JP4656489B2 - Microbial concentration method - Google Patents

Description

  The present invention relates to a method for concentrating microorganisms that can easily concentrate microorganisms such as bacteria, yeasts, molds, and viruses in fields such as food, medicine, pharmaceuticals, and the environment.

Concentrating and separating microorganisms such as bacteria, yeasts, molds, and viruses is important for obtaining information on microorganisms, collecting and using microorganisms, or eliminating (sterilizing) microorganisms.
For example, in the case of pathogenic microorganisms, concentration / separation of microorganisms is used for diagnosis and treatment. In the case of useful microorganisms, the concentrated microorganisms can be separated and removed to obtain useful components in the supernatant. In addition, concentrated useful microorganisms themselves can be obtained.

  On the other hand, conventionally, in microbial tests in the food field, the environmental field, and the like, in order to confirm the presence of microorganisms in a sample, separation detection by culture, detection by biochemical tests, detection of nucleic acids, and the like are performed. However, in these tests, for the sample to be tested for microorganisms, bacteria in a part of the sample are extracted and cultured, or partial characteristics of the sample are only amplified and detected. That is, only the partial characteristics of the sample are inspected, and the properties of the entire sample cannot be grasped as a whole.

For this reason, in order to test a large amount of samples, the concentration of microorganisms in the samples has been performed. As a method for concentrating microorganisms, centrifugation, filtration using a filter of organic polymer fibers such as cellulose, nylon, and polyester have been performed for a long time.
However, these classic microbial concentration methods cannot provide a high concentration factor. Furthermore, expensive equipment such as a centrifuge and a polymer fiber filter is required, which is expensive.

As a solution to such a problem, the bacterial cell concentration method and the bacterial cell concentration reagent shown in Patent Document 1 have been proposed. In this method, first, an anion and a cation are present in an aqueous solvent containing microorganisms to produce a poorly water-soluble substance. Next, the microorganisms are adsorbed on the poorly water-soluble substance to collect the microorganisms. Furthermore, the microorganisms are recovered by dissolving a poorly water-soluble substance adsorbed by the microorganisms with a solubilizer.
JP 2000-14380 A

However, the conventional method has the following problems.
(1) By dissolving a poorly water-soluble substance adsorbed by microorganisms with a solubilizer, the volume of the phase containing the microorganisms increases, and it can be concentrated only about several tens of times.
(2) The recovery rate of microorganisms is about 90%, and about 10% is lost.
(3) Since there is no specificity in adsorption, impurities other than microorganisms are also concentrated.
The present invention solves such a problem, and has a high magnifying power, a high recovery rate, and has a specificity that water-soluble contaminants such as starch and proteins and contaminants having a high affinity for water are not concentrated. Is to provide. The present invention is particularly suitable for bacteria (VBNC, viable but nonculturable) that are alive but difficult to culture by conventional methods. Bacteria in such a state did not form colonies on the medium and could not be detected by conventional culture methods. Therefore, until now, it was treated as “non-existent” and excluded from hygiene management.

  In order to achieve the above object, the present invention is a method for concentrating microorganisms, comprising adding a surfactant and a hydrophilic organic solvent to a solution containing microorganisms to form a uniform solution, and then lowering the pH of the solution. The solution is separated into two phases, a precipitation phase in which microorganisms, a surfactant, a hydrophilic organic solvent, and water are mixed, and a supernatant phase, and the microorganisms are concentrated in the precipitation phase.

  In one embodiment of the microorganism concentration method according to the present invention, the surfactant is a fluorocarboxylic acid.

  In another embodiment, the method for concentrating microorganisms according to the present invention is characterized in that the hydrophilic organic solvent contains at least one of alcohol, ether, ketone, amide, nitrile and sulfoxide.

According to the present invention, there is provided a microorganism concentration method having specificity that does not concentrate water-soluble contaminants such as starch and protein, and high-affinity contaminants such as starch and protein at a high magnification.
As will be described later, according to the microorganism concentration method of the present invention, the following effects can be expected.
1. Since a large-capacity sample can be concentrated to a very small volume, microorganisms can be concentrated tens of thousands of times.
2. Microorganisms in the sample can be concentrated with a recovery rate of about 100%.
3. Water-soluble contaminants such as starch and protein and contaminants with a high affinity for water are not concentrated and can be separated from microorganisms.

  Hereinafter, the microorganism concentration method according to the present invention will be described in more detail with reference to the embodiment thereof.

The microorganism concentration method according to the present invention is carried out using a surfactant, a hydrophilic organic solvent, and a reagent for lowering pH, and includes at least the following steps.
1. A step of adding a surfactant and a hydrophilic organic solvent to a solution containing microorganisms to form a uniform solution (step 1).
2. Lowering the pH of the solution and separating the solution into two phases, a precipitated phase in which microorganisms, a surfactant, a hydrophilic organic solvent, and water are mixed, and a supernatant phase (step 2);
In the microorganism concentration method according to the present invention, bacteria, yeasts, molds, viruses and the like are targeted, and the above steps are sequentially executed to concentrate microorganisms in the precipitated phase. Each step will be further described below.

Step 1
In Step 1, a surfactant and a hydrophilic organic solvent are added to a solution containing microorganisms to form a uniform solution.
Such a “homogeneous solution” is a concept that includes not only a complete solution but also a pseudo-homogeneous solution in which micelles are formed. It is preferable that the surfactant and the hydrophilic organic solvent have compatibility. In Step 1, the solution containing the microorganism, the surfactant, and the hydrophilic organic solvent form a single liquid phase as a whole.
As the surfactant, a fluorocarboxylic acid is preferable, and it is preferable to use a carboxylic acid in which at least one hydrogen atom bonded to each carbon atom is substituted with a fluorine atom.

  Fluorocarboxylic acids are more prone to dissociate protons than the corresponding carboxylic acids and are hydrophobic. Therefore, unless the pH of the fluorocarboxylic acid is significantly lower than that of the corresponding carboxylic acid, the proton is easily dissociated and dissolved in water, and except for some lower ones, a mineral acid is added thereto to adjust the pH. When it decreases, it will be easily dissociated and separated from the aqueous phase. Such a tendency becomes more remarkable as the degree of fluorination increases. Therefore, in the present invention, it is preferable to use a completely fluorinated carboxylic acid such as perfluorodecanoic acid or perfluorooctanoic acid.

  Among the above surfactants, for example, perfluorooctanoic acid is solid at room temperature. Therefore, when the pH of the aqueous solution containing perfluorooctanoic acid is lowered, perfluorooctanoic acid is precipitated as a solid. However, if a hydrophilic organic solvent having compatibility with acetone or tetrahydrofuran or the like is allowed to coexist in an aqueous solution containing perfluorooctanoic acid, perfluorooctanoic acid forms a precipitated phase together with the hydrophilic organic solvent and water. Separate from.

  In the case where the surfactant used in the present invention is a fluorocarboxylic acid represented by the following general formula (1), if it is made acidic by adding a mineral acid or an organic acid to a pH of 6 or less, it will react with microorganisms. The microorganisms can be concentrated as a precipitation phase in which the agent, the hydrophilic organic solvent, and water are mixed. That is, if fluorocarboxylic acid is used as a surfactant, the pH is reduced to about 6 or less and does not need to be lowered much. Therefore, it is suitable for concentrating microorganisms alive or concentrating genes without damaging them.

Here, R is a fluoroalkyl group, and at least one hydrogen atom bonded to each carbon atom is substituted with a fluorine atom. In general, X is a divalent organic group containing no fluorine. Specifically, it is an alkylene group having about 1 to 4 carbon atoms, an oxyalkylene group, or an alkylene group having a heteroatom such as oxygen, nitrogen or sulfur in the carbon chain. For example, —CH ₂ CH ₂ SCH ₂ CH ₂ —, —CH ₂ —O—CH ₂ — and the like can be mentioned.

  As the hydrophilic organic solvent, one or more of alcohol, ether, ketone, amide, nitrile and sulfoxide are suitable. For example, ethanol, methanol, tetrahydrofuran, dioxane, acetone, dimethylformamide, acetonitrile, dimethyl sulfoxide and the like are suitable.

  When the hydrophilic organic solvent is selected from ethers, amides, alcohols and ketones, it is possible to concentrate the microorganisms to the precipitated phase while maintaining their form. As the ether, tetrahydrofuran is particularly suitable, as the amide, particularly dimethylformamide, as the alcohol, particularly ethanol, and as the ketone, acetone is particularly suitable.

Step 2
In this step, the pH of the solution obtained in Step 1 is lowered, and this solution is separated into two phases: a precipitation phase in which microorganisms, a surfactant, a hydrophilic organic solvent, and water are mixed, and a supernatant phase. To do.

As a reagent for lowering the pH, for example, hydrochloric acid, nitric acid, and sulfuric acid are preferably used.
According to the microorganism concentration method of the present invention, when a water-soluble substance and a hydrophilic substance coexist with a microorganism, the microorganism can be separated into a precipitation phase, and the water-soluble substance and the hydrophilic substance can be separated into a supernatant phase. Thereby, microorganisms can be concentrated.

  When adding a reagent for lowering pH to a solution composed of a surfactant, a hydrophilic organic solvent, a microorganism and water, it is generally not left as it is after the addition, but it is allowed to stand after it is gently stirred. Is preferred. That is, if mixing is not performed by stirring, it takes time until the acid is uniformly diffused, and the reaction is delayed by that amount, so that phase separation is delayed. Therefore, it is preferable to provide a stirring process.

The stirring is preferably performed by shaking or using a stirrer, vortex mixer or the like.
In step 2, when the precipitation phase is formed, if the temperature of the entire reaction solution is lowered, the precipitation rate is increased. This is presumably because the solubility of the hydrophilic organic solvent that dissolves in water decreases as the temperature decreases.

  In addition, when the pH of a solution composed of a surfactant, a hydrophilic organic solvent, a microorganism and water is lowered and then centrifuged, the precipitated phase can be formed in a shorter time than the time for allowing it to stand to form a precipitated phase. Can be formed.

As shown in FIG. 1 (a), 10 μL of Escherichia coli (ATCC: 10798) (generic name: E. coli) as a test strain was shake-cultured in an LB medium at 37 ° C. for 18 hours (the number of E. coli was about 10). ⁷ ) 2.5 mL of 0.1 M perfluorooctanoic acid 2 was added to pure water 1 (769 ml). Next, 38 mL of tetrahydrofuran 3 was added. Further, 192 mL of 6.0 M hydrochloric acid 4 was added, the lid of the container was closed, and the mixture was shaken up and down 30 times, and stirred so that the solution became uniform. This was left to stand at room temperature for 3 hours to form a precipitated phase 5 on the bottom of the container as shown in FIG. After measuring the volume of the precipitated phase using a pipette, take 5 μL each of the supernatant phase 6 and the formed precipitated phase 5, place each on a slide glass, and further cover with a cover glass. The number of bacteria was measured by counting the number of bacteria.

As a result, number of E. coli in the supernatant phase whereas 0 / was 5 [mu] L, E. coli number of precipitated phase was 5.8 × 10 ⁶ cells / 5 [mu] L. Since the volume of the formed precipitated phase was about 20 μL, the number of E. coli in the precipitated phase was determined by the following calculation formula.

  The number of E. coli in the precipitation phase was almost the same as the number of E. coli added at the beginning, and E. coli was not found in the supernatant phase, indicating that the recovery rate of E. coli was almost 100%.

Moreover, the concentration rate of the present Example was calculated | required with the following formula | equation.

It is a conceptual diagram explaining one Example of the microorganism concentration method which concerns on this invention.

Explanation of symbols

1 Mixed solution of E. coli and pure water 2 Perfluorooctanoic acid 3 Tetrahydrofuran 4 Hydrochloric acid 5 Precipitation phase containing E. coli 6 Supernatant phase

Claims (3)

Add a surfactant and a hydrophilic organic solvent to a solution containing microorganisms to form a uniform solution,
Then lower the pH of the solution,
The solution is separated into two phases, a precipitate phase in which microorganisms, a surfactant, a hydrophilic organic solvent, and water are mixed, and a supernatant phase,
A method for concentrating microorganisms characterized by concentrating microorganisms in the precipitated phase ,
Here, the method for concentrating microorganisms, wherein the surfactant is a fluorocarboxylic acid and the hydrophilic organic solvent is compatible with the fluorocarboxylic acid . 2. The microorganism concentration measuring method according to claim 1, wherein the surfactant is perfluorooctanoic acid . The method for concentrating microorganisms according to claim 1, wherein the hydrophilic organic solvent is tetrahydrofuran, dimethylformamide, ethanol or acetone .

Images