Analysis And Identification Of Antioxidant Peptides From Olive Kernel Protein

Olive nucleolus is rich in protein,which is one of the plant proteins with high nutrition.Most olive kernels are used to extract oil,and the residue is often discarded as a by-product of processing.In this paper,olive kernel was selected as raw material,olive kernel protein was extracted,and olive kernel polypeptide was prepared by enzymatic hydrolysis,and it was identified and analyzed.The main experimental results are as the following:(1)Using defatted olive kernel as raw material,olive kernel protein was extracted by alkali dissolving and acid precipitation method.The extraction process of olive kernel protein was optimized by single factor experiment and orthogonal experiment.The optimum extraction conditions were as follows: ratio of material to liquid 1:30(g/m L),p H=11.0,temperature 55 ℃,time 100 min.Under these conditions,the extraction rate of olive kernel protein was 76.52 %,and the physical and chemical properties of protein(freeze-dried powder)under different conditions were determined.(2)Using defatted olive kernel as raw material,four component proteins were extracted by Osborne method,which were albumin,globulin,gliadin and glutenin.The results of SDS-PAGE electrophoresis showed that the molecular weight distribution of albumin subunit was14-28 k Da,the molecular weight distribution of globulin was about 14-45 k Da,the molecular weight of glutenin subunit was 18-60 k Da,and gliadin had no band.The secondary structures of the four proteins were analyzed.the results showed that albumin was dominated by α-helix structure,globulin and gliadin were dominated by β-corner structure,and glutenin was dominated by β-fold structure.Compared with the physical and chemical properties of the four proteins,globulin had the highest water holding capacity,glutenin had the best oil retention and foaming ability,but poor foam stability and the best emulsifying property of gliadin.(3)Six kinds of single enzyme were selected to hydrolyze olive kernel protein.the results showed that papain was suitable for preparing antioxidant peptides of olive kernel.On the basis of the single factor experiment of papain hydrolysis of olive kernel protein,response surface analysis was again optimized to determine the best conditions of enzymatic hydrolysis: enzyme bottom ratio 3.3 %,temperature 61 ℃,time 88 min.Under this condition,the DPPH free radical scavenging rate of olive kernel polypeptide was 69.53 %.Among the six in vitro antioxidant models,olive kernel peptides showed good antioxidant activity in other models,except for their low scavenging ability to hydroxyl radicals and superoxide ions,and had a significant dose-effect relationship.(4)The olive kernel polypeptide powder was identified by nano-HPLC-MS/MS,all peptides below 3k Da were identified,proteins and peptides of known sequences were removed,brand-new peptides with confidence greater than 95% were screened,and the peptides of different lengths(4-10 amino acids)were selected,WLSF,TSVLR,LVYLVR,AGGKPFQ,YYPFKGFA,AFNVDERLAR.The peptides were synthesized by rapid Fmoc method to verify their antioxidant activity,and the structural properties and safety of six antioxidant peptides were predicted by means of biological information.it was found that these six peptides had high antioxidant activity and safe and non-toxic.