JPS6167477A - Seed cultivation of microorganism - Google Patents

Abstract

PURPOSE:To enable the mass-culture of seed microorganism useful for a liquid culture, in a short time, by impregnating a liquid medium in a porous substance inert to the microorganism, inoculating the desired microbial cells in the medium, and culturing the cells in stationary state. CONSTITUTION:A polyol having polyoxyethylene structure (e.g. polyethylene glycol) is made to react with a polyisocyanate (e.g. tolylene diisocyanate) in the presence of a peptide such as collagen, albumin, gelatin, etc. to obtain a polyurethane containing a peptide matrix in the molecule. A liquid medium is impregnated in the porous substance composed of the foam of the above polyurethane, and desired microorganism (e.g. mold) is inoculated and cultured in stationary state. The microorganism can be proliferated rapidly by this process, and the obtained seed microorganism can be utilized economically as the seed for the main culture.

Description

Detailed Description of the Invention (Technical Field) The present invention relates to a method for seed culture of microorganisms, in particular, a method for substantially growing microorganisms, especially filamentous fungi, in a porous material that is dispersed in a culture medium and is not subject to metabolism by microorganisms. This article relates to a method for culturing seed microorganisms used in liquid culture.

(Prior art) It has already been possible to produce primary metabolites such as glycerin and alcohol, or secondary metabolites such as penicillin and streptomycin, by liquid culturing various microorganisms including molds and other filamentous fungi and actinomycetes. Are known.

When microorganisms are cultured in liquid, depending on the type of microorganism.

Microbial cells grow in the form of pellet growth or pulpy growth. "Bellet growth" refers to a growth mode in which microbial hyphae become entangled with each other during culture to form a granular cell mass and grow. Pere-throat proliferation is observed, for example, in the production process of penicillin. Pulpy growth refers to a growth mode in which microbial cells grow while being uniformly distributed in a culture solution. Pulpy growth is observed, for example, in the production process of streptomycin. When pellet proliferation occurs, the cell proliferation rate is extremely low.

When pulpy multiplication occurs, the proliferated microbial cells exist in a uniformly mixed state in the culture solution.

The viscosity of the culture solution becomes extremely high. Therefore, a large amount of power is required to stir the culture solution. This is because microbial cells exist in a mixed state in the culture solution in both pellet growth and pulpy growth.

Separation and extraction of metabolites from culture fluid is difficult.

Monitoring of the culture fluid also becomes cumbersome and time consuming.

In order to overcome these drawbacks of pellet growth or pulpy growth, the present inventors have proposed a method of culturing microorganisms by dispersing in a culture medium a porous material that is not metabolized by microorganisms (particularly (Gan No. 59-71515)
. According to this method, the microorganisms to be cultured grow within the pores of this porous material 1, for example a polyurethane foam.

Since the inner surface of the porous material has a large contact area with air, sufficient air is supplied to the microorganisms existing within the pores of the porous material.

Proliferates extremely quickly. Furthermore, since there are almost no bacterial cells in the culture solution, microbial cells do not undergo pellet or pulpy growth. therefore.

No particularly large power is required to stir the culture solution.

Separation of the target metabolite and monitoring of the culture solution can also be easily performed. When performing a liquid culture method using such a porous material on a large scale, the seed microorganisms used therein are prepared, however, by static culture, aeration-stirring culture, shaking culture, etc. in a liquid medium. be exposed. Seed preparation by static culture.

The resulting microbial cells exhibit pellet growth morphology,
Since microbial cells grow by forming a spore layer on the surface of the culture solution, the supply of oxygen to microbial cells is insufficient.

Growth rate is slow. Increasing the amount of medium does not increase the growth rate. Therefore, if a large amount of seeds are required for double culture, it is necessary to perform preculture several times based on these seeds. Also.

In seed preparation by aerated agitation culture or shaking culture.

Microbial cells grow more efficiently than static culture.

Power for aeration and stirring must be supplied externally. Equipment for this purpose is also required.

(Object of the Invention) The object of the present invention is to produce seeds for liquid culture in a short time in large quantities that allow microbial cells to substantially grow in a porous material that is dispersed in a liquid culture medium and is not subject to microbial metabolism. The object of the present invention is to provide a method for preparing the same. Another object of the present invention is to provide a static culture method for obtaining a large amount of seed microorganisms using a large amount of medium.

(Structure of the Invention) The present invention is directed to the cultivation of seed microorganisms used in liquid culture in which a porous material that is not metabolized by microorganisms is dispersed in a liquid culture medium, and microbial cells are substantially grown in the porous material. In this method, the porous material is impregnated with a liquid medium, the desired microbial cells are inoculated into the porous material, and the desired microbial cells are cultured stationary, thereby achieving the above object.

Examples of porous materials used in the present invention include:

Natural products such as synthetic resin foams and sponges can be used. As the synthetic resin foam, for example, foams such as polyvinyl alcohol and polyurethane are used. In particular, polyurethane foams having a polyoxyethylene structure are preferably used because they have excellent hydrophilicity and water retention ability.

Such polyurethane is obtained by reacting a polyol having a polyoxyethylene structure with a polyisocyanate. Examples of polyols having a polyoxyethylene structure include polyethylene glycol; a mixture of polyethylene glycol and polypropylene glycol;
Examples include polyethylene glycol-polypropylene glycol, which is a copolymer of ethylene oxide and propylene oxide. Basic polyols can also be used.

The basic polyol here is ethylene diamino.

diethylenetriamine, methylamine, butylamine,
piperazine, ethanolamine, propatoolamine,
It is obtained by adding several units of ethylene oxide to an amine such as N-methyljetanolamine in the form of polyoxyethylene. As the polyol having a polyoxyethylene structure, polyethylene glycol having a molecular weight of about 400 to 1,000 is preferably used. When the polypropylene component is added to polyethylene glycol, its content is
! Preferably, it is less than 365 parts by weight. If it is in excess, the hydrophilicity of the resulting foam will decrease.

The above polyisocyanates include 3, for example, aromatic,
Various polyisocyanates such as aliphatic and polyether are used. For example, tolylene diisocyanate
Examples include diphenylmethane diisocyanate, diphenyl diisocyanate, naphthalene diisocyanate, xylene diisocyanate, butane diisocyanate, triphenylmethane-4-4'4'-1-diisocyanate.

A polyol without a polyoxyethylene structure can be used alone or in combination with the polyol having a polyoxyethylene structure. Examples of such polyols include glycerin, trimethylolethane,
Trimethylolpropane, 1,2,6-hexandriol, pentaerythritol, sorbitol, sucrose.

α-methyl glucoside is mentioned. Natural polymers such as cellulose, carboxymethylcellulose, and hydroxymethylcellulose and derivatives thereof can also be used as the polyol.

During the reaction between polyols and polyisocyanates.

When a bebutide such as collagen, albumin, or gelatin is allowed to coexist in the reaction system, a polyurethane in which a pebutide matrix is formed within two molecules can be obtained. When a polyurethane foam with a peptide matrix formed within its molecules is used as a porous material, the moisture content of the porous material is high (
This results in good wetting between the porous material and the culture medium. As a result, the affinity between microorganisms and the porous material becomes higher, and the microorganisms grow more rapidly.

As the foam, both semi-open foam and open foam can be used. It is preferable to use open foam because the wider the air-liquid surface where the culture medium and air come into contact, the better.

The average pore diameter of the porous material is 1 μm to 10 mm, preferably 100 μm to 3 mm. If the pore diameter is too small, it becomes difficult for microorganisms to penetrate into the porous material, and if it is too large, the internal surface area of the porous material becomes small.

The apparent specific gravity of such porous materials is between 0.01 and 0.01.
1, and it is possible to absorb 0.5 to 1.2 g of water per 1 cffl. The size of the porous material is 175% of the volume of the culture tank when performing main culture using the obtained seed microorganism.
It is preferably 1/20 or less. For example, a plurality of cubic urethane foams each having a negative side of 5 mm are used.

If the size of the porous substance is too large, the stirring efficiency during culture will be poor.

The above-mentioned porous material medium is placed in an incubator such as an Erlenmeyer flask, and after sterilization, desired microorganisms are inoculated.

Seed culture is performed by static culture. Because the surface area of porous materials is extremely large and air exists within the pores,
Large amount of oxygen supplied to microorganisms.

Therefore, microorganisms do not exhibit the pellet growth or pulpy growth seen in conventional static culture, agitation culture, etc., and rapidly grow inside and on the surface of the porous material. The amount of the medium is suitably 50 to 200% of the amount of the medium that can be supported by the porous material used, most preferably around 90%. If the microorganism to be cultured is, for example, a filamentous fungus,
If the seed medium liquid volume is around 90%, the seeds will grow within and on the surface of the porous material without producing spores.Generally,
During periods of high growth, bacterial cells proliferate without producing spores. Therefore, when culturing is performed using seeds grown in such a form, the metabolism and growth of microorganisms can occur rapidly. When the volume of the medium for seed culture exceeds 100%, spores are produced on the surface of the porous material and the surface of the culture medium and proliferate.

However, 1. Considering that the water in the culture solution evaporates during seed culture, the amount of medium should be 100 to 200% of the amount of medium that can be supported by the porous material, as long as there is no problem with the growth of microorganisms.
It is preferable that Even if the amount of the medium is 200% or more, the growth rate of microorganisms is much faster than when no porous material is used.

The porous material in which the seed microorganisms have grown is placed in the main incubator and stirred or shaken. The total amount of porous material is medium 1
100cnl per 00m1 (apparent volume) or less,
Preferably it is 10-5QcJ. When it exceeds 100 d, the stirring efficiency decreases. In contrast, a porous material in which microorganisms do not grow is dispersed in a medium.

When culturing is performed by adding seed microorganisms that have been separately cultured in a liquid medium, there is a lag time (lag period) of several days before the target metabolite is produced in the culture medium. exist. However, according to the seed culture method of the present invention, since the microorganisms are grown within the porous material, such a lag period is absent or shortened, and as a result, metabolites by the microorganisms are advantageously produced. In a culture method using such a porous material, as described above, there are substantially no microbial cells in the culture solution. Therefore.

During the main culture period, an appropriate amount of culture solution is removed as appropriate.

It is possible to continue the culture by supplying a new liquid medium. As a result, two metabolites are obtained successively.

In order to preserve seed microorganisms, it is desirable to prepare them so that they form spores and store them at room temperature. However, seed microorganisms prepared without forming spores can be stored as they are at low temperatures (e.g., -80°C). can be stably stored.

Since the stored seed microorganisms are in a state where the microorganisms have grown sufficiently within the porous material, the necessary amount of seed microorganisms can be grown without going through the conventional process of growing the stored microorganisms through several stages of pre-culture. can be used as is as a seed for main culture.

The seed culture method of the present invention can be applied to bacteria, as well as filamentous fungi such as molds and actinomycetes.

(Example) The present invention will be explained by examples.

1st family in flames (8) Preparation of seed culture stock solution: 40 g of lactose.

Corn steeple force - 20g, sodium nitrate 3g.

0.5 g of potassium dihydrogen phosphate and 0.25 g of magnesium sulfate were added to 1N water and mixed to prepare a medium.

In this medium Penicillium chrysogenum (Penicillium chrysogenum)
cillium chrysogenum) was cultured for 4 days.

32 ml of the culture solution was collected and added to 320 ml of the same fresh medium as above to obtain a seed culture stock solution.

(B) Preparation of seeds: Prepare four 100ml Erlenmeyer flasks and add Ig (approx.
1c++1) each. The average pore diameter of the polyurethane foam is about 0.5 mm, and it can support 0.9 g of the medium in item (A) above per lci. The shape is a cube with each side approximately 5 mm. These Erlenmeyer flasks were dry heat sterilized at 180°C for 1 hour. Add the seed culture stock solution obtained in section (A) to 4 flasks at +10m6゜20m j!
, 30 ml 1 and 40 ml were added at 25°C for 10
Static culture was performed for 1 day. The obtained seeds were designated as Seed (I), Seed (■), Seed (I[[), and Seed (IV), respectively. 6 days after the start of culture, 8 days later and 1
After 0 days, lactose in the culture solution was quantified.

The results are shown in FIG. In the figure, ○ indicates a seed (I).

indicates the change in the quantitative value of the seed (■), the number 2 indicates the seed (■), and the symbol ◇ indicates the change in the quantitative value of the seed (I'/). The same applies to FIGS. 2 and 3. In Seed (1), the polyurethane foam absorbed all of the seed culture stock solution, and the bacterial cells did not form spores but grew only inside the foam. Seeds (n) to (IV) grew by forming a green spore layer on the surface of the polyurethane foam and the surface of the seed culture solution.
A spore layer was formed on the seed (n) from the 2nd day, and on the 3rd day on the seeds (I[[) and seed (IV), and on the 6th day, the entire surface of the culture solution was covered with the spore layer.

Comparative example 1 (A) Preparation of seed culture stock solution: Same as Example 1.

(B) Preparation of seeds: Same as Example 1 (B) except that polyurethane foam was not used.

Seed the obtained seeds (■) and Seed (■), respectively.
, Seed (■) and Seed (■).

The respective lactose quantitative values are shown in FIG. In the figure, ・ is a seed (V), M is a seed (■), ■ is a seed (■),
And ◆ indicates a change in the quantitative value of seeds (■). The same applies to FIGS. 2 and 3. Seed (V) ~ (
In all cases (2), a spore layer was formed from the 6th day after the start of culture.

From the observation results of Example 1 and Comparative Example 1 and FIG. 1,
It is clear that particularly rapid proliferation occurred in seeds (1) and (1). The amount of culture liquid for seeds (1) and (II) is in the range of 50 to 200% of the amount of liquid that can be supported by the porous material.

Example 2 Seed (1), seed (■), and seed (■) after 6 days of culture in Example 1 and Comparative Example 1.

Seed (■), Seed (VI) and Seed (■) were used as seeds in this example.

The medium Room 1 prepared in Example 1 (A) was placed in a 500 ml Sakalo flask.
) and then cultured with shaking for 7 days. For Seed (n) and Seed (I[l), half of the polyurethane foam was added to a separate medium, and about half of the formed spore layer was inoculated. Seed (n) and seed (I[I) were also cultured with shaking for 7 days. After the start of culture.

After 2 days, 3 days, 4 days, and 7 days, the amounts of lactose and penicillin contained in each culture were measured. The results are shown in FIGS. 2 and 3.

A culture medium was prepared in the same manner as in Example 2. The entire seed (V) was mixed uniformly, 5 ml of it was collected and planted in a medium, and cultured under the same conditions as in Example 2. Seed (Vl
) and seeds (■) were also treated in the same manner. The results are shown in FIGS. 2 and 3.

From Figures 2 and 3, seeds (I) to (III) using porous materials for sugar metabolism and penicillin production.
It can be seen that it is preferable to use

Soutachimasu A - 100 ral of the same medium as in Example 1 and 1 g of the same polyurethane foam as in Example 1 were placed in a 500 ml Sakaro flask, and seeds (I) cultured for 4 days were added thereto at 225°C. Shaking culture was performed at 1100 rpm for 9 days. Sampling was performed every day for 6 days after the start of culture.

The penicillin produced was quantified. The results are shown in FIG. 4 as a solid line.

Same as Example 3 except that no M" dynamic polyurethane foam was used. The results are shown in FIG. 4 as a broken line.

Comparative Example 4 50 mff of the same medium as in Example 1 was placed in a 500 m 12 Sakaro flask, and Penicillium chrysogenum was cultured with shaking (100 rpm) for 4 days to obtain a seed culture solution.

100 ml of culture medium and 2 g of the same polyurethane foam as in Example 1 were placed in a 500 mβ Sakaro flask.

Add 2 m6 of the above seed culture solution to this and heat at 25°C for 9 hours.
Shaking culture was performed at 1100 rpm for 1 day. 2 after starting culture
The penicillin produced was quantified every 4 hours.

The results are shown in FIG. 4 by the dashed line.

From Figure 4, it can be seen that by using seeds with porous materials,
It can be seen that the lag time in penicillin production becomes extremely short. (Effects of the Invention) According to the seed culture method using static culture of the present invention.

Microorganisms multiply rapidly compared to conventional seed culture.

Therefore, a large amount of seed microorganisms can be cultured in a large amount of medium in a short period of time. Seed microorganisms can be prepared at a speed comparable to or faster than these conventional methods without requiring special power such as one rotation of shaking and three ventilations as in conventional stirring culture or shaking culture. The seed microorganism thus obtained is advantageously used as a seed for main culture in which the microorganism is grown by dispersing a porous substance in the culture solution. When this seed is used, microorganisms grow more rapidly in the main culture medium than when using conventional liquid culture seeds, and the target metabolite can be produced with no lag time or with a significantly short lag time. It becomes possible to produce it.

The obtained seed microorganisms can be preserved semi-permanently by freezing them at a low temperature such as -80°C. Since this seed microorganism is in a state where microorganisms have grown sufficiently within the porous material, microorganisms can be grown using the required amount of seeds without the need for several stages of pre-cultivation using preserved microorganisms as in the conventional method. It can be used as is for large-scale main culture.

[Brief explanation of drawings]

Figure 1 is a graph showing changes in the amount of lactose in the culture solution in the seed cultures of Example 2 and Comparative Example 1, and Figure 2 is a graph showing changes in the amount of lactose in the culture solution in Example 2 and Comparative Example 2. , FIG. 3 is a graph showing changes in the amount of penicillin in the culture solution in Example 2 and Comparative Example 2, and FIG. 3 is a graph showing changes in the amount of penicillin in the culture solution in Comparative Example 3 and Comparative Example 4. that's all

Claims (1)

[Claims] 1. A method for cultivating seed microorganisms used in liquid culture, in which a porous material that is not subject to metabolism by microorganisms is dispersed in a liquid culture medium, and microbial cells are substantially grown in the porous material. A method for seeding microorganisms, comprising: impregnating the porous material with a liquid medium, inoculating desired microorganism cells therein, and culturing them statically. 2. The seed culture method according to claim 1, wherein the amount of the liquid medium for seeds is 50 to 200% of the amount of the medium that can be supported by the porous substance. 3. The seed culture method according to claim 1, wherein the microorganism is a filamentous fungus. 4. The seed culture method according to claim 1, wherein the porous material is a polyurethane foam. 5. The seed culture method according to claim 4, wherein a peptide matrix is formed within the polyurethane molecules. 6. The seed culture method according to claim 5, wherein the peptide forming the peptide matrix is collagen, albumin, or gelatin.