KR20130117033A - Ovotrasferrin hydrolysate having with antioxidant activity and method for preparation thereof - Google Patents

Abstract

PURPOSE: A production method of hydrolysate of albumen over transferrin having an improved antioxidative function is provided to use tris(2-carboxyethyl)phosphine hydrochloride for the hydrolysis of the over transferring. CONSTITUTION: Over transferring is processed with tris(2-carboxyethyl)phosphine hydrochloride (TCEP) to be reduced. The obtained hydrolysate is dialyzed with a dialyzing film to remove TCEP. The hydrolysate is centrifugal separated to obtain supernatant, and the supernatant is freeze-dried.

Description

Hydrolyzate of egg white protein overtransferin with antioxidant activity and preparation method thereof {Ovotrasferrin hydrolysate having with antioxidant activity and method for preparation}

The present invention provides a functional overtransferrin hydrolyzate by cleaving the disulfide bond of the overtransferin through Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) that selectively cleaves 15 disulfide bond sites contained in the overtransferrin. It relates to the development of an antioxidant material using the hydrolyzate as an active ingredient.

Antioxidant is a property of inhibiting the oxidation of a substance, and in the food industry, it is a function of preventing or delaying changes in flavor and color of food caused by oxidation and rancidity of fats and oils. The antioxidant function prevents diseases such as cancer, arteriosclerosis, and diabetes caused by free radicals not only in the food system but also in the living body, and thus, there is a growing interest in antioxidants (hands, food science and industry). , 30: 22-29, 1997).

Generally, the antioxidants currently used are classified into synthetic antioxidants and natural antioxidants. A representative synthetic antioxidant, butylated hydroxytoluene (BHT), has been reported to be involved in teratogenic or cancerous development in animal experiments (Branen et al., Amer.Oil Chem. Soc., 52; 59-63, 1975) While its superior antioxidant effects, side effects are also problematic. On the other hand, natural antioxidants have almost no side effects compared to synthetic antioxidants, but α-tocopherol is currently used as a food additive or drug, but due to the constraints of high price and fat solubility, development of alternative natural antioxidants is required. This research is being actively conducted (Lee et al., Korean J. Food Preserv., 11; 126-129, 2004, Lee et al., J. Life Sci., 18; 1106-1114, 2008).

Eggs, which are well known as natural whole foods, have been found to play a role in the physiological functions and interactions of each ingredient, and their importance is further emphasized in that they can be used as the main ingredients of functional health foods (Jeon et al., J. Food Sci.Ani.Resour., 22; 267-273, 2002). Overtransferrin, the iron-binding protein of eggs, is the second most common monosaccharide glycoprotein after albumin, with 15 disulfide bonds per molecule and a type of transferrin capable of binding two iron ions (Wu and Acero-Lopez, Food Res. Int., 46; 480-487, 2012). As such, it has been known to have an antimicrobial activity by binding to iron, which is an essential nutrient for microbial growth, by having a high binding force to iron atoms (Ibrahim. Et al., Bioch. Biophys. Acta., 1523; 196-205, 2000 ) In addition, anticancer (Ibrahim and Kiyono, J. Agric. Chem. 57; 11383-11390, 2009), immunopotentiation (Hirota et al., J. Vet. Med. Sci., 57; 825-829, 1995), Antihypertension (Lee et al., J. Food Sci. Ani. Resour., 30; 286-290, 2010) is known.

Food proteins are known to cause degeneration during digestion or fermentation and ripening processes in the human body, which can exhibit various biological activities in vivo (Moller et al., 2008). Therefore, proteins as well as proteins with chemical and physical denaturation are recognized as valuable materials.

Korean Patent Application Publication No. 10-2011-40018 discloses an activity for lowering blood pressure of protein overtransferin hydrolyzate present in egg white, and the Republic of Korea Patent Publication No. 10-2004 -48650 discloses a seasoning method containing hydrolyzate of egg white protein. In addition, the Republic of Korea Patent Publication No. 10-2002-67203 discloses a method for producing a thrombolytic peptide isolated from egg string.

Accordingly, it is an object of the present invention to induce useful hydrolysates of the egg white protein, overtransferin, through a hydrolysis reaction using TECP.

Another object of the present invention is to provide a functional overtransferin hydrolyzate having an enhanced antioxidant function.

The object of the present invention is to hydrolyze the overtransferrin by treating Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) with high-purity overtransferrin (purity: 98%); Dialysis of the hydrolyzate with a dialysis membrane to remove TCEP; Centrifuging the TCEP-removed over-transferrin hydrolyzate to obtain a supernatant, and lyophilizing the supernatant; It was achieved through the step of measuring the lipid peroxidation inhibitory activity of the lyophilized sample.

The present invention has an effect of obtaining a hydrolyzate of the overtransferin, which is an egg white protein, through a hydrolysis reaction using TECP, and providing a hydrolyzate of the overtransferrin having enhanced antioxidant function.

1 is a schematic diagram for the production of hydrolyzate using overtransferin Tris (2-carboxyethyl) phosphine hydrochloride (TCEP).
Figure 2 is a graph of the lipid peroxidation inhibitory activity using the TBA test of the overtransferin hydrolyzed via TCEP of the present invention; Control (◆), overtransferrin 2 mg / mL (▲), inventive hydrolyzed ovotrasferrin 2 mg / mL (●), inventive hydrolyzed ovotrasferrin 1 mg / mL (Δ), inventive hydrolyzed ovotrasferrin 0.5 mg / mL (○).
Figure 3 using the FTC method of the overtransferin hydrolyzed via the present invention TCEP Graph of lipid peroxidation inhibitory activity; (◆), overtransferin 2 mg / mL (▲), inventive hydrolyzed ovotrasferrin 2 mg / mL (●), inventive hydrolyzed ovotrasferrin 1 mg / mL (△), inventive hydrolyzed ovotrasferrin 0.5 mg / mL (○).

In the present invention, tris (2-carboxyethyl) phosphine Hydrochloride (TCEP) was used to hydrolyze pure overtransferin protein to develop a functional peptide with excellent antioxidant activity.

The obat transferrin hydrolyzate of the present invention can be used by adding a food composition. It is preferable that the present invention ovatransferrin hydrolyzate is added to the food composition in the form of powder or powder form of 5 to 20%.

Functional food composition that can be used by adding the present invention the barat transferrin hydrolyzate is a food, beverage, or beverage additives for health, the functional composition is mixed with water in 0.1% to 20% by weight relative to 100% by weight of food It is preferable to use in the form of a soft or hard capsule filled with a functional beverage for the purpose of the antioxidant effect or the powder of the present invention ovatransferrin hydrolyzed to dry powder.

The composition comprising the present invention barat transferrin hydrolyzate as an active ingredient may be used in various ways, such as supplements for the antioxidant effect. Examples of the food to which the present composition can be added include various foods, gums, candy, confectionery, tea, vitamin complexes, and health functional foods.

The beverage composition containing the obat transferrin hydrolyzate of the present invention may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary beverages, although there is no particular limitation as an essential ingredient in the indicated ratio. Examples of such natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And sugars such as conventional sugars such as polysaccharides such as dextrin, cyclodextrin and the like and xylitol, sorbitol, erythritol. As flavoring agents other than those described above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. .

In addition to the above, the ovatrantransfer hydrolyzate of the present invention includes various nutrients, vitamins, minerals (electrolytes), dietary ingredients, synthetic flavors and natural flavoring agents, colorants and neutralizing agents (cheese, chocolate, etc.), pectic acid and its Salts, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks and the like. In addition, the obat transferrin hydrolyzate of the present invention may contain pulp for producing natural fruit juice and fruit juice beverage and vegetable beverage. The present invention can be used as a mixed agent such as lactic acid bacteria preparation beverages or pastes such as yogurt. These components may be used independently or in combination. Preferably, the inventive obatransferin hydrolyzate is selected from the range of 0.01 to about 20 parts by weight per 100% by weight.

Hereinafter, the specific contents of the present invention will be described in detail through preferred examples and experimental examples, but the scope of the present invention is not limited by the following examples and experimental examples.

< Example  1> invention Overtransferrin Hydrolysis of Peptides  Produce

Overtransferrin (purity: 98%) was used by Ahn Dong-wook of Iowa State University, Ames. Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) reagent used for the reduction of the overtransferin was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan).

Hydrolysates of overtransferrin were prepared by the following method (Ibrahim et al., 2009). 0.2 mM TCEP was dissolved in 20 mM citrate-phosphate buffer of pH 4.0 together with overtransferin and treated with 0.1% SDS and reacted at 37 ° C. for 6 hours, and then the pH was adjusted to 7.0 using an aqueous ammonium solution. This was dialyzed for 48 hours with a cut off size of 3,500 Da dialysis membrane to remove TCEP. This was centrifuged at 3,000 rpm for 20 minutes (Combi-514R, Hanil, Gyunggi-do, Korea) and the supernatant was freeze-dried and used in the experiment.

< Example  2> invention Overtransferrin  Hydrolyzate of Antioxidant ability  Measure

Ammonium thiocyanate and Iron (II) chloride tetrahydrate were used as Samchun Pure Chemical Co., Ltd. (Gyunggi-do, Korea), and ascorbic acd and butylated hydroxyanisole are manufactured by Sigma Chemical Co., Ltd. Purchased from St. Louis, Mo., USA.

Experimental Example  One Thiobarbituric acid ( TBA The present invention by the method Overtransferrin  Hydrolyzate of  Measurement of Lipid Peroxidation Inhibitory Activity

Lipid peroxidation using thiobarbituric acid was measured by Osawa. et al. (1981) and Kim et al. (2011) was measured by modifying the method.

To 100 mL of tris-maleate buffer (pH 6.8), 1.0 g of corn oil and 0.1 mL of Tween 20 were added and homogenized with a homogenizer (Ika ultra-turrax T25, Staufen, Germany) for 4 minutes to form an oil emulsion stock solution. 0.5 mL of 0.2% ascorbic acid and 200 ppm FeCl ₃ were added to 8 mL of this solution, and 1 mL of unhydrolyzed overtransferrin and overtransferrin were added at each concentration to react at 37 ° C at regular intervals. The absorbance was measured by. First, 50 mL of Thiobarbituric acid / trichloroacetic acid (20 mM TBA / 15% TCA, w / v) solution and 10% butylated hydroxyanisole solution (10% BHA in 90% ethanol) were added to 1 mL of sample solution for each concentration. Samples were allowed to develop for 15 minutes at ° C. After cooling in flowing water, the supernatant was measured by absorbance at 532 nm (Optizen 2120 UV plus, Mecasys, Seoul, Korea) by centrifugation (3,000 rpm, 15 min).

TBA analysis, one of the aldehydes measurement methods for measuring the degree of peroxidation of lipids of the present invention, colorizes the red color resulting from the reaction of representative malonaldehyde and TBA among carbonyl compounds secondaryly produced by oxidation of polyunsaturated fatty acids. It is a method to quantify and measure the degree of rancidity of oils and fats.

In the present invention, the antioxidant effect on the components of the overtransferrin extracted from egg whites and the overtransferin hydrolyzate of the present invention was compared with the absorbance value of the control and antioxidant activity in the oil emulsion, and the absorbance measurement results are summarized in FIG.

As shown in FIG. 2, the overtransferin hydrolyzate of the present invention exhibited a low TBA value as compared to the control to which distilled water was added instead of the sample. In the case of the reaction for 24 hours, the absorbance value was 0.419 for the control group and 0.315 for the overtransferin (2 mg / mL). On the other hand, in the case of the overtransferin hydrolyzate of the present invention, low absorbance was observed at 0.024 and 0.039 at concentrations of 1 and 2 mg / mL, respectively. The results indicate high fat rancidence inhibitory activity.

In the control and overtransferin up to 48 hours of reaction, the maintenance rancidation continued to show a final absorbance value of 0.600 or more, but the change in absorbance was not so large as shown in FIG. In the case of the overtransferin hydrolyzate of the present invention, the absorbance value was about 0.3 at the concentration of 1 and 2 mg / mL.

Experimental Example  2 Ferric thiocyanate  ( FTC The present invention by the method Overtransferrin  Hydrolyzate on  Measurement of lipid peroxidation inhibitory activity

Lipid peroxidation by ferric thiocyanate was measured by modifying Kim et al. (2004). 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 with 0.1 mL of TCEP-using hydrolyzate by concentration, and then incubated at 37 ° C. Absorbance was measured every 24 hours. First, 4 mL of 70% ethanol, 0.1 mM of 20 mM ferrous chloride, and 30% ammonium thiocyanate were added to 0.1 mL of the stock solution, and the absorbance was measured at 500 nm after reaction for 3 minutes.

Ferric thiocyanate (FTC) method is used to measure the degree of oxidation by measuring the amount of peroxide produced at the beginning of lipid oxidation (Haraguchi et al., 1992). The formed peroxide reacts with ferrous chloride to produce a red ferric chloride pigment. (Jeong et al., 2007). In the present invention, the concentrations of the overtransferin and TCEP hydrolyzate used as the control were 0.5, 1, and 2 mg / mL and reacted with linoleic acid to measure absorbance at 24 hour intervals to investigate peroxide production in FIG. 3. Indicated. In the case of the control measured only with distilled water as a solvent without adding a sample, the absorbance was measured to be 0.825 due to the formation of peroxide by lipid oxidation on the 5th day of the reaction, but 0.608 for the overtransferin (2 mg / mL) In the case of absorbance by the hydrolyzate treatment of the invention overtransferin it was found that the low level of lipid oxidation progressed to 0.130, 0.164, 0.161, respectively. As a result of calculating the lipid oxidation inhibition rate of each treatment based on the absorbance values of the control group measured on the 5th day of the reaction, the overtransferin showed a low oxidation inhibition rate of 26%, but in the case of the hydrolyzate of the overtransferin of the present invention, the inhibition rate of 80% or more. Showed.

As described above, the present invention not only has the effect of providing the hydrolyzate of overtransferin as a natural antioxidant, but also is very useful for poultry and food industries.

Claims (3)

Reducing overtransferrin by treating tris (2-carboxyethyl) phosphine hydrochloride (TCEP) with overtransferrin (overtransferrin); Dialysis of the obtained hydrolyzate with a dialysis membrane to remove the TCEP; Centrifuging the overtransferin hydrolyzate from which the TCEP has been removed to obtain a supernatant, and then freezing and drying the method. An overtransferin hydrolyzate having lipid peroxidation inhibitory activity prepared by the method of claim 1 An antioxidant functional supplement composition comprising the overtransferin hydrolyzate of claim 2 as an active ingredient.

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