Study On The Inducement,Design And Emulsification Properties Of Egg White Peptide Aggregation Behavior

This work was funded by the National Key R&D Program of China(2022YFD2101002).Bioactive peptide is an active ingredient with higher nutritional value.It has many physiological functions such as antioxidant,immune regulation,anti-inflammatory,anticancer,antibacterial,hypotensive,and so on.However,there is a common problem of low added value in the application of biological active peptides.Fully exploring the dual functional characteristics of bioactive peptides in nutrition and processing,and developing dual functional ingredients are conducive to improve the added value of bioactive peptide products and expand the application field of bioactive peptides.In this paper,egg white peptide(EWP)was used as a template,and proanthocyanidins(PC)were used to induce the formation of EWP aggregates and make them have good emulsification properties.Further,pH-treated was used to further obtain dual functional components with "antioxidant+emulsifying properties".During the study,it was found that the transformation of the β-sheet might be beneficial for improving emulsification properties.For this reason,the peptide sequences with βsheet tendency in EWP were screened to construct a "sequence-β-sheet-emulsification properties "relationship network.Finally,the self-assembly and emulsification mechanism of EWP based on β-sheet wereanalyed.The main research results are as follows:(1)PC induced EWP to form aggregates with a binding ratio of 1:2.The binding mode was that EWP combined with PC through hydrogen bond and hydrophobic interaction,and then EWP combined with EWP on PC outer layer via hydrogen bond.PC acted as a bridge to induce the aggregation of PC-EWP-EWP monomers to form EWP-PC aggregates.The addition of PC will made the secondary structure of EWP change from β-turn to β-sheet.Compared with EWP,the formation of aggregates could significantly improve the interfacial properties and emulsifying properties.Among them,the surface tension of aggregates formed in 1:4 ratio decreased by 5%,and the three-phase contact angle increased by 53%.In addition,the addition of PC made the EWP-PC emulsion droplets disperse evenly,increased the viscosity of emulsion.Therefore,EWP-PC emulsion showed superior storage stability and physical stability.(2)The functional properties of EWP-PC aggregates were further improved by pH-treated.pH did not change the binding mechanism of EWP and PC,but changed the binding strength,structure of aggregate and main driving force between EWP and PC.Specifically,the binding mode between EWP and PC was always static quenching,but the binding strength increased with the increase of pH value.Due to the change of surface charge,with the increase of pH value,the content of β-sheet increased gradually.Under acidic conditions,hydrogen bond was the main driving force between PC and EWP.Under neutral and alkaline conditions,the balance of electrostatic repulsion and hydrophobic interaction was the main way to form aggregates.In addition,EWP-PC-9 has the largest antioxidant activity in vitro,anti-hydrogen peroxide damage ability and emulsifying activity index(EAI)/emulsifying stability index(ESI),which indicated the potential of EWP-PC-9 as a dual-functional component.(3)The 4 peptide sequences that could form β-sheet structure were obtained in EWP(<1 kDa),namely ESIINF(EF-6),VPIEGII(VI-7),YLNKCIKIN(YN-9)and MIIHDRREF(MF-9).When the concentration was higher than critical aggregation concentration,EF-6 showed antiparallelism β-sheet and formed nanotube structure.Ⅵ7,YN-9 and MF-9 were also formed β-sheet structure,but the micromorphology appeared as short rod or fine fiber structure.The 4 peptides showed good emulsifying performance,of which EF-6 had the highest EAI and MF-9 had the most significant ESI.Correlation analysis showed that the amino acid composition of the peptide sequence mainly affected the emulsification performance of the peptide compared with other factors.Hydrophobic amino acids were beneficial to the improvement of EAI,and charged amino acids were beneficial to the improvement of ESI.On the premise of similar amino acid properties,the formation of β-sheet is conducive to the improvement of the emulsification performance of peptides,especially the improvement of EAI.(4)EF-6 with the strongest β-sheet structure was used as the research object,and was used to further analysis the self-assembly mechanism and emulsification mechanism of peptide based on β-sheet.Phenylalanine and glutamic acid were two key amino acids in the self-assembly process of EF-6.The aromatic side chain of phenylalanine could improve the stability of β-sheet structure and promote the formation of nanofiber structure.As an acidic amino acid,glutamic acid realized the pH response of EF-6.EF-6 formed an antiparallel β-sheet structure at pH 3-4 and transformed into a twisted antiparallel β-sheet structure at pH 2 and pH 5-8.The mutual transformation of electrostatic repulsion and π-π stacking of phenylalanine residues has an important influence on the transformation of β-sheet structure.β-sheet structure played an important role in improving emulsification performance.Nanofibers formed a film at the oil-water interface to capture the suspended oil droplets in the high-viscosity peptide solution,which improve the emulsion stability.EF-6 has a 20%oil-phase loading capacity at a concentration of 2 mM.Compared with individual EWP(<1 kDa)and EWP-PC aggregates,EF-6 self-assembly based on β-sheet showed better emulsification capacity.