing the target dead bacteria on the membrane filter can be shortened.
The dust removal treatment can be performed using a normal filter for dust removal purposes, for example, a depth type filter such as a stoma filter. The filter that can be used for the dust removal treatment blocks the permeation of large residues that could not be removed by centrifugal treatment and some viable bacteria, but the dead bacteria recovered by the present invention with a small particle size can permeate. It can be selected as appropriate.

By going through the above steps, dead bacteria whose cell shape has changed significantly can be adsorbed on the membrane filter. Then, by fixing the adsorbed dead bacteria to the membrane filter and collecting the dead bacteria together with the fixed membrane filter, the desired dead bacteria can be recovered.
Further, in the method for recovering dead bacteria of the present invention, the dead bacteria are recovered together with the membrane filter. Therefore, by observing the membrane filter with an electron microscope such as SEM, it is possible to easily image the desired dead bacteria.

There is no particular limitation on the method of fixing the dead bacteria adsorbed on the membrane filter. For example, a membrane filter on which the target dead bacteria are adsorbed is immersed in a fixing solution such as glutaraldehyde, and the dead bacteria are fixed on the membrane filter. Then, the dewatered aqueous solution is dropped to dehydrate the dead bacteria fixed on the membrane filter. After dehydration, collect the membrane filter.
More specifically, 2% glutaraldehyde is passed through the membrane filter after filtration as pre-fixation (pre-fixation), and then the membrane filter and adsorbed dead bacteria are washed with 100 mM HEPES. After that, the membrane filter is installed so that the surface on which the bacterial cells are adsorbed on the disposable dish is on the top and the dead bacteria are not peeled off as quietly as possible so that the lower surface of the membrane filter does not entrain air. If air is caught in, bleed the air with tweezers as appropriate. Then, 2% glutaraldehyde cooled to 4 ° C. is gently dropped by 4 to 5 mL so as to hit the wall surface of the disposable dish so that the bacteria on the surface of the membrane filter do not come off. Then, fix at 4 ° C for 18 hours or more. At the time of fixing, the membrane filter may float and the cells (dead bacteria) may not be fixed on the membrane filter, so check whether the membrane filter is completely immersed in the fixing solution (glutaraldehyde) at regular intervals. .. If the membrane filter is raised, press the outer periphery of the membrane filter with tweezers and completely immerse it in the fixing liquid. Then, a dehydrated aqueous solution (preferably an ethanol-increasing system) having a concentration group of 50 to 100% by volume was sequentially dropped from a low concentration to the killed bacteria fixed on the membrane filter, and the cells were fixed on the membrane filter. Dehydrate the fungus. After dehydration, collect the membrane filter.

When the suspension is centrifuged, the dead bacteria that are killed by the conventional sterilization method are transferred to pellets. On the other hand, in the method for recovering dead bacteria of the present invention, the killed bacteria are recovered by filtering the supernatant after centrifugation of the suspension and recovering the membrane filter on which the dead bacteria are adsorbed. Therefore, the membrane filter on which the specific dead bacteria are adsorbed and fixed, which is recovered by the method for recovering the dead bacteria of the present invention, can be subjected to imaging with an electron microscope by performing a predetermined treatment. According to the method for imaging dead bacteria of the present invention, it is possible to image dead bacteria that cannot be imaged by the conventional method.
The imaging method using an electron microscope is not particularly limited, and can be performed by a conventional method. Hereinafter, the method of imaging dead bacteria will be specifically described. However, the present invention is not limited to this.
For example, the dehydrated membrane filter is transferred to a disposable dish, completely immersed in a critical point drying solution such as t-butanol (special grade reagent), and frozen. After that, the frozen membrane filter is installed in the critical point dryer together with the disposable dish to perform critical point drying. By immersing the membrane filter in the critical point drying solution before the critical point drying, the cells can be dried while protecting or maintaining the fine structure (flagella, cilia, etc.) of the cell surface layer of the dead bacteria. In the present invention, the critical point drying can be performed by a conventional method. In the present invention, "critical point drying" is a method in which a sample after dehydration is placed in a pressure vessel together with liquefied carbon dioxide gas and heated, and can be performed by a conventional method.
After the critical point dry solution is completely volatilized, the membrane filter is placed on the SEM sample table with the observation surface facing up, and platinum vanadium is vapor-deposited by ion sputtering. After that, the dead bacteria can be imaged by a normal temperature SEM, a cryo (FIB) SEM, or the like.

With respect to the above-described embodiment, the present invention further discloses the following method for recovering dead bacteria and a method for imaging dead bacteria.

<1> The sterilized bacteria are suspended in a solvent, the suspension is centrifuged, the supernatant after centrifugation is filtered using a membrane filter, the dead bacteria are adsorbed on the membrane filter, and the cells die on the membrane filter. A method for recovering dead bacteria, in which the bacteria are fixed and the dead bacteria are collected together with the membrane filter.

<2> The sterilized bacteria are suspended in a solvent, the suspension is centrifuged, the supernatant after centrifugation is filtered using a membrane filter, the dead bacteria are adsorbed on the membrane filter, and the cells die on the membrane filter. Bacteria are fixed, dead bacteria are collected together with the membrane filter, the recovered membrane filter is immersed in a critical point drying solution, the membrane filter is frozen, the frozen membrane filter is dried at the critical point, and the membrane filter is dried at the critical point. A method for imaging dead bacteria, which is used for imaging using an electron microscope.
<3> The method according to <1> or <2> above, wherein the critical point dry solution is t-butanol.

<4> The membrane filter is treated with a fixing solution to fix the target dead bacteria, the dead bacteria fixed on the membrane filter are dehydrated using a dewatering aqueous solution, and the dead bacteria are collected together with the membrane filter. The method according to any one of <3>.
<5> The above item 1 of <1> to <4>, wherein the supernatant after centrifugation is subjected to dust removal treatment, and the supernatant after dust removal treatment is filtered using the membrane filter. the method of.
<6> The method according to <5>, wherein the dust removal treatment is performed using a depth type filter.
<7> The above-mentioned <5> or <6>, in which the dead cells whose cell shape contained in the supernatant is significantly changed by the dust removal treatment is separated from the viable and dead cells having a large cell diameter, cell fragments of microorganisms, and the like. > The method described in the section.
<8> The method according to any one of <1> to <7>, wherein the pore size of the membrane filter is 0.1 to 0.45 μm.
<9> Any of the above <1> to <8>, wherein the suspension is centrifuged at 7000 to 15000 G for 5 to 15 minutes, preferably 10000 to 15000 G for 10 to 15 minutes, and more preferably 15000 G for 15 minutes. The method described in item 1.
<10> The solvent is selected from the group consisting of polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, monopalmitate monostealto and monostealto monooleart. The method according to any one of <1> to <9>, which is an aqueous solution containing at least one surfactant.
<11> The method according to any one of <1> to <10>, wherein the concentration of the solvent is 0.05 to 0.2% by mass.
<12> The item according to any one of <1> to <11>, wherein the shape of the dead bacteria is significantly different from the shape of the dead bacteria killed by another sterilization method (preferably heat sterilization or alcohol sterilization). Method.
<13> The method according to any one of <1> to <12>, wherein the killed bacterium is a roughened killed bacterium having a cell surface in the form of pores and having a protrusion structure on the cell surface.
<14> The method according to <13>, wherein the size of the pores on the cell surface of the killed bacterium is about 0.01 to 0.5 μm.
<15> The method according to any one of <1> to <14>, wherein the killed bacterium has pleated protrusions on the cell surface.
<16> The method according to any one of <13> to <15>, wherein the killed bacterial pore-like and protrusion structure and / or fold-like protrusions are present over the entire cell surface layer.
<17> The method according to any one of <1> to <16>, wherein the cells are fragmented by the sterilization treatment and pores are present on the cell surface layer.
<18> The method according to <17>, wherein the intracellular substance of the killed bacterium leaks out of the cell from the pore.
<19> The method according to any one of <1> to <18>, wherein the cell diameter of the dead bacterium is about 0.4 to 1.0 μm, preferably about 0.4 to 0.5 μm.
<20> The method according to any one of <1> to <19>, wherein the killed bacterium is a killed bacterium killed by an electric field sterilization treatment.

Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited thereto.

(1) Preparation of test bacteria Staphylococcus aureus NBRC 12732 and Escherichia coli ATCC 8739 were each inoculated into 10 mL of liquid medium (nutrient broth) in 1 loopful volume, and cultured with shaking at 37 ° C. and 120 rpm for 24 hours. Then, 100 μL of the culture solution was inoculated into 10 mL of a liquid medium (nutrient broth), and shake culture (main culture) was carried out at 37 ° C. and 120 rpm for 2 to 4 hours.
The bacterial solution was prepared by adjusting the bacterial count to 1 to 3 × 10 ⁵ CFU / mL using nutrient broth obtained by diluting the main culture bacterial solution 20 times.

(2) Sterilization test When a knit made of piezoelectric polylactic acid is stretched, an electric field is generated. It is known that this electric field can kill bacteria.
A knit made of piezoelectric polylactic acid was placed on a pulling machine with a load of 150 g. After installation, 200 μL of the bacterial solution was inoculated on a 10 to 15 spot test cloth and a normal cloth designated by JIS L 1902. After that, the piezoelectric polylactic acid knit was cultured with a pulling machine at a stroke of 10 mm, and the ordinary cloth was cultured at 37 ° C. for 18 hours under static conditions.
For each test cloth after culturing, the bacteria on the cloth were collected using an extract, and the viable cell count was measured by the culture method.

(3) Recovery of dead bacteria
The bacteria after the electric field sterilization treatment were suspended in 20 mL of a 0.2-0.05 mass% Tween80 (polyoxyethylene sorbitan monooleate) aqueous solution. Then, using a high-speed cooling centrifuge (Yamato Kagaku) and an angle rotor F-35-6-30, the suspension was centrifuged at 15000 G for 15 minutes while cooling at 4 ° C.
The supernatant after centrifugation was collected, filtered with a stoma filter (AS ONE), and dust-removed. Then, the whole amount was filtered using an isopore GTTP04700 having a pore size of 0.2 μm and a disposable syringe, and the dead bacteria were adsorbed on the membrane filter.
After filtration, 1 mL of 0.1 MHEPES was passed through the membrane filter to wash the membrane filter, and 1 mL of 2% glutaraldehyde was passed through the membrane filter for pre-fixation. The membrane filter after passing the liquid was installed in a disposable dish so that the filtration surface was on top. 4 mL of 2% glutaraldehyde was dropped on the wall surface of the disposable dish so that the fixative would not come into direct contact with the membrane filter, and the membrane filter was fixed at 4 ° C for 18 hours while being pressed with tweezers so that it would not come out.
After that, after collecting and discarding the entire amount of the fixative, 30 each with an ethanol rising system (50, 70, 80, 90, 95, 100 (1st time), 100 (2nd time)%) while avoiding direct contact with the filter surface. Dehydrated at 4 ° C for a minute.

(4) Imaging of dead bacteria The membrane filter after dehydration treatment was completely immersed in 4 ml of t-butanol and frozen at -20 ° C for 30 minutes or more together with the disposable dish. After freezing, the disposable dish and the membrane filter were dried at the critical point using the critical point dryer ES-2030 (Hitachi). The dried membrane filter was fixed on an SEM sample table (Nisshin EM) and platinum-deposited at 30 mA for 80 seconds using an ion sputter E-1030 (Hitachi). The membrane filter after platinum deposition was observed and photographed using SEM S-4300E (Hitachi).

The results are shown in FIG. As shown in FIG. 1, in the bacteria after electrosterilization collected from the supernatant after centrifugation, cells are fragmented regardless of whether they are gram-positive or gram-negative. In addition, the cell surface is roughened, and fold-like structures (projections) are exposed so as to cover the cells. Furthermore, pores on the cell surface layer, which are not observed in viable bacteria, are observed, and intracellular substances are leaked to the outside of the cell. The fold-like structure of the cell surface is in a raised state as compared with other parts, and exists as a cytoskeleton-like structure.
Furthermore, the dead Staphylococcus aureus no longer has tufted connections of grapes (see FIGS. 1A and 1B).

As described above, according to the present invention, it is possible to recover dead bacteria that have been killed by electric field sterilization or the like, which could not be collected by the conventional method.
Further, since the target dead bacteria are collected together with the adsorbed and fixed membrane filter, the target dead bacteria can be easily imaged by subjecting the collected membrane filter to imaging with an electron microscope.

Claims (13)

Suspend the bacteria after electrosterilization in a solvent and
Centrifuge the suspension and
By filtering the supernatant after centrifugation using a membrane filter, dead bacteria killed by electric field sterilization are adsorbed on the membrane filter.
Fix the dead bacteria on the membrane filter and
Collect the fixed killed bacteria together with the membrane filter.
A method for recovering dead bacteria that have been killed by electric field sterilization . Suspend the bacteria after electrosterilization in a solvent and
Centrifuge the suspension and
By filtering the supernatant after centrifugation using a membrane filter, dead bacteria killed by electric field sterilization are adsorbed on the membrane filter.
Fix the dead bacteria on the membrane filter and
The fixed killed bacteria were collected together with the membrane filter and collected.
Immerse the recovered membrane filter in a critical point drying solution.
Freeze the membrane filter and
Dry the frozen membrane filter to a critical point and
The membrane filter dried at the critical point is used for imaging using an electron microscope.
A method of imaging dead bacteria killed by electric field sterilization . The method according to claim 2, wherein the critical point dry solution is t-butanol. Any one of claims 1 to 3, wherein the membrane filter is treated with a fixing solution to fix the target dead bacteria, the dead bacteria fixed on the membrane filter are dehydrated using a dewatering solution, and the dead bacteria are collected together with the membrane filter. The method described in item 1. The method according to any one of claims 1 to 4, wherein the supernatant after centrifugation is subjected to a dust removal treatment, and the supernatant after the dust removal treatment is filtered using the membrane filter. The method according to any one of claims 1 to 5, wherein the pore size of the membrane filter is 0.1 to 0.45 μm. The method according to any one of claims 1 to 6, wherein the suspension is centrifuged at 7000 to 15000 G for 5 to 15 minutes. The method according to any one of claims 1 to 7, wherein the killed bacterium is a roughened killed bacterium having a pore-like cell surface and a protruding structure on the cell surface. The method according to claim 8, wherein the size of the pores on the cell surface of the killed bacterium is about 0.01 to 0.5 μm. The method according to any one of claims 1 to 9, wherein the killed bacterium has pleated protrusions on the cell surface. The method according to any one of claims 8 to 10, wherein the pore-like and protrusion-like structure and / or fold-like protrusion of the dead bacterium is present over the entire cell surface layer. The method according to any one of claims 1 to 11, wherein the cells are fragmented by the electric field sterilization treatment and pores are present on the cell surface layer. The method according to any one of claims 1 to 12, wherein the cell diameter of the killed bacterium is about 0.4 to 1.0 μm.

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