JPH0523193A - Production of high-viscosity cultured product derived from filamentous fungi - Google Patents

Abstract

PURPOSE:To efficiently and economically obtain high-viscous cultured product such as beta-glucan without exerting adverse influence to hypha by aerobically subjecting a filamentous fungi belonging to the genus Macrophomopsis to liquid culture while stirring the cultured product with a paddle type agitator. CONSTITUTION:A filamentous fungi strain KAB 55 (FERM P-9366) of the genus Macrophomosis, etc., is cultured in a medium for 3 day and planted in a jar fermentor to which a paddle type agitator is fitted and aerobically cultured at 25 deg.C for 4 day to produce highly viscous cultured product and then the cultured product is centrifuged to remove a microbial fungal cell and an organic precipitating agent such as ethanol is added to the supernatant. Thereby the product is subjected to precipitation separation to provide the objective cultured product containing high-viscous beta-glucan, wherein all D-goucopyranosyl residues of the main chain have beta-1,3 bond and branched at C-6 position and D- glucopyranosyl residues of the side chain have beta-1,6 bond in a ratio of 1 based on 4 D-glucopyranosyl residues.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a macrophomops which produces highly viscous β-glucan.
The present invention relates to a method for producing a filamentous fungus culture belonging to the genus sis).

[0002]

BACKGROUND OF THE INVENTION In order to efficiently liquid-culture filamentous fungi belonging to the genus Macrophomopsis, it is necessary to maintain a large amount of aeration and homogeneity in the culture system. . As described in Japanese Patent Application No. 2-413440, the present inventors have developed a highly viscous β by shaking culture or deep aeration culture.
-Found and proposed a method for producing glucan.

However, in general, the amount of aeration is often restricted by the capacity of the device itself, and excessive aeration is not preferable because it causes scattering and evaporation of the culture solution. In particular, as in the present invention, when the culture has a high viscosity, the homogenization of the culture system cannot be maintained and the highly efficient culture cannot be performed by stirring only by aeration (bubbling). Therefore, it is conceivable to use a stirring blade to uniformly stir, but depending on the form of the stirring blade, a shearing force is generated due to the rotation, which damages the hyphae and negatively affects the growth of bacteria. For example, Macro Phomopsis (Macr
When stirring a highly viscous culture of a filamentous fungus belonging to the genus Ophomopsis) with a turbine-type stirring blade, there was a problem that high-speed rotation caused damage to the hyphae due to shearing force, resulting in a decrease in the growth rate.

The inventors of the present invention have conducted extensive studies in view of the above problems, and as a result, using a paddle type stirring blade not only can stir mycelia uniformly without damaging the mycelium, but also can produce β-glucan per bacterial cell amount. The present invention has been completed by finding that the production amount is significantly increased, and the purpose is to:
It is to establish an efficient method for producing a highly viscous culture derived from a filamentous fungus belonging to the genus Macrophomopsis.

[0005]

Means for Solving the Problems The present invention, when a filamentous fungus belonging to the genus Macrophomopsis is liquid-cultured to obtain a highly viscous culture, the culture solution is stirred with a paddle type stirring blade,
A method for producing a highly viscous culture derived from a filamentous fungus, which comprises performing aerobic culture.

The details of the configuration of the present invention will be described below. The microorganism used in the present invention is, for example, Macrophomopsis KA deposited as Micromachine Research Deposit No. 9366.
Examples include those named B55 strain.

Examples of the carbon source used for culturing the filamentous fungus include glucose, maltose, starch, sucrose, fructose, molasses, and a mixture thereof.
When the above-mentioned saccharides are used, β-glucan and α-glucan are simultaneously accumulated in the culture, but if a saccharide containing galactose as a skeleton is used as a carbon source, a microorganism belonging to the genus Macrophomopsis has β -It is more preferable because only glucan can be selectively produced and accumulated in the culture, and β-glucan can be recovered and utilized more easily and inexpensively.

Examples of sugars containing galactose as a skeleton include galactose, lactose, melibiose,
Raffinose, stachyose and the like can be mentioned, but lactose is particularly preferable in terms of yield.

As the nitrogen source, it is possible to use all organic and inorganic nitrogen sources generally used for culturing microorganisms, such as absorbent cotton seed flour (Pharmamedia), corn steep liquor, yeast extract, dry yeast, various Peptone, oatmeal meat extract, casein hydrolyzate,
Examples thereof include ammonium salts and nitrates.

Other additives include inorganic salts such as sodium chloride, magnesium, calcium and phosphoric acid. Further, a trace amount of metal salts such as iron, copper and manganese may be added to the medium, if necessary.

The culture conditions are preferably pH 3.5 to 9.0, more preferably pH 5.0 to 8.0, and the culture temperature is 1.
0-40 degreeC is preferable, More preferably, it is 20-35 degreeC.
The culture is usually carried out for 3 to 7 days.

The ventilation amount is preferably 0.5 to 4 vvm, more preferably 1 to 3 vvm. 0.5 vvm
If it is less than 4, the productivity of β-glucan is poor due to insufficient ventilation, and if it exceeds 4 vvm, the culture solution tends to be scattered and evaporated, which is not preferable.

The paddle type stirring blade related to the present invention may have any of the configurations shown in FIGS. 1, 2, 3 and the like, or may be attached to other commercially available jar-famenters.

The stirring conditions are preferably 10 to 100 rpm when the stirring is carried out under constant conditions from the beginning. If it is less than 10 rpm, uniform stirring is insufficient, and if it exceeds 100 rpm, the shear force tends to damage the hyphae.
Also, as the viscosity of the culture solution rises to several 100 CPS or more, the stirring condition is continuously or stepwise adjusted to 70 to 250 r.
You may raise gradually in the range of pm.

Thus, a highly viscous culture derived from a filamentous fungus, which is the objective substance of the present invention, can be obtained, and β-glucan can be obtained by purifying it as follows.

The culture is treated with an appropriate method such as filtration or centrifugation to remove the microbial cells. Then, the obtained filtrate or supernatant is mixed with a suitable precipitating agent such as ethanol, methanol, isopropanol, propanol,
Add about 10 to 35% by weight of organic precipitant such as acetone,
-Precipitate the glucan. This precipitate is separated by an appropriate method such as filtration or centrifugation to obtain β-glucan. After re-dissolving in water, precipitation with a precipitant is repeated, followed by dialysis and freeze-drying to obtain purified β-glucan.

The present invention will be described in detail below with reference to examples and comparative examples. Example 1 A strain KAB55 belonging to the genus Macrophomopsis (Micromachine Lab. No. 9366) was used in a medium having the composition shown in Table 1 below.
After pre-culturing for 150 days, 150 ml of this was put in a 5 l jar fermenter (manufactured by Mitsuwa Bio Co., Ltd.) containing 3 l of the same composition medium equipped with a paddle type stirring blade (Mitsuwa Bio Co., Ltd., FIG. 1).
(Type: KMJ-5B-3U type) and cultured at 25 ° C. for 4 days. Aeration rate is 1 vvm, stirring speed is 70
Main culture was performed at rpm.

[0018]

[Table 1]

The quantification of β-glucan in the obtained culture broth was analyzed by HPLC under the following conditions. Then, the sugar remaining in the medium was examined using a digital sugar content meter PR-1 (manufactured by Atago Co.). The amount of bacterial cells was examined by drying the wet bacterial cells obtained by centrifugation at 120 ° C. for 17 hours.

[0020]

[Table 2]

Comparative Example 1 In place of the paddle type stirring blade, two turbine type stirring blades (manufactured by Mitsuwa Bio Co., FIG. 4, (A), (B)) 10c below the liquid surface were used.
The same procedure as in Example 1 was carried out except that the device was mounted at the positions of m and 18 cm.

After the second day of main culture, the rotation of the stirring blade was not transmitted to the entire culture solution, and apparently the flow of the culture solution was stopped. The β-glucan was quantified, the residual sugar was quantified, and the bacterial cell amount was examined in the same manner as in Example 1.

Comparative Example 2 A strain KAB55 belonging to the genus Macrophomopsis (Microtechnical Laboratory Acceptance No. 9366) was pre-cultured for 3 days in a medium having the same composition as in Example, and 150 ml of this was stirred with a turbine-type stirring blade (Mitsuwa Bio Co., Ltd.). 5 liter jar fermenter (manufactured by Mitsuwa Bio Co., type: KM) containing 3 liters of the same medium equipped with the product (Fig. 4).
J-5B-3U type) and cultured at 25 ° C. for 4 days. The main culture was performed at an aeration rate of 1 vvm and a stirring rotation speed of 120 rpm.

After the third day of main culture, the rotation of the stirring blade was not transmitted to the entire culture solution, and the flow of the culture solution was apparently stopped. The β-glucan was quantified, the residual sugar was quantified, and the bacterial cell amount was examined in the same manner as in Example 1.

Control Example Strain KAB55 belonging to the genus Macrophomopsis (Microtechnical Laboratory Acceptance No. 9366) was pre-cultured for 3 days in a medium having the same composition as that of Example, and 150 ml of this was added to 3 l of the same medium in 5 l. Yong Jar Fermenter (Mitsuo Bio, Model: KMJ
-5B-3U type) and cultured at 25 ° C. for 4 days. The main culture was performed with an aeration rate of 1 vvm. The amount of β-glucan, the amount of residual sugar and the amount of cells were determined in Example 1.
It investigated by the method similar to.

[0026]

[Table 3]

As can be seen from Table 3, in Example 1 using the paddle type stirring blade, the amount of β-glucan produced per bacterial cell amount was about 1.8 times that of the control example in which stirring was not performed. . On the other hand, when the turbine type stirring blade is used (Comparative Example 1)
The production amount of β-glucan per cell amount was almost the same as that of the control example without stirring, and when the rotation number was increased, the mycelium was damaged (Comparative Example 2), and the production amount was rather decreased.

[0028]

INDUSTRIAL APPLICABILITY As described above, according to the present invention, macrophomopsis (Macrophophl) that produces highly viscous β-glucan is produced.
The present invention provides a method for producing a filamentous fungus culture belonging to the genus Omopsis), which does not adversely affect mycelia by using a paddle-type stirring blade, and evenly maintains the entire culture system even in a highly viscous state. The yield of the highly viscous culture can be greatly improved, and the production can be performed efficiently and economically.

[Brief description of drawings]

FIG. 1 is a view showing a paddle type stirring blade used in Example 1.

FIG. 2 is a diagram showing a form example of a paddle type stirring blade.

FIG. 3 is a view showing a form example of a paddle type stirring blade.

FIG. 4 (A) is a diagram showing a turbine-type stirring blade used in Comparative Examples 1 and 2, and FIG. 4 (B) is a diagram showing the stirring blade as viewed from the direction a.

Continuation of front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display area C12R 1: 645)

Claims (2)

[Claims] 1. Macrophomopsis (Macropho)
When a filamentous fungus belonging to the genus Mopsis) is liquid-cultured to obtain a highly viscous culture, the culture is agitated by a paddle type stirring blade to perform aerobic culture, which is a production of a highly viscous culture. Method. 2. The highly viscous culture contains a highly viscous β-glucan, and the binding mode of the highly viscous β-glucan is such that all main chain D-glucopyranosyl residues are β-1,3.
C- of the main chain D-glucopyranosyl residue which is a bond
At a ratio of one for every four β-1,3-bonded D-glucopyranosyl residues that are branched at the position 6 and are the main chain,
The method for producing a highly viscous culture derived from a filamentous fungus according to claim 1, wherein a D-glucopyranosyl residue which is a side chain is bound to β-1,6.