Research On The Preparation And Antibacterial Activity Of Antimicrobial Peptide From Ovotransferrin

Over the years, many scholars have been working on the antibacterial peptides, the results achieved will be more, not only discovered their antibacterial activity, also announced a partial amino acid sequence, Tomita and other scholars have been discovered that the antibacterial function of the active peptides which come from the hydrolyzate of the bovine Lactoferrin was degraded by protease; Evan F. Haney et al. discovered a new sequence of cationic antibacterial peptides from the N-terminal of bovine lactoferrin, was named as lactoferrampin; because of Ovotransferrin(OVT) and bovine lactoferrin(Lf) are the same nature protein, thus, Ovotransferrin was hydrolyzated by the appropriate protease, we will acquired the peptide with antibacterial activity. The current study on antibacterial peptide isolated from Ovotransferrin has been founded by Hisham R. Ibrahim, which obtained by the enzymatic methods, it's made of 92 amino acid residues.The main contents of this paper are:(1)The optimum hydrolysis conditions of preparing the Ovotransferrin antibacterial peptides by pepsin digests Ovotransferrin are under the temperature 40℃, pH value is 2.0, the concentration of substrate is 4%, the ratio of enzyme and substrate is 1:100, the hydrolysis time is 2h, its antibacterial activity is better; according to the variance analysis of orthogonal analysis, the significance of the concentration of substrate and pH value on the experimental results were 0.05, the significance of the ratio of enzyme and substrate on the experimental results was 0.1, the effect of time on the experimental results was not significant;On the experimental study of preparing the Ovotransferrin antibacterial peptides by trypsin digest Ovotransferrin, examining the influence of the changes of the concentration of substrate, pH value, reaction time, temperature, of four factors on the enzymatic experiment, the results are shown that the antibacterial activity of peptide which comes from hydrolyzate by trypsin was not obvious, and under some conditions, there is no activity;The experimental studies on double enzymes to digest Ovotransferrin to prepare antibacterial peptides have shown that, after digesting by pepsin, used the trypsin to secondary hydrolysis can not improved the antibacterial activity, and even some conditions will decrease the activity, thus, the methods of single enzyme of trypsin and double-enzymes hydrolysis are also not suitable for the preparation of antibacterial peptides from Ovotransferrin.(2)By high performance liquid chromatography, using TSK2000 gel column, determined the molecular weight range of antibacterial activity peptide mixture which are from Ovotransferrin was digested by pepsin, measured the peak time of standard bacitracin, bovine insulin, cytochrome C at 19.990min, 17.669min and 14.00min respectively, according to the relationship between molecular weight and retention time, acquired the regression equation is: Y =-0.1354X + 6.0129, the relative coefficient of R2 is 0.9941. according to the regression equation, calculated the molecular weight range of antibacterial activity peptide mixtures which are from Ovotransferrin was digested by pepsin, and after initial separation by ultrafiltration membrane is about 3000-9000 Da; this article study mainly on the separation,purify and antibacterial activity of peptides with the molecular weight range is about 2300-4800 Da.(3)Using the method of column chromatography, choosing the Sephadex G-50 as medium to separate Ovotransferrin antibacterial peptide mixture, according to investigating distilled water and different gradient concentrations of NaCl as the mobile phase, and different concentration of the sample and different volume of the sample impact on the separation processes, through the exploration can be drawn, the best purified conditions are: the distilled water as the mobile phase, determine wavelength of 220nm, the flow rate of 1.5mL/min, the concentration of sample is 0.2mg/mL, the sample volume 0.2mL, the isolated effect to the sample is best, and the sample was isolated from three single peaks.(4)By the high performance liquid chromatography, measured the molecular weight range of the three components were purified by Sephadex G-50. Drawing the standard curve between the molecular weight of standards and peak time, the peak time of standard bacitracin, bovine insulin, cytochrome C at 20.178min, 15.796min and 13.056min respectively, the regression equation of standard curve is: Y =- 0.1327X +5.8333, the relative coefficient of R2 is 0.9989; according to the equation, calculated the molecular weight range of the three peaks were purified by Sephadex G-50;The experiment of antimicrobial activity showed that the purified Ovotransferrin antibacterial activity peptide have the activity against gram-negative bacteria and gram-positive bacteria activity, and more sensitive to gram-positive bacteria; and antibacterial activity of the peak 1 is higher than the peak 2 and peak 3; we known by HPLC spectrum, the molecular weight of the peak 1 is about 3015 Da, the purity is high;By the analysis of the spectrum of amino acid showed that, the purified peak 1 with positive charges, and with a cationic nature, in consistent with the nature of antibacterial peptides;According to the study on the stability of peak1, It has better thermal stability, pH value within the range of 1-10 also has good adaptability, still has good activity; in boiling water heated to 100℃about 30min, the activity only slightly reduced; the antibacterial activity after repeated freezing and thawing several times did not decline;According to the experiment of the minimum inhibitory concentration, we known that, the MIC of the peak 1 on S.aureus is 60μg/mL; the MIC of the peak 1 on E.coli is 100μg/mL.The study on antibacterial peptides as one of the research hotspots, while it has few studies on Ovotransferrin as the raw material is extracted from eggs to preparing the antibacterial peptides, only a few foreign scholars have carried out; this article will use the enzymatic technology, separate the antibacterial peptides by column chromatography, screen the new peptides with antibacterial activity, provide the basis for the comprehensive development and utilization of egg white protein, lay the research basis for the further applications of antibacterial peptides on the field of food and medicine.