JPH0790182A - Silk fibroin excellent in shelf stability and its production - Google Patents

Abstract

PURPOSE:To industrially advantageously produce an aqueous solution of silk fibroin and a water-soluble silk fibroin powder which are of high quality and excellent in transparency of the aqueous solution and have good shelf stability. CONSTITUTION:An aqueous solution of silk fibroin or a water-soluble silk fibroin powder, which contains urea and/or thiourea. It is produced by mixing an aqueous solution of silk fibroin with urea and/or thiourea, and freeze drying or spray drying the mixture as required.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a silk fibroin aqueous solution of high quality and excellent in storage stability, or a water-soluble silk fibroin powder which is a dried product thereof, and a method for producing the same.
Particularly, the present invention relates to a silk fibroin aqueous solution or a water-soluble silk fibroin powder suitable for use in industrial materials and a method for producing the same.

[0002]

2. Description of the Related Art Silk fibroin (silk protein) has properties such as proper hygroscopicity and moisturizing property, excellent affinity for skin and hair, and protective action, and therefore has been conventionally used in silk fibroin aqueous solutions and Silk fibroin powder has been used for applications such as cosmetic bases. In addition, recently, research is being conducted to apply this to applications such as industrial materials and medical bases.

Generally, an aqueous silk fibroin solution is disclosed in Japanese Patent Publication No.
7-4723, copper-ethylenediamine aqueous solution, copper hydroxide-ammonia aqueous solution, copper hydroxide-alkali-glycerin aqueous solution, lithium bromide aqueous solution, calcium or magnesium or zinc hydrochloride or nitrate, or It is produced by a method in which a scoured silk raw material is dissolved in at least one solvent selected from the group consisting of an aqueous solution of thiocyanate and an aqueous solution of sodium thiocyanate and dialyzed. Also, Japanese Patent Publication No. 59-31520 proposes a method for producing a silk fibroin peptide, which comprises hydrolyzing the thus obtained 0.5 to 20% by weight aqueous silk fibroin solution with an enzyme, an acid or an alkali. Has been done.

In the case of these methods, unlike the case where a strong acid or a strong alkaline aqueous solution is used as the solvent, the solvent is mild, so that the useful protein structure of silk fibroin is not damaged, and dialysis is carried out by the membrane surface area ( Since a multilayer membrane structure or a hollow fiber bundle structure satisfying cm ² ) / priming capacity (cm ³ ) ≧ 10 is used, a transparent and uniform high-quality silk fibroin aqueous solution can be stably produced. it can.

By the way, the quality stability of these silk fibroin aqueous solutions for long-term storage is somewhat insufficient, and there are various problems and restrictions in use. For example, when it is stored for less than 1 month, it turns dark brown, becomes turbid, or, in a remarkable case, a large amount of gel is precipitated.

As a countermeasure against this, the present inventors have hitherto added a chelating agent (JP-A 63-92671), a polyhydric alcohol (JP-A 2-113066), an organic amide compound (JP-A 2-113066) to the aqueous solution. No. 2-240165) and a water-soluble heterologous protein (Japanese Patent Laid-Open No. 2-281079) have been proposed.

On the other hand, as silk fibroin and silk fibroin peptide powder, Japanese Patent Publication No. 40-24920, Japanese Patent Publication No. 26-4947 and Japanese Patent Publication No. 58-3844.
No. 9 discloses a fibrous silk fibroin powder obtained by crushing silk thread as it is or embrittled by a chemical treatment, and a colloid solution obtained by dissolving and dialysis silk fibroin in an appropriate concentrated neutral salt or the like. Granular silk fibroin powder obtained by pulverizing gel silk fibroin produced by mist drying, and a colloidal solution obtained by dissolving silk fibroin in a suitable inorganic neutral salt or alkaline aqueous solution and then dialyzing Is added, air is blown, isoelectric point coagulation, ultrasonic treatment or stirring at a high shear deformation rate is used to coagulate and precipitate silk fibroin, which is dehydrated, dried, and then pulverized, followed by finely powdered silk fibroin.

The above-mentioned silk fibroin powder is essentially insoluble in water. On the other hand, as the water-soluble silk fibroin powder, a colloidal solution obtained by dissolving silk in a suitable inorganic neutral salt or alkaline solvent is hydrolyzed. After decomposition, spray drying (Japanese Patent Laid-Open No. 56-40695) and freeze drying a colloidal solution of silk fibroin having an average degree of polymerization of 3 to 600 (Japanese Patent Laid-Open No. 61-18).
No. 0800), a method for producing flaky silk powder by freeze-drying an aqueous silk fibroin solution having an average degree of polymerization of 650 or more (Japanese Patent Application No. 5-147080) has been proposed.

Among the substances to be mixed as the stabilizer of the silk fibroin aqueous solution, the chelating agent is ineffective when the average degree of polymerization of silk fibroin is 600 or more.
Since the water-soluble heterologous protein is generally translucent in its own aqueous solution, it reduces the transparency of the silk fibroin aqueous solution when mixed. Further, organic amide cannot be used as a cosmetic base, and if it is used incorrectly, it destabilizes the aqueous solution. Polyhydric alcohol is also a gelling agent for the silk fibroin aqueous solution, so that it should be used with caution.

The above is the same as in the case of silk fibroin or silk fibroin peptide powder, and the storage stability of an aqueous solution obtained by dissolving this in water is not good, especially when the average degree of polymerization is 650 or more. The re-dissolvability itself deteriorates and it takes a long time to completely dissolve it.

[0011]

The inventors of the present invention have completed the present invention as a result of earnest research on improvement of product properties and quality of silk fibroin. The purpose of the present invention is to
An object of the present invention is to provide a silk fibroin aqueous solution and a water-soluble silk fibroin powder having high quality, excellent transparency of the aqueous solution, and good storage stability. Another object is to provide a method for industrially producing such an aqueous silk fibroin solution and water-soluble silk fibroin powder.

[0012]

The present invention relates to an aqueous silk fibroin solution or a water-soluble silk fibroin powder characterized by containing urea and / or thiourea, and the method of the present invention is to dissolve it in an aqueous medium. The obtained silk fibroin aqueous solution is mixed with urea and / or thiourea, and if necessary, freeze-dried or spray-dried.

The effect of adding urea and / or thiourea to the silk fibroin aqueous solution and water-soluble silk fibroin powder of the present invention is remarkable, and in the silk fibroin aqueous solution, the transparency of the solution is remarkably improved, The liquid can be stored for several months or longer without causing cloudiness or gel-like substances. A particularly excellent storage stabilizer for urea and / or thiourea is that it improves the transparency of the aqueous solution and silk fibroin does not gel even if the mixing concentration is increased. Urea is particularly suitable for use as a cosmetic base. There is no limitation and it is easy to use. On the other hand, the effect of addition to the water-soluble silk fibroin powder is also good, and especially the re-solubility of silk fibroin powder having an average degree of polymerization of 650 or more is remarkably improved. Of course, the storage stability and the transparency improving effect of the aqueous solution obtained by dissolving the powder are excellent.

The present invention is effective for an aqueous silk fibroin solution having an average number of monomers of 2 or more, and is particularly effective when the average number of monomers is more than 600 (reference material, JP-A-63-9).
2671).

The raw materials for the silk fibroin used in the present invention include eyebrows, raw silk, eyebrows scraps, raw silk scraps, screws, fried cotton, silk cloth scraps, burettes, etc. in the presence of an activator, if necessary, according to a conventional method.
The dried product is prepared by removing sencin in warm water or in warm water in the presence of an enzyme.

The solvent of silk fibroin applied to the present invention is copper-ethylenediamine aqueous solution, copper hydroxide-ammonia aqueous solution (Schweiser reagent), copper hydroxide-alkali-
Glycerin aqueous solution (Lohe's reagent), lithium bromide aqueous solution, calcium or magnesium or zinc hydrochloride or nitrate or thiocyanate aqueous solution, and sodium thiocyanate aqueous solution may be mentioned, but from the viewpoint of cost and use, hydrochloric acid of calcium or magnesium. Salts or nitrates are preferred. The concentration of these aqueous solutions varies depending on the type of solvent used, temperature, etc., but the concentration of the metal salt or the like is usually 10 to 80% (weight), preferably 20 to 40% (weight). Although it dissolves even at 80% (weight) or more, there is no substantial difference in the silk fibroin aqueous solution produced, which is a problem in terms of economy.

The silk raw material after scouring is added to the solvent consisting of the above aqueous solution, and the temperature is 60 to 95 ° C, preferably 70 to 85 ° C.
Although it dissolves uniformly in a device such as a kneader, the liquid ratio is usually 2 to 50, preferably 3 to 30.

In order to obtain a highly pure silk fibroin aqueous solution from the obtained silk fibroin solution, dialysis is subsequently performed. For dialysis, a dialysis membrane typified by a cellophane membrane or a dialysis machine using hollow fibers is used to almost completely remove the salts and the like. In this case, it is necessary to specify the dialysis amount and the dialysis membrane area in order to narrow the molecular weight distribution of the target silk fibroin as much as possible and to adjust the ratio of the α-structured silk fibroin to 50% by weight or more. That is, desalting is performed using a multilayer membrane structure or a hollow fiber focusing structure that satisfies the following formula. Membrane surface area (cm ² ) / priming capacity (cm ³ ) ≧ 10 (Here, the priming capacity means the inner volume between dialysis tubes or membranes)

When the above numerical value is less than 10, the membrane separation is not performed rapidly and the residence time in the dialyzer becomes long, and the obtained aqueous fibroin solution often has already started to decompose. In that case, the fibroin protein becomes insoluble in water (β structure) due to denaturation due to putrefaction, and it is difficult to make it soluble in cold water again. In particular, in order to carry out the present invention smoothly and economically, the above numerical value is preferably 30 or more, and particularly preferably 50 or more. In order to satisfy this condition, for example, in the case of a hollow fiber bundle structure, the hollow fiber diameter needs to be 4 mm or less.

The dialysate obtained by the method of the present invention has a very low residual salt concentration of 0.003 to 0.06% (by weight), and especially when the hollow fiber diameter is about 0.2 mm, the membrane surface area is (Cm ² ) / priming capacity (cm ³ ) ≈200, and this can be achieved with a residence time in the dialyzer of several 10 minutes, whereby an extremely high-quality silk fibroin aqueous solution can be obtained.

In the present invention, the protein concentration of the silk fibroin aqueous solution is not essential, but is usually 1 to 30%.
(Weight), preferably 2-20% (weight), optionally concentrated. If it is 1% (weight) or less, it is uneconomical because it needs to be concentrated in a later step, and if it is 30% (weight) or more, the viscosity becomes high and reaction and operation are difficult.

When silk fibroin having an average molecular weight of several hundred to several thousand is to be obtained, it is hydrolyzed using an enzyme, an acid or an alkali.

Examples of the hydrolytic enzyme applied to the present invention include ordinary proteolytic enzymes such as pronase obtained from actinomycetes, an enzyme group considered to be a mixture of several proteinases such as prolase obtained from papaya, promerin and the like. These may be used alone or in admixture of two or more. The amount of the enzyme used varies depending on the type of enzyme, the purity, the reaction conditions, the average degree of polymerization of the target silk fibroin, etc., but is usually 0.01 to 10.0% (weight) with respect to the silk fibroin, preferably 0.02-
It is 5.0% (weight). The conditions of hydrolysis in this case are usually 5 to 9, preferably 6 to 8.5, and a temperature of 20 to 70 ° C., although they vary depending on the type and concentration of the enzyme used.
It is preferably carried out at 30 to 45 ° C. for 0.1 to 72 hours, preferably 0.5 to 12 hours.

Examples of the acid used for hydrolysis include inorganic acids such as hydrochloric acid and sulfuric acid, and organic acids such as citric acid, tartaric acid, malonic acid, succinic acid and maleic acid. As the alkali, sodium hydroxide, potassium hydroxide, lithium hydroxide, an inorganic alkali such as aqueous ammonia, an organic alkali such as methylamine can be used, but the reactivity, economy,
From the viewpoint of stability, sodium hydroxide and potassium hydroxide are preferable.

The condition of hydrolysis with an acid or alkali varies depending on the type of acid or alkali used, the average degree of polymerization of the desired peptide and the degree of polymerization distribution, but is usually 0.03.
~ 3.0N, preferably 0.3N or less at a temperature of 2
0-110 ° C, preferably 30-100 ° C, 0.5-
The reaction is carried out for 48 hours, preferably 1 to 24 hours, and then an alkali or an acid is added to neutralize.

In the present invention, in order to impart long-term storage stability to the silk fibroin aqueous solution, urea or thiourea or a mixture is added to the solution after dialysis or the solution after hydrolysis, and further if necessary. Accordingly, a fungicide / preservative and a chelating agent are added.

The amount of urea or thiourea added is preferably 1 to 200% (by weight), more preferably 10 to 100%, based on the pure silk fibroin in the aqueous solution. If it is 1% or less, almost no addition effect is recognized, while if it is added in excess of 200%, the effect remains the same as 200% or less and it is not economical.

The addition of a fungicide / preservative and a chelating agent is preferable for more stable storage of the silk fibroin aqueous solution, and the addition amount is 0.01 to 1.0 for the mold / preservative.
% (Weight), and the chelating agent is 0.01 to 1.0% (weight). The fungicides and preservatives are not particularly limited, and examples thereof include benzoic acid, sorbic acid, dehydroacetic acid, propionic acid, salts thereof, and P-oxybenzoic acid ester. As the chelating agent, any ordinary chelating agent can be applied if there is no problem in the use of silk fibroin, but EDTA or sodium tripolyphosphate or sodium hexametaphosphate is preferable from the economical point of view. EDTA is pH
In view of the above, 2Na salt or 3Na salt is more desirable.

In the present invention, in order to obtain a water-soluble silk fibroin powder characterized by containing urea or thiourea, freeze-drying or spraying the silk fibroin aqueous solution containing urea or thiourea as described above. To dry.

Freeze-drying can be carried out by an ordinary freeze dryer, but the silk fibroin concentration in the aqueous solution is 2%.
When it is less than (weight), the yield of the obtained dried product is extremely small compared to the amount of the aqueous solution in process, which is not practical.

For freeze-drying, for example, first, an aqueous silk fibroin solution is placed in a shallow vat-shaped container at a depth of 5 to 1.
Inject it to 0 mm and once the whole is -20 to -40
Quench to ℃ and freeze. Insert this into multiple shelves in the chamber of the freeze-dryer, and at the beginning, about 0.5 torr,
At the end, it is dried under reduced pressure of about 0.05 torr. During vacuum drying, the heater embedded in the shelf replenishes the heat of vaporization,
Adjust the temperature of the frozen product to an appropriate range (eutectic point).

Examples of the spray dry treatment include a method using a nozzle rotating at a high speed, a method using a high-speed air stream, and the like. Then, the silk fibroin aqueous solution is dried and pulverized in a zero atmosphere at 40 to 150 ° C., preferably 70 to 120 ° C. for a short time. The spray drying conditions can be appropriately selected to obtain the silk fibroin powder having the desired performance.

The dried product obtained by freeze-drying or spray-drying is pulverized with a pulverizer into a desired size. The crushed product is a soft powder with a brilliant, silky luster and a bulk specific gravity of 0.05 to 0.5 depending on the size of the powder.
It is a very light powder with g / cm ³ .

[0034]

EXAMPLES The present invention will be specifically described below with reference to examples.
In addition, the transparency in the examples was measured by JISK-0102 (industrial drainage test method).

EXAMPLE 1 Raw silk waste was used as a raw material for silk fibroin and
9 parts of a solution of 30 parts of Marcel soap and 3000 parts of water
Stir-refined for 3 hours at 5-98 ° C to remove residual glue 0.1%
The content was reduced to the following, washed with water, and dried with hot air at 80 ° C. On the other hand, 100 parts of water was mixed with 100 parts of calcium chloride (CaCl ₂ .2H ₂ O) to prepare 200 parts of a 38 wt% calcium chloride aqueous solution, and the mixture was heated to 110 ° C. 40 parts of scoured raw silk scraps were added to this with stirring with a kneader for 5 minutes, and further stirred for 30 minutes to completely dissolve it.

Next, inner diameter 200 μ, film thickness 20 μ, length 5
Bundling 2000 00mm regenerated cellulose hollow fibers,
Using a hollow fiber type dialysis device in which both ends of the solution are focused and fixed (sealed) without blocking the hollow hole, the solution is introduced at a rate of 0.2 l / hour and dialyzed with deionized water. Then, an aqueous silk fibroin solution was obtained. The fibroin concentration of the aqueous fibroin solution was 10.0% (weight), the average degree of polymerization was 1200, and the residual calcium chloride was 0.001% (weight).

The obtained silk fibroin aqueous solution was divided into 2 minutes,
Urea 1.0% (by weight) in solution concentration, EDTA
2Na salt 0.2% (weight), sodium benzoate 0.
3% (by weight) was mixed, the other was the same and the urea alone was not mixed. The results are shown in Table 1 as the transparency of the aqueous solution.

[0038]

[Table 1]

As is clear from Table 1, the addition of urea has a remarkable effect in improving the transparency of the silk fibroin aqueous solution, and also has an excellent effect in improving the storage stability.

Example 2 The procedure of Example 1 was repeated except that the urea was replaced with thiourea. The results are shown in Table 2.

[0041]

[Table 2]

As is clear from Table 2, the transparency improving effect and storage stabilizing effect of thiourea with respect to the silk fibroin aqueous solution are remarkable.

Example 3 In accordance with Example 1, the concentration of urea added, the transparency improving effect and the storage stability effect were tested. The effect is shown in Table 3.

[0044]

[Table 3]

As is clear from Table 3, the transparency improving effect and the storage stability effect increase as the urea addition amount increases in the range of 1.0 to 200%. However, 200%
If added over the range, the effect is not changed and it is not economical.

Example 4 An aqueous silk fibroin solution was produced in the same manner as in Example 1. The concentration of silk fibroin was 10.0% (by weight), and the average degree of polymerization was 1000. Urea is added to this at a solution concentration of 2.0%
(Weight), EDTA.2Na salt 0.2% (weight), and sodium benzoate 0.3% (weight) were mixed. The obtained silk fibroin aqueous solution was rapidly cooled to −30 ° C. and frozen. This was dried by a usual freeze-drying method in which the initial stage of drying was 0.5 torr and the end point was about 0.05 torr, and this was pulverized with a Henschel mixer to obtain a laminated mica plate-like silk fibroin powder. The powder contains 20% urea based on pure silk fibroin.

Next, 125 parts of the obtained silk fibroin powder was redissolved in 875 parts of cold water. Re-dissolution was extremely easy and completely dissolved within 10 minutes. The transparency of the aqueous solution obtained by re-dissolution was 22 degrees. Further, the transparency when left for one month was 20 degrees.

On the other hand, as a comparative example, an aqueous silk fibroin solution was prepared in the same manner as in Example 4 except that urea was not added, and freeze-dried in the same manner. The obtained silk fibroin powder 105
Parts were redissolved in 895 parts cold water. In this case, complete redissolution was difficult even after stirring for 24 hours, and about 10% of silk fibroin powder remained undissolved. The transparency of the aqueous solution obtained by re-dissolution was 8 degrees. Moreover, what was left for one month was completely gelled.

As described above, the effect of adding urea to the silk fibroin powder is remarkable in the effect of improving the re-solubility of the powder, improving the transparency of the re-dissolving solution, and improving the storage stability.

[0050]

INDUSTRIAL APPLICABILITY As described above, the silk fibroin aqueous solution and the water-soluble silk fibroin powder obtained by the method of the present invention suppress the turbidity of the liquid or the formation of gel-like substances for a long time due to the effect of the added urea. Can be saved in the state. In addition, re-solubility of water-soluble silk fibroin powder can be remarkably improved by adding urea. The aqueous silk fibroin solution and the water-soluble silk fibroin powder obtained in the present invention are useful as a film-forming agent, a basic cosmetic, a makeup cosmetic, a cosmetic base such as a bath agent, a fiber treatment agent, and a pharmaceutical base.

Claims (2)

[Claims] 1. A silk fibroin aqueous solution or water-soluble silk fibroin powder, which contains urea and / or thiourea. 2. A silk fibroin aqueous solution characterized by mixing urea and / or thiourea with an aqueous silk fibroin solution obtained by dissolving in an aqueous medium, and further optionally freeze-drying or spray-drying the mixture. Alternatively, a method for producing a water-soluble silk fibroin powder.