Separation,Purification And Active Evaluation Of Anti-inflammatory Peptides From Whey Protein Hydrolysate

Whey protein is a kind of mixed protein with high nutritional values,which is extracted from milk.Whey peptides,the hydrolysates of whey protein,are easily digested and absorbed by human body.Many of them showed a variety of physiological functions such as antioxidant,anti-hypertension,anti-inflammatory activity,regulation of body metabolism,and are well known as the "best of the best".This thesis focused on preparation of anti-inflammatory peptides obtained from whey protein hydrolysate by alcalase,and optimization of the conditions of ultrafiltration,Sephadex G-25 gel column chromatography and semi-preparative HPLC.From the fraction with MW < 1 kD of whey protein hydrolysate,eight anti-inflammatory peptides were identified by HR-MS,including two new peptides Asp-Tyr-Lys-Lys-Tyr(DYKKY)and Asp-Gln-Trp-Leu(DQWL).Eight synthetic peptides significantly suppressed the expression of IL-1?,COX-2,and TNF-? mRNAs in the lipopolysaccharide-induced RAW 264.7 mouse macrophage cells,indicating their potential anti-inflammatory activities.Among them,DQWL showed the highest inhibitory ability on IL-1?,COX-2 and TNF-? expression both on mRNA and protein levels at the concentration of 10 and 100 ?g/mL.In addition,DQWL treatment significantly inhibited NF-?B activation by suppressing the nuclear translocation of NF-?B p65 and blocking IKK ?/? phosphorylation and I?B ? degradation together with p38 MAPK activation.Our results show that peptide DQWL has the potential anti-inflammatory effects.Isolation of peptide with high purity through multi-step purification steps,which provides a good sample for the extraction,separation and composition analysis of peptides in the future.Moreover,the discovery of new peptides sequence has provided theoretical basis for the development of functional foods,dietary supplements and cosmetics.