JPS58316B2 - Seikintai no Hifukuhouhou - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for coating viable bacterial cells in the form of particles or threads with a semipermeable membrane.

Conventionally, a method for coating microparticles by forming a semipermeable membrane in a liquid involves injecting an aqueous sol of a polymer compound in which an enzyme is dissolved into a coagulation bath whose pH is adjusted to make the polymer compound insoluble. A method of coagulating and solidifying the polymer compound (Japanese Patent Publication No. 42-6054), or a method of physically or chemically injecting an organic solvent solution of a charged synthetic polymer in which fine particles are dispersed into an organic solvent of an oppositely charged synthetic polymer. A method of coating dispersed particles by binding them to make them insoluble (Japanese Patent Publication No. 42-60
55), but the method of the present invention provides a novel coating method that is fundamentally different from these methods.

Specifically, as a result of research on methods for coating viable bacterial cells, the present inventors discovered that a polymer that forms a semipermeable membrane in water was dissolved in dimethylformamide (hereinafter referred to as DMF) or dimethyl sulfoxide (hereinafter referred to as DMSO). However, if this is dropped into water, the polymer will coagulate and precipitate, and at this time, if live bacterial cells are dispersed in DMF or DMSO in the polymer solution, the polymer will gel around the powder. , DMF or DMSO permeates the gel membrane and leaks into the water, while the surrounding water permeates into the membrane, and the polymer coagulates and precipitates, and the living bacteria are coated with the polymer, and the living bacteria used We discovered the fact that the body is not deactivated.

The present invention was completed based on the above findings, and consists of dispersing live bacterial cells in a solution of DMF or DMSO in which a polymer that forms a semipermeable membrane in water is dissolved, and then submerging this in water. This is a method for coating viable bacterial cells, which is characterized by coagulating and solidifying the polymer around the viable bacterial cells by dropping or extruding the polymer, thereby covering the viable bacterial cells in the form of granules or filaments with a semipermeable membrane.

In the method of the present invention, a polymer forming a semipermeable membrane in water is first dissolved in DMF or DMSO.
In this case, if the concentration of the polymer is too low, it is undesirable because it will scatter on the water surface when it is dropped into water later, and the concentration will vary slightly depending on the type of polymer used, but it is about 5 (W/
V) It is preferably 5 or more.

On the other hand, if this concentration is too high, it will be difficult to drip into the coagulation bath, and it will also have an adverse effect on the water permeability after coating, so it is preferable to use it at an upper limit of about 30 (W/V)%.

Further, the polymer used is not limited to any type as long as it is soluble in DMF or DMSO and insoluble in water and can form a semipermeable gel film in water.
Those that produce appropriate viscosity when dissolved in DMF or DMSO are preferred, such as cellulose polymers such as cellulose acetate, ethyl cellulose, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, cellulose acetate dibutylamino hydroxypropyl ether, and polyvinyl polymers such as polyvinyl chloride. Polymer, polyvinyl chloride
Copolymers of polyvinyl-based polymers such as polyvinyl acetate copolymers, polyvinyl chloride-polyvinyl propionate copolymers, other polystyrene,
Polyester or polyacrylonitrile is effectively used.

Then, DMF or DMS in which the above polymer was dissolved
The living cells are dispersed in a solution of O, but in this case, if the living cells contain too much water, the polymer will immediately precipitate, which is undesirable. Like lower alcohols D
It is necessary to use active bacterial cells that have good miscibility with MF and DMSO and have been washed to remove water with a hydrophilic organic solvent that does not precipitate polymers.

In addition, in this case, if the viscosity of the polymer is high and it is difficult to uniformly disperse the live bacteria, it is possible to disperse the live bacteria in DMF or DMSO before dissolving the polymer. Needless to say.

The viable bacterial cells to be used may be those that have the activity of not passing through the membrane after being coated with the polymer, and
Anything that is insoluble in MF or DMSO is acceptable for use.

Moreover, it goes without saying that two or more types of living microorganisms may be mixed and used, and further, other fine particles may be mixed with the living microorganisms.

Next, by dropping or extruding this dispersion into water, the living bacteria are coated with the polymer in the form of granules or filaments.When coating in the form of granules, it is sufficient to simply drop the dispersion into water; The particle size can be controlled to any desired size depending on the size, but if the particles are to be made even finer, known means used for microencapsulation using an in-liquid curing method, such as spraying from a nozzle, may be used as appropriate. I can do it.

In addition, when coating in the form of threads, the dispersion liquid may be extruded into water through a nozzle, and hollow fibers can also be formed depending on the shape of the nozzle.

Water is used as the coagulation bath in this case, but this water may be buffered if necessary, and substances necessary for preserving the viable bacterial cells after coating may be dissolved in the water as appropriate. It's okay to stay.

Since the live bacterial cells coated in granular or filamentous form by the method of the present invention are surrounded by a semipermeable membrane, they have good water permeability or permeability to low-molecular substances in aqueous solutions. By selecting the bacterial cells, it is possible to provide coated living bacterial cells that have extremely high industrial utility value.

Next, the present invention will be specifically explained with reference to reference examples and examples, but the present invention is not limited thereto.

Reference Example 1 100 ml of potato glucose medium was placed in a 500 ml Erlenmeyer flask and steam sterilized at 120°C for 20 minutes.
No.) was inoculated and cultured with shaking at 26°C for 72 hours. Three plants were collected, washed with acetone, and dried under reduced pressure to obtain 1.5 g of dried bacterial cells.

Reference example 2 Glucose 3%, yeast extract 1%, k2HPO40.1
%, MgSO4,7H2O0.05%, KCl0.05
%, liquid medium containing FeSO40,001% (pH 7,
0) After steam sterilizing 100ml at 120°C for 20 minutes,
Achromobacter strain B-402-2 (Feikoken Bacterial Serial No. 1095) was inoculated, cultured with shaking at 26°C for 72 hours, and the bacterial cells were collected, washed with acetone, and dried under reduced pressure. Obtain 20 g of bacterial cells.

Example 1 1.5 g of cellulose acetate is dissolved in 20 ml of DMSO, and 1.5 g of dried Fusarium solani cells obtained in Reference Example 1 are uniformly dispersed therein.

1 Next, this dispersion was dropped into 11 0.05M acetate buffer (pH 5.5) from a capillary with an inner diameter of approximately 1.0 mm, and the resulting bacterial cells coated with a cellulose acetate film were separated by a furnace to remove Fusarium. - Obtain 20 g of Solani water-containing capsules (particle size 2-4 mm).

This water-containing capsule body 20.9 has a cyan concentration of 1100 pp.
of cyanide in 150 ml of aqueous cyanide solution, continue shaking at 26°C, and measure the cyanide concentration using the silver nitrate method.
It becomes less than ppm and becomes undetectable in 24 hours.

Example 2 Dissolve 20 g of cellulose acetate in 200 ml of DMSO, uniformly disperse 20 g of dried bacterial cells of Achromobacter strain B-402-2 obtained in Reference Example 2, and then rotate this dispersion. Using a plate (atomizer cup), droplets were dropped into 101 0.1M phosphate buffer (pH 6.0), and the cells coated with the cellulose acetate film produced were separated from each other to isolate Achromobacter. Water-containing capsule of B-402-2 strain of the genus Bacterium (particle size 1-2 mm)
Obtain 300g.

3 g of this water-containing capsule was added to a 0.1M phosphate buffer (pH 6.0) containing 50 mg of 6-aminopenicillanic acid and 500 μm of D-phenylglycine methyl ester hydrochloride.
) and reacted by shaking at 30°C for 60 minutes, and the reaction filtrate was subjected to silica gel thin layer chromatography (developing solvent: n-butanol:ethanol:water=
4:1:1) and color development by the hydroxylamine method showed a spot around RL=0.44, indicating the formation of α-aminobenzylpenicillin.

Example 3 1 g of polyacrylonitrile was dissolved in 15 d of DMSO, and 1 g of dried Fusarium solani cells obtained in Reference Example 1 was uniformly dispersed therein. Then, this dispersion was poured into an annular orifice (spinelet). Polyacrylonitrile hollow fibers containing Fusarium solani cells (outer diameter 0.5 mm
, inner diameter 0.1 mm and wall thickness 0.2 mm).

The hollow fibers obtained by the above method are, for example, cut into a certain length and installed as a hollow fiber bundle in a housing that supports the fiber bundle with both ends open, and a cyan-containing liquid is poured into the fiber bundle from both ends. By allowing the cyanide-containing liquid to flow in and applying a constant pressure to it, the cyanide-containing liquid is decomposed in the process of permeating the wall membrane of the hollow fibers, and as a result, a cyanide-free liquid can be obtained from the outlet provided in the housing. can.

Claims (1)

[Claims] 1. Disperse live bacteria in a solution of dimethylformamide or dimethyl sulfoxide in which a polymer that forms a semipermeable membrane in water is dissolved, and then drop or extrude the solution into water to create a solution around the live bacteria. A method for coating live microbial cells, which comprises coagulating and solidifying the polymer, thereby covering the viable microbial cells in the form of granules or filaments with a semipermeable membrane.