KR101415367B1 - Harp seal collagen peptide, a method for manufacturing said halp seal collagen peptide and use of the same - Google Patents

Abstract

The present invention relates to a half seal collagen peptide, and a manufacturing method and a use thereof. More specifically, the present invention is provided to prevent and treat diseases related to an oxidative stress and diabetes and be used as a cosmetic composition by: separating oil from half seal skin using a supercritical device; and manufacturing a half seal collagen peptide by performing a hydrolysis by using alkalase and flavourzyme.

Description

Harp seal collagen peptide, a method for manufacturing the same, and use of the same,

The present invention relates to a half seals collagen peptide, a method for producing the same, and a method for producing the same, and more particularly, to a method for producing a half seals collagen peptide by separating an oil from a half sealskin using a supercritical apparatus and performing hydrolysis using alkalase and flavonoid, And to utilize it as an antioxidant, antidiabetic and cosmetic composition.

Recently, interest in skin cosmetics has increased, and various cosmetics and health supplements have been actively studied. Among them, collagen peptide is one of the most important biomaterials as a protein material composing a living organism, and it is widely used in pharmaceuticals, foods, and cosmetics.

The market for collagen peptides is rapidly growing both domestically and internationally, which is a very promising area for economy and industry. However, the related research is being actively carried out abroad rather than domestic. Especially, collagen peptide products mainly using pork and fish scales as raw materials are mainly made, and collagen peptides based on harp seal have not been studied yet. In addition, studies on omega-3 fatty acids have focused on the application and application of omega-3 fatty acids and on the technical aspects, and studies on its production methods have not been conducted properly.

Collagen is a very important fibrous protein in the body of humans and other animals. It is the main component of the skin's dermis and connective tissues, and it also makes up 90% of the proteins that make up the bones. Collagen, which is used for medical and cosmetic purposes, is a macromolecular protein extracted from bone and skin of animals and composed of three strands of polypeptide chain with molecular weight of about 100,000. Collagen has long been used for edible purposes. Collagen and its metabolites, such as gelatin, are digested by digestive enzymes in the digestive tract and absorbed in the form of amino acids, just like other proteins. Collagen enhances immune function, promotes cell regeneration, strengthens joints, stimulates metabolism of skin, maintains moisturizing power, and has an excellent effect on skin beauty. Collagen is synthesized in the human body as a young age, but after 20 years, the amount of production is reduced, so the elasticity of the skin is reduced with age, symptoms such as gum collapse, muscle pain, damage to the blood vessel wall appears. Especially in the face exposed to the sun by the free radicals, which are harmful oxygen in the air, pigment called lipofusin appears and black spots and stains occur. In order to prevent and treat these diseases, collagen-rich foods, collagen-processed foods, and collagen cosmetics should be used. Collagen peptide refers to collagen which is currently used in the food industry by removing the gelation ability of gelatin by increasing digestion absorption rate by hydrolyzing collagen or gelatin with enzymes or the like to lower molecular weight.

The inventors of the present invention completed the present invention while studying the half seals in order to produce collagen peptides having various effects as described above.

1. Nanjo, F., Goto, K., Seto, R., Suzuki, M., Sakai M., Hara, Y., 1996. Scavenging effects of tea catechins and their derivatives on 1,1, -diphenyl-2 -picrylhydrazyl radical. Free Radic. Bio. Med. 21, 895-902.

It is an object of the present invention to provide a production method for producing collagen peptides from halved seals.

Another object of the present invention is to provide a composition for preventing and treating oxidative stress-related diseases comprising half-fish collagen peptide produced by the above-described method as an active ingredient, and a health functional food for preventing and improving antioxidative diseases .

It is still another object of the present invention to provide a composition for preventing and treating diabetic-related diseases, which comprises the half seal collagen peptide produced by the above-described method, as an active ingredient, and a health functional food for preventing and improving antioxidative diseases.

It is still another object of the present invention to provide a cosmetic composition comprising the half seal collagen peptide produced by the above production method as an active ingredient.

In order to solve the above-mentioned problems, the present invention provides a method for producing a half seals collagen peptide comprising the step of hydrolyzing a half sealshaft using alcalase and / or flavorzyme do.

In one embodiment of the present invention, the concentration of the alcalase or flavorzyme may be 0.3 to 1.5 wt%, respectively, relative to the weight of the half-beaten leather.

In one embodiment of the present invention, the step of hydrolyzing may be carried out at 50 to 60 DEG C for 30 minutes to 5 hours.

In one embodiment of the present invention, the step of hydrolyzing may be a first hydrolysis with an alcalase followed by a second hydrolysis with flavozyme.

In one embodiment of the present invention, the method for manufacturing a half-seals collagen peptide may further comprise separating oil from the half seals using a supercritical extraction device prior to the hydrolysis step.

In addition, the present invention provides a half seal collagen peptide produced by the above method.

In addition, the present invention provides a composition for preventing or treating oxidative stress-related diseases comprising half-fish collagen peptide as an active ingredient.

The present invention also provides a health functional food for preventing or ameliorating an oxidative stress-related disease, which comprises a half seal collagen peptide as an active ingredient.

In addition, the present invention provides a composition for preventing or treating diabetes-related diseases, which comprises a half sealant collagen peptide as an active ingredient.

In addition, the present invention provides a health functional food for preventing or ameliorating a diabetes-associated disease, which comprises a half sealant collagen peptide as an active ingredient.

In addition, the present invention provides a cosmetic composition comprising a half sealant collagen peptide as an active ingredient.

The collagen peptide according to the present invention is a collagen peptide capable of replacing the conventional pit collagen peptide and fish scale collagen peptide and exhibits antioxidant activity and antidiabetic activity and is useful for oxidative stress related diseases and diabetes related diseases And may be used as a cosmetic composition.

1 is a schematic diagram of a supercritical apparatus used for extracting oil from a half sealskin.
FIG. 2 is a scanning electron microscope (SEM) photograph for comparing the particle shape of the purified half-seals collagen peptide (A) with the particle shape of the donkey collagen peptide (B) and the fish scale collagen peptide (C).
FIG. 3 compares the DSC analysis curves of the half seals collagen peptide (A), the ponytail collagen peptide (B), and the fish scale collagen peptide (C).
Figure 4 compares the TGA analysis curves of the half seals collagen peptide (A), the donkey collagen peptide (B), and the fish scale collagen peptide (C).
FIG. 5 is a graph comparing the zeta potentials of a 10% aqueous solution of a half seals collagen peptide (A), a donkey collagen peptide (B), and a fish scale collagen peptide (C).
FIG. 6 is a graph comparing the emulsifiability of a halftone collagen peptide (A), a donkey collagen peptide (B), and a 10% aqueous solution of a fish scale collagen peptide (C).
FIG. 7 is a mass spectrometry (MALDI-TOF) analysis result showing the molecular weight distribution of the half-seal collagen peptide.
8 is a graph showing the effect of pH on the solubility of the half seal collagen peptide.

The present invention relates to a half seal collagen peptide, a method for producing the same and a use thereof, and a method for producing a collagen peptide from a half seal skin, A composition for preventing or treating diabetes-associated diseases, a health functional food, or a cosmetic composition.

The harp seal is a carnivorous animal belonging to the seals of the seals, which has a size of 180cm and a weight of about 130kg inhabit the seas around the South Atlantic and the Arctic Ocean. The hair color has a half pattern on the silver background and the head is black.

The inventors of the present invention have found that such a collagen peptide having high purity can be obtained by treating the leather of harp seals with alcalase and flavoba, and these collagen peptides have antioxidant activity and antidiabetic activity.

Therefore, the half sealant collagen peptide according to the present invention can effectively prevent or treat oxidative stress or diseases related to diabetes.

Such oxidative stress-related diseases may include cancer, autoimmune diseases, liver disease, neurodegenerative diseases, coronary artery disease (CAD), or cardiovascular disease (CVD). The coronary artery disease prevented or treated by the composition of the present invention may be used for the treatment of myocardial infarction, angina pectoris, other chronic coronary artery disease, atherosclerosis, acute coronary syndrome (e.g., non-ST-elevation myocardial infarction) Or STEMI (ST elevation myocardial infarction), peripheral arterial occlusive disease (PAOD), or cerebrovascular accidents.

In the present invention, "treatment ", as used herein, unless otherwise indicated, refers to reversing, alleviating, inhibiting, or preventing the disease or condition to which the term applies, or one or more symptoms of the disease or disorder , And the term "treating ", as used herein, refers to the act of treating when" treatment "is defined as above.

Thus, in a mammal, "treatment" or "treatment " of an oxidative stress or diabetes related disorder may include one or more of the following:

(1) inhibiting the development of oxidative stress or diabetes-related diseases,

(2) prevent the spread of oxidative stress or diabetes related diseases,

(3) alleviating oxidative stress or diabetes related diseases,

(4) prevent the recurrence of oxidative stress or diabetes-related diseases and

(5) palliating symptoms of oxidative stress or diabetes-related diseases;

The harp collagen peptide or oil according to the present invention can be obtained by extracting and isolating from the nature using the extraction and separation methods known in the art. In the present invention, the extraction is carried out by using a suitable solvent, And includes, for example, all of Half Seabass extract, polar solvent-soluble extract, and non-polar solvent-soluble extract. However, peptides separated and purified without using any other solvent as described above may also be utilized as the functional material of the present invention.

As an appropriate solvent for extracting the extract from the harp seal, any pharmaceutically acceptable organic solvent may be used, and water or an organic solvent may be used. Examples of the solvent include, but are not limited to, purified water, methanol alcohol having 1 to 4 carbon atoms, acetone, ether, benzene, chloroform (including methanol, ethanol, propanol, isopropanol, and butanol) various solvents such as chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane may be used alone or in combination.

As the extraction method, any one of the methods such as hot water extraction method, cold extraction method, reflux cooling extraction method, solvent extraction method, steam distillation method, ultrasonic extraction method, elution method and compression method can be selected and used. In addition, the desired extract may be further subjected to a conventional fractionation process or may be purified using a conventional purification method. There is no limitation on the production method of the half extract of the present invention, and any known method can be used.

In the present invention, the harp extract is a concept that includes all the extracts, fractions and tablets obtained in each step of extraction, fractionation or purification, their diluted solutions, concentrates or dried products.

The composition of the present invention may be a pharmaceutical composition or a food composition.

The pharmaceutical composition of the present invention can be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the above-mentioned active ingredients. Examples of the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, A lubricant or a flavoring agent can be used.

The pharmaceutical composition may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described active ingredients for administration.

The pharmaceutical composition may be in the form of granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, natural and synthetic gums such as corn sweeteners, acacia, tracker candles or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like. Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

In one embodiment of the present invention, the half sealant collagen peptide of the present invention may be contained in the composition at a concentration of 10 to 200 μM, and may be included in an amount of 0.1 to 95% by weight based on the total weight of the composition.

The composition of the present invention may also be a food composition. The food composition may contain, as an active ingredient, a half bee collagen peptide, as well as various flavors or natural carbohydrates, .

Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. The above-described flavors can be advantageously used as natural flavorings (tau martin), stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.).

The food composition of the present invention can be formulated in the same manner as the above pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolates, foods, confectionery, pizza, ram noodles, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes, .

In addition, the above-mentioned food composition may further contain other ingredients such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and flavors such as natural flavors, coloring agents and aging agents (cheese, chocolate, etc.) Alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the food composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks.

Since the half-seal bum collagen peptide as an active ingredient of the present invention is a natural substance and has almost no side effects such as chemical agents, it can be safely used for long-term use for the prevention or treatment of oxidative stress or diabetes-related diseases.

The present invention also provides a health functional food for preventing or ameliorating oxidative stress or diabetes related diseases comprising half-fish collagen peptide as an active ingredient.

The health functional food of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and circles for the purpose of preventing or improving oxidative stress or diabetes related diseases.

In the present invention, the term "health functional food" refers to a food prepared and processed by using raw materials or ingredients having useful functions in accordance with Law No. 6727 on Health Functional Foods, and the nutritional control on the structure and function of the human body Or for the purpose of obtaining a beneficial effect for health use such as physiological action.

The health functional foods of the present invention may contain conventional food additives and, unless otherwise specified, whether or not they are suitable as food additives are classified according to the General Rules for Food Additives approved by the Food and Drug Administration, Standards and standards.

Examples of the food items included in the above food additives include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as persimmon extract, licorice extract, crystalline cellulose, high color pigment and guar gum; L-glutamic acid sodium preparations, noodle-added alkalis, preservative preparations, tar coloring preparations and the like.

For example, the health functional food in the form of tablets may be prepared by granulating a mixture of an active ingredient of the present invention, which is an active ingredient of the present invention, with an excipient, a binder, a disintegrating agent and other additives in a usual manner, Compression molding, or direct compression molding of the mixture. In addition, the health functional food of the tablet form may contain a mating agent or the like if necessary.

The hard capsule of the capsule type health functional food can be prepared by filling a normal hard capsule with a mixture of a half sealant collagen peptide of the present invention and an additive such as an excipient and the soft capsule is prepared by mixing an acupuncture extract with an excipient And filling the mixture with a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative and the like, if necessary.

The ring-shaped health functional food may be prepared by molding a mixture of the active ingredient of the present invention, which is an active ingredient of the present invention, with a half-fish collagen peptide, an excipient, a binder, a disintegrant, etc. by a conventionally known method. The surface can be coated with a material such as starch, talc or the like.

The granular health functional food may be prepared by granulating a mixture of the active ingredient of the present invention, which is an effective ingredient of the present invention, with a collagen peptide of the present invention, an excipient, a binder, a disintegrant, etc., And the like.

The health functional food may be a beverage, a meat, a chocolate, a food, a confectionery, a pizza, a ramen, a noodle, a gum, a candy, an ice cream, an alcoholic beverage, a vitamin complex and a health supplement food.

In addition, the present invention provides the use of a composition comprising, as an active ingredient, a half seal collagen peptide for the manufacture of a medicament or food for the prevention or treatment of oxidative stress or diabetic-related diseases. The composition of the present invention comprising the above-mentioned half sealant collagen peptide as an active ingredient can be used for the preparation of medicines or foods for the prevention or treatment of oxidative stress or diabetes related diseases.

The present invention also provides a method of preventing or treating an oxidative stress or a diabetes related disease comprising administering a half seal collagen peptide to a mammal.

The term "mammal " as used herein refers to a mammal that is the subject of treatment, observation or experimentation, preferably a human.

The term "therapeutically effective amount " as used herein refers to the amount of active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as contemplated by a researcher, veterinarian, physician or other clinician, The amount that induces the relief of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dose and the number of administrations of the active ingredient of the present invention will vary depending on the desired effect. Thus, the optimal dosage to be administered can be readily determined by those skilled in the art and will vary with the nature of the disease, the severity of the disease, the amount of active and other ingredients contained in the composition, the type of formulation, and the age, The age, body weight, sex, diet, time of administration, route of administration and fraction of the composition, duration of treatment, concurrent medication, and the like. In the treatment method of the present invention, in the case of an adult, it is preferable to administer the half sealant collagen peptide of the present invention at a dose of 0.01 mg / kg to 250 mg / kg once or several times a day.

In the treatment method of the present invention, the composition comprising the half sealant collagen peptide of the present invention as an active ingredient can be administered orally, rectally, intravenously, intraarterially, intraperitoneally, intramuscularly, intrasternally, transdermally, topically, ≪ / RTI > can be administered in a conventional manner.

Meanwhile, the half seal collagen peptide according to the present invention can be manufactured from a cosmetic composition. When the composition of the present invention is prepared with a cosmetic composition, the composition of the present invention may contain not only the above-mentioned half-coat collagen peptide but also components commonly used in cosmetic compositions, such as antioxidants, stabilizers, Vitamins, pigments and flavoring agents, and carriers.

In addition, the composition of the present invention may be mixed with an organic UV blocking agent that has been used in the past so long as it does not deteriorate the skin protecting effect by reacting with the half seals collagen peptide in addition to the half seals collagen peptide described above.

Organic UV protectors include, but are not limited to, glyceryl paraben, drometrizol trisiloxane, drometrizole, dipaloyyl trioleate, disodium phenyldibenzimidazole tetrasulfonate, diethylhexylbutamidotriazone, Benzoylhexyl benzoate, di-methoxy cinnamate, a mixture of Rawson and dihydroxyacetone, methylenebis-benzotriazolyltetramethylbutylphenol, 4-methylbenzylidene camphor, menthyl anthranilate, benzophenone- Benzophenone-3 (oxybenzone), benzophenone-4, benzophenone-8 (dioxyphenylbenzene), butylmethoxydibenzoylmethane, bishexylhexyloxyphenol methoxyphenyltriazine, synoxate, Octyl-p-methoxycinnamate, polysilicon-15 (dimethicone-ethylbenzylphthalate, ethylhexylmethoxycinnamate, ethylhexylmethoxycinnamate, ethylhexylsalicylate, ethylhexyltriazone, isoamyl- Yeah Bit), terephthalic ylidene dikaem peoseol sulphonic Acid and their salts, tea yieyi - may be used salicylate and at least one member selected from the group consisting of amino-benzo ripening Acid (paba).

In addition, the cosmetic composition of the present invention may be prepared in any form conventionally produced in the art, and examples thereof include solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactants- Containing cleansing, oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, the cosmetic composition of the present invention can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

When the formulation of the present invention is a paste, cream or gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .

When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In the case of a spray, in particular, / Propane or dimethyl ether.

When the formulation of the present invention is a surfactant-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide ether Alkylamido betaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester.

When the cosmetic composition of the present invention is a soap, a surfactant-containing cleansing formulation or a surfactant-free cleansing formulation, it may be applied to the skin and then wiped off, washed or rinsed with water. The surfactant-containing cleansing formulation may be a cleansing foam, a cleansing water, a cleansing towel and a cleansing pack. The surfactant-free cleansing formulation may be a cleansing cream, , Cleansing lotion, cleansing water and cleansing gel, but is not limited thereto.

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>

Experimental Materials and Devices

In order to prepare the collagen peptide from the harp seal of the present invention, a halved seaweed (Nutricna) was provided as a raw material and stored in a refrigerator. CO ₂ and n-hexane (SK chemical Co.) were used as extraction solvents to remove fat from the harp seals, and hydrolytic enzymes were Alcalase and Flavourzyme (Novozyme Co ) Were used. As a control for the characterization of the collagen peptide of the half seals, donkey collagen peptide and fish scale collagen peptide (CNA Biotech) were used.

The R-401 series Supercritical Fluid Extraction System (hereinafter referred to as "R-401 SFE system", Reaction Engineering, Inc.) was used as an oil extraction device for separating oil from the half seal.

< Example  2>

Harp seal  Collagen peptide of  Produce

<2-1> Harp seal  Separation of oil from leather

In order to prepare the half-seal collagen peptide, the non-collagenous oil was first separated from the half seals. For this purpose, 50 g of halved seals were cut into pieces, washed with water to remove impurities, put into an extraction tank of a supercritical extraction apparatus, and then pressurized to 2,000 psi by supplying carbon dioxide. At this time, to prevent cavitation of the carbon dioxide injection part, a cooling tank was installed to prevent the carbon dioxide from being vaporized, and the pressure of the pressurized carbon dioxide was regulated by the back pressure regulator (BPR). When the pressure reached 1,900 psi, the temperature of the extraction tank was adjusted to 60-80 ° C using a thermocouple thermometer and the half seal oil was separated by extraction for 6 to 8 hours.

<2-2> Harp seal  Hydrolysis of leather

200 ml of distilled water was added to the oil-removed half-boiled leather according to Example 2-1, and then the protein hydrolytic enzymes Alcalase and Flavourzyme were used to change the concentration of the enzyme and the number of hydrogens The hydrolysis efficiency was measured according to the decomposition time.

As a result, as shown in Tables 1 and 2, the optimal concentrations of Alcalase and Flavourzyme were 1.0 wt% based on the weight of each of the two enzymes, And the hydrolysis efficiency was the highest when the hydrolysis time was 3 hours, respectively.

Table 1 below shows the hydrolysis rates according to the alkaline treatment conditions, and Table 2 shows the hydrolysis rates according to the flavonous treatment conditions.

Accordingly, the inventors of the present invention treated primary alcalase with 0.3-1.5 wt% of alcalase in relation to the mass of the half seals, and then subjected to primary hydrolysis at 55 ° C for 30 minutes to 5 hours to obtain 0.3 to 1.5 wt% Flavourzyme was added and the mixture was subjected to secondary hydrolysis by stirring at 55 ° C for 30 minutes to 5 hours.

The hydrolyzed sample was subjected to primary filtration under reduced pressure to separate the hydrolyzate and the residue, and the primary filtrate was filtered under reduced pressure to remove impurities. The filtrate was agitated at 85 캜 for 1 hour to inactivate the remaining enzyme to produce collagen peptide from the half beef skin. 2 wt% of activated carbon was added to the prepared collagen peptide, and the mixture was stirred at 55 ° C for 20 minutes to discolor and deodorize. The collagen peptide decolorized and deodorized was filtered under reduced pressure to separate activated carbon and subjected to ultrafiltration to obtain an aqueous solution of a collagen peptide of the half coat. The aqueous solution was lyophilized to prepare a high purity half - water collagen peptide.

< Example  3>

Half-water collagen peptide of  Character analysis

<3-1> Structure analysis using infrared spectrum

The structure of the half-seals collagen peptide according to the present invention was compared with the results of FT-IR analysis of the commercialized ponyo collagen peptide and fish scale collagen peptide (see Table 3).

As a result, in the case of a half-seal collagen peptide according to the invention As can be seen in Table 3, the bending vibration peak of which can be rep a specific action of the collagen peptide amine NH group 1536 cm ^{- 1,} the stretching vibration of CN group The peak was observed at 1241 cm ^{- 1} , and the stretching vibration peak of OH group of carboxyl group was confirmed at 3319 cm ^{- 1} , and the stretching vibration peak of C = O group was confirmed at 1651 cm ^{- 1} . On the other hand, the fish were donpi to read each of the functional groups on the same wavelength in the infrared spectrum of scale collagen peptide, bending vibration peak of the NH groups of the amine are respectively 1534 cm ^-1 and 1536 cm ^{- 1,} the stretching vibration peak of the CN group are each 1237 cm ^-1 and 1243 cm ^- was available on the ^first, the stretching vibration peak of the OH group is a carboxyl group, respectively 3334 cm ^-1 and 3384 cm ^{- 1,} respectively in, the stretching vibration peak of the C = O group is 1649 cm ^-1 And 1648 cm ^{- 1} , respectively.

<3-2> Analysis of compositional amino acid composition

The PICO-tag method was used to analyze the composition of amino acids constituting the half-seal collagen peptide, which was compared with the composition of the control peat pork and fish scale collagen peptide amino acids (see Table 4).

As a result, as shown in Table 4, it was confirmed that hydroxy-proline (OH-proline), which is an amino acid which plays a role of restoration of tissue in the half-seals collagen peptide according to the present invention, and glycine (glycine) content was confirmed. In addition, histidine (histidine), which plays an important role in body balance and skin nutrition and plays a role in the formation of auditory nerve cells, is involved in growth and development and plays an important role in the metabolism of threonine threonine) was contained in an amount of 5.09 wt%. On the other hand, 5.41 wt% of valine, which is essential amino acid to promote muscle and brain activity, which helps to relieve emotions, 2.54 wt% of methionine, which is an amino acid that activates hepatic function and prevents hair loss, and other collagen peptide Isoleucine, which is an essential amino acid that is not contained in the body, plays a role in lowering blood sugar and promoting the transfer of sugar into the cells, 2.71wt% of leucine, 6.67wt% of hemoglobin, , 3.47wt% of phenylalanine, an amino acid that promotes the secretion of hormones from the thyroid gland, and 2.50wt% of lysine, which is an essential amino acid for growth. Compared to other collagen peptides, Was much higher.

Accordingly, it is expected that the content of essential amino acids can be increased in the production of processed foods using the half seals collagen peptide of the present invention, and thus it is expected to have higher nutritional value than processed foods using other collagen peptides.

<3-3> Using a scanning electron microscope Harp seal  Particle analysis of collagen peptides

In order to confirm the particle shape of the collagen peptide of the present invention, it was observed through a scanning electron microscope.

As a result, it was confirmed that the half-seal collagen peptide particles according to the present invention had a spherical shape as shown in Fig. 2 (A). On the other hand, both the case of the donkey collagen peptide (FIG. 2 (B)) and the fish scale collagen peptide (FIG. 2 (C)) of the control group showed spherical particle shapes, I could. This difference in surface was produced by lyophilization in the case of the present invention, but it was judged that the difference was caused by the difference in the preparation method by spray-drying in case of ponik collagen peptide and fish scale collagen peptide.

<3-4> Harp seal  Measurement of weight change according to denaturation temperature and temperature of collagen peptide

The denaturation temperature of the half seal collagen peptide of the present invention was analyzed by DSC.

As a result, the denaturation temperature of the half-seaweed collagen peptide was about 5.8 ° C, the denaturation temperature of the ponchoclagen peptide was about 14.4 ° C, and the denaturation temperature of the fish scale collagen peptide was about 15.1 ° C. And low values.

On the other hand, the present inventors measured the weight change of the half seal collagen peptide according to the temperature through TGA.

As a result, a weight reduction of 12.9 mg, which is 82.5% of the total collagen peptide weight, was observed in the temperature range of 209.7 to 360 ° C for the half-seal collagen peptide, whereas for the pork collagen peptide, 1.2.3 mg And the weight loss of 9.5 mg, which is 73.9% of the total weight, appears secondary at 273.3 ~ 382.4 ℃. On the other hand, fish scale collagen showed a weight loss of 0.7 mg, which is 8.3% of the total collagen peptide weight at 92.3 ° C, and a weight loss of 6.6 mg, which is 72.7% of the total collagen peptide weight at 266.2 to 390.7 ° C, .

As a result of measuring the weight change according to the denaturation temperature and temperature, it was found that the half-seal collagen peptide of the present invention had a relatively low denaturation temperature and a weight change with temperature was less stable to deep-sea heat. And the other. Generally, the amino acid structure of the collagen peptide is characterized by repeating the sequence of Gly-XY. When the Gly-XY is Gly-Pro-Hyp, the hydroxyl group of hydroxyproline causes the thermal stability of the collagen peptide The higher the content of hydroxyproline, the more stable is the heat stability. The content of hydroxyproline contained in the half seals collagen peptide of the present invention is 5.54 wt%, which is considerably lower than that of 9.50 wt% of the content of the pork collagen peptide and 15.76 wt% of the fish scale collagen peptide . Based on these results, it was confirmed that the larger the content of hydroxyproline, the better the thermal characteristics of the collagen peptide.

<3-5> Harp seal  Collagen peptide Isoelectric point  Measure

The isoelectric point refers to the pH of the solution when the algebraic sum of charges possessed by amphoteric electrolytes or colloidal particles is zero. The collagen peptide has a property of increasing solubility and decreasing viscosity near the isoelectric point The equipotentiality of the half-seal collagen peptide of the present invention was measured. That is, to determine the isoelectric point of the half - water collagen peptide according to the pH change, an isoelectric point was measured by preparing a 10 wt% aqueous solution of collagen peptide of the half - water collagen peptide.

As a result, as shown in FIG. 5, the isoelectric point of the half sealant collagen peptide rapidly decreased and the value of zeta potential decreased to about 5 at pH 5. The pit collagen peptide and the fish scale collagen peptide had a zeta potential value near pH 6 And it was confirmed that it showed a zero value. In general, when the pH of the collagen peptide is lower than the lower limit of the isoelectric point, the positive charge and the higher pH are negatively charged. In this case, the collagen peptide of the present invention is relatively positively charged.

<3-6> Harp seal  Of collagen peptide aqueous solution Emulsifying property

Collagen peptides are often used as cosmetics due to wrinkle improvement and antioxidant effects. One of the important characteristics required for use as cosmetics is the emulsifying ability to mix liquids and the like that do not fuse with each other. The present inventors have analyzed the emulsifiability of the half seals collagen peptide.

As a result, as shown in FIG. 6, the half-seal collagen peptide aqueous solution exhibited the lowest emulsifiability at 23.2 vol% at pH 5, the isoelectric point. In contrast, the aqueous solution of Ponch collagen peptide showed 32.7 vol% at pH 6, and the aqueous solution of fish scale collagen peptide showed 33.1 vol% at pH 6, the isoelectric point. The reason for the lowest emulsification at the isoelectric point is that it is because the electric repulsion between the molecules is minimized at the isoelectric point and the solubility is lowered by preventing the collagen peptide from being denatured.

In general, the emulsifying properties of protein hydrolysates are affected by various factors such as the molecular weight, the degree of exposure of the hydrophobic groups in the molecular structure, the pH and the temperature, and so on. It is known to be affected.

Based on the above experimental results, it was found that, in the production of a cosmetic formulation containing a half sealant collagen peptide improved in emulsification by changing these factors, a safe formulation in which a half sealant collagen peptide acts as a natural emulsifier to reduce the content of an emulsifier is manufactured It is expected to be able to do.

<3-7> Harp seal  Of collagen peptide aqueous solution Contact angle  Measure

 The contact angle refers to the angle formed when the liquid equilibrates thermodynamically on the solid surface. When the contact angle is measured, the substance exhibiting 40 ° or less is referred to as a hydrophilic substance.

The contact angle was found to be 35.5 ° in the 10 wt% aqueous solution of the collagen peptide of the present invention. The contact angle was 38.7 °, the contact angle of fish scale collagen peptide was 34.3 °, and the contact angle was 35.5 °. .

<3-8> Harp seal  Determination of content and purity of collagen peptide

The content and purity of the half seal collagen peptide of the present invention were determined by examining the ratio of the hydroxyproline content to the glycine content used in the synthesis of the collagen peptide through analysis of the amino acids shown in Table 4 above and confirming the collagen peptide components and using a nitrogen analyzer As a result, it was shown that the half seal collagen peptide had a high collagen peptide content of 98.93 ± 4.70%.

<3-9> Harp seal  Molecular weight measurement of collagen peptide

  A MALDI-TOF mass spectrometer was used to measure the molecular weight of the half-seal collagen peptide of the present invention, and the measurement results are shown in FIG.

The molecular weight distribution of the half-seal collagen peptide of the present invention has a distribution range of 750 to 2500 Da and an average molecular weight of about 1,645 Da. Generally, the molecular weight absorbable to the skin is reported to be 3,000 Da. The half-collagen peptide of the present invention has a small molecular weight and is easy to penetrate into the skin and can easily be decomposed into amino acids in the body. Therefore, It can be used as a potential material that can demonstrate skin-improving efficacy.

<3-10> Harp seal  Solubility measurement of collagen peptide

In general, the solubility of collagen peptides with respect to pH is strongly influenced by the isoelectric point. That is, when the pH of the collagen peptide is higher or lower than the isoelectric point, the solubility increases while the repulsive force increases between residues charged with the collagen peptide molecule. On the contrary, the mutual bonding between the hydrophobic group and the hydrophobic group increases and the solubility decreases due to precipitation and aggregation of the collagen peptide at the isoelectric point where the total charge of the collagen peptide molecule becomes zero.

As shown in FIG. 8, the solubility of the harp collagen peptides according to the present invention, measured by pH change, was drastically decreased at pH 5 near the isoelectric point, and the solubility was increased in acidic and alkaline regions there was.

<3-11> Harp seal  Analysis of components of collagen peptide

The nutritional composition and calorie of carbohydrate, crude protein, crude fat, ash and moisture of the half seal collagen peptide of the present invention were analyzed.

As a result, the results as shown in Table 5 were obtained. It was confirmed that the half sealant collagen peptide did not contain carbohydrate and crude lipid, and the protein contained 91.4 wt%. On the other hand, the ash content was found to contain 4.3 wt% and the water content was 4.3 wt%. In addition, it was confirmed that the half sealant collagen peptide had a calorific value of 380.9 Kcal per 100 g.

< Example  4>

Harp seal  Antioxidant activity measurement of collagen peptide

<4-1> Measurement of polyphenol content

Polyphenolic materials are aromatic compounds with two or more phenolic hydroxyl (-OH) groups in one molecule, and are mainly composed of flavonoids and tannins. It is known that it has various physiological activities such as prevention of tooth decay, hypertension inhibition, anti-AIDS, antioxidant, and anti-cancer.

The present inventors utilized the Dewanto method to measure the polyphenol content of the half sealant collagen peptide. That is, when the Folin-Ciocalteu reagent is reduced by the polyphenolic compound of the half-coat collagen peptide, it is analyzed that the color develops to blue of molybdenum.

As a result, the total polyphenol content of the harp collagen peptides was 22.3 ± 0.31 mg / g. Based on the above results, the present inventors have found that a pharmaceutical composition or a health food supplement exhibiting an effect of preventing or ameliorating the circulatory system diseases such as lowering the blood cholesterol level and increasing the HDL-cholesterol level through foods containing the half seals collagen peptide It could be expected to be available.

<4-2> DPPH Radical Scatters  And ABTS cation decolorization assay On by Antioxidant power  Measure

Radical scavenging activity is an index of antioxidant activity by phenolic substances such as phenolic acid and flavonoids. It is known that the electron donating ability (EDA%) increases with the reducing power.

DPPH (1,1-diphenyl-2-picrylhydrazyl) analysis is a method of measuring antioxidative capacity by reducing the amount of purple color by an aromatic compound and an aromatic amine due to the electron donating ability of the antioxidant as an index. The present inventors utilized the method disclosed in the prior art document 1 to measure 1,1-diphenyl-2-picrylhydrazide (DPPH) radical scavenging activity. That is, the sample solution (60ul) was mixed with 60uL of DPPH (60uM / L) dissolved in methanol for 10 seconds, and then the solution was transferred to a 100uL Teflon capillary tube and analyzed with an ESR spectrometer (JES-FA machine, After 2 minutes elapsed, the spin adduct on the ESR spectrometer was measured under the conditions of a center field of 3475 G, a modulation frequency of 100 kHz, an amplitude modulation of 2 G, a microwave power of 5 mW, and a gain of 6.3 × 10 ⁵ Respectively.

As a result, as shown in Table 6, the electron donating ability of the half sealant collagen peptide was 17.29 ± 3.02% at a concentration of 12.5 mg / mL, and the antioxidant activity was 9.64 ± 0.08 mg AA eq / g there was.

< Example  5>

Harp seal  Collagen peptide Anti-diabetic  Effect measurement

Α-Glucosidase is an enzyme present in the brush-border membrane of small intestine epithelium. It serves to hydrolyze disaccharides and polysaccharides into monosaccharides, which are the state for carbohydrate digestion and absorption. The inhibitory effect on α-glucosidase can be used as a measure of anti-diabetic activity since it can inhibit the increase of blood glucose after carbohydrate diets.

The inventors of the present invention measured the inhibitory effect on the α-glucosidase of the half seal collagen peptide of the present invention and found that the half seal collagen peptide exhibited activity of 35.72 ± 1.79% at a concentration of 50 mg / mL I could.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (11)

And hydrolyzing the halftone leather with an alcalase and / or a flavorzyme. The method according to claim 1,
Wherein the concentration of the alcalase or flavorzyme is in the range of 0.3 to 1.5 wt%, based on the weight of the half seals. The method according to claim 1,
Wherein the step of hydrolyzing is carried out at 50 to 60 DEG C for 30 minutes to 5 hours. The method according to claim 1,
Wherein the hydrolyzing step is a step of primary hydrolysis with an alcalase followed by a secondary hydrolysis with flavozyme. The method according to claim 1,
And separating oil from the half seals using a supercritical extraction device prior to the step of hydrolyzing the hydro-hydrolyzate. A hydrolyzate of a half seal prepared by the method of any one of claims 1 to 5. delete An antioxidant functional health functional food comprising the hydrolyzate of the half-seal of claim 6 as an active ingredient. A composition for preventing or treating diabetes mellitus comprising the hydrolyzate of the half seal of claim 6 as an active ingredient. A health functional food for preventing or ameliorating diabetes, which comprises the hydrolyzate of the half seals of claim 6 as an active ingredient. A cosmetic composition comprising the hydrolyzate of the half seal of claim 6 as an active ingredient.

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