Study On Highly Efficient Extraction And Quantitative Determination Of Lysozyme In Chicken Egg White

In this paper, the lysozyme was extracted and purified from chicken egg white through modern separation techniques. And the separation efficiency of different process was compared. In addition, a new rapid method for determination of lysozyme using Resonance Rayleigh scattering techique has been developed. Finally, the enzymological properties and vitro antibacterial activity of lysozyme were analyzed. The main investigated results are listed as follows:The chicken egg white lysozyme was abstracted using ion-exchange chromatography. Effects of the resin type and its consumption, adsorption time, ammonium sulfate eluent concentration and elution time on the yield of lysozyme were studied respectively. Optimize the extraction process using orthogonal response surface combination design with three variables of cation exchange resin consumption (X1), adsorption time (X2) and ammonium sulfate concentration (X3).The regression equation is Y=0.339667+0.041 X1+0.038375 X2-0.021375 X3+0.00925 X1X2-0.00425 X1X3-0.0105 X2X3-0.051583 X12-0.068833 X22-0.039833 X32.The optimum extraction conditions were:resin accounts for 32% of the volume of egg white solution, adsorption for 6.5 h and the mass concentration of ammonium sulfate elution 9.3%.Under such conditions, it could produce lysozyme with yield of 0.324% and activity of 16300 U/mg.The lysozyme, which was extracted from chicken egg white using ion-exchange chromatography, was further desalted and purified by ultrafiltration technique. The 10 kDa polyether sulfone (PES) membrane was chosen for this process with transmembrane pressure 0.2 MPa, stirrer speed 200 r/min. The relationship between concentrate volumes, permeate flux and desalination rate was studied. As a result, when the volume of the concentrate solution was 10% of the original feed, more than 90% of (NH4)2SO4 could be removed. And the lysozyme yield was 0.275%;the enzymatic activity was 21500 U/mg.In addition, this article examines the separation of lysozyme directly from natural chicken egg white solution using ultrafiltration technique. The influence of transmembrane pressure, stirring speed, feed solution dilution multiple and pH value on permeate flux and protein permeability were discussed. Finally, the optimum conditions for ultrafiltration were obtained:diluting the egg white solution with 0.1 mol/L NaCl solution with the ratio of 1:1,pH 6.5,transmembrane pressure 0.2 MPa and stirrer speed 200 r/min. Separated lysozyme from chicken egg white under these conditions, the lysozyme yield was 0.301%;enzymatic activity was 18200 U/mg.A new method for determination of lysozyme based on Resonance Rayleigh scattering with gold nanoparticles as probe was proposed in this experiment. The Resonance Rayleigh scattering (RRS) spectrum, nonlinear scattering (SOS and FDS) spectra and absorption spectrum of the gold nanoparticles-lysozyme system have been analyzed. And the effects of several factors on scattering intensities were investigated, including pH value of solution, amount of gold nanoparticles, mixing sequence of each reagent and the coexisting substances, etc.The results showed that the coexisting substances have little influence on the RRS intensities of the systems. Moreover, the possible mechanism for the RRS enhancement of gold nanoparticles and lysozyme binding products was preliminary discussed. The RRS method for determination of lysozyme has good sensitivity and selectivity with the detection limits 30.1 ng/mL.And it was proved that the method established in this study is suitable for the determination of lysozyme in synthetic and natural chicken egg white samples.The studies of the enzymological properties of lysozyme indicated that it has the highest enzyme activity when pH was 6 and temperature was 55℃.In pH 6-7 and temperature range of 20-60℃,the activity of lysozyme could be well maintained and will not change greatly along with the changes of temperature. Most of metal ions, which can be well combination with lysozyme, have little effects on the enzyme activity. And the lysozyme could be activated by Na+,Ca2+,Mn2+ ions.Several surfactants have a certain inhibitory effect on lysozyme activity, but the inhibition is relatively weak.The vitro antimicrobial experiment showed that the lysoyme 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 0.313 mg/mL and 0.625 mg/mL for E. coli,1.25 mg/mL and 5.0 mg/mL for S. aureus,0.625 mg/mL and 1.25 mg/mL for Salmonella, respectively.