KR100420824B1 - Silk peptide and silk fibroin powder materials made by silk and manufacturing method for those materials - Google Patents

Abstract

The present invention is a method for producing a low molecular weight silk peptide and a high molecular weight water insoluble fibroin powder using fibroin obtained by refining to remove sericin through a selection process and washing process using sub-nausea and lung dog. Alkyl hydrolysis of the refined silk with calcium hydroxide or barium hydroxide, followed by filtration, the filtrate is washed with water and dried, the filtrate is precipitated by filtration of the alkali in the solution, the filtrate is concentrated, dried Characterized in that.

Silk peptides prepared by the production method of the present invention can be used for food, high molecular weight water insoluble fibroin powder can be used for industrial purposes.

Description

Silk peptide and silk fibroin powder materials made by silk and manufacturing method for those materials

The present invention is to wash-refined by using a cocoon and other sub-nax, hydrolyzed refined fibroin to an alkaline solution to prepare amino acids and low molecular weight fibroin peptide-based material and fine water insoluble high molecular weight fibroin powder material And a low molecular weight silk peptide and a high molecular weight fibroin powder produced by the method.

Silk has long been widely used as a garment because of its aesthetic excellence. In recent years, focusing on the fact that silk is composed of almost 100% protein, developments to utilize it as a functional material, especially a food material, have been actively developed.

Examples of patents and scholarly literature known to date are as follows. The development of using silk as a food material is divided into two categories. In other words, silk fibroin is used and silk sericin is used. Among them, the development contents to utilize actual fibroin include food (Japanese Patent Laid-Open Publication No. 1-256350), nutritional supplement (Japanese Patent Laid-Open Publication No. 7-31417), and hangover inhibitors (Japanese Patent Laid-Open Publication No. 1-256352). , Manufacturing method of the cholesterol rising inhibitory food (Japanese Patent Laid-Open Publication No. 4-210577), blood sugar rising inhibitory food (Japanese Patent Laid-Open Publication No. 4-210576), functional food (Korean Patent Publication No. 96-582), and its functionality are disclosed. It is.

As introduced above, many researches and developments have been made for the development and commercialization of a manufacturing method for utilizing silk fibroin as a food material, but has shown various problems and limitations in food manufacturing. For example:

In order to use the macromolecule silk fibroin (molecular weight of 100,000 or more) as a functional food, low molecular weight is required. As a means, two manufacturing methods using chemicals have been applied.

The first manufacturing method is a method of hydrolysis using an acid such as hydrochloric acid, followed by neutralization with alkali, followed by desalination-concentration-drying. In this manufacturing method, it is necessary to hydrolyze with high concentration of acid for a long time, and remove high concentration of salt generated as a result of neutralization, so it is impossible to avoid the use of enormous facilities and excessive chemicals. It is restricted. In addition, the color of the produced low molecular weight silk fibroin was dark brown, it was inevitably restricted to use as a food. Therefore, a manufacturing method using ion exchange resins has been proposed to remove the color, but in this case, the yield is reduced to 40% or less (Japanese Society for Sampling, 61 (1), 32-35, 1992). ).

The second manufacturing method is a two-step method of dissolving silk fibroin with a salt such as calcium chloride, and then hydrolyzing it again with an acid, an alkali, an enzyme, or the like. Since this method uses a high concentration of salt solution (40% w / v or more), ultra-filtering or dia-filtering method is introduced to remove it later. To do this, a huge amount of facility costs must be put into place, which is subject to manufacturing restrictions.

In addition, U.S. Patent No. 5,853,764 discloses a method for producing silk fibroin powder by decomposing silk fibroin using an aqueous alkali solution and neutralizing it with an acid, but this method is neutralized with an acid such as sulfuric acid after alkali decomposition. Because it had a problem that it can not be used for food.

Therefore, in order to solve the various problems described above, the present inventors have studied the method of decomposing refined silk fibroin by chemical hydrolysis using a special alkali, followed by precipitation of alkali to remove silk amino acids and low molecular weight. Silk peptides can be prepared and used as food materials, and high molecular weight water-insoluble silk fibroin powders prepared in the same process can be usefully used for industrial purposes.

Accordingly, an object of the present invention is to provide a production method capable of producing high-molecular-weight water-insoluble silk fibroin powder, which is a silk amino acid and peptide suitable for food materials, and an industrial material at a minimum process and at a minimum cost.

1 is a micrograph (40 times magnification) of a low molecular weight silk peptide obtained in Example 1 of the present invention.

Figure 2 is a micrograph (40x magnification) of a low molecular weight silk peptide from which color was removed in Example 2 of the present invention.

3 is a micrograph (40 times magnification) of a high molecular weight water insoluble fibroin powder obtained in Example 3 of the present invention.

Figure 4 is a micrograph (40 times magnification) of the low molecular weight silk peptide obtained in Example 4 of the present invention.

5 is a micrograph (40-fold magnification) of the high molecular weight water insoluble fibroin powder obtained in Example 5 of the present invention.

6 is a manufacturing process chart of the present invention.

The above object of the present invention is obtained by alkali hydrolysis of the refined silk with calcium hydroxide and barium hydroxide, followed by filtration and precipitation of alkali present in the solution, followed by filtration, concentration and drying. It is achieved by the process for the preparation of molecular weight fibroin powder.

Hereinafter, the present invention will be described in detail.

1st process

In order to use as a raw material of the present invention, silkworm cocoons and other sub-silsa are removed through fine selection and washing to remove contaminants and foreign matter and finely cut.

2nd process (high pressure refining process)

In order to remove sericin from the silkworm cocoon or sub-sasa provided in the first step, 5-20 times of water is added to the raw material and heated at 100-130 ° C. for 20 to 120 minutes to remove sericin.

Third process (hydrolysis of fibroin)

Alkali hydrolysis of the fibroin obtained from the said 2nd process is carried out. For example, fill water with liquid ratio 10-40 based on fibroin. The alkali used was calcium hydroxide [Ca (OH) 2] or barium hydroxide [Ba (OH) 2] solution, and the concentration was adjusted to 0.4 to 2.0 w / v% (11.5 to 12.4 based on the pH of the solution). , 8 to 48 hours at 90 to 100 ℃ and then cooled to precipitate. At this time, the precipitate contains a high molecular weight water insoluble fibroin and an alkali, which is separated by filtration to obtain a hydrolyzed low molecular weight silk peptide solution. Fibroin solubility at this time is shown in Table 1.

Table 1 Hydrolysis Conditions, Solubility and Average Polymerization

Processing time Conc (%.) Solubility (%) Average degree of polymerization 12 14 12 14 0.2 37.40 37.60 30.5 13.4 0.4 52.30 53.10 11.1 6.60 0.6 63.55 64.12 2.05 1.43 0.8 63.69 64.12 1.41 1.40 1.0 64.06 64.40 1.65 1.18 1.2 64.21 66.20 1.64 1.23

4th process (high molecular weight water insoluble fibroin powder manufacturing process)

A sufficient amount of water is added to the precipitate separated in the third step and the filtration separation process is repeated until alkali is no longer eluted. The solid thus obtained is dried and pulverized to obtain a high molecular weight water insoluble silk powder material 1.

5th process (hydrolysis process of high molecular weight water insoluble fibroin)

The water insoluble fibroin contained in the precipitate separated in the third step is hydrolyzed again under the same conditions as in the third step to obtain a low molecular weight silk peptide solution.

6th process (alkali removal process in silk peptide solution)

In order to remove dissolved alkali in the silk peptide solution separated in the third and fourth processes, chemicals capable of producing alkali and poorly soluble salts used (for example, sulfuric acid, oxalic acid, liquefied carbon dioxide, Dry ice, ammonium bicarbonate) is added as much as the equivalent amount of dissolved alkali and left to heat, and precipitates are precipitated with calcium salt or barium salt. At this time, the precipitate is separated by filtration to obtain an aqueous solution of silk peptide from which alkali is removed. In addition to the method of precipitation and removal, it may be neutralized with citric acid and left in the form of citric acid salt.

7th process (concentration process of a silk peptide solution)

The silk peptide aqueous solution recovered in the 6th process is concentrated in vacuo at the temperature of 40-70 degreeC, or it is concentrated so that a density | concentration may be 10 to 40% using an ultrafiltration membrane.

8th process (removal of dyestuff material)

The concentrated silk peptide aqueous solution obtained in the seventh step is passed through an ion exchange resin, or a powdery activated carbon or hydrogen peroxide treatment to obtain a colorless silk peptide aqueous solution.

9th process (drying and decomposition process)

The colorless silk peptide solution concentrated in the above process is dried and pulverized to obtain substance 2 having a final tripeptide (tri-peptide).

Through the manufacturing process as described above to obtain a low molecular weight silk peptide material 2 and a high molecular weight water insoluble fibroin powder material 1 for the purpose of the present invention.

Next, the present invention will be described in more detail with reference to Examples.

(Example 1)

First step: 500g was prepared by removing fines from a clean slice dog and cutting it finely.

Second step: 500 g of pure keratin dogs were put together with 10 L of water in an autoclave, heated at 125 ° C. for 50 minutes, removed sericin and water-soluble impurities, and then washed once with hot water and dried. At this time, the weight was 350g.

Third step: 350 g of pure fibroin was added to 7 L of a 0.7% solution of Ca (OH) 2 and hydrolyzed at 95 ° C. for 12 hours.

4th process: The solution obtained at the said process was filtered by the filter paper, and the insoluble material was removed.

5th step: 54 g of ammonium bicarbonate was added to the aqueous solution obtained by the said process, it was left to stir well, the calcium carbonate produced was precipitated, and the deposit was isolate | separated with the filter paper.

6th step: The aqueous solution obtained by the said process was vacuum-concentrated to ¼ at 60 degreeC, and then lyophilized, and 205g of low molecular weight silk-peptide substance 2 was obtained. In order to determine the molecular weight of the peptide obtained from the end group quantification method, the average degree of polymerization was found to be 2.09, indicating that substance 2 was composed of a low molecular weight peptide. In addition, the color measured by the color difference meter (Minolta) to find the degree of coloration of this material was 82.20. The micrograph (40 times magnification) of the obtained low molecular weight silk peptide was shown in FIG.

(Example 2)

Since the low molecular weight silk peptide material obtained in Example 1 was slightly yellow, the concentrated solution of the sixth process was passed through a cation exchange resin and then powder dried for the purpose of removal thereof. The amount of powder obtained at this time was about 30% as compared with Example 1. The average degree of polymerization of the powders obtained was 2.38 and the whiteness was 83.43. The micrograph (40 times magnification) of the low molecular weight silk peptide from which the obtained color was removed was shown in FIG.

(Example 3)

The precipitate in the fourth process of Example 1 was collected, poured into 5 L of water, stirred well and filtered five times to completely remove the alkali, and then dried to obtain 130 g of a high molecular weight water insoluble fibroin substance 1. It was. A micrograph (40 times magnification) of the obtained high molecular weight water insoluble fibroin powder is shown in FIG. 3.

(Example 4)

The precipitate recovered in the fourth step of Example 1 was added to 2.5 L of a 0.7% solution of Ca (OH) 2 and then hydrolyzed at 95 ° C. for 12 hours. Subsequently, 84.5 g of substance 2 was obtained in the same manner as in the fifth and sixth steps of Example 1. At this time, the amount of ammonium bicarbonate added was 20 g, and the whiteness was slightly yellowish brown at 60.99. In order to remove the color, powdered carbon was added with 10% w / w peptide to remove the pigment material, and the whiteness was 73.4, and the average degree of polymerization of the low molecular weight silk peptide thus obtained was 2.96. The micrograph (40 times magnification) of the obtained low molecular weight silk peptide was shown in FIG.

(Example 5)

The precipitate recovered in Example 4 was obtained in the same manner as in Example 3, obtaining 34.4 g of a high molecular weight water insoluble fibroin powder and having a whiteness of 72.11. The micrograph (40 times magnification) of the obtained high molecular weight water insoluble fibroin powder was shown in FIG.

Table 2 shows the amino acid composition of the silk peptide obtained by the method of the present invention.

TABLE 2 Amino Acid Composition (%) of Low Molecular Weight Silk Peptides Obtained in the Present Invention

Amino acid A B C Asp. 3.47 1.05 2.32 Ser. 5.75 6.77 0.95 Glu. 2.70 0.76 2.02 Pro. 5.58 3.44 3.81 Gly. 42.30 46.63 46.17 Ala. 21.58 24.58 24.73 Val. 2.64 1.88 3.51 Met. 0.39 0.30 0.59 Ile. 0.70 0.39 0.73 Leu. 0.76 0.58 0.90 Tyr. 7.37 1.66 3.61 Phe. 0.95 0.38 0.54 His. 0.44 0.36 0.26 Lys. 0.70 0.96 0.90 Amm. 4.30 9.87 8.29 Arg. 0.39 0.39 0.66 Total 100.02 99.64 99.99

A: peptide material obtained in Example 1 of the present invention

B: peptide material obtained in Example 2 of the present invention

C: peptide material obtained in Example 4 of the present invention

As described above, in the present invention, in the preparation of low molecular weight silk peptides, since the hydrolysis (0.1 N concentration) is performed under alkaline conditions under mild conditions, such as hydrochloric acid hydrolysis (1 N to 3 N concentration), In addition to reducing the amount of use, it was possible to avoid browning due to oxidation during hydrolysis as much as possible, and to reduce production facilities by precipitating and removing used alkalis. It is expected to be applicable to functional foods such as hangover prevention, hepatocyte protection, and cholesterol level suppression, which have demonstrated the efficacy of silk peptides.

In addition, high molecular weight water-insoluble fibroin powder is expected to be developed for textile processing, cosmetic raw materials and the like.

Claims (9)

Alkyl hydrolysis of the refined silk with calcium hydroxide or barium hydroxide, followed by filtration, the filtrate is washed with water and dried, the filtrate is precipitated by the precipitation of alkali in the solution to remove the filtrate, the filtrate is concentrated and dried Silk peptide and a method of producing a water-insoluble high molecular weight fibroin powder. The method of claim 1, wherein the alkali is removed by precipitation using at least one compound selected from the group consisting of sulfuric acid, oxalic acid, liquid carbon dioxide, dry ice, and ammonium bicarbonate. The method of claim 1, further comprising passing the alkali-depleted silk peptide solution through a cation exchange resin or removing the pigment material by powdered activated carbon or hydrogen peroxide treatment. The method according to claim 1, wherein the water-insoluble fibroin powder is again subjected to alkali hydrolysis, filtered, and precipitated and removed, followed by concentration and drying. The method according to claim 1, wherein the calcium hydroxide or barium hydroxide is added at an amount of 10 to 40 times the weight of fibroin in an aqueous solution of 0.2 to 2.0%. The method according to claim 1, wherein the alkali hydrolysis is performed at 90 to 100 ° C for 8 to 48 hours. Alkali hydrolysis of the refined silk with calcium hydroxide or barium hydroxide, followed by filtration, the filtrate is washed with water and dried, the filtrate is concentrated and dried by neutralizing the alkali in the solution with citric acid and dried ( A method for producing an insoluble high molecular weight fibroin powder. delete delete