JP5066720B2 - Quinone profile method using quinone compounds extracted using compressed carbon dioxide - Google Patents

Description

  The present invention relates to a quinone profile method, which is a technique for analyzing environmental microbial communities, and relates to a simple and rapid method using compressed carbon dioxide (particularly supercritical carbon dioxide).

Research on microorganisms in the environment as a community, analysis on the amount and type of microorganisms at a certain point in time, and dynamic analysis on how these microorganisms change with time is progressing. This research not only grasps the environmental pollution status, but also characterizes and evaluates environmental water, soil, compost, agricultural land or activated sludge, environmental remediation using microorganisms, environmental purification (bioremediation), biological It can also be applied to control activated sludge and methane fermentation in biological water treatment. Moreover, it becomes an effective method for the primary screening at the time of identifying microorganisms.
Conventionally, it is known that 99% or more of microorganisms present in the environment (for example, in soil) are difficult-to-cultivate microorganisms that cannot be cultured in a laboratory. For this reason, the classical method of isolating and culturing microbial communities can only analyze 1% or less of the total microbial community. In order to solve this difficulty, a method of directly extracting nucleic acids from environmental microorganisms has been developed (Patent Document 1). However, this method is expensive and cumbersome, and it is difficult to perform a quick analysis on site.

  In order to analyze microbial communities, there is a quinone profile method in which a quinone compound is analyzed in addition to the nucleic acid analysis method. This is a method proposed based on the fact that quinone compounds such as ubiquinone, menaquinone and plastoquinone are mainly used for each microorganism. That is, this is a method for analyzing the amount and type of microorganisms by extracting various quinone compounds from microorganisms in the environment and quantitatively analyzing each quinone compound.

  In order to carry out the quinone profile method, an organic solvent such as chloroform / methanol is added to a sample (for example, soil, water, air) collected from the environment, followed by an extraction process such as shaking / extraction, followed by pretreatment, An analysis process using a liquid chromatograph was performed. However, this method required a long time of about 2 hours for the extraction step, about 0.5 hour for the pretreatment and about 2 hours for the analysis step. For this reason, it was difficult to carry out analysis easily and quickly at the site. Furthermore, since a large amount of organic solvent is used in the extraction process, the load on the environment is large. In addition, since the operation is complicated, the quantitative property is poor, and the sensitivity is not good, it is not suitable for the determination of a small amount of sample and the analysis of microorganisms in the atmosphere. In addition, it has been difficult to develop a device that can be smoothly implemented while achieving automation and downsizing.

  In conventional research, a method for extracting a quinone compound using compressed carbon dioxide has been developed (Patent Document 2). However, there has been no research using the extracted sample obtained by this method for the quinone profile method. In addition, Dr. White, University of Tennessee, an American researcher, has reported to academic societies the results of extracting only three types of ubiquinones from environmental samples using the SFE (Supercritical fluid extraction) method. However, this report does not describe the extraction efficiency and has not been applied to the quinone profile method. According to the inventor's interview, Dr. White said the study was stopped because the SFE method did not work.

In recent years, the effect of coenzyme Q10 (ubiquinone 10) is being recognized, and the amount of its use is rapidly increasing. In order to cope with this, it is desired to establish a simple and rapid method for extracting and purifying ubiquinone 10.
The present invention has been made in view of the above-mentioned problems, and its purpose is to extract a quinone compound from an environmental sample simply and quickly, and to investigate a microbial community using this quinone compound. It is in providing the method etc. which can implement. Another object is to provide a method for extracting and purifying ubiquinone 10 simply and quickly.
JP-A-2005-65605 German Patent Publication No. 294280 (DD294280 A5)

By using compressed carbon dioxide (especially supercritical carbon dioxide), the present inventors can easily and rapidly extract a quinone compound derived from a microorganism from an environment-derived sample, and the quinone compound thus extracted is used in the quinone profile method. By providing, it was found that the microbial community in the environment could be investigated, and the present invention was basically completed.
That is, the method for extracting a quinone compound according to the first invention includes an extraction step of bringing compressed carbon dioxide into contact with an environment-derived sample.

A “quinone compound” is a substance that exists as a fat-soluble component in a cell membrane of a microorganism and is involved in respiratory chain / photosynthetic electron transfer as one of electron transfer substances. Examples of quinone compounds include ubiquinone (UQ), menaquinone (MK), and plastoquinone (PQ). However, according to the present invention, vitamins such as vitamin K1 (VK1) are well extracted. Therefore, in the present invention, the quinone compound can include various vitamins in a broad sense.
The quinone compound has a different structure depending on the length of each skeleton and isoprene side chain. Ubiquinone and menaquinone having n isoprene side chains are referred to as UQ-n and MK-n, respectively. Further, they are referred to as UQ-n (Hx) and MK-n (Hx) depending on the difference in hydrogen saturation x (see FIG. 1). Each quinone compound exhibits a specific oxidation-reduction potential depending on differences in skeleton type, number of isoprene side chains, side chain saturation, and the like, and therefore quinone molecular species differ depending on differences in energy metabolism in microorganisms. For this reason, by separating and quantifying each quinone, it is possible to analyze the characteristics of the microbial community (that is, the amount and type of microorganisms) in the environment-derived sample. This analysis method is called a quinone profile method. In addition, some quinone compounds have industrial applicability alone. For example, ubiquinone 10 is manufactured and sold in large quantities as coenzyme Q10. In the present invention, such a quinone compound can be extracted quickly and easily. For this reason, in the present invention, the quinone compound is preferably ubiquinone 10.

“Environment-derived sample” means a sample collected from soil, water, air, or a microbial fermentation layer.
More specifically, examples of soil include coastal areas of rivers, seas, lakes, ponds, river bottoms, lake bottoms, tidal flats, farmland, forests, wetlands, grasslands, compost, biological wastewater treatment sludge, etc. Is done. In order to apply the present invention to soil, the soil itself can be used as an environment-derived sample. A sample (liquid or solid) extracted from soil using an appropriate extract (water, organic solvent, separation column, etc.) can also be used as an environment-derived sample.

Examples of water include rivers, lakes, tidal flats, agricultural land, forests, wetlands, grasslands, and biological wastewater. In order to apply the present invention to water, water itself can be used as an environment-derived sample. A sample (liquid or solid) extracted from water using an appropriate extract (organic solvent, separation column, etc.) can also be used as an environment-derived sample.
As the atmosphere, in addition to the general atmosphere, the atmosphere in a closed space (for example, exhaled breath, building, underpass, cave, etc.) is exemplified. In order to apply the present invention to the atmosphere, in addition to using the atmosphere itself as an environment-derived sample, the microorganism is concentrated by passing it through a filter or column that captures microorganisms while compressing the atmosphere with a compressor. It can be used as a derived sample.

  The microorganism fermentation layer means a fermentation layer cultured for extracting a ubiquinone compound of a microorganism containing a specific ubiquinone compound (for example, ubiquinone 10). Examples of such microorganisms include microorganisms isolated from those naturally containing ubiquinone 10, or microorganisms incorporating a gene for producing ubiquinone 10 (for example, JP 2002-345469, JP 2002-191367, JP 2001-61478, JP-A 2000-228987, WO 2002/040682).

“Compressed carbon dioxide” means supercritical carbon dioxide or liquid carbon dioxide. Carbon dioxide is a substance having a critical temperature (Tc) of 31 ° C. and a critical pressure (Pc) of 7.39 MPa. Temperature and pressure regions beyond Tc and Pc are called supercritical states and exhibit both liquid and gas characteristics. In the present invention, it has been found that supercritical carbon dioxide or liquid carbon dioxide acts as a good solvent for extracting a quinone compound. In the present invention, of the compressed carbon dioxide, supercritical carbon dioxide can be preferably used.
“Contacting” means that the quinone compound can be extracted from an environment-derived sample by using compressed carbon dioxide. Specific examples include a method in which an environment-derived sample is added to compressed carbon dioxide and extracted by stirring, a method in which compressed carbon dioxide is passed through the environment-derived sample, and the like. In addition, ultrasonic waves or microwaves can be used for extraction.

  As a condition for extracting the quinone compound, carbon dioxide may be in a compressed state (that is, in a liquid or supercritical state). More specifically, when the temperature is about −56 ° C. and the pressure is about 5 MPa or more, liquid carbon dioxide is obtained, and therefore, it can be implemented. However, it is preferable to carry out under conditions that satisfy a supercritical state in which the temperature is about 31 ° C. or higher and the pressure is about 7.39 MPa or higher. In this case, if the temperature becomes too high, there is a concern that the stability of the quinone compound may be reduced. Therefore, the extraction is preferably performed at about 70 ° C. or less.

In the present invention, the extracted quinone compound may be limited due to the polarity of compressed carbon dioxide. In order to avoid this, it is possible to extract various quinone compounds having different polarities by adding an appropriate organic solvent in addition to compressed carbon dioxide. Examples of the organic solvent used at this time include methanol, ethanol, acetone, chloroform, hexane, diethyl ether and the like. Of these, methanol, ethanol, acetone, and chloroform are preferable, methanol, ethanol, and acetone are more preferable, and methanol is most preferable. The mixing ratio of the organic solvent is 1% or more, preferably 3% or more, and more preferably 5% or more with respect to the total amount of the compressed carbon dioxide and the organic solvent.
Further, when ubiquinone 10 is extracted as a quinone compound, since administration to humans is considered, it is preferable to use ethanol in consideration of safety. However, if sufficient consideration is given to safety, it is preferable to use methanol from the viewpoint of extraction efficiency.

In the present invention, it is preferable to provide an adsorption step for adsorbing the extracted quinone compound after the extraction step of the quinone compound using compressed carbon dioxide. This is because the quinone compound can be quickly separated and purified.
The adsorption step means adsorbing to a suitable adsorbent material in order to separate the quinone compound from compressed carbon dioxide and other compounds. Here, the adsorbing substance means a substance having an ability to adsorb a quinone compound under an appropriate condition, and for example, an inorganic adsorbent having an adsorbing ability such as silica and alumina can be used.

  Moreover, it is preferable to apply to the quinone profile method which monitors the microbial community in an environment using the quinone compound extracted by the method of this invention. The quinone profile method is one of the methods for analyzing the microbial community in the environment. The quinone compound called ubiquinone, menaquinone and plastoquinone is mainly used for each microorganism. Based on the proposed method. By extracting various quinone compounds from microorganisms in the environment and performing quantitative analysis on each quinone compound, the amount and type of microorganisms can be analyzed. As for the quinone profile method, a redundant extraction method of quinone compounds using an organic solvent has been conventionally used, and thus it has not been used for rapid microbial community analysis in the field. According to the study by the present inventors, an extraction sample to be applied to the quinone profile method has been successfully obtained by applying a method for extracting a quinone compound using compressed carbon dioxide. In addition, the quinone profile method according to the method of the present invention not only provides a good degree of dissimilarity with the results of the quinone profile method obtained by the conventional extraction method, but also can distinguish a smaller difference in microbial flora. I also understood that.

The quinone compound extraction apparatus according to the second invention includes an extraction container into which an environment-derived sample can be introduced, a liquid feed line through which compressed carbon dioxide is inserted into the extraction container, and a pump that sends compressed carbon dioxide to the liquid feed line. And a pressure regulator for keeping the inside of the extraction container at a predetermined pressure, and an adsorbing member for adsorbing the extracted quinone compound.
In the second aspect of the present invention, a plurality of extraction containers and a plurality of adsorption members are provided in parallel to the liquid feed line, and the environment is controlled while moving and controlling so that one adsorption member corresponds to one extraction container. It is preferable to adopt a configuration in which extraction and adsorption from a derived sample are performed. With such a configuration, extraction and adsorption of a quinone compound from a plurality of environment-derived samples can be automated, so that a simple and rapid quinone compound extraction apparatus can be provided.

According to the present invention, by using compressed carbon dioxide (particularly supercritical carbon dioxide), the operation is simple and quick (about 1/5 of the conventional method) with almost no organic solvent and in the environment. A quinone compound derived from a microorganism can be extracted. Since the quinone compound thus obtained can be applied to the quinone profile method, it is also possible to conduct a local microbial community survey that was impossible with the conventional method.
Further, according to the present invention, it is possible to develop an apparatus that can be smoothly implemented while achieving automation and downsizing.

Next, embodiments of the present invention will be described in detail with reference to the drawings. However, the technical scope of the present invention is not limited by the following embodiments, and various changes can be made without changing the gist thereof. It can be carried out with modification. Further, the technical scope of the present invention extends to an equivalent range.
<Activated sludge sample>
The activated sludge sample was collected from the aeration tank of the wastewater treatment plant at Toyohashi University of Technology. The sludge sample was lyophilized for 24 hours, and fine particles of 500 μm or less were collected by sieving.

<Extraction process using supercritical carbon dioxide>
In FIG. 2, the outline | summary of the quinone compound extraction apparatus used for this embodiment was shown. A carbon dioxide cylinder 1 is provided in the left direction (upstream) in the figure. A cooler 2 that maintains the liquid state by cooling carbon dioxide is connected to the downstream side of the cylinder 1 via a pump 3 (SCF-201 pump (manufactured by JASCO Corporation)). Further downstream, an organic solvent tank 4 is connected via a pump 5 (SCF-201 pump (manufactured by JASCO Corporation)). In the middle of the liquid feed line 6, a joint portion 7 for mixing liquid carbon dioxide and an organic solvent is provided. The mixed solvent passes through the mixing column 8 and is sent to the extraction container 9. A heater 10 (353B GC oven (GL Sciences)) is provided outside the mixing column 8 and the extraction container 9 to keep the mixing column 8 and the extraction container 9 at a predetermined temperature. Can do. A multi-wave type UV detector 13 (MD-1510 (manufactured by JASCO Corporation)) having a high-pressure cell is provided downstream of the extraction container 9, and the presence or absence of a substance extracted from the sample can be detected. A back pressure regulator 11 (880-81 back pressure regulator (manufactured by JASCO Corporation)) is provided downstream of the UV detector 13 so as to control the internal pressure of the extraction container 9. Further, an adsorption cartridge 12 is mounted on the most downstream side of the line in the right direction in the figure, and a quinone compound is adsorbed here.

In order to carry out the extraction process, about 0.1 g of sludge sample is put into the extraction container 9 and then the pumps 3 and 5 are driven while the heater 10 is controlled to a predetermined temperature, and the dioxide is removed from the line 6. It was performed by inserting carbon and an organic solvent at a predetermined flow rate.
The extraction process conditions were as follows. The extraction vessel temperature was 25 ° C. to 65 ° C., the pressure was 10 MPa to 35 MPa, the total flow rate of carbon dioxide flow and organic solvent was 3.0 mL / min. Further, the extraction time of the quinone compound from the sludge sample was set to 15 minutes unless otherwise specified. At the most downstream of the apparatus, a silica gel column (two Sep-Pak Plus Silica cartridges, manufactured by Waters) is attached as a cartridge 12 for carrying out the adsorption step, and the extracted quinone compound is added. Captured.

  After the extraction step and the adsorption step were completed, the captured quinone compound was eluted by washing the silica gel column with acetone. Further, after the quinone compound in acetone was adsorbed on another silica gel column (two Sepppack columns), the menaquinone compound was eluted and separated by first passing through a 2% diethyl ether / hexane solution. Next, the ubiquinone compound was eluted by passing a 10% diethyl ether / hexane solution through a Sepppack column. Each elution solution was evaporated and concentrated with an evaporator, and then washed and collected with acetone. Thus, the menaquinone compound and the ubiquinone compound were separated and purified and analyzed by HPLC.

<Extraction process using organic solvent: conventional method>
For comparison, an extraction process by a conventional organic solvent method was performed (J. Biosci. Bioeng., 87, 378-382). The extraction process was as follows. That is, an organic solvent (chloroform: methanol = 2: 1 (v / v)) is added to about 0.1 g of sludge sample, shaken with a shaker for 30 minutes, centrifuged at 9000 rpm for 10 minutes, and then contains a quinone compound. The chloroform layer was separated. This extraction operation was repeated three times.

  Next, a mixture of hexane and water (hexane: water = 25: 15 (v / v)) was added to the chloroform layer and shaken, and then centrifuged at 7000 rpm for 10 minutes, and the quinone compound was re-applied from the chloroform layer to the hexane layer. Extracted. After the hexane layer was appropriately evaporated and concentrated using an evaporator, the hexane layer was passed through a sep pack cartridge to adsorb the quinone compound. Subsequently, the menaquinone compound was eluted and separated through a 2% diethyl ether / hexane solution, and the ubiquinone compound was eluted through a 10% diethyl ether / hexane solution. Each solution was evaporated and concentrated with an evaporator, and then washed and collected with acetone. Thus, the menaquinone compound and the ubiquinone compound were separated and purified and analyzed by HPLC.

<Analysis by HPLC>
The separated and purified quinone compound was analyzed by HPLC (manufactured by Shimadzu Corporation). The column is an ODS column (Zorbax-ODS, 4.6 mm I.D.x250 mm, Agilent Technologies, USA), and the detector is a UV-Vis detector (Model SPD-10A, manufactured by Shimadzu Corp.) and photodiode detection. A vessel (SPD-M10A, manufactured by Shimadzu Corporation) was used. The column oven was maintained at 35 ° C. Methanol: isopropanol = 9: 2 (v / v) was used as the mobile phase, and the flow rate was 1.0 mL / min. Each quinone compound was identified by the retention time and the UV spectrum pattern of each peak. Ubiquinone 10 (UQ-10) and vitamin K1 were used for the determination of each ubiquinone and menaquinone. Absorbance data at 275 nm was used to quantify the ubiquinone compound, and absorbance data at 270 nm was used to quantify the menaquinone compound.

<Test results>
1. Results when the extraction solvent was changed FIG. 3 shows the concentration of the quinone compound extracted when the extraction solvent was changed. The temperature at the time of extraction was 35 ° C., and the pressure was 25 MPa. First, when the extraction solvent was carbon dioxide only (flow rate: 3.0 mL / min), the quinone compound could be extracted from the activated sludge sample as shown at the left end of the graph. However, the concentration of the extracted quinone compound was low. At this time, minor quinone compounds (for example, MK-10, MK-5 (H4), MK-10 (H4), and MK-10 (H8)) were not extracted. From this result, it was considered that carbon dioxide alone was not sufficient to dissolve polar substances. Then, it tried to improve the extraction efficiency of a quinone compound by mixing a polar organic solvent (namely, methanol, ethanol, acetone, and chloroform) with a carbon dioxide.

  The mixing ratio of each organic solvent was 10% of the total extraction solvent. That is, the carbon dioxide flow rate was 2.7 mL / min, and the organic solvent flow rate was 0.3 mL / min. As shown in FIG. 3, the amount of quinone extracted increased by mixing a polar organic solvent. At this time, the total amount of the extracted quinone compound decreased in the order of methanol, acetone, ethanol, and chloroform.

  Next, the change of the extraction amount of the quinone compound when changing the mixing ratio of methanol was confirmed. FIG. 4 shows changes in the extraction amount of the quinone compound when the mixing ratio of methanol is 1%, 5%, 10%, and 20%. In all cases, the total flow rate of carbon dioxide and methanol was 3.0 mL / min. When the mixing ratio was 5% or more, the quinone compound could be extracted sufficiently satisfactorily. However, when the mixing ratio was up to 10%, the amount of quinone extracted increased as the mixing ratio increased. Further, when the mixing ratio was 20% and 10%, no difference was found in the results. Therefore, in the following experiment, extraction of the quinone compound was performed with a methanol mixing ratio of 10% (that is, a carbon dioxide flow rate of 2.7 mL / min and a methanol flow rate of 0.3 mL / min).

2. Result when the pressure at the time of extraction was changed The change of the extraction amount of the quinone compound when the pressure at the time of extraction was changed was confirmed. FIG. 5 shows changes in the extraction amount of the quinone compound when the pressure is 10 MPa, 15 MPa, 20 MPa, 25 MPa, and 30 MPa. As other conditions, the temperature was 35 ° C., the carbon dioxide flow rate was 2.7 mL / min, the methanol flow rate was 0.3 mL / min, and the treatment time was 15 minutes.

  The quinone compound could be extracted sufficiently satisfactorily at any pressure. However, when the pressure was between 10 MPa and 25 MPa, the amount of extracted quinone gradually increased as the pressure increased. Moreover, when the pressure was 30 MPa and 25 MPa, no difference was observed in the results. Therefore, in the following experiment, extraction of the quinone compound was performed at a pressure of 25 MPa.

3. Results when the temperature during extraction was changed Changes in the amount of quinone compound extracted when the temperature during extraction was changed were confirmed. FIG. 6 shows changes in the amount of quinone compound extracted when the temperature was 25 ° C., 35 ° C., 45 ° C., 55 ° C., 65 ° C., and 75 ° C. As other conditions, the pressure was 25 MPa, the carbon dioxide flow rate was 2.7 mL / min, the methanol flow rate was 0.3 mL / min, and the treatment time was 15 minutes.

  The quinone compound could be extracted sufficiently satisfactorily at any temperature. However, when the temperature was between 25 ° C. and 55 ° C., the amount of extracted quinone gradually increased as the temperature increased. Moreover, the tendency for the extraction amount of a quinone compound to reduce a little was recognized by temperature 65 degreeC and 75 degreeC. Since some quinone compounds are vulnerable to heat, we thought that decomposition might occur. Therefore, in the following experiment, the quinone compound was extracted at a temperature of 55 ° C.

4). Result when changing extraction time The change of the extraction amount of the quinone compound when changing extraction time was confirmed. FIG. 7 shows changes in the extraction amount of the quinone compound when the extraction time is 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, and 30 minutes. As other conditions, the pressure was 25 MPa, the temperature was 55 ° C., the carbon dioxide flow rate was 2.7 mL / min, and the methanol flow rate was 0.3 mL / min.
In any extraction time, the quinone compound could be extracted sufficiently satisfactorily. However, when the extraction time was between 5 minutes and 15 minutes, the amount of quinone extracted gradually increased as the time increased. In addition, no difference was observed in the extracted quinone compounds when the extraction time was 15 minutes, 20 minutes, 25 minutes, and 30 minutes.

5. Comparison of quinone profiles between the method of the present embodiment and the conventional method (1)
Next, the difference of the quinone profile when the quinone compound was extracted from the activated sludge sample was confirmed between the method of the present embodiment (hereinafter sometimes referred to as “SFE method”) and the conventional method. The extraction conditions in the method of this embodiment were a pressure of 25 MPa, a temperature of 55 ° C., a carbon dioxide flow rate of 2.7 mL / min, a methanol flow rate of 0.3 mL / min, and an extraction time of 15 minutes.

  8 and 9 show HPLC patterns after extraction of ubiquinone and menaquinone in each of the method of this embodiment and the conventional method. Peaks 1 to 4 in the chart of FIG. 8 indicate UQ-7, UQ-8, UQ-9, and UQ-10, respectively. Moreover, peaks 1 to 12 in the chart of FIG. 9 are MK-6, MK-7, MK-8, MK-8 (H2), MK-8 (H4), MK-9, MK-9 (H2), respectively. ), MK-9 (H4), MK-10, MK-10 (H2), MK-10 (H4), and MK-10 (H6). By comparing these charts, the method of the present embodiment and the conventional method seemed to obtain qualitatively equivalent results for the type and amount of the extracted quinone compound.

  Table 1 shows the results of confirming the extraction reproducibility of the above 16 types of quinone compounds (4 types of ubiquinone and 12 types of menaquinone) using the method of the present embodiment by 5 tests.

This result shows that according to the method of this embodiment, any quinone compound can be extracted with good reproducibility.
FIG. 10 is a graph comparing the total quinone compound amount, the total menaquinone compound amount, and the total ubiquinone compound amount extracted from the activated sludge sample by the method (SFE Method) of the present embodiment and the conventional method (Conventional Method). showed that. Comparing the data of both methods, it was shown that the quinone compound was well extracted in both cases. Also, the total quinone amount and the total menaquinone amount / total ubiquinone amount seemed to have good similarity in both methods.

  In FIG. 11, the graph which compared the molar ratio of each quinone compound extracted from the activated sludge sample by the method of this embodiment and the conventional method was shown. As shown in the figure, the ratio of each quinone compound extracted by both methods seemed very similar. In both methods, among the extracted menaquinone compounds, the main ones were MK-10 (H4), MK-9 (H2), MK-6, and MK-7. Of the ubiquinone compounds, the main ones were UQ-8, UQ-10, and UQ-9.

Next, a dissimilarity index D was defined in order to quantitatively compare the results of both methods. That is, the obtained quinone profile (that is, the ratio of each quinone compound (i and j)) data was applied to the following formula (1), and the numerical value D was obtained.

  In the formula, fki and fkj indicate the ratio of each quinone in the quinone compound k (that is, ubiquinone compound or menaquinone compound). The larger D, the more different the two quinone profiles are. Moreover, if D is 0, it has shown that both quinone profiles correspond. In the conventional method and the method of the present embodiment, the D value was 0.09, which was sufficiently small. This indicates that both methods show substantially the same results. Furthermore, microbial diversity of quinone profiles (microbial diversity of quinone profiles) DQ was defined by the following formula (2).

式中、ｆk はキノン化合物kの割合を示し、ｎは微生物の割合が０.００１以上であったものの数を示している。本実施形態の方法の結果では、ＤＱ値は１１.９９であった。一方、従来法の結果では、ＤＱ値は１１.９５であった。このことより、両方法は、よく似た微生物多様性度を示すことがわかった。

In the formula, fk represents the ratio of the quinone compound k, and n represents the number of microorganisms having a ratio of 0.001 or more. As a result of the method of the present embodiment, the DQ value was 11.99. On the other hand, in the result of the conventional method, the DQ value was 11.95. This indicates that both methods show similar microbial diversity.

6). Comparison of quinone profiles between the method of this embodiment and the conventional method (2)
Next, the activated sludge after the university waste water treatment and the activated sludge after the machine factory waste water treatment (aerobic tank) are subjected to the quinone compound extraction operation by the conventional method and the method of this embodiment, and the extracted sample is subjected to the quinone profile method. Went. The extraction conditions of the method of this embodiment are the same as described above. By each extraction method, the quinone compound was extracted 3 times from each activated sludge, and the quinone compound was measured.

The results are shown in FIGS. As shown in FIG. 12, in the two types of activated sludge, there was no significant difference in the total amount of quinone compound extracted from the cells between the method of this embodiment and the conventional method. Moreover, as shown in FIG.13 and FIG.14, in both methods, the kind and the number of the extracted quinone compound were also the same.
The dissimilarities of the results of the quinone profile method using quinone compounds extracted by both methods are 0.057 (activated sludge after university wastewater treatment) and 0.068 (activated sludge after machine factory wastewater treatment, respectively) )Met. That is, since the results of the quinone profile method performed with the quinone compound obtained by the conventional method and the method of this embodiment were comparable, it was found that the method of this embodiment can replace the conventional extraction method. It was.

7). The quinone profile based on the method of this embodiment for various sludges Next, sludge after a university wastewater treatment (aerobic tank), sludge after a general sewage wastewater treatment (aerobic tank and anaerobic tank), after machine factory wastewater treatment About seven kinds of sludge (aerobic tank and anaerobic tank) of sludge (aerobic tank and anaerobic tank) and sludge (aerobic tank and anaerobic tank) after wastewater treatment of food processing plant, the quinone compound is extracted by the method of this embodiment, and the extraction is performed. The sample was subjected to the quinone profile method.

  The results are shown in FIG. As shown in FIG. 15, for any sludge, it was possible to extract the quinone compound from the cells well. Moreover, about the quinone profile obtained about the sludge of the aerobic tank and the anaerobic tank, the difference of dissimilarity was hardly seen. This result was consistent with existing research results obtained by conventional methods. In addition, since the microbial community structure differs depending on the drainage characteristics, it was found that for each drainage, an appropriate sludge treatment operation based on each characteristic must be used. From these things, it turned out that the method of this embodiment can be applied suitably for the quinone profile method for analyzing the microbial cell in activated sludge.

  FIG. 16 shows the result of analyzing each sludge sample three times in the result of FIG. 15 and showing each dissimilarity for one sample. From this figure, it was found that the variation in dissimilarity was very small for each sample, and good reproducibility was exhibited. The maximum value of the obtained dissimilarity was sludge (anaerobic tank) after wastewater treatment at the machine factory, and was 0.066.

  This result will be further described with reference to FIG. In FIG. 16, it is assumed that the dissimilarity of the quinone profile method obtained from the sample obtained by the extraction method of the present embodiment follows a lognormal distribution. In FIG. 17, the cumulative probability density of each dissimilarity value in that case is plotted. This figure shows the cumulative density of dissimilarity when the quinone profile method is performed on the extraction samples obtained by the extraction method of the present embodiment and the conventional method. The cumulative distribution function of the conventional method has been obtained by the conventional method so far (Dept. of Materials Engineering, Graduate School of Engineering, Yokohama National University, Ph.D. paper “Performance analysis of aerobic biofilter” 180, December 1993). From the figure, in the conventional method, the cumulative probability density reached 97% when the dissimilarity was 0.1 (Akira Hiraishi; Population Dynamics and Environmental Purification, Journal of Water Environment Society, 15 (9 ), Pp.558-563). On the other hand, in the extraction method of the present embodiment, the cumulative probability density reached 97% when the dissimilarity was 0.07.

  That is, in the conventional extraction method, it was difficult to compare the difference in microflora having a dissimilarity of about 0.1 (it is considered that they are the same microbiota). It has been found that by using it, it is possible to distinguish between smaller microbiota differences. In other words, in the conventional method, if the dissimilarity is 0.1 or less, the microbial flora in the two samples must be judged to be the same. On the other hand, in the method of the present embodiment, it was found that if the dissimilarity is not 0.07 or less, it cannot be determined that they are the same microflora.

  FIG. 18 shows the result of cluster analysis of data obtained by performing the quinone profile method on the samples extracted by the method of the present embodiment for the above seven types of sludge. Cluster analysis is an analysis method that calculates dissimilarity for each sample, takes dissimilarity on the horizontal axis, and combines low values (that is, it is judged that the microbiota similarity is high). It is. When FIG. 18 is seen with reference to FIG. 17, it is determined that the microflora is the same if the dissimilarity is 0.1 or less when the conventional method is used. It was difficult to examine the difference between the microflora of the anaerobic tank and the anaerobic tank, and the microflora of the anaerobic tank and the aerobic tank in the mechanical wastewater treatment plant. However, when the extraction method of the present embodiment is used, it is not determined that the microflora is the same unless the dissimilarity is 0.07 or less. Therefore, the microbiota of the aerobic tank and the anaerobic tank in general sewage drainage Can be determined not to be the same. That is, since the accuracy of analysis is improved from 0.1 of the conventional method to 0.07 of the method of the present embodiment, the microflora of the aerobic tank and the anaerobic tank in general sewage drainage is not the same. It can be judged.

8). Comparison of Conventional Extraction Method and Extraction Method of Present Embodiment Next, a comparison between the conventional method and the method of the present embodiment was performed for the extraction method of the quinone compound while integrating the results of 1 to 7 above. FIG. 19 shows a comparison of the characteristics of both methods (the SFE method is the extraction method of the present embodiment).
The SFE method requires a high-pressure apparatus and uses carbon dioxide gas, but the apparatus can be easily automated and the amount of organic solvent used is reduced. In particular, do not use chloroform, which is designated as a specified chemical substance by the Industrial Safety and Health Act. Further, the extraction time can be shortened to 1/6 of the conventional method. Thereby, even when there are a large number of samples, it is possible to cope with the SFE method. In addition, since it was difficult to process many samples rapidly by the conventional method, it was difficult to utilize the quinone profile method in various fields.

9. Test Results on Compost Sample Next, using the compost sample, the influence of the drying process method and extraction time before extraction on the extraction of the quinone compound in the microbial cells by the method of this embodiment was examined. Prior to extraction of the quinone compound, the sample was not dried, the sample was dried for 24 hours at 37 ° C. using an oven, and the sample was freeze-dried for 24 hours. In each sample, the extraction results of the quinone compound obtained in two extraction times of 30 minutes and 60 minutes are shown in FIG. Thereby, the influence which the quantity (water content) of the water contained in a sample has on extraction can be examined. From the figure, it was found that if the drying process is different, the extraction efficiency of the quinone compound by the method of the present embodiment is affected. It was found that the extraction amount of the quinone compound increases when the extraction time is extended from 30 minutes to 30 minutes.

10. Comparison of extraction of quinone compounds from compost and soil Next, using the conventional method and the method of this embodiment (SFE method), (A) compost and (B) extracting quinone compounds from microorganisms in the soil, The results were compared. At this time, each sample was dried at 37 ° C. for 24 hours before extraction. The extraction conditions of the method of this embodiment were 55 ° C., 25 MPa, and 60 minutes.

  The results are shown in FIG. From the figure, quinone compounds could be extracted from compost and soil by the SFE method as in the conventional method. At this time, the number of extracted quinone compounds was also the same. However, it was confirmed that the extraction amount of the quinone compound obtained by the SFE method is lower than that of the conventional method in each sample. However, this is because the extraction conditions of the quinone compound when using the SFE method are not optimized for compost and soil. When the extraction conditions were optimized, it was considered that almost the same amount of quinone compound was extracted between the conventional method and the SFE method as in the case of activated sludge. Moreover, the dissimilarity when the sample obtained by the conventional method and the SFE method is applied to the quinone profile method is 0.054 for (A) compost and 0.031 for (B) soil. there were. Since the obtained dissimilarity was sufficiently low, it was confirmed that the SFE method could replace the conventional method for compost and soil.

11. Measurement of quinone compound using UPLC Next, the quinone compound extracted from the activated sludge by the SFE method was separated and measured using UPLC (Ultra Performance Liquid Chromatography, manufactured by Waters). FIG. 22 shows a chromatogram chart. From this figure, it was found that by using UPLC, ubiquinone and menaquinone can be separated simultaneously in a shorter time than the conventionally used HPLC.

  In HPLC, as shown in FIGS. 8 and 9, about 20 minutes (ubiquinone) and about 50 minutes (menaquinone) are required for the separation of each quinone. In addition, in this case, it is difficult to measure ubiquinones and menaquinones simultaneously. However, by using UPLC, it was possible to separate all of them in less than 30 minutes with ubiquinones and menaquinones flowing at the same time. Thus, it was found that if UPLC is used, it is not necessary to perform a rough fractionation treatment of a quinone compound using a Sep-pak, so that the time required for quinone analysis can be greatly reduced.

As described above, according to the present embodiment, by using supercritical carbon dioxide and a polar organic solvent (particularly methanol), it is possible to easily and quickly form environmental microorganisms with only a very small amount of organic solvent as compared with the conventional method. The derived quinone compound can be extracted. When the extracted sample obtained by this method was applied to the quinone profile method, a result substantially equal to or higher than the conventional method was obtained. For this reason, the method of the present embodiment is a simple and quick method that can replace the conventional method. This method also makes it possible to conduct on-site microbial community surveys that were impossible with conventional methods.
Moreover, according to the method of this embodiment, the quinone compound can be extracted and purified easily and quickly from a microorganism containing a large amount of a specific quinone compound (for example, ubiquinone 10).

  In the present embodiment, only one cartridge 12 is provided. However, according to the present invention, a plurality of extraction containers and a plurality of adsorption members are provided in parallel to the liquid feed line, and one extraction container is provided. Therefore, it is possible to perform the extraction and adsorption from the environment-derived sample while controlling the movement so that one adsorption member is associated. By doing so, extraction and adsorption of the quinone compound from a plurality of environment-derived samples can be automated, so that a simple and rapid quinone compound extraction apparatus can be provided. Further, the apparatus can be downsized.

It is a chemical formula which shows the structure of ubiquinone (UQ-n (Hx)) and menaquinone (MK-n (Hx)). In the figure, (A) shows a ubiquinone compound, and (B) shows a menaquinone compound. It is a figure which shows the outline | summary of the supercritical carbon dioxide extraction apparatus in this embodiment. It is a graph which shows the total amount of the quinone compound extracted when the polar organic solvent was mixed with the carbon dioxide, and the ratio of each quinone compound. From the left, the result of extraction with a mixed solvent of carbon dioxide alone, methanol 10%, ethanol 10%, acetone 10%, chloroform 10% is shown.

It is a graph which shows the change of the extraction amount of a quinone compound when the mixing ratio of methanol is 1%, 5%, 10%, and 20%. It is a graph which shows the change of the extraction amount of a quinone compound when the pressure at the time of extraction is 10 MPa, 15 MPa, 20 MPa, 25 MPa, and 30 MPa. It is a graph which shows the change of the extraction amount of a quinone compound when the temperature at the time of extraction is 25 degreeC, 35 degreeC, 45 degreeC, 55 degreeC, 65 degreeC, and 75 degreeC. It is a graph which shows the change of the extraction amount of a quinone compound when extraction time is 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, and 30 minutes.

It is a chart figure which shows a HPLC pattern when a ubiquinone compound is extracted using the method of the conventional method and this embodiment. (A) shows the HPLC pattern when extracted by the method of the present embodiment, and (B) shows the HPLC pattern when extracted by the conventional method. It is a chart figure which shows a HPLC pattern when a menaquinone compound is extracted using the method of the conventional method and this embodiment. (A) shows the HPLC pattern when extracted by the method of the present embodiment, and (B) shows the HPLC pattern when extracted by the conventional method. It is the bar graph which compared the total quinone compound amount extracted from the activated sludge sample, the menaquinone compound amount, and the ubiquinone compound amount by the conventional method and the method of this embodiment. The left bar shows the result by the conventional method, and the right bar shows the result by the method of the present embodiment. It is the bar graph which compared the density | concentration of each quinone compound extracted from the activated sludge sample by the conventional method and the method of this embodiment. The left bar shows the result by the conventional method, and the right bar shows the result by the method of the present embodiment.

It is a graph which shows a result when a quinone compound is extracted from university wastewater sludge and machine factory wastewater sludge using the conventional method and SFE method, and it applies to a quinone profile method. It is the graph which compared the extraction amount for each quinone about the result of having extracted the quinone compound using the conventional method and SFE method about university wastewater sludge, and applying it to the quinone profile method. It is the graph which compared the extraction amount for each quinone regarding the result of having extracted the quinone compound using the conventional method and SFE method about machine factory wastewater sludge (aerobic tank), and applying to the quinone profile method.

It is a graph which shows a result when extracting a quinone compound using SFE method about various sludge, and applying to a quinone profile method. It is the graph which performed 3 times SFE method extraction about various sludge, and confirmed dissimilarity. It is a graph which shows the relationship between dissimilarity and a cumulative probability density in the conventional method and SFE method. It is a figure which shows the result of having performed the cluster analysis about the quinone profile obtained from seven types of sludge. A dotted line drawn to 0.07 on the horizontal axis indicates a reference value for determining whether or not the microflora is the same in the SFE method.

It is a table | surface which compares the characteristic of the conventional method and SFE method. It is a graph which shows the result when a quinone compound is extracted from the compost after performing various drying processes, and it applies to a quinone profile method. It is a graph which shows the result when extracting a quinone compound from (A) compost and (B) soil, and applying to a quinone profile method about the conventional method and SFE method. It is a chart figure when measuring by UPLC in order to apply the quinone compound extracted from activated sludge by SFE method to a quinone profile method.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Carbon dioxide cylinder 3, 5 ... Pump 6 ... Liquid feeding line 9 ... Extraction container 10 ... Heater 11 ... Back pressure regulator (pressure regulator)
12 ... Suction cartridge (suction member)

Claims (1)

A method for extracting quinone compounds used in a quinone profile method for monitoring microbial communities in the environment,
An extraction step of extracting a quinone compound by contacting compressed carbon dioxide, to which an organic solvent of 5% to 20% is added to an environment-derived sample, under conditions of 25 ° C. to 65 ° C., 10 MPa to 35 MPa, and 5 minutes to 30 minutes; An adsorption step of adsorbing the quinone compound to the first silica gel column; and after the first silica gel column is washed with acetone to elute the quinone compound, the quinone compound is adsorbed to the second silica gel column, The eluate eluted from the second silica gel column with a 2% diethyl ether / hexane solution is used as a menaquinone compound sample, and then the eluate eluted from the second silica gel column with a 10% diethyl ether / hexane solution is used as a ubiquinone compound. as a sample, obtaining said menaquinone compound samples and ubiquinone compound sample key A method for extracting the emission compound,
The method for extracting a quinone compound, wherein the organic solvent is at least one selected from the group consisting of methanol, ethanol, and acetone.

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