JP2946491B2 - Method for producing micellar aqueous solution of keratin - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing a micelle aqueous solution of keratin without irreversible modification of disulfide bonds. The micelle aqueous solution obtained by the method of the present invention is used for manufacturing products of various industries such as keratin polymer membranes, films, fibers, sponges and the like.

[Related Art] Keratin, which is present as a structural protein in animal tissues such as hair, animal hair, and feathers, has been attracting attention as a raw material for industrial materials such as membranes and fibers.

However, keratin is insoluble or hardly soluble in ordinary solvents. Therefore, in order to utilize keratin in a natural raw material, it is necessary to significantly reduce the molecular weight by hydrolysis, or to perform irreversible protection by reducing the disulfide bond of keratin or chemically treating the generated thiol group. That is, a hydrolyzate produced by treating a natural product containing keratin with a concentrated acid or alkali; a reducing agent and a protein denaturing agent such as a high-concentration urea aqueous solution are used in combination to reductively cleave disulfide bonds of keratin to form thiol. Aqueous keratin solution; keratin derivative chemically modified irreversibly with monoiodoacetic acid or sodium sulfite / sodium tetrathionate to prevent recombination of the thiol group of the keratin; or reduced in molecular weight by reductive cleavage and proteolytic enzyme It is used as a form of keratin aqueous solution.

[Problems to be Solved by the Invention] However, in order to recover keratin from a protein obtained by the oxidation treatment, a complicated chemical treatment is required, and the recovery yield is extremely low. On the other hand, in the reduction treatment, keratin is dissolved in a solvent such as methanol, ethanol, or amide containing water as a main component in the presence of a solubilizing agent composed of a reducing agent such as thiol and a protein denaturing agent such as urea under alkaline conditions. After reducing and solubilizing, the solubilizing agent is removed by dialysis, ultrafiltration or the like. It has also been proposed to add a surfactant to a keratin solution after a reduction treatment in order to prevent keratin from being insolubilized during dialysis (JP-A-63-301809).
Such a reduction treatment is a simpler method of preparing keratin than an oxidation treatment.However, when a hard keratin-containing material such as hair, wool or horn is used as a raw material, the extraction rate of keratin is slow and the keratin recovery rate is high. Low.

Furthermore, a method has 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, and the modified keratin substance is separated as a precipitate (Japanese Patent Application Laid-open No. Sho.
53-23999). However, this method relates to the production of a modified keratin substance having a heavy metal adsorption capacity used in the treatment of industrial wastewater, and does not disclose production of a micellar aqueous solution of keratin or improvement of the extraction rate of keratin.

[Means for Solving the Problems] Accordingly, the present inventors have intensively studied a method for efficiently extracting keratin from a natural product containing keratin. As a result, the keratin-containing substance is stirred and heated in water containing a surfactant in addition to the reducing agent and the protein denaturing agent, and then is subjected to dialysis, whereby keratin can be efficiently extracted, and a micellar aqueous solution of keratin can be obtained. Was obtained.

That is, the present invention provides a keratin-containing substance in an aqueous medium,
It is intended to provide a method for producing a micellar aqueous solution of keratin, comprising reducing in the presence of a surfactant and dialyzing the resulting micelle aqueous solution. The present invention also provides a method for producing a micelle aqueous solution of keratin, which comprises reducing a keratin-containing substance in an aqueous medium in the presence of a surfactant under ultrasonic irradiation. In addition, the keratin powder itself obtained by the conventional method can be dissolved in the aqueous medium by stirring and heating together with the reducing agent to easily obtain a keratin solution.

In the production method of the present invention, a keratin-containing substance is first reduced in an aqueous medium in the presence of a surfactant. For this reduction, a reducing agent that reduces the disulfide bond of keratin present in the keratin-containing substance to a thiol group is generally used. This reaction is usually performed by adding a protein denaturing agent.

The keratin-containing substance may be a substance containing intrinsic keratin, and for example, human hair, sheep, animal hair of horses and cows, feathers of birds such as chickens are preferably used, but nails, horns and hooves of cows are also used. . Keratin powder can be prepared by various known methods (for example, TT Sun and H. Green, J. Biol. Chem., 253, 205).
3-2060 (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 the water-miscible organic solvent include lower fatty alcohols such as methanol and ethanol.

Examples of the reducing agent include thiols such as 2-mercaptoethanol, thioglycolic acid, toluene-ω-thiol, dithiothreitol, and dithioerythritol; trialkylphosphines such as tripropylphosphine and tributylphosphine; sodium hydrogen sulfite; And the like.

The amount of the reducing agent used is 0.0 per 10 g of the keratin-containing substance.
Although it is 5 to 0.50 mol, it is preferable to use 0.05 to 0.20 mol per 10 g of the keratin-containing substance from the viewpoint of reaction efficiency and economy.

Examples of the surfactant include anionic surfactants such as alkyl sulfates (for example, sodium dodecyl sulfate), alkyl sulfates, fatty acid alcohol phosphates, and sulfosuccinates; [Wherein, ¹ to ^{2 of} R ¹ , R ² , R ³ and R ⁴ are a linear or branched alkyl group having 8 to 20 carbon atoms or a hydroxyalkyl group, and the rest is 1 to 3 carbon atoms. Wherein X is a halogen atom, an alkyl sulfate group having 1 to 2 carbon atoms or an aromatic quaternary amine salt such as an alkylpyridium halide, etc.] A betaine-based amphoteric surfactant such as an N-carboxymethyl compound, N-sulfoalkylated product of fatty acid amine, or imidazoline sulfonic acid (a hydrophobic group is mainly an alkyl group or an acyl group having 12 to 14 carbon atoms, and a counter ion is an alkali. Metal, etc.); polyoxyethylene alkyl ether type, fatty acid ester type, polyethylene imine type, polyglycerin ether type, ester Nonionic surfactants such as (hydrophobic groups are primarily alkyl or acyl group of 12 to 14 carbon atoms); and the like. Of these, anionic surfactants are particularly preferred. The addition amount of these surfactants is 10 to 50% by weight, preferably 10 to 25% by weight of the keratin-containing substance.

The protein denaturing agent breaks a hydrogen bond in keratin, and specific examples thereof include urea and thiourea. When using a keratin-containing substance having a hard tissue, an alkali such as sodium hydroxide or ammonia having a dissolving effect on proteins or an inorganic salt such as zinc chloride, sodium iodide, or lithium bromide is used as a dissolution aid. Is preferred. The amount of the protein denaturing agent used is appropriately determined in consideration of the solubility of the keratin-containing substance or the efficiency of the dialysis treatment in the next step, but is usually 3 to 20 times by weight, preferably 5 to 5 times the keratin-containing substance.
15 weight times. For example, when urea is used, usually 3 to 15
It is by weight, preferably 5 to 12 times by weight.

In the production method of the present invention, a specific operation of the reduction step is performed, for example, as follows. That is, the keratin-containing substance is usually immersed in an aqueous solution of a protein denaturant of 4 to 8 mol /, for example, in the case of urea, 5 to 8 mol / of an aqueous solution of urea so that the entire amount of the keratin-containing substance is soaked, This is performed by sealing the container and stirring at room temperature to 100 ° C. for 1 to 24 hours.

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

The reaction solution thus obtained is subjected to the following dialysis treatment after removing insolubles by centrifugation or filtration.

The dialysis treatment can be performed by a conventionally known treatment means. For example, a reaction solution from which insolubles have been removed as described above,
That is, the keratin filtrate is put into a container having a semi-permeable membrane such as cellophane, for example, and immersed in the container containing the external solution.
As the external solution, an aqueous medium containing 0.1 to 0.5% of a reducing agent capable of reducing a disulfide bond to a thiol group can be used. For example, a mixture of the aqueous medium and the reducing agent used in the above reduction step can be used. it can. The external solution is usually used at 20 to 40 times the volume of the keratin filtrate. The temperature can be room temperature and the time is usually between 12 and 36 hours. By performing such dialysis treatment 2 to 4 times, the protein denaturing agent and the surfactant in the keratin filtrate can be removed, and the reducing agent can be reduced to the same concentration as the external solution. can get.

The micellar aqueous solution of keratin obtained in this manner is
When calibrated by the wry protein determination method, it varies depending on the keratin-containing substance as a raw material, but is about 2 to 4% by weight. According to the amino acid analysis, cysteine and cystine in the keratin vary depending on the type of the keratin-containing substance, but generally 4 to 10 cysteines and 100 cysteines per 100 amino acid residues,
It has 0.5 to 2 pieces. In addition, it can be seen from the electrophoretic analysis that a protein having a molecular weight of 15,000 to 130,000 is mainly used.

The mechanism of such keratin solubilization is to convert insoluble keratin to a thiol group by reducing the disulfide bond while increasing the affinity with water molecules by the hydrogen bond interaction of a protein denaturant such as urea at a high concentration. According to it. However, in the reduction step (keratin extraction step) of the present invention, the yield of keratin is increased by 10 to 20% compared to the absence of a conventional surfactant (for example, JP-A-63-301809) (described later). (See Tables 1 and 2)
It is remarkably improved to 70% (see Table 2 below).

Further, in the present invention, keratin is efficiently extracted in a short period of time without sufficient conversion of the disulfide bond of keratin to a thiol group by reduction. For example, when hair is used as a keratin-containing substance, the keratin extraction rate at 50 ° C. for 5 hours reaches 80% or more, and the molecular weight of the extracted keratin is about 45,000 to 120000 by polyacrylamide electrophoresis. 12000-45000 each accounted for about 40% and 60% of the total weight. 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 45,000 or less occupying about 70% of the total weight, and a higher molecular weight keratin accounts for about 30% of the total weight. It is.

When the reaction solution of the present invention was examined by a light scattering method, dispersion mainly having an associated molecular weight of 25,000 to 60,000 was observed. Such an aggregate was not observed in the reaction solution in the absence of the conventional surfactant, and is considered to be caused by the surfactant.The surfactant probably enters the hydrophobic keratin-rich peptide site and is stable. It seems reasonable to think that the micelles are formed. That is, it is considered that the keratin in which the disulfide bond was reduced to be converted to a thiol group was efficiently extracted as a micelle dispersion of keratin by incorporating a coexisting surfactant. In fact, according to the nuclear magnetic resonance spectrum and the protein analysis, the dialyzed aqueous keratin solution contains keratin protein and the surfactant as main components.
Table 1 below shows that such an extraction method, a so-called micelle extraction method, is particularly effective for keratin from hard keratin-containing substances such as nails and hooves.

The keratin aqueous solution obtained by the ultrasonic irradiation in the present invention has an amino acid composition and a protein composition substantially equivalent to those of the keratin aqueous solution obtained under non-ultrasonic irradiation if the keratin-containing raw materials are the same. Amino acid analysis and polyacrylamide gel electrophoresis.

The obtained micelle aqueous solution is formed into various high molecular weight films, films, fibers, sponges, and the like by a known film forming method and a forming method.

[Examples] Next, the present invention will be described more specifically based on examples and comparative examples.

Example 1 20 g of wool (collected from Collidale species) was 370 m in 8M urea aqueous solution
Then, 12 g of sodium dodecyl sulfate and 35 ml of 2-mercaptoethanol were added. Then close the container and seal at 60 ℃
Ultrasonic irradiation was performed at an output of 80 W while stirring for 8 hours.
After allowing the reaction to return to room temperature, insolubles were removed by filtration. Put the filtrate in a cellophane tube and add 0.3% by weight
The solution was dialyzed twice against an aqueous mercaptoethanol solution (10). 470 ml of the obtained colorless and transparent dialysate is a micelle aqueous solution of the target keratin containing a small amount of 2-mercaptoethanol and the surfactant.

When the protein content of this solution was determined by the Lowry method, it contained 0.27 g of keratin, the keratin concentration was 2.7%, and the keratin yield was 63%. In addition, keratin powder obtained by freeze-drying the aqueous solution was subjected to amino acid analysis, and it was found that 7.8 cysteines and 0.1 cystine per 100 amino acid residues.
There were seven. When examined by polyacrylamide gel electrophoresis, a protein having a molecular weight of 15,000 to 70,000 was the main component.

Example 2 Wool 20 g was treated in the same manner as in Example 1 except that the reaction time was changed to 24 hours without performing ultrasonic irradiation, and keratin was obtained as a colorless and transparent dialysate containing a small amount of 2-mercaptoethanol and a surfactant. (480 ml) was obtained. When this solution was subjected to protein quantification in the same manner as in Example 1,
At a keratin concentration of 2.5%, the keratin yield was 60%. Amino acid analysis of keratin powder obtained by freeze-drying this aqueous solution showed 7.5 cysteines and 0.9 cystine per 100 amino acid residues. When examined by polyacrylamide electrophoresis, the molecular weight was 1
5,000 to 70,000 proteins were the major components.

Comparative Example 1 A micelle aqueous solution of keratin was prepared in the same manner as in Example 2 except that a surfactant was added. The results are shown in Table 1.

Example 3 Human hair was washed with a neutral detergent and then washed with hexane. 5 g of this human hair was immersed in 120 ml of an 8M aqueous urea solution. To this, 5 g of sodium dodecyl sulfate and 8 ml of 2-mercaptoethanol were added, the vessel was sealed, and the mixture was stirred at 50 ° C for 5 to 35 hours.
After 5, 10, and 35 hours, 10 ml of each reaction product was collected, and insolubles were removed by filtration. The obtained filtrate was put into a cellophane tube and dialyzed twice against a 0.3% by weight aqueous solution of 2-mercaptoethanol 2 to obtain a target keratin as a colorless and transparent dialysate containing a small amount of 2-mercaptoethanol and a surfactant. Was obtained. 10 g of this solution is L
As a result of protein quantification by the owry method, keratin yields shown in Table 2 were obtained. Amino acid analysis of keratin powder obtained by freeze-drying the aqueous solution showed that amino acid 100
There were 8 cysteines and 1.2 cystine per residue. Examination by polyacrylamide electrophoresis showed that proteins containing proteins with molecular weights of 45,000 to 120,000 and 12,000 to 45,000 were contained, and the ratios were 40% and 60%, respectively.

Comparative Example 2 A micelle aqueous solution of keratin was prepared in the same manner as in Example 3 except that the surfactant was not added. The results are shown in Table 2.

Example 4 10 g of chicken wings were immersed in 180 ml of a 60% by weight aqueous solution of lithium bromide, and after adding 5 g of sodium dodecyl sulfate and 10 ml of 2-mercaptoethanol, the container was tightly sealed and shaken at 45 ° C for 2 hours at an output of 80 W. Ultrasonic irradiation was performed. The reaction solution from which insoluble matter was removed by filtration was put into a cellophane tube and dialyzed twice against 10% by weight of a 0.3% by weight aqueous solution of 2-mercaptoethanol. 230 ml of the desired aqueous keratin micelle solution was obtained as a colorless and transparent dialysate containing a small amount of 2-mercaptoethanol and the surfactant. At a keratin concentration of 3.3%, the keratin yield was 76%.

Amino acid analysis of keratin powder obtained by freeze-drying the aqueous solution showed that 6.1 cysteines and 1.2 cystine were present per 100 amino acid residues. When examined by polyacrylamide electrophoresis, the molecular weight was 1
5,000 to 70,000 proteins were the major components.

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

Comparative Example 3 A micelle aqueous solution of keratin was prepared in the same manner as in Example 4, except that the treatment time was changed to 24 hours without adding a surfactant. The results are shown in Table 1.

Example 6 10 g of ground cow's horn was immersed in 150 ml of an 8M aqueous urea solution, 80 g of lithium bromide, 5 g of sodium dodecyl sulfate and 15 ml of 2-mercaptoethanol were added.
Ultrasonic irradiation at an output of 80 W while shaking for 8 hours.
After returning to room temperature, insolubles were removed by filtration. The filtrate was placed in a cellophane tube and dialyzed twice against 10% by weight of a 0.3% by weight 2-mercaptoethanol solution. 185 ml of the obtained colorless and transparent dialysate is a micelle aqueous solution of the desired keratin containing a small amount of 2-mercaptoethanol and the surfactant. The keratin concentration of this solution is 3.0%,
The keratin yield was 55%. Amino acid analysis of keratin powder obtained by freeze-drying the aqueous solution showed that cysteine was 4.5 and cystine was 0.6 per 100 amino acid residues. Further, when examined by polyacrylamide electrophoresis, a protein having a molecular weight of 20,000 to 60,000 was the main component.

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

Comparative Example 4 A micellar aqueous solution of keratin was prepared in the same manner as in Example 7 except that no surfactant was added. The results are shown in Table 1.

Next, in Examples 1, 2 and Comparative Example 1 (raw material: wool), the same operation was performed except that the reaction time was changed as shown in Table 2 below, and Test Nos. 1 to 4 and Test No. Five~
8. Test mice No. 9 to 12 were prepared. The results of Example 3 (raw material: human hair) are shown in Test Nos. 13 to 15, and the results of Comparative Example 2 are shown in Test Nos. 16 to 18.

As shown in Table 2 and FIG. 1, the method of the present invention can efficiently obtain a high keratin yield in a short time.

[Effects of the Invention] According to the production method of the present invention, keratin is efficiently extracted in high yield, and the solubilizing agent can be removed by dialysis without causing insolubilization. The operation is easy because no alkali is required, and the obtained micelle aqueous solution has high stability at room temperature.

[Brief description of the drawings]

FIG. 1 is a graph showing the relationship between the reaction time of the reduction treatment and the keratin yield.

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-63-301809 (JP, A) JP-A-2-51533 (JP, A) JP-A-3-11099 (JP, A) 51095 (JP, A) JP-A-56-30909 (JP, A) JP-A-53-23999 (JP, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) C08H 1 / 00-1 / 06 A61K 7/00-7/50

Claims (2)

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