Antioxidative Effects Of Plastein Reaction-Stressed Hydrolysates Introduced With Extrinsic Amino Acids

Natural proteins have a lot of biological activities, such as antioxidative, antibacterial,&antihypertensive activities, which can be widely used in many kinds of food. But the primary structure of active peptides derived from a part of the material proteins, therefore their activities were also limited by the primary structures of material proteins. Because the active peptides obtained by enzymatic hydrolysis of the proteins could not achieve the demand of the biological activity, researchers focused on the modification of peptides to obtain active peptides which were of higher activity.Firstly, hydrolysates were prepared by hydrolyzing soybean proteins and casein with different proteases, and the extrinsic amino acids, tyrosine, phenylalanine or tryptophane, were introduced into the above hydrolysates through plastein reaction. Secondly, the ethanol-water solvents (3:7, v/v) were used to fractionate the modified hydrolysates with plastein reaction. The antioxidative activity of modified hydrolysates and fractionated products were analyzed, and hydrolysates of soybean protein and casein were used as controls. The peroxidation lesion model of human fetal hepatocytes was built, and the protecting effects of fractionated hydrolysates to the H2O2-induced injury cells were estimated. The results obtained in this study were as followed:(1) Soybean protein and casein were hydrolyzed with alcalase and papain respectively to prepare protein hydrolysates, and then the hydrolysis time was determined by estimating their degree of hydrolysis (DH), peptide recovery and antioxidant activities. Therefore, after soybean protein and casein were hydrolyzed for6h and2h, the hydrolysates were optimal substrates of plastein reaction, and the DH of soybean protein and casein were10.12%and9.42%, and DPPH radical of soybean protein and casein were29.18%and38.71%, separately.(2) Extrinsic amino acids (tyrosine phenylalanine or tryptophane) were added into the soybean protein and casein hydrolysate on the level of0.60mol/mol and0.74mol/mol that were determined with single factor, separately.(3) The ethanol-water solvents (3:7, v/v) were used to fractionate the modified hydrolysates which have higher antioxidant. The supernate of the fractionated production had higher DPPH radical activity, superoxide radical and reducing power compared with the sediment, the obtained supernatant or precipitate had an enhanced or decreased antioxidant activity. These results demonstrated that fractionation can effectively extract antioxidant peptides with better activity.(4) The peroxidation lesion model of human fetal hepatocytes was employed to assess hepatoprotective effect of the hydrolysates against H2O2-induced injury. Human fetal hepatocytes of logarithmic phase were preincubated with antioxidant peptides (0.5,1,2mg/mL) at37℃for24h,48h and72h, separately. And then were oxidative injuried by H2O2(5mmol/L), for1.5h to establish the model, while the control group was not added with antioxidant peptides or H2O2. Cell viability was measured by the MTT assaying. The survival rates of lesion cells were significantly improved which had pretreated with antioxidant peptides. It was suggested that damaged human liver cells induced by H2O2were protected by hydrolysates from plastein reaction modification.(5) Human fetal hepatocytes were preincubated with antioxidant peptides lmg/mL at37℃for48h. As a result of that modification products can decrease of intracellular MDA formation and increase of the GSH and CAT activity, it had a protective effect on cells.