JPH0721061B2 - Method for producing water-soluble keratin protein - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial field of application) The present invention relates to a hard protein which is a main component of a substance constituting wool, feathers, hair, body hair such as cows and pigs, horns, nails and hoofs. Keratin (hereinafter referred to as "keratin protein")
The present invention relates to a method for producing a keratin protein having a desired desired molecular weight by subjecting to, after being subjected to an alkali treatment, partial hydrolysis with an acid or alkali, enzymatic degradation, oxidative degradation or reductive degradation. The keratin protein produced by the present invention is used for foods, cosmetics, industrial products and the like.

(Prior Art) Conventionally, various researchers have proposed various methods for solubilizing keratin proteins.

The conventional method is basically such that the disulfide bond (-S-S-) existing in the cystine residue in the keratin protein is cleaved with a reducing agent to give a thiol group (-SH) (first step), and then the liquid. It consists of two steps in which an enzyme or the like is caused to act in a medium to cleave the peptide bond of the main chain (second step). In the first step of this method, first, keratin protein is swollen by adding aqueous urea, and then mercaptans such as thioglycolic acid, mercaptoethanol, thioglycerin and thiosalicylic acid or sodium sulfide, potassium sulfide, calcium sulfide, sulfide. The disulfide bond is cleaved using a reducing agent such as a sulfide such as triethanolamine, diethanolamine sulfide and monoethanolamine sulfide. In addition, in the second step, generally pH 1-
Acidic enzymes such as pepsin in the region 3 and neutral enzymes such as promeline in the region of pH 5-8 are allowed to act for a long time to cleave the peptide bond. Such a two-step method is currently the center of research on a method for producing water-soluble keratin.

(Problems to be Solved by the Invention) However, such a conventional method has a problem peculiar to its configuration.

The ease of peptide cleavage in the second step of the conventional method is
It depends on the conditions of the first step. Therefore, what kind of contents and conditions of the first step is still an important issue, and various studies have been made by those skilled in the art.
However, in order to make the reducing agent work efficiently in the first step,
There is a limitation that the pH must be in the alkaline region, and it is not always easy to study the conditions.

Further, both the first step of the conventional method and the second step using an enzyme or the like are complicated in operation and the control of the reaction is relatively difficult. Further, there is a problem that the time required for each process is long and the cost is high. Further, the conventional method has a problem that the yield is poor because the loss in each step is large.

(Means for Solving the Problems) The present invention solves the problems of the conventional method, and reduces the treatment time of a water-soluble keratin protein having a desired molecular weight according to the intended use of foods, cosmetics, industrial products and the like. The present invention provides a method for obtaining a good yield in a simple and easy process.

The keratin to which the present invention is applied may be any of the keratin proteins constituting wool, feathers, hair, body hair of cattle, pigs, etc., horns, nails, hoofs and the like.

The present invention is a method which essentially comprises two steps: alkali treatment and partial decomposition. The main feature of the present invention is that the keratin protein is treated with an alkali to produce a water-soluble keratin protein through a mechanism that is fundamentally different from the conventional method.

The alkaline treatment of the present invention is performed by immersing the keratin protein in a solution of alkaline salt. The alkaline salt of the present invention includes a wide range of salts which show alkalinity when made into a solution, and among them, it is preferable to use calcium hydroxide, sodium hydroxide or potassium hydroxide. Further, calcium hydroxide is particularly preferable and general.

By dipping the keratin protein in a solution of such an alkaline salt, the disulfide bond (-
S-S-) is partially converted into a thioether bond (-S-). Cleavage of disulfide bond to thiol group (-SH)
Unlike the conventional method described above, the present invention has a novel feature in that a thioether bond is partially formed in a keratin protein. Specifically, it is characterized by changing a cystine residue having a disulfide bond in a keratin protein to a lanthionine residue having a thioether bond. The thioether bond is so strong that it is not cleaved in the peptide decomposition step after alkali treatment and remains in the keratin protein to the end. Therefore, it becomes possible to control the molecular weight of the final product, the water-soluble keratin protein, by controlling the production of lanthionine residues during the alkaline treatment (Test Example 2).

The production of lanthionine residue may be controlled by changing any one of the conditions such as the concentration of the alkaline salt solution, the time and temperature of the alkali treatment, or a combination of these conditions. Good. Specifically, when calcium hydroxide is used as the alkaline salt, a concentration of 0.1% by weight to a saturated solution (3-4% by weight) can be used, the pH is in the range of 11-13, and the treatment temperature is Is
The immersion time can be changed within 40 hours at 40 ° C or less. When sodium hydroxide or potassium hydroxide is used, it can be used up to a concentration of 0.001 to 0.1 N, pH is in the range of 11 to 13, and treatment temperature is 40 ° C or lower.
The immersion time can be changed within 24 hours. During the alkaline treatment, the alkaline salt solution may be stirred. Also,
Before and after the alkali treatment, the keratin protein is appropriately washed with water by a usual method.

For the keratin protein to be treated, it is possible to efficiently obtain a water-soluble keratin protein having a desired molecular weight by clarifying the relationship between the alkaline treatment conditions and the amount of lanthionine residue produced in advance.

For example, regarding the keratin used in Test Example 1, the conversion rate from cystine residue to lanthionine residue increases as the alkali treatment time increases, and the specific relationship is as shown in Table 1. By clarifying this relationship also with respect to other conditions such as processing temperature, it becomes possible to appropriately select the conditions for obtaining water-soluble keratin having a desired molecular weight. Furthermore, by combining with the conditions for peptide degradation after alkali treatment, it becomes possible to more accurately control the molecular weight. As described above, since the present invention has many conditions under which the present invention can be changed, the present invention is also characterized in that the molecular weight can be adjusted with higher accuracy.

It is also clear from Test Example 1 that the alkaline treatment according to this test example does not substantially change amino acid residues other than the cystine residue and the lanthionine residue. Therefore, the alkaline treatment of the present invention selectively acts on cystine residues and does not involve undesirable side reactions. Further, the alkali treatment of the present invention is very simple in that the keratin protein is immersed in the solution of the alkaline salt, and the treatment time is short, so that it is extremely practical.

The keratin protein is treated with alkali and then subjected to partial decomposition of peptide bonds. For such partial decomposition, a commonly used method such as acid hydrolysis, alkali hydrolysis, enzymatic decomposition, oxidative decomposition or reductive decomposition can be used as it is. In the above-mentioned conventional method, since the acid or alkali partial decomposition cannot be carried out because the decomposition proceeds to the amino acid level, the present invention has a wider range of choices for the peptide decomposition method. When the acid hydrolysis is carried out according to the present invention, for example, keratin protein is added at a ratio of 1-2 kg to 4-8 kg of 10-30% by weight of hydrochloric acid and the decomposition is carried out at 80-100 ° C for 1-10 hours. After partial decomposition, it is deoxidized with an anion exchange resin. When alkali hydrolysis is performed, for example, 0.1-10% sodium hydroxide aqueous solution 4
Keratin protein was added at a ratio of 1-2 kg to -8 kg.
Decompose at -100 ° C for 1-5 hours. After partially decomposing, it is deoxidized with a cation exchange resin.

The method for producing a water-soluble keratin protein of the present invention may include steps other than the above-mentioned alkali treatment and partial decomposition of peptides. For example, desalting after partial decomposition of the peptide,
Purification such as filtration, deodorization and decolorization may be performed. Further, it may be concentrated and dried after partial decomposition and purification. Further, a preservative and the like may be added in the form of a solution.

The present invention will be further specifically described by the following examples and test examples, but the scope of the present invention is not limited to these examples and test examples.

Example 1 1 kg of keratin protein was washed with water and then immersed in a 1% by weight calcium hydroxide aqueous solution for 6 hours. Thereafter, the keratin protein was washed with water, 30% by weight hydrochloric acid acidic solution 4 was added, and the mixture was boiled at 100 ° C. for 2 hours. The solution was decolorized and deodorized with activated carbon to obtain light yellowish brown oligokeratin. The molecular weight of the obtained oligokeratin was found to be about 1000 as a result of measurement by gel permeation method.

Example 2 1 kg of keratin protein was washed with water and then immersed in a 1 wt% calcium hydroxide aqueous solution for 2 hours. Then, wash the keratin protein with water, add 10% by weight hydrochloric acid acidic solution 4 and
It boiled for an hour. The solution was decolorized and deodorized with activated carbon to obtain light yellowish brown oligokeratin. The molecular weight of the obtained oligokeratin was found to be about 400 as a result of measurement by gel permeation method.

Example 3 1 kg of keratin protein was washed with water and then immersed in a 0.5 wt% calcium hydroxide aqueous solution for 24 hours. Then, the keratin protein was washed with water, 50% of formic acid 5 was added, and oxidative decomposition was carried out at 35 ° C. for 24 hours. The solution was decolorized and deodorized with activated carbon to obtain light yellowish brown oligokeratin. The molecular weight of the obtained oligokeratin was found to be about 1000 as a result of measurement by gel permeation method.

Test Example 1 After washing the keratin protein with water, it was immersed in a calcium hydroxide solution at a final concentration of 0.5% by weight at room temperature. The composition of keratin protein not soaked and keratin protein extracted 1, 2, 6 and 24 hours after soaking was washed with water, and then the composition of amino acid residues such as cystine residue and lanthionine residue in the keratin protein was determined. Examined. The result is
It is as shown in Table 1.

Test Example 2 The keratin protein was washed with water and then immersed in a 2.0 wt% calcium hydroxide aqueous solution at 28 ° C. for 1, 6 and 24 hours. afterwards,
1 kg of keratin protein washed and neutralized was put into a 2.9 wt% sodium hydroxide aqueous solution 8 and hydrolyzed at 80 ° C. for 3 hours. After the keratin protein after hydrolysis is desalted, it is detected with UV light having a wavelength of 280 nm, and Sephadex (Sephadex) is detected.
dex) G-75 column was used to measure the change in molecular weight.
FIG. 1 is a chromatogram of each of the samples immersed for 1, 6 and 24 hours. From the comparison of the peak of each sample and the calibration curve of the standard substances such as ovalbumin, bovine albumin and cytochrome C, the immersion time of 1, 6 and
The molecular weights of the 24-hour sample peaks are 9,700 and 19,00, respectively.
Estimated to be 0 and 36,000. This example shows that the molecular weight of the final product increases as the lanthionine residue is increased by increasing the immersion time in the alkaline salt.

[Brief description of drawings]

FIG. 1 is a chromatogram of Sephadex G-75 of keratin protein which was subjected to alkaline hydrolysis after being immersed in a calcium hydroxide solution under the conditions of Test Example 2.

Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display area C07K 1/12 8318-4H

Claims (1)

[Claims] 1. A method for producing a water-soluble keratin protein by immersing the keratin protein in a solution of an alkaline salt and then partially hydrolyzing, enzymatically decomposing, oxidatively decomposing or reductively decomposing it with an acid or an alkali.