Study On Separation Of Egg White Proteins And Preparation Of ACE Inhibitory Peptides

Egg white contains abundant proteins,including lysozyme(LYZ),ovalbumin(OVA)and ovotransferrin(OVT).There are commonly methods for separating these proteins such as salting out method,ultrafiltration and ion exchange chromatography,but these methods are low separation efficiency,relatively complex operation,expensive equipment needs and other shortcomings.The purpose of this study was to study the regularity of precipitation of OVA,OVT and LYZ by polyethylene glycol(PEG),and to establish a new process for stepwise separating LYZ,OVA and OVT based on PEG method.Meanwhile,LYZ with high purity was obtained by using aqueous two-phase extraction(ATPE)and OVA was purified by anion-exchange chromatography.After isolation of OVA and LYZ,the possibility was explored that ACE inhibitory peptides was explored by enzymatic hydrolyzing egg white by-product.The reaction conditions were optimized by response surface method.The contents and experimental results were as follows:1、Firstly,the influence of precipitation of OVA,OVT and LYZ was researched by divers PEG in different condition of various concentration and pH.The results showed that PEG 4000,6000,8000 and 10000 could precipitate OVA,OVT and LYZ effectively.The precipitation rate was affected by mass fraction of PEG and pH significantly.The difference of precipitation rates of OVA,OVT and LYZ was the highest when pH was 7.5 and the mass fraction of PEG was 12%,the precipitation rates were 1.8%,54.6% and 68.3% respectively.When the pH was adjusted from 7.5to 5.5,the amplitude of variation of the three protein precipitation rates was also the largest,the precipitation rates of OVA,OVT and LYZ increased by 7.9%,43.5% and21.9% respectively.According to the above rules,the process for separating OVA was established based on PEG precipitation method: PEG 4000 was added to the egg white of pH 7.5 to 12% by mass.The supernatant was collected by centrifugation to obtain OVA with a purity of 88.1% and a recovery of 95.1%;when the pH ofresulting supernatant was adjusted to 5.5,the purity of OVA increased to 99.7%,the recovery was 87.3%.2、Secondly,the process route of stepwise separating OVA,OVT and LYZ was established by PEG method.The results showed that when PEG 4000 was added to the egg white of pH 5.0 to the final mass fraction to 16%,after centrifugation,supernatant I was collected to obtain LYZ(recovery 74.7%)with a purity of 58.2%.In the meantime,20 mM Tris-HCl(pH 8.0)dissolved the precipitation I,the quality of 31.6% of the 50% PEG 4000(w/w)was added continuously.After centrifugation,supernatant II was collected to obtain OVA(recovery 92.8%)with a purity of 83.41%,OVT with a purity of 89.53%(recovery 90.7%)was getted in the precipitation II.3、Thirdly,the processes of purification of the above LYZ and OVA by ATPE and ion exchange chromatography were studied.The results showed that PEG-(NH4)2SO4 aqueous two-phase extraction could be structured by adding(NH4)2SO4 solution with 10% mass ratio to the supernatant I which contained LYZ.After centrifugation,LYZ with a purity of 96.34% was obtained in the supernatant(recovery 70.2%),the specific enzyme activity of LYZ was 25000 U/mg.Whereafter,OVA which existed in the supernatant II was purified by anion resin.The results showed that the static adsorption capacity of per gram of FPA 98 resin for OVA was4.42 mg/g,the elution effect of eluent which contained NaAc,NaCl and MgCl2 on OVA was studied,a fractional elution process which contained 50 mM NaAc in the eluent was established to remove impure protein such as OVT.Step elution process which contained 50 m M MgCl2 in the eluent was adopted to elute OVA,an electrophoretic pure OVA with a recovery of 66.2% could be obtained in the MgCl2 eluant.4、Fourthly,the processes of preparing high activity ACE inhibitory peptides were also studied by using residual egg white byproducts after separation of OVA and LYZ as the raw materia which hydrolyzed by Bromelain.The enzymolysis processwas optimized by response surface analysis method based on the single factor experiment that had three investigate factors of enzymolysis time(h),enzyme ratio([E]/[S])and initial pH.The results showed that the optimum conditions for the preparation of ACE inhibitory peptides were as follows: enzymolysis time 8.02 h,enzyme ratio([E]/[S])7%,initial pH 6.87,and The ACE inhibitory rate of the hydrolyzate can reach 56.00%.In this paper,the regularity of precipitation of OVA,OVT and LYZ by PEG method was explored,a new process of stepwise separation of OVA,OVT and LYZ based on PEG method was established.The process of purifying LYZ using PEG-(NH4)2SO4 aqueous two-phase extraction was established,the process of purification of OVA by ion exchange chromatography of FPA 98 resin was explored.High activity ACE inhibitory peptides were prepared using egg white byproducts after isolating of OVA and LYZ.The above results not only help to make full use of Chinese surplus egg resources,but also help to improve the level of deep processing of eggs in China.It also provided a theoretical basis for the development of new functional foods.