JPS62115274A - Cultivation of microorganism - Google Patents

Abstract

PURPOSE:To efficiently form pellets with a small amount of spores, by inoculating conidia of a microorganism having conidium-forming ability into a liquid culture medium, stationarily cultivating and then aerobically cultivating the microorganism. CONSTITUTION:Conidia of a microorganism having conidium-forming ability are inoculated into a liquid culture medium and stationarily cultivated during a time of forming mycelia. The culture fluid after the stationary cultivation is directly or transferred to a new liquid culture medium and cultivated by an ordinary aerobic cultivation method. The ratio of numbers of spores required for forming one pellet is reduced to a value as low as <=1 as compared with a conventional one of about 10,000 and a given amount of pellets can be obtained even with a small amount of inoculated spores.

Description

DETAILED DESCRIPTION OF THE INVENTION <Object of the Invention> The present invention relates to a culture method for efficiently forming mycelium of a microorganism capable of forming conidia.

<Industrial Application Fields> Microorganisms capable of forming conidia are used to produce amino acids, organic acids, vitamins, cancer drugs, antibiotics, enzymes such as protease, glucoamylase, and glucose oxidase, pigments, and enzyme inhibitors. It is widely used in

<Prior Art> Much research has been conducted on methods for culturing microorganisms capable of forming conidia.

When a microorganism capable of forming conidia is cultured aerobically in a liquid medium, the mycelium takes two forms: spore-like and pellet-like. If the mycelium takes on a somewhat lug-like shape during culture, there are disadvantages such as a decrease in the productivity of the desired product and a decrease in the separation of the mycelium and filtrate. There is. Therefore, when the microorganism is industrially cultured aerobically in a liquid medium, it is desirable to form pelleted mycelium.

For example, to get 102/- takulets, 106/-(
It requires a large amount of spores (inoculated spores/formed pellets) = 10". If this culture is carried out in a large industrial culture tank, it is practically impossible as it requires an extremely large amount of spores. Furthermore, even if it were possible to mass-produce spores using a large-scale solid culture method, this method requires culturing in a solid medium, so culture control such as temperature and humidity is not easy, and the overall equipment Since it cannot be kept under complete sterile control, there is a high risk of contamination with various bacteria, and pure culture is extremely difficult.

Problems to be Solved by the Present Invention The problem to be solved by the present invention is to establish a method for culturing the microorganisms on an industrial scale to efficiently form pellets using a small amount of spores.

<Means for Solving the Problems> The present inventors have conducted extensive research in order to solve the above problems.

In order to measure the germination rate of microorganisms capable of forming conidia in a liquid medium, the present inventors inoculated an appropriately diluted spore solution and observed the germination status of spores under electrostatic conditions. The hyphae that germinated over time became light, cotton-like mycelium in areas with a large number of inoculated spores, but in areas with a small number of spores, in addition to filamentous hyphae, hyphae with ciliated and circular surroundings were formed. Flat mycelium was observed with the naked eye.

We thought that if this disk-shaped mycelium grew, it would turn into pellets, so we transferred it to normal aerobic culture and continued culturing, and the desired pellets were obtained.The present invention was completed based on this knowledge. It is what was done.

That is, the present invention inoculates conidia of a microorganism capable of forming conidia into a liquid medium, performs static culture for a certain period of time, and then performs aerobic culture using this culture solution as it is or by transferring it to a new liquid medium. The present invention relates to a method for culturing a microorganism capable of forming conidia, which is characterized by the following.

The microorganism used in the present invention may be any microorganism having the ability to form conidia, such as filamentous fungi, actinobacteria, and basidiomycetes.

A specific example is Kratos Irium colocache (
Cladosporium colocasiae)
AJ 6667 (IFO6698), Gonatobotryum ap
IFO9098, mycophenolic acid producing bacterium Penicillium brevl Compa
ctum) AJ 117096 [FERM-P569
P-chrysogenum AJ 7343, a penicillin-producing bacterium
ATCC10002], a glucose oxidase producing bacterium Nicilium...! - Zoro r nam (P・pa'r
progenum) AJ 17045 [FERM-
P1846], as a cephalosporin C-producing bacterium, Cephalum poulia 4 A (Cephalo[Iporum Po
lyaleurum) A J 6993 [ATCC2
0359), grotease-producing bacteria include Aspergillus phenisis (Aspergillus Phenlc)
i s ) AJ 17063 [ATCC 1433
2], Aspergillus niger (A-niger) A J 7 as gelcoamylase and citric acid producing bacteria
172 [ATCC9642), the fosfomycin-producing bacterium Stregutomyces viridochromodanes (St
reptomyces Vlridochromoge
nes) ATCC21240 etc. are used.

The liquid medium used in the present invention can be any liquid medium in which the microorganism can grow.

For example, as a medium for producing phenylalanine ammonia-lyase, the medium shown in Table 1 is used, and as a medium for producing mycophenolic acid, known media such as W, L, and MuthetaLA are used.
ntimicrob, Agenta Chemther
The culture medium described in A.P., +8+3-2l-327 (1975) is used. As a medium for penicillin fermentation, Joh
Synthetic media such as nson (Ronsuke Sumiki, “Antibiotics”, P, 1
77.1961) is used. Examples of cephalosporin C fermentation media include A, L, DEMAIN,
J, F, NEWKIRK, and D, HEND
LIN.

J, Bacteriol, 85.339 (1963)
A modified medium of the described medium is used.

~, Table 11 f as a medium for glucoamylase production
.

A medium with the composition shown in ... is used.

Leo 7,,, - I, Gao□. , Wow 1. A medium having the composition shown in Table 4 is used.

Conidia can be produced by culturing the microorganism on a potato dextrose agar medium, for example, and reducing the number of spores produced by 0.1
It may be prepared by suspending it in a solution containing a surfactant such as % Tween 80.

In the static culture method, conidia inoculated into a liquid medium may be left as is at a temperature where the microorganisms can grow until hyphae are produced, but a small amount of aeration may also be applied.

The length of time for leaving varies depending on the bacterial species, but it is usually sufficient as long as it is 12 hours or longer to allow observation with the naked eye.

The culture solution that has been statically cultured is transferred to a new medium and cultured using normal aerobic culture methods, such as rotation or shaking, or aeration with aeration and aeration in a bubble column culture tank. Just do it.

The temperature at which the 4-lets are formed may be any temperature as long as this microorganism can grow, but it is preferable to use the optimum culture temperature that produces useful substances.

Various useful substances produced by the method of the present invention can be quantified and collected by each known method.

Mycophenolic acid in the culture solution was analyzed and quantified by high-performance liquid chromatography, using the penicillin G growth inhibition method.

Fosfomycin quantification was performed using Eseherichia Co.
The test was carried out using the growth inhibition method using 1tK-12 and ATCC10798.

The measurement of glucoamylase activity was based on the saccharification power measurement method ("I Experimental Chemistry Course #24, p. 272, 1961).

As explained above, the present invention, prior to normal aerobic culture,
The present invention relates to a method for culturing microorganisms capable of forming conidia, which is characterized by static culture.

<Functions and Effects> With the completion of the present invention, the ratio of the number of spores required to form one beret (hereinafter abbreviated as C/P) was 10.00 compared to the conventional one.
It is now possible to reduce the value from about 0 to 1 or less. The method of the present invention can produce a predetermined amount of spores even with an extremely small amount of spore inoculation compared to the conventional spore inoculation amount.As a result, the production amount of useful substances can be improved, and the amount of bacterial cells and culture solution can be increased. The industrial value is extremely large because the separability of the molecules is also improved.

Examples will be described below.

Example 1 Cladosporium colocachea A J 6667 (I
FO6698) i Potato dextrose agar medium 6 Eiken was inoculated, and after culturing at 2.6°C for 7 days, spores 1 and 0.
It was suspended in an aqueous solution containing 1% Tvreen 80, and a certain volume thereof was inoculated into the culture medium shown in Table 1 (300-mL Erlenmeyer flask filled with 50-mL medium), statically cultured at 26°C, and then Rotational culture was carried out at 26°C for 40 hours at 220 rpm. To determine the number of inoculated spores, the spore suspension was
The spore count was measured using Mr. A's hemocytometer and diluted appropriately to change the number of spores.

Table 1 Polybegton 1.0 Yeast extract 1.0 to 2HPO40.3K) (2PO40.l MgSO4・7H200.05 Fealalanine 0.15 (PH6.0, 120°C, sterilized for 20 minutes) Bacterial growth is oriental It was expressed as a dry weight after suction filtration using filter paper shop 5 and dried at 105° C. for 18 hours.The number of islets was determined by sampling an appropriate amount and counting with the naked eye, and expressed as a value based on the total amount of the flask.

The results are shown in Table 2.

Example 2 Spores 2.8X of spore liquid prepared in the same manner as Example 1
10@f, 500 d triangular 7 Lasco K 100 tM was inoculated into the medium shown in Table 1, and static culture was performed at 26°C.

3.0 in which the entire volume (100 d) of the stationary culture finished solution was filled with the medium 1.41 shown in Table 1! small jar fermenter
aerated (1/4 V, V, M) and stirred (700
rpm) culture was performed. In parallel, 2.8 x 10' spores were directly inoculated into a small jar fermenter and cultured without static culture. The results are shown in Table 3.

Table 3 48 40 8.4xlO' 0.41 0.3 Example 3 Nicilium brevi conbactum A J 1170
96 (FERM-P 5693) ft inoculated into the natural slant medium shown in Table 4, and after culturing at 27°C for IO days, the spores were collected and added to 0.1M phosphate buffer (TJ16.8').
of the medium shown in Table 5 (300-
Inoculate 50 ml of culture medium into an Erlenmeyer flask for 27 hours.
Rotational culture was carried out at 226 rpm for 5 days at °C.

The experimental plot in which spores were rotary cultured without static culture was designated as (A).
The plot in which rotary culture was performed after static culture for 48 hours was designated as (B), and the results are shown in Table 6.

Table 4 Ingredients Concentration (Shika) Glucose 1.0 4deton 0.2 Malt extract 0.1 Yeast extract 0.1 Agar 1.5 (pH 7.0, sterilized at 120°C for 20 minutes) Table 5 Ingredients Concentration Glucose 5.0 (g/dg) Glycine
1.46 1 Methionine 0.05 #KH2PO40,3# MgSO4・7H200,1# FeSO4'7I(202, OpprnCuSO40,
3' Zn80a O,25'MnSO4'4H200,16tK2MoO40,02# Example 4 Penicillium chrysogenum A, a penicillin G-producing bacterium
J 7343 (ATCC 10002) was cultured in exactly the same manner as in Example 3, except that the production medium shown in Table 7 was used.

The results are shown in Table 8.

Table 7 Ingredients Concentration (gAit) Lactose 3.0 Glucose 1.0 Ammonium lactate 0.55 Ammonium acetate 0.35 KH2PO40,3 MgSO4・7H200,025 FeSO4'7H200,1 CuSO4・5H200,005'ZnSO4'7H200, 002 Na 2 SO40,05 MnSO4'4H200,002 (P)17.0, sterilized at 120°C for 20 minutes) Example 5 Cephalosporium, a cephalosporin C producing bacterium
Polyaleurum A J 6993 (ATCC2035
Example 3 except that 9) was performed using the production medium shown in Table 9.
Culture was carried out in the same manner as described above.

The results are shown in Table 10.

Table 9 Ingredients Concentration (IiA) Sucrose 3.6 Glucose 2.7 Ammonium sulfate 0.75 DL-methionine 0.3 L-cystine hydrochloride 0.16 to 2HPO4
2,10 KW2PO41,53 Na2804 0.075MgSO
4.7H200,018 Fe(804)2'(NH4)26H200,015C
a C100,OO53 MnSO4' 4H200,003 ZnSO4・7H200,0003 CuSO4' 5H200,00035Example 6 Culcoamylase producing bacterium Asvercillus nikah AJ7172 (ATCC9642)'iExecuted except for using the production medium in Table 11 Culture was carried out in the same manner as in Example 3, and the results are shown in Table 12.

Table 11 Soluble starch 5D Flavor liquid 3.0 Ammonium sulfate 0.5 KH2PO40,3 CaCO3*, 0.5 (pH 6.0, sterilized at 120℃ for 15 minutes) * Separately sterilized using dry heat method.

Example 7 Streptomyces pyridochromogenes ATCC2 cultured at 27°C for one week on the slanted agar medium shown in Table 13.
1,240 spores were inoculated into the culture medium shown in Table 14 (20 volumes in a 500-capacity shoulder flask), and cultured at 27°C for 3 days.

In experimental area A, shaking culture was performed immediately, and in experimental area B, shaking culture was performed after 30 hours of quiet culture.

The results are shown in Table 15.

131st! Ingredients Concentration (mi/111) Glucose
1.0 Yeast extract 0.3 Malt extract 0.3 Polybegtone 0.5 Agar 1.5 Table 14 Ingredients Concentration (gAit) Soluble Dengun 5.0 Polypeptone 1.0 Glutamic acid 2.0 Dry yeast 0.5 MgSO4・7H201,0 KH2PO41,0

Claims (1)

[Claims] It is characterized by inoculating conidia of a microorganism capable of forming conidia into a liquid medium, culturing it statically for a certain period of time, and then culturing the culture solution as is or by transferring it to a new liquid medium for aerobic culture. Method for culturing microorganisms capable of forming conidia