JP2853950B2 - Rotary shaking culture vessel with baffle - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a culture vessel equipped with a baffle, and more particularly, to a baffle which, when used in a rotary shaking culture, has a good oxygen supply and mass transfer, and has extremely little bubbling and splashing of a culture solution. The present invention relates to an attached culture vessel.

[0002]

2. Description of the Related Art Containers conventionally used for rotary shaking culture of fungi such as aerobic bacteria include culture vessels without baffles and baffles that are not inclined with respect to the generatrix on the side of the vessel. There is a culture vessel with a baffle provided with a vertical baffle.

[0003] Examples of the culture vessel include an Erlenmeyer flask and a beaker.

However, in a culture vessel without a baffle, during culture, turbulence in the flow of the culture solution is difficult to produce, resulting in insufficient oxygen supply and mass transfer. It was easy to become. In particular,
When the culture product is fibrous, the tendency is remarkable because fibers are entangled. If the cultivated product becomes a large mass during the cultivation, oxygen supply and mass transfer become further insufficient, and there is a disadvantage that the productivity of the cultivated product deteriorates.

[0005] In order to improve the above-mentioned drawbacks, a baffled culture vessel has been developed in which turbulence is likely to occur in the culture solution flow. However, a conventional baffled culture vessel equipped with a so-called vertical baffle has been developed. Has another problem. For example, the culture solution tends to adhere to the container wall, cotton plug, and the like in the gas phase due to foaming and splashing during the rotation shaking culture. The fungus grows on the container wall and the like to which the culture solution has adhered, and a culture product is produced. This product further adsorbs the cells and culture product in the culture solution, and is cultured and produced on the container wall and the like in the gas phase. Things become big chunks. Due to such a phenomenon, in a conventional rotary shaking culture using a baffled culture vessel, the culture result varies (poor reproducibility of culture), and it is difficult to accurately evaluate productivity in shaking culture. there were. In particular, when the viscosity of the culture medium is high or when the viscosity of the culture solution increases with the culturing time, this disadvantage becomes remarkable.

[0006] In some cases, foaming of the culture solution can be suppressed by adding an antifoaming agent. However, it is desirable to avoid adding extra reagents.Moreover, many antifoaming agents have the property of decreasing the oxygen transfer rate due to their addition, and this causes another problem. Is not preferred.

[0007] Spattering of the culture solution can be avoided to some extent by lowering the rotation speed. However, the problem that the shearing force of the culture solution flow is weakened and oxygen supply and mass transfer are deteriorated, which is an essential solution. It doesn't work.

[0008]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a disadvantage of a culture vessel without a baffle (oxygen supply,
In addition, the productivity of the culture product is poor due to insufficient mass transfer) and the disadvantages of the conventional baffled culture vessel having a so-called vertical baffle (foaming of the culture solution and splashing of the droplets) are also improved. An object of the present invention is to provide a culture vessel equipped with a baffle for rotating shaking culture that has been eliminated.

[0009]

That is, the culture vessel of the present invention is a vessel characterized in that the baffle is inclined with respect to the generatrix on the side surface of the vessel.

In the present invention, the baffle is inclined with respect to the generatrix on the side surface of the culture vessel. The inclination is either forward or backward in the direction of the culture solution flow. Can be achieved. However, more preferable is when the inclination exists forward in the culture solution flow direction.

The baffle of the present invention is inclined with respect to the generatrix on the side surface of the culture vessel. If the angle of inclination of the baffle with respect to the generatrix is reduced, oxygen supply and mass transfer increase, but the bubbling of the culture solution and Splashes increase. Also,
Increasing the tilt angle decreases oxygen supply and mass transfer, but reduces foaming and splashing of the culture solution.

Therefore, the above-mentioned inclination angle can be appropriately determined according to the purpose of use of the culture vessel, etc., but in terms of both securing oxygen supply and mass transfer, and suppressing foaming of the culture solution and scattering of droplets, In order to simultaneously exhibit excellent effects, the inclination angle is preferably from 15 ° to 75 °, more preferably from 30 ° to 60 °, and most preferably about 45 °.

In the present invention, the number of baffles per culture vessel can be appropriately selected depending on the capacity of the culture vessel, culture conditions, and the like, but is preferably 2 to 4. A particularly preferred number of baffles is three.

The shape of the cross section in the short direction of the baffle of the present invention and the height of the baffle can be appropriately selected. For example, a triangular shape with rounded corners, a semicircular shape, a semi-elliptical shape, a thin plate shape, or the like can be used. Is desirable.

The baffle may have a linear shape, or a downward or upward gentle curve along its length.

The culture vessel with a baffle of the present invention can be processed by various methods well known to those skilled in the art, in particular, using a processing technique for producing a conventional so-called vertical culture vessel with a baffle.
For example, a baffle can be formed by pressing a predetermined mold from the outside of the Erlenmeyer flask to the conical surface of the Erlenmeyer flask and providing a concave groove in the inner direction of the Erlenmeyer flask during the manufacturing process of the Erlenmeyer flask made of glass.

The function of the present invention will be described below with reference to FIGS. FIG. 1 is a front view of an Erlenmeyer flask as one embodiment of the present invention, FIG. 2 is a right side view of the Erlenmeyer flask of the present invention, and FIG. 3 is a plan view of the Erlenmeyer flask of the present invention. The Erlenmeyer flask 1 of the present invention includes a baffle 2 that is inclined with respect to the busbar 3 instead of the conventional vertical baffle that is present without being inclined with respect to the busbar 3 on the conical side surface of the Erlenmeyer flask. As a result, the culture solution collides with the baffle 2 obliquely instead of at a right angle during the rotation shaking culture, so that the intensity of the collision is weakened and foaming and scattering of droplets can be suppressed. Further, since the culture solution is in an appropriate turbulent state, oxygen supply and mass transfer are sufficiently performed, and the productivity of the culture product is improved. Such a function applies to all of the containers of the present invention.

[0018]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the examples.

The present invention and its effects are illustrated by the production of bacterial cellulose (BC) by rotary shaking culture of acetic acid bacteria. BC is a fibrous culture product, and it is easy to form large lumps, and it is easy for foaming and splashing of the culture solution to occur during culture, and acetic acid bacteria are aerobic,
This is because it is suitable for the description of the present invention.

1. Medium Composition A medium having the following components was used for BC production by acetic acid bacteria.

Sucrose 40.0 g / l KH ₂ PO ₄ 3.0 g / l MgSO ₄ .7H ₂ O 2.4 g / l (NH ₄ ) ₂ SO ₄ 1.0 g / l Corn steep liquor 2 1.0% Antifoam 0.01% 2. Production method of BC by acetic acid bacteria (1) Seed culture 750 ml Roux filled with 100 ml of the above medium
The flask was inoculated with 1 ml of the stored bacterial solution, and was incubated at 28 ° C. for 3 days.
Seed culture was performed by standing.

(2) Main culture After the seed culture, the Roux flask was thoroughly shaken, and the contents were filtered under a sterile condition with gauze, and the filtrate was 7.5.
of the medium was inoculated into a 300-ml baffled conical flask with 67.5 ml of the medium. Using a shaking incubator (BR-3000L manufactured by Taitec), an amplitude of 2.5 cm,
Culture was performed for 4 days while rotating and shaking at a rotation speed of 180 rpm and a temperature of 28 ° C.

3. Quantitative method of BC The solid in each flask was washed with water to remove the medium components, and then treated in a 1% NaOH solution at 110 ° C for 20 minutes to remove cells. Further, the cellulose was washed with water until the washing liquid became nearly neutral, and then dried under vacuum at 80 ° C., and the dry weight was measured.

4. Baffled Erlenmeyer Flask of the Present Invention Used A 300 ml baffled Erlenmeyer flask shown in FIGS. 1 to 3 was used. The inclination angle A of the baffle with respect to the generatrix on the conical side surface of the flask is about 45 °, the short-side section of the baffle has a triangular shape with a rounded tip, and the baffle has a substantially straight shape along its long direction. , Its length is 80 mm. One Erlenmeyer flask has three baffles.

[5] Example of culture The following example was performed under the above conditions.

Embodiment 1 FIG. [Purpose] A. xylinum ATCC 23770,
A. pasteurianus FERM P-128
Rotational shaking culture was performed on the two strains of No. 84, and it was determined whether there was a difference between the attachment of BC to the flask wall and the foaming of the culture solution during the culture between the conventional baffled Erlenmeyer flask and the baffled Erlenmeyer flask of the present invention. Examined.

[Results] As shown in Table 1, it was revealed that BC did not attach to the flask wall at all in the baffled Erlenmeyer flask of the present invention. In conventional Erlenmeyer flasks with baffles, the culture solution foams during culturing,
It is probable that the scattering of the droplets was remarkable but did not occur at all in the baffled Erlenmeyer flask of the present invention.

[0028]

[Table 1]

Embodiment 2 FIG. [Purpose] A. xylinum ATCC 23770,
A. pasteurianus FERM P-128
84; sp. Rotational shaking culture was performed on the three strains of BPR 2001, and it was examined whether there was a difference in BC productivity between the conventional baffled conical flask and the baffled conical flask of the present invention.

[Results] As shown in FIG. 4, in the culture vessel with baffle of the present invention, the BC productivity was greatly improved as compared with the conventional culture vessel with baffle.

Embodiment 3 FIG. [Purpose] The baffle has various inclination angles (forward inclination with respect to the culture solution flow) with respect to the generatrix on the side of the Erlenmeyer flask.
A. Erlenmeyer flask with a baffle having a capacity of pasteur
Example 2 was carried out using ianus FERM P-12883 strain. Rotational shaking culture was performed for 4 days in the same manner as described above, and it was examined how the tilt angle affects BC productivity.

[Results]

[0033]

[Table 2]

As can be seen from Table 2, the BC productivity was greatly improved in the baffled Erlenmeyer flask of the present invention, particularly in the baffle Erlenmeyer flask with a 45 ° tilt angle, as compared with the conventional baffle Erlenmeyer flask. .

Embodiment 4 FIG. [Purpose] A 300 ml baffled Erlenmeyer flask having various inclination angles with respect to the generatrix on the side of the Erlenmeyer flask. Rotational shaking culture was performed for 1 week in the same manner as in Example 1 using xylium ATCC 23770 strain, and how the tilt angle affects the adhesion of BC to the wall of an Erlenmeyer flask and the productivity of BC during the culture. I checked.

[Results] As can be seen from Table 3, the Erlenmeyer flask with baffle of the present invention has a higher BC productivity than the Erlenmeyer flask without baffle and the conventional flask with baffle having a tilt angle of 0 °. The BC was greatly improved, and the adhesion of BC to the wall of the Erlenmeyer flask was also greatly improved as compared with the conventional Erlenmeyer flask with a baffle.

[0037]

[Table 3]

[0038]

EFFECTS OF THE INVENTION By using the culture vessel with a baffle of the present invention for rotary shaking culture, the drawbacks (foaming of a culture solution and splashing of a culture solution) of a conventional culture vessel with a baffle are reduced, and a culture vessel without a baffle is used. (The productivity of the culture product is poor due to insufficient oxygen supply and mass transfer) can be simultaneously improved.

[Brief description of the drawings]

FIG. 1 shows a front view of an Erlenmeyer flask with a baffle which is one embodiment of the present invention.

FIG. 2 shows a right side view of an Erlenmeyer flask with a baffle which is one embodiment of the present invention.

FIG. 3 is a plan view of an Erlenmeyer flask with a baffle which is one embodiment of the present invention.

FIG. xylinum ATCC 23770,
FIG. 3 is a graph in which the relationship between the number of days of culture and the amount of accumulated cellulose is plotted for three strains of FERM P-12884 and BPR 2001 using a conventional baffled conical flask and the conical flask with baffle of the present invention.

[Explanation of symbols]

 1: Erlenmeyer flask with baffle 2: Baffle 3: Generating line on conical side of flask A: Inclination angle to generatrix on conical side of baffle

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Tohru Koda 309 Imainakacho, Nakahara-ku, Kawasaki-shi, Kanagawa Prefecture Ajinomoto Co., Ltd. 1-110 Imai Ryo Co., Ltd. (72) Inventor Takayasu Tsuchida 1730 Kamikuratacho, Totsuka-ku, Yokohama-shi, Kanagawa Prefecture. −13 (56) References JP-A-1-82800 (JP, U) (58) Fields investigated (Int. Cl. ⁶ , DB name) C12M 1/00-3/10

Claims (8)

(57) [Claims] 1. A method for rotating and shaking culture, characterized in that a baffle is inclined with respect to a generatrix on a side surface of a container.
Container of culture. 2. The container according to claim 1, wherein the culture container is an Erlenmeyer flask. 3. Each baffle is divided above and below the container.
2. The baffle of claim 1, wherein the baffle is continuous.
Or the container according to 2. 4. The inclination angle of the baffle with respect to the generatrix is 15
The container according to any one of claims 1 to 3, wherein the angle is 75 °. 5. An inclination angle of the baffle with respect to the generatrix is 30.
The container according to claim 4, wherein the angle is about 60 °. 6. The inclination angle of the baffle with respect to the generatrix is about 4
The container according to claim 5, wherein the angle is 5 °. 7. The number of baffles per container is 2 to 4.
The container according to any one of claims 1 to 6, wherein the container is a unit and the baffles are arranged at equal intervals on a side surface of the container. 8. The container according to claim 7, wherein the number of baffles per container is three, and the baffles are arranged at equal intervals on the side surface of the container.