Preparation Of Egg-yolk Phosvitin Phosphopeptide And Research On Its In-vitro Anti-inflammatory Activity

Phosvitin phosphopeptides(PPPs)from hen-egg yolk,which have been found to possess antimicrobial,antioxidant activity and calcium absorption promotion capacity,as well as potential anti-inflammatory ability.In this paper,technologcial conditions for the preparation of Phosvitin and its phosphorpeptides were determined,and also anti-inflammatory ability of PPPs has been evaluated at cellular level.Anion exchange chromatography was performed to further purify phosvitin(PVs)from egg yolk,and NaAc/HAc buffer(20 mmol/L,pH 5.0)was used as starting buffer,followed by the step-by-step elution with 0.3,0.5,0.6 mol/L NaCl solution.The established method has a high recovery rate(almost 57.6%)and putity(95.43%)with N/P ratio being 2.78 and protein content 85.66%.The final product was composed of ?-PVs,?1-PVs and the ?2-PVs with their molecular weight being 44.4 Ku,37.5 Ku and 42.0 Ku respectively and the relative content 67.95%,4.17% and 23.30% respectively.Then,with enzymatic hydrolysis(DH)and inhibition activity on LPS-indued RAW264.7 cell secretion of NO(inhibition rate,IA)as the response value,the enzymatic conditions of PVs were determined by single factor experiment and response surface methodology.Among them: the single-factor test determined the substrate concentration of(2%)and hydrolysis time(3 h).NaOH pretreatment concentration,pH and temperature as the response surface methodology were selected to further optimize the factors,but also it was proved that the appropriate control of alkali concentration of NaOH pretreatment could control dephosphorization rate of PVs.PVs with different dephosphorization rate has different performance in suppression of RAW264.7 cells producing the amount of NO.central composite response surface methodology rotation regression model was used to optimize the value of the NaOH pretreatment concentration,pH and the reaction temperature,and these three factors were 0.26 mol/L,8.12,40.39°C;the end products PPPs under this contions has high IA level(61.25%),which ash content was 2.03%,moisture content 1.50% and P/N 11.97.Finally,the expression level of cytokines(TNF-?,IL-6 and IL-8)and NO was studied in the LPS indued Caco-2 and RAW264.7 cells after pretrement of PPPs.PPPs significantly inhibited the synthesis amout of NO in the both cell lines in the dose-dependented manner,and the inhibition rate on RAW264.7 cells was higher than the Caco-2 cell: when the concentration of PPPs was 1000 ?g/ml,the IA on RAW264.7 cells was 70%,while on the Caco-2 cell inhibition rate was only 47%;PPPs significantly inhibited LPS-induced secretion of TNF-? in both cell lines in the dose-dependted manner,and the inhibition effect reached the best when PPPs concentration was 1000?g/ml;PPPs significantly inhibited IL-6 secretion of RAW264.7 cells in a dose-dependent manner within a concentration range of 0.1-100 ?g/ml;PPPs with concentration higher than 10 ?g/ml significantly inhibited IL-8 secretion of Caco-2 cells,and in the concentration range of 10-1000 ?g/ml,this inhibition effect porformed in a dose-dependent manner.The results showed that PPPs played anti-inflammatory effects on cells in vitro through lowering the levels of expression of inflammatory factors.