JP2797308B2 - Method for producing microbial cellulose - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Use) The present invention combines Acetobactor aceti subsp. Xylinum with a bubble column culture (proliferation of bacteria) method and a stationary plate culture method (production of microbial cellulose). More specifically, the present invention relates to a method for mass-producing sheet-form microbial cellulose by seed culturing Acetobactor aceti subsp.xylinum, inoculating the seed in a bubble column culture tank without a stirrer, and containing oxygen. The present invention relates to a method for producing a sheet-shaped microbial cellulose, which comprises culturing in a bubble column while aerating gas, dispensing a culture solution in which the obtained bacteria have grown into a tray incubator, and culturing the microorganism in a stationary state.

(Prior art) Acetobactor aceti subsp. Xylinum produces extracellular cellulose (microbial cellulose) in sheet or gel form. This microbial cellulose is very excellent in physical properties such as elasticity and breaking strength, and is expected to be used in a wide range of applications such as dessert foods, acoustic plates, special sheets, and cosmetics.

Such production of microbial cellulose is performed by a static culture method, and a sheet-like film of microbial cellulose is formed on the surface of the culture solution. The conventional static culture method is called Ac
This is a method in which etobactor aceti subsp. xylinum is seed-cultured, and the cultured inoculum is inoculated into a container medium and allowed to stand and cultured for about 20 days.

Accordingly, there is a need for mass production of microbial cellulose and for greatly shortening the culture period.

Acetobactor aceti
A method of introducing an aeration and stirring culture step between the seed culture step and the stationary culture step of subsp. xylinum was considered.

However, if aeration and agitation culture is adopted, the culture period is shortened, but as the culture proceeds, microbial cellulose produced in the stirrer is entangled or microbial cellulose gel adheres to the culture tower, and the culture tower is washed. There are problems that require a lot of time and effort.

As a result of intensive studies by the present inventors to solve the above-mentioned drawbacks and mass-produce microbial cellulose, microbial cellulose can be produced in high yield by culturing using a bubble column culture tank instead of an aeration stirring culture tank. It has been found that they can be obtained and that the washing of the culture tank can be easily and easily performed, and that the culture time can be greatly shortened, and the present invention has been accomplished based on this finding.

(Means for Solving the Problems) The method for producing the sheet-form microbial cellulose of the present invention comprises: seed culturing Acetobactor aceti subsp. Xylinum; inoculating the seed into a bubble column culture tank without a stirrer; It consists of culturing in a bubble column while passing an oxygen-containing gas, dispensing the obtained culture solution into a tray incubator, and culturing the mixture in a stationary state.

The medium used in the seed culture and bubble column culture of the present invention is sucrose or sugar; yeast extract or synthetic amino acids and phytic acid; (NH ₄ ) ₂ SO ₄ ; KH ₂ PO ₄ ;
₄ and sterilized at 110-120 ° C for 10-30 minutes. The pH of the medium used for seed culture is 3.5 to 7.5, and the pH of the medium used for bubble column culture is 3.5 to 7.5.

The preferred composition of the medium used is 2-10 g / dl sucrose or 2-10 g / dl sugar; 0.1-1.0 g / d yeast extract.
l or synthetic amino acids 0.1-1.0 g / dl and fistic acid 0.01
(NH ₄ ) ₂ SO ₄ 0.1 to 2.0 g / dl; KH ₂ PO ₄ 0.1 to 1.0 g / dl and MgSO ₄ 0.01 to 0.5 g / dl.

Sheet culture was performed using the above sterilized medium
r aceti subsp.xylinum, inoculate and shake on a shaker for 20-35
C. for 24 to 96 hours.

In order to prevent the gel from adhering to the inside of the culture tank due to the growth of bacteria, the bubble column culture tank does not have a stirring device such as a stirring rod or a stirring blade inside the culture tank. An oxygen-containing gas introduction mechanism is provided for the purpose of supplying oxygen and stirring the contents during the culture. An outlet for the introduced gas is provided at the top of the culture tank, and an outlet for the culture solution is provided at the bottom. In addition, in order to easily dismantle and clean the culture tank,
An opening mechanism is provided at the bottom or on the wall surface of the bubble column culture tank.

The oxygen-containing gas introduction mechanism may have at least one gas outlet directly on the bottom or bottom wall of the bubble column culture tank, or at least one gas outlet on the part protruding inside from the lower wall of the bubble column culture tank. What is necessary is just to have two blowing holes. In the case of a structure protruding into the tank, it is preferable that the gel adhered during the culture is easily washed.

Fill the above-described medium into the above-mentioned bubble column culture tank, and
0.1 ~ 50% after sterilizing at 10 ℃ for 10 ~ 30min,
Preferably, 0.5 to 10% is inoculated, and oxygen-containing gas is passed at a rate of 0.01 to 2 v.vm, preferably 0.1 to 0.5 vvm, and 20 to 35 v.
° C, preferably 25-33 ° C for 24-120 hours, preferably 24
Incubate for ~ 72 hours.

Examples of the oxygen-containing gas to be aerated here include oxygen gas, a mixed gas of oxygen and nitrogen, and sterilized air.

There is no agitating blade inside the culture tank such as bubbles, and the liquid is mixed only with the oxygen-containing gas to be introduced.If the oxygen-containing gas aeration is less than 0.01 vvm, the mixing of the culture medium in the bubble column culture tank is not possible. Insufficient and insufficient supply of oxygen is not preferable. Also, the oxygen-containing gas ventilation rate
If it exceeds 2v.vm, the stirring of the culture medium becomes too vigorous, which is not preferable.

The tray-stage static incubator of the present invention is a fermenter in which trays made of a heat-resistant resin that does not deform at 120 ° C., for example, polycarbonate or metal, are stacked stepwise on a shelf frame. Each tray is stacked with a gap so that the oxygen-containing gas can be passed through the culture solution surface at a rate of 0 to 5000 ml / min, preferably 0.2 to 2000 ml / min. Alternatively, trays having vent holes on the upper side surface may be stacked so that the oxygen-containing gas vents at the above-described ratio. The trays stacked in the form of a shelf are housed in a box provided with an oxygen-containing gas introduction mechanism and a discharge mechanism.

The stationary culture in the present invention is a method of dispensing a culture solution that has been subjected to bubble column culture into each tray of the above-mentioned tray-stage static culture device that has been sterilized, and the culture solution surface of the tray has an oxygen-containing gas of 0 to 5000 ml.
Introduce the gas to ventilate at a rate of 20/35 ° C / min.
For 3 to 25 days.

Sterilization treatment of the stationary shelf incubator is 0.3 ~ at the temperature of 110 ~ 120 ℃.
It is carried out for 2 hours, preferably in the presence of water at a rate of 10 to 100 ml / l.

(Effects of the Invention) The method of the present invention is suitable for mass production of microbial cellulose sheet, and can provide microbial cellulose sheet with a short culture period and high yield.

For example, compared to the conventional culture method comprising a seed culture step and a stationary culture step using a container, the method of the present invention can obtain at least a 20% increase in yield in about 2/3 of the culture days.

Further, according to the method of the present invention, washing the bubble column culture tank,
It is possible to greatly reduce time and labor in assembling and cleaning a stationary culture vessel.

Hereinafter, the present invention will be described specifically with reference to Examples.
The present invention is not limited to these examples.

Example A sucrose 5 g / dl, yeast extract 0.5 g / dl, (NH ₄ ) ₂ SO ₄ 0.5 g / dl, KH ₂ P were placed in a 500 ml shoulder shake flask with a shoulder.
_{O 4 0.3g / dl, MgSO 4} 7H 2 O0.05g / dl TMA-821 medium containing (Toshiba Silicone Co.) 0.001 ml / dl of (pH = 5.0) 400 ml
Dispensed, sterilized in an autoclave at 120 ° C for 20 minutes,
Acetobactor aceti subsp.xylinu refreshed in advance
m Inoculate ATCC 10821 strain, shaker (100 rpm), 30 ℃
For 48 hours.

An oxygen-containing gas introduction mechanism is provided at the bottom of the culture tank, an introduction gas outlet is provided at the top of the culture tank, and a culture solution outlet is provided at the bottom thereof, so that the bottom is open. 150 l of the same medium (pH = 4) as the seed culture was placed in a bubble column culture tank having an inner volume of 0.175 m ³ and batch-sterilized at 120 ° C. for 20 minutes.

After sterilization, the seed solution obtained by seed culture (5.
7 × 10 ⁷ cells / ml) 1% inoculated, sterilized air 0.2
The cells were cultured at 30 ° C. for 72 hours with aeration at a rate of 5 v. Vm (the number of cells after 69 hours of culture was 1 × 10 ⁸ cells / ml).

Each tray (vertical x horizontal x height = 530 mm x 420) of a stationary tray incubator sterilized by an autoclave at 120 ° C for 60 minutes
mm × 80 mm). The number of trays in the tray standing incubator is nine. Sterilized air was introduced so that the air flow rate on the surface of the culture medium in the tray of the standing tray incubator was 1 / min, and the cells were cultured at 31 ° C. for 10 days.

The amount of gel per tray after 10 days of culture is 3.12 kg, and the gel thickness is
The yield (weight / filling liquid amount) was 78.0%, and the gel amount per tray culture vessel was 28.08 kg.

Operations such as washing the bubble column culture tank, washing the stationary culture vessel, dispensing the culture solution, and observing the culture state were easy, and the processing time was greatly shortened and labor was saved.

Comparative Example The seed obtained in the example was inoculated into the same medium (5 l) as in the example, which was inserted into each container (length × width × height = 645 mm × 385 mm × 150 mm).

Sterilized air was aerated so that the amount of aeration on the surface of the medium became 150 ml / min, and culture was performed at 31.5 ° C. for 20 days. After 22 days of culture, the amount of gel per container is 3.1 kg, gel thickness is 10 to 1
3 mm, and the yield (weight / amount of the filling solution) was 62%.

──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Tadashi Otomo ▲ ▼▼ 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa Prefecture Ajinomoto Central Research Laboratory Co., Ltd. (72) Wataru Nakamatsu Wataru Nakamatsu Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa 1-1 Ajino Central Research Laboratory Co., Ltd. (72) Inventor Koji Shimazaki 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa Prefecture Ajino Central Research Laboratory Co., Ltd. (58) Field surveyed (Int.Cl. ⁶ , DB name ) C12P 19/04 CA (STN)

Claims (4)

(57) [Claims] 1. A seed culture of Acetobactor aceti subsp. Xylinum, inoculation of the seed in a medium in a bubble column culture tank not equipped with a stirrer, and bubble column culture with aeration of an oxygen-containing gas. A method for producing a sheet-shaped microbial cellulose, which comprises dispensing a culture solution into a tray incubator and culturing the solution in a stationary manner. 2. The method according to claim 2, wherein the seed culture is sucrose or sugar; yeast extract or synthetic amino acid and phytic acid; (NH ₄ )
_{2 SO 4; KH 2 PO 4} ; 20~35 ℃ in medium containing and MgSO _4, 2
2. The method of claim 1, comprising performing for 4 to 96 hours. 3. A bubble column culture comprising sucrose or sugar; yeast extract or synthetic amino acid and phytic acid; (NH ₄ )
A medium containing ₂ SO ₄ : KH ₂ PO ₄ ; and MgSO ₄ is inoculated at 0.1 to 50%, and subjected to oxygen-containing gas at a rate of 0.01 to 2 v. Vm and aerated at 20 to 35 ° C. for 24 to 120 hours. The method of claim 1 comprising: 4. The stationary culture is carried out by adding oxygen-containing gas at 0 to 5000 ml /
20-35 ° C, 72-
The method of claim 1 comprising performing for 600 hours.