JPH0380467B2 - - Google Patents

Description

[Detailed description of the invention] (Industrial application field) The present invention relates to a method for culturing aerobic filamentous fungi.

(Prior art) The production of primary metabolites such as glycerin and alcohol or secondary metabolites such as penicillin and streptomycin has been carried out at an industrial level by growing and culturing cells such as molds and actinomycetes in a culture solution. It is well known that there are.

When cells are cultured in a culture medium, the cells proliferate by pellet growth or pulpy growth, depending on the type of cells being cultured. Pellet growth refers to a growth form in which hyphae of cultured cells become entangled during culture, forming granular cell clusters and multiplying, and occurs in the production of penicillin, etc. Pulpy proliferation refers to a mode of proliferation in which proliferating cells are uniformly distributed in a culture medium, and occurs in the production of streptomycin, etc. However, these conventional propagation modes have had various problems. It is well known that when pellet proliferation occurs, the cell proliferation rate is extremely low and cells cannot be efficiently proliferated. Furthermore, in the case of cells that undergo pulpy culture, the cultured cells are present in a uniformly mixed state in the culture solution, so the viscosity of the culture solution becomes extremely high, and the power required to stir the culture solution becomes extremely high. Furthermore, in both pellet growth and pulpy growth, cells exist in a mixed state in the culture medium, so when separating and extracting metabolites from the culture medium, it is necessary to collect a small sample from the culture medium during cell growth. When analyzing culture fluid and metabolites, it is necessary to remove solid cells, and separating and extracting metabolites from the culture fluid or monitoring the culture fluid is extremely troublesome and time-consuming. It was hot.

(Objective of the Invention) An object of the present invention is to provide a method for efficiently culturing aerobic filamentous fungi by multiplying aerobic filamentous fungi that produce pellet growth using a growth form different from pellet growth according to conventional culture methods. There is a particular thing. It is also an object of the present invention to allow aerobic filamentous fungi to grow without the presence of proliferating aerobic filamentous bacteria in the culture solution, and to achieve this through a step of removing solid aerobic bacteria from the culture solution. It is an object of the present invention to provide a method for culturing aerobic filamentous fungi, which allows easy separation and extraction of metabolites from a culture solution or analysis of a culture solution without causing any damage. Furthermore, another object of the present invention is to make it possible to stir the culture solution with low power without increasing the viscosity of the culture solution during the growth of aerobic filamentous fungi, and to improve oxygen supply conditions. An object of the present invention is to provide a method for culturing aerial filamentous fungi.

(Structure of the Invention) The method for culturing aerobic filamentous fungi of the present invention is characterized by culturing aerobic filamentous fungi in a plurality of foam carriers dispersed in a culture solution.

As the foamed carrier used in the present invention, any of foamed hydrophilic materials and foamed hydrophobic materials can be used, but examples include foamed polyvinyl alcohol, urethane foam, etc. I can do it. As the urethane foam, for example, one manufactured by a continuous foaming method from polyethylene glycol diisocyanate and polypropylene glycol diisocyanate can be used. Pore diameter of foamed carrier is 10μm to 10mm
A foamed carrier is preferable because it allows aerobic filamentous fungi to grow efficiently within the confines of the foamed carrier. When using a foamed carrier dispersed in a culture solution, the size of the foamed carrier should be 1/5 or less of the size of the culture tank, preferably 1/2
It is desirable to set the value to 0 or less from the point of view of improving stirring efficiency, and the maximum volume of the foamed carrier (apparent volume including the volume of foam inside the foamed carrier) is 100ml of culture medium.
It is preferable to set it as 100 cm ^<3> , preferably 50-10 cm ^<3> . Examples of aerobic filamentous fungi that can be grown by the method of the present invention include molds and actinobacteria.

(Effects of the Invention) The present invention allows aerobic filamentous fungi to be cultured within the pores of a foam carrier, and by using the aerobic filamentous fungi culturing method of the present invention, conventional culturing methods can be used. Cells proliferate in a different manner than in the case of cell growth.

Specifically, the method of the present invention allows aerobic filamentous fungi, which grow in pellets according to the conventional method, to grow by pulpy growth or only within the foam carrier, and the culture solution It is grown by a new growth form in which aerobic filamentous fungi are not present. In addition, aerobic filamentous fungi that caused pulpy growth according to the conventional method, but according to the method of the present invention, the number of aerobic filamentous fungi in the culture solution is further reduced, resulting in pulpy growth, or aerobic fungi only within the foam carrier. The filamentous fungi are grown in a new growth form in which no aerobic filamentous fungi are present in the culture solution.

Therefore, by using the present invention, aerobic filamentous fungi, which conventionally caused pellet growth, can be efficiently grown by a growth mode different from pellet growth. In addition, if aerobic filamentous fungi are grown only within the foamed carrier, and aerobic filamentous fungi are grown in a new growth form where aerobic filamentous fungi are not present in the culture solution, it is considered a solid material. Since the step of separating and extracting aerobic filamentous fungi from the culture solution can be omitted, the separation and extraction of metabolites from the culture solution and the analysis of the culture solution can be carried out easily and in a short time. Even when pulpy propagation is performed, the amount of aerobic filamentous fungi in the culture solution is smaller than in the case of conventional methods, so solid matter can be easily separated. Furthermore, although there are almost no aerobic filamentous bacteria in the culture solution, even if they do exist, the amount is smaller than in the case of conventional methods, so the viscosity of the culture solution can be kept low, and the culture solution can be stirred with low power. It can be done efficiently. Furthermore, since oxygen supply conditions can be improved, aerobic filamentous fungi can be efficiently cultured.

(Example) The present invention will be explained below using examples.

Example 1 In 1 part water, 40 g lactose, 20 g corn steep liquor, 3 g NaNO ₃ , 0.5 g KH ₂ PO ₄ and
Culture solution 100 prepared by adding and mixing 0.25g of MgSO ₄
ml into a 300 ml Erlenmeyer flask, and add Penicillium chrysogenum to this culture solution.
chrysogenum) was added, and 2 g (22 cm ³ ) of urethane foam for artificial soil made by Nitto Denki Kogyo Co., Ltd., approximately 5 mm square and having an average pore diameter of approximately 0.5 mm, was placed in the Erlenmeyer flask. The temperature of the culture solution in the Erlenmeyer flask was maintained at 24° C., and the Erlenmeyer flask was stirred at a stirring speed of 200 rpm to culture, and this was used as Example 1.

Aerobic filamentous fungi were cultured in the same manner as in Example 1 above, except that urethane foam was not mixed, and this was used as Comparative Example 1.

In the case of Comparative Example 1, the growing aerobic filamentous fungi were uniformly mixed in the culture solution and multiplied in a pulpy manner, but in the case of Example 1, the aerobic filamentous fungi grew in the foam carrier, resulting in aerobic growth. Even after the growth of filamentous fungi, no aerobic filamentous fungi were present in the culture solution, and the culture solution continued to maintain a transparent state as at the time when aerobic fungus growth started.

FIG. 1 shows the lactose concentration in the culture solution and the production amount of penicillin in Example 1 and Comparative Example 1.

Although the lactose concentration decreased faster in Comparative Example 1 than in Example 1, the production amount of penicillin reached the same maximum amount on the 7th day in both Example 1 and Comparative Example 1. The quantity unit γ of penicillin produced is μg/
means.

Example 2 100 ml of the same culture solution as in Example 1 was added to a volume of 300 ml.
Penicillium chrysogenum (Penicillium chrysogenum) was added to this culture solution.
chrysogenum) was added, and then placed in an Erlenmeyer flask with an average pore diameter of approximately 0.5 mm.
1 g (30 cm ³ ) of polypropylene glycol (RPG) urethane foam was added. In the same manner as in Example 1, the temperature of the culture solution in the Erlenmeyer flask was maintained at 24° C., and the Erlenmeyer flask was stirred using a rotary shaker at a stirring speed of 200 rpm to culture, and this was used as Example 2.

Aerobic filamentous fungi were cultured in the same manner as in Example 2, except that urethane foam was not mixed, and this was used as Comparative Example 2.

In the case of Comparative Example 2, the growing aerobic filamentous fungi were uniformly mixed in the culture solution and multiplied in a pulpy manner, but in the case of Example 2, the aerobic filamentous fungi grew in the foam carrier, resulting in aerobic growth. Even after the growth of filamentous fungi, no aerobic filamentous fungi were present in the culture solution, and the culture solution continued to maintain a transparent state as at the time when aerobic fungus growth started.

FIG. 2 shows the lactose concentration in the culture solution and the production amount of penicillin in Example 2 and Comparative Example 2.

Although the lactose concentration decreased faster in Comparative Example 2 than in Example 2, the maximum value of penicillin production was shown one day later in Example 2 than in Comparative Column 2; were almost the same.

Example 3 1 part water, 30 g satsucrose, 5 parts yeast extract
g, K ₂ HPO ₄ 1g, NaNO ₃ 3g, MgSO ₄・
Czapek culture solution made by adding and mixing 0.5 g of 7H ₂ O, 0.5 g of KCl, and 0.01 g of FeSO ₄ 7H ₂ O
Pour 100ml into a 300ml Erlenmeyer flask, and add Aspergillus oryzae to this culture solution.
Add 1 ml of pre-culture solution of M. oryzae), and then add 0.5 g of urethane foam for artificial soil manufactured by Nitto Electric Industry Co., Ltd. into an approximately 5 mm square with an average pore diameter of approximately 0.5 mm in an Erlenmeyer flask.
(5.5cm ³ ). Next, the temperature of the culture solution in the Erlenmeyer flask was maintained at 24° C., and the Erlenmeyer flask was stirred at a stirring speed of 200 rpm using a rotary shaker to obtain Example 3A. This example 3A
Example 3B was obtained by culturing in another Erlenmeyer flask in exactly the same manner as described above.

Aerobic filamentous fungi were cultured in two separate Erlenmeyer flasks in the same manner as in Examples 3A and 3B above, except that urethane foam was not mixed, and these were used as Comparative Examples 3A and 3B.

In the case of Comparative Examples 3A and 3B, aerobic filamentous fungus growth was carried out by pellet growth that creates a mass of aerobic filamentous fungi in the culture solution, but in Examples 3A and
In the case of 3B, aerobic filamentous fungi were carried out by pulpy growth.

Figure 3 shows Examples 3A and 3B and Comparative Examples 3A and 3B.
The lactose concentration in the culture solution and the production amount of α-amylase activity in 3B are shown.

Embodiment 3A of the present invention, as shown in FIG.
The production amount of α-amylase activity in Comparative Examples 3A and 3B is much higher than that of Comparative Examples 3A and 3B. Also,
Glucose decomposition was also faster in Examples 3A and 3B than in comparative rows 3A and 3B. The quantitative unit of α-amylase activity, DP, is the number of milligrams of amylose that is reduced by 10% in blue iodine coloring at 40°C for 30 minutes in the bluevalue method.

Example 4 1 water, 25 g of glucose, 25 g of soybean flour, 3 g of dry yeast, 2 g of (NH ₄ ) ₂ SO ₄ , 2 g of NaCl,
Pour 100 ml of a culture solution prepared by adding and mixing 0.1 g of KH ₂ PO ₄ , 2 g of CaCO ₃ , and 2.4 ml of soybean oil into a 300 ml Erlenmeyer flask, and add Streptomyces griseus to this culture solution as a preculture solution. 1 ml of the solution was added, and 0.5 g (5.5 cm ³ ) of urethane foam for artificial soil manufactured by Nitto Electric Industries, Ltd., having an average pore diameter of about 0.5 mm and a square shape of about 5 mm, was placed in the Erlenmeyer flask. Next, the temperature of the culture solution in the Erlenmeyer flask was maintained at 24° C., and the Erlenmeyer flask was stirred with a rotary shaker at a stirring speed of 200 rpm to culture, and this was used as Example 4A. Example 4B was cultured in another Erlenmeyer flask in exactly the same manner as in Example 4A.
And so.

Aerobic filamentous fungi were cultured in two different Erlenmeyer flasks in the same manner as in Examples 4A and 4B above, except that urethane foam was not mixed, and these were used as Comparative Examples 4A and 4B.

In both Examples 4A and 4B and Comparative Examples 4A and 4B, aerobic filamentous fungi grew by pulpy growth in which aerobic filamentous fungi were uniformly mixed in the culture solution, but after the culture in Examples 4A and 4B, The viscosity of the culture solution was lower than that of the culture solution after culturing in Comparative Examples 4A and 4B.

FIG. 4 shows the glucose concentration in the culture solution and the production amount of streptomycin in Examples 4A and 4B and Comparative Rows 4A and 4B.

Embodiment 4A of the present invention, as shown in FIG.
The amount of streptomycin produced in Comparative Examples 4A and 4B was greater in a short period of time than in Comparative Examples 4A and 4B.

Example 5 100 ml of the same culture solution as in Example 1 was added to a volume of 300 ml.
Penicillium chrysogenum (penicillium
chrysogenum) was added, and then placed in an Erlenmeyer flask with an average pore diameter of approximately 0.5 mm.
Corner Nitto Denko urethane foam for artificial soil 0.5g
(5.5cm ³ ). Next, the temperature of the culture solution in the Erlenmeyer flask was maintained at 24°C in the same manner as in Example 1.
This Erlenmeyer flask was cultured by stirring at a stirring speed of 200 rpm using a rotary shaker, and this was used as an example.
It was set to 5A. Example 5B was obtained by culturing in another Erlenmeyer flask in exactly the same manner as in Example 5A.

Aerobic filamentous fungi were cultured in two different Erlenmeyer flasks in the same manner as in Examples 5A and 5B above, except that urethane foam was not mixed, and these were used as Comparative Examples 5A and 5B.

In the case of Comparative Examples 5A and 5B, aerobic filamentous fungus growth was carried out by pellet growth that creates a mass of aerobic filamentous fungi in the culture solution, but in Examples 5A and
In the case of 5B, aerobic filamentous fungi were grown by pulpy growth in which the growing aerobic filamentous fungi were uniformly mixed in the culture medium.

FIG. 5 shows the lactose concentration in the culture solution and the production amount of penicillin in Examples 5A and 5B and Comparative Examples 5A and 5B.

Embodiment 5A of the present invention, as shown in FIG.
The amount of penicillin produced in Comparative Examples 5A and 5B was greater in a short period of time than in Comparative Examples 5A and 5B. In addition, the decomposition of lactose was also observed in Comparative Example 5A and
Examples 5A and 5B were faster than 5B.

Next, a method for culturing aerobic filamentous fungi at an industrial level using the present invention will be explained.

FIG. 6 is a schematic diagram of one embodiment of an aerobic filamentous fungus culturing apparatus using the method of the present invention. Inside the culture tank 1, the culture solution 2 described in each of the above embodiments is held. A plurality of foamed carriers 3 are dispersed and mixed in the culture solution 2, and aerobic filamentous fungi to be cultured are mixed in the culture solution 2. The culture solution 2 is stirred by a stirring device 4. Further, the culture solution 2 is maintained at a substantially constant temperature by a temperature controller. The culture solution is sequentially dripped into the culture tank 1 from an upper drip device 5, and nutrients are supplied to the growing aerobic filamentous fungi. The culture tank 1 is equipped with probes 7 such as a PH sensor, a dissolved oxygen concentration sensor 7, and a temperature sensor 8 for measuring the state of the culture solution.
Since the probes 6, 7, and 8 are constantly washed by the foamed carrier 3 as the culture solution 2 is stirred, the state of the culture solution 2 can be accurately measured. When taking out the culture solution 2 in order to sample the culture solution 2 or accumulate metabolites, the culture solution 2 is removed from the outlet 9 provided at the bottom of the culture tank 1.
However, if the foamed carrier 3 is dispersed and mixed in the culture solution 2, the growth of aerobic filamentous fungi takes place exclusively inside the pores of the foamed carrier 3, so there is no growth favorite in the culture solution 2. The aerobic filamentous fungi will be in a state where they are almost non-existent or in a state where they have been reduced further, so that separation of the solid aerobic filamentous fungi is not required at all, or the separation work becomes extremely easy.

[Brief explanation of drawings]

FIGS. 1 to 5 are graphs showing the experimental results of Examples 1 to 5, respectively, and FIG. 6 is a schematic diagram illustrating a cell culture method at an industrial level using the method of the present invention. DESCRIPTION OF SYMBOLS 1... Culture tank, 2... Culture solution, 3... Foam carrier, 4... Stirring device, 5... Drip device, 6...
...PH sensor, 7...Dissolved oxygen temperature sensor, 8
...Temperature sensor, 9...Outlet.

Claims (1)

[Scope of Claims] 1. A method for culturing aerobic filamentous fungi, which comprises culturing aerobic filamentous fungi in a plurality of foam carriers dispersed in a culture solution. 2. The method for culturing aerobic filamentous fungi according to claim 1, wherein the foam carrier has a pore diameter of 10 μm to 10 mm. 3. The method for culturing aerobic filamentous fungi according to claim 1 or 2, wherein the size of the foam carrier is 1/5 or less of the size of the culture tank in which the culture solution is held. 4 The maximum volume of the foamed carrier is the same as the culture solution.
The method for cultivating aerobic filamentous fungi according to any one of claims 1 to ³ , wherein the amount is 100 cm 3 or less per 100 μ.