KR20170037545A - Manufacturing method of oyster hydrolysate and peptides purified from oyster hydrolysate - Google Patents

Abstract

The present invention provides a production method of an oyster hydrolysate and a peptide isolated therefrom. The oyster hydrolysate produced by the method reduces the number of melanocytes and reduces the distribution of melanin granules present in a skin layer of the skin of around the melanocytes, thereby being able to be helpfully used as a health functional food for skin whitening or a cosmetic composition.

Description

Technical Field [0001] The present invention relates to a method for preparing oyster hydrolyzate and a peptide isolated therefrom,

The present invention relates to a method for producing oyster hydrolyzate and a peptide isolated therefrom.

Marine organisms with various species have different kinds of compounds exhibiting physiological activity depending on the species. In relation to the functional characteristics of compounds derived from marine organisms, seaweed polysaccharides, minerals, vitamins, antioxidants, enzymes and physiologically active peptides And so on. In particular, peptides not only have nutrition, dissolution and emulsification properties but also exhibit a wide range of physiological activities such as anticancer efficacy and immunity enhancement. Peptides with such physiological activities can be obtained from plant and fish extracts, and can be obtained through chemical synthesis or by protein hydrolysates and microbial fermentation processes by protein enzyme hydrolysis.

Much research has been conducted on functional peptides derived from natural products based on the specific primary binding of proteins in marine organisms. Abel studied the functional properties and separation techniques of peptides from sponges, molluscs and seaweeds. Guadalupe et al. Studied the antioxidative and anticancer effects of hydrocracked sponges and molluscs. In addition, we have isolated Kahalalides, a cyclic depsipeptides with anticancer efficacy and antibacterial activity, from Sacoglossan mollusk and Elysia rufescens.

The elderly population of Korea is expected to reach 12.7% in 2014 and 20.8% in 2026 (Statistics Korea, Future Population Projection, 2012). The interest in drugs that can delay aging due to the increase in the elderly population has greatly increased, and the development of cosmetics to keep the skin young has also progressed rapidly. Studies to prevent or delay the aging of the skin include studies on moisturizing factors to control the skin's moisture balance, and substances that help to improve whitening and wrinkles.

Oysters are oysters and mollusks that live in the sea. It is a delicious and nutritious seafood that many people around the world have been enjoying for a long time. Oysters consist of proteins in 60-70% of solids content and contain large amounts of functional ingredients such as vitamins A, B1, B2, C and minerals such as iron, calcium, phosphorus, zinc, selenium and copper. Oyster hydrolysates obtained by hydrolyzing protein-rich oysters with proteolytic enzymes are rich in free amino acids and contain large amounts of low molecular weight peptides. The low molecular weight compound is not only good in penetration of the skin, but also has increased solubility in water, and thus it is possible to apply various cosmetic materials as a water-soluble raw material.

1. Korean Patent No. 10-0810134 2. Korean Patent Publication No. 10-2013-0023326

An object of the present invention is to provide a method for producing oyster hydrolyzate comprising the step of putting a proteolytic enzyme in a oyster and performing a hydrolysis reaction.

In order to accomplish the above object, the present invention provides a method for preparing oyster hydrolyzate comprising the step of putting a proteolytic enzyme in a oyster and performing a hydrolysis reaction.

In one embodiment of the present invention, the protease is protamex, neutrase or AMG (Glucoamylase).

In one embodiment of the present invention, the protamex is subjected to a hydrolysis reaction at 35 ° C to 45 ° C for 30 minutes to 2 hours, and the neutraase is incubated at 45 ° C to 55 ° C for 30 minutes To 2 hours, and the AMG (Glucoamylase) may be subjected to a hydrolysis reaction at 55 ° C to 65 ° C for 1 hour to 3 hours.

In one embodiment of the present invention, the method may further include the step of inactivating the protease.

The present invention also provides a health functional food for skin whitening comprising an oyster hydrolyzate as an active ingredient.

In one embodiment of the present invention, the oyster hydrolyzate may be one prepared by preparing oyster hydrolyzate.

The present invention also provides a cosmetic composition for skin whitening comprising an oyster hydrolyzate as an active ingredient.

In one embodiment of the present invention, the oyster hydrolyzate may be one prepared by preparing oyster hydrolyzate.

In addition, the present invention relates to a method for producing a peptide having an amino acid sequence selected from the group consisting of S (Ser) -S (Ser) -D (Asp) -N (Asn) peptide, D (Asp) -G (Gly) Ala) -K (Lys) peptide as an active ingredient.

In addition, the present invention relates to a method for producing a peptide having an amino acid sequence selected from the group consisting of S (Ser) -S (Ser) -D (Asp) -N (Asn) peptide, D (Asp) -G (Gly) Ala) -K (Lys) peptide as an active ingredient.

The oyster hydrolyzate and the peptide isolated therefrom according to the present invention have an effect of reducing the number of melanocyte cells and reducing the distribution of melanin granules present in the skin layer of the skin around the melanocyte or skin, The separated peptides can be usefully used as skin whitening health functional foods or cosmetic compositions.

Figure 1 shows the results of analysis of cytotoxicity of oyster hydrolyzate (PNA) to melanocytes.
Fig. 2 shows the results of inhibiting melanin biosynthesis against oyster hydrolyzate (PNA).
Figure 3 shows the results of tyrosinase inhibitory effect on oyster hydrolyzate (PNA).
Figure 4 shows the chromatogram after performing a Q-Sepharose column from oyster hydrolyzate (PNA).
Figure 5 shows the chromatogram after Superdex 30 column was run on the Q-31_35 fraction (Q-31_35 Fr).
Figure 6 shows the chromatogram after performing the Source 5 RPC ST column in the Q31-35_S20 fraction (Q31-35_S20 Fr).
Figure 7 shows the results for LCMS (Liquid Chromatography Mass Spectrometry) on purified peptides.
Figure 8 shows information on purified peptides.
Figure 9 shows information on purified peptides.
Figure 10 shows the results for MS / MS (Mass Spectrometry / Mass Spectrometry) on purified peptides.
Fig. 11 shows the result of DOPA staining of mouse tissues after oral administration of oyster hydrolyzate (PNA) to C57bl / 6J mice (x100).
12 shows the results of oral administration of ovarian hydrolyzate (PNA) at various concentrations (35 mg / kg, 70 mg / kg, 140 mg / kg) to mice and then the ear tissues of mice were isolated at 3, 6 and 9 weeks for DOPA staining and This is the result of Fontana-Masson staining.
FIG. 13 shows the results of oral administration of ovarian hydrolyzate (PNA) at various concentrations (35 mg / kg, 70 mg / kg, and 140 mg / kg) 9.1. And the number of melanocytes was measured using a program.
14 shows the results of oral administration of each concentration (35 mg / kg, 70 mg / kg, 140 mg / kg) of oyster hydrolyzate (PNA) to mice and then 3, 6 and 9 weeks, 9.1. The distribution of melanin granules was measured using a program.

The food composition containing the oyster hydrolyzate of the present invention as an active ingredient may further comprise suitable carriers, excipients and diluents conventionally used in the production thereof.

As used herein, the term " food " means a natural product or a processed product containing one or more nutrients. Preferably, it means that the food can be directly eaten through a certain degree of processing. Food, food additive, health functional food and beverage, preferably gum or candy.

Foods to which the peptide of the present invention can be added include, for example, various foods, beverages, gums, candies, tea, vitamin complexes, and functional foods. In addition, in the present invention, the food may contain special nutritional foods (e.g., crude oil, spirits, infant food, etc.), meat products, fish products, tofu, jelly, noodles (Such as soy sauce, soybean paste, hot pepper paste, mixed sauce), sauces, confectionery (eg snacks), dairy products (eg fermented milk, cheese), other processed foods, kimchi, pickled foods But are not limited to, fruits, vegetables, beverages, beverages, fermented beverages, ice creams, etc.), natural seasonings (eg, ramen soup, etc.), vitamin complexes, alcoholic beverages, alcoholic beverages and other health supplement foods. The food, beverage or food additive may be prepared by a conventional production method.

In the present invention, the functional food refers to a food group which is imparted with added value to function and express the function of the food by using physical, biochemical, biotechnological techniques and the like, the regulation of the biological defense rhythm of the food composition, The functional food of the present invention is preferably a food product which is capable of sufficiently expressing a biological control function for improving skin wrinkles and a whitening effect on a living body Means food. The functional food may include a food-acceptable food-aid additive, and may further comprise suitable carriers, excipients and diluents conventionally used in the production of functional foods.

In the present invention, beverage is a generic term for drinking or enjoying a taste, and is intended to include functional beverages. The beverage is not particularly limited as long as the beverage contains the peptide of the present invention as an active ingredient as an essential ingredient at the indicated ratio, and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages . Examples of such natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrins, cyclodextrins and the like, and Xylitol, sorbitol, and erythritol. Natural flavors (tau martin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate may be generally about 1 to 20 g, preferably 5 to 12 g per 100 ml of the composition of the present invention. In addition, the composition of the present invention can be used for the production of natural fruit juice, fruit juice drink, Can be added.

In addition to the above-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. These components can be used independently or in combination. The additive may be added in an amount of 0 to 100,000 parts by weight, preferably 0.00001 to 10,000 parts by weight, per 100 parts by weight of the peptide of the present invention, but is not limited thereto.

The functional beverage according to the present invention can be used to control the bio-defense rhythm of the beverage group or beverage composition to which the added value is imparted so that the function of the beverage acts on the specific purpose by physical, biochemical or biotechnological techniques, Means a beverage which has been designed and manufactured so that the biological control function relating to recovery and the like is fully expressed in living bodies.

The functional beverage is not particularly limited to the other ingredients except that it contains the oyster hydrolyzate of the present invention as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Examples of such natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrins, cyclodextrins and the like, and xylitol , Sorbitol, and erythritol. Natural flavors (tau martin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The natural carbohydrate may be included in an amount of 0 to 20 parts by weight, preferably 1 to 18 parts by weight, more preferably 5 to 12 parts by weight per 100 parts by weight of the composition of the present invention.

In addition, in the food composition for the purpose of improving skin wrinkles or whitening, the amount of the oyster hydrolyzate of the present invention may be in the range of 0.00001% by weight to 50% by weight of the total food, but is not limited thereto.

Examples of products to which the cosmetic composition of the present invention can be added include cosmetics such as astringent lotion, softening longevity lotion, nutrition lotion, various creams, essences, packs, foundation and the like, cleansing, cleanser, soap, .

Specific formulations of the cosmetic composition of the present invention may include, but are not limited to, softening lotion, gel, water-soluble liquid, milk lotion, nutritional cream, massage cream, essence, oil in water emulsion, water in oil type emulsion, facial anhydrous product, A lipid, a lipid, a lipid, a lipid, a lipid, a lipid, a lipid, a lipid, a lipid, a lipid, Lipstick, lip gloss, face powder, two-way cake, eye shadow, mascara, cheek color, and eyebrow pencil.

According to a preferred embodiment of the present invention, the content of the active ingredient (oyster hydrolyzate) of the present invention is 0.0001-40 wt%, preferably 0.5-40 wt%, more preferably 1.0-30 wt% Weight%. If the content of the active ingredient (oyster hydrolyzate) is less than 0.0001% by weight, the skin wrinkle improvement or whitening effect is greatly reduced, and if it exceeds 40% by weight, skin irritation may be caused, .

The cosmetic composition according to the present invention may be formulated by stabilizing the active ingredient (oyster hydrolyzate) contained in the nanoliposome. When the active ingredient (oyster hydrolyzate) is contained in the inside of the nanoliposome, the active ingredient is stabilized to solve problems such as precipitation formation, discoloration, and deterioration upon formulation, and the solubility and transdermal absorption rate of the ingredient can be increased. The expected efficacy can be maximized.

In the present invention, nanoliposome refers to a liposome having a typical liposome form and having an average particle diameter of 10 to 500 nm. According to a preferred embodiment of the present invention, the average particle diameter of the nanoliposome is 50 to 300 nm, more preferably 100 to 200 nm. When the average particle diameter of the nanoliposome exceeds 500 nm, improvement of skin penetration and improvement of formulation stability is very weak among technical effects to be achieved in the present invention.

The nanoliposomes used to stabilize the extract according to the present invention may be prepared by a mixture comprising a polyol, an oily component, a surfactant, a phospholipid, a fatty acid and water.

The polyol used in the nanoliposome of the present invention is not particularly limited and is preferably a polyol such as propylene glycol, dipropylene glycol, 1,3-butylene glycol, glycerin, methylpropanediol, isopropylene glycol, pentylene glycol, erythritol, , Sorbitol, and mixtures thereof. The amount thereof is 10 to 80% by weight, preferably 30 to 70% by weight, based on the total weight of the nanoliposome.

The oil component used in the preparation of the nanoliposome of the present invention may be selected from a variety of oils known in the art and is preferably a hydrocarbon oil such as hexadecane and paraffin oil, Vegetable oils such as silicon oil such as dimethicone and cyclomethicone, sunflower oil, corn oil, soybean oil, avocado oil, sesame oil and fish oil, ethoxylated alkyl ether oils, propoxylated alkyl ether oils ₄₀ is a fatty alcohol, and mixtures thereof -, phytosphingosine, sphingosine, and scan non-ping the same scan pinggo cannabinoid lipid, cholesterol side-by celebrity, sitosterol cholesteryl sulfate, cholesteryl sulfate, cytokines, C _10. The amount thereof may be 1.0 to 30.0% by weight, and preferably 3.0 to 20.0% by weight based on the total weight of the nanoliposome.

Any surfactant known in the art may be used as the surfactant used in the preparation of the nanoliposome of the present invention. For example, anionic surfactants, cationic surfactants, amphoteric surfactants and nonionic surfactants can be used. Preferred are anionic surfactants and nonionic surfactants. Specific examples of anionic surfactants include alkyl acyl glutamates, alkyl phosphates, alkyl lactylates, dialkyl phosphates and trialkyl phosphates. Specific examples of nonionic surfactants include alkoxylated alkyl ethers, alkoxylated alkyl esters, alkyl polyglycosides, polyglyceryl esters and sugar esters. Particularly preferred surfactants are polysorbates belonging to nonionic surfactants. The amount thereof may be 0.1 to 10% by weight, preferably 0.5 to 5.0% by weight, based on the total weight of the nanoliposome.

The phospholipid, another component used in the preparation of the nanoliposomes of the present invention, is a lipophilic lipid that is used with both affinity lipids and is composed of natural phospholipids (e.g., egg yolk lecithin or soy lecithin, sphingomyelin) Dipalmitoyl phosphatidylcholine or hydrogenated lecithin), preferably lecithin. In particular, unsaturated lecithin or saturated lecithin derived from natural origin extracted from soybean or egg yolk is preferable. Normally, the amount of phosphatidylcholine is 23 to 95% and the amount of phosphatidylethanolamine is 20% or less in natural derived lecithin. In the production of the nanoliposome of the present invention, the amount of the phospholipid to be used is 0.5 to 20.0% by weight, preferably 2.0 to 8.0% by weight based on the total weight of the nanoliposome.

Fatty acid to be used in nano-liposome preparation of the present invention is a higher fatty acid, preferably a C ₁₂ - ₂₂ as a saturated or unsaturated fatty acid of the alkyl chain, e.g., lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid and linoleic acid . The amount thereof may be 0.05 to 3.0% by weight, preferably 0.1 to 1.0% by weight, based on the total weight of the nanoliposome.

The water used in the preparation of the nanoliposome of the present invention is generally deionized distilled water, and the amount thereof may be 5.0-40 wt% based on the total weight of the nanoliposome.

The preparation of nanoliposomes can be accomplished through a variety of methods known in the art, but most preferably is made by applying a mixture comprising the ingredients to a high pressure homogenizer. The preparation of a nanoliposome by a high pressure homogenizer can be carried out under various conditions (for example, pressure, number of times, etc.) depending on a desired particle size, and preferably at a high pressure homogenizer So that the nanoliposome can be produced.

The cosmetic composition of the present invention may be used alone or in combination, or it may be used in combination with other cosmetic compositions other than the present invention. The cosmetic composition having an excellent wrinkle-reducing effect according to the present invention can be used according to a conventional method of use and can be used in a number of times depending on the skin condition or taste of the user.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example  1. Materials and Methods

1.1. material

Crassostrea gigas were purchased from a farm in Tongyoung, Kyungsang Province and frozen rapidly frozen in the form of oolgungs (Daewon Foods, Tongyoung) for 1 ~ 2 years. Protamex (Protease (Subtilisin)) and Neutrase (Protease (neutral)), which are recognized as food additives, were purchased from Busan, Korea for proteolytic enzymes for hydrolysis of oysters. B16-F10 cells (mouse melanoma cell, ATCC, USA) were used in the experiment and the medium was Hyclone (Thermo Fisher Scientific Inc. Kansas, USA).

1.2. Statistical processing

All the experimental results of this study are shown as the mean of the measured values obtained by repeating more than 3 times. Statistical analysis was performed using Student's t- test, and statistical significance was tested at the P <0.05 level for the control group.

Example  2. Oyster enzyme Of the hydrolyzate (PNA)  Produce

To prepare the cucumber enzyme hydrolyzate, first 100 kg of frozen oysters are thawed, washed with water, and then heated (blanching). The blanched oysters (37.3 kg) were pulverized by a pulverizer, and 24 kg of water, which is three times the amount of the pulverized oysters, was added to 8 kg of the pulverized oysters. 1% Protamex (80 g) was added to the pulverized oysters, Hydrolysis reaction is carried out for a period of time, 1% of neutrase (80 g) is added, and hydrolysis reaction is carried out at 50 ° C for 1 hour. After incubation at 100 ° C for 30 minutes, the temperature was adjusted to 60 ° C, and 0.8% AMG (Glucoamylase, 64g) was added. After reacting at 60 ° C for 2 hours, enzyme inactivation RTI ID = 0.0 &gt; 100 C &lt; / RTI &gt; The hydrolyzate was filtered through a 200 mesh filter (brix 4.8) and the filtrate was concentrated at 90 ° C for 5 hours (brix 28.3). Thereafter, the concentrate was lyophilized, and finally 1.2 kg of oyster hydrolyzate (termed PNA in the present invention) was prepared.

Example  3. in vitro  Experiment result

3.1. oyster The hydrolyzate (PNA)  Cytotoxicity analysis

To determine the cytotoxicity of oyster hydrolysates (PNA), B16-F10 cells, which are mouse melanoma cells, were treated with oyster hydrolysates at different concentrations to determine cell viability using Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto , Japan). As a result, it was confirmed that no cytotoxicity was observed with oyster hydrolyzate (PNA) (FIG. 1).

3.2. oyster Of the hydrolyzate (PNA)  Melanin biosynthesis Low performance  effect

Experiments have been conducted to analyze whether oyster hydrolyzate (PNA) has an activity effect inhibiting melanin biosynthesis. B16-F10 cells were treated with oyster hydrolyzate (PNA) at various concentrations (25, 50 and 100 μg / ml) for 3 days. Cells were harvested and lysed in 5 mM EDTA, 0.1 M SPB pH 6.8), 1% Triton X-100), centrifuged, and washed with alo-ol. The cells were dissolved in 1 N NaOH solution containing 10% DMSO for 1 hour at 405 nm Absorbance was measured.

As a result, it was confirmed that the oyster hydrolyzate (PNA) had an effect of inhibiting the biosynthesis of melanin (FIG. 2).

3.3. oyster Of the hydrolyzate (PNA)  Tyrosinase activity Low performance  effect

Experiments have been conducted to analyze whether oyster hydrolyzate (PNA) inhibits tyrosinase activity. Tyrosinase is known as an oxidase that regulates the production of melanin. B16-F10 cells were treated with oyster hydrolyzate (PNA) according to the respective concentrations (25, 50 and 100 μg / ml) and cultured for 3 days. Cells were harvested and rapidly frozen at -70 ° C and thawed The cell membrane was destroyed three times. After centrifugation, the protein was quantitated using the bio-rad protein kit in the supernatant, and 10 mM L-dopa, 0.1 M PBS (pH 6.8) and supernatant were added, followed by reaction at 35 ° C for 1 hour Absorbance was measured at 475 nm.

As a result, it was confirmed that the oyster hydrolyzate (PNA) had the effect of inhibiting tyrosinase activity related to the production of melanin, similarly to the result of Example 4 (Fig. 3).

Example  4. Oyster From the hydrolyzate (PNA)  Available for whitening Of peptide  refine

4.1. Of peptide  Refining method

Experiments have been conducted to purify peptides that appear to be effective for whitening in oyster hydrolyzate (PNA). Ethanol was added to remove proteins from the oyster hydrolyzate (PNA), and the mixture was incubated at room temperature for 1 hour. The supernatant was obtained by centrifugation at 6000 xg for 20 minutes, and the supernatant was obtained at 40 ° C or less using a rotary evaporator Lt; / RTI &gt; Then, it was dissolved in 25 mM Tris-Cl (pH 7.5), filtered at 0.45 μm, and chromatographed using a Q-Sepharose column (4.6 × 30 cm, GE Health Life Science) , And the result is shown in Fig.

&Lt; Q-Sepharose column condition >

sample injection vol: 5 mL

solvent A: 25 mM Tris-Cl (pH 7.5)

solvent B: 1 M NaCl-25 mM Tris-Cl (pH 7.5)

gradient: A 100% (0-75 min), 0-100% (75-230 min), A 0% (230-32 0 min), A 100% (320-985 min)

flow rate: 1 mL / min

detection: 254 nm

fraction volume: 5 mL

The Q31-35 fraction (Q31-35 Fr) was concentrated using a rotary evaporator at 40 ° C or lower, dissolved in distilled water, and then subjected to Superdex 30 column (1.6 × 60 cm, GE Healthcare Life Science) The chromatographic conditions were as follows, and the results are shown in FIG.

<Superdex 30 column condition>

sample injection: 1 mL

eluent: distilled water

flow rate: 1.0 mL / min

detection: 254 nm

fraction volume: 5 mL

The Q31-35_S20 fraction (Q31-35_S20 Fr) was concentrated using a rotary evaporator at 40 ° C or lower, dissolved in 0.1% TFA / DW, and then purified using a Source 5 RPC ST column (4.6x150 mm, 5 μm, GE Healthcare Life Science). The results are shown in FIG. 6.

<Source 5 RPC ST column condition>

sample injection: 50 μL

solvent A: 0.1% TFA / DW

solvent B: 0.1% TFA / Acetonitrile

gradient: 5-55% (0-50 min), 55-95% (50-62.5 min), 95-5% (62.5-67.5 min), 5% (67.5-82 min)

flow rate: 1 mL

detection: 220 nm

fraction volume: 1 mL

The above-mentioned Q31-35_S20_R27 fraction was concentrated using a rotary evaporator at 40 ° C or lower, dissolved in 0.1% formic acid, and then purified using an ACQUITY UPLC® BEH 130 C18 column (Thermo (Dionex) UHPLC Ulitmate 3000) The chromatographic conditions were as follows.

<ACQUITY UPLC @ BEH 130 C18 column condition>

solvent A; 0.1% formic acid / DW

solvent B: 0.1% Formic acid / ACN

sample injection: 10 μL

gradient: B% 10-90% (0-12 min), 90% (12-13 min), 90-10% (13-15 min)

flow rate: 300 μL / min

The amino acid sequence of the purified peptide was analyzed, and the results are shown in FIGS. 7 to 10.

4.2. Refined To peptides  Whitening effect

The amino acid sequence of an effective peptide is S (Ser) -S (Ser) -D (Asp) -N (Asn) -N (Asn) -D (Asp) -G (Gly) -K (Lys). (A) -K (Lys) peptide and D (Asp) -G (Gly) -E (Glu) The number of hydrogen acceptors, number of rings, number of rotatable bonds, polar surface area, LogS (pH = 7.0), LogP and melanin production inhibition (%) were measured for each of them. The results are shown in Table 1.

Peptides Molecular weight, Da Number of hydrogen donors Number of hydrogen acceptors Number of rings Rotatable Coupling Polarity surface area LogS, pH = 7.0 LogP Melanin production inhibition (%) SSDN 421.36 11 15 0 13 271.47 0.38 -7.94 55.7 DEG 319.27 7 11 0 10 196.12 0.5 -3.94 40.5 AK 217.27 6 6 0 7 118.44 0.66 -2.1 68.0

The toxicity of each peptide was predicted, and the results are shown in Table 2.

Peptides LD50 (rat) Ames test, probability (%) Mutation, probability (%) Clastogenicity DNA damage (probability) Carcinogenicity
(Probability) Repeatability (probability) OR (mg) RI Chromosome Probability (Microncleus) SSDN 2.09 0.3803 0.24 0.30 0.32 0.25 0.18 0.07 0 DEG 3.54 0.4794 0.02 0.06 0.10 0.08 0.11 0.04 0 AK 3.83 0.7686 0.09 0.23 0.12 0.09 0.13 0.05 0

Example  5. Animal test results

5.1. oyster Of the hydrolyzate (PNA)  Setting dose

Experiments were conducted to determine the amount of oyster hydrolyzate (PNA) after UVB irradiation for 10 days using C57bl / 6J mice to induce pigmentation. The ovarian hydrolyzate (PNA) of the present invention was set at a dose of 140 mg / kg in consideration of the fact that ascorbic acid (ascorbic acid) widely used as a positive control was 70 mg / kg. (PNA) was orally administered for 9 weeks, respectively, and the degree of whitening effect was confirmed by DOPA (3,4-dihydroxylphenylalanine) staining of the skin layer.

As a result, the number of melanocytes and melanin granules were increased in the induction group irradiated with UVB only, and the number of melanocytes and melanin granules in the group treated with oyster hydrolyzate (PNA) And granules were remarkably reduced to a similar extent to the normal group (Fig. 11).

Based on the above results, the dose of oyster hydrolyzate (PNA) was set at a low (35 mg / kg), medium (70 mg / kg) and high (140 mg / kg) kg).

5.2. oyster Hydrolyzate  Whitening efficacy verification

To confirm the whitening efficacy of oyster hydrolyzate (PNA), mouse oyster hydrolyzate (PNA) was orally administered. After 3 weeks, 6 weeks and 9 weeks, ear tissues were separated and subjected to DOPA staining and Fontana-Masson Staining was performed. As a result, it was confirmed that the number of melanocytes and peripheral melanin granules decreased in a concentration-dependent manner. Through the Fontana-Masson staining in the same tissue, it was confirmed that melanin was generated in the whole skin layer in the induction group compared with the normal group, and melanin production was reduced in a concentration-dependent manner when oyster hydrolyzate (PNA) was treated 12).

To verify the degree of whitening effect of oyster hydrolyzate (PNA), DOPA stained photographs were quantitatively analyzed using Image pro premier 9.1 (media cybernetic inc., USA) program. We measured the number of melanocyte cells by administration period and compared the activity of melanocytes with that of peripheral melanin granules. As a result, it was confirmed that oyster hydrolyzate (PNA) decreased melanocyte cell number and peripheral melanin granule in a concentration dependent manner.

In the third week, the induction group was 114% higher than that of the normal group, and the oyster hydrolyzate (PNA) was decreased by 9% in medium (35 mg / kg) % And high (140 mg / kg) dose, respectively, and the positive control group showed a 40% decrease. Positive controls were found to have efficacy between the medium (70 mg / kg) and high (140 mg / kg) dose groups. In the high dose group (140 mg / kg), the number of melanocyte cells was decreased to the same level as that in the normal group (FIG. 13).

In addition, at 6th week, induction group showed 100% increase compared with normal group, and oyster hydrolyzate (PNA) decreased in low dose by 19%, medium dose by 43% and high dose by 51% The positive control group showed a 40% decrease. Positive controls showed efficacy between low dose and medium dose groups. In the high dose group, as in the third week, the number of melanocyte cells in the same level as the normal group was decreased (FIG. 13).

In addition, at 9th week, the induction group showed 95% increase compared to the normal group, and the oyster hydrolyzate (PNA) decreased by 15% in low dose, 41% in medium dose and 48% in high dose, The positive control group showed a 37% decrease. Positive controls showed efficacy between low dose and medium dose groups. In the high-dose group, the number of melanocyte cells decreased to the same level as that of the normal group as in the case of the third and sixth weeks (Fig. 13).

In order to verify the degree of whitening efficacy of oyster hydrolyzate (PNA) and to compare the tendency with DOPA staining, Fontana-Masson staining photographs were quantitatively analyzed using Image pro premier 9.1 (media cybernetic inc., USA). As a result, oyster hydrolyzate (PNA) was found to decrease melanin granules throughout the skin layer in a concentration-dependent manner (Fig. 14).

Specifically, in the third week, the distribution of melanin granules was increased to 126% in the induction group compared with the normal group, and the oyster hydrolyzate (PNA) was 3% in the low dose, 40% in the medium dose, 58% in the control group, 43% in the control group, and between the moderate and high dose groups in the distribution of melanin granules in the positive control group. The distribution of melanin granules was consistent with the tendency of quantitative results of DOPA staining at 3 weeks (Figure 14).

In addition, at 6th week, the distribution of melanin granules in the induction group was increased by 281% compared with that of the normal group, and oyster hydrolyzate (PNA) was 32% in the low dose group, 54% %, And the positive control group showed a 46% decrease. Positive controls showed efficacy between the low and medium doses in the distribution of melanin granules. The distribution of melanin granules was consistent with the tendency of the DOPA staining results at 6 weeks (Fig. 14).

In addition, at 9th week, the distribution of melanin granules in the induction group was increased by 316% compared with that of the normal group, and the oyster hydrolysates (PNA) were 33% at the low dose, 60% at the medium dose, 75 And 41% in the positive control group, respectively. Positive controls showed efficacy between the low and medium doses in the distribution of melanin granules. The distribution of melanin granules was consistent with the tendency of the results of DOPA staining of 9th week (Fig. 14).

Quantitative analysis of DOPA staining and Fontana-Masson staining showed better efficacy in the high-dose group than in the positive control group in the third week, but overall efficacy in the low-dose and high-dose groups was better than in the positive control group It looked. In particular, the low-dose group was significantly improved in efficacy compared to the third week, and the whitening efficacy was greatly improved as the administration period was prolonged. In the 9th week, the whitening efficacy was almost the same as that of 6th place in all dose groups, and saturation was observed at the highest efficacy value that each dose group could exhibit.

Through such in vivo experiments, oyster hydrolyzate (PNA) inhibits the activation of melanocytes in the epidermal layer and inhibits the formation of melanin granules around it, as well as inhibiting the production of melanin granules throughout the skin layer It was confirmed that there is an excellent whitening effect. It was confirmed that it is about 1.5 times more effective than ascorbic acid as a positive control.

Claims (10)

And adding a proteolytic enzyme to the oyster to carry out a hydrolysis reaction. The method according to claim 1,
Wherein the protease is protamex, neutrase or AMG (Glucoamylase). 3. The method of claim 2,
The protamex is subjected to a hydrolysis reaction at 35 ° C to 45 ° C for 30 minutes to 2 hours and the neutrase is hydrolyzed at 45 ° C to 55 ° C for 30 minutes to 2 hours , And the AMG (Glucoamylase) is subjected to a hydrolysis reaction at 55 ° C to 65 ° C for 1 hour to 3 hours. The method according to claim 1,
Further comprising the step of inactivating the protease. Health functional food for skin whitening containing oyster hydrolyzate as active ingredient. 6. The method of claim 5,
Wherein said oyster hydrolyzate is produced by a method according to any one of claims 1 to 4. 5. A health functional food, A cosmetic composition for skin whitening comprising an oyster hydrolyzate as an active ingredient. 8. The method of claim 7,
Wherein the oyster hydrolyzate is produced by a process according to any one of claims 1 to 4. The composition according to any one of claims 1 to 4, D (Asp) -G (Gly) -E (Glu) peptide or A (Ala) -K (Ser) Lys) peptides as an active ingredient. D (Asp) -G (Gly) -E (Glu) peptide or A (Ala) -K (Ser) Lys) peptide as an active ingredient.

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