JPH0767686A - Production of silk fibroin peptide having low molecular weight - Google Patents

Abstract

PURPOSE:To provide a process for producing a silk fibroin peptide having low molecular weight and useful for foods, cosmetics, etc., at a low production cost. CONSTITUTION:A silk fibroin constituting silk fiber, etc., and having high molecular weight is decomposed with a neutral inorganic salt to produce a silk fibroin peptide having low molecular weight and useful for foods, cosmetics, etc. This process is carried out by decomposing the high-molecular weight silk fibroin with a neutral inorganic acid, hydrolyzing the decomposition product with a proteinase to obtain an aqueous solution of low-molecular weight silk fibroin peptide and desalting the solution by electrodialysis.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a low molecular weight silk fibroin peptide, and more particularly to a method for producing a low molecular weight silk fibroin peptide used for foods, cosmetics and the like.

[0002]

2. Description of the Related Art In recent years, low molecular weight silk fibroin peptides obtained by decomposing high molecular weight silk fibroin forming silk fibers and the like are used for applications such as food and cosmetics. As a method for producing such a low molecular weight silk fibroin peptide, it was obtained in Japanese Patent Publication No. 59-31520, etc. by decomposing high molecular weight silk fibroin forming silk fibers with a neutral inorganic salt such as calcium chloride. A production method has been proposed in which an aqueous solution is passed through a cellophane tube or a fibrous hollow permeable membrane to be dialyzed, and then hydrolyzed by a proteolytic enzyme. In addition, JP-A-6
In JP-A-183298, there is also proposed a production method in which a high-molecular-weight silk fibroin is hydrolyzed in one step with an acid, an alkali, or an enzyme to obtain an aqueous solution, which is desalted by electrodialysis.

[0003]

According to such a production method, it is possible to obtain an aqueous silk fibroin peptide solution having a low molecular weight which can be used for cosmetics and the like. However, in the production method of performing hydrolysis with a proteolytic enzyme after dialysis, during the hydrolysis of the enzyme, it is usually necessary to add an acidic or alkaline compound to adjust the pH value of the aqueous solution. It is necessary to perform desalting. Therefore, there is a drawback that the manufacturing process becomes complicated. On the other hand, according to the manufacturing method that employs electrodialysis, it is possible to eliminate the need for performing the desalting step again without performing hydrolysis or the like again after electrodialysis.
There are various problems with the one-step hydrolysis due to alkalis or enzymes.

That is, the decomposition due to acid is caused by, for example, J.
0 (5), 358-362 (1991), high molecular weight silk fibroin was added to 1N in 1N hydrochloric acid.
Since the harsh condition of performing heat treatment at 10 ° C. is required, most of the obtained decomposition products are likely to be amino acids. For this reason, it is difficult to control the molecular weight of the decomposed product, and an acid-resistant special container is required in terms of equipment. Further, the decomposition due to alkali may cause racemization of the obtained decomposition product, which may be unsuitable for food. Further, it is very difficult to decompose by an enzyme into a low molecular weight suitable for foods, that is, a low molecular weight that can be digested and absorbed in the human body.

As described above, various problems exist in the conventional method for producing a low molecular weight silk fibroin peptide, and to provide a low molecular weight silk fibroin peptide used for foods and cosmetics at a low cost. I couldn't. Therefore, an object of the present invention is to be able to inexpensively supply low molecular weight silk fibroin peptides used for foods and cosmetics,
It is intended to provide a method for producing a low molecular weight silk fibroin peptide.

[0006]

Means for Solving the Problems In order to achieve the above-mentioned object, the present inventors firstly decomposed high molecular weight silk fibroin with a neutral inorganic salt such as calcium chloride to obtain a decomposed product, and An enzymatic hydrolysis was carried out to obtain an aqueous solution in which a low molecular weight silk fibroin peptide was dissolved, and then the aqueous solution was passed through a cellophane tube or a fibrous hollow permeable membrane for dialysis to desalt.
According to such a production method, since high-molecular weight silk fibroin can be decomposed under mild conditions, a special container is not required in terms of equipment, and the molecular weight of the decomposed product can be controlled by a proteolytic enzyme. It is possible by adjusting the addition amount and the hydrolysis time. However, since the yield of the low molecular weight silk fibroin peptide was reduced to about 50%, it was found that it could not be industrially adopted as a method for producing a low molecular weight silk fibroin peptide. Therefore, the present inventor further studied to improve the yield of the low-molecular weight silk fibroin peptide, as a result, desalting from the aqueous solution in which the low-molecular weight silk fibroin peptide was dissolved, by performing electrodialysis, The inventors have found that the yield of low-molecular-weight silk fibroin peptide can be significantly improved and have reached the present invention.

That is, according to the present invention, when a high molecular weight silk fibroin forming a silk fiber or the like is decomposed by a neutral inorganic salt to produce a low molecular weight silk fibroin peptide used for foods, cosmetics and the like. A hydrolyzate obtained by decomposing the high molecular weight silk fibroin with a neutral inorganic salt is hydrolyzed by a proteolytic enzyme to obtain an aqueous solution in which a low molecular weight silk fibroin peptide is dissolved, and then desalting from the aqueous solution The method for producing a low-molecular-weight silk fibroin peptide is characterized in that In the present invention having such a constitution, it is preferable to use protease, nuclysin, papain, or trypsin as a proteolytic enzyme for the degradation of silk fibroin peptide. Further, by using the silk fiber from which sericin has been removed as the high molecular weight silk fibroin, the high molecular weight silk fibroin can be easily decomposed. Further, by dehydrating the desalted aqueous solution to form a powder, it is possible to easily handle the low-molecular weight silk fibroin peptide.

[0008]

In the present invention, the degradation product obtained by decomposing high molecular weight silk fibroin with a neutral inorganic salt is hydrolyzed by a proteolytic enzyme to give high molecular weight silk fibroin under mild conditions. It can be decomposed, and the molecular weight of the decomposed product can be controlled by adjusting the addition amount of the proteolytic enzyme and the hydrolysis time. In addition, since the high molecular weight silk fibroin is previously decomposed by the neutral inorganic salt during the hydrolysis by the proteolytic enzyme, it can be easily decomposed by the proteolytic enzyme to a predetermined molecular weight. Further, the silk fibroin peptide aqueous solution decomposed to a predetermined low molecular weight is desalted by an electrodialysis method, and compared with desalting by a cellophane tube or a fibrous hollow permeable membrane, the yield of the silk fibroin peptide of a low molecular weight is increased. Can be significantly improved. Thus, according to the present invention, high-molecular-weight silk fibroin can be decomposed under mild conditions while controlling the molecular weight of the decomposed product, and the yield of low-molecular-weight silk fibroin peptide is significantly improved. As a result, you can
A low molecular weight silk fibroin peptide can be provided at low cost.

[0009]

SUMMARY OF THE INVENTION In the present invention, first, high molecular weight silk fibroin forming silk fibers and the like is decomposed by a neutral inorganic salt. By such decomposition, it can be easily decomposed to a desired molecular weight in the subsequent enzymatic decomposition step. As the high molecular weight silk fibroin used as a raw material in this step, it is preferable to use silk fiber from which sericin has been removed. As the silk fiber, kneaded silk from which sericin has been removed by refining, silk spun cotton scrap, silk spun scrap, or the like can be used, and in particular, a buret generated in the silk spinning step is easily available and is most suitable. The term "bullet" refers to waste cotton generated in the silk spinning process, which has been boiled in an alkaline solution such as an aqueous sodium carbonate solution to remove sericin.

As the neutral inorganic salt, an alkali metal salt, an alkaline earth metal salt, or copper sulfate can be used, and lithium bromide or calcium chloride is particularly preferable. Here, the addition amount of the neutral inorganic salt is 0.005 to 0.
05 mol / high molecular weight silk fibroin (g), especially 0.
01-0.04 mol / high molecular weight silk fibroin (g)
It is preferable that Addition amount of neutral inorganic salt is 0.00
If the amount is less than 5 mol / high molecular weight silk fibroin (g), the decomposition rate of the high molecular weight silk fibroin tends to decrease, while if it exceeds 0.05 mol / high molecular weight silk fibroin (g). However, the effect of improving the decomposition rate of high molecular weight silk fibroin has almost reached saturation. The decomposition temperature by the neutral inorganic salt is preferably about 60 to 95 ° C., and the bath ratio is preferably 2 to 50 times that of the high molecular weight silk fibroin as a raw material.

In the present invention, the decomposition product of high molecular weight silk fibroin decomposed by a neutral inorganic salt is further hydrolyzed by a proteolytic enzyme. As described above, since the high-molecular-weight silk fibroin has been decomposed in advance, it can be easily decomposed to a desired molecular weight by a proteolytic enzyme.
Therefore, the molecular weight of the silk fibroin peptide can be easily controlled to a desired value by adjusting the addition amount of the proteolytic enzyme, the decomposition time, and the like. Such proteolytic enzymes include protease, nuclesin, papain,
Alternatively, one or more trypsin may be mixed and used. When using these enzymes, the pH value of the aqueous solution containing the degradation product is adjusted to the optimum value at which the enzymatic activity is exhibited by adding an acidic or alkaline compound as needed.

As described above, desalting from an aqueous solution containing a low molecular weight silk fibroin peptide decomposed to a desired molecular weight is performed by an electrodialysis method. In the electrodialysis method, a DC electric field is applied from the outside of a passage surrounded by a cation exchange membrane and an anion exchange membrane, and ions are ionized in an aqueous solution passing through the passage surrounded by an ion exchange membrane. It is a method of discharging inorganic components through each ion exchange membrane. According to this electrodialysis method, an organic substance having a larger molecule than an inorganic component generally cannot pass through an ion exchange membrane, so that even silk fibroin peptide whose degree of decomposition has remarkably progressed remains in the desalted aqueous solution. Therefore, the yield of low molecular weight silk fibroin peptide can be improved. On the other hand, in desalination using a cellophane tube or a fibrous hollow permeable membrane, the molecular weight cutoff of the cellophane tube or the fibrous hollow permeable membrane is high.
The silk fibroin peptide whose degree of decomposition has remarkably advanced flows out together with the inorganic component, and the yield of low-molecular-weight silk fibroin peptide decreases.

The desalted aqueous solution containing the low-molecular weight silk fibroin peptide may be subjected to decolorization / deodorization treatment with hydrogen peroxide water or the like, if necessary. The hydrogen peroxide solution used is decomposed after decolorization and deodorization. Further, an aqueous solution containing the silk fibroin peptide may be freeze-dried or the like and pulverized into a powder form. Prior to this freeze-drying, it is preferable to carry out preliminary freezing.
The obtained low molecular weight silk fibroin peptide having a molecular weight of 500 or less can be provided for food and has a molecular weight of 3
About 000 to 2000 silk fibroin peptides can be provided for cosmetics and other industrial materials.

[0014]

EXAMPLES The present invention will be described in more detail by way of examples. Example 1 In 300 ml of 40% aqueous solution of calcium chloride dihydrate,
The mixture obtained by gradually adding 150 g of the boulette generated in the silk spinning process was heated at 100 ° C. for 1 hour to dissolve the boulette. The obtained solution was filtered to remove impurities, and water was added to make 1000 ml. Nucleicin (protease activity: 50,000 units) 0.6 g / l and protease "Amano" A (protease activity: 10,000 units) 0.4 g / l were added to the obtained aqueous solution, while stirring. It was kept at 50 ° C. for a predetermined time to carry out enzymatic decomposition. The aqueous solution, which has undergone a predetermined enzymatic decomposition after a predetermined time has passed, is once heated to 90 ° C. to inactivate the enzyme and then filtered, and the filtrate is electrodialyzer [Asahi Kasei Co., Ltd., G-3
It was desalted by electrodialysis with a cartridge AC-220-400, electrode solution: sodium nitrate 0.5% aqueous solution). Further, hydrogen peroxide water was added to the desalted aqueous solution to perform decolorization / deodorization treatment, and then excess hydrogen peroxide water was decomposed. Then, the decolorized and deodorized aqueous solution was pre-frozen in a freezer [Tokyo Rika Co., Ltd., CA-11.
5], freeze-dried by a freeze dryer [Tokyo Rika Co., Ltd., FD-5N], and pulverized into a powder. The obtained powder was white and odorless, and was well soluble in cold water.

The yield, desalting ratio, and molecular weight distribution of the obtained powder were calculated or measured by the following methods. Yield (amount of powder obtained) / (amount of dissolved burette) × 10
0 (%) Desalination rate (chlorine concentration before dialysis-chlorine concentration after dialysis) / chlorine concentration before dialysis x 100 (%) The chlorine (Cl) concentration is measured by a salinity analyzer [Toa Denpa Co., Ltd., SAT-2A]. )It was used. Molecular Weight Distribution The molecular weight distribution of the obtained powder was measured by gel filtration chromatography using a TSK gel G2500PW XL column.

In this example, an experiment was carried out with various enzymatic decomposition times, and the results are shown in Table 1 below.

[Table 1] As is clear from Table 1, the obtained powder has a molecular weight of 5
Low molecular weight compounds of 00 or less are also included, and the yield is 85
It can be at least%. Moreover, powders having different molecular weight distributions can be obtained by changing the enzymatic decomposition time. Therefore, the molecular weight distribution of the obtained powder can be controlled by the enzymatic decomposition time.

Example 2 In Example 1, the amount of enzyme added was nuclein (protease activity: 50,000 units) 0.3 g / l, protease "Amano" A (protease activity: 10,000)
Table 2 shows the results obtained by carrying out in the same manner as in Example 1 except that the amount was changed to 0.2 g / l.

[Table 2] Comparing Table 1 and Table 2, the decomposition rate can be controlled also by the addition amount of the decomposing enzyme. Therefore, the molecular weight distribution of the obtained powder can be controlled also by the amount of enzyme added.

Comparative Example 1 Instead of the desalting method using the electrodialyzer of Example 1, a cellulose tube [VISKASE SALES CORP., Uc-36 was used.
32-100], the filtrate is caused to flow through the cellulose tube immersed in the running water of distilled water, and the distilled water flowing out of the outside of the cellulose tube is 0.1%.
The same procedure as in Example 1 was carried out except that a desalting method was adopted in which desalting was performed until the solution did not become cloudy even when an aqueous silver nitrate solution of N was added.
The results obtained are shown in Table 3.

[Table 3]

Comparative Example 2 Instead of the desalting method using the electrodialyzer of Example 1, a hollow fiber module [Asahi Kasei Kogyo KK, uF module, SE] was used.
P-1013], the filtrate is circulated in the hollow fiber by a pump, and the liquid flowing out from the outside of the hollow fiber is desalted until it does not become cloudy even when 0.1N silver nitrate aqueous solution is added. Example 1 was repeated except that the method was adopted. The results obtained are shown in Table 4.

[Table 4] As is clear from Tables 3 and 4, the powders contained in the obtained powder have a molecular weight of 500 or less in a trace amount, and the yield is 63.9% at most.

[0020]

INDUSTRIAL APPLICABILITY According to the present invention, the molecular weight of the low molecular weight silk fibroin peptide contained in the finally obtained aqueous solution can be easily controlled, and the yield of the low molecular weight silk fibroin peptide can be improved. It can be significantly improved. Therefore, a silk fibroin peptide having a molecular weight suitable for various uses can be provided at low cost.

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Claims (4)

[Claims] 1. A high molecular weight silk fibroin forming a silk fiber or the like is decomposed with a neutral inorganic salt to produce a low molecular weight silk fibroin peptide used for foods, cosmetics, etc. Degradation product of silk fibroin of No. 1 with neutral inorganic salt is hydrolyzed by proteolytic enzyme to obtain an aqueous solution in which low molecular weight silk fibroin peptide is dissolved, and then desalting from the aqueous solution is performed by electrodialysis. A method for producing a low-molecular-weight silk fibroin peptide, which is carried out by 2. The method for producing a low molecular weight silk fibroin peptide according to claim 1, wherein the proteolytic enzyme is protease, nuclysin, papain, or trypsin. 3. The method for producing a low-molecular-weight silk fibroin peptide according to claim 1, wherein silk fiber from which sericin has been removed is used as the high-molecular-weight silk fibroin. 4. The method for producing a low molecular weight silk fibroin peptide according to claim 1, wherein the desalted aqueous solution is dehydrated to give a powder.