Study On The Interaction Mechanism Between Epsilon-polylysine And Milk Proteins

ε-polylysine is a secondary metabolite derived from Streptomyces albulus,and as an antibacterial peptide is composed of 25-35 L-lysine.Due to its broad-spectrum antimicrobial efficacy and non-toxic property,EPL has been used as a safe and natural food preservative worldwide.In recent years,the complexes formed byε-polylysine and biological macromolecules have attracted the interest of many researchers due to their unique structure and properties.Most of the relevant research focused on the description of phase separation and the construction of nano-or micro-complexes,but the underpinning of the interaction is a lack of understanding.This dissertation expected to elucidate the interaction mechanism betweenε-polylysine and three milk proteins,β-casein,β-lactoglobulin and bovine serum albumin,respectively.Therefore,it elucidated the influence of the conformation of milk proteins on the interaction mechanism through multiscale characterizations and illustrated the discontinuous multiscale aggregation betweenε-polylysine and protein driven by non-covalent interaction.This dissertation paved the way to understand the complicated system formed byε-polylysine and proteins and to design the controllableε-polylysine/protein complexes.The details are as follows:Multiscale characterization of the phase transition ofε-polylysine/milk proteins systems.The influence of external and internal parameters,such as p H,ionic strength,and molar ratio,on the macroscopic phase transitions of theε-polylysine/milk protein systems have been investigated.Firstly,the macroscopic interaction behavior and intensity of theε-polylysine/milk protein systems were measured by using the turbidimetric contour maps.Secondly,the mesoscopic transition of theε-polylysine/milk protein systems induced by p H was monitored on the basis of hydrodynamic diameter(D_h)andζ-potential.Then the electrostatic potential contours of the milk proteins were adopted to visualize the heterogeneity of surface charge distribution of different milk proteins induced by p H and to explain the difference from their macroscopic transition on the molecular level.When equal molar or a large amount ofε-polylysine existed in the milk proteins solution,an“overcharge”effect was imposed byε-polylysine to reverse the net surface charge of the protein and prevent its self-aggregation near the isoelectric point.Meanwhile,the molecular weight and the surface charge anisotropy that determines the properties of the scale-spanning of the p H-inducedε-polylysine/proteins systems.Multiscale characterization ofε-polylysine/milk protein systems structure.Theε-polylysine/milk proteins systems,dissolving in 5 m M phosphate buffer solution(p H7.0),were separated into metastable mesophase and unstable precipitated phase through ultracentrifugation to characterize their hierarchical structure.The composition,morphology and structure of the mesophase dispersions and precipitates were analyzed by polyacrylamide gel electrophoresis(PAGE),liquid chromatography-mass spectrum(LC-MS),Dynamic light scattering(DLS),spectroscopy and microscopy.These results showed that the mesophases are all nano-scale compact spherical or ellipsoidal,with polydisperse characteristics,while the precipitated phases are all micro-scale amorphous structures with sponge-like porosity.In conclusion,the states of the protein multimers were dissociated byε-polylysine through non-covalent interactions and then formed the hierarchical systems with the scale-spanned from the complexes to the precipitates.The interaction mechanism betweenε-polylysine and milk protein.The driven force and the underpinning mechanism for the evolution of the scale-spannedε-polylysine/milk proteins system have been discussed through experiments and molecular dynamics simulations.The"nucleation-growth"theory was adopted to explain the kinetics of phase separation when the transition from the mesophase to the precipitated phase.Then the influence of the accessed-solvent on the proteins by adding either 1 M sodium chloride or 8 M urea have been investigated to illustrate the formation mechanism of the complex,mesophase and precipitated phase indirectly by using DLS.Moreover,the molecular dynamics simulations were employed to investigate the mechanism of the formation of the complex.These results showed thatε-polylysine adapted its conformation to the negative surface charge patches of milk proteins by electrostatic attraction.Furthermore,the hydrophobic interaction and intermolecular hydrogen bonding are the main forces that drive the formation of the mesophase and precipitated phase.In conclusion,the synergy and competition among the non-covalent forces with different length scales leads to the discontinuous and the properties of the scale-spannedε-polylysine/milk proteins systems.Evaluation of the affinity betweenε-polylysine and milk proteins.The affinity betweenε-polylysine and the three milk proteins mixed in equal quality were evaluated.PAGE and LC-MS were employed to monitor the quality of each protein during titration.Furthermore,we evaluated the affinity betweenε-polylysine and skim milk and two dairy ingredients(sodium caseinate and whey protein isolate).These results indicated thatε-polylysine have strong affinities with either casein micelles or caseinate,however,subtle selective with individual casein components were not observed.Meanwhile,a relative strong affinity betweenε-polylysine andβ-lactoglobulin than that of bovine serum albumin was observed without significant phase separation.We concluded that the hydrophobic patches of proteins determined their affinity toε-polylysine because of the distinctness of the hydrophobic terminal inβ-casein.Further,we proposed the electrostatically dynamic equilibrium whenε-polylysine exist in the heterogenetic proteins’solution,the magnitude of hydrophobic interaction drives the inversed phase separation corresponding to the energy minimization.The characteristics and applications of"solid-liquid"phase separation.The attribution of the molecular weight of casein hydrolysates in broths on the antibacterial efficacy of the multiscale of theε-polylysine/milk proteins systems have been studied.These results showed that the mesophase could maintain the antibacterial efficacy,but the phase separation impaired the antibacterial ofε-polylysine.Furthermore,this phenomenon has been explained by the irreversible competition theory and extended to the microorganisms subsequently.The compact complexes from theε-polylysine/sodium caseinate precipitated phase were prepared by ultracentrifugation.The compact complexes,which could be considered safe and non-toxic,have an excellent swelling property,plasticity,and porosity.The ability of compact complexes to absorb the hydrophobic compounds have been considered.These results indicated that the absorption of volatile compounds could preparation the template materials with different pore sizes and morphologies,and the absorption of nutritious compound,curcumin,which could be considered as the controlled-release matrix for these compounds.