Study On Separation, Purification And Identification Of Embryonated Eggs Antioxidant Peptide

The human body will produce some reactive oxygen and other free radicalsproducts during human metabolic process, The excessive accumulation of thesesubstances will bring about oxidative damage to DNA, proteins and other biologicalmacromolecules, and cause harm to human health. Therefore, it is necessary todevelop some exogenous oxidants resistance to remove ROS and these free radicals.As bioactive activity peptides, antioxidant peptides are always extracted or producedfrom vegetable proteins，animal proteins, and microbial fermentation products. Andthey have become one of study hotspots in biological field, due to the features of highactivity, small molecular weight, easy absorption,natural and security.At the incubation temperature, fertilized eggs can develop into embryos and theembryos can survive from oxidative damage in high concentrations of oxygenenvironment. This biological phenomenon indicates that the embryonic eggs someantioxidant substances in embryonic eggs can protect the normal development of theembryo. During incubation, the proteins of embryonic eggs can be transformed intoabsorbable nutrients for the embryo, and low molecular weight peptides or aminoacids are always formed at first step. It is favorable to prepare antioxidant peptidesfrom embryonic egg. However, the preparation of antioxidant peptides fromembryonic eggs is rarely reported at domestic and abroad.The paper selected embryonated eggs white of San Huang chicken as rawmaterials to research the change of physicochemical properties and the antioxidantactivity of water-soluble protein (peptide) in the incubation period. The result showedthat the content of moisture, fat and pH value of egg white presented an upwardtrendduring the incubation period, while the content of ash and protein showed anincreasing trend, the polypeptide content raised at first, then decreased, andthe maximum (6.61mg/g) appeared at6th day. The water-soluble protein (peptide)has strong antioxidant activity, and Its DPPH and OH scavenging capacity and totalantioxidant capacity were enhanced with extended incubation time and the increasing sample concentration, respectively. The total antioxidant capacity of the6th sampleenhanced sharply with the increasing concentration of sample.In this dissertation，6days of fertilized eggs white was hydrolyzed by five typesof proteases. According to the DPPH radical scavenging rate of hydrolysates, andalkaline protease was chosen as the optimum enzyme to hydrolyze fertilized eggswhite for antioxidant peptides. The response surface analysis based on Box-Behnkendesign was employed to optimize hydrolysis conditions of antioxidant peptidesprepared by hydrolyzing fertilized eggs white, The result showed under the conditionof hydrolysis temperature46℃, pH9.1, substrate concentration4.28%, enzymeamount21000U/g, the DPPH radical scavenging rate was maximum and reached to84.97%.The protein hydrolysis was fractionated consecutively by ultrafiltrationmembranes with small tangential-flow ultrafiltration system.30kD,10kD, and5kD ofmolecular weight cut-off (MWCO) were used. Four fractions with more than30KD,30-10kD,10-5kD, less than5KDof MWCOs were seperated, and they werelabeled MWCOⅠ, MWCOⅡ, MWCOⅢ, MWCOⅣ, respectively. Theantioxidantactivities of these fractions and protein hydrolysis samples were evaluatedusing different antioxidant assays in vitro. The results indicated that MWCOⅠ hasthe strongest scavenging activities on DPPH radical, and when the concentration is30mg/ml, the scavenging rate reaches83.94%; however, MWCOⅣ has the strongestscavenging activities on hydroxyl radical, superoxide anion, total antioxidant capacityand reducing power, and when the concentration is10mg/mL, the hydroxyl radicalscavenging rate is47.91%, the superoxide anion scavenging capacity of69.23%, thelight absorption values are0.136and0.537at the wavelength of695nm and700nm,respectively, which revealed that MWCOⅣ has strong antioxidant activity.The fraction MWCOⅣ was separated and purified into14fractions bypreparative RP-HPLC and their antioxidantactivitywas evaluated by differentantioxidant assays in vitro.The results showed that the fraction13,10and7havestrong antioxidant activity. The purities of these three fractions were determined byanalytical RP-HPLC. It was found that the fraction10and7both displayed high purities. And they identified by Q-TOF-MS/MS. The result showed that the fraction10molecular weight is204Da, and its amino acid sequence is Ser-Val.