KR100335702B1 - Silk peptide and process for the preparation thereof - Google Patents

Abstract

The present invention uses a high concentration of acid, alkali, or inorganic salts in the production of silk peptides in a conventional manner, such as corrosion of the device, excessive neutralizer demand, removal of a large amount of salt, excessive waste water, as well as deterioration of product quality or yield Many problems are on the rise. The present invention relates to a method for producing silk peptides with good yield by simplifying complicated processes such as corrosion of a device, which is a problem in hydrolysis by existing acids and bases, and removal of a production salt, which is a problem in enzymatic hydrolysis. In the present invention, silkworm cocoons, silkworms, and the like, which have been removed from raw silk, pupa and foreign substances, can be used as raw materials for hydrolysis without any separate process. Fibroin and sericin refining solution, unrefined silkworm cocoon or sub-naxa were hydrolyzed using a different method of hydrolysis different from the conventional method, namely the method of short fiberization by known swelling, in particular heating under a nitrogen atmosphere in a high pressure reactor. The pressure is 3-7Kg / ㎠ with water, swells for 10-60 minutes, rapidly depressurizes to short fiber, and then hydrolyzes with a small amount of hydrochloric acid, that is, hydrochloric acid of 0.05 N or less, or 10 times or less with respect to this short fiber. The product is separated by dissolving with calcium chloride and hydrolyzing with enzyme. The product is separated by activated carbon when there is odor or coloring component and removed by using ultrafiltration membrane with molecular weight of 50,000 ~ 100,000 in the presence of unhydrolyzate and impurities. Fibroin peptides with an average molecular weight of 200-2,000 prepared by separating small amounts of salts and low-molecular substances and drying them with a freeze dryer or a powder dryer. A simple and novel process for the preparation of tide or mixed silk peptides.

Description

SILK PEPTIDE AND PROCESS FOR THE PREPARATION THEREOF}

The present invention is the hydrolysis of fibroin, unrefined silkworm cocoon or sub-naxa, which is different from the conventional method, that is, hydrolysis using a method of short-fiberization by known swelling (Japanese Patent Application Laid-Open No. J3-35024). After treatment with water vapor of 3 ~ 7Kg / ㎠ in a pressure vessel with water vapor, prefibrillation with pretreatment called swelling, followed by hydrolysis with a small amount of hydrochloric acid, or dissolution with a small amount of calcium chloride and hydrolyzed with an enzyme to hydrolyze low molecular weight proteins and polypeptides. And a simple process for the preparation of fibroin peptides and mixed silk peptides containing amino acids and the like, which solve the problem of corrosion of the device, which is a disadvantage in the conventional chemical hydrolysis and hydrolysis of enzymes, and the complex process of removing excess salt. Avoid and use it in food, beverages, cosmetics, and detergents.

Sisa was attached to sericin around fibroin in the fibrillated area. Sericin was removed by secondary treatment such as refining, and was used in clothing as silk fibers.However, recently, the excellent properties of silk fibers were used to make membranes, powders, gels and aqueous solutions. Attention has been drawn to direct or indirect use in food, cosmetics, medical supplies, and the like.

These dogs are composed of natural protein containing about 18 kinds of amino acids, and are used in many fields. For example, glycine and serine, which are contained in many components, lower blood cholesterol level, alanine can reduce hangover, and tyrosine is used for dementia. It has been found to be effective, and other amino acids have been shown to be effective one after another.

These silk fibers have been treated as high-quality clothing materials for a long time. However, due to the development of chemical fibers, they are gradually losing their place, and the recession of the latent business is becoming more severe. The development and development of Cordyceps sinensis is expected to revitalize the latent business, but there is no market yet. The manufacture of powder using silkworm cocoon is commercialized by some companies, but the wastewater problem in the manufacturing method In the current situation where there is a limitation of excessive manufacturing costs, the new simple manufacturing method may contribute to the activation of the potential business in developing high-performance products.

In the conventional case, using hydrolyzed fibroin in the preparation of fibroin peptides, the reaction yield is low and the molecular weight is difficult to control when hydrolyzed by acid, and the corrosion of the device using a high concentration of acid is also a major problem in commercialization.

Hydrolysis by bases requires the generation of toxic substances and the process of neutralization and desalination with bases after the reaction, causing problems such as racemization of products.

Therefore, in recent years, enzymatic hydrolysis is preferred, but a large amount of neutral salt such as calcium chloride is required for solubilization of silk, which requires considerable time for desalination, and there is concern about degeneration of the product and commercialization using expensive enzymes. Problems such as the complexity of the apparatus and the increase in manufacturing cost have arisen.

In the conventional case, dogs are widely used as basic materials in all of you, and in recent years, development for the use of such proteins as functional foods has been actively developed. It is.

Therefore, it is required to develop a method for easily producing silk peptides by solving the problems of desalination, device corrosion by hydrolysis by minimizing the use of acid and minimizing the use of calcium chloride.

The present invention is to solve the problems described above, it is an object to adopt a method different from the conventional hydrolysis method to produce a high-quality silk peptide at a low cost by a simple method.

In addition, there is another object to make the material used to provide amino acids, polypeptides and proteins that are likely to be lacking in the human body to food, cosmetics, detergents and the like.

The raw material for the production of silk peptides according to the present invention is characterized by hydrolysis by changing the reaction conditions in two cases.

First, when fibroin obtained by chemical refining, enzymatic refining, or high pressure refining is used, the second is characterized by hydrolysis using unrefined cocoons, sub-silsa and the like.

More specifically, when hydrolyzing fibroin, it is heated to 3 ~ 7Kg / ㎠ steam used in Japanese Patent Application Laid-Open No. J3-35024, rapidly depressurized, swelled, dried and pulverized to induce short fiber, and then hydrolyzed using 0.05 N hydrochloric acid or less. This invention was completed by the method of advancing and the method of hydrolyzing with an enzyme after melt | dissolution using 10 times or less of calcium chloride in said short fiber.

That is, in the case of hydrolysis by hydrochloric acid, the silk fiber is shortened by heat steam treatment and then hydrolyzed using a small amount of hydrochloric acid to obtain silk peptides. It was found to be decomposed.

In addition, in the case of enzymatic hydrolysis, the ease of dialysis was confirmed by minimizing the generation of salt by inducing short fiberization and dissolving enzyme hydrolyzed by using calcium chloride of 10 times or less for short fiber.

When odor and coloring occurred in the product during hydrolysis, it was possible to obtain a silk peptide hydrolyzed with good yield simply by treatment with activated carbon, alumina, silica gel, and the like.

In addition, in order to prevent oxidation of the reactants during the short fiber, the inside of the reactor was placed in a nitrogen atmosphere, and the used water is better to use ionized water.

At this time, in case of remaining high molecular weight, unreacted substances and impurities, it was possible to separate and remove by ultrafiltration membrane with an average molecular weight of 50,000 to 100,000.

There is no need to separate neutralization after the reaction, but when neutralization was performed, the salt could be easily removed by reverse osmosis membrane with 95% salt excretion.

With the production of silk peptides, the incidence of adult diseases due to improved eating habits, ie, irregular eating, excessive drinking, etc. of modern people is increasing. In other words, when searching for and establishing various functions, it is possible to develop products for cosmetics, adult disease prevention products and textile processing.

Hereinafter will be described a novel manufacturing method of a fibroin peptide or a mixed peptide that can be used as a material of the functional food, cosmetics, detergent according to the present invention.

Example 1

Hydrolysis of Fibroin

40 grams of silk fibroin fibers were placed in a high-pressure reactor, and the inside of the reactor was placed in a nitrogen atmosphere. The internal pressure of the reactor was increased to 4 Kg / cm 2 by heating steam using ionized water to perform short fiberization.

Hydrochloric acid was added to short fibrinated fibroin and hydrolysis was carried out at 95 ° C. for 3 hours to carry out the decomposition reaction.

After completion of the decomposition, the solution was neutralized and filtered to remove undegraded products. Ultrafiltration membranes with a molecular weight of 50,000 to 100,000 were used to completely remove high molecular weight and unreacted materials, followed by dialysis for 4 hours using a reverse osmosis membrane with a 95% salt excretion. A clear liquid was obtained.

27 grams of white powder having a sweet taste was obtained by drying with a freeze-dryer, and the molecular weight was measured by GPC to obtain an average molecular weight of 700 and a fibroin peptide having the composition of Table 1 using an amino acid analyzer.

Amino Acid Composition of Fibroin Peptides amino acid Furtherance(%) amino acid Furtherance(%) Alanine 29.3 Arginine 0.5 Aspartic acid - Cysteine 0.2 Glutamic acid 1.0 Glycine 44.5 Histidine 0.2 Isoleucine 0.7 Leucine 0.5 Lysine 0.3 Methionine 0.1 Phenylalanine 0.6 Propine 0.3 Serine 12.1 Threonine 0.9 Tryptophan 0.2 Tyrosine 5.2 Varin 2.2

Comparative Example 1

Enzymatic Hydrolysis of Fibroin

10 times of calcium chloride was added to 40 grams of the short fiber obtained in Example 1, and the salt was removed by dialysis for 5 hours after dissolving the short fiber, and 0.8 gram of enzyme having activity in the neutral region was added and decomposed for 1 hour at the optimum temperature. The reaction was carried out to obtain a slightly yellow solution.

Lyophilized digested peptide gave 23 grams of white powder.

Example 2

Hydrolysis of Kochi

After removing the foreign matter of the cocoon and cut into about 10 equal parts, 40 grams were added to the high-pressure reactor without the refining process, followed by the short fiber, hydrolysis and separation process as in Example 1.

After drying by lyophilizer to obtain 25 grams, the molecular weight was measured by GPC and the average molecular weight 600, analyzed by an amino acid analyzer to obtain a mixed silk peptide of the amino acid composition as shown in Table 2.

Amino Acid Composition of Mixed Silk Peptides amino acid Furtherance(%) amino acid Furtherance(%) Alanine 10.41 Arginine 0.07 Aspartic acid 0.50 Cysteine 10.16 Glutamic acid 0.51 Glycine 71.59 Histidine 0.19 Isoleucine 0.09 Leucine 0.20 Lysine 0.12 Methionine 0.06 Phenylalanine 0.28 Propine 0.84 Serine 0.48 Threonine 0.18 Tryptophan 0.76 Tyrosine 2.46 Varin 1.07

Comparative Example 2

Enzymatic Hydrolysis of Kochi

10 times of calcium chloride was added to 40 grams of short fibers obtained in Example 2, and the salts were removed by dialysis for 5 hours after dissolving the short fibers. The reaction was carried out to obtain a slightly yellow solution.

The digested peptide was lyophilized to obtain 22 grams of white powders.

Functional foods come in many varieties, but peptide proteins are known to be effective in relation to adult diseases.

Silk protein, which has been treated as a noble material from the past, is composed of more than 98% of amino acids.

Specifically, functional foods for adult diseases such as diabetes, hypertension, etc. have been developed and marketed by the production of fibroin peptides, and are being developed and applied to various materials such as wood modification, synthetic fiber, contact lenses, and dentures.

However, there are many problems in the production of peptides by degradation of this protein. That is, there are many problems in manufacturing, such as the use of excess hydrochloric acid, removal of salt by dialysis for several days due to corrosion of the device, generation of excessive wastewater, or use of excess neutral salt, and generation of wastewater by refining by hydrolysis pretreatment.

The present invention enables the preparation of peptides using a minimum amount of hydrochloric acid, and facilitates dialysis with the use of a minimum amount of neutral salts, as well as the development of a method of hydrolyzing the cocoon itself, excluding the process of refining, which is a pretreatment. Unlike the method, the silk peptide can be prepared simply, and thus, a low-cost, high-quality product having excellent business performance unlike the conventional method can be obtained.

In addition, there is considerable value in terms of environmental revival and the revival of the sheep wool industry, which is being eroded by expanding the availability of silkworm cocoons, increasing the value-added by-products of silk yarn, or using waste from silk textile companies.

Claims (9)

Fibroin peptide or mixed silk prepared by refining fibroin or sub-silsa and cocoon or green sand in a high-pressure reactor under a nitrogen atmosphere to 3-7 Kg / ㎠ pressure with heated steam to hydrolyze with hydrochloric acid of 0.01-0.05N Peptides. The method of claim 1, Fibroin peptides or mixed silk peptides prepared using ultrafiltration membranes having molecular weight of 50,000-100,000 for treatment with activated carbon when there are odor components, coloring components and impurities and for removing high molecular weight. The method of claim 1, Fibroin peptide or mixed silk peptide prepared by dialysis for 2 to 6 hours with ultrafiltration membrane of 95% salt excretion to remove salt. The method of claim 1, Fibroin peptide or mixed silk peptide having an average molecular weight of 200 to 2,000. The method of claim 1, Fibroin peptide or mixed silk peptide prepared by dissolving with calcium chloride of 10 wt% or less with respect to short fibers and hydrolyzing with enzyme. delete A method of preparing fibroin peptides using hydrochloric acid of 0.05 N or less after short-fiberization of the refined fibroin fiber or sub-naxa to a pressure of 3-7 Kg / cm 2 with heated steam. A method of producing mixed silk peptides using hydrochloric acid of 0.05 N or less after short-fiberization of unrefined cocoons or raw silk to a pressure of 3-7 Kg / cm 2 with heated steam. delete