KR100680066B1 - Isolation of antioxidative peptide from mussel digests hydrolyzed in gastrointestinal digestion system - Google Patents

Abstract

A method for isolating a mussel antioxidative peptide from mussel hydrolysate is provided to separate only peptides showing most strong anti-oxidative activity from mussel protein by two step hydrolysis process, centrifugal process, dialysis process, freeze-drying process and three-step purification. The method comprises the steps of: (a) reacting pepsin with a first substrate, which is prepared by mixing 20 grams of mussel with 100 ml of water and then adjusting a pH thereof to be 1.8-2.2, at a temperature of 35-40 deg.C for 1.5-2 hours, where the ratio of the first substrate to the pepsin is 250:1, so as to obtain a first hydrolysate; (b) reacting a mixture of trypsin and alpha-chymotrypsin with a second substrate, which is prepared by mixing the first hydrolysate, a bile salt mixture, 1M CaCl2, 0.25M bistris, and 0.1% porcine pancreatic lipase and then adjusting a pH to be 8.0, at a temperature of 35-40 deg.C for 2-3 hours, where the ratio of the second substrate to the mixture is 250:1, so as to obtain a second hydrolysate; (c) after centrifuging the second hydrolysate under 10,000Îg at a temperature of 4 deg.C for 15 minutes to recover supernatant therefrom, dialyzing it using an electrodialysis device having a membrane transmitting molecules with less than 100 Da and then freeze-drying it to prepare a mussel hydrolysate; and (d) purifying the mussel hydrolysate through ion exchange liquid chromatography(LC), gel filtration LC, and C18 reversed phase LC to isolate single peptide.

Description

 Isolation of antioxidative peptide from mussel digests hydrolyzed in gastrointestinal digestion system}

       1 is a flow chart of a method for separating mussel antioxidant peptides from the hydrolyzate using the gastro-intestinal digestion system of the present invention

     Figure 2 shows the results of electrophoresis on denaturing polyacrylamide gel (SDS-PAGE) of the mussel hydrolyzate of the present invention

       Figure 3 is a graph of lipid peroxidation inhibitory effect according to the purification of the mussel hydrolyzate of the present invention

     Figure 4a is the primary ion peak graph of the final purified mussel antioxidant peptide of amino acid sequencing using the electrospray ionization tandem mass spectrometer (ESI-QTOF tandem mass) of the present invention active fraction

     Figure 4b is an amino acid sequence identification graph through the secondary ionization and molecular ion analysis of amino acid translation analysis using the electrospray ionization tandem mass spectrometer (ESI-QTOF tandem mass) of the active fraction of the present invention

Figure 5 is a radical scavenging activity graph of the antioxidant peptide finally purified from the mussel gastrointestinal hydrolyzate of the present invention

      The present invention relates to a method for separating mussel antioxidant peptides from a hydrolyzate using a gastro-intestinal digestion system, and more specifically, by mixing mussel proteins and water to adjust the pH to the same digestive conditions of the stomach (胃) 1 For teasing, Pepsin is first hydrolyzed under the same conditions as the digestive conditions of the stomach; The hydrolyzate obtained through primary hydrolysis was mixed with bile salt mixture, 1M calcium chloride, 0.25M Bistris, and 0.1% porcine pancreatic lipase to adjust the pH of the secondary substrate, which was adjusted to trypsin ( trypsin) and alpha-chymotrypsin are secondary hydrolyzed to small molecule peptides in an environment similar to the digestive conditions of the small intestine; The hydrolyzate obtained through secondary hydrolysis was subjected to centrifugation and dialysis and then lyophilized to obtain a mussel hydrolyzate obtained by gastric-intestinal digestion system, followed by ion exchange liquid chromatography (Ion exchange LC: Liquid Chromatography, Gel filtration LC, and C18 Reversed Phase LC for three successive purification steps; It is characterized by the separation of a single peptide showing the strongest antioxidant power, which not only shows superior lipid peroxidation inhibitory activity than conventional natural antioxidants, but also effectively alleviates or eliminates the toxicity of reactive oxygen species, radicals and peroxides. -Isolation method of mussel antioxidant peptide from hydrolyzate using small intestine digestion system.

      In general, antioxidants are commonly referred to as substances that prevent oxidation, free radical scavengers that control the chain reaction of autooxidation according to the mechanism of action, peroxide decomposers that deactivate the peroxides by non-radicals, Metal deactivator to deactivate the oxidation promoting action of trace metals, synergist to increase antioxidant activity when co-existing with radical inhibitor in automatic oxidation, and singlet oxygen scavenger to eliminate various active oxygen system Are classified. Ingold (1968) divided antioxidants into two broad groups: first, as a primary or chain-reactive antioxidant, a group that reacts with lipid radicals to produce more stable products, and second, as a second or delayed antioxidant as a different mechanism of action. It was divided into groups that delayed automatic oxidation. Primary antioxidants block the prolongation of the chain reaction by providing hydrogen atoms to the lipid free radicals to make relatively stable radicals, preventing automatic oxidation. Antioxidants of this type are mostly phenolic compounds, such as tocopherol, gallic acid and derivatives thereof, quercetin, rhamnetin, camperol, rutin, quercitrin and caffeic acid. Flavonoids such as acid) are known.

      Secondary antioxidants do not have antioxidant effects on pure lipids but increase the effectiveness of or inhibit the effects of primary antioxidants. Antioxidants in this form include phospholipids, synergistic citric acid chelates with oxidative metal ions, and vitamin C, which are synergistic with primary antioxidants such as tocopherol. The oxidation of lipids, which is a typical oxidation reaction, is a reaction in which the formation and decomposition of peroxides are mainly caused by lipid radicals (L ·) or singlet oxygen produced by high temperature or light. In the presence of phenolic hydroxy groups act as free radical acceptors of fats and oils, the free radicals produced in the early stages of fat and oil have a stable resonance hybrid to act as an antioxidant. Such antioxidants are present in various plant extracts, flavorings, fermentation products, etc. as flavonoids or phenolic compounds. It has also been reported that oligopeptides produced by hydrolyzing proteins and plants using an acid or various enzymes also exhibit antioxidant activity (未 綱 et al., 1989; 本 井, 1989). Synthetic antioxidants include phenolic compounds, sulfur compounds, citric acid and derivatives thereof, phosphoric acid and derivatives thereof (Macrae et al., 1993), which have a large substituent indicating steric hindrance to form stable radicals and exhibit antioxidant properties.

      The current research direction of antioxidants is the role of scavengers of oxygen radicals and oxide radicals that cause aging and carcinogenesis in the limited study as a conventional food antioxidant (大 澤, 1985) and the in vivo lipid peroxidation reaction. Extensive research has been conducted on effects such as mutagenicity (大 澤, 1982) and cancer-induced expression suppression (大 澤, 1983), which are presumed to be caused by inhibition or radical involvement.

      Synthetic antioxidants affect incidence and various enzyme groups in animal experiments. In particular, BHT has been reported to be involved in teratogenic or cancerous development (Branen, 1975). Natural antioxidants have little side effects and are now widely used as food additives or drugs. However, since tocopherol, which is the most widely used natural antioxidant, has limitations in terms of high price and fat soluble, development of alternative natural antioxidants is desired.

      Conventional natural antioxidants are food protein hydrolysates, peptides containing leucine or valine at the N terminus isolated from beta conglycinin, a soy protein, and albumin (egg of egg white). Peptides containing histidine derived from albumin and peptides isolated from bovine skin hydrolysates are known to have antioxidant effects.

 However, the fact that fish consumption is inversely inversely proportional to cancer incidence and mortality, although it is widely known by epidemiological statistics from advanced countries such as the United States and Japan, lacks scientific evidence to support him. In the same hydrolysis system as the physiological digestive processes of the stomach and small intestine, scientific evidence of suppressing intractable diseases such as aging, cancer, and diabetes has not been found in any of the documents including the above statistical data.

Therefore, the object of the present invention was devised to improve the various problems as described above, two-stage hydrolysis process, centrifugation process, dialysis process to separate only a single peptide showing the strongest antioxidant power in mussel protein , Freeze-drying process, three-stage purification process, not only shows superior lipid peroxidation inhibition activity than conventional natural antioxidants, but also effectively alleviates or eliminates the toxicity of reactive oxygen species, radicals and peroxides. It is an object of the present invention to provide a method for separating mussel antioxidant peptides from hydrolysates using a gastro-intestinal digestion system.

      The present invention relates to a method for separating mussel antioxidant peptides from a hydrolyzate using a gastro-intestinal digestion system, and more specifically, by mixing mussel proteins and water to adjust the pH to the same digestive conditions of the stomach (胃) 1 For teasing, Pepsin is first hydrolyzed under the same conditions as the digestive conditions of the stomach; The hydrolyzate obtained through primary hydrolysis was mixed with bile salt mixture, 1M calcium chloride, 0.25M Bistris, and 0.1% porcine pancreatic lipase to adjust the pH of the secondary substrate, which was adjusted to trypsin ( trypsin) and alpha-chymotrypsin are secondary hydrolyzed to small molecule peptides in an environment similar to the digestive conditions of the small intestine; The hydrolyzate obtained through secondary hydrolysis was subjected to centrifugation and dialysis and then lyophilized to obtain a mussel hydrolyzate obtained by gastric-intestinal digestion system, followed by ion exchange liquid chromatography (Ion exchange LC: Liquid Chromatography, Gel filtration LC, and C18 Reversed Phase LC for three successive purification steps; It is characterized by the separation of a single peptide showing the strongest antioxidant power, which not only shows superior lipid peroxidation inhibitory activity than conventional natural antioxidants, but also effectively alleviates or eliminates the toxicity of reactive oxygen species, radicals and peroxides. -Isolation method of mussel antioxidant peptide from hydrolyzate using small intestine digestion system.

      When explaining the configuration of the present invention through an embodiment showing a separation method of the mussel antioxidant peptide, and an experimental example for measuring the antioxidant properties are as follows.

[ Example ] Gastrointestinal digestive system From hydrolyzate Mussel antioxidant Peptide  Separation method

[Production of Hydrolyzate]

     To 20 g of mussel protein, 100 ml of water is added, and pepsin (Pepsin, EC 3.4.23.1) is 250: 1 for a primary substrate adjusted to pH 1.8-2.2, which is the same as gastric digestion conditions. The reaction time was 1 hour 30 minutes to 2 hours, and the first hydrolysis was carried out in an environment similar to the above conditions. Bile salt mixture (0.125 M taurocholate, 0.125 M glycodeoxycholic acid), 1 M calcium chloride (CaCl), 0.25 M Bistris (pH 6.5), 0.1% (w / v) porcine pancreas in primary hydrolysate After mixing with lipase (porcine pancreatic lipase, EC 3.1.1.3), trypsin (EC 3.4.21.4) and alpha-chymotrypsin (α EC 3.4. .21.1) was subjected to secondary hydrolysis at 250: 1, temperature 35-40 ° C., and reaction time 2 hours to 3 hours in an environment similar to the intestine conditions. After secondary hydrolysis, centrifugation (10,000 × g, 15 min, 4 ° C.) was used to recover only the supernatant, followed by an electrodialyzer equipped with a molecular weight permeation membrane of 100 Da or less (electrodialyzer, Micro Acilyzer model G3, manufactured by Asahi Chemical Co., Ltd.). , Tokyo, Japan), followed by lyophilization and use as a mussel hydrolyzate via a gastro-intestinal digestion system.

      As a result of the first and second hydrolysis of mussels through the same process as the physiological conditions of the stomach and small intestine of the human body, most of the mussels were hydrolyzed to low molecular weight peptides having a molecular weight of 15 kDa or less (see FIG. 2). .

      Denatured conditions of the mussel hydrolyzates of Figure 2 electrophoresis results on polyacrylamide gel (SDS-PAGE), G is the gastric digestion (gastric digestion) in the stomach, D represents the duodenal digestion (duodenal digestion) in the duodenum.

[Isolation and Purification of Antioxidant Peptides]

       The fraction obtained by separating the mussel hydrolyzate from the gastro-intestinal digestion system into a Hireprep 16/10 DEAE FF anion exchange column equilibrated with 20 mM sodium acetate buffer (pH 4.0). Dialysis (product name spectraper membrane tubing, manufacturer sartorius, Germany) removed the salt and lyophilized. After measuring the fractional antioxidant properties of the lyophilisate, the highly antioxidative fraction was eluted using a reversed-phase LC column 10.0 × 250 mm on a high performance liquid chromatography (HPLC) system. The elution rate was 1.0 ml / min, and the absorbance was measured at 215 and 280 by linear phase gradient using distilled water and 0.1-40% (v / v) acetonitrile as the mobile phase.

      Three steps of purification, Ion exchange LC (Liquid Chromatography), gel filtration liquid chromatography, to separate only the single peptide with the strongest antioxidant power from the mussel hydrolyzate after gastric-intestinal digestion (Gel filtration LC) and C18 Reversed Phase LC were used to isolate and quantify each lipid peroxidation inhibitory effect compared with natural antioxidants, tocopherol (vit E) and ascorbic acid (vit C). (See FIG. 3).

      After lyophilization of the fraction containing the peptide with excellent antioxidant properties in the fraction of anion chromatography, high performance liquid chromatography (HPLC) equipped with an octadecylsilane (ODS) octadecylsilane (ODS) C18 reversed phase column (HPLC) The mixture was separated by melting (1 ml) with an acetonitrile solution by linear concentration gradient method (0.1-40% acetonitrile, 40 min) and divided into three fractions. As a result of measuring the antioxidant properties of the three fractions, the molecular weight and amino acid sequence were confirmed by the electrospray ionization tandem mass spectrometry (ESI-QTOF tandem mass). As a result, a single peptide having a molecular weight of 1.5 kDa, It was confirmed that it was leucine-valine-glycine-glutamate-glutamine-alanine-valine-proline-alanine-valine-cystine-valine-proline (LVGEQAVPAVCVP) (see FIG. 4A, FIG. 4B).

[ Experimental Example ] Antioxidant Measurement

[Manufacture of linoleic acid emulsion]

      Linoleic acid emulsion was prepared according to the method of Osawa (1981). That is, 0.13 ml of linoleic acid, 10 ml of ethanol, and 10 ml of 50 mM phosphate buffer solution (pH 7.0) were mixed, and hydrolyzate was added to the mixture at a concentration of 1% (w / w) for linoleic acid, respectively. Storage in a thermostat controlled at 40 ± 1 ° C. promotes automatic oxidation.

[Ferric thiocyanate method]

   Antioxidant activity was measured by Ferric thiocyanate according to the method of Mitsuda (1966) et al. That is, after adding 0.1 ml of 75% ethanol, 0.1 ml of 30% ammonium thiocyanate and 0.1 ml of 2 × 10 M ferrous chloride / 3.5% hydrochloric acid (HCl) to 0.1 ml of the oil-based turbidity solution, Absorbance was measured at 500 nm after being set at room temperature for minutes.

[Measurement of free radical scavenging activity]

       Significant activity of free radical scavenging activity measured by electron spin resonance spectroscopy of the DPDP radicals, cupoxide oxides, and hydroxy radicals was measured using electron spin resonance spectroscopy. Shown (see FIG. 5).

        The calculation of the radical concentration percentage of fraction scavenging ability is as follows.

1. Measurement of DPPH radical scavenging activity

       DPPH radical scavenging was performed according to the method of human name Nanjo (1996). That is, after mixing 60 μl of 60 μM DPPH ethanol solution and 60 μl hydrolyzate solution, the mixed solution was transferred to a quartz capillary tube and measured by electron spin resonance spectroscopy after 2 minutes. The spectrum is 336.5 ± 5 mT, power: 5 Hz, amplitude: 1 × 1000, modulation width: 0.8 mT. It was recorded under conditions of a sweep width of 10 mT and a sweep time of 30 s. DPPH radical scavenging ability was calculated using the following equation.

       DPPH radical scavenging ability,% = [100-(electron spin resonance signal area at sample treatment / electron spin resonance signal area at no sample treatment)] × 100.

2. Superoxide radical scavenging activity

      Superoxide radical scavenging ability was measured according to the method of Gue (1999). That is, 20 μl of 0.3 mM riboflavin, 20 μl of 5.0 mM EDTA, 20 μl of 0.1 M DMPO and 20 μl of a hydrolyzate solution are mixed, followed by 1 minute with a 365 nM UV lamp, and the solution is added to a quartz capillary. tube), and after 2 minutes, it was measured by electron spin resonance spectroscopy (electron spin resonance spectroscopy). Spectrum is based on a field of 336.5 ± 5 mT, power: 10 ㎽, amplitude: 1 × 1000, scan width: 10 mT, sweep time: 30 s Recorded. Superoxide radical scavenging activity was calculated using the following equation.

      Superoxide radical scavenging ability,% = [100-(electron spin resonance signal area in sample treatment / electron spin resonance signal area in no sample treatment)] × 100.

3. Measurement of Hydrogen Peroxide Radical Scavenging Activity

      Hydrogen peroxide radical scavenging function was measured according to the method of Rosen G.M (1984). 0.2 ml of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) 0.2 ml 10 mM iron sulfate (FeSO4) in 0.2 ml of the hydrolyzate solution. ) Was mixed with 0.2 ml of 10 mM HO and 0.2 ml of 10 mM HO, and then left at room temperature for 2.5 minutes, and then transferred to a quartz capillary tube, which was measured by electron spin resonance spectroscopy. The spectrum is 336.5 ± 5 mT, power: 1 ㎽, amplitude: 1 × 200, modulation width: 0.1 mT. It was recorded under conditions of a sweep width of 10 mT and a sweep time of 30 s. Hydroxy peroxide radical scavenging activity was calculated using the equation below.

 Hydrogen peroxide radical scavenging ability = [100 − (electron spin resonance signal area when sample treated / electron spin resonance signal area when sample not treated)] × 100.

        Referring to the effects of the above-described configuration and operation in detail as follows.

 Separation method of mussel antioxidant peptide from hydrolyzate using gastric-small intestine digestion system of the present invention is a human body through two steps of hydrolysis and centrifugation, dialysis, freeze-drying, three steps of purification process of mussel protein It provides a single peptide having the effect of effectively alleviating or eliminating the harmful toxins of free radicals, radicals and peroxides.

Claims (2)

         In the method for separating the antioxidant peptide,         20 ml of mussel protein and 100 ml of water were mixed with Pepsin (Pepsin) under conditions of reaction time of 1 hour 30 minutes to 2 hours at 35-40 ° C, such as body temperature, to adjust the pH to 1.8-2.2. Primary hydrolysis by reaction at 250: 1;         The pH was 8.0 by mixing a bile salt mixture, 1M calcium chloride (CaCl), 0.25M Bistris, and 0.1% porcine pancreatic lipase to the hydrolyzate obtained through primary hydrolysis. Reaction time at 35 to 40 ° C. was controlled by 250: 1 reaction mixture of trypsin and alpha-chymotrypsin at a molecular weight of 15 kDa under conditions of 2 to 3 hours. Secondary hydrolysis with the following low molecular weight peptides;        The hydrolyzate obtained through secondary hydrolysis was centrifuged at 10,000 x g for 15 minutes at 4 ° C to recover only the supernatant, followed by dialysis using an electrodialysis apparatus equipped with a molecular weight permeation membrane of 100 Da or less and lyophilized. To prepare mussel hydrolyzate;  Ion exchange LC (Liquid Chromatography), Gel filtration LC, and C18 Reversed Phase LC on mussel hydrolysate prepared through gastric-intestinal digestion system Separation method of the mussel antioxidant peptide from the hydrolyzate using a gastro-intestinal digestion system, characterized in that to separate a single peptide through a purification process.          In the method for separating the antioxidant peptide,         20 ml of mussel protein and 100 ml of water were mixed with Pepsin (Pepsin) under conditions of reaction time of 1 hour 30 minutes to 2 hours at 35-40 ° C, such as body temperature, to adjust the pH to 1.8-2.2. Primary hydrolysis by reaction at 250: 1;        The pH was 8.0 by mixing a bile salt mixture, 1M calcium chloride (CaCl), 0.25M Bistris, and 0.1% porcine pancreatic lipase to the hydrolyzate obtained through primary hydrolysis. Reaction time at 35 to 40 ° C. was controlled by 250: 1 reaction mixture of trypsin and alpha-chymotrypsin at a molecular weight of 15 kDa under conditions of 2 to 3 hours. Secondary hydrolysis with the following low molecular weight peptides;         The hydrolyzate obtained through secondary hydrolysis was centrifuged at 10,000 x g for 15 minutes at 4 ° C to recover only the supernatant, followed by dialysis using an electrodialysis apparatus equipped with a molecular weight permeation membrane of 100 Da or less and lyophilized. To prepare mussel hydrolyzate;         Ion exchange LC (Liquid Chromatography), Gel filtration LC, and C18 Reversed Phase LC on mussel hydrolysate prepared through gastric-intestinal digestion system Mussel antioxidant peptide from the hydrolyzate using a gastro-intestinal digestion system, characterized in that to separate a single peptide through a purification process using.

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