JPH07322874A - Method for membrane surface liquid culture, apparatus and system threfor - Google Patents

Abstract

PURPOSE:To improve the productivity of both cells and useful substances by enabling the culturing of various organic cells according to single method for culturing such as a method for membrane surface liquid culturing. CONSTITUTION:This method for membrane surface liquid culture is to divide the interior of a case 3 into a gas substance chamber (S1) and a liquid culture medium chamber (S2) with a porous membrane 4, constitute an apparatus 1 for the membrane surface liquid culture, connect a circulating line for taking out a liquid culture medium in the liquid culture medium chamber (S2) and refeeding the taken out liquid culture medium into the liquid culture medium chamber (S2) to the apparatus 1 for the membrane surface liquid culture and provide a preparing tank 2 for the liquid culture medium in the course of the circulating line.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention directly contacts a liquid culture medium on one side and a gas body on the other side with a porous membrane sandwiched between porous membranes, and allows biological cells to be present on the surface of the membrane in contact with the gas body. A method and an apparatus for culturing.

[0002]

2. Description of the Related Art Aspergillus oryzae
In the liquid culture method of gills oryzae),
A membrane surface liquid culture method has recently been developed as a culture method having a completely different concept from the conventional method (Kazuhiro Nakanishi et al., 1992 Annual Meeting of the Society of Biotechnology, Proceedings, p. 146).

As shown in FIG. 10, the apparatus used for the liquid culture on the membrane surface is a porous medium in which a liquid medium 101 is allowed to stand in a case 100, and a porous membrane 102 is fixed to an upper opening of the case 100. Liquid medium 10 on the back surface of the membrane 102
1 is contacted, and the surface of the porous membrane 102 in this state is inoculated with Aspergillus oryzae cells 103, and the culture is carried out while directly contacting with the atmosphere.

[0004]

According to the above-mentioned membrane surface liquid culture method, Aspergillus oryzae can be cultured in a natural state to produce neutral protease, and aeration with a conventional liquid medium is possible. Unlike agitation culture, it has the advantage that it does not require stress such as agitation on cells. However, as the culture proceeds in the membrane surface liquid culture method, the concentration of neutral protease increases at the portion in contact with the lower surface of the porous membrane, the nutrients supplied to Aspergillus oryzae cells are insufficient, and a sufficient amount of neutral Inability to produce protease.

[0005]

The object of the present invention is to provide a method for culturing various organism cells on a membrane surface by a single culture method called a membrane surface liquid culture method, producing the cells, or culturing with the cells. It is to improve the productivity of the production of useful substances in liquid.

The inventors of the present invention have conducted extensive studies to achieve the above-mentioned object, and as a result, in the above-mentioned membrane surface liquid culture method, by flowing a liquid medium in contact with one membrane surface by a predetermined method, The first invention of the present application is based on the finding that it is possible to culture various organism cells on the membrane surface and to markedly improve the productivity of the cells or the productivity of useful substances into the culture medium by the cells. Was completed.

Here, as the flow of the liquid medium, a method of circulating the liquid medium, a method of stirring the liquid medium, or the like is adopted, and the pressure of the gas body contacting the porous membrane is at least on the liquid side. It is preferable that the pressure is not less than the pressure. By doing so, the liquid medium does not pass through the membrane and overflow from the membrane surface. The degree of pressurization of the gas body is such that it does not hinder the liquid medium from osmotic pressure passing through the pores of the porous membrane and coming into contact with the organism cells. That is, the osmotic pressure of the liquid medium is not suppressed.

Further, in the membrane surface liquid culture apparatus according to the second aspect of the present invention, which is configured to allow the liquid medium to flow, the case is divided into a gas body chamber and a liquid medium chamber by a porous membrane, A stirring device was provided in the liquid medium chamber.

Further, a system incorporating the membrane surface liquid culture device according to the third aspect of the present invention, which is designed to enable the flow of the liquid medium, is a liquid medium in a liquid medium chamber which is a part of the membrane surface liquid culture device. A circulation line was taken out and fed again into the liquid medium chamber, and a liquid medium preparation tank was provided in the middle of this circulation line. A system incorporating the membrane surface liquid culturing device according to the fourth aspect of the invention is configured by arranging a plurality of membrane surface liquid culturing devices in series, and comprises a liquid medium chamber and a downstream side of the upstream membrane surface liquid culturing device. The liquid culture chamber of the membrane culture device on the side was connected with a liquid culture medium supply line, and a liquid culture medium preparation tank was provided in the middle of this liquid culture medium supply line. Also,
A system incorporating a membrane surface liquid culture device according to a fifth aspect of the invention is configured with a plurality of membrane surface liquid culture devices, and a plurality of membrane surface liquid culture devices are arranged in parallel on at least one of an upstream side and a downstream side. The liquid medium chamber of the upstream membrane surface liquid culture device and the liquid medium chamber of the downstream film surface liquid culture device are connected by a liquid medium supply line, and the liquid medium in the middle of the liquid medium supply line. A preparation tank was provided.
In the fourth or fifth invention, the liquid medium chamber of the most downstream membrane surface liquid culture device and the liquid medium chamber of the most upstream membrane surface liquid culture device are connected by a liquid medium return line, and this liquid A liquid culture medium preparation tank may be provided in the middle of the culture medium return line.

By the way, the liquid medium in the present invention includes:
Conventional culturing methods for various cells, for example, standing, stirring, continuous,
Those commonly used in fed-batch culture can be preferably used, and in addition to the liquid medium, the culture medium obtained by using the liquid medium is also included in the liquid medium.

The porous membrane which can be used in the present invention is made of a known organic or inorganic material, and preferably has a pore size of 0.01 to 5.0 μm,
It has a thickness of 0.05 to 10 mm and can be sterilized by autoclave under normal conditions. For example, as organic materials, nylon, PTFE (polytetrafluoroethylene), polypropylene, cellulose nitrate, polysulfone, PVDF (polyvinylidene dichloride), etc. Can be mentioned. Moreover, the thing made from those mixtures can also be used.

The organism cells that can be cultured in the present invention are those of microorganisms, plants and animals. Those cells that can be cultured under the conventional conditions of conventional solid and liquid culture methods are preferably used. Examples of the microorganisms include bacteria, actinomycetes, filamentous fungi, yeasts, monocotyledonous and dicotyledonous plants in plants, algae, known normal cells in animals, cancer cells, hybridoma cells and the like. it can. The pore diameter of the porous membrane that can be used for culturing these cells is within the above range, but is appropriately selected depending on the type of the living organism cell to be cultured. For example, 0.01 to 0.4 μm for bacterial cells, 0.1 to 0.5 μm for actinomycete cells, filamentous fungal cells, and yeast cells.
m, algal cells, plant cells, 0.1 to 10 μm, and animal cells, 0.1 to 5 μm.

In the present invention, the biological cells inoculated on the surface of the porous membrane come into direct contact with the gas body. As the gas body, atmosphere, and a gas body of oxygen, carbonic acid, nitrogen, or a mixture thereof is used. The type of these gas bodies or the mixing ratio of the gas bodies depends on the living organism cells to be cultured. For example, in the case of aerobic organism cells, the atmosphere, oxygen gas or carbon dioxide gas mainly composed of them, a mixture with nitrogen gas, in the case of anaerobic organism cells nitrogen and carbon dioxide gas bodies or a mixture thereof, plants For the body, air, a mixture of carbon dioxide gas or nitrogen gas, oxygen gas, and for animal cells, a mixture of carbon dioxide gas and oxygen gas or the atmosphere is preferably used. The concentration of the main body gas body, the type of gas body,
Depending on the type of organism cells and the type of useful substance to be produced, it is, for example, any concentration up to 100%, preferably 5 to 30%.

The culture temperature of the above-mentioned organism cells may be that used in the conventional culture method (0 to 90 ° C.), and specifically depends on the type of the organism cells. For example, 0 to 90 ° C is suitable for microbial somatic cells, 5 to 50 ° C for plant somatic cells, and 10 to 45 ° C for animal somatic cells.

[0015]

The liquid medium passes through the pores of the porous membrane at an osmotic pressure, contacts the organism cells on the membrane surface of the porous membrane, and supplies the nutrients necessary for growth and production of useful substances. Since the liquid medium is circulated or agitated, fresh liquid medium is always supplied to the biological cells on the membrane surface.

[0016]

Embodiments of the present invention will be described below with reference to the accompanying drawings. Here, FIG. 1 is a flow chart of a membrane surface liquid culture method for flowing a liquid medium according to the circulation method of the present invention, and FIG.
FIG. 1 is an overall system diagram of a membrane surface liquid culture system that performs liquid medium flow by the same circulation system. The membrane surface liquid culture system includes a membrane surface liquid culture device 1 and a liquid medium preparation tank 2, The device 1 has two case halves 3a, 3b.
The porous membrane 4 is sandwiched between the flange portions provided at the periphery of the opening of the gas and is tightened with the bolt 5 via the O-ring, so that the gas membrane chamber S1 and the liquid culture medium chamber S are formed in the case 3 by the porous membrane 4.
It is divided into two. It should be noted that the membrane surface liquid culture device 1 is not made by combining the two case halves 3a and 3b,
The porous membrane 4 may be inserted into one case to define the gas body chamber S1 and the liquid culture medium chamber S2. Here, the arrangement of the porous membrane 4 of the membrane surface liquid culture device is not limited to horizontal and may be vertical.

Case half 3 forming the gas body chamber S1
A gas body supply port 6 and a gas body discharge port 7 are provided in a, a gas body supply pipe 8 is connected to the gas body supply port 6, and a blower 9 is provided.
Thus, the gas body is sent into the gas body chamber S1 through the heater 10 and the sterilizing filter 11. The gas body chamber S1
A temperature sensor 12 is provided inside, and the temperature detected by the temperature sensor 12 is sent to a control device 13, and the heater 10 is controlled by a signal of the control device 13 so that the gas body reaches a predetermined temperature.

A gas body discharge pipe 14 is connected to the gas body discharge port 7. By opening a valve 15 of the gas body discharge pipe 14, the gas body in the gas body chamber S1 is passed through the sterilizing filter 16. Is discharged to the outside.

The temperature sensor 12 is installed as described above,
Based on that, the gas body is controlled to a predetermined temperature by the heater 10 and is supplied to the gas body chamber S1 from one port, and the gas body discharged by the respiration of the biological cells is discharged from the other port. It is suitable to increase the production amount of useful substances. In addition, it is suitable to install a sterilizing filter at the gas body supply port and the gas body discharge port in order to keep the inside of the gas body chamber S1 aseptic.

On the other hand, the case half 3b forming the liquid culture medium chamber S2 is provided with a liquid culture medium discharge port 17 and a liquid culture medium supply port 18, and is circulated between the liquid culture medium discharge port 17 and the liquid culture medium supply port 18. The liquid culture medium discharge pipe 19 and the liquid culture medium supply pipe 20 constituting the line are connected, and the preparation tank 2 is provided in the middle of the circulation line.
A pump 21 and a heater 22 are provided in the middle of the process, a temperature sensor 23 facing the inside of the liquid culture medium chamber S2 detects the temperature, and the detected temperature is input to a controller 24.
Are trying to control.

The pH sensor 2 is provided in the preparation tank 2.
5, the valve of the acid solution tank 27 or the alkaline solution tank 28 is opened / closed by a signal from the controller 26 according to the detection value of the pH sensor 25 to control the pH of the liquid medium.

In order to operate the membrane surface liquid culture system by the above circulation system, first, the liquid medium sterilized by the autoclave is aseptically placed in the liquid medium preparation tank 2. Then, the liquid medium heated to a predetermined temperature is supplied by the pump 21 to the liquid medium chamber S2 from the supply port 18 via the liquid medium supply pipe 20. On the other hand, the liquid medium supplied to the liquid medium chamber S2 is taken out from the discharge port 17 and returned to the liquid medium preparation tank 2 again via the liquid medium discharge pipe 19. After adjusting the composition and pH of the liquid medium in the preparation tank 2, the liquid medium chamber S2 is again adjusted.
Feed liquid medium into. That is, the liquid medium is circulated to flow the liquid medium. In such a state, the biological cells C are uniformly inoculated on the membrane surface of the porous membrane 4. In addition, depending on the case, it is possible to circulate intermittently. In this case, the flow rate is 0.1 to 300 / hour, preferably 1 to 30 / hour. The flow rate is defined as the ratio of the amount of circulating liquid medium per hour to the amount of liquid medium contained in the culture tank.

Next, the gas body G is sent to the heater 10 through the gas body supply pipe 8 by using the blower 9 and heated to a predetermined temperature. The heated gas body G is passed through the gas body supply pipe 8
The gas is supplied from the gas body supply port 6 to the gas body chamber S1 through the sterilizing filter 11. The gas body G that has entered the gas body chamber S1 is discharged from the gas body discharge port 7 to the outside of the gas body chamber S1 through the gas body discharge pipe 14 and the sterilizing filter 16.

The useful substances that can be produced in the liquid medium according to the present invention include protein-related substance degrading enzymes such as proteases and peptidases, starch-related substance degrading enzymes such as amylase, and chitin-related substance degrading enzymes. , Pectinases, cellulases, glucosidases, enzymes such as creatinase used in various clinical tests, and enzymes such as nucleolytic enzymes, amino acids, organic acids, saccharides, vitamins, nucleic acid-related substances, alcohol , Fatty acids, alkaloids, antibiotics,
Various substances such as antibacterial substances can be mentioned. Also, these various useful substances can be produced in various organism cells grown on the porous membrane. The various useful substances produced are collected by operations such as extraction, separation, fractionation, purification and crystallization according to a conventional method. Incidentally, the above-mentioned organism cells,
In the production of useful substances, the purpose may be achieved simultaneously by one culture, but may be achieved individually.

The method for exfoliating the biological cells from the surface of the porous membrane 4 is not particularly limited. For example, a scraping plate or the like may be used for scraping, or a pumping method may be used. The obtained cells may be used for any purpose, for example, extraction of enzymes, physiologically active substances, separation, purification, or conversion into food by known drying means, pharmaceutical preparation, and the like.

FIG. 3 is a cross-sectional view showing another embodiment of the membrane surface liquid culturing apparatus. The same members as those in the membrane surface liquid culturing apparatus are designated by the same reference numerals and the description thereof will be omitted. In this embodiment, a stirring device 30 is attached to the liquid medium chamber S2. Specifically, a motor 31 is fixedly provided on the outer wall of the liquid culture medium chamber S2, and a stirring blade 32 rotated by the motor 31 is arranged in the liquid culture medium chamber S2.

The stirrer 30 may be a magnetic stirrer in order to improve the sealing property, and the linear flow velocity is 0.1 to 1000 m / hour, preferably 0.5 to 800 m / hour. The flow linear velocity in this case is defined as the maximum linear velocity (m / hour) of the liquid medium per hour. Further, the membrane surface liquid culture device provided with the stirring device 30 can be operated as a membrane surface liquid culture system by itself.

In the case of the agitation method, a temperature sensor is installed in the liquid medium chamber S2 so that the temperature of the liquid medium is maintained at a predetermined value, and a heater or cooler for adjusting the temperature of the liquid medium based on the temperature sensor is installed. It is preferable to attach an operating liquid medium temperature controller. Further, in order to keep the pH of the liquid medium at a predetermined value, it is preferable to install a pH sensor in the liquid medium in the liquid medium chamber S2, and to attach a pH controller for supplying acid or alkali based on the pH sensor. Is. Further, it is convenient to connect and install a substrate replenishing device in order to keep the substrate concentration such as glucose constant. It is preferable that a pH sensor and a pH adjusting device, a temperature sensor and a liquid medium heating device, and a substrate replenishing device such as glucose are connected and installed in the liquid medium chamber S2 and the like.

FIG. 4 shows a flow of two-stage connection (fluid medium flow: one-way system) in the system of the present invention, which is used for culturing cells of plant and animal bodies, or It is preferably used for the purpose of producing only somatic cells. The flow rate has the same meaning as in the case of the circulation system and is defined as the ratio of the amount of liquid medium supplied per hour to the amount of liquid medium contained in the liquid medium chamber S2. In this case, the flow rate is 0.01 to 1 / hour, preferably 0.05 to 0.2 /
It's time.

In the above one-way system, the liquid medium chamber of the upstream membrane surface liquid culture device 1a and the liquid medium chamber of the downstream film surface liquid culture device 1b are connected by a liquid medium supply pipe, A liquid culture medium preparation tank 2 is provided in the middle of the liquid culture medium supply pipe. In the case of this method, the liquid medium is supplied from a liquid medium storage tank (not shown) to the liquid medium chamber of the upstream membrane surface liquid culture device by a pump via the liquid medium supply pipe. It is desirable to connect and install a liquid culture medium warming device that adjusts the temperature of the liquid culture medium to a predetermined temperature between the membrane surface liquid culture device on the upstream side. In addition, in order to adjust the temperature, the entire culture device may be placed in a room having a predetermined temperature.

FIG. 5 shows a two-stage connection flow (flow of liquid culture medium: circulation system) in the system of the present invention, in which the liquid of the upstream membrane surface liquid culture apparatus 1a is used. Medium chamber and downstream membrane-surface liquid culture device 1b
And a liquid medium supply line, and a liquid medium preparation tank 2 is provided in the middle of the liquid medium supply line.
Further, the liquid medium chamber of the membrane surface liquid culture device 1b on the downstream side and the liquid medium chamber of the membrane surface liquid culture device 1a on the upstream side are connected by a liquid medium return line, and the liquid medium is prepared in the middle of this liquid medium supply line. A tank 2 is provided.

FIG. 6 shows a flow of two-stage connection using the parallel system and the serial combination of the system of the present invention. In this system, the membrane surface liquid culture apparatus 1a, 1a is provided on the upstream side.
Are arranged in parallel, and upstream membrane surface liquid culture devices 1a, 1a
The liquid medium chamber and the liquid medium chamber of the membrane surface liquid culture device 1b on the downstream side are connected by a liquid medium supply line, and a liquid medium preparation tank 2 is provided in the middle of the liquid medium supply line.

FIG. 7 shows a flow of two-stage connection using the parallel system and the serial combination in the system of the present invention. In this system, the membrane surface liquid culture devices 1b, 1b are provided on the downstream side.
Are arranged in parallel, and the liquid medium chamber of the upstream membrane surface liquid culture device 1a and the liquid medium chambers of the downstream membrane surface liquid culture devices 1b and 1b are connected by a liquid medium supply line. A liquid culture medium preparation tank 2 is provided midway.

In the culture of the forms shown in FIGS. 4 to 7, alcohol fermentation with yeast is performed in the upstream membrane surface liquid culture device 1a, and acetic acid fermentation with acetic acid bacteria is performed in the downstream membrane surface liquid culture device 1b. Or biotin production by bacteria in the upstream membrane surface liquid culture device 1a, and then in the downstream membrane surface liquid culture device 1b, biotin-requiring Corynebacterium (Corynebacterium).
m) Bacteria such as genus can be cultured to produce useful substances such as amino acids and nucleic acid-related substances. In these culture forms, it is possible to produce organism cells or useful substances or their conversion by combining microbial cells with each other, combining microbial cells with animal cells or plant cells, combining animal cells with plant cells, etc. is there.

Next, specific comparative examples and experimental examples will be described. The system shown in FIGS. 1 and 2 was used for the experiment.

Experimental Example 1 The experimental conditions are as follows. The culture device used was the same as that used for circulating the liquid medium by the circulation system. However, a porous membrane SE22 (pore diameter, 0.22 μm; diameter, 9 cm; material, porsulfone; manufactured by Fuji Photo Film Co., Ltd.) is used as the porous membrane.
The liquid medium chamber S2 has a capacity of 20 ml, the gas body tank has a capacity of 30 ml, the liquid medium preparation tank has a capacity of 80 ml, and the slow bacterial membrane 17 has a pore diameter of 0.2 μm.
I used the one. However, the pH adjuster was not operated. Air is used as the gas body, and filamentous fungus A is used as the organism cells.
spergillus oryzae var. ory
Kojic acid was produced using the zae IFO30113 strain. Further, the temperature of the liquid medium in the liquid medium chamber S2 and the temperature of the gas body in the gas body chamber S1 are 30 ° C., the circulation speed, that is, the flow rate of the liquid medium is 1 / hour, and the supply speed of the gas body is 30.
It was ml / min. The composition of the liquid medium used in the experiment is as follows: glucose, 10.0% (w / v); yeast extract, 0.5
% (W / v); K ₂ HPO ₄ , 0.1% (w / v); Na
Cl, 0.05% (w / v); FeSO ₄ .7H ₂ O,
0.05% (w / v); MgSO 4 · 7H 2 O, 0.00
1% (w / v); pH, 6.0 (prepared with 1N HCl). The liquid medium was autoclaved at 120 ° C for 10 minutes.

First, 50 ml of a liquid medium sterilized by an autoclave was aseptically added to the liquid medium preparation tank 2, and the liquid medium was circulated as described above. When the circulation reached equilibrium, the pump was stopped, and 200 μl of the spore suspension liquid of the filamentous fungus (total number of spores: 5000) was uniformly placed on the membrane surface of the porous membrane 3. The atmosphere was sent to the gas body chamber S1 as described above using the pump 9, and was then discharged to the outside of the gas body chamber S1.

In this state, the filamentous fungus was cultured for 4 days. The hyphae of the filamentous fungus covered the entire membrane surface of the porous membrane 4.
At this point, the pump 21 was operated again to circulate or flow the liquid medium between the liquid medium preparation tank 2 and the liquid medium chamber S2. Liquid medium circulation was continued for another 5 days. At this point, the glucose concentration in the liquid medium became zero. In the liquid medium in the liquid medium preparation tank 2, autoclave sterilization was performed so that the final glucose concentration of the total liquid medium was 10%.
10 ml of 0% glucose solution was added. After that, the culture was continued while further circulating. FIG. 8 shows the change with time in the production of koji acid. On the 9th and 18th days of culture, the production of koji acid was 0.7 and 2.9 mg / ml, respectively, and the total production was 15
It was 0 mg. The dry cell weight of the filamentous fungus was finally 450 mg. In addition, p of liquid medium
H was not controlled.

(Experimental example 2) Aspe
rgillus oryzae ATCC20386,
Aspergillus oryzae IAM270
4, Aspergillus soya ATCC20
Each of the 388 filamentous fungi was cultured to produce a neutral protease. Then, the spore suspension of each strain was inoculated on the upper surface of the porous membrane 4 and cultured for 4 days without moving the liquid medium until the hypha covered the entire upper surface of this membrane. After this,
While moving the liquid medium, it was cultured for 21 days at a liquid medium moving speed of 1 / hour. At this point, the glucose concentration in the liquid medium became zero. While manually controlling the glucose concentration in the liquid medium to 0.01% in the liquid medium preparation tank 2,
It was cultured for 17 days. The pH in the liquid medium was kept at 7.0 after the liquid medium was started to move.

As a result, the total amount of neutral protease produced was Aspergillus oryzae ATCC20.
386 is 280000 units, Aspergillus
oryzae IAM2704 is 700000 units,
Aspergillus soya ATCC2038
8 was 250,000 units.

The following liquid medium was used in Experimental Example 2. Glucose, 0.2% (w / v); casein,
0.4%; K ₂ HPO ₄ , 0.1% (w / v); NaC
1, 0.05% (w / v); FeSO ₄ .7H ₂ O, 0.
05% (w / v); MgSO 4 · 7H 2 O, 0.001%
(W / v); pH, 7.0 (prepared with 1N NaOH).
The method for measuring neutral protease is as follows. Substrate 0.6% Casein (0.05M potassium phosphate buffer, pH 7.0) 1.25 ml to sample 0.25
Add ml and carry out the reaction at 30 ° C. for 60 minutes or 120 minutes. A sample not reacted by adding 1.25 ml of 0.44 M TCA (trichloroacetic acid) solution before the reaction was used as a control.
The reaction was stopped by adding 1.25 ml of 0.44 M TCA (trichloroacetic acid) solution to the reaction solution. After standing at room temperature for 30 minutes, it was filtered with a paper and the small fragmented peptide in the liquid was measured by the Lowry-Folin method.

Comparative Example The experimental conditions in the comparative example were the same as those in the experimental example 1 except that the liquid medium was allowed to stand without flowing.

In the comparative example, when the temperature of the liquid medium reached 30 ° C., the spores of the filamentous fungus were inoculated on the entire membrane surface,
Cultured. As described above, on the 4th day, the hypha covered the entire membrane surface. After further culturing, the glucose concentration became 0 on the 15th day.
Became. At this point, the membrane fixing bolt 4 was aseptically removed, then the culture tank 1, the porous membrane 3 and the gas body tank 2 were removed, and the liquid medium was replaced with 20 ml of the fresh liquid medium. Further, the culture device was set back to the original state and the culture was continued. The change with time of the production of koji acid is shown in FIG. Culture 1
On the 5th and 25th day, the production of malic acid was 0.4 and 1.2 mg /
In ml, the total yield was 32 mg. The cell mass was finally 500 mg.

From FIGS. 8 and 9, the production of koji acid by the culture method of the present invention was about 4.5 times higher than that of the control in total yield.
It was also found that the amount was about 5 times higher per unit cell amount. As described above, the method of the present invention remarkably improves the productivity of koji acid by filamentous fungi.

Various measuring methods are as follows. Glucose concentration: Performed by the glucostat method. That is, 1.5 N of N was added to 1 ml of an appropriately diluted sample.
0.435 ml of aOH solution was added, and the mixture was allowed to stand at room temperature for 15 minutes. Then 0.565 ml of a 1.5 N acetic acid solution was added (this operation brings the pH to 5.0). To this, 1 ml of 50 mM sodium acetate buffer (pH 5.0) was added. Furthermore, glucostat reagent (Diacolor GC, TO
1 ml of YOBO) was added and the absorptiometer (SHIMAZ)
U UV-160) was used to measure the rate of increase in absorbance at a wavelength of 550 nm. The glucose concentration in the sample was calculated from the calibration curve of the standard sample. Dry cell weight: After scraping the cells directly from the membrane surface, the cells were washed three times with distilled water by centrifugation or by careful consideration. The washed cells were dried at 100 ° C. for 2 days, and the weight was measured. Koji acid: Beatly method (Methods)
Enzymol. Volume 3, 238-241, 1957
Year). That is, the appropriately diluted sample solution 1
1 ml of 1% FeCl ₃ dissolved in 0.1N HCl
1 ml of 6H ₂ O solution was added, and the mixture was left standing at room temperature for 30 minutes.
Then, after measuring the absorbance at 540 nm, the concentration of koji acid was calculated from a calibration curve obtained by using a solution of koji acid having a known concentration.

In each of the above embodiments, the circulation of the liquid medium itself and the stirring of the liquid medium itself have been described as means for flowing the liquid medium, but instead of circulating or stirring the liquid medium, a membrane is used. You may move one.

[0047]

EFFECT OF THE INVENTION Since the culture method of the present invention is constructed as described above, it is possible to eliminate the difference in the concentration of nutrients or products generated in the vicinity of the membrane surface of the liquid medium due to the growth of organism cells, and Inhibition of growth of organism cells by the high-concentration product produced in the liquid medium near the surface is eliminated. Therefore, it is possible to cultivate various kinds of biological cells by one culturing method called the membrane surface liquid culturing method, and it is possible to remarkably improve the productivity of the biological cells and useful substances.

[Brief description of drawings]

FIG. 1 is a flow chart of a membrane surface liquid culture method for flowing a liquid medium by the circulation method of the method of the present invention.

FIG. 2 is an overall system diagram of a membrane surface liquid culture system that performs liquid medium flow by the same circulation method.

FIG. 3 is a sectional view showing another embodiment of the membrane surface liquid culture device.

FIG. 4 is a flow of two-stage connection by a serial method in the system of the present invention (flow of liquid medium: one-way method)

FIG. 5 is a flow of two-stage connection by a serial method in the system of the present invention (fluid medium flow: circulation method)

FIG. 6 is a flow chart of a two-stage connection using a parallel system of the system of the present invention and a serial combination (the upstream membrane surface liquid culture device is in parallel).

FIG. 7 is a flow of two-stage connection using a parallel system and a serial combination of the system of the present invention (downstream membrane surface liquid culture device is parallel).

FIG. 8 is a graph showing changes with time in the production of koji acid by the method of the present invention.

FIG. 9 is a graph showing changes over time in the production of koji acid by a conventional method.

FIG. 10 is an overall system diagram of a membrane surface liquid culture device for carrying out a conventional method.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Membrane surface liquid culture device, 1a ... Membrane surface liquid culture device on the upstream side, 1b ... Membrane liquid culture device on the downstream side, 2 ... Liquid culture medium preparation tank, 3 ... Case, 3a, 3b ... Case half body, 4 ... Porous membrane, 9, 21 ... Pump, 10, 22 ... Heater, 11,
16 ... Sterilization filter, 19 ... Liquid medium discharge pipe, 20 ... Liquid medium supply pipe, 30 ... Stirrer, C ... Inoculated cells, S1 ...
Gas body chamber, S2 ... Liquid culture medium chamber.

Claims (10)

[Claims] 1. A biological medium is brought into direct contact with a porous membrane on one side and a gas body on the other side with a porous membrane sandwiched between them, and the organism on the membrane side of the porous membrane on the gas body side while flowing the liquid medium. A membrane surface liquid culture method, which comprises culturing cells. 2. The membrane surface liquid culture method according to claim 1, wherein the flow of the liquid medium is due to the circulation of the liquid medium. 3. The membrane surface liquid culture method according to claim 1, wherein the liquid medium flows by stirring the liquid medium. 4. The membrane liquid culture method according to claim 1, wherein the gas body is brought into direct contact with the porous membrane under pressure, and the pressure on the gas body side is equal to or higher than the pressure on the liquid side. Membrane surface liquid culture method. 5. A membrane surface liquid culture device for directly contacting a liquid medium on one side with a porous membrane and a gas body on the other side with a porous membrane sandwiching a porous membrane, and culturing biological cells on the surface in contact with the gas body. The membrane surface liquid culture apparatus is characterized in that the inside of the case is divided into a gas body chamber and a liquid medium chamber by a porous membrane, and a stirring device is provided in the liquid medium chamber. 6. The membrane surface liquid culture apparatus according to claim 5, wherein the case is formed by abutting and coupling the openings of two case halves with a porous membrane interposed therebetween. Liquid culture device. 7. A membrane surface liquid culture system incorporating a membrane surface liquid culture device in which a case is defined by a porous membrane into a gas body chamber and a liquid medium chamber, wherein the membrane surface liquid culture device has a liquid medium. A membrane surface liquid culture system, characterized in that a circulation line for taking out the liquid medium in the chamber and feeding it again into the liquid medium chamber is connected, and a liquid medium preparation tank is provided in the middle of this circulation line. 8. A membrane surface liquid culture system incorporating a membrane surface liquid culture device in which a gas membrane chamber and a liquid medium chamber are defined by a porous membrane in the case, wherein the membrane surface liquid culture system comprises a plurality of membranes. The surface liquid culture device is arranged in series, and the liquid medium chamber of the membrane liquid culture device on the upstream side and the liquid medium chamber of the membrane liquid culture device on the downstream side are connected by a liquid medium supply line. A membrane surface liquid culture system comprising a liquid culture medium preparation tank provided in the middle of a supply line. 9. A membrane surface liquid culture system in which a membrane surface liquid culture device having a gas body chamber and a liquid medium chamber defined by a porous membrane inside a case is incorporated, wherein the membrane surface liquid culture system comprises an upstream side and A plurality of membrane surface liquid culture devices are arranged in parallel on at least one of the downstream sides, and the liquid medium chamber of the upstream membrane surface liquid culture device and the liquid medium chamber of the downstream membrane surface liquid culture device are the liquid medium. A membrane surface liquid culture system, characterized by being connected by a supply line, and a liquid medium preparation tank being provided in the middle of the liquid medium supply line. 10. The membrane surface liquid culture system according to claim 7 or 8, wherein a liquid medium chamber of the most downstream membrane surface liquid culture device and a liquid medium chamber of the most upstream membrane surface liquid culture device are provided. Are connected by a liquid culture medium return line, and a liquid culture medium preparation tank is provided in the middle of the liquid culture medium return line.