Study On The Composition Of Whey Protein And The Separation And Purification Of Antioxidant Peptide From Fresh Horse Milk

Objective: Composition differences by column chromatography and SDS-PAGE electrophoresis separation and identification of horse milk,milk,donkey milk,camel milk whey protein,and the separation and purification of fresh milk in Ruyuan horse of bioactive peptides,their biological activity assay,provide the basis for the study of fresh horse milk function.Method:(1)the 4 kinds of fresh milk in high speed centrifugation,whey protein isolate,by ion exchange chromatography and SDS-PAGE gel electrophoresis,four different kinds of milk whey protein components were identified,revealing the differences of protein.(2)fresh milk whey protein hydrolysate Ma were enriched and determine its antioxidant activity by 3Kd,10 Kd and 30 Kd ultrafiltration;choose active peptides with SephadexG-75 and RP-HPLC were purified,identified MALDI-TOF/TOF,obtained antioxidant peptide.(3)inhibition by MTT method for the determination of fresh horse milk antioxidant peptide on the proliferation of human lung cancer cell line A549,detect the cell cycle and apoptosis by flow cytometry,GSH,SOD,MDA through the detection of oxygen index,reveal its antioxidant ability.Results:(1)compared with fresh milk protein ingredients of fresh horse milk protein,found that there are three kinds of known protein,molecular weight were 69.60 KD,46.22 KD and26.35KD;compared with fresh donkey milk protein ingredients,found that there are three kinds of known protein,molecular weight were 100.92 KD,69.60 KD and 76.31KD;compared with the fresh camel milk protein ingredients,found that there are four kinds of known protein,molecular weight were 100.92 KD,95.56 KD,26.35 KD and 76.31 KD.(2)the horse milk whey hydrolysate by ultrafiltration and Sephadex G-75 gel Sephadex and semi preparative separation and purification of RP-HPLC MALDI-TOF/TOF mass spectrometry identification,preliminary determination of antioxidant peptide molecular weight is 1594.89 KD,the amino acid composition of VAPFPQPVVPYPQR.(3)MTT method.The experimental results show that the antioxidant peptides can inhibit theproliferation of A549 cells,the arrest in the G1 phase and increase cell apoptosis,compared with the model group,high dose group of antioxidant peptides increase GSH content and SOD activity,reduce the secretion of MDA,significant difference,statistically significant(P<0.05).Conclusion:(1)through the detection,horse milk and fresh camel milk,donkey milk,milk whey protein composition have certain differences.(2)through separation and purification and structural identification of molecular weight of 1594.89 KD,VAPFPQPVVPYPQR peptides consisting of antioxidant peptide of fresh mare's milk in Ruyuan.(3)antioxidant peptide can inhibit the proliferation of A549 lung cancer cells,increase the content of GSH,increase the activity of SOD,decrease the secretion of MDA,and has a certain antioxidant activity.