JPS6027680B2 - Method for producing keratin hydrolyzate - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a keratin hydrolyzate useful as a hair cold wave agent and a hair setting agent.

In general, cold waving of hair consists of a reducing agent such as thioglycolic acid, and the pH is adjusted to approximately 9 to 10 with an alkali.The first liquid is applied to the hair and allowed to penetrate, and then the hair is moderately wound to create a lot. The disulfide bonds of cystine in the keratin of the hair are cut with the reducing agent to give curls to the hair, and then a second liquid consisting of an oxidizing agent such as sodium bromate or hydrogen peroxide is applied. This is done in such a way that new disulfide bonds are formed at the set positions, thereby giving the hair elasticity again.

However, the problem with this method is that some side reactions occur during oxidation, producing Ker-S-SCH2COO, and as a result, disulfide bonds are not completely regenerated, resulting in a large amount of hair loss. The disadvantage is that it can cause severe damage.

Therefore, there is a demand for a drug that can provide a waving effect that does not damage the hair and is less transparent than conventional cold waving drugs.

Therefore, the present inventors have conducted various studies in order to meet such demands, and have reduced keratin with mercaptans or sulfides in an alkaline environment, and then hydrolyzed it with an enzyme, thereby adding a mercapto group to each molecule. It was discovered that a keratin hydrolyzate containing two or more of the following can produce excellent waving and setting effects without damaging the hair, and has previously filed a patent application for the same (Japanese Patent Application No. 124289/1989). ).

Based on the above findings, the present inventors have conducted further research and found that keratin has an average molecular weight of less than 200 degrees Celsius, which can be obtained by reducing keratin with mercaptans or sulfides in an alkaline environment and then hydrolyzing it with an enzyme. A keratin hydrolyzate having less than two mercapto groups in one molecule on average does not damage the hair, and can produce excellent waves similar to those of a keratin hydrolyzate having two or more mercapto groups in one molecule. We have discovered a method that can provide both effective and set effects, and have completed the present invention. That is, the keratin hydrolyzate obtained by the method of the present invention is similar to the keratin hydrolyzate having two or more mercapto groups in one molecule, and is applied onto hair in the form of a dilute aqueous solution. Apply or spray;
When the hair is wrapped around a lot and the moisture is dried, the mercapto groups in the hydrolyzate are oxidized by oxygen in the air and crosslinked with the mercapto groups of other molecules in the keratin hydrolyzate that are in contact with the layer. to form a disulfide bond,
It leaves the hair curled and forms a high molecular weight coating on top of it. As mentioned above, in order for the mercapto groups in a keratin hydrolyzate to crosslink with the mercapto groups of other keratin hydrolyzate molecules to form disulfide bonds through oxidation, a certain amount of mercapto groups in the keratin hydrolyzate must be present in the keratin hydrolyzate. A mercapto group is required, and therefore the average molecular weight of the keratin hydrolyzate must be above a certain level, and the lower limit of the average molecular weight of the keratin hydrolyzate obtained in the present invention is as shown in Example 3 below. To, 820
It is appropriate to

Moreover, since the keratin hydrolyzate of the present invention has an amino group and a carboxyl group in its molecule, these combine with the carboxyl group and amino group in the hair to form a salt, so the bond with the hair is strong. and does not come off easily even after washing with water. In this way, the keratin hydrolyzate obtained by the method of the present invention can impart a suitable waving effect without damaging the hair, and can maintain this effect for a long period of time.

In addition, the keratin hydrolyzate of the present invention has a chemical structure similar to that of hair, so when it is applied to hair, it does not give a strange feeling unlike conventional resin-based setting agents, and it also has peptide bonds. Therefore, it is breathable and does not make your hair stuffy.

Furthermore, the keratin hydrolyzate obtained by the method of the present invention has the property of cleaving the disulfide bonds in the keratin of the hair when the pH is adjusted to 7 to 10 with an alkali and it is applied to the hair and allowed to penetrate. It can be used in the same way as thioglycolic acid, cysteine, etc., and has a wave-imparting effect due to the formation of a film due to the bonding of the keratin hydrolysates and the cleavage and regeneration of disulfide bonds in the hair. , it is possible to give suitable waves to the hair. In this case, unlike thioglycolic acid, cysteine, etc., the keratin hydrolyzate of the present invention has a chemical structure similar to that of hair, so it will not damage hair. In carrying out the present invention, any keratin that constitutes wool, feathers, hair, horns, claws, hooves, etc. can be used, but wool is particularly preferred from the viewpoint of easy availability.

Examples of mercaptans used as a reducing agent in the present invention include thioglycolic acid, mercaptoethanol, thioglycerin, and thiosarcic acid.

Examples of the sulfide include sodium sulfide, potassium sulfide, calcium sulfide, triethanolamine sulfide, jetanolamine sulfide, and monoethanolamine sulfide. Examples of enzymes used for hydrolysis in the present invention include acidic proteolytic enzymes such as pepsin, and neutral proteolytic enzymes such as promelain, thermolysin, trypsin, and chymotrypsin.

When carrying out the method of the present invention, first, keratin is placed in an aqueous solution of a reducing agent adjusted to an alkaline state, and placed under a rope.
Preferably, the air in the system is replaced with an inert gas such as nitrogen, and the disulfide bond of cystine in keratin is reductively cleaved at a temperature of 0 to 40 oo to form a mercapto group.

Note that when using an alkaline reducing agent such as sulfide, it is not necessary to add an alkaline substance to keep the reaction solution alkaline, but when the reducing agent is acidic such as thioglycolic acid, it is not necessary to add an alkaline substance to keep the reaction solution alkaline. It is desirable to adjust the reaction solution to an alkaline range by adding an alkaline agent such as caustic soda or caustic potash to the reaction solution. The pH of the reaction solution is preferably adjusted to 8 to 11. It is preferable to add urea to the reaction solution because urea swells keratin and facilitates the action of the reducing agent. In addition, as the keratin of wool and other materials dissolves, the liquid nature of the reaction solution becomes acidic, so to prevent this, tris(hydroxymethyl)-aminomethane, ammonia, sodium bicarbonate, etc. are added as a buffer. It is preferable to leave it there. After the reduction reaction, the reaction mixture was passed through a vacuum filter to concentrate the reactants, and the solution was roughly filtered through an ultraviolet filter to reduce the volume by about 1/2 to 1/2.
The concentrated liquid is subjected to dialysis to remove the remaining reducing agent and to prepare a solution suitable for the next enzymatic decomposition.
Either the pH is adjusted to be HI or the reaction product is precipitated by placing the passed liquid in an acidic aqueous solution of hydrochloric acid without filtration, and the resulting precipitate is washed.

Then, an enzyme is added to the reaction product to perform hydrolysis.

The pH during enzymatic decomposition is preferably adjusted to a range of pH 3 to 3 for acidic enzymes such as pepsin, and pH 3 for neutral enzymes such as promelain.
It is preferable to adjust to a range of H5 to H8. Further, the reaction temperature is preferably 30 to 450°, and the reaction time is usually 3 to 2 feeding hours. The amount of enzyme used as well as the reaction time and temperature have a large effect on the molecular weight of the hydrolyzate.

Therefore, to determine how much enzyme to use and how to set the reaction time and temperature, it is necessary to examine the molecular weight distribution of the obtained hydrolyzate by gel filtration method, or to determine whether the average molecular weight of the obtained keratin hydrolyzate is relatively high. Small (average molecular weight 1
If the gel filtration method cannot be used (less than 000), by measuring the total amount of nitrogen and the amount of amino nitrogen, we can empirically determine the optimal conditions according to the desired molecular weight of the hydrolyzate. All you have to do is decide. In the present invention, the average molecular weight of the resulting keratin hydrolyzate is set to less than 200 because if the average molecular weight is too large, it will not have the ability to cleave disulfide bonds in hair, and keratin generally contains amino acids. Cystine is contained at a ratio of 1 to 1 q solids, and the average molecular weight of amino acids in keratin is about 100, so if the average molecular weight of the keratin hydrolyzate is 2000 or more, the hydrolyzate Since two or more mercapto groups are contained in one molecule of the hydrolyzate, the purpose is different from the present invention, which aims to contain an average of less than two mercapto groups in one molecule of the hydrolyzate. become. Then, the obtained keratin hydrolyzate is further subjected to reduced pressure concentration, if necessary, to be appropriately concentrated.

As is clear from the above explanation, the keratin hydrolyzate obtained by the method of the present invention does not damage the hair, gives a suitable waving effect to the hair, and maintains the effect, so it is suitable for cold treatment. Although it is extremely useful as a waving agent and a setting agent, its uses are not limited to these, for example, it can be used as a base for hair cosmetics such as hair treatment agents, hair straighteners, and hair straighteners. It is also useful as a solidifying agent for textile products.

Next, the method of the present invention will be explained with reference to Examples.

Example 1 1. Into the beaker, put 360 ml of urea, 4 ml of tris-(hydroxymethyl)-aminomethane, and 4 ml of EDTAI, add distilled water to make a total volume of about 800 M, and stir to dissolve most of the added reagents. , 20 mercaptoethanol was added.

Next, a 20% (wt%) aqueous solution of caustic soda was added to adjust the solution to pHIO, and distilled water was added to bring the total volume of the solution to 1. After adding 20 g of degreased wool to this solution and removing the bubbles generated, the container was covered with a lid and left at room temperature for 3 days with occasional lighting.

Next, the resulting reaction mixture was subjected to vacuum treatment to remove unreacted wool. Approximately 820 volumes of the obtained nutrient solution were passed through an ultrafilter (
By ultrafiltration using Amicon's Model 402 Cell, Diaflow Membrane UMI0 (molecular weight 10,000), the concentration of the reaction product is increased and the solvent containing urea and reducing agent is removed. I left.

The concentrated solution obtained was packed into a cellophane dialysis tube and dialyzed against 5 sleeves of 0.1N formic acid for 8 hours.
Furthermore, dialysis was repeated twice with 5 sleeves of 0.1N formic acid for 8 hours each, and then with 3 sleeves of distilled water for 3 hours.

The concentrated solution after dialysis was transferred to a beaker above the 500, and a solution of 50 parts of pepsin dissolved in 5 parts of 0.1N acetic acid was added thereto.

While maintaining the reaction solution at 370° C. in a hot water bath, the reaction solution was thoroughly clarified using an electromagnetic evaporator, and keratin was hydrolyzed over a period of 2 feedings. After the reaction was completed, a 20% aqueous caustic soda solution was added to adjust the pH of the reaction solution to 7, which was kept at 7000 in a steam bath and left to stand for 30 minutes to inactivate pepsin. After cooling, the obtained reaction solution was filtered under reduced pressure, and the filter solution was transferred to a 500' eggplant type kolben with a stopper, and concentrated under reduced pressure using a rotary evaporator to hydrolyze keratin with a dry residue of 20%. I got something.

A portion of the obtained keratin hydrolyzate was taken and diluted to a 0.5% solution with 0.1N acetic acid, and one part of it was filtered through a gel filter (Agarose Gel G-5U4 manufactured by Pharmacia).
0, column length 30 arcs, 1 fraction 1.0 arc), the results shown in FIG. 1 were obtained.

The peptide concentrations in each fraction shown in FIG. 1 were determined by measuring the absorbance of the skin at a wavelength of 23 using an ultraviolet partial photometer. It is known that in the gel filtration method, the logarithm of the molecular weight of a sample has a linear relationship with the outflow volume of that sample.

The molecular weight cutoff for G-25 is between 1000 and 5000, but when blue dextran (molecular weight 200) is run to determine the fraction with the maximum cutoff molecular weight, it appears in fraction 13, and when salt is run, it appears in fraction 29. From this, a graph as shown in Figure 2 was obtained.
In FIG. 1, the fraction with the highest peptide concentration is fraction 24, and it was estimated from FIG. 2 that this fraction had a logMW of 3.2 liters, or a molecular weight of about 1,600.

As a precaution, the total nitrogen content and the amino nitrogen content of the keratin hydrolyzate with a dry residue of 20% (the ash content was 1% or less) were measured using the Kjeldahl method and the Bansley method, respectively. (VanSlyke) method, the total nitrogen content was 3265%, and the amino nitrogen content was 0.
．． It was 207%.

Since the ratio of the two, 15.77, is almost comparable to the number of amino acids per peptide, assuming the molecular weight per amino acid to be 105, the average molecular weight of the peptides in the obtained keratin hydrolyzate is determined to be 1660. Met. This approximately agrees with the value determined by the previous gel filtration method. Next, cysteine residues in the obtained keratin hydrolyzate were quantified by the following experiment.

First, a portion of the obtained hydrolyzate was diluted to a 1% solution with 0.1N acetic acid, and the sample was filtered through a gel filter using the method described above.
Only fractions 23 to 25 on the hillside having a molecular weight of 160G were detected, and the peptide concentration of these was measured by the bullet method shown below.

Four portions of pureed reagent were added to sample 1, and after 30 minutes, the absorbance of the skin at wavelength 55 was measured using a visible partial photometer.

Separately, an aqueous solution of crystalline albumin with a known concentration (approximately 0
．． The calibration curve shown in Figure 3 was obtained by performing the same operation for 4 samples of 1-0.4% aqueous solution) and distilled water.
Based on this, the peptide concentration in the sample was determined, and from Figure 3, the peptide concentration in this sample was 1.06.
It turned out to be secretion.

On the other hand, the cysteine concentration in this sample was determined by the Ellman method.

That is, 5,6-dithiobis(2-nitrobenzoic acid)
9 of 10 in 0.09M phosphate buffer (pH 7.0)2.
Prepare a solution dissolved in addition to [5] and 0.2M hard sodium carbonate, and add 50 grams of the 5,5-diothiobis(2-nitobenzoic acid) solution and 0.2M hard sodium carbonate to sample 0.5'. 2 parts of sodium carbonate was added, and 10 minutes later, the absorbance at wavelength 41 was measured using an ultraviolet partial photometer. Separately, a cysteine hydrochloride aqueous solution of known concentration (approximately 0.1-2.
The calibration curve shown in Figure 4 was obtained by performing the same operation on 4 samples of 0 M aqueous solution), and the mercapto groups in the cysteine residues in the sample were determined based on the calibration curve. The cysteine residue concentration of is 1.1
It was found to be 8mM.

From the above measurement results, it was found that the obtained keratin hydrolyzate contained a mercapto group corresponding to 13.4 cysteine per 100 peptides with a molecular weight of about 1600. As a result, it was found that an average of 1.8 mercapto groups were contained per molecule of peptide having a molecular weight of 1,600.

Further, a 2% aqueous solution of the obtained keratin hydrolyzate was applied to the hair, the hair was wound into a rod, and the hair was dried with a hair dryer to evaporate water.

Thereafter, the lot was removed from the hair, but the hair remained appropriately wavy. After 2 hours, the hair was washed with water, but the waves remained unchanged.

Example 2 25% of wool was adjusted to pHIO with caustic soda.
8M sodium thioglycolate overnight (0.1% ED
After removing generated bubbles, the mixture was allowed to stand at room temperature for 2 days with occasional stirring.

Next, the resulting reaction mixture was subjected to vacuum treatment to remove unreacted materials, and the resulting solution was added with an aqueous solution containing 80% of concentrated hydrochloric acid, and left to stand for 5 hours to precipitate the reaction product.

In order to wash the resulting precipitate of the reaction product, after removing the top lighting solution, two portions of water were added, and after standing for 30 minutes, the top lighting solution was removed again, and this operation was repeated two more times.

The obtained precipitate was transferred to a 500°C beaker, and a solution of 40 parts of pepsin dissolved in 4 parts of 0.1N acetic acid was added thereto, and the reaction solution was heated at 370° C. in a hot water tank.
It was hydrolyzed for a while.

Thereafter, in the same manner as in Example 1, a keratin hydrolyzate with a dry residue of 20% was obtained.

The obtained keratin hydrolyzate was subjected to gel filtration in the same manner as in Example 1, and the average molecular weight was determined to be about 1300.

In addition, in the same manner as in Example 1, the total nitrogen amount was reduced to 3.
．． The average molecular weight of peptides in the resulting keratin hydrolyzate was determined to be 1270. Furthermore, the concentration of cysteine residues was determined by the Ellman method in the same manner as in Example 1, and it was found that in a peptide with a molecular weight of approximately 1300, the concentration of mercapto groups corresponding to 1.12 cysteine residues per 100 molecules was determined. As a result, it was found that each peptide molecule having a molecular weight of 1300 contained an average of 1.1 mercapto groups.

A 2% aqueous solution of the obtained keratin hydrolyzate was applied to the hair, the hair was wound into a rod, and the hair was dried with a hair dryer to evaporate the moisture.

Thereafter, the lot was removed from the hair, but the hair remained moderately wavy. Example 3 35 kg of wool was mixed with 1 ml of 0.9M sodium sulfide (0.1% EDT).
After removing the bubbles generated, the container was allowed to stand for 2 hours while occasionally holding a candle.

Next, the obtained reaction mixture was passed under reduced pressure to remove unreacted substances, and the obtained solution was passed through an ultrafiltration filter in the same manner as in Example 1, and concentrated until the reaction solution was 1/2. did.

The obtained concentrate was packed into a cellophane dialysis tube, and dialysis was repeated three times for 6 hours each with three sleeves of distilled water.

The concentrated solution after dialysis was transferred to a beaker, and the pH was adjusted to 5 by adding acetic acid using an FH meter.

Add 500 parts of promeline (50 units/night) and 50 parts of cysteine hydrochloride to this, and boil the reaction solution in a steam bath for 40 parts.
Hydrolysis was carried out for 2 hours while maintaining the temperature at 0.00. After the reaction was completed, the reaction solution was heated to 70 qo and left to stand for 30 minutes to inactivate promeline. The obtained reaction solution was sieved under reduced pressure, and the same procedure as in Example 1 was carried out until the dry residue was 2.
A 0% keratin hydrolyzate was obtained.

When the obtained hydrolyzate was subjected to gel analysis in the same manner as in Example 1, the main component appeared in fractions 29 and onwards, so it was estimated that the average molecular weight of this product was 1000 or less. In addition, when the total nitrogen content and the amino nitrogen content of this keratin hydrolyzate (the dry residue was 20% and the ash content was 1% or less) were determined using the same method as in Example 1, the total nitrogen content was determined by the same method as in Example 1. It was found that the elemental amount was 3.256% and the amount of amino nitrogen was 0.415%. The ratio between the two is 7.8.
5, the average molecular weight of peptides in the keratin hydrolyzate was determined by calculation in the same manner as in Example 1, and the average molecular weight was 820. In addition, the concentration of cysteine residues was determined by the Ellman method in the same manner as in Example 1, and the dry residue of the obtained keratin hydrolyzate was 1
It was found that mercapto groups corresponding to 12.3 cysteines were contained per 100 units, and as a result, it was found that 1 molecule of peptide contained an average of 0.8 mercapto groups.

A 2% aqueous solution of the resulting hydrolyzate was mixed with a 0.1% iron gluconate solution and immediately applied to the hair, which was wound into a lot and dried with a hair dryer to evaporate the moisture.

Thereafter, the lot was removed from the hair, but the hair remained appropriately wavy.

[Brief explanation of the drawing]

Figure 1 shows the effluent fraction and absorbance (23 plates m
), Figure 2 is a graph showing the relationship between the fractionated liquid when a standard substance is passed through a gel and the logarithm of molecular weight, and Figure 3 is a graph showing the relationship between albumin concentration and absorbance (55 blood). FIG. 4 is a graph showing the relationship between cysteine concentration and absorbance (41 degrees). Figure 1 Figure 2 Figure 3 Figure 4

Claims (1)

[Claims] 1. A keratin hydrolyzate having an average molecular weight of 820 or more and less than 2,000 and an average of less than 2 mercapto groups per molecule, which is characterized by reducing keratin with mercaptans or sulfides in an alkaline region and then hydrolyzing it with an enzyme. Production method.