JPH0648977B2 - Method of culturing microorganisms - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for culturing bacteria and yeast. More specifically, the present invention relates to a method for producing a high concentration and efficient production of microbial cells and their metabolites by mixing gelled natural polysaccharides with a liquid medium and culturing a microorganism in the liquid medium.

[Conventional technology and problems]

When industrially producing metabolites, enzymes, or bacterial cells using bacteria, yeast, etc., how high the concentration of the microorganism to be used is cultivated is an important factor. For this reason, methods such as dialysis culture and sulphation culture have been developed and are highly effective. However, these methods are disadvantageous in that they require special equipment and require high equipment investment and complicated control.

Further, since the obligate anaerobic bacterium cannot grow in the presence of oxygen, it is necessary to perform all the culture operations under anoxic environment (anaerobic condition). In addition to the difficulty of culturing due to this, in general, obligate anaerobic bacteria are often lower in growth rate and maximum bacterial cell concentration than other microorganisms such as aerobic bacteria, and thus high concentration of obligate anaerobic bacteria Obtaining anaerobic bacterial cells is often difficult. For this reason, obligate anaerobic bacteria, aerobic bacteria,
The current situation is that research is lagging behind in other basic fields such as biochemistry and physiology and in applied fields such as fermentation engineering, compared to other microorganisms.

[Means for solving problems]

The present inventors have conducted a series of studies on high-concentration culture of microorganisms such as bacteria and yeast. As a result, by contacting the natural polysaccharide gel with a liquid medium and growing the microorganism in the liquid medium, it was found that it is effective in improving the growth rate and the maximum cell yield, and based on this, the present invention is completed. Came to. That is, the present invention is agar, carrageenan,
Provided is a novel method for culturing a microorganism that gels a gel-forming natural polysaccharide typified by alginic acid or the like and cultivates another organism such as bacteria or yeast in a liquid medium while contacting with a commonly used liquid medium. It is a thing.

Bacteria used in the present invention include aerobic bacteria and anaerobic bacteria, anaerobic bacteria that grow under anoxic conditions are facultative anaerobic bacteria that can grow in the presence of oxygen, and in the presence of oxygen. There are obligate anaerobic bacteria that cannot grow.

The obligate anaerobic bacterium, among so-called anaerobic bacteria that grow under anoxic conditions, indicates bacteria that cannot grow in the presence of oxygen, for example, Clostridium (Clostridium).
Genus, Eubacterium, Acetobacterium, Selenomonu
s) genus, bacteria belonging to the genus Butyribacterium, methane bacteria, sulfate-reducing bacteria, and most photosynthetic bacteria.

The natural polysaccharides used in the present invention include agar,
Any natural polysaccharide having a gel-forming property such as carrageenan, alginic acid, xanthan gum and locust bean gum can be used. Of these, agar, carrageenan and alginic acid are preferable. These natural polysaccharides can be used alone or in combination of several kinds. Furthermore, the effect on the growth rate and maximum cell yield can be further improved by adding and using a medium component and other specific components in the gel. The method for forming the natural polysaccharide gel in the present invention may be a known method depending on the type of natural polysaccharide to be used. For agar, for example, after heating an agar-containing solution having an appropriate concentration, coagulation is carried out. Thus, in the case of alginic acid, a gel can be obtained by bringing an alginic acid-containing solution having an appropriate concentration into contact with a divalent or trivalent metal salt solution such as a calcium salt, an iron salt or an aluminum salt. The natural polysaccharide concentration of the gel can be arbitrarily selected as long as it is at least the minimum concentration capable of forming a gel, and an appropriate concentration may be selected depending on the type of natural polysaccharide used. The volume ratio of the gel and the liquid medium can be arbitrarily selected, but usually the gel volume is 1 to 5 with respect to the liquid medium.
It is preferably doubled. The shape of the gel is arbitrary, and in addition to the method of forming a gel layer at the bottom of the incubator and stacking the liquid medium, it is used by suspending the gel molded in any shape such as spherical or rectangular shape in the liquid medium. You can also do it.

The liquid medium used in the present invention may be any medium as long as it can grow the bacteria or yeast to be cultured. For example, among those classified as obligate anaerobes, acetobacterium (Acetobacter
bacterium, genus Eubacterium, genus Butyribacterium, and the like, known media such as M. Archives of Microbiology by M. Braun et al.
Crobiology) 120, 201-204 (1979) and the like are used. The culturing temperature may be any temperature as long as the bacterium and yeast to be cultivated can grow, but it is preferable to use the optimum growth temperature for each bacterium and yeast. Further, other culture conditions are also arbitrary, depending on the type of bacteria to be cultured, yeast, under conditions suitable for each growth, standing, stirring,
Culture can be performed by a method such as gas aeration and shaking, and continuous culture can also be performed.

The present invention does not allow the cells to exist in the gel, and relates to a method of growing the cells to a high concentration only in a liquid medium, and the immobilization method of encapsulating the cells in the gel, Purpose·
It is also different from the method.

〔The invention's effect〕

By using the present invention, the maximum cell yield in a liquid medium reaches about 1.5 to 13 times that in a normal liquid culture. Further, according to the present method, it is possible to easily and inexpensively perform high-concentration culturing of bacterial cells as compared with the conventional high-concentration culturing method without requiring a special device.

〔Example〕

The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

Example 1 Bacillus subtilis IFO3007, Staphylococcus aureu (Staphylococcus aureu)
s) IFO3060, Lactobacillus
leichmannii) IFO3376, Escherichia coli
hia coli) IFO3301, Propionibacterium shermanii IFO12391, Acetobacterium woody DSM2396
6 genera and 6 species of bacteria and Saccharomyces cerevisiae (Sac
Charomyces cerevisiae) IFO0203, one genus and one yeast, was used and cultured in the combination of the media shown in Table 1. The composition of the medium components of each medium is shown in Tables 2, 3, 4, and 5.

Put 100 ml of each medium in a 500 ml Erlenmeyer flask, add 4 g of agar, cap with silicone foam, and heat at 120 ° C for 20 minutes.
After heat sterilization for a while, the mixture was allowed to cool and solidify at room temperature to form an agar gel layer containing medium components on the bottom of the flask. 12 to this
100 ml of a liquid medium heat-sterilized at 0 ° C. for 20 minutes was added, and the liquid medium was overlaid on the agar gel layer. About 0.3 ml of the above-mentioned bacteria and yeast, which had been pre-cultured for a suitable period under each culture condition, was inoculated into the liquid medium, and the culture was performed under the conditions shown in Table 1. All the above operations were performed under anaerobic conditions for the obligate anaerobic bacterium Acetobacterium woody. In addition, as a control experiment, culturing with only a liquid medium containing no agar gel was simultaneously performed under the same conditions. The cell concentration in the culture was measured with a spectrophotometer at 610
The absorbance was measured using a 1 cm cell at a wavelength of nm. The maximum cell yield for each strain is shown in Table 6. In each case, the maximum cell yield was higher than that in normal liquid culture due to the presence of the agar gel, which was about 1.5 times higher for Bacillus subtilis and about 1.9 times higher for Lactobacillus reichmannii.
Escherichia coli increased about 1.6 times, Propionibacterium shermani increased about 2.8 times, Saccharomyces cerevisiae about 2.3 times, and Acetobacterium Woody about 5.2 times.

Example 2 As a result of performing in the same manner as in Example 1 except that Propionibacterium shamani was used and k-carrageenan was used instead of agar as the gelling natural polysaccharide, the maximum cell yield was k-carrageenan. 13.2 in medium containing gel
This was about 3.8 times that of ordinary liquid culture.

Example 3 As a result of the same procedure as in Example 1 except that Propionibacterium and Shamani were used and an agar gel containing no medium components was used, the maximum cell yield was 5.35 in the medium containing the agar gel. This is about 1.4 times that of normal liquid culture.

Example 4 As a result of carrying out in the same manner as in Example 1 except that Propionibacterium shermani was used and the volume ratio of the agar gel containing the medium components and the liquid medium was 4: 1, the maximum cell yield was
In the medium containing the agar gel, it was 47.6, which was about 12.4 times that of the normal liquid culture.

Example 5 100 ml of a solution prepared by adding 4 g of agar to "methanol / D medium" shown in Table 5 was placed in a 500 ml flask, and nitrogen 6 was added.
After being anaerobic in an atmosphere of 7% and carbon dioxide 33%, sealed with a butyl rubber stopper, sterilized under pressure for 20 minutes at 120 ° C., and allowed to cool and solidify at room temperature to form an agar gel layer containing medium components on the bottom of the flask. Let 100 ml of "Methanol / D liquid medium" which was separately anaerobized and sterilized.
Was added in the glove box, and the liquid medium was overlaid on the agar gel layer. This was pre-cultured for 3 days in "methanol / D liquid medium" beforehand.
Woody (Acetobacter im woodii) DSM1030, Acetobacter woody (Acetobacterium wood-ii) DSM239
6, Butyribacterium methylotrophicum (Butyribact
erium methylotrophicum) ATCC33266, Euba-cterium limosum DSM20543, Clostridium formicoaceticum (Clostridium for
About 0.3 ml of obligate anaerobic bacterial strain of 4 genera, 4 species and 5 strains of mico-aceticum) DSM92 was inoculated under anaerobic conditions of about 0.3 ml, and each culture was started at 30 ° C. under static conditions. The culture medium for Clostridium formicoaceticum was prepared by adding 10 g of fructose instead of methanol. In addition, as a control experiment, culturing with only a liquid medium containing no agar gel was simultaneously performed under the same conditions. The cell concentration in the culture is
Absorbance was measured using a 1 cm cell at a wavelength of 610 nm using a spectrophotometer. Table 7 shows the growth rate and maximum cell yield of each strain. In both cases, the growth rate and the maximum cell yield were clearly higher than those in the normal liquid culture due to the presence of the agar gel. Particularly, in the case of Acetobacterium Woody DSM1030, the growth rate was about 2.2.
The maximum cell yield reached about 5.2 times.

Example 6 Acetobacterium Woody DSM2396 was used, and except that k-carrageenan was used instead of agar as the gelling natural polysaccharide, the same procedure as in Example 5 was performed. As a result, the growth rate and the maximum cell yield were k- In the medium containing carrageenan gel, the amounts were 3.65 and 12.4, which were about 1.9 times and about 6.7 times that of the normal liquid culture, respectively.

Example 7 Acetobacterium Woody DSM2396 was used and the same procedure as in Example 5 was performed except that an agar gel containing no medium components was used. As a result, the growth rate and the maximum cell yield were 3 in the medium containing the agar gel. The values were 0.27 and 4.05, which were about 1.7 times and 2.2 times that of the usual liquid culture, respectively.

Example 8 Acetobacterium Woody DSM2396 was used, except that the volume ratio of the agar gel containing the medium components to the liquid medium was 5: 1. As a result, the growth rate and the maximum cell yield were the same. , 4.3 and 3 for medium containing agar gel
It was 0.9, which was about 2.3 times and about 16.6 times that of the normal liquid culture, respectively.

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Claims (3)

[Claims] 1. A method for culturing a microorganism, which comprises contacting a gelled natural gel-forming polysaccharide with a liquid medium and culturing bacteria or yeast in the liquid medium. 2. The method for culturing a microorganism according to claim 1, wherein the bacterium or yeast is an obligate anaerobic bacterium. 3. The method for culturing a microorganism according to claim 1, wherein the gel-forming natural polysaccharide is at least one selected from agar, carrageenan and alginic acid.