JPS6257319B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention uses structural proteins composed mainly of keratin (referred to as "keratin substances" in the present invention) such as wool, feathers, and hair as raw materials, and has a molecular weight range of 200 to 200. 10,000 soluble low molecular weight keratin (herein referred to as "oligokeratin").

It is well known that low molecular weight proteins (oligopeptides) obtained by partial hydrolysis of fibrous proteins such as collagen and keratin are useful as cosmetic base materials for hair etc. [Reference: I .Bonadeo et Cosmetics and
Toiletries, 92 , 45 (1977)], methods for producing these oligopeptides include partial hydrolysis with acid or alkali, decomposition with protein hydrolase, and reflux extraction (pyrolysis) using a liquid medium at high temperature. is used. In particular, the usefulness of methods using hydrolases is well known in the field of protein chemistry because the molecular weight of the resulting oligopeptide can be controlled relatively easily.

However, when keratin materials are used as raw materials, they are difficult to decompose with enzymes due to their chemical structure and advanced tissue structure. Since this method requires manipulation, the cost of the product is high, and it is not practically satisfactory. That is, keratin is insoluble because it has a natural crosslinked structure (disulfide bonds, ionic crosslinks, hydrogen bonds, hydrophobic interactions, etc.), and is characterized by being difficult to be hydrolyzed by enzymes.
In the case of wool, the proportion of untreated wool degraded by pronase is less than 20%, the proportion degraded by trypsin is at most 10%, and the proportion degraded by pepsin is about 5% [Reference: PH Springel, Aust. J.
Biol. Sciy, 16 , 272 (1963), WGCrewther et al.
Advan.Protein Chem., 20 , 191 (1965), WG
Crewther et al. J. Biol. Chem., 242 , 4310 (1967)].
Therefore, the conventional method for preparing oligokeratin using hydrolase is to cleave the disulfide bonds that make keratin insolubilized by the action of a reducing agent in the presence of a solubilizing agent such as urea. A method is known in which an enzyme is allowed to act once dissolved in a liquid medium. but,
According to this method, in order to separate and recover oligokeratin, it takes a lot of effort to separate the insolubilized part during reduction by filtration, and to remove solubilizing agents such as urea and reducing agents by ultrafiltration, dialysis, etc. I need.

As a result of intensive studies to solve these problems, the inventors of the present invention found that the present inventors can manipulate the raw material by simply applying a protein hydrolase to fibrous reduced keratin (keratein), which has been reduced without changing the form of the raw material, such as the original hair. The inventors have discovered that oligokeratin that is easily soluble in water and organic polar solvents can be obtained in a high yield very easily, and have arrived at the present invention.

That is, in the present invention, when reducing and then hydrolyzing a keratin material, the keratin material is subjected to a reduction reaction while maintaining the form of the raw material in the reaction medium before and after the reduction reaction, and then a protein hydrolase is added to the keratin material. The present invention provides a method for producing a low molecular weight keratin material, characterized in that a hydrolysis reaction is carried out in water using the above method.

The purpose of the present invention is to provide a new method for producing oligokeratin having excellent performance as a cosmetic base material, and the advantages and characteristics of the present invention will become clear from the following description.

The characteristics of the present invention are as follows: (1) A natural substance composed mainly of keratin is reduced to cleave the disulfide bonds in the keratin, but the reduction reaction also leaves the keratin substance as a raw material in the reaction medium after the reaction. (2) A swelling agent for the keratin material, which has been conventionally used in the enzymatic hydrolysis treatment of keratin materials, is used for the reduced keratin material that retains the form of the above-mentioned raw material. Alternatively, the objective is to produce oligokeratin at a high yield by directly allowing only a hydrolase to act without using a solubilizing agent or the like.

The advantages of the present invention are that (1) the reduced keratin substance exists in the reducing reaction medium while retaining its raw material form, so that it is extremely easy to separate the reduced keratin from the reducing reaction medium; 2) The enzymatic hydrolysis reaction can be carried out in an aqueous solution containing only a small amount of acid or alkali, which is added to adjust the pH to the optimum level for the hydrolytic enzyme used. In the separation and recovery of dissolved oligokeratin, it becomes possible to omit the complicated steps of ultrafiltration and dialysis, and the separation and recovery of oligokeratin is extremely easy.

The oligokeratins provided by the present invention generally have a molecular weight in the range of 200 to 10,000, and under suitable conditions may have a molecular weight in the range of 500 to 5,000. In addition, the above-mentioned oligokeratin has a thiol side chain generated as a result of cleavage of at least a portion of the disulfide bonds that form the crosslinks of the polypeptide chain of keratin, but the amino group originally possessed by the polypeptide chain In addition to functional groups such as carboxyl groups, alcoholic and phenolic hydroxyl groups, it has an amino group and a carboxyl group at the end of its molecular chain, making it highly functional and having excellent performance as a cosmetic base material. .

The method of the present invention will be explained in more detail below.

Keratin is a type of structural (tissue) protein that exists in large amounts in the skin of vertebrates and tissues that develop outward from it, and therefore, the keratin materials used as raw materials in the method of the present invention include: Examples include horns, hooves, claws, body hair, feathers, etc. of vertebrate animals, preferably wool and feathers, and these can be used as they are or after being crushed or cut into appropriate sizes as required.

In addition, these keratin substances can be used as needed.
Pretreatment such as cleaning and degreasing can also be performed.

According to the invention, the keratin material is first reduced. Reduction can generally be carried out in a liquid medium. The liquid medium that can be used must be stable against reduction, and the selection of the reducing medium is extremely important in order for the keratin material to retain its raw material form even after the reduction reaction. That is, the liquid medium used in the present invention must be selected so as not to dissolve or swell the keratin before and after the reduction reaction. Suitable examples of such a medium include water that does not contain a solubilizing agent such as urea; alcohols such as methanol, ethanol, n-propanol, and isopropanol; or a mixture of water and alcohols.

During reduction, the raw keratin material described above is immersed in the liquid medium. In this case, there is no strict limit on the weight ratio of keratin material to liquid medium;
Depending on the type and shape of the keratin material used,
Advantageously, it is at least 1:20 or more, usually in the range 1:30 to 1:100.

As reducing agent, any reducing agent is used which is capable of cleaving disulfide bonds (-SS-) in keratin materials into thiol groups (-SH).

As reducing agents that can be used, organic and inorganic reducing agents of the type that act nucleophilically on disulfide bonds (-S-S-) are generally preferred, and as organic reducing agents, thiol derivatives and phosphorus compounds are preferred. Suitable agents, in particular mercaptoethanol, thioglycolic acid, tributylphosphine, triphenylphosphine, etc., are advantageously used, and inorganic reducing agents include, for example, sodium bisulfite (NaHSO ₃ ) and sodium bisulfide ( NaSH) etc. are suitable.

The reduction can be carried out by methods known per se [Reference: WGCrewther et al. Advan.
Protein Chem., 20 , 191 (1965), JA
Maclaren, Aust.J.Chem., 15 , 824 (1962), J.
A. Maclaren et al. Aust. J. Chem., 19 , 2355
(1966)]. For example, when using the above-mentioned thiol derivatives or inorganic reducing agents, generally an excess equivalent amount, relative to the disulfide bonds in the keratin material used,
It is usually advantageous to use at least 2 equivalents per disulfide bond, preferably 4 to 10 times the equivalent, and when using the phosphorus compound, it is advantageous to use it in an amount of at least 2 times the equivalent, preferably 4 to 10 times, per disulfide bond. Generally, it is sufficient to use an equivalent to slightly excess equivalent amount of the phosphorus compound.

The amount of disulfide bonds in keratin materials can be determined by amino acid analysis, and the method is known [M. Friedman, AT
Noma, Textile Res. J., 40 , 1073 (1970)].

Reduction can be either acidic, neutral or alkaline.
It can also be carried out under PH conditions. However, under too high or low pH conditions, swelling or dissolution of the keratin material occurs, and the reduced keratin material is unable to retain its raw form.
It is usually advantageous to carry out the reduction at a pH in the range of 1 to 8.

There are no particular restrictions on the temperature and pressure during reduction;
The reduction temperature can be varied within a wide range depending on the type of reducing agent used, the type of raw materials, etc., but room temperature is sufficient as the reduction temperature. Although normal pressure is sufficient for the pressure during reduction, the reaction can also be carried out under reduced pressure or increased pressure, if necessary.

The reduction can also preferably be carried out in an inert gas atmosphere, for example nitrogen.

Although the disulfide bonds in the keratin material are cleaved by the reduction as described above, the reduced keratin material remains in the reducing medium in the form of the untreated raw material. Therefore, the reduced keratin material can be easily separated and recovered by means such as filtration or centrifugation, and after thorough washing, it can be subjected to the enzyme treatment step described below. The separation and washing operations must be performed with care so that the thiol side chains generated by cleavage in the keratin material are not oxidized during the separation operation; for example, they may be carried out in an inert gas atmosphere, or Alternatively, it is preferable to adjust the pH of the reducing medium and washing liquid to 6 or less to perform the separation and washing operations in a stable state against oxidation of the thiol side chain.

To adjust the pH, use a water-soluble acidic substance, such as hydrochloric acid,
This can be easily carried out by adding an inorganic acid such as sulfuric acid, nitric acid or phosphoric acid; or an organic acid such as acetic acid, propionic acid or p-toluenesulfonic acid. In particular, volatile acids, particularly hydrochloric acid, are preferred for reasons such as ease of separation of the acid from the oligokeratin produced.

The reduced keratin material obtained as described above, which retains the form of the raw material, is then subjected to enzymatic hydrolysis treatment. In the present invention, the enzymatic hydrolysis treatment is carried out in an aqueous solution adjusted to optimize the activity of the hydrolase used, but a small amount of acid is added to the aqueous solution to adjust the pH of the aqueous solution. Alternatively, one of the features and advantages of the present invention is that it contains only alkali and the hydrolytic enzyme used. As the hydrolase, acidic enzymes such as pepsin and neutral enzymes such as papain, pronase, trypsin, etc. can be used. However, the thiol side chain of reduced keratin is unstable and easily oxidized when the pH of the aqueous medium is 6 or higher, and easily forms disulfide bonds [References: Shigeru Daijo, "Chemistry of Organic Sulfur Compounds (Part 1)", Kagaku Doujin, Chapter 2, 1968]. Therefore,
It is desirable to carry out the hydrolysis treatment preferably using an acidic enzyme such as pepsin. Furthermore, the temperature of the enzymatic hydrolysis treatment must be controlled so that the enzyme used exhibits optimal activity. For example, in the case of pepsin, a temperature range of 35-40°C is preferred. The molecular weight of the obtained oligokeratin can be adjusted by appropriately selecting the type and amount of enzyme used, reaction time, temperature, etc. If the enzyme used needs to be inactivated, this can be done by methods known in the field of enzyme chemistry, such as heat treatment or pH adjustment.

In the separation and recovery of oligokeratin from the oligokeratin aqueous solution obtained as described above, if there are insoluble foreign substances or insoluble solids that adhere to keratin, these are removed by means such as filtration or centrifugation. Thereafter, it can be carried out by a conventional method, for example, by concentrating the oligokeratin aqueous solution and then freeze-drying it.

The oligokeratin thus obtained is a white to pale yellow powder and can be used as a cosmetic base material, particularly for hair.

As mentioned above, purification processes such as ultrafiltration and dialysis have traditionally been indispensable when producing oligokeratin. It required a lot of time, but
The present invention has made it possible for the first time to produce oligokeratin through an extremely simple process that does not require such a purification process, does not require a large plant, and does not require a long time.

The present invention will be explained in more detail with reference to Examples below.

Example 1 10 g of wool fiber is immersed in 1000 c.c. of 20% (W/W) n-propyl alcohol aqueous solution, and sufficient nitrogen gas is passed through the solution. And 2.5ml of tri-n-
Add butylphosphine and shake gently at room temperature for 1 day. At this point, the wool retains its original fiber state, which is filtered and washed with ethanol, then dried under reduced pressure (approximately 10 mmHg) at 30°C.
Fibrous reduction treated wool was obtained. 10 g of the fibrous reduced wool thus obtained was immersed in 1000 c.c. of 0.075N hydrochloric acid (PH1.3), 200 mg of pepsin was added, and the mixture was shaken gently at 40°C for 1 day, followed by adding water. Carry out a decomposition reaction. As the hydrolysis reaction progresses, the wool fibers dissolve in the hydrochloric acid, and after 3 to 4 hours, the insoluble portion is almost completely gone. After filtering the oligokeratin hydrochloric acid solution obtained in this way, it was concentrated in an evaporator to about 300 c.c., and then freeze-dried to give 9.8 g of pale yellow powder oligokeratin (solubilization rate 98%).
I got it. The oligokeratin was carboxymethylated with iodoacetic acid, and its molecular weight was determined to be 1100 by gel filtration (using Sephadex G-50). In addition, the molecular weight was similarly calculated|required also in the following example.

Reference example 1 10g of untreated wool fiber without reduction treatment
It was immersed in 1000 c.c. of 0.075N hydrochloric acid and gently shaken at 40°C for one day to perform a hydrolysis reaction. At this point, most of the fibrous wool remained, and when this was filtered, washed with ethanol, and dried, it weighed 9.2 g. In other words, the solubilization rate is 8%.

Example 2 In Example-1, 15 ml of thioglycolic acid was used instead of tri-n-butylphosphine as the reducing agent, and 1000 c.c. of water was used instead of the 20% n-propyl alcohol aqueous solution as the reduction reaction dispersion medium. Except using
The same operation as in Example 1 was performed. As a result, pale yellow powder oligokeratin was obtained with a solubilization rate of 93%. The molecular weight of this product was 1000.

Example 3 In Example 1, 15 ml of 2-mercaptoethanol was used instead of tri-n-butylphosphine as the reducing agent, and 1000 c of water was used instead of the 20% n-propyl alcohol aqueous solution as the reduction reaction dispersion medium. The same operation as in Example 1 was performed except that c. As a result, pale yellow powder oligokeratin was obtained with a solubilization rate of 90%. The molecular weight of this product was 1000.

Example 4 In Example-1, sodium hydrogen sulfite 20 was used instead of tri-n-butylphosphine as the reducing agent.
The same operation as in Example 1 was carried out, except that 1000 c.c. of water was used instead of the 20% n-propyl alcohol aqueous solution as the reduction reaction dispersion medium. As a result, pale yellow powder oligokeratin with a solubilization rate of 92% and a molecular weight of 1100 was obtained.

Example 5 The same procedure as in Example 1 was performed except that 200 mg of papain was used instead of pepsin as the hydrolytic enzyme and the pH of the hydrolysis reaction solvent was adjusted to 6.8. As a result, pale yellow powder oligokeratin with a molecular weight of 1500 was obtained with a solubilization rate of 50%.

Example 6 The same procedure as in Example 1 was carried out except that 200 mg of pronase P was used instead of pepsin as the hydrolase and the pH of the hydrolysis reaction solvent was adjusted to 7.2. As a result, pale yellow powder oligokeratin with a solubilization rate of 85% and a molecular weight of 1300 was obtained.

Claims (1)

[Claims] 1. When reducing and then hydrolyzing a keratin material, the keratin material is subjected to a reduction reaction while retaining the form of the raw material in the reaction medium before and after the reduction reaction, and then the keratin material is subjected to a reduction reaction in water using a protein hydrolase. A method for producing a low molecular weight keratin material, which comprises performing a hydrolysis reaction.