KR20120049042A - Antioxidant composition comprising enzymatic hydrolysates of mytilus coruscus - Google Patents

Abstract

PURPOSE: An anti-oxidative agent composition which includes mussel hydrolysate is provide to prevent and treat all kinds of diseases and aging which can be caused by active oxygen. CONSTITUTION: An anti-oxidative agent composition includes peptide which is separated from hydrolysate of mussel proteins as an active ingredient. The mussel protein hydrolysate is manufactured by the falling stages: dissolving lyophilized mussels in buffering solution; performing hydrolysis by adding protease in the solution; heating the hydrolysate in hot water; and filtering, concentrating, and drying the heated hydrolysate. The buffer solution is phosphate buffer or glycine-HCl buffer.

Description

Antioxidant composition comprising mussel hydrolyzate as active ingredient {Antioxidant composition comprising enzymatic hydrolysates of Mytilus coruscus}

The present invention relates to an antioxidant composition containing mussel hydrolyzate as an active ingredient, and more particularly, to an antioxidant composition and a mussel protein hydrolyzate comprising a peptide isolated from the mussel protein hydrolyzate and a pharmaceutically acceptable carrier. The present invention relates to a method for preparing and separating and purifying a peptide from the hydrolyzate, and to a primary structure sequence of a mussel protein-derived peptide having antioxidant efficacy.

Marine organisms contain chemicals and biological metabolites that cannot be retained and produced by existing terrestrial organisms due to species diversity and specificity of the habitat environment. Factors also exist, attracting attention as materials for new drugs and new physiologically functional substances. Moreover, as the exploration and development of leading materials, which have been actively conducted on land, began to reach their limits, the interests naturally turned to marine life. A large number of pharmacologically active substances have been reported that show anti-cancer, anti-inflammatory, anti-fungal, antimicrobial, antiviral and anti-oxidant properties derived from marine organisms, which have been discovered to date by advanced marine countries such as the United States, Japan and Europe. The importance of research on the production of food resources from marine resources is also increasing.

In general, shellfish have been used as important food resources since ancient times, due to the regional characteristics of Korea surrounded by the sea on three sides, which have been used as important food resources since the 1990s. As the share of production exceeds 90% and its output increases every year, in addition to the income increase of fish prices and the job creation effect in the fishing villages, it also provides a more hygienic and savory raw material and provides the unique flavor and nutritional characteristics of shellfish. High value-added processing technology that utilizes this technology has become a major concern for fishery development.

In particular, recently, studies have been conducted to examine their physiological activities by separating and purifying hydrolysates obtained by hydrolyzing animal and plant proteins using various enzymes. These hydrolysates have been found to have antioxidant and blood pressure lowering effects, and have been reported to be used as anti-aging agents for skin aging and hypertension. In addition, protein hydrolysates are used in a variety of other fields such as processed foods, seasonings, shampoos, cosmetics.

However, in Korea, acid hydrolyzate of protein is almost used, and when hydrolyzing protein with acid or alkali, essential amino acids such as tryptophan and cysteine are lost, as well as lignolanine Toxic by-products such as lysinoalanine have been produced, which has raised the issue of the safety of acid hydrolysates of proteins in developed countries, including Japan.

Oxygen is the most abundant element on earth (53.8%), accounting for 21% of the dry air, and all living things use oxygen to generate the energy needed to sustain life. However, the ground state triplet oxygen, an oxygen but stable molecular state that is absolutely necessary to maintain life, has various physical, chemical, and environmental factors such as enzyme system, reduction metabolism, chemicals, pollutants, and photochemical reactions. Factors such as superoxide radical (O ₂ ), hydroxyl radical (HO), hydrogen peroxide (H ₂ O ₂ ), singlet oxygen (HO) When converted to active oxygen, it has a double sided effect that causes fatal oxygen toxicity to the living body. In other words, these free radicals have non-selective and irreversible destruction effects on cell components such as lipids, proteins, sugars, and DNA. It is known to cause various diseases such as skin diseases, digestive diseases, inflammation, rheumatism and autoimmune diseases (Halliwell B. 1991. Drug antioxidant effects. Drugs 42: 596-605). In addition, various peroxides in the body, including lipid peroxides resulting from lipid peroxidation by these free radicals, also cause various functional disorders due to oxidative destruction of cells, thereby causing aging and disease.

On the other hand, even in normal cells, some free radicals and other free radicals and peroxides are generated during metabolism, but in vivo, SOD (superoxide dismutase), peroxidase, Along with antioxidant enzymes such as catalase, antioxidants such as vitamin E, vitamin C, glutathione, ubiquinone (ubiquinone, coemzyme Q10), and uric acid are present to protect themselves. However, oxidative stress is caused when abnormality is caused in such a bioprotective device or the generation of active oxygen exceeds the capacity of the bioprotective system by various physical and chemical factors. Accordingly, antioxidants such as compounds capable of scavenging such free radicals or inhibitors of peroxide production are expected as agents for inhibiting or treating aging and various diseases caused by these oxides.

The study of antioxidants began in earnest when McCord and Fridovich discovered SOD, an enzyme that eliminates superoxide radicals, in 1969. Recently, diseases such as aging and adult diseases are known to be due to the presence of free radicals. Research into antioxidants that can regulate free radicals is actively underway. Antioxidants include SOD and catalase enzymes, synthetic antioxidants such as tert-butylhydroxytoluene (BHT) and tert-butylhydroxy-anisol (BHA), tocopherol, ascorbic acid, tannin, and carotenoids. There are some natural antioxidants such as (carotenoids), flavonoids, etc. In addition, various kinds of antioxidants are being developed (Kitahara K., Matsumoto Y., Ueda HA and Ueoka R. 1992. Chem. Pharm. Bull 40: 2208-2209; Hatano T. 1995. Natural Medicines 49: 357-363).

However, these antioxidants are limited to use due to various problems such as toxicity, low activity and limit of use.

Accordingly, the present inventors have made intensive efforts to develop antioxidants derived from natural products without toxicity and side effects, and have confirmed the excellent antioxidant effect of the mussel protein hydrolyzate according to the present invention and completed the present invention.

After all, the main object of the present invention is to contain a peptide isolated from the mussel protein hydrolyzate as an active ingredient, and to provide a safe and effective natural antioxidant composition for the suppression of aging and various diseases caused by the oxides produced by active oxygen will be.

Still another object of the present invention is to provide a method for preparing a mussel protein hydrolyzate and a method for separating and purifying peptides from the hydrolyzate.

In order to achieve the above object, the present invention provides a method for preparing an antioxidant composition and a mussel protein hydrolyzate comprising a peptide isolated from the mussel protein hydrolyzate and a pharmaceutically acceptable carrier and a method for separating and purifying the peptide from the hydrolyzate It is about.

The present invention provides an antioxidant composition containing the peptide isolated from the mussel protein hydrolyzate as an active ingredient.

The mussel protein hydrolyzate is a finely pulverized lyophilized mussels dissolved in a buffer, the protein hydrolase is added to the solution and stirred to proceed the hydrolysis reaction, and then heated in hot water and filtered, concentrated and It is characterized by being manufactured by drying.

In addition, the buffer is a phosphate buffer (glycine-HCl buffer) or glycine-HCl buffer (protease), the proteolytic enzyme Flavorzyme (Neutrase), Papain (Papain), pepsin ( pepsin), Trypsin, Alpha-chymotrypsin, α-Chymotrypsin, Alcalase, and Protamex.

In addition, in the present invention, the hydrolysis step is characterized in that the hydrolysis of the reaction temperature of 25 ~ 70 ℃, reaction time 6 ~ 10 hours in a buffer of pH 1.0 ~ 10.0, preferably the pH of the buffer is 2.0 ~ 8.0, the reaction temperature is heated to 30 ~ 60 ℃ reaction time 8 hours, concentrated with a reduced pressure evaporator is preferably lyophilized.

In addition, the peptide isolated from the mussel protein hydrolyzate comprises the steps of: (1) separating the mussel protein hydrolyzate by molecular weight by TFF (Tangetial Flow Filtration) system to measure the physiological activity of each fraction; (2) separating the fraction having the most physiological activity among the separated fractions by chromatography; (3) measuring the biological activity of each of the separated fractions; And (4) purifying the fraction having the highest physiological activity among the separated fractions. The method is prepared by the method comprising the steps of: (2) to (4) by repeating steps to separate and purify into a single substance. do.

In addition, in the step (2), the antioxidant peptide is separated by protein liquid chromatography equipped with an ion exchange column (DEAEFF 16/10), and then preparative C ₁₈ column (GROM-SIL 120 C ₁₈ , 20 High Performance Chromatography with X 250 mm), High Performance Chromatography with analytical C ₁₈ columns (GROM-SIL 120 C ₁₈ , 4.0 X 250 mm) and GPC column (GPC-SB 802.5) for easy separation by size It is characterized by having a strong antioxidant capacity through a purification process using a high performance chromatography.

In addition, the present invention is characterized by analyzing the amino acid sequence using the PTH analysis column by the Edman degradation method of the separated and purified peptide.

The present invention also provides antioxidants and health functional foods containing the composition as an active ingredient.

In addition, the mussel protein hydrolyzate of the present invention and the functional peptide separated therefrom have excellent free radical scavenging activity, and thus, medicines, health supplements, and the like for suppressing or treating aging and various diseases caused by oxides produced by active oxygen, It may be usefully used in cosmetics, food, food additives, and the like, but is not limited thereto.

In addition, when the mussel protein hydrolyzate of the present invention and the functional peptide separated therefrom are used as a medicine, it may further include appropriate carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions, It can be prepared in a pharmaceutically acceptable formulation, including the pharmaceutical composition made in this scope. In addition, the pharmaceutical use of the pharmaceutical composition as an antioxidant is also included in the scope of the present invention.

The carrier or excipient includes water, dextrin, calcium carbonate, lactose, propylene glycol, liquid, paraffin, physiological saline, dextrose, sucrose, sorbitol, mannitol, ziitol, erythritol, maltitol, starch, gelatin, calcium phosphate, calcium Silicates, cellulose, methyl cellulose, polyvinylpyrrolidone, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and minerals, which may be used one or more, but are not limited thereto. Both carriers and excipients can be used. In addition, when the antioxidant composition is formulated, conventional fillers, extenders, binders, disintegrants, surfactants, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers or preservatives may be further included.

In addition, when the mussel protein hydrolyzate of the present invention and the functional peptide separated therefrom are used as cosmetics, wrinkle improvement and whitening functional cosmetics, softening lotion, nourishing cosmetics, eye cream, nourishing cream, massage cream, cleansing cream, essence, etc. In the cosmetic composition of each formulation, it can be prepared in the formulation of the formulation, including the protein hydrolyzate and functional peptide, other ingredients as wrinkles and whitening raw materials or conventional cosmetic raw materials according to the formulation or purpose of use of cosmetics, etc. It can be selected and combined without difficulty.

According to the present invention as described above, the mussel protein hydrolyzate, the antioxidant peptide isolated and purified from the hydrolyzate, and the composition comprising the hydrolyzate exhibit an excellent antioxidant effect on free radical scavenging, and thus are produced by active oxygen. It is effective in suppressing or treating aging and various diseases caused by them.

In addition, the mussel protein hydrolyzate of the present invention can be used safely without toxicity and side effects as a substance derived from natural products.

1 is a schematic diagram showing a process for producing mussel protein hydrolyzate.
Figure 2 shows the hydroxyl radical scavenging activity of mussel protein hydrolysates (1, flavozyme; 2, neutrases; 3, protamax; 4, alcalase; 5, pepsin; 6, alpha-chymotrypsin 7, trypsin; 8, papain).
Figure 3 shows the hydroxyl radical scavenging activity of the mussel protein hydrolyzate (papaine) by molecular weight (> 30,000 Da; 10,000 ~ 30,000 Da; 5.000 ~ 10,000 Da; 5,000 Da <).
Figure 4 shows the separation of the protein corresponding to the molecular weight of more than 30,000 Da of mussel protein hydrolyzate (papaine) by protein liquid chromatography equipped with an anion exchange column.
Figure 5 is a protein liquid chromatography equipped with an anion exchange column separated by a protein corresponding to a molecular weight of 30,000 Da or more of mussel protein hydrolyzate (papaine) using high performance chromatography equipped with a preparative column It shows that separated by.
FIG. 6 is a protein liquid chromatography equipped with an anion exchange column to separate proteins corresponding to a molecular weight of 30,000 Da or more of mussel protein hydrolyzate (papaine) using high performance chromatography equipped with a preparative column. After separation by fractions proved efficacy was shown to be separated by high performance chromatography equipped with an analytical column.
7 is a protein liquid chromatography of mussel protein hydrolyzate (papine) of the present invention is separated by a protein liquid chromatography equipped with an anion exchange column and separated by high performance chromatography equipped with a preparative column. After the fraction proved to be effective by high performance chromatography equipped with an analytical column, the fractions were separated by high performance chromatography equipped with a GPC column for easy separation by size.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example  1. Protein of mussels Hydrolyzate  Produce

Mussel samples used in this experiment were purchased from Yeosu fish market, and stored at -20 ° C to prepare hydrolyzate. The finely ground lyophilized mussels were dissolved at a ratio of 0.1 M buffer (buffer, pH 2.0-8.0) and mussels: buffer = 1:50 (w / w), then preheated to 30-60 ° C. with stirring at 150 rpm for 30 minutes. Then, the proteolytic enzyme: mussel = 1: 50 (w / w) ratio of the proteolytic enzyme, flavozyme, neutrase, papain, pepsin, trypsin, alpha-chymotrypsin, alkalase and prota Max was added and stirred at 180 rpm for 8 hours to hydrolyze at 30 to 60 ° C, and then heated at 100 ° C for 10 minutes to terminate the hydrolysis reaction.

After the reaction was completed, the solution was filtered using Whatman filter No. 41 (Whatman filter No. 41), concentrated under a reduced pressure evaporator, and lyophilized at −80 ° C. to prepare a mussel protein hydrolyzate. The composition and reaction conditions of the enzyme used at this time are shown in Table 1 below.

However, PB in the above means phosphate buffer (Phosphate Buffer), GHB means glycine-HCl buffer (Glycine-HCl Buffer).

Experimental Example  1. Mussel Protein Hydrolyzate  Antioxidant Activity Measurement

Free radicals are generally unstable and have very high reactivity because they have two pairs of electrons in opposite directions of spin and have active electrons that are not paired differently from ordinary molecules in a stable state. Although it acts as a defense in the human body, it is known to promote the oxidation of our body when excessive production causes aging and various diseases. Antioxidative effect of mussel-derived protein hydrolyzate was measured by scavenging the hydroxyl radical (ESR spectrometer, JEX-PX 2000-300 model, JEOL, Japan).

The hydroxyl radical scavenging ability is generated by DMPO (5,5-dimethyl-1-pyrroline N-oxide) by generating hydroxyl radicals based on the Fenton reaction in which iron (Fe) ions and hydrogen peroxide (H ₂ O ₂ ) react. The amount of) -OH was determined to determine the hydroxyl radical scavenging rate.

Mussel hydrolysates using eight enzymes (1, flavozyme; 2, neutrases; 3, protamax; 4, alcalase; 5, pepsin; 6, alpha-chymotrypsin; 7, trypsin; 8, papain), 0.2 ml of DMPO, 0.2 ml of 10 mM FeSO _{4 and} 0.2 ml of 10 mM H ₂ O ₂ were added sequentially, followed by vigorously vortexing for 10 seconds, and then reacting for 2 minutes 30 seconds. The reaction mixture was transferred to a capillary tube and the amount of hydroxyl radicals was measured on an ESR spectrometer. [ESR measurement conditions-central field: 3475G, modulation frequency: 100 Hz, modulation amplitude: 2G, microwave power: 1 Hz, gain: 6.3 × 10 ⁵ , Temperature: 298 K].

The results are shown in Figure 2, it was confirmed that papain hydrolyzate of the mussel protein hydrolyzate of the present invention has the highest hydroxyl radical scavenging activity.

Experimental Example 2. Mussel-derived enzyme From hydrolyzate  Antioxidant Peptide Material Isolation and Purification I

In order to isolate and purify the antioxidant peptide of the hydrolyzate hydrolyzed mussels with papain, the TFF system is used to separate and purify proteins according to the membrane size, and less than 5,000 Da, 5,000 to 10,000 Da, 10,000 to 30,000 Da, and 30,000 Da The hydroxyl radical scavenging rate was measured after lyophilization by dividing into four fractions by molecular weight. As a result, it was confirmed that the highest activity in the size of more than 30,000 Da of the four fractions (see Figure 3).

Experimental Example 3. Mussel-derived enzyme From hydrolyzate  Antioxidant Peptide Material Isolation and Purification II

This experimental example is the second process of separating and purifying antioxidant peptides from mussel papain hydrolysates. Protein liquid chromatography equipped with anion exchange column was used for the fractions of 30,000 Da or more, which showed the best antioxidant activity in the TFF method. It was isolated and purified using.

Protein Liquid Chromatography is a device that specializes in the purification of proteins by applying high-performance chromatography technology. It is a fully automatic separation device using a tube (separation tube) uniformly containing a packing material suitable for ion exchange or size exclusion. In this experiment, protein liquid chromatography was performed by mounting a column suitable for anion exchange, and finally 5 fractions were obtained (see FIG. 4). As a result of measuring the antioxidant activity by lyophilizing each fraction, as shown in Table 2, the hydroxyl radical scavenging activity was the highest in the D-1 fraction and the IC ₅₀ value was 0.251 mg / ml.

Experimental Example 4. Mussel-derived enzyme From hydrolyzate  Antioxidant Peptide Material Isolation and Purification III

This experimental example is a third step of separating and purifying the antioxidant peptide from the mussel papain hydrolyzate. The fraction D-1, the fraction showing the best antioxidant activity in the above Experimental Example 3 using protein liquid chromatography, was used for preparative column. It was separated and purified using the equipped high performance chromatography.

High performance chromatography is a method in which a gas sample or a gasified liquid or a solid sample is developed by a carrier gas in a uniformly packed tube (separation tube), but separated by passing through a gaseous state without decomposition. In this experiment, a preparative column was mounted and chromatographed. Finally, six fractions were obtained (see FIG. 5).

As a result of measuring the antioxidant activity of each fraction by lyophilization, as shown in Table 3, the hydroxyl radical scavenging activity was the highest in the DI-ii fraction and the IC ₅₀ value was 0.238 mg / ml.

Experimental Example 5: Mussel-derived enzyme From hydrolyzate  Antioxidant Peptide Material Isolation and Purification IV

This Experimental Example is a fourth step of separating and purifying the antioxidant peptide from the mussel papain hydrolyzate. The fraction DI-ii obtained in Experimental Example 4 was isolated and purified using high performance chromatography equipped with an analytical column. .

Finally, two fractions were obtained (FIG. 6). As a result, fraction DI-ii-a had the highest hydroxyl radical scavenging activity as shown in Table 4, and the IC ₅₀ value was 0.151 mg / ml.

Experimental Example 6: Mussel-derived enzyme From hydrolyzate  Antioxidant Peptide Material Isolation and Purification V

This experimental example is a fifth step of separating and purifying an antioxidant peptide from mussel papain hydrolyzate. The high performance chromatography equipped with a GPC column capable of separating the fraction DI-ii-a obtained in Experimental Example 5 by size Finally separated and purified one more time, and finally, one fraction was obtained (FIG. 7). As a result, as shown in Table 5, the IC ₅₀ value representing the 50% hydroxyl radical fraction of the fraction is 0.118 mg / ml.

Experimental Example 7: Antioxidant Peptide  Confirm Amino Acid Arrangement

In this experimental example, in order to confirm the amino acid sequence of the antioxidant peptide obtained in Experimental Example 6, Edman degradation method (Edman, P., Acta Chemica Amino acid sequence was analyzed from N-termina according to Scandinavica , 4 , 283-293, 1950).

As a result, the amino acid sequence of the antioxidant peptide was confirmed as Ser-Leu-Pro-Ile-Gly-Leu-Met-Ile-Ala-Met, respectively, set forth in SEQ ID NO: 1 (see Table 6).

As described above, specific portions of the contents of the present invention have been described in detail, and for those skilled in the art, these specific techniques are merely preferred embodiments, and the scope of the present invention is not limited thereto. Will be obvious. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> Konkuk University Industrial Cooperation Corp. <120> Antioxidant composition comprising enzymatic hydrolysates of          Mytilus coruscus <130> P10-E423 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 10 <212> PRT <213> Mytilus coruscus <400> 1 Ser Leu Pro Ile Gly Leu Met Ile Ala Met   1 5 10

Claims (10)

Antioxidant composition containing a peptide isolated from the mussel protein hydrolyzate as an active ingredient.
The method of claim 1,
The mussel protein hydrolyzate is
(1) dissolving lyophilized mussels in buffer;
(2) hydrolysis by adding proteolytic enzyme to the solution;
(3) heating the hydrolyzate in hot water;
(4) filtering, concentrating and drying the heated hydrolyzate; antioxidant composition characterized in that it was prepared by a method comprising a.
The method of claim 2,
The buffer of step (1) is an antioxidant composition, characterized in that any one of phosphate buffer (Phosphate buffer) or glycine-HCl buffer (Glycine-HCl buffer).
The method of claim 2,
Antioxidant composition, characterized in that the pH of the buffer of step (1) is 1.0 ~ 10.0.
The method of claim 2,
The proteolytic enzymes are Flavorzyme, Neutrase, Papain, Pepsin, Trypsin, Alpha-Chymotrypsin, Alcalase And Protamex (Protamex) antioxidant composition, characterized in that at least one member selected from the group consisting of.
The method of claim 2,
The protein hydrolysis of step (2) is an antioxidant composition, characterized in that the enzyme treatment with a reaction time of 6 to 10 hours at a reaction temperature of 25 ~ 70 ℃.
The method of claim 1,
The peptide is
(1) separating the mussel protein hydrolyzate by molecular weight by TFF (Tangetial Flow Filtration) system and measuring the physiological activity of each fraction;
(2) separating the fraction having the most physiological activity among the separated fractions by chromatography;
(3) measuring the biological activity of each of the separated fractions; And
(4) purifying the fraction having the highest physiological activity among the separated fractions; prepared by the method comprising a, characterized in that the separation and purification to a single material by repeating the steps (2) to (4) Antioxidant composition.
The method of claim 7, wherein
Step (2) is a protein liquid chromatography equipped with an ion binding column (DEAEFF 16/10), high performance chromatography equipped with a preparative C ₁₈ column (GROM-SIL 120 C ₁₈ , 20 X 250 mm), for analysis. Purification using high performance chromatography with C ₁₈ columns (GROM-SIL 120 C ₁₈ , 4.0 X 250 mm) and high performance chromatography with GPC columns (GPC-SB 802.5) Antioxidant composition, characterized in that.
Antioxidant containing the composition of any one of Claims 1-8 as an active ingredient.
Health functional food containing the composition of any one of Claims 1-8 as an active ingredient.

Images