JP4382202B2 - Fat content measurement method - Google Patents

Description

[0001]
[Industrial application fields]
The present invention measures the dielectric constant and / or conductivity of an electrolyte solution by utilizing the dielectric relaxation phenomenon of a particle suspension in the case of particles in which an electrolyte is present in an electrolyte solution surrounded by an insulator film. This relates to a method for measuring the concentration of the non-electrolyte in the electrolyte solution. More specifically, the present invention relates to a simple and rapid method for measuring the amount of oil and fat present in cells surrounded by a cell membrane that is an insulator film and filled with a cell substrate that is an electrolyte solution.
[0002]
[Prior art]
Since most of the fats and oils obtained from plants and microorganisms accumulate in the cells, the measurement of the fat and oil content is very important in the acceptance inspection of the fats and oils raw material, the production process control, and the improvement of the process. Usually, in order to measure the fat content, the fat must first be extracted. As a method for extracting the fat, a method using an organic solvent such as hexane or chloroform, or a method of extracting by crushing or pressing the raw material Etc. are known. After extraction of fats and oils, impurities and other solvents are further removed, and the weight and fat weight measurement method and chromatographic method for fat and oil content measurement are often used, but these methods are complicated and require a lot of time. There is a problem of requiring labor.
[0003]
For example, in a conventional method for measuring the fat content in microbial cells (see, for example, JP-A-6-153970), filtration and drying for obtaining dry cells from the culture solution takes about 3 hours. Cost. Next, it takes about 1 hour to extract total lipids by the extraction method of Bligh & Dyer. It takes 3 hours to methyl esterify this with anhydrous methanol-hydrochloric acid. About 4 hours are required to extract the fatty acid methyl ester and remove the solvent. Furthermore, in order to dissolve the methyl ester concentrate again in a predetermined amount of solvent and analyze it by gas chromatography, it takes about 1 hour when taking into account the time for eluting all fatty acid components by chromatography. . In this way, the time required for the analysis is about 12 hours in total. However, since the analysis is generally performed by daytime work, the analysis is completed on the next day after the sample is collected. For this reason, it has been practically impossible with the prior art to use the amount of fatty acid produced as a process control item because the time difference between the actual process progress and the end of analysis is large.
[0004]
On the other hand, the dielectric constant obtained by electrical measurement of microbial suspension is known to have a high correlation with the cell concentration of the microbial suspension, and electrical measurement of dielectric constant is quick and simple. The dielectric constant is used for measuring the bacterial cell concentration. The features of dielectric constant measurement are (1) easy measurement and continuous automatic measurement, (2) measurement of turbid samples and colored samples that are difficult with optical measurement, and (3) direct measurement of cell concentration. Therefore, it is more accurate than indirect measurement from the amount of metabolism of cells, and (4) it can measure only living cells because it uses the barrier ability against ions of cell membranes.
[0005]
For example, electrical measurement of dielectrics can be performed in wastewater treatment (Japanese Patent Publication No. 4-25800), cultured yeast ("Instrumentation" (1996), Vol. 39, No. 3, pages 51-55), animal and plant culture. Cells ("Bioprocess Engineering" (1994), No. 11, pp. 213-222), immobilized microorganisms in culture ("J. Fermentation and Bioengineering" (1992), Vol. 72, No. 4, 291-291). 295)), and attempts to measure the concentration of cells or cells have been reported. In the above (), an example of a document reporting such an attempt is given.
[0006]
In addition to attempts to measure the concentration of these cells or cells, attempts to measure changes in the volume of intracellular vacuoles have also been reported ("biochimica et Biophysica Acta" (1995), No. 1245, No. 99- (See page 105). However, this is an example measured by analyzing the dielectric relaxation of the vacuolar insulator film (vacuum membrane), not the dielectric relaxation by the cell membrane.
[0007]
As described above, there have been many reports on the use of dielectric measurement. However, an attempt to easily and quickly measure the content of non-electrolytes such as fats and oils in an electrolyte solution surrounded by an insulator film such as a cell membrane has been reported. Not reported until.
[0008]
For this reason, for example, when fats and oils such as highly unsaturated fatty acids are produced by microorganisms, a method for easily and rapidly measuring the content of fats and oils produced by the microorganisms and accumulated in the cells is strongly desired. It was.
[0009]
[Problems to be solved by the invention]
The present invention has been proposed in view of the above-described problems of the prior art, and an object of the present invention is to simply and easily reduce the amount of fats and oils present in a cell substrate surrounded by a cell membrane that is an insulator film. It is to provide a method for measuring quickly.
[0010]
[Means for Solving the Problems]
In light of the present state of the oil content measurement technique described above, the present inventors have intensively studied various methods, and as a result, the fats and oils in a cell surrounded by a cell membrane that is an insulator film and filled with a cell substrate. It has been found that there is a correlation that can be approximated by a straight line between the volume of the cell and the dielectric relaxation frequency and / or conductivity inside the cell. The present invention has been proposed on the basis of such findings, and in order to measure the amount of fats and oils in a cell substrate surrounded by a cell membrane that is an insulator film, the dielectric relaxation frequency and / or cell substrate by the cell membrane is measured. It is characterized by measuring the electrical conductivity. On the other hand, by obtaining in advance a calibration curve indicating the correlation between the volume of fats and oils in the particle and the dielectric relaxation frequency and / or conductivity inside the particle, the measured dielectric relaxation frequency or conductivity can be obtained. By applying to a calibration curve, the fat content of the cell can be determined.
[0011]
For example, biological cells are composed of a cytoplasm and a cell membrane surrounding the cytoplasm, and the cell membrane is mainly composed of a lipid bilayer, so that it can be regarded as an insulator with extremely low conductivity. On the other hand, since the cell substrate that fills the cytoplasm is composed of water and various ions dissolved in this water, it can be regarded as a conductive electrolyte solution, and fats and oils in the cytoplasm are regarded as non-electrolytes. be able to.
[0012]
Therefore, the present invention is based on the knowledge that there is a predetermined correlation between the amount of fat and oil in the cytoplasm surrounded by the cell membrane and filled with the cell matrix and the dielectric constant and / or conductivity of the cytoplasm. Provided is a method for measuring the fat content in the cytoplasm by measuring the conductivity of the cell substrate from the dielectric relaxation frequency.
[0013]
According to the present invention, a method for measuring the fat content in a cell surrounded by a cell membrane that is an insulator film and filled with a cell substrate that is an electrolyte solution is, for example, the fat content in a microbial cell. It can be used for measurement. In the case where the specimen used for the measurement is a microorganism having the ability to accumulate oils and fats, it is desirable that the specimen is a culture solution or suspension of the microorganism.
[0014]
Examples of the above-mentioned microorganisms capable of accumulating fats and oils include, for example, the genus Conidiobolus, the genus Entomophthora, the genus Absidia, the genus Cunninghamella, the genus Mortierella, the genus Morizella, h, Mucor, Pythium, Aspergillus, Fusarium, Pericularia, Penicillium, Cladosporium, Porisporum Or the fungus of the genus Claviceps, the genus Candida Cryptococcus genus, Endomyces genus, Galactomyces genus, Williopsis genus, Waltmyces genus, Lipomyces genus, Rhododrum genus, Rhodospodium p Rhodotorula genus, Trichosporon genus, or Yarrowia genus yeast, and Spirulina genus, Chlorella genus, Crypthecodium genus, Isochris cis (Nanochloropsis) genus, Phaeodactylum Algae of the genus, Porphyridium genus, Ulkenia genus, or Schizochytrium genus.
[0015]
DETAILED DESCRIPTION OF THE INVENTION
In order to carry out the oil content measurement method according to the present invention, first, an alternating electric field whose frequency changes is applied to at least one location of the suspension of the measurement object. The application of an alternating electric field to the suspension of the measurement object can be achieved by installing a pair of electrodes and placing the suspension of the measurement object between the electrodes, or by applying an electric field generated by electromagnetic induction around the coil. A method of applying to the suspension of the measurement object (for example, see “Instrumentation” (1996), Vol. 39, No. 3, pages 51-55) can be used. The frequency of the alternating electric field is swept in the range of 5 Hz to 100 MHz to obtain a series of measured values of the dielectric constant and conductivity of the measurement object, thereby measuring the frequency characteristics of the dielectric constant and conductivity of the measurement object. The magnitude of the AC electric field to be applied should be selected so as not to damage the cells to be measured. In the case of microbial cells, the voltage is preferably 2 V or less, more preferably 1 V or less.
[0016]
Next, the conductivity ka (unit S / m) of the particle (cell) medium is determined from the series of measured conductivity values obtained above. When the particle (cell) concentration is low, the conductivity measurement value on the lowest frequency side, the conductivity convergence value on the low frequency side or its predicted value can be used as an approximation of the conductivity ka.
[0017]
Further, the relaxation frequency fc (unit: Hz) is obtained from the frequency characteristic of the dielectric constant obtained above. The relaxation frequency fc means “frequency at which the midpoint between the low-frequency-side convergence value and the high-frequency-side convergence value of the dielectric constant is observed”. One method for obtaining the relaxation frequency fc is that the measured frequency characteristic value of the dielectric constant is the Cole-Cole equation (see “J. Chemical Physics” (1941), No. 9, pages 341-351),
[0018]
[Expression 1]
ε = ε _h + Re {(ε _l −ε _h ) / (1+ (jf / fc) β)} (1)
The parameter fc that best fits the curve is obtained by a non-linear least square method using a computer. In Equation (1), ε is the dielectric constant, ε _h is the high frequency convergence value of the dielectric constant, ε _l is the low frequency convergence value of the dielectric constant, f is the frequency of the applied AC electric field, and fc is relaxation. Frequency, β is a Cole-Cole parameter, and j is an imaginary unit (= √ (−1)). However, the method of calculating the relaxation frequency fc is not limited to this. Usually, the relaxation frequency fc in the present invention falls within the frequency band of 100 kHz to 10 MHz.
[0019]
Based on the above measurement, the content of the non-electrolyte in the target electrolyte solution, that is, the fat content in the cytoplasm can be measured as follows.
(1) When the conductivity ka obtained above is constant and hardly changes:
For example, when cells are suspended or dispersed in a certain concentration of electrolyte solution for measurement, a straight line approximates between the relaxation frequency fc and the fat content in the electrolyte solution inside the insulator film. There is a correlation that can be. Therefore, by obtaining a calibration curve showing such a correlation in advance, the fat and oil content can be obtained easily and quickly using the relaxation frequency fc obtained from the frequency characteristics of the dielectric constant.
[0020]
(2) When the conductivity ka obtained above changes:
For example, when the electrolyte concentration in the culture solution changes with the progress of culture in a culture solution of microorganisms, the fat content in the cytoplasm can be determined by the following method. The relationship between the conductivity ki (unit S / m) of the cytoplasm surrounded by the cell membrane, the conductivity ka of the particle medium and the relaxation frequency fc is as follows. [0021]
[Expression 2]
1 / fc = A [(1 / ki) + (1 / ka)] (2)
In the case of an ellipsoid, a rod-shaped body and a fibrous body whose major axis is sufficiently longer than the minor axis,
[Equation 3]
1 / fc = B [(1 / ki) + (1 / ka)] (3)
Can be approximated by However, in Formula (2), A is a constant equal to πDCm, D is a cell diameter (unit m), Cm is a capacitance per unit cell membrane area (unit F / m ² ), and in Formula (3), B is a constant equal to πTCm, T is the short axis diameter of the cell in the case of an ellipsoid, the diameter of the ridge or fiber in the case of rods and fibers (unit m), and Cm is the capacitance per unit area of the cell membrane (unit) F / m ² ).
[0023]
In the expressions (2) and (3), the values of the constants A and B are values inherent to the electrolyte solution surrounded by the cell membrane. Moreover, it has been found that there is a correlation that can be approximated by a straight line between the conductivity ki and the fat content in the cytoplasm. Therefore, a calibration curve showing such a correlation and the values of constants A and B are obtained in advance for the cell, and the conductivity ki of the measurement symmetrical cell is measured. The oil and fat content can be determined.
[0024]
When the values of the constants A and B of the cells to be measured are not obtained in advance, for example, the change of 1 / fc relative to 1 / ka when an electrolyte such as an inorganic salt is added to the cell suspension. The values of the constants A and B can be obtained from the plots. However, the method for obtaining the constants A and B is not limited to this.
[0025]
By using the method described above, the fat content of microbial cells can be measured. This method can be carried out quickly and easily without destroying the measurement object, and can also be applied to online measurement of changes in the fat content of microbial cells during liquid culture. Therefore, the present invention is extremely useful when applied to process control and process control of fats and oils production by microorganisms.
[0026]
【Example】
Hereinafter, specific examples of the method for measuring oil content according to the present invention will be described with reference to some examples. However, the technical scope of the present invention is not limited to these examples.
[0027]
Example 1
The filamentous fungus Mortierella alpina CBS754.68 was cultured in a 50 L culture tank in a medium mainly composed of soy protein and glucose. The culture solution was collected over time in a 500 mL beaker, an electromagnetic induction probe was placed near the center of the solution, and the dielectric constant and conductivity of the culture solution were measured in the frequency range from 100 kHz to 30 MHz. Among the obtained dielectric constant data, using the dielectric constant data in the frequency range from 400 kHz to 30 MHz, the relaxation frequency fc is obtained by curve fitting to the equation (1) by the nonlinear least square method, and the conductivity ka is 100 kHz. Conductivity values were used.
[0028]
Since the strain used is a filamentous fungus, the formula (3) applied when the particles are fibrous is used. In order to obtain the value of constant B, 0.5 mL of KCl aqueous solution (concentration 25%) was added to the culture solution 6 times in total, and each time the addition was performed, the relaxation frequency fc and conductivity ka were obtained, and the reciprocals of both were plotted. The value of the constant B was determined from the slope and determined to be 1.35 × 10 ⁻⁷ [F / m]. Using the relaxation frequency fc, the conductivity ka, and the constant B thus determined, the conductivity ki was determined from Equation (3). On the other hand, the fatty acid content per dry cell of the filamentous fungus was determined using a known method described in JP-A-6-153970 for extracting fats and oils from the cell.
[0029]
The above procedure was repeated for the same filamentous fungus every time the fatty acid content was different, and the relationship between the obtained fatty acid content and the conductivity ki was plotted, and FIG. 1 was obtained. FIG. 1 shows that there is a linear approximation between the conductivity ki of the filamentous fungi and the fatty acid content. Therefore, when the fatty acid content of the filamentous fungus is unknown, the fatty acid content is obtained by calculating the conductivity ki of the filamentous fungus according to the above procedure when the graph of FIG. 1 is obtained in advance as a calibration curve. be able to.
[0030]
Example 2
Yeast Lipomyces starkeyi IFO10385 strain was cultured in a 10 L culture tank in a medium mainly composed of yeast extract and glucose. The culture solution was collected over time, dielectric measurement was performed in the same manner as in Example 1, and the conductivity ki was calculated using Equation (2) applied to the sphere. Thereafter, the culture broth was centrifuged to collect yeast cells, and washing with water was repeated three times to obtain washed yeast cells, which were dried and the fatty acid content per cell was measured in the same manner as in Example 1. The above procedure was repeated for the same yeast every time the fatty acid content was different, and the relationship between the obtained fatty acid content and conductivity ki was plotted, and FIG. 2 was obtained. FIG. 2 shows that there is a linear approximation between the electrical conductivity ki and the fatty acid content of yeast. Therefore, by obtaining the graph of FIG. 2 in advance as a calibration curve, when the fatty acid content of the yeast is unknown, the fatty acid content can be obtained by calculating the electrical conductivity ki of the yeast according to the above procedure. it can.
[0031]
Example 3
The filamentous fungus Mortierella alpina CBS754.68 was cultured in a 50 L culture tank in a medium mainly composed of soy protein and glucose. Bacteria were obtained from the culture solution by centrifugation, washed with 10 mM KCl solution, and centrifuged to obtain bacteria. The same washing operation was repeated twice more, then suspended in a 10 mM KCl solution and filled in a platinum electrode cell, an AC electric field having a frequency band of 5 Hz to 10 MHz was applied between the electrodes, and the frequency characteristics of the dielectric constant were measured. However, relaxation due to dielectric relaxation of the cell membrane is observed in two places, and the convergence value on the low frequency side at that time is observed in a frequency band of approximately 10 kHz or less, and the convergence value on the high frequency side is observed in a frequency band from 100 kHz to 10 MHz. It was done. Therefore, after subtracting the influence of electrode polarization, the relaxation frequency fc was determined as the midpoint between these two convergence values, and the fatty acid content per cell was measured in the same manner as in Example 1.
[0032]
The above procedure was repeated for the same filamentous fungus every time the fatty acid content was different, and the relationship between the resulting fatty acid content and the relaxation frequency fc was plotted, and FIG. 3 was obtained. FIG. 3 shows that there is a relationship that can be approximated by a straight line between the relaxation frequency fc of the filamentous fungus and the fatty acid content. Therefore, the fatty acid content is obtained by calculating the relaxation frequency fc of the filamentous fungus according to the above procedure when the fatty acid content of the filamentous fungus is unknown by obtaining the graph of FIG. 3 in advance as a calibration curve. be able to.
[0033]
【The invention's effect】
As described above, as can be understood from the description of the method for measuring oil content according to the present invention based on some examples, the present invention can quickly and easily measure the oil content of microorganism cells such as filamentous fungi and yeast. There is a special effect that you can. Therefore, the present invention is extremely useful when applied to process control and process control of fat and oil production by microorganisms.
[Brief description of the drawings]
FIG. 1 is a graph showing the relationship between the conductivity ki of filamentous fungi and the fatty acid content per dry cell.
FIG. 2 is a graph showing the relationship between the electrical conductivity ki of the yeast cytoplasm and the fatty acid content per dry cell.
FIG. 3 is a graph showing the relationship between the relaxation frequency fc of filamentous fungi and the fatty acid content per dry cell.

Claims (5)

A method for measuring the content of fats and oils present inside a cell surrounded by a cell membrane that is an insulator film and filled with a cell substrate that is an electrolyte solution,
A measurement method comprising: measuring a dielectric relaxation frequency of the cell suspension system and / or measuring a conductivity of the cell substrate. further,
Obtaining in advance a calibration curve indicating the relationship between the fat content of the cells and the dielectric relaxation frequency of the cells;
Obtaining a fat content corresponding to the measured dielectric relaxation frequency in the calibration curve;
The measurement method according to claim 1, further comprising: further,
Obtaining in advance a calibration curve indicating the relationship between the fat content of the cells and the conductivity of the cells;
Obtaining a fat content corresponding to the measured conductivity in the calibration curve;
The measurement method according to claim 1, further comprising: The measurement method according to any one of claims 1 to 3, wherein the cells are ellipsoidal, rod-shaped and fibrous microorganisms. The measurement method according to claim 1, wherein the cell is a spherical microorganism.

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