Extraction Of Dairy Cow Derived Polypeptides And Analysis & Identification For The Reductive Component

To prepare reductive polypeptides from dairy cows' placentae with high efficiency and investigate their molecular construction.This study used Ultrasonication,trypsin,papain,and pepsin hydrolysis to prepare the freeze-dried dairy cows placental polypeptides,measured the extraction rates and reducibility(via Prussian blue method).Then acquired the optimal extract conditions by orthogonal and response surface models.Furthermore,established the integrated extraction rate,which combined the extraction rates and reducibility,as an evaluation index,then re-optimize extract conditions by response surface model.Then screened the antioxidative placental polypeptides by Label-free methodology and BIOPEP database,analyzed the bioactive residuals of those polypeptides.Provide the theological basis for the utilization and further research of dairy cows' placenta.Experiment 1: Establishment and optimization of the extraction of placental reductive polypeptides from the dairy cow Result showed that:(1)natural reductive polypeptides exist in the placenta of dairy cows.The optimal extract conditions were: 50 %concentration,1800 w and 7.5 min for Ultrasonication,corresponding extraction rate 0.95 %.Vit C equivalent 2.80 ?g/mg;hydrolysis 5.78 h,6.18 h and 6.22 h,substrate concentration30.21 %,34.78 % and 30.71 %,enzyme/substrate ratio 3.24 %,5.00 % and 3.18 % for trypsin,papain,and pepsin can acquire highest extraction rate: 5.55 %,6.97 % and 6.14 % respectively;hydrolysis 5.13 h,3.95 h and 4.83 h,substrate concentration 35.59 %,34.78 % and 35.82 %,enzyme/substrate ratio 4.19 %,3.64 % and 3.57 % for trypsin,papain,and pepsin can acquire highest reducibility: 39.32 ?g/mg,70.54 ?g/mg and 58.59 ?g/mg respectively.(2)Nonreductive proteins and be enzymatically hydrolyzed into reductive peptides by trypsin,papain,and pepsin,in which,papain has the highest efficiency.In addition,apart from trypsin,polypeptides prepared by papain and pepsin showed no significant correlations between reducibility and extraction rate.The extraction rate of placental polypeptides showed a positive correlation with all factors,but reducibility performed parabola-like trends with those factors.(3)Optimal extract condition evaluated by integrated extraction rate were: hydrolysis 5.8 h,4.7h and 5.49 h,substrate concentration 34.96 %,34.03 % and 35.74 %,enzyme/substrate ratio3.33 %,3.66 % and 3.92 %,corresponding integrated extraction were 2037.92 ?g/g,4414.09?g/g and 3490.41 ?g/g for trypsin,papain,and pepsin respectively.Experiment 2: Analysis and identification of reductive placental polypeptides based on Label-free methodology Result showed that:(1)Excluded repetitions,727 proteins and4321 polypeptides were identified in total.541,86,137 proteins;2766,129 and 1171 peptides were identified from trypsin,papain and pepsin groups,corresponding 74.40%,11.80% and18.8% of total protein numbers,and 64.0%,2.90% and 27.10% of total protein numbers respectively.Indicated the papain have the highest hydrolysis efficiency.(2)The reducibility and antioxidative functions are provided by amino acid residuals like Ile-Arg,Glu-Leu,and Leu-Lys.Conclusion:(1)Natural reductive polypeptides exist in the placenta of dairy cows.The optimal extract condition for trypsin,papain,and pepsin were 5.80 h,4.70 h and 5.49 h,substrate concentration 34.96 %,34.03 % and 35.74 %,enzyme/substrate ratio 3.33 %,3.66 %and 3.92 %,corresponding integrated extraction rate were 2037.92 ?g/g,4414.09 ?g/g and,3490.41 ?g/g respectively.In which,papain has the highest hydrolysis efficiency;(2)The reducibility and antioxidative functions are provided by amino acid residuals like Ile-Arg,GluLeu,and Leu-Lys.