KR100763938B1 - Hydrolysates from Alaska pollack frame - Google Patents

Abstract

The present invention relates to a peptide having antihypertensive and antioxidant activity isolated from pollock, and more particularly to a peptide having antihypertensive and antioxidant activity that inhibits ACE activity, which is one of the factors involved in blood pressure control from pollock. It is an object of the present invention to provide an antihypertensive and antioxidant composition containing the same as an active ingredient.

Pollack Frame, Antihypertensive Peptide, Antioxidant Peptide

Description

Peptides isolated from pollock frame protein {Hydrolysates from Alaska pollack frame}

1 is a schematic diagram showing a process of extracting coenzyme from the mackerel gut according to the present invention.

2 is a graph comparing MICE activity in natural substrates (A, casein: B, hemoglobin).

3 is a graph comparing MICE activity in synthetic substrates (A, BAPNA; B, BAEE; C, BTEE; D, ATEE).

Figure 4 is a graph showing the pH effect on pollock hydrolyzate by MICE.

5 is a graph showing the temperature effect on pollock hydrolyzate by MICE.

6 is a graph showing the substrate concentration effect on pollock hydrolyzate by MICE.

7 is a graph showing the substrate-to-enzyme ratio effect on pollock hydrolyzate by MICE.

8 is a graph showing the reaction time effect on pollock hydrolyzate by MICE.

9 is a graph showing the results of separation and purification of pollock hydrolyzate by ion chromatography.

10 is a graph showing the gel chromatography results of the hydrolyzate separated from Sephadex G-25.

11 is a graph showing the results of separation and purification by HPLC of the hydrolyzate separated from Sephadex G-25.

12 is a graph showing the results of rechromatography on a Capcell Pak C ₁₈ -VG column.

13 is a graph showing a Lineweaver-Burk plot of ACE inhibitory activity in the presence of inhibitory peptides.

14 is a graph showing the results of observing blood pressure change of SHR by administering an ACE inhibitory peptide derived from pollock hydrolyzate (* P <0.05, ** P <0.01).

Figure 15 shows the results of fractionation of antioxidant peptides with SP-Sephadex C-25 column (bottom panel) and the results showing antioxidant activity (top panel).

FIG. 16 shows the results of gel chromatography (bottom panel) and antioxidant activity of the active fractions obtained from SP-Sephadex C-25 (top panel).

FIG. 17 shows the results of separation and purification by reverse phase HPLC on a Primesphere 10 C ₁₈ column (bottom panel) and results showing antioxidant activity (top panel).

Figure 18 shows the results of separation and purification by reverse phase HPLC on Capcell Pak C ₁₈ -VG column (bottom panel) and the results showing antioxidant activity (top panel).

Figure 19 shows the results of measuring the antioxidant activity of pollen frame hydrolase in the linoleic acid automatic oxidation system by Ferric thiocyanate method.

20 is a graph showing the results of measuring the antioxidant activity of pollack MICE hydrolyzate in DPPH radicals.

21 is a graph showing the antioxidant activity of the active fractions in linoleic acid autooxidation system.

22 is a graph showing DPPH radical scavenging activity of active fractions at various concentrations.

Figure 23 is a graph showing the synergistic effect of α-tocopherol and APH in linoleic acid automatic oxidation system.

24 is a graph showing the results of measuring the toxicity of antioxidant peptides cultured in human normal hepatocytes.

25 is a graph showing the effect of hydrolyzate on cell necrosis by t- BHP concentration.

FIG. 26 is a graph showing the effect of concentration of hydrolyzate on cell necrosis of t- BHP. FIG.

The present invention relates to a peptide having antihypertensive and antioxidant activity isolated from pollock, and more particularly to a peptide having antihypertensive and antioxidant activity that inhibits ACE activity, which is one of the factors involved in blood pressure control from pollock. It is an object of the present invention to provide an antihypertensive and antioxidant composition containing the same as an active ingredient.

The sea area of Korea is 4.5 times the land area, and the biological resources are about 300,000 species, which is 7 times the land life, and its economic value is estimated to be 100 trillion won per year. Currently, the search for functional and bioactive substances in terrestrial organisms has reached its limit. In general, the most anticancer leading substances are found in the ocean, and because of the diversity of the species and the environment that forms a unique ecosystem different from the land, it contains a variety of substances with new functions that terrestrial life does not exhibit. However, despite the diversity and abundance of these potentially bioactive species, research on the discovery of bioactive and functional substances from marine organisms is a rudimentary level. In the case of developed countries, the focus of attention is already inclined toward marine life, but in Korea, basic research on them is still insufficient. As the biological and biotechnology industry is developing at a very rapid growth rate, the development of bioactive and functional materials using marine organisms is most suitable as a breakthrough in the provision of new materials and technological limits, and the value of products generated from marine biotechnology is As it is estimated to be higher than other industrial sectors, research on this is urgently needed in Korea.

The annual total catch of Korean fishery products is 3,244 thousand tons (2001), and the total supply of imports and the previous year's inventory is 4,420 thousand tons. According to the trend of increasing the preference of fish and shellfish of Korean people, the processing rate of fish processing factory increases every year, accounting for more than 85% of the total supply, so that the residue of fish processing residues such as fish processing residue, fish bone, fish, dark, intestine, scales, etc. The amount is also increasing. Most of these residues are used or disposed of as feed, which effectively causes the poor utilization of resources and causes environmental pollution. In addition, even in the decorative part other than the processed residue of the fish, many water-soluble proteins, flotation products, fish oil, etc. are washed out with the waste liquid by a process such as washing with water. Therefore, there is a need to develop new functional materials with high added value using useful components contained in fishery processing by-products. In addition, since bioactive substances derived from marine organisms have a small amount and economic effects are insignificant, development of bioactive substances using fishery by-products containing a large amount of potential bioactive substances, such as sugars, proteins, and lipids, has been difficult. The economic effect is expected to be enormous.

Recently, as the demand for protein having functionalities increases, studies to improve the protein's functionality have been actively conducted. In addition, researches have been conducted to search for bioactive peptides present in protein hydrolysates (Whitaker et al., 1977; Kinesella). Et al., 1987; Quaglia et al., 1987; Casteels et al., 1989; Benson et al., 1990; Uesaka et al., 1991).

The history of bioactive peptide research was first demonstrated in 1889 that the onset of diabetes was caused by a deficiency of a substance present in the pancreas, resulting from ablation of the dog's pancreas. It has also been proven to contain substances that exhibit antidiuretic effects (Fujino et al., 1986). Subsequently, a chemical study of these substances proceeded to explain the chemical structures of vasopressin and oxytosin for the first time as peptide-based hormones, followed by adrenocorticotropic hormone (adrenocorticotropic hormone) ACTH) has been elucidated, and the structure of angiotensin as a blood pressure raising substance has been revealed. In parallel with chemical structural elucidation, these peptide-based hormones were first synthesized as oxytocin (Vigneaud et al., 1953) and vasopressin (Vigneaud et al., 1954). In the 1960s, the biological activity of these chemically synthesized peptides showed that vasopressin was weak but exhibited uterine contraction and milk secretion of oxytocin. The bioactivity, which was understood to be due to the mixture of trace substances, was found to be due to the intrinsic structure of these peptides. That is, it was confirmed that not all amino acid residues constituting the peptides exhibited physiological activity in the peptide of a specific residue in expressing different bioactive activities depending on the type of peptide.

Since hypertension is involved in many factors in the onset and maintenance of blood pressure, the mechanisms that regulate blood pressure in the human body, the Renin-angiotensin system and It is known that blood pressure control problems occur when the homeostasis of the kallikrein-kinin system is not maintained (Saxena, 1992). Currently, the study of inhibitors of angiotensin I converting enzyme (ACE) involved in these two systems has been suggested as a potential development for the treatment of hypertension. In other words, ACE cleaves the C-terminal dipeptide of the angiotensin I of the renin-angiotensin system and converts it to angiotensin II, an octapeptide, which has a strong vasoconstrictive action. It acts to increase blood pressure by inactivating bradykinin, a nonapeptide with vasodilation (Ondetti et al., 1982; Ehlers et al., 1990). In 1965, Ferreira reported that some of the venomous venoms contained a substance that enhances bradykinin activity, and in 1968, Vane was identified in the same snake venom. After that, the structure of the two substances was confirmed to be the same peptide. As a result, researches on inhibitors of ACE have been actively conducted, and Squibb's Ondetti et al. (1977) developed a powerful oral blood pressure enhancer, captopril, but found fatal side effects such as proteinuria and agranulocytosis. Therefore, various captopril derivatives have been developed to overcome this, but it was difficult to completely overcome them. In order to solve these problems, researches on the enzymatic hydrolysates of proteins have recently been actively conducted. Casein digests (Maruyama et al., 1982), zein (Miyoshi et al., 1991a), and soybeans (Kawamura, 1989) ), Sardine meat (Suetsuna et al., 1986), bonito (Kohama et al., 1991), and viscera (Fujii et al., 1993), and microorganism-derived ACE inhibitors, such as Streptomyces K-26 discovered by Yamato et al. (1986); (1986) reported that Actinomadura spiculosospora extracts were found to have ACE inhibitory activity, and Streptomyces K-26 showed similar activity to captopril's ACE inhibitory activity in vitro .

In Korea, it is limited to studies on miso (Shin et al., 1995; Hwang, 1997), whey (Pak et al., 1996), anchovy meat hydrolyzate (Lee, 1998), and squid (Suh et al., 1997).

Antioxidants are substances that inhibit or retard antioxidants. Since the study of autoxidation in the 1940's, research has been conducted in terms of anti-oxidation and human aging. Davies (1986) divided oxidative defense mechanisms in the cellular system into a primary defense system and a secondary defense system. Primary defense is a mechanism that prevents the continuous oxidation of lipids by converting free radical-type lipids produced in the course of lipid peroxidation into more stable products, also known as chain-breaking antioxidants. Tocopherol, gallic acid and its derivatives, flavonoid-based and antioxidant enzyme superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), etc. This belongs here. In contrast, secondary defense is a repair system that removes the components of molecules or cells that have been damaged by oxidants or free radical reactions beyond the primary antioxidant defenses. Phospholipid enzyme (phospholipase A ₂ ) and fat acyl transferase (enzyme involved in phospholipid repair of damaged membranes), catalytic / multifunctional proteinase complex (Rivett, 1989) and oxidative damage Mouths include DNA glycosylase and DNA ligase (Halliwell and Gutteridge, 1989) that repair DNA. Generally, antioxidants are classified into synthetic antioxidants and natural antioxidants. Synthetic antioxidants BHT and BHA have been reported to affect the biological enzyme system and cause malformation or cancer (Branen, 1975; Fujimoto, 1980). On the other hand, α-tocopherol, which is a natural antioxidant, has almost no side effects and is widely used as a food additive or a medicine. However, it is required to develop an alternative natural antioxidant due to the use of high price and fat soluble. Therefore, research on the development of natural antioxidants to replace synthetic antioxidants and α-tocopherol has been actively conducted. Among them, studies on antioxidants from foods include casein (Yamaguchi et al., 1980), soy protein (Pratt et al., 1972), Bovine serum albumin (Yukami et al., 1972), oil seed protein (Rhee et al., 1979), wheat gliadin (Iwami et al., 1987), beach pea (Chavan et al., 1999) and zein (Wang et al., 1991).

In addition, the hydrolyzate of protein is essentially used in various processed foods, seasonings, samfu, cosmetics, and many other fields, the domestic situation is almost using acid hydrolyzate of protein. However, hydrolysis of proteins with acids or alkalis leads to the loss of essential amino acids such as tryptophan and cysteine, as well as the generation of toxic by-products such as lysinoalanine. The safety of acid hydrolysates of proteins is on the rise. In addition, little research has been done to remove the bitter components indicated as a disadvantage of enzymatically digested soy.

In order to solve this problem, the present inventors developed a highly active complex enzyme in the mackerel gut, and using this complex enzyme to decompose the protein of processed pollack residues, efficiently recovering protein hydrolysates from membrane reactors to prevent antioxidant and The present invention has been completed by isolating and purifying hypertension peptides.

Therefore, an object of the present invention relates to an antihypertensive peptide isolated from pollock frame and an antihypertensive composition containing the same as an active ingredient.

The present invention also relates to an antioxidant peptide isolated from pollack pollen and an antioxidant composition containing the same as an active ingredient.

The present invention relates to a peptide having antihypertensive activity isolated from a pollock frame, which is a by-product processed seafood.

The peptide according to the present invention relates to a peptide having antihypertensive activity of SEQ ID NO: 1.

The present invention also relates to a peptide having the antioxidant activity of SEQ ID NO: 2 isolated from the pollock frame which is a by-product processed by-product.

The present invention also relates to an antihypertensive composition and an antioxidant composition containing a peptide having the antihypertensive and antioxidant activity as an active ingredient.

The composition of the present invention is formulated as an external skin preparation or oral dosage form, such as a dry powder which is in the form of a solution, a suspension or an emulsion in an oil or an aqueous medium, or dissolved in sterile, water-free water before use. It may be formulated as a parenteral formulation such as subcutaneous injection, intravenous injection or intramuscular injection.

In the case of oral dosage forms, for example, tablets, troches, sugar-containing tablets, aqueous or oily suspensions, dispersible powders, etc., in a known manner using a pharmaceutically acceptable carrier or forming agent. Or in the form of oral dosage forms in the form of particles, emulsions, soft or hard capsules, syrups, elixirs, which may be suitably prepared according to unit dosages, forms and the like.

Parenteral formulations may be injected as a sterile injectable solution or as a suspension in which the active ingredient is suspended in a solvent such as a non-toxic usable diluent or 1,3-butanediol. Excipients or solvents that can be used include water, Ringer's solution, and isotonic saline solution. Cosolvents such as ethanol, polyethylene glycol, and polypropylene glycol can be used. In addition, sterile, nonvolatile oils can be customarily used as solvents or suspending solvents. The suppository form is formulated by mixing the drug with a suitable non-irritating excipient, such as cocoa butter or polyethylene glycol, which is solid at room temperature but liquid at the rectal temperature and will melt in the rectum to release the drug and then enter the rectum. Administration.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are intended to illustrate the invention and the scope of the invention is not limited to these examples.

Pollack frame (Alaska pollack frame) used in the embodiment of the present invention was stored after freezing at -20 ℃ received the remaining frame after the fillet (filleting) from Daelim Fisheries processing plant of Seogu, Busan. In order to extract the coarse enzyme derived from mackerel guts, the mackerel guts were recovered from the fish market in Nam-gu, Busan, and stored at -70 ° C.

Hammarsten casein, a natural substrate for coenzymes, is available from Merck Co. Product, hemoglobin is manufactured by Sigma Chemical Co. The product was used. In addition, the synthetic substrate _{BTEE (N-benzoyl- L -tyrosine ethyl} ester), ATEE (N-acetyl- L -tyrosine ethyl ester), BAPNA (N-α-benzoyl- DL -arginine- p -nitroanilide-naphthylamide), BAEE (N-α-benzoyl- _L -arginine ethyl ester) is obtained from Sigma Chemical Co. The product was used.

Peptide purification resins such as SP-Sephadex C-25, Sephadex G-50, and Sephadex G-25, which are used to separate and purify physiologically active peptides from the hydrolyzate obtained in the membrane reactor, are available from Sigma Chemical Co. The acetonitrile for HPLC required to separate and purify peptides by product, HPLC (Thermo Separation Products, Model Spectra System P2000), was purchased from Fisher Scientific and used as a reverse-phase ODS column (Macherey-Nagel). , Nucleosil 100-7 C ₁₈ , size: φ10 × 250mm) was used for final purification.

Ultrafiltration membrane devices for the production of hydrolysates were purchased from Millipore Co. (Bedford, USA) and used a molecular weight cut-off (MWCO) of 10,000, 5,000, 3,000 and 1,000 Da, respectively. It was.

Angiotensin I converting enzyme (ACE), Hippuryl-Histidyl-Leucine (HHL), and captopril, which were used to measure ACE inhibitory activity, were purchased from Sigma. Ammonium thiocyanate and diphenyl-picryl-hydrazyl (DPPH) used to measure antioxidant activity were purchased from Sigma and used linoleic acid as a fat or oil. %) Was purchased from Sigma, tetramethoxypropane and the control antioxidant BHT (butylated hydroxytoluene, 99% purity) were purchased from Fluka, and α-tocopherol (95% purity) was purchased from Influence Chemical. In addition, normal hepatocytes (Chang) used to measure the antioxidant activity in the cell culture system was used at the Department of Pharmacology, College of Medicine, Dongguk University, and CCK-8 cell counting kits were purchased from Dojindo, Japan. . T- BHP ( tert -butyl hydroperoxide), MTT (4,5-dimethyl tiazol-2-yl-2,5-diphenyl tetrazolium bromide), DMEM medium (Dulbecco's modified eagle medium), L-glutamine, DMSO (dimethyl sulfoxide, ethylenediamine tetraacetic acid (EDTA) and nitro blue tetrazolium (NBT) are from Sigma Chemical, and fetal bovine serem (FBS) and antibiotic / antimycotics are from Gibco (Gibco BRL, Life). Techno. Inc., NY, USA), and all other reagents were purchased and used as an express reagent for analysis.

Example 1 Coenzyme Extraction from Mackerel Intestines

The protease derived from the mackerel gut was extracted according to the method of Kim et al. (1997), the process is shown in FIG.

After the chopped mackerel was chopped, about _twice as much buffer solution (20 mM Tris-HCl buffer solution containing 5 mM CaCl ₂ , pH 7.0) was added to the mackerel, and homogenized twice at 9,500 × g for 2 minutes with a homogenizer. . After activating this in a 40 ° C. constant temperature water bath for 3 hours, the same amount of acetone was added to the supernatant obtained by centrifugation (9,500 × g, 20 min) and left for 6 hours in a 2 ° C. cold room to obtain a precipitate. This was again centrifuged (2,370 × g, 10 min) and the precipitate was obtained again.

In order to remove the insoluble protein from the recovered protein, the same amount of distilled water was added and centrifuged (9,500 × g, 10 min), and the supernatant was lyophilized and stored at -20 ° C. : mackerel intestine crude enzyme).

Test Example 1 Determination of Coenzyme Activity from Mackerel Intestines

1. Determination of activity on natural substrates

The specific activity of the coenzyme against the natural substrate casein was measured according to the method of Anson (1938). That is, the substrate solution was prepared by adding 50 ml of 0.1 M sodium phosphate buffer (pH 7.0) to 2 g of Hemmarstein casein and heating it for 5 minutes in a constant temperature water bath at 100 ° C. to prepare 100 ml with the same buffer. . 0.5 ml of enzyme solution was added to the reaction solution (1.5 ml of buffer solution and 0.5 ml of substrate solution), followed by cultivation (40 ° C., 20 min), and then 2.5 ml of 5% TCA solution was added to stop the reaction and left at room temperature for 30 minutes. Then, centrifuged (1,500 x g, 10 min). Take 1 ml of this centrifuged supernatant, add 2.5 ml of 0.55M Na ₂ CO ₃ , add 0.5 ml of 3-fold diluted Folin-phenol reagent, and let stand for 30 minutes at room temperature. Then, at 660 nm Activity was measured by measuring absorbance. The activity of the enzyme was expressed as 1 unit (U) and 1 U (mg) of protein, which releases 1 μmole of tyrosine per minute of reaction time under a given reaction condition. . In the present invention, the intrinsic activity was calculated by the following equation.

In the above, 0.4128 is the slope obtained from the relation between the amount of free tyrosine and the absorbance (660 nm).

The intrinsic activity of the mackerel gut-derived coenzyme (MICE) on natural substrates casein and hemoglobin is shown in FIG. 2. The intrinsic activity of MICE was 0.65 U / mg protein for casein and 0.24 U / mg protein for hemoglobin.

This is not only true of casein and hemoglobin activity of coenzyme (TICE) extracted from tuna rye water reported by Kim et al. (1997) higher than 0.54, 0.197 U / mg protein, but also of casein degradation activity of coenzyme extracted from tuna rye water. 0.5 L of the commercial enzyme Neutrase, 0.6 L of Alcalase, α-chymotrypsin and pronase E, which are lower in activity than papain and similar to trypsin Reported.

The reason why TICE is lower than commercial enzymes is that TICE is a state of coenzyme that is only partially purified from tuna rye water, and therefore contains some fat that can inhibit the enzyme reaction. Marishita (1964) measured the activity of proteolytic enzymes in the organs of aquaculture, eel, rainbow trout, and sweetfish. It has been reported to be active. As described above, almost all alkaline protease is present in the viscera of fish.

2. Determination of activity on synthetic substrates

<Determination of Synthetic Substrate Activity against Trypsin>

Reactivity to the nitroanilide substrate was expressed as reactive activity against BAPNA, and was measured by the method of Erlanger et al. (1966). That is, the amount of p-nitroanilide released when the reaction mixture was mixed with 0.95 ml of 0.5 mM BAPNA made from 100 mM Tris-HCl (pH 8.0) solution and 50 μl of enzyme solution for 5 minutes at 30 ° C. was measured using a spectrophotometer. Wavelength: 410 nm). In this case, 1 μmole of p-nitroanilide released per reaction time (min) at 30 ° C. was expressed as 1 unit of enzyme activity.

In the above, 8800 is the extinction coefficient of p -nitroanilide.

Degradation activity for the ester substrate was measured for BAEE. 0.95 ml of 0.5 mM BAEE made of 100 mM Tris-HCl (pH 8.0) solution and 50 μl of enzyme solution were added to the reaction mixture, followed by reaction at 30 ° C. for 5 minutes, followed by free benzoyl-L-arginine. The amount of was measured at a wavelength of 253 nm to calculate its specific activity.

In the above, 1048 is the extinction coefficient of benzoyl-L-arginine.

<Determination of Activity of Synthetic Substrate on α-chymotrypsin>

Reactivity to the ester substrate was determined by the degradation activity for BTEE and ATEE. Activity against BTEE was measured according to Hummel (1959) method. In other words, 0.95 ml of 0.5 mM BTEE made with 100 mM Tris-HCl (pH 8.0) solution containing 25.6% methanol and 50 μl of enzyme solution were used as the reaction mixture and reacted for 5 minutes at 30 ° C. The amount of tyrosine (benzoyl-L-tyrosine) was measured at a wavelength of 256 nm to calculate its specific activity.

In the above, 964 is an extinction coefficient of benzoyl-L-tyrosine.

The activity against ATEE was measured under the same reaction conditions as the method for measuring activity of BTEE. The degradation activity against ATEE was measured at 238 nm by measuring acetyl-L-tyrosine released during the reaction. Was determined.

In the above, 2250 is an extinction coefficient of acetyl-L-tyrosine.

Activity of the synthetic substrate of MICE is shown in FIG. The highest activity (6.456 U / mg protein) against BAEE (benzol-L-arginine ethyl ester), trypsin, and BAPNA (benzoyl-DL-arginine-p-nitroanilide-) naphthylamide) activity was relatively low (0.124). The activity of MICE against BTEE (benzoyl-L-tyrosine ethyl ester) and ATEE (acetyl-L-tyrosine ethyl ester), the synthetic substrates of α-chymotrypsin, was 5.660 and 0.856 U / mg.

Pyeun et al. (1993) reported that BAPNA activity of coenzyme extracted from anchovy viscera and yellowfin rapeseed water was 0.05 and 0.02 U / mg protein, respectively, and Uchida et al. (1973) showed trypsin, chymotrypsin and A precursor of carboxypeptidase was reported, Kolodzeiskaya et al. (1988) reported on trypsin distributed in the pyloric waters of the Far Eastern salmon, and Genicot et al. (1987) reported on trypsin in the Antarctic pyloric. Therefore, it can be seen that the coenzyme of the mackerel gut contains a large amount of proteolytic enzymes that exhibit specific activity on the ester synthesis substrate of trypsin and α-chymotrypsin.

Test Example 2 Examination of Optimal Hydrolysis Conditions for Pollack Frame Protein

General component analysis of pollock frame

General components were measured according to the conventional method. Moisture was measured by atmospheric pressure drying, crude fat by Soxhlet method, crude protein by semi-micro Kjeldahl method, and crude ash by dry painting method.

Pollack processing residue used as a raw material was filled with the remaining residue after filleting from Daelim Fisheries Processing Plant (water: 78.13%, bone: 8.51%, meat: 13.36%) at -20 ℃ It was stored in a frozen place and used in an experiment. Table 1 shows the results of the general component analysis of the entire meat portion of pollack pollen.

Ingredient Content (% by weight) moisture 81.52 (-) protein 13.55 (73.32) Geology  3.29 (17.80) Ash  1.64 (8.88)

      (), Percent by dry weight

As can be seen in Table 1, the components of pollack frame was 81.52%, 13.55%, 3.29% and 1.64% of moisture, protein, lipid and ash content, respectively.

2. Hydrolysis degree measurement

The degree of hydrolysis (DH) of pollack frame using Mackerel gut-derived coenzyme (MICE) was obtained by dispersing 1 g of each frame in 100 ml of buffer solution to make the substrate-to-enzyme ratio 100: 1. After reacting at 40 ° C. for 1 hour. When the reaction is completed, take 2 ml from the reaction mixture, add 20% TCA solution to the same amount, centrifuge (2,370 × g, 5 min), take a certain amount of supernatant, and measure 10% TCA soluble nitrogen by Lowry method. After the hydrolysis degree was calculated as follows.

3. pH effect

To examine the pH conditions for hydrolyzing the processed pollock residue protein, 0.1 M glycine-HCl buffer (pH 3.0 to 4.0), 0.1 M sodium acetate-acetic acid buffer (pH 5.0 to 6.0), 0.1 M disodium hydro PH using genphosphate-sodium dihydrogen phosphate buffer (pH 7.0), 0.1 M Tris-HCl buffer (pH 8.0-9.0) and 0.1 M sodium carbonate-sodium bicarbonate buffer (pH 10.0-12.0), respectively The effects were examined and the results are shown in FIG. 4.

The hydrolysis rate of pollack processed residue was high at pH 10.0 and 11.0, especially about 40% at pH 10.0. The mackerel gut coenzyme used showed strong activity of alkaline protease. Able to know. Murakami and Noda (1981) reported that the alkaline proteolytic enzymes extracted from sarcoid pyloric water were at pH 10.0, and Kim et al. (1986) showed that the optimal pH of protease distributed in the gut tissue of mackerel and sardines was 9.0 and 9.8 and 9.4 and 10.0 for pyloric origin.

However, according to the optimum pH of alkaline proteinase extracted from fish muscle, both carp and shrimp showed maximum activity at pH 8.0. These results indicate that the optimum pH of the protease extracted from fish muscle is usually around 8.0, but the protease extracted from the pyloric or pancreas is most active near 10.0.

4. Effect on temperature

The optimum temperature conditions were examined while changing the temperature from 30 to 60 ° C. under the optimum pH condition for the hydrolysis degree of pollack pollen frame, and the results are shown in FIG. 5. As shown in FIG. 5, the maximum activity was shown at 50 ° C., and thereafter, the peak activity was rapidly decreased.

Doke and Ninjoor (1987) reported that alkaline proteolytic enzymes derived from shrimp muscle showed the maximum activity at 60 ° C. Iwata et al. (1974) reported that alkaline proteins derived from muscles of 21 marine fish and 4 freshwater fishes. Hydrolytic enzymes have generally been reported to exhibit maximum activity at 60-65 ° C. Pyeun and Kim (1986) reported that the coenzyme extracted from mackerel pyloric water showed the maximum activity at 45 ° C but rapidly decreased after 50 ° C and almost inactivated at 70 ° C.

However, the temperature effects of the purified proteolytic enzyme (Ooshiro, 1971) derived from mackerel pylori and purified trypsin (Simpson et al., 1990) derived from cod pyloric rally showed maximum activity near 40 ° C and then 50 ° C. Reported reduced activity by 75% and 60%, respectively. This fact suggests that hydrolyzed proteolytic enzymes are more effective at higher temperatures than when they are purified. Thus, comparing the activity of alkaline proteolytic enzymes derived from viscera and muscles, it was found that the viscera showed the maximum activity at about 40-50 ° C., which is usually lower than the muscles of 60-65 ° C.

5. Effect on substrate concentration

The optimum substrate concentration effect on hydrolysis was examined while varying the substrate concentration from 0.5 to 3% under the optimum pH and temperature conditions for the hydrolysis degree of pollack pollen frame, and the results are shown in FIG. 6.

As shown in FIG. 6, the highest efficiency was found at the substrate concentration of 1%, and the hydrolysis rate was about 42%. In general, the substrate concentration in the protein hydrolysis reaction was about 1-5%. In this experiment, the optimum effect on the substrate concentration was 1%, and at low substrate concentration, hydrolysis did not occur efficiently. The reason for this result is that the processed pollock residue protein is insoluble in water.

6. Effect on substrate-to-enzyme ratio

The degree of hydrolysis was examined while varying the substrate-to-enzyme ratio under optimum pH, temperature, and substrate concentration conditions for the degree of hydrolysis of pollack pollen, and the results are shown in FIG. 7.

In the effect of substrate-to-enzyme ratio, hydrolysis proceeded slowly until substrate-to-enzyme ratio was 500: 1, and then hydrolysis proceeded rapidly. Therefore, considering the productivity of the hydrolyzate according to the amount of enzyme addition, it is judged that 50: 1 is an optimal substrate to enzyme ratio.

7. Effect on reaction time

The coenzyme derived from the mackerel gut was analyzed for the degree of hydrolysis according to the change of time (1, 2, 4, 8, 12, 24, and 48) with respect to the pollock processed residue protein. The coenzyme derived from the mackerel gut showed a hydrolysis rate of about 67% due to the increase in the reaction time of processed pollock residue protein up to 12 hours, and the increase rate was decreased up to 24 hours thereafter, but continued to increase up to about 70%. Reached.

Hale (1969) compared the relative activity of commercially available enzymes in the hydrolysis of fish proteins and reported that the hydrolysis rate after 24 h hydrolysis was about three times higher than that of papain. In addition, Kim et al. (1997) showed that hydrolysis rates of pro-enzyme E and α-chymotrypsin were 10% and 5% lower than papain, respectively. Reported high. On the other hand, when the casein was used as a substrate, the degree of hydrolysis with the time change rapidly increased to about 75% until 8 hours, but gradually increased until 24 hours, indicating a hydrolysis efficiency of about 81%.

Ooshiro (1971) compared the degree of hydrolysis of casein hydrolyzed proteinase from mackerel rye water with casein-derived trypsin for 4 hours. The degree of hydrolysis was reported, but bovine trypsin reported only about 15% hydrolysis due to substrate specificity. Ramakrishna et al. (1987) also compared the activity of fish chymotrypsin with bovine chymotrypsin over time, using a collagen substrate for 8 hours at 35 ° C and using an insoluble protein portion of fish as a substrate. When the reaction was performed at 37 ° C. for 5 hours, the fish chymotrypsin was reported to have about 2 times higher activity. These results suggest that it is more efficient to use enzymes derived from fish to hydrolyze fish proteins.

8. Fraction by Molecular Weight of Pollack Frame Protein Hydrolysates

After thawing the pollock frame, the meat portion was separated and hydrolyzed to 100 g (dry weight) of the hydrolysis under optimum conditions of hydrolysis. After hydrolysis, the enzyme was inactivated at 100 ° C., and the extract was centrifuged (4,650 × g) to obtain a molecular weight cut-off (MWCO) range of 10 using an ultrafiltration membrane system. , 5, 3, 1 kDa through the membrane in order to each fraction and lyophilized and used as a sample of the bioactive material.

Example 2 Isolation and Purification of Antihypertensive Active Peptides

In order to determine the characteristics of the hydrolysates fractionated by molecular weight, which showed antioxidant and antihypertensive properties, screening and purification of bioactive substances were performed. In other words, by measuring the physiological activity at each purification step using ion exchange chromatography, gel filtration chromatography, and HPLC to separate and purify the functional material from the most active fraction, the biochemical characteristics, structure and function of the physiological functional material It was used as a sample to grasp.

Table 2 shows the results of measuring the antihypertensive activity of hydrolysates obtained by hydrolysis of pollock frame protein by MICE. As shown in Table 2, the inhibitory activity in APH V (1 kDa or less) was the highest antihypertensive activity (87.62%), and the antihypertensive substance was separated and purified using this.

Hydrolyzate ACE inhibitory activity (%) APH V (MW 1 kDa or less) APH IV (MW 1-3 kDa) APH IV (MW 3-5 kDa) APH IV (MW 5-10 kDa) APH IV (MW 10-30 kDa) 87.62 78.95 73.84 68.24 59.60

1. Isolation by ion chromatography

A hydrolysis sample was injected into a previously equilibrated SP-Sephadex C-25 column (φ2.5 × 45 cm), and the nonadsorbed portion was eluted with 250 ml of the same buffer solution, and the adsorbed portion was a buffer solution and 1 M NaCl. The solution was fractionated and eluted by linear concentration gradient method. Protein content was measured by Lowry method and the protein content of each fraction was diluted to the same, and the antioxidant and antihypertensive activity of each fraction was measured and compared in the same manner as described above. The fraction with the highest activity among the eluted fractions was continuously purified.

Among the fractions passed through the membrane, the hydrolyzate (APH V) that passed 1 kDa having the best antihypertensive activity was separated by ion chromatography. In other words, 5 ml of a 1 mg / ml hydrolysis solution was injected into a column (φ 1.0 × 25 cm) filled with SP-Sephadex C-25 resin equilibrated with 20 mM sodium acetate buffer (pH 4.0). After eluting the nonadsorbed portion with 500 ml of the same buffer solution, the adsorbed portion was fractionated by linear concentration gradient using 300 ml of buffer and 300 ml of 2 M NaCl solution (flow rate, 2 ml / min; collection volume, 5 ml) and separated into five fractions. After desalting these fractions and lyophilizing, the antihypertensive activity was measured. As a result, the IC ₅₀ value was 0.11 mg / ml in the IV fraction (Fig. 9, Table 3).

Fraction IC ₅₀ value (mg / ml) I 0.98 II 0.76 III 0.32 IV 0.11 V 0.45

2. Gel Filtration Chromatography

Among the fractions separated from the column packed with SP-Sephadex C-25, each highly active fraction was injected into a Sephadex G-25 column (φ2.5 × 98 cm) equilibrated with a buffer solution and eluted with deionized water. Each fraction was measured for absorbance at 280 nm and 220 nm, and then lyophilized and then squared to determine the antioxidant and antihypertensive activity of each fraction. The fraction with the highest activity among the eluted fractions was continuously purified.

Among the fractions separated from the cation exchange resin SP-Sephadex C-25, the fraction showing the highest antihypertensive activity was eluted in a column (φ2.5 × 98 cm) filled with Sephadex G-25 gel resin. Got it. After lyophilization, the ACE inhibitory activity was measured. As a result, the IC ₅₀ value of Fraction D was 0.035 mg / ml (FIG. 10, Table 4). Therefore, fraction D was continuously collected by gel chromatography, concentrated in a vacuum rotary concentrator, and lyophilized, and re-separation was performed using HPLC to separate the sample into a single substance most effective for ACE inhibitory activity.

Fraction IC ₅₀ value (mg / ml) A 0.134 B 0.066 C 0.078 D 0.035

3. Final separation and purification of physiologically active substances using HPLC

The lyophilisate of the fraction having the highest antioxidant and antihypertensive activity among the fractions separated by gel chromatography was dissolved in 0.1% trifluoroacetic acid (TFA) solution and injected into HPLC. ODS reverse phase column (Shodex Co.) was used in HPLC (Dionex Co.), and the mobile phase consisted of 0.1% TFA / H ₂ O and 0.1% TFA / acetonitrile in a linear phase gradient (0-50% acetonitrile, 40 min), and then each fraction was separated. Antioxidant and antihypertensive activity was measured on the separated fractions, and the following assays were performed on the fractions with the highest activity among the eluted fractions.

The lyophilisate of the fraction with the highest antihypertensive activity among the fractions separated from the column packed with Sephadex G-25 was dissolved in deionized water containing 0.1% TFA and separated again by HPLC. In other words, eluted with linear phase gradient using deionized water containing 0.1% TFA and acetonitrile (0 to 70%, 50min) as a mobile phase using HPLC equipped with a reverse phase column. As a result of measuring their antihypertensive activity, the second fraction had the highest activity as 0.023 mg / ml (FIG. 11, Table 5). This was again separated by HPLC adjusting the concentration of deionized water and acetonitrile to 85:15 to obtain a single peak (Fig. 12, Table 6).

Fraction IC ₅₀ value (mg / ml) a 0.424 b 0.023 c 0.058 d 0.042 e 0.078 f 0.194

Fraction IC ₅₀ value (mg / ml) aI 0.145 bI 0.013

4. Amino Acid Sequencing of Bioactive Peptides

The amino acid sequence was digested by Edman's method of cleaving amino acids from the N-terminus, and analyzed by gas phase auto sequencer (Perkin Elmer, New Jersey, U.S.A.). That is, the amino acid that inhibited dissociation under basic conditions was attached to phenylisothiocyanate (PICT) to remove excess reagents and reaction derivatives with phenylthiocarbamyl (PTC) peptide, and then cleaved under acidic conditions to anilinothiozoline (anilinothiozoline). , ATZ) derivatives and the more stable derivatives, phenylthiohydantoin (PTH) amino acid was identified by transformation. The sequence was determined by repeating the above procedure for the N-terminus of the remaining peptide.

Lineweaver-Burk plots were used to determine the inhibition mechanism of the final purified peptides. That is, 13, 26 ㎍ / ml of the inhibitory peptide was added and the inhibition rate according to the change in the concentration of the substrate was measured, showing a non-competitive inhibition mechanism (Fig. 13). Ondetti and Cushman (1982) reported that three peptides at the C-terminus have a strong effect on ACE inhibitory activity, because the C-terminal amino acid reacts with the active site of ACE, Cheung et al. (1980) It showed that ACE inhibitory activity was excellent when tryptophan, tyrosine, phenylalanine, and proline were present at the C-terminus, and dipeptide was an aliphatic amino acid at the N-terminus, which effectively binds to the active site of ACE. Reported. Nakamura et al. (1995) found that proline residues in proteins affect ACE inhibitory activity most, while Arg at the C-terminus affects the inhibitory activity of guanidine inversion.

After the final purified material was separated by HPLC, the amino acid sequence was analyzed by gas-phase auto sequencer using Edman digestion method. The purified peptide was 8 amino acids of Phe-Gly-Ala-Ser-Thr-Arg-Gly-Ala. The final ACE inhibitory activity was 1.47 μM.

In general, the report of antihypertensive activity, Kim et al. (2001) separated and purified two peptides of the ACE inhibitory peptide of the woo gelatin hydrolysate, the sequence of which is Gly-Pro-Val and Gly-Pro-Leu The activity was 4.67 μM and 2.55 μM, respectively. Byun et al. (2001) isolated two ACE inhibitory peptides from pollock lysate, identified as Gly-Pro-Met (17.13 μM) and Gly-Pro-Leu (2.55 μM), respectively. Wako et al. (1996) determined the amino acid sequence by separating and purifying peptides with high ACE inhibitory activity from extracts obtained by autolysis of squid liver and torso. Try-Ala-Leu-Pro-His-Ala (IC ₅₀ = 9.8 μM) And Gly-Tyr-Ala-Leu-Pro-His-Ala (IC ₅₀ = 27.3 μM), Maruyama et al. (1985) reported that the amino acid sequence of the peptide with high ACE inhibitory activity from the enzymatic hydrolysate of casein was Ala-Val- Reported as Pro-Tyr-Pro-Gln-Arg (IC ₅₀ = 15.0 μM) and Phe-Phe-Val-Ala-Pro (IC ₅₀ = 6.0 μM). Yokoyama et al. (1992) also found that the sequences of peptides with high ACE inhibitory activity from the enzymatic hydrolysates of bonito are ILe-Lys-Pro-Leu-Asn-Tyr (IC ₅₀ = 43.0 μM) and Asp-Tyr-Gly-Leu -Tyr-Pro (IC ₅₀ = 62.0 μM), but the ACE inhibitory activity of the partially truncated forms of Ile-Lys-Pro, Leu-Asn-Tyr, Asp-Tyr-Gly and Leu-Tyr-Pro, respectively It was reported to be 1.7 μM, 81 μM, 2700 μM and 6.6 μM. Meanwhile, Kawakami et al. (1995) synthesized a peptide similar to Leu-Lys-Tyr (IC ₅₀ = 9.78 μM) and measured the ACE inhibitory activity. As a result, Leu-Lys-Pro (IC ₅₀ = 2.8 μM) and Val- It has been reported that the activity of Lys-Pro (IC ₅₀ = 2.6 μM) is excellent.

In view of the above results, the structure of the peptide showing ACE inhibitory activity has been thought to be deeply related to Pro-Pro and Ala-Pro, which are the C-terminal portions of the snake venom-derived peptides known as potent ACE inhibitors in recent years. Other combinations of amino acids such as zein (Leu-Arg-Pro, IC ₅₀ = 0.27 μM) have been reported to show strong ACE inhibitory activity, as well as the length of the peptide may be an important factor in the inhibitory activity. Judging. Therefore, ACE inhibitory activity is thought to be somewhat affected by the composition of the constituent amino acids, but is more likely to be affected by the type of the constituent peptide and the amino acid sequence. Compared with the above results, the ACE inhibitory peptide found in this study was composed of Arg-Gly-Ala similar to the three amino acid sequences at the C-terminus, and the activity was 1.47 μM.

Test Example 3 Antihypertensive Activity Measurement

1. Determination of antihypertensive activity through ACE inhibitory activity

ACE inhibitory activity was measured according to the method of Cushman and Cheung (1971). 50 μl of 25 mU / ml ACE solution was added to 50 μl of the hydrolyzate dissolved in 0.1 M sodium borate buffer (pH 8.3, containing 300 mM NaCl) and then incubated at 37 ° C. for 30 minutes. Treated. 150 μl of 4.15 mM Hip-His-Leu (107.4 mg / 10 ml sodium borate buffer) was added thereto and reacted at 37 ° C. for 60 minutes. Then, 0.25 ml of 1 N HCl solution was added to stop the reaction. 0.5 ml of ethyl acetate was added to the reaction solution, followed by stirring. The mixture was centrifuged (2,500 × g, 5 min), and 200 μl of the supernatant was aliquoted. The supernatant was completely dried and then dissolved by adding 1 ml of distilled water, and the absorbance was measured at 228 nm. The concentration of ACE inhibitor was defined as IC ₅₀ (mg / ml) by calculating the concentration of hydrolyzate required to inhibit the inhibitory activity of ACE by 50%.

2. In vivo Of blood pressure lowering activity

Angiotensin I was orally administered to 10 mg / kg (SHR body weight) of an ACE inhibitory peptide in spontaneously hypertension rats (SHR), 180-220 kg, females, 6 to 8 weeks of age. The inhibitory effect of the test solution on elevated pressure was examined. The test solution dissolved in physiological saline was 0.45 ml (including physiological saline used for washing after administration). Blood pressure was measured by non-invasive blood pressure measurement device over time. Captopril, a commercially available blood pressure lowering agent, was used as a control. At this time, the comparison was made based on the peak vessel pressure, that is, SBP.

3. Measurement of Inhibition Pattern for ACE

Inhibition patterns for ACE were measured according to the method of Bush et al. (1984). That is, various concentrations of peptide were added to the reaction solution, and ACE inhibitory activity was measured by varying the substrate concentration. After adding the peptide, the reaction rate of ACE was determined by using the Lineweaver-Burk plot.

<Determination of Blood Pressure Drop Activity in Vivo of ACE Inhibitory Peptides>

Angiotensin I was administered orally with 10 mg / kg (SHR body weight) of an ACE inhibitory peptide in spontaneously hypertension rats (SHR), 180-220 g, female, 6-8 weeks of age. As a result of examining the inhibitory effect on the pressure, it was slightly lower than the effect of the commercially available blood pressure reducing agent captopril, but showed the highest blood pressure drop of 23 mmHg at 3 hours after administration (FIG. 14). Recently, many ACE inhibitory peptides have been isolated from proteins and their antihypertensive effects using SHR have been investigated. Fujita et al. (1999) suggest that Leu-Lys-Pro-Asn-Met obtained from fish protein hydrolysates is a prodrug type ACE inhibitory peptide. This means that Leu-Lys-Pro-Asn-Met is hydrolyzed by ACE to Leu-Lys-Pro. Leu-Lys-Pro is an 8-fold higher ACE inhibition than Leu-Lys-Pro-Asn-Met. The effect was reported.

After oral administration to SHR, Leu-Lys-Pro-Asn-Met had the highest blood pressure drop at 6 hours, while Leu-Lys-Pro showed the maximum blood pressure drop at 4 hours. Some antihypertensive drugs have been reported to have side effects such as abnormal blood pressure rise or cough after oral administration, but the peptides used in this experiment are expected to be used because they do not show any side effects after oral administration.

Example 3 Isolation and Purification of Antioxidant Peptides

Among the hydrolyzates separated in each stage of the membrane reactor, only single peptides showing the strongest antioxidant power in the high antioxidant power were separated by ion exchange chromatography, gel chromatography and HPLC.

1. Ion Chromatography

First, we measured the antioxidant activity of each fraction fractionated by eluting 5 ml of the sample solution on a column (φ4 × 40 cm) filled with SP-Sephadex C-25 resin equilibrated with 20 mM sodium acetate buffer solution (pH 4.0). It is shown in 15. Elution of 1 L of 20 mM sodium acetate buffer (pH 4.0) in a column filled with cation exchange resin and separation by linear concentration gradient using 300 ml of the same buffer as 300 ml of 1.0 M NaCl solution (flow rate: 60 ml) / hr, collection volume: 5 ml), separated into 6 fractions. After desalting and lyophilizing each fraction, the antioxidant properties were measured by Ferric thiocyanate method, and the most excellent antioxidant effect was obtained in fraction IV (73 ~ 82).

2. Gel Chromatography

In the fraction of the cationic chromatography, the freeze-dried product of fraction IV containing the peptide having excellent antioxidant property was dissolved in distilled water, and then packed with Sephadex G-25 resin having a separation range of 1,000 to 5,000 Da (φ2.5 × 90cm). ) And equilibrated with distilled water to obtain the four main fractions using the GradiFrac System (Pharmacia Biotech., Sweden), the antioxidant properties of the fractions showed the highest antioxidant activity in the second fraction (Fig. 16) . It was lyophilized and re-separated by HPLC.

3. Separation by HPLC

Using HPLC equipped with an ODS column, eluting (2 ml / min) with H ₂ O and acetonitrile solution in a linear gradient (0 to 35%, 40 min), as shown in FIG. The three main fractions were fractionated, and their antioxidant properties were measured. The first fraction showed the best antioxidant activity, and the fractions were purified by H ₂ O and acetonitrile using a HPLC equipped with a Capcell Pak C ₁₈ -VG column. As a result of separation and purification by linear phase gradient method (0-15%, 30 min), it was separated into three main fractions but showed the best antioxidant activity in the P3 fraction (FIG. 18).

4. Amino Acid Composition and Structure Analysis of Antioxidant Peptides

Amino acid sequencing of the antioxidant peptides included Val at the N-terminus and included Gly, Thr, Glu, Pro, His and Asn residues (SEQ ID NO: 2). Murase et al. (1993) reported that histidine-containing peptides have high antioxidant activity due to metal chelating and lipid radical trapping of histidine imidazole rings. Peptides, including histidine, also showed better antioxidant activity than histidine itself, because histidine increases the hydrophobicity of the peptide. Saito et al. (2003) synthesized three tripeptides containing histidine and tyrosine (Tyr-His-Tyr, Try-Lys-Tyr and Try-Arg-Tyr) to examine the antioxidant activity. Showed the highest antioxidant activity.

Yamaguchi et al. (1975) reported differences in antioxidant capacity according to the sequence of peptides. For dipeptides, the antioxidant powers of Ala-His, Ala-Met, Ala-Tyr and Ala-Try were good when alanine was N-terminally. This is because the antioxidant power is greatly influenced by the specific amino acid located at the N- or C-terminus.

Chen et al. (1995) found that among the hydrolysates hydrolyzed by β-conglycinin, a soy protein, antioxidant peptides contain hydrophobic amino acids valine or leucine at the N-terminus, and have proline, histidine and tyrosine in the array. It has been reported that it consists of 5 to 16 amino acid residues.

In this study, His residue was included in the purified amino acid sequence, which showed better antioxidant activity than α-tocopherol.

Test Example 4 Antioxidant Activity of Hydrolyzate

Different enzymes have different sites to break down protein substrates, and thus, the peptide has different functional properties. Therefore, as a result of examining various physiological activities such as antioxidant, antihypertensive, and anticancer, the activity of anti-pollution and antihypertensive were examined for the processed hydrolyzate hydrolyzed from the optimum condition of enzyme.

1. Ferric thiocyanate law

Antioxidant activity of the protein hydrolyzate was measured according to the method of Mitsuda et al. (1966). The linoleic acid emulsion was mixed with 0.13 ml of linoleic acid, 10 ml of ethanol, and 10 ml of 50 mM phosphate buffer (pH 7.0) according to the method of Osawa (1981), and the hydrolyzate was added to the mixture with 1% of linoleic acid. added at a concentration of w / v) to promote automatic oxidation while stored in a thermostat controlled at 40 ± 1 ° C. After adding 4.7 ml of 75% ethanol to 0.1 ml of linoleic acid, 0.1 ml of 30% ammonium thiocyanate was mixed.

Next, 0.1 ml of 20 mM FeCl ₂ /3.5% HCl solution was added thereto, followed by color development at room temperature for 3 minutes, and the activity was determined by measuring absorbance (wavelength: 500 nm) with a spectrophotometer.

From the above results, hydrolysis was performed under optimal conditions of hydrolysis in order to efficiently recover pollen frame protein by MICE. That is, the substrate concentration was 1%, the substrate-to-enzyme ratio 50: 1, pH 10, temperature 50 ° C., and hydrolysis for 12 hours. After hydrolysis, the hydrolysates were fractionated by the molecular weight size through the ultrafiltration membrane, and the final five fractions were obtained using membranes having a molecular weight limit of 10, 5, 3 and 1 kDa (APH I: 10 kDa or more). , APH II: 10-5 kDa, APH III: 5-3 kDa, APH IV: 3-1 kDa and APH V: 1 kDa or less (peptide of SEQ ID NO: 2)). As a result of examining the antioxidant activity of the hydrolyzate obtained by Ferric thiocyanate method, it showed the highest antioxidant activity in APH V (peptide of SEQ ID NO: 2), which was 10.37% higher than the commercially available natural antioxidant α-tocopherol. (FIG. 19).

2. Measurement of Radial Scavenging Activity (RSA)

RSA was measured according to the method of Hatano et al. (1988). Dissolve 50 mg of each hydrolyzate in 4 ml of distilled water, mix it with 1 ml of 1.5 × 10 ^-4 M DPPH / MeOH, leave for 30 minutes at room temperature, and measure the absorbance at 517 nm to determine the amount of DPPH remaining. The results are shown in FIG. 20. Commercially available natural antioxidant α-tocopherol and synthetic antioxidant BHT were used as controls.

As a result, all the hydrolyzates showed scavenging activity in DPPH, and especially, APH V (peptide of SEQ ID NO: 2) had the highest DPPH scavenging activity but lower than commercially available natural antioxidant α-tocopherol and synthetic antioxidant BHT (FIG. 20). .

3. Measurement of antioxidant power according to concentration

Antioxidant activity according to hydrolyzate concentration was determined by Ferric thiocyanate method and RSA value. That is, the hydrolyzate was added to 0.1-2% of the holding weight to measure the activity of each.

Antioxidant activity of APH V (SEQ ID NO: 2 peptide) with the highest antioxidant capacity was measured by Ferric thiocyanate method and radical scavenging activity (RSA). Ferric thiocyanate was more effective than α-tocopherol when the concentration of APH V (peptide of SEQ ID NO: 2) was greater than 1%, and lower than that of α-tocopherol at 0.1 and 0.5%, but was slightly higher (FIG. 21).

For RSA, the concentration-dependent radical scavenging effect of APH V (peptide of SEQ ID NO: 2) on DPPH gradually increased up to 1% and was nearly constant above that (FIG. 22). These results indicate that, when the concentration of the peptide is low, the degree of oxidation of the peptide itself does not significantly affect the antioxidant activity of the peptide. However, when one or more of the peptides are added, the degree of oxidation of the peptide affects the antioxidant effect. It is thought that this is because it acts as a urea. In this study, the optimal concentration for the antioxidant activity of the hydrolyzate was found to be 1%.

Ji et al. (1992) reported that the spice extracts showed the highest antioxidant activity in edible soybean oil at the concentration of 1% of fat or oil, and Saiga et al. (2003) reported that the pork muscle fiber protein was derived from papain and liquid. When the hydrolyzate hydrolyzed with actinase E was 2%, the antioxidant activity was higher than that of vitamin E, and papain hydrolyzate was somewhat higher. Park et al. (2001) reported that the concentrations of hydrolyzate extracted from egg yolk showed antioxidant effects without significant difference between 1% and 2%. These reports show that the source of each hydrolyzate is different, but the optimum concentration is at similar concentrations.

4. Measure synergy

The synergistic effect of the hydrolyzate was determined by Ferric thiocyanate method. That is, the ferric thiocyanate value was measured while adding α-tocopherol to 0.1% of linoleic acid and adding hydrolyzate to 1% of the holding weight to the oil-and-water suspension in a thermostat controlled at 40 ± 1 ° C. As a control, compared with α-tocopherol without hydrolyzate.

The synergistic effect of the hydrolyzate was determined by Ferric thiocyanate method. That is, the ferric thiocyanate value was measured while adding α-tocopherol to 0.1% of linoleic acid and adding hydrolyzate to 1% of the holding weight to the oil-and-water suspension in a thermostat controlled at 40 ± 1 ° C. Compared with α-tocopherol without hydrolyzate as a control, it showed only slightly lower antioxidant activity than α-tocopherol in the group with APH I, and in all other groups, especially APH V ( In the group to which the peptide of SEQ ID NO: 2) was added, it showed the highest antioxidant capacity (FIG. 23). From the above results, it can be seen that when the pollock frame protein hydrolyzate is used in combination with a commercially available antioxidant α-tocopherol, it shows a very high synergistic effect.

Yamaguchi et al. (1979) reported that soy protein hydrolysates enhance the antioxidant effects of α-tocopherol. Saito et al. (2003) reported that Tyr-His-Tyr, Tyr-Lys-Tyr and Tyr-Arg-Tyr Peptides having a sequence were synthesized and their antioxidant activity was measured. Tyr-His-Tyr showed the best activity, and in the presence of BHA and δ-tocopherol, the synergistic effect was more than 30 times higher than that of the control, but citric acid and No synergistic effect was reported in the presence of α-tocopherol. However, Tyr-Lys-Tyr and Tyr-Arg-Tyr did not show a synergistic effect with any compound. Yamaguchi (1989) examined the synergistic effects of hydrolysates such as soy protein, milk casein, gelatin, albumin and α-tocopherol, and all of the hydrolysates had a relatively synergistic effect.Kawashima et al. (1979) found that commercially available gelatin It was reported that the hydrolyzate showed a very excellent synergistic effect by the combined use of vitamin C and citric acid.

Saiga et al. (2003) reported that the antioxidative activity against pH of hydrolysates hydrolyzed pork muscle fiber with papain and actinase E showed the highest antioxidant activity at pH 7.1. However, Krogull et al. (1987) Proteins, peptides and amino acids oxidize on their own and have reported differences in antioxidant activity depending on pH, temperature, water activity, and the presence or absence of an antioxidant or inhibitor. In other words, the antioxidant activity of peptides is highly dependent on their structure and measurement system. Chen et al. (1996) found that the antioxidant activity of Leu-Leu-Pro-His-His isolated from soy protein hydrolysates was determined by Pro-His-. His was called, but the activity of Pro-His-His was slightly decreased when the measurement conditions were changed. In addition, as a result of synthesizing and studying various peptides based on these sequences, it was reported that histidine-containing peptides have high activity because histidine acts as a metal ion chelate and an active oxygen scavenger. Hatate et al. (1997) also found that the Asp-Thr-His-Lys peptide isolated from the enzymatic hydrolysates of bovine serum albumin enhances the antioxidant activity of α-tocopherol and sodium ascobate. Although the exact mechanism for the antioxidant activity of the peptide is not clear, it is known that histidine residues play an important role in activity.

The antioxidant power of the hydrolyzate is influenced by each amino acid, that is, high antioxidant amino acids that act as radical scavengers and inhibit the formation of peroxides, but the peptides affect the antioxidant properties depending on the composition and position of the constituent amino acids. I think. In addition, the synergistic effect of protein hydrolysates and phenolic antioxidants is believed to result from reduction and regeneration of oxidized antioxidants such as amino acids.

5. Antioxidant Effect in Cell Culture System

<Cell Culture>

Normal liver cells (Chang) were cultured in DMEM medium containing 10% fetal calf serum (FBS). In a 75-cm2 plastic flask (Falcon Co., England), Chang cells were placed in DMEM medium containing 10% FBS, 7.5% NaHCO ₃ 150 μg / ml, glutamine 58.4 μg / ml and antibiotic / antifungal 4.4 μl / ml. The cells were incubated at 37 ° C. under 5% CO ₂ . Cell lines were maintained by secondary culture once every 2-3 days.

<Cell Quantification>

Remove medium from cells grown in 75-cm ² plastic flasks, wash with CMF-PBS (calcium magnesium free-phosphate buffered saline, pH 7.2), remove cells from flask bottom with 0.25% trypsin / EDTA. After centrifugation (80 × g, 3min) by neutralization with cell culture medium. Culture medium is added to the pellet of the remaining cells, followed by repeated suction with a sterile pipette to form a single cell suspension, and then trypan blue is mixed with the cell suspension in a ratio of 9: 1 and hemocytometer on an optical microscope It was measured using a hemocytometer.

<Determination of Cytotoxicity of Antioxidant Active Substances>

Toxicity of the hydrolyzate was determined by CCK-8 quantification. That is, normal cells (Chang cells) were dispensed to 5 × 10 ⁴ cells / well in a 96-hole plate. This was incubated in an incubator at 37 ° C., 5% CO ₂ , followed by incubation for 48 hours with the addition of 10 μl of hydrolyzate at various concentrations. Cytotoxicity of the hydrolyzate was determined by using the CCK-8 kit for 48 hours incubation time of Chang cells.

24 shows the results of measuring cytotoxicity in cultured normal hepatocytes of pollock frame-derived protein hydrolysates. As shown in FIG. 24, when the cell viability of the hydrolyzate was not added at 100%, the hydrolyzate added group was 100% or more. Particularly, APH V (peptide of SEQ ID NO: 2) (1 kDa or less) hydrolyzate was shown. When added, the survival rate was about 119% at a concentration of 5 mg / ml. Therefore, the hydrolyzates used in this experiment did not show any toxicity to cultured hepatocytes, and it was confirmed that they were stable.

<t Effects of Hydrolysates on Cell Necrosis at Different B-Concentrations>

In order to observe the effect of hydrolyzate on cell necrosis by concentration of t- BHP, first put the hepatocytes into 5 × 10 ⁴ cells / well in a 96-well plate and incubate for 2 hours in a CO ₂ incubator. After the concentration was added 5 mg / ml per well, the total volume was adjusted to 100 μl with 10% FBS-DMEM medium and incubated for 18 hours at 37 ℃, 5% CO ₂ conditions. The culture solution was washed with CMF-PBS, and SFM (serum free medium) and t- BHP were added in a total volume of 200 μl from 0 μM to 300 μM by concentration, followed by incubation for 120 minutes, followed by CCK-8. The absorbance was measured by the kit to determine the effect.

the viability of normal hepatocytes in accordance with the concentration of t -BHP After 2 hours, the t -BHP processing the measurement result, because in the 225 μM t -BHP treatment group showed a cell viability of about 50% 225 μM t -BHP the IC ₅₀ (Fig. 25). As a result of measuring the antioxidant activity of the hydrolyzate on the cell death by t- BHP concentration, the cell survival rate of the hydrolyzate added group was increased by 15-28% compared to the non-added group. To date, two mechanisms have been reported to explain the necrosis of hepatocytes caused by t- BHP, one of which is related to lipid peroxidation of cell membranes reported by Masaki et al. (1989), DPPD (N, N'-diphenyl). Antioxidants such as -p-phenylene diamine) can prevent cell necrosis caused by t -BHP, a lipid peroxidation inducer. The other mechanism of cell necrosis is lipid peroxidation without being affected by antioxidants. Is to lose the function of the mitochondria. Frankel (1984) also reported that lipid secondary products, such as MDA, produced by the oxidation of lipids are powerful crosslinkers that react with amino groups of enzymes, proteins, and DNA. Pascoe et al. (1987) reported that OH (hydroxyl radical) Active oxygen-based systems such as LOO · (lipid radical) have been reported to reduce the protective effect of cells by inducing the consumption of antioxidants associated with hydrophobic membranes such as α-tocopherol. Fariss et al. (1984) reported that reduction of reduced glutathione (GSH), the most abundant nonprotein thiol in hepatocyte cytoplasm, affects cell survival, and Masaki et al. (1989) reported that iron chelates against the cytotoxicity of t- BHP. Jane deferoxamine delays cell death by inhibiting GSH consumption, and antioxidants DPPD and catechol are produced by t -butyl alkoxy radicals regardless of glutathione metabolism or calcium intracellular homeostasis. It has been reported to delay cell death by eliminating. Benjamin et al. (1993) reported that a 6 kDa thymus peptide isolated from calf thymus reduces hepatic MDA production and prevents hepatic glutathione and is endogenous in vascular endotherial cells. Increasing antioxidants have been reported to delay cell death.

In this study, histidine removes lipid radicals from amino acids and inhibits the production of lipid peroxide, MDA.

<t Effects of Hydrolysates on Cell Necrosis of -BHP>

In order to observe the concentration-dependent effects of hydrolyzate on cell necrosis of t- BHP, firstly, put the hepatocytes into 5 × 10 ⁴ cells / well in a 96-plate plate and incubate for 2 hours in a CO ₂ incubator. After the concentration was added at 0.5 mg / ml per well at 7.5 mg / ml, the total volume was adjusted to 200 μl with 10% FBS-DMEM medium and incubated for 18 hours at 37 ° C. and 5% CO ₂ . Wash the culture with CMF-PBS, add SFM (serum free medium) and 225 μM t -BHP so that the total volume is 200 μl by concentration, incubate it for 120 minutes, and absorb the absorbance with CCK-8 kit. The effect was determined by measuring.

In order to observe the antioxidant effect according to the concentration of the hydrolyzate, the hydrolyzate was pretreated in the cultured hepatocytes, and the survival rate of the cells was measured by inducing the oxidation of the cells with t -BHP. When 2.0, 5.0 and 7.5 mg / ml were added, 1.0 mg / ml showed the highest cell viability of about 75% (FIG. 26). Pascoe et al. (1987) reported that adriamycin (ADR), an anticancer drug, causes myocardial infarction by consuming GSH to generate reactive oxygen species, which is associated with the generation of free radicals and consumption of GSH during ADR oxidation-reduction. As a result of measuring the effect of vitamin E on this, the content of intracellular α-tocopherol was increased and the cell viability was the highest at the concentration of 0.6-1.0 nmole / cells, which acted as a free radical scavenger. It was estimated. In addition, we measured the effects of α-tocopherol in hepatocytes treated with ethacrynic acid, which directly depleted ADR and GSH. Because of the close relationship with the decrease in α-tocopherol present, it was reported that decreasing the thiol group content weakens the cellular defense mechanism.

As described above, the peptide isolated from the pollock frame according to the present invention has antihypertensive effect by inhibiting ACE activity, which is one of the factors involved in blood pressure control, and histidine removes lipid radicals to remove lipid radicals. Inhibition of production was found to have antioxidant efficacy to increase the survival rate of cells.                     

<References>

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Claims (4)

delete delete Peptide extracted from pollock frame and having the amino acid sequence of SEQ ID NO: 2 and having antioxidant activity. Antioxidant composition containing the peptide according to claim 3 as an active ingredient.

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