KR20230098939A - Composition for Anti- Inflammation Comprising Cocoon Hydrolysate Produced by Enzyme Treatment - Google Patents

Abstract

The present invention relates to an anti-inflammatory composition containing a silkworm cocoon enzyme hydrolysate. Since the silkworm cocoon enzyme hydrolysate according to the present invention exhibits a significant anti-inflammatory effect, it can be applied not only as a treatment for inflammatory diseases but also as a health functional food, thereby being expected to be used in various fields to increase the income of farmers.

Description

Composition for Anti-Inflammation Comprising Cocoon Hydrolysate Produced by Enzyme Treatment}

The present invention relates to an anti-inflammatory composition comprising silkworm cocoon enzyme hydrolysate.

In modern society, the functional food industry uses excellent raw materials in terms of nutrition by searching for new materials and isolating only the beneficial ingredients essential to the human body. Among them, silkworm cocoons contain serisin and fibroin proteins, among which fibroin proteins have recently been actively developed for use as a material for functional foods and beverages.

In the prior art using silkworm cocoons, silk proteins collected from silkworm cocoons are used as they are without undergoing any special treatment process, or a method of purifying the dissolved fibroin aqueous solution by dialysis is used to increase production efficiency in mass production. Problems such as uneconomical aspects such as reduced yield or reduced value as a functional food material due to delay in degradation in the human body or difficulty in degradation due to the structural characteristics of the fiber and amino acid sequence of silk protein, and reduced absorption rate contains

In addition, in order to solve the above problems, recently, a process of hydrolyzing silkworm cocoons using hydrochloric acid at a high temperature of 100 ° C or higher has been developed to produce silk amino acids or peptides with excellent absorption by the human body. By using it, a neutralization process is necessarily required, and the salt generated from it induces a salty taste. Therefore, in order to use it as food, desalination, decolorization, and deodorization processes are required, which lowers the production yield.

On the other hand, silk hydrolysates differ in amino acid composition and oligopeptide molecular weight depending on the silk hydrolysis method and the method of filtering the residue, and the unique physiological activity of silk hydrolysates also varies considerably. Silk hydrolysates currently being developed are mostly hydrolyzed silkworm cocoons with acids and alkalis.

Thus far, a variety of methods have been disclosed, such as a fermentation method, an enzymatic decomposition method, or a method using solvent extraction as a technology using silkworm cocoons.

Nevertheless, there is still a need in the art to improve a method for obtaining a silkworm cocoon extract with a high content of active ingredients by solving the problems of the prior art and eliminating unnecessary steps while ensuring sufficient extraction in a short time. am.

On the other hand, most of the currently developed inflammation improving or therapeutic agents are mainly synthetic compounds, which have a problem of causing side effects in the body and lack an improvement effect on skin inflammation, so that new anti-inflammatory agents or therapeutic agents can be developed from natural materials with high stability in the body. It is in need of development.

Korean Patent Publication No. 2012-0056138

Under these circumstances, the present inventors prepared silk protein hydrolysates from silkworm cocoons using various methods and, while studying their physiological activities, identified that silk protein hydrolysates prepared by a specific method had high anti-inflammatory efficacy and the present invention The invention was completed.

Accordingly, one object of the present invention is to provide a method for producing cocoon-derived silk protein hydrolysate and a cocoon-derived silk protein hydrolyzate prepared according to the method.

Another object of the present invention is to provide an anti-inflammatory composition comprising silkworm cocoon-derived silk protein hydrolysate.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory diseases, including a hydrolyzate of cocoon-derived silk protein.

Another object of the present invention is to provide a food composition for preventing or improving inflammatory diseases, including silkworm cocoon-derived silk protein hydrolysate, and health functional food containing the same.

Another object of the present invention is to provide a cosmetic composition for anti-inflammatory, comprising silkworm cocoon-derived silk protein hydrolyzate.

Another object of the present invention is to provide a feed composition for preventing or improving inflammatory diseases, including silkworm cocoon-derived silk protein hydrolysate.

The terms used herein are used for descriptive purposes only and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as "include" or "have" are intended to designate that there is a feature, number, step, operation, component, part, or combination thereof described in the specification, but one or more other features It should be understood that the presence or addition of numbers, steps, operations, components, parts, or combinations thereof is not precluded.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiment belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and unless explicitly defined in the present application, they should not be interpreted in an ideal or excessively formal meaning. don't

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

According to one aspect of the present invention, the present invention provides a method for producing a cocoon-derived silk protein hydrolysate and the cocoon-derived silk protein hydrolysate produced thereby, comprising the following steps:

(a) scouring the silkworm cocoon to remove sericin;

(b) dissolving the refined product prepared in step (a) with a neutral salt; and

(c) obtaining a silk protein hydrolyzate by hydrolyzing the lysate prepared in step (b) with an enzyme.

In an embodiment of the present invention, the silkworm cocoon is preferably a sericin-free cocoon, that is, a scouring dog.

Any neutral salt known in the art may be used as the neutral salt, as long as the object of the present invention can be achieved, and calcium chloride, sodium chloride, potassium chloride, sodium nitrate, potassium nitrate, sodium sulfate, potassium sulfate, ammonium chloride and sulfuric acid It may be one or more selected from the group consisting of ammonium, but is not limited thereto.

Preferably, according to one embodiment of the present invention, the neutral salt is an aqueous solution of calcium chloride (CaCl ₂ /EtOH/H ₂ O) is used.

In addition, as long as the object of the present invention can be achieved, any enzyme known in the art may be used as the enzyme, and Sumizyme, Papain, Pepsin, Trypsin, α-Chymotrypsin, Kojizyme, pancreatin, alkaline protease, Protamex, Flavorzyme, kallikrein, Cathepsin, thermolisin, Subtilisin, Bromelain, Ficin, Actinidin, Peptidase and Transglutaminase ( Transglutaminase) may be one or more selected from the group consisting of, but is not limited thereto.

Preferably, according to one embodiment of the present invention, the enzyme uses Sumizyme.

In addition, the production method of the present invention may further include a step of inactivating the enzyme by raising the temperature to 90 ° C. after the hydrolysis of step (c) is completed.

According to one embodiment of the present invention, the method for producing a cocoon enzymatic hydrolyzate of the present invention includes an enzymatic decomposition step as a second step.

The enzyme is characterized in that it is added in an amount of 0.1 to 10 parts by weight per 100 parts by weight of the lysate. This is because when the enzyme is added in less than 0.1 parts by weight, the enzyme reaction is not performed well, so that the anti-inflammatory effect desired by the present invention cannot be obtained, and when added in excess of 10 parts by weight, health function is rather caused by the over-added enzyme There is a concern that it will be difficult to apply as a food or cosmetic material.

Enzymes used in the present invention include sumizyme. For example, Sumizyme is administered in an amount of 0.1 to 10 parts by weight, preferably 1 to 10 parts by weight, per 100 parts by weight of the lysate, and then stirred at 37 to 70 ° C, preferably 50 ° C for 1 day to hydrolyze the cocoon. Upon completion of the inactivation, it is preferable to raise the temperature to 90° C. for inactivation for 15 minutes.

In addition, the present invention is a cocoon hydrolyzed product obtained after filtering the enzymatically hydrolyzed cocoon hydrolyzate to remove unreacted residues, or obtained by high-pressure sterilization of the cocoon hydrolyzed product of the present invention. As long as it is liquid, it is possible to provide a cocoon enzyme hydrolyzate.

The filtration is preferably performed one or more times using a 0.1 to 3 μm filter. If the pore size of the filter is less than 0.1 ㎛, the filter is ultra-precise and ultra-expensive (ultrafiltration method), so there is a problem of poor economic feasibility. There is a problem that is likely to be.

The cocoon enzyme hydrolyzate of the present invention obtained as described above is used interchangeably with 'cocoon-derived silk protein hydrolysate' or 'cocoon hydrolyzate' in the present specification.

The silkworm cocoon enzyme hydrolyzate prepared according to the manufacturing method of the present invention is not subjected to strong acid treatment or long-term high temperature treatment, so it has little destruction of physiologically active substances and does not have a salty taste due to neutralization of strong acid, so it can be used directly as a health food. , By inhibiting the production or expression of matrix metalloprotease 1 (MMP1) and matrix metalloprotease 1 (MMP3), and the production or expression of nitric oxide (NO) and interleukin-6 (IL-6), an excellent anti-inflammatory effect is achieved.

In addition, according to another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases, including the cocoon-derived silk protein hydrolyzate.

The pharmaceutical composition of the present invention may be formulated and used in various forms according to conventional methods. For example, it can be formulated into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions and syrups, and can be formulated and used in the form of external preparations, suppositories and sterile injection solutions.

The composition of the present invention may contain one or more known active ingredients having preventive or therapeutic effects on inflammatory diseases together with silkworm cocoon hydrolyzate.

In an embodiment of the present invention, the inflammatory disease is degenerative arthritis, inflammatory bowel disease, peritonitis, osteomyelitis, cellulitis, pancreatitis, trauma-induced shock, bronchial asthma, allergic rhinitis, cystic fibrosis, acute bronchitis, chronic bronchitis, acute bronchiolitis, Chronic bronchiolitis, osteoarthritis, gout, spondyloarthropathy, ankylosing spondylitis, Reiter's syndrome, psoriatic arthropathy, enteropathic spondylitis, juvenile arthropathy, juvenile ankylosing spondylitis, reactive arthropathy, infectious arthritis, post-infectious arthritis, gonococcal arthritis , tuberculous arthritis, viral arthritis, fungal arthritis, syphilitic arthritis, Lyme disease, arthritis associated with vasculitic syndrome, polyarteritis nodosa, hypersensitivity vasculitis, Lougenic granulomatosis, polymyalgia rheumatica, articular cell arteritis, calcium crystal deposition arthropathy , pseudogout, non-articular rheumatism, bursitis, tenosynovitis, epicondylitis (tennis elbow), neuropathic joint disease, hemarthrosic, Henoch-Schenlein purpura, hypertrophic osteoarthropathy, multicentric Reticular histiocytoma, scoliosis, hemochromatosis, hemoglobinopathy, hyperlipoproteinemia, hypogammaglobulinemia, familial thalassemia fever, Behat's disease, systemic lupus erythematosus, relapsing fever, multiple sclerosis, sepsis, septic shock, acute Respiratory distress syndrome, multiple organ failure, chronic obstructive pulmonary disease, rheumatoid arthritis, acute lung injury, bronchopulmonary dysplasia, asthma, periodontitis , stomatitis, peritonitis, gastritis, enteritis, arthritis, nephritis, hepatitis, and preferably at least one selected from the group consisting of inflammatory skin diseases, if the disease caused by inflammation is not limited thereto.

The composition of the present invention may further include pharmaceutically acceptable additives, wherein the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, and lactose. , Mannitol, Taffy, Gum Arabic, Pregelatinized Starch, Corn Starch, Powdered Cellulose, Hydroxypropyl Cellulose, Opadry, Sodium Starch Glycolate, Carnauba Wax, Synthetic Aluminum Silicate, Stearic Acid, Magnesium Stearate, Aluminum Stearate, Calcium Stearate, White sugar and the like may be used. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.

The composition of the present invention can be administered in various oral or parenteral formulations during actual clinical administration. When formulated, commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants are used. It can be prepared by, and suitable formulations known in the art are preferably those disclosed in literature (Remington's Pharmaceutical Science, recently, Mack Publishing Company, Easton PA).

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient, for example, starch, calcium carbonate, sucrose or It is prepared by mixing lactose and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. In addition, the liquid formulations for oral administration include suspensions, solutions for oral administration, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, preservatives, etc. this may be included.

Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.

The dosage of the pharmaceutical composition of the present invention may vary depending on the formulation method, administration method, administration time and/or route of administration of the pharmaceutical composition, and the type and degree of response to be achieved by administration of the pharmaceutical composition. , various factors, including the type of subject to be administered, age, weight, general health condition, symptoms or severity of disease, sex, diet, excretion, drugs used simultaneously or at the same time in the subject, and other components of the composition, and the like It can be varied according to similar factors well known in the medical field, and those skilled in the art can easily determine and prescribe an effective dosage for the desired treatment.

The dose of the pharmaceutical composition of the present invention is preferably administered at a concentration of, for example, 1 to 500 mg/kg, more preferably 200 to 450 mg/kg, and even more preferably 300 to 400 mg/kg. , even more preferably 360 mg/kg, but the dosage is not limiting the scope of the present invention in any way.

The administration route and administration method of the pharmaceutical composition of the present invention may be each independent, and are not particularly limited in the method, and any administration route and administration method as long as the pharmaceutical composition can reach the target site can follow

The pharmaceutical composition may be administered orally or parenterally. The parenteral administration method includes, for example, intravenous administration, intraperitoneal administration, intramuscular administration, transdermal administration, or subcutaneous administration.

The pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers for the prevention or treatment of diseases caused by calcium deficiency.

In addition, according to another aspect of the present invention, the present invention provides a food composition for preventing or improving inflammatory diseases, including the above-described silkworm cocoon-derived silk protein hydrolyzate, and a health functional food containing the same.

The silkworm cocoon hydrolyzate of the present invention can be used as a health functional food, food additive or dietary supplement. When the cocoon hydrolyzate is used as a food additive, the cocoon hydrolyzate may be added as it is or mixed with other foods or food ingredients and used as appropriate according to a conventional method.

As a specific example, when producing food or beverage, the cocoon hydrolyzate of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw material. However, for long-term intake for health and hygiene purposes or health control purposes, it may be added in an amount below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range. there is. The type of food is not particularly limited, but examples of food to which the silkworm cocoon hydrolyzate of the present invention can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, and ice cream Dairy products including dairy products, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, etc., include all health foods in a conventional sense.

When the food composition of the present invention is prepared as a beverage, it may contain additional ingredients such as various flavoring agents or natural carbohydrates, like conventional beverages. Examples of the natural carbohydrate include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; natural sweeteners such as dextrins and cyclodextrins; Synthetic sweeteners such as saccharin and aspartame may be used. The natural carbohydrate is included in an amount of 0.01 to 10% by weight, preferably 0.01 to 0.1% by weight, based on the total weight of the food composition of the present invention.

The food composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonic acid It may include a carbonation agent used in beverages, and the like, and may include natural fruit juice, fruit juice beverages, and fruit flesh for the manufacture of vegetable beverages, but is not limited thereto. These components may be used independently or in combination. The proportion of the additives is not particularly limited, but is preferably included within the range of 0.01 to 0.1% by weight based on the total weight of the food composition of the present invention.

In the case of long-term intake for the purpose of health and hygiene or health control, the food composition of the present invention can be taken for a long period of time because there is no problem in terms of safety.

In addition, according to another aspect of the present invention, the present invention provides a cosmetic composition for anti-inflammatory, comprising the silk protein hydrolyzate derived from silkworm cocoon described above.

The cosmetic composition of the present invention can be easily prepared according to a method known in the art, including one or more excipients and additives generally used in the field of manufacturing cosmetic compositions together with the silkworm cocoon hydrolyzate of the present invention.

More specifically, the cosmetic composition of the present invention contains the silkworm cocoon hydrolyzate of the present invention as an active ingredient, together with a dermatologically acceptable excipient, and a basic cosmetic composition (toilet, cream, essence, face wash such as cleansing foam and cleansing water, pack , body oil), color cosmetic compositions (foundation, lipstick, mascara, makeup base), hair product compositions (shampoo, rinse, hair conditioner, hair gel) and soap. The excipient is not limited thereto, but may include, for example, a skin softener, a skin penetration enhancer, a colorant, a fragrance, an emulsifier, a thickening agent, and a solvent. In addition, it may additionally include flavoring agents, pigments, bactericides, antioxidants, preservatives, moisturizing agents, and the like, and thickeners, inorganic salts, synthetic polymer materials, and the like may be included for the purpose of improving physical properties.

For example, in the case of preparing a facial cleanser and soap containing the cocoon hydrolyzate of the present invention, it can be easily prepared by adding white-spotted radishes, powder, extract, or suspension to a common facial cleanser and soap base.

When preparing a cream, it can be prepared by adding mace lignan or nutmeg extract to a general oil-in-water (O/W) cream base. Herein, synthetic or natural materials such as flavors, chelating agents, pigments, antioxidants, preservatives, and proteins, minerals, and vitamins for the purpose of improving physical properties may be additionally added.

In addition, according to another aspect of the present invention, the present invention provides a feed composition for preventing or alleviating inflammatory diseases, comprising a silkworm cocoon-derived silk protein hydrolysate.

The feed composition of the present invention may be a feed additive or formulated feed.

In the present invention, 'feed additive' refers to a substance added to feed in small amounts for nutritional or specific purposes. They can be mixed in any proportion. When formulated, the cocoon hydrolyzate may be applied in an amount of 0.01 to 99% by weight based on the total weight of the feed additive.

The feed composition of the present invention includes 0.1 to 5 parts by weight of a feed additive containing silkworm cocoon hydrolyzate as an active ingredient based on 100 parts by weight of the formulated feed. As an example of the compound feed, 53.71% by weight of corn, 23.2% by weight of soybean meal, 1.0% by weight of rapeseed meal, 2.0% by weight of corn meal, 3.0% by weight of ginseng, 2.7% by weight of wheat hull, 0.3% by weight of potato starch, 2.8% by weight of oil 9.9% by weight of calcium carbonate, 0.68% by weight of dibasic calcium phosphate, 0.23% by weight of sodium chloride, 0.03% by weight of phosphorus enzyme, 0.05% by weight of choline chloride, 0.2% by weight of methionine, 0.1% by weight of mineral mix, and 0.1% by weight of vitamin mix is formed In addition, it goes without saying that conventional poultry formulated feeds can be used. In addition, it goes without saying that compounded feeds such as cows, pigs, ducks, etc. may be used according to the type of livestock to be raised.

Livestock may be raised by feeding the feed composition, and the cocoon hydrolyzate may be diluted in water and provided as drinking water during breeding of livestock.

The feed composition of the present invention may further include other nutritional components within the range expected by those skilled in the art.

The silkworm cocoon hydrolyzate of the present invention can be incorporated into commercially common animal feed compositions, and can be fed, for example, to cattle, swine, sheep, poultry, and the like. To this end, the silkworm cocoon hydrolyzate of the present invention can be mixed with common animal feed ingredients and, if necessary, processed into a purified form. Common animal feed ingredients are, for example, corn, barley, oats, soybeans, fishmeal, bran, soybean oil, minerals, trace elements, amino acids and vitamins.

In addition, since the composition of the present invention includes the cocoon hydrolyzate of the present invention described above, duplicate descriptions are omitted to avoid excessive complexity of the present specification.

Since the silkworm cocoon enzyme hydrolyzate according to the present invention exhibits a remarkable anti-inflammatory effect, it is expected to be used in various fields to increase the income of farmers as it can be applied as a health functional food as well as a treatment for inflammatory diseases.

1 shows a method for producing a silkworm cocoon silk protein hydrolyzate according to the present invention.
Figure 2a shows the results of acid/alkali and neutral salt dissolution of silkworm cocoons. Figure 2b shows the shape of the solution before and after silk protein hydrolysis.
Figure 3 shows the results of FPLC chromatograms of silk proteins for each preparation method (absorbance change at 280 nm according to elution time).
4 shows the results of FTIR analysis of silk proteins by manufacturing method.
5 shows the CD spectrum analysis results of silk proteins for each manufacturing method.
Figure 6 shows the MMP1 and MMP3 inhibitory effects by silk protein treatment for each manufacturing method in an arthritis-inducing environment.
Figure 7 shows the MMP1 and MMP3 inhibitory effect by treatment with the concentration of the enzyme hydrolyzate of sumizyme in an arthritis-inducing environment.
Figure 8 shows the NO inhibition effect by silk protein treatment for each manufacturing method in an inflammation-inducing environment.
Figure 9a shows the IL6 inhibitory effect by treatment with sumizyme enzymatic hydrolysate in an inflammatory environment. Figure 9b shows the IL6 inhibitory effect by treatment with a concentration of sumizyme enzyme hydrolyzate in an inflammation-inducing environment.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Preparation of silkworm cocoon hydrolysate containing cocoon-derived silk protein

As shown in FIG. 1, the present inventors have used various methods, that is, dissolution and hydrolysis methods using enzymes, to prepare a degradation product containing cocoon-derived silk proteins using silkworm cocoons, Alkaline and neutral salt dissolution samples (three kinds of silk lysates) and proteolytic enzyme hydrolysates samples (ten kinds of silk enzyme hydrolysates) were prepared.

First, 20 g dried silkworm cocoons were purified using a Soxhlet system for 5 hours (occurring at least 5 rotations of the solvent) using 40 times the volume of ethyl ether. A small amount of wax and lipid components present in the cocoon were removed to maximize the protein content of the cocoon.

The silk dissolution was performed using an acid/alkali and neutral salt dissolution method in which a solvent was added to the silkworm cocoon refining material from which sericin was removed according to the volume ratio (yoke ratio) shown in Table 1.

As a result, when three kinds of silk lysates were prepared using hydrochloric acid, sodium hydroxide, and calcium chloride, the yields were acid (26.4%) < alkali (32.0%) < neutral salt (calcium chloride) dissolution (86.1%) in order. high (Fig. 2a).

In addition, as shown in Table 2, the neutral salt lysate was treated with 4 kinds of digestive enzymes, 5 kinds of microbial-derived proteolytic enzymes, and 1 kind of plant-derived proteolytic enzymes, respectively, to obtain 10 kinds of silk protein enzyme hydrolysates. was prepared (Fig. 2b).

Example 2. Characterization of silkworm cocoon hydrolysates containing cocoon-derived silk proteins

The present inventors analyzed the characteristics of silk proteins by manufacturing method using high-speed protein liquid chromatography, infrared spectroscopy, and far-dichroism spectroscopy to investigate the physiological activity of hydrolysates containing cocoon-derived silk proteins. .

First, fast protein liquid chromatography (FPLC) analysis can separate and analyze protein mixtures by size, and the conditions are as follows:

- Equipment used: AKTA purifier 100 (GE healthcare)

- Column: SuperdexTM 30 Increase 10/300 GL

- Mobile phase: 4M urea

- Flow rate: 0.5 mL/min

- Sample concentration and volume: 0.1 %, 300 uL

- detection wavelength: 280 nm.

As a result of FPLC analysis, as shown in Figure 3 and Table 3 (result of calculating the molecular weight (Da) of each peak shown in the FPLC chromatogram of silk protein by manufacturing method), it was confirmed that the molecular weight distribution of silk protein varies depending on the dissolution method . In addition, the acid-alkali lysate has a very low molecular weight compared to the neutral salt lysate, and their main molecular weight was calculated to be 12 kDa for the acid-alkali lysate and 48 kDa for the neutral salt lysate (Table 3).

In addition, in the case of enzymatic hydrolysates treated with neutral salt lysate, different molecular weight distributions were shown depending on the type of enzyme, and high molecular weight silk proteins of about 30 kDa were present in Pepsin, Trypsin, a-Chymotrypsin, Sumizyme, and Papain hydrolysates. It was found that the low molecular weight of 1.5 kDa or less was predominantly present in the hydrolysates of Pancreatin, Protease, Alkaline protease, Protamex, and Flavourzyme (Table 3). This difference is presumed to be due to differences in enzymatic activity for silk protein substrates for each enzyme.

The molecular weight unit of the peak is Da.

In addition, Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) analyzes a sample using the characteristic of absorbing light differently according to the vibration and rotational movement of the molecular skeleton when white light in the infrared region is irradiated to the sample. It is an analysis method that can obtain information on chemical bonding within, and a Fourier transform spectrometer S100 (Perkin Elmer) was used.

As a result of FTIR analysis, as shown in FIG. 4, it was confirmed that the spectrum of silk protein varies depending on the manufacturing method. Through the formation of broad absorption peaks between 3200 - 2500 cm-1 in acid/alkaline lysates containing large amounts of low-molecular-weight proteins, and hydrolysates of Pancreatin, Protease, Alkaline protease, Protamex, and Flavourzyme, bound water was found in these samples. It was found that a large amount of was present (Figs. 4A and 4C).

1800 - 1200 cm ^-1 region for detailed structural analysis was enlarged and observed (FIGS. 4B and 4D), Amide I-III region (I: 1650 - 1600 cm -1; II: 1550 - 1500 cm ^-1 ; III : 1280 - 1230 cm ^-1 ) It was found that the peptide bond of the protein is present through the absorption peak at.

In addition, an absorption peak was observed at 1390 cm ^-1 in acid/alkali lysates and enzymatic hydrolysates excluding Pepsin, which seems to be due to symmetric vibration of the carboxyl group (COO-) generated by cleavage of peptide bonds. In the neutral salt lysate in which enzymatic degradation did not occur, the peak did not appear at the corresponding position, and in the case of the Pepsin enzyme hydrolysate in which degradation occurred in an acidic environment of pH 2, the peak did not appear at the corresponding position due to protonation.

In addition, circular dichroism spectroscopy (CD) is an analysis method that can obtain information on the structural characteristics of a sample by using the principle that a material with an asymmetric structure absorbs left or right polarized light differently, The protein shows a typical CD spectrum in the region of 190 - 240 nm depending on its secondary structure (beta-sheet: negative peak at 218 nm; alpha-helix: two negative peaks at 222 and 208 nm, positive at 193 nm). peak; beta-turn: positive peak at 207 nm; random coil: negative peak at 195 nm)

- Equipment used: Circular dichroism spectropolarimeter J-1500 (Jasco)

- Used cuvette: 1 cm pathlength cuvette

- Sample concentration: 0.002 %

As a result of the CD analysis, as shown in FIG. 5 , it was confirmed that the CD spectrum of the silk protein varied depending on the preparation method. Neutral salt lysates form a negative peak at 218 nm and form a β-sheet secondary structure, while acid lysates and enzyme hydrolysates have a strong negative peak in the range of 195 - 198 nm. From the formation of peaks, it was found that a random coil secondary structure was predominantly present. On the other hand, in the case of alkaline lysate, it was found that the beta-turn secondary structure was dominantly present through the appearance of positive peaks in the region between 200 and 205 nm of the protein. Among several enzymatic hydrolysates, when digested with Sumizyme, the negative peak, meaning random coil between 195 and 198 nm, moved to 200 nm, and the size of the negative peak at 218 nm, meaning beta-sheet structure, compared to other samples. It can be inferred that the beta-sheet structure content is higher than that of other enzymatic hydrolysates through the large appearance.

Example 3. Effect of cocoon silk protein on collagenase and MMP3 production

The present inventors confirmed the effects of silk protein (fibroin) for each manufacturing method confirmed in Examples 1 and 2 on collagenase and MMP3 production of human-derived chondrocyte cell line SW1353 cells.

Collagenase (Matrix metalloprotease 1, MMP1) and MMP3 are enzymes used to decompose cartilage tissue due to over-secretion by cartilage cells in the environment of degenerative arthritis. To investigate, SW1353 cells were treated with IL1beta, an arthritis-inducing substance, and the effect of inhibiting the production of MMP1 and MMP3 of silk proteins was confirmed.

Briefly, it is as follows: SW1353 cells were dispensed in a 24-well plate at a density of 2x10 ⁵ cell/well, and after 24 hours, IL1beta (40 ng/mL), a degenerative arthritis inducer, was added along with silk protein sample (1000 μg/mL). Incubated for 24 hours. Then, the cell medium supernatant was obtained and used for analysis of MMP1 and MMP3 production. The amount of MMP1 and MMP3 produced was measured using an enzyme-linked immunosorbent assay (ELISA).

As a result, as shown in FIG. 6, when IL1beta, an arthritis-inducing substance, was treated alone, the secretion of MMP1 and MMP3 was 12.5 times (FIG. 6A) and 8.2 times (FIG. 6B), respectively, compared to the untreated group (control). increased. Excessive secretion of tissue-degrading enzymes such as MMP1 and MMP3 threatens cartilage health and may lead to degenerative arthritis.

Interestingly, most of the silk proteins treated with IL1beta did not inhibit the production of MMP1 and MMP3, but Sumizyme enzyme hydrolysate significantly reduced the increased secretion of MMP1 and MMP3 by one-third of IL1beta treatment. .

Therefore, it can be seen that Sumizyme enzyme hydrolyzate can effectively inhibit inflammation.

Therefore, Sumizyme enzyme hydrolyzate was selected as the active ingredient of the present invention, and an experiment was conducted to confirm the effective concentration by treating Sumizyme enzyme hydrolyzate at different concentrations (100, 200, 500, 1000 μg/mL).

As a result, as shown in FIG. 7, at a low concentration of 200 μg / mL or less, MMP1 and MMP3 production inhibitory effects are not shown, and a weak effect at a concentration of 500 μg / mL and an excellent effect at a concentration of 1000 μg / mL are found. could

Example 4. Effect of cocoon silk protein on the production of inflammatory mediators in RAW264.7 cells

The present inventors confirmed the effect of the enzyme hydrolyzate of Sumizyme confirmed in Example 3 on the production of inflammatory mediators in mouse-derived macrophage cell line RAW264.7 cells.

Excessive inflammatory reactions in the body are known to cause destruction of normal tissues and promote various aging reactions. To investigate the anti-inflammatory effect of silk protein, RAW264.7 cells were treated with LPS, an inflammation-inducing substance, and the inhibitory effect of silk protein on the production of inflammatory mediators (Nitric oxide and IL6) was investigated.

Briefly, it is as follows: RAW264.7 cells were dispensed in a 24-well plate at a density of 2x10 ⁵ cell/well, and after 24 hours, LPS (1 μg/mL), an inflammation-inducing substance, was mixed with silk protein sample (1000 μg/mL). Incubated together for 24 hours. Then, the cell medium supernatant was obtained and used for analysis of the amount of inflammatory mediators produced. Nitrix oxide (NO) content in the cell culture medium was measured using Griess reagent. IL6 production was measured using an enzyme-linked immunosorbent assay (ELISA).

As a result, as shown in FIG. 8, when comparing the NO production inhibitory effect of silk protein for each manufacturing method, it was found that the enzyme hydrolyzate of Sumizyme effectively inhibited NO production.

In addition, as shown in FIG. 9a, when comparing the inhibitory effect of interleukin 6 (IL6) production of silk protein for each manufacturing method, the neutral salt lysate and the enzyme hydrolyzate of Sumizyme showed excellent inhibitory effects on inflammatory mediators compared to other samples.

However, in the case of the neutral salt lysate, a problem of low solution stability appeared due to the high molecular weight.

Therefore, Sumizyme enzyme hydrolyzate was selected as the active ingredient of the present invention, and an experiment was conducted to confirm the effective concentration by treating Sumizyme enzyme hydrolyzate at different concentrations (100, 200, 500, 1000 μg/mL).

As a result, as shown in FIG. 9b, Sumizyme exhibited an inhibitory effect on IL6 production in a concentration-dependent manner.

Overall, the present inventors prepared hydrolysates containing cocoon silk proteins obtained through various methods, and found that among them, cocoon hydrolysates obtained by treating silkworm cocoons with Sumizyme enzyme showed remarkable anti-inflammatory effects. . Therefore, the selected silkworm cocoon Sumizyme enzymatic hydrolyzate of the present invention can be applied as a health functional food as well as a treatment for inflammatory diseases, and thus, it is expected to increase farm household income.

Hereinafter, the present invention will be described in more detail through formulation examples. The formulation examples are only for exemplifying the present invention, and the scope of the present invention is not construed as being limited by the formulation examples.

Formulation Example 1. Preparation of food composition

1-1. Manufacture of health functional food

Silkworm cocoon hydrolysate 100 mg

Appropriate amount of vitamin mixture

70 g Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

Vitamin B12 0.2 g

Vitamin C 10 mg

10 g of biotin

Nicotinamide 1.7 mg

50 g folic acid

Calcium Pantothenate 0.5 mg

Appropriate amount of mineral mixture

Ferrous sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium Carbonate 25.3 mg

Potassium Phosphate Monobasic 15 mg

Dibasic Calcium Phosphate 55 mg

Potassium citrate 90 mg

Calcium Carbonate 100 mg

Magnesium Chloride 24.8 mg

Although the composition ratio of the above vitamin and mineral mixture was prepared by mixing ingredients suitable for relatively healthy food in a preferred embodiment, the mixing ratio may be arbitrarily modified. , Granules can be prepared and used in the preparation of health food compositions according to conventional methods.

1-2. Manufacture of health drinks

Silkworm cocoon hydrolysate 100 mg

15 g of vitamin C

100 g of vitamin E (powder)

19.75 g iron lactate

Zinc oxide 3.5 g

Nicotinamide 3.5 g

Vitamin A 0.2 g

Vitamin B1 0.25 g

Vitamin B2 0.3 g

water quantity

After mixing the above ingredients according to the usual health drink manufacturing method, stirring and heating at 85 ° C. for about 1 hour, the resulting solution is filtered and collected in a sterilized 2 L container, sealed and sterilized, and then refrigerated. It is used for preparing the health drink composition of the present invention. Although the composition ratio is a mixture of ingredients suitable for a relatively favorite beverage in a preferred embodiment, the mixing ratio may be arbitrarily modified according to regional and ethnic preferences such as the class of demand, the country of demand, and the purpose of use.

Formulation Example 2. Preparation of pharmaceutical composition

2-1. manufacture of powders

Silkworm cocoon hydrolysate 20 mg

Lactose 100 mg

Talc 10 mg

A powder is prepared by mixing the above ingredients and filling them in an airtight bag.

2-2. manufacture of tablets

Silkworm cocoon hydrolysate 10 mg

Corn Starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above ingredients, tablets are prepared by tableting according to a conventional tablet manufacturing method.

Formulation Example 3. Manufacture of Feed Additives

Silkworm cocoon hydrolyzate 2.0%

Glucose 2.0%

Peptone 1.0%

Yeast extract 1.0%

Dibasic Phosphate 0.2%

Magnesium Sulphate 0.05%

Cysteine 0.05%

Purified water to 100%

Appropriate amount of excipient degreasing steel

In the above, specific parts of the present invention have been described in detail, and for those skilled in the art, it is clear that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (16)

A method for producing a cocoon-derived silk protein hydrolysate comprising the following steps:
(a) scouring the silkworm cocoon to remove sericin;
(b) dissolving the refined product prepared in step (a) with a neutral salt; and
(c) obtaining a silk protein hydrolyzate by hydrolyzing the lysate prepared in step (b) with an enzyme.
According to claim 1,
The neutral salt of step (b) is at least one selected from the group consisting of calcium chloride, sodium chloride, potassium chloride, sodium nitrate, potassium nitrate, sodium sulfate, potassium sulfate, ammonium chloride and ammonium sulfate Method.
According to claim 2,
Characterized in that the neutral salt is an aqueous calcium chloride solution.
According to claim 1,
The enzymes in step (c) include Sumizyme, Papain, Pepsin, Trypsin, α-Chymotrypsin, Kojizyme, and Pancreatin , Alkaline protease, Protamex, Flavorzyme, kallikrein, Cathepsin, thermolisin, Subtilisin, bromelain (Bromelain), ficin (Ficin), actinidin (Actinidin), peptidase (Peptidase) and transglutaminase (Transglutaminase) characterized in that at least one member selected from the group consisting of, method.
According to claim 4,
The method, characterized in that the enzyme is Sumizyme.
According to claim 1,
Characterized in that it further comprises the step of inactivating the enzyme by raising the temperature to 90 ℃ after the hydrolysis of step (c) is completed.
According to claim 1,
Characterized in that the cocoon-derived silk protein hydrolysate inhibits the production or expression of Matrix metalloprotease 1 (MMP1) and Matrix metalloprotease 1 (MMP3).
According to claim 1,
The cocoon-derived silk protein hydrolyzate is characterized in that it inhibits the production or expression of nitric oxide (NO) and interleukin-6 (IL-6).
A cocoon-derived silk protein hydrolysate prepared according to the method of claim 1.
An anti-inflammatory composition comprising the cocoon-derived silk protein hydrolyzate of claim 9.
A pharmaceutical composition for preventing or treating inflammatory diseases, comprising the cocoon-derived silk protein hydrolyzate of claim 9.
According to claim 11,
The inflammatory diseases include degenerative arthritis, inflammatory bowel disease, peritonitis, osteomyelitis, cellulitis, pancreatitis, trauma-induced shock, bronchial asthma, allergic rhinitis, cystic fibrosis, acute bronchitis, chronic bronchitis, acute bronchiolitis, chronic bronchiolitis, osteoarthritis, gout, Spondyloarthropathy, ankylosing spondylitis, Reiter's syndrome, psoriatic arthropathy, enteropathic spondylitis, juvenile arthropathy, juvenile ankylosing spondylitis, reactive arthropathy, infectious arthritis, post-infectious arthritis, gonococcal arthritis, tuberculous arthritis, viral arthritis , fungal arthritis, syphilitic arthritis, Lyme disease, arthritis associated with vasculitis syndrome, polyarteritis nodosa, hypersensitivity vasculitis, Lougenic granulomatosis, polymyalgia rheumatica, articular cell arteritis, calcium crystal deposition arthropathy, pseudogout, non-joint Rheumatism, bursitis, tenosynovitis, epicondylitis (tennis elbow), neuropathic joint disease, hemarthrosic, Henoch-Schenlein purpura, hypertrophic osteoarthropathy, multicentric reticulocytoma, scoliosis ( scoliosis), hemochromatosis, hemoglobinopathy, hyperlipoproteinemia, hypogammaglobulinemia, familial thalassemia fever, Behat's disease, systemic lupus erythematosus, relapsing fever, multiple sclerosis, sepsis, septic shock, acute respiratory distress syndrome, multiple organ failure , chronic obstructive pulmonary disease, rheumatoid arthritis, acute lung injury, bronchopulmonary dysplasia, asthma, periodontitis, stomatitis, peritonitis, gastritis, Enteritis, arthritis, nephritis, characterized in that at least one member selected from the group consisting of hepatitis and inflammatory skin diseases, the composition.
A food composition for preventing or improving inflammatory diseases, comprising the cocoon-derived silk protein hydrolyzate of claim 9.
Health functional food containing the food composition for preventing or improving inflammatory diseases of claim 13.
An anti-inflammatory cosmetic composition comprising the cocoon-derived silk protein hydrolyzate of claim 9.
A feed composition for preventing or improving inflammatory diseases, comprising the cocoon-derived silk protein hydrolyzate of claim 9.

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