JPH082302B2 - Cell culture member and cell culture method using the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a cell culture member having a polymer membrane having a pore diameter of 2 μm or less, which is difficult for permeation of bacteria to penetrate, as a wall membrane, and a cell culture method using the same. is there.

[Prior art]

In order to stabilize the enzyme activity and enable repeated use despite being water-soluble, a technology was developed to use the enzyme in the form of a solid catalyst by insolubilizing the enzyme in water by a physical or chemical method. Are used for industrial production of L-amino acids, isomerized sugars, 6-aminopenicillanic acid, malic acid and the like. Furthermore, by advancing the technique of immobilized enzymes and utilizing microbial cells (immobilized microorganisms) that still contain the enzyme, extraction and purification operations can be omitted, stability at the time of immobilization or operation is good, and multi-step enzyme system It is advantageous for use and has the advantage of being able to supply coenzymes and ATP. In addition, a method of immobilizing live cells can be used for the production of secondary metabolites, and it is also possible to improve the yield by using immobilized proliferating microorganisms.

For the above reasons, the establishment of immobilization technology is basically important, but there are carrier binding method, cross-linking method and encapsulation method, etc. (Ichiro Chibata, Tetsuya Tosa, Yuka, 31 , 414 (1982), Tatsuo Tsunono, Yuki Nakamura, Yasuto Ohtake,
Naomori Mori, PPM, No.6, 28 (1987), edited by Saburo Fukui, "Microorganisms as biocatalysts", Kyoritsu Shuppan (1979)).

[Problems to be solved by the invention]

In the fermentation production technology of aerobic bacteria, the diffusion rate of nutrients and enzymes affects the production rate of the desired product.
Generally, the utilization efficiency of nutrients is low, and the growth of cells and the production of products increase the viscosity of the culture broth to inhibit the biosynthetic reaction and reduce the production rate of the desired product. In the case of filamentous fungi, in particular, deficiency of nutrients and enzymes is likely to occur due to formation of bacterial mass having a three-dimensional network structure and thickening of the culture solution. Also,
There is a lot of room for improvement in that it takes time to separate the product from the culture solution.

Next, a technique for applying the bacterial cells / proliferated bacterial cells immobilized in the gel to a bioreactor has been developed, and it has been reported that productivity of alcohol fermentation is improved. However, it requires complicated operations such as preparation of gel containing bacteria, adjustment of gel size and strength, and sterilization. In addition, the gel is destroyed due to the growth of viable cells, and this makes it difficult to collect the product due to turbidity with the culture solution.

[Means for solving problems]

As a result of intensive studies conducted by the present inventors on a method for easily and inexpensively culturing bacterial cells, a bag of a polymer membrane having a large number of micropores having a sealable opening and a size of 2 μm or less through which a culture solution can pass. By using a bacterial cell culture member composed of a bacterial body and a bacterial cell support that adheres and supports the bacterial cells to be used by being housed in the bag, it is possible to generate a large surface area, overgrowth of gas, and generation of bacterial mass accompanying it. Since it does not contain nutrients and enzymes, it is durable and can be used repeatedly over a long period of time.In addition to proliferating cells, resting cells can be used for high productivity. It was found that sterilization of the culture solution is not necessary because there are bacterial cells in the bag body and that complicated gel adjustment operations such as size and strength adjustment and sterilization of the gel are not required, and the present invention is completed based on this finding. I arrived.

That is, according to the present invention, a bag of a polymer membrane having a sealable opening, a pore size of 2 μm or less, and a large number of micropores permeable to a culture solution, and a bacterium used by being contained in the bag. There is provided a bacterial cell culture member comprising a bacterial cell support for attaching and supporting a body.

Further, according to the present invention, the bacterial cell and its support are housed in a bag of a polymer membrane having a pore size of 2 μm or less having a sealable opening and a large number of micropores permeable to a culture solution. After that, an opening of the bag is sealed, and the obtained sealed bag is immersed in a culture solution for culturing, to provide a cell culture method.

As the support for immobilizing the bacterial cells in the present invention,
For example, non-woven fabric, polymer gel, polyurethane foam and the like can be mentioned. As the polymer film forming the bag, a plastic film such as polyethylene can be used.

〔Example〕

 Next, the present invention will be described in more detail with reference to Examples.

Example 1 Gelaguard 3201 (stretched polyethylene film, pore size
0.4 × 0.04μm) 50 (length) x 40 mm (width), 3 pieces of non-woven bags, 45 (width) x width (35 mm) of non-woven fabric (soaked and dried in 1% Tween80 aqueous solution), then N / 1000 It was immersed in a hydrochloric acid aqueous solution for 1 hour or more.

After this, Penicllium spiculiisporum Lehman No.10
About 100 mg of the -1 cell was put in a bag and heated with a sealer to seal the entrance of the bag. Ten bags made in this way were placed in a 1-volume wide-mouthed polybin containing 300 ml of non-sterile medium of the following composition, the mouth was covered with aluminum foil, and a reciprocating shaking incubator with aeration (amplitude 70 mm, 120th floor / minute) (Reciprocating) and cultured at 30 ° C. for 6 days. The culture solution foamed remarkably as the fermentation proceeded. After culturing, when the bag was cut open, bacterial cells having a thickness of about 0.5 mm adhered to both sides of the non-woven fabric in a film form and weighed 1.1 g, indicating that the bacterial cells had proliferated sufficiently. No germs were found to propagate in the culture solution outside the bag. (For comparison, when only the same culture solution was shaken under the same conditions, black mold of various bacteria propagated.) In addition, leakage of the cultured cells to the outside of the bag was not observed. The glucose content in the culture was 2.4% according to the glucose meter. Viscosity of the culture medium at the time of adjustment is 10 cp
Hasn't changed since. Then add ethanol to the fermentation broth
After adding 100 ml of 1N sodium hydroxide for neutralization, 100 ml of 1N hydrochloric acid was added for neutralization, and spiculisporic acid (4, 4,
The ring-opened form of 5-dicarboxy-4-pentadecanolide (1,3,4-tetradecanetricarboxylic acid) is the culture solution 1
1.3 g was produced per unit.

[According to Tabuchi et al., J. Ferment. Technol., 55, 37; 43 (1977)] Next, Table 1 shows the progress of the experiment using the bacterial cell culture member.

Example 2 Duraguard 3501 (0.4 × 0.04 μm) the same as in Example 1
Penicllium spicurisporum L in a bag sandwiched with nonwoven fabric
Three bags of 100 mg of ehman No. 10-1 sealed were prepared. These 3 in a 1-volume wide-mouthed bottle containing 300 ml of culture solution.
Put the bag, cover the mouth with aluminum foil, and at 30 ° C., Example 1
It culture | cultivated similarly to. As shown in Experiment No. 2 in Table 1, the cell weight after 6 days was twice as much as No. 1 per bag.
Reached 2200 mg. In this case, bacterial growth is remarkable and the thickness is about
It adhered to a 0.7 mm film. Compared with Example 1, the production efficiency of spiculisporic acid decreased.

Comparative Example 1 5 of the same Gurard 3501 (0.4 × 0.04 μm) as in Example 1
A bag of 0 × 40 mm was made, and 100 mg of the bacterial cell was sealed in the bag without the non-woven fabric. When opened after 6 days, the amount of cells was almost the same as that at the start of culture. Therefore, it was found that it is necessary to put the non-woven fabric in the bag in order to support and grow the bacterial cells.

Example 3 As can be seen from the comparison between Example 1 and Comparative Example 1, it is preferable to place a support of bacterial cells such as a non-woven fabric in a bag made of Juraguard. It was also possible to support the cells of Penicllium spicurisporum by using calcium alginate gel instead of the non-woven fabric. That is, 0.8% alginic acid aqueous solution or Penicllium spicurisporum cells were suspended.
0.8% alginic acid aqueous solution was dropped from a syringe into 1.5% calcium chloride aqueous solution with stirring at room temperature to obtain calcium alginate beads (with a diameter of about 2 mm) with or without cells. The gel containing the bacterial cells was put in the bag of Jeragard as it was, and the empty beads were put in the bag of Gelagard together with the bacterial cells to be sealed, and then cultured in the same manner as in Example 1. After 6 days, cell growth was observed, indicating that the gel is useful for supporting the cells. Further, polyurethane foam may be used as the support, and 6 days after the inoculation of the bacterial cells, the bacterial cell amount was tripled.

Example 4 Ten polymer film bags, in which the bacterial cells obtained in Example 1 were grown and fixed on a non-woven fabric, were taken out, placed on a glass funnel, the culture solution was naturally dropped and removed, and then repeatedly washed with water. It was Using the enzyme system of mycelium immobilized in these bags, spiculisporic acid biosynthesis (bioconversion) was performed using a 1-volume wide-mouth plastic bottle containing 300 ml of the medium containing the substrate shown in the table below. went. The cells were made to produce spiculisporic acid from α-ketoglutaric acid in a reciprocating shaking culture device (amplitude 70 mm, reciprocating 120 times / min) at 30 ° C. for 5 days. After completion of the reaction, the reaction solution was cooled to 7 ° C., washed with filtered water, and the obtained residue was dried to obtain ethyl ether.
It was dissolved in an ethanol (1: 1) solution. Thin-phase chromatography was performed using benzene as the developing solution to confirm the formation of spiculisporic acid.

Example 5 Torulopsis bombicola ATCC 22214 is sophorolipid: 7-L-[(2'-O-β-D-glucopyranosyl-β-D-glucopyranosyl) by utilizing glucose and oleic acid as carbon sources in yeast. Oxo]
It is known to produce a homologue of octadecenoic acid-1,4 ″ -lactone as a main component and related compounds thereof [N. Kosaric et.al., J.am. Oil Chem. Soc., 61,1735
(1984)]. According to the same procedure as in Example 1, 5 bags in which bacterial cells were carried in the polymer membrane were placed in a 1-necked plastic bottle containing 300 ml of non-sterile culture solution, and shaken while aerated at 30 ° C. for 6 days. And cultured. The amount of bacterial cells was eight times that at the time of inoculation (100 mg).

Claims (2)

[Claims] 1. A bag of a polymer membrane having a pore size of 2 μm or less, which has a sealable opening, and a large number of micropores through which a culture solution can permeate, and a bacterium used by being housed in the bag are attached. A bacterial cell culture member comprising a bacterial cell support for supporting. 2. A bacterium and its support are housed in a bag of a polymer membrane having a pore size of 2 μm or less, which has a sealable opening, and a large number of micropores permeable to a culture solution. A method for culturing bacterial cells, which comprises sealing the opening of the bag and immersing the obtained sealed bag in a culture solution for culturing.