KR20190055624A - Method of preparing Cocoon Sericin extract, and composition including the same - Google Patents

Abstract

The present invention provides a sericin extract obtained by extracting sericin from cocoons using deep seawater or thermal water; a method of producing the same; and a pharmaceutical composition, a cosmetic composition, and a cleaning composition, comprising the extract. More specifically, mineral-sericin of the present invention has antioxidant, antibacterial, and whitening effects that are five times or more stronger than that of pre-existing sericin.

Description

[0001] The present invention relates to an eco-friendly preparation method of sericin extract and a composition containing the extract,

The present invention relates to an eco-friendly method for preparing a sericin extract and a composition containing the extract. More particularly, the present invention relates to a method for extracting sericin from a silkworm cocoon using deep sea water or hot spring water, Extracts, extracts containing mineral components, sericin and herbal extracts from deep seawater or hot spring water from cocoon cocoons and corymbs using deep sea water or hot spring water, their preparation, and pharmaceutical compositions, cosmetic compositions and cleansing compositions comprising the extracts .

Silk is synthesized in silk gland cells, stored in silk gland cell lumen, and secreted in liquid silk to produce cocoon silk gland. The structure of the silk fiber cocoon silk fiber is composed of the fibroin protein in the core part and the sericin protein surrounding it. Sericin acts as a natural cross-link (natural rubber) in the silk polymer to form a cocoon. Silk degumming is an important process for elegant luster and soft touch by removing sericin from the fibroin area. It is mainly purified water or soap (marseille soap; Svilokos et al. 1992) or soda alkali method (Na ₂ CO ₃ , NaOH, Na ₂ S ₂ O ₄ , Na ₂ P ₃ O ₁₀ , and CH ₃ COOH, Rangam et al., 2010).

In the silk industry, large amounts of sericin-containing solutions have been treated with waste water because only fibroin fibers are needed. However, recent reports on sericin's efficacy and functionality have shown that offshoot, by-product, is recycled. However, refinement by soap / soda alkaline solution contains many impurities and chemical reagents even after the step of removing and separating the chemical used to increase the sericin solubility in the refining process, so that pure sericin extraction is almost impossible and the neutralization step, A dialysis step and the like are required.

Sericin extracted from silk refining through high temperature and high pressure (HTHP; Sheetal et al., 1994), estimation method (Gulrajani et al., 1992) and enzyme purification method (Sheetal et al., 1994) , And wastewater.

Conventional methods of refining soap, alkali, acid, etc. are effective in removing sericin, but they cause hydrolysis of sericin protein structure and loss of active ingredient. Therefore, it is necessary to develop cericin extraction method with high energy efficiency without using chemical reagents and minimizing sericin damage. During the sericin purification process, sericin is hydrolyzed partially or totally and dissolved in the purification buffer. Extraction temperature and time affect the effect, quality and quantity of sericin and need to be controlled to obtain effective substances.

As known, sericin has a composition similar to natural moisturizing factor of human skin, so it has been reported that it has high ability of moisturizing ability, strong binding ability to skin horny, UV blocking and anti-wrinkle function and antioxidant function. Are not reported.

Recent environmental and psychological influences have shown that skin and scalp are excessively active oxygen, which causes skin aging, collagen and keratin to decrease, resulting in hair loss, and thus contains antioxidants, anti-aging, moisturizing and antibacterial functions for scalp health It is necessary to develop technology for separation and purification of raw materials.

An object of the present invention is to provide a sericin extract obtained by extracting sericin from silkworm mucilage using deep sea water or natural carbonate of hot spring water (hereinafter referred to as " minerals - sericin, Marine mineral-Sericin, MS), a process for its production, and pharmaceutical compositions, cosmetic compositions and cleaning compositions comprising the extracts.

Disclosure of the Invention The present invention has been proposed in order to solve the problems of the conventional art described above, and it is an object of the present invention to provide a method of removing natural carbonate ions (CO ₂ + H ₂ O ⇔ H ₂ CO ₃ ⇔ H ⁺ + HCO ₃ ^- ⇔ H ⁺ + CO ₃ ^{2 -} ).

An example of the present invention relates to a mineral-sericin (MS) extract containing minerals and sericin obtained by extracting sericin from a cocoon using deep-sea water or hot spring water, and a method for producing the same.

Another example of the present invention is an extract (hereinafter referred to as mineral-Sericin-Morus alba (MSM) extract) containing minerals, sericin and bark extracts (hereinafter referred to as mineral-Sericin-Morus alba (MSM) extract) obtained from cocoon cocoons and corynebacterium using deep sea water or hot spring water. And a method for producing the same.

An example of the present invention is to minimize or reduce the change in the amino acid composition and the molecular weight of the silkworm cocoons, the incorporation of fibroin and chemical reagent components, and the change in the amino acid composition and the molecular weight, which are caused by the treatment of sericin with a high temperature alkali aqueous solution for a long time, . In addition, the present invention aims to provide an efficient process for safety and environment-friendly (use of all wastewater) in the sericin extraction process using deep sea water.

It is another object of the present invention to provide an extracting process for extracting bark of alfalfa from the sericin extraction process using deep seawater for further improvement of functions such as antioxidation, whitening, anti-wrinkle, antibacterial and stability.

It is still another object of the present invention to provide a cosmetic composition comprising an extract of deep sea mineralized water of sea water or freeze-dried powder prepared according to the present invention as an active ingredient.

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

An example of the present invention is a mineral-sericin extract comprising sericin prepared by using a deep seawater or hot spring water as an extraction raw material, a method for producing the mineral-sericin extract, and a pharmaceutical comprising the mineral-sericin extract Compositions, food compositions or cosmetic compositions. In a preferred embodiment, the sericin extract according to the present invention may be a sericin extract prepared from a silkworm cocoon using deep sea water, and is hereinafter referred to as an example of a mineral-sericin (MS) extract.

Another example of the present invention relates to a method for producing a mixed extract (mineral-sericin-mangabbukus extract) prepared by extracting cocoon cocoons, corynebacterium or upper limb using deep sea water or hot spring water, And to a pharmaceutical composition, food composition or cosmetic composition comprising the extract. The extract of Corynebacterium or Corynebacterium may be prepared by preparing a mixed extract material by mixing Corynebacterium or Corynebacterium with cocoon cocoa, extracting it with corysine extract prepared from cocoon cocoa, or extracting it with sericin prepared from cocoon The extract may be prepared by separately adding an extract extracted from the bark or the upper limb. In a preferred embodiment, the mixed extract according to the present invention may be one obtained by extracting the sericin extract prepared from the silkworm cocoon with the addition of the bark or the upper bark, and more preferably using the deep sea water, adding the bark bark to the sericin extract prepared from the silkworm cocoons And is hereinafter referred to as an example of a mineral-sericin-mars bark (MSM) extract.

The extract according to the present invention can be used in the form of an extract itself, a concentrate and / or a powder. The dried powder may be dried by a lyophilization method.

A method for producing a mineral-sericin (MS) extract according to an embodiment of the present invention comprises the steps of (1) preparing a cocoon cocoon, (2) extracting cocoon with deep sea water or hot spring water containing minerals, And (3) removing the fibroin contained in the extract. Preferably, the method comprises the steps of (4) concentrating the extract obtained in the step (3), and / Or (5) drying the concentrate.

It is expected that the sericin recovery using the natural-friendly method using the deep sea water without the use of the chemical reagent in the method of producing the extract according to the present invention will be applied to the environmental protection aspect of the future industry and the economic ripple effect. The refining of silk and silk using deep sea water containing marine minerals enables minerals-sericin extract to be obtained with minimal energy cost and less time, while minimizing the incorporation and damage to silk fibroin, It is possible.

(CO ₂ + H ₂ O ⇔ H ₂ CO ₃ ⇔ H ⁺ + HCO ₃ ^- H ₂ O) as a solvent that can replace a solvent such as soap, soda water, etc. used in refining silkworm cocoon in the above- ⇔ H ⁺ + CO ₃ ^{2 -} ). Conventionally, removal of the alkaline component removing step, which occurs at the time of recovery after soap for obtaining sericin, soda alkali method (Na ₂ CO ₃ , NaOH, Na ₂ S ₂ O ₄ , Na ₂ P ₃ O ₁₀ , CH ₃ COOH, etc.) It is dissolved using natural carbonate ion of deep sea water or hot spring water to obtain pure sericin including marine minerals.

Carbon dioxide is dissolved in seawater and chemical reaction occurs in seawater. It reacts with minerals in seawater to produce carbonates. There are various kinds of them, one of which is sodium carbonate. In water are the carbonate ion _{CO 2 + H 2 O ⇔ H} 2 CO 3 ⇔ H + + HCO 3 - is present forms a ballast in a variety of forms, such as Scheme ^{_{- ⇔ H + + CO 3 2}} . A commonly used sodium carbonate reagent reacts with water and exists in the form of Na ₂ CO ₃ + H ₂ O ⇔ NaOH + NaHCO ₃ .

According to the present invention, the mineral content of the MS extract or the MSM extract prepared using deep sea water is higher than that of sodium carbonate, magnesium, potassium, boron, and strontium. In a specific example, the MS extract or MSM extract according to the present invention prepared using deep sea water has a sodium concentration of 20,000 to 50,000 ug / g, specifically 35,000 to 45,000 ug / g, magnesium 15,000 to 30,000 ug / g, G, in particular from 5,000 to 17,000 ug / g, and strontium from 40 to 250 ug / g, in particular from 70 to 200 ug / g. In addition, MSM extracts have higher sodium, calcium, potassium and boron contents than MS extracts.

Specifically, MSM extract according to the present invention has antimicrobial activity against Bacillus and Staphylococcus, as compared with conventional extracts using sodium carbonate and MS extract (see FIG. 7). The MSM extract has antimicrobial activity against acnes and dandruff, and the MS extract has antibacterial activity against dandruff (see FIG. 8).

The present invention further provides a method for producing an extract comprising sericin protein of a deep sea water or hot spring water of the present invention and a sericin protein of silkworm cocoon, further comprising an extract of Morus alba L. for the purpose of further enhancing functions such as antioxidation, whitening, anti- To provide a method for preparing the extract.

The present invention relates to a method for extracting and purifying sericin without loss of sericin-derived components or using antioxidant, whitening, or antibacterial function using a natural carbonate of deep sea water without using conventional soap or alkaline water. Is prepared by extracting and dissolving sericin, which is high in solubility, simple process (neutralization process is omitted), low cost and long-term storage, compared with soap or alkaline water using natural carbonate of deep sea water or spring water of the East Sea and dried. The marine minerals-sericin extract of the present invention is characterized by containing a silk amino acid, a silk peptide, a marine mineral, and an extract of Phytophthora barkifolia. The composition can be used for cosmetics, scalp cleaner, skin application agent, skin cleanser, mouthwash have.

In addition, the extract according to the present invention may be a mineral component contained in deep sea water or hot spring water, which is an extraction solvent. The minerals contained in the extract according to an embodiment of the present invention are in the range of 80,000 to 88,000 mg / L, and the sodium, magnesium, sodium, and calcium contained therein are main minerals and trace amounts of boron, silicon, zinc, strontium, It was contained as an element and there were no harmful factors.

In an exemplary embodiment, a marine mineral composition comprising serine (24.3 mg / g), aspartic acid (21.8 mg / g), glycine (11.6 mg / g), and other minerals including sodium, magnesium, calcium, Lt; RTI ID = 0.0 > amino acid < / RTI > composition protein. In an exemplary embodiment, an MSM extract or powder having superior antioxidative, antimicrobial, whitening and anti-wrinkle effects than sericin extract or powder extracted with water or soda water is provided.

In addition, since the existing sericin protein which has been isolated may have a fear of bacterial proliferation in preservation, it is necessary to add a preservative such as sodium azide and thus it is difficult to apply it to medicines, foods, cosmetics, etc. However, Extracts can be stored, distributed, and provided for long-term stability by acting as a preservative in the sodium chloride contained in deep sea water.

In addition, the mineral-sericin powder according to the present invention can be used for various functions and efficacy analysis of sericin. Specifically, the mineral-sericin of the present invention has an effect of five times or more as much as the antioxidation, antibacterial, and whitening of existing sericin. In addition, moisturizing, regenerating, and nutrition, it is effective for hair, skin and scalp care.

(Na), skin elasticity (K), skin metabolism (Mg, Ca), calcium (Ca), magnesium (Mg) ). In the case of extracting sericin according to the present invention, when the deep sea water is used, the yield is higher than that of the sodium carbonate reagent. In the case of the aqueous solution of the crude extract, concentrated extract and extract powder according to the present invention, The 1% (w / v) aqueous solution has pH 5.5 to pH 7.5, preferably pH 6.0 to 7.0, and is suitable for skin protection, disinfection, sterilization and the like. Specifically, the 1% (w / v) aqueous solution of the extract powder according to the present invention has a pH of 6.8 and a salinity of 5.7, which is advantageous for the production of neutral or acidic products such as hair loss and atopic treatment.

Therefore, the mineral-sericin obtained in the present invention is stable in properties, has excellent efficacy, can be used in cosmetics or food fields, and is extracted and separated by a deep sea water. It can be usefully applied to other fields. As a result, the present invention is expected to contribute to a new sericin extraction separation technology and product development effect.

The extract according to the present invention may be an extract containing sericin protein and an extract containing minerals and mulberry extract. The sericin or a fragment thereof contained in the extract according to the present invention has a molecular weight of 10 to 250 kDa, and the sodium carbonate extract has been decomposed into a low molecular state, while the MS and MSM extracts have various ranges of protein distribution (Fig. 2). In addition, sericin extract, a glycoprotein conjugated with mannose and galactose, is effective in maintaining activity and efficacy.

The extract according to the present invention contains sericin protein and a fragment thereof. Specifically, as a result of analyzing the kinds and contents of amino acids, 15 kinds of constituent amino acids were analyzed. Among them, hydrophilic serine and aspartic acid are the main components. The amino acid composition is similar to the natural moisturizing factor of human epidermis (Table 5). In addition, the composition of this hydrophilic amino acid acts to increase the hygroscopicity about 50 times as compared with glycerin.

Methods and components of the extract of the present invention will be described in detail below. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram illustrating a process for producing powders of a mineral-sericin extract (MS) or a mineral-sericin-cauliflower extract (MSM) according to an embodiment of the present invention.

The subject to be extracted for producing the extract according to the present invention may be a mixture of (i) silkworm alone, or (ii) one or more mulberry snakes selected from the group consisting of silkworm, cortex and upper limb.

In the method for producing an extract according to the present invention, the step (1) of preparing the cocoon cocoons may be carried out by removing impurities in the cocoon cocoons and cutting them. For example, in step (1), the cocoon can be removed by putting it in a 95 ° C washing solution for 3 minutes to remove the impurities. The content ratio of the extraction solvent (w / v, or w / w) is from 1:10 to 1:100, 1:20 to 1:100, 1:13 to 1:100, 1:14 to 1:100, 1:50 to 1:100, 1:20 to 1: 90, 1:30 to 1:70, or 1:40 to 1:60. Through the above process, it is possible to remove impurities, for example, substances other than cocoons, bacteria and the like.

Silkworm (Bombyx mori), Japanese oak silkworm (Antheraea yamamai), and chinese oak silkworm (Antherae pernyi), which belong to Bombycidae. Of these, silkworm silkworms may be either artificial flesh or artificial feed. For example, it is possible to use a cocoon cocoon made by a silkworm before a pupa, and cutting this cocoon layer. The smaller the cut size of the cocoon is, the higher the solubility is, and it is desirable to divide approximately 1 to 8 cocoons into cocoons.

98% of the silk contained in the cocoon is a silk protein, consisting of 25-30% by weight of sericin and 70-75% by weight of fibroin. Sericin is a type of silk protein in the silkworm cocoon, which together with the fibrous fibroin constitutes the main silk protein. Fibroin is an insoluble fibrous protein and sericin is an amorphous matrix of solubilized spherical proteins and has a gum to hold fibroin filaments. Sericin is distributed from 17.8 wt% to 33 wt% from the inside to the outside of cocoon, and fibroin is distributed from 79.1 wt% to 65 wt%, and sericin is distributed widely in the outer thick part.

The water absorption is about 50 times higher than that of glycerin because about 80% of the amino acids constituting sericin have a hydrophilic lateral group. Therefore, sericin has a composition similar to the natural moisturizing factor (NMF) of the skin, so it has high moisturizing ability, strong binding ability to the skin's keratin, sunscreen and wrinkle preventing function, antioxidant function and tyrosinase inhibiting action Have been reported, and the high UV blocking effect shows excellent characteristics in the development of cosmetics.

Mori Radicis Cortex is a moth (Morus alba L.) and a mulberry tree. It is made by removing the cork layer from the root of the root of the root of the plant. In the winter, it ishes the roots cleanly, peels off the cork layer when it is fresh, And the skin was separated, and only the white skin was taken. On the other hand, the upper limb (Mori Ramulus) is the dried branch of the mulberry plant. Morus alba, Linne, Morus alba for Peldulls DIPPEL, Morus bombycis, Koidzumi, Morus bombycis var. Caudatifolia, Morus bombycis var. maritima KOIDZ, Morus bombycis for Kase VYKEI, Mongolian mulberry, King mulberry and Morus tiliaefolia, Makino. These are deciduous broad-leaved arboreous trees, which are distributed at altitudes between 500 and 1,400 m They are horizontally distributed.

In the combined extracts of the silkworm mucilage and mulberry according to the present invention, the upper and the upper bark may be used alone or as a mixture for extraction. The mixing ratio is not particularly limited. For example, the weight ratio of the upper bark: 9: 1 or 2: 8 to 8: 2, specifically 1: 1 by weight. In addition, the upper and upper bark can be reused several times, for example, three times, after being used for producing the extract.

In the method for producing an extract according to the present invention, the step (2) of extracting cocoon cocoons with deep seawater or hot spring water containing minerals to prepare an extract containing sericin may be carried out by mixing the cocoon prepared in step (1) Or by immersion in hot water and heating. For example, sericin can be extracted by immersing 50 g (2%, w / v) of cocoon into 2.5 L of deep sea water at 98 캜 for 120 minutes.

In one embodiment of the present invention, the cocoon prepared in step (1) is boiled in the deep seawater at a rate of about 2% (w / v) at 95 to 98 캜 for 120 minutes. For example, about 5 g of cocoon cocoon cut into 2.5 L of deep seawater is allowed to stand at 98 ° C or higher, and is eluted for 90 minutes to 120 minutes. The elution time can be appropriately selected in consideration of solubility of sericin and inflow or outflow of fibroin.

The amount of deep seawater or hot spring water used for the dissolution of the sericin can be appropriately selected for the sericin extraction yield. For example, the amount of the deep seawater or hot spring water to be used is 100 mL to 10,000 mL , 2000 mL to 9000 mL, 3000 mL to 7000 mL, or 4000 mL to 6000 mL may be used. The extract according to the present invention can obtain a protein comprising about 10 to 35 parts by weight, or 20 to 30 parts by weight of sericin or a fragment thereof based on 100 parts by weight of the raw material cocoon.

About 5 to 20% by weight, 7 to 18 parts by weight, based on 100% by weight of the extract powder obtained by heat extraction, concentration and drying of the sericin extract according to the present invention (i.e., based on the solid content of the extract) 8 to 15 parts by weight of sericin or a fragment thereof and 1 to 10% (w / v) or 2 to 8% (w / v) of a mineral content, for example, 5% . ≪ / RTI > The water used in the conventional silk protein refining process and 0.02 M Na ₂ CO ₃ Compared to an alkaline solution, when the deep sea water or hot spring water is used according to the present invention, the solubility of the sericin extract is high, the pH is in a weak acidity range, and the salt can be controlled by sodium. In the case of a 1% aqueous solution of mineral-sericin powder, the pH was 6.4 to 6.8 and the salinity was found to be 0.7 to 0.87 (Table 2).

The extraction solvent used in the preparation of the extract according to the present invention may be minerals-containing water, for example, deep sea water or hot spring water, preferably deep sea water. The deep ocean water includes raw water, concentrated water, and high mineral water. Specifically, the deep sea water can be obtained from the water taken from the Ulleungdo, East Sea, Goseong, and other 200m depths on the east coast.

The present invention relates to a method of extracting sericin from a silkworm cocoon by using deep sea water or hot spring water, thereby causing a loss of function such as antioxidation and whitening of sericin which occurs when the silkworm is treated with a high temperature alkali aqueous solution for a long time, , Or to minimize or reduce the problems such as changes in the constituent amino acids and the molecular weight, and to provide an efficient sericin extraction method that is safe and natural in the sericin extraction process (using the entire waste water). In addition, by using deep sea water or hot spring water as an extraction solvent, it is possible to obtain an extract having the minerals contained therein as it is, and it has advantages of skin cleansing, skin moisture control, promotion of skin metabolism, removal of waste pores and sebum, have.

Deep Sea Water refers to seawater that does not reach the water depth of 200 m or less. The Deep Sea Water Conference of the Fisheries Agency of Japan has no organic matter production due to photosynthesis and seawater that does not cause vertical mixing in winter. It is. Marine deep seawater is a useful marine resource with low temperature (0-17 ℃), cleanness, stability, eutrophic, maturation and so on. It is utilized in various fields.

Mineral components of deep sea water are found to be good for skin, and they are also used in cosmetic products such as lotion, conditioner and shampoo. Deep sea water is divided into mineral water, high mineral water, and demineralized water depending on raw water and refining process. The raw water is used in its own state without diluting or concentrating the deep seawater. Concentrated water is concentrated by various methods such as heating the deep sea water, vacuuming, concentration, freezing, or membrane filtration, And the desalted water is obtained by reducing the sodium chloride content in the salt of the raw water or adding the mineral concentrated water to the demineralized water and processing the hardness lower than the raw water to the liquid phase.

The deep sea water has a salinity of 3 ‰ (per mill), an alkaline solution having a pH of 7.7 to 8.2, a large amount of effective minerals such as sodium (Na), potassium (K), calcium (Ca) and magnesium It is possible to suppress the loss of the sericin-derived component and the incorporation of the fibroin-derived component by using the carbonate generated by the reaction with CO ₂ in the fermentation broth, and it is possible to use the dissolved whole amount by minimizing sericin disappearance without wastewater, As shown in FIG.

An example of the deep ocean water of the present invention is an aqueous solution containing 9,000 to 13,000 mg / L of sodium (for example, 11,000 mg / L), 900 to 1,400 mg / L of magnesium (for example, 1,288 mg / L, such as 394 mg / L, and potassium 300 to 500 mg / L, such as 414 mg / L, and the weight ratio of sodium, magnesium, calcium and potassium is 25-28: For example, 11,000 mg / L of sodium, 1,288 mg / L of magnesium, 394 mg / L of calcium and 414 mg / L of potassium, in a ratio of 1: 0.5 to 1.5: 0.5 to 1.5, And may include marine minerals such as silicon, boron, strontium, zinc, aluminum and the like.

As an example of deep ocean water applied to the present invention, it may be one having the mineral composition shown in Table 1 below.

Hot spring water is groundwater that has been heated to above the average temperature due to geothermal heat and has been released into the surface. It usually has a water temperature of 25 ℃ or higher and contains certain mineral components, water vapor, and gas. As a hot spring, a wide range of hot springs means water that is physically and chemically different from ordinary water and whose natural special water comes out from the earth as an indicator. The classification of hot springs varies according to the scholar or country, but is generally divided according to temperature, dissolution type of hot spring, liquid ion concentration, solubility of minerals, or development status of hot springs. In the present invention, a hot spring water means hot mineral water which is eluted from underground and whose components are not harmful to the human body. Preferably, the hot mineral water is sodium, fluorine, potassium, calcium, iron, carbonic acid And the like. In one embodiment, the hot spring water of the present invention may be a Deokgu hot spring water in the Eungbongsan area on the hot spring in Deokgu-gu, Uljin-gun, Gyeongbuk province.

In another embodiment, the hot spring water of the present invention comprises 0.40 to 0.60 mg / L of potassium, 40.0 to 43 mg / L of sodium, 3.00 to 3.50 mg / L of calcium, 0.01 to 0.05 mg / L of magnesium, 30.00 to 35.00 mg / L, 0.05 to 0.10 mg / L of lithium, 0.01 to 0.05 mg / L of strontium, 0.01 to 0.05 mg / L of iron, 0.01 to 0.05 mg / L of manganese, 0.01 to 0.05 mg / L of copper, 0.01 to 0.05 mg / L may be 0.01 to 0.05 mg / L, 8.0 to 12.0 mg / L of fluorine, 4.00 to 5.00 mg / L of chlorine ion and 5.00 to 6.00 mg / L of sulfuric acid ion.

In the present invention, the hot spring water can be used as it is or can be processed and used, for example, as a concentrate or a solid obtained by evaporation, concentration and drying. Alternatively, a concentrate obtained by evaporating the natural hot water or by artificially desalting and drying (or evaporating) the hot water can be used. The method of evaporation, concentration and drying is not particularly limited, but can be obtained, for example, by evaporating underground hot spring water into primary, secondary and tertiary evaporators, filtering and concentrating and drying.

As an example of the hot spring water applied to the present invention, it may be one having the mineral composition shown in Table 2 below. The results of analysis of the components of Deokgu hot spring water are presented in the table. The unit is mg / L.

ingredient Content (mg / L) Potassium (K) 0.47 Sodium (Na) 42.3 Calcium (Ca) 3.1 Magnesium (Mg) 0.02 Silicon oxide (SiO ₂₎ 32.7 Lithium (Li) 0.08 Strontium (Sr) 0.03 Iron (Fe) 0.02 Manganese (Mn) 0.01 Copper (Cu) 0.03 Lead (Pb) 0.05 Zinc (Zn) 0.03 Aluminum (Al) 0.05 Fluorine (F) 9.8 Chlorine ion (Cl ^- ) 4.9 Sulfate ion (SO ₄ ^{2 -} ) 5.82 Total solids (TS) 160

In the method for producing an extract according to the present invention, the step (3) of removing the fibroin contained in the extract is obtained from the cocoon, and the removal of the fibroin contained in the extract containing sericin may be performed by various methods, for example, / ≪ / RTI > or filtered with a filter. Specifically, the extract is filtered on a glass or plastic filter with a cotton cloth, and purified by separation from the extraction residue. The purified solution contains sericin having a molecular weight of 10 to 250 kDa while the fibroin is completely removed. When fibroin is incorporated into the extract according to the present invention, the fibroin contained in the extract should be removed because of the problems such as lowered solubility and allergy. When fibroin is removed by the filtration method, the precipitate, which is an impurity contained in the extract, can be removed together, which is more effective.

The removed fibroin may be washed with water and dried to be used in silk fabrics, regenerative medicine, cosmetics, and the like.

Conventionally, in the method for producing sericin extract using soap or alkaline water, the extract is neutralized or removed after the removal of the fibroin due to high pH (9.0 or more) buffer, incorporation of fibroin fragment due to high temperature and high pressure, Buffer exchange or dialysis or enzyme removal is essential. However, according to the present invention, there is no need for a dialysis process in a method for producing sericin extract using deep sea water or hot spring water as an extraction solvent. The process of dialysis of deep ocean water or mineral concentrated water channel may cause problems such as deterioration of antioxidant function, pH increase, Na overload, and microbial contamination. In addition, since the sericin extract is filtered to remove the fibroin, the dialysis process is omitted, which saves time, effort and economy.

The method for producing an extract according to the present invention may further comprise at least one step selected from the group consisting of (4) concentrating the obtained extract and (5) drying the concentrate, wherein step (4) ) To obtain a concentrate, or the concentrate can be obtained by drying the concentrate by performing steps (4) and (5).

The extract obtained in the above step (3) can be used as it is for various purposes, but it can be used in the form of powder by performing (4) concentration step and concentration step, (4) concentration step and (5) have. The concentrate can be stored for 1 to 2 months at a refrigeration temperature (for example, 4 ° C), and can be used depending on the application. The dried product can be stored for 1 year or more at a freezing temperature condition (for example, -20 ° C to 0 ° C) Do. In addition, solutions or soluble powders of sericin extract can be used in various fields such as various functional analysis of sericin.

The step (4) of concentrating the extract can be carried out by heating the extract. The heating temperature is, for example, a lower limit value of 85 ° C or higher, preferably 90 ° C, 93 ° C or 95 ° C or higher, 90 ° C or lower, 93 ° C or lower, 95 ° C or lower, 97 ° C or lower, or 98 ° C or lower. Therefore, the concentration heating temperature according to the present invention is 85 to 90 DEG C, 85 to 93 DEG C, 85 to 95 DEG C, 85 to 97 DEG C, 85 to 98 DEG C, 90 to 93 Or 90 ° C to 97 ° C or 90 ° C to 98 ° C or lower, 93 ° C to 95 ° C or 93 ° C to 97 ° C or 93 ° C to 98 ° C or lower, or 95 ° C to 95 ° C, 97 deg. C, or 95 deg. C to 98 deg. C or lower. The time required for the concentration step may be appropriately selected according to the required concentration of the final concentrate, and may be, for example, 30 to 120 minutes, 30 to 90 minutes, or 50 to 60 minutes.

The drying step (5) may be carried out by various drying methods which can be pulverized without impairing the activity of the extract, and may be carried out, for example, by a freeze-drying method. The above-described freeze-drying method is substantially free from the freeze-drying conditions of a solution containing a conventional protein.

A method for producing an extract according to one embodiment of the present invention comprises the steps of (1) preparing a cocoon by cutting a cocoon to remove a pupa and cutting a cocoon, (2) deep seawater containing carbonate ions and minerals And (3) removing the fibroin contained in the extract. Preferably, the method comprises the steps of: (4) culturing the extract in the step ( Concentrating the extract obtained in step (3), and / or (5) drying the concentrate. For example, the step (1) of preparing the cocoon for cocoon is a step of cutting and cocooning the cocoon and adding 1 to 50% (w / v), preferably 5% v) to 20% (w / v), for example, 10% (w / v), at 85 ° C or higher, for example, at 99 ° C to remove impurities.

The step of extracting the step 2) may include adding 0.5 to 30% (w / v), for example, 2% (w / v) of cocoon to deep seawater or hot spring water at 85 to 98 ° C, For 60 to 180 minutes, for example, for 120 minutes, and sericin extraction can be performed.

The method for producing a mineral-sericin-mallow (MSM) extract of the present invention comprises the steps of (1) preparing cocoon cocoons, (2) extracting cocoon cocoons with deep seawater or hot spring water containing minerals, And (3) removing the fibroin contained in the extract. Preferably, the method for producing the extract comprises (4) concentrating the extract obtained in the step (3), and / or (5) drying the concentrate.

Step 3) After removing the fibroin fiber, the extract of cornicin and the upper body of the sericin solution are extracted and concentrated to prepare a marine mineral-sericin-mucilage extract.

In addition, Morus alba is added to the extracts containing deep seawater and sericin to heat and concentrate the extracts by evaporation of water and to extract the extract of Morus albacci. Can be further enhanced.

According to still another aspect of the present invention, there is provided a composition comprising as an active ingredient of the above-mentioned Marine mineral Sericin (MS) extract or powder. According to another aspect of the present invention, there is provided a composition comprising the above-mentioned marine mineral water sericin mers bark (MSM) with Mors bark extract added thereto. The composition may be a pharmaceutical composition, a cosmetic composition and a food composition.

The cosmetic composition according to the present invention has whitening, antioxidant or antimicrobial activity. Specifically, the antimicrobial activity may be an antimicrobial activity against gram-negative bacteria, dandruff and acne bacteria, and may be used for preventing or improving acne, May be a cosmetic composition.

The cosmetic composition comprising the mineral-sericin (MS) extract or the mineral-sericin-plant (MSM) extract according to the present invention can be administered extracutaneously, transdermally or subcutaneously, or hair- External application for skin, or transdermal application, external application for hair, more preferably skin, hair.

The cosmetic composition may be applied percutaneously to the skin, scalp, or hair. Specific examples thereof include a hair care product, a skin care product, a cleanser, a skin toner, a lotion, a mask pack and the like. Refers to a composition that can be used in the manufacture of all cosmetic products such as basic cosmetics, makeup cosmetics, body products (body cleansers, body lotions, etc.), shaving products, hair products (shampoo, rinse, hair essence, etc.) It may be formulated in the form of a warning agent, a spray agent, a suspension, an emulsion, a cream, a gel, a foam, etc., but there is no particular limitation on its form. For example, it can be used in the form of lotion, cream, essence, cosmetic lotion, spray, gel, pack, sunscreen, makeup base, foundation (solid type, liquid type or spray type), powder, , Cleansing oil, etc.) and a detergent (cleansing foam, soap, body wash, etc.). In addition, the cosmetic composition according to the present invention may be a tooth care product (oral product). Preferably, the cosmetic product includes a scalp shampoo, a hair conditioner, a hair conditioner, a hair protector, a shaving soap, a soap, a skin cleanser, a hand soap, a facial lotion, a skin toner, a mask pack, a tooth cleaner, do.

An example of the present invention relates to a pharmaceutical composition comprising the mineral-sericin (MS) extract or the mineral-sericin-plant (MSM) extract. Specifically, the pharmaceutical composition has an antibacterial activity, May be an antimicrobial activity against gram-negative bacteria, dandruff and acne bacteria, and may be a pharmaceutical composition for preventing, improving or treating acne, or a pharmaceutical composition for preventing, improving or treating dandruff. Mineral-sericin (MS) extracts or mineral-sericin-plant (MSM) extracts have been described above.

In addition to the above-mentioned cosmetic composition or pharmaceutical composition active ingredient, any kind of ingredient which can be used for ordinary commercialization or formulation such as a surfactant, emulsifier, soap acid, solvent, binder, diluent, lubricant, stabilizer, Preservatives, antibiotics, antioxidants, antifoaming agents, antimicrobial agents, enzymes, plant or mineral oils, fats, fluorescent substances, fungicides, hygroscopic substances, moisturizing agents, fragrances, fragrance carriers, preservatives And may contain one or more cosmetically acceptable further components or pharmacologically acceptable excipients selected from the group consisting of a protein, a silicone, a solubilizing agent, a saccharide derivative, a sunscreen agent, a vitamin, a plant extract and a wax, The kind and content thereof are not limited to the use of the final product and the cosmetic composition may contain a solvent normally contained in the form It may be to.

The present invention relates to an eco-friendly method for producing sericin extract and a composition containing the extract. More particularly, the present invention relates to a method for extracting sericin from a silkworm or a silkworm or mulberry using deep sea water or hot spring water, An extract comprising sericin, or an extract comprising a mineral component, sericin and mulberry extract, a process for producing the extract, and a pharmaceutical composition, a cosmetic composition and a cleaning composition comprising the extract.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram showing a process for producing a powder of marine mineral-sericin extract (MS) or marine mineral-sericin-mucilage extract (MSM) according to an embodiment of the present invention.
FIG. 2 is a photograph showing the result of performing CBB (Coomassie brilliant blue) after 10, 12, and 15% SDS-PAGE electrophoresis according to an example of the present invention, wherein lane M is a protein marker, Lanes 2 to 5 are 1%, 2.5%, 5% and 10% aqueous solutions of the marine mineral-sericin extract powder of Example 1 dissolved therein.
FIG. 3 is a graph showing the results of immunohistochemical staining for the presence of several lectin antibodies ConA (Canavalis ensifomis, α-linked mannose, and the like) for identifying periodic acid chiff (PAS) ) DBA (Dolichos biforus, α 1,3-N-acetygalactose) RCA120 (Ricinus communis, β-galactose), M is a marker and CS is 0.02 M carbonate (Na2CO3, sodium carbonate ), MS is 2.5% sericin dissolved in deep sea water, and MSM is 2.5% sericin added to MS.
FIG. 4 is a graph showing the results of the measurement of the concentration of sericin extract powder in water, Comparative Example 1 (0.02 M carbonate) and the deep sea water obtained in Examples 1 and 2 for 6 months at 4 ° C, Staphylococcus aureus, listeria, yeast, microorganisms other than fungi.
FIG. 5 shows results of analysis of antioxidant activity of sericin extracts using water, Comparative Example 1 (0.02 M carbonate) and deep sea water obtained in Examples 1 and 2, according to an example of the present invention.
Figure 6 shows the EC ₅₀ (half maximal effective concentration) of the antioxidant effect of Figure 5.
Fig. 7 shows results of antibacterial activity of sericin extracts using water, Comparative Example 1 (0.02M carbonate), and deep sea water obtained in Examples 1-1 and 1-2, according to an example of the present invention.
FIG. 8 shows results of antibacterial experiments on acylcholine and dicotyledonous ginseng extracts of sericin using the deep sea water obtained in Comparative Example 1 (0.02 M carbonate) and Examples 1-1 and 1-2, according to an embodiment of the present invention.
Fig. 9 shows the results of toxicity test on mouse skin cell B16F10 cells.
Fig. 10 shows toxicity test results on human skin cell hs895.sk cells.
11 shows the results of analysis of reactive oxygen species (ROS) removal ability in mouse skin cell B16F10 cells.
FIG. 12 shows the results of analysis of whitening function of sericin extract in mouse skin cell B16F10 cells.
FIG. 13 shows the results of total antioxidant capacity (TAC) analysis in cells according to Example 10. FIG.

Hereinafter, the present invention will be described in more detail with reference to examples. The examples described below are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these examples.

Example  1: Preparation of mineral-sericin extract (MS)

In order to prepare an extract containing marine minerals and sericin by extracting sericin from silkworm cocoon using deep seawater, the cocoon derived from the silkworm silkworm, which is the target of extraction, was cut off and cut into 8 to 10 pieces. The crushed silkworm cocoons were washed with water at a temperature of 95 ° C for 3 to 5 minutes to remove impurities and bacteria, and washed with running water to prepare a silkworm cocoon.

The purified silkworm silkworm was added to deep sea water having a temperature of 95 to 98 캜 and stirred for 90 to 120 minutes to extract sericin. The amount of the above-mentioned cocoon was added at a ratio of 1:50 based on the weight ratio of deep sea water (silkworm: deep sea water). The fibroin from the extract was removed and removed to obtain a marine mineral-sericin extract. The removed fibroin may be washed with water and dried to be used in silk fabrics, regenerative medicine, cosmetics, and the like.

The marine mineral-sericin extract was concentrated by heating at 95 to 98 DEG C for 50 to 60 minutes.

The concentrated extract was filtered through a glass or plastic filter two to three times so as to prevent the precipitate or the remaining fibroin from being contained. The filtration was carried out for about 5-10 minutes in the case of heated 2.5-3 L concentrated extract. The filtered concentrated extract was stored in a liquid state or lyophilized to obtain an extract, which was then used in the experiment. In 2.5 L of deep seawater, 130 g of marine mineral-sericin extract powder containing pure sericin protein was obtained from 50 g of silkworm cocoon. Of these, sericin or its fragment was 13 g, and its content was about The extraction yield of 10% (w / w) sericin protein is shown (see Table 3).

The amount of extracted solvent, amount of sample, and the time required for extraction of MS extract according to this embodiment are shown in Table 3 below. In addition, 2,500 mL of deep sea water was added to 50 g of silkworm cocoons, 2,000 mL was obtained. The crude extract was heated for 60 minutes to concentrate and 1,400 mL of concentrated extract was obtained. The concentrated extract was lyophilized to prepare a final 130 g of MS extract powder.

The extraction temperature and time conditions used in this example, the extraction yield during the extraction process, the pH of the obtained extract, and the salinity in the 1% solution are shown in Table 4 below. Table 4 discloses the extracts of Examples 1 and 2 using the extracts (Comparative Example 1) using water as the four extraction solvents, the extract using the carbonate aqueous solution (Comparative Example 1), and the deep sea water, Compared to extracts from aqueous solutions, extracts with eco-friendly deep sea water in accordance with the present invention yield sericin proteins or fragments thereof at an equivalent or higher level than the extractive raw materials.

According to the present invention, the sericin extraction method using deep sea water has advantages such as securing pure sericin, omission of neutralization step, long-term storage, efficacy and efficiency improvement compared with the conventional extraction method (Table 3). In the case of extracts of sericin, the yield was higher than that of the sodium carbonate reagent. In the case of the 1% extract solution, the pH was about 6.8 and the salinity was about 0.87, and the pH and salinity were suitable for skin protection, disinfection and sterilization (Table 4) . The yield of the sericin protein shown in Table 4 is expressed by the content (weight) of sericin protein contained in the extract on the basis of 100 g of the raw silkworm cocoa used.

Example  2: Marine Minerals - Sericin - Eucalyptus extract ( MSM )

A silkworm cocoon cut in substantially the same manner as in the production method of the marine mineral-sericin (MS) extract of Example 1 was prepared, the above-mentioned silkworm cocoons were extracted using a deep sea water, fibrous fibroin was firstly removed To obtain a mineral-sericin extract.

A 1: 1 (w / w) mixture of Morus bark and Morus branch was added to the obtained mineral-sericin extract at 1:50 (w / v) For 60 minutes to obtain a marine mineral-sericin extract containing the extracts of Manganese and Thyme. At the same time, the extract was concentrated to obtain a marine mineral-sericin-manganese (MSM) extract containing the extracts of Manganese and Thinner.

The concentrated MSM extract was filtered through a glass or plastic filter two to three times in order to prevent the sediment or the remaining fibroin from being contained. The filtration was carried out for about 5-10 minutes in the case of heated 2.5-3 L concentrated extract. The filtered concentrated extract was stored in a liquid state or lyophilized to be soluble sericin powder, which was then used in the experiment.

Extraction of MSM Extract According to this Example The amount of extracted solvent, the amount of sample, and the time required for each step are shown in Table 3 below. In addition, 2,500 mL of deep sea water was added to 50 g of silkworm cocoons, 2,000 mL was obtained. A total of 50 g of mulberry obtained by mixing the above extract and a mixture of top and bottom bark at a weight ratio of 1: 1 was added and heated for 60 minutes to extract and concentrate the mulberry tree to obtain 1,400 mL of concentrated extract. Was lyophilized to prepare a final 130 g of MSM extract powder. In 2.5 L of deep sea water, 130 g of marine mineral-sericin powder containing pure sericin protein was obtained from 50 g of silkworm cocoon. It is more effective than general extraction method in securing pure sericin, omission of neutralization step, long-term storage, efficacy, efficiency improvement (Table 3).

The extraction temperature and time conditions used in the examples, the extraction yield during the extraction process, the pH of the obtained extract and the salinity in the 1% solution are shown in Table 4 below. Table 4 discloses the extracts of Examples 1 and 2 using the extracts (Comparative Example 1) using water as the four extraction solvents, the extract using the carbonate aqueous solution (Comparative Example 1), and the deep sea water, Compared to extracts from aqueous solutions, extracts with eco-friendly deep sea water in accordance with the present invention yield sericin proteins or fragments thereof at an equivalent or higher level than the extractive material.

In addition, when using deep sea water for sericin extraction, the yield was relatively higher than that of sodium carbonate reagent. In the case of 1% solution, the pH was about 6.4 and the salinity was about 0.7, which was suitable for pivot protection, disinfection and sterilization 4)

Comparative Example  One: Sodium carbonate salt  Preparation of extract using aqueous solution

Conventionally, in order to extract silkworm cytochisin using sodium carbonate, the cocoon originated from the silkworm silkworm, which is the object of extraction, was firstly cut out, and the pupa was removed and cut into 8 to 10 pieces. The cut silkworm cocoons were washed with water at 95 캜 for 3 to 5 minutes to remove impurities and bacterium, and washed with running water to prepare a silkworm cocoon.

The purified silkworm silkworm was added to a 0.02 M sodium carbonate solution having a temperature of 95 to 98 캜 and stirred for 90 to 120 minutes to extract sericin. The addition amount of the above-mentioned pulverized cocoons was used as 1:50 (weight ratio of silkworm: deep seawater) to the weight ratio of the deep seawater. The fibroin from the extract was removed and removed to obtain a sericin extract.

The sericin extract was concentrated by heating at 95 to 98 DEG C for 50 to 60 minutes.

The concentrated extract was filtered through a glass or plastic filter two to three times so as to prevent the precipitate or the remaining fibroin from being contained. The filtration was carried out for about 5-10 minutes in the case of heated 2.5-3 L concentrated extract. The filtered concentrated extract was stored in a liquid state or lyophilized to be soluble sericin powder, which was then used in the experiment.

According to this comparative example, the extraction temperature and time conditions in the process of preparing sericin from cocoon, the extraction yield in the extraction process, the pH of the obtained extract and the salinity in the 1% solution are shown in Table 4 below.

Comparative Example  2: Preparation of sericin extract using water

A silkworm cocoon cut in a substantially same manner as in Comparative Example 1 was prepared and extracted with water instead of the sodium carbonate solution to prepare a sericin water extract.

According to this comparative example, the extraction temperature and time conditions in the process of preparing sericin from cocoon, the extraction yield in the extraction process, the pH of the obtained extract and the salinity in the 1% solution are shown in Table 4 below. When sericin was extracted with water, it was confirmed that the extraction yield of sericin protein was lower than that of extracts using carbonate and deep seawater.

Example  3: Analysis of sericin extract

3-1: Electrophoretic analysis of mineral-sericin extract

To analyze the proteins contained in the mineral-sericin extract prepared in Example 1, the extract solution was subjected to electrophoresis on three types of SDS-PAGE gels having respective 10%, 12%, and 15% gel contents Coomassie Brillaiant Blue (CBB) was stained to confirm the molecular weight distribution of the protein. To obtain an accurate distribution, an aqueous solution of 1%, 2.5%, 5%, and 10% concentrations of the MS extract powder obtained in Example 1 was prepared and electrophoresed. The results are shown in FIG.

FIG. 2 is a photograph showing the results of analysis using CBB (Coomassie brilliant blue), showing SDS-PAGE gel electrophoresis analysis of the protein contained in the marine mineral-sericin extract prepared in Example 1. FIG. Lane M is a protein marker, lane 1 is the sericin crude extract prepared in Example 1 (no concentration process is performed), lane 2 is a 1% aqueous solution of marine mineral-sericin (MS) powder, lane 3 is marine mineral- 2.5% aqueous solution of powder, lane 4 is a 5% aqueous solution of marine mineral-sericin powder, and lane 5 is a 10% aqueous solution of marine mineral-sericin powder. The protein contained in the marine mineral-sericin extract obtained in Example 1 was confirmed in a molecular weight range of approximately 10 to 250 kDa (FIG. 2).

3-2: N- glycan Sugar chain formula  Confirm

The sericin protein (CS) extracted from the silkworm cocoate using the carbonate according to Comparative Example 1 and the sericin protein contained in the two sericin extracts (MS, MSM) extracted from the silkworm cocoa using the deep sea water according to Examples 1 and 2 , And lectin reacting with specific sugar chains was used to predict the sugar chain structure.

Specifically, in order to confirm the N-glycan sugar chain modification of the sericin protein according to the buffer for separation, the SDS-PAGE electrophoresis gel obtained in Example 3-1 was applied to a band using Periodic Acid Schiff (PAS) staining solution And the results of the experiment are shown in Fig.

FIG. 3 is a graph showing the results of determination of periodic acid chiff (PAS) staining for identifying N-glycan formulas of isolated sericin proteins and several lectin antibodies ConA (Canavalis (1). In the present study, we found that DBA (α-linked mannose), DBA (Dolichos biforus, α 1,3-N-acetygalactose) and RCA120 (Ricinus communis, β-galactose) M is a marker, CS is 2.5% sericin dissolved in 0.02 M carbonate (Na2CO3) according to Comparative Example 1, MS is 2.5% sericin solution according to Example 1, MSM is 2.5 % Sericin solution.

From the results of FIG. 3, according to the results of electrophoresis and Coomassie Brillaiant Blue (CBB) staining on SDS-PAGE gel, the sericin extract obtained by using Na ₂ CO ₃ (Sodium Carbonate, CS) And 20 kDa molecular weight. In addition, the MS and MSM extracts prepared in Examples 1 and 2 showed protein positions in the molecular weight range of 10 to 250 kDa.

Further, the analysis using the three lectins (ConA, DBA, RCA120) reacting with N-glycans showed that the extraction using Conventional Carbonate (Comparative Example 1) and the present invention using ConA in response to mannose The extracts from the deep seawater extracts were expected to contain mostly mannose.

In the case of O-glycan, it was confirmed that RCA120 reacted with N-acetylgalactosamine (GalNAc) -dependent DBA and β-galactose (β-gal) with sericin isolated from deep seawater according to the present invention 3).

3-3: Amino acid analysis of extract

6 M hydrochloric acid containing 0.05% (w / v) 2-mercaptoethanol was added to the test sample in a volume ratio of 1000, and then hydrolyzed in a vacuum state at 110 ° C for 24 hours. The obtained hydrolyzate was frozen with dry ice and ethanol, mounted in a degasser, and subjected to thorough degassification by repeating melting and freezing.

The degassed hydrolyzate was placed in a test tube and hydrolyzed at 110 ° C. for 22 to 24 hours. The test tube was opened and reduced in pressure, and dried at 40 DEG C to remove hydrochloric acid. A test solution was prepared by adding 0.2 N sodium citrate buffer (pH 2.2) or 0.02 N hydrochloric acid solution to the test tube, followed by filtration through a membrane filter, followed by analysis of amino acid composition using liquid chromatography (Agilent LC system, USA) Respectively. The amino acid component analysis results are shown in Table 5 below.

amino acid Sericin Comparative Example 2 Comparative Example 1 Example 2 Asp 17.8 20.5 23.4 20.0 Thr 8 6.7 5.2 7.4 Ser 31 23.9 21.3 28.1 Glu 4.4 9.2 7.9 6.3 Pro 0.4 1.2 1.1 1.3 Gly 19.1 11.3 11.6 11.4 Ala 3.8 4.5 5.2 3.5 Cys ≪ 0.05 - - - Val 3.1 3.2 4.4 2.9 Met ≪ 0.05 - - - Ile 0.4 0.9 1.3 0.8 Leu 0.8 1.5 2 1.3 Tys 3.3 2.8 3.4 4.5 Phe 0.2 0.8 0.9 0.5 Lys 1.0 5.9 4.3 4.0 His 2.7 1.8 2.3 2.4 Arg 3.9 4.8 5.2 4.9 Sum 99.9 99 99.5 99.3

In Table 5 above, sericin is commercially available from Abhilasha, 2015 (Abhilasha Rangi and Lalit Japura (2015) The Biopolymer Sericin: Extraction and Applications Department of Fashion Technology, BPS Mahila Vishwaavidyalaya, Sonepat, India) and TingTing, 2016 (Ting-TingCaoYu-QingZhang 2016) Processing and characterization of silk sericin from Bombyx mori and its application in biomaterials and biomedicines. Silk Biotechnology Laboratory, School of Biology and Basic Medical Sciences, Soochow University, RM702-2303, No. 199, Renai Road, Dushuhu Higher Edu. , Sericin (Comparative Example 2) extracted with water, sericin (Comparative Example 1) extracted using a sodium carbonate reagent, and deep seawater obtained in Example 2, The amino acids of sericin MSM extracts were serine, aspartic acid and glycine, and the composition of other amino acids was similar.

3-4: Mineral analysis of the extract

In order to analyze inorganic elements as minerals, powders of the extracts obtained in Examples 1 and 2 were dissolved in water to prepare a 1% (w / w) solution, and an inductively coupled plasma atomic emission spectrometry (ICP- AES) (Korea Polymer Testing Laboratory, KOPTRI).

In order to measure 21 kinds of inorganic elements Na, Mg, Ca, K, B, Sr, Tl, Fe, Mn, Al, Cd, Co, Cr, Cu, Ga, In, Li Ni, Pb, Zn, 10 ml of the standard product (1,000 ppm) was added and 20% (v / v) nitric acid solution was added to make 100 ml and diluted. The standard solution and the blank test solution were injected into the inductively coupled plasma emission spectrometer, and the emission intensity of the standard solution was measured. The regression equation was calculated by preparing a calibration curve from the linear function between concentration and emission intensity.

The powders of the extracts obtained in Examples 1 and 2 were dissolved in water and each solution of 1 wt% was injected into an inductively coupled plasma luminescence spectrometer to measure the luminescence intensity of 21 elements and assigned to the regression equation of the calibration curve To determine the concentration of the element of interest. The concentrations of the elements of interest in the sample were calculated according to the following equation. The results of the mineral analysis obtained according to the above method are shown in Table 6.

(Ug / ml) x dilution / sample (g)

The samples used in the following Table 6 are the sericin extracts extracted with sodium carbonate reagent according to Comparative Example 2 and the MSM extracts obtained from Example 2. In Table 4, trace elements are As, Ba, Be, Cd, Co, Cr, Cu, Ga, Ge, In, Li, Mn, Mo, Nb, Ni, Pb, Rb, Re, Sb, , Ta, Ti, Tl, V, W, Y and Zr. The element concentrations in the samples of Table 6 are expressed in units (ug / g).

ingredient Comparative Example 1
(Sodium carbonate extract) Example 1
(MS extract) Example 2
(MSM extract) Na 17,000 39,500 40,400 Mg 1,730 28,740 28,600 Ca 4,980 3,841 6,470 K 362 8,145 12,700 B 0 51 98.6 Sr 26.3 101 171 Al 102 8 9 Si 257 101 114 Zn 80.4 7 8 Other trace elements Non-detection Non-detection Non-detection Sum 24.648 80,502 88,580

As shown in Table 6, it was confirmed that the total minerals content contains about 4 times or more essential minerals than the sodium carbonate solution using sericin extraction using deep sea water. Especially, the contents of sodium, magnesium, potassium, boron and strontium of the extracts of Examples 1 and 2 were much higher than those of Comparative Example 1. Compared to the extract of Example 1, the extract of Example 2 contained a large amount of potassium, boron and strontium. Sodium (Na) is effective for skin cleansing and pH control, potassium (K) is for controlling skin moisture, calcium (Ca) for amino acid and protein production, magnesium (Mg) for skin metabolism promotion, boron (B) (Sr) have an effect of assisting enzyme activity and the like, and the extract according to the present invention is effective as a skin protecting agent, a scalp protective agent and a body care agent.

Example  4: Microbial Storage Stability Test

For the presence and quantitative analysis of the sericin extract obtained in the present invention, the freeze-dried powder of the sericin extract obtained in Examples 1 and 2 was dissolved in water, and the sample was dissolved in 1% or 10% . In addition, the extract powder of Comparative Example 1 was dissolved in water to prepare a sample with a 1% homogeneous solution.

1 mL of the sample solution was mixed with Escherichia coli, E. coli, Staphylococcus aureus, S. aureus, Environmental Listeria, yeast, Fungi, and rapid aerobic inoculated to ^{count) petrifilm TM (3M, USA} ) for measurement and incubated for 24 hours at 37 ℃. Also, Escherichia coli, Staphylococcus aureus and Listeria were quantitatively analyzed. Quantitative analysis was carried out using 3M Petrifilm. After incubation at 37 ° C for 24 hours, the number of colonies formed was confirmed. The strains showing typical colonies were counted as x dilution factor, and the result of culturing the microorganisms using the 1% sample solution is shown in Fig. Fig. 4 is a graph showing the results of the measurement of the concentration of water, 0.02 M carbonate, and the sericin powder of Example 1 at 4 占 폚 for 6 months. Then, the microorganisms other than Escherichia coli, Staphylococcus aureus, Listeria, The results of the analysis of the degree of contamination are shown.

As shown in FIG. 4, the mineral-sericin (MS) extract and the mineral-sericin-mallow (MSM) extract obtained in Examples 1 and 2 according to the present invention are effective against Escherichia coli, Staphylococcus aureus, Listeria, And confirmed safety. However, according to the comparative example, sericin extracted with a sodium carbonate reagent confirmed the culture of other microorganisms.

Example  5: Antioxidant activity analysis of extract

Free radical scavenging activity was analyzed using DPPH (2,2-diphenyl-1-picrylhydrazyl), one of the free radicals.

Specifically, the extract powder obtained in Examples 1 and 2 was dissolved in sterilized water to prepare sample solutions at various concentrations, and stock solution was prepared by dissolving DPPH in 1 mM of methanol or water. And diluted. 100 uL of the 0.1 to 3% sample solution was dispensed into a 96-well plate and ascorbic acid (vit C) was used as a positive control. 100 uM DPPH was added to the well plate at 100 uL and allowed to stand at room temperature for 10-30 minutes after shading, and the absorbance was measured at 517 nm. As a negative control group, methanol or water was treated with 100 uL DPPH to measure the absorbance and perform the zero point treatment. The absorbance was measured and then calculated according to the following equation. The results of the experiment are shown in FIG.

scavenging activity (%) = (1-OD sample / OD blind) x 100

FIG. 5 is a graph showing the antioxidant activity of sericin extract prepared using 0.02M carbonate according to Comparative Example 1 and sericin extract using deep seawater according to Examples 1 and 2. From the above analysis results, the antioxidant activity of the deep seawater sericin extract obtained in Example 1 was higher than that of the sericin extract by conventional carbonates. In particular, the extract of Example 2, in which the extract was added with corn syrup, exhibited an antioxidative activity value of 88.4% Showed excellent antioxidative effect and increased antioxidative activity in a concentration dependent manner.

Further, the EC ₅₀ value was obtained on the basis of the above-described analysis result, and it was shown in FIG. FIG. 6 shows the half maximal effective concentration (EC ₅₀ ) of the antioxidant effect shown in FIG.

As shown in the EC ₅₀ value of 6, the EC ₅₀ value of MSM extract obtained in Comparative Example in accordance with the first obtained in the sericin extract in Example 1 is extracted with sodium carbonate MS extract of Example 2, 2.3%, 1.8% or 0.08% And that the MSM extract of Example 2 could be expected to have a higher antioxidative effect in a smaller amount.

Example  6: Antimicrobial activity analysis of extract

Bacillus cereus, Bacillus megaterium, ATCC 14579, Staphylococcus aureus (St. Aureus, ATCC) were used to test the antimicrobial activity of the MS extract of Example 1 and the MSM extract of Example 2, 12600), Listeria monocytogenes (L. monocytogenes, ATCC 15313), Pseudomonas flurorescens (ATCC 13525), Escherichia coli (E. coli, ATCC 11775), Salmonella enteritidis (S. enteritidis, ATCC 31194), Vibrio Parahaemolyticus (V. parahaemolyticus, ATCC 17802) was used as a test strain.

Propionibacterium acnes (ATCC 6919) and Malassezia furfur (ATCC 14521) were used as test strains.

Specifically, the test strain was pre-cultured in LB medium at 37 占 폚 for 24 hours. 1% LB agar was dispensed in 20 mL aliquots to prepare a plate culture medium. 200 μL of the pre-cultured test bacteria was added thereto, and the coated microbial cells were uniformly coated on a culture medium with a sterilized glass rod, and the applied test bacteria were dried for about 30 minutes . A sample solution was prepared with a 1% or 10% homogeneous solution of the extract powder of Examples 1 and 2 by placing a paper disk (8 mm, Toyo Roshi Kaicha Ltd., Japan) on the medium on which the test bacteria were coated and dried, , And incubated at 37 ° C for 24 hours. The diameter of the inhibition clear zone around the paper disk was measured, and the antibacterial activity was analyzed.

The antimicrobial activity analysis results are shown in FIG. 7 and FIG. FIG. 7 is an analysis of the antibacterial activity of the sericin extract prepared using 0.02 M carbonate according to Comparative Example 1 and the sericin extract using the deep sea water according to Examples 1 and 2. As shown in Fig. 7, the MSM extract of Example 2 had antimicrobial activity against Bacillus and Staphylococcus aureus, as compared with the conventional extract using sodium carbonate.

FIG. 8 shows an antimicrobial test for acne and dandruff. The MSM extract of Example 2 had an antimicrobial effect against acnes and dandruff. The MS extract of Example 1 had antimicrobial activity against dandruff.

Example  7: Cell line toxicity experiment

Cell line toxicity tests were performed using Cell counting kit-8 (CCK-8, Dojindo). (ATCC CRL-6475) and human skin cells hs295T (ATCC CRL-7233) and hs895.sk (ATCC CRL-7636) and human mast cell 3T3-L1 (ATCC CRL-3242 ) Was used. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 ° C in a 5% CO ₂ incubator.

A sample solution of 0.1%, 1% and 5% concentrations of sericin extract prepared using 0.02M carbonate and sericin extract using deep seawater according to Examples 1 and 2 was prepared according to Comparative Example 1, respectively.

The cell line was cultured for 24, 48, or 72 hours at a concentration of 5 x 10 ³ cells / well in a 24-well plate, followed by culturing for 12-24 hours. CCK-8 was added to the culture, and after 2 to 4 hours of reaction, the absorbance at 540 nm was measured, and the cell viability of the untreated group was determined as 100% And Fig. FIG. 9 shows toxicity tests on mouse skin cells B16F10 cells, and FIG. 10 shows results of toxicity tests on human skin cells hs895.sk cells.

In mice toxicity test of the skin cells of B16F10 cell shown in Figure 9, but in both the sample solution is a 5% concentration 72 hours determined that there is no toxicity, sericin extract with Na ₂ CO ₃ in 0.2 M according to the Comparative Example 1 The toxicity was confirmed at 5% concentration. In the toxicity test of human hs295T cells, the sericin extract using 0.2 M Na ₂ CO _{3 according} to Comparative Example 1, the MS extract obtained from Example 1, and the MSM extract solution obtained from Example 2 were treated with 0.1%, 1% and 5% Concentrations were not toxic at all, these samples were found to be non-toxic up to 5% concentration (Figure 10)

Example  8: Antioxidant activity analysis using cell line

8-1: Superoxide  radical scavenging activity

In the first analysis of antioxidant activity, Superoxide radical scavenging activity was measured by Nishikimi et al. (1972). The cell line used in the experiment was the melanoma cell line B16F10 (ATCC CRL-6475) of the rat used in Example 6.

 To 20 uL of diluted sericin-treated or untreated cells, 800 uL of 20 mM Tris solution (pH 8.0) containing 60 uM nitro blue tetrazoliu (NBT) and 98 uM β-nicotinamide adenine dinucleotide (NADH) After mixing, 80 uL of 20 mM Tris solution and 100 uL of 33 uM phenazine methosulfate (PMS) were added, respectively. Formazan measurements of superoxide radicals induced by PMS / NADH nonenzymatically were performed by measuring the absorbance of the reactants at 560 nm for 10 minutes and calculating the following equation.

Superoxide radical scavenging ability = [(A-B) / A] x 100

A, absorbance of not added sample

B, absorbance of added sample

The results are shown in FIG. 11. Antioxidant activity of rat melanoma cell line B16F10 (ATCC CRL-6475) was confirmed to have an antioxidative effect similar to albutin 1% at 5% MSM (FIG. 11).

8-2: Total antioxidant capacity

The second antioxidant activity assay was performed using a total antioxidant capacity assay kit (ab65329, USA). The cell line used was the mouse melanoma cell line B16F10 (ATCC CRL-6475) used in Example 6 and the human skin cell hs295T (ATCC CRL-7233).

The total antioxidant capacity (TAC) was determined by diluting 2 × 10 ⁶ cells of mouse B16F10 cells and human hs 295T cells in 6 well plates with the Erel method modified for Trolox equivalent antioxidant capacity (TEAC) After treatment, cells are collected by time, washed with PBS, resuspended in 100 μl of distilled water, pipetted, and left on ice for 10 minutes. Cells were spun down, the supernatant was transferred to a new tube, CU ^{2 +} solution was added, left at room temperature for 1 hour, and absorbance was measured at 570 nm.

Specifically, rat B16F10 cells and human hs 295T cells prepared in the same manner as in Example 7 were cultured in DMEM medium containing 10% FBS. 2 × 10 ⁶ cells were placed in a 96-well plate, and the sample was cultured for 12-24 hours. Cells were harvested by centrifugation in 10% sodium phosphate buffer (PBS, pH 6.8), and 100 μL of distilled water was added to each well. Add 100 μL of Positive Control (Vitamin C) and 100 μL of each sample. Place the Trolox in 96 well and place Cu ^{2 +} 100 μL on top. After mixing, the mixture was incubated at room temperature for 1.5 hours and absorbance was measured at 570 nm.

Example  9: Quantification of melanin in cell line

For melanin quantitative analysis, rat B16F10 cells and human hs 295T cells used in Example 7 were cultured in DMEM medium containing 10% FBS.

6, the well plate 3 x 10 ⁵ cells in a 12-volume dispensing and obtained after 24 hours of incubation, the comparative example embodiment MS extract, obtained from the extract of sericin in Example 1, using 0.2 M of Na ₂ CO ₃ according to the first example 2 MSM extract solution and 1% solution of Arbutin solution were treated and cultured for 24, 48 or 72 hours. Cells were collected at each time point and dissolved in 1 N NaOH supplemented with 10% DMSO. The cells were incubated at 80 ° C for 1 hour and absorbance was measured at 475 nm. The results are shown in FIG.

Analysis of the whitening effect of B16F10 cells in rats showed that 1% MS and MSM had an anti-melanin effect such as albutin 1% (Fig. 12).

Example  10: Intracellular  Total antioxidant capacity (TAC) analysis

For measurement of antioxidative activity in cells, the mouse B16F10 cells and human hs 295T cells used in Example 7 were cultured in a DMEM medium containing 10% FBS. 6 well plate ⁵ x 10 5 dose dispensing, and after 12 to 24 hours incubation of the sample, obtained in Comparative Example exemplary MS extract, obtained from sericin extract of Example 1 with 0.2 M of Na ₂ CO ₃ according to the first example 2 MSM extract solution and control were treated with 5% solution of Arbutin solution with antioxidant and whitening function. After 48 hours after the treatment, it was dissolved by collecting cells from 10% sodium phosphate buffer (PBS, pH 6.8), Cu 2 + was mixed with a solution (abcam, USA) incubated for 1 to 2 hours at 37 ℃ and 540 nm absorbance was measured. The antioxidant activity (%) was obtained after substitution with a calibration curve using Trolox.

The sericin extract (MS, MSM) using deep sea water in rat B16F10 and human hs295T cells showed similar antioxidative effects to arbutin but not with sodium carbonate reagent (Fig. 13).

Claims (18)

An extract comprising minerals and sericin, which is prepared by extracting cocoon cocoons to be extracted with deep sea water or hot spring water containing carbonate ions and minerals as an extraction solvent. The extract according to claim 1, wherein the extract further comprises at least one mulberry extract in a group consisting of an alfalfa and an upper limb. [Claim 2] The method according to claim 1, wherein the extract of the bark or the upper bark is obtained by mixing at least one mulberry with moss cocoa in the group consisting of the moss and the upper bark, or extracting the minerals and sericin, And extracting the extract. The extract according to claim 3, wherein the mixed weight ratio of (i) silkworm cocoa used in the preparation of the extract and (ii) at least one mulberry in the group consisting of the bark and the upper bark is 1: 9 sowl 9: 1. The extract according to claim 1, wherein the extract is a fibroin contained in a cocoon. The extract of claim 1, wherein the extract comprises 20,000 to 50,000 ug / g of sodium, 15,000 to 30,000 ug / g of magnesium, 2,000 to 20,000 ug / g of potassium, and 40 to 250 ug / g of strontium. The extract of claim 1, wherein the extract comprises sericin or a fragment thereof having a molecular weight range of 10 to 250 kDa. Preparing a cocoon cut material,
Extracting sericin in a deep sea water or hot spring water containing carbonic acid ions and minerals as an extraction solvent in a temperature range of 95 to 98 캜 to obtain a sericin extract,
And removing fibroin from the extract. ≪ RTI ID = 0.0 > 11. < / RTI > 9. The method according to claim 8, further comprising, after the step of extracting the sericin, heating and concentrating the extract at a temperature ranging from 95 to 98 占 폚. 9. The method according to claim 8, wherein at the step of obtaining the sericin extract, at least one mulberry is added and extracted from the group consisting of the mulberry and the upper mushroom, or at least one mulberry is added to the sericin extract, 98 < 0 > C to produce an extract further comprising mulberry extract. 10. The method of claim 9, wherein the extraction solvent is deep ocean water or hot spring water that has not undergone dialysis to remove minerals. 10. The method of claim 9, further comprising lyophilizing the extract to produce a powder. 9. A cosmetic composition comprising an extract according to any one of claims 1 to 7. 14. The cosmetic composition according to claim 13, wherein the cosmetic composition has whitening, antioxidant or antibacterial activity. 14. The cosmetic composition according to claim 13, wherein the antibacterial activity is antimicrobial activity against gram-negative bacteria, dandruff and acne bacteria. 14. The cosmetic composition according to claim 13, wherein the cosmetic composition is a hair care product, a skin care product, a cleanser or a tooth care product. A pharmaceutical composition for preventing, improving or treating acne, comprising the extract according to any one of claims 1 to 7. 7. A pharmaceutical composition for preventing, improving or treating dandruff comprising the extract according to any one of claims 1 to 7.

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