JPH0622768A - Fermenting production of mold - Google Patents

Abstract

(57) [Summary] [Object] To provide a fermentation production method using an immobilized filamentous fungus. [Structure] Contains a filamentous fungus growth medium impregnated in liquid form,
Propagating filamentous fungi in an oxygen-containing gas atmosphere on a porous support made of fibers with an apparent density of 0.01 to 0.07 g / cm ³ consisting of fibers having an average fineness of 20 to 150 denier to which filamentous fungus is attached. The filamentous fungus is fixed to the above-mentioned support, and subsequently, the support on which the proliferated filamentous fungus is immobilized is immersed in a fermentation medium to carry out fermentation culture of the filamentous fungus, and a fermentation product of the filamentous fungus is separated. [Effects] High productivity can be achieved, filamentous fungi can be used repeatedly, and the process of separating filamentous fungi from the fermentation product is unnecessary.
Fermentation culture process can be carried out under mild conditions, and complicated condition control is not required.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for fermentative production of filamentous fungi, which uses the filamentous fungus immobilized on a porous support.

[0002]

2. Description of the Related Art In fermentation production in which a useful product is obtained by utilizing the metabolism of a microorganism while proliferating the microorganism, the microorganism is generally dispersed in a production medium in a free state to grow the microorganism. The products secreted from the body are separated from the culture product solution and collected. The reason why the bacterial cell dispersion liquid is used is that the bacterial cells in the free state exhibit high growth and metabolic activity. However, the fermentation production method using the bacterial cell dispersion liquid has a drawback that it is difficult to separate the product from the bacterial cells and the bacterial cells cannot be used repeatedly. Furthermore, it is difficult to control the culture conditions. In particular, when aerobic bacteria are used, aeration is necessary, but there are problems that the air outlet is clogged with the bacterial cells and foaming occurs.
In order to eliminate the above-mentioned drawbacks, it is possible to use the cells by immobilizing them on an appropriate support, but the immobilized cells have a low proliferative activity, and the productivity of metabolites decreases. . Even if the cells can be sufficiently grown on the support, the conventional support is immediately clogged with the cells,
There is a drawback that productivity is reduced because the growth space is limited and the permeability of the culture solution is deteriorated. Therefore, the fermentation production using the immobilized cells is difficult, and the conventional use of the immobilized cells was to carry out the enzyme reaction of the intracellular enzyme or the enzyme secreted from the cells without the proliferation of the cells. ,
It was limited to bioreactors and biosensors.

[0003]

DISCLOSURE OF THE INVENTION The present inventors have found that in a fermentation production method involving the growth of bacterial cells when obtaining a metabolite,
As a result of intensive studies to simultaneously solve the drawbacks of the method using the bacterial cell dispersion solution and the problems when using immobilized bacterial cells, by adopting a specific fiber porous body as a support, It has been found that the bacterial cells can be immobilized without substantially impairing the activity of the bacterial cells, and the fermentation production can be efficiently carried out using such a bacterial cell-immobilized support. The present invention is based on these findings.

[0004]

Therefore, according to the present invention, an apparent density of 0.01, which comprises a filamentous fungal growth medium impregnated in a liquid state and which has filamentous fungus adhered thereto and has an average fineness of 20 to 150 denier, is used. ^On a porous support made of fiber of about 0.07 g / cm ^{3 in} the oxygen-containing gas atmosphere, the filamentous fungi are grown and fixed on the support, and subsequently the support on which the proliferated filamentous fungi is fixed The present invention relates to a method for fermentative production by a filamentous fungus, characterized in that the filamentous fungus is immersed in a fermentation medium to carry out fermentation culture of the filamentous fungus, and a fermentation product of the filamentous fungus is separated.

The method of the present invention comprises (1) a gas phase culturing step for immobilizing filamentous fungi on a support, (2) a fermentation culturing step using the support obtained in step (1), and (3) the above And a step of separating the target product from the fermentation product liquid obtained in the fermentation culture step (2). In the gas phase culture step (1),
First, a filamentous fungus is inoculated into a wet support impregnated with a filamentous fungus growing culture solution, or a dry support is impregnated with a filamentous fungus growing culture solution containing dispersed spores or hyphae of the filamentous fungus. Then, a support containing the spores or mycelia of the filamentous fungus and the culture solution is prepared. Next, this support is put under water in an oxygen-containing gas atmosphere (for example, in air) at a temperature (for example, 25 to 37 ° C.) preferable for the growth of filamentous fungi, and preferably water is put in an incubator, When placed under conditions such that water vapor is supplied by spontaneous evaporation, the support has a three-dimensional porous structure, so that the filamentous fungus is not only the outer surface of the porous body but also the surface inside the pores. Proliferate while being entangled in
The grown bacterial cells are reliably fixed on the fiber-made porous support.

Since filamentous fungi are aerobic microorganisms and require oxygen for growth, filamentous fungi grow only on the surface of the medium in the conventional culture on a medium. Further, even when the growth culture is performed while the porous body is immersed in the liquid medium, the filamentous fungi only propagate near the interface between the medium and air. That is, in liquid culture, it is difficult to immobilize filamentous fungi so that they are distributed throughout the porous structure. On the other hand, since the fiber porous support is used in the gas phase in the method of the present invention, the liquid medium and oxygen necessary for growth can coexist even inside the pores of the three-dimensional porous body. The growth of filamentous fungi proceeds to the inside of the pores, the filamentous fungus can be uniformly distributed throughout the porous support, and a support carrying a large amount of immobilized filamentous fungus can be prepared. .

The support that can be used in the method of the present invention is not particularly limited as long as it is a porous body made of fibers having an average fineness of 20 to 150 denier and an apparent density of 0.01 to 0.07 g / cm ³ . A preferred support is a fibrous woven or knitted fabric having a three-dimensional porous structure, particularly a non-woven sheet. When the apparent density is less than 0.01 g / cm ³ , the handling strength of the support is lowered and it becomes difficult to use it in the method of the present invention. Also, the apparent density is 0.07
If it exceeds g / cm ³ , clogging occurs due to the growth of filamentous fungi. Further, the fineness of the fibers constituting the support is 20 to 15
It must be in the 0 denier range. If it is thinner than 20 denier, it is difficult to form a large space in which filamentous fungus can grow, and if it is thicker than 150 denier, filamentous fungus is less likely to be entangled, and the fixation of filamentous fungus becomes unstable.

The non-woven fabric support is preferably a fiber entanglement treatment such as needle punching or hydroentanglement.
This is because it is easy to maintain a three-dimensional porous structure and the contact area including the surface of the constituent fibers inside the nonwoven fabric becomes large. Further, a non-woven fabric whose strength is improved mainly by adhering fiber intersections with a resin binder and a non-woven fabric made of fibers having heat resistance that can withstand autoclave treatment are also preferable.

A known growth medium suitable for the growth of filamentous fungi can be used and is not particularly limited. The concentration of the growth medium is also not particularly limited, but the concentration can be made higher than usual and the growth step (1) can be performed in the presence of steam,
Particularly when a bulky support is used, it is difficult to attach and retain the medium, so that the concentration may be, for example, about 3 times higher than usual. Alternatively, the support may be impregnated with a medium having a concentration similar to the usual concentration, and added to the support by spraying or the like during the gas phase culture step (1). Completion of the growth of the filamentous fungus inside the support, when the nutrients in the medium held by the support are consumed, the filamentous fungus makes spores, which can be determined by the spores settlement, At this point, the gas phase culture step (1) can be completed. In addition, to be precise, prepare multiple same supports, take out the supports at regular intervals and measure the amount of proliferating cells, determine the growth curve from the relationship between the culture time and the amount of proliferating cells, and grow It is advisable to determine the culture time until the growth of the cell mass stops in a preliminary experiment. Usually, this culture time and the time until spore settlement begins are almost the same.

In the next fermentation culture step (2), the fixed support obtained in the gas phase culture step (1) is immersed in a fermentation production medium. In the fermentation culturing step (2), a normal medium selected depending on the types of the fixed filamentous fungus and the target product is brought into contact with the support by a batch method or a continuous method. Since a large amount of filamentous fungi are immobilized on a wide surface in the porous support used in the method of the present invention, there is sufficient space for efficient contact with the culture solution and the growth of filamentous hyphae in the porous support. Therefore, high productivity can be maintained for a long period of time. Further, since the filamentous fungus is immobilized, it is not detached together with the medium when the medium is replaced. In this fermentation culture step (2), without moving the support itself,
It is preferable that the culture solution pass through the support. It is preferable that the stirring is carried out to the extent that no bubbles are formed and an oxygen-containing gas (particularly air) is supplied into the culture solution.

In the product separation step (3), the target fermentation product is separated from the fermentation product liquid and collected. At this time, since the filamentous fungus is immobilized on the support, the fermentation product liquid does not substantially contain the fungus, and unlike the conventional method, the step of separating from the fungus is unnecessary. Also, the cells can be reused. For other separation and recovery operations, the operations of the conventional method can be used as they are.

In the method of the present invention, filamentous fungi (fungi) such as Aspergillus genus, Mucor genus, Rhizopu genus are used.
Various fermentation products can be obtained using a microorganism belonging to the genus s) or the genus Penicillium. Specific examples of these filamentous fungi, main raw materials contained in the fermentation production medium, and fermentation products are as follows (fermentation products / filamentous fungi / main raw materials are described in this order). When the fermentation product is an organic acid: citric acid / A. Niger / sucrose, gluconic acid / Aspergillus niger / glucose, kojic acid / Aspergillus flavus / glucose, kojic acid / Aspergillus oryzae (A. ory
zae) / glucose, pyruvic acid / mucor mandshuricus / glucose, fumaric acid / rhizopus niglycans (R. ni)
gricans) / starch. Where the fermentation product is an enzyme: glucoamylase / R. Niveus / glucose, lipase / R. Delemar /
Peptone and glucose. Where the fermentation product is an antibiotic: penicillin / penicillium chrysogenum (P. chrysogenu)
m) / Lactose and corn steep liquor. The present invention is particularly preferably used for the production of kojic acid.

[0013]

The present invention will be described in detail below with reference to examples, but these do not limit the scope of the present invention. Example 1 Needle punch felt (15) of polyester fiber (Oyama ester; fineness = 30 denier; fiber length = 76 mm)
Nonwoven fabric prepared by spray impregnating 0 g / m ² ) with a urethane binder (Toyo Polymer; Mercy 590) (area weight = 200 g / m ² ; thickness = 7 mm; apparent density =
0.03g / cm ³ ) is cut into a size of 4cm × 15cm,
Cylindrical stainless steel wire mesh support (diameter = about 40m
It was wrapped around m) and fixed. The non-woven fabric-supported wire mesh support was placed in a tall beaker (300 ml), autoclaved (121 ° C, 15 minutes), and a filamentous fungus growth medium (glucose 30 g / l, yeast extract 9 g / l, malt extract 9 g).
/ L, polypeptone 15 g / l) and a concentration of 1 × 10 ⁶ cells / ml of Aspergillus oryzae (Aspergillus).
soryzae) (IFO30113) After immersing in a suspension of spores for about 5 minutes, the suspension is removed, and the filamentous fungus is grown by leaving it in the atmosphere of saturated steam at 30 ° C. for about 72 hours in the air. , Propagated. Subsequently, a medium for kojic acid production (glucose 93 g / l, bonito extract 2.8 g
(1 / l, pH 4.0) was added, and fermentation culture was carried out for 20 days while gently stirring with a magnetic stirrer in a constant temperature water bath at 30 ° C., and then the medium was recovered. Water was evaporated from the collected medium and then cooled to 4 ° C. to obtain kojic acid crystals. The produced amount of kojic acid was 20 g / l per unit dry cell.

Comparative Example 1 Needle punch felt (150 g / m ² ) of polyester fiber (Toray; Tetron (registered trademark); fineness = 6 denier; fiber length = 76 mm) was spray impregnated with a urethane binder (Toyo Polymer; Mercy 590). The non-woven fabric prepared by the above method (Basis weight = 200 g / m ² ; Thickness = 2.4 mm;
The same operation as in Example 1 was repeated except that a support obtained by cutting apparent density = 0.08 g / cm ³ ) into a size of 4 cm × 15 cm was used. The production amount of kojic acid was 3 g / l per unit dry cell.

Comparative Example 2 Polypropylene Fiber (Chisso; Fineness = 200 Denier;
Thermal fusion felt with fiber length = 76 mm (Basis weight = 900 g /
m ² ; thickness = 10 mm; apparent density = 0.09 g / cm ³ )
The same operation as in Example 1 was repeated except that a support cut into a size of 4 cm × 15 cm was used. The amount of kojic acid produced was 5 g / l per unit dry cell.

Comparative Example 3 Felt prepared by subjecting polyester fibers (Toray; Tetoron; fineness = 3 denier; fiber length = 51 mm) to needle punching [area weight = 163 g / m ² ; thickness = 2.4 mm;
The same operation as in Example 1 was repeated except that a support obtained by cutting an apparent density = 0.07 g / cm ³ ] into a size of 4 cm × 15 cm was used. The amount of kojic acid produced was 2.7 g / l per unit dry cell.

[0017]

According to the method of the present invention, high productivity per unit cell can be realized. Further, the filamentous fungus can be repeatedly used, the step of separating the filamentous fungus from the fermentation product liquid is not necessary, and the fermentation culture step can be carried out under mild conditions, and it is necessary to perform complicated condition control. Nor.

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Claims (1)

[Claims] 1. An apparent density of 0.01 to 0.07 g / cm ^{3 comprising a} filamentous fungal growth medium impregnated in a liquid state, and comprising filamentous fungi attached thereto and having an average fineness of 20 to 150 denier.
On a fiber-made porous support of, under an oxygen-containing gas atmosphere, the filamentous fungi are grown and fixed to the support,
Next, a fermentation production method using a filamentous fungus, which comprises immersing the support on which the grown filamentous fungus is immobilized in a fermentation medium to perform fermentation culture of the filamentous fungus, and separating a fermentation product of the filamentous fungus.