Research On Ovotransferrin Purification, Structure Characterization And Antimicrobial Tasts In Vitro

Ovotransferrin have important physiological functions, such as Fe3+ transferrin, antimicrobial properties, inhibit the formation of free radicals and so on. Ovotransferrin in egg white is rich, however, relatively slow progress have been maken on ovotransferrin isolation and puriication.In this paper, the ovotransferrin was extracted and purified from chicken egg white respectively through ultrafiltration and iron exchange chromatography. Firstly, the different impact factors were studied during ovotransferrin extraction and purification. And then the physical and chemical properties were discussed, what's more, the structural characterization of iron-bind ovotransferrin was studied. Finally, the vitro antibacterial activity of ovotransferrin were analyzed. The main investigated results are listed as follows:1. The chicken egg white ovotransferrin was abstracted using ultrafiltration. Effects of ways to reducing the viscosity of liquid egg white was studied, and the results is diluting 15 times, shearing rate of 7 kr/min and the temperature is 45℃. The conditions during ultrafiltration is diluting 15 times, stirring rate of 125 r/min, operating pressure 0.15 MPa and the pH value is 7. Based on the single-factor test, selecting the operating pressure, dilution ratio, and the stirring rate as the factors of orthogonal test, optimizing the conditions of ultrafiltration process and the result is A3B2C2. Owing to A3B2C2 is not in L9(34) orthogonal design table, compared A3B2C2 with A3B2C1 and determined the optimal condition is A3B2C2. By SDS-PAGE gel electrophoresis and HPLC, confirming that the isolated protein is ovotransferrin and the purity is 29.6%.2. After ovomucoid was removed from fresh egg white, fresh egg white was purified by ammonium sulfate fractionation. The method consists of a preliminary step of frontal chromatography, also called displacement chromatography, on a Q Sepharose Fast Flow column. This simple and powerful chromatographic step enables the pre-purification of ovotransferrin that is further purified by a second conventional preparative chromatography performed on the same Q Sepharose FF column. The ovotransferrin obtained was detected by sodium dodecyl sulfate polyacrylamide gel electropheresis and high-performance liquid chromatography. Its purity rate is calculated at 98.3%.3. The iron binding capacity of ovotransferrin was studied as well as impact factors of iron binding capacity. In neutral or alkaline environment, the iron binding capacity of ovotransferrin was good; 0,40,80,120 and 160 mmol/L sodium bicarbonate were added respectively and found 120 mmol/L concentration of HCO3- on the binding of iron is more effective; keeping ovotransferrin at 55℃,60℃,65℃for 30 min respectively and capillary storing at 70℃for 20 s, then rapid cooling to 25℃, finding that sterilization at 60℃can maintain the iron binding capacity of ovotransferrin; CD analysis revealed that with the increase of iron binding degree, a-helix was increased,β-shee decreased corresponding,β-turn had a slight increase, whlie random on the contrary; ANS fluorescence probe measured the surface hydrophobicity index was 70.35; Zeta potential and average particle size measurements show that with the increase of iron binding degree, positively charged iron and the negatively charged amino acid residues of ovotransferrin binding closely, which makes the orginal stretch spatial structure of ovotransferrin becoming much closer; AR 1000 rheometer measured the critical gel concentration of ovotransferrin was 20%.4. The vitro antimicrobial experiment showed that the ovotransferrin has better inhibitory effect on E.coli and Salmonella, It also has a certain inhibitory effects on S.aureus at higher concentration. The minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) are 2.5 mg/mL and 10.0 mg/mL for E.coli, 5.0 mg/mL and 15.0 mg/mL for Salmonella,3.5 mg/mL and 12.5 mg/mL for S.aureus, respectively. The antibacterial effect of different iron-binding degree of ovotransferrin against E.cili, Salmonella, Staphylococcus aureus become weak with the iron-binding degree increase. At the same time, Fe3+-binding ovotransferrin have the bese antibacteral against E.cili, Salmonella and Staphylococcus aureus.Ovotransferrin can improve the immune capacity, which has important significance to enhance the body resistance to disease. Breast-fed infants have stronger antibacterial resistance than milk-fed, because breast milk contains more lactoferrin. Ovotransferrin develop as a biological function has broad prospects.