JPH0578577B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing chitin molded bodies, and more particularly to various chromatography methods, carriers for immobilized enzymes and immobilized bacterial cells, and microcarriers for culturing animal cells. The present invention relates to a method for producing granular chitin that can be used for rears, etc.

Chitin is widely distributed in the living world, from animals such as crustaceans and insects to microorganisms, and forms the skeletal structure of these organisms. Its chemical structure is that N-acetyl-D-glucosamine is It is a polysaccharide that is connected in a linear chain through bonds. In the case of cellulose, glucose is linked by β-1,4 bonds, and the binding mode of chitin and cellulose is the same. Furthermore, these polysaccharides have in common that they are abundant in the natural world, and cellulose is used in various fields by being formed into paper, fibers, films, etc., and has become an essential resource in daily life.

On the other hand, chitin is not utilized as a useful resource despite its abundance. However, the unique properties of chitin have long attracted attention, and various industrial applications have been proposed.
Despite this, the reason why chitin is being discarded without being used effectively is that the technology for molding chitin has not been established. The industrial use of chitin currently in practical use includes the use of chitosan powder obtained by N-deacetylation of chitin as a polymer flocculant. The molding technique in this case is only pulverization, which is technically extremely easy. Most of the methods for using chitin that have been proposed so far have been investigated using chitin powder, and there are few examples of evaluating the properties of chitin after molding it into a form that can actually be used.

As a molding technique for chitin, molding into fibrous or film forms has been considered, but granulation processing has not been studied so far. for example,
JP-A-55-167048 proposes a method for producing spherical chitin molded bodies, but this method uses chitosan obtained by N-deacetylation of chitin as a raw material, and a chitosan solution is dispersed in a dispersion solution. This is a method for producing granular chitin by dispersing, granulating, and acylating. Therefore, this method not only does not involve granulating chitin, but also involves the steps of converting chitin into chitosan and acylating chitosan in addition to granulating, and is a method for industrially producing granular chitin in large quantities and at low cost. It is not advantageous as such.

The present inventors focused on the fact that chitin molding technology has not yet been established, which is why the utilization of chitin is delayed despite the abundance of chitin as mentioned above. As a result of extensive research into a method for producing granular chitin that can be used as a carrier for immobilized bacterial cells or as a microcarrier for culturing animal cells, it was surprisingly possible to drop chitin in solution into a coagulation bath. The present invention was achieved by discovering that granular chitin can be obtained by this method.

That is, the gist of the present invention is a method for producing a granular chitin molded article, which is characterized by dropping chitin in solution into a coagulation bath and coagulating it.

The term "granular" as used in the present invention means shapes such as spherical, rice grain-like, cylindrical, flat spherical, and other irregular shapes. According to the production method of the present invention, granular chitin having a substantially uniform shape and size can be obtained. Further, according to the production method of the present invention, granular chitin having a diameter or major axis of 0.001 to 10 mm, particularly 0.001 to 5 mm can be easily obtained.

In the present invention, chitin refers to chitin that has a degree of deacetylation of 50%, in addition to chitin obtained by purifying natural products.
Also includes chitin within. It also includes various chitin derivatives obtained by chemically modifying chitin. Examples of chitin derivatives include etherified chitins such as carboxymethylated chitin and hydroxyethylated chitin, and esterified chitins such as acetylated chitin and sulfonated chitin. Examples of esterified products include carboxylic acids such as formic acid, acetic acid, propionic acid, butyric acid, valeric acid, isopropionic acid, isobutyric acid, isovaleric acid, benzoic acid, cinnamic acid, salicylic acid, anthranilic acid, and phthalic acid; sulfuric acid; Examples include sulfonic acids such as toluenesulfonic acid and sulfanilic acid, carbonates, and esterified products of their anhydrides.

In the present invention, chitin in solution refers to a homogeneous solution in which chitin or various derivatives thereof are dissolved in a solvent. Examples of the solvent include methylene chloride containing trichloroacetic acid, dimethylacetamide containing lithium chloride, or N- containing lithium chloride.
Examples include methylpyrrolidone. Chitin can be dissolved in these solvents by a known method, and the chitin concentration is usually 0.01 to 10 w/w%, although it depends on the degree of polymerization of the chitin used. The viscosity of the solution chitin at 30°C is preferably 5000 cps or less, more preferably 2000 cps or less, and optimally
It is less than 1000cps.

As the coagulation bath in the present invention, any liquid can be used as long as it does not dissolve chitin, but it must be a liquid that can be mixed with the solvent for chitin in solution and has a specific gravity that is lower than that of chitin in solution. The desired property of the coagulation bath is that it is small. Further, the coagulation bath does not need to be a single type of solvent, but may contain a mixture of two or more different solvents, or an inorganic salt, a surfactant, a crosslinking agent, etc. as necessary. Specific examples of solvents used in the coagulation bath include water, alcohols such as methanol, ethanol, propanol, and butanol, halogenated hydrocarbons such as methylene chloride, carbon tetrachloride, and 1,2-dichloroethane, acetone, ether, tetrahydrofuran, Examples include dimethyl sulfoxide. The concentration of the coagulation bath is related to the coagulation rate of chitin in solution, so it is desirable to keep it as constant as possible.The temperature varies depending on the type of coagulation bath used, but is generally preferably -10 to 80%.
℃, more preferably -5 to 50℃, optimally 0 to 50℃
It is sufficient that the temperature is within the range of 40℃. Furthermore, when producing granular chitin, it is desirable that the coagulation bath be gently stirred.

The chitin molded article having a granular form according to the present invention can be produced by dropping the chitin solution as described above into a coagulation bath. This means that the liquid is pushed out from the tip and falls discontinuously into the coagulating liquid drop by drop.

When producing granular chitin by the method of the present invention, the size of the chitin particles is defined by the size of the droplets of chitin in solution. That is, the size of the chitin particles can be controlled by the diameter of the nozzle that discharges the chitin solution and the viscosity of the chitin solution.

The granular chitin coagulated in the coagulation bath according to the method of the present invention may be further washed with another solvent or dried by a commonly known method, depending on the purpose of use.

Furthermore, when producing granular chitin, it goes without saying that by dispersing or dissolving various substances in solution chitin, granular chitin containing those substances can be obtained. For example, if a solution of chitin in which enzymes or microorganisms are dispersed is formed into granules by the method of the present invention, enzymes or microorganisms entrappingly immobilized on granular chitin can be obtained, which can also be used for pharmaceuticals, agricultural chemicals, etc.
It is also possible to produce granular chitin containing fragrances and the like.

In the method for producing a chitin molded article of the present invention, the principle of the process in which chitin in solution solidifies into granules is gelation of chitin by simple solvent exchange. Unlike conventional methods, this method is characterized by the fact that granular chitin can be obtained with extremely easy operation and under mild conditions.

The chitin molded body obtained by the present invention can be used for various chromatography, carriers for immobilized enzymes and immobilized bacterial cells, microcarriers for culturing animal cells, etc., and furthermore, it has biocompatibility, which is a feature of chitin. It can also be used as a sustained-release base material for pharmaceuticals and as a medical adsorbent.

The present invention uses chitin, for which molding technology has not yet been established, as a direct raw material and provides a technology for processing and molding chitin into granules, thereby greatly contributing to the active and effective use of chitin.

EXAMPLES The present invention will be explained in more detail with reference to Examples below.

In the following examples, "solution viscosity of chitin" means 0.2 w/w% of chitin to a dimethylacetamide solution in which LiCl is dissolved at a concentration of 8w/w%.
This is the value measured at 30°C using a B-type viscometer to measure the viscosity of a chitin solution dissolved at a concentration of .

Example 1 Chitin coarse powder prepared from red snow crab (manufactured by Kyowa Yushi Co., Ltd.) was pulverized into 100 mesh pieces using an impact type pulverizer (Hosokawa Micron Victory Mill VP-10). Approximately 100g of this powder is mixed with 1 part of 0.5N-HCl
Suspended in aqueous solution, treated at 65℃ for 30 minutes, and cooled.
It was neutralized using a 10N-NaOH aqueous solution, washed with water, and dried. The solution viscosity of the purified chitin thus obtained was 50 cps.

This purified chitin was purified as follows:
It was dissolved according to the method described in Japanese Patent No. 134101. That is, 8.0 g of purified chitin was added to 992 g of a dimethylacetamide solution containing 8w/w% LiCl that had been ice-cooled in advance, and stirred for about 30 minutes while cooling on ice to swell the chitin. A clear solution was obtained after stirring for 30 minutes.
This solution was filtered using a 1480 mesh stainless steel net to obtain chitin in solution. The viscosity of the chitin solution at 30° C. measured using a B-type viscometer was 425 cps.

100 g of the chitin solution thus obtained was dropped into the coagulation bath from a glass pipette with a tip diameter of 1.2 mm. 500 ml of methanol was used as a coagulation bath, and it was gently stirred with a magnetic stirrer. The distance from the pipette tip to methanol was 10 cm, and the temperature of methanol was about 20°C. The granular chitin coagulated in the coagulation bath was stirred in the coagulation bath for about 30 minutes, then transferred to 500 ml of methanol, stirred for about 30 minutes, and the same operation was repeated two more times.
Repeated times.

The methanol-containing granular chitin produced in this way was perfectly spherical and had a diameter of about 2.6 mm.

This spherical chitin containing methanol was placed in a 500ml glass beaker, the top of the beaker was covered with nylon mesh, and compressed air was blown to dry it.
Further vacuum drying was performed for about 12 hours to obtain 750 mg of dry granular chitin. The diameter of the dry granular chitin was approximately 0.6 mm.

Example 2 In Example 1, 100 g of powdered chitin was suspended in a 0.5N-HCl aqueous solution of 1 and heated at 70°C for 30
After cooling, it was neutralized using a 10N-NaOH aqueous solution, washed with water, and dried. The solution viscosity of chitin thus obtained was 25 cps. 10 g of this chitin powder was added to 1990 g of N-methylpyrrolidone in which a saturated amount of LiCl had been dissolved and dissolved according to the method described in Example 1. The viscosity of this chitin solution at 30°C measured using a B-type viscometer was 120 cps. This chitin solution was made into fine particles using a sprayer and dropped into methanol which was being gently stirred. The fine chitin coagulated in methanol was washed with ion-exchanged water, and the diameter was 0.02 to 0.15 mm.
450 ml of finely divided chitin swollen with water was obtained.

Reference Example 1 The inside of granular chitin is thought to be porous, and the presence or absence of a molecular sieving effect was investigated as a utilization of this property.

That is, the fine particulate chitin obtained in Example 2,
A separation test between blue dextran (molecular weight: 2 million) and glucose (molecular weight: 180) was performed using a column with a diameter of 1 cm and a length of 40 cm, and using ion-exchanged water as the eluent. Blue dextran was measured by absorbance at 640 nm, and glucose was measured by the Anthrone-sulfuric acid method. As a result, blue dextran was eluted in an eluate volume of around 16 ml, and glucose was eluted in a volume of around 26 ml. In other words, it was confirmed that granular chitin has a molecular sieving effect and is useful as a carrier for gel chromatography.

Reference Example 2 Since chitin has excellent biocompatibility, its use as a microcarrier when culturing animal cells was considered, and its qualifications were investigated. For this purpose, add non-essential amino acids and nucleosides to 5 ml of culture medium (Dulbecco's modified Eagle medium), and
When 2.5 x 10 ⁵ human fibroblasts were inoculated and cultured in Petri blood containing 10 v/v % fetal bovine serum), the effect of adding granular chitin on cell yield was investigated.

That is, the fine particulate chitin obtained in Example 2 was heat sterilized in advance at 120°C for 30 minutes, and then washed with a culture solution.
It was added at the same rate as the granular chitin-free group.
The cells were cultured for 5 days using a CO ₂ incubator.
After culturing, the cells were collected by treatment with a trypsin-EDTA solution and the number of cells was counted. When granular chitin was added, the number of cells proliferated to an average of 6.2 x 10 ⁶ cells, and in the area without the addition, the average number was 3.0 x 10 cells. There were ⁶ pieces. That is, the cell yield increased approximately twice by the addition of granular chitin, confirming that granular chitin is useful as a microcarrier.

Claims (1)

[Claims] 1. A method for producing a granular chitin molded article, which comprises dropping chitin in solution into a coagulation bath and coagulating it.