JP3094181B2 - Microcapsule using recycled natural keratin as wall material and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to microcapsules containing regenerated natural keratin as a wall material, suitable for inclusion of dyes, fragrances, pharmaceuticals, agricultural chemicals, enzymes and other drugs or for immobilization of enzymes and the like, and production thereof. About the method. As used herein, "regenerated natural keratin" refers to reducing disulfide bonds without adding hydrolysis treatment of peptide bonds by enzymes or the like to natural keratin and without adding any other irreversible chemical treatment. A regenerated polymer in which keratin is once solubilized as a thiol group and then insolubilized again by disulfide bonds between the thiol groups.

[0002]

2. Description of the Related Art Conventionally, microcapsules have been developed for the purpose of improving stability, release characteristics and other various properties by including a drug or the like.

[0003] The material used as the wall material of the microcaps is required to be easy to handle, for example, microcapsules can be manufactured by a simple apparatus and method, and the obtained microcapsules are not suitable for the quantity of the capsules themselves, such as drugs. It is required that the inclusion amount is large. Furthermore, microcapsules used for pharmaceuticals, agricultural chemicals, etc. are particularly characterized by being excellent in biocompatibility, being able to be manufactured without using a cross-linking agent, which is a problem in toxicity in particular, and having biodegradability. Is required. For this purpose, it is preferable to use a biological substance in a natural or very close state as a raw material of the wall material of the microcapsules that can be used for pharmaceuticals and the like.

Various methods are known for producing microcapsules depending on the properties of the raw material of the wall material of the microcapsules. It is necessary to use a method that does not impair the biocompatibility of the material, and therefore it is required that the microcapsule wall material is also a material to which such a method can be applied.

On the other hand, keratin exists as a structural protein in animal hair and feathers such as nails, hair and wool, but microencapsulation using keratin itself as a wall material has not been studied. As a slightly related technique, a microcapsule using keratin (hereinafter referred to as "keratin hydrolyzate") which has been subjected to a hydrolysis treatment of a peptide chain with a protease such as pepsin has been disclosed (Japanese Patent Publication No. 59-1984).
No. 33017). However, this technique does not show any repair treatment of a peptide chain once degraded by treatment with proteolytic enzymes, and therefore, the keratin hydrolyzate is polymerized by cross-link formation by disulfide bonds between chain fragments. Even after the capsule wall has been constructed, each cut keratin chain remains unrepaired and does not regenerate native keratin.

[0006]

Under such circumstances,
The present invention uses a regenerated natural keratin as a wall material without a treatment for reducing the molecular weight by cleavage of a peptide bond or other irreversible chemical modification, and is preferable in terms of biocompatibility and biodegradability and includes a drug and the like. It is intended to provide a microcapsule having a large amount and a method for producing the microcapsule.

[0007]

In order to achieve the above object,
The present inventor has conducted various studies on a method for producing microcapsules by extracting natural keratin from a keratin-containing substance without subjecting to peptide bond decomposition treatment. As a result, microcapsules made of regenerated natural keratin can be obtained by extracting and treating keratin from a keratin-containing substance by a method described below without performing a hydrolysis treatment with a protease and other treatments that cause irreversible denaturation. Was successfully manufactured. In addition, it was confirmed that the microcapsules produced by this method had remarkably superior properties as compared with the known microcapsules using the above-mentioned keratin hydrolyzate.

Hereinafter, the present invention will be described in order of a step [I] of producing water-soluble keratin used as a raw material for a wall material of a microcapsule and a step [II] of producing microcapsules using the water-soluble keratin. I do. [I. Keratin extraction method and result without hydrolytic treatment or other irreversible chemical modification] In this step, the keratin-containing substance was reduced by solubilization and extraction by stirring with a reducing agent in a liquid medium, and the obtained keratin-containing substance was obtained. Insoluble matter is removed from the extract, and the reducing agent is removed by dialysis or other appropriate means in the presence of a surfactant.

As the keratin-containing substance, any substance containing genuine keratin such as human hair, wool or other animal hair, feathers, hooves and the like can be used.

As the liquid medium, for example, a solvent which is stable against reduction and has an affinity for keratin-containing substances can be used. For example, water or alcohols or amides or a mixture thereof can be used. It is preferably used. The used amount of the liquid medium may be any amount that can immerse the keratin-containing substance, but is preferably 10 to 40 times the weight of the used amount of the keratin-containing substance for processing.

The above-mentioned liquid medium is not essential, but is preferably urea, thiourea or the like for the purpose of increasing the efficiency of reductive solubilization of hardly solubilizable materials such as animal hair, hair, horns, claws, and hooves. , Alcohols such as methanol, ethanol and propanol, inorganic salts such as zinc chloride, sodium iodide and lithium bromide, ammonia, sodium hydroxide and the like can also be added as dissolution aids.
The addition amount of these solubilizers is appropriate. For example, in the case of urea, it is usually 3 to 15 times by weight, preferably 5 to 12 times by weight based on the keratin-containing substance.

For the reduction, any reducing agent that can reduce the disulfide bond of keratin present in the keratin-containing substance to a thiol group can be generally used. For example, mercaptoethanol, thioglycolic acid, toluene-ω-
Thiol derivatives such as thiol, dithiothreitol and dithioerythritol, phosphorus-containing compounds such as triphenylphosphine, tripropylphosphine and tributylphosphine, and inorganic reducing compounds such as sodium hydrogen sulfite are preferably used. The amount of the reducing agent to be used is preferably 0.01 to 0.50 mol, more preferably 0.05 to 0.25 mol, per 10 g of the keratin-containing substance.

The reduction solubilization is preferably carried out on the alkaline side in order to promote the reaction.
It is particularly preferred to be in the range of 10 to 11. The reaction is carried out by heating if desired. The reaction temperature and reaction time can be appropriately set according to the difficulty of solubilizing the keratin-containing substance, and for example, stirring can be performed at room temperature to 100 ° C. for 1 to 24 hours.

The above-mentioned surfactant which needs to be present in the liquid medium during the operation of removing the reducing agent is usually obtained after reducing and solubilizing a keratin-containing substance into a solution. Insoluble matter is removed by filtration or the like, and the solution is added to the solution before the dialysis step is started. Examples of the surfactant include (1) anionic surfactants such as alkyl sulfates such as sodium dodecyl sulfate, alkyl sulfates or sulfates such as polyoxyethylene alkyl ether sulfates, and fatty acid alcohols. Acid salt, sulfosuccinate salt or formalin condensate of naphthalene sulfonic acid, etc. (2) amphoteric surfactant, betaine surfactant, etc., (3) nonionic surfactant, polyoxyethylene alkyl ether (4) A quaternary ammonium salt can be used as the cationic surfactant, such as a type, a fatty acid ester type, a polyethylene imine type, a polyglycerin ether type, an ester type and the like. Of these, anionic surfactants are particularly preferred. Addition of the surfactant prevents turbidity and precipitation from occurring during subsequent removal of the reducing agent by dialysis or the like, and makes it possible to obtain a desalted and purified keratin solution.

The amount of the surfactant to be added may vary depending on the concentration of the keratin solution and the type of the keratin-containing substance used as a raw material, and is not necessarily limited, but is usually 0.01 to 5% by weight, preferably 0.1 to 5% by weight. ~ 2% by weight
It is.

The removal of the reducing agent is carried out by an appropriate means such as dialysis, electrodialysis, ultrafiltration or the like, and can be carried out until the excess surfactant is removed. The external dialysis solution can be, for example, ion-exchanged water. If a small amount of a reducing agent (a reducing agent capable of preventing the conversion of a thiol group into a disulfide bond) (for example, 0.1 to 0.5% in the case of 2-mercaptoethanol) is added to the outer dialysis solution, It is possible to prevent keratin chains from being recombined due to reoxidation of thiol groups of keratin chains. Therefore, when coexistence of oxygen and other oxidizing agents is considered during dialysis, it is usually preferable to add a small amount of a reducing agent.

The aqueous solution of keratin obtained by desalting and purification is used as it is, or after appropriately adjusting the concentration by ultrafiltration or the like, or once stored as a dry product by freeze-drying or the like until use, and then dissolved again in water. Can be used in the subsequent microencapsulation stage. The water-soluble keratin is water-soluble even after being dried by freeze-drying or the like, and has 1 to 5 cysteines per 100 amino acid residues,
Contains 0.5 to 3 cystine, average molecular weight 30,000
To 70000.

If the surfactant used in the above is added at the time of reduction solubilization instead of after the reduction solubilization, the reduction solubilization is promoted to increase both the yield and the extraction rate. Was found. Therefore, in order to perform reduction solubilization more efficiently, it is more preferable to perform reduction solubilization in the presence of a surfactant. Further, as the surfactant used for this, an anionic surfactant such as sodium dodecyl sulfate is particularly preferable. Such methods typically increase the yield by about 10 to 20% and increase the extraction rate by about 20 to 70% compared to adding the surfactant after reductive solubilization.

In this case, among the above-mentioned liquid media, it is particularly preferable to use, for example, an aqueous solvent, for example, water or a mixture of water and a water-miscible organic solvent such as methanol and ethanol. In the reduction solubilization in the presence of a surfactant, since the reaction is sufficiently promoted, the reduction solubilization is usually performed without particularly adjusting the pH of the reaction solution to the alkaline side. Other reaction conditions may be the same as in the case where the surfactant is added after reduction solubilization.

Water-soluble keratin obtained by reduction solubilization in the presence of a surfactant is also analyzed, and cysteine is usually found to be 4 to 1 per 100 amino acid residues by amino acid analysis.
0, usually 0.5 to 2 cystine, and electrophoretic analysis confirmed that the protein had a molecular weight of 15,000 to 130,000 as a main component.

The reduction solubilization reaction can be performed under ultrasonic irradiation. Ultrasonic irradiation can further increase the keratin yield (eg, from 45% to 55% in cow horn) and increase the keratin yield in the reductive solubilization reaction in the presence of a surfactant. It has the effect of shortening the reaction time required to obtain it (for example, from 24 hours to 8 hours for cow horn). Therefore, it is more advantageous to carry out the reduction solubilization under ultrasonic irradiation. Ultrasonic irradiation can be performed using an appropriate ultrasonic irradiation device, and the output can be set as appropriate. For example, the output can be 50 to 200 W per 1 L of the reaction solution.

Hereinafter, an example of producing water-soluble keratin will be described. [Water-soluble keratin production example 1] 20 g of wool (charcoal noyl)
With 0.8M aqueous potassium thioglycolate solution (pH 1
0.5) It was immersed in 300 mL and stirred at 5 ° C. for 36 hours. The insolubles were removed from the reaction product by filtration, and diluted to 600 mL with ion-exchanged water. To this solution, 10 g of sodium dodecyl sulfate (SDS) was added and dissolved, and the solution was placed in a cellophane tube and dialyzed twice against ion-exchanged water (10 L) to obtain a colorless and transparent keratin aqueous solution (650 mL).

When the protein content of this solution was determined by the Lowry method, the keratin concentration was 1.2%. Amino acid analysis of keratin powder obtained by freeze-drying the aqueous solution revealed that cysteine was 3.3 and cystine was 1.2 per 100 amino acid residues. According to the polyacrylamide-SDS electrophoresis,
A protein having a molecular weight of 30,000 to 70000 was the main component.

[Production Example 2 of Water-Soluble Keratin] 10 g of defatted wool (Merino sp.), 6.0 g of sodium dodecyl sulfate, 16 g of sodium bisulfite and 300 mol of urea 300 mol
mL of the mixture was sealed and treated with a bath-type ultrasonic device at 50 to 55 ° C. for 1 hour. The insolubles were removed by filtration, and the filtrate was placed in a cellophane tube, and dialyzed against a 0.2% by weight aqueous sodium hydrogen sulfite solution (3 L) as an external solution. About 330 mL of a colorless and transparent aqueous solution obtained by removing a small amount of insoluble matter from the dialysate by centrifugation contained 1.4% by weight of keratin (by protein analysis by the Lowry method). According to amino acid analysis, this keratin has 7.6 cysteines and 0.8 cystine per 100 amino acid residues.
According to DS electrophoresis, the molecular weight was about 40,000 and 600.
00 proteins (30 to 40%, 50 to 60% respectively)
Was the main component.

[II. Method for producing microcapsules of the present invention and characteristics of obtained microcapsules] Method for producing microcapsules using recycled natural keratin as a wall material using water-soluble keratin obtained above, and characteristics of microcapsules obtained thereby Will be described below. The keratin aqueous solution obtained above can be used as it is, or the concentration can be appropriately adjusted by ultrafiltration or the like, or once dried by freeze-drying or the like, and used again as an aqueous solution in the following steps.

The water-soluble keratin obtained above is
Since the peptide has not been subjected to a peptide chain cleavage treatment with a protease or the like, the ability to form a membrane is significantly higher than that of a keratin hydrolyzate (see Test Example 1), which is extremely advantageous for efficient production of microcapsules. is there. Moreover, the microcapsules produced from the water-soluble keratin obtained as described above have much better stability than the microcapsules produced from the keratin hydrolyzate (see Comparative Example 1).

In order to produce microcapsules from the water-soluble keratin obtained as described above, for example, any of various methods known as a method for producing microcapsules, such as a phase separation method, a spray coagulation granulation method, and the like. Can be used, and any method can easily produce a microcapsule that is stable and is preferable in terms of biocompatibility. In particular, when the purpose is to manufacture microcapsules having a special feature of having a fine and uniform particle size, an extremely thin capsule wall, and high stability, the ultrasonic method described below is extremely difficult. This is particularly preferable because it can easily achieve this object.

[1. Outline of each preferred method] (1)
Out of the various methods for producing microcapsules of the present invention, preferred ones are outlined in (4) to (4).

(1) Ultrasonic irradiation method: A mixture of an aqueous keratin solution and an organic solvent or the like insoluble or hardly soluble in water (for example, an organic solvent such as toluene or hexane or an oily drug) (aqueous keratin solution / organic solvent, etc.) Varies depending on the production purpose of the microcapsule, but is usually 0.1%.
To 10) in a temperature range of 0 ° C. to 50 ° C., for example.
For example, ultrasonic irradiation is performed for 10 seconds to 10 minutes. Of the amino acid residues of keratin, the mercapto group of the cysteine residue is changed to a disulfide bond to form a crosslink between keratin chains.As a result, water-soluble keratin becomes insoluble regenerated natural keratin in water. An extremely thin stable film is formed on the surface of fine particles of a solvent or the like, and microcapsules of a uniform particle size obtained by efficiently confining the solvent or the like as a core substance can be obtained (see Examples 3 and 4). .

(2) Vibration / stirring method: An oxidizing agent capable of oxidizing an SH group such as hydrogen peroxide and sodium periodate into the mixture of the above (1) (aqueous keratin solution and solvent) to convert it into a disulfide bond. After adding, vigorously shake and stir with a vortex mixer or a stirring motor. Microcapsules using regenerated natural keratin as a wall material can be produced by the same simple operation as the ultrasonic method (Example 5).
See).

(3) Modification of the above two methods: The mixture of the above (1) is violently vibrated and stirred by an ultrasonic irradiation device, a vortex mixer, a stirring motor or the like in a gas atmosphere lacking oxidizing ability such as nitrogen gas. Then, an emulsion is prepared, and the oxidizing agent described in the above (2) is added and stirred (see Example 6).

(4) A method in which another wall material component is added: A mixture of an aqueous keratin solution and another protein aqueous solution, or a mixture of a keratin aqueous solution and an aqueous solution of a non-protein compound having an SH group or a disulfide bond is used as a wall material raw material. To produce microcapsules containing regenerated natural keratin as a wall material by the method described in the above (1) to (3) (Examples 7 and 8). The properties of the obtained microcapsules can be changed according to other components to be mixed.

In the above (1) to (4), if a substance in which a substance such as a dye, a fragrance, a drug or the like is dissolved in an organic solvent or the like in advance is used, these are efficiently contained in a microcapsule as a core substance. (Examples 8 and 9).

[2. Known components] Hereinafter, known components used for producing the microcapsules of the present invention will be described in detail. (I) Keratin-containing aqueous solution: The following keratin aqueous solution (ia) alone, a mixture of the keratin aqueous solution (ia) and a substance described in the following (ib) or (ic), or It is a mixture of the aqueous keratin solution (ia) and (ib) and (ic).

(Ia) Keratin aqueous solution: An aqueous solution of keratin produced by the above-mentioned method using keratin such as wool, human hair, chicken wings, dog hair, and cow horn as a keratin raw material.

(Ib) Other proteins or peptides mixed with the aqueous keratin solution: collagen, gelatin,
Proteins having a mercapto group or a disulfide bond, such as fibrinogen, silk, egg white lysozyme, and insulin; glycyl-glycyl-cysteine (Gly-Gly-Cy)
s) and (glycyl-glycyl-cystine) ₂ [(Gly-Gly-C
yt) ₂ ]. Or those in which all or a part of a plurality of disulfide bonds present in these are reduced to a mercapto group.

(Ic) Non-protein having a mercapto group or a disulfide group: a polymer such as a polyvinyl alcohol carrying an SH group (for example, 1 to 20 SH groups with an average molecular weight of 2,000) Aqueous solution, glutathione, 2-mercaptoethanol and the like.

(Ii) Organic solvent and the like: As the organic solvent used in the present invention, hydrocarbon solvents such as toluene, xylene, hexane, decane and cyclohexane, which are hardly soluble in water, are most preferable, and ethers such as diethyl ether are preferable. Halogen-containing hydrocarbons such as a mold solvent, fluorocyclohexane and Freon 113 can also be used. However, the solvent is not limited to these, and any solvent having low solubility in water can be used. In addition to the solvent, other liquid substances insoluble or hardly soluble in water, such as vitamin E acetate and other oily drugs, can be used.

(Iii) Oxidizing agent: When using an oxidizing agent, a mercapto group such as air, oxygen, hydrogen peroxide, sodium periodate, sodium perbromate, ammonium persulfate and potassium iodate is bonded to a disulfide bond. It is preferable to use a material that can be oxidized. Further, together with these oxidizing agents, for example, iron ions can be used in combination as oxidation promoters or catalysts.

[3. Examples of specific method of forming microcapsules] As a method for forming microcapsules, various known microcapsule manufacturing methods can be used as follows, but the particle size is fine and uniform, the capsule wall is extremely thin and The ultrasonic method is particularly preferred in that a microcapsule having high stability can be easily produced.

(3-i) Ultrasonic method: The ultrasonic irradiation device may be any device as long as it can irradiate the sample with ultrasonic waves. To increase the efficiency of microcapsule formation, titanium or the like is used. A probe-type device that generates ultrasonic waves from the tip of a metal probe is preferable. The ultrasonic irradiation conditions are appropriately adjusted according to the components and volumes of the sample, but generally, the total volume of the keratin-containing aqueous solution and the organic solvent is 10 mL
The irradiation may be performed at 30 to 50 W for 10 seconds to 5 minutes.

When a halogen-containing hydrocarbon or the like is used as an organic solvent, the efficiency of microcapsule formation and encapsulation may be reduced. In this case, a small amount of hydrogen peroxide may be added prior to ultrasonic treatment. By adding an oxidizing agent such as the above, it is possible to increase the production efficiency of microcapsules.

Alternatively, an oxidizing agent may be added to a mixture of the resulting emulsion by ultrasonic treatment in an atmosphere of a gas lacking oxidizing ability such as nitrogen gas. When the core substance is easily oxidized, this method has an effect of microencapsulation while preventing oxidation of the core substance, and is particularly useful. The usage fee of the oxidizing agent is generally an amount corresponding to 1 to 6 times the number of oxidizing agent molecules per SH group in the raw material.

Keratin and wall material other than keratin [
The mixing ratio of (ii) and (ic)] can be 1 to 500% by weight based on keratin. For example, collagen, gelatin and fibrinogen have a mixing ratio of 3 to 500% by weight.
0 to 500% by weight, 1 to 100% by weight for silk,
10 to 200% by weight of SH group-supporting polyvinyl alcohol is used. The amount of the organic solvent varies depending on the solubility of the core material, but it is used in an amount of 0.1 to 5 times, usually 0.5 to 2 times the volume of the total amount of the aqueous solution of keratin and the above-mentioned wall material.

(3-ii) Stirring method: The raw material for the wall material is the same as that of the ultrasonic method, but instead of the ultrasonic operation, the raw material is stirred while being vigorously vibrated by a vortex mixer, or vigorously stirred by a stirring motor. Before the treatment, a small amount of oxidizing agent (1 to 6 times the number of oxidizing agent molecules per SH group)
Alternatively, the oxidizing agent may be added to the emulsified mixture formed by stirring, and then the mixture may be stirred gently so that the oxidizing agent is well mixed.

(3-iii) Method of adjusting pH: The organic solvent or the like as a core substance is dispersed in an aqueous keratin solution, and an acid such as citric acid or acetic acid is added thereto to adjust the pH to about 4 to 5. As a result, the water-soluble keratin reaches the isoelectric point, aggregates and deposits around the core substance as a nucleus, and surrounds it to form a microcapsule prototype. Then the air,
By introducing and adding oxygen and other oxidizing agents, mercapto groups of each molecule of water-soluble keratin are oxidized to form a disulfide bond, polymerize and form an insoluble film, and microcapsules are formed.

(3-iv) Spray-drying method: A water-soluble or water-insoluble core substance is dissolved or dispersed in an aqueous keratin solution, sprayed with a spray drier and brought into contact with hot air to evaporate and dry the water. Thereby, microcapsules are formed. The method has the advantage that both water-soluble and water-insoluble substances can be microencapsulated as a core substance, and the capsule wall can be easily insolubilized.

(3-v) Method by forming coacervate: An aqueous solution of a negatively charged polyanion, for example, gum arabic, is added to an aqueous keratin solution adjusted to pH 5 or higher, regardless of the pH, to obtain a diluted aqueous solution. A core material that is hardly soluble or insoluble in water is dispersed therein. By adding an acid such as acetic acid or citric acid to the system to lower the pH, only the charge of the keratin molecule is changed from negative to positive, and the core coacervate of water-soluble keratin and polyanion with the core substance as the core. A film is formed. Subsequently, by appropriately performing an oxidation treatment, the film becomes insoluble in water, and microcapsules are formed. Other examples of polyanions capable of forming such a complex coacervate include sodium alginate, agar, carboxymethylcellulose, polyvinyl methyl ether maleic anhydride copolymer, polyvinyl benzene sulfonic acid, condensates of formalin and naphthalene sulfonic acid, and the like. Examples thereof include a polymer having an acid group in the molecule and a surfactant.

Further, by dispersing a core substance in an aqueous keratin solution and adding an alcohol or an inorganic salt thereto, a simple coacervate or salt coacervate film can be formed with the core substance as a nucleus. The microcapsules are formed by treating and insolubilizing.

[4. Isolation of microcapsules]
The isolation of the microcapsules obtained in -i) to (3-iii) and (3-v) is preferably performed as follows. (4-i) The treatment liquid is directly concentrated or dried (freeze-dried, etc.).

(4-ii) The treatment liquid is centrifuged to separate and fractionate the microcapsules. In this state, the wall material and the oxidizing agent which did not contribute to the production of the microcapsules may remain as impurities outside the microcapsules, and in order to further modify the microcapsules, water or water was added to the microcapsule fraction. Add buffer, stir after centrifugation,
The microcapsules are separated and fractionated again. After repeating this operation several times, the microcapsule dispersion is used as it is, or concentrated or dried (freeze-dried, etc.).

(4-iii) Using a semipermeable membrane such as a cellophane membrane, the treatment liquid is treated with water, a buffer, a fragrance, a dye,
Dialysis is performed on an aqueous solution in which a bioactive drug or the like is dissolved. The solution after dialysis is used as it is, or concentrated or dried (eg, by freeze-drying).

The diameter of the microcapsules obtained as described above varies depending on the type of the keratin-containing aqueous solution, the volume ratio of the organic solvent to the keratin-containing aqueous solution, the method of applying vibration or stirring, and the time, and cannot be specified unconditionally.
When a 1: 1 volume mixture (20 mL) of a 2.5% by weight keratin aqueous solution and toluene is subjected to ultrasonic treatment at room temperature for 3 minutes at 50 W, it may be microspheres mainly composed of 1 to 3 μm. Obtained by a light scattering method, and when the same sample was observed with a transmission electron microscope, the wall thickness was about 0.02 μm.
Was very thin, like a paper balloon.

[0054]

The present invention will be described in more detail with reference to the following examples.
The present invention is not limited to these. (Example 1) Microencapsulation by pH adjustment: 5 g of vitamin E acetate was uniformly dispersed in 500 mL of the keratin aqueous solution (keratin concentration: 1.2% by weight) obtained in Production Example 1, and 5% of the dispersion was stirred. A citric acid aqueous solution was added dropwise, and keratin was aggregated at pH 4 to form a microcapsule prototype around the dispersed vitamin E acetate. This was separated by centrifugation, air oxidized while drying by blowing air, and further dried under reduced pressure to complete microcapsules. The resulting microcapsules were insoluble in water at 20 ° C. regardless of the pH.

(Example 2) Microencapsulation by spray drying: To 500 mL of the keratin aqueous solution (keratin concentration 1.2% by weight) obtained in Production Example 1, 2 g of methylene blue was added and dispersed, and this was sprayed with a spray drier. Then, a water-insoluble keratin film was formed around methylene blue by contacting with hot air to evaporate and dry the water, thereby performing microencapsulation.

Example 3 10 mL of the keratin aqueous solution (keratin concentration 1.2% by weight) obtained in Production Example 1 was placed in a wide-mouth test tube.
And 10 mL of toluene, and the mixture was irradiated with ultrasonic waves at 25 ° C. for 3 minutes at an output of 50 W while the mixture was indirectly stirred with a magnet bar. The resulting white suspension was centrifuged at 3000 rpm for 15 minutes to separate a cloudy solid substance,
0 mL), stirred, and then centrifuged in the same manner. After repeating the same washing operation twice more, it was freeze-dried. The obtained white powdery substance (about 0.10 g) was a relatively uniform microcapsule according to observation with a transmission electron microscope, and had a wall thickness of about 0.02 μm and a diameter of 1.2 to 1.5 μm. . The light scattering measurement of the white powdery substance showed almost the same diameter distribution.

Comparative Example 1 A keratin hydrolyzate (average molecular weight 2200) according to the method described in JP-B-59-33017 was used in the same manner as in Example 3 in place of the keratin aqueous solution obtained in Production Example 1. Tried microencapsulation. However, the particulate matter obtained from the water-soluble keratin hydrolyzate was extremely fragile, and the dispersion in water disintegrated only by standing at room temperature to release the internal toluene.

(Example 4) Aqueous keratin solution obtained in Production Example 2 (keratin concentration: 1.4% by weight) (10 m
L) and toluene (10 mL) were added, and the mixture was irradiated with ultrasonic waves at 25 ° C. for 3 minutes at an output of 50 W while the mixture was indirectly stirred with a magnet bar. The resulting white suspension was added to 300
Centrifuge at 0 rpm for 15 minutes to separate cloudy solids,
Water (20 mL) was added, and after stirring, the mixture was centrifuged in the same manner. After the same washing operation was further repeated twice, it was freeze-dried. According to transmission electron microscope observation, the obtained white powdery substance (about 0.11 g) is a relatively uniform microcapsule, the wall thickness is about 0.02 μm, and the diameter is 1.2 to 1.5 μm.
m. The light scattering measurement of the white powdery substance showed almost the same diameter distribution. Amino acid analysis of keratin powder obtained by freeze-drying a keratin aqueous solution as a raw material revealed that
The cysteine content was about 8 in the microcapsules, although 7.5 cysteines and 0.9 cystine were present per residue.
Had increased to pieces. Other amino acid residues almost coincided with the values in the raw material.

(Example 5) Aqueous keratin solution obtained in Production Example 2 (keratin concentration: 1.4% by weight) (10 m
L), 30% aqueous hydrogen peroxide (0.05 mL) and toluene (10 mL) were added, and the mixture was vigorously stirred at 25 ° C. for 5 minutes with a vortex mixer. The resulting white suspension is 200
Centrifuge at 0 rpm for 15 minutes to separate cloudy solids,
Water (20 mL) was added, and after stirring, the mixture was centrifuged in the same manner. After the same washing operation was further repeated twice, it was freeze-dried. According to transmission electron microscopic observation of the obtained white powdery substance (about 0.11 g), the diameter of the resulting microcapsules is 3 to 10 μm, although it varies slightly.

(Example 6) The keratin aqueous solution obtained in Production Example 2 (keratin concentration: 1.4% by weight) (10 m
L) and toluene (10 mL) were added, and ultrasonic treatment was performed at 25 ° C. for 5 minutes in a nitrogen gas atmosphere. A 30% aqueous hydrogen peroxide solution (0.07 mL) was added to the resulting white suspension, and the mixture was shaken gently and allowed to stand for 15 minutes. Then, at 2000 revolutions / minute,
Centrifuge for 5 minutes to separate the cloudy solid, and add water (20 mL)
, And the mixture was centrifuged in the same manner after stirring. After the same washing operation was further repeated twice, it was freeze-dried immediately. According to transmission electron microscopic observation of the obtained white powdery substance (about 0.11 g), the diameter of the resulting microcapsules is 2 to 5 μm, although it varies slightly.

Example 7 Collagen (type I) (0.5%, I-PC, manufactured by Koken Co., Ltd.) (10 m
L) was added, and the mixture was made weakly alkaline with 28% aqueous ammonia.
Then, the keratin aqueous solution (keratin concentration: 1.4% by weight) (6 mL) obtained in Production Example 2 was added, and the mixture was shaken at 50 ° C. for 10 to 15 minutes. After the initially gelled mixture turned into a fluid, toluene (10 mL) was added, and the mixture was irradiated with ultrasonic waves at 25 ° C. for 3 minutes at a power of 35 W while indirectly stirring the mixture with a magnet bar. The resulting white suspension was
Centrifuge at 00 rpm for 15 minutes to separate white solids,
Water (20 mL) was added, and after stirring, the mixture was further centrifuged. After repeating the same washing operation twice more, the white suspension was freeze-dried. According to transmission electron microscopic observation, the obtained white powdery substance (about 0.10 g) was a relatively uniform microcapsule, the wall thickness was about 0.02 μm, and the diameter was 1.5 to 3 μm.
μm. The light scattering measurement of this white powdery substance showed almost the same diameter distribution.

Example 8 Polyvinyl alcohol carrying a mercapto group (molecular weight: 2,000, SH
A 2.5% aqueous solution (3 mL) of 1 to 3 groups) was added, and then the keratin aqueous solution obtained in Production Example 2 (keratin concentration 1.
(4% by weight) (20 mL), and the mixture was sufficiently shaken with stirring.
Next, 2% by weight of silicone oil (ALDRICH) was dissolved in 1
-Fluorocyclohexane (10 mL) and the mixture was stirred at 25 ° C. for 5
Ultrasonic treatment was performed at 0 W power for 3 minutes. The resulting white suspension was centrifuged at 2000 times / min for 15 minutes to separate a cloudy solid, which was dispersed and stored in pure water (5 mL). The dispersion was freeze-dried to obtain about 0.28 g of powder. The dispersion contained relatively uniform particles according to electron microscopic observation, and showed a main diameter distribution of 2 to 8 μm according to light scattering measurement. About 1 organic solvent layer separated by the centrifugation
It is clear that silicon is included in the microcapsules because the silicon amount of the residue obtained by the concentration is 5 to 20% of the used amount, in addition to the small amount of 2 to 2 mL.

Example 9 Microcapsules were obtained in exactly the same manner as in Example 4 except that a toluene solution (10 mL) of 3% by weight of vitamin K was used instead of toluene (0.30 g). UV absorption spectrum measurement of the ethanol dispersion of the microcapsules revealed that 70% of the vitamin K used was contained in the microcapsules.

Test Example 1 Amount of Keratin Peeled from Keratin Film: The aqueous solution of keratin obtained by the method of Production Example 2 and the water-soluble keratin hydrolyzate obtained by the method described in JP-B-59-33017 were applied to the glass surface. Keratin thin film (thickness: about 100 μm) was prepared by using the concentration (average molecular weight: 2200). This is immersed in Tris-HCl buffer pH 7.4,
Shake at 40 ° C. A certain amount of the aqueous solution was collected, the amount of dissolved protein was quantified by the Lowry method, and the detached amount was produced.
Table 1 shows the results.

[0065]

[Table 1]

It was also attempted to prepare a keratin thin film using a water-soluble keratin hydrolyzate having an average molecular weight of 15,000 by the method described in JP-B-59-33017, but it was difficult to form a keratin thin film.

[0067]

As described above, according to the present invention, a microcapsule having natural keratin as a wall material, having a small wall thickness and high stability, and having an extremely fine and uniform particle size, can be easily obtained. Can be manufactured. Further, the microcapsules of the present invention are significantly superior in microcapsule production efficiency, stability, and the like, as compared with microcapsules of a known technique using keratin hydrolyzate as a wall material. Furthermore, the microcapsules of the present invention are made of a regenerated natural keratin obtained by once separating a cross-link between natural keratin chains and forming the same again as a wall material, and irreversible such as cleavage of the keratin chains themselves. Since it does not involve chemical modification, it is preferable in terms of biocompatibility, and can be used in various fields as described above.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int.Cl. ⁷ , DB name) B01J 13/02 A61K 9 / 50,47 / 42

Claims (8)

(57) [Claims] 1. A microcapsule comprising recycled natural keratin as a wall material. 2. The regenerated natural keratin is obtained by insolubilizing water-soluble keratin obtained by treating a keratin-containing substance with a reducing agent in a liquid medium to extract keratin, in the form of a wall of a microcapsule. The microcapsule according to claim 1, which is: 3. A microcapsule having a wall material comprising a water-soluble keratin obtained by treating a keratin-containing substance with a reducing agent in a liquid medium to extract keratin, and a protein or polypeptide having a mercapto group or a disulfide bond. And / or a mixture of polyvinyl alcohol carrying a mercapto group or a disulfide bond, and the mixture is insolubilized in the form of a wall of the microcapsule. 4. A method of treating a keratin-containing substance with a reducing agent in a liquid medium to extract keratin, thereby insolubilizing water-soluble keratin obtained by coating the substance around the core substance as a wall material. A method for producing microcapsules using recycled natural keratin as a wall material. 5. A water-soluble keratin obtained by extracting a keratin by treating a keratin-containing substance with a reducing agent in a liquid medium, a protein or polypeptide having a mercapto group or a disulfide bond, and / or a mercapto group or a disulfide. A method for producing microcapsules, comprising coating a mixture with polyvinyl alcohol carrying a bond around a core material as a wall material and insolubilizing the mixture. 6. The method according to claim 4, wherein the aqueous solution containing the wall material is mixed with a core material which is a water-insoluble or hardly-soluble liquid material, and this is subjected to ultrasonic treatment and / or vigorous stirring. Or the production method according to 5. 7. The method according to claim 6, wherein an oxidizing agent capable of converting a thiol group into a disulfide bond is added and stirred before the ultrasonic treatment and / or vigorous stirring. 8. The method according to claim 1, wherein the sonication and / or vigorous stirring is performed in a gas atmosphere lacking oxidizing ability, and then an oxidizing agent capable of converting a thiol group into a disulfide bond is added and stirred. The method according to claim 6.