KR20200079713A - Agent simulant composition of gram positive bacterial and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a gram-positive bacterial agent simulant composition to provide samples for a military experiment and training including a nontoxic and pathogen-free strain composition, and a manufacturing method thereof. According to the present invention, the gram-positive bacterial agent simulant composition comprises at least one strain selected from a group consisting of Bacillus atrophaeus, Bacillus subtilis (KCTC 1028), and Bacillus thuringiensis (KCTC 3452), wherein the number of live bacteria of the strain is 1.0x10^12 cfu/ml or less and is a spore or a nurse cell in terms of morphology.

Description

Gram-positive bacteria mock agonist composition and method for manufacturing the same {AGENT SIMULANT COMPOSITION OF GRAM POSITIVE BACTERIAL AND MANUFACTURING METHOD THEREOF}

The present invention relates to a composition for simulating Gram-positive bacteria and a method for manufacturing the same.

At a time when biological warfare and the threat of terrorism are rapidly increasing due to the spread of biological weapons worldwide, it is absolutely necessary to establish a protection system for biological weapons to minimize human damage from attacks by biological agents. Currently, there is an increasing need for an early detection and response system for the use of terrorism and war on biological weapons worldwide, and the anti-corrosion prediction and timely response to pollution and other timely reactions to North Korea's nuclear asymmetric power. Management system development is needed. To this end, standards for bio-weapon detection and identification verification necessary for the identification of the type, origin and existence of biological agents such as viruses, bacteria, and toxins are developed, and it is necessary to progress production standardization. In addition, the standardized high concentration for the establishment of battlefield management system such as electric power transfer in the battlefield and the simulation of related biological explorers through the test of extinction characteristics in the environment using a custom-made similar biologic agent with similar genetic origin of the bioagent. There is a need for a method of manufacturing a mock agent.

Of the microorganisms used as biological simulation agents, Bacillus and Clostridium microorganisms form spores under certain conditions, which are the most difficult to destroy among all living organisms and continue to survive under adverse conditions. It is said to be possible. High resistance to stress such as ultraviolet rays, heat of about 100°C, and drying, which most common microorganisms kill, requires strong special treatment such as autoclaving to effectively kill these spores.

When used as a biological pesticide or mock agent, Bacillus thuringiensis is usually sprayed outdoors in the form of spores or used in a laboratory. As mentioned earlier, spores are the most difficult to destroy among the forms of living organisms. In the case of unintentionally leaking outdoors, it has a problem that it persists for a very long time in nature.

The gram-positive biological agent of the present invention belongs to the category A biological agent category A defined by the American Centers for Diseases Control and Prevention (CDC) (Non-patent Document 0001). Bacillus atropeus is currently used as a gram-positive mock-anthropogenic mock-acting agent, and has already been used abroad as a mock-acting agent, but it is very expensive and difficult to mass-produce and use (Non-Patent Document 0002) 1×10 ¹⁰ There is an increasing demand for products containing high purity and high concentrations of live bacteria over cfu/g.

Therefore, in order to use as a mock agent for evaluating the environmental properties of biological agents, microorganisms capable of realizing the properties of morphological, molecular biological, and morphological real agents must be selected, and designed and manufactured for mass production with selected mock agents It is necessary to establish an optimal manufacturing process through standardization, etc.

Centers for Disease Control and Prevention (CDC). (2014) Bioterrorism Agents/Deseases. Forney, T.L. Et al. (1997). Evaluation of Erwinia herbicola as a surrogate biological warfare agent (BW) aerosol. Batelle memorial institute. Columbus, OH. Ana M. et al. (2009). Assessment of Bioaerosol generation and sampling efficency based on Pantoea agglomerans. Aerosol Science and Technology. 43:620-628.

The present invention is to solve the above-mentioned problems, the object of the present invention, Bacillus atrophus (Bacillus atrophaeus), Bacillus subtilis (Bacillus subtilis, KCTC 1028) and Bacillus thuringiensis (Bacillus thuringiensis, KCTC 3452 ) Is a composition comprising one or more strains selected from the group consisting of, wherein the number of viable cells of the strain is 1.0×10 ¹² cfu/ml or less, to provide a gram-positive fungus simulation agent composition.

However, the problems to be solved by the present invention are not limited to those mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

Gram-positive bacteria mock agonist composition according to an embodiment of the present invention, consisting of Bacillus atrophaeus (Bacillus atrophaeus), Bacillus subtilis (Bacillus subtilis, KCTC 1028) and Bacillus thuringiensis (Kacc 3452) As a composition comprising a strain comprising at least one selected from the group, the number of viable cells of the strain is 1.0×10 ¹² cfu/ml or less, and is morphologically spores or feeder cells.

According to an embodiment of the present invention, the gram-positive bacteria mock agonist composition may be a lyophilized powder or a liquid suspension.

According to an embodiment of another aspect of the present invention, a method for preparing a gram-positive bacteria mock agonist composition comprises: selecting an anthracnose mock agonist candidate strain, culturing the selected strain, and after culturing, separating the cells Obtaining, washing and suspending the cells, concentrating the washed cells, and performing lyophilization of the concentrated cells, liquid suspension, or both.

According to an embodiment of the present invention, the step of culturing the selected strain may include a pure culture step, a species culture step, and a main culture step in a liquid medium.

According to one embodiment of the invention, the liquid medium, TSB (tryptic soy broth) medium, LB (Luria-Bertani broth) medium, NB (Nutrient broth) medium and YM (Marine broth and 0.5% tryptone mixed) medium It may be any one selected from the group consisting of.

According to an embodiment of the present invention, the culture temperature and the stirring speed in the step of culturing the selected strain may be 28 ℃ to 32 ℃ and 150 rpm to 250 rpm.

According to an embodiment of the present invention, in the step of culturing the selected strain, the pH of the culture solution may be 7.0 to 7.5.

According to an embodiment of the present invention, the washing solution in the step of washing and suspending the cells may include distilled water, saline (Saline, 0.85% NaCl), TBS, PBS, or a mixture of two or more thereof. .

According to an embodiment of the present invention, the lyophilization may be performed by using an excipient comprising trehalose, sucrose or lactose at a concentration of 4% to 6%.

According to one embodiment of the present invention, the excipient may be a 5% concentration of trehalose (Trehalose) having a cell recovery rate of 70% or more.

According to an embodiment of the present invention, the liquid suspension may be performed using saline (Saline, 0.85% NaCl) or distilled water.

The present invention, a composition comprising at least one strain selected from the group consisting of Bacillus atrophaeus, Bacillus subtilis (KCTC 1028) and Bacillus thuringiensis (KCTC 3452) As, the number of viable cells of the strain is 1.0×10 ¹² cfu/ml or less, and morphologically may be spores or feeder cells, to provide a composition for simulating Gram-positive bacteria.

More specifically, it is possible to mass-produce a bacterial composition having a genetically close relationship with a biological agent and having no toxicity and pathogenicity, thereby providing a sample for military experiment and training including the toxic and pathogenic strain composition.

1 is a flow chart showing a method for preparing a military test and training sample according to the present invention.
2 is a graph showing the absorbance (OD) value when the mock agent strains are cultured in different media.
3 is a graph showing the OD value when the mock agent strains are cultured at different temperatures.
4 is a graph showing the OD value when the mock strains are cultured at different stirring speeds.
5 is a graph showing changes in acidity, dissolved oxygen and cell mass when culturing a mock strain.
Figure 6 is a graph showing the viable cell yield when the mock agent strains are washed with different washing solutions.
7 is a graph showing the viable cell yield when the mock agent strains are lyophilized using side solutions.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, various changes may be made to the embodiments, and the scope of the patent application right is not limited or limited by these embodiments. It should be understood that all modifications, equivalents, or substitutes for the embodiments are included in the scope of rights.

The terms used in the examples are for illustrative purposes only and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly indicates otherwise.

Throughout the specification, when one member is positioned "on" another member, this includes not only the case where one member is in contact with the other member, but also the case where another member is present between the two members. When an element or layer is indicated as being “on”, “connected to”, or “coupled to” another element or layer, it is directly a different component or layer. It can be understood that it can be in, can be connected to, can be combined, or there can be intervening elements and layers.

Throughout the specification, when a part "includes" a component, it does not exclude other components, but means that other components can be further included, such as "include" or "have". The term is intended to indicate that a feature, number, step, operation, component, part, or combination thereof described on the specification exists, and that one or more other features or numbers, steps, operation, component, part, or combination thereof. It should be understood that it does not preclude the existence or addition possibility of ones.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person skilled in the art to which the embodiment belongs. Terms, such as those defined in a commonly used dictionary, should be interpreted as having meanings consistent with meanings in the context of related technologies, and should not be interpreted as ideal or excessively formal meanings unless explicitly defined in the present application. Does not.

In addition, in the description with reference to the accompanying drawings, the same reference numerals are assigned to the same components regardless of reference numerals, and redundant descriptions thereof will be omitted. In describing the embodiments, when it is determined that detailed descriptions of related well-known technologies may unnecessarily obscure the subject matter of the embodiments, detailed descriptions thereof will be omitted.

Hereinafter, the gram-positive bacteria mock agonist composition of the present invention will be described in detail with reference to examples and drawings. However, the present invention is not limited to these examples and drawings.

Hereinafter, the present invention will be described in more detail by examples and comparative examples.

However, the following examples are only for illustrating the present invention, and the contents of the present invention are not limited to the following examples.

Gram-positive bacteria mock agonist composition according to an embodiment of the present invention, consisting of Bacillus atrophaeus (Bacillus atrophaeus), Bacillus subtilis (Bacillus subtilis, KCTC 1028) and Bacillus thuringiensis (Kacc 3452) As a composition comprising at least one strain selected from the group, the number of viable cells of the strain is 1.0×10 ¹² cfu/ml or less, and includes morphologically spores or feeder cells.

According to one side, the gram-positive bacteria mock agonist composition is a Gram-positive bacterium of the genus Bacillus, which is harmless to the human body and has been widely used worldwide as a bio pesticide for harmful insects due to its insecticidal effect. There is no harm to the human body, but the size of anthrax and spores are similar, and various properties are preferable as a simulant of anthrax, so it may be a simulated anthrax for use in the biodefense field in the United States and the United Kingdom.

According to one side, “mock agonist” as used herein refers to a highly enriched microbial viable or dead cell using a safe strain that has no toxicity and pathogenicity compared to a real biological agonist, and a similar biological agonist is used as a synonym for the mock agonist. Can.

According to one side, the strains of the genus Bacillus may be spore-producing strains, such as Bacillus subtilis, Bacillus thuringensis, Bacillus atropeus, and preferably Bacillus atropeus, Bacillus. It may include one or more selected from the group consisting of subtilis and Bacillus thuringensis.

According to one side, spores used in the present specification may have the same meaning as endospores formed in microorganisms so as to have resistance in extreme environments such as high temperature, low temperature, ultraviolet light, drying and high pressure.

According to one side, the Bacillus thuringiensis (Bacillus thuringiensis, KCTC 3452) is a Gram-positive bacterium of the genus Bacillus, which is harmless to the human body and may produce an insecticidal effect on Cry protein produced during spore formation.

According to one side, the genome information of the Bacillus thuringensis strain may be a GenBank CP001903.1 sequence known from GenBank of the National Center for Biotechnology Information (NCBI), a known database.

According to one side, at least 700,000 when spraying dozens of Kg in Seoul using electric power projection systems such as missiles or radiation guns because of the absence or absence of vaccines or therapeutics in Korea, in relation to biological weapons including anthrax Deaths may occur, and the extent of casualties due to secondary infection is unpredictable. The characteristics, treatment and decontamination methods of biological weapons possessed by North Korea are shown in Table 1 below.

Biological weapons Characteristic Treatment and decontamination Anthrax
(anthrax) -Anthrax (Bacillus anthracis) infection
-Respiratory type, skin type, digestive type
-Respiratory malformations are cold-like symptoms such as initial fever and cough, and afterwards, severe symptoms such as dyspnea and discoloration occur within 2-3 days. Most deadly with infections above 90%.
-The skin type has a mortality rate of 20% of the swelling and discoloration of the skin, and the digestive type has a mortality rate of 25-60% with symptoms such as pain and hemorrhage diarrhea. -Antibiotic treatment: High concentration of ciprofloxacin antibiotics and antidote should be administered for more than 60 days.

According to an embodiment of the present invention, the size of the strain may be 1 x 3 μm to 1.5 x 5 μm.

According to one side, in the case of strains conforming to the size, it may be most suitable as a mock agent.

According to an embodiment of the present invention, the biological safety level of the strain may be that of BSL-1.

According to one side, the biological safety grade BSL-1 is a grade determined to be harmless to the human body, and in general, a strain used in the present invention, unlike a strain having a BSL grade of about 3 in biological agents is used. Can also be used in microbiological laboratories to improve fitness for military training.

According to one side, in the case of the above BSL-3 class, human-to-human transmission is a faint and easy-to-treat microorganism, but it causes disease to humans or animals, requires complete containment, restricts access to hallways, and is a high-performance filter It may be necessary.

According to an embodiment of the present invention, the shape of the strain may be a rod.

According to one side, the shape of the strain may be an elongated rod shape, a chain rod shape arranged in a row like a chain, a spiral, etc., but the shape of the strain most suitable for the mock agent composition is an elongated membrane It may be of a rod type.

According to an embodiment of the present invention, the gram-positive bacteria mock agonist composition may be a lyophilized powder or a liquid suspension.

According to one side, the Gram-positive bacteria mock agonist composition may be a lyophilized powder or a liquid suspension, and in particular may further contain a modifying additive for commercialization of the lyophilized powder form, in the case of a liquid suspension It may be in the form of diluting the freeze-dried powder by a serial dilution method after making a suspension in saline or the like.

According to one side, the gram-positive bacteria mock agonist composition may be in a pure form concentrated to a high concentration after washing steps in both lyophilized powder or liquid suspension.

According to an embodiment of another aspect of the present invention, a method for preparing a gram-positive bacteria mock agonist composition comprises: selecting an anthracnose mock agonist candidate strain, culturing the selected strain, and after culturing, separating the cells Obtaining, washing and suspending the cells, concentrating the washed cells, and performing lyophilization of the concentrated cells, liquid suspension, or both.

According to an embodiment of the present invention, the step of culturing the selected strain may include a pure culture step, a species culture step, and a main culture step in a liquid medium.

According to one side, the pure culture step, the species culture step and the main culture step, may include a step of performing a culture of the culture and the culture medium for the pure cultured strain.

According to one side, the main culture step in the liquid medium may be performed twice in the same medium.

According to one side, the pure culture step may mean culturing only one type of microorganism, and using a smear plate culture method or an injection plate method, the microorganisms in the sample may form independent colonies, respectively. In order to find a colony of microorganisms, a pure separation step may be performed.

According to one side, the pure culture step, the colony shape and size may optionally include strains to further include Gram characteristics, cell shape, oxidase test, catalase test, and endogenous spore formation.

According to one side, in the culture step of Bacillus bacteria, identification of Bacillus bacteria can be performed by a gene analysis method that analyzes the gene sequence of cells, and Gram-positive endogenous spores among strains showing typical Bacillus colony patterns in Bacillus medium. Rule-forming bacteria may be purely cultured.

According to one side, the seed culture and the main culture can be carried out through known conventional medium and culture conditions. Preferably, in the seed culture, a colony with high growth rate and high productivity is collected, and then, The cells of the superior colonies collected in the step of culturing the species can be activated several times, and the activated cell culture medium can be cultured by inoculating the main culture medium.

According to one embodiment of the invention, the liquid medium, TSB (tryptic soy broth) medium, LB (Luria-Bertani broth) medium, NB (Nutrient broth) medium and YM (Marine broth and 0.5% tryptone mixed) medium It may be any one selected from the group consisting of.

According to one side, the liquid medium is not particularly limited as long as it is a liquid medium for culturing a bacterial agent and is usually used for culturing bacterial microorganisms. Preferably, tryptic soy broth (TSB) medium, LB (Luria- Bertani broth) medium, NB (Nutrient broth) medium and YM (Marine broth mixed with 0.5% tryptone) medium may be any one selected from the group consisting of, and most preferably, TSB (tryptic soy broth) medium days Can.

According to one side, in order to select the most suitable medium among the liquid medium, after activating the strain, incubate for 1 day at 37°C in TSB, NB, LB, YM liquid medium, and measure the OD value of the culture solution for each strain By selecting the strain having the best cell growth, the OD value of the culture medium for each medium can be measured to select a smooth medium with the best cell growth.

According to one side, the liquid medium can be used to cultivate feeder cells in a culture period of 1 to 3 days and spore cells in a culture period of 5 to 7 days using the liquid medium.

According to one side, the cells included in the gram-positive bacteria mock agonist composition may be prepared in both nutrient cells and spore cells, respectively.

According to one side, the feeder cell means a cell that actively metabolizes and proliferates, and the spore cell may be an endogenous spore occurring in the feeder cell.

According to one side, the spore cells may be used as a survival means to endure difficult times when the nutrients are depleted or the environment is difficult to survive or grow.

According to an embodiment of the present invention, the culture temperature and the stirring speed in the step of culturing the selected strain may be 28 ℃ to 32 ℃ and 150 rpm to 250 rpm.

According to one side, in the culture step, the step of obtaining only by separating the cells may proceed, wherein the separation of the cells may be performed through centrifugation, and may be performed in a sterile state in consideration of cross contamination from the outside. have.

According to one side, in order to confirm the culture temperature, a variety of culture temperatures can be administered at the same time and under the same conditions by administering a predetermined inoculation amount in an Erlenmeyer flask, and as a result, preferably 28°C to 32°C, Most preferably, the culture at 30° C. may be most suitable.

FIG. 3 is a graph showing OD values when the mock strains are cultured at different temperatures. Referring to FIG. 3, in a specific medium condition, various culture temperatures are inoculated with 1% inoculation in several 100 ml volumes of Erlenmeyer flasks (v /v) as a result of incubation at the same condition for 24 hours, the OD (600 nm) value at 30°C can be confirmed to be closest to 1.5.

According to an embodiment of the present invention, in the step of culturing the selected strain, the pH of the culture solution may be 7.0 to 7.5.

According to one side, within the pH range, culture optimization may be achieved, and if it is outside the corresponding numerical range corresponding to neutrality, the cells in the culture medium may be killed by acidity or basicity.

According to an embodiment of the present invention, the washing solution in the step of washing and suspending the cells may include distilled water, saline (Saline, 0.85% NaCl), TBS, PBS, or a mixture of two or more thereof. .

According to one side, the washing and suspension solution may be used to obtain a microbial cell from the culture medium through centrifugation of the main culture medium and to not degrade the number of live cells while washing foreign substances other than cells.

According to one side, the washing and suspending solution is not particularly limited, but preferably, may include distilled water, saline (Saline, 0.85% NaCl), TBS, PBS, or a mixture of two or more thereof, Most preferably, when using saline (Saline, 0.85% NaCl), it is possible to obtain the purest cells and not degrade the viable cells.

According to an embodiment of the present invention, the lyophilization may be performed by using an excipient comprising trehalose, sucrose or lactose at a concentration of 4% to 6%.

According to one side, the excipient may be a crystallization excipient used in a lyophilized product, and is not particularly limited, but is preferably of Trehalose, Sucrose or Lactose. It can be a disaccharide.

According to one side, with respect to the above excipients, bulk additives such as saccharides and polyols used to increase the distance of protein killing to prevent agglomeration should not be easily re-allocated or remain amorphous in the solid state, especially in the case of reducing sugars. May react with proteins and may not be suitable as excipients.

According to one side, the disaccharide used as the excipient may preferably have a concentration of 4% to 6%, and the excipients within the concentration range, unlike conventional stabilizers, stabilizers reduce the loss of activity upon freeze drying And is used to increase the activity recovery during restoration, it can be suitably used as an excipient for the purpose of dilution or increase.

According to one embodiment of the present invention, the excipient may be a 5% concentration of trehalose (Trehalose) having a cell recovery rate of 70% or more.

According to one side, the excipient may most preferably be trehalose, and trehalose is more stable, and furthermore, it can be most effective when bacteria can live and reach the effect without dying in the stomach. It can be very effective because it shows a recovery rate of 70% or more compared to a conventional excipient having a cell recovery rate of less than 70%.

According to an embodiment of the present invention, the liquid suspension may be performed using saline (Saline, 0.85% NaCl) or distilled water.

According to one side, the liquid suspension in the saline or distilled water may be most efficient in not degrading the viable cell count while washing foreign substances other than cells.

Hereinafter, the present invention will be described in more detail by examples and comparative examples.

However, the following examples are only for illustrating the present invention, and the contents of the present invention are not limited to the following examples.

Example. Preparation of Gram-positive bacteria mock agonist composition

One. Selection of candidate agents for mock agonists

Based on the biological properties and morphological and genetic similarities with the anthrax actual agent, a candidate group of simulated agents for each actual agent was selected. In addition to these criteria, there is a possibility of mass production due to excellent growth of bacteria through culture. Stable strains with low biosafety levels with non-pathogenic bacteria with biological stability were selected as priority. The results are shown in Table 2 below.

Real agent Mock agent candidate strain Selection Anthrax
Bacillus anthracis KCTC 3701 Bacillus atrophaeus KCTC 3452 Bacillus thuringiensis KCTC 1028 Bacillus subtilis O

2. Selection of excellent strains and optimal media

Candidate strains of three different types of Gram-positive bacteria mock agonists are biological and morphological similarities and mock agonists, based on biological stability. ).

The selection criteria are shown in Table 3 and Table 4 below.

division Biological agents Mock agonist (candidate) Bacillus anthracis Bacillus atrophaeus
(KCTC 3701) Bacillus thuringiensis
(KCTC 3452) Bacillus
subtilis
(KCTC 1028) Gram stain + + + + Shape rod rod rod rod Motility - - - - Spore + + + + Size 1~1.5 x 3~5 ㎛
(spore: 1~1.5 ㎛) 1~1.2 X 3~5 ㎛ 1 X 3 μm 1~1.5 X 3~5 ㎛ Air condition Aerobic Aerobic Aerobic Aerobic BSL rating 3 One One One Type strain ○ ○

Biological agents Mock agonist Bacillus anthracis
(Anthrax) Bacillus atrophaeus
(KCTC 3701) Bacillus thuringiensis
(KCTC 3452) Bacillus subtilis
(KCTC 1028)

After activating the strain, it was incubated for 1 day at 37°C in TSB, NB, LB, YM liquid medium. Bacillus subtilis (KCTC1028), the strain having the best cell growth, was selected by measuring the OD value of the culture medium for each strain as shown in FIG. 2 below, which is advantageously applied to mass production. Proceeded.

By measuring the O.D value of the culture medium for each medium of Bacillus subtilis (KCTC1028), the smooth medium having the best cell growth was TSB (BD, USA) medium.

As a result, strains of KCTC 1028 purchased from KCTC were selected, and cultured using TSB medium using selected KCTC 1028 strains from the subsequent culture.

3. Optimizing the culture of selected Gram-positive bacteria mock agents

In the excellent medium conditions obtained in step 1 (1), first, various culture temperatures such as 23°C, 30°C, and 37°C were cultured in the same condition of 24 hours at an inoculation amount of 1% (v/v) in a few 100 ml volumes of Erlenmeyer flasks. As a result, as shown in Fig. 3, the optimum culture temperature of this strain was 30°C.

In addition, as a result of culturing at 150 rpm, 200 rpm, 250 rpm under the same culture conditions for 24 hours at a culture temperature of 30° C. and an inoculation amount of 1% (v/v) at an Erlenmeyer flask 100 ml volume, as shown in FIG. 4 below It was found that the optimum stirring speed of the strain was 200 rpm.

4. Test culture of selected Gram-positive bacteria mock agonist

The strains of Bacillus subtilis KCTC 1028, which were activated by culturing TSB nutrient agar in pure water for 1 day, were applied to the 5 L culture tank (Korea fermenter) for optimal culture conditions of the flasks in steps 2 (2) and 3 (3) above. And cultured (see FIG. 5 below).

The culture conditions are the optimum culture conditions identified in Step 1 (1) and Step 2 (2) above, and the culture temperature is 30°C, the initial pH is 7.2, and 3% of TSB medium (BD company) 3% (v/v) of the culture medium. ), and incubation conditions such as 0.5 vvm aeration.

After incubation for 24 hours, a part of the culture solution was collected, and the number of viable cells in the culture solution was analyzed using a step dilution plate plating method.

As a result, the number of live bacteria Bacillus subtilis KCTC 1028, which is a gram-positive bacterium simulation agent, showed excellent number of live bacteria of 1×10 ⁹ cfu/ml.

5. Optimization of the washing process for the selected Gram-positive bacteria mock agent

Gram-positive bacteria simulated by the method of step 4 (4) Bacillus subtilis KCTC 1028 strain Acquired the cells from the culture medium by centrifugation and washed with saline as a washing solution so as not to degrade the viable cell count while washing foreign substances other than cells (Saline , 0.85% NaCl), TBS, PBS, etc., were used.

Among them, the saline solution showed the highest viable cell count (see FIG. 6 below).

6. Optimization of selection of freeze-dried excipients for Gram-positive bacteria

After lyophilization, lyophilization was performed using various saccharides such as Trehalose, Sucrose, and Lactose to select excipients showing a high number of live bacteria, and then the number of live bacteria of the live bacteria was analyzed.

Bacillus subtilis KCTC 1028 cells obtained through cultivation and washing in the previous 3 steps (3) and 4 steps (4) were suspended and uniformly suspended in a cell suspension after 100 ml of an excipient solution using 5% of each saccharide was sterilized. After the same amount of 10 ml was bottled in a 20 ml glass vial prepared for washing and sterilization, it was frozen in an ultra-low temperature freezer at -70 °C (-70 °C deep freezer), and then freeze-dried for 3 days.

The lyophilisate in the glass vial thus obtained was suspended by injecting 10 ml of 0.85% NaCl (Saline solution) as a diluent for analysis of viable cell count, and then the viable cell count for each excipient was measured using a step dilution plate plating method.

Among them, trehalose showed a cell recovery rate of 73.8% compared to other excipients (see FIG. 7 below).

Here, the cell recovery rate is the number of living cells after lyophilization compared to the number of living cells in a solution in which cells are suspended in an excipient solution before lyophilization, and means the recovery rate. As a result, the number of viable cells of the anthracnose sham freeze-dried preparation of the KCTC 1028 strain using the culture according to the process of the above Example and the trehalose after the manufacturing process as an excipient is 1.8×10 ¹⁰ cfu/g, and a high concentration of 10 ¹⁰ cfu/g or more. A mock drying agent was prepared.

As described above, although the embodiments have been described by a limited embodiment and drawings, those skilled in the art can make various modifications and variations from the above description. For example, even if the described techniques are performed in a different order than the described method, and/or the described components are combined or combined in a different form from the described method, or replaced or replaced by another component or equivalent Appropriate results can be achieved. Therefore, other implementations, other embodiments, and equivalents to the claims are also within the scope of the following claims.

Claims (11)

A composition comprising at least one strain selected from the group consisting of Bacillus atrophaeus, Bacillus subtilis (KCTC 1028) and Bacillus thuringiensis (KCTC 3452),
The number of viable cells of the strain is 1.0×10 ¹² cfu/ml or less, and morphologically is spores or feeder cells.
Gram-positive bacteria mock agent composition. According to claim 1,
The gram-positive bacteria mock agent composition is a lyophilized powder or a liquid suspension.
Gram-positive bacteria mock agent composition. Selecting an anthracnose mock candidate strain;
Culturing the selected strain;
After the culture, the step of separating the cells to obtain;
Washing and suspending the cells;
Concentrating the washed cells; And
Lyophilizing the concentrated cells, performing liquid suspension or both;
Containing,
Method for producing gram-positive bacteria mock agent composition. According to claim 3,
The step of culturing the selected strain,
Pure culture step;
Species culture step; And
Culturing as seen in liquid medium;
That includes,
Method for producing gram-positive bacteria mock agent composition. According to claim 4,
The liquid medium is any one selected from the group consisting of TSB (tryptic soy broth) medium, LB (Luria-Bertani broth) medium, NB (Nutrient broth) medium, and YM (mixed of marine broth and 0.5% tryptone) medium. That,
Method for producing gram-positive bacteria mock agent composition. According to claim 3,
Incubation temperature and agitation speed in the step of culturing the selected strain is 28 ℃ to 32 ℃ and 150 rpm to 250 rpm,
Method for producing gram-positive bacteria mock agent composition. According to claim 3,
In the step of culturing the selected strain, the pH of the culture solution is 7.0 to 7.5,
Method for producing gram-positive bacteria mock agent composition. According to claim 3,
The washing solution in the step of washing and suspending the cells, distilled water, saline (Saline, 0.85% NaCl), TBS, PBS or a mixture of two or more thereof,
Method for producing gram-positive bacteria mock agent composition. According to claim 3,
The lyophilization is performed by using an excipient comprising trehalose, sucrose or lactose at a concentration of 4% to 6%,
Method for producing gram-positive bacteria mock agent composition. The method of claim 9,
The excipient is a 5% concentration of trehalose (Trehalose) having a cell recovery rate of 70% or more,
Method for producing gram-positive bacteria mock agent composition. According to claim 3,
The liquid suspension, which is carried out using saline (Saline, 0.85% NaCl) or distilled water,
Method for producing gram-positive bacteria mock agent composition.

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