Abstract:

【Objective】The objective of this study was to investigate the effects of malondialdehyde (MDA), a final product of lipid peroxidation, on the structure and emulsifying properties of walnut protein isolate (WPI). Understanding the molecular mechanism, by which the oxidative modification of WPI influences its functional property, could provide a theoretical basis for mitigating oxidative deterioration of WPI-stabilized emulsion.【Method】The walnut protein was isolated by alkali dissolution and acid precipitation. MDA was generated by adding 1,1,3,3-tetraethylcyclopropane (final concentrations of 0, 0.1, 1, 5, 10 and 20 mmol·L^-1) to react with the WPI at room temperature for 24 h. Excess MDA was removed by dialysis, and MDA-oxidized WPI was freeze-dried prior to analysis. Oxidative stress markers (thiol, disulfide bond, amine and carbonyl group), physicochemical properties (solubility, turbidity, hydrophobicity, particle size, and potential), and emulsifying properties of the oxidized WPI were analyzed.【Result】Oxidation induced by MDA, particularly at 5-20 mmol·L^-1, significantly reduced the solubility of WPI from 68.74% to 11.88%, but had no noticeable effect on the turbidity of protein solutions (the values for all samples maintained both at approximate 0.32). MDA had little effect on the total thiol group, disulfide bond, free amino group and carbonyl group at 0-1 mmol·L^-1, but significantly decreased the total thiol group and free amino group and increased disulfide bond and carbonyl content at higher concentrations. SDS-PAGE results confirmed that MDA with a concentration above 5 mmol·L^-1promoted the formation of intra/inter-molecular disulfide bonds and other covalent bonds between the walnut proteins. MDA did not alter the secondary structure of WPI at below 0.1 mmol·L^-1, but significantly reduced the content of α-helix, β-sheet and β-turn and increased the content of random coil at above 1 mmol·L^-1. The decline in fluorescence intensity of WPI with the increase of MDA concentration was parallel to the disordered change in protein secondary structure, especially for MDA concentrations above 10 mmol·L^-1. Oxidation by <1 mmol·L^-1 MDA had no remarkable effect on the reduction, but >1 mmol·L^-1 MDA significantly reduced protein hydrophobicity to as little as 1/10 of the control. MDA at less 1 mmol·L^-1 had no impact on the protein particle sizes and their change, whereas MDA at above 1 mmol·L^-1 significantly increased protein particle size and reached a maximum particle size of approximate 1 160 nm at 10 mmol·L^-1 MDA. Meanwhile, the oxidation significantly reduced the charge of the protein with the increase of MDA concentration. Oxidation by 0.1 mmol·L^-1 MDA significantly reduced protein emulsifying activity, while oxidation by 1 mmol·L^-1 MDA significantly reduced emulsifying stability. The maximum loss of 2/3 of the total emulsifying index was observed when the MDA concentration reached 20 mmol·L^-1. 【Conclusion】As the degree of oxidation increased in the oxidation system, MDA as byproducts derived from lipid peroxidation, significantly modified the protein structure (including residue groups), promoted protein cross-linking and formation of large aggregates, and changed the physical and chemical properties of WPI. This resulted in a significant loss of the emulsifying properties of WPI.

Key words: walnut isolate protein, malondialdehyde, oxidation, protein structure, emulsifying properties