JP2746328B2 - Method for analyzing dissolved components of culture solution, analyzer, culture method, and culture device - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an analysis method and an analysis device for analyzing components dissolved in a culture solution, and further relates to a method and an apparatus for culturing microorganisms or animal and plant cells.

[Conventional technology]

In recent years, components dissolved in the culture solution in cell culture,
For example, there is an increasing demand for quantitative measurement of cell metabolites and substrates to be supplied in real time. For example, in culturing Escherichia coli, there is a problem that acetic acid metabolized by the cells inhibits cell growth, and it is desired to rapidly measure the acetic acid concentration in the culture solution.

Conventionally, as a method of measuring the components dissolved in the culture solution,
There are titration method, enzymatic method, thin layer chromatography, gas chromatography and liquid chromatography, etc.
These analyzers require more than 15 minutes for a single measurement.

In addition, if the culture solution is directly introduced into the analyzer, components other than the target volatile components and its decomposed products may be detected, which may make analysis difficult. It is preferable to use a GC (gas chromatograph) mass spectrometer,
LC (liquid chromatograph) mass spectrometers have been developed, but it is difficult to measure continuously in a short time.

[Problems to be solved by the invention]

The prior art described above does not take into account the point of continuous analysis of dissolved components with high accuracy in a short period of time, and has a problem that it is difficult to analyze dissolved components for efficient cell culture.

An object of the present invention is to provide a method and apparatus for continuously analyzing dissolved components in a culture solution collected from a culture tank in a short time with high accuracy when culturing microorganisms or animal and plant cells, and for efficiently performing cell culture. It is to provide a culture method and apparatus.

[Means for solving the problem]

In order to achieve the above object, the present invention provides a culture method comprising: adjusting the pH of a culture solution to a set value, vaporizing dissolved components in the culture solution, and analyzing the vaporized dissolved components. This is an analysis method for liquid dissolved components. The analysis of the dissolved components is preferably performed by a mass spectrometer, and the vaporization of the dissolved components of the culture solution can be preferably performed by bringing the culture solution into contact with a carrier gas.
The analysis method of the present invention is useful when the analysis target is an organic acid, particularly acetic acid.

The analyzer used for the analysis method is a means for collecting a culture solution from a culture tank, a means for adjusting the pH of the collected culture solution,
Means for evaporating dissolved components from the adjusted culture solution, and
An apparatus comprising means for analyzing vaporized dissolved components. The means for analyzing vaporized dissolved components is preferably a mass spectrometer. In the analyzer, a means for evaporating dissolved components from the pH-adjusted culture solution may be provided in the means for adjusting the pH of the collected culture solution.

Furthermore, the present invention provides a method for culturing microorganisms or animal and plant cells, wherein after adjusting the pH of the culture solution to a set value, the dissolved components of the culture solution are vaporized, and the vaporized dissolved components are analyzed. A method for culturing microorganisms or animal and plant cells, wherein the culture conditions are controlled based on the method, and a means for collecting a culture solution from a culture tank, a means for adjusting the pH of the collected culture solution, and a component dissolved from the pH-adjusted culture solution. A culturing apparatus comprising: means for evaporating water; means for analyzing vaporized dissolved components; and control means for controlling culturing conditions based on the analysis value of the dissolved components in the culture solution.

 Hereinafter, details of the present invention will be described.

The volatile dissolved components to be analyzed in the culture solution to which the analysis method and the analysis device of the present invention can be applied include, specifically, organic acids, carbon dioxide, ammonia and the like. In the case of acetic acid, ammonia and the like.

If the target component to be analyzed is a highly volatile component such as methanol or ethanol, it easily diffuses into the culture exhaust gas, so measurement can be performed by directly introducing the exhaust gas into analytical means such as a mass spectrometer. It is. In contrast,
When an organic acid, particularly acetic acid, is used as the target component, it hardly diffuses into the exhaust gas under the common culture conditions of Escherichia coli at 30 to 40 ° C and around pH 7, so the exhaust gas is used as it is in an analytical method such as a mass spectrometer. Is difficult to measure simply by introducing

Therefore, in the present invention, in order to measure volatile dissolved components such as acetic acid, which is a growth inhibitory substance, after adjusting the culture solution or the culture supernatant from which cells have been removed to a set pH value, the dissolved components in the culture solution are adjusted. Is quickly and continuously measured by evaporating and analyzing.

An analyzer for that purpose is a means for collecting the culture solution from the culture tank, a means for adjusting the pH of the collected culture solution, a means for evaporating dissolved components from the pH-adjusted culture solution, and analyzing the vaporized dissolved components. Means is provided.
Hereinafter, each means will be described.

Means for collecting the culture solution from the culture tank include a method of extracting the culture solution with a pump or pressure, and a method of collecting the culture solution using a filtration device provided with a centrifugal separator or a ceramic tube in order to remove cells from the extracted culture solution. Can be adopted.

As a means for adjusting the pH of the collected culture solution, a method using a pH controller, a method of automatically injecting a certain amount of acid or alkali, or the like can be adopted. The set value of pH varies depending on the target measurement component.For example, when acetic acid is used as the measurement component, the pH is set so that acetate in the culture solution is converted to acetic acid.
Is 6 or less, preferably 2-3. The amount of liquid withdrawn from the culture tank to adjust the pH is 10 ml / min or less, desirably 1 m, in order to reduce the effect on the amount of liquid in the culture tank.
l / min or less is good.

As a means for evaporating dissolved components from a pH-adjusted culture solution, there are a heating method, a method of vaporizing with a carrier gas, and a method of using both methods in combination.To increase the vaporization efficiency, use both methods. Is good. The higher the heating temperature, the better, but a range of 50 to 90 ° C can be adopted from the viewpoint of temperature control and analysis accuracy. As the carrier gas, a gas having a different molecular weight from the target component is used, but an inert gas such as helium gas or argon gas is preferable.

FIG. 2 shows an example of a vaporization container constituting a vaporization apparatus using a method of volatilizing with a carrier gas.

(A), a culture solution whose pH has been adjusted is flowed from a liquid inlet 1 to a liquid outlet 2 by an overflow method, a carrier gas is passed through the gas supply inlet 3 into the liquid, and the gas supply port 4 is supplied from the gas phase above the liquid. It is a container that passes.

(B) is a liquid flow system similar to (A), except that the bottom of the liquid phase is constituted by a porous plate 5 composed of, for example, a hydrophobic porous membrane or a glass filter plate, and from below the porous plate 5 upward. A container through which a carrier gas is ventilated.

(C) is a container that allows the liquid to flow from the liquid inlet 1 to the liquid outlet 2 and to flow from the air supply inlet 3 to the air supply outlet 4 so as to flow in the opposite direction.

(D) is a container in which plates are arranged in a multistage manner in the same container as in (C).

(E) is a container in which an insert 6 such as a cylindrical tube made of ceramics or stainless steel or a particulate molded product such as glass beads is placed in the same container as in (C).

Further, the pH adjusting device and the dissolved component vaporizing device may be separately provided, but the pH adjusting and the dissolved component vaporizing may be performed in the same container.

As a means for analyzing the vaporized dissolved components, a mass spectrometer is preferable in terms of sensitivity, measurement time, and the like. However, when only one kind of the target component is used, it is also possible to use a semiconductor sensor having a very high sensitivity.

The method for culturing microorganisms or animal and plant cells of the present invention is characterized by culturing microorganisms or animal and plant cells while controlling the culturing conditions based on the analysis values obtained by the above-described dissolved component analysis method. Further, the apparatus for culturing microorganisms or animal and plant cells of the present invention includes the above-mentioned analyzer and means for controlling the culturing conditions based on the analysis values obtained thereby.

As means for controlling the culture using the analysis value of the dissolved component in the culture apparatus of the present invention, a method of analyzing the supply amount of the culture substrate from the analysis value by a minicomputer or the like, for example, the flow of a feed medium using the acetic acid concentration as an index. The acetic acid concentration in the culture solution is controlled by increasing or decreasing the amount, and the culture control method for preventing the growth inhibition by acetic acid, or when the analytical value of the dissolved component reaches a certain set value, the medium is centrifuged or filtered. A method of performing exchange or the like is adopted.

In addition, it is desirable that the liquid sending pipe and the air sending pipe of the apparatus composed of the above-mentioned units be as short and narrow as possible in order to minimize the time required from the collection of the culture solution to the analysis of the dissolved components.

(Operation)

In the analysis method of the present invention, by collecting the culture solution from the culture tank, it is possible to create a condition environment suitable for analyzing the dissolved components without evaporating the culture conditions by removing the target dissolved components out of the culture system. Of the harvested culture
The dissolved component can be dissociated from the culture solution by adjusting the pH, and the dissolved component can be separated from the culture solution by vaporizing the dissolved component from the pH-adjusted culture solution, and introduced into the analyzer.

At that time, by setting a pH and a temperature suitable for the target component, introduction of components other than the target component into the mass spectrometer can be reduced, and interference by decomposition products of other components can be reduced. In addition, since the sample can be introduced into the analysis means in a short time and continuously, it is possible to measure the dissolved component of the culture solution in real time. Therefore, by measuring a growth inhibitory substance in a culture solution in real time using the analysis method of the present invention and controlling the culture conditions based on the measurement, growth inhibition can be prevented and efficient culture can be achieved.

〔Example〕

An embodiment of the present invention will be described with reference to FIG. 1 showing a system diagram of a culture apparatus, but the present invention is not limited thereto.

As a means for collecting the culture solution from the culture tank 7, a culture supernatant is collected by a culture solution sampling pump 8 and a cross-flow type filtration device 9. PH adjuster equipped with a device that can output signals
The pH of the supernatant liquid is adjusted to a set value by 10 and the supernatant liquid whose pH has been adjusted in the vaporization apparatus 11 is provided with a flow rate control apparatus capable of outputting a gas flow rate as an electric signal as a means for vaporizing dissolved components. The dissolved component is separated from the supernatant liquid into the carrier gas by contact with the gas, and the above process is kept at a set temperature by, for example, a warming device 12 provided with a temperature controller, and the analysis value is analyzed as a means for analyzing the vaporized component. The vaporized dissolved component is measured by the mass spectrometer 13 that can output an electric signal. Means for controlling culture using the analysis value of dissolved components include pH
The culture control device 14 provided with a device capable of analyzing the measurement value output from the mass spectrometer 13 using the signal output from the control device 10 or the vaporization device 11 allows the supply amount of the culture substrate from the analysis value of the dissolved component. The determination is performed and output to the substrate supply device 15 by an electric signal to control the supply amount of the substrate.

〔The invention's effect〕

According to the present invention, the analysis of the components dissolved in the culture solution can be performed continuously in a short time with high accuracy, and the analysis in real time becomes possible.
In the culture of microorganisms or animal and plant cells, efficient culture can be performed by controlling the culture conditions based on this analysis value.

[Brief description of the drawings]

FIG. 1 is a system diagram showing an apparatus according to an embodiment of the present invention.
(A) to (E) are cross-sectional views of a vaporization container in the apparatus of the present invention. 1 ... liquid inlet 2 ... liquid outlet 3 ... air supply inlet 4 ...
Air supply outlet, 5 ... pore plate, 6 ... insert, 7 ... culture tank, 8 ... sampling pump, 9 ... filtration device, 10 ... pH adjustment device, 11 ... vaporization device, 12 …… Insulation device, 13 …… Mass spectrometer, 14 …… Culture control device, 15 …… Substrate supply device

Claims (12)

(57) [Claims] 1. A method of culturing by collecting a portion of a culture solution from a culture solution and adjusting the pH of the portion of the culture solution to a set value so that a predetermined dissolved component is vaporized from the portion of the collected culture solution. Vaporizing the dissolved components of the part of the liquid,
Next, a method for analyzing dissolved components of a culture solution, comprising analyzing the vaporized dissolved components. 2. The method according to claim 1, wherein the analysis of the vaporized dissolved components is performed by a mass spectrometer. 3. The method for analyzing a dissolved component of a culture solution according to claim 1, wherein vaporization of the dissolved component of the culture solution is performed by bringing the culture solution into contact with a carrier gas. 4. The method for analyzing a dissolved component in a culture solution according to any one of claims 1 to 3, wherein the analysis target of the dissolved component in the culture solution is an organic acid. 5. The method according to claim 4, wherein the organic acid is acetic acid. 6. A means for collecting a part of the culture solution from the culture tank, a means for adjusting the pH of the part of the culture solution so that a predetermined dissolved component is vaporized from the part of the collected culture solution, and the pH is adjusted. An apparatus for analyzing a dissolved component of a culture solution, comprising: means for evaporating a dissolved component from the part of the culture solution; and means for analyzing the vaporized dissolved component. 7. An apparatus according to claim 6, wherein the means for analyzing the vaporized dissolved components is a mass spectrometer. 8. The means for adjusting the pH of a collected culture solution,
8. The apparatus for analyzing a dissolved component of a culture solution according to claim 6, further comprising means for vaporizing the dissolved component from the pH adjusted culture solution. 9. A method for culturing microorganisms or animal and plant cells, wherein a portion of the culture solution is collected from the culture solution, and the pH of the portion of the culture solution is adjusted so that a predetermined dissolved component is vaporized from the portion of the collected culture solution. By adjusting to the set value,
A method for culturing microorganisms or animal and plant cells, comprising: evaporating the dissolved component of the part of the culture solution, analyzing the vaporized dissolved component, and controlling the culturing conditions based on the obtained analysis value. 10. The method for culturing microorganisms or animal and plant cells according to claim 9, wherein the control of the culture conditions is performed by controlling the supply amount of the culture substrate. 11. A means for collecting a part of the culture solution from the culture tank, a means for adjusting the pH of the part of the culture solution so that a predetermined dissolved component is vaporized from the part of the collected culture solution,
means for evaporating dissolved components from the part of the pH-adjusted culture solution, means for analyzing the vaporized dissolved components, and control means for controlling the culture conditions based on the analysis value of the culture solution dissolved components. A culturing device for microorganisms or animal and plant cells. 12. The apparatus for culturing microorganisms or animal and plant cells according to claim 11, wherein the control means for controlling the culture conditions controls the supply amount of the culture substrate.