KR100933962B1 - Control method of bacteria motion and method for producing aligned bacterial cellulose thereby - Google Patents

Abstract

PURPOSE: A method for controlling bacteria movement is provided to produce bacteria cellulose membrane with fiber structure of even arrangement and uniform thickness. CONSTITUTION: A method for controlling bacteria movement comprises: a step of treating bacteria to attach a reaction unit of charged particle or magnetic particle on the bacteria surface; a step of inoculating the treated bacteria to culture device; and a step of controlling the movement of inoculated bacteria by operating reaction source according to controlling purpose. The used bacteria strain is Acetobacter xylinum.

Description

Control method of bacteria motion and method for producing aligned bacterial cellulose using the control method

 The present invention relates to control of bacterial behavior, and more particularly to producing high-quality bacterial cellulose using a bacterial behavior control method for controlling the movement of bacteria in order to achieve a desired purpose in bacterial culture and the bacterial behavior control method. It is about how to.

 Bacteria (ie, bacteria) belong to the Monera family, the most prosperous of all living things. They live in the outer environment like soil and water, but they also live in other organisms, such as the stomach and intestines of animals. Most pathogenic bacteria are bacteria. They range in size from 0.5μm to 0.5mm, and they have cell walls just like plant cells and fungal cells, except that peptidoglycan, not cellulose, is the main ingredient.

Bacterial cellulose is a polymer produced by β-1,4 binding of glucose, which is produced by the cultivation of a large number of bacteria, in particular acetobacter genus, among others, Acetobacter xylinum, which is derived from plants. It is a broad category of polysaccharides that has essentially the same chemical structure as cellulose but exhibits very desirable properties.

These bacterial celluloses have a very unique size and aspect ratio (a diameter of about 40 to 100 nm and a length of 0.1 to 15 microns, respectively) in bundle form (averages of 0.1 to 0.2 micrometers in diameter). It is composed of very fine cellulose fibers with high purity and crystallinity, uniform and fine network structure, high mechanical stability and low density. Due to these characteristic structures and properties, bacterial cellulose is being developed as a new industrial material.

As an example, when compared to artificial membranes using polypropylene or polyethylene terephthalate, artificial membranes made of bacterial cellulose show improved mechanical strength.

Acetobacter, which produces bacterial cellulose, especially Acetobacter xylium, is well known as a cellulose-producing strain. Gram with a rod form of 0.6-0.8 micrometers x 1.0-4 micrometers. It is a gram-negative bacterium. Strictly speaking, it is an aerobic organism; In other words, metabolism is respiratory and not fermentable. The bacterium is further distinguished by its ability to produce multiple poly β-1,4-glucan chains that are chemically identical to cellulose. Microcellulose chains, or microfibers, of reticular bacterial cellulose are synthesized at the bacterial surface, the outer site of the cell membrane. Such microfibers generally have a cross-sectional dimension of about 1.6 nm x 5.8 nm.

On the other hand, in static or standing culture conditions, the microfibers on the bacterial surface combine to form fibrils having a cross-sectional dimension of generally about 3.2 nm x 133 nm.

As described above, in order to produce bacterial cellulose, acetobacters are passaged in a test tube and inoculated in HS medium known as a representative medium for bacterial cellulose production. The microfibers secreted from each cell and secreted from different cells form a cellulose film having a shape as shown in FIG. 1 in which the microfibers cross each other to form a network structure with an uneven thickness.

Thus far, the method for producing bacterial cellulose cellulose of the network structure as shown in Fig. 1 is known, but the method for producing high-quality bacterial cellulose having an even thickness and uniform arrangement is known. none.

The present inventors have made efforts to develop a production method for obtaining high-quality bacterial cellulose, and as a result, a method of controlling the behavior of bacteria according to the intention of cultivation, and using the method to produce a relatively uniform and evenly arranged bacterial cellulose The method was developed to complete the present invention.

Accordingly, it is an object of the present invention to provide a bacterial behavior control method capable of intentionally controlling the movement of bacteria in accordance with the purpose of culture in bacterial culture.

In addition, another object of the present invention is to provide a method for producing bacterial cellulose membranes having a relatively uniform thickness and evenly arranged fiber structure by controlling the behavior of the bacteria in acetobacter culture to produce bacterial cellulose.

In addition, another object of the present invention is to provide a bacterial incubator having a structure that can easily control the bacterial behavior to easily achieve the purpose of culture.

The objects of the present invention are not limited to the above-mentioned objects, and other objects that are not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention comprises the steps of treating the bacteria to include a reaction unit; Inoculating the treated bacteria in an incubator to which an action force acting on the reaction unit is applied; And controlling the behavior of the inoculated bacteria by operating the agent generating the action force according to the control intention.

In a preferred embodiment, the incubator further comprises a behavior limiting unit to limit the movement of bacteria.

In a preferred embodiment, the behavior limiting unit is a microstructure according to the control intention to physically limit the behavior of the bacteria by the microstructure of the structure to move in the intended direction.

In a preferred embodiment, the reaction unit has a constant directivity with respect to the direction of action of the action force.

In a preferred embodiment, the action force is an electric field if the reaction unit is a charged particle, the action force is a magnetic field if the reaction unit is a magnetic particle.

In a preferred embodiment, the action force is changed in the action direction, action time or action intensity according to the control intention.

In addition, the present invention provides a method for producing bacterial cellulose, comprising: treating the passaged Acetobacter xylinum strain to include a reaction unit; inoculating the treated strain into an incubator in which a pH-adjusted culture solution is accommodated and an action force acting on the reaction unit is applied; And controlling the action force applied to the incubator through the operation of an agent that generates the action force while culturing the inoculated strain to produce bacterial cellulose of even thickness or arrangement. To provide.

In a preferred embodiment, the operating interval, size or direction of the action force applied to the incubator is adjusted according to the cellulose production rate of the inoculated strain.

In a preferred embodiment, if the reaction unit contained in the strain is a magnetic particle, the action force applied to the incubator is a magnetic field, and if the reaction unit is charged particles, the action force is an electric field.

In a preferred embodiment, a behavior limiting unit having a micro-shape which physically limits the behavior of the strain so that the bacterial cellulose produced by the strain becomes a uniform arrangement is further installed in the incubator.

In addition, the present invention includes a main body including a culture solution receiving portion; An action source built in the main body or installed outside the main body to generate an action force applied to the accommodation portion; And it provides a bacterial behavior control incubator comprising a control module for controlling the operation of the incubator including the operation of the agent.

In a preferred embodiment, it further comprises an oxygen supply module for supplying oxygen to the receiving portion.

In a preferred embodiment, it further comprises a behavior limiting unit which is a microstructure that physically limits the behavior of bacteria that are installed in the receiving portion and cultured.

The present invention also provides bacterial cellulose produced by the production method of any one of claims 7 to 10 having a fiber structure of uniform thickness and arrangement.

The present invention has the following excellent effects.

First, according to the bacterial behavior control method of the present invention, it is possible to intentionally control the movement of bacteria in accordance with the purpose of culture during bacterial culture.

In addition, according to the bacterial cellulose production method of the present invention, by controlling the behavior of the bacteria in the culture of acetobacter producing bacterial cellulose, a bacterial cellulose membrane having a relatively uniform thickness and an evenly arranged fiber structure can be produced.

In addition, according to the bacterial incubator of the present invention, it is possible to easily control the bacterial behavior to easily achieve the purpose of culture.

The terms used in the present invention were selected as general terms as widely used as possible, but in some cases, the terms arbitrarily selected by the applicant are included. In this case, the meanings described or used in the detailed description of the present invention are considered, rather than simply the names of the terms. The meaning should be grasped.

The bacterial cellulose used in the present invention may refer to a microfiber produced by Acetobacter and / or a membrane structure formed by combining the microfibers.

Hereinafter, with reference to the preferred embodiments shown in the accompanying drawings will be described in detail the technical configuration of the present invention.

However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Like numbers refer to like elements throughout the specification.

First, the bacterial behavior control method of the present invention is one step further from the method of controlling the behavior of bacteria by using the main nature of the bacteria (daylight, main nature, chemotaxis, etc.) intentionally according to the culture purpose And artificially control irrespective of the bacterial hostility.

However, the artificial control of these bacteria is not possible by culturing the bacteria as they are, so that the bacteria respond to a specific action force on the inner or outer surface of the bacteria so that the bacteria can be induced in the direction of the action force in response to an external action force. A procedure must be performed to treat the reaction unit so that the bacteria contain the reaction unit.

Here, the reaction unit included in the bacteria is not limited at all to the material and structure as long as it has a certain directivity with respect to the direction of action of a particular action force. Preferably, the reaction unit may be charged particles or magnetic particles, in which case the action force for inducing the movement of the reaction unit is an electric field or a magnetic field.

Bacterial control in the case where the action force is an electric field is a method of utilizing the potential difference between the cell surface and its surroundings (culture medium). In general, the isoelectric point (pI) of the cell surface is about 3.5-5.0 because of the functional groups (such as carboxyl groups) possessed by proteins. If the pH of the acetobacter xylium is 4.0, if the pH of the culture medium is controlled to 6.0 (typical culture pH), a net potential difference of 2.0 is negatively generated during the culture. Will be moved to. If the direction of movement is to be changed again, the polarity of the electrode is reversed (negative to positive) to move the cells moved to the anode to the cathode. By properly controlling the strength and conversion speed of the voltage and current, bacterial cellulose in the desired arrangement can be obtained.

For example, when the force is a magnetic field, the magnetic particles used in the present invention are ferromagnetic nano-sized particles of iron oxide (Fe2O3, Fe3O4), Ferrite (Fe3O4 in which Fe is changed to another magnetically related atom, ex: CoFe2O4 , MnFe2O4)), alloys (oxidation problems caused by magnetic atoms, alloying with precious metals to increase conductivity and stability, ex: FePt, CoPt, etc.), especially bacteria that want to control the surface of magnetic particles Organic nanoparticles or magnetic nanoparticles coated with specific functional groups are preferable in order to bond functional groups capable of bonding with specific proteins.

More specifically look at the treatment step of including the reaction unit in the bacteria. For example, if the reaction unit is a nano-size very fine charged particles or the nature of the bacteria to be controlled as a magnetic particle can be easily introduced into the particles by treatment by a known method to introduce the particles inside the bacteria The reaction unit can be included inside the bacteria. At this time, the more reaction units introduced into the bacteria, the easier it may be to control, but the concentration should be determined in consideration of the effect on the growth of bacteria.

In addition, if the reaction unit is not suitable for introduction into the bacteria due to the size of the reaction unit and / or the nature of the bacteria to be controlled, a specific reaction of the reaction unit is required after conjugating a reactor specific to the surface protein of the bacterium to be controlled to the reaction unit. The reaction unit may be attached to the outer surface of the bacteria by a known method such as inducing binding of the coagulant to the bacterial surface protein so that the reaction unit is included in the bacteria.

Thereafter, the bacteria containing the reaction unit are inoculated into the incubator to which the action force acting on the reaction unit is applied, and then the agent that generates the action force is operated according to the control intention, thereby culturing the bacteria according to the culture purpose. The acting force for intentionally controlling the behavior may change the direction of action, the time of action or the intensity of action depending on the control intention.

Looking at the control of the behavior of bacteria after the bacteria containing the reaction control unit is inoculated in the incubator with reference to Figure 2, Figure 2a is a state in the incubator has not yet been operated and Figure 2b operates the source of Figure 2a It is shown that the state can intentionally control the behavior of bacteria from left to right.

Thus, by applying the action force acting on the reaction unit contained in the bacteria it is possible to artificially control the behavior of the bacteria.

On the other hand, in order to more easily control the behavior of the bacteria containing the reaction unit may be further installed a behavior limiting unit to limit the movement of the bacteria in the incubator.

The behavior limiting unit is a microstructure according to the control intention to physically limit the behavior of bacteria by the microstructure of the structure and to move it in the intended direction. The behavior limiting unit may be manufactured using a MEMS processing technique based on a material compatible with the biocompatible material or the bacteria to be controlled or easily separated from the bacterial product depending on the culture purpose.

That is, when the purpose of cultivation is to use the finished product by adding the products produced by the bacteria to be controlled to the behavior limiting unit, it is preferable that the behavior limiting unit is a biocompatible material including cellulose, but the purpose of the cultivation is limited. If it is desired to obtain only the bacterial product completely separated from the unit, it is preferable that the material is incompatible with the bacterial characteristics or easily separated from the bacterial product.

Next, looking at the configuration of the incubator to more easily perform the above-described bacterial behavior control method as shown in Figure 3 main body 110, the source 120 and the culture medium receiving unit 140, oxygen supply ( 130, a control module (not shown), which may further include an alkali supplier 160 and a pH sensor 170 in some cases.

The main body 110 includes a culture medium accommodating part 140 and a heater for maintaining the optimal culture temperature of bacteria cultured in the accommodating part 140 on the wall of the main body 110 constituting the culture medium accommodating part 140. H) is preferably embedded.

In FIG. 3, the upper surface of the main body 110 is not shown, but the entire upper surface may be formed in a plate shape in order to insert a culture solution into the receiving portion 140 or to facilitate bacterial inoculation. It may be configured to form a door. At this time, the upper surface is preferably sealingly coupled using the sealing 150. In some cases, the upper surface may be formed of a transparent body, and when configured in this way, it is easy to observe the bacterial culture state and the control state in the accommodation unit 140.

In addition, the action source 120 may be embedded in the main body 110 or may be installed outside the main body 110, and preferably, the action source 120 may be installed to be uniformly applied with the action force over the entire receiving portion 140. The source 120 may be a magnetic field generator or electric field generator of known configuration. For example, as an electric field generating device, a plurality of electrodes in contact with a culture solution accommodated in the receiving unit 140, and a circuit for generating a potential difference between the electrodes in a range in which the culture solution does not undergo electrolysis, the circuit may be provided. A circuit that sweeps and applies a voltage in a range in which the culture solution does not electrolyze to a plurality of electrodes in a constant direction in turn is effective for moving bacteria in a certain direction.

In addition, the control module may set the heater (not shown) built in the main body 110, the oxygen supply module 130, whether the action source 120 is driven, the driving time, the driving interval, and the like according to the control intention. Preferably, the configuration includes a setting input unit and a control state display unit for automatically controlling the set state.

In some cases, it may include an oxygen supply module 130 for supplying oxygen in the case of culturing aerobic bacteria in the receiving portion 140. In addition, it may include an alkali supply module 160 and the pH sensor 170 that can confirm this to supply alkali in case the bacteria cultured in the receiving unit 140 is sensitive to the concentration of pH.

In addition, although not shown, the behavior limiting unit, which is a micro-structured structure installed in the receiving unit 140 to physically limit the behavior of the bacteria cultured in the receiving unit 140 in order to more easily control the behavior of the bacteria during the culturing It may be further included.

The shape and material of the behavior limiting unit may vary depending on the shape and control intention of the receiving portion 140 and is formed by MEMS technology.

Example

Production of bacterial cellulose with uniform thickness and / or arrangement

As a bacterium for producing bacterial cellulose, Acetobacter xylinum strain, a kind of acetic acid bacterium, was selected and passaged in vitro, and the strain controlled by the controller was prepared at a concentration of 10 ⁶ CFU / ml or more. The strain applied to the magnetic field controller was treated to include magnetic particles as a reaction unit.

The acetobacter xylium strains then prepared were prepared by the incubator of the present invention (see FIG. 3) having 500 parts of receptacle per 100 parts of bacterial cellulose production medium (HS medium, CSL medium, Glu-Fru medium, etc.). One side (i.e. starting point of action) was inoculated. The medium is composed of monosaccharides, organic nitrogen, inorganic phosphorus, other inorganic salts, distilled water as representative medium for bacterial cellulose production, and is adjusted to pH 4.5 to 6.

Inoculated bacteria were incubated at 30 ° C. for 3-5 days to prepare bacterial cellulose, and the acetobacter xyllium was a magnetic field by applying an electric or magnetic field as a starting point from which the bacteria were inoculated (one side of the receiving part). The bacterial cellulose was produced by inducing movement to the functioning end point (the other side of the receptacle), and when the bacteria reached the other side of the receptacle, the bacteria were reapplied by reapplying the magnetic field changed in the opposite direction of the original magnetic field. Again control to move toward one side of the receiving portion. This control was repeated to obtain bacterial cellulose that was more uniform in thickness and arrangement.

The bacterial cellulose thus obtained forms a high-quality film that is not only thicker than the conventional bacterial cellulose but is more uniform in arrangement. Therefore, the bacterial cellulose is in contact with the human body (clothes, blankets, etc.) or a medical device (eg, inserted into the human body). Catheter) or a variety of medical devices, such as medical devices in contact with the human body.

As described above, the present invention has been illustrated and described with reference to preferred embodiments, but is not limited to the above-described embodiments, and is provided to those skilled in the art without departing from the spirit of the present invention. Various changes and modifications will be possible.

1 is a photograph of a typical bacterial cellulose produced by acetobacter,

Figure 2a and Figure 2b is a schematic diagram to intentionally control the bacterial behavior according to the culture purpose in the culture of bacteria,

Figure 3 is a schematic diagram showing an embodiment of an incubator that can easily implement the bacterial behavior control method of the present invention.

110: incubator body 120: source 130: oxygen supply (O ₂ Port)

140: culture medium containing portion 150: sealing (Sealing)

160: Alkali Port

170: pH sensor

Claims (14)

Introducing a reaction unit, which is a charged particle or a magnetic particle, into or adheres to the bacterial surface; Inoculating the treated bacteria in an incubator to which an action force which is an electric or magnetic field is applied such that the reaction unit has a constant directivity according to the type of the reaction unit; And Controlling the behavior of the inoculated bacteria by operating the agent generating the action force according to the control intention. The method of claim 1, Bacterial behavior control method characterized in that the incubator further comprises a behavior limiting unit for limiting the movement of bacteria. The method of claim 2, The behavior limiting unit is a microstructure according to the control intention, the bacterial behavior control method characterized in that the movement of the bacteria in the intended direction by physically limited by the microstructure of the structure. delete delete The method of claim 1, The action force is a bacterial behavior control method, characterized in that the action direction, action time or action intensity is changed according to the control intention. In the method of producing bacterial cellulose, A step of introducing a reaction unit which is charged or magnetic particles into or adheres to a bacterial surface into the passaged Acetobacter xylinum strain; inoculating the treated strain into an incubator in which a pH-adjusted culture solution is accommodated and an action force, which is an electric or magnetic field, is applied such that the reaction unit has a constant directivity according to the type of the reaction unit; And Controlling the action force applied to the incubator through the operation of the action to generate the action force while culturing the inoculated strain to produce bacterial cellulose of an even thickness or arrangement; uniform fiber structure bacterial cellulose production method comprising a. The method of claim 7, wherein The operation interval, size or direction of the action force applied to the incubator is uniform fiber structure bacterial cellulose production method characterized in that it is adjusted according to the cellulose production rate of the inoculated strain. delete The method of claim 7, wherein Method for producing a homogeneous fibrous structured bacterial cellulose, characterized in that the behavior limiting unit having a fine shape to physically limit the behavior of the strain so that the bacterial cellulose produced by the strain is uniformly arranged in the incubator. delete delete delete A bacterial cellulose produced by the production method of any one of claims 7, 8 and 10, having a uniform thickness and arrangement of the fibrous structure.

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