KR102107819B1 - Silk fibroin hydrolyzate production method for improving cognitive function and memory with improved productivity and silk fibroin hydrolysis prepared using the same - Google Patents

Abstract

The present invention relates to a method for producing a silk fibroin hydrolysate with improved productivity. According to the present invention, the method for producing a silk fibroin hydrolysate with improved productivity comprises: a step of preparing a silk fibroin powder; a step of dissolving the silk fibroin powder; a step of performing hydrolysis by treating the silk fibroin lysate with a mixed enzyme containing a first proteolytic enzyme comprising Ficin and a second proteolytic enzyme containing any one selected from the group consisting of Delvorase, Chymotrypsin, Subtilisin, Lactoferrin, Kokulase P, Keratinase, and a mixture thereof; and an enzyme removal step of removing the proteolytic enzyme.

Description

Method for manufacturing silk fibroin hydrolyzate for improved cognitive function and memory improvement with improved productivity and silk fibroin hydrolyzate manufactured using the same TECHNICAL PRODUCT

The present invention relates to a method for producing a silk fibroin hydrolyzate having improved productivity and a silk fibroin hydrolyzate prepared using the same. In more detail, the present invention generally uses a large amount of a solvent in which calcium chloride is dissolved to produce a silk-derived peptide prepared by hydrolyzing silk fibroin, and the process for removing the calcium chloride is additionally performed. It is possible to reduce the size of the process compared to the same amount of production by reducing the amount of, and by reducing the amount of calcium chloride, the time required for the process of removing calcium chloride is reduced, the productivity is improved. It is to provide a hydrolyzate of silk fibroin prepared using.

Hydrolysis (加 水 分解) refers to a reaction in which a large molecule, which was originally one, decomposes into several ions or molecules during a chemical reaction. That is, the silk itself cannot be digested in the body, but the fibroin derived from silk produced by hydrolysis of silk is water-soluble and has an excellent absorption rate in the body, and has been studied in various fields along with its functionality.

In more detail, silk refers to cocoon produced by releasing silk thread into a sediment tube after repeating stripping while the larvae of silkworms eat mulberry leaves and grow, and the main components are fibroin and sericin. It corresponds to two types of natural proteins.

When the natural protein silk is hydrolyzed, various kinds of amino acids are generated, and the amino acids produced by the hydrolysis are water-soluble and have excellent absorption in the body. Accordingly, silk proteins are hydrolyzed in various ways to be used as foods that can exhibit certain functions. For example, fibroin derived from silk is known to suppress oxidative stress and lower blood sugar. Accordingly, various hydrolytic enzymes or hydrolysis methods have been studied to increase the functionality of fibroin derived from silk or to find new functionality.

Silk fibroin hydrolyzate, that is, when one example is illustrated as a method for producing a silk-derived peptide, selects cocoon, dissolves the fibroin obtained by removing sericin in a calcium chloride solution, purifies with water, and then filters to remove salt. . The salt-removed silk fibroin lysate is hydrolyzed by treatment with a hydrolase or acid in a hydrolysis reactor. Thereafter, the peptide derived from silk is prepared through a filtration and drying process.

In the process of producing peptides derived from the stomach and silk, the amount of solution of calcium chloride used is quite large, and in the process of removing salt, a considerable amount of water must be used and the capacity of the reactant is inevitably dissolved or hydrolyzed. The capacity of the reactor used in steps and the like is inevitably large.

In addition, there is a problem that the amount of water used to remove the salt of calcium chloride is considerable, and it takes a long time to process.

Accordingly, the present inventors studied a process capable of miniaturizing the process equipment by reducing the capacity of the reactor compared to the same production amount of the final product in the hydrolysis process for silk fibroin, and as a result, the content of calcium chloride and the amount of water can be reduced, The present invention was completed by modifying the order of the steps.

In the case of the present invention, it is possible to obtain the same final product as a conventional large-scale facility as a miniaturized process facility. In addition, it is possible to improve the process efficiency by reducing the amount of water and salt removal time required for the process.

KR 10-2018-0101802 A KR 10-0881210 B1

It is an object of the present invention to provide a method for producing a hydrolyzate of silk fibroin with improved productivity that can reduce the time required for the process, miniaturization of existing process equipment compared to the same final product production amount in the production of silk-derived peptides. It is for.

According to the present invention, a silk-derived peptide having a high yield in a short time can be produced in a smaller process.

An object of the present invention is to provide a method for producing a silk-derived peptide that can more effectively produce a silk-derived peptide using a hydrolase. In addition, it is to provide a method for producing a silk-derived peptide having an excellent effect of improving cognitive function, in particular, an initial improvement effect.

Another object of the present invention is to provide a silk-derived peptide prepared according to the method for preparing the silk-derived peptide.

In order to achieve the above object, a method for preparing a hydrolyzate of silk fibroin having improved productivity according to an embodiment of the present invention includes preparing a silk fibroin powder; Dissolving the silk fibroin powder; In the silk fibroin lysate, a first proteolytic enzyme containing Ficin and delvorase, chymotrypsin, Subtilisin, Lactoferrin, and Cokrase P ( Hydrolysis by treatment with a mixed enzyme comprising a second proteolytic enzyme comprising any one selected from the group consisting of Kokulase P), keratinase and mixtures thereof; And an enzyme removal step of removing the proteolytic enzyme.

In the method for producing a silk fibroin hydrolyzate with improved productivity, the silk fibroin powder may be produced by mixing silk fibroin and glycerin, crushing, washing with water, filtering and drying.

In the method for producing a silk fibroin hydrolyzate having improved productivity, the silk fibroin may be subjected to a low temperature plasma treatment.

The silk fibroin hydrolyzate according to another embodiment of the present invention may be produced by the method for manufacturing a silk fibroin hydrolyzate having the above productivity.

On the other hand, the silk fibroin hydrolyzate referred to in the present invention is defined to include a silk peptide or a silk-derived peptide.

The food for improving cognitive function and memory according to another embodiment of the present invention may be produced by the method for manufacturing a hydrolyzate of silk fibroin in which the productivity is improved.

The mixed enzyme may further include cathepsin K.

In the method of manufacturing the silk-derived peptide, the enzyme removal step may be to remove the activity of the enzyme by heat treatment.

The silk-derived peptide may be for preventing or improving cognitive dysfunction.

The cognitive dysfunction is dementia, learning disability, real certification, forgetfulness, aphasia, agonism, delirium, AIDS-induced dementia, Vincewaner's disease, Lewy body dementia, frontotemporal dementia, mild cognitive impairment, multiple infarct dementia, pick disease, meaning It may be selected from the group consisting of dementia, Alzheimer's dementia or vascular dementia.

Hereinafter, the present invention will be described in more detail.

A method for producing a hydrolyzate of silk fibroin having improved productivity according to an embodiment of the present invention includes preparing a silk fibroin powder; In the silk fibroin lysate, a first proteolytic enzyme containing Ficin and delvorase, chymotrypsin, Subtilisin, Lactoferrin, and Cokrase P ( Hydrolysis by treatment with a mixed enzyme comprising a second proteolytic enzyme comprising any one selected from the group consisting of Kokulase P), keratinase and mixtures thereof; And an enzyme removal step of removing the proteolytic enzyme.

In the method for producing a silk fibroin hydrolyzate with improved productivity, the silk fibroin powder may be produced by mixing silk fibroin and glycerin, crushing, washing with water, filtering and drying.

In the method for producing a silk fibroin hydrolyzate having improved productivity, the silk fibroin may be subjected to a low temperature plasma treatment.

When using the silk fibroin powder, in the step of dissolving the silk fibroin, a much smaller amount of calcium chloride and water can be used compared to the prior art, and the silk fibroin can be dissolved at a relatively low temperature. Accordingly, not only can the process required to use the silk-derived peptide be miniaturized, but also the content of water used can be reduced.

Specifically, the conventional technique dissolves silk fibroin in a calcium chloride solution and then proceeds to hydrolysis. Therefore, the calcium chloride solution used to dissolve the silk fibroin is usually about 5 to 10 times the number of solvents compared to the silk fibroin. In addition, the reaction temperature used to dissolve is also carried out at a relatively high temperature of 80 to 110 ℃.

However, when the silk fibroin powder is used, the capacity of the calcium chloride solvent can be mixed by 0.8 to 1.3 times the mass of the silk fibroin powder to dissolve the silk fibroin powder. In addition, the reaction may proceed at a relatively low temperature of 40 to 60 ℃. As a result, the capacity of the reactants can be reduced, so that the process can be miniaturized. In addition, since the amount of the calcium chloride solution used is reduced, the amount of water used to remove the salt and the time required to remove the salt can be shortened. Therefore, it is possible to produce silk-derived peptides in a short time with a much smaller reactor to obtain the same amount of the final product, thereby having excellent process efficiency.

Specifically, the silk fibroin powder mixes glycerin with silk fibroin from which sericin has been removed. The ground mixture is then washed with water, filtered and dried. The silk fibroin powder produced by the above method can have a certain solubility in itself.

In addition, the sericin-removed silk fibroin is treated with a low-temperature plasma at atmospheric pressure to modify the surface of the silk fibroin material, and after mixing the glycerin, the washing, filtration and drying processes have better solubility.

In the case of general silk fibroin, it is insoluble in water, but the silk fibroin powder prepared by the above method has a solubility of about 30% or more in water. In addition, unlike the case of using a normal silk fibroin, even if a much smaller amount of calcium chloride solution is used, the silk fibroin can be completely dissolved.

The mixed enzyme may further include cathepsin K.

Silk (silk) refers to the fibers obtained from cocoons made by silkworms, and collectively refers to products made from silk yarns made from these. In a broad sense, it also refers to Yajamsa, such as Yakjamsa, and silk products from raw silk. The true silk, commonly called the dog, is also called the cultivated or domestic silk. Meanwhile, the cocoon in the present invention is defined as a concept including silk.

Silk fibers are composed of fibroin and sericin, and contain a small amount of brain, fat and inorganic salts. Silk consists of fibroin and sericin, which are fibrous proteins. It is composed of biopolymers in its natural state. Compounds with a molecular weight of more than 10,000 are usually fibers, membranes or tubers. Silk is a high-molecular protein, protein is made of amino acids, and amino acids are composed of simple alpha amino acids by combining amino groups (-NH ₂ ) and carboxyl groups (-COOH) on carbon.

In addition, a polymer having many chains of amino acids linked together in a rosary shape is called a polypeptide. Eventually, -NH ₂ and -COOH of the two amino acids react, and water (H ₂ O) escapes, and fiberization is achieved by arranging them in parallel.

The amino acids that make up fibroin have many glycine (side chain -H) and alanine (-CH ₃ ). These amino acids are characterized by a short side chain of the molecule. And the sum of serine (side chain-CH ₂ OH) and tyrosine (side chain-CH (OH) CH ₂ ) is more than 90%. The difference between the amino acid composition of sericin is that the content of acidic amino acids (aspartic acid, glutamic acid) and excitatory amino acids (lysine, alanine, histitin) is low. When these amino acids are combined in different linkage combinations, a polypeptide is formed. When hydrolyzed fibroin, it becomes an amino acid, and an alpha amino acid having a molecular weight of from 300 to 500,000 to 3,000 to 3,500 forms a single molecule.

The silk-derived peptide referred to in the present invention refers to a product processed by hydrolysis of silk fibroin derived from the silk to a size sufficient to be absorbed by the body to have a certain amino acid sequence.

Preferably, the method for preparing a silk-derived peptide may further include a sericin removal step of removing the sericin by mixing and heat-treating the cocoon and purified water and dissolving the silk fibroin before the dissolving step of dissolving the silk fibroin. .

The sericin removal step refers to a step of removing the sericin surrounding the silk fibroin. Since sericin contains polar amino acids, it is common to remove it while performing heat treatment on purified water. Particularly, as the sericin removal step proceeds at a high temperature, the process efficiency of sericin removal increases, but there is a problem that the temperature cannot be increased indefinitely because the loss of fibroin occurs at a temperature exceeding 97 ° C.

In the method for producing the silk fibroin-derived peptide, the sericin removal step includes a thickener and an additive, and may be heat-treated at 30 to 70 ° C.

The sericin removal step may include a thickener and an additive, and heat treatment is performed at 30 to 70 ° C while heat treatment, and at the same temperature, the sericin removal process may be much faster than when the viscosity is not increased. In particular, when the viscosity is increased, there is an advantage that fibroin is not lost.

In the method for preparing the silk fibroin-derived peptide, the mixture of cocoon and purified water in the sericin removal step may have a viscosity of 1.2 to 120 cP.

According to the viscosity range, in the sericin removal process, not only can the sericin be quickly removed, but also has the advantage that there is little loss of fibroin.

Preferably, in the method for producing the silk fibroin-derived peptide, the sericin removal step is a mixture of cocoon and purified water having a viscosity of 1.3 to 40 cP, a first heat treatment step of heat treatment at 98 to 100 ° C for 2 to 10 minutes, and It may be to include a second heat treatment step of heat treatment to 30 to 40 ℃.

The first heat treatment step is the temperature at which fibroin is dissolved, but when the heat treatment is started by adjusting the viscosity of the cocoon and purified water to 1.3 to 40 cP, loss of fibroin may not occur.

Subsequently, if a relatively low secondary heat treatment step is performed, sericin may be rapidly removed.

In the first heat treatment step, when the viscosity of the mixture of cocoon and purified water is less than 1.3 cP, there is a problem that rapid loss of fibroin occurs. On the other hand, if the viscosity exceeds 40 cP, the process improvement effect for sericin removal may not appear despite the first heat treatment step.

Therefore, in the case of the primary heat treatment and the secondary heat treatment process while maintaining the viscosity range, the sericin can be removed more effectively, but loss of fibroin does not occur, thereby improving the efficiency of the sericin removal process.

When the additive is included, it is possible to minimize the loss of skin loin, as well as to increase the yield of fibroin hydrolysates, that is, silk fibroin-derived peptides, which exhibit the effect of improving memory and cognitive function in a later step.

Preferably, the additive contains xylase, and based on 100 parts by weight of xylase, sorbitol 10 to 100 parts by weight; Mannitol 1 to 10 parts by weight; Calactose may be included in 0.1 to 5 parts by weight.

In the case of the above range, the loss of fibroin in the sericin removal step is reduced, and the yield of the peptide derived from silk fibroin having a cognitive function improvement effect in the hydrolysis step is increased, and the efficiency of improving cognitive function and memory can be improved.

The dissolving step refers to preparing silk fibroin to be mixed with purified water, an organic solvent, and other liquids.

The hydrolysis step is a silk fibroin lysate, delvorase, chymotrypsin, subtilisin, lactoferrin, Kokulase P, keratinase And a hydrolase containing a proteolytic enzyme containing any one selected from the group consisting of mixtures thereof.

Preferably, the hydrolase may be any one selected from the group consisting of ficin, cocrase P, keratinase and mixtures thereof. According to the above range, it is possible to produce a silk fibroin peptide having a high effect of improving cognitive function and memory.

More preferably, the hydrolase may include physin, and may be a mixture of 5 to 20 parts by weight of cocrase P and 5 to 20 parts by weight of keratinase with respect to 100 parts by weight of physin. According to the above range, the effect of improving cognitive function and memory can be excellent, and the effect of improving cognitive function and memory can be rapidly increased within a short period. Therefore, it is possible to produce a silk fibroin peptide capable of improving cognitive function and memory in a relatively short period of time.

The enzyme removal step of removing the proteolytic enzyme refers to removing the enzyme or removing the activity of the enzyme after the hydrolysis step, and is defined to include all forms that can be used by those skilled in the art. .

In addition, the enzyme removal step may be to remove the activity of the enzyme by heat treatment.

In the method for preparing the silk-derived peptide, the proteolytic enzyme may further include cathepsin K. When the umizyme is included, the absorption rate of the hydrolyzate and the activity of the active ingredient for improving the cognitive function are increased, so that the cognitive function and memory improvement effect can be exhibited with a small amount in a shorter time.

Preferably, the hydrolase contains physin, and 5 to 20 parts by weight of cocrase P, 5 to 20 parts by weight of keratinase, and 1 to 5 parts by weight of cathepsin K are mixed with respect to 100 parts by weight of physin. It may be used. In the case of the above range, a silk-derived peptide having a significantly increased activity of an active ingredient for improving the absorption rate and cognitive function of a hydrolyzate can be prepared.

The enzyme removal step may be to remove the activity of the enzyme by heat treatment.

In the method of manufacturing the silk-derived peptide, the silk-derived peptide may be for improving memory.

In the method for preparing the silk-derived peptide, the silk-derived peptide may be for preventing or improving cognitive dysfunction.

The cognitive dysfunction is dementia, learning disability, real certification, forgetfulness, aphasia, agonism, delirium, AIDS-induced dementia, Vincewaner's disease, Lewy body dementia, frontotemporal dementia, mild cognitive impairment, multiple infarct dementia, pick disease, meaning It may be selected from the group consisting of dementia, Alzheimer's dementia or vascular dementia.

The food for improving cognitive function and memory according to another embodiment of the present invention may include a silk-derived peptide prepared according to the method for preparing the silk-derived peptide.

The cognitive function and memory improvement referred to in the present invention are not only the meaning that the cognitive function and the memory slowdown are recovered or improved. It is defined as including the phenomenon of cognitive function and memory decline caused by brain neurological disease being prevented or improved.

A drug for preventing and improving brain neurological diseases according to another embodiment of the present invention may include silk-derived peptides prepared according to the method for preparing the silk-derived peptides.

The brain neurological disease referred to in the present invention is dementia, Huntington's disease, Parkinson's disease and neurodegenerative diseases such as amyotrophic lateral sclerosis, ischemic or reperfusion diseases such as ischemic stroke, depression, schizophrenia and post-traumatic stress disorder It is defined as including all mental disorders such as.

The method for manufacturing a silk fibroin hydrolyzate with improved productivity according to the present invention provides a method for producing a peptide derived from silk, miniaturizing existing processing equipment compared to the same final product production amount, and reducing the time required for the process. do.

According to the present invention, a silk-derived peptide having a high yield in a short time can be produced in a smaller process.

The present invention provides a method for producing a silk-derived peptide that can more effectively produce a silk-derived peptide using a hydrolase. In addition, it is possible to provide a method for producing a silk-derived peptide having an excellent effect of improving cognitive function, in particular, an initial improvement effect.

1 is a flowchart of a method for manufacturing a hydrolyzate of silk fibroin with improved productivity according to an embodiment of the present invention.
Figure 2 relates to the results of a cognitive memory evaluation test for silk peptides prepared according to an embodiment of the present invention.
Figure 3 is for the results of the effect experiment on the cerebral infarction site by ischemia of the silk peptide prepared according to an embodiment of the present invention.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art to which the present invention pertains can easily practice. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein.

[Production Example 1: Preparation of silk fibroin powder]

Silk fibroin powders P2 to P4 were prepared by treating sericin-removed silk fibroin (P1) as shown in Table 1 below.

P1 Silk fibroin with sericin removed P2 Powder production by mixing alcohol with silk fibroin, grinding and drying P3 Glycerin is mixed with silk fibroin, crushed, washed with water and dried to prepare powder P4 Preparation of the powder in the same manner as P3 in atmospheric fibrin-treated silk fibroin

[ Experimental example  One: silk  Preparation of fibroin powder]

In Table 1, while maintaining 50 ° C for the fibroin powders P2 to P4 fished, the calcium chloride solution required to completely dissolve the silk fibroin is hydrolyzed while mixing the calcium chloride solution with respect to the silk fibroin of P1 to P4. The amount of was evaluated. The amount of the calcium chloride solution was expressed as a ratio to the weight of silk fibroin used as a solute.

P1 P2 P3 Calcium chloride solution 830% 130% 80%

Referring to [Table 2] above, it can be seen that in the case of P2 and P3, silk fibroin can be completely dissolved even when a relatively small amount of calcium chloride solution is used. In particular, it can be confirmed that it can have excellent solubility even when the reaction temperature is low compared to the prior art.

Therefore, in the case of the silk fibroin powder, since the amount of the calcium chloride solution used as a solvent can be greatly reduced, the process efficiency can be increased by miniaturizing the reactor used in the step of dissolving and hydrolyzing the silk fibroin. In addition, it can be seen that since the amount of calcium chloride used can be reduced, it is possible to reduce the amount and time of water required to remove the salt, thereby achieving process efficiency.

[Production Example 2: Preparation of silk-derived peptide]

The dissolved silk fibroin was mixed with purified water, and the proteolytic enzymes S1 to S11 according to the composition as shown in [Table 3] were treated at 1w%, followed by hydrolysis for 5 hours. After that, heat treatment was performed for 10 minutes to remove the activity of the enzyme, followed by freeze-drying to prepare a powdery form as shown in [Table 4].

S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 Flavozyme 100 - - - - - - - - - - Protamax - 100 - - - - - - - - - Escape - - 100 100 100 100 100 100 100 100 100 Cokrase P - - - One 5 10 20 30 10 10 10 Keratinase - - - One 5 10 20 30 10 10 10 Card Depsin K - - - - - - - - 0.5 3 6

(Unit: parts by weight)

NF1 NF2 TN1 TN2 TN3 TN4 TN5 TN6 TN7 TN8 TN9 Processing enzyme S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11

[Experimental Example 2: Experiment on the effect of improving memory and cognitive function]

Experimental Example 1: Animal model experiment for improving cognitive function

A. Preparation of experimental animals

The experimental animals are male white paper (Sprague Dawley, 140-180 g, Korea Laboratory Animal Center), and they are put in 4 animals per breeding box for a week in a constant environment (room temperature 25 ± 1 ℃, relative humidity 60 ± 10%). Water was provided without restriction, and at this time, limited feeding was performed to maintain 80% of the weight. A total of 50 animals were used in this experiment. From 3 days before training, the test was conducted by adapting to the maze for 5 minutes.

B. delayed alternation test

There are a total of three passages (left, right, and center), and each passage entrance is prepared with a “ㅠ” shaped maze with partitions to allow the experimenter to control the entry and exit of animals.

In the first trial, the starting box was adapted for 1 minute. The first trial was a 'forced selection trial', where one passage was blocked with a partition and the back was blocked with a partition when an animal entered the other passage. Animals were considered to have entered when the limbs of the animals had passed through the entrance to the passage. Animals were fed for 60 seconds in the passage.

From the next trial, the 'free selection trial' allowed the animals to freely select the direction of the passage (left or right) starting from the starting box. Animals were compensated for prey only when they entered the passage from the previous trial and into the other.

The interval between trials was 5 seconds and 20 seconds with a delay, and the cut-off time for each trial was 60 seconds. The number of times the animal entered the passage through which the reinforcement was given and the time taken to enter the passage were measured. The same procedure was performed for 9 days of 10 trials a day to evaluate.

C. Method of administration

The silk peptides NF 1, 2 and TN 1 to TN 9 prepared according to the preparation method of the present invention were prepared as a solution of 100 mg / ml and administered orally. All samples were dissolved in distilled water and administered orally.

D. Results of cognitive memory evaluation

The results of the T-maze experiment for the groups administered with silk peptides NF 1, 2 and TN 1 to TN 9 are shown in [Fig. 2] below. [Fig. 2] shows that the experiment was conducted by configuring the trial interval as 5 seconds, and the performance gradually improved during the training period of 9 days.

 The present inventors were able to derive similar tendency results when using a mixed enzyme centered on papain in other experiments conducted simultaneously, referring to [Fig. 2] below, by TN3 to TN5, TN7 to TN9 In this case, it can be confirmed that the improvement effect on cognitive function is apparent. In addition, it was found that in the case of the above production example, the early improvement effect was also excellent.

In particular, it can be seen that TN8 exhibits an excellent effect of improving initial cognitive function compared to other preparation examples. It can be understood that the peptide having a specific sequence in the silk fibroin hydrolyzed by the mixed hydrolase is mixed in a certain range and accelerates the effect of improving cognitive function, thereby exhibiting a rapid effect at the initial administration.

As a result of this analysis, a peptide comprising an amino acid sequence represented by the following [General Formula 1a] or [General Formula 1b]; It is believed that the above-described acceleration effect may occur when peptides containing an amino acid sequence represented by the following [General Formula 2] are simultaneously included.

[Formula 1a]

a ₁ -Gly-Gly-a ₁

(In the above general formula, a ₁ is Ala, Tyr, or Val)

[General Formula 1b]

a ₁ -Gly-Gly-a ₁ -Gly-a ₂

(In the above general formula, a ₁ is Ala, Tyr, or Val, and a ₂ is Ala, Tyr, Ile or Val.)

 [Formula 2]

Ile-Gly-Z ₁ -Z ₂

(In the above general formula, Z ₁ is Ala, Tyr, Val or Gln, and Z ₂ is Ala, Tyr, Val or Gly.)

Therefore, in the case of the above range, it is possible to provide a peptide derived from silk having an effect of improving cognitive function and memory, and in particular, it is possible to accelerate an initial synergistic effect on improvement of cognitive function and memory, and the effect of memory and cognitive function is further improved. Silk peptides can be prepared.

Experimental Example 2: Local ischemic induction animal model

Evaluation of the effect on the area of cerebral infarction due to ischemia using a local ischemia-induced animal model to confirm the degree of improvement in memory and cognitive function for silk peptides prepared using the hydrolytic enzymes of NF1, NF2, TN5 and TN8 The experiment was conducted.

A. Drug treatment and construction of a local ischemic induction animal model

A local ischemic induction animal model was constructed using a male white paper (Sprague-Dawley rat) weighing 200-250 g. The drug was administered orally with 1 g / kg of NF1, NF2, TF5 and TF8 1 hour before induction of ischemia or 1 hour after induction of ischemia (oral administration group, n = 6), and ischemia control group (n = 6). BG101 was administered in the same dose as the silver drug treatment group. Experimental animals were anesthetized by intramuscular injection of ketamine (30 to 40 mg / kg), followed by skin incision in the neck in the supine position, common carotid artery, external carotid artery, and internal carotid. artery) and separated from surrounding tissue. First, electrocauterize the upper thyroid gland and larynx and arteries, which are branches of the external carotid artery, electrocauterize the pterygopalatine artery, which is the branch of the internal carotid artery, and cut the external carotid artery to remove the 4-0 nylon thread (ETHICON, INC), etc. After inserting into the internal carotid artery through the carotid artery, the nylon cerebral artery was occluded by placing the nylon thread 16 to 18 mm in the carotid artery branch.

B. Experiment of reduction effect of cerebral infarction due to ischemia

After 6 hours of induction of ischemia, the animals were isolated and the brain was isolated. The extracted brain was sectioned at 2 mm intervals from the anterior pole, reacted with 2% triphenyl tetrazolium chloride (TTC, Sigma) solution at 37 ° C for 30 minutes, and then 4% paraformaldehyde ( Sigma) solution and fixed. After taking the stained tissue sections, the area of the area that turned white due to the occurrence of cerebral infarction was measured using the MCID image processing system (Imaging Research Inc.) and dyed red, and the area of the entire brain section was measured. After calculating the ratio of the area of contrast infarction, the average value was calculated. In addition, the ratio of the volume occupied by the cerebral infarction area in the total volume in each brain section was measured. The results are shown in [Fig. 3].

Referring to FIG. 3, it can be confirmed that the case of the silk glass fibroin provided by the present invention can reduce the cerebral infarction.

Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements of those skilled in the art using the basic concept of the present invention defined in the following claims are also provided. It belongs to the scope of rights.

Claims (5)

Preparing a silk fibroin powder;
Dissolving the silk fibroin powder;
Mixed enzyme containing 5 to 20 parts by weight of Kokase P (Kokulase P), 5 to 20 parts by weight of keratinase (Keratinase), based on 100 parts by weight of the ficin, in the silk fibroin lysate Hydrolysis by treatment with; And
And an enzyme removal step of removing the proteolytic enzyme,
The silk fibroin powder
Silk fibroin and glycerin are mixed, crushed, washed with water, filtered and dried.
Method for manufacturing silk fibroin hydrolyzate with improved productivity. delete According to claim 1,
The silk fibroin
Low temperature plasma treatment
Method for manufacturing silk fibroin hydrolyzate with improved productivity. delete delete

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