JPS641169B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing collagen beads,
The collagen material is shaped into spheres by dropping an aqueous liquid of the collagen substance into an organic medium that is sparingly miscible or immiscible with water, or by emulsifying it into the organic solvent using an emulsifier, and then into spheres. It consists of hardening the shaped collagen material by using a hardening agent or by irradiating it with chemically active radiation such as ultraviolet rays or gamma rays.
The present invention relates to a method for producing collagen beads.

BACKGROUND OF THE INVENTION Various molded articles made from collagen, such as films, threads, tubes, or rods, such as medical films, surgical threads, and artificial casings for sausages, have been developed. However, no collagen bead-shaped molded product has been developed so far.

The present inventors intended to produce collagen beads as a collagen molded article that could be used as a carrier for biologically active substances by being packed into a column, and researched various conditions to provide various physical properties required as a carrier for column filling. The present invention was completed.

Collagen substance aqueous liquid (hereinafter sometimes simply referred to as collagen liquid) that can be used as a raw material in the present invention includes a collagen aqueous solution solubilized by the action of an enzyme or an alkali (Japanese Patent Publication No.
11037, Japanese Patent Publication No. 46-15033), collagen fiber aqueous dispersions, denatured collagen aqueous solutions obtained by subjecting these collagen solutions to ultrasonic treatment, ultraviolet irradiation treatment, heat treatment, etc., and mixtures thereof. The concentration of the collagen material in the aqueous solution is such that it provides the necessary surface tension and viscosity to disperse the aqueous solution of the collagen material into spherical shapes in an organic solvent that is substantially immiscible with water. The concentration is usually between 3 and 20% by weight.

According to the method of the present invention, a spherical aqueous liquid is prepared by dropping the collagen liquid into an organic solvent that is substantially immiscible with water or emulsifying it in this organic solvent, which is then further processed into a spherical aqueous liquid. Submit to the process of

The organic medium used in the present invention must be insoluble or sparingly soluble in water as described above, and must also have a specific gravity similar to that of the collagen solution, and a surface tension lower than that of water or the collagen solution. It has to be something. When droplets of a suitable size are dropped into such an organic medium, the collagen liquid becomes spherical and floats in the organic medium. Desirable organic media have a specific gravity of 0.9 to 1.1 and a surface tension of less than 40 dynes/cm at room temperature.

Any substituted or unsubstituted aromatic hydrocarbon compound, substituted or unsubstituted aliphatic hydrocarbon compound, other organic compound, silicone oil, etc. that are liquid at room temperature and satisfy the above conditions can be used in the present invention.

The surface tension and viscosity of the collagen liquid change depending on the state and concentration of the collagen substance contained therein. Generally, increasing the concentration of collagen material reduces surface tension and increases viscosity. In order to facilitate molding, it is preferred that the surface tension of the collagen liquid is not reduced too much, such as below 50 dynes/cm at room temperature, and the viscosity is not increased above 70 poise at room temperature.

Collagen solubilized by enzymes is soluble at a pH of about 3.0 or less, but the pH of the aqueous solution
A low viscosity emulsion-like aqueous dispersion of collagen fibers can be obtained by increasing the ratio from about 4.5 to about 10.5. In addition, the denatured collagen aqueous solution obtained by ultrasonication or ultraviolet irradiation of a collagen aqueous solution solubilized with an enzyme or an alkali at a temperature of 10°C has the collagen molecules cleaved, especially in an aqueous solution with a concentration of 20% or less. The viscosity of the solution is approximately 1/10 that of the original aqueous solution. Furthermore, a denatured collagen aqueous solution obtained by heat-treating an enzyme- or alkali-solubilized collagen aqueous solution has a viscosity approximately 1/1000 of the original undenatured collagen solution when the collagen concentration is 20% or less.

The low-viscosity collagen liquid produced in this way can of course be used alone as a collagen raw material, or it can be blended with other high-viscosity collagen liquids, especially undenatured collagen liquid, to adjust the viscosity and surface tension of the raw collagen liquid as appropriate. can do.

For example, when mixing the emulsion-like collagen fiber dispersion into an alkali-solubilized collagen aqueous solution,
It is preferred that the former be 5 to 30 parts by weight and the latter 95 to 70 parts by weight. Further, when adding any of the above-mentioned modified collagen aqueous solutions to the emulsion-like collagen fiber dispersion, it is preferable that the amount of the former is 30 to 90 parts by weight, and the latter is 70 to 30 parts by weight. When mixing any of the above denatured collagen aqueous solutions with an enzyme or alkali solubilized collagen aqueous solution, 15 to 60 parts by weight of the former and 85 parts by weight of the latter.
It is preferable to mix between 40 and 40 parts by weight.

In addition, if heat-treated denatured collagen is blended in an amount of 10% or less of the total collagen amount in all cases, this raw collagen liquid is formed into a spherical shape in an organic medium and then gelled by cooling. This causes the collagen solution to solidify, making subsequent operations easier.

The particle size of the beads can be freely determined by changing the droplet size, collagen liquid concentration, etc. For example, from a capillary with an outer diameter of 0.5 mm, 3
When a collagen solution with a concentration of 30% to 30% by weight is dropped into an organic medium, the particle size of the beads can vary from 0.5 to 2 mm depending on the concentration of the collagen solution. Similarly, when dropping from a tube with an outer diameter of 5 mm, the bead size can vary from 3 to 8 mm.

Microscopic beads can be produced by emulsifying collagen liquid in an organic medium using an emulsifier. In this case, the particle size of the beads can be freely controlled by the type of emulsifier, the shearing force applied to the liquid during emulsification, and the collagen concentration of the raw material liquid. For example, using a nonionic surfactant with HLB4 or less,
By stirring with a homogenizer at various speeds, 100 to 300 meshes of collagen beads can be obtained. Furthermore, fine collagen beads of 30 to 100 meshes can be obtained by emulsifying using a nonionic surfactant of HLB 4 to 9 under the same conditions as above. In addition to nonionic surfactants, cationic, anionic and amphoteric surfactants can also be used.

The spherical collagen liquid formed by dropping or emulsifying the collagen liquid is then hardened.
For curing, the aldehyde is transferred into the spherical collagen liquid by adding aldehydes that are soluble in the organic medium used, or by dispersing the aldehyde dissolved in water or alcohol in the organic medium. It can crosslink and harden the collagen liquid. Examples of aldehydes that can be used include formaldehyde, acetaldehyde, glutaraldehyde, glyoxal, and Kuhn's liquid.

Furthermore, instead of aldehyde, various metal salts effective in curing collagen, such as chromium complex salts, aluminum sulfate, ferric sulfate, etc., can be used as an aqueous solution to effect curing in the same manner.

Alternatively, the spherical collagen liquid can be dispersed in an organic medium and irradiated with ultraviolet rays or gamma rays to crosslink the collagen molecules and harden the collagen liquid. In addition, when performing ultraviolet irradiation treatment, it is necessary to use an organic solvent that does not substantially absorb ultraviolet rays.

Prior to hardening, the spherical collagen liquid is dehydrated and solidified by transferring it to a dehydrating medium such as a concentrated aqueous solution of salts, acetone, or alcohol, and then treated with the hardening agent or Curing can also be performed by ultraviolet gamma ray irradiation treatment. The hardening treatment may be carried out in a dehydrating medium. By dehydrating and coagulating before hardening, the collagen concentration in the spherical collagen dispersion increases, so that the physical properties of the resulting collagen beads can be changed.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 Enzyme solubilized collagen aqueous solution (PH3.0, concentration 12
%) was adjusted to pH 8.0 and stirred well to obtain a milky collagen fiber dispersion. 20 parts by weight of an alkali-solubilized collagen aqueous solution (PH 8.0, concentration 6%) was mixed with 70 parts by weight of the emulsion-like collagen fiber dispersion and stirred well. To this mixed solution was added 10 parts by weight of an aqueous alkali-solubilized denatured collagen solution with a pH of 8.0 and a concentration of 18% that had been heat-treated at 40°C for 10 minutes, and the mixture was thoroughly mixed and defoamed at 30°C. This was placed in a syringe container maintained at 30°C and dropped into a 1:1 benzene-monochlorobenzene solution at 15°C through a syringe needle with an outer diameter of 2 mm to form a spherical collagen liquid. Next, this spherical collagen dispersion was transferred to a saturated aqueous solution of sodium sulfate and left overnight to perform dehydration and concentration.
After the 11.4% collagen solution was dehydrated, the concentration of collagen material was approximately doubled. Furthermore, the spherical particles were transferred to a saturated aqueous solution of sodium sulfate with a pH of 8.0 containing 0.3% glutaraldehyde, and heated at 25℃30.
Tanning treatment was carried out for a minute, followed by thorough washing with water. Diameter with satisfactory physical properties
Uniform collagen beads of 4.6±0.1 mm were obtained.

Example 2 50 parts of enzyme-solubilized collagen with a PH3.0 concentration of 4%
Fifty parts of alkali-solubilized denatured collagen with a pH 3.0 concentration of 12% that had been heat-treated at 40°C for 10 minutes was mixed at 30°C, and the mixture was defoamed.

Separately, benzene-monochlorobenzene (1:
1) Add 0.5% (W/V) of 25% glutaraldehyde aqueous solution to the mixed solution and uniformly disperse it using a nonionic surfactant to prepare a shaping organic medium, which was kept at 20 °C. . The collagen liquid is dropped into this medium as a droplet from an injection needle, and is made into a spherical shape.
It was left to stand overnight for hardening treatment. Thereafter, the spherical particles were collected and thoroughly washed with water. Uniform collagen beads with a diameter of 5.8±0.1 mm and good hardness and elasticity were obtained.

Example 3 Enzyme-solubilized collagen aqueous solution at pH 3.0 and concentration 2.0
%, placed in a quartz flask, and irradiated with an ultraviolet lamp at 5°C for 10 hours to cleave collagen molecules. Purify and concentrate this liquid to pH
3.0, the concentration was 15%. 30 parts of an alkali-solubilized collagen aqueous solution having a pH of 3.0 and a concentration of 7% and 70 parts of the ultraviolet-treated denatured collagen were thoroughly mixed at 10°C.
The above collagen solution was mixed into methylene chloride in which 1% (W/V) of a nonionic surfactant had been dissolved with vigorous stirring at 10°C, and was uniformly dispersed.
Fine spherical collagen particles were separated by filtration, placed in ethyl alcohol, dehydrated, and dried by evaporating the alcohol. Furthermore, the dehydrated fine particles were transferred to a saturated aqueous salt solution (PH2.0) containing 1.0% (W/V) of High Neochrome (Nippon Kagaku Kogyo KK) with stirring, and left overnight at room temperature for hardening treatment. Summer. Thereafter, the particles were collected and thoroughly washed with water, and approximately 200 meshes of uniform particles were obtained. When it was packed in a column and water was run through it, no clogging occurred and it showed good physical properties as a column carrier.

Example 4 Enzyme-solubilized collagen was adjusted to a PH3.0 concentration of 2%, and treated with ultrasound at 10°C for 5 hours using a 10KHz ultrasound generator to reduce the collagen molecules by approximately 1/2.
Cut into 4 lengths. This liquid is purified and concentrated.
The pH was 3.0 and the concentration was 7%. 30 parts of alkali-solubilized collagen with a pH of 3.0 and a concentration of 15% and 70 parts of the sonicated denatured collagen were well mixed at 10°C.

The above collagen solution was mixed into cyclohexane in which 1% (W/V) of a nonionic surfactant had been dissolved with vigorous stirring at 10° C., and uniformly dispersed. This was transferred to a quartz flask and irradiated with ultraviolet rays for 5 hours using an ultraviolet lamp while stirring and feeding nitrogen gas. Fine collagen particles were collected, washed with ethyl alcohol, and then thoroughly washed with water. As in Example 3, fine collagen beads of about 100 meshes were obtained.

Claims (1)

[Scope of Claims] 1. A collagen substance aqueous liquid having a collagen substance concentration of 3 to 20% is dropped into an organic medium that is poorly miscible or immiscible with water, or is emulsified in the organic medium. A method for producing collagen beads, which comprises forming the aqueous collagen material into spheres, and then hardening the spherical aqueous collagen material. 2. The method according to claim 1, wherein the collagen substance aqueous solution is a solubilized collagen aqueous solution. 3. The method according to claim 1, wherein the collagen substance aqueous solution is a collagen fiber dispersion. 4. The method according to claim 1, wherein the collagen material aqueous solution is a denatured collagen aqueous solution. 5. Claim 1, wherein the collagen substance aqueous liquid is a mixture of two or more of the group consisting of a solubilized collagen aqueous solution, a collagen fiber dispersion, and a denatured collagen aqueous solution.
The method described in section. 6. The method according to claim 1, wherein the spherical collagen liquid is hardened by including a collagen hardening agent in advance in the organic medium. 7. The method according to claim 1, wherein the spherical collagen liquid is dehydrated by using a dehydrating agent before hardening. 8. The method according to claim 7, wherein the dehydrating agent is ethanol, methanol or accent. 9. The method according to claim 7, wherein the dehydrating agent is a concentrated aqueous inorganic salt solution. 10. The method of claim 1, wherein the hardening of the spherical collagen liquid is carried out by the use of a hardening agent selected from aldehydes, metal salts or combinations thereof. 11. The method according to claim 1, wherein the curing of the spherical collagen liquid is performed by irradiation with ultraviolet rays and/or gamma rays.