Directional Preparation Of Hazelnut Protein-derived Antioxidant Peptides And Their Inhibition Against Oil Oxidation

Oil oxidation always causes a big nutrition loss and furtherly deteriorate the quality of the emulsified foods.Any attempt able to stop oil from oxidation has been attractive for food industry.It is well-known that the utilization of antioxidant additives is an effective approach in this aspect.Bioactive peptides have become an ideal option for antioxidants due to wide material source,excellent antioxidant activity and potential intoxication.However,the labor-consuming preparation in manufacturing bio-peptides as well as the uncertain relationship between structure and activity bring a big challenge for their industrialized promotion.In this study,hazelnut protein-original biopeptides through in silico technology were prepared according to the “A-M-G” theory.The characterizations of hazelnut-original peptides were analyzed by evaluating their roles in improving the chemical and physical status of the tested emulsion system.Furtherly,how to improve the antioxidant capacity of peptide by alkylation modification was discussed.The following are main results.(1)Establishment of a virtualization tool for screening oil oxidation-inhibiting peptides.This work firstly discussed the possibility of amino acid residues from 20 amino acids and 22 artificial-made dipeptides in influencing the antioxidant capacity of peptides via chemical experiments and electron density distribution analysis.The relationship between the structural information of each synthesized dipeptide and its antioxidant activity was then evaluated.It was seen that only the presence of Tyr,Trp,and Met residues or the synthesized dipeptides containing these active amino acids showed an ability to inhibit linoleic acid oxidation.Tyr,Trp,and Met residues are found to be active sites of the potential antioxidant dipeptides.In addition,the big data collected from BIOPEP database and published articles was integrated to analyze the structural properties of oil oxidation inhibiting peptides.It is seen that the antioxidant capacity of each candidate peptide depends largely upon its molecular weight(Mw),Grand average of hydropathy(GRAVY),and position of the active amino acid residues in sequence.It is clear that the peptides with properties: with Mw ranging from 200 to 800 Da,with GRAVY ranging from-2 to1 and containing Tyr,Trp,or Met at C/N-terminal,might act as a virtualization filter for selecting the wanted bioactive peptides.This rule was known as the “A-M-G” theory.(2)Presentation of a bioinformatic-based technique for screening hazelnut-original peptides against oil oxidation.Based on the “A-M-G” theory,the hydrolysates of hazelnut protein purposely cleaved by alkaline protease were harvested,and then the desirable antioxidant peptides were selected.Next,several synthesized peptides were examined for verifying the antioxidant activity.It was seen that the 5 peptides FSEY,QIESW,SEGFEW,IDLGTTY,and GEGFFEM,derived from hazelnut protein,showed good inhibition activity against linoleic acid oxidation.(3)Determination of Active sites and free radical scavenging mechanisms of antioxidant peptides.To confirm the active sites of hazelnut-original oligopeptides,and explain the scavenging free radical mechanism of Tyr containing peptide,Density Functional Theory(DFT)was introduced to calculate the structural information of these bio-peptides.It is observed that active sites of the four antioxidant peptides,FSEY,QIESW,SEGFEW and IDLGTTY,are located at Tyr,Trp,Trp,and Tyr residues,respectively.Fukui functions indicated that the phenolic hydroxyl group in the structure of peptide FSEY act as both electron-giving and free radical attack site.Inaddition,two mechanisms for peptide FSEY to scavenge lipid free radical are presented.The first is One step H-atom transfer(HAT)model,and another is Sequential Proton Loss-Electron Transfer(SPLET)model.(4)Inhibition of the hazelnut-original oligopeptide FSEY against oil oxidation.To investigate the potential of oligopeptide FSEY as an antioxidant additive,this peptide was added to an emulsion system incubated for a period of time.The formation of oxidative products and physical stability of the emulsion during a given period of accelerated oxidation experiments were analyzed in the presence or absence of peptide FSEY.It was found that peptide FSEY maintained the chemical stability of the emulsion very well by scavenging free radicals and then inhibiting the generation of primary and secondary oxidation products.As well,peptide FSEY kept the emulsion’s physical stability better by inhibiting the accumulation of hydroperoxides and by steric hindrance effect,preventing the increase in particle size,stabilizing the original color,and alleviating the acidification of the emulsion.(5)Improvement of oil oxidation-inhibiting capacity of oligopeptide FSEY via lipidation modification.To evaluate the effects of lipidation modification on the antioxidant activity of hazelnutoriginal peptide,oligopeptide FSEY was modified via alkylation.Data from antioxidant test,water phase distribution and critical micelle concentration showed that a modification of alkyl significantly increased the antioxidant ability of FSEY against oil oxidation.Especially,the modification of 4 carbons to peptide,i.e.,C4-FSEY,leaded this peptide to perform the best antioxidant activity.The nonlinear phenomenon is attributed to the mechanisms of “reduced mobility” and “internalization”.In summary,our work presents a practical route for successfully screening the desirable active antioxidant peptides from hazelnut protein hydrolysates in terms of the importance of active amino acids in antioxidant peptide sequence.It reveals the free radical-scavenging mechanisms of peptide FSEY,and also illustrates the transfer rules of Cx-FSEY in water-interface-oil phases.Importantly,this work not only innovates a theoretical guide for the directional preparation of antioxidant peptides from any sources,but offers a novel insight to the structural modification of oil oxidation-inhibiting peptides as well.