JPH04189832A - Production of aqueous micellar solution of keratin - Google Patents

Abstract

PURPOSE:To remove a solubilizing agent without causing insolubilization and obtain the subject aqueous solution, useful as keratinous polymer membranes, films, fibers, sponges, etc., by reducing a keratin-containing substance in the presence of a surfactant in an aqueous medium and then dialyzing the resultant micellar aqueous solution. CONSTITUTION:A keratin-containing substance (e.g. human hair, animal hair or feather) is reduced in the presence of a surfactant (preferably an anionic surfactant such as sodium dodecylbenzene sulfate) in an aqueous medium preferably while being irradiated with ultrasonic waves and the resultant micellar aqueous solution is dialyzed to afford the objective aqueous solution.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for producing a micellar aqueous solution of keratin without irreversible denaturation of nosulfide bonds. The micellar aqueous solution obtained by the method of the present invention is used in the production of products for various industries such as keratin polymer membranes, films, fibers, and spononons.

``Prior art: Keratin, which exists as a structural protein in animal tissues such as hair, animal fur, and feathers, has long attracted attention as a raw material for industrial materials such as membranes and fibers.

However, keratin is insoluble or sparingly soluble in common solvents. In order to utilize keratin from natural raw materials, it is necessary to significantly shorten the molecular weight by hydrolysis, reduce the sulfite bonds of keratin, or irreversibly protect the generated thiol groups by chemical treatment. . In other words, the nosulfide bonds of keratin are reduced using a hydrolyzate 1 reducing agent produced by treating natural products containing keratin with alkali and a protein denaturing agent such as a highly concentrated aqueous urea solution. A keratin aqueous solution that has been cleaved to form a thiol group, a keratin derivative irreversibly chemically modified with monoiodoacetic acid, sodium sulfite/sodium tetrathionate, etc. to prevent the recombination of the thiol group of the keratin, or a keratin derivative that has been chemically modified irreversibly by reductive cleavage and proteolytic enzymes. It is used in the form of an aqueous solution of keratin with a reduced molecular weight.

[Problems to be Solved by the Invention] However, in order to restore keratin from protein obtained by oxidation treatment, complicated chemical treatment is required and the recovery yield is extremely low. On the other hand, in the reduction treatment, keratin is removed under alkaline conditions in a solvent such as methanol, ethanol, or amide, whose main component is water, in the presence of a solubilizing agent consisting of a reducing agent such as thiol and a protein denaturing agent such as urea. is reduced and solubilized, and the solubilizing agent is removed by dialysis, ultrafiltration, etc. It has also been proposed to add a surfactant to the keratin solution after the first reduction treatment to prevent keratin from becoming insolubilized during dialysis (Japanese Patent Laid-Open No. 63-30180
No. 9). This type of reduction treatment is a simpler method for preparing keratin than oxidation treatment, or when hard keratin-containing materials such as hair, wool, and horn are used as raw materials, the extraction rate of keratin is slow and the keratin recovery rate is low. low.

Furthermore, a method has also been proposed in which a metal salt is added to a liquid obtained by reducing a keratin-containing substance in an aqueous or organic medium to separate a modified keratin substance as a precipitate (Japanese Patent Laid-Open No.
3-23999). However, this method relates to the production of modified keratin materials with heavy metal adsorption capacity used in the treatment of industrial wastewater, and does not include the production of keratin micellar aqueous solutions and the improvement of the extraction rate of keratin (1). Not yet.

[First Means to Solve the Problems] Therefore, the present inventor conducted extensive studies on a method for efficiently extracting keratin from natural products containing keratin. As a result, keratin can be efficiently extracted by stirring and heating a keratin-containing substance in water containing a reducing agent, a protein denaturing agent, and a surfactant, followed by dialysis treatment, resulting in a keratin micellar aqueous solution. We obtained the knowledge that this can be obtained.

That is, the present invention provides a method for producing a keratin micellar aqueous solution, which comprises reducing a keratin-containing substance in an aqueous medium in the presence of a surfactant, and dialyzing the obtained micellar aqueous solution.

First, the present invention provides a method for producing a keratin micellar aqueous solution, which comprises reducing a keratin-containing substance in an aqueous medium in the presence of a surfactant and under ultrasonic irradiation. In addition, a keratin solution can be easily obtained by stirring and heating nickeratin powder obtained by a conventional method and dissolving it together with the reducing agent in the aqueous medium.

In the production method of the present invention, first, a keratin-containing substance is reduced in an aqueous medium in the presence of a surfactant.

For this reduction, a reducing agent that reduces the nosulfide bonds of keratin present in the keratin-containing material to thiol groups is generally used. Further, this reaction is usually carried out by adding a protein denaturing agent.

The keratin-containing substance may be any material as long as it contains true keratin; for example, human hair, sheep, horse or cow hair, chicken and bird feathers are preferably used, but nails, cow horns and hooves can also be used. It will be done. Keratin powder can also be prepared using various known methods (
For example, T, T, Sun and H, Green,
J. Biol. Chem., 253.2053-2
060 (1978)) is used.

The aqueous medium may be water alone or a mixture of water and a water-miscible organic solvent, and a solvent having a water content of 50% by weight or more, preferably 80% by weight or more is used. Examples of water-miscible organic solvents include lower aliphatic alcohols such as methanol and ethanol.

In addition, the reducing agents include thiols such as 2-mercaptoethanol, thioglycolic acid, toluene-ω-thiol, notothiothreitol, and dithioerythritol; trialkylphosphines such as nitripropylphosphino and tributylphosphine; and sodium bisulfite. Examples include inorganic reducing compounds such as.

The amount of reducing agent used is 0 per 10g of keratin-containing material.
．． 05 to 0.50 mole, but from the viewpoint of reaction efficiency and economy, it is 0.05 to 0.2 per 10 g of keratin-containing material.
Preferably, 0 mol is used.

The above-mentioned surfactants include anionic surfactants such as alkyl sulfates (for example, sodium dobnol sulfate), alkyl sulfate ester salts, fatty acid alcohol phosphate ester salts, and sulfosuccinate ester salts: where R1, R2, R3, and R4 One or two of them are straight chain or branched alkyl groups having 8 to 20 carbon atoms or hydroquine alkyl groups, and the remainder are alkyl groups having 1 to 3 carbon atoms or hydroquine alkyl groups. A cationic surfactant represented by a benol group, X is a halogen atom, an alkyl sulfate group having 1 to 2 carbon atoms, or an aromatic quaternary amine salt such as an alkyl pyridium halide;
- Betaine-based amphoteric surfactants such as carboquine methyl derivatives, N-sulfoalkylated products, and imidacillin sulfonic acid (hydrophobic groups are mainly alkyl groups or anlu groups having 12 to 14 carbon atoms, counter ions are alkali metals, etc.) ); polyokine ethylene alkyl ether type, fatty acid ester type,
Nonionic surfactants such as polyethyleneimine type, polyglycerin ether type, and ester type (hydrophobic group is mainly an alkyl group having 12 to 14 carbon atoms, such as an anol group):
Nato is mentioned. Among these, anionic surfactants are particularly preferred.

The amount of these surfactants added is 10 to 10% of the amount of keratin-containing substances.
500-50% by weight or 10-25% by weight.

The protein denaturing agent is one that breaks hydrogen bonds in keratin, and specific examples thereof include urea and thiourea. When using hard keratin-containing materials, use alkalis such as sodium hydroxide or ammonia, or inorganic salts such as zinc chloride, sodium iodide, or lithium bromide, to help dissolve proteins. Preferably, the amount of the protein denaturing agent to be used as a keratin-containing agent is determined as appropriate considering the solubility of the keratin-containing substance or the efficiency of the dialysis treatment in the next step, or it is usually 20 times by weight, preferably 5
~15 times the weight. For example, when using urea, the amount is usually 3 to 15 times by weight, preferably 5 to 12 times by weight.

In the production method of the present invention, the specific operation of the reduction step is carried out, for example, as follows. That is, the keratin-containing material is immersed in an aqueous solution of a protein denaturant, usually 4 to 8 mol/Q, such as 5 to 8 mol/ρ in the case of urea, so that the entire amount of the keratin-containing material is immersed, and a reducing agent and a surfactant are added. After that, the container is tightly stoppered and stirred at room temperature to 100°C for 1 to 24 hours.

In the above reduction step, the reaction system can also be irradiated with ultrasonic waves. For ultrasonic irradiation, a known ultrasonic irradiation device such as a probe type or a bathtub type can be used. The intensity of ultrasonic irradiation varies depending on the size of the reaction system; for example, when the size of the reaction system is 1 a or less, an output of 50 to 200 W is sufficient. The reduction reaction can be promoted by ultrasonic irradiation, and an aqueous keratin micelle solution can be obtained in a shorter time and with higher yield.

The reaction solution thus obtained is subjected to the next dialysis treatment after removing insoluble matter by centrifugation or filtration.

Dialysis treatment can be performed by conventionally known treatment means. For example, the reaction solution, ie, the keratin filtrate after removing insoluble matter as described above, is placed in a container made of a semipermeable membrane such as cellophane, and this is immersed in a container containing an external solution. As the external liquid, an aqueous medium containing 0.1 to 0.5% of a reducing agent capable of reducing disulfide bonds to thiol groups can be used.For example, a mixture of an aqueous medium and a reducing agent used in the above reduction step can be used. Can be used. The volume of the external solution is usually 20 to 40 times that of the keratin filtrate. The temperature may be room temperature, and the time is usually 12 to 36 hours. By performing such dialysis treatment 2 to 4 times, the protein denaturing agent and surfactant in the keratin filtrate can be removed, and the reducing agent can be reduced to the same concentration as the external solution.
An aqueous micellar solution of keratin is obtained.

The keratin micelle aqueous solution obtained in this way was
When calibrated using WRY's protein assay method, it is approximately 2 to 4% by weight, depending on the keratin-containing material used as the raw material.

In addition, according to amino acid analysis, /staino and 7-stine in the keratin vary depending on the type of keratin-containing a substance, but are generally 4 to 1 each per 100 amino acid residues.
0 pieces, 05 to 2 pieces. However, electrophoretic analysis reveals that the main component is protein with a molecular weight of 15,000 to 130,001].

The mechanism of keratin solubilization is that insoluble keratin is converted into thiol groups by reducing nosulfide bonds while increasing its affinity with water molecules through hydrogen bond interaction with high concentrations of protein denaturing agents such as urea. Possibly. However, the reduction step (keratin extraction step) of the present invention
In the absence of conventional surfactants (for example, JP-A-6
3-301809)), the yield of bekeratin increases by 10 to 20% (see Tables 1 and 2 below), and the extraction rate also increases significantly by 20 to 70% (see Table 2 below).

In addition, in the present invention, keratin can be efficiently extracted in a short time without sufficiently converting the nosulfide bonds of keratin into thiol groups by reduction.Secondly, the obtained keratin contains a large amount of high molecular weight substances. It can be done. For example, when hair is used as a keratin-containing substance, the keratin extraction rate reaches 80% or more in 5 hours at 50°C, and the molecular weight of the extracted keratin is determined by polyacrylamide electrophoresis. From this molecular weight 45000 to +20000.1200
0 to 45,000 were about 40% and 60% of the total weight, respectively. On the other hand, even under the same conditions, keratin extracted from hair in the absence of a surfactant has a molecular weight of about 450.
Those with a molecular weight of 0.0 or less account for about 70% of the total weight, and those with a higher molecular weight account for 30%.

When the above-mentioned reaction solution of the present invention was examined by a light scattering method, a dispersion mainly having an associated molecular weight of 25,000 to 60,000 was observed. Since such aggregates were not observed in conventional reaction solutions in the absence of surfactants, they are thought to be caused by surfactants, and are probably caused by surfactants entering the highly hydrophobic VeptiF site of keratin. It seems reasonable to think that stabilized micelles are formed. That is, it seems that the nosulfite bonds were reduced and converted to thiol groups, and the keratin was efficiently extracted as a micellar dispersion of keratin by incorporating the coexisting surfactant. In fact, according to nuclear magnetic resonance spectroscopy and protein analysis, the dialyzed aqueous keratin solution is mainly composed of keratin protein and the surfactant. It can be seen from Table 1 below that such an extraction method, the so-called micelle extraction method, is particularly effective for removing keratin from hard keratin-containing materials such as nails and hooves.

In addition, the keratin aqueous solution obtained by ultrasonic irradiation in the present invention has an amino acid composition and a protein composition that are essentially between those of the keratin aqueous solution obtained without ultrasonic irradiation if the keratin-containing raw materials are the same. This was suggested by amino acid analysis and polyacrylamide electrophoresis.

The obtained micelle aqueous solution is formed into various high molecular weight membranes, films, fibers, subonnos, etc. by known film forming methods and forming methods.

[Example] Next, the present invention will be explained in more detail based on Examples and Comparative Examples.

Example 1 20g of wool (collected from Cot 1idale species) is 8M
It was immersed in 370 mQ of urea aqueous solution, and 12 g of sodium todenol sulfate and 35 mA of 2-mercaptoethanol were added. Then, the container was tightly stoppered, stirred at 60°C for 8 hours, and then irradiated with ultrasonic waves at an output of 80W. After the reaction mixture was returned to room temperature, insoluble materials were removed by filtration. The filtrate was placed in a cellophane tube and dialyzed twice against 0.3% by weight aqueous 2-mercaptoethanol solution (+00.470 mQ of the resulting colorless and transparent dialysate contained a small amount of 2-mercaptoethanol and the surfactant. This is a micellar aqueous solution of keratin.

Protein determination of this solution by the Lowry method revealed that it contained 0.27 g of keratin, the keratin concentration was 27%, and the keratin yield was 63%.

In addition, amino acid analysis of keratin powder obtained by freeze-drying the aqueous solution revealed that per 100 amino acid residues,
The number was 7.8 for protein and 07 for nostin. In addition, when examined by polyacrylamide electrophoresis, it was found that 70,000 proteins were produced from 15,000 molecules per molecule.

Example 2 20 g of wool was treated in the same manner as in Example I except for one thing, except that the reaction time was 24 hours without ultrasonic irradiation, and a small amount of 2-
A keratin micelle aqueous solution (480 m □) was obtained as a colorless and transparent dialysate containing mercaptoethanol and a surfactant. When this solution was subjected to protein quantification in the same manner as in Example 1, the keratin yield was found to be 2.5% at a keratin concentration of 2.5%. is 60%.In addition, amino acid analysis of the keratin powder obtained by freeze-drying this aqueous solution revealed that, per 100 amino acid residues, there were 7.5 nstins and 069 nsstins. When examined by polyacrylamide electrophoresis, it was found that proteins with molecules fi 15,000 to 70,000 were the main components.

Comparative Example 1 A micelle aqueous solution of keratin was prepared in the same manner as in Example 2 except that no surfactant was added.

The results are shown in Table 1.

Example 3 Wash human hair with a neutral detergent and then with hexane.

5g of this person's hair and 120mf of 8M urea aqueous solution! f: immersed. Add 5 g of sodium todesol sulfate and 8 m of 2-merkabutoethanol to this, seal the container, and heat to 50°C.
The mixture was stirred for 5 to 35 hours. 5. After 1O and 35 hours, each reaction product was collected and the insoluble matter was removed by filtration.The obtained filtrate was put into a cellophane tube and
Dialysis was performed twice against a 3% by weight aqueous 2-mercaptoethanol solution 2Q to obtain the desired micelle aqueous solution of keratin as a colorless and transparent dialysate containing a small amount of 2-mercaptoethanol and a surfactant. The protein content of this solution log was determined by the Lowry method, and the keratin yields shown in Table 2 were obtained. In addition, amino acid analysis of keratin powder obtained by freeze-drying the aqueous solution was conducted, and it was found that amino acids 10
There were 8 nilinosteins and 12 nostins per 0 residues. Furthermore, when polyacrylamide electrophoresis was performed, molecular weights of 45,000 to 120,000 and 12
,000 to 45,000 proteins, which were suggested to be 40% and 60%, respectively.

Comparative Example 2 A keratin micelle aqueous solution was prepared in the same manner as in Example 3 except for the two sides except that no surfactant was added.

The results are shown in Table 2.

Example 4 10g of chicken wings in 60% by weight lithium bromide aqueous solution 180mρ
After adding 5 g of sodium dodenle sulfate and 2-mercaptoethanol IOmC, the container was sealed and irradiated with ultrasonic waves at an output of 80 W while shaking at 45° C. for 2 hours.

The reaction solution from which insoluble matter had been removed by filtration was placed in a cellophane tube and dialyzed twice against a 3% by weight aqueous solution of 2-mercaptoethanol (10%). 230ml (!) of the desired keratin micelle aqueous solution was obtained as a dialysate.The keratin concentration was 3.3% and the keratin yield was 76%.

Amino acid analysis of the keratin powder obtained by freeze-drying the aqueous solution showed the presence of 1 amino acid per +00 residues, 61 nosteine and 12 stino residues. In addition, when examined by polyacrylamide electrophoresis,
Proteins with a molecular weight of 15,000 to 70,000 were produced.

Example 5 An aqueous keratin micelle solution was prepared in the same manner as in Example 4, except that ultrasonic irradiation was not performed and the treatment time was 15 hours. The results are shown in Table 1.

Comparative Example 3 A keratin micellar aqueous solution was prepared in the same manner as in Example 4, except that no surfactant was added and the treatment time was 24 hours. The results are shown in Table 1.

Example 6 10 g of crushed cow horn was immersed in 150 mQ of 8M urea aqueous solution, and 80 g of lithium bromide and 5 g of sodium todenol sulfate were added.
After adding 15 mQ of 2-mercaptoethanol and 2-mercaptoethanol, the container was tightly stoppered and shaken at 60'C for 8 hours.
Ultrasonic waves were irradiated with W. After returning to room temperature, insoluble matter was removed by filtration. 11! Put the solution into a cellophane tube and add O3% by weight 2-mercap)/ethanol solution 10% (
The patient underwent dialysis twice. Obtained f2 colorless and transparent dialysate 1
85 mρ is a micellar aqueous solution of keratin containing a small amount of 2-mercaptoethanol and the surfactant. The keratin concentration of this solution was 30%, and the keratin yield was 55%. The keratin powder obtained by freeze-drying the aqueous solution was analyzed for amino acids, and it was found that there were 45 f-linosteins and 06 linostinoins per 100 amino acid residues. In addition, when examined by polyacrylamide electrophoresis, the molecular weight was 20,000 to 60,000.
The main component is protein.

Example 7 A keratin micelle aqueous solution was prepared in the same manner as in Example 6, except that the treatment time was 24 hours without ultrasonic irradiation. The results are shown in Table 1.

Comparative Example 4 A micellar aqueous solution of 1-nickeratin was prepared in the same manner as in Example 7 except that no surfactant was added.

The results are shown in Table 1.

Table 1) □2z'SDS None 602460 Comparative Example 1 None None 60 24
43 Example 48 Feather SDS Yes 45276/
/ 5 nol SDS no 4
5 15 65 Comparative Example 3 None
None 45 24 51 Example 6 Cow horn SD
S Yes 608557no 7”
SDS None 60 24 45 Comparative Example 4 Nol None None 60 24 2
8 Next, in Example I, Example 2, and Comparative Example 1 (raw material: wool), the reaction times were changed as shown in Table 2 below, and the same operations were carried out except for the reaction time. Test No., 1 to 4, and Test No.
Micelle aqueous solutions of Test No. 1, No. 5-8, and Test No. 9-12 were prepared. In addition, the results of Example 3 (raw material human hair) were tested as No. 1.
3 to 15, and the results of Comparative Example 2 are shown in Test Nos. I6 to 18.

Table 2 SDS:)'Sodium Denor Sulfate As shown in Table 2 and Figure 1 above, the method of the present invention allows a high keratin yield to be obtained efficiently in a short period of time.

[Effects of the Invention j According to the production method of the present invention, keratin can be efficiently extracted in a high yield, and the solubilizing agent can be removed by dialysis without causing insolubilization. Further, since no alkali is particularly required, the operation is easy, and the obtained one-comicelle aqueous solution is highly stable at room temperature.

[Brief explanation of drawings]

FIG. 1 is a graph showing the relationship between the reaction time of the reduction treatment and the keratin yield. Patent applicant: Takeda Pharmaceutical Company Limited

Claims (2)

[Claims] (1) A method for producing a keratin micelle aqueous solution, which comprises reducing a keratin-containing substance in an aqueous medium in the presence of a surfactant and dialyzing the obtained micelle aqueous solution. (2) A method for producing a keratin micellar aqueous solution, which comprises reducing a keratin-containing substance in an aqueous medium in the presence of a surfactant and under ultrasonic irradiation.