Fabrication Mechanism And Structural Characteristics Of The Ternary Aggregates By Lactoferrin, EGCG And Pectin

Polyphenol may interact with protein and polysaccharide, respectively, reversibly by usually non-covalent forces such as hydrogen bonding, hydrophobic bonding and van der Waals forces involved. The ternary complexes containing protein, polyphenol and pectin can be applied in many fields, such as food, pharmaceutical and cosmetic industries.The molecular interaction between bovine lactoferrin (LF) and (-)-epigallocatechin-3-gallate (EGCG) were investigated in aqueous solution at pH6.0. The presence of EGCG did not change the size and turbidity of LF-EGCG complex in aqueous solution until the LF/EGCG molar ratio was over a critical value of1:25. The fluorescence spectra revealed that both tryptophanyl and tyrosyl groups of LF were associated with the interaction with EGCG. The infrared spectra of freeze-dried LF-EGCG complexes showed that they were different from those of LF and EGCG alone, FTIR and far-UV CD results indicated that EGCG induced a progressive increase in the proportion of a-helix structure at the cost of/β-sheet structure of LF. The near-UV CD data testified that LF tertiary conformation was altered in the presence of EGCG. Isothermal titration calorimetry (ITC) analysis implied that EGCG was spontaneously bound to LF by a two-stage mechanism, about31EGCG molecules were integrated with1molecule LF and hydrogen bonds were always involved in the assemble process.The interaction between native, thermally modified lactoferrin (LF) and (-)-epigallocatechin-3-gallate (EGCG) at pH3.5,5.0and6.5was investigated. Turbidity, particle size and charge of LF-EGCG complexes were mainly dominated by pH value and secondary structure of protein. At pH3.5and5.0, LF-EGCG complexes were nano-particles which had the high ζ-potential, the small size and soluble state. At pH6.5, they were sub-micrometer particles which had the low ζ-potential, the large size and insoluble state. The infrared spectra of freeze-dried LF-EGCG complexes showed that they were different from those of LF and EGCG alone. Far-UV CD results indicated that the heat treatment might irreversibly alter the secondary structure of LF and EGCG induced a progressive increase in the proportion of a-helix structure at the cost of β-sheet and unordered coil one of LF at pH3.5,5.0and6.5. EGCG exhibited strong affinity for native LF but weak one for thermally modified LF at pH5.0and6.5. An opposite result was observed at pH3.5.The aggregation of lactoferrin and (-)-epigallocatechin gallate (EGCG) was inhibited by polyphenols, oligosaccharides and collagen peptide in this study. Polyphenols, oligosaccharides or collagen peptide could effectively prevent the formation of lactoferrin-EGCG aggregates, respectively. The addition sequence of lactoferrin, polyphenols (oligosaccharides or collagen peptide) and EGCG could affect the turbidity and particle size of the ternary complexes in the buffer solution, however, it hardly impacted the ζ-potential and fluorescence characteristics. With either positive or negative charge, polyphenols and collagen peptide disrupted the formation of lactoferrin-EGCG aggregate mainly through the mechanism of its competition with EGCG molecules which were surrounded lactoferrin molecule surface with weaker binding affinities, forming polyphenols or collagen peptide-lactoferrin-EGCG ternary complex, neutral oligosaccharides, the ternary complex was generated mainly through steric effects, accompanied by the change of lactoferrin secondary structure induced by gallic acid, chlorogenic acid and xylo-oligosaccharide.The ternary aggregates were fabricated by lactoferrin (LF), pectin (high methylated pectin (HMP)/low methylated pectin (LMP)) and (-)-epigallocatechin gallate (EGCG) through three different fabrication methods at pH5.0. The turbidity, particle size and ζ-potential of ternary aggregates were influenced by the types of pectin, the concentration of EGCG and fabrication methods. The fluorescence intensity of LF was decreased with the increase of EGCG concentration for all ternary aggregates. Far-UV circular dichroism results indicated that EGCG could alter the secondary structure of LF with an increase in the proportion of β-sheet structure at the cost of unordered coil structure. According to Near-UV circular dichroism results, EGCG could also modulate the tertiary structure of LF at the presence of pectin. In addition, EGCG could increase the viscoelasticity of the ternary aggregates with HMP, leading to better stability of the ternary aggregates. An opposite result was observed for the ternary aggregates with LMP.The ternary complex fabricated by LF, EGCG and HMP could improve the stability of β-carotene emulsion, inhibit theβ-carotene degradation, and improve its chemical stability; spray dried ternary aggregate provide a better protection against β-carotene degradation compared with the ternary aggregate in solution.