JPH037595A - Production of keratin hydrolyzate - Google Patents

Abstract

PURPOSE:To obtain a keratin hydrolyzate in high yield with slight discoloration and smell by hydrolyzing keratin with hydrochloric acid having a given concentration of hydrogen chloride, neutralizing the prepared reaction mixture and hydrolyzing the neutralized mixture with a protease. CONSTITUTION:Keratin such as wool is blended with 50-300wt.% hydrochloric acid having 20-38wt.% hydrogen chloride concentration and optionally 2-20wt.% thioglycolic acid or cysteamine to give a reaction mixture. Then the mixture is hydrolyzed at 10-50 deg.C to give a reaction product, which is neutralized with NaOH to give a neutralized reaction product having pH7-10. Then the reaction product is reacted with a protease such as subtilisin at 30-60 deg.C for 3-48 hours to give a keratin hydrolyzate having 300-3,000 average molecular weight.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a method for producing keratin hydrolyzate.

(Prior Art) Conventionally, protein hydrolysis has been carried out using acids, alkalis, or enzymes (for example, as disclosed in Japanese Patent Application Laid-open No. 61-69T).
(IT Publication).

[Problem to be solved by the invention]

However, when hydrolyzing keratin from wool or hair as a protein raw material, it cannot be easily hydrolyzed with ordinary protein hydrolases, and alkaline hydrolysis can remove cystine, one of the amino acids that make up keratin. is destroyed, and the resulting hydrolyzate becomes useless when used as a formulation for hair cosmetics. Furthermore, although acid hydrolysis does not destroy cystine,
Since it is not easily hydrolyzed, it is necessary to use harsh conditions, which makes it difficult to control the molecular weight of the resulting hydrolyzate, and also causes the resulting hydrolyzate to be colored and odorized.

That is, keratin, such as wool and hair, has a large amount of cystine among its constituent amino acids, and in addition to the peptide structure, the peptide chains are firmly connected by SS bonds caused by cystine. Therefore, under normal conditions, there is no way to dissolve it without changing its chemical structure. Therefore, keratin cannot be hydrolyzed by proteolytic enzymes that can only employ mild hydrolysis conditions. Furthermore, hydrolysis with alkali or acid has problems such as destruction of cystine and difficulty in controlling molecular weight.

Therefore, the present invention uses keratin from wool, hair, etc.
The molecular weight of the resulting keratin hydrolyzate can be easily controlled without destroying cystine, and the yield is high.
Moreover, it is an object of the present invention to provide a method for producing a keratin hydrolyzate that can easily obtain a keratin hydrolyzate with little coloring and odor.

[Means for Solving the Problems] In the present invention, when hydrolyzing keratin, hydrochloric acid with a hydrogen chloride concentration of 20 to 38% by weight is added to the keratin from 50 to 38% by weight.
31) After hydrolyzing keratin at 10-50°C using 0% by weight, the reaction mixture is neutralized to pH 7-10,
The above objective was achieved by further hydrolyzing the keratin using a protein hydrolase.

That is, in the present invention, the peptide bonds of amino acids that are relatively easily hydrolyzed are first heated at 10 to 50°C with hydrochloric acid.
Because the hydrolysis is carried out under such mild conditions, the molecular weight of the resulting hydrolyzate can be easily controlled, and the coloring and
Less odor.

Next, hydrolysis with a protein hydrolase is performed, but since the keratin has become water-soluble due to the previous partial hydrolysis with hydrochloric acid, the hydrolysis with the enzyme proceeds sufficiently even under mild conditions, and the resulting product is obtained. A highly stable keratin hydrolyzate that can easily control the molecular weight of the keratin hydrolyzate, can be hydrolyzed in high yield, has little coloration and odor, and does not increase coloration or odor over time. Decomposition products can now be obtained.

In carrying out the present invention, keratin is first hydrolyzed with hydrochloric acid, but this hydrochloric acid has a hydrogen chloride concentration of 20 to 3.
In the present invention, in which 8% (wt%) hydrochloric acid is used, hydrochloric acid with such a high concentration is used because hydrolysis proceeds more smoothly with higher concentration hydrochloric acid. The amount of hydrochloric acid used is 50 to 300% of the keratin to be hydrolyzed, but this is because if the amount of hydrochloric acid used is less than the above range, especially in the early stage of the reaction, the reaction mixture is Because it is homogeneous, it is difficult to control the hydrolysis, and the yield of the hydrolyzate obtained is poor.In addition, if the amount of hydrochloric acid used is greater than the above range, it is not only unnecessary, but also Desalination after decomposition and i! This is because it requires a great deal of effort to shrink, which is undesirable. In addition, the amount of hydrochloric acid used is 50 to 300% of the keratin, which means that
This means that the above-mentioned hydrochloric acid is used in a weight ratio of 0.5 to 3.0 times as much as keratin.

The above hydrolysis of keratin with hydrochloric acid is carried out at 10 to 50°C, but this is because if the temperature during hydrolysis is lower than lOoC, the progress of hydrolysis will be slow, and if the temperature during hydrolysis is 50"C This is because as the grain size increases, the hydrolysis reaction becomes more intense, making it difficult to control the molecular weight of the resulting hydrolyzate, and increasing coloration and odor.

Additionally, if thioglycolic acid or cysteamine is used together with hydrochloric acid during the hydrolysis of keratin with hydrochloric acid, the thioglycolic acid or cysteamine will reduce and cleave the disulfide bond of cystine, making the cystine moiety water-soluble, and During hydrolysis, the cystine part, which is the most difficult to hydrolyze, becomes easily hydrolyzed.
Hydrolysis proceeds uniformly, making it easier to control the degree of hydrolysis and improving yield. Cystine contained in keratin is reduced to cysteine by thioglycolic acid or cysteamine, but this cysteine is contained in hydrolysates and is oxidized by oxygen in the air or oxidizing agents such as hydrogen peroxide. Regenerates disulfide bonds to become cystine. Therefore, the resulting hydrolyzate has little reduction in cystine and can be effectively used as a raw material for hair cosmetics. In addition, thioglycolic acid or cysteamine inhibits the oxidation and decomposition of impurities such as sugars and fats contained in keratin, reducing odor and coloring of the hydrolyzate and improving the purity of the hydrolyzate. do.

The amount of thioglycolic acid or cysteamine used is 2 to 2
It is appropriate to set it at 0%, and thioglycolic acid and cysteamine may be used together, and the total amount of both should be 2 to 20% of the keratin to be hydrolyzed.
It is appropriate to Further, cysteamine may be used in the form of a salt such as cysteamine hydrochloride.

As the protein hydrolase, for example, neutral protein hydrolases such as papain, promelain, thermolysin, trypsin, pronase, and chymotrypsin, and domestically produced neutral protein hydrolases such as sptilisin and staphylococcus protease are used. It will be done.

However, if thioglycolic acid or cysteamine is used together with hydrochloric acid hydrolysis, enzymatic hydrolysis will also be carried out in the presence of thioglycolic acid or cysteamine, so even under such conditions, the activity will not be maintained. It is preferable to use an enzyme that does not cause loss, is economical, and has an optimum pH (pH at which the enzyme is most active) of 7 to 10, particularly 8 to 9. From this point of view, protein hydrolysis in the present invention As the enzyme, sptilisin is particularly suitable.

That is, subtilisin does not lose its activity even in the presence of thioglycolic acid or cysteamine, and its optimal pH is 8.
~9, it can easily hydrolyze a keratin hydrolyzate partially hydrolyzed with hydrochloric acid, the molecular weight can be easily controlled, and the amount used can be small, making it economically suitable. .

This enzymatic hydrolysis is usually carried out at 30-60'C.
The reaction time is ˜48 hours.

The final keratin hydrolyzate preferably has an average molecular weight of 300 to 3,000. That is, those in this range have good adsorption to hair, are suitable as formulations for hair cosmetics, and are easily soluble in water and easy to handle.

The raw material for keratin is animal hair such as wool.

Hair, feathers, claws, horns, hooves, etc. are used. In particular, wool is preferred because it is easily available and can be easily hydrolyzed.

(Example) Next, the present invention will be explained in more detail by giving Examples.

Example 1 75% hydrochloric acid with a hydrogen chloride concentration of 30% to 500g of crushed wool
0 g was added, stirred at 30°C for 72 hours to hydrolyze, and then neutralized by adding 20% aqueous sodium hydroxide solution.
I made it H8.

Next, 0.2 g of sptilisin (a domestically produced neutral protein hydrolase) was added to the above reaction solution, and hydrolysis was carried out at 50° C. for 24 hours with stirring. During hydrolysis, 20%
Add sodium hydroxide aqueous solution appropriately to adjust the pH of the reaction solution to 8.
I kept it. After hydrolysis with subtilisin, the reaction solution was filtered, and the iIt solution was adjusted to pH 2 with hydrochloric acid, heated to 50°C, and stirred to deactivate subtilisin. After the reaction solution was adjusted to pH 6, it was desalted by electrodialysis using the electrodialysis device described below, decolorized with activated carbon, and then the concentration was adjusted to obtain an aqueous solution of keratin hydrolyzate with a concentration of 25%. The color, yield, average molecular weight and amount of cystine of the obtained keratin hydrolyzate are shown in Table 1 below. In addition,
The electrodialyzer used for desalting the reaction solution is as follows.

Model: DO-Cb (manufactured by Kijin Engineering ■) Membrane name: Selemion CMV and AMV (manufactured by Asahi Glass ■, product name) Membrane dimensions: 18cn (approximately 5% as anhydrous sodium sulfate) Catholyte: Aqueous sodium sulfate solution (approximately 5% as anhydrous sodium sulfate) Example 2 During hydrolysis with hydrochloric acid in Example 1, thioglycolic acid 5
Hydrolysis was carried out in the same manner as in Example 1, except that 0 g was added, and thereafter the steps up to decolorization with activated carbon were carried out in the same manner as in Example 1.

Next, 5 g of 35% hydrogen peroxide was added to the decolorized reaction solution, stirred, and allowed to react overnight.

Next, the remaining thioglycolic acid and dithioglycolic acid were removed by passing the solution through a column filled with 300 ml of anion exchange resin Diaion WA20 (trade name, manufactured by Mitsubishi Chemical Industries, Ltd.), and then the concentration was adjusted to 25. % keratin hydrolyzate aqueous solution was obtained. The color, yield, average molecular weight and amount of cystine of the obtained keratin hydrolyzate are shown in Table 1 below.

Example 3 Cysteamine hydrochloride 5 during hydrolysis with hydrochloric acid in Example 1
Hydrolysis was carried out in the same manner as in Example 1, except that 0 g was added, and thereafter the steps up to decolorization with activated carbon were carried out in the same manner as in Example 1.

Next, 5 g of 35% hydrogen peroxide was added to the decolorized reaction solution, stirred, and allowed to react overnight.

Next, cation exchange resin Amberlite IRC-50 (
The remaining cysteamine hydrochloride was removed by passing the solution through a column packed with 300 r+l (trade name, manufactured by Organo ■), and the concentration was then adjusted to obtain an aqueous solution of keratin hydrolyzate with a concentration of 25%. The color, yield, average molecular weight and amount of cystine of the obtained keratin hydrolyzate are shown in Table 1 below.

Comparative Example 1 Hydrolysis was carried out using hydrochloric acid at a concentration of 70°C and a hydrolysis time of 8 hours, and the same procedure as in Example 1 was carried out, except that hydrolysis with subtilisin and the accompanying deactivation operation were not performed. % of keratin hydrolyzate was obtained. The color, yield, average molecular weight and amount of cystine of the obtained keratin hydrolyzate are shown in Table 1 below.

Comparative Example 2 A keratin hydrolyzate with a concentration of 25% was prepared in the same manner as in Example 1, except that hydrolysis with hydrochloric acid was carried out in the same manner as in Example 1, and hydrolysis with sptilisin and accompanying deactivation operations were not performed. I got it. The color, yield, average molecular weight, and amount of cystine of the obtained keratin hydrolyzate were measured in accordance with Section 1 below.
Shown in the table.

Comparative Example 3 Add 700 g of a 6% aqueous sodium hydroxide solution to 500 g of wool, perform hydrolysis for 1 hour while stirring at 80°ct', cool the reaction solution, and then proceed as in Example 1 to obtain a concentration of 2.
A 5% aqueous solution of keratin hydrolyzate was obtained.

Table 1 shows the results of examining the color, yield, average molecular weight, and amount of cystine of the 25% keratin hydrolysates of Examples 1 to 3 and Comparative Examples 1 to 3.

Note that the colors in Table 1 are based on the measurement results by the Gardner method, and the larger the value, the darker the color.

The yield indicates the result obtained by measuring the total nitrogen amount (Kelpool method).

The molecular weight was measured by a gel filtration method, and the gel filtration conditions for the molecular weight measurement were as follows.

Etoma A moxibustion column: TSK get G3000PWXL diameter 7
．． 8 m + w x length 30 cm Solvent: 0.05% trifluoroacetic acid, 45% acetonitrile - water Flow rate: 0.3 m j! /min Detection = Ultraviolet absorbance detector (wavelength 210n...) Standard material: Aprotinin (MW6500) α-MSH (M
W1665) Bradykinin (MW1060) Glutathione (MW307) The amount of cystine can also be measured using an amino acid automatic analysis needle (JEOL JEOL
As shown in Table 1, the keratin hydrolysates of Examples 1 to 3 were compared to the keratin hydrolysates of Comparative Example 1 (corresponding to the conventional acid hydrolysis method). The color is lighter in comparison (i.e.
The numerical value representing the intensity of the color was small), and the increase in color over time was also small.

In addition, the keratin hydrolysates of Examples 1 to 3 were compared to Comparative Example 1.
The hydrolysis of Examples 1 to 3 is easier to control than the hydrolysis of Comparative Example 1, and the average molecular weight is higher than that of Comparative Example 1. This indicates that a hydrolyzate can be easily obtained.

Furthermore, regarding the amount of cystine in the keratin hydrolyzate, the keratin hydrolyzate of Examples 1 to 3 has a larger amount of cystine than the keratin hydrolyzate of Comparative Example 1.
The amount of cystine in the raw material wool was 9.4 mol%, indicating that the decrease in the amount of cystine due to hydrolysis was small.

In addition, at the same time as the color measurement, Examples 1 to 3 and Comparative Example 1
The keratin hydrolyzate of Comparative Example 1 had a stronger amino acid odor over time, but the keratin hydrolyzates of Examples 1 to 3 had almost no odor at the back. There was no increase in depth over time.

In addition, the keratin hydrolyzate of Comparative Example 2 was obtained by simply hydrolyzing easily hydrolyzed amino acids with hydrochloric acid under mild conditions, so the yield was extremely low, and the molecular weight was too large, making it unstable. does not dissolve in the pH
There was a problem that some parts of the liquid precipitated due to fluctuations in the water content and storage.

In addition, since the keratin hydrolyzate of Comparative Example 3 was obtained by alkaline hydrolysis, cystine was destroyed and the amount of cystine decreased to 0.4%, and there was also a hydrogen sulfide odor produced by the destruction of cystine. Ta.

〔Effect of the invention〕

As explained above, according to the present invention, keratin can be hydrolyzed in a controlled manner (and in a high yield).Furthermore, according to the present invention, keratin can be hydrolyzed without causing a significant decrease in the amount of cystine. It can be easily hydrolyzed, and the resulting hydrolyzate has little coloring and odor, and also has little increase in color and odor over time, and has a high amount of cystine, so it is suitable for cosmetic formulations, especially hair cosmetics. It is very useful as a compounding agent.

Claims (4)

[Claims] (1) When hydrolyzing keratin, add 50 to 30% hydrochloric acid with a hydrogen chloride concentration of 20 to 38% by weight based on the keratin.
A keratin hydrolyzate characterized in that the keratin is hydrolyzed at 10 to 50°C using 0% by weight, the reaction mixture is neutralized to pH 7 to 10, and then the keratin is further hydrolyzed with a protein hydrolase. manufacturing method. (2) The method for producing a keratin hydrolyzate according to claim 1, wherein the keratin is wool. (3) The method for producing a keratin hydrolyzate according to claim 1, wherein 2 to 20% by weight of thioglycolic acid and/or cysteamine is used based on the keratin together with hydrochloric acid. (4) The method for producing a keratin hydrolyzate according to claim 1, wherein the protein hydrolase is sptilisin.