Structure-activity Relationship Of Antioxidative Peptides Derived From Whey Protein

Synthetic antioxidants have been commonly used to preserve food products. Because of their potential health hazard, interest in utilizing natural antioxidant peptides has increased substantially. Recently, studies have found there are sequences with antioxidant activity in whey protein. However, less structure-acitivity relationship information was available. In this study, whey protein was hydrolyzed and purified to obtain peptides with different antioxidant activities. The structure-activity relationship of perified peptides was elucidated by the theoretical calculation. The aim was to expand scope of application of cheese by-products, as well as provide theoretical reference for the research on antioxidant peptide.Initially, the preparation conditions of WPH were researched. Six types of proteases were selected through comparing hydrolysis degree and DPPH·scavenging-activity. We found the optimum protease was Alcalase 2.4L, and 2 h was selected as the hydrolysis time.With 60% ethanol as desorption agent, the adsorption rate and the adsorption capacity were 70.80% and 28.15 mg/g, respectively; the desorption rate and the reclaim rate were 93.21% and 65.74%, respectively. After desalination, the IC50 of DPPH·and O2-·decreased to 3.92 mg/mL and 5.00 mg/mL from 4.21 mg/mL and 8.18 mg/mL. The lipid peroxidation inhibition was impoved, increased from 41.98% to 56.49%. The content of hydrophobic amino acid increased from 29.04 g/100g to 35.34 g/100g.Moreover, after isolated by gel filtration chromatography, semi-preparative RP-HPLC and analytic RP-HPLC, eight compenents with different antioxidant activities were achieved. Identified by Q-TOF MS, the amino acid sequences were D3a(Arg-Val-Tyr), D4a (Ala-His-Leu-Trp), D4b(Gly-Thr-Ser-Val), D5a(Val-Phe), D5b(Try-Ser-Leu), D6b (Leu-Phe),D7a (Val-Ala-Gly-Thr-Ser-Val-Tyr), D9a (Trp-Tyr-Ser-Leu)。The last peptide displayed the highest superoxide radical-scavenging activity (87.90±4.00% at 500μmol/L).The structure-activity relationship of antioxidant activity peptides with the amino acid sequence of VF,LF,RVY,YSL,WYSL and GSTV was elucidated by the theoretical calculation. Using the calculation of molecular mechanics and quantum chemistry, the chemical structures of the six peptides were simulated. The energies of frontier orbital and the population of charges were also studied. The results show that the frontier orbital energy gap and antioxidant activity of the peptide is related, the frontier orbital energy gap can be used to characterize the size of the biological activity of peptide molecules. It was conjectured that the situs of reactivity scavenging radical is located at the N9-H49 of the indole ring of WYSL. The dihedral angle of the indole ring shows that the dihedral angle is very small, highly mobile charge, which will help dehydrogenation reaction.