JP2005312398A - Microbe preservation gel - Google Patents

Abstract

PROBLEM TO BE SOLVED: To stably store microorganisms for a long period of time at room temperature without requiring an expensive and large-scale apparatus, and to check whether the stored microorganisms are alive or dead, and whether other harmful bacteria are mixed. Provided is a gel for preserving microorganisms that can be confirmed with the naked eye.
SOLUTION: Long-term storage has been made possible by forming a biofilm on microorganisms. An example of a medium for forming a biofilm is a gel that contains an inorganic salt compound and a carbon source and is gelled by adding a gelling agent. In this case, it is preferable not to mix a nitrogen source as much as possible, and it is desirable to make the gel highly transparent.
[Selection] Figure 1

Description

  The present invention relates to a microorganism storage technique for storing microorganisms such as bacteria, and more particularly to a microorganism storage gel suitable for storing microorganisms for a long period of time at room temperature.

  Conventionally, as techniques for preserving microorganisms such as bacteria, (1) planting method, (2) cryopreservation method, and (3) freeze-drying preservation method are known. The planting method of (1) is a method in which an agar slant medium or a solid medium without a slant is made with a fast-growing nutrient medium and planted by streaking or puncturing. In the cryopreservation method (2), a microorganism is suspended as a cryopreservation solution containing 10 to 20% glycerol and frozen at −80 ° C. The freeze-drying preservation method (3) is a method in which a microorganism suspended in a dispersion is contained in an ampoule, and this is vacuum-treated with a freeze-dryer to directly sublimate moisture in a frozen state.

  Among these methods, JP-A-6-292564 discloses a cryopreservation method corresponding to the above (2) (Patent Document 1). According to the cryopreservation method described in this publication, a cell or tissue of an organism is treated with a frost damage protection solution comprising an aqueous solution containing 20 to 70% glycerin and 10 to 70% sucrose, and is frozen at a cryogenic temperature. It has become.

JP-A-6-292564

  However, the method (1) described above requires frequent planting and propagation of microorganisms, requires a lot of labor, and increases the frequency of contaminating microorganisms, resulting in a short life span. Moreover, since it is in a state in which a replica always accompanied by a replication of DNA (DeoxyriboNucleic Acid) is continuously created, a different replication may be created for some reason. In this case, there is a problem that the character of the stock strain changes. In addition, if the lethal properties are accumulated, there is a problem that the proliferation ability is lost.

  In addition, in the methods (2) and (3) including the invention described in JP-A-6-292564, since the microorganisms are not grown and stored as they are, the cells start to be damaged. However, the microorganism itself cannot repair or recover it, and the possibility of dying increases. In addition, an expensive ultra-low temperature freezer or freeze dryer is required, and the presence or absence of microorganisms during storage cannot be observed. On the other hand, thawed microorganisms usually cannot be stored again. Therefore, microbial conservants must store without knowing whether the microorganisms are alive or not, especially in the case of environmental microorganisms with little data on storage conditions, the stored strains will be lost unexpectedly. There is a problem. Furthermore, the method (3) has a problem that microorganisms are highly likely to be contaminated during the freeze-drying operation unless a very special technique is acquired.

  Especially in tropical countries, it is difficult to preserve bacteria, there are few skilled people in planting techniques, and it is not possible to purchase expensive ultra-low temperature freezers or other storage devices. There are cases where valuable useful bacteria such as those that have been lost are lost, which is a serious problem.

  The present invention has been made to solve such problems, and does not require an expensive and large-scale apparatus, and can stably store microorganisms over a long period of time at room temperature. It is an object of the present invention to provide a gel for preserving microorganisms that can be confirmed with the naked eye, such as the viability of dead microorganisms and the presence or absence of other harmful bacteria.

  The feature of the gel for microorganism storage according to the present invention is that the microorganism is stored by forming a biofilm having a three-dimensional membrane structure.

  In the present invention, the gel for preserving microorganisms contains at least an inorganic salt compound and a carbon source, and is gelled with a gelling agent. In this case, it is preferable not to mix the nitrogen source as much as possible into the microorganism-preserving gel.

  Furthermore, in the present invention, the gelling agent is preferably one that can form a gel with as high a transparency as possible so that the state of the microorganism can be accurately observed from the outside visually.

  Further, in the present invention, when microorganisms form a biofilm, in order to prepare an environment in which this matrix is easily recognized as if it is part of a microbial population instead of xenobiotics, Saccharides are preferably used, and one of them is gellan gum.

  Furthermore, in the present invention, the pH value of the microorganism-preserving gel is preferably in the range of 3.5 to 7.2.

  Moreover, in this invention, it is preferable to comprise the said gellan gum 0.2 to 0.6% (w / v) addition with respect to the aqueous solution which melt | dissolved the said inorganic salt compound and the said carbon source.

  Moreover, in this invention, it is preferable to contain the acicular crystallization prevention material which prevents the acicular crystallization of the said gellan gum, and especially a porous cellulose sphere is desirable.

  According to the present invention, microorganisms can be stably stored over a long period of time at room temperature without the need for expensive and large-scale equipment, and the presence or absence of contamination of other microorganisms or the state of the stored microorganisms Etc. can be confirmed with the naked eye.

  Hereinafter, an embodiment of a gel for preserving microorganisms and a method for producing the same according to the present invention will be described with reference to the drawings.

  As a result of intensive research on the above-mentioned problems, the inventors of the present application have 1) high-permeability so that the stored microorganism can be visually observed from the outside. 3) Inventing a medium for storing microorganisms having characteristics such as being easily liquefied by applying vibration from a gel state and easily removing microorganisms, and 4) being easily re-storable. It came to do. The medium according to the present invention is a gel-like medium that allows a microorganism to form a biofilm, and includes, for example, an inorganic salt compound and a carbon source, and is formed into a gel by a gelling agent.

  The composition of one embodiment of the gel for preserving microorganisms according to the present invention will be described in detail. The inorganic salt compound contains an inorganic salt necessary for the initial growth of microorganisms. However, this inorganic salt compound does not contain a component that can be a nitrogen source of microorganisms. Normally, microorganisms cannot live without nitrogen. However, many microorganisms produce ammonia as a metabolite using a nitrogen source as a raw material, and there is a risk that their own growth environment will be deteriorated and killed. On the contrary, the presence of an excessive nitrogen source is not preferable. For example, medium compositions such as high ammonia, high protein, and high amino acid are not preferable from the viewpoint of long-term storage. Therefore, it is desirable that the inorganic salt compound in this embodiment does not contain at least an ammonium salt, nitrate, or nitrogen-containing organic substance, and does not have a nitrogen source used by microorganisms.

As an example of the above-described nitrogen-free inorganic salt compound, in the present embodiment, a so-called Winogradsky nitrogen-free inorganic salt medium (hereinafter referred to as “Winogradsky medium”) for azotobacter culture is used. The composition of the Winogradsky medium shown in this embodiment is potassium phosphate (KH ₂ PO ₄ ) 50.0 g / l, magnesium sulfate (MgSO ₄ .7H ₂ O) 25.0 g / l, and sodium chloride (NaCl ) 25.0 g / l, ferrous sulfate (FeSO ₄ · 7H ₂ O) 1.0 g / l, sodium molybdate (Na ₂ MoO ₄ · 2H ₂ O) 1.0 g / l, manganese sulfate ( MnSO ₄ .4H ₂ O) 1.0 g / l is used as a stock solution, and it is composed of 5 ml of this stock solution and 1000 ml of ultrapure water.

In this embodiment, calcium carbonate (CaCO ₃ ) is separately added for the purpose of adding calcium, which is an inorganic metal element essential for microorganisms. The reason why calcium carbonate is separately mixed in this manner is that when mixed with other inorganic salt compounds at a high concentration, extremely insoluble calcium nitrate is formed and other metal salts are coprecipitated. Further, if this calcium carbonate is not added, it may not be gelled by the gelling agent. For this reason, in this embodiment, fine powdery calcium carbonate is added in the final step of a one-fold dilution of the Winogradsky medium to produce a complete inorganic salt solution.

  Next, the carbon source is an energy source of microorganisms and plays a role of constituting cells. In this embodiment, an amount is added only to give the microorganism being preserved the minimum growth ability. In other words, it is to maintain a semi-dormant state where microorganisms do not grow unnecessarily, and to create an environment that hardly changes the character. Examples of the carbon source used in the present embodiment include various sugars such as glucose (Glucose), sucrose (Saccharose), and mannitol, organic acid sodium salts, organic acid potassium salts, citric acid, malic acid, and the like. Can be mentioned. In particular, sucrose, mannitol, and the like are preferable because they are good crystals that do not contain impurities, and do not rapidly change the pH value of the inorganic salt solution and the medium pH value during the growth of microorganisms.

  Next, the gelling agent will be described. The purpose of adding the gelling agent in this embodiment is to first buffer the impact received by the microorganisms to be stored. That is, when a microorganism is stored in a solid, the microorganism is directly damaged by an external impact and easily damaged. In addition, when microorganisms are stored in a liquid, the microorganisms are easily damaged because they collide with the wall of the container or sink to the bottom to form aggregates. On the other hand, when microorganisms are stored in a semi-fluid gel, the microorganisms move slowly due to the action of the gel and do not collide strongly with the wall surface, and the impact received from the outside is also buffered by the gel. Therefore, it is possible to reduce the probability that the microorganisms are killed or the gene is damaged and changes its character. For this reason, it is preferable that the gel has a hardness, viscosity, and elasticity suitable for preserving microorganisms and is not easily decomposed by microorganisms.

  In addition, the second object of the gelling agent added in the present embodiment is an action that microorganisms stored in the gelated storage gel can form a biofilm to temporarily suspend metabolic activity. It is to have. In this embodiment, gellan gum which is a polysaccharide derived from bacteria is used as a gelling agent. A gel for preserving microorganisms is produced by adding this gellan gum, the microorganisms are preserved and allowed to elapse for a predetermined period, and then extracted with ethyl acetate. Many were extracted. This is thought to be because gellan gum is derived from bacteria, so that it may be easily recognized by microorganisms as a part of the microbial population, not as a xenobiotic.

  In addition, as a gelling agent used in this embodiment, in order to prevent killing during storage of microorganisms, it does not contain a cationic species that shows toxicity at a high concentration, and permeates and absorbs nutrients and phosphates. What has the property which does not prevent is preferable.

  Furthermore, a gelling agent that has the ability to maintain the pH value around the microorganism to be stored for a long period of time, eliminates radicals, or keeps the dissolved oxygen concentration constant is considered to be more preferable. It is done.

  In this embodiment, gellan gum is used as a gelling agent, but is not limited to this, and any gelling agent that has good compatibility with microorganisms and that forms a biofilm can be used. It may be an artificial gelling agent provided. Agar, carrageenan, and alginic acid, which are well known as gelling agents, have carboxy groups and sulfate groups, and chelate strongly with specific metals, or concentrate a large amount of sodium in the gel. It has the property to do. For this reason, it causes imbalance of metal ions and inhibits the growth of microorganisms, so that it cannot be used as a medium for preservation as in the present invention.

  Moreover, when a porous insoluble material such as porous cellulose spheres is mixed in the gel for preservation of microorganisms of this embodiment and freeze-dried, the preservation period is greatly extended and permanent preservation is possible. More specifically, gels gelled with gellan gum have the property of producing pseudo crystals with needle-like orientation when dried rapidly. Since this pseudocrystal is like a bundle of fine fibers, there is a possibility that microorganisms stored inside will be crushed or protruded from the fibers and released. In order to avoid such a phenomenon, it is necessary to mix the gellan gum with a matrix having an interaction that maintains a three-dimensional structure, thereby losing the directionality of the gellan gum fibers.

  Therefore, in this embodiment, the formation of a needle-like crystal is inhibited by constructing a three-dimensional space by adding porous cellulose spheres. As a result, microorganisms confined in the microorganism storage gel of this embodiment are gently stored without being damaged because they are included in the polymer matrix lattice in a three-dimensional space even when freeze-dried. Is done.

  The porous insoluble material is not limited to porous cellulose spheres, and it is considered possible to use activated carbon or celite. However, activated carbon has a strong surface field adsorptivity, which may cause inconvenience compared to porous cellulose spheres. Celite has a weak adsorbing power, but has a drawback that the effective pore volume is small.

  Next, a method for producing the gel for preserving microorganisms of this embodiment will be described with reference to FIG.

  In this embodiment, first, the above-described Winogradsky medium is generated as an inorganic salt compound not containing a nitrogen source, and sodium hydroxide (4M NaOH) is added thereto to adjust the pH value to 7.2. A suspension which is an inorganic salt solution is generated (step S1). At this time, the pH value is adjusted to 7.2 in order to maintain the stability of the inorganic salt in the Winogradsky medium. That is, if the pH value is set to about 7.2, each inorganic salt exists as a fine insoluble matter, but when the pH value is inclined to acidity, it precipitates as an insoluble and crystalline lump like gypsum. This is because it cannot be suspended at all.

Next, 5 ml of the suspension, 0.2 to 1.0% of sucrose as a carbon source, and 100 mg of fine powdered calcium carbonate are added to 1 liter of ultrapure water and dissolved to dissolve the basic aqueous solution. Generate (step S2). Then, the sparingly soluble inorganic salt is dissolved in the basic aqueous solution by applying ultrasonic waves for about 15 to 30 minutes (step S3). Thereafter, the basic aqueous solution is adjusted to a pH value of 6.2 using sulfuric acid (2M H ₂ SO ₄ ), and then filtered through a membrane filter having a pore diameter of 0.45 μm (step S4). Thereby, insoluble fine particles are removed and high transparency is obtained.

  The pH value 6.2 of the basic aqueous solution is pH 6.0 to pH 7.6, which is maintained in a neutral green color when bromothymol blue (BTB), a pH indicator, is added to the gelled medium at 40 ppm. Set to be within range. As a result of repeated experiments by the inventors of the present application, within the above range, the reproducibility of the semi-fluidity of the gel is good, and the pH value of the gel is rapidly lowered by the color of bromothymol blue. This is because it can be easily distinguished from bacteria that are not.

  Subsequently, a gellan gum solution is produced by adding 0.3% (w / v) gellan gum to the basic aqueous solution after filtration and preheating (step S5). At this time, the amount of gellan gum to be added is preferably in the range of 0.2 to 0.6% (w / v) depending on the purpose, with 0.3% (w / v) as a standard. If it is within this range, microorganisms can spread uniformly in the gel after gelation, and the hardness is such that it can be easily liquefied if vibration is applied. On the other hand, if the gellan gum is less than 0.2% (w / v), it is difficult to gel the base aqueous solution, and if it is more than 0.6% (w / v), the gel is excessively cured.

  Then, an appropriate amount of gellan gum solution is dispensed into a test tube with a screw cap, sterilized by autoclaving at 120 ° C. for 20 minutes (step S6), and then cooled at room temperature for 3 to 12 hours (step S7). Through the above process, a gel having semi-fluidity and high transparency is generated.

  According to the microorganism storage gel of the present embodiment described above, when the microorganism to be stored is confined in the microorganism storage gel, since the gel does not contain a nitrogen source, the microorganism fixes the necessary amount of nitrogen to the nitrogen. It is thought that it will be reused rather than being obtained by degrading bacterial protein and exhausting excess nitrogen outside the body.

  In addition, since the gel contains the minimum carbon source and gellan gum, microorganisms do not grow unnecessarily as a group, but form a biofilm to temporarily suspend their metabolic activity and make a semi-dormant It becomes a state. As a result, the variation of the trait is reduced, and fresh cells having a proliferation ability are constantly updated in a limited population density.

  Therefore, the gel for preserving microorganisms of this embodiment is not limited to the cryopreservation method or freeze-drying preservation method that completely stops the growth as in the conventional preservation method, or conversely, the interval growth, that is, gene replication is always performed. It is completely different from the planting method that keeps creating replicas with, and can be said to be a preservation method that slowly grows under self-control by limiting the environment and ability of the microorganisms to replicate to a minimum. In other words, the microorganisms in the microorganism storage gel of this embodiment maintain a semi-dormant state that does not grow unnecessarily as a population of microorganisms, have few variability in traits, and are always limited to fresh cells having a proliferation ability. It is in a state of being continuously updated within the set population density. Thus, for example, even if the microorganisms being stored are damaged, the microorganisms are resistant to being stored at room temperature for a long period of time in order to surely repair the wounds generated in the DNA, although slowly. .

  In addition, many bacteria scatter the transmitted light because they have a spherical shape or a similar elliptical spherical shape while they are alive. Therefore, many viable bacteria can be observed with the naked eye as clear turbidity or white particles. When bacteria die, self-lysis called lysis occurs, so light scattering often disappears and becomes transparent. For this reason, it is difficult to determine from the outside whether the gel with low permeability is dead or not, but since the gel for microbial storage of this embodiment using gellan gum is highly transparent, Even if it exists, the color of microorganisms can be permeated, and the presence or absence of microorganisms and contamination with other bacteria can be visually confirmed from the outside.

  Next, more specific examples of the gel for preserving microorganisms in the present embodiment will be described.

  The gel for preserving microorganisms used in this Example 1 is 0.5% (v / v) stock solution for Winogradsky medium as an inorganic salt compound and 0.3% sucrose as a carbon source with respect to ultrapure water. (W / v), 100 mg of fine powdery calcium carbonate as calcium carbonate, and gellan gum 0.3% (w / v) as a gelling agent. Then, “Herbaspirillum sp.” Separated from the inside of the stem of Shinjugaya was confined in this microbe-preserving gel, stored at room temperature (about 20 ° C.), and the survival time was measured.

  On the other hand, as a comparison object of Example 1, prepared with Winogradsky medium 0.5% (v / v), sucrose 1.0% (w / v), yeast extract 0.005% (w / v) A sample obtained by confining and storing Hervaspyriram in the agar slope medium was used as Comparative Example 1, and the survival time was measured. Further, as Comparative Example 2, a 10% glycerol-containing cryopreservation solution was used, and Hervas pyrilum was frozen at −80 ° C., and its survival time was measured.

  The results of Example 1 and Comparative Examples 1 and 2 are shown in FIG. As shown in FIG. 2, in Comparative Example 1, the hervaspyriram was almost killed in about 2 months. In Comparative Example 2, some survived for more than one year, but almost died after two years. On the other hand, it was confirmed that in the gel for preserving microorganisms of Example 1, Hervaspyriram did not die even after 2 years or more, and the long one survived after about 3 and a half years.

  According to the present Example 1 as described above, it was confirmed that the herbuspyriram confined in the microorganism storage gel of the present embodiment survives for a long time at room temperature.

  Next, in Example 2, the microorganism storage gel used in Example 1 is used, and Rhizobium sp. Is confined in the microorganism storage gel and stored at room temperature. did.

  On the other hand, as a comparison target of Example 2, prepared with Winogradsky medium 0.5% (v / v), sucrose 0.005% (w / v), and yeast extract 1.0% (v / v) Comparative Example 3 was prepared by confining and storing Rhizobium in the agar slope medium. Moreover, what preserve | saved Rhizobium in the bouillon culture medium (1x Nutrient broth agar) 30g / L by Nippon Suisan Co., Ltd. was made into the comparative example 4, and what diluted the bouillon agar medium used in this comparative example 4 10 times. The sample containing Rhizobium was used as Comparative Example 5, and the sample obtained by storing Rhizobium in Difco's potato-dextrose agar medium was used as Comparative Example 6. The survival time at each room temperature in Comparative Examples 3-6 was as follows. Each was measured.

  The results of Example 2 and Comparative Examples 3 to 6 are shown in FIG. As shown in FIG. 3, in Comparative Example 3, Rhizobium almost died out in about one month. Further, in Comparative Example 4 and Comparative Example 5, almost one week and in Comparative Example 6 were almost dead in two weeks. On the other hand, in the gel for preserving microorganisms of Example 2, it was confirmed that Rhizobium survived even after 1 year or more.

  According to Example 2 as described above, it was confirmed that the Rhizobium trapped in the microorganism-preserving gel of the present embodiment survives for a long time at room temperature.

  In addition, the gel for preserving microorganisms according to the present invention is not limited to the above-described examples, and can be appropriately changed.

For example, in the present embodiment, the hardness of the gel after gelation is set to such an extent that microorganisms can spread uniformly and can be easily liquefied if vibration is applied. However, depending on the microorganisms to be stored, it is necessary to strengthen elements or adjust to an arbitrary pH according to the environment in which they lived. For this reason, if there is a factor that decreases the pH value or reduces the gelation ability such as Al ³⁺ (aluminum cation), the pH value setting can be changed or the amount of various matrices can be increased or decreased. The gel hardness should be adjusted.

It is a flowchart figure which shows one Embodiment of the manufacturing method of the gel for microorganisms storage which concerns on this invention. It is a figure which shows the lifetime of the herbus pyrilum in the present Example 1 and Comparative Examples 1 and 2. FIG. It is a figure which shows the lifetime of the lysobiium in the present Example 2 and Comparative Examples 3-6.

Claims (10)

  A gel for preserving microorganisms, which is preserved by forming a biofilm on microorganisms.   2. The microorganism storage gel according to claim 1, wherein the microorganism storage gel contains at least an inorganic salt compound and a carbon source, and is gelled by a gelling agent.   3. The microorganism-preserving gel according to claim 1 or 2, wherein a nitrogen source is not mixed.   The gel for preserving microorganisms according to any one of claims 1 to 3, wherein a gelling agent that forms a transparent gel is used as the gelling agent.   5. The microorganism-preserving gel according to claim 1, wherein a polysaccharide derived from bacteria is used as a gelling agent.   The gel for preserving microorganisms according to claim 5, wherein gellan gum is used as the gelling agent derived from bacteria.   The gel for preserving microorganisms according to claim 6, wherein the pH value is set in a range of 3.5 to 7.2.   The microorganism according to claim 6 or 7, wherein 0.2 to 0.6% (w / v) of the gellan gum is added to an aqueous solution in which the inorganic salt compound and the carbon source are dissolved. Gel for storage.   The gel for preserving microorganisms according to any one of claims 6 to 8, further comprising an acicular crystallization preventing material for preventing acicular crystallization of the gellan gum.   The gel for preserving microorganisms according to claim 9, wherein porous cellulose spheres are used as the acicular crystallization preventing material.

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