KR101631657B1 - Ovotrasferrin hydrolysate having with antioxidant activity and method for preparation thereof - Google Patents

Abstract

The present invention has an excellent effect of providing a hydrolyzate of overtransferrin having an increased antioxidant function by preparing a functional peptide by hydrolyzing an over-transferrin, an egg white protein, with TECP.

Description

TECHNICAL FIELD The present invention relates to a hydrolyzate of an ovarian protein overtransferrin having an antioxidant activity and an antioxidant activity and a method for preparing the same,

The present invention relates to a method for producing a functional overtransferrin hydrolyzate by cleaving a disulfide bond of the overtransferrin through Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) which selectively cleaves 15 disulfide bond sites contained in overtransferrin, And to the development of antioxidative materials containing the hydrolyzate as an active ingredient.

Antioxidant is a property that inhibits the oxidation of a substance. In the food system, it refers to a function of preventing or delaying changes in flavor and color of food caused by oxidation, rancidity of the oil, and the like. The above-mentioned function of inhibiting the oxidation can be prevented not only in the food system but also in the living body, such as cancer caused by active oxygen, atherosclerosis and diseases such as diabetes, , 30: 22-29, 1997).

In general, the currently used antioxidants are classified into synthetic antioxidants and natural antioxidants. Butylated hydroxytoluene (BHT), a representative synthetic antioxidant, has been reported to be involved in teratogenesis or cancer development in animal experiments (Branen et al. Amer. Oil Chem. Soc., 52; 59-63, 1975). In addition to its excellent antioxidant effect, the side effects are also questionable. On the other hand, natural antioxidants are currently used as food additives or medicines because they have few side effects when compared with synthetic antioxidants. However, the development of alternative natural antioxidants is required due to limitations on the use of expensive and fat- Lee et al., J. Life Sci., 18, 1106-1114, 2008). In addition, there are many studies that have been conducted in Korea.

Eggs, well known as natural complete foods, have been shown to play a role in the physiological functions and interactions of the components, and their importance is being emphasized in that they can be used as a major ingredient in functional health foods (Jeon et al. J. Food Sci. Ani Resour., 22: 267-273, 2002). Over-transferrin, an iron-binding protein in eggs, is a monosaccharide protein that occupies a heavier weight next to albumin. It has 15 disulfide bonds per molecule and is a kind of transferrin capable of binding two iron ions (Wu and Acero-Lopez, Food Res. Int., 46: 480-487, 2012). In this way, it has been known that iron has high binding capacity to iron atoms, and thus has an antibacterial activity by binding and removing iron, an essential nutrient for microbial growth (Ibrahim et al., Bioch. Biophys. Acta., 1523, 196-205, 2000 In addition, anti-cancer (Ibrahim and Kiyono, J. Agric. Chem. 57: 11383-11390, 2009), immune enhancement (Hirota et al., J. Vet. Med. Sci., 57: 825-829, The physiological activities such as anti-hypertension (Lee et al., J. Food Sci., Ani. Res. 30, 286-290, 2010) are known.

It is known that food proteins can denature in the body during digestion or during fermentation and aging process during food production, and can exhibit various physiological activities in vivo (Moller et al., 2008). Therefore, proteins that have undergone chemical and physical denaturation are recognized as functional materials.

Korean Patent Laid-Open No. 10-2011-40018 discloses the activity of lowering the blood pressure of the protein over-transferrin hydrolyzate present in the egg white for the development of a functional material through physical and chemical modification of the egg white protein. Korean Patent Laid- -48650 discloses a process for preparing seasonings containing a hydrolyzate of egg white protein. Korean Patent Laid-Open No. 10-2002-67203 discloses a method for preparing a thrombolytic peptide separated from egg yolk.

Accordingly, it is an object of the present invention to derive a useful hydrolyzate from an egg white protein, overtransferrin, through a hydrolysis reaction using TECP.

Another object of the present invention is to provide a functional overtransferrin hydrolyzate having the above-mentioned antioxidant function.

The above object of the present invention is achieved by a method for purifying an overtransferrin, comprising: hydrolyzing an over-transferrin by treating Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) to an over-transferrin (purity: 98%) having high purity; Dialyzing the hydrolyzate to a dialysis membrane to remove TCEP; Centrifuging the TCEP-removed over-transferrin hydrolyzate to obtain a supernatant, and lyophilizing the supernatant; And measuring lipid peroxidation inhibiting activity of the lyophilized sample.

The present invention provides a hydrolyzate of over-transferrin, which is an egg white protein, through hydrolysis using TECP, and provides a hydrolyzate of over-transferrin with enhanced antioxidant function.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic diagram for preparing a hydrolyzate using over-transferrin with Tris (2-carboxyethyl) phosphine hydrochloride (TCEP).
FIG. 2 is a graph showing inhibitory activity against lipid peroxidation by TBA assay of over-transferrin hydrolyzed through TCEP of the present invention; The ovotrasferrin hydrolyzed ovotrasferrin of the present invention (mg), the ovotrasferrin hydrolyzate of the present invention (mg), the hydrolyzed ovotrasferrin of the present invention (mg), the hydrolyzed ovotrasferrin 0.5 of the present invention mg / mL (O).
Figure 3 is a graph showing the results of the FTC assay of the over-transferrin hydrolyzed through the TCEP of the present invention Lt; RTI ID = 0.0 > lipid < / RTI > (O), ovotrasferrin (mg) of the present invention hydrolyzed, ovotrasferrin of the present invention (mg), ovotrasferrin of the present invention (mg) of 2 mg / / mL (O).

In the present invention, a functional peptide having excellent antioxidant activity was developed by hydrolyzing pure overtransferrin protein using Tris (2-carboxyethyl) phosphine Hydrochloride (TCEP).

The overtransferrin hydrolyzate of the present invention can be used by adding a food composition. The above-mentioned overtransferrin hydrolyzate of the present invention is preferably added to the food composition in the form of a concentrate of 5 to 20% or powder.

The functional food composition which can be used by adding the overtransferrin hydrolyzate of the present invention is a food, beverage, or beverage additive for health and the like. The functional composition is mixed with 0.1 to 20 wt% It is preferable to use a functional beverage for the purpose of antioxidative effect or a soft or hard capsule filled with the overtransferrin hydrolyzate of the present invention as a dry powder.

The composition comprising the overtransferrin hydrolyzate of the present invention as an active ingredient can be used variously for an auxiliary food for antioxidative effect. Foods to which the present composition can be added include, for example, various foods, gums, candies, confectionery, tea, vitamin complexes, and health functional foods.

The beverage composition containing the overtransferrin hydrolyzate of the present invention is not particularly limited as an essential ingredient in the indicated ratio, but may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Examples of such natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above .

In addition to the above, the overtransferrin hydrolyzate of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), dietary components, synthetic flavors and natural flavors, colorants and intermediates such as cheese, Salts of alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the overtransferrin hydrolyzate of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. The present invention can be used as a mixer such as a lactic acid bacterium preparation drink or paste such as yogurt. These components may be used independently or in combination. Preferably, the overtransferrin hydrolyzate of the present invention is selected from the range of 0.01 to about 20 parts by weight per 100% by weight.

Hereinafter, the present invention will be described in detail with reference to preferred embodiments and experimental examples. However, the scope of the present invention is not limited by the following examples and experimental examples.

<Example 1> Production of hydrolysis peptide of over-transferrin

Over-transferrin (purity: 98%) was supplied by Professor Andong Wook of Iowa State University, Ames, USA. Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) reagent used for the reduction of the overtransferrin was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan).

Hydrolysates of overtransferrin were prepared as follows (Ibrahim et al., 2009). 0.2 mM TCEP was dissolved in 20 mM citrate-phosphate buffer (pH 4.0) with overtransferrin, treated with 0.1% sodium dodecyl sulfate (SDS) and reacted at 37 ° C for 6 hours. Respectively. TCEP was removed by dialysis with a cut-off size of 3,500 Da for 48 hours. The supernatant was centrifuged at 3,000 rpm for 20 minutes (Combi-514R, Hanil, Gyunggi-do, Korea) and the supernatant was lyophilized and used in the experiment.

<Example 2> Measurement of antioxidant activity of the over-transferrin hydrolyzate of the present invention

Ammonium thiocyanate and Iron (II) chloride tetrahydrate were used in the experimental examples of the present invention as described in Samchun Pure Chemical Co., Ltd. (Gyunggi-do, Korea), ascorbic acid and butylated hydroxyanisole were purchased from Sigma Chemical Co., Ltd. (St. Louis, MO, USA).

EXPERIMENTAL EXAMPLE 1 Measurement of lipid peroxidation inhibitory activity of the present invention over-transferrin hydrolyzate by Thiobarbituric acid (TBA) method

Measurement of lipid peroxidation using thiobarbituric acid. et al. (1981) and Kim et al. (2011) was modified and measured.

1.0 mL of corn oil and 0.1 mL of Tween 20 were added to 100 mL of Tris-maleate buffer (pH 6.8) and homogenized for 4 minutes with a homogenizer (Ika ultra-turrax T25, Staufen, Germany) to form an oil emulsion stock solution. Add 0.5 mL of 0.2% ascorbic acid and 200 ppm FeCl ₃ to 8 mL of this solution, add 1 mL of the hydrolyzate of non-hydrolyzed overtransferrin and overtransferrin to each concentration, react at 37 ° C, Absorbance was measured. First, add 2 mL of Thiobarbituric acid / trichloroacetic acid (20 mM TBA / 15% TCA, w / v) solution and 50 μL of 10% butylated hydroxyanisole solution (10% BHA in 90% ethanol) The samples were allowed to develop for 15 minutes at &lt; RTI ID = 0.0 &gt; After cooling in running water, the supernatant was measured by absorbance at 532 nm (Optizen 2120 UV plus, Mecasys, Seoul, Korea) by centrifugation (3,000 rpm, 15 min).

The TBA assay, which is one of the methods for measuring the degree of peroxidation of lipid of the present invention, is a method for measuring the degree of red color which is a result of reaction of TBA with malonaldehyde, which is a secondary carbonyl compound produced by oxidation of polyunsaturated fatty acid And measuring the degree of rancidity of the retention.

In the present invention, the antioxidant effect of the over-transferrin extracted from egg white and the over-transferrin hydrolyzate of the present invention was compared with that of control and antioxidant activity in the oil emulsion, and the results of the absorbance measurement were summarized in FIG.

As shown in FIG. 2, the over-transferrin hydrolyzate of the present invention showed a low TBA value as compared with the control to which distilled water was added instead of the sample. The absorbance value of the control group was 0.419 for the 24 hour reaction and 0.315 for the overtransferrin (2 mg / mL). On the other hand, in the case of the over-transferrin hydrolyzate of the present invention, low absorbance was observed at 0.024 and 0.039 at 1 and 2 mg / mL, respectively. The above results indicate a high inhibition of lipid peroxidation.

In the case of the control and the overtransferrin until 48 hours of the reaction, persistent rancidity occurred and the absorbance value was 0.600 or more at the end, but the change of the absorbance was not significant as shown in Fig. In the case of the over-transferrin hydrolyzate according to the present invention, the absorbance value was 0.3 at a concentration of 1 or 2 mg / mL, indicating that the hydrolyzate of the present invention over-transferrin inhibited lipolysis by about 46-48%.

EXPERIMENTAL EXAMPLE 2 Measurement of inhibitory activity of lipid peroxidation by the over-transferrin hydrolyzate of the present invention by ferric thiocyanate (FTC) method

Measurement of lipid peroxidation by ferric thiocyanate was performed by modifying the method of Kim et al. (2004). To the 0.1 mL of TCEP hydrolysates by concentration 0.4 mL of 40 mM phosphate buffer (pH 7.0), 0.2 mL of linoleic acid (25 mg / mL in ethanol), and 0.2 mL of distilled water were mixed to prepare a stock solution, which was incubated at 37 ° C And the absorbance was measured every 24 hours. First, 4 mL of 70% ethanol, 20 mL ferrous chloride, and 0.1 mL of 30% ammonium thiocyanate were added to 0.1 mL stock solution, and the absorbance was measured at 500 nm for 3 minutes.

Ferric thiocyanate (FTC) method measures the degree of oxidation by measuring the amount of peroxide produced at the beginning of lipid oxidation (Haraguchi et al., 1992). The peroxide formed reacts with ferrous chloride to produce a red ferric chloride dye (Jeong et al., 2007). In the present invention, peroxidase production was measured by measuring absorbance at 24 hour intervals after reacting with linoleic acid at concentrations of 0.5, 1, and 2 mg / mL of the over-transferrin and TCEP hydrolyzate used as comparators, . In the case of the control without the addition of the sample, the absorbance was measured as 0.825 due to the production of peroxide by lipid oxidation on the 5th day of the reaction but 0.608 in the case of overtransferrin (2 mg / mL) In the case of the absorbance of the hydrolysates of the invented overtransferrin, 0.130, 0.164 and 0.161, respectively, showed low levels of lipid oxidation. Based on the absorbance value of the control measured on the 5th day of the reaction, the inhibition rate of lipid oxidation of the treatment groups was calculated. As a result, overtransferrin showed a low oxidation inhibition rate of 26%, but in the case of the hydrolysates of overtransferrin of the present invention, Respectively.

INDUSTRIAL APPLICABILITY As described above, the present invention not only provides a hydrolyzate of overtransferrin as a natural antioxidant, but also is a very useful invention for the poultry industry and the food industry.

Claims (1)

Overtransferrin was dissolved in citrate-phosphate buffer (pH 4.0) with tris (2-carboxyethyl) phosphine hydrochloride (TCEP) and reacted at 37 ° C for 6 hours to reduce the overtransferrin; Removing TCEP from the hydrolyzate obtained above; In the method for purifying the hydrolyzate of the overtransferrin by centrifuging the TCEP-removed hydrolyzate,

Adding 0.1% sodium dodecyl sulfate (SDS) in the overtransferrin reducing step, and then adjusting the pH to 7.0 with an aqueous ammonia solution; And removing the TCEP by dialyzing the dialyzed membrane at a cut-off size of 3.5 KDa for 48 hours. The method for purifying an overtransferrin hydrolyzate having increased lipid peroxide inhibiting activity.

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