KR102227125B1 - Method for storing bacteria and dry power produces by the same method - Google Patents

Abstract

The present invention provides a method for storing bacteria comprising preparing a suspension in which bacteria and nanoparticles having a porous structure are suspended after drying, and atomizing the suspension into droplets and drying the atomized droplets.

Description

Bacteria storage method and dry powder produced thereby {METHOD FOR STORING BACTERIA AND DRY POWER PRODUCES BY THE SAME METHOD}

The present invention relates to a method of storing bacteria and a dry powder produced thereby, and more particularly, to a method of storing live bacteria in a dry powder form, and to a dry powder produced thereby.

In the distribution of biological resources for domestic and overseas research, it is most common to move bacteria in frozen storage conditions. Therefore, the method of storing bacteria in liquid or gel form for cryopreservation is currently being implemented first in the pre-sale procedure. Other storage methods have been studied continuously, but practically usable methods are still insufficient.

Among the similar techniques in the related art, Patent Document 1 discloses a technique for producing a dry powder through a bacterial extract, and finally encapsulating the extract of a specific bacteria for the purpose of cancer treatment into a powder through a suspension. However, in Patent Document 1, the focus of the powder component is on the extract, not the bacteria, and furthermore, since the peptide components produced by the bacteria in the suspension culture step are the main purpose of dry powdering, the survival of the bacteria in the powder is largely irrelevant. The survival effect is also insufficient.

Therefore, it is necessary to present a technique for solving this problem.

KR10-2013-0137637A

An object of the present invention is to provide a method for storing bacteria that can control the survival rate or preservation period of bacteria in the form of dry powder, and a dry powder produced thereby.

In order to solve the above problems, the present invention provides a suspension of bacteria and nanoparticles having a porous structure after drying, and a step of atomizing the suspension into droplets and drying the atomized droplets. Provide storage methods.

According to an embodiment, the nanoparticles may be any one selected from the group consisting of carbon, glass, zeolite, aluminum oxide, polyaniline, and polystyrene polymer, or a combination thereof.

According to an embodiment, the suspension may have a size ratio of 1:0.1 to 1:10 between nanoparticles and bacteria.

According to an embodiment, the atomized droplet may be 3 μl to 10 μl.

According to an embodiment, the drying may be performed by dispensing the atomized droplets on a superhydrophobic surface.

According to an embodiment, the drying may be performed in air by spraying the atomized droplets using a spraying device.

In addition, the present invention provides a dry powder produced by the method for storing bacteria.

According to the present invention, the survival rate or storage period of bacteria is controlled by the porous structure generated by suspending and drying nanoparticles and bacteria together, thereby improving the survival rate of bacteria to extend the storage period, or the maximum survival time and storage period. Can be controlled.

Compared with the conventional liquid or gel type of bacteria storage method, the temperature sensitivity can be lowered. Since there is no need to keep the bacteria frozen during storage and transport, the method of storing bacteria can be simplified, storage costs can be reduced, and there is an effect of preventing a decrease in the survival rate due to the inability to control the temperature. That is, by changing the storage form from liquid or gel to a solid powder form, it is possible to increase the stability when moving bacteria and reduce the cost of environmental control.

1 is a step-by-step flowchart of a method for storing bacteria according to an embodiment of the present invention.
2 is a view showing a process of drying a suspension on a superhydrophobic surface according to an embodiment of the present invention.
3 is an SEM image of a residue made of nanoparticles having a porous structure according to an embodiment of the present invention.
4 is an image photographing a process of culturing a residue according to an embodiment of the present invention.
5 and 6 are graphs showing the survival rate of bacteria in residues containing nanoparticles according to an embodiment of the present invention.

Hereinafter, the present invention will be described in detail through preferred embodiments. Prior to this, terms or words used in the specification and claims should not be construed as being limited to their usual or dictionary meanings, and the inventors appropriately explain the concept of terms in order to explain their own invention in the best way. Based on the principle that it can be defined, it should be interpreted as a meaning and concept consistent with the technical idea of the present invention. Therefore, the configuration of the embodiments described in the present specification is only the most preferred embodiment of the present invention, and does not represent all the technical spirit of the present invention, and various equivalents and modifications that can replace them at the time of the present application It should be understood that there may be. In addition, throughout the specification, when a certain part "includes" a certain component, it means that other components may be further included rather than excluding other components unless otherwise stated.

How to store bacteria

1 is a step-by-step flowchart of a method for storing bacteria according to an embodiment of the present invention.

As shown in Figure 1, the bacteria storage method according to an embodiment of the present invention, the step of preparing a suspension in which bacteria and nanoparticles are suspended (S10) and the step of atomizing and drying the suspension into droplets (S20). Including, it is possible to produce a residue including porous structures in an agglomerated state after drying.

Hereinafter, each step will be described in detail.

First, it is possible to prepare a suspension in which bacteria and nanoparticles having a porous structure are suspended (S10).

Bacteria not only play an important function in the material cycle of the ecosystem, but are also used in the production of useful substances in industrial fields such as biotechnology. Bacteria are well known to have the ability to survive even in extreme environmental conditions, but the environmental tolerance of individual bacterial species is very limited.

Bacteria are particularly sensitive to high temperatures, and even when exposed to a temperature slightly higher than an appropriate temperature, the growth rate is significantly reduced and the viability is greatly reduced, so that the bacteria are conventionally stored in a frozen state to store the bacteria.

The present invention is to lower temperature sensitivity, and according to an embodiment of the present invention, the bacteria to be stored are living things and are not particularly limited to their type, and representative examples are Staphylococcus aureus, Pseudomonas E. Pseudomonas aeruginosa), Bacillus subtilis, Candida albicans, Aspergillus brasiliensis, Escherichia coli, etc. It may be any one or a combination thereof.

In addition, nanoparticles are porous nanoparticles, and particles having a porous structure may be generated after drying the aqueous solution in which the nanoparticles are dispersed. Nanoparticles are materials having a porous structure, in which a pore structure having a size of 0.4 to 100 nm (or 1 to 100 nm) is arranged or arranged in an orderly manner, and may be a powder, a thin film/thick film, or a bulk material.

In the case of powder, the shape of nanoparticles may be spherical, hollow sphere, fiber, tube, or the like, and a film or bulk shape may be prepared as a starting material.

The nanoparticles dispersed in the suspension may be one type, but are not limited thereto, and a plurality of types of nanoparticles may be dispersed in the suspension in order to diversify the growth rate of bacteria when water is introduced into the dry powder.

Here, the material of the nanoparticles may be a polymer such as carbon, glass, zeolite, aluminum oxide, polyaniline, and polystyrene capable of controlling the size of porosity.

Such, the nanoparticles can be dispersed in the bacteria and distilled water to prepare a suspension, and the size ratio between the nanoparticles and the bacteria of the suspension is not particularly limited in order to facilitate controlling the survival rate of bacteria due to a dry porous structure after suspension, It is preferably 1:0.1 to 1:10.

The survival rate of bacteria may be determined by factors such as the relative concentration ratio of bacteria and nanoparticles, the relative size ratio of bacteria and nanoparticles, and the size of the total porous material. The survival rate or duration of storage can be controlled.

Meanwhile, a solution in which the bacteria and nanoparticles are suspended together may be atomized into droplets, and the atomized droplets may be dried (S20).

There are various methods of atomizing and drying the suspension, but according to an embodiment, a droplet dropping device for dropping droplets may be used to drop the droplets onto the superhydrophobic surface to dry them.

2 is a view showing a process of drying a suspension on a superhydrophobic surface according to an embodiment of the present invention.

As shown in Fig. 2(a), a suspension of bacteria-nanoparticles on a plate treated with a superhydrophobic surface can be dispensed into micronized droplets at predetermined intervals.

At this time, the volume of the dispensed droplets is preferably 3 to 10 μl in order to maintain the shape of a sphere, and the superhydrophobic surface maintains a state of maintaining minimal contact with the spherical droplets in the air, but with the superhydrophobic surface In order to minimize the contact between the droplets, an oily substance may be displayed at the location where the droplets are settled on the superhydrophobic surface.

It is preferable that the distance between the dispensed droplets be kept at least 10 to 15 mm to prevent evaporation interference between each other.

After the liquid was evaporated, as shown in FIG. 2(b), a residue made of nanoparticles having a porous structure was generated as shown in FIG. 3, and as shown in FIG. 3, bacteria are present in the porous structure of the nanoparticles. Will be accepted.

As a result of drying (a part of FIG. 3) a droplet including a 100 μm sphere and E. coli on the superhydrophobic surface, it appeared as shown in parts b and c of FIG. 3. Here, part b of FIG. 3 is a photographed image of the residue when the concentrations of E.coli and microspheres are 0.03% and 0.03%, respectively, and part c of FIG. 3 shows the concentrations of E.coli and microspheres are 0.003% and 0.003%, respectively. If it is 0.03%, it is the image of the residue.

When the dried powder, that is, the residue was dip in an agarose medium and cultured, as shown in FIG. 4, colonies were generated by living bacteria. Part a of FIG. 4 shows that 9 small droplets of solution on a hydrophobic surface are positioned to prevent interference with each other in the evaporation rate, and part b of FIG. 4 is dried while maintaining an appropriate temperature and humidity inside the environmental chamber for a specific time. In Figure 4, part c is a state in which the residues on the superhydrophobic surface have been transferred to the agar culture dish, and in Figure 4, part d is that the agar culture dishes are stored in the incubator to cultivate the cells in the residue, and the colony Is growing on a medium.

According to the present invention, as a result of culturing at regular time intervals as the time elapsed after drying the medium elapsed, when the experiment was conducted under the same conditions, when nanoparticles were contained than the residue of the solution containing only bacteria, As shown in Figure 5, it was confirmed that the bacteria survive longer on average. In other words, it can be seen that as time passes, the survival rate of bacteria does not decrease significantly when nanoparticles are included.

Unlike the previous embodiment, the droplets are not dispensed on the superaqueous surface, and the suspension can be dried by spraying the atomized droplets in the air in a high temperature dry environment using a spray device such as a nebulizer.

Likewise, the dried residue has a powder form, and there is an effect that the dried powder can be produced in a larger amount than in the previous example.

At this time, the degree of uniformity of the particles sprayed by the spraying device is proportional to the precision when controlling the bacterial survival rate of the generated powder, and the higher the uniformity of the spraying particles is preferable to precisely control the bacterial survival rate.

According to the present invention, there is a high utility value for effective bacteria transfer into an environment where temperature control is difficult, for example, the body. In particular, it is more efficient in taking or administering microorganisms that have a positive medical effect in vivo, such as lactic acid bacteria.

In addition, according to the present invention, it can be applied to the development of natural medicines and cosmetics using biological resources that are recently spotlighted by using various microorganisms that are effective for anti-cancer and anti-aging, and furthermore, nanoparticles that are harmless to the human body or have other benefits are used. Therefore, due to the nature of the nanoparticles, an increase in the surface area relative to the volume can bring about an effect of increasing the absorption power, so it can produce excellent pharmaceutical and cosmetic effects.

Experimental example

The nanoparticle suspension was used by diluting polystyrene nanoparticles (or microspheres) having a diameter of 10 μm, 1 μm, and 0.1 μm, respectively, by Polybead to a volume percentage of 0.064%.

Specific resources for the nanoparticle suspension used in this experimental example are shown in Table 1.

[Table 1]

E. coli was used as the bacteria, and the size of the E. coli was (2~4)㎛ × (0.4~0.7)㎛, but the E. coli with a short long axis took the form of cocci, and its diameter was about 1㎛. The ratio is shown in Table 2 below.

[Table 2]

The suspension for each concentration of bacteria (E. coli) and the nanoparticle suspension were mixed at a ratio of 1:1, so that 0.032% of the volume percentage of the nanoparticles was maintained as shown in Table 3.

[Table 3]

Thereafter, the suspension of bacteria and nanoparticles was atomized into droplets of 3 to 10 µl, and after being dispensed and dried by maintaining 10 to 15 mm on a superhydrophobic surface-treated plate, the survival rate of bacteria with time was confirmed.

When nanoparticles of different diameters (0.1 μm, 1 μm, 10 μm) were used for OD600 standard bacteria concentrations of 0.05A, 0.1A, 0.2A, and 0.4A using a spectrophotometer, the bacterial survival rate over time was also shown. It was the same as 6.

Eventually, by changing the concentration of the bacteria and/or the diameter of the nanoparticles, it was possible to control the survival rate of the bacteria over time.

In particular, when using nanoparticles having a diameter of 1 μm for an OD600 standard bacterial concentration of 0.1A, it was confirmed that the survival rate was significantly improved compared to the same bacteria when there were no nanoparticles.

In the above, preferred embodiments of the present invention have been described in detail with reference to the drawings. The description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that other specific forms can be easily modified without changing the technical spirit or essential features of the present invention.

Therefore, the scope of the present invention is indicated by the claims to be described later rather than the detailed description, and all changes or modified forms derived from the meaning, scope and equivalent concepts of the claims are included in the scope of the present invention. It must be interpreted.

10: superhydrophobic surface 11: droplet
12: oily substance 13: residue

Claims (7)

Preparing a suspension in which bacteria and nanoparticles having a porous structure are suspended after drying; And
Atomizing the suspension into droplets and drying the atomized droplets;
Including,
The drying step,
The atomized droplets are settled on the oily substance displayed on the superhydrophobic surface and dried, but the atomized droplets are 3 μl to 10 μl in the form of a sphere,
The residue consisting of nanoparticles having the porous structure, the bacteria are accommodated in the porous structure of the nanoparticles,
The survival rate of the bacteria is controlled according to the relative concentration ratio and size ratio between the nanoparticles and the bacteria, and the suspension is characterized in that the size ratio between the nanoparticles and the bacteria is 1:0.1 to 1:10. How to store bacteria. The method of claim 1,
The nanoparticles are any one selected from the group consisting of carbon, glass, zeolite, aluminum oxide, polyaniline, and polystyrene polymer, or a combination thereof. delete delete delete delete Dry powder produced by the method of storing bacteria according to any one of claims 1 to 2.

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