JPH09121843A - Culture of microorganism and apparatus therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a high-quality cultured product such as koji with no concern for sundry germ contamination, by holding microorganisms and a substrate in a closed vessel and suppressing the temperature rise during culture process by promoting evaporation through pressure reduction to culture the microorganisms under simplified temperature control. SOLUTION: Microorganisms such as bacteria or yeast and a substrate are held in a closed vessel, the temperature rise during culture process is suppressed by promoting evaporation by pressure reduction and scavenging heat in the form of the latent heat involved in the evaporation. It is preferable to conduct the culture in such a way that the concentration of the carbon dioxide generated from the microorganisms is measured, and when the measurement result exceeds a specified value, the vessel is depressurized to decrease the amount of the carbon dioxide. Furthermore, it is preferable that the culture be conducted using an apparatus equipped with a control unit 11 to control the pressure reducing device based on the data from a temperature detector 8, a carbon dioxide detector 9 and a thermostat.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method and an apparatus for culturing microorganisms.

[0002]

2. Description of the Related Art Solid culture of microorganisms will be briefly described by taking sake production using koji mold as an example. The process of producing sake is rice polishing, washing, soaking, steamed rice, koji making, and fermentation. Among these, the method according to the present invention is a koji making step, and each step will be briefly described here in order. First, brown rice is whitened, washed with water, made to absorb water, and steamed in a pot. This completes steamed rice. For the next koji making, spread the steamed product, cool it, sprinkle the seed koji, and stir it well so that spores are evenly formed. The seed koji is rice or the like in which the koji mold has propagated, but there are also powdery forms such as spores of the propagated fungus.

Then, the bacteria are cultured in a shallow wooden box in the culture room. This culture is called koji making. At this time, control the temperature and humidity by manually stirring or blowing air. This is because the temperature of koji gradually rises due to respiratory heat as it grows. Management during this culture is extremely difficult, and has been performed only by Mr. Mori, who has been skilled in the past.

The temperature of cultivating koji is 3 in relation to the culturing time.
It must be strictly controlled above 0 ° C and below 42 ° C. This is because the growth rate is remarkably reduced when the temperature is 30 ° C or lower, and the bacteria are almost dead when the temperature is 42 ° C or higher. The purpose of blowing air during culturing is to supply oxygen for respiration because the koji mold itself is aerobic, and to evaporate the water contained in the koji, thereby removing the latent heat of vaporization to lower the temperature. The purpose of putting in the above-mentioned shallow wooden box is to ensure that the air blows uniformly.

Further, a method is also practiced in which, instead of simply sending air above the koji, the air is sent from above and below the koji and the wind is applied (passed) to the koji stacked below.
In this case, since the wind needs to pass through, the bottom has a net shape.

[0006]

Among the above methods, in the case of putting it in a wooden box and sending the wind in the lateral direction, the substrate (rice, etc.) usually has a deposition thickness of 2 to 4 cm and cannot be deposited thickly. It was very space-consuming and difficult to arrange. In particular, because the wind hits different places, it was very skillful to move the wooden box itself, mix the koji by hand, and measure the temperature of each koji by hand. This is because the temperature must be controlled within a small range as described above.

Further, in the method of passing the wind in the vertical direction, the deposition thickness of the substrate can be about 20 to 30 cm. However, since the resistance when passing the substrate is different depending on the place, the amount of passing wind is different and The temperature may drop too much or the rice may crack in places where it often passes. Further, the growth of the mycelium of koji increases the resistance of the substrate, which makes ventilation difficult as the growth progresses.

[0008]

In view of the above circumstances, the present inventor has completed the method and apparatus of the present invention as a result of earnest research, and the feature is that the method is In the solid culture of aerobic microorganisms, the microorganisms and the substrate are kept in a closed container, and the temperature rise in the culture process is suppressed by reducing evaporation by promoting evaporation and reducing the heat by evaporation. Then,
An airtight container, temperature detector, thermostat, depressurizer,
And a control device for controlling the pressure reducing device based on the data from the temperature detector.

Microorganisms generally refer to a group of microscopic organisms such as bacteria, yeasts and filamentous fungi. The koji mold used above is a kind of filamentous fungus, and Bacillus amyloliquefaciens F. described later is a kind of bacteria. Similarly, these are those which are heated by breathing and are cooled by evaporation of water, and the present invention can be applied to all of them. The present invention relates to aerobic and solid-cultured ones. The koji mold described in the above sake production is a general term for filamentous fungi of the genus Aspergillus for producing koji used in the brewing industry of Japan, Aspergillus oryzae, Aspergil.
There are lus tamarii and Aspergillus niger.

The closed container is a container that can be depressurized, and its shape and size are not particularly limited. Further, decompression can be performed by using an ordinary vacuum pump, and no special method is required. The degree of pressure reduction may be such that the temperature can be maintained within the above range. Usually, as is clear from the saturated vapor pressure diagram, it is 20 to 30 mmHg at 30 to 40 ° C.
Of course, since there is a time lag, the pressure may be reduced further. When the pressure is reduced, the temperature naturally lowers so as to reduce the saturated vapor pressure to that pressure. That is, in terms of energy, heat is taken from the koji by latent heat of vaporization.

In the automatic operation method of the present method, the upper and lower limits of the temperature of the material to be cultured are set in relation to the culture time. Then, when the culture progresses and the temperature reaches the upper limit, the pressure is reduced to lower the temperature. And when the temperature drops and reaches the lower limit,
The depressurization is stopped and fresh air at a given temperature is introduced. It is better to raise the temperature of this air to the lower limit temperature (or a temperature slightly higher than it). This depressurization and air introduction are repeated to advance the culture. In the case of automatic operation, all the above operations are performed by automatic valves and vacuum pumps,
No manpower is required. However, everything may be done manually.

In this method, unlike the conventional air blowing method, there is no unevenness in the passage of air because the pressure in the closed vessel is uniformly reduced. Since the pressure is uniform in the container, the degree of evaporation is also uniform, which causes the temperature to drop uniformly. This point is extremely superior to the conventional ventilation method.

Further, since the degree of depressurization and the temperature are strictly linked, and the pressure in the container can be automatically and uniformly controlled in the operation of the vacuum pump and the opening / closing of the valve. The worker who did it is unnecessary. In addition, it is not necessary to always look around the temperature rise of koji. That is, by the data from the temperature sensor (s),
There is no room for human cans to work simply by operating the vacuum pump. Of course, a person may operate the vacuum pump by observing the measured value from the temperature sensor without using a computer or automatic control.

Further, in the present invention, it is useful to measure not only the temperature but also the amount of carbon dioxide (carbon dioxide gas) with a sensor. Carbon dioxide is generated as the culture progresses, and this is due to the increase in respiration volume accompanying the growth of koji. As with oxygen-breathing animals, increased carbon dioxide in the surrounding atmosphere affects respiration, so carbon dioxide around the koji hinders growth above a certain level. Therefore, even if the temperature rise does not reach the predetermined value, when the amount of carbon dioxide gas increases, the pressure is reduced to reduce the carbon dioxide gas. Of course,
It is natural to supply fresh air thereafter. In this case, the degree of pressure reduction is small so that the temperature is not lowered so much. That is, it is about 300 to 600 mmHg.

Further, a culture experiment was conducted on oxygen deficiency due to reduced pressure. The depressurization is temporary, that is, when the temperature drops, clean air at a predetermined temperature is immediately introduced. I investigated the effect of this temporary lack of oxygen,
There was no effect. This introduced air is passed through a filter to filter out germs, dust, etc.
The temperature has been raised to about 42 ° C).

Therefore, since only clean air is introduced into the airtight container, the quality of the product is not deteriorated by various bacteria, and dust is not mixed in, so that a high quality product can be manufactured with high yield.

Next, the device of the present invention will be described. First, the main body is the closed container described above. There is a large opening for taking in and out raw materials (such as steamed rice) and a lid for closing it. As for the size, the diameter is 1-2 m and the height is 2-4 m.
The degree is appropriate, but is not particularly limited.

The temperature detector is preferably an ordinary thermocouple type, and it is easy to connect a computer or the like. However, if automatic operation is not used, mercury or alcohol can be used. The carbon dioxide detector may be a commercially available one.

The thermostatic device has the meaning of heat insulation that does not bring a change in the outside temperature into the inside, and has the function of raising the temperature and keeping the temperature when the outside is low temperature. This is preferably an electric heater around the body or a hot water jacket. When the outside air temperature is 30 ° C or lower, the temperature is raised to about 30 ° C to promote the growth of bacteria, and when the outside air temperature is 40 ° C or higher, it is cooled with cold water. However, since it is hard to think, there is no jacket and only an electric heater may be used. Of course, only the jacket may be used. The hot water jacket only needs to have a double structure around the main body of the apparatus and circulate hot water in the double structure.

The decompressor means a vacuum pump and a gas-liquid separator (drain separator) installed in front of it. These may also be ordinary ones. The gas-liquid separator is preferably one that can quantify the liquid (water) accumulated therein. There is almost a correlation between the growth of the koji mold and the evaporation amount of water contained in rice, and when the rice is cultured at a normal temperature, the koji making is completed when the evaporated water amount reaches a predetermined amount. In the past, the amount of evaporated water and the like could not be measured and was ignored, but in the present invention, this can be done, so that the final management can be performed by the amount of water.

The control device is a device for controlling the operating state of the vacuum pump, the opening and closing of valves, etc. based on the measured values from the temperature sensor and the carbon dioxide sensor, and is usually a computer.

INDUSTRIAL APPLICABILITY The method and apparatus of the present invention can be used not only for cultivating koji for sake brewing described above but also for culturing microorganisms for various purposes.
It is obvious that the heat generation and the cooling effect by the reduced pressure are the same.

[0023]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The device of the present invention will be described in more detail based on the embodiments shown in the drawings. FIG. 1 shows one embodiment of the present invention.
It is a schematic sectional drawing which shows embodiment. The main body 1 which is a closed container is composed of a lower substrate housing portion 2 and an upper lid 3, both of which are connected by a flange 4, and a sealing packing is provided therebetween. The housing portion 2 is provided with a sun plate 5 that serves as a bottom for housing the substrate. A jacket portion 6 is provided around the main body and is connected to a hot water circulating device 7. A temperature sensor 8, a carbon dioxide gas sensor 9, and a pressure gauge 10 are provided in the housing portion, and a control device (measuring device and personal computer) 1
The measurement value is introduced in 1. Control device 1
The opening degree of the pressure reducing line valve 12 (usually a check valve) and the air introduction valve 13 are adjusted by the control signal from 1. The decompression device comprises a vacuum pump 14, a gas-liquid separator 15 and a condenser 16, and is connected to the main body by a pipe having a decompression line valve 12. The condenser 16 is for liquefying water vapor, and may be of any type such as a refrigeration type or a compression type.

Air is introduced from the air introducing device 17 through a pipe having the air introducing valve 13. The air introducing device 17 is composed of an air filter and a temperature controller (usually a heater or a heater with a humidifier). A drain valve 18 for draining wash water is provided at the bottom of the main body.

Next, the operating condition will be described. There is 190 liters between the substrate storage upper limit of the main body 1 and the bottom sana plate 5, and steamed rice (Nihonbare,
100 kg of rice polishing degree 70%, product temperature 32 ° C.) (converted to white rice weight) is put, the lid 3 is connected with the flange 4, and the hot water circulation device 7 is set to 33 ° C. The main body is closed with the air introduction valve 13, the pressure reducing line valve 12, and the drain valve 18 in order to avoid evaporation of steamed rice moisture. In this state, the temperature is kept for 18 hours, and the koji mold is germinated. After 18 hours, set the hot water circulation device 7 to 3
By setting the temperature to 9.5 ° C, the control device 11 which controls the whole makes the upper limit of the koji culture temperature 38.5 ° C and the lower limit 37.5 ° C.
Set to. The upper limit of carbon dioxide concentration was set to 6%, and the lower limit was not specified.

The product temperature increased as the culture time passed, and the carbon dioxide concentration increased with the product temperature. After 19 hours of culturing, the carbon dioxide concentration reached the set upper limit value (6%), and carbon dioxide discharge was started. To discharge carbon dioxide, open the decompression line valve 12 and press the vacuum pump 1
4 is activated and the pressure gauge indicates 360 mmHg, the decompression line valve 12 is closed, the pump is stopped, the air introduction valve 13 is opened, 38 ° C. clean air is sucked into the device, and the inside of the body is returned to normal pressure. Then, the air introduction valve 13 was closed. Thereafter, this operation was intermittently repeated until the culture was completed. The number of operations was 24.

On the other hand, the product temperature is 25 hours after culturing,
The upper limit of 38.5 ° C was reached. Therefore, the decompression line valve 12 was opened, the vacuum pump 14 was operated, the pressure gauge 10 was decompressed until it reached 30 mmHg, and the depressurized state was continuously maintained until the product temperature reached 37.5 ° C. When the product temperature reaches 37.5 ° C, the decompression line valve 12 is closed, the pump is stopped, the air introduction valve 13 is opened, and 38 ° C clean air is sucked into the device to return it to normal pressure, and then air is introduced. The valve 13 was closed. During this time, it was about 3 minutes. This operation was repeated intermittently during the culture period. The number of operations was 45 times. Of course, carbon dioxide gas is discharged even under the reduced pressure for adjusting the temperature.

After 40 hours of culturing, the gas-liquid separator 1
The water volume of 5 reached 11.3 liters. At this point, the koji making was completed, and the koji was continuously stored in a depressurized state. After 4 hours, the flange 4 was opened and the koji was taken out. The quality of the koji was a little dry, and it was a breakthrough-type fair-white koji with a good texture and good taste. The titer of this koji is as shown in Table 1. Compared with the enzyme titer of standard koji, the α-amylase and glucamylase titers are high, and it is of excellent quality as a koji for sake. there were.

[Table 1]

Next, using the device of the present invention, Bacillus amylo
Enzyme production was carried out using liquefaciens F. Body 1
65 kg of wheat bran and 35 liters of 8% soybean meal leaching solution were mixed with each other, heat sterilized by a conventional method, and then allowed to cool and inoculated with the above bacteria. The culture was started at a product temperature of 37 ° C, and the upper limit of the culture temperature was set to 37.5 ° C and the lower limit was set to 36.5 ° C.

After 20 hours of culture, the substrate temperature reached the upper limit. Therefore, the same depressurization operation as in the above example was performed, and the lower limit temperature was reached 5 minutes later, so clean air at 37 ° C. was introduced. This depressurization operation was repeated about every 16 minutes, and after 60 hours of culture, the culture was completed and a substrate sample was taken to measure the enzyme titer. The results are shown in Table 2, which was almost the same as the conventional enzyme titer.

[Table 2]

[0031]

The present invention has the following great effects. There is a difference between the decompression for temperature control and the decompression for carbon dioxide concentration control, and it is possible to manage according to the set numerical values. Efficient because there is no work such as moving, turning, and transshipping steamed rice in the conventional method. Since filtered air is introduced into the airtight container, there is no concern about contamination of various bacteria. The thickness of the steamed rice can be 100 to 200 cm, and the koji can be efficiently produced in a relatively small incubator. If hot water is passed through the outer jacket of this incubator to control the temperature, the conventional koji room, large-scale heat-insulating device is not required. The size of the heat-retaining space is about 1.5 to 2 times that of the steamed rice contained, and the space may be smaller than the conventional 20 to 200 times.

[Brief description of the drawings]

FIG. 1 is an overall schematic sectional view showing an example of a device of the present invention.

[Explanation of symbols]

 1 Device Main Body 2 Substrate Storage Section 3 Lid 4 Flange 5 Sana Plate 6 Jacket 7 Hot Water Circulator 8 Temperature Sensor 9 Carbon Dioxide Gas Sensor 10 Pressure Gauge 11 Controller 12 Pressure Reduction Line Valve 13 Air Inlet Valve 14 Vacuum Pump 15 Gas-Liquid Separator 16 Condenser 17 Air introduction device 18 Drain valve

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location // (C12N 1/20 C12R 1:07)

Claims (4)

[Claims] 1. In the solid culture of aerobic microorganisms, the microorganisms and the substrate are held in a closed container, and the temperature rise during the culture process is suppressed by reducing the pressure to accelerate evaporation and take heat as the latent heat of evaporation. A method for culturing a microorganism, which comprises: 2. The method for culturing a microorganism according to claim 1, wherein the concentration of carbon dioxide gas generated from the microorganism is measured, and when the measured value exceeds a predetermined value, the inside of the container is depressurized to reduce the amount of carbon dioxide gas. 3. An apparatus for culturing microorganisms, which is a closed vessel, and has a temperature detector, a thermostatic device, a decompression device, and a control device for controlling the decompression device based on data from the temperature detector. 4. The microorganism culture device according to claim 3, which is equipped with a carbon dioxide detector.