JPH07177876A - Culture management method - Google Patents

Abstract

(57) [Summary] [Purpose] To provide a method for controlling the culture of microorganisms. In the method for controlling the culture of microorganisms (including cells), the method for controlling the culture of microorganisms (including cells) is used to control the culture using the osmotic pressure value of the culture solution in the culture step as an index. Furthermore, the correlation between the osmotic pressure value of the culture solution in the culture step and at least one value of the substrate concentration, the metabolite concentration, or the microorganism (including cells) concentration of the culture solution is obtained in advance, and this is calculated. A method for controlling the culture of microorganisms (including cells) that controls the culture as an index. [Effect] It is possible to easily and quickly perform monitoring and control of a culture state in processes such as substance production by culturing microorganisms (including cells) and cell proliferation.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for controlling the culture of microorganisms and cells of animals, plants and the like. More specifically, the field of foods represented by lactic acid fermentation, alcoholic fermentation, amino acid fermentation and the like, In addition, fermentation of antibiotics, pharmaceutical fields represented by fermentation of physiologically active substances, animal cells, agricultural fields represented by proliferation of plant cells, and other fields of microorganisms, and cells of animals, plants, etc. The present invention relates to a method for controlling culture of a microorganism (including cells), which is useful for monitoring and controlling processes such as substance production by culture and cell growth.

[0002]

2. Description of the Related Art In recent years, various substance production techniques by culturing cells of microorganisms, animals, plants and the like have been developed and put into practical use, and process monitoring and control of the substance production can be accurately and accurately performed. Prompt operation has become extremely important in production technology.

Conventionally, various methods have been developed for performing process monitoring and control such as production of substances by culturing cells of such microorganisms and cells of animals, plants, etc., but any of these methods has been developed. In the culturing process, most of the things are performed while sequentially measuring the substance concentration and the like of the culture broth and monitoring changes and transitions of the substance concentration and the like in the culture broth, and generally, for example, at an appropriate timing of the culturing process. The culture broth is collected and filtered in advance, deproteinized,
After performing appropriate pretreatment such as dilution treatment and extraction treatment with an organic solvent, the substrate concentration and metabolite concentration etc. of the culture broth can be determined by an analytical method such as liquid chromatography, gas chromatography, etc. or an enzymatic method. The measurement was performed, and the culture process was monitored and controlled based on the measurement result.

However, in such a method, as described above, it is necessary to collect the culture broth and further subject it to pretreatment such as filtration treatment, deproteinization treatment, and dilution treatment in advance. Since it is a method that requires a considerable amount of labor and time, for example, it is necessary to sequentially measure the concentration of a specific substance in the culture medium with an instrument, so monitoring and control of processes such as substance production and cell proliferation It has a problem that it is difficult to perform the procedure in a timely, convenient, and rapid manner. Therefore, in the art, the concentration of a substance in a culture solution as described above is measured, and the culturing process is performed. There has been a strong demand to develop a new culture management technique that replaces the conventional method of performing monitoring and control.

[0005]

Based on such a situation, the present inventors can certainly solve the above-mentioned problems of the prior art, and at the same time, determine the concentration of substances in the culture solution as described above. Accurate, simple and easy to use
As a result of intensive research aimed at developing a completely new culture control method capable of rapidly and rapidly controlling culture control such as monitoring and control of the culture process, It was found that the intended purpose can be achieved by using the value as an index, and the present invention has been completed.

That is, the present invention does not employ means for measuring the substance concentration of the culture solution, which has been required as an essential means in the conventional method for controlling the culture of microorganisms and cells of animals, plants and the like. Another object of the present invention is to provide a new culture management method capable of accurately and promptly carrying out monitoring and control of processes such as production of substances by culturing the microorganisms and cell proliferation.

Further, the present invention provides a simple culture management method which does not require various pretreatment operations which are indispensable for measuring the concentration of a substance in a culture solution which is required in the conventional culture management method. The purpose is.

Further, according to the present invention, by simply measuring the osmotic pressure of the culture solution in the culturing step, it is possible to accurately, easily and rapidly monitor and control the processes such as the production of substances by culturing and cell proliferation. It is intended to provide a possible new culture management method.

[0009]

The present invention which achieves such an object comprises the following technical means (1) and (2). (1) In a method for controlling the culture of a microorganism (including cells), the method for controlling the culture of a microorganism (including cells) is characterized in that the culture is controlled by using an osmotic pressure value of a culture solution in the culture step as an index. .

(2) In the culture management method described in (1) above, a value of the osmotic pressure of the culture solution in the culture step is previously set,
Correlation with at least one value of substrate concentration, metabolite concentration, or concentration of microorganisms (including cells) in the culture solution is obtained, and the correlation is used as an index to control the culture. Culture management method). Microorganisms (including cells) characterized by using an osmotic pressure value of a culture solution as an index in a method for controlling the culture of microorganisms (including cells)
Culture management method.

Next, the present invention will be described in more detail. The culture management method of the microorganism (including cells) of the present invention comprises:
As mentioned above, lactic acid fermentation, alcohol fermentation, amino acid fermentation and other food fields, antibiotic fermentation production,
Pharmaceutical field typified by fermentative production of physiologically active substances, agricultural field typified by mass growth of animal cells and plant cells,
And microorganisms in other fields, and substance production in the culture of cells such as animals and plants, monitoring and control of processes such as cell growth, and the like, the microorganisms, cell types, fermentation methods, and culture. It is possible to use without being limited to the type of method. Also,
The term "microorganisms (including cells)" as used in the present invention means microorganisms and cells such as animals and plants,
Regardless of its type and morphology, it is defined as including appropriate microorganisms, cells of animals and plants.

Further, the culture medium as used in the present invention means
The microorganisms, cells of animals, plants and the like are cultured in a certain liquid medium to mean a culture solution of the microorganisms and cells in a culture process for carrying out substance production, cell proliferation, etc., so-called ordinary culture. Any liquid may be used, and the kind, composition, etc. are not limited.

Next, in the present invention, it is necessary to measure the osmotic pressure of the culture solution in the culturing step, and the method of measuring the osmotic pressure is, for example, an osmometer using a freezing point depression method or the like. Desirably, the osmotic pressure of the culture solution in the culture tank may be measured with an online osmometer capable of continuous measurement, and the specific method thereof may be any suitable means, and is not particularly limited.

According to the research conducted by the present inventors, the osmotic pressure value of the culture solution at an appropriate stage of the culture process has been used as an index in the conventional culture control method in the research process so far. That there is a certain correlation with the so-called substance concentration in the culture medium such as substrate concentration, metabolite concentration, or microorganism (including cell) concentration, and that the osmotic pressure value can be used as an index for culture control Is found as a new finding, and as a result, in culturing microorganisms and cells, by measuring the osmotic pressure of the culture solution in the culturing step, the value of the osmotic pressure is used as an index, or by a previously determined relational expression, It was clarified that the substrate concentration, metabolite concentration, or cell concentration was calculated from the osmotic pressure value, and the condition of the culture could be monitored using the calculated concentration as an index.

That is, a conventional method for directly measuring the substance concentration in a culture solution by measuring the osmotic pressure of the culture solution in the culturing step using an osmometer, preferably an online osmometer capable of continuous measurement. It has become clear that the state of the culture solution can be known more simply and quickly.

In this case, for example, the above relational expression is
It can be created by determining the correlation between the measured value of the osmotic pressure of the culture solution in the culturing step and the numerical value of at least one of the substrate concentration, metabolite concentration, or microorganism and cell concentration of the culture solution. Yes, the condition of culture can be monitored using this as an index. Further, in this case, for example, the dilution rate of the continuous culture method in the continuous culture facility, the control of the flow acceleration of the fresh medium by the fed-batch culture method, etc.
It is also possible to appropriately use the above osmotic pressure value or other state variable calculated from it for controlling any manipulated variable in the culture process.

The specific contents such as the substance concentration in the culture medium, which is compared with the osmotic pressure value of the culture medium, include the types of microorganisms and cells used, the types of substances produced, the types of culture medium,
Although it may be set individually depending on the culture conditions, the culture form, etc., some suitable examples thereof will be exemplified. In the case of management, it was found that there is a certain correlation in the relationship between the osmotic pressure of the culture solution and the residual glucose concentration, ethanol concentration, and microbial cell concentration in the culture solution. If the relational expression is obtained, the osmotic pressure of the culture solution can be measured at an appropriate time during the culture process, and the value of the osmotic pressure can be used as an index to accurately, simply, and rapidly culture the culture state of the culture solution. It was found that the management of

Further, for example, in the case of controlling the culture in the lactic acid fermentation culture process of lactic acid bacteria, according to the study of the present inventors, the osmotic pressure of the culture solution, the lactic acid concentration of the culture solution, the bacterial growth, and the residual glucose It has been found that there is a constant proportional relationship with the concentration under certain conditions. Therefore, if the relational expression between the factors is obtained in advance, the appropriate timing of the culture process can be obtained. It was found that by simply measuring the osmotic pressure of the culture solution, the culturing state of the culture solution can be controlled accurately, simply and quickly by using the value of the osmotic pressure as an index.

Further, for example, in the case of culture management in the culture process by the fed-batch culture method of Chlorella in a continuous fresh medium supply culture facility, according to the studies of the present inventors,
It was found that there is a high correlation between the osmotic pressure of the culture and the glucose concentration remaining in the culture, and thus
In advance, if you obtain the relational expression of both, just by measuring the osmotic pressure of the culture solution at an appropriate time of the culture process, using the value of the osmotic pressure as an index, the culture state of the culture solution is accurate, simple, It can be quickly and rapidly managed, which allows glucose to be fed in synchronism with bacterial cell growth, and it has been found that the control of the culture state by the fed-batch culture method can be performed easily and quickly. It was

As described above, according to the present invention, as described above, the culture control of the microorganisms in the culture process of the microorganisms such as yeast, lactic acid bacterium, and chlorella can be performed by simply measuring the osmotic pressure in the culture solution. Accurate, convenient, and fast, but the result is that other microorganisms
It is clear from the comprehensive and systematic experimental results of the present inventors that the same can be obtained in the case of animal cells and plant cells. Therefore, the present invention is not limited to these microorganisms, As in the case of the microorganism, other microorganisms,
The same applies to cells such as animals and plants.

[0021]

The present invention, as described above, is a microorganism (including cells).
The osmotic pressure value of the culture solution measured at an appropriate time in the culture process of the culture is the value of the substance concentration such as substrate concentration, metabolite concentration, cell concentration, etc. used as an index of the conventional culture control method. Further, it was developed on the premise that there is a certain correlation under certain conditions between the values such as cell metabolism and growth rate, and in the present invention, the culturing step The value of the osmotic pressure of the culture solution in is used as an index for culture control. Therefore, according to the present invention, the substrate concentration, the metabolite concentration, the cell concentration in the culture solution, the cell metabolism and growth rate, and other culture conditions can be accurately determined from the value of the osmotic pressure of the culture solution in the culture process. Therefore, it is possible to monitor easily and quickly. Furthermore, based on these values, the culture temperature is adjusted in synchronization with the bacterial growth, the feeding of the medium, and the optimization of the dilution rate in continuous culture. It is possible to easily carry out the control of various operational variables according to the culture state such as the determination of.

[0022]

EXAMPLES Next, the present invention will be described more specifically by showing examples of the present invention, but the present invention is not limited to the examples. Example 1 Culture management of ethanol fermentation by yeast Saccharomyces cerevisiae Saccharomyces cerevisiae IFD10217 as a microorganism for the purpose of obtaining and monitoring residual glucose concentration, ethanol concentration, and bacterial cell concentration (OD660) from the osmotic pressure value of the culture solution.
Using a modified YM medium (yeast extract 0.3%, malt extract 0.3%, peptone 0.5%, glucose 4.
5%) and static culture was carried out under the condition of 30 ° C. The ethanol and glucose concentrations were measured by an enzymatic method, and the osmotic pressure was measured by a freezing point depression method, and converted to 273 ° K.
The result is shown in FIG. As is clear from FIG. 1, the osmotic pressure had a positive proportional relationship with the bacterial cell growth and the ethanol concentration, and had an apparent negative proportional relationship with the residual glucose concentration.

The consumption of nutrients other than glucose and the change in osmotic pressure due to metabolites other than ethanol were smaller than the effects of glucose and ethanol, and it was found that they change with glucose or ethanol in parallel with time. Was not examined. The relationship between osmotic pressure, residual glucose concentration, ethanol concentration, and bacterial cell concentration is shown in FIG. When the residual glucose concentration (g / l) is Gr, the ethanol concentration (g / l) is E, the cell concentration (OD660) is X, and the osmotic pressure (0sm / kg) is π, it changes during culture. Three
In the osmotic pressure range of 0 to 0.530 sm / kg, the following relational expressions (1) to (3) were obtained by the method of least squares.

Gr = -185.9 × π + 102.0 (r = -0.996) (1) E = 105.2 × π-31.3 (r = 0.972) (2) X = 24.4 × π-7.4 (r = 0.982) (3)

In this case, the change with time of the osmotic pressure of the culture medium shows a high correlation with all of the residual glucose concentration, the alcohol concentration and the bacterial cell concentration, and the osmotic pressure of the culture medium during the culture should be measured. It was found that the state of the culture process can be grasped. Therefore, it was clarified that by measuring the osmotic pressure of the culture solution in the ethanol fermentation culture process using yeast, it is possible to monitor changes over time in the target substrate concentration, metabolite concentration, and cell concentration.

Example 2 Culture Management of Lactic Acid Fermentation by Lactobacillus Lactobacillus casei Considering the dissociation rate of acid affected by pH, the culture monitoring by osmotic pressure during the culture of lactic acid bacteria was examined. Rogosa medium (J.
Inf. Dis., 110, 258-267, 1962) in Lactobacillus ca.
The sei was statically cultured at 37 ° C. Glucose concentration,
The time-dependent changes in the lactic acid concentration, bacterial cell concentration (OD660), osmotic pressure, and pH are shown in FIG. The osmotic pressure was converted and shown at 273 ° K. As is clear from FIG. 3, the osmotic pressure showed a positive proportional relationship with the bacterial cell growth and the lactic acid concentration, and had an apparent negative proportional relationship with the residual glucose concentration.

The osmotic pressure is π _atm (atm), the molar concentration of solute is C (mole / kg), the absolute temperature at the thermal equilibrium of the solution and the pure solvent is T (° K), and the gas constant (0.082 kg.atm. ° K ^-1 · mol
If e ⁻¹ ) is R, the following equation (4) of Van't Hoff π _atm = RTC (4) is established in a range in which the concentration C does not become too large.

Therefore, by measuring the osmotic pressure and the temperature, the solute concentration can be calculated from the above equation (4). The main factor involved in osmotic pressure change in homolactic acid bacterium culture is lactic acid which increases contrary to the decrease of glucose-based nutrient source. Furthermore, lactic acid has a small dissociation rate due to the influence of the pH of the culture solution, which decreases over time with the culture. Therefore, although it is possible to calculate each state variable from the osmotic pressure by an approximate expression based on the least squares method based on the experimental value as in Example 1, it is more accurate to use the theoretical expression considering the dissociation rate. Becomes

Therefore, assuming that the pH when the dissociation rate of the electrolyte is 50% is pKa, the following (5) Henderson-Hass
Elbalch's formula pH = pKa + log [Dissociation concentration] / [Non-dissociation concentration] (5) is modified to obtain the dissociation rate (%) by the following equation (6) ^: Dissociation rate = {10 ^{[pH- It can be calculated as pKa]} / (10 ^[pH-pKa] +1)} × 100 (6).

It is impossible or not possible to measure the changes with time of nitrogen sources other than glucose and nutrient sources such as trace components that affect changes in osmotic pressure. However, these are proportional to the bacterial cell concentration and can be expressed as the product of the bacterial cell concentration and a constant k. From the above, osmotic pressure (0sm / kg) is π, osmotic pressure at the start of culture (0sm / kg) is π _i ,
When the glucose concentration (g / l) consumed is G, the bacterial cell concentration (OD660) is X, and the produced lactic acid concentration (g / l) is L, the osmotic pressure value is
From the equation (6), the following equation is obtained. π = -G / 180 + k ・ X + L / 90 ・ {10 ^[pH-pKa] / (10 ^[pH-pKa] +1) +1} + π _i (7)

The pKa of lactic acid is 3.86, and when a constant k is obtained such that the actually measured value and the calculated value of the osmotic pressure are the same, −
0.0057 is obtained, and by substituting these into the above equation, π = -G / 180-0.0057 ・ X + L / 90 ・ {10 ^[pH-3.86] / (10 ^[pH-3.86] +1) +1 } + Π _i (8) was obtained.

Lactic acid yield L / G based on glucose consumed
(G-lactic acid / g-glucose) and cell yield X / G (OD
660 / g-glucose) is L / G = 0.85 (9) X / G = 0.51 (10) from the experimental values, so simultaneous equations (8) to (10) Solving the equation for glucose concentration, lactate concentration, and bacterial cell concentration, the following (1) with osmotic pressure and pH as parameters
The relational expressions 1) to (13) were obtained.

G = (π-π _i ) / [0.0094 · {10 ^[pH-3.86] / (10 ^[pH-3.86] +1) +1} -0.0085] (11) L = (π-π _i ) /[0.0111 ・ {10 ^[pH-3.86] / (10 ^[pH-3.86] +1) +1} -0.0099] (12) X = (π-π _i ) / [0.0185 ・ {10 ^[pH-3.86] / (10 ^[pH-3.86] +1) +1} -0.0160] (13)

Under the condition of π ₁ = 365 mOsm / kg, the numerical values of each state variable calculated by this formula are plotted in solid lines and the measured values are plotted in dots in FIG. The calculated value and the measured value are in good agreement, and it was revealed that by measuring the osmotic pressure and the pH, it is possible to monitor the changes over time in the target substrate concentration, metabolite concentration, and cell concentration. That is, in this case, the change with time of the osmotic pressure of the culture medium shows a high positive or negative correlation with any of the residual glucose concentration, the lactic acid concentration, and the bacterial cell concentration, and the culture liquid during the culture of lactic acid bacterium fermentation by lactic acid bacteria. It was found that the state of the culturing process can be grasped by measuring the osmotic pressure of.

In the culture using milk instead of the Rogosa medium, the same result was obtained when the viable cell count was measured instead of OD660 as the cell amount, and it was also useful for monitoring the fermented milk production process.

Example 3 Culture Management in Fed-Batch Culture Process of Chlorella and Chlorella regularis In dark culture of Chlorella in a fermenter, growth inhibition by glucose as a substrate is observed, and therefore the substrate mass is controlled from the osmotic pressure value of the culture solution. Fed-batch culture was investigated. Chlorell
a regularis was subjected to aeration and agitation culture in a medium containing glucose and urea as main components at 35 ° C (Agr. Bio
l. Chem., 38, 1, 9-18, 1974). In the same manner as in Example 1 described above, glucose was also converted into glucose concentration Gr (g / l) and 273 ° K during chlorella culture using glucose as a representative state variable, including other substrates that change in association with glucose. The relationship between the osmotic pressure π (0 sm / kg) was examined. As a result, it was found that there is a high correlation (FIG. 5), and the following relational expression was obtained by the least squares method. Gr = 101.6 × π − 4.8 (r = 0.995) (14)

Using this relational expression, the concentration of glucose remaining in the culture broth was calculated from the osmotic pressure value, and fed-batch culture in which the glucose concentration was controlled to 3% or less was examined. Immediately before glucose depletion, the osmotic pressure at 5g / l is 88m0sm / kg.
Since it is 338m0sm / kg at 30g / l, start the addition of the feed medium containing glucose at the time when the osmotic pressure reached 88m0sm / kg as the culture control solution, and continue to add it until 338m0sm / kg. The medium was fed again when the osmotic pressure reached 88 m0sm / kg again. The result of continuing the culture by repeating this method is shown in FIG.

The chlorella concentration was expressed by packed cell volume (PCV ml / l). The calculated value and the measured value of glucose concentration in the culture system based on the osmotic pressure value are in good agreement, and the glucose is controlled to 3% or less without depletion, and can be fed exponentially in synchronization with cell growth. It was Fed-batch culture control based on osmotic pressure was found to be a simple, rapid and excellent method.

In the case of chlorella, glucose as a substrate is consumed during the culturing process, but the glucose concentration that can be added at the initial stage of culturing is limited, so it is necessary to add it appropriately during culturing. In addition, since it was found that there is a high correlation between osmotic pressure and glucose concentration, the culture management of chlorella should be
It has been found that this can be done by measuring the osmotic pressure.

[0040]

INDUSTRIAL APPLICABILITY As described above in detail, the present invention relates to a method for controlling the culture of microorganisms (including cells), wherein the osmotic pressure value of the culture solution in the culture step is used as an index. The present invention relates to a method for managing culture of cells (including cells), and according to the present invention, it is possible to easily and quickly monitor and control the culture state in processes such as substance production by culturing microorganisms (including cells) and cell proliferation. It can be carried out.

Further, according to the present invention, the osmotic pressure value of the culture solution in the culturing step and the value of at least one of the substrate concentration, the metabolite concentration or the microorganism (including cells) concentration of the culture solution are previously set. It is possible to accurately, easily and swiftly control the process monitoring and control of the culture of the microorganism (including cells) by obtaining the correlation of the above.

[Brief description of drawings]

FIG. 1 shows the time course of each state variable in the yeast culture process.

FIG. 2 shows the relationship between osmotic pressure and each state variable in the yeast culture process.

FIG. 3 shows changes with time of each state variable in the lactic acid bacterium culture process.

FIG. 4 shows changes with time in calculated values and measured values of each state variable.

FIG. 5 shows the relationship between osmotic pressure and glucose concentration in the chlorella culture process.

FIG. 6 shows changes with time of each state variable in fed-batch culture using glucose concentration calculated from osmotic pressure as an index.

Claims (2)

[Claims] 1. A method for controlling the culture of microorganisms (including cells), wherein the culture is controlled using the osmotic pressure value of the culture solution in the culture step as an index. Management method. 2. The culture management method according to claim 1, wherein at least the osmotic pressure value of the culture solution in the culturing step and the substrate concentration, metabolite concentration, or microorganism (including cells) concentration of the culture solution are previously set. Find the correlation with one value,
A method for controlling the culture of microorganisms (including cells), characterized by controlling the culture using this as an index.